Lactic Acid Bacteria: Omics and Functional Evaluation [1st ed. 2019] 978-981-13-7831-7, 978-981-13-7832-4

Table of contents :
Front Matter ....Pages i-v
Introduction (Fengwei Tian)....Pages 1-33
Genetic Operation System of Lactic Acid Bacteria and Its Applications (Haiqin Chen, Chen Chen, Chunqing Ai, Chengcheng Ren, He Gao)....Pages 35-76
Comparative Genomic Analyses of Lactic Acid Bacteria (Wei Chen, Hongchao Wang)....Pages 77-95
Transcriptomics of Lactic Acid Bacteria (Zhennan Gu, Guozhong Zhao)....Pages 97-129
Proteomics of Lactic Acid Bacteria (Yue Xiao, Yanjun Tong, Wei Chen)....Pages 131-165
Metabolomics of Lactic Acid Bacteria (Wanqiang Wu, Nan Zhao)....Pages 167-182
Functional Evaluation Model for Lactic Acid Bacteria (Qixiao Zhai, Wei Chen)....Pages 183-237
Lactic Acid Bacteria and Gut Health (Haitao Li, Zhifeng Fang)....Pages 239-260
Lactic Acid Bacteria and Host Immunity (Linlin Wang, Zhao He, Peijun Tian, Gang Wang)....Pages 261-296
Commercial Strains of Lactic Acid Bacteria with Health Benefits (Xin Tang, Jichun Zhao)....Pages 297-369
Safety Evaluation of Lactic Acid Bacteria (Wei Chen, Leilei Yu, Ying Shi)....Pages 371-409

Citation preview

Wei Chen Editor

Lactic Acid Bacteria Omics and Functional Evaluation

Lactic Acid Bacteria

Wei Chen Editor

Lactic Acid Bacteria Omics and Functional Evaluation

Editor Wei Chen Jiangnan University Wuxi, China

The print edition is not for sale in The Mainland of China. Customers from The Mainland of China please order the print book from: Science Press. ISBN 978-981-13-7831-7    ISBN 978-981-13-7832-4 (eBook) https://doi.org/10.1007/978-981-13-7832-4 © Springer Nature Singapore Pte Ltd. and Science Press 2019 This work is subject to copyright. All rights are reserved by the Publishers, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publishers, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publishers nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publishers remain neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Contents

1 Introduction����������������������������������������������������������������������������������������������    1 Fengwei Tian 2 Genetic Operation System of Lactic Acid Bacteria and Its Applications ��������������������������������������������������������������������������������   35 Haiqin Chen, Chen Chen, Chunqing Ai, Chengcheng Ren, and He Gao 3 Comparative Genomic Analyses of Lactic Acid Bacteria��������������������   77 Wei Chen and Hongchao Wang 4 Transcriptomics of Lactic Acid Bacteria ����������������������������������������������   97 Zhennan Gu and Guozhong Zhao 5 Proteomics of Lactic Acid Bacteria��������������������������������������������������������  131 Yue Xiao, Yanjun Tong, and Wei Chen 6 Metabolomics of Lactic Acid Bacteria ��������������������������������������������������  167 Wanqiang Wu and Nan Zhao 7 Functional Evaluation Model for Lactic Acid Bacteria������������������������  183 Qixiao Zhai and Wei Chen 8 Lactic Acid Bacteria and Gut Health ����������������������������������������������������  239 Haitao Li and Zhifeng Fang 9 Lactic Acid Bacteria and Host Immunity����������������������������������������������  261 Linlin Wang, Zhao He, Peijun Tian, and Gang Wang 10 Commercial Strains of Lactic Acid Bacteria with Health Benefits��������������������������������������������������������������������������������  297 Xin Tang and Jichun Zhao 11 Safety Evaluation of Lactic Acid Bacteria ��������������������������������������������  371 Wei Chen, Leilei Yu, and Ying Shi

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Chapter 1

Introduction Fengwei Tian

1.1  S  ection I Overview and Definition of Lactic Acid Bacteria 1.1.1  Overview of Lactic Acid Bacteria Lactic acid bacteria (LAB) is a general term for a class of bacteria that use the metabolism of carbohydrates in the external environment to produce lactic acid. Lactic acid bacteria are widely distributed in nature and exist in a variety of habitats. Extremely rich in biodiversity, they are closely related to human production and life and have important social and economic value. They are valuable biological resources for human beings. The utilization of lactic acid bacteria by human beings has a very long history. According to reliable archaeological evidence, it can be dated back to more than 10,000 years ago. In the long historical process, the utilization of lactic acid bacteria has made outstanding contributions to the development and practice of human society. In the great development of human natural science and engineering science in the late nineteenth century and early twentieth century, along with the rapid development of biological science, microbiology, and other related disciplines, the science and technology of lactic acid bacteria also achieved unprecedented development. On the one hand, in terms of basic science, lactic acid fermentation is adopted as a model metabolic method. Lactic acid bacteria are mode research organisms of basic biological sciences such as microbiology, biochemistry, genetics, and molecular biology, which have important theoretical research significance. On the other hand, in practical application, lactic acid bacteria have a wide range of application significance in industrial biomanufacturing, food production and processing, high-efficiency agriculture, high-efficiency livestock breeding, F. Tian (*) Jiangnan University, Wuxi, China e-mail: [email protected] © Springer Nature Singapore Pte Ltd. and Science Press 2019 W. Chen (ed.), Lactic Acid Bacteria, https://doi.org/10.1007/978-981-13-7832-4_1

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Fig. 1.1  Number of incomplete statistics for science and technology of lactic acid bacteria related research publications

medical health, and other important fields closely related to human beings. In recent years, the application of new technologies and new methods such as microbiome, proteomics, bioinformatics, and big data analysis technology has further revealed the great importance of lactic acid bacteria in related fields and attracted the researchers from different research fields. In Fig. 1.1, the number of publications on the research of lactic acid bacteria science and technology has grown significantly during the last 10 years of the twentieth century to the first 10 years of the twenty-­ first century. It has become a major research and application development hotspot of modern science and application technology.

1.1.2  The Definition and Membership of Lactic Acid Bacteria Lactic acid bacteria are an important class of prokaryotic microorganisms. However, lactic acid bacteria do not have a clear definition, nor is it a taxonomic term of microbiology and bacteriology. Different researchers have different understandings and definitions of lactic acid bacteria and their range in different development periods. In the early twentieth century, when the lactic acid bacteria science and technology system began to form, the lactic acid bacteria mainly refer to the bacteria that can acidify the milk (milk-souring bacteria) (Beijerinck 1901; Orla-Jensen 1919). Later, different researchers successively proposed different definitions and

1 Introduction

3

descriptions of lactobacillus from the aspects of cell morphology characteristics and physiological metabolic reactions. For example, Kandler (1983) believed that Lactobacillus was “a kind of gram-positive, non-spore-producing, micro-aerobic bacteria whose main products of carbohydrate fermentation was lactic acid.” Wood and Holzapfel (1995) defined Lactobacillus as “a kind of bacterial group that metabolizes carbohydrates and takes lactic acid as the only or main metabolite.” Carr et al. (2002) considered that lactic acid bacteria are a class of heterogeneous bacteria that are Gram-positive, do not form spores, are negatively contacted with enzymes, and are metabolites of lactic acid. Axelsson et al. (2004) considered that lactic acid bacteria are a type of bacillus and cocci which are Gram-positive, do not form spores, do not breathe, and ferment carbohydrates with lactic acid as the main product. It can be seen from these definitions that lactic acid bacteria do not have a clear definition and boundary but are a general term for a class of bacteria with similar physiological characteristics. Many definitions suggest that lactic acid bacteria do not form spores; however, some spore-forming bacteria such as Sporolactobacillus and Bacillus bacteria can also form lactic acid by homolactic fermentation. Therefore, we believe that the production of lactic acid by metabolism is the most important core metabolic characteristic of lactic acid bacteria. Lactic acid bacteria are a general term for a variety of heterogeneous bacteria with different taxonomic status and morphological physiology, which are positive in Gram staining, generally do not produce spores, usually do not produce energy by breathing, and mainly metabolize carbohydrates to produce lactic acid. At the beginning of the research of lactic acid bacteria, mainly Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus bacteria formed the core of lactic acid bacteria, and later with the discovery of more lactic acid bacteria and the evolution and correction of bacterial classification, the current lactic acid bacteria have already included about 41 genera (Bergey’s Manual Trust 2015). Among them, lactic acid bacteria with important scientific and application value are mainly concentrated in Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, and Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weissella for 12 genera. Bifidobacterium adopts a bifidus metabolic pathway different from that of general lactic acid bacteria. Although it belongs to the Actinomycete class in microbial taxonomy, Bifidobacterium bacteria generates lactic acid and acetic acid through heterolactic fermentation. Therefore, relevant researchers traditionally believe that Bifidobacterium belongs to lactic acid bacteria. In addition, there are some human and animal pathogenic bacteria, such as Listeria bacteria, which are in line with the description of the characteristics of lactic acid bacteria and also belong to the generalized member of lactic acid bacteria family. The species involved in lactic acid bacteria are described in Chap. 2 of this book.

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1.2  S  ection II Development History and Applications of the Science and Technology of Lactic Acid Bacteria 1.2.1  S  tage Division of Development Course and Applications of the Science and Technology of Lactic Acid Bacteria In the long history of human civilization, lactic acid bacteria have been widely used for a long time. However, until modern times, with the development of science and technology systems, the development of science and technology systems of lactic acid bacteria has been formed and developed. The development course of scientific research and technical application of lactic acid bacteria can be divided into five stages: The first stage, the original stage of lactic acid bacteria development (from prehistoric period to 1857 in which Pasteur discovered lactic acid bacteria and associated it with lactic acid fermentation); the second stage, the initial stage of the formation of lactic acid bacteria science and technology application system (from 1857 to 1919 in which Orla-Jensen established lactic acid bacteria system in 1919); the third stage, the formal formation and development stage of the lactic acid bacteria science and technology application system (from 1919 to 1960); the fourth stage, the mature stage of the lactic acid bacteria science and technology application system (from 1960 to 2001 in which the sequencing of the strain of lactic acid bacteria is completed ); and the fifth stage, the gradual development stage of the lactic acid bacteria science and technology application system (the whole genome sequencing of the first strain of lactic acid bacteria since 2001). Figure 1.2 shows some milestones in the history of lactic acid bacteria science and technology. The important representative events in the development of lactic acid bacteria research and technology application are shown in Table 1.1.

1.2.2  D  evelopment Course of the Practical Application of Lactic Acid Bacteria The practical application of lactic acid bacteria can also be divided into two stages: the first stage is the unconscious use of lactic acid bacteria derived from empirical knowledge; the second stage is the practical application of lactic acid bacteria based on science.

Fig. 1.2  Important events in the development of science and technology of lactic acid bacteria.

1 Introduction 5

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Table 1.1  Development history of practice application and technology research of lactic acid bacteria Time 8000 BC to 5000 BC 6000 BC to 4000 BC 5000 BC to 3000 BC 3000 BC 2000 BC to 1300 BC 1000 BC 76 BC 540 in Anno Domini AD 800 AD 1000 AD 1145 1719 1780

1847

1857

1873

1880 1881

1884

Events and comments North African residents began to consume natural acidified fermented milk and cheese and other dairy products (Dunne et al. 2012) Ancient India mastered the production process of yogurt (dahi), cheese, and sour cream Ancient Egyptians used pottery to make natural yogurt and mastered the production process of different series of fermented dairy products (Farnworth 2008a) Thracians made yogurt products by natural fermentation of goat milk (prokish or kvasenomliako) (Chomakow 1973; Tamang and Kailasapathy 2010) Hellenes and Chaldaic began to make cheese, and natural fermented sour cream appeared in Mesopotamia Chinese people started making kimchi (salted fermented vegetables) (Chen Gong 2011) In ancient Rome, Pliny described a method for making white cabbage into sour kimchi using a ceramic container (McElhatton and Marshall 2007) The book of Qimin Yaoshu recorded the production method of yogurt and soaked vegetables (Qimin Yaoshu, volume ninth, li lao eighty-fifth) Turks started making yogurt In the mountains of North Caucasus, goat milk was placed in the sheepskin sac and naturally fermented to form kefir fermented milk The prehistory books such as Samkuksaki recorded that the production of kimchi in the Korean peninsula was made by fermenting vegetables in stone containers Levinhoek discovered microbes found in many natural samples, including feces and oral food debris Carl Wilhelm Scheele, Swedish medical chemist, first isolated and identified lactic acid (acid of milk) from sour milk and described its chemical properties (Scheele 1780) C. Blondeau determined that lactic acid was a product of the fermentation process of a microorganism (then considered to be a certain fungus Penicillium) (Blondeau 1847) French scientist Pasteur discovered lactic acid bacteria (then called yeast lactis) under the microscope for the first time in the study of rancidity in wine drinks, linking lactic acid production to microorganisms (Pasteur 1857) For the first time, a series of dilution methods were used to separate the pure culture strain of lactic acid bacteria, Streptococcus lactis, named Lactococcus lactis, from lactated milk (Lister 1873) Danish Chr. D. A. Hansen began to produce commercial lactic acid bacteria starter French scientist Fremy produced lactic acid by fermentation. In 1881, the first industrial fermentation of lactic acid was carried out in the United States (Ghaffar et al. 2014) Hueppe used Bacterium acidilactici to name the bacteria that made the milk sour, and for the first time, the name “yogurt bacteria” was named “lactic acid bacteria” (Milth 1884) (continued)

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Table 1.1 (continued) Time 1885

1892

1899

1895–1910

1900 1901

1901 1905

1905

1911

1915

1919

1917 1919

1920

Events and comments Cantani A G attempted to use lactic acid bacteria to produce antagonistic substances for bacterial biotherapy against Mycobacterium tuberculosis (Cantani 1885; Florey 1945) German doctor Albert Döderlein first described a bacterium (Doderlein bacillus) derived from vaginal secretions of healthy pregnant women, which antagonizes staphylococci by producing lactic acid (Lash and Kaplan 1926) Henry Tissier, a doctor from the Pasteur Institute, isolated a Bacillus bifidus from breast-fed healthy baby feces and supplemented this food with a Mycobacterium to treat intestinal flora imbalance (Tissier 1906) Different Lactobacillus members such as Lactobacillus delbrueckii, Lactobacillus casei, Streptococcus, and Bifidobacterium were isolated and discovered successively Ernst Moro, an Austrian pediatrician, discovered Bacillus acidophilus n. spec., which is now Lactobacillus acidophilus (Moro 1900) The Dutchman Martinus Willem Beijerinck promoted the understanding of the production of lactic acid bacteria in kefir and yogurt and proposed the term Lactobacillus (genus Lactobacillus Beijerinck 1901) as the official name of lactic acid bacteria (van Iterson et al. 2013) Cahn (1901) studied bacilli in infant feces and studied the ecology of infant feces Stamen Grigoroff, a physiologist and microbiologist from Bulgaria, isolated a Bacillus bulgaricus from yogurt at the age of 27, namely, Bacillus bulgaricus (Grigoroff 1905) Pasteur Institute’s Metchnikoff published the Longevity: An Optimistic Study based on his observations of diet and health during his travels in Bulgaria, proposing the hypothesis that the intake of fermented yogurt contributes to health and longevity (Metchnikoff and Mitchell 1908) Loudon M. Douglas published the book The Bacillus of Long Life, which further discussed the role of bacilli in yogurt and yogurt in health and longevity (Douglas 1911) Daviel Newman first used intravesical infusion of lactobacillus culture to reduce the probability of urinary tract infection in women with cystitis, laying the foundation for the clinical application of lactic acid bacteria (Newman 1915) The Danish bacteriologist Orla-Jensen firstly classified lactic acid bacteria based on culture and physiological and biochemical characteristics systematically. For the first time, he wrote a monograph on lactic acid bacteria, marking the initial formation of lactic acid bacteria science and technology system (Orla-Jensen 1919) Lactobacillus drug lactasin (lactasin, “Biofermin”) named active Lactobacillus powder preparation (including Enterococcus faecalis) put on the market as a drug Dr. Isaac Carasso used strains introduced in the Balkans and strains isolated in the Mei Laboratory as a starter to produce yogurt in Barcelona and sold it through pharmacies Yale University’s Rettger et al. proved that B. bulgaricus, which is derived from yogurt, cannot survive in the gut of model animals and humans, thus questioning Michnikov’s hypothesis and promoting further exploration of lactic acid bacteria and health (continued)

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Table 1.1 (continued) Time 1921~1922

1928

1930~1935

1935

1942

1940~1970

1945

1970

1953–2001 1962

1970

1974

1983

1984 1987

Events and comments Rettger et al. reported the clinical efficacy of Lactobacillus acidophilus-­ fermented yogurt, especially for digestion (Cheplin and Rettger 1920; Rettger and Cheplin 1921) Roger and Whittler of the US Department of Agriculture’s Dairy Industry Bureau firstly reported Lactococcus lactis-produced antibacterial peptide, nisin (Rogers and Whittier 1928) Minoru Shirota (Da Tianyu) isolated a Lactobacillus casei Shirota and in 1935 realized the commercial production of Lactobacillus casei-fermented lactic acid bacteria beverage Eggerth and Gagnon used anaerobic separation techniques to isolate intestinal anaerobic bacteria such as Bacteroides and Eubacterium (Eggerth and Gagnon 1933; Eggerth 1935) Five strains of lactic acid bacteria were isolated from sour milk by Tang Tenghan et al., and one strain with high lactic acid production ability was obtained. The acid production rate reached 88.44%, and the industrial production of lactic acid products was realized Common media for lactic acid bacteria culture, such as MRS medium, SL medium, M17 medium, and BL medium, were developed successively, which promoted the development and research of lactic acid bacteria culture technology The development of antibiotic application, aseptic animal model technology, and anaerobic microbial culture technology has promoted the development of lactic acid bacteria and intestinal microecology Tomotari Mitsuoka began the study of intestinal microflora, established the classical analytical method of intestinal microflora, and made a systematic analysis of intestinal microflora The concept and related products of probiotics represented by lactic acid bacteria were gradually formed, developed and maturity. Three glycopeptides with anticancer activity were isolated from the cell wall of Lactobacillus bulgaricus. The antitumor effect of lactic acid bacteria was reported for the first time. The research on the function of lactic acid bacteria is deeper Woese established a bacterial identification method based on 16S rRNA oligonucleotide sequence analysis and constructed a life tree of phylogenetic of prokaryotic microorganisms and promoted the development of bacterial taxonomy CHR-HANSEN Company introduced direct vat set starter to the market (DVS® cultures or Direct Vat Set), significantly improving the production conditions of fermented dairy products LGG (Lactobacillus rhamnosus) isolated from healthy human has the characteristics of strong activity and gastric acid resistance and can be colonized in the intestines for up to 2 weeks (Gorbach and Goldin 1989) CHR-HANSEN Company introduced probiotics of Bifidobacterium BB-12 Zhang Hao and Xu Benfa started work on the development and application of bifidobacteria in China (continued)

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Table 1.1 (continued) Time 1989

1999

2001

2001

2001

2002 2003

2003

2008

Events and comments In 1989, the US Food and Drug Administration (FDA) and the Association of American Feed Control Officials (AAFCO) published a list of 42 microbial strains that can be directly fed and generally considered safe, involving 30 species of lactic acid bacteria Document No. 105 issued by the Ministry of Agriculture of the People’s Republic of China, Catalogue of Allowable Feed Additives, covered 12 kinds of microbial additives, of which 7 are lactic acid bacteria The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) have introduced the definition of probiotics, “a type of living microorganism that has a beneficial effect on the host after sufficient intake” The Ministry of Health of the People’s Republic of China promulgated the “Regulations on the Evaluation of Probiotics for Health Foods” and published the “List of Probiotics for Health Foods” (Wei Fa Jian Fa [2001] No. 84), giving the official definition of probiotics in China: “Microecological preparations that promote the ecological balance of the gut microbiota and have a beneficial effect on the human body” France’s Alexander Bolotin et al. published the complete genome-wide sequence of the first strain of lactic acid bacteria (Lactococcus lactis ssp. lactis IL1403), marking the study of lactic acid bacteria into the era of omics In the Cities of London, Ontario, and Canada, the FAO/WHO Joint Expert Working Group drafted Guidelines for the Evaluation of Probiotic in Food Document No. 84 of the Ministry of Health of the People’s Republic of China approved Lactobacillus reuteri as a probiotic strain that can be used in health foods, making the number of probiotic bacteria increase to ten species for health food Notice 318 of the Ministry of Agriculture of the People’s Republic of China announced that 14 kinds of microorganisms are allowed to be used in feed, of which 10 are lactic acid bacteria The World Gastroenterology Organisation (WGO) identifies the specific and possible functions of lactic acid bacteria

1.2.2.1  U  nconscious Use of Lactic Acid Bacteria Derived from Empirical Knowledge In the practice of human society, lactic acid bacteria are mainly used in the production of food and their products for disease prevention and treatment. The traditional fermented foods with lactic acid fermentation as the main features include various types of fermented dairy products, fermented cereals, and fermented fruit and vegetable products. These fermented foods appear in the historical development of ancient civilizations in all different regions, reflecting an important role of lactic acid bacteria in the practice of human society and economic development.

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 ifferent Types of Lactic Acid Bacteria-Fermented Dairy Products Are D Important Practical Applications of Lactic Acid Bacteria Fermented dairy products made by fermentation of lactic acid bacteria have a long history. As early as prehistoric times, more than 8000 years ago, residents living in the Sahara area of North Africa began to raise animals such as cows and sheep and began to consume dairy products such as milk, naturally acidified fermented milk, and cheese (Dunne et al. 2012). It is the earliest record of human use of lactic acid bacteria supported by archaeological evidence. In ancient India, from 6000 to 4000 years ago, humans mastered the production process of yogurt, cheese, and sour cream and produced lactic acid bacteria-fermented dairy products such as dahi, chakka, and cheese. These fermented dairy products are widely recorded in Buddhist works and Sanskrit literature. For example, as the Great Nirvana Sutra records, “best Tihu (the scream part of the dairy products) makes best effect. If you can take it, all diseases will be removed, for every medicine contains in it.” “From the cow to get the milk, from the milk to make the cheese, from the cheese to make the raw cake, from the raw cake to make the boiled cake, from the boiled cake to get the “Tihu”, and Tihu is the best.” Nirvana Sutra, volume 14, records “just as we get cheese from milk and milk is thin while cheese is thick.” Besides, yogurt is listed among the “eight good things” in Tibetan Buddhism. Today, fermented dairy products are still the main food for people in these areas. Furthermore, the content of lactic acid bacteria-fermented yogurt is also recorded in the chapter of Genesis of the Holy Bible. The Jewish ancestor Patriarch Abraham treated three angels with yogurt and sweet milk and by doing so, they get longevity. In the 5000 BC to 3000 BC, the ancient Egyptians began to make natural yogurt with earthenware vessels and mastered the different varieties of production process of fermented dairy products including Zabadi/Zabady yogurt, Laban zeer/Khad cheese, and Kishk/Karish cheese. In 3000 BC, the Thracian people living in the current Bulgarian nomads found that goat’s milk poured on the skin often became sour under the effect of temperature and body temperature and formed a good odor and mouthfeel and poured the sour milk into the boiled milk to make a yogurt product (prokish or kvasenomliako), which later evolved into a modern yogurt product; in the ancient Thrace language, “yog” means thick, and “urt” means milk. Ancient Greece and Babylon began making cheese as early as 1550 BC. Cheese has become an important part of the diet of the people at that time. In 750 BC, ancient Rome began to make cheese. Around 1300 BC, fermented butter was produced in the Babylon region of Mesopotamia. Around 800 AD, Turkey began to produce yogurt. It is said that yogurt is a tribute to the ancient Islamic saints, so it is called yogurt. From a linguistic point of view, yogurt is imported into English from the Turkish (Turkey Dictionary) language. Around 1000 AD, people living in the mountains of the North Caucasus put goat’s milk in a sheepskin sac and formed a kefir after natural fermentation. Kefir is an alcoholic fermented milk by yeast and lactic acid bacteria. The product is considered to be a magical food given to people by the Prophet Muhammad of Islam.

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There are also many records of lactic acid bacteria-fermented dairy products in China. Qimin Yaoshu is a book written by Jia Sixie, a famous agronomist in the Northern Wei Dynasty. (Qimin Yaoshu was finished around the middle of the sixth century.) In the Volume 6: 57th of sheep breeding mentioned the production method of lactic acid bacteria-fermented milk, “as for the methods of cheese, cattle and goat milk are both available, and they also can be mixed.” “Cow’s or goat’s milk is both available. The milk should be boiled for four or five times, and it should be filtered into a jar with a bag. The cheese is as warm as the temperature of the human body, and a half spoon of fragrant should be added to one liter of the boiled milk. Then stir vigorously to spread the added fragrant cheese in the boiled milk and to get the mature cheese the next day.” This is the earliest written record of using lactic acid bacteria to produce fermented dairy products in China. Later, the method of making fermented cheese was also briefly mentioned in the Compendium of Materia Medica.  ermentation of Fruits and Vegetables and Fermented Grains by Lactic Acid F Bacteria Is a Practical Application of Lactic Acid Bacteria Fermented dairy products are a typical example of human use of lactic acid bacteria. In addition, people have also explored the use of lactic acid bacteria to make other types of fermented foods, such as fermented vegetables and fermented grains. In China, as early as the third century BC, people began to unconsciously use lactic acid bacteria to make fermented vegetables. Chinese (salted) kimchi began more than 3000 years ago during the Shang and Zhou dynasties. “There are radishes and miscellaneous melons in the fields. Peeled the radish and marinated the melon, which is dedicated to the ancestor. The pickled melon is the Chinese sauerkraut.” Zhou Li Tian Guan records that “the next soup will not have five flavors, and salt dishes will be added, and a soup is a soup made with meat or pickles.” Later, the fermented vegetable production process originated in China was introduced to other parts of Asia and even Europe with Genghis Khan’s expedition in Eurasia. The ancient Roman historian and naturalist Pliny first described the use of ceramic containers and advocated a method of making white cabbage into sour kimchi and promoting the use of fermented dairy products to treat gastroenteritis in his book Natural History (McElhatton and Marshall 2007). In 1145, in the Korean historical work Samkuksaki, it was mentioned that the Korean peninsula had begun to ferment vegetables with stone containers to make kimchi. In addition to fruits and vegetables, humans also unconsciously use lactic acid bacteria for the production of grain fermented foods. As early as 7000 BC, cereal crops such as wheat and barley were used as human staples. Human beings have also begun to unconsciously use the life metabolic activities of microorganisms such as ethanol fermentation of yeast, lactic acid fermentation of lactic acid bacteria, and acetic acid fermentation of acetic acid bacteria to produce all kinds of

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fermented cereals, including sour bread, beer, steamed bread, and so on. Around 3000 BC, the ancient Egyptians discovered that after the dough was put on for a period of time, it would inflate and form a wine and sour taste. After roasting, a soft and delicious fermented wheat food was obtained. Therefore, the processing technology of bread was grasped. In China, the important fermented cereal food involved in the action of lactic acid bacteria is steamed buns. The history of steamed buns can be traced back to at least the Warring States Period. Shi wu gan zhu records “The king of qin made steamed cakes”; Xiao Zixian recorded in the qi Shu the use of “mianqi cakes” at the time of Temple sacrifices. “Mianqi cakes” can be seen as the prototype of steamed buns. In Africa, the working people use lactic acid bacteria to ferment sour porridge, which can be used as a weaning food for young children. It is still widely eaten in some countries in Africa today.  ong-Term Practical Application Makes People Realize that Lactic Acid L Bacteria-Fermented Products Have Good Disease Prevention and Treatment Effects In the long-term production practice, people have gradually realized that lactic acid bacteria-fermented products have good disease prevention and treatment effects. In the first century AD, the ancient Roman historian and naturalist Pliny advocated the use of fermented dairy products to treat gastroenteritis and related diseases (Natural History). In the fifteenth century, the first record of European functions related to fermented dairy products originated from France: Francis the First suffered from a serious gastrointestinal disease (probably is dysentery); when the French doctors were helpless, the king of the king, Suleiman the First, sent a doctor who used fermented goat milk to cure his disease. In China, there are also some records of using lactic acid bacteria fermentation products for disease treatment. In the fourth volume of The Secret History of the Mongols, sour milk for the treatment of Genghis Khan’s injury is mentioned: “Genghis Khan’s neck was injured. He felt so thirsty but he couldn’t find horse milk in the carriage except a bucket of cheese. He ordered others to find water to dilute the cheese and then drank the dilute cheese for three times. After it Genghis Khan said, ‘I feel better, and my life has been saved’” (Zhang Heping 1994). Li Shizhen once mentioned in his Compendium of Materia Medica, “Cheese, can detoxify, quench thirst, remove the heat in the chest, treat heat sores and muscle sores on the body, stop the thirst and heat, and can be laxative, enhance the color and spirit” (Heping Zhang 1994). 1.2.2.2  Practical Application of Lactic Acid Bacteria Based on Science With the development of microbiology represented by the research of Pasteur and Koch, the application of lactic acid bacteria in social practice has also been greatly developed, thus entering the second stage of its development. Lactic acid bacteria from different species are widely used in industrial production practices such as

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food, light industry, medicine, and feed, that is, conscious and rational application based on the science of lactic acid bacteria. The representative events at this stage are as follows.  ommercial Production of Yogurt and Other Lactic Acid Bacteria-Fermented C Foods In 1919, in view of the digestive and intestinal diseases of young children at that time, inspired by the works of Mechnikov, Isaac Carasso (1874–1939), a Spanish Jewish businessman and doctor, used strains introduced in the Balkans and isolated in the Mei Laboratory as a starter to produce yogurt products and sold in pharmacies to help treating children diarrhea. The Danone yogurt business is the predecessor of Danone Group, which now is the world’s largest dairy producer. After Koch, with the understanding of microbial pure culture and the application of related technologies, around 1890, Germany, the United States, and Denmark began to use lactic acid bacteria starter culture for the industrial production of yogurt and cheese, which greatly improved lactic acid bacteria fermentation products like the yogurt and cheese production. Besides, it is also one of the important events in the history of modern biological industrial technology, laying the basis for the development of the industrial microbiology and modern biological engineering technology. Entering the 1980s, with the application of the freeze-drying technology in the preparation of lactic acid bacteria starter, a highly active directed vat set lactic acid bacteria starter appeared, which avoided the cumbersome preparation process of the starter and greatly promoted the development of lactic acid bacteria-related products. Application of Lactic Acid Bacteria Fermentation Products In addition to the lactic acid bacteria starter, useful metabolites of lactic acid bacteria have also been developed. As early as 1891, the United States began industrial production of lactic acid. In 1895, Germany’s Boehringer Ingelheim succeeded in industrializing the production of lactic acid by fermentation. In 1901, in Japan, Lactobacillus delbrueckii was inoculated with sweet potato as a raw material to produce lactic acid by industrial fermentation of lactic acid bacteria. In addition, another important event in the development and utilization of useful metabolites of lactic acid bacteria is the commercial production of nisin, a biological preservative for foods using lactic acid bacteria. Nisin is a polypeptide-type bacteriocin produced by Lactococcus lactis and has a broad and good inhibitory effect on Gram-­ positive bacteria. It was commercialized by the British company Aplin & Barrett in 1959 (Hall 1963) and was approved by FAO/WHO as a food additive in 1961. A high-yield mutant strain of nisin was also selected from the Institute of Microbiology of the Chinese Academy of Sciences. In 1994, it was commercialized at Zhejiang Yinxiang Bioengineering Co., Ltd.

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Drugs and Medical Applications of Lactic Acid Bacteria In the medical application of lactic acid bacteria, Cantani (1885) proposed microbial-­ based bacterial therapy based on the observed in vivo antagonism. In 1915, Daviel Newman used lactobacillus to treat bladder infection for the first time because of its anti-infection ability based on lactic acid produced by lactobacillus, laying a foundation for its clinical application in medicine. As early as 1917, Takeda Pharmaceutical Co., Ltd. of Japan began to use Enterococcus faecalis to produce lactobacillus preparations for treatment of gastrointestinal diseases. China began to use fecal bacillus to produce lactobacillus preparations such as lactase in the 1950s. In addition, there are drugs such as Lactobacillus Lactobacillin produced by Lactobacillus acidophilus. In addition to treatment for digestive diseases, there are also some lactic acid bacteria preparations for the treatment of reproductive tract infections in women.

1.2.3  T  he Development of Scientific Research on Lactic Acid Bacteria Although lactic acid bacteria have been widely used in human society practice, the scientific and technological research on lactic acid bacteria has been developed systematically with the development of modern science. Several important nodes and landmark events in the development of lactic acid bacteria research are shown in Table 1.1. The following will focus on the history of important events in the development of lactic acid bacteria science and technology. 1.2.3.1  Pasteur’s Contribution to Lactic Acid Bacteria The founder of modern microbiology who has made great contributions to the development of lactic acid bacteria and the development of microbiology is the French chemist and microbiologist Louis Pasteur (1822–1895). Prior to Pasteur, the Dutch amateur scientist Antonie van Leeuwenhoek, who liked to sample and observe, used a self-­made microscope to observe a variety of samples, such as feces and food residue attached to the teeth, and found that they existed in different environments. The kind of microbes (micro-movements) opened the door to the world of wonderful microscopic organisms but did not specify the role of microorganisms; Sweden’s Scheler discovered the lactic acid found in milk. However, neither of these individuals linked microbes to the formation of lactic acid. The chance of discovering the formation of lactic acid by lactic acid bacteria finally fell on a wellprepared and well-­known person who entered the kingdom of science. Pasteur insisted on the perfect combination of pure science and practice and insisted on experimentation as a method and the means to carry out scientific discovery and solve the basic strategies of practical problems and made great contributions to the

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development of related science. For lactic acid bacteria, Pasteur’s contribution is linking lactic acid formation to microorganisms for the first time. From 1854 to 1856, ethanol fermentation and brewing were the main industries in the Lille region where Pasteur lived. M. Bigo, the father of one of Pasteur’s students, worked in the wine industry. He found that in the production of alcoholic beverage from beetroot, the problem of rancidity which formed acid liquid rather than ethanol was easy to appear. Pasteur analyzed and compared the normal sample by field sampling and microscope observation technique. Two kinds of microorganism were found. One is yeast which exists in the normal fermentation sample and can make the beet juice carry on the normal ethanol fermentation. The other is the rod-shaped “lactic acid yeast” (lactic yeast), which is present in the acidic fermentation sample. It is the existence and activity of the “lactic acid yeast” that makes the fermentation of sugar in the beetroot to the direction of producing lactic acid. It is Pasteur’s research that stems from the practice of production that makes lactic acid-forming microorganisms enter the human eye for the first time. In practice, Pasteur told Bigo that if the rod-shaped lactic acid yeast was found in the fermentation broth, it would be discarded. This extremely simple method solved the problem faced by the brewing industry at that time. It can be said that it saved the wine industry at that time. In terms of basic theory, according to this study, Pasteur submitted a title entitled “Report on Lactic Acid Fermentation” to the Science Society of Lille (English translation titled Report of the Lactic Acid Fermentation, German translation titled Mémoiresur la fermentation Appeléelactique), published in the French journal Chemical Progress exhibition (Pasteur 1857, 1995). The scientific discovery of “living microbes for lactic acid fermentation” marks the birth and beginning of modern microbiology and has become a milestone in the history of microbiology (Pasteur 1857). 1.2.3.2  Discovery of Some Important Lactic Acid Bacteria As early as 1847, C.  Blondeau studied the fermentation process of lactic acid, butyric acid, acetic acid, and urea and determined that lactic acid is the end product of the fermentation process of a certain microorganism (then he considered to be a fungus Penicillium). In 1857, after Pasteur discovered the relationship between microbes and lactic acid fermentation, under the impetus of pure bacterial culture technology, during the period from the end of the nineteenth century to the beginning of the twentieth century, different scholars discovered and isolated various lactic acid bacteria including important members such as Lactobacillus, Bifidobacterium, Streptococcus, Leuconostoc, etc. First, in 1873, Joseph Lister first used a series of dilutions to acidify cow’s milk in an attempt to confirm Pasteur’s theory. The first pure culture strain of lactic acid bacteria, Streptococcus lactis, was isolated from the milk, and Liszt named it Bacterium lactis (Streptococcus Lactis, the current Lactococcus lactis subsp.) (Lister 1873). Lactobacillus is the most abundant strain of lactic acid bacteria.

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The Dutch microbe scientist and botanist Martinus Willem Beijerinck proposed the term Lactobacillus as the official form of Lactobacillus genus (genus Lactobacillus; Beijerinck 1901) (van Iterson et al. 2013). In 1896, Leichmann and Lafar discovered and isolated Lactobacillus delbrueckii from sour grain mash, and L-lactic acid was the main metabolite. The Austrian pediatrician Ernst Moro discovers Bacillus acidophilus n. spec., which is named Lactobacillus acidophilus now, while studying infant digestive physiology (Moro 1900); In 1899, Henry Tissier, a pediatrician at the Pasteur Institute in Paris, France, isolated a dominant Y-shaped bacterium from the feces of breast-fed healthy infants and named it Bacillus bifidus (Tissier 1906). In 1905, a 27-year-old Bulgarian physiologist and microbiologist Stamen Grigoroff (1878–1945) isolated Lactobacillus bulgaricus (Grigoroff 1905) from acidified milk. In 1906, Andrewes and Horder isolated Enterococcus faecalis from endocarditis patients (then named Streptococcus faecalis); Orla-Jensen isolated Lactobacillus casei from cheese in 1916. Through the discovery of these lactic acid bacteria, it is recognized that lactic acid bacteria represent a very important physiological metabolic group of bacteria in nature and it is necessary to systematically classify lactic acid bacteria, which laid the foundation for Orla-Jensen to establish a systematic lactic acid bacteria system. 1.2.3.3  E  stablishment of the Basis and Application System of Lactic Acid Bacteria In addition to his contribution to basic biology, bacterial taxonomy, Orla-Jensen’s contribution to lactic acid bacteria science and technology is that he established a complete system of lactic acid bacteria, laying the foundation for the development of lactic acid bacteria technology and applications. Orla-Jensen has been adhering to a highly rigorous scientific attitude. After 10 years of unremitting experimentation, observation, and analysis, he systematically described a variety of lactic acid bacteria and determined the most stable indicator system that can be used as a classification standard for lactic acid bacteria and wrote the book Dairy Bacteriology in 1919. In addition, he conducted in-depth research on the theoretical and practical applications related to lactic acid bacteria. In his doctoral thesis (1904), he studied the types and formation of volatile fatty acids in cheese, and in his later years, he developed lactic acid bacteria nutrition. In his later years, he carried out research on nutritional requirements of lactic acid bacteria and published some pioneer papers on nutritional physiology of lactic acid bacteria (Orla-Jensen et  al. 1936; OrlaJensen and Snog-kjaer 1940). In addition, Orla-Jensen is also concerned with dairy hygiene. The methylene blue reduction test was developed as a method of milk grading, which is still widely used today, and the use of lactic acid bacteria to make silage was explored. It can be said that the work of Orla-Jensen laid the foundation for the sustainable development of lactic acid bacteria science and technology in both basic theory and practical application.

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1.2.3.4  A  nalysis of Basic Physiological Metabolism of Lactic Acid Bacteria The physiological metabolism of lactic acid bacteria mainly involves nutrition physiology, cell membrane transport, environmental physiology (interaction between environmental factors and lactic acid bacteria), carbohydrate metabolism, and protein metabolism. Since the beginning of the twentieth century, the people’s understanding of lactic acid bacteria has made great progress. However, limited to the level of understanding at that time, the understanding of the physiology of lactic acid bacteria was mainly limited to the apparent physiological characteristics such as acid production and gas generation (Orla-Jensen 1919). Although Eduard Buchner et al. in Germany discovered the phenomenon of ethanol fermentation of sucrose by cell-free yeast extract as early as 1897, its research really paid attention to it after winning the Nobel Prize in 1907 (Buchner 1907). Beginning to understand the physiological metabolic activity of microorganisms at the subcellular level marks the birth of the modern “enzymology theory” of life sciences and also promotes the study of physiological metabolism of lactic acid bacteria at the subcellular level and enzyme molecular level. In terms of carbohydrate metabolism, after almost 50 years of exploration from the Buchner period (1897) to the 1940s, people realized the whole details of the glycolysis pathway (EMP pathway, Embden-­ Meyerhof-­Parnas pathway), which is a basic metabolic pathway shared by different types of life. Regarding lactic acid bacteria, in theory, Kluyver and Donker proposed the terms of homofermentation and heterofermentation in 1924. After that, key enzymes related to carbohydrate metabolism of lactic acid bacteria and key enzymes derived from physiological metabolism of different lactic acid bacteria, such as lactate dehydrogenase, were successively isolated, identified, and characterized (Stephenson 1928; Garvie 1980), and related metabolic pathway networks have been discovered and confirmed. A model of lactic acid fermentation based on glycolytic pathway for carbohydrate metabolism of lactic acid bacteria was developed. The classical model of heterogeneous lactic acid fermentation (Nelson and Werkman 1935; DeMoss et al. 1951) based on pentose phosphate pathway and the bifid fermentation model (de Vries and Stouthamer 1968) based on fructose-6-­ phosphate phosphoketolase (F6PPK). By the 1960s and 1970s, the reaction mechanism of carbohydrate metabolism in lactic acid bacteria was well-understood. In addition to carbohydrate metabolism, microbial nutrition is also the main content of lactic acid bacteria physiological metabolism research. Orla-Jensen is a pioneer in the study of microbial nutrition in lactic acid bacteria (Orla-Jensen et  al. 1936; Orla-Jensen and Snog-kjaer 1940). This work began in the mid-1930s with microbial nutrition studies of lactic acid bacteria. The development mainly occurred in the 1940s and 1950s. Lactobacillus is a typical growth factor heterotrophic bacterium, and its nutritional requirements are very demanding. At first, people only realized the nutritional requirements of lactic acid bacteria at different raw material level (Davis 1939). Later, people gradually realized the nutritional requirements of lactic acid bacteria from the compound level. During this period, the key nutritional

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factors affecting the growth and metabolism of lactic acid bacteria were gradually discovered. These nutritional factors mainly included fatty acids-like growth factors, amino acid-like growth factors, vitamin-like growth factors, base-like growth factors, and bifido cytokines that specifically promote the growth of Bifidobacteria, etc. (Snell 1945; Kitay and Snell 1950; Tittsler et al. 1952). To sum up, people began to form a systematic understanding of the nutritional requirements of lactic acid bacteria. At the same time, the culture technology of lactic acid bacteria has also been developed, and many scholars have found different types of mediums (such as the MRS medium, the M17 medium, and the SL medium) suitable for lactic acid bacteria culture and culture techniques (like anaerobic culture technology, etc.), which strongly promoted the development of lactic acid bacteria science and application technology. Solute transport and environmental physiology are important aspects of microbial physiology. Microbial environmental physiology mainly refers to the interaction of environmental factors such as pH and temperature with microbial cells. Environmental factors such as pH, oxygen, osmotic pressure, and nutrients play an important role in the metabolic process of lactic acid bacteria (Thompson 1987). The metabolic modes and product types of lactic acid bacteria change significantly under different environmental conditions. For example, oxygen causes Lactococcus lactis to change from homogenous lactic acid fermentation mode to heterotypic lactic acid fermentation mode (Thomas et  al. 1979). Hemoglobin in the external environment can transform the metabolic patterns of certain lactic acid bacteria such as Enterococcus faecalis, Lactococcus, and Leuconostoc from fermentation to respiration, which is also an important part of the physiological metabolism research of lactic acid bacteria (Bryan-Jones and Whittenbury 1969; Sijpesteijn 1970). Microbial cell solute transport is an important physiological process in which cells absorb nutrients from the external environment by a specific means or mechanism and transport them into the cytoplasm through transmembrane. The research work in this area is mainly carried out around Lactococcus lactis. It is the main content of research on lactic acid bacteria in the 1970s and 1990s. We mainly carried out some research on the basic laws of transport and absorption of important solutes such as carbohydrates, amino acids, protein short-peptides, and some anions/ cations of lactic acid bacteria and analyzed the solute transport involving lactic acid bacteria. We have analyzed some mechanisms of action involving lysate transport and energy metabolism in lactic acid bacteria, such as proton potential (PMF) cotransport, precursor-product reverse transport, and group translocation based on phosphotransferase systems. 1.2.3.5  Molecular Biology Period of Lactic Acid Bacteria Research With the analysis of the double-helix structure of DNA molecules in the 1960s, the people’s understanding of biological sciences and life phenomena has entered a new period of molecular biology, which has a greater impact on the classification of lactic acid bacteria (as mentioned above). It also greatly promotes people’s

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understanding of the metabolic process of lactic acid bacteria. People began to try to reveal the mechanism of physiological metabolism and product formation and genetic regulation model of lactic acid bacteria at the nucleic acid molecular level and found the material basis of life activity phenomenon of lactic acid bacteria such as plasmids, chromosomes, and so on. Genetic transfer patterns such as conjugation, transduction, transformation, transfection, protoplast fusion, and transposon were found in different lactic acid bacteria. The gene cloning and expression systems of Lactococcus and Lactobacillus were established. The application of molecular biology theory and technology in the field of lactic acid bacteria not only enables people to understand the life metabolism of lactic acid bacteria at the molecular level but also provides the theoretical basis and powerful tools for bioengineering and utilization of lactic acid bacteria. The understanding and application have reached an unprecedented height. 1.2.3.6  E  xploration and Application of the Function of Lactic Acid Bacteria Before the development of lactic acid bacteria science and technology, people have gradually realized the beneficial functions of lactic acid bacteria and their products on humans and animals in production practice. For example, ancient Roman naturalists advocate the use of fermented dairy products to treat gastrointestinal diseases. The fermented yogurt made with goat’s milk in the century treated the diarrhea disease of the French emperor Francis I. In the Chinese Yuan Dynasty, Hui Sihui mentioned in the yinshanzhengyao that the “cow cheese” obtained by fermentation of lactic acid bacteria has “sweet, non-toxic, stop the thirst and heat, get rid of the heat in the chest ” and other functions. In the Ming Dynasty, Li Shizhen recorded in the fiftieth volume of Materia Medica of the Compendium of that “cheese” was used to “be laxative, eliminate swelling, enhance the color and spirit.” However, the scientific excavation and rational understanding of the beneficial functions of lactic acid bacteria began in the late nineteenth and early twentieth centuries, when people have noticed that certain lactic acid bacteria may have functions to promote health and disease treatment. Cantani and Döderlein proposed bio-­ antagonism based on lactic acid bacteria to attempt biotherapy for different infectious bacteria (Cantani 1885; Döderlein 1892). Henry Tissier, a pediatrician at the Pasteur Institute in Paris, France, isolated a dominant Y-shaped bacterium from breast-fed healthy baby feces and named it Bacillus bifidus. Tissier noted that breast-fed infants have a relatively low probability of developing diarrheal disease and found that supplementing this Bacillus bifidus in food can treat infantile diarrhea due to intestinal flora imbalance (hyperproliferation of hydrolyzed bacteria) (Tissier 1906). Yale University’s Rettger reported the clinical efficacy of Lactobacillus acidophilus yogurt in 1920 and 1921, especially for digestion (Cheplin and Rettger 1920; Rettger and Cheplin 1921). However, the work of really promoting the discovery of lactic acid bacteria comes from the theoretical hypothesis of lactic acid bacteria and human health pro-

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posed by several scientists represented by Metchnikoff, which believes that lactic acid bacteria and their fermented products are beneficial to human health. These theoretical hypotheses have greatly promoted the development of lactic acid bacteria science and technology. Meichenikov (1845–1916) was a Russian-born Jewish zoologist. He shared a Nobel Prize in Physiology or Medicine with Paul Ehrlich in 1908 for discovering the macrophage immune defense mechanism that exists in innate immunity. Known as the “Father of innate immunity,” his work and writings have had a tremendous impact on immunology, lactic acid bacteria, and health, respectively. The sensible scientist who had committed suicide twice because of his two wives who died of illness (death) had traveled to Bulgaria during his tenure at the Pasteur Institute in France. Meichenikov, meanwhile, noticed that there were many long-lived centenarians in Bulgaria. The main diet of these long-lived people is a fermented yogurt (yahourth), made by so-called Bulgarian Bacillus (isolated from fermented yogurt by Bulgarian scientists). Meichenikov believes that intestinal bacteria can cause intestinal damage and is harmful to the body. By ingesting fermented yogurt containing lactic acid bacteria, it can inhibit the occurrence of intestinal decay and make people prolong life. Based on this, he published a far-­ reaching book in 1908, The Prolongation of Life: Optimistic Studies (Metchnikoff and Mitchell 1908), which first proposed the role of lactic acid bacteria in human health in the form of speculation and hypothesis through epidemiological investigations. In 1911, the British dairy nutritionist Loudon M. Douglas (1864–1944) published The Bacillus of Long Life, which treats yogurt as a natural therapy. Eating yogurt can alleviate the toxic factors produced by the human body and promote human health (Douglas 1911). The theoretical contribution of Mitsuoka (1990) is as follows: Mitsuoka from the University of Tokyo, Japan, in the 1960s established an analytical method based on selective culture of intestinal flora species (Mitsuoka et al. 1965). To understand the composition and relative proportions of intestinal bacteria, he tried to correlate the functions of different members, which promoted people to explore and understand the healthy beneficial function of lactic acid bacteria. In theory, Mr. Mitsuoka proposed a concept of health that the intestinal tract and gastrointestinal dysfunction are the root cause of different diseases of the body and gastrointestinal health is the basis and core of the body’s health. The beneficial effects of Lactobacillus and Bifidobacterium were pointed out; and Bifidobacterium was considered to be an important index to characterize the health degree of the body. The microecological balance of gastrointestinal tract by dietary supplementation of Bifidobacterium or by dietary regulation to increase the level of Bifidobacterium is helpful to prevent and alleviate a series of diseases and the aging process (Mitsuoka 1978,1990). Although these theories do not have sufficient and definitive scientific evidence, they have greatly promoted the in-depth exploration of the relationship between lactic acid bacteria and human health. In the twentieth century, people used various methods and techniques of function excavation and evaluation to deeply explore the beneficial physiological functions of lactic acid bacteria. The position of lactic acid bacteria also expanded from the intestinal tract to all aspects of the body, extending from the initial beneficial

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function of the intestine to systemic metabolism, neurodevelopment, mental cognition, and so on. In 2009, the World Gastroenterology Organisation (WGO) published guidelines for the use of probiotics and prebiotics. For the first time, international authoritative medical organizations have identified the functions and possible functions of lactic acid bacteria (Farnworth 2008b; World Gastroenterology Organisation 2009). Nowadays, with the development of the National Human Microbiome Project, people are more aware that the intestinal flora, including lactic acid bacteria, plays an important role in the physiological metabolism and the occurrence and development of diseases. The further research on the physiological function of lactic acid bacteria will continue.

1.3  S  ection III Application of Lactic Acid Bacteria in Human Social Practice In the previous section, we briefly reviewed the development of lactic acid bacteria science and technology. Regardless of the history of development or the current situation, lactic acid bacteria play an important role in human social practice. This section describes the social practice of lactic acid bacteria from the following aspects.

1.3.1  Lactic Acid Bacteria and Food Manufacturing Industry Fermented food is the precious wealth given by nature to mankind, and it is also an important part of human main food. The preparation of fermented food involves many microorganisms recognized as safe (GRAS) and edible directly. Lactic acid bacteria (lab) is one of the most important microorganisms in food production. Different countries and regions have their own characteristics of traditional lactic acid fermented food based on lactic acid fermentation principle. On the one hand, lactic acid bacteria as the main fermentation agent, through lactic acid fermentation to form short-chain fatty acids acidification and the accompanying role of biological metabolism, improves the sensory characteristics of food matrix profile. The nutritional value of fermented products was improved by releasing amino acids and forming vitamins. The lactic acid bacteria bio-antagonism substances such as short-­ chain fatty acids, bacteriocins, and hydrogen peroxide have significantly improved the preservation and safety of the products and are typical models for the natural green processing of foods. On the other hand, lactic acid bacteria are an important component of functional probiotics and, as a supplemental starter, can impart specific health-promoting functional properties to the fermented product. In addition, some antagonistic lactic acid bacteria can also be directly used as biopreservatives for commercial use in food processing, manufacturing, and biological control of pathogenic bacteria in food. In recent years, with people’s attention to health and the

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need for functional properties of products, lactic acid bacteria fermentation products with specific functional properties have received widespread attention. Although there are still a series of related legislative management issues, lactic acid bacteria-fermented foods have achieved rapid development. The food produced by lactic acid bacteria fermentation includes fermented dairy products, fermented meat products, fermented aquatic products, fermented cereal foods, fermented fruits and vegetables products, edible lactobacillus preparations, and so on. According to incomplete statistics, at present, there are as many as 120 kinds of food products concerning lactic acid bacteria fermentation (involving primary fermentation and secondary fermentation) (Steinkraus 1995; Reed and Nagodawithana 1996; Hui et al. 2004). Lactic acid bacteria-fermented food has become an important part of food biomanufacturing. The economic output value of related products has reached about 50 billion US dollars, which is an important component of human social and economic activities.

1.3.2  L  actic Acid Bacteria and Farming and Aquaculture Industry Lactic acid bacteria are also widely used in the field of modern agriculture. Firstly, lactic acid bacteria (lactic acid bacteria), as the important microorganisms of direct feeding microorganisms (direct-fed microbial, DFM), are widely used in the field of feed. Feed lactobacillus preparation can improve the growth efficiency and disease resistance of farmed animals by inhibiting the growth of pathogenic bacteria, regulating the micro-ecological balance of animal intestines, forming vitamins and beneficial enzymes, and so on. At the same time, beneficial lactic acid bacteria and other direct feed microorganisms can also replace or reduce the extensive use of growth-promoting antibiotics in animal breeding, which is an effective solution to the growing problem of microbial drug resistance. Lactic acid bacteria can be used as a microbial inoculant for silage production, inhibiting pathogenic bacteria and toxin-producing fungi in feed by bio-metabolism of lactic acid bacteria, improving the palatability of animal feed, and improving the nutritional value and safety of feed. Lactic acid bacteria can also be used as an effective microorganism (EM) preparation for the production of fermented feeds using cereals, agricultural product processing by-products, and crude plant biomass. Lactic acid bacteria have prominent inhibitory effects on some aquaculture species such as prawns, and some lactic acid bacteria preparations such as Pediococcus can improve the ability of aquaculture species to resist aquatic pathogenic bacteria and viral infections. In addition, lactic acid bacteria are endosymbiotic bacteria and epiphytes commonly found on plants. The bacteriocin produced by lactic acid bacteria plays an important role in the rhizosphere micro-ecological balance of plants. The lactic acid bacteria produce some antibacterial compounds through lactic acid fermentation and have inhibitory effects on plant pathogenic bacteria such as Xanthomonas, Erwinia, and Pseudomonas. The lactic acid bacteria inoculant has a certain protec-

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tive effect on plant tissues. Based on this, lactic acid bacteria can be used as preparations of biological fertilizers and biocontrol agents. For the pathogenic bacteria of crops, lactic acid bacteria can be used as an inoculant to achieve biological control of certain crop pathogenic bacteria such as Fusarium and have biosuppressive effects on certain mold-forming mycotoxins. Lactic acid bacteria can also be used in the fermentation of bio-fertilizer and the safe treatment of fertilizer, which has a good application prospect in the field of eco-agricultural planting.

1.3.3  Lactic Acid Bacteria and Medical Health Industry Lactic acid bacteria can give food a rich and beneficial effect. People have long noticed the application of lactic acid bacteria and their products in medicine and health, including dysfunction, disease prevention and treatment, and specific health promotion. First, despite the lack of a clear mechanism of action, based on extensive evidence-based medical evidence, lactic acid bacteria live/dead preparations have a good therapeutic and recovery effect on dysfunction and disease of the digestive system of the gastrointestinal tract. Some commonly used lactic acid bacteria include Enterococcus, Lactobacillus, Bifidobacterium, and Streptococcus. These lactic acid bacteria preparations can effectively correct the gastrointestinal flora disorder caused by various factors such as bad diet, antibiotic use, food-borne pathogen infection, radiotherapy and chemotherapy, surgery, etc. and inhibit the production and absorption of intestinal toxic factors. They have been clinically proven and widely used. Secondly, based on the biological antagonism of lactic acid bacteria producing lactic acid to reduce pH, hydrogen peroxide, and bacteriocin, lactobacillus preparations made from specific lactic acid bacteria are used to control infection of body system or specific body parts. For example, lactobacillus preparations (Lactobacillus delbrueckii, Lactobacillus rhamnosus, etc.) were used to control the imbalance of reproductive tract flora and inflammatory infections in women, and Lactobacillus bulgaricus cream was used to treat skin burns. Lactobacillus acidophilus and Lactobacillus bulgaricus freeze-dried capsules were used for the treatment and prevention of oral infection and dental caries. Thirdly, antibiotics play an important role in the treatment and control of infectious diseases. However, the excessive use of antibiotics has also brought about the growing problem of microbial resistance, because of the great potential of beneficial lactic acid bacteria and probiotics for infection control and disease treatment. Due to the great potential of beneficial lactic acid bacteria and probiotics for infection control and disease treatment, after antibiotics, biotherapy based on beneficial lactic acid bacteria and probiotics is expected to be an effective alternative medicine program for infectious diseases, which has been widely concerned by people. Finally, different levels of model research and clinical practice have proved that lactic acid bacteria and its preparation have a good health promotion effect by strengthening the biological barrier of the body, synthesizing of essential vitamins, promoting the absorption of nutrients, and having the beneficial regulation of specific immunity and non-specific immunity.

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In summary, it has been confirmed that certain lactic acid bacteria and probiotic strains have outstanding clinical application effects, including alleviating lactose intolerance and treating diarrhea caused by different causes. There are also many studies to prove that lactic acid bacteria have potential disease treatment effects, such as antitumor, alleviating hypertension, adjuvant treatment for Helicobacter pylori infection, regulating blood lipids and lowering cholesterol levels, preventing diabetes, etc., but more clinical validation is needed. In recent years, some studies on microbiology have also shown that beneficial lactic acid bacteria, probiotic bacteria, and certain gastrointestinal functional bacteria can exert important intervention effects on the whole body through the brain-gut axis. In addition to the digestive system, lactic acid bacteria and probiotics also have important effects on mental, emotional control, nerves, and endocrine. It can be foreseen that with the deepening of research, functional lactic acid bacteria and probiotics have very broad application prospects in the medical and health industry.

1.3.4  L  actic Acid Bacteria and Industrial Manufacturing Industry In addition to its wide application in the food and pharmaceutical fields, the application of lactic acid bacteria in the industrial sector mainly includes biofermentation of lactic acid, biorefinery of biomass, and bioengineering of various valuable products using lactic acid bacteria as a cell factory. Lactic acid, chemically known as 2-hydroxypropionic acid, is a natural organic acid having two optical isomers, D and L, in the form of a colorless or yellow transparent syrupy liquid. Lactic acid is an important industrial raw material with many important uses. It is widely used as a fine chemical in food, chemical, medical, pharmaceutical, textile, agricultural, and environmental protection fields. The use of lactic acid bacteria for lactic acid fermentation to prepare lactic acid is one of the main methods of industrial lactic acid production. After saccharification of starchy raw materials, Lactobacilli are mainly inoculated for fermentation production. The strains currently used for industrial lactic acid fermentation production mainly include certain strains of Lactobacillus delbrueckii subsp. bulgaricus, Bacillus coagulans, and Lactobacillus sp. Biorefinery is a bioprocessing method that produces agricultural chemicals, fuels, and bio-based materials from agricultural waste, plant-based starch, and lignocellulose. It can be seen from this definition that biorefinery is a very broad concept in the field of bioengineering, covering biotransformation with microbes as the main body and core. Lactic acid bacteria are one of the suitable microbial groups for biorefinery. The fermentation of lactic acid also belongs to the content of biorefinery. In addition, the metabolic pathway of “cell factories” of lactic acid bacteria was genetically modified to produce a variety of high value-added metabolites, such as sugar alcohol, vitamins, functional exopoly-

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saccharide, γ-aminobutyric acid, food flavor substances, short-chain organic acids, conjugated linoleic acid, active peptides, and nutritional drugs (nutraceutics).

1.4  S  ection IV Lactic Acid Bacteria Science and Technology System and Development Frontier 1.4.1  C  omposition of Lactic Acid Bacteria Science and Technology System Around the basic science and application of lactic acid bacteria, a system of science and technology involving microbiology, fermentation engineering, food manufacturing, nutrition, and health science has been formed, although it is somewhat far-­ fetched and intersects with each other in many fields, such as microbiology, fermentation engineering, food manufacturing, nutrition and health science, and so on. However, we can still generalize the connotation and scope of “lactic acid bacteria science and technology”: “lactic acid bacteria science and technology” is a whole set of research on lactic acid-producing bacteria at the level of cells, molecules, or specific ecological groups as well as the basic laws of life activities. Besides, it explores the interaction among lactic acid bacteria and non-lactic acid bacteria, and other organisms such as plants, animals, and humans, and then, applied it to science and technology in the fields of industrial fermentation, food manufacturing, agriculture, animal husbandry, medicine, health, bioengineering, and environmental protection. Specifically, the science and technology of lactic acid bacteria include the biological basis of lactic acid bacteria, mainly related to the basic microlife activities of lactic acid bacteria, with emphasis on the basis. The application technology of lactic acid bacteria includes industrial fermentation of lactic acid bacteria, food bio-production based on lactic acid fermentation and biotransformation, etc. These parts are relatively independent of each other, but there is a tight or sparse relationship.

1.4.2  D  evelopment Frontier in Research and Application of Lactic Acid bacteria From the history of the development of lactic acid bacteria, lactic acid bacteria is a multidisciplinary cross-field; its hot issues and development front are also involved in a number of different fields. Some new theories, techniques, and methods, such as histology and systems biology, provide a powerful tool for the study of lactic acid

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bacteria and accumulate a great deal of information about the biological behavior of lactic acid bacteria, which also makes the scientific understanding and application of lactic acid bacteria more in depth. Here, based on the editor’s own understanding, the development frontiers of lactic acid bacteria research and application are briefly reviewed, which is inevitably biased and generalized, only for readers’ reference. 1.4.2.1  Research on Basic Microbiology of Lactic Acid Bacteria Under the guidance of macrogenomic information of environmental microorganisms, we should further optimize and improve the culture methods and techniques suitable for lactic acid bacteria, isolate and culture new lactic acid bacteria from specific natural habitats, and explore the biodiversity resources of lactic acid bacteria. We should perfect and enrich the existing resource system of lactic acid bacteria, enrich and perfect the theory and technology of lactic acid bacteria taxonomy, continue to discover the information molecules with taxonomic significance, and make the taxonomic classification and intraspecific classification of lactic acid bacteria more reasonable and further straighten out the phylogeny of Lactobacillus. We use modern omics techniques such as metagenomics, genomes, transcriptomes, proteomics, and metabolomes to carry out the physiology, carbohydrate metabolism, organic acid metabolism, protein metabolism, environmental physiological response and adaptation, genetic and genetic recombination of lactic acid bacteria, as well as the activity process and detailed regulation mechanisms based on metabolite-controlled protein A (CcpA) and transcription factor (TF). Besides, we carried out a global phenotypic characterization analysis of lactic acid bacteria microbial cells to further improve people’s understanding of the phenomenon of apparent life activities of lactic acid bacteria, by doing so to form a systematic understanding of lactic acid bacteria. 1.4.2.2  H  igh-Efficiency Biomanufacturing Research of Lactic Acid Bacteria Cell Factory Lactic acid bacteria are ideal model organisms for biomanufacturing and biorefinery. At present, the main research on biomanufacturing and biorefinery by using lactic acid bacteria as a cell factory focuses on the construction of gene efficient expression elements and gene efficient expression systems, modification of metabolic pathways, and reconstruction of new metabolic pathways. First of all, in the context of understanding the genomic information, we use bioinformatics methods and techniques to further explore the genetic structural elements and regulatory elements of valuable lactic acid bacteria that carry specific biological traits, and to construct and improve exogenous gene efficient expression systems (such as foodgrade expression systems) and surface display systems for valuable lactic acid

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bacteria for specific applications such as bio-manufacturing, food, and medicine. Besides, we explore the use of lactic acid bacteria as a carrier to produce and prepare biological products such as biological vaccines, and use metabolic engineering techniques, genome editing techniques, synthetic biology techniques, etc. to modify, transform, and create metabolic pathways of valuable lactic acid bacteria and to realize valuable products such as organic acids, functional sugar alcohols, vitamins, fatty acids, and flavor substances based on biomass raw materials. 1.4.2.3  S  tudy on Environmental Physiology and Cell Response of Lactic Acid Bacteria for Practical Application Lactic acid bacteria are the most commonly used industrial fermentation microorganisms. Food biomanufacturing and other bioengineering applications are subject to a variety of stresses from bioprocessing conditions and external physical, chemical, and biological environmental factors, such as acid, oxygen, starvation, low temperature, osmotic pressure, dryness, and bacteriophage, which affect the physiological function and activity of lactic acid bacteria cells. For the purpose of improving and guiding the biomanufacturing of lactic acid bacteria, the characterization and molecular mechanism exploration and optimization research of bio-­ processing conditions, external environmental factors, and lactic acid bacteria are a prominent hotspot in the application field of lactic acid bacteria, including research on the effects of environmental factors on the functional properties of lactic acid bacteria and the physiological stress mechanism of lactic acid bacteria to various environmental factors. Metabolic engineering techniques and strategies were used to modify the signal transduction network, transcription, key enzymes, and metabolic pathways in environmental response of lactic acid bacteria cells. We use metabolic engineering techniques and strategies to directionally modify important regulatory points at the level of signal transduction networks, transcription, key enzymes, and metabolic pathways of lactic acid cell environmental response, and to improve the resistance ability of lactic acid bacteria to environmental stress during biological manufacturing and processing. Besides, from the perspective of cell physiology and metabolic damage, we systematically studied the physiological damage and functional weakening in the whole process of lactic acid bacteria starter preparation, determined the key factors affecting the reduction of lactic acid bacteria activity and weakening the function, and carried out targeted protection strategy research to improve lactic acid bacteria ability to resist environmental stress.

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1.4.2.4  S  tudy on the Interaction Between Lactic Acid Bacteria and Other Life Species and the Function Mining and Development of Lactic Acid Bacteria Species in different forms of natural ecosystems depend on each other, and there are complex interactions between them. The relationship between lactic acid bacteria and other life species is a prominent hotspot in the research of lactic acid bacteria in recent years. Related life species involve humans, animals, and plants, and related fields include medical health, animal science, and plant science. People began to explore the specific interaction mode and regulation mechanism between lactic acid bacteria and other organisms at different micro and macro levels, such as molecular, cell, tissue, organ, system metabolic network, population, and the effects of this interaction. Taking lactic acid bacteria and humans as examples, in the early twentieth century, people have noticed the role of lactic acid bacteria in the health of the body. With the advancement of the Human Genome Project and the Human Microbiome Project, it was discovered that human symbiotic microbiota, including lactic acid bacteria, may play a huge role in the body’s physiological metabolism, health maintenance, and disease development. Specifically, people began to explore the relationship between lactic acid bacteria (and other human commensal microorganisms) and hosts by using different levels of research models such as model in vitro, cell models, tissue model in vivo, animal models, and population experiments. Microecological balance, gastrointestinal health, physiological metabolism, immune balance, neuropsychological, aging, and the effects of the overall body system homeostasis and intervention effects were explored and analyzed. The function of lactic acid bacteria was excavated in order to form a systematic theoretical system of the interaction between lactic acid bacteria and the body and to optimize the practical application based on lactic acid bacteria. Studies on the interaction between lactic acid bacteria and other living species such as animals are similar to that between lactic acid bacteria and human beings. 1.4.2.5  T  he Development of Lactic Acid Bacteria for Food and Animal Use with Vitality and Function as Their Core Features, as well as the Preparation and Application of Starter Cultures As mentioned above, lactic acid bacteria as a starter (starter culture) have a high value of industrial applications, of which the key is the vitality and function of lactic acid bacteria. Therefore, on the basis of the development of microbiology and functional exploitation of lactic acid bacteria, the research on the preparation and application of lactic acid bacteria-fermented food and animal feed with the core characteristics of vitality and function is a research hotspot in the field of application technology of lactic acid bacteria. For the preparation and application of fermented food such as cheese, fermented milk, fermented vegetables, and fermented

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grain food, natural or recombinant lactobacillus strains that can significantly improve product quality, flavor, manufacturing process, and food safety were developed by using classical and new genetic breeding technologies. On the basis of understanding the environmental physiology of lactic acid bacteria, the preparation technology and related starter products of lactic acid bacteria with high activity and suitable for industrial application were developed. The application and development of new starter product of lactic acid bacteria in different food base materials, such as fruits and vegetables, cereals, milk, meat, aquatic products, are also being developed.

1.5  Section V Organization Structure of the Book Lactobacillus science and technology is a rapidly growing field. The author’s research team tried the basic biology of lactic acid bacteria (involving system classification, ecological distribution, physiological metabolism, genetic recombination, phage), experimental methods of lactic acid bacteria (involving separation and identification, culture preservation, genetic manipulation, omics analysis), lactic acid bacteria environmental physiology and ecology (involving acid, osmotic pressure, temperature, oxidation, phage, bile salts, hunger, and other environmental stresses and cellular responses), lactic acid bacteria bioengineering (involving organic acids, extracellular polysaccharides, vitamins, bacteriocins, sugar alcohols, substance, functional fatty acids, drugs, and biological products), lactic acid bacteria function mining (involving functional mining methodology, intestinal health, immunity, food safety, typical cases, lactic acid bacteria safety evaluation, etc.), lactic acid bacteria industrial applications (involving fermented milk, fermented fruits and vegetables, fermented grains, alcoholic beverages, starter preparation, pharmaceutical preparations, animal feed), lactic acid bacteria-related regulations, and other major levels to carry out the organization of the entire book and strived to present new theories, technologies, functions, and applications of lactic acid bacteria science and technology to readers based on the existing knowledge system of lactic acid bacteria. The organization structure and relationship of the whole book are shown in Fig. 1.3. The first is introduction; the second is basic biology of lactic acid bacteria; the third is histology of lactic acid bacteria; the fourth is environmental physiology of lactic acid bacteria; the fifth is bioengineering of lactic acid bacteria; the sixth is functional excavation and evaluation of lactic acid bacteria; the seventh is the industrial application of lactic acid bacteria; the eighth is the regulation and management of lactic acid bacteria; and the ninth is the experimental methodology of lactic acid bacteria.

Fig. 1.3  The organization and chapters of the book

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References Andrewes FW, Horder TJ (1906) A study of the streptococci pathogenic for man. Lancet 168(4333):708–713 Axelsson L, Salminen S, Von Wright A et al (2004) Lactic acid bacteria: classification and physiology. In: Dillon VM (ed) Lactic acid bacteria microbiology & functional aspects. Marcel Dekker/CRC Press, New York Beijerinck M (1901) Sur les ferments lactiques de l’industrie. Archives Néerlandaises des Sciences Exactes et Naturelles 6:212–243 Bergey’s Manual Trust (2015) Bergey’s manual of systematics of archaea and bacteria. Wiley, in association with Bergey’s Manual Trust, New York Blondeau C (1847) Des fermentations. J Pharm 312:244–261 Bryan-Jones DG, Whittenbury R (1969) Haematin-dependent oxidative phosphorylation in Streptococcus faecalis. J Gen Microbiol 58(2):247–260 Buchner E (1907) Cell-free fermentation. Nobel Lecture: 103–120 Buchner E, Rapp R (1897) Alkoholische gährung ohne hefezellen. Ber Dtsch Chem Ges 30(3):2668–2678 Cahn D (1901) Über die nach Gram färbbarenBacillen des Säulingsstuhles Bacilli of infant stools stainable according to Gram. CentralblattfürBakteriologie I Abteilung Originale 30:721–726 Cantani A (1885) Un tentativo di batterioterapia. Gior Int Sci Med 7:493 Carr FJ, Chill D, Maida N (2002) The lactic acid bacteria: a literature survey. Crit Rev Microbiol 28(4):281–370 Chen Gong (2011) Chinese Kimchi processing technology. China Light Industry Press, Beijing Cheplin HA, Rettger LF (1920) Studies on the Transformation of the intestinal flora, with special reference to the implantation of Bacillus acidophilus: II. Feeding experiments on man. Proc Natl Acad Sci USA 6(12):704–705 Chomakow H (1973) The dairy industry in the People’s Republic of Bulgaria. Center for Scientific, Technical and Economic Information in Agriculture and Forestry, Agricultural Academy, Bulgaria Davis J (1939) The nutritional requirements of the lactic acid bacteria. J Dairy Res 10(02):186–195 de Vries W, Stouthamer AH (1968) Fermentation of glucose, lactose, galactose, mannitol, and xylose by Bifidobacteria. J Bacteriol 96(2):472–478 DeMoss RD, Bard RC, Gunsalus IC (1951) The mechanism of the heterolactic fermentation: a new route of ethanol formation. J Bacteriol 62(4):499–511 Döderlein A (1892) Uber Scheidensekrete und Scheidenkeime [Vaginal secretions and vaginal microbes]. Die Verhandlungen der deutschen Gesellschaftfür Gynäkologie 4:35–50 Douglas LMQ (1911) The Bacillus of long life. G. P. Putnam’s Sons, New York Dunne J, Evershed RP, Salque M et al (2012) First dairying in green Saharan Africa in the Fifth Millennium BC. Nature 486(7403):390–394 Eggerth AH (1935) The Gram-positive non-spore-bearing anaerobic bacilli of human feces. J Bacteriol 30(3):277–299 Eggerth AH, Gagnon BH (1933) The bacteroides of human feces. J Bacteriol 25(4):389–413 Farnworth ER (2008a) The evidence to support health claims for probiotics. J  Nutr 138(6):1250S–1254S Farnworth ER (2008b) Handbook of fermented functional foods, 2nd edn. Taylor & Francis, London Florey HW (1945) Use of micro-organisms for therapeutic purposes. Br Med J 2(4427):635 Garvie EI (1980) Bacterial lactate dehydrogenases. Microbiol Rev 44(1):106–139 Ghaffar T, Irshad M, Anwar Z et  al (2014) Recent trends in lactic acid biotechnology: a brief review on production to purification. J Radiat Res Appl Sci 7(2):222–229 Gorbach SL, Goldin BR (1989) Lactobacillus strains and methods of selection, Google Patents

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Grigoroff S (1905) Etude sur le laitfermenté comestible: le Kissélo-mléko de Bulgarie. Revue Médicale de la Suisse Romande (in French). Libraires-éditeurs. Librairie de L’Université, Genéve Hall RH (1963) Production of nisin: US, US 3093551A Holzapfel WHN, Wood BJB (1995) The genera of lactic acid bacteria. Springer, New York Hui YH, Meunier-Goddik L, Josephsen J et al (2004) Handbook of food and beverage fermentation technology. Taylor & Francis, London Kandler O (1983) Carbohydrate metabolism in lactic acid bacteria. Antonie Van Leeuwenhoek 49(3):209–224 Kitay E, Snell EE (1950) Some additional nutritional requirements of certain lactic acid bacteria. J Bacteriol 60(1):49–56 Kluyver A, Donker H (1924) The unity in the chemistry of the fermentative sugar dissimilation processes of microbes. Proc Akad v Wetenschappen Amsterdam 28:297–313 Lash AF, Kaplan B (1926) A study of Doderlein’s vaginal Bacillus. J Infect Dis 38(4):333–340 Lister J (1873) On the lactic fermentation and its bearings on pathology. Trans Pathol Soc Lond 29:425–467 McElhatton A, Marshall R (2007) Food safety: a practical and case study approach. Springer, New York Metchnikoff E, Mitchell PC (1908) The prolongation of life: optimistic studies. G. P. Putnam’s Sons, New York Milth (1884) Milth. a. d. kaiserl. Gesundh. Amt 2:309 Mitsuoka T (1978) Intestinal bacteria and health: an introductory narrative. Harcourt Brace Jovanovich, California Mitsuoka T (1990) Bifidobacteria and their role in human health. J Ind Microbiol 6(4):263–267 Mitsuoka T, Sega T, Yamamoto S (1965) Eine verbesserte Methodik der qualitativen und quantitativen Analyse der Darmflora von Menschen und Tieren. Zentralbl Bakteriol Orig 195(4):455–469 Moro E (1900) Ueber den Bacillus acidophilus. Jahrb Kinderh 52:38–55 Nelson ME, Werkman CH (1935) Dissimilation of glucose by heterofermentative lactic acid bacteria. J Bacteriol 30(6):547–557 Newman D (1915) The treatment of cystitis by intravesical injections of lactic Bacillus cultures. Lancet 186(4798):330–332 Orla-Jensen S (1919) The lactic acid bacteria. A.F. Host &Son, Copenhagen Orla-Jensen S, Otte NC, Snog-kjaer A (1936) The vitamin and nitrogen requirements of the lactic acid bacteria. Zentralbl Bakteriolii 6(5):1–52 Orla-Jensen S, Snog-kjaer A (1940) Factors which promote or inhibit the development of lactic acid bacteria. Reprinted [in full] from: K. danskevidensk. Selsk biol Skr 12:5–19 Pasteur L (1857) Mémoiresur la fermentation appeléelactique. C R Chim 45:913–916 Pasteur L (1995) Mémoiresur la fermentation appeléelactique Extraitparl’auteur. Mol Med 1(6):599–601 Reed G, Nagodawithana TW (1996) Biotechnology, Enzymes, Biomass, Food and Feed. Wiley, New Jersey Rettger LF, Cheplin HA (1921) A Treatise on the Transformation of the Intestinal Flora, with Special Reference to the Implantation of Bacillus acidophilus. Yale University Press, New Haven Rogers LA, Whittier EO (1928) Limiting factors in the lactic fermentation. J  Bacteriol 16(4):211–229 Scheele CW (1780) Om Mjölkochdesssyra, about milk and its acid. Kongliga Vetenskaps Academiens Nya Handlingar New Proceedings of the Royal Academy of Science, vol. 1, pp 116–124 Sijpesteijn AK (1970) Induction of cytochrome formation and stimulation of oxidative dissimilation by hemin in Streptococcus lactis and Leuconostoc mesenteroides. Antonie Van Leeuwenhoek 36(3):335–348

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Snell EE (1945) The nutritional requirements of the lactic acid bacteria and their application to biochemical research. J Bacteriol 50(4):373–382 Steinkraus K (1995) Handbook of Indigenous fermented foods, 2nd edn, revised and expanded. Taylor & Francis, London Stephenson M (1928) On lactic dehydrogenase: a cell-free enzyme preparation obtained from bacteria. Biochem J 22(2):605–614 Tamang JP, Kailasapathy K (2010) Fermented foods and beverages of the world. CRC Press, Florida Thomas TD, Ellwood DC, Longyear VMC (1979) Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures. J Bacteriol 138(1):109–117 Thompson J (1987) Regulation of sugar transport and metabolism in lactic acid bacteria. FEMS Microbiol Lett 46(3):221–231 Tissier H (1906) Traitement des infections intestinalespar la mthode de la transformation de la florebactrienne de lintestin. CR SocBiol 60:359–361 Tittsler RP, Pederson CS, Snell EE et al (1952) Symposium on the lactic acid bacteria. Bacteriol Rev 16(4):227 vanIterson G, de Jong LDD, Kluyver AJ (2013) Martinus Willem Beijerinck: his life and his work. Springer, New York Wood BJB, Holzapfel WH (1995) The genera of lactic acid bacteria. Blackie Academic and Professional, Glasgow World-Gastroenterology-Organisation (2009) World gastroenterology organisation practice guideline: probiotics and prebiotics. Arab J Gastroenterol 10(1):33–42 Zhang Heping (1994) Ancient Chinese dairy products. Zhonggue Rupin Gongye 22(4):161–167

Chapter 2

Genetic Operation System of Lactic Acid Bacteria and Its Applications Haiqin Chen, Chen Chen, Chunqing Ai, Chengcheng Ren, and He Gao

Lactic acid bacteria (LAB), a class of commonly existing microorganisms in nature, are important components of gut commensal microflora in humans and animals. Previous studies suggested that LAB exerted specific physiological and biochemical functions on the host such as improving intestinal microbial balance, immunomodulation, inhibiting tumor growth, lowering cholesterol levels, as well as regulating blood pressure and are therefore widely used in food manufacturing and functional food development. Due to the continuous development of modern molecular biology techniques, studies regarding exploiting LAB as expression hosts in addition to fermentation starter cultures and probiotics have received increasing attention from both academia and industry. In the 1980s, some researchers initiated molecular genetic research for LAB. They characterized lactose metabolism-related genes and proteins in LAB and established preliminary DNA delivery systems for LAB. Over the past decades, owing to the advances in modern DNA sequencing and gene characterization techniques, structures and functions of LAB genomes and plasmid-related genes have been further elucidated, which lays a solid theoretical foundation for the further development of LAB-based gene expression systems (Bolotin et al. 2001; Altermann et al. 2005).

H. Chen (*) · C. Ren · H. Gao Jiangnan University, Wuxi, China e-mail: [email protected]; [email protected]; [email protected] C. Chen Shanghai Institute of Technology, Shanghai, China e-mail: [email protected] C. Ai Dalian Polytechnic University, Dalian, China © Springer Nature Singapore Pte Ltd. and Science Press 2019 W. Chen (ed.), Lactic Acid Bacteria, https://doi.org/10.1007/978-981-13-7832-4_2

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2.1  LAB-Associated Gene Expression Systems LAB expression systems were developed lately as compared to traditional Escherichia coli, Bacillus, and yeast expression systems. Generally, there’re still many limitations in LAB-based gene expression systems such as low gene expression efficiency, complicated procedures, and low transformation efficiency. Nevertheless, genetic engineering of LAB offers remarkable advantages over other traditional expression systems due to the intrinsic properties of LAB strains, thereby exhibiting great potential for applications in antigen screening, protease expression, and immunotherapy. The advantages of LAB-based expression systems are listed below: (1) LAB are safe and edible food-grade microorganisms granted by WHO and FAO and have been applied in food products for thousands of years; (2) LAB are inherent enteric microflora in humans and animals and play a key role in the establishment and maintenance of host immune system; (3) some LAB strains adhere tightly to the gut mucosa; and (4) LAB culture supernatants can be directly consumed without the necessity of purifying expressed heterologous proteins. LAB-based gene expression systems comprise host strains, expression vectors, and heterologous genes. This chapter will briefly describe host strains and cloning vectors in LAB-based gene expression systems in terms of their compositions and features.

2.1.1  Host Strains in Expression Systems for LAB LAB strains of different genera or species and even different strains within the same species differed greatly in terms of their biochemical, ecological, and molecular immune properties (Meijerink et al. 2012; Ai et al. 2015). This strain diversity creates extensive host strain options in LAB but also causes difficulty of selecting proper strains as expression hosts since high-level production of heterologous proteins in a specific strain-based expression system cannot always be achieved in other strains (Rigaux et al. 2009). Currently, several genera including Lactococcus, Enterococcus, and Lactobacillus have been most widely applied as host strains (Table 2.1) owing to their extraordinary features such as good resistance to the harsh conditions in the digestive tract, low immunogenicity, relatively high electro-transformation efficiency, and long retention time in the gastrointestinal tract. Due to the progress in molecular genetics research on LAB, LAB as food-grade delivery vehicles have received increasing attentions. In order to further facilitate the screening of recombinant LAB strains, host strains can be modified based on specific requirements such as lacF-deficient strains and strains with nisRK integrated into their chromosomes.

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Table 2.1  Host strains commonly used in LAB Host strain Lactobacillus plantarum Lactococcus lactis Lactobacillus acidophilus Lactobacillus casei Lactobacillus cremoris Lactobacillus johnsonii Enterococcus faecalis Lactobacillus sakei Lactobacillus helveticus Lactobacillus reuteri Lactobacillus pentosus

Expression vector pGIT032

References Corthesy et al. (2005)

pTREX1, pP16pip pNZ123

Lee et al. (2001), Mannam et al. (2004), and Robinson et al. (2004) Kim et al. (2005)

pPGS1 pFBYC04

Lee et al. (2005) Biet et al. (1998)

pNZ124

Scheppler et al. (2002)

hBD2-Cy3 pSIP, pMG36c pCI

Kandaswamy et al. (2013) Jimenez et al. (2015) Oliveira et al. (2006)

pNIES pG

Wu and Chung (2007) Liu et al. (2011)

2.1.2  Vectors in LAB-Based Gene Expression System 2.1.2.1  Plasmids in LAB Since Chassy and Flickinger (1987) detected plasmids in LAB, researchers have further studied plasmids that are present in LAB. The distribution of plasmids has been suggested to be highly uneven and strain-specific in LAB. More plasmids were found in LAB strains such as Lactobacillus reuteri, Lactobacillus helveticus, and Lactobacillus acidophilus when compared with strains belonging to other species. Moreover, the size (1–150 kb) and amounts of plasmids differed greatly in different LAB strains (Chassy et al. 1976; Vescovo et al. 1981; McKay and Baldwin 1990). Overall, plasmids in LAB are characterized by the following features: (1) Most plasmids in LAB are cryptic plasmids, and only a few of them are associated with the host’s specific phenotypes such as bacteriocin synthesis, sugar metabolism, and antibiotic resistance (Smiley and Fryder 1978; Fortina et al. 1993). (2) The copy number of the LAB plasmids is correlated with their size. Smaller plasmids are replicated at a higher number. (3) Plasmids from LAB, which have a broad host range, can replicate in a wide range of host bacteria. Since most of plasmids from LAB are cryptic, their functions on gene transcription, translation, and protein secretion are still not completely clear, which might be one of causative factors for delayed development in LAB molecular biologics study. Nevertheless, owing to the advances in genetic engineering techniques, vectors for cloning, expression, and integration in LAB were successively developed through

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studying the regulatory elements within plasmids isolated from LAB. At present, cloning and expression vectors are the most extensively applied vectors in LAB. 2.1.2.2  Cloning Vectors for LAB Genetic cloning techniques, one of the effective means of resolving the complexity of genetics, allow isolation of single desired genes and construction of new LAB starter strains carrying these isolated genes. Cloning vectors are used for amplification and propagation of foreign DNA inserts and have certain copy numbers within host cells. Cloning vectors have the following essential features: (1) suitable cloning sites, into which foreign DNA can be inserted, (2) autonomous replication or replication along with chromosomal DNA once plasmids are integrated into the host chromosome, and (3) selectable markers which facilitate the selection of transformed cells harboring DNA insert-containing plasmids. To date, various types of cloning vectors including plasmids, viral vectors/bacteriophages, and artificial vectors that incorporate segments from the plasmids, bacteriophage, or genomic DNA have been constructed. These artificial vectors might only have an origin of replication but not promoters for expression. Even though Escherichia coli is the most frequently applied host in molecular cloning, other microorganisms sometimes are also used as hosts. Thus, shuttle vectors containing a second replication origin, which assures their replication in other types of microorganisms (e.g. LAB), are needed. The enterococcal plasmid pAMβ1, a θ-type-replicating plasmid with a broad host range, is the first plasmid used for constructing LAB cloning vectors. It is also one of the prototype cloning vectors for lactococci and lactobacilli. Several plasmids such as pIL252 and pIL253, which are based on pAMβ1, were established. Other common plasmid replicons such as pWV01 and pSH71 can propagate and replicate in various LAB strains. Moreover, there are other low-copy and high-copy derivative plasmids such as pGK1 and pGK12. In order to screen the right transformants, one or multiple resistance genes are ligated into vectors as selectable markers. Since a large number of wild-type LAB strains are resistant to ampicillin, kanamycin, and tetracycline, erythromycin and chloramphenicol resistance genes serve as the common selection markers. Of note, since the transfer and dissemination of antibiotic resistance genes in the environment are potentially deleterious to the environmental ecosystem, resistance genes are not suitable selection markers in the food industry. Therefore, several food-­ grade expression systems for LAB based on the sugar utilization, sensitivity to PH and temperature, and bacteriocin resistance of host strains have been developed. Apart from single-component cloning vectors, some researchers developed several two-component cloning systems. Emond et  al. (2001) constructed a two-­ component food-grade cloning vector pVEC1, which is a pCD4 derivative and carries the functional pCD4 replicon. Another pCD4-derived plasmid pCOM1 was also constructed as a companion vector. Plasmid pCOM1, in which an erythromycin resistance gene serves as the dominant selection marker, is deficient of repB gene.

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After the selection of recombinant strains carrying both plasmids, recombinant bacterial cells will lose plasmid pCOM1 when grown under antibiotic-free conditions owing to the incompatibility between pVEC1 and pCOM1. By using this cloning system, Lactococcus lactis MG1363 and industrial strains were shown to exhibit stable phage-resistant phenotype. 2.1.2.3  Expression Vectors for LAB To ensure effective expression of genes of interest, expression vectors must have elements for constitutive or inducible protein expression besides the basic elements in cloning vectors such as origins of replication and heterologous genes. In terms of the cellular location of expressed proteins, there’re three types of protein expression in LAB as described below: 1. Cytoplasmic expression: it can effectively protect produced protein from the external environment. But cell lysis is required for intracellular protein release. 2. Secreted expression: the secretion of recombinant protein into extracellular medium is directed by signal peptide. 3. Cell surface displaying of target protein: it can be achieved by anchoring recombinant proteins to the cell wall. In recent years, a large number of expression vectors in LAB have been constructed for different applications in the fields of food, medicine, and life sciences.

2.1.3  Intracellular Expression Systems for LAB Intracellular expression of heterologous proteins in LAB is achieved by inserting foreign genes into LAB expression vectors without signal peptides and introducing recombined vectors into LAB hosts via electroporation. Plasmids for intracellular expression in LAB comprise promoters, multiple cloning sites for insertion of foreign genes, terminators, selection markers, and replicons. Thus far, most commonly applied promoters in LAB expression systems are lacA, lacR, lacF, T7, xylA, lacS, nisA/nisZ, and nisF (Kleerebezem et  al. 1997). Based on the carried resistance gene, expression vectors in LAB can be divided into antibiotic resistance-based vectors and food-grade expression vectors. Traditional expression vectors for LAB carry one or multiple antibiotic resistance-encoding genes such as erythromycin and chloramphenicol resistance genes, and transformants are selected via antibiotic selection pressure. The plasmids pNZ8037 and pNZ8048 carrying chloramphenicol resistance genes were developed on the basis of the food-grade NICE system and are standard expression vectors for L. lactis. These two plasmids contain the pSH71 replicon and nisin-inducible promoter nisA and can be utilized as expression vectors in both Escherichia coli and L. lactis. To overcome plasmid pNZ8048-induced low-­ level expression in LAB, plasmids pNZ8148 and pNZ8150 carrying

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chloramphenicol resistance genes were constructed based on pNZ8048. These two upgraded plasmids are capable to drive highly efficient expressions in LAB.

2.1.4  Secreted Expression Systems for LAB 2.1.4.1  Features of Secretion Vectors in LAB Exploiting LAB as expression hosts or cell factories to produce antigenic proteins, pharmaceutical molecules and other functional factors have become a new research filed on LAB.  To achieve secretory expression of protein in LAB, heterologous genes are inserted expression vectors with signal peptides, which direct the secretion of intracellular recombinant proteins to extracellular environment after transforming LAB hosts with recombinant vectors via electroporation. Signal peptides, a vital determining factor for secretory expression, are peptides residing on recombinant precursor proteins, which can be targeted to the secretory pathway by signal peptides. Through the cleavage of signal peptidase, signal peptides are separated from the mature proteins, which are translocated to extracellular compartment afterward (Nielsen et al. 1997). Compared with intracellular expression, secreted expression holds the advantage of synthesizing heterologous proteins in an active form without aggregation within the host cells, which effectively prevents the loss of proteins during protein recovery. Moreover, secreted recombinant proteins can be separated from other cellular proteins, thus simplifying the downstream purification procedures for target proteins. However, not all proteins are suitable for secretory expression, especially for those naturally nonsecreted proteins, whose secretion is determined by multiple factors such as their size, structure, charges, and signal peptide. For one specific protein, different signal peptides can result in differential secretion efficiencies (Meazza et  al. 1997). Zhang et  al. (2010) showed that modifying signal peptide structure pronouncedly improved the secretion efficiency of recombinant protein in LAB. Therefore, selection of proper signal peptides is of great importance for secretory expression system for LAB or other hosts. 2.1.4.2  Secretion Systems Based on Usp45 Signal Peptide At present, the signal peptide of Usp45 protein isolated from L. lactis is the most extensively applied signal peptide for secretory expression in LAB and was identified in the genome of L. lactis MG1363 by van Asseldonk et al. (1990). LAB secretion systems can efficiently recognize Usp45 signal peptide, which resulted in enhanced secretion of heterologous proteins such as antigens (Ribeiro et al. 2002; Bermudez-Humaran et  al. 2003b; Zhang et  al. 2011) and antibodies (Bermudez-­ Humaran et al. 2003a; Zhang et al. 2010). To date, extensive studies have suggested that enhanced heterologous protein expressions via secretory expression are closely

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correlated with the abilities of signal peptides to stabilize variable proteins, inhibit the proteolysis by intracellular proteases, and improve protein secretion efficiency. In the study by Enouf et al. (2001), bovine rotavirus NSP4 protein was intracellularly and extracellularly expressed in L. lactis, but mature NSP4 protein could not be efficiently secreted. In addition to the Usp45-NSP4 precursor and mature NSP4 protein, two degradation products of mature NSP4 protein were also detected in the intracellular compartment of the NSP4-secreting LAB strain. This indicates that mature NSP4 protein might be partially degraded by intracellular proteases, which could be inhibited by infusion with the Usp45 signal peptide. 2.1.4.3  Secretion Systems Based on the Signal Peptide of S-Layer Protein Signal peptide sequences exert crucial influences on the secretion efficiency of heterologous proteins in LAB. Due to the complex relation between protein secretion and signal peptides, it is not realistic to apply one specific signal peptide for the secretory expression of all different types of heterologous proteins in a specific LAB strain. Thus, searching for novel signal peptides to establish a diverse signal peptide library is an effective approach to elevate protein secretion expression levels in LAB. S-layer protein, whose relative molecular weights are in the range from 40,000 to 200,000, is a layer of bioactive macromolecules present on the cell wall surface of many bacteria and archaea. Most S-layer proteins consist of a single species of protein or glycoprotein. S-layer proteins account for 10–15% of the total bacterial proteins, and the gene expression machinery for their synthesis and secretion is quite strong. Many researchers consider abundant signal peptide options as one of the effective means to enhance the secretion efficiency in LAB. S-layer protein-encoding genes are highly efficiently expressed and secreted in LAB, which are closely linked to the high transcription efficiency of their promoters and high secretion efficiency of their signal peptides. Based on this characteristic of S-layer proteins in LAB, their one major application in LAB is to develop highly efficient (secretion) expression systems for LAB (Zhang et al. 2010). Kahala and Palva (1999) introduced the promoter of a S-layer protein SIpA in two different LAB hosts and found that this promoter significantly enhanced beta-glucuronidase (gusA) and aminopeptidase N (pepN) expression in L lactis and L. plantarum, respectively. Expression levels of gusA and pepN account for 15% and 28% of the total cellular proteins in L. lactis and L. plantarum, respectively. In addition, Sibakov et  al. (1991) increased secretion of β-lactamase in a recombinant L. lactis strain by using the strong promoter and signal peptide of lactobacilli S-layer protein-encoding gene. Even though promoter and signal peptide have been suggested to be closely related to the expression and secretion efficiency of heterologous proteins in LAB, the characteristics of the expression hosts also affect the transcription efficiency of S-layer protein promoter. It was shown that using the same S-layer protein promoter yielded differential protein expression efficiencies in different LAB hosts (Kahala and Palva 1999), which further facilitates the research on signal peptide sequences of LAB expression systems.

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2.1.4.4  Secretion Systems Based on Other Types of Signal Peptides In addition to LAB-originated signal peptides, signal peptides derived from other bacteria have also been applied to construct secretion expression vectors for LAB. In a study by Le Loir et  al. (2001), staphylococcal nuclease (nuc) was secreted by recombinant L. lactis with the native signal peptide of nuc gene, whose secretion efficiency was lower than when applying the Usp45 signal peptide. This might be attributed to the effects of signal peptides on protein conformation (Kajava et al. 2000). In addition to screening suitable native signal peptides, many researchers focused on modifying native signal peptide sequences to elevate the secretion efficiency of recombinant proteins in LAB.  In L. lactis, insertion of a nine-residue synthetic propeptide (LEISSTCDA) after the Usp45 signal peptide sequence achieved a secretion efficiency of up to 80% for nuc, the yield of which was upregulated 2–4-folds (Le Loir et al. 2005).

2.1.5  T  he Surface Expression System of Lactic Acid Bacteria (LAB) 2.1.5.1  S-Layer Protein Expression System In 1985, G. P. Smith took the advantage of major coat protein P3 of the filamentous bacteriophage to establish a molecular genetic system for phage, which plays various important roles in some fields, such as interactions between protein-protein and DNA-protein, analysis of antigen peptides, protein directed evolution, and signal transduction. However, the nature characteristics of the phage make it hard to express high molecular weight (HMW) protein, which limits the application of the phage expression system. To solve the above problem, some researchers did some efforts to develop new system by using bacteria that can fully express heterologous HMW protein. Based on the safety feature of LAB, its expression system received lots of attention and exhibited a high potential value in live vaccines, diagnosis, enzyme immobilization, and so on. Recent studies indicate that some proteins that anchor on the cell surface of LAB play key roles in cell adhesion, immune responses, signal transduction, and other life activities. Based on the structural characteristics of surface protein, some were used to establish new expression system. It was apparent that the monomolecular crystalline array of proteinaceous subunit, which was termed as S-layer, was considered as one of the most common surface structures on bacteria. Sequence analysis shows that S-layer protein of LAB has two conserved domains (N-terminal secretion signal peptide and C-terminal anchoring peptide) and an intermediate variable region that participates in the protein refolding and crystallization process. Based on the above structure property of S-layer protein, the expression gene of heterologous protein can be inserted into S-layer protein coding gene, and then it was expressed on the cell surface of LAB following the S-layer protein expression.

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There is a growing body of evidence showing that bacterial S-layer protein expression system could exert great application potential in the field of microbiology, molecular biology, immunology, and biological catalysis. Due to its safety property, the expression system in LAB has received more attention from different study fields, such as recombinant vaccines, bacterial adhesion, and antibody. 2.1.5.2  Cell Wall Anchoring Expression System To date, some enzymes that anchor surface proteins to the cell wall are proven to be used as cell wall anchoring domain of heterologous protein in the surface expression system based on LAB, especially cell wall hydrolases and the aggregation factor. Due to the difference in anchor position, the surface expression system can be divided into cell wall anchoring form, cell membrane anchoring form, and surface layer-associated proteins anchoring form. According to the bonding form between the anchoring protein and cell, the surface expression system is divided into covalent bond and non-covalent bond formation. On account of the binding site between heterologous protein and anchoring protein, it can be divided into two main forms: N-terminal and C-terminal formation. For the former, the expressed heterologous protein locates in the middle of the signal peptide and the anchoring region, such as the M6 protein from Streptococcus pyogenes, protein A of Staphylococcus aureus, and other surface protein in the Gram-positive bacteria. For the latter, the expressed protein locates the downstream of the anchoring region, such as proteinase PrtP from L. lactis, lysozymes from Bacillus phages, extracellular hydrolase from Lactobacillus strain, and peptidoglycan hydrolase of Enterococcus strain. The LPXTG motif contained a hydrophobic domain and a positively charged tail, which was termed as a marker sequence of proteins which was anchored to the bacterial cell wall (Kuczkowska et  al. 2015). After translocation, LPXTG motif would be firstly cleaved and then cross-linked at the threonine residue to a nucleophile, i.e., an active amino group of the peptidoglycan stems peptide or the lysine residue of the pilin motif (Kuczkowska et al. 2015). García-Mantrana et al. (2016) showed that two phytases from bifidobacteria could be cloned in L. casei under the control of a nisin-inducible promoter, and they were able to produce, export, and anchor to the cell wall. Kuczkowska et  al. (2015) indicated that recombinant L. plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag-derived antigen causes increased recruitment of T cells, in which the heterologous proteins were expressed in the cell surface. LysM is widely distributed in more than 4000 proteins in both prokaryotes and eukaryotes, and this protein was firstly discovered in lysozyme of Bacillus phage φ29 acting as a C-terminal repeat comprising of 44 amino acids with seven amino acids inserted (Buist, Kok et al. 1995). The best-characterized LysM containing protein is the N-acetylglucosaminidase AcmA of L. lactis, which is required for cell separation and cell lysis during the stationary phase of L. lactis (García-Mantrana et al. 2016). The composition of Lysm domain family is highly abundant, which

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could be attributed to gene diversity, varied amount, and combinational diversity with other protein domain, and thus some of them were used as an anchor to display heterologous proteins on the surfaces of LAB. Hu et al. (2010) showed that a Novel LysM domain was isolated from L. fermentum bacteriophage endolysin and used as an anchor to display protein in the surfaces of LAB. To date, there is a growing body of evidence indicating that LAB could be used as a potential vehicle although some disadvantages need to be solved, such as thicker cell walls, less heterologous protein production, and so on.

2.1.6  Applications of LAB-Based Gene Expression System 2.1.6.1  LAB as Vaccine Delivery Vehicles (1) Applications of LAB vaccine delivery system In recent years, due to the progress in developing LAB as mucosal vaccine delivery vectors, the application of recombinant LAB-based vaccines has been extensively expanded. Vaccines based on genetically engineered LAB strains have been exploited for disease management or as nutritional supplements. Herein, we will describe in detail the current status of preclinical laboratory research regarding diverse applications of recombinant LAB vaccines. 1. Prevention and treatment of infectious diseases At the early stage, the primary recombinant LAB-based mucosal vaccines against infectious diseases were genetically modified LAB strains expressing key antigen proteins (or fragments). Recombinant LAB vaccines are applied in two distinctive forms: immunoprophylaxis and immunotherapy. These two immunization forms differ in their timing of intervention. For immunoprophylaxis animals are administered with recombinant LAB strains prior to establishing experimental disease models, while for immunotherapy immunization animals administered with recombinant vaccines are carried out after animal models are developed. LAB vaccines against infections will be described below based on the types of pathogenic microorganisms involved. (A) Bacterial and fungal infections Thus far, a large number of genetically modified LAB strains producing crucial antigens (fragments) of common human or animal pathogens have been successfully constructed and confirmed to confer immune protection against infections in various animal infection models (Wells et  al. 1993; Norton et  al. 1997; Maassen et al. 1999; Grangette and Muller-Alouf 2001; Robinson et al. 1997, 2004; Cheun et al. 2004; Corthésy et al. 2005; Hanniffy et al. 2007). In order to optimize the immune efficacy of recombinant vaccines, researchers attempted to construct recombinant LAB strains expressing different antigen proteins or fragments derived from one specific pathogenic microorganism for immunization

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(Lee et al. 2001; Wu and Chung 2007; Wei et al. 2010; Hongying et al. 2014). Wu and Chung (2007) constructed a recombinant Lactobacillus reuteri strain producing the fusion protein of heat-stable enterotoxin and heat-labile enterotoxin B of enterotoxigenic Escherichia coli (ETEC) as a mucosal vaccine against ETEC infections. Wei et al. (2010) exploited Lactobacillus casei as a carrier for fimbriae protein of ETEC K99 in mucosal immunization. Through certain evolved strategies, pathogens are capable to evade the host immune defense system so that they can continuously colonize, proliferate, and migrate in the hosts to cause infections. These strategies including specific cell surface components (e.g., surface capsules and pili) and synthesis of virulence factors (e.g., toxins and proteases) are unitized by pathogens to interfere or subvert host immune surveillance. Thus, in order to promote the recognition of specific pathogens by the host immune system and to trigger specific defensive immune responses, it is of vital importance to select essential virulence factor of pathogens as the target of vaccine development. Besides, developing recombinant LAB strains as mucosal delivery vectors for antibody fragments to enhance host immunity against pathogens is another effective strategy against infections. Beninati et  al. (2000) constructed recombinant Streptococcus gordonii strains expressing a microbicidal single-chain antibody (H6), which were found to inhibit Candida albicans-induced vaginal inflammation via vaginal immunization. (B) Viral infections So far, researchers have successfully constructed genetically modified LAB strains against many common pathogenic viruses such as rotavirus, hepatitis B virus, and influenza virus and evaluated their efficacy in experimental animal models of viral infection (Xin et al. 2003; Ho et al. 2005; Perez et al. 2005; Lee et al. 2005; Poo et al. 2006; Lei et al. 2011; Liu et al. 2011; Zhang et al. 2011). It is promising that Xin et al. (2003) engineered a recombinant L. lactis strain to express and anchor the envelope protein of the human immunodeficiency virus (HIV) on its cell surface. They found that oral immunization of mice with this genetically modified LAB strain induced HIV-specific cellular (high level of IFN-γ-secreting lymphocytes in the spleen and intestinal lymph node) and humoral immune responses (high level of HIV-specific serum IgG and fecal IgA antibodies). Furthermore, expressed HIV antigen fragments were shown to be presented to T lymphocytes by dendritic cells, thereby mounting adaptive immune reactions (Xin et al. 2003). (C) Parasitic infections Parasites, another major group of infectious organisms, can cause severe organ or tissue damage in the hosts. However, due to the difficulty in their in  vitro ­cultivation, the development of anti-parasitic vaccines has been greatly hindered. Therefore, synthesis of recombinant parasite antigens using molecular biology techniques for immunization is of great significance for facilitating anti-parasitic vaccines development. Thus far, there have been some promising results with regard to applying LAB as vaccine vehicles against parasitic infections (Zhang et al. 2005;

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Ramasamy et al. 2006; Lee et al. 2009; Yam et al. 2011). Zhang et al. (2005) genetically modified Lactococcus lactis to express Plasmodium yoelii antigen fragment MSP-119 and orally immunized two strains of mice (BALB/c and C57BL/6) with this recombinant strain. They observed that this recombinant LAB strain effectively enhanced host immunity against malaria parasites in both strains of mice. 2. Inflammatory bowel disease (IBD) management IBD is group of chronic, recurrent gastrointestinal inflammatory disorders caused by intestinal dysfunction. IBD is often concomitant with parenteral complications and needs long-term therapy, thus leading to a marked decline in IBD patients’ quality of life. In spite of the similar clinical symptoms such as abdominal pain and diarrhea, IBD should be discriminated from pathogen-induced gastroenteritis. Ulcerative colitis (UC) and Crohn’s disease (CD) are two main forms of IBD. In recent years, the prevalence of IBD has been increasing worldwide; effective IBD prevention and treatment are desperately needed. So far, researchers have carried out a range of studies on mucosal vaccination with genetically engineered LAB against IBD based on differential IBD pathogenesis and therapeutic directions and obtained some promising results. The first attempt to apply genetically modified LAB strains against IBD was performed by Steidler and Hans (2000). They constructed a recombinant L. lactis strain expressing anti-inflammatory cytokine interleukin-­ 10 (IL-10). Intragastric administration with this recombinant strain pronouncedly attenuated dextran sulfate sodium (DSS)-induced colitis in mice by 50% and also effectively suppressed the development of IL-10 gene deficiency-­ caused colitis in mice. More encouragingly, an “upgraded” recombinant IL-10-­ expressing L. lactis strain has been tested in a phase I clinical trial and demonstrated to be safe in CD patients and biologically contained (Braat et al. 2006). This is also the first clinical trial performed with genetically modified LAB (Braat et al. 2006), which greatly promotes research on recombinant LAB vaccines. Apart from IL-10, some other anti-inflammatory molecules such as trefoil factor (TFF) and neuropeptide α-melanocyte-stimulating hormone (α-MSH) have also been expressed in genetically modified LAB strains for controlling IBD. TFF are small polypeptides with beneficial effects on mucosal protection and repair, while α-MSH is a neuroendocrine peptide with anti-inflammatory properties (Ren et al. 2005; Zhu et al. 2014). Vandenbroucke et al. (2004) observed pronounced alleviation of inducible acute colitis and spontaneous chronic colitis in mice after intragastric administration with TFF-expressing L. lactis. Yoon et al. (2008) found that oral immunization of mice with a α-MSH-secreting L. casei strain significantly ameliorated DSS-induced acute colitis in mice. Neutralizing the inflammatory cytokine tumor necrosis factor (TNF)-α is another effective therapeutic strategy for IBD.  In accordance with this strategy, several recombinant mucosal vaccines based on genetically modified LAB have been developed. A study by Vandenbroucke et al. (2009) confirmed the protective efficacy of recombinant L. lactis secreting anti-TNF-α nanobodies on both DSS- and IL-10 deficiency-induced colitis in mice after oral immunization. Furthermore, the affibody against TNF-α has been surface displayed in L. lactis and recombinant

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affibody was shown to exhibit TNF-α-binding capability. These results indicate that this affibody-expressing strain has a potential in binding intestinal TNF-α and might be used to mitigate gut inflammation in IBD (Ravnikar et al. 2010). Since oxidative stress plays a key role in the pathogenesis of inflammatory disorders, antioxidant enzymes become another type of potential IBD therapeutic agents. Thus far, researchers have engineered LAB strains as mucosal delivery vectors for antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) to achieve the immunomodulation of IBD (Carroll et al. 2007; LeBlanc et al. 2011). LeBlanc et al. (2011) constructed recombinant L. casei expressing SOD or CAT and assessed their protective efficacy in mice with trinitrobenzenesulfonic acid (TNBS)induced CD. It was observed that mice that received these SOD or CAT-producing LAB strains displayed less weight loss, less bacterial translocation to the liver, and alleviated colonic tissue damage. Although the precise pathogenesis of IBD is still obscure, it has been proposed that insufficient innate antigenic stimulation can result in immune dysfunction, which triggers excessive immune reactions against innocuous intestinal antigens and thereby leads to inflammation and tissue damage. Therefore, utilization of anti-­ inflammatory agents to suppress excessive inflammatory responses in IBD patients is a major clinical treatment option for IBD. A study conducted by Foligne et al. (2007) offers new therapeutic targets for IBD management. It is known that some pathogens can effectively circumvent the host immune surveillance, stimulate anti-­ inflammatory cytokine production, and resist the host immune responses against them. Based on this theory, the low-calcium response V (LcrV) protein, one virulence factor of enteropathogenic Yersinia pseudotuberculosis was expressed in genetically engineered L. lactis (Foligne et al. 2007). The protective properties of this recombinant strain were demonstrated in TNBS- and DSS-induced murine colitis models. It was shown that this strain effectively induced IL-10 secretion and dampened colonic inflammation. Intriguingly, the protective effects of this recombinant strain in mice with TNBS-induced colitis were IL-10-dependent. Moreover, similar protective efficacy in TNBS-induced colitis model was observed by immunization with this LcrV-expressing L. lactis strain or an IL-10-expressing L. lactis strain (Foligne et al. 2007). 3. Management of allergic disorders Type I allergy, also known as immediate hypersensitivity reactions, is an IgE-­ mediated, immune disorder characterized by Th2-skewed immune responses. Clinical symptoms of type I allergy occur immediately upon allergen exposure. Food allergy, dust mite allergy, and pollen allergy are all immediate hypersensitivity disorders. In recent years, there has been accelerating incidence of type I allergy worldwide. However, there are still great difficulties in its effective clinical ­prevention or treatment. Traditional type I hypersensitivity treatment is allergenspecific immunotherapy, in which immune tolerance to specific allergens is developed. In the traditional desensitization therapy, patients are exposed to or injected with allergen extracts via non-mucosal routes for administration such as subcutaneous injection. Due to the limitation in the availability of highly pure allergen extracts,

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severe adverse reactions often occur in allergic patients receiving traditional desensitization therapy. Therefore, genetic engineering of LAB strains for producing and mucosal delivering recombinant allergens can effectively circumvent the aforementioned problems. The generally recognized safe status and immunomodulatory properties of LAB render them ideal vectors for presenting allergens to the host mucosal surface. So far, a number of allergen-expressing recombinant LAB strains have been successfully applied for type I allergy management (Adel-Patient et al. 2005; Charng et al. 2006; Cortes-Perez et al. 2007; Huibregtse et al. 2007; Rigaux et al. 2009; Schwarzer et al. 2011). Allergic reactions are triggered upon the binding of allergen-specific IgE antibodies to the IgE binding epitopes on allergens. Allergen T cell epitopes is the molecular basis for the recognition of allergens by T cells, thereby conferring immunomodulatory effects. The progress in identifying allergen epitopes in recent years greatly facilitates the development of recombinant hypoallergenic allergens or T cell epitope peptide-based allergy therapies. T cell epitope peptides have been successfully applied to modulate cat and insect venom (Bohle 2006). To summarize, it is promising to apply recombinant LAB strains expressing novel allergen mutants as mucosal vaccines for the prophylaxis and alleviation of allergic diseases. In recent years, some researchers have applied cytokine-expressing genetically engineered LAB against allergy, indicating a new research direction for managing allergic diseases (Frossard et al. 2007). Frossard et al. (2007) found that oral administration of mice with an IL-10 expressing recombinant L. lactis strain effectively inhibited allergic reactions and reduced serum Th2-type antibody (IgE and IgG1) production. Cytokine-expressing recombinant LAB strains were also used to boost the immunomodulatory effects of allergen (or allergen fragments)-expressing LAB in allergy management. Cortes-Perez and Ah-Leung (2007) showed that an IL-12-­ producing recombinant L. lactis strain enhanced the protective efficacy of a genetically engineered LAB strain expressing cow’s milk allergen in a cow’s milk allergy mouse model. Cross-linking of allergen-specific IgE antibodies with cell surface receptors on basophils or mast cells is a prerequisite for triggering the release of inflammatory mediators such as leukotriene and histamine and mounting allergic reactions. Therefore, using humanized anti-human IgE antibodies to bind free IgE to prevent the cross-linking of IgE with mast cells is an effective approach for allergy prevention or alleviation. Based on this, Scheppler et  al. (2005) genetically modified L. johnsonii to express and anchor either an IgE mimotopes or an anti-idiotypic single-­chain fragment variable (scFv) mimicking an IgE epitope on the cell surface. This recombinant stain was shown to be recognized by anti-human IgE monoclonal antibody and induce systemic IgG antibodies against human IgE through both systemic and mucosal immunization. These results indicate that these recombinant strains can potentially induce anti-IgE response, preventing or relieving allergic symptoms.

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(2) Clinical applications of recombinant LAB as vaccines As stated above, preclinical studies on recombinant LAB-based mucosal vaccines have achieved remarkable results, promoting their clinical research. Nevertheless, there are still some concerns regarding the clinical effectiveness and safety of recombinant LAB-based vaccines, which hinders the launch of their clinical trials. Steidler and Hans (2000) confirmed the remarkable protective efficacy of mucosal immunization of an IL-10-expressing genetically engineered L. lactis strain in two different mouse models of colitis. This research group developed a thymidine-deficient expression system (ActoBiotics™) for L. lactis to facilitate its clinical research. The thymidylate synthase-encoding gene thyA in L. lactis chromosome was replaced with the human IL-10 gene so that this modified recombinant strain cannot survive under thymidine- or thymine-free conditions. This modification prevents the release of this genetically engineered strain into the environment, thus eliminating the biosafety concerns toward this strain raised by its potential replication and pervasion (Bahey-El-Din 2012). Based on this thyA-deficient expression system, Braat et  al. (2006) launched the first phase I clinical trial on recombinant LAB-based mucosal vaccines. After obtaining promising results in CD patients during its phase I clinical trial, its phase IIA clinical trial has also been conducted. Although the phase IIA clinical trial confirmed its biosafety status, it was not effective on mucosal repair (Bermúdez-Humarán et al. 2011). Clinical studies have also been performed with another genetically engineered LAB strain (AG013), which was constructed to express human TFF1 based on ActoBiotics™ expression system. Its phase Ib trial demonstrated its safety and tolerability in participants as well as its efficacy in improving chemotherapy-induced ulcerative oral mucositis in patients with locally advanced head and neck cancer (Limaye et al. 2013). 2.1.6.2  Recombinant LAB in Enzyme Preparations In addition to being developed as mucosal vaccines, genetically engineered LAB also exhibit great potential in the production of catalytically active enzymes. Relevant studies have also made some progress. We will describe their applications below based on their specific purposes. Some researchers constructed enzyme-expressing LAB strains to regulate relevant biochemical reactions in food manufacturing, thereby improving food attributes such as flavor and texture. Yao et al. (2010) developed a recombinant L. lactis NZ9000 strain, which carries a secreted expression plasmid inserted with the bovine trypsin gene. Recombinant precursor protein with signal peptide was detected in the protoplast fraction of recombinant bacteria. The recombinant bovine trypsin was also shown to be biologically active. It avoids the biosafety risk from extracting bovine trypsin from cow pancreas, strengthens the proteolytic systems in LAB, and enhances bioactive peptide production during the manufacturing of fermented dairy products, thereby boosting the potential health benefits of fermented dairy products (Yao et al. 2010). Alpha-amylase has also been expressed in different cellular locations of genetically engineered LAB. Native α-amylase gene was introduced and

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expressed in two L. plantarum host strains (strain WCFS1 and a food-grade strain TGL02) with a secretion efficacy of 90% (Kanpiengjai et al. 2015). Notably, the properties of native wild-type α-amylase such as broad pH tolerability and maltose-­ producing activity were also found in secreted α-amylase by the recombinant TGL02 strain (Kanpiengjai et al. 2015). Furthermore, alpha-amylase was also effectively expressed on the cell surface of L. casei by using the Bacillus subtilis anchor protein PgsA (Narita et al. 2006). This α-amylase-displaying strain was shown to exhibit strong hydrolytic ability on soluble starch and to hydrolyze 36.3 g/L of soluble starch, yielding 21.8 g/L of lactic acid within 24 h (Narita et al. 2006). Another application of enzyme-expressing recombinant LAB is to deliver recombinant enzymes with biocatalytic activities to host mucosa in order to modulate the host immune functions. Furthermore, significant progress has been made in related research in this field, which has been drawing great attention. Oxidative stress is known to cause damage in cells or tissues, thereby triggering inflammatory diseases and accelerating host aging. Antioxidants have been a popular research topic in multiple fields in recent years owing to their capabilities of eliminating oxygen free radicals. Thus far, in the field of recombinant LAB-based mucosal vaccines, LAB have been genetically engineered to express antioxidant enzymes such as SOD and CAT, and their potential in managing gut inflammatory disorders has also been demonstrated in animal studies. Carroll et  al. (2007) observed that genetically modified L. gasseri expressing Streptococcus thermophilus-­ derived SOD significantly alleviated intestinal inflammation in IL-10-deficient mice. Moreover, genetically engineered LAB (e.g., L. plantarum and L. casei) as delivery vectors for SOD or CAT were shown to prevent or mitigate TNBS- or DSS-­ induced gut inflammation in mice or rats (Rochat et al. 2007; Watterlot et al. 2010; LeBlanc et al. 2011). In addition to antioxidant enzymes, some researchers also developed recombinant LAB as mucosal delivery vehicles for certain enzymes to improve clinical symptoms induced by the deficiency of these enzymes. Drouault et  al. (2002) applied a Staphylococcus hyicus lipase-expressing L. lactis strain to improve pancreatic insufficiency-elicited defective lipid metabolism in pigs. 2.1.6.3  Recombinant LAB in Metabolic Regulation In recent years, researchers have directionally modified or regulated certain metabolic pathways in LAB using genetic and biochemical engineering techniques, thereby altering the substrate utilization spectra of LAB, facilitating their substrate utilization, and directionally augmenting the production of desired metabolites. To a certain extent, these studies have greatly improved the production efficiency of LAB in the food industry. Diacetyl, a natural by-product during dairy and alcoholic fermentations, has been widely exploited as a flavoring agent in the food industry due to its strong buttery flavor. LAB can utilize citric acid that is minorly present in milk to synthesize an intermediate α-acetolactate (α-AL), which is subsequently converted to diacetyl

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via oxidative decarboxylation. In order to enhance diacetyl production by LAB, researchers have attempted to regulate the production of crucial enzymes involved in diacetyl synthesis in LAB (Platteeuw and Hugenholtz 1995; Hugenholtz and Kleerebezem 2000). A lactate dehydrogenase (LDH)-deficient L. lactis NZ2700 strain was genetically modified to overexpress α-ALS, achieving a high production of the diacetyl analog acetoin with lactose as substrates. However, no high diacetyl production was found by this strain. Afterward, Hugenholtz and Kleerebezem (2000) constructed a genetically engineered α-AL decarboxylase (ALDB)-deficient L. lactis strain overexpressing NADH oxidase. This recombinant strain cannot convert α-AL to acetoin due to its α-ALDB deficiency. Moreover, high expression of NADH oxidase in this strain imparts it efficient synthesis of diacetyl. Apart from diacetyl, regulating L-alanine synthesis is another classic case of utilizing genetic engineering tools for metabolic engineering in LAB. By genetically modifying a L-LDH- and alanine racemase-deficient L. lactis strain to express the alanine dehydrogenase (L-AlaDH) gene from B. sphaericus, Hols and Kleerebezem (1999) successfully altered the carbon flux in the sugar metabolism of wild-type L. lactis from homolactic fermentation to homoalanine fermentation. Besides, by using the LDH promoter from S. thermophilus, the B.subtilis AlaDH was overexpressed in recombinant L. lactis NZ9000, whose alanine production was upregulated 26 folds as compared to the untransformed L. lactis strain (Ye et al. 2010). To date, a large number of studies have suggested the beneficial effects of LAB-­ produced exopolysaccharide (EPS) during milk fermentation on the hosts. EPS from LAB have been substantially applied as thickeners in the food industry because of its specific textural properties (Tong et al. 2015). However, limited production levels of EPS in many LAB strains hinder their industrial application. Therefore, some research teams have successfully applied genetic engineering tools to enhance EPS production by LAB (Levander and Svensson 2002; Boels et al. 2003; Svensson et al. 2005). By regulating the expression levels of key enzymes in the central pathways of carbohydrate metabolism, EPS expression levels were elevated in S. thermophilus (Levander and Svensson 2002; Svensson et al. 2005).

2.2  Food-Grade Expression System for Lactic Acid Bacteria 2.2.1  Basic Requirements of Food-Grade Expression System The food-grade expression system is an expression system that maximizes the production of food and food-related products. Food-grade genetic expression system must contain the following characteristics: first, genetic expression vectors in the system are food-grade, consisting of DNA from known safe microbes and cannot contain non-food-grade DNA fragments. The lactic acid bacteria (LAB) vectors that are currently used generally carry one or more antibiotic (such as erythromycin, chloramphenicol) resistance genes to maintain a certain selection pressure. However, these resistance factors will drift, and if these carriers are used in daily life, they will

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have serious consequences for biosafety. Therefore, the use of food-grade selection markers that are harmless to the human body in place of antibiotic resistance markers is one of the effective means to solve this problem. Second, the expression host must be a safe, well-characterized, and stable food-grade microorganism. Lactobacillus, Lactococcus lactis, and Bifidobacteria are generally regarded as safe (GRAS) food-grade microorganisms. In addition, the host bacteria also need to be stable enough in food or in the body. Third, the inducer used in the expression system is also food-grade, such as sucrose, lactose, pyrimidine, nisin, etc.

2.2.2  Selective Marker of Food-Grade Food-grade vectors of lactic acid bacteria require the vector is free of non-food-­ grade functional fragments. According to the difference of screening methods, selective marker of food-grade about LAB can be divided into two categories: complementary selection markers and dominant selection markers. A complementary selection marker requires a deletion mutation in the host chromosome, and then the vector’s selective marker is used to compensate for the deletion mutation, thereby restoring the host to a certain characteristic. Complementary markers often use genes that encode important proteins involved in metabolic transformation. The drawback of defective marker is that it can only be used for specific vectors – the host system. Dominant selection markers are mainly used to provide new phenotypic characteristics by taking advantage of the characteristics of the host bacteria themselves, and do not depend on the expression genes of the host. Therefore, such markers can be applied to other bacteria of the same genus or even to other lactobacillus. However, there are not enough food-grade dominant selection markers applied at present, mainly due to the following: (1) the selective process is complicated; and (2) sometimes the labeling system is too large because it needs to contain several genes. Selective markers of food-grade that have been applied to LAB can be classified into four categories: saccharide utilization selective markers, auxotrophic ­complementary selection markers, bacteriocin resistance markers, and heavy metal resistance markers. 2.2.2.1  Saccharide Utilization Selective Markers Sugar is an important raw material for industrial fermentation. Different LAB are different in using the types and efficiencies of sugar. Most LAB generally cannot use xylose, inulin, sucrose, and melibiose. Therefore, cloning genes associated with the utilization of these sugars into non-fermenting strains give them the ability to ferment a certain sugar. The current research on lactose operons is the most in-depth. In the lactose operon, the lacF gene encodes the key enzyme IIA of the lactose phosphotransfer-

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ase system in L. lactis. First, the complete lactose operon was integrated into the chromosome of L. lactis, and then the lacF deletion was made by double crossover homologous recombination to construct a lac− type receptor strain. At the same time, the LAB replicon pSH71, the promoter P32, and the lacF gene were used to construct the vector pFI846. When a plasmid containing the lacF gene is introduced into the lac− strain, the bacteria will restore the lac+ phenotype, and positive colonies can be screened on plate medium containing bromocresol purple (Platteeuw et al. 1996). Platteeuw et al. (1996) also constructed a food-grade vector by using the lacF gene as a selective marker, which includes the lacA promoter and the transcription terminator of the aminopeptidase N gene pep N. It enhances the stability of the vector, and the gusA gene was successfully expressed using this vector. Domestic Hesong et  al. (2010) cloned the β-galactosidase gene from the L. acidophilus genome and expressed it in L.lactis. Positive clones were screened by 5-bromo-4-­chloro-3-indolyl β-D-galactopyranoside (X-gal). The recombinant L. lactis was passaged for 60 generations by X-gal color development screening method, and the β-galactosidase enzyme activity and specific activity were measured. The results showed that positive clones could be successfully screened by expressing active β-galactosidase and X-gal color development. The β-galactosidase specific activity assay was performed on recombinant L. lactis after 60 generations by X-gal. There was no significant difference compared with the second generation of X-gal screening (P = 0.592 > 0.05) and no significant difference compared with the erythromycin screening (P = 0.882 > 0.05). Thus, the β-galactosidase gene has good activity and stability as a screening marker. Posno and Heuvelmans (1991) cloned the xyl gene for xylose ferment of the L. pentosus MD353 into the E. coli-lactobacillus shuttle vector pLP3537 to obtain a recombinant pLP3537-xyl plasmid. Further, the plasmid was transferred to L. casei ATCC393 which could not utilize xylose. As a result, the transformant obtained the ability to utilize xylose, thereby obtaining xylose fermentation as a food-grade selective marker of Lactobacillus. 2.2.2.2  Auxotrophic Complementary Selection Markers The auxotrophic complementary selection marker is a commonly used method for constructing food-grade expression systems. In bacteria, some products encoded by genes can catalyze the basic metabolic reactions of bacteria. When these genes are deleted or mutated, the bacteria will not be able to synthesize the corresponding products, thus causing the bacteria to fail to grow normally in the original growth environment. Only by supplementing the corresponding substrate will the bacteria return to its original phenotype, which is the auxotrophy of the bacteria. These genes are cloned into a plasmid and introduced into an auxotrophic strain, which is complementary to the host bacteria, and the bacteria can restore a certain characteristic. Therefore, you can choose according to this feature. Food-grade expression vectors have been successfully constructed with using the thyA, arl, thr, supB, and supD genes.

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The thyA gene encodes a thymidylate synthase, which plays a key role in DNA synthesis. If the gene is missing, the strain cannot grow on the basic medium. The thyA gene is a safe food-grade selection marker that can be used to construct food-­ grade expression systems. At the same time, the content of thymine or thymidine in dairy products and the gut of animals (including humans) is very small, providing favorable conditions for the use of such carriers. Ross and O’Gara (1990) first constructed a food-grade vector with the thyA gene as a screening marker. However, since LAB lacking thy A gene were not constructed, the application of the gene in LAB was limited. Wang Chunfeng et al. (2001) of China Agricultural University obtained recombinant vectors by replacing the erythromycin resistance gene on plasmid pW425e with thyA. At the same time, thyA-deficient Lactobacillus acidophilus was screened as a recipient strain, and a food-grade carrier receptor system with thyA as a screening marker was obtained. The Eimeria tenella SO7 gene was introduced into the vector and expressed in LAB and E. coli. Xiong Yanwen et al. (2004) constructed a vector pSH91 applied to L. lactis. With a total length of 2337 bases, it is a food-grade vector composed of selective marker thyA gene, replicator of pWV01, and polyclonal site of plasmid pUC18. After transforming the vector into thyA-deficient L. lactis, the host bacteria returned to the wild type. Alanine is a structural component of the Gram-positive bacteria wall, and D-alanine is an indispensable component of the bacterial cell wall peptidoglycan. When it is absent, the cell wall synthesis is blocked, causing the bacteria to die. D-Alanine is usually not contained in the raw materials of industrial fermentation, and L-alanine in the medium needs to be converted into D-alanine. Alanine racemase catalyzes the conversion of D-alanine and L-alanine. Therefore, the alr gene can serve as a food-grade complementary marker. Bron and Benchimol (2002) first used homologous recombination to mutate the alr gene on the chromosome to construct a mutant strain of the alr gene, providing a receptor strain. Bron and Benchimol (2002) obtained a mutant strain of L. plantarum and L. lactis arl gene as a recipient strain by using the deletion mutation. After introducing the vector of cloned alr gene into the host bacteria, the two alr gene mutant host bacteria can grow on the medium without D-alanine. Lu Wenwei (2014) established a selective marker for the alanine racemase gene (alr). First, the alanine racemase gene of L. lactis NZ9000 and L. casei BL23 was knocked out by the temperature-sensitive plasmid pG+host9 and the suicide integration vector pRV300, respectively. The host cell itself became D-Ala auxotrophy, and then the alanine racemase gene acts as a complementary selection marker to achieve functional complementation. In the process of prokaryotic ruthenium synthesis, if the related genes are meteorite mutation, the strain will be purine auxotrophy. The product encoded by the nonsense suppressor gene supB can make up for the auxotrophy. Similarly, when the gene in the pyrimidine synthesis pathway undergoes an amber mutation, the nonsense suppressor gene supD gene product can compensate for this defect. Thus, food-grade vectors can be constructed using supB and supD as selective markers. Sorensen and Larsen (2000) constructed a new food-grade expression vector pFG200 based on the vector pFG1. The pFG200 expression vector uses supD as a selection marker and a pyrimidine auxotrophic strain as a host strain. Moreover, the

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pFG200 vector has no significant effect on the growth rate of the host bacteria and the acidification rate of the milk. However, a major drawback of such complementary systems is that specific mutations must be introduced into each mature host in a food-grade manner prior to application of the complementary plasmid. 2.2.2.3  Bacteriocin Resistance Markers Since bacteriocin is a food-grade product, its resistance genes and immune genes can be used as food-grade selection markers. The bacteriocin resistance genes that have been utilized now include the nisin resistance gene (nsr), the immune gene (nis I), and the immunogenic gene of Lactobacillus F (laf I). A strain containing the nisin resistance gene (nsr) or the immunogenic gene Nis I can grow normally on a substrate containing a certain concentration of nisin. Therefore, the nisin resistance gene or immune gene is an ideal food-grade selection marker. The nsr gene contains an open reading frame of 957 bases that encodes a protein of 318 amino acids. Von Wright et al. (1990) inserted a fragment containing the nsr gene in the cloning vector pVS34 and eliminated the chloramphenicol resistance gene originally carried in the vector and constructed a food-grade vector with NSR as a selective marker. Hughes and Mc Kay (1992) constructed a food-grade cloning vector pFM011 with NSR as a selection marker and cloned a sequence encoding bacteriophage resistance in this vector to obtain a plasmid with both nisin resistance and phage resistance, named pFM012. The vector was introduced into L. lactis LM0230, which has phage resistance. This indicates that the nsr gene can be used to directly screen transformants, thereby replacing traditional antibiotic resistance markers. The laf I gene is an immune gene of Lactobacillus F produced by L. johnsonii VPI 11088. Studies have shown that if laf I is destroyed, the strain is sensitive to Lactobacillus F. The vector pTRK434 carrying laf I was introduced into the Lactobacillus F-sensitive strain Lactobacillus johnsonii. The host bacteria restored the immunity of the bacteriocin and increased the immune tolerance by 64-fold compared with the non-transformant cells. Allison and Klaenhammer (1996) ­transformed the plasmid pTRK434 containing the laf I gene into the fermentative lactobacillus NCDO 1750. The transformant was selected using a medium containing Lactobacillus F to achieve selective labeling using laf I. 2.2.2.4  Selection Markers for Heavy Metal Resistance Some plasmids of LAB contain resistance genes of metal ions such as cadmium (Cd) and copper (Cu), and food-grade carriers have been successfully constructed using these resistance genes. Liu et  al. (1996) isolated a copper resistance gene (cuR) from L. lactis plasmid pND306 to construct a food-grade vector pND968. Wong et al. (2003) linked the cadmium ion resistance genes cadA and cadC to the S. thermophilus vector pND913 and removed the non-food-grade fragments to

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obtain the food-grade cloning vector pND919, which was successfully applied to the food-grade expression of S. thermophilus. In addition, a dual plasmid selection marker system has great potential for use in food microbial applications and has been developed and utilized by researchers. The system comprises two plasmids, one is a vector carrying a functional replicon and the foreign gene to be expressed, but no selection marker, and the other is a concomitant plasmid, and the plasmid system with the antibiotic resistance selection marker is also applied to the food-grade selectable marker of LAB. Emond et al. (2001) successfully constructed a dual plasmid system in which two plasmids dissociate the major antibiotic markers of the vector plasmid. The vector consists entirely of L.lactis and complements the phenotypic characteristics of the associated plasmid. After transformation screening, the associated plasmid is readily removed in antibiotic-free medium and remains highly stable without selective pressure.

2.2.3  Food-Grade Inducer In the process of cloning and expression of LAB, certain inducers are required. In food expression systems, it is required that the inducer must be food-grade and edible for humans, such as lactose, sucrose, nisin, and the like. Among them, nisin belongs to the wool sulfur bacteriocin, which is produced by L. lactis. The mature nisin molecule contains 34 amino acids with a molecular weight of 3510 Da. Nisin is a polypeptide substance, which is non-toxic and does not produce antigens in humans. After consumed, nisin can be inactivated by protease action in the digestive tract, so it will not change the intestinal flora structure. Nisin has been accepted as a food additive by the FAO/WHO expert committee in 1969 and is now used in more than 50 countries around the world. These properties of nisin determine that it can act as an inducer to induce the production of heterologous proteins in food-­ grade expression systems without any toxic effects on the human body and thus is a food-grade inducer. In addition to nisin, mutant derivatives of nisin and certain nisin analogs can also be used as inducers to induce the nisA promoter and can even be induced by the fermentation supernatant of nisin or its homologs or even nisin-­ producing bacteria. The using concentration of nisin is significantly lower than its minimum inhibitory concentration (MIC), ranging from 0.01 to 10  ng/mL.  This concentration does not inhibit the growth of microorganisms, even if the host does not contain the NisI and NisFEG immune systems. In the process of inducing expression, the inducing agent needs to be added in the log phase, and the expression level can be controlled within a power range of 1000 times, and there is a linear dose-response relationship between the induced concentration and the protein production level in this range. The highest production rate arrives 2 h after induction, and the yield of the target protein can reach 60% of the soluble protein.

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2.2.4  F  ood-Grade Expression System of LAB and Its Application 2.2.4.1  NICE System Eleven genes related to nisin biosynthesis are clustered into a DNA fragment about 14 kb in the order of nisA/Z, nisB, nisT, nisC, nisI, nisP, nisR, nisK, nisF, nisE, and nisG, in which nisR and nisK are two components of the regulatory system and the promoter nisA and nisF can be induced by nisin. In 1995, the Dutch Dairy Research Institute (now NIZO Food Research Institute) invented the food-grade expression system NICE (nisin-controlled gene expression system) by studying the self-­ regulating biosynthesis mechanism of nisin. Based on the promoter nisA of the nisin biosynthesis gene cluster and the two-component regulatory system gene nisRK, this system regulates gene expression through the induction of nisin. A valid nisin-induced NICE system consists of three parts: (1) Gram-positive bacteria containing the nisRK gene as host bacteria; (2) nisin, nisin analogs, or nisin mutants as inducers; (3) nisA or plasmid for the nisF promoter. (1) The working mechanism of the NICE system In the process of nisin autonomic regulation in NICE system, the histidine kinase NisK acts as a sensor for nisin, and the NisR protein acts as a regulator of transcription, which activates the transcription of the target gene. Once the extracellular nisin exists, nisin binds to the receptor NisK. Subsequently, NisK transfers the phosphate group to NisR by autophosphorylation, and the activated NisR induces the nisin operon at the nisA promoter, which induces a regulatory process shown in Fig. 2.1.

Fig. 2.1  NICE system and its mechanism

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(2) Host Gram-positive bacteria, including Lactobacillus, Streptococcus, Enterococcus, Bacillus, Leuconostoc, and particularly any of the NisR and NisK proteins that can express a certain level in Lactococcus can be used as a host for the NICE system. So far, researchers have built two series of host cells: (1) L. lactis that can produce nisin, such as L. lactis FI5876 and NZ9700; they are all modified from wild bacteria by plasmid elimination or phage elimination. Other examples include that L. lactis NZ9800 is obtained by reducing the four bases of the nisA gene; FI7332 integrates the erythromycin resistance gene into the nisA gene of FI5876. (2) Host cells that cannot produce nisin. These host cells integrate the nisRK gene into the genome, such as the commonly used host strains L. lactis NZ900 and L. lactis NZ3900. For nisin-producing bacteria, nisin not only induces its own production but also induces the expression of its target gene. For non-nisin-producing bacteria, nisin only induces the expression of the target gene. In addition to integrating nisK and nisR into the genome, the researchers designed multiple plasmids such as pNZ9520 and pNZ9530 to apply the NICE system to other hosts based on the plasmid pAMβ1 for wide host applications. (3) Plasmid vector The plasmid vector requires a promoter comprising nisA or nisF for nisin-­induced expression, and, to date, more than 20 related plasmid vectors have been constructed for expression of Gram-positive bacteria, even E. coli. Commonly used plasmid vectors are divided into five categories: One type is a transcriptional fusion vector, such as the common pNZ8020. The vector comprises a promoter of nisA, a NisR binding site, and the ribosome binding site. The polyclonal site is located behind the promoter region, and the gene expression needs its own initiation codon. The second type is a translational fusion vector, such as the commonly used pNZ8048. After the Nco I restriction endonuclease is inserted into the promoter region, since it contains ATG as a starting site, the inserted target gene can achieve high transcription efficiency and ultimately guarantee the expression level of target protein. Studies have shown that the use of this type of fusion vector to express β-glucosidase, its expression activity is six times higher than that of the ­transcriptional fusion vector. In the third mode, the promoter of Nisin A and the target gene were ligated in vitro and then cloned into the target vector together. The vector was used to successfully express germicidin PA-1 and Escherichia coli V. The fourth mode is a two-plasmid NICE system consisting of two compatible plasmids: one plasmid carries the nisRK regulatory gene and the other carries the target gene under the control of the nisA promoter. Its inducibility is the same as in L. lactis, but the time to reach the highest protein production level is significantly longer than L. lactis. The fifth mode is a vector for secretion expression, such as pNZ8110, which uses a signal peptide secreting the protein Usp45 for secretion expression of the protein of interest. (4) Application of NICE system The NICE system has the characteristics of high-efficiency expression and the value of practical application. It can be applied to the high-efficiency expression of

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important proteins in biotechnology, such as bacteria, viruses and eukaryotic antigens, cytokines, bacteriocins, and membrane proteins in various LAB. In the cellular metabolic pathway, it is also possible to precisely modulate the activity of key enzymes and control the production of metabolites of interest. 1. Overexpression of protein Using the NICE system, proteins from different sources can be overexpressed to help people study the properties of proteins or enzymes. At the same time, because it is derived from Gram-positive bacteria, there is no inclusion body when expressing proteins with close relationship. For example, de Ruyter and Kuipers (1996) used the NICE system to homologously express the pepN gene, and its protein expression reached 50% of that of soluble cells, and no inclusion bodies were produced. 2. Expression of membrane protein Under normal physiological conditions, the natural expression level of membrane proteins is relatively low, and a large number of its heterologous expression has become a restrictive condition for the study of function and structure. Combined with the difficulty of purification and crystallization of membrane proteins, heterologous expression of membrane proteins has become a bottleneck in membrane protein-related research due to its hydrophobic properties and toxicity to host cells. The NICE system has certain advantages for overexpressing membrane proteins: (1) the proteolytic ability of L. lactis is weak; (2) the nisA promoter is a strong and rigorous promoter, which can make the expression of membrane proteins under reasonable control; and (3) the expressed membrane protein can be dissolved by a surfactant. The membrane proteins of many prokaryotic cells have been expressed by the NICE system, such as ABC transporter, MFS transporter, peptide transporter, etc., and the expression level can reach 10% of membrane protein. Bernaudat and Frelet-Barrand (2011) expressed several eukaryotic membrane proteins derived from Arabidopsis thaliana in L.lactis, including polycopper oxidase and naphthoquinone oxidoreductase. Some scholars have tried to promote the expression of eukaryotic membrane proteins through systematic transformation, such as the addition of fusion proteins, but the related research is generally relatively difficult and lagging. 3. Secretory protein expression and surface display system Food-grade lactobacillus is used in the production of protein in industrial fermentation but is also a suitable candidate for the delivery of heterologous proteins in food or the digestive tract. Antigens, vaccines, drugs, and the like can be expressed in LAB by secreting protein expression and surface display systems. Enouf et al. (2001) expressed the recombinant immunogen protein NSP4 using the NICE system. The results showed that the NICE system can produce rNSP4 protein with antigen and immunogenic properties and has the same immunogenic properties as viral proteins. Bermudez-Humaran and Langella (2002) used this system to produce IL-12. Experiments show that the activity of IL-12 produced by the system is

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similar to that of commercial IL-12, which can be used in mass production and in vivo application of IL-12. (1) Other bacteriocin-induced food-grade expression systems In addition to the NICE system, some other LAB have a similar mechanism for bacteriocin production. But unlike the NICE system, the main function of peptide secretion is not the bacteriocins but the expression of the whole operating unit stimulated by external hormones. Sorvig et al. found a bacterial production operon in L. sakei and used it to construct a series of expression vectors. β-Glucuronidase and aminopeptidase were used as expression genes, respectively, and the expression effects of different vectors were compared. It was found that using pSH71 vector and sakacin P promoter, and using sakacin P as the inducer, the expression level of proglucuronidase was ten times that of the wild-type strain. However, the expression level of aminopeptidase accounted for half of the intracellular soluble protein (Sorvig and Gronqvist 2003; Sorvig and Mathiesen 2005). Nguyen and Nguyen (2012a, b) induced the expression of β-galactosidase (LacZ) of L. bulgaricus DSM 20081 in L. plantarum using the plasmids pSIP403 and pSIP409 constructed above. By adding the corresponding inducing peptide IP-673, the expression level of LacZ reached 53,000  U/L, accounting for 63% of intracellular soluble protein. In the same year, the author used these systems to express the chitosanase of B. licheniformis in L. plantarum with an expression level of 5 mg recombinant protein per liter of medium. Therefore, the above system is very valuable for the expression of LAB, particularly the expression of Lactobacillus proteins. (2) Sugar-induced food-grade expression system Through genome-wide sequencing of a large number of LAB, it can be known that the LAB genome contains a plurality of sugar metabolism-related gene clusters, which can ensure that LAB grow on a medium containing various sugars as a carbon source. In the sugar-induced expression system of lactobacillus, the sugar selective marker is mainly used. As a food-grade inducer, sugar can be used as a marker for non-antibiotic resistance. Since the lactose operon is well studied, most ­expression systems use lactose as a selective marker to induce gene expression. Many lactose-induced food-grade expression systems were constructed by lacA promoter. Eaton et al. (1993) introduced the reported gene luxAB behind the promoter lacA and expressed L.lactis as the host bacteria. When lactose was used as the carbon source, the activity of the promoter was increased by seven times. Platteeuw et al. (1996) constructed a vector containing the lac A promoter with the lacF gene as a selection marker and inserted a transcription terminator of pep N into the vector to increase the stability of the vector and cloned DNA. The β-glucuronidase gene (gusA) was cloned downstream of the lac A promoter and introduced into the plasmid-­free L. lactis NZ3000 receptor. The strain could be grown in lactose-­ containing medium, and the gus A gene was highly expressed with lactose induction. The food-grade plasmids pLEB590 and pLEB600 with L casei as host bacteria and pSH71 replicator and repA gene as the skeleton, respectively, carrying P45 and PpepR promoter, and screening with lactose selection markers. The proline imino-

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peptidase gene pep I was successfully expressed with these two plasmids (Takala and Saris 2002). Payne and MacCormick (1996) first inserted the lactose operon into the chromosome of L. lactis MG5267 and then integrated the lysin gene derived from Listeria phage LM-4 into the lac promoter, and its expression was controlled by the lac promoter. And ultimately the expression of the LM-4 lysin gene is controlled by lactose. In addition, there are carbon sources such as xylose sucrose as selection markers. Using D-xylose as a selective marker, the e-lactobacillus lactobacillus shuttle vector pLP3537 was cloned and expressed the xylose reductase gene cluster xyl on the chromosome of L. pentosaceus, which enabled the recombinant lactobacillus to have the ability to utilize D-xylose. The possibility of using xylose as a selective marker was confirmed. Lokman et al. (1991) linked the gene of chloramphenicol acetyltransferase to the downstream of xylose promoter and introduced it into Lactobacillus pentosaceus, which were cultured in the medium containing xylose and glucose, respectively. The results showed that the activity of chloramphenicol acetyltransferase was 60~80 times that of glucose culture under the induction of xylose. Xylose-inducible expression system (XIES) is a sugar-dependent expression system. By using xylose-inducible promoter PxylT, intracellular or secreted proteins can be produced. The first use of this expression system was the heterologous expression of the S. aureus nuclease gene (intracellular and extracellular expression using the signal peptide of the Usp45 protein) in L. lactis NCDO2118. When the system is induced with an appropriate inducer, such as xylose or glucose, a large amount of the target protein can be expressed. The system can be a good complement to the NICE system, as its induction is tightly regulated. At the same time, when the system is used for protein secretion expression, the secreted protein is not observed to decompose (de Azevedo and Karczewski 2012). Leenhouts and Bolhuis (1998) constructed a food-grade expression system with sucrose as the selective marker by the integration of lactobacillus. The gene scrA/ scrB encoding sucrose and hydrolase was cloned into lactococcal plasmid pWV01. By using the copy of this plasmid, lactobacillus expression vector pINT124 pINT125 with sucrose as the screening marker was successfully constructed. Mahmoud and Sameh (2011) used this expression vector to express the bacteriocin gene pctA in L. lactis. (3) Zinc-induced expression system Llull and Poquet (2004) designed a new expression system PZNZitR-driven heterogeneous expression system based on the promoter of zit operator. The zit operon is primarily involved in zinc regulation. When zinc is absent, such as when the cell is starving, the operator’s transcriptional repressor leaves its repressor site, and RNA polymerase can bind to the PZN promoter to induce subsequent protein expression. By expressing two target proteins, it was shown that this expression system is highly inducible, but the amount of protein obtained is lower than that of the NICE system. Recently, a new zinc-induced expression system, Zirex, can induce protein expression in L. lactis. The promoter is induced by zinc with a concentration below

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toxicity, and the SczA repressor regulates the activation of PczcD and leads to the induced expression of subsequent fluorescent proteins. In addition, the NICE system and the Zilex system are combined to produce different proteins at different times in one microorganism. (4) Environmentally induced food-grade expression system When LAB express proteins in vitro, they can induce protein expression by adding specific inducers. However, in  vivo, the relevant inducers cannot be supplemented to the microorganisms at the appropriate concentration to achieve the induced concentration. To compensate for this deficiency, researchers have successively studied the stress-inducible expression system (SICE) and P170, which induce the expression of specific proteins in the gut. Benbouziane and Ribelles (2013) found a stress-inducible expression system in L. lactis and studied it. The system is controlled by the pGroESL promoter and expresses the target protein only under specific stress conditions such as high salt, high temperature, and low pH. In addition, the system has great potential for the expression and transport of vaccine proteins at specific sites in the body for the treatment of inflammatory bowel disease (IBD) and human papillomavirus. Another major feature of LAB is the production of lactic acid during growth. Based on this feature, a pH-induced expression system can be constructed. Madsen et al. (1999) found that the promoter P170 regulated by pH value was induced in the culture environment with a pH value of 6.5~6.0 and the bacteria entering the stable period. Through genetic manipulation, the deletion of 72 bases at the front end of P170 mrna does not affect the promoter regulation, but increases the pH induction effect of the promoter by 150 times and the gene expression level accordingly. Temperature is also an important factor influencing protein expression. Nauta et al. (1997) designed a thermo-unstable Rro repressor variant Rrol2 using lac Z as a reporter gene to construct a temperature-controlled expression system. When the temperature was increased from 24 to 42 °C, the amount of β-glucosidase expression was increased by 500 times.

2.3  Gene Knockout System in Lactic Acid Bacteria 2.3.1  T  he Mechanisms and Characteristics of Gene Knockout Vector A gene knockout is a genetic technique in which foreign gene is inserted into site-­ specific position, and its purpose is to make the target gene have a directed change. This technique conquered the defect of random mutation (blindness and contingency) and is an ideal method for genetically modified organisms. Compared with other organisms, the development of gene knockout in LAB is relatively low speed owing to thicker cell wall, low conversion rate, and less vectors. The gene knockout

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technique can be used to modulate the metabolic responses, decrease ferment cost, and enhance the production and purity of the aimed product. Moreover, it could be used to study the structure and function of genes. So far, the most common ways used in gene knockout depend on a traditional homologous recombination, and a few studies adopt means of site-specific recombination, homologous recombination involving single-stranded DNA substrate, and CRISPR/Cas system. 2.3.1.1  Gene Knockout Based on Homologous Recombination The theoretical basis of classic gene knockout technique is based on homologous recombination. Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Based on the above theory, the targeted gene deletion, insertion, and missense mutation could be reached via homologous recombination between a recombinant vector and host genome in the host cell. Considering the difference in the number of homologous recombination, the gene knockout can be simply divided into two types: one-time homologous recombination and twice homologous recombination. Leenhouts and Bolhuis (1998) showed that the targeted gene of L. lactis was inactivated via insertion of foreign gene by the single-crossover integration system. In addition, prolyl dipeptidyl aminopeptidase gene was inactivated in the chromosomal of L. helveticus via recombination between the pepXP-derived repeat (Bhowmik and Steele 1993). Compared with one-time homologous recombination, two recombination reactions were observed in the latter, and it caused two different results, in which the targeted gene was completely deleted in one result and the gene was disrupted in other. Ferain et al. (1996) demonstrated that two lactate dehydrogenases played a major impact on peptidoglycan precursor synthesis in L. plantarum via two recombination reactions. L. citreum is an important lactic acid bacterium in fermented foods, but dextran production often causes undesired ropiness. To prevent this side effect, a dextransucrase knockout mutant was constructed by Jin and Li (2014), and it indicated that L. citreum dextransucrase not only synthesizes dextran for cell protection but also provides fructose as an important carbon source for cell growth.

2.3.2  G  ene Knockout Based on Improved Homologous Recombination 2.3.2.1  H  omologous Recombination with a Counterselectable Selection Marker Homologous recombination can be used for gene disruption, but gene targeting is inefficient because of low homologous recombination frequency in the secondary reaction, which leads to some difficulty in screening the mutants. To solve the above

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gene X mutant strain

Fig. 2.2  Gene deletion through two-hybrid system

problem, the use of a reasonable counterselectable selection marker is helpful in screening the correct mutant (Fig. 2.2). It means that the mutant containing this counterselectable selection marker can be died in an exact selection condition, and the mutant without this selection marker is able to survive in the same condition. The use of selection marker could reach the aim of rapid screening of exact mutant without introducing new gene. The fourth intermediate in the pathway was identified to be orotate, which was also confirmed that it could not be utilized as a pyrimidine source by most L. lactic strains due to lack of the special transporter (Kilstrup, Hammer et  al. 2005). Furthermore, the gene responsible for utilizing orotate as a sole pyrimidine source by an auxotrophic mutant has been already identified to be oroP, which also lead to its sensitivity to its toxic analog 5-fluoroorotate. Based on the above property, a new selection vector pCS1966 was constructed by Solem and Defoor (2008), considered as an efficient tool for special strain construction, which was proper for sequence-­ specific integration based on homologous recombination and also that in a bacteriophage attachment site. In addition, another counterselectable selection marker gene that was always used in LAB is the upp gene that encodes uracil phosphoribosyltransferase results in resistance to 5-fluorouracil (Martinussen and Hammer 1994). The main drawbacks of using the upp is that this gene is found in almost every organism and that 5-fluorouracil may be toxic even in a upp mutant (Martinussen and Hammer 1995). Previous study showed that the upp gene deletion did not affect pyrimidine metabolism and biological characteristics. In 2009, a 3.0-kb pOR-based counterselectable integration vector, which beared a upp expression cassette, called pTRK935, was constructed by Goh et al., and this tool was identified to be very valuable to determine how L. acidophilus performed its probiotic function (Goh and Azcarate-Peril

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2009). Furthermore, a temperature-sensitive replicon called pWV01 was also adopted by Song and Cui (2014) in order to construct two integration plasmids with chloramphenicol-resistant using upp gene as a counterselective marker for L. casei ATCC393 and L. lactis MG1363. Many results have showed that no significant difference existed between the wild-type and mutant lactic acid bacteria except for 5-FU resistance through determining the genetic stability, growth curve, carbon utilization, and also scanning electronic microscopy. Although the upp gene was widely used in the screening of resistant strains, some disadvantages still exist in the gene knockout process. Because the upp gene widely exists in some bacteria and may participate in some important metabolic responses, the upp gene deletion in the host cell could cause some influence in the growth. 2.3.2.2  H  omologous Recombination with a Temperature-Sensitive Type System The replicon of a thermosensitive plasmid is a thermosensitive replicon, which can make the plasmid replicate at lower temperature and shut off at elevated temperature. This genetic system for homologous recombination allows the thermosensitive plasmid massive replication at lower temperature and increases the transformation frequency. Maguin et  al. (1992) isolated a replication-thermosensitive mutant pVE6002 and used it for the construction of a thermosensitive plasmid. The result showed that its transposition frequencies were about 1%, which were higher than wild type. Biswas et  al. (1993) constructed a thermosensitive broad host range rolling-­circle plasmid, pG+ host5, which contains a pBR322 replicon for propagation in E. coli at 37 °C, and developed a protocol for gene replacement. Cultures were first maintained at 37 °C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 °C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. Atiles et al. (2000) disrupted IlvE activity in L. lactis LM0230 via a thermosensitive plasmid, indicating that IlvE is the only enzyme capable of synthesis of Ile and Val from their biosynthetic precursors. To study the role of propanediol dehydrogenases pdh30 during glycerol metabolism, the gene disruption mutant L. brevis INIA ESI38::pORI28-pdh30 was constructed by Langa and Arques (2015) by site-­ specific integration of plasmid pORI28 and the temperature-sensitive plasmid pVE6007. HPLC analysis of the glycerol fermentation products showed an involvement of the pdh30  in the 3-hydroxypropionic acid (3-HP) biosynthesis. The temperature-­sensitive plasmid is a valuable tool in the gene knockout system, and it could be used in some Gram-positive bacteria. However, this genetic system is unstable, and its high-temperature condition also limits its application in LAB.

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2.3.3  G  ene Knockout System Based on Site-Specific Recombination It is known for us that site-specific recombination possesses significant conservative characteristics, and this recombination would occur in these DNA strands with segments possessing at least a certain degree of sequence homology. A recombinase enzyme and also the corresponding recombination sites are enough for some certain systems to carry out all the reactions; however, many accessory proteins and/or the corresponding sites would be necessary. Cre/loxp and TP901–1/att family are considered as the two most common site-specific systems according to the amino acid sequence homology and mechanistic relatedness. 2.3.3.1  cre/loxp Recombination System cre/loxp recombination system just included a single enzyme, called cre recombinase, recombining a pair of short sequences, also known as lox sequence, in which the enzyme and the lox site are separated from bacteriophage P1. There are 4 subunits and 2 domains (C-terminal and N-terminal domains) among Cre protein with 343 amino acids in total. The structure of C-terminal domain is similar to that of integrase family protein in lambda phage (Sternberg and Hamilton 1981). LoxP is a special site on bacteriophage P1, which consisted 34 bp, including two sets of 13-bp symmetric sequences with asymmetric 8-bp sequence in the middle of the sequence, in which the symmetric 13-bp structure is palindromic and the middle 8-bp sequence is not. This special structure provided a certain direction for loxP. Generally, the loxP site existed in pairs when genetic manipulation was performed. The behavior of floxed sequence depended on the orientation of loxP sites, while it will be excised with loxP site in the same direction and it will be inverted with loxP sites in the opposite orientation. The Cre recombinase can not only recognize wild LoxP sequence but also recognize the mutant LoxP sequence in which a set of symmetric 13-bp sequences or asymmetric 8-bp sequences have slight changes, which further expand its application range. Lambert and Bongers (2007) constructed an effective mutagenesis vector that contains Cre/Lox cassette and disrupted bile salt hydrolase bsh gene and α-galactosidase melA gene in L. plantarum WCFS1. Remus and Kranenburg (2012) studied the effect of four clusters of genes on surface polysaccharide production by using the Cre/LoxP system. The role of selection marker gene is easy to screen the exact mutants, but its existence could also cause some unnecessary changes in the expression of its upstream and downstream genes in the genome. Considering the characteristics of the Cre/LoxP system, it is always used to remove the market gene in the genome. Zhu and Zhao (2014) constructed the thymidylate synthase gene (thyA)-deficient strain derived from L. lactis NZ9000 using the Cre-loxP recombination system and used it as a food-grade selection marker for L. lactis in food and industry applications.

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2.3.3.2  TP901-1/att Recombination System TP901-1/att recombination system can stably replicate, and site-specific integrate their DNA into the host chromosome, which is constructed by integrating TP901-1, lactococcal temperate bacteriophage, into the chromosome of Lactococcus subspecies. The phage attachment site (attP) and the chromosomal attachment site (attB) can be integrated into new hybrid sites: attL and attR. Zhu and Zhao (2014) used the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in L. lactis. A genetic tool special for repetitive, marker-free, and site-specific integration was constructed by Petersen and Martinussen (2013) in L. lactis. In this tool, a vector with nonreplicating plasmid, called pKV6, included a phage attP, useful to be integrated into a bacterial attB. i pKV6 would be integrated into the chromosome of the host with being flanked by attL and attR hybrid attachment sites in high frequency just when the corresponding vector was transformed into L. lactis with the ability to express the phage TP901-1. loxP recombinants would be selected based on the 5-fluoroorotic acid just when a plasmid responsible for expressing cre recombinase lacks the replicating ability in L. lactis. The gene involved in controlling xylose utilization was usefully adopted to be integrated into the chromosome of L. lactis strain MG 1363 in two steps in order to determine whether the constructed system would perform its function. 2.3.3.3  Linear Transformation Knockout System Linear DNA, containing the mutated or deleted gene flanked by homologous regions of the chromosome, is transformed into recombination-proficient strains, which is one of the hot spots on study of gene knockout method. It’ s reported that a recombinant E. coli strain was constructed through replacing the RecBCD function with phageλ’s Red function via recombining the chromosome with short linear DNA fragments at a greatly elevated rate by Murphy (1998), with at least 70-fold higher than that by arecBC, sbcBC, or recD strain. The rate is at least 70-fold higher than that exhibited by arecBC sbcBC or recD strain. Compared with double crossover homologous recombination, the homologous arm of the above method is about 35~50  bp, and it has a high recombination efficiency. However, this method is always used in Gram-negative bacteria, and few studies were found in LAB. Recently, single-stranded DNA recombination is always used in the gene knockout system in LAB. The single-stranded DNA binding proteins produced by redβ and redT gene are always used as recombinases that can promote annealing of complementary DNA strands. Van Pijkeren and Britton (2012) constructed the plasmid pJP042 and pJP005 to assess the ability of the L. reuteri RecT protein to support recombineering. It showed that the intrinsic vancomycin resistance of L. reuteri was significantly lower in the mutations, and the minimum inhibitory concentration of vancomycin was reduced from >256 to 1.5 mu g/mL by creating a single AA change in the d-Ala-d-Ala ligase enzyme. Compared with classic double crossover

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homologous recombination, ssDNA recombineering exhibits more advantages, such as easy operation, high recombination efficiency, not restricted by restriction enzyme cutting site, shorter homologous arms, and acquisition by direct PCR.

2.3.4  New Gene Knockout Method With the development of molecular biology, some new gene knockout methods have attracted lots of attention, such as RNA interference, transcription activator-­ like effector nuclease technique, zinc finger nuclease gene targeting technique, CRISPR/Cas system, and so on. Among them, CRISPR/Cas system could be used in the gene knockout of lactic acid bacteria in the future. The exact mechanism of CRISPR/Cas system is that CRISPR-derived RNA form a compound of tracrRNA/ crRNA with trans-activating RNA, which guides the Cas 9 nuclease cut the DNA target specified by the guide RNA. By designing the above two RNA, it can form a leading indicator short guide RNA that guide Cas nuclease cut the targeted DNA. As a RNA-oriented dsDNA binding protein, Cas9 nuclease is one of the first known unifying factors that collectively recognize RNA, DNA, and protein. The compounds of protein and Cas9 nuclease-null can specifically bind any DNA sequence together with moderate amount of sgRNA. The terminal of sgRNA can bind with the target DNA without affecting the combination between Cas9 and the target DNA. Therefore, Cas9 can deliver any compounds of protein and RNA to any DNA sequence (Liu and Xu 2015). CRISPR-Cas9 selection system has been identified to be the most ideal system used to edit some genes in lactic acid bacterium for its high efficiency (Oh and van Pijkeren 2014), in which the efficiency in recovering subtle changes in the genome would range from 90% to 100%. CRISPR-Cas9 has been successfully used in recombineering, for example, the codon saturation mutagenesis in L. reuteri chromosome and also identification of such low-efficiency events as oligonucleotidemediated chromosome deletion. CRISPR-Cas 9 would be also useful in identifying the recombinant bacterial cells with low recombineering efficiency and also eliminating the need for ssDNA recombineering optimization procedures.

2.3.5  The Common Vectors and Their Application The gene knockout can be used to modulate the metabolic flux, interfere with the production of unnecessary secondary metabolism, reduce ferment cost, and raise the production and purity of target product. In addition, it can also be used to study the structure and function of genome of LAB and its beneficial mechanisms. Increasing reports have focused on the peptidoglycan-degrading enzyme involved in a range of bacterial processes and also the interaction between the host and the microbe; however, the function of this enzyme in lactobacilli is still unknown.

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Systematic gene deletion system has been adopted by Rolain and Bernard (2012) in exploring the functional role of the peptidoglycan hydrolase (PGH) complement located in the genome of L. plantarum. The role of N-acetylglucosaminidase Acm2 and NplC/P60 D, L-endopeptidase LytA, as key determinants in the morphology of L. plantarum has been shown in that study. eps gene cluster has been reported in L. johnsonii requiring for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 which was useful in constructing a capsular layer. epsA is the first gene of the cluster with putative function as transcriptional regulator, and the function has been identified by the result that deletion of epsA gene would result in complete loss of the ability of growing the EPS-1 and EPS-2 on the cell surface, and this ability would be fully restored just when this gene was complemented. All these results showed that epsA gene could regulate the EPS production positively. Reutericyclin is a unique antimicrobial tetramic acid produced by some strains of L. reuteri, but its synthesized mechanism is still unclear. Lin and Lohans (2015) showed that deletions of rtcNRPS or rtcPhlABC in L. reuteri TMW1.656 abrogated reutericyclin production but did not affect reutericyclin resistance. To raise the production of diacetyl in LAB, Platteeuw and Hugenholtz (1995) cloned the als gene for alpha-acetolactate synthase of L. lactis MG1363 in a multicopy plasmid under the control of the inducible L. lactis lacA promoter. Furthermore, the effect of alpha-acetolactate synthase overproduction on the formation of end products in various L. lactis strains was also determined under different fermentation conditions. These metabolic engineering studies suggest that more than 80% of the lactose can be converted via the activity of the overproduced alpha-acetolactate synthase in L. lactis.

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applied AG013  in subjects with locally advanced head and neck cancer receiving induction chemotherapy. Cancer 119(24):4268–4276 Lin XB, Lohans CT (2015) Genetic determinants of reutericyclin biosynthesis in Lactobacillus reuteri. Appl Environ Microbiol 816:2032–2041 Liu B, Xu H (2015) CRISPR/Cas: a faster and more efficient gene editing system. J  Nanosci Nanotechnol 153:1946–1959 Liu CQ, Leelawatcharamas V, Harvey ML et al (1996) Cloning vectors for lactococcus based on a plasmid encoding resistance to cadmium. Curr Microbiol 33:35–39 Liu DQ, Qiao XY, Ge JW et al (2011) Construction and characterization of Lactobacillus pentosus expressing the D antigenic site of the spike protein of transmissible gastroenteritis virus. Can J Microbiol 57(5):392–397 Llull D, Poquet I (2004) New expression system tightly controlled by zinc availability in Lactococcus lactis. Appl Environ Microbiol 70(9):5398–5406 Lokman BC, Santen PV, Verdoes JC et al (1991) Organization and characterization of three genes involved in d -xylose catabolism in Lactobacillus pentosus. Mol Gen Genomics 230(1):161–169 Maassen CB, Laman JD, den Bak-Glashouwer MJ et  al (1999) Instruments for oral disease-­ intervention strategies: recombinant Lactobacillus casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis. Vaccine 17(17):2117–2128 Madsen SM, Arnau J, Vrang A et al (1999) Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis. Mol Microbiol 32(1):75–87 Maguin E, Duwat P, Hege T et al (1992) New thermosensitive plasmid for gram-positive bacteria. J Bacteriol 174(17):5633–5638 Mahmoud KT, Sameh EM (2011) Heterologous expression of pctA gene expressing propionicin T1 by some lactic acid bacterial strains using pINT125. Alexandria University, Netherlands Mannam P, Jones KF, Geller BL (2004) Mucosal vaccine made from live, recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes. Infect Immun 72(6):3444–3450 McKay LL, Baldwin KA (1990) Applications for biotechnology: present and future improvements in lactic acid bacteria. FEMS Microbiol Rev 7(1–2):3–14 Meazza R, Gaggero A, Neglia F et al (1997) Expression of two interleukin-15 mRNA isoforms in human tumors does not correlate with secretion: role of different signal peptides. Eur J Immunol 27(5):1049–1054 Meijerink M, Wells JM, Taverne N et al (2012) Immunomodulatory effects of potential probiotics in a mouse peanut sensitization model. FEMS Immunol Med Microbiol 65(3):488–496 Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180(8):2063–2071 Narita J, Okano K, Kitao T et al (2006) Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch. Appl Environ Microbiol 72(1):269–275 Nauta A, van den Burg B, Karsens H et al (1997) Design of thermolabile bacteriophage repressor mutants by comparative molecular modeling. Nat Biotechnol 15(10):980–983 Nguyen HA, Nguyen TH (2012a) Chitinase from Bacillus licheniformis DSM13: expression in Lactobacillus plantarum WCFS1 and biochemical characterisation. Protein Expr Purif 812:166–174 Nguyen TT, Nguyen HA (2012b) Homodimeric beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization. J Agric Food Chem 607:1713–1721 Nielsen H, Engelbrecht J, Brunak S et al (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10(1):1–6 Norton PM, Wells JM, Brown HW et al (1997) Protection against tetanus toxin in mice nasally immunized with recombinant Lactococcus lactis expressing tetanus toxin fragment C. Vaccine 15(6–7):616–619

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Schwarzer M, Repa A, Daniel C et al (2011) Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization. Allergy 66(3):368–375 Sibakov M, Koivula T, von Wright A, Palva I (1991) Secretion of TEM beta-lactamase with signal sequence isolated from the chromosome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol 57(2):341–348 Smiley MB, Fryder V (1978) Plasmids, lactic acid production, and N-acetyl-D-glucosamine fermentation in Lactobacillus helveticus subsp. Appl Environ Microbiol 35(4):777–781 Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228(4705):1315–1317 Solem C, Defoor E (2008) Plasmid pCS1966, a new selection/counterselection tool for lactic acid bacterium strain construction based on the oroP gene, encoding an orotate transporter from Lactococcus lactis. Appl Environ Microbiol 7415:4772–4775 Song L, Cui H (2014) Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid. J Microbiol Methods 102:37–44 Sorensen KI, Larsen R (2000) A food-grade cloning system for industrial strains of Lactococcus lactis. Appl Environ Microbiol 664:1253–1258 Sorvig E, Gronqvist S (2003) Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum. FEMS Microbiol Lett 2291:119–126 Sorvig E, Mathiesen G (2005) High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors. Microbiology 151(Pt 7):2439–2449 Steidler L, Hans W (2000) Treatment of murine colitis by Lactococcus lactis secreting interleukin­10. Science 2895483:1352–1355 Svensson M, Waak E, Svensson U et al (2005) Metabolically improved exopoly- saccharide production by Streptococcus thermophilus and its influence on the rheological properties of fermented milk. Appl Environ Microbiol 71(10):6398–6400 Takala TM, Saris PE (2002) A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI. Appl Microbiol Biotechnol 594–5:467–471 van Asseldonk M, Rutten G, Oteman M et al (1990) Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 95(1):155–160 van Pijkeren JP, Britton RA (2012) High efficiency recombineering in lactic acid bacteria. Nucleic Acids Res 4010:e76 Vandenbroucke K, Hans W, Van Huysse J et al (2004) Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology 127(2):502–513 Vandenbroucke K, de Haard H, Beirnaert E et al (2009) Orally administered L. lactis secreting an anti-TNF nanobody demonstrate efficacy in chronic colitis. Mucosal Immunol 3(1):49–56 Vescovo M, Bottazzi V, Sarra PG et  al (1981) Evidence of plasmid deoxyribonucleic acid in Lactobacillus. Microbiologica (Bologna) 4(4):413–419 von Wright A, Wessels S, Tynkkynen S et al (1990) Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin resistance determinant. Appl Environ Microbiol 56:2029–2035 Watterlot L, Rochat T, Sokol H et al (2010) Intragastric administration of a superoxide dismutase-­ producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice. Int J Food Microbiol 144(1):35–41 Wei CH, Liu JK, Hou XL et  al (2010) Immunogenicity and protective efficacy of orally or intranasally administered recombinant Lactobacillus casei expressing ETEC K99. Vaccine 28(24):4113–4118 Wells JM, Wilson PW, Norton PM et al (1993) Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge. Mol Microbiol 8(6):1155–1162

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Chapter 3

Comparative Genomic Analyses of Lactic Acid Bacteria Wei Chen and Hongchao Wang

3.1  Interspecific Evolution of the Lactobacillus Genome Genetic diversity is the sum of the genetic variation between different species and within the same population. The genetic information of an organism is stored in the DNA sequences of chromosomes and organelle genomes. Lactobacillus keeps its traits stable by accurately duplicating its DNA and passing on the genetic information generation by generation. However, many factors can affect the accuracy of DNA replication, including external factors and internal factors, which together lead to different levels of genetic variation. These accumulated genetic variations enrich the genetic diversity. Genetic polymorphism is not only the result of evolution, but also the premise of evolution.

3.1.1  E  volution of Lactobacillus Based on Comparison of 16S rRNA Genes In bacterial classification, 16S rDNA sequence analysis is a necessary and most commonly used identification and analysis method, and is now the most basic detection index for studying bacterial evolution. Prokaryotes mainly contain 23S rRNA, 16s rRNA and 5S rRNA, which contain about 2900 bases, 1540 bases and 120 bases respectively. Since the 16S rRNA sequence is the most conserved, it has become the most fundamental molecular indicator for the study of bacterial classification, identification and evolution. Besides highly conserved regions, 16S rRNA sequences also contain relatively variable regions, and the homology of conserved regions is W. Chen (*) · H. Wang (*) Jiangnan University, Wuxi, China e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. and Science Press 2019 W. Chen (ed.), Lactic Acid Bacteria, https://doi.org/10.1007/978-981-13-7832-4_3

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the basis to determine the classification status of bacteria. This foundation reflects the relationship between bacteria, while the specific sequences of the variable regions reveal the differences between bacteria. By comparing the highly conserved and relatively variable 16S rDNA sequences, the taxonomic position of bacteria and the evolutionary relationship between bacteria can be determined. Different bacterial classification results in changes in the sequence of the 16s rRNA variable region, but the constant region is basically conserved, so this method can not only reflect the differences between different species, but also make it relatively easy to obtain the sequence by sequencing technology, which has been accepted by scientists. After isolation, purification and culture of lactic acid bacteria, total DNA and ribosomal RNA were extracted. Then16S rDNA sequence was amplified by polymerase chain reaction (PCR) through probe hybridization or 16S rRNA specific primer. The sequence information of 16S rDNA gene was compared with the sequence data in the database to determine its position in the evolutionary tree, and then the species of lactobacillus was identified. However, the public database contains public and unpublished sequence information (Olsen et al. 1991; Cole et al. 2009), also contains a lot of poor quality and inaccurate sequence information, and even incorrect comments, which will have a negative impact on the application of the method. Since the 1880 s, 16 s rRNA sequence analysis as the reference of bacterial taxonomy was gradually applied to classification and classification greatly influenced the professional term. The variable region analysis of 16S rRNA sequences has been widely used in the classification study of lactobacillus, which has led to the classification of some undefined taxonomic groups based on phenotypic characteristic systems, and also found many homologous mutant lactobacillus groups (Holzapfel et al. 2001). Zheng Huajun (2010) constructed a phylogenetic tree of 11 strains of 9 strains of lactobacillus, and found that the 16S rRNA sequence could not distinguish the sequences of different strains between the same lactobacillus, and all species were divided into 3 groups of lactobacillus, lactobacillus and streptococcus (Zheng Huajun 2010). Due to differences in mutation rate and gene level transfer, differences in 16S rRNA gene sequences cannot accurately reflect the phylogenetic relationship (Bakker et  al. 2007). So scientists hope to reconstruct phylogenetic trees by encoding gene sequences.

3.1.2  E  volution of Lactobacillus Based on Butler Gene Comparison In past studies, scientists have used protein-coding genes as phylogenetic markers to classify and evolve lactobacilli. Single-locus sequence analysis (SLSA) of protein-­ coding genes can obtain higher taxonomic resolution and analysis speed is higher than 16S rRNA gene analysis. Steward-type gene sequence analysis is an important component of lactobacillus SLSA method (Bruyne et  al. 2009; Ehrmann et  al. 2009), which is superior to 16S rRNA gene sequence analysis in terms of taxonomic

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resolution. When the genetic sequence of sodA(manganese superoxide dismutase) was analyzed, the Streptococcus infantarius subsp. Coli in the genus Streptococcus was reclassified as a new species Streptococcus lutetiensis, while other groups of Streptococcus pasteurianus (Poyart et  al. 2002) were proposed. However, subsequent studies by Schlegel et al. (2000) proved that neither streptococcus Paris nor parabiosa were new species. Obviously, the difference between the inside and outside of the protein-coding gene can identify the phylogenetic relationship between the individual species and the strain. However, the level of gene transfer will change the development of the system, so there are many defects in the analysis of individual genes. (Konstantinidis et al. 2006). At least five housekeeping genes from different chromosomal loci and widely distributed in taxonomic groups are analyzed to provide sufficient information for identification of bacterial species from related groups (Stackebrandt et al. 2002). Zeigler (2003) argued that the “set” type prediction of candidate genes is a limited data set. He also suggested that the five genes actually corresponded to or even exceeded the DNA hybridization capacity required by the distribution-related bacterial strains for other species (Zeigler 2003).

3.1.3  E  volution of Lactobacillus Based on Whole-Genome Comparison The phylogenetic relationships of lactobacillus which are analyzed by single or several genes cannot accurately reflect the taxonomic evolution. The study of genomics based on whole genome sequence analysis can lay a solid foundation for the study of lactobacillus classification and evolution relationship. (Makarova et al. (2006a) for the first time conducted comparative genomics study on 9 different strains of lactobacillus by using the method of interspecific comparative genomics analysis, and found that the lactobacillus showed gene acquisition and deletion in the evolutionary process. When lactobacillus is in a nutrient-rich environment, the protein-­ coding genes related to nutrient substance metabolism in the genome will be missing in different circumstances (Makarova et al. 2006a). It is because of the continuous development of this mutation that the nutritional deficient strain is finally formed. Gao (2014), Zhong (2015) compared 138 strains of Streptococcus pneumoniae gene groups to illustrate the evolution of different kinds of streptococcus. Phylogenetic trees were constructed based on the protein sequences encoded by 278 core genes. The results showed that two genetic lineages existed in the evolution process of streptococcus, and the evolution path of each group was the same as that of genus. Zheng Huajun (2010) found that the three genera of lactobacillus, lactobacillus and streptococcus had similar classification conditions in the evolutionary tree and 16S rRNA sequence tree, but their topological structures were significantly different. At the species level, the differentiation of the three genera was no longer as clear as that of the 16S rRNA sequence tree. And the evolutionary tree based on the method of comparative genomics shows that there are significant differences between different

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strains from the same subspecies (Zheng Huajun 2010). In traditional research, scientists use 16S rRNA sequences to construct phylogenetic trees to understand the evolutionary relationships between lactic acid bacteria. However, the 16S rRNA gene sequence analysis is difficult to distinguish lactobacillus which is closely related to evolution. The evolution of lactobacillus can be explained by studying the number of variation at genomic level through phylogenetic analysis. The 16S rRNA system development tree is the basic research method to understand the basic evolutionary relationship between lactic acid bacteria. Genomic phylogenetic tree and 16S rRNA phylogenetic tree are considered as references and supplements. Sun et al. (2015) constructed a phylogenetic tree by comparing the protein nucleic acid sequences of 16 strains of lactic acid bacteria with 452 genera of lactic acid bacteria in the phylum, in which the evolution of all model strains can be divided into two large branches. The results showed that lactobacillus genus is a paraphyletic taxa (paraphyletic), which evolved continuously by a ancestors. In other words, the facultative heterozygous lactobacillus was differentiated into obligate homozygous and heterozygous fermentation to adapt to its living environment. At the same time, the progenitor of this genus was the lactobacillus mode strain belonging to facultative heterozygous fermentation, while the progenitor was the lactobacillus mode strain belonging to strictly homozygous fermentation and strictly heterotypic fermentation. The animal lactobacillus in the first branch of evolution had niche movement, while the animal lactobacillus in the second branch of evolution had parallel evolution in the animal habitat and other habitats (Zhong Zhi 2015). Zhong Zhi et  al. (2015) constructed a comparative genomic phylogenetic tree based on the analysis of 37 strains of Enterococcus. By comparing and analyzing the phylogenetic tree of 16S rRNA sequences, the whole topology is more similar, but there are large differences in deep branches. Based on the core genome, the guiding value of the branch nodes of most phylogenetic trees is 100%, and the phylogenetic tree can more accurately reflect the phylogenetic position of the strain. The analysis also revealed the relationship between evolution and the environment in which enterococci may have first been hosted by plants and birds, then passed on to mammals, and accelerated evolution in the environment (Zhong Zhi 2015). The differences in the branches of the enterococcus genome show that the differentiation of enterococcus is accompanied by different levels of gene transfer. Strains in the younger branch had larger genomes and more genes than those in the older branch, suggesting that gene acquisition may have dominated the evolution of the strain, and that its environment at the time may have determined the direction of evolution (Zhong Zhi 2015). The above results indicate that genome-wide sequence analysis can truly reflect the phylogenetic relationship and taxonomic status of strains, and is a powerful tool for studying the classification and evolution of lactic acid bacteria.

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3.2  The Microevolution About the Lactic Acid Bacteria Microevolution focus on character impacted by gene and genovariation influenced by selective pressure, which is the evolution of the same or similar species and a process that caused intraspecific differentiation (Song et al. 2015). Microevolution includes gene mutation, gene recombination, gene transfer and natural selection (Siezen et al. 2008). Gene mutations include nucleotide substitution, transcriptional deletion, recombination, and gene conversion, which are the absolute driving forces of evolution. Genetic recombination can't change the frequency of allele, but it can increase inheritance change in a certain extent. Genetic shift pay a important role in fixation of mutants with neutral mutations in small populations. The positive selection of natural selection weed out the genovariation, however the balance seleciton can increase the genovariation (Siezen et al. 2008).The heritable variation and heritable basis is the research emphasis in biology, that can retrospect the rule of their development process. The analysis of microevolution research the phylogeny and the evolution process in molecular level, it is the heritable basis which can reveal the biological function and the evolutionary mechanism about lactic acid bacteria, which offer the theoretical basis which can guide how to apply the lactic acid bacteria among the food industry.

3.2.1  I ntraspecies Evolution of Lactic Acid Bacteria Based on DNA Fingerprinting We only can research the microevolution of the lactic acid bacteria with the aid of the conserved sequence dut to they are prokaryote. We can explain the possible microevolution process about the lactic acid bacteria by the distribution of the variant population, which rely on the difference population structure of different strain by the high-resolution mark. There are many DNA finger-print analysis technology and analysis methods base on DNA sequence which can reveal the change ofunknown population structure , such as randomly amplified polymorphic DNA, RAPD (Solieri and Giudici 2010), restriction fragment length polymorphism, RFLP (Yu et al. 2011), amplified fragment length polymorphism, AFLP (Tanigawa and Watanabe 2011) and pulsed-field gel slectrophoresis, PFGE etc. (Picozzi et al. 2010; Sawadogo-Lingani et al. 2007). RAPD is the molecular marker technology which can classify and authenticate unknown strain, which it adopt random primer and as a template base on genome DNA and get DNA fragment by PCR and analysis the finger-print produced by gel electrophores (Doria et  al. 2013). This method is convenient and efficient and already proceed the classification and evolution research about lactobacillus and Bifidobacterium successfully. RFLP and fluorescence labeling often use jointly, it

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detect the size of DNA fragment which is Restriction Enzyme Digestion DNA amplify product, but the recognition and cutting site of restriction enzyme will change because of replacement、deficiency and recombination among the individual allele, which can cause the difference length of restriction fragment among the different genotype (Bokulich and Mills 2012). This method has stable analysis result and can be used for the classification and recognition of a bulk of samples, however it's consumption of time and labor is more and has lower sensitivity because the transportation of cytomembrane and the Southern hybridization is necessary for RFLP.  AFLP is a kind of molecular marker based on PCR.  At present has been widely used in construction of genetic map, genetic diversity analysis, important gene location, molecular marker assisted breeding and classification of biological systems, etc. (Pothakos et  al. 2014). Its drawback is that the cost is higher, and stronger technical, because its are dominant marker, is not conducive to analysis of population genetic structure. PFGE technique separable 10 KB ~ 10 MB of large molecules of DNA, its principle is by changing electric field direction, make the DNA molecules in the agarose gel swimming party to subsequently corresponding change, small molecules with DNA electric field conversion can quickly change the direction of movement, although large molecules and DNA in pulse field for use under the will to move, but more difficult in the gel. Different sizes of DNA molecules in gel will present different mobility, DNA band spectrum to a certain extent, reflects the DNA content and the molecular size, can be used as the basis for classification. PFGE is a kind of important for separation of oita sub quality linear DNA electrophoresis, is currently the polymorphism of lactic acid bacteria research one of the widely used methods. PFGE stable results, high resolution, good repeatability and easy standardization etc, and therefore are bacteria molecular classification of the “gold standard” (Gonzalez-Arenzana et  al. 2014). The ideal DNA molecular typing technology has the following characteristics: the genome contains multiple polymorphic sites and has a characteristic distribution; it can generate multiple independent and reliable molecular markers; high resolution, simple, fast, economical and efficient; DNA samples Less demand; correlation with phenotypic characteristics; no need for genomic DNA sequence information (Zhou Ning 2012). Although there are many methods for taxonomic identification and polymorphism research, no molecular typing technology can fully satisfy the taxonomic identification of all lactic acid bacteria due to the difference of genomic information of lactic acid bacteria and genotyping techniques in genome abundance, polymorphism detection, specific loci, repeatability and technical requirements. Therefore, scientists need to determine the research method according to the purpose of the experiment. Yang Jixia (2013) obtained the fingerprints of 39 strains of bacteria from yak milk by RAPD technology. The clustering of strains showed that the strains of different strains clustered into one or more groups. Meanwhile, it also shows that there is a certain correlation between strains and regions. (Yang Jixia 2013) For example, the strains from Yunnan and Tibet are mostly distributed in a group, while the strains from Xinjiang are far away. This may be related to their geographical location, and also reflects that there is a certain relationship between strains and their original

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origin. Psoni et al. (2007) analyzed the diversity of 40 Lactococcus lactis strains isolated from Greek goat cheese by PFGE, and compared them with RAPD and plasmid typing. The results showed that the 40 strains were abundant in polymorphism, which were consistent with those obtained by RAPD and plasmid typing (Zhou Ning 2012). Dimitrov et al. (2008) identified and analyzed the polymorphism of lactic acid bacteria isolated from healthy human body by AFLP, PFGE and RAPD. The results show that the RAPD technology is the easiest and quickest to operate, but the resolution is general. PFGE method is more stable and has high resolution, but it takes a long time. AFLP technology has the highest resolution in strain typing, relatively simple operation, and strong stability and resolution (Zhou Ning 2012). Because the above techniques are not practical in identification of closely related strains, limited by laboratory conditions and difficult to achieve data exchange and comparison, a more convenient, reliable and higher resolution method is needed for identification of closely related strains. Genomic short sequences, distributed on different sites of bacterial genome and separated at different distances, present differences in strain and species levels. Rep-­ PCR takes the bacterial genome DNA as template, and uses genomic short sequence as primer to carry out PCR, amplifying DNA sequences among repeated sequences. After gel electrophoresis, the amplified products can form a series of bands, that is, DNA fingerprint. The fingerprints of different strains of bacteria or subspecies are specific, so the identification and diversity of bacteria can be studied (Zhou Ning 2012). Rep-PCR is more and more widely used in the classification, identification and polymorphism of lactic acid bacteria because of its simple operation, high resolution, economy, rapidity and applicability. de Urraza et  al. (2000) identified 37 thermophilic lactic acid bacteria isolated from dairy products by Rep-PCR and analyzed their diversity. Adıguzel et al. (2009) used Rep-PCR to analyze 76 strains of lactic acid bacteria isolated from fermented sausages in Turkey and identified them at the level of species and strains. Gao Yang (2013) used Rep-PCR to analyze 24 strains of lactic acid bacteria in the intestines of cold water fish from different sources and found that the fingerprints of low-temperature resistant lactic acid bacteria produced more bands, which can reflect the differences in genome levels of different strains. The dendrogram of fingerprint cluster analysis showed that all strains could be divided into six groups at 70% similarity level. At the same time, the genetic differences between Lactococcus and Enterococcus at species and strain level could be clearly seen from the dendrogram (Gao Yang 2013).

3.2.2  I ntraspecies Evolution of Lactic Acid Bacteria Based on DNA Sequence Differences In order to study bacterial genotypes and microevolution more deeply, a series of methods based on DNA sequence difference analysis technology emerged as the times require. The 16S rRNA intergenic spacer region(ISR) (Sun 2006) based on a single gene and the nucleotide sequence analysis based on partial fragments of

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multiple housekeeping genes were early used for genotyping of lactic acid bacteria (Yu et  al. 2012), such as multiple-locus variable-number tandem-repeat analysis(MLVA) (Chiou et  al. 2010) and multilocus sequence typing (MLST) (Picozzi et al. 2010; Maiden et al. 2013). MLST, once the most mature and standardized analytical method, has been widely used in the research of genetic diversity, population structure and micro-evolution of lactic acid bacteria because of its high resolution, simplicity, and reproducibility. Maiden et al. (1998) improved the multilocus enzyme electrophoresis (MLEE) to obtain a new molecular typing method, MLST.  By studying 11 housekeeping genes of 107 strains of Neisseria meningitidis, they analyzed about 470 base pairs of each gene, and compared the diversity of alleles. At the same time, they took each group of different alleles as a genotype to construct allele profile. Finally, the phylogenetic tree was constructed by clustering analysis, and it was found that each unique genotype corresponded to a sequence typing ST (Maiden et al. 1998). MLST technology can be used to compare ST to find correlations between different strains, and strains with high correlation have the same ST or ST with only a few individual loci and STs of unrelated strains differ in at least 3 or more loci (Taylor et al. 1999). MLST technology can also be used to analyze the genetic relationship between strains from different sources, especially to monitor gene recombination events between different populations. Therefore, it can make up for the shortcomings of traditional typing methods when analyzing the evolution relationship of strains and judging isolates. With the rapid development and wide application of sequencing technology, MLST technology is being widely used in laboratories all over the world (Maiden et al. 1998). The technology can be standardized, the results are easy to verify and preserve, and the genetic information of strains can be transmitted through the Internet. The selection of housekeeping genes is the key to the multi-site sequence typing method. The housekeeping genes that exist in almost all prokaryotes are highly conserved, and there will be differences between the strains. Konstantinidis et al. (2006) confirmed that 3 genes were the least number to be used in MLSA to predict gene horizontal transfer (Konstantinidis et al. 2006). Rivas et al. (2004) selected five housekeeping genes from 18 different strains of Leuconostoc cerevisiae and used MLST technology for multi-site sequence typing analysis for the first time. The results showed that MLST could effectively identify Leuconostoc alcoholicus isolates and gene recombination was an important reason for the increase of genetic diversity of Leuconostoc alcoholicus (De et al. 2005). Cai et al. (2007) analyzed the alleles of 40 Lactobacillus casei strains by MLST technology. The results showed that the specific living environment caused the specific genetic evolution of the strain, and the genetic characteristics were closely related to the living environment. At the same time, it was pointed out that the evolution process of lactic acid bacteria under the action of different natural environments was completely traceable (Tanganurat et al. 2009). Calmin et al. (2008) studied five genes (recA,rplB, pyrG, leuS and mle) of Pediococcus parvulus and Pediococcus damnosus in wine using MLST technology and found that MLST effectively completed the molecular typing of these two cocci, which laid the foundation for screening wine starter(Calmin

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et  al. 2008). In 2008, scientists used MLST technology to study Lactobacillus ­plantarum isolated from fermented fruits and vegetables, and completed analysis of genetic diversity and strain evolution, and found that carbon plays an important role in the evolution of L. plantarum (Tanganurat et al. 2009). Diancourt et al. (2007) studied the population structure of Lactobacillus casei using MLST technology. Seven housekeeping genes were selected to distinguish 12 alleles and 31 ST types. Only ST1 appeared in 17 Lactobacillus casei, and each of the other strains represented 1 ST type alone. This indicated that MLST technology was effective in typing Lactobacillus casei (Diancourt et al. 2007). Dan et al. (2014) used MLST technology to analyze the population structure of 50 strains of L. lactis isolated from natural fermented dairy products from China and Mongolia. The results showed that there was a lateral transfer of genes in Leuconostoc cerevisiae and two subgroups with high similarity were formed (Tong et al. 2013). Sun Zhihong (2014) selected eight housekeeping genes of 305 Lactobacillus deltoides Bulgaria subspecies for MLST study, which were divided into 121 ST types and consisted of 14 homologous complexes (Sun Zhihong 2014). Five possible major ancestor groups were predicted, and it was inferred that the Bulgarian subspecies of Lactobacillus delbrueckii isolated from Mongolia were closer to the earliest ancestor group. At the same time, the progenitor strain of Lactobacillus delbrueckii Bulgaria subspecies was the closest to Mongolian isolates, showing a trend of spreading from Mongolia to Russia and then to China, which indicated that different sequence types are directly related to their isolation sources and isolation sites (Zhong Zhi 2015). Delorme et  al. (2009) applied MLST technology to illustrate the evolution of Streptococcus thermophilus, and found that the relationship between S. thermophilus and Streptococcus salivarius was the closest. The genetic relationship between the strains of S. thermophilus was analyzed and it was found that ST3 is the most primitive ST type, which is the ancestral type. It has also been found that some genes have metastasized and lost during the evolution of S. thermophilus (Delorme et  al. 2009). Yu Jie (2013) performed MLST analysis on 10 target genes in 260 strains of Streptococcus thermophilus isolated from different countries and found that these strains were divided into 119 ST types, with the largest number of ST-29 strains, followed by ST-57 and ST-106. Six strains isolated from Russia were divided into four different ST types, 106 strains of Streptococcus thermophilus isolated from China were divided into 50 different ST types, and 148 strains of Streptococcus thermophilus isolated from Mongolia were divided into 71 different ST type. The ST types of Streptococcus thermophilus isolates isolated from different regions are different. The number of alleles of 10 target genes is 7–17, which may be the result of natural environment selection (Yu Jie 2013). There was a significant linkage disequilibrium in these strain populations, and there was no genetic recombination between the alleles of the regional strains. Isolation of strains from the same region will form corresponding clonal complexes, indicating that the evolution of Streptococcus thermophilus in different environments is entirely different. The main factors affecting evolution are environmental climate and geographical location, and the milk system may not be a major factor.

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Therefore, MLST technology is a powerful tool to study the genetic evolution of lactic acid bacteria. However, because MLST site carry very limited genetic ­information and the genetic evolution relationship revealed by MLST loci is limited by population structure and population information, it is difficult to analyze microevolution.

3.2.3  I ntraspecies Evolution of Lactic Acid Bacteria Based on Genome Resequencing Due to the continuous development of high-throughput sequencing technology, DNA sequence difference analysis technology based on genome-wide information has been continuously applied to the study of microevolution of bacterial populations. This technology has the advantages of low cost, high throughput and short time-consuming, which provides a strong support for the study of microevolution of lactic acid bacteria (Li et al. 2009). Genomic sequencing of different individuals for species of known genomic sequences is called genomic resequencing technology, which analyzes genetic evolution by detecting differences in genes or structures of populations or individuals. The principle of this technique is to find single nucleotide polymorphic loci, insertion/deletion loci, structural variation loci and copy number variation by comparing the known genome sequences with the results of re-sequencing, so as to find genetic differences to analyze genetic evolution. The continuous completion of lactic acid bacteria genome sequencing not only clarifies the biological metabolic pathway, but also provides a rich reference sequence for studying the evolution of lactic acid bacteria at the genome-wide level. More and more scientists are planning to study the species differentiation and population inheritance of lactic acid bacteria at the genome-wide level. Whole genome sequencing can comprehensively analyze the genetic information of the genome and systematically clarify the complexity and diversity of the genome, which can provide powerful technical support for the study of micro-evolution of lactic acid bacteria. Cai et al. (2009a) used the Lactobacillus casei ATCC 334 genome as a reference for comparative genomic hybridization analysis of 21 genomes of Lactobacillus casei isolated from human, plant and cheese from different regions. The analysis showed that the Lactobacillus casei strain isolated from different environments had a special functional gene compared with the reference strain, indicating that the strain had different degrees of gene insertion/deletion when adapting to the environment, and this result coincided with MLST analysis. The same results not only confirmed the accuracy of MLST technology, but also proved that genome resequencing is an effective technical means to study the micro-evolution of lactic acid bacteria (Cai et  al. 2009a). Smokvina et  al. (2013) completed the genome re-­ sequencing of 34 strains of Lactobacillus casei with reference to the Lactobacillus casei Zhang genome sequence, and systematically analyzed the differences in sugar metabolism between different strains of Lactobacillus casei. The results showed

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that Lactobacillus casei contains multiple phosphoenolpyruvate transport systems (PTS), which allows these strains to utilize more sugars and adapt to different ­environments. It was also found that Lactobacillus casei isolates from milk source encoded relatively few PTS, which may be due to the adaptation of Lactobacillus casei to the nutritional environment of milk and the abandonment of part of the transport system that had not been used for a long time in the evolution process, indicating that Lactobacillus casei has a oriented micro-evolutionary process in order to adapt to different environments (Smokvina et al. 2013). The above results fully confirm that the genome re-sequencing technology can completely reflect the genetic information of the genome, so as to fully reveal the complexity and diversity of the genome, analyze the inheritance and evolution of the lactic acid bacteria population. In addition, genome re-sequencing technology can also provide a powerful technical means for clarifying the microevolution of lactic acid bacteria and revealing the influence of environment on the evolution of lactic acid bacteria. The study of microevolution of bacteria can deeply analyze the influence of environment on the evolution of microbial population. The genetic background and evolution of lactic acid bacteria are the primary basis for the research and development of lactic acid bacteria and their application in the food industry. By studying the micro-evolution of lactic acid bacteria, we can confirm the relatives and evolutionary relationship among various genus of lactic acid bacteria, which is conducive to the reconstruction of phylogenetic tree, and provide a solid theoretical basis for establishing a more accurate and rapid classification and identification system of lactic acid bacteria. Although scientists have been discovering the probiotic properties of lactic acid bacteria in recent years, many unknown functions of lactic acid bacteria still need to be explored urgently. Therefore, the study of the microevolution mechanism of lactic acid bacteria can provide molecular support for the analysis of the formation and evolution of its biological functions. In-depth discovery of key genes with probiotic characteristics and basic research on related genes can further exploit the resources of lactic acid bacteria. Among the methods described above, the 16S rRNA gene sequence of a single gene cannot accurately reflect the evolution of lactic acid bacteria in nature. Nucleic acid sequence analysis based on multiple housekeeping loci has many advantages, but it is limited to individual loci in the whole genome, which will miss important genetic information and result in more errors between the constructed population structure and the real structure. Scholars can analyze the structure and composition of Lactobacillus genome by exploring the complete genome data in depth, thus providing new ideas for studying the evolution mechanism of lactobacillus.

3.3  Core Genome and Pan Genome of Lactic Acid Bacteria With the development of microbial genome sequencing technology, new genes are always found in different strains of the same strain after genome sequencing. Therefore, the whole genetic information of a strain cannot be represented by the

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genome information of a single strain. Medini et al. 2005 put forward the concept of core genome (Medini et al. 2005), which called the genes existing in all strains as the core genome of the strain. Pan genome refers to the entire genome of a species, including the core genome (Lefebure and Stanhope 2007). The core genome is essential to maintain the basic survival and phenotype of the strain, which retained in evolution because of the high selective pressure. The diversity of pan-genome can explain the difference of genome genetic information among different strains (Wang Yajun 2012). Bacteria with low pan-genomic diversity live in stable environments, while bacteria with high pan-genomic diversity have strong adaptability to the environment. According to the genome, genes and the degree of variability are different. Pan genomes and core genomes are commonly used to identify gene families rather than single genes. Once a genome is added, the gene family is redefined and the number of gene families in the core genome and pangenome is recalculated.

3.3.1  The Core Genome and Pan Genome of Lactobacillus Cai et  al. (2009b) analyzed the microarray hybridization of Lactobacillus casei ATCC344, which indicated that 1941 core genes existed in 2678 predicted genes of all 21 strains analyzed (Cai et al. 2009b). The comparative genome analysis of 11 strains of 9 Lactobacillus species showed that it was difficult to identify homology at the nucleic acid level, so it was necessary to compare them at the protein level. When a protein whose matching length exceeds 40% of the minimum protein length and whose homology is also over 40% is defined as a homologous protein, the number of core genes decreased gradually with the increase of strains. Only 529 core genes were found in 11 strains of Lactobacillus, mainly in energy metabolism, transport and binding protein, transcription, cell process, replication and protein synthesis (Zheng Huajun 2010). The core genome and pangenome analysis of 66 Lactobacillus showed that there were 16 663 gene families and 365 gene families in the pangenome and core genome of 37 Lactobacillus genus, respectively. When the genome of Lactococcus was increased, the number of pangenomes increased greatly (18,232), because the increased Lactococcus gene did not exist in Lactococcus, while the core genome decreased substantially. The pangenome of 66 Lactobacillus genomes contains 29,247 gene families, while the core genome contains only 261 gene families. Lactobacillus is a large genus, containing a wide variety of species,and the difference between core genome and pan genome can be used as a criterion for determining intraspecific differences (Schillinger and Endo 2014). For Lactobacillus, comparative genomic analysis of 37 Lactobacillus strains from 17 species showed that the difference was 16,298 gene families (16 663 pangenomes subtracted 365 core genomes) (Schillinger and Endo 2014), which was much larger than the difference observed in 27 genomic analysis of 7 Vibrio strains (Vesth et al. 2010). Sun et al. (2015) sequenced the genome of 149 Lactobacillus model strains and obtained the accurate genome map of the strain. The core gene family was determined based on the principle that the amino acid similarity of coding proteins was more than

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50% and it was found that the genome of each model strain encoded an average of 2200 genes, and the core genome contained 72 core genes which encoded proteins related to cell-based growth and replication. The pangenome of these strains is expanding with the increase of genome number, which contains more than 70,000 genes, while the core genome is decreasing with the increase of genome number (Sun et al. 2015). (Siezen et al. (2010) compared the microarray data of 42 strains of fermented food with Lactobacillus plantarum WCFS1,discovering that the core genome of 2049 genes existed in all strains and 121 genes had no homology compared with other sequenced lactic acid bacteria (Siezen et al. 2010).Through comparative genomics analysis of Lactobacillus bulgaricus, it was found that the 265 KB gene sequence was unique to 2038 strains of genome. Among the three strains, 1301 genes are highly homologous at the nucleic acid level, which is the core gene of Lactobacillus bulgaricus, while the pangenome contains 2199 genes.112 genes in the core genome are identical in nucleic acid sequence, 214 genes only have synonymous mutations in coding region, and the remaining genes have differences in coding protein level. The ancestors of Lactobacillus bulgaricus may have about 2200 genes, losing 15–30% in the course of evolution and the main source of genomic variability is the slow spread of core genes (Zheng Huajun 2010). Makarova and Koonin (2007) identified the core genomes of 12 sequenced genomes of Lactobacillus with 567 gene clusters in the direct homologous group. Major genes in the core genome are associated with replication, transcription and translation, but about 100 genes have not been identified. An interesting phenomenon is that there are no two core genomes that are directly homologous outside the genus Lactobacillus. One of these unique genes encodes proteins that contain the peptidoglycan-binding LysM domain, and the other contains no identified domain, located in the conserved genome region and containing two enzymes associated with tRNA 4-thiourea nucleoside modification (Makarova and Koonin 2007).

3.3.2  The Core Genome and Pan Genome of Enterococcus Enterococci are normal constituents of the gastrointestinal flora of humans and other animals (Willems and van Schaik 2009; Willems et al. 2011). Enterococcus faecalis is the most common enterococcal species recovered from infections. Data show that from 1990 to 2010, infections with E. faecalis have been on the rise in the United States, Europe, and South America (Boyd et al. 2002; Leavis et al. 2006; Hidron et al. 2009; Willems and van Schaik 2009). The comparative genomic analysis of Enterococcus from different sources showed that their genomic sequences were quite different. The genomic differences of Enterococcus strains could be analyzed by studying the pan-genome and core genome. Enterococcus faecium has a large genome, strong adaptability to the environment, and easy to obtain the sequence of foreign genes. Scientists have found that only about 50% of the genes in the Enterococcus genome belong to the core genome and share it with other genomes. This indicates that there are more specific genes in

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E. faecium strains from different sources, while the other 50% genes are included in the pangenome, which also indicates that the genome of E. faecium from different sources is more diverse. Wang Yajun (2012) made comparative genomics analysis of 28 strains of E. faecium,discovering that the pangenome of E. faecium increased with the addition of new strains and new gene families were added to the pangenome of each new strain.The pan genome and core genome contain 5743 genes and 1440 genes respectively.Only about half of the genes in E. faecium genome belong to the core genome, which indicates that there are more specific genes in Enterococcus faecium strains from different sources (Wang Yajun 2012). Qin et al. (2012) studied the comparative genome of 22 strains of E. faecium and found that the pangenome and core genome contained 3169 genes and 1652 genes, respectively (Qin et al. 2012). The estimated number of core genomes is similar to that reported by Leavis et al. (2007) by microarray method (Leavis et al. 2007). Zhong Zhi (2015) conducted a comparative genomics study on 37 Enterococcus sp. strains in order to analyze the genetic relationship and species evolution of Enterococcus. The pan genome of Enterococcus contains 29,545 genes, and the core genome contains 605 genes.Enterococcus genome contains an average of 3062 genes, with only 20% of the core genome and 28% of the smallest Enterococcus genome, indicating that the diversity of Enterococcus genome is very strong.Most of the genes in the core genome are essential for maintaining life and reproduction, and are essential components of the Enterococcal genome (Zhong Zhi 2015). In addition to the core genome and pan-genome, some strain-specific genes have been found in the genome of Enterococcus, which only exist in one strain of Enterococcus. Through comparative analysis of 37 strains of Enterococcus, 368 strain-specific genes were found, with an average of less than 10 per strain. Compared with the large pan-genome, the strain-specific gene is only 1% of the pan-genome, indicating that although the genome of Enterococcus is more complex and diverse, the gene exchange between species within the genus is much more than that between strains within the genus and those outside the genus (Zhong Zhi 2015).

3.3.3  The Core Genome and Pan Genome of Streptococcus The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. Lefebure and Stanhope (2007) analyzed the pangenome of 1898 genes and the core genome of 1487 genes of Streptococcus thermophilus based on two completed genome sequences and one preliminary completed genome sequence (Lefebure and Stanhope 2007). The comparison of all complete and preliminary genomic sequences of Streptococcus showed that the pangenome of Streptococcus could exceed 6000 genes. Rasmussen et  al. (2008) analyzed three complete genome sequences and gene information on GenBank, and constructed a pangenome of more than 2200 genes for S.thermophilus. By molecular hybridization of DNA from 47 strains, the core genomes of 1271 genes can be identified. However, these data suggest that adding additional strains to the analysis

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will reduce the number of genes in the core genome (Rasmussen et  al. 2008). Tettelin et  al. (2005) sequenced the whole genome of 8 strains of Streptococcus lactis and found that there was a core genome in 1800 genes, and the pangenome of each strain increased to a critical value of about 33 genes (Tettelin et  al. 2005). Similar data were also found in Streptococcus pyogenes. Specific aspects of the environmental interaction-potential and the metabolic capacity of 6 small-intestinal Streptococcus strains were investigated through analysis of their genome sequences. The results of classification of isolates showed that three different strains of Streptococcus mutans were isolated from one ileal sample, which belonged to Streptococcus mitis group, Streptococcus bovis group and Streptococcus salivarius group. Compared with 58 strains of Streptococcus pneumoniae in the public database, the pangenome and core genome of S.pneumoniae were composed of 12,403 genes and 574 genes, respectively.

3.3.4  The Core Genome and Pan Genome of Bifidobacterium Bifidobacteria are commonly found as part of the microbiota in the human gastrointestinal tract (GIT) (Ventura et al. 2007), where their presence has been positively correlated with the health status of their host (Ventura et al. 2009a, b). Bifidobacteria have been claimed to elicit several health-promoting or probiotic effects, such as strengthening of the intestinal barrier, modulation of the immune response and exclusion of pathogens (Marco et al. 2006; O’Hara and Shanahan 2007). Whole-­ genome sequencing efforts have revolutionized the study of bifidobacterial genetics and physiology. Unfortunately, the sequence of a single genome does not provide information on bifidobacterial genetic diversity and on how genetic variability supports improved adaptation of these bacteria to the environment of the human gastrointestinal tract (GIT). Lukjancenko et al. (2012) completed the comparative genome analysis of nine Bifidobacterium strains, and determined that the pan-genome of Bifidobacterium contains 5000 genes, and the core genome contains 506 genes. These conserved core genes can encode most of the core housekeeping functions, such as replication, transcription, translation, and adaptation and interaction with specific environments, such as sugar metabolism, cell biosynthesis and signal transduction. The difference in the number of genes between pan-genome and core genome indicates that there are some genetic differences between strains. The study of the functions of specific genes and conservative genes will provide a strong basis for the study of the commonness and characteristics of Bifidobacterium adapting to intestinal environment (Lukjancenko et al. 2012). In order to study the complexity of Bifidobacterium genome, Bottacini et al. (2010) carried out comparative genome analysis of bifidobacterium and determined the total number of genes of Bifidobacterium pangenome and core genome. The genome analysis of 14 strains of Bifidobacterium showed that the total number of genes in the core genome and pangenome was 521 and 5125 respectively, which was twice the average number of genes in the Bifidobacterium genome for the number of Bifidobacterium genomes

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is limited and the whole genome of Bifidobacterium cannot be fully understood (Makarova et  al. 2006b). After completing the comparative genomic analysis of Bifidobacterium longum JDM301 and other bifidobacteria, Wei Yanxia (2012) found that the core genome of Bifidobacterium was composed of 1265 genes, playing an important role in amino acid transport and metabolism, carbohydrate transport and metabolism, ribosome structure and synthesis, DNA replication, recombination and repair. 21% of the core protein was associated with carbohydrate, amino acid transport and metabolism. (Wei Yanxia 2012)

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Tanigawa K, Watanabe K (2011) Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii. Microbiology 157(3):727–738 Taylor JW, Geiser DM, Burt A, Koufopanou V (1999) The evolutionary biology and population genetics underlying fungal strain typing. Clin Microbiol Rev 12(1):126–146 Tettelin H et al (2005) Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial “pan-genome”. Proc Natl Acad Sci USA 102(39):13950–13955 Tong D et al (2013) A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia. BMC Microbiol 14(1):1–9 Ventura M et al (2007) Genomics of Actinobacteria: Tracing the evolutionary history of an ancient phylura. Microbiol Mol Biol Rev 71(3):495–49+ Ventura M et al (2009a) Genome-scale analyses of health-promoting bacteria: probiogenomics. Nat Rev Microbiol 7(1):61–U77 Ventura M et al (2009b) The Bifidobacterium dentium Bd1 Genome Sequence Reflects Its Genetic Adaptation to the Human Oral Cavity. PLoS Genet 5(12) Vesth T et al (2010) On the origins of a Vibrio species. Microb Ecol 59:11–13 Wang Y (2012) Genome-based insights into the evolution and function of Enterococcus faecium. (Doctoral dissertation, Shanghai Jiao Tong Unverisity) Wei Y (2012) Studies on genome and funvtional genes of Bifidobacterium longum JDM301. (Doctoral dissertation, Shanghai Jiao Tong Unverisity) Willems RJL, van Schaik W (2009) Transition of Enterococcus faecium from commensal organism to nosocomial pathogen. Future Microbiol 4(9):1125–1135 Willems RJL, Hanage WP, Bessen DE, Feil EJ (2011) Population biology of Gram-positive pathogens: high-risk clones for dissemination of antibiotic resistance. FEMS Microbiol Rev 35(5):872–900 Yang J (2013) Phenotypic, genotypic and probiotic characterization of lactic acid bacteria isolated from Chinese yak milk cheeses. (Doctoral dissertation, Southwest University) Yu J (2013) Multiocus sequence typing of Streptococcus thermophiles from traditional fermented dairy products in China, Russia and Mongolia. (Doctoral dissertation, Inner Mongolia Agricultural University) Yu J et al (2011) Phenotypic and genotypic characteristics of lactic acid bacteria isolated from sour congee in Inner Mongolia of China. J Gen Appl Microbiol 57(4):197–206 Yu J et al (2012) Phylogenetic study of Lactobacillus acidophilus group, L. casei group and L. plantarum group based on partial hsp 60, phe S and tuf gene sequences. Eur Food Res Technol 234(6):927–934 Zeigler DR (2003) Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int J Syst Evol Microbiol 53(6):1893–1900 Zheng H (2010) Genomic analysis of Lactobacillus delbrueckii subsp.bulgaricus strain 2038. (Doctoral dissertation, Fudan University) Zhong Z (2015) Comparative genomic analysis of the type strains of genus Enterococcus and multilocus sequence typing of Enterococcus faecalis isolated from fermented foods. (Doctoral dissertation, Inner Mongolia Agricultural University) Zhou Ning et al (2012) Advances in molecular approaches and their applications in lactic acid bacteria. Sci Technol Food Ind (5):69–73

Chapter 4

Transcriptomics of Lactic Acid Bacteria Zhennan Gu and Guozhong Zhao

4.1  Summary of Transcriptomics 4.1.1  Concept of Transcriptomics Transcriptomics is a frontier subject in the post-genomics era and an important part of functional genomics, the study of the species, structure, function, and transcription regulation that occurred in the cells (the study of gene expression on RNA level). The purpose to study transcriptome is to understand all the genetic bases of organisms, including the expression regulation system, as well as the function and interaction of proteins expressed. Transcriptome is a bridge to link genetic information of the genome with the biological function of proteome. Thereby, regulation on transcriptional level is the most widely studied and most important method of organism regulation currently. Transcriptomics is one of the most important tools for large-scale analysis of biological gene expression processes at present and is more efficient compared to the analysis on genome level (genomics). Different with genomics, transcriptomics is also influenced by time phase and cell environment. Because of its capacity to study any moment of cell life cycle, transcriptomics can not only be successfully used to interpret the functional components of the genome, or to predict molecular components, but also be applied to study biological processes and the pathogenesis of disease. There are two definitions in the transcriptome, which are distinguished from broad sense and narrow sense, respectively. The definition of transcriptome in broad

Z. Gu (*) Jiangnan University, Wuxi, China e-mail: [email protected] G. Zhao Tianjin University of Science & Technology, Tianjin, China e-mail: [email protected] © Springer Nature Singapore Pte Ltd. and Science Press 2019 W. Chen (ed.), Lactic Acid Bacteria, https://doi.org/10.1007/978-981-13-7832-4_4

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sense refers to species. A particular case is the total RNA transcribed from all the genetic information of the cell or tissue of the species, including the RNA and noncoding RNA encoding the protein of this species (such as rRNA, tRNA, snoRNA, snRNA, and microRNA). These RNAs have a wide range of functions and play an extremely important role in the growth and development of species and various biological processes (Lewin 2004). Based on the above definition of the transcriptomics, we know that the transcriptome has its specific time and space constraints. Thereby, the transcriptome is different in different growth conditions and different growth stages in its life stage in the same cell or a tissue. This difference is not only reflected in the difference in the type and number of genes expressed but also in the same cell at different times and in different physiological states. At the same time, the existence of mRNA splice variants is also a significant difference between the transcriptome and the genome, that is, the same mRNA precursor is processed into a plurality of normal mRNA isoforms with different exon compositions, so that the gene regulates regulation. Expression products are more diverse and complex. In addition, the transcriptome has an extremely complex regulatory network, including the regulation of genes and genes between genes and proteins, and the microenvironment of cells, the different physiological and biochemical reactions of cells, and cells. The signal transduction pathway in the body constitutes a complex transcriptional regulatory network mechanism in the body. Under the guidance of the principles of the genetic center, the genetic information of cells is precisely regulated by genes, which can be transcribed from mRNA into mRNA, and then translated to reach proteins, which ultimately determines the structure and function of the species cells. The biological macromolecules associate and coordinate different cells to further constitute tissues, organs, and biological individuals. Therefore, RNA is a bridge between gene-linked proteins, and the identity of all expressed genes and their transcription levels are collectively referred to as transcription (Wang et al. 2009; Costa et al. 2010).

4.1.2  S  ignificance and Current Status of Transcriptomics Study The study of gene sequences and gene structures on the genome of a species is only part of the genomics study, and genomics research alone cannot explain all the mysteries in the life system. The functions of gene sequences, the life processes involved, the way of expression regulation, and the gene expression levels of specific genes under different environmental conditions need to be resolved. Transcriptomics is an important technology based on genomics. Transcriptomics can study the structure and function of specific genes at the overall level of the species and reveal the mechanisms of specific biological processes and diseases. Functional genomics studies of various genes and their transcriptional expression products can provide new ideas

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for food microbial fermentation and resource development and improvement and provide new methods for humans to solve health, food, energy, and environmental problems (Velculescu et al. 1997). Through the study of transcriptomics, information on the expression of specific genes under specific conditions can be found to speculate on the special function of the unknown gene, thereby revealing the regulatory mechanism of the specific gene. The molecular tag of a specific gene expression profile can not only discriminate the gene phenotype of the cell but also be used for the diagnosis of major diseases. Transcriptomics research can be applied to production practices to efficiently mine key genes and enhance their function. Through the analysis of transcriptome differential expression profiles of specific genes, it is possible to predict the adverse factors of fermentation in the production process in more detail.

4.2  Methodologies of Transcriptomics 4.2.1  Hybridization-Based Microarray (EST) The concept of EST is derived from the computer chip, also know as a microarray. Gene chip is a gene-based technique developed in recent years, which is used to rapidly detect differential gene expression, large-scale genome expression profiling, and differential identification of disease-causing genes, or research on diseaserelated genes (Schena et al. 1998). The principle of gene chip technology is using a series of techniques (photoconductive chemical synthesis, solid surface chemical synthesis, and photolithography) to synthesize many oligonucleotide probes (including cDNA, EST, or gene-specific oligonucleotides) on solid supports (such as silicon wafers, polypropylene, glass sheets, and nylon membranes), which are then hybridized to the first-strand cDNAs generated by reverse transcription of mRNA derived from different cells, tissues, or organs, and the cDNA is radioactively isotoped (P32 or P33) or fluorescent (cy3 or cy5) labeled, and the gene expression levels of target material were detected and analyzed using autoradiography and laser confocal technology. Gene chip technology analyzes gene expression and has the characteristics of high information quantity, high capacity, high accuracy, etc. It is the most commonly used technique in transcriptomics research. Its applications include gene expression detection, mutation detection, genomic polymorphism analysis, gene library mapping, and hybridization sequencing. However, there are also many problems that hinder its development, such as high cost, complicated technique, low detection sensitivity, poor repeatability, narrow analysis range, etc., mainly found in probe synthesis, probe immobilization, molecular labeling, reading, and efficient and reasonable analysis of large amount of data in the later stage.

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4.2.2  Sequencing-Based Transcriptome Analysis 4.2.2.1  Expressed Sequence Tag (EST) The expressed sequence tag (EST) technique originated from the Human Genome Project. The first report on the application of EST technology by Adams and colleagues proposed the EST concept in 1991 (Adams et al. 1991). They first randomly picked 609 clones from a cDNA library of human brain tissue and then sequenced to obtain a set of expressed sequence tags and finally aligned the sequence homology between the database and the expressed sequence tags and concluded that only 36 of the expressed sequence tags represent known genes and another 337 represent unknown genes. Some very short sequences, ranging from 300 to 500 bp, generated from cDNA libraries, the expressed sequence tags, are essentially small stretches of cDNA fragments corresponding to mRNA. This small segment of genes represents a specific recognition site on the genome and is expressed in cells, tissues, or individuals at a particular tissue or developmental stage. It is useful by using each tag to represent a gene to examine the type and amount of transcripts as a whole. A sufficient number of ESTs from a certain tissue can represent the expression of the gene of the tissue. The number of ESTs can reflect the expression level of a gene. The more copies of a gene, the more abundant its expression and the corresponding ESTs that can be detected. Therefore, it is possible to understand the expression and abundance of genes in the organisms using the technology of expressing sequence tag (Ewing and Green 2000; Nagaraj et al. 2007). 4.2.2.2  Sander Sequencing (SAGE) The serial analysis of gene expression (SAGE) is a rapid detection technique for the detection of mRNA and gene expression levels in cells (Velculescu et al. 1995). It is well known that the types and amounts of mRNA are very large in different cells, and it is very difficult to completely sequence and measure them individually. However, if a short nucleotide sequence can be used to represent each mRNA, the workload will be reduced dramatically. There are two reasons for SAGE: First is a nine- to ten-base sequence containing enough information. A short nucleotide sequence tag can uniquely identify a transcript. For example, the human genome can only encode about 80,000 transcripts, and a nine-base sequence can resolve 262,144 different transcripts (49). So theoretically it can be said that a nine-base tag can represent a transcript sequence uniquely. Second, when the nine-base tags in a clone are sequenced and the data from these short sequence nucleotide sequences are input into a computer for processing in a continuous data format, thousands of mRNA transcripts can be analyzed. So the 9  bp sequence length was originally selected for construction of nucleotide tags. SAGE technology is characterized by its high throughput, fastness, and high sensitivity, which contribute to its wide applications in various fields (Boon et al. 2002). When the nature of the gene is not known in advance, it can also study the biological phenomenon caused by the transcriptional changes of the cell and can

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complete almost all mRNA detection in the cell in a short time, obtain its copy number, and discover the potential unknown gene. This is the biggest advantage of SAGE technology. In addition, this technique can also be used to distinguish different variants and antisense transcripts of transcripts, providing a very large data platform for the study of transcriptomes. SAGE technology provides a powerful tool for the study of quantitative classification and expression of genes in various states by identifying mRNA transcripts with specific short sequence tags on the transcript (CDNA). Multiple tags are randomly ligated into a large number of concatemers and cloned into vectors, and then each clone was sequenced. The initial promotion of SAGE technology has certain difficulties, mainly because of some defects in the previous SAGE method, including difficulty in label identification, large workload, complicated technical process, and huge cost. However, with the development of science and technology, this technology has been widely used in many countries and regions. Nowadays, the development of SAGE method is toward to simplify the operation steps, reduce the sample size, increase the label length, and improve the specificity between the tag and the target gene and other aspects (Ye et al. 2000). 4.2.2.3  Massively Parallel Signature Sequencing (MPSS) In 2000, American scientist Brenner et al. invented a new gene expression analysis technology based on gene microarray—massively parallel signature sequencing (MPSS)— which is an efficient and rapid detection method (Brenner et al. 2000). The technology has been improved on the basis of SAGE, adding a universal linker to the cDNA, which is a pioneer in the development of next-generation sequencing technology. The principle of MPSS: There is a sequence tag site in the base sequence of cDNA that can specifically recognize transcript information, and the length is 10–20b, so the tag sequence is linked together with an unknown long contiguous base sequence. The analysis of the base sequence can be performed on the basis of molecular cloning. The measured gene expression level is a digital expression system implemented on the basis of quantitatively determining the expression level of the corresponding transcript. First, a base sequence is detected at one end of the mRNA, that is, a tag sequence containing 10–20 bases; secondly, the copy number of the tag sequence is calculated, that is, the copy number of the mRNA; and finally, corresponding gene expression levels are calculated by the number of copies of the mRNA. MPSS technology can be used for detection and analysis regardless of whether the gene sequence is known or not and regardless of the level of gene expression. Moreover, this method utilizes powerful statistical techniques to provide an unprecedented depth of analysis for discovering functional relationships between genes (2008). 4.2.2.4  High-Throughput RNA-Seq Technology With the development of next-generation sequencing platforms, high-throughput RNA-Seq technology has been widely used in transcriptomics research in recent years based on deep sequencing technology. RNA-Seq, also known as whole

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transcriptome sequencing, uses a second-generation high-throughput sequencing technology to sequence cDNA libraries that are reverse transcribed from total RNA in tissues or cells and align the sequencing sequences with reference sequences, obtain information quickly and comprehensively on the entire transcript of a tissue or cell under certain conditions, and provide epoch-making new technologies for transcriptomics research. High-throughput RNA-Seq technology opened a new era of genome-wide transcriptomics research. The advent of RNA-Seq has altered our understanding of the variety and complexity of transcriptomes, as well as a way to more accurately evaluate transcripts and their homologs. Accurate and comprehensive information on transcripts of the sample is the most versatile feature of the high-throughput RNA-Seq technology, in addition to its high detection sensitivity, low background signal, accurate accuracy, and good repeatability. The sample consumption is small, the experimental operation is relatively simple, and the detection cost is relatively low. The advantages of RNA-Seq technology are significant compared to the several other sequencing techniques described above. First, RNA-Seq technology not only detects the overall transcriptional level of multiple species at the single nucleotide level but also finds unknown and rare transcripts and can identify and analyze gene fusions and alternative splicing; second, RNA-Seq can be performed on different platforms (e.g., Illumina’s genomic analysis platform) for different experimental purposes. Of course, the biggest advantage of RNA-Seq technology is that it can accurately identify and screen different tissues or cells in the same condition (time) or different conditions (time) in a short time with only a small amount of samples. All differential transcripts are transcribed, as well as comprehensive information annotation and analysis of the genomes corresponding to these transcripts (Wang et al. 2009; Robinson and Oshlack 2010).

4.3  A  pplication of Transcriptomics in the Study of Environmental Stress Responses of LAB During the growth cycle of lactic acid bacteria, the pH will decrease with the change of its growth environment or the gradual consumption of nutrients, resulting in the accumulation of its metabolites. At this time, the survival and growth of lactic acid bacteria will be affected by acid stress, salt stress, oxygen stress, and hunger stress. Therefore, understanding of the stress-resistant mechanism of lactic acid bacteria will be helpful in relieving the damage caused by stress, thereby improving the growth efficiency of lactic acid bacteria. Transcriptomics is a good tool for this purpose. It can study the transcription and transcriptional regulation of lactic acid bacteria under different environmental conditions. Studying the gene expression levels of lactic acid bacteria in different stresses from the RNA level is helpful for comprehensive understanding of various stress response mechanisms. So, it is important, yet hard, to find the anti-stress measures to reduce the effects of stress factors on the survival, growth, and metabolism of lactic acid bacteria.

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4.3.1  Heat Shock Response The heat stress response is a stress response in which microorganisms resist high temperatures in a normal physiological temperature environment. Although lactic acid bacteria are not subject to heat stress in the normal intestinal environment, lactic acid bacteria suffer from some high temperature stress during the production of many products, such as spray drying, pasteurization, etc. (Chen et al. 2013). After being stimulated by lactic acid bacteria, lactic acid bacteria must regulate their expression and metabolic pathways through their own defense systems and produce a series of heat shock proteins to reduce or eliminate the damage caused by high temperature stress. Heat stress response is often accompanied by transient induction of specific proteins and changes in cellular physiological structure, thereby enhancing the resilience of microbial cells to the thermal environment. Bifidobacterium is a strain that naturally resides in the human intestine and has been applied widely in various functional foods. However, during the production and processing of these functional foods, Bifidobacterium is often exposed to a high temperature environment, and the heat stress will have a greater impact on its growth and accumulation of its metabolites. Therefore, in order to better understand the response of Bifidobacterium under high temperature stress in depth, Rezzonico et al. used microarray technology to study the transcriptome of Bifidobacterium NCC2705 under stress condition of different high temperatures (50 °C treatment for 3, 7, and 12 min). The level has been studied in depth. The study showed that the expression of Bifidobacterium genome was significantly changed under high temperature treatment conditions and 46% of the gene expression levels were changed. Under the high temperature stress, the metabolic activity of Bifidobacterium was reduced and the high temperature stress stimulating factor was increased. At the same time, several genes with unknown functions were significantly induced, while the expression levels of three of these genes have remained stable under the high temperature stress condition from other strains, which is sufficient to demonstrate their multifaceted role in high temperature stress response. The final results indicate that the induction of the gene encoding the bifidobacteria and SmpB protein may be related to the high temperature stress response of bifidobacteria (Rezzonico et al. 2007). It was showed by studies on heat stress of Bifidobacterium longum NCC2705 that 46% of genes had changes on transcriptional levels and the metabolic activity-­ related genes of Bifidobacterium longum are downregulated. Genes with significant changes in expression levels include chaperone genes DnaJ, DnaK, GroEL, and GroES, transcriptional activator ClgR, heat shock transcription factor HspR, cold shock protein CspA, transcriptional repressors Lex4 and HreA, endopeptidase ClpB, DNA repair protein RecA, etc. (Rezzonico et al. 2007). The Bifidobacterium breve UCC2003 genome contains a clpB gene belonging to the Clp proteolytic system, similar to the proteolytic system-related gene clpB in actinomycete which induces heat stress and osmotic stress in Bifidobacterium breve (Ventura et al. 2005). When the growth temperature of Streptococcus thermophilus changed from 42 to 50 °C, the expression level of 196 genes (10.4% of total genes) changed, of which

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102 were upregulated and 94 were downregulated. The expression of heat stress-­ related genes such as dnaK, groESL, and clpL is elevated. The gene expression of transcriptional regulators such as the HrcA, CtsR, Fur, MarR, and MerR family protein changed as well. The expressions of genes related to signal transduction, cell wall, ion homeostasis, ABC transporter, and restriction modification system are also induced. There are many alterations of gene expression from a number of genes coding for the proteins with unknown functions (Li et al. 2011).

4.3.2  Cold Shock Response The preparation of the lactic acid bacteria starter is often carried out by vacuum freezing-drying, but the lactic acid bacteria are largely killed at a low temperature of 4 °C (Marceau et al. 2004). Therefore, to study the effect of cold stress on lactic acid bacteria during the preparation of lactic acid bacteria fermenting agent has become more important. When lactic acid bacteria grow below their optimal growth temperature, many physiological and morphological changes occur, such as the effect of low temperature on the cell membrane, low temperature affecting the activity of related enzymes, low temperature causing changes in fatty acids, and low temperature stress on gene expression. Different types of cold-induced proteins produced by different lactic acid bacteria are different. Under cold stress conditions, lactic acid bacteria are affected by two major problems, decreased cell membrane fluidity and disruption of DNA and RNA second structural stability, and further affect their transport systems and DNA transcription, translation, and repair. Some cold stress proteins are also induced by heat shock, and many pressure-related proteins and HSP proteins are inhibited under cold stress (Dominguez and O’Sullivan 2013). The cspL, cspP, and cspC genes of Lactobacillus plantarum are associated with cold stress. Under low temperature conditions, overexpression of cspL gene can accelerate the growth of Lactobacillus plantarum. cspP can reduce the lag phase of Lactobacillus plantarum in the fresh medium. cspC can increase the tolerance of Lactobacillus plantarum to cold stress (Derzelle et al. 2003). Li et al. found that low temperature caused a decrease in the enzyme activity of the lactic acid bacteria and a delay in the action of the enzyme, which may cause a change in the metabolites of the lactic acid bacteria. Growth of lactic acid bacteria under low temperature stress can also reduce the sensitivity of certain metabolic regulation processes, leading to metabolic imbalances and even growth arrest. Although freezing has little effect on hexokinase and pyruvate kinase, it has a significant effect on lactate dehydrogenase. During the freezing process, the expression of the strain lactate dehydrogenase gene was inhibited, which was a major factor in the damage of lactic acid bacteria (Li 2011). Propionibacterium faecalis is a safe probiotic identified by the US Food and Drug Administration. It is usually used to produce Swiss cheese. When matured at room temperature of 30 °C, the cheese was immediately transferred to a 4 °C cold storage and stored for about 9 days. During this low temperature period of 9 days, Propionibacterium freudenreichii did not die, but continued to survive. Dalmasso et al. used transcriptomics, proteomics, and RT-qPCR methods to explore and study

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the survival rate and metabolism of the probiotics at low temperatures (Dalmasso et al. 2012). A transcriptome microarray analysis of 565 genes in this strain revealed that 25% of the genes had differences in gene expression at 30 and 4 °C. At 4 °C, gene expression in the cell structure of Propionibacterium freudenreichii decreased. In the process of starch and glycogen synthesis, in the link of lactic acid, alanine, and serine to pyruvate, the related genes are overexpressed. Therefore, it is concluded that Propionibacterium freudenreichii plays an important role in the formation of cheese flavor. Even at low temperatures, P. faecalis still maintains good cellular metabolic activity. It has been reported that the cold stress of L. lactis is often induced by lower temperatures in a transient way (Panoff et al. 1994).

4.3.3  Acid Stress Response Lactic acid bacteria produce a large amount of organic acids during their growth. With the accumulation of various organic acids, lactic acid bacteria are in a low pH acidic environment, causing most of the growth and metabolism of lactic acid bacteria to be carried out in this low pH acidic environment. Thus, the lactic acid bacteria have an acid stress response. The acid stress response mechanism of lactic acid bacteria is a complicated network of regulation system, involving many aspects, among which the more important regulation includes the regulation of gene and protein expression (Wu et al. 2007). Acid stress often induces various stress response mechanisms of lactic acid bacteria, including regulating the homeostasis of pH in lactic acid bacteria cells, inducing expression of stress-regulated proteins, and maintaining various physiological functions of cell membranes (Wu et al. 2012a, b). Broadbent et al. (2010) used a combination of transcriptomics and physiological analysis to analyze the mechanism of acid tolerance in Lactobacillus casei ATCC334. In physiological studies, cell membrane fatty acids such as saturated fatty acids and cyclopropane fatty acids were significantly higher in acid-adapted cells than in control cells. It was also found that the percentages of C14:0, C16;1n(9), C16:0, and C19:0(1lC) in the acid-adapted cells were significantly higher than those in the control group, while the percentages of C18:1n(9) and C18:1n(11) were significantly decreased (Broadbent et  al. 2010). The transcriptome analyses showed that acid adaptation induced a stress response by comparing the acid-adapted group (pH 4.5 for 20 min) with the control group (pH 6.0 growth condition), which promoted the acid-resistant response of cells, including fermentation of malic acid and lactic acid and accumulation of intracellular histidine. The microarray dataset shows that in an acidic environment at pH 2.5, the induction of chemical stress, by adding malic acid or 30 mmoL histidine, can increase the survival rate of C. sinensis by nearly 100-­ fold. Organic acid lactate is the main fermentation product of Lactobacillus plantarum. Lactic acid in free state has a greater inhibitory effect on living cells. In general, organic acid stress is difficult to study because its toxicity is highly dependent on its environment, including free acid and pH.  Pieterse studied the lactic acid stress mechanism of Lactobacillus plantarum by culturing Lactobacillus plantarum in different concentrations of lactate and different pH and osmolality to observe the

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changes in the transcriptome triggered by stress factors of different lactic acids (Pieterse et al. 2005). Microarray analyses showed that the genes encoding transketolase (lp_3538) and transaldolase (lp_3539) were upregulated under the lactic acid stress conditions and both of them were inevitably associated with the pentose phosphate cycle. At the same time, the expression of the gene encoding phosphotransferase (lp_3531) was increased by fivefolds; the genes encoding phosphoenolpyruvate synthase (lp_l9l2) and phosphoenolpyruvate hydroxylase (lp_3418) were significantly increased as well. Both enzymes are essential enzymes on the pathway for the conversion of pyruvate to oxaloacetate. The subsequent conversion to malic acid by malate dehydrogenase can produce NAD+, and this pathway is further activated by downregulation of both pyruvate hydroxylase gene (lp_2136) and pyruvate kinase gene (lp_l897). Koponen et al. used a combination of transcriptomics and proteomics to study the phosphorylation and protein expression of Lactobacillus rhamnosus LGG after acid stress with two physiological conditions most relevant to their physiology were selected, pH 48 and pH 58 (Koponen et al. 2012). The results from both microarray transcriptomics and proteomics were consistent. The results of transcriptomics revealed that the most important enzyme against strains of acid stress is F0F1-­ ATPase. F0F1-ATPase can maintain a relatively stable pH in the lactic acid bacteria producing cells and pump out the protons (H+) from inside of the cells consuming the ATP energy in the cells. The study found that the expression of F0F1-ATPase gene was upregulated under acidic conditions in LGG strains, while the expression levels of a large number of genes related to nucleic acid biosynthesis and protein synthesis were dramatically reduced. It was also proved that LGG strain can regulate pyruvate metabolism under the influence of pH. In addition, pH changes lead to changes in the protein phosphorylation of the strain. Based on the pH change during the fermentation of skim milk with Streptococcus thermophilus, Streptococcus thermophilus at different fermentation time points were collected, and 1962 genes were compared by transcriptomics. The first time period was used as a control group, and the expression level of 115 genes in the second time period was changed. Among them, 65 genes were upregulated and 50 genes were downregulated. In the third period, the expression levels of 114 genes changed, of which 33 genes were upregulated and 81 genes were downregulated. In the fourth time period, the expression levels of 125 genes changed, of which 43 genes were upregulated and 82 genes were downregulated. Through the analysis of different expressions of genes, it was found that ptsG plays a certain role in the utilization of glucose by Streptococcus thermophilus and may also be involved in the regulation of certain metabolism. Its encoded protein plays an important role in acid tolerance. Two-component systems, Nudix family hydrolase and κRE family regulators, also play important regulatory roles in acid tolerance (Li 2014). Fernandez et  al. used a combination of proteomics and transcriptomics techniques to reveal the cellular changes in Lactobacillus bulgaricus during acid adaptation (Fernandez et  al. 2008). Under acid-stimulated conditions, two proteins associated with carbohydrate and pyruvate metabolism, PoxI and Fba, were induced by Lactobacillus bulgaricus. Fatty acid biosynthesis-related genes accC, fabH, and fabl were also induced. The chaperone protein genes grpE, groES, groEL, hrcA,

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dnaK, dnaJ, clpE, clpL, and clpP were induced by acid, while the synthesis of ClpC involved in stress response was inhibited. It is clear that when the Lactobacillus bulgaricus is subjected to acid stress, the pyruvate metabolism changes, and the biosynthesis of fatty acids is further strengthened, thereby affecting the fluidity of the cell membrane and improving the acid resistance of Lactobacillus bulgaricus.

4.3.4  Oxidative Stress Response The effect of oxygen on the physiological metabolism of lactic acid bacteria has two sides. The biomass of lactic acid bacteria grown under aerobic conditions is higher than that of lactic acid bacteria grown under anaerobic conditions, because in the case of aerobic conditions, the energy released by conversion of lactic acid to acetic acid can be utilized after the end of glucose consumption. However, in the conversion process of lactic acid and acetic acid, H2O2 is produced under the action of NADH oxidase and pyruvate oxidase (Fu 2013). H2O2 is a non-free radical oxygen derivative of glucose reactive oxygen species (ROS). These reactive oxygen species, non-radical oxygen derivatives, can cause some degree of damage to the organism (Messner and Imlay 1999). Reactive oxygen species can destroy the biofilm, protein, and iron-dependent enzymes of lactic acid bacteria cells and also cause DNA oxidative damage and metabolic dysfunction and expression disorder. The reduction of reactive oxygen species will reduce the damage to cells, and reducing such damage is one of the key factors affecting the biomass and the survival of lactic acid bacteria. The regulation mechanism of lactic acid bacteria on oxidative stress is mainly divided into two types, namely, scavenging reactive oxygen species and body protection mechanisms to reduce the negative effects caused by oxidative stress. Zuo and colleagues used physicochemical structural analysis and transcriptomics techniques to study the oxidative stress response mechanism of Bifidobacterium longum subsp. longum BBMN68 (Zuo 2014). The morphological and surface properties under oxidative stress showed that the aggregation and surface h­ ydrophobicity of Bifidobacterium were stronger with the increase of oxidative stress and time and polyphosphate particles were gradually produced in the cells. Bifidobacterium can adapt to oxidative stress by altering the structure of its cell surface and simultaneously produce polyphosphate as a molecular chaperone to protect its protein. In addition, they also used RNA-Seq technology to determine the expression profile of Bifidobacterium longum subtype BBMN68 under oxidative stress conditions and analyzed the functions of differential genes and the metabolic pathways involved in these genes by bioinformatics. The results showed that the response of Bifidobacterium longum subtype BBMN68 to oxidative stress is a comprehensive and complex process involving the whole genome and 20% of genes had more than two times difference in the level of gene expression. Responses to oxidative stress include a range of defense and adaptive mechanisms, such as promoting the degradation of reactive oxygen species and maintaining redox balance, inducing general stress response and adaptive adjustment of physiological processes such as protein repair and central metabolic pathways. By overexpressing the differentially

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expressed genes in the oxidative stress response of Bifidobacterium longum BBMN68, the genes of interest, such as nfB1, nfnB2, trxB1, nox, and ahpC, were functionally verified. The results indicate that AhpC is the main enzyme in Bifidobacterium that clears endogenous peroxides produced by aerobic metabolism. AhpC also interacts with other antioxidant stress genes to promote or activate different electron acceptors, such as thioredoxin (Trx), or other NADPH-based ROS clearance pathways to cope with oxidative stress. By recombinant co-expression of LpKatL and SSodA4 in Bifidobacterium, the accumulation of peroxide in the cells was greatly reduced, the nucleic acid is protected from free radical damage, and the viability of Bifidobacterium cells was greatly improved under lethal oxidative stress conditions. It is worth noting that the introduction of LpKatL and StSodAa effectively alleviated the stress of the cell’s own antioxidant system, including TrxB1 and HenN involved in reactive oxygen scavenging, Fe-S protein complex, Fe2+ chelated Dps protein, protein repair associated DnaK and Lpd2, etc. Serrano et al. (2007) used transcriptomics techniques to study the role of thioredoxin (TRX) in the anti-oxidative stress of Lactobacillus plantarum WCFS1 (Serrano et al. 2007). Sulfuroreductin is an important disulfide oxidoreductase that catalyzes a series of redox reactions in cells. trxBI-encoded thioredoxin reductase is an important enzyme expressed by Lactobacillus plantarum WCFS in response to oxidative stress, which can help the cells to resist the damage caused by oxidative stress. It was found that the activity of thioredoxin reductase activity in the trxBI gene-overexpressing strain was three times higher than that of the wild-type strain. At the same time, as the intracellular thioredoxin reductase activity is increased, a series of antioxidant stress reactions occur. The transcriptome analysis of wild-type and txB gene-overexpressing strains under oxidative stress conditions showed that 267 genes were upregulated due to oxygen stress and the production of a large number of thioredoxin reductase. In addition, the expression of 27 genes in the trxBI-­ overexpressing strain was also confirmed to be significantly changed by gene expression analysis. The overexpression of trxBI gene can activate repair DNA repair, stress response, and sulfur and sulfur-containing amino acid related amino acids biosynthesis pathways. There are 16 genes that respond to the large-scale production of thioredoxin reductase and oxidative stress, including genes related to sputum metabolism, energy metabolism (gapB), stress-related response (groEL, npr2), and manganese transport (mntH2). All of the above results indicate that thioredoxin reductase encoded by trxBI can increase the tolerance of Lactobacillus plantarum to oxidative stress, and this response simultaneously induces transcription of 16 genes involved in anti-oxidative stress.

4.3.5  Osmotic Stress Response During the fermentation and preservation of lactic acid bacteria-fermented foods, lactic acid bacteria are often in an environment of high salt concentration. However, the high concentration salt causes the change of osmotic pressure to trigger the

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water inside of the cells flow to the outside and the cytoplasmic separation which leads to bacteria cells to stop growing or even die. Therefore, the ability to adapt to salt stress is a very important factor for lactic acid bacteria to survive and grow in natural living environment and industrial production. Lactic acid bacteria use many strategies to cope with changes in external osmotic pressure to maintain normal osmotic pressure within the cell. Some outer membrane proteins of lactic acid bacteria can directly or indirectly regulate the permeability of cell membranes to salt ions to participate in the regulation of osmotic pressure. The balance of Na+ on lactic acid bacteria cell membrane is achieved by the efflux of Na+ via the N+a/H+ reverse transporter on the plasma membrane. In addition to the efflux of Na+, it usually increases its absorption of K+ to restore the balance of Na+/K+, thereby restoring the relationship between water and ions in the lactic acid bacteria cells. K+ can promote the recovery of morphology and osmotic pressure of lactic acid bacteria cells and function as an enzyme activator and messenger of gene expression (Glaasker et al. 1996). Lactic acid bacteria are Gram-positive bacteria, but their ion balance is very similar to Gram-­ negative bacteria, such as E. coli (Calderon et al. 2004). The primary response of E. coli to external osmotic pressure is to absorb a large amount of K+ from the outside and then absorb the compatible soluble solutes to protect its osmosis. Some scholars have studied the salt adaptation of Halophilic tetrazolium and Phytophthora sphaeroides and found that there is no significant relationship between intracellular K+ concentration and changes in extracellular salt concentration (Glaasker et al. 1996; Robert et al. 2000). Thus, K+ plays a relatively small role in the osmotic balance of lactic acid bacteria. A large amount of compatible solutes that accumulate in cells may be the main strategy for lactic acid bacteria to cope with the increase of external osmotic pressure (Le Marrec 2011). Under the condition of hyperosmotic concentration, decomposing metabolic compatible solutes are inhibited in the lactic acid bacteria cells by intracellular enzymes. A high concentration of compatible solutes can be accumulated in bacteria cells, thereby exerting the function of osmotic protection. At present, researchers have found that compatible solutes that can accumulate in bacterial cells are mainly amino acid derivatives, amino acids, small peptides, methylamines, and their sulfonic acid analogs, sulfates, and polyols (Roberts 2005). Many species of lactic acid bacteria, such as Lactobacillus casei, Lactococcus lactis, and Lactobacillus plantarum, are able to absorb certain compatible solute from the outside of the cell to protect the cells (Hutkins et al. 1987; Molenaar et al. 1993; Kets and de Bont 1994; Glaasker et al. 1996). Many lactic acid bacteria cells, after salt stress, respond to increased osmotic pressure by altering the lipid composition of their cell membranes. Changes in the lipid composition of the cell membrane, particularly the ability to modify changes in the lipid head group, can affect the activity of the compatible solute transporter. Studies have shown that when salt stress occurs, the most important change in the fatty acid composition of L. lactis NCDO 763 cell membrane is the increase in the content of cyclopropanol fatty acid, and the ratio of unsaturated fatty acid to saturated fatty acid in the cell membrane does not change. In addition, the amount of

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cardiolipin has also changed when the osmotic pressure in the environment has increased, and this change is also one of the most important factors for bacteria to adapt to salt stress (Romantsov et al. 2009). Studies have shown that when the extracellular osmotic pressure of lactic acid bacteria increases, the expression levels of some genes in lactic acid bacteria will change accordingly. These genes do not participate in the absorption of compatible solute, such as genes encoding universal stress response proteins. These universal stress response proteins include molecular chaperones and some proteases such as DnaK, GroEL, and GroES. Through transcriptomics and proteomics studies, it has been found that lactic acid bacteria induce increased expression of stress-responsive proteins under conditions of high osmotic pressure (Flahaut et al. 1996). Transcriptomics studies have shown that the high salt environment greatly affects the transition between citric acid and succinic acid, hindering the progress of the lactic acid degradation cascade. Citric acid is more involved in the transport process between cell membranes in response to the effects of high concentrations of osmotic pressure. It also affects some of the functions of cell membranes including the synthesis of teichoic acid (Bron et al. 2006). Under the induction of high concentration of salt, the expression level of genes involved in carbohydrate metabolism in lactic acid bacteria cells will also change accordingly. For example, when Lactobacillus rhamnosus is subjected to 3.5% salt stress, glycolysis-related enzymes were all significantly altered, such as glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose phosphate isomerase (Prasad et al. 2003). Lactobacillus sakei showed a downward trend in the expression of fructose kinase converting fructose-6-phosphate to fructose-­1,6-phosphate in a growth environment with a salt concentration of 4% (Marceau et al. 2004). It can be explained that when the lactic acid bacteria respond to changes in the external osmotic pressure, their own metabolic regulation has not stopped, and corresponding changes have occurred. Zhao analyzed the functional role of glycine betaine in salt tolerance of Lactobacillus plantarum ST-III and the salt tolerance response of Lactobacillus plantarum ST-III using RNA-Seq transcriptomics sequencing technology (Zhao 2014). The salt-tolerant gene cluster ProU of Lactobacillus plantarum ST-III was verified functionally. The results showed that the transcription level of Lactobacillus plantarum ST-III was affected to some extent by the amount of salt added, including the balance of inorganic ions, carbohydrate transport and metabolism, amino acid transport and metabolism, cell wall/membrane/encapsulated biosynthesis and DNA replication, recombination, repair, etc. Compared to those cultured at low salt (2% NaCl) concentration, when cultured with Lactobacillus plantarum ST-III at high salt concentration (6% NaCl), more compatible solute transporters were found, and the genes coding for inorganic ion transporters and DNA repair have changed. In addition, in three samples with different salt concentrations (control, 2% NaCl, 6% NaCl), a total of 146 ncRNAs were predicted, of which 33, 54, and 10 ncRNAs were in the control group, 2% NaCl and 6% NaCl, respectively. Of the 146 ncRNAs, 37 ncRNA target genes were successfully predicted. When glycine betaine was present in the environment, the expression level of the compatible solute transporter did not

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change significantly. Carbohydrate transport and metabolism, ribosomal structure and synthesis, cell wall/membrane/membrane biosynthesis, and inorganic ion transport and metabolism are all altered by the addition of glycine betaine.

4.3.6  Bile Stress Response Bile is the second stress other than acid that lactic acid bacteria face when entering the human gastrointestinal tract. It is composed of a mixture of electrolytes, bile salts, phospholipids, cholesterol, bilirubin, and proteins. Bile can be secreted into the duodenum to promote the emulsification and absorption of fat-soluble nutrients (Begley et al. 2005). The presence of a certain proportion of bile can cause stress on the lactic acid bacteria cells, resulting in DNA damage, protein structure changes, and prolonged protein transport time. Lactic acid bacteria cells play an important role in bile salt stress by lipid composition and membrane egg yolk and cell membrane function. The tolerance of intestinal commensal microorganisms to bile salt exposure is an important feature of their survival and colonization of the intestinal environment. Ruiz et al. studied the bile salt protection system of Bifidobacterium breve UCC2003 by means of transcriptomics and found a large number of bile salt-inducing genes with specific functions, such as the Bbr_0838, Bbr_0832, and Bbr_1756 genes that assist the transporter MFS superfamily, and three genes that direct the ABC transporter Bbr_0406–0407, Bbr_l804–1805, and Bbr_1826–182 (Ruiz et  al. 2012). Bron et  al. applied gene hybridization microarray technology to study the whole gene transcription of Lactobacillus plantarum WCFS1 under 0.1% porcine bile salt stimulation conditions (Bron et  al. 2006). Transcriptome measurements revealed that 28 genes were upregulated and 62 genes were downregulated in the presence of bile salts. Among them, seven genes involved in oxidative stress and acid stress are also involved in the bile salt stress response of Lactobacillus plantarum WCFS1, including glutathione reductase gene and glutamate decarboxylase gene. It was also found that 14 genes localized on the cell membrane changed, including the dlt operon, F0F1-ATPase enzyme, etc. Koskenniemi et  al. combined transcriptomics and proteomics to study the response of Lactobacillus rhamnosus LGG under different concentrations of bile salt (Koskenniemi et al. 2011). The results showed that under the influence of 0.2% of bovine bile salts, there were 316 genes that changed significantly at the transcriptional level, while the expression of 42 proteins changed at the protein level, including proteins in the cell and on the cell surface. Among them, changes of 14 proteins correspond to the genes changes at transcriptional level. These results indicate that under normal bile salt stress, common stress responses and cell membrane-related functionalities occur in a variety of ways, including effects on fatty acid composition, cell surface charge, and extracellular polysaccharide thickness. These changes may enhance the ability of cell membranes to resist bile salt stress. It is worth noting that there is a significant decrease in the protein that catalyzes the synthesis of extracellular polysaccharides, whereas a protein specifically designed to remove bile

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salts from cells is upregulated. All these indicate that cell can alleviate bile-triggered damage by regulating the protein expression. Lactobacillus is constantly secreting bile salt hydrolase, and whether bile salt hydrolase affects bile salt tolerance requires further investigation. Most of the bile salt hydrolase gene bsh1 of Lactobacillus salivarius is located on the mitochondria of the strain, and small number of Lactobacillus salivarius has an additional bile salt hydrolase gene bsh2 on the chromosome. The bsh2 gene tends to have a higher level of bile salt tolerance. By exposing Lactobacillus salivarius to bile and bile salt medium, respectively, transcriptomics was used to study the gene expression level; it was found that the expression of proteins related to pressure and promoting substance outflow changes significantly, while the gene expression of bile salt hydrolase has no significant change. Thus, the changes in bile salt hydrolase of Lactobacillus salivarius do not determine the tolerance level of bile salt tolerance, and other biological effects may exist in bile salt hydrolase (Fang et al. 2009).

4.4  A  pplication of Transcriptomics in the Analyses of LAB Diversity of Fermented Food Lactic acid bacteria-fermented food usually refers to a microbial fermentation process mainly consisting of lactic acid bacteria, which degrades macromolecules such as proteins and carbohydrates into small molecules, and the main metabolite is lactic acid, with a taste of acidic, aromatic, and fresh flavor. As one of the main microbial groups of traditional lactic acid-fermented foods, lactic acid bacteria have the isotypic and heterotypic lactic acid fermentation. In the isotypic lactic acid fermentation process, carbohydrate products are degraded into the lactic acid only. In addition to lactic acid, volatile compounds such as alcohols and aldehydes are also produced in the heterotypic lactic acid fermentation process, giving the unique taste and aroma of fermented foods. Meanwhile, the growth of lactic acid bacteria via interspecific competition forms an acidic environment and produces antagonistic metabolites, which preferably inhibits spoilage microorganisms and pathogenic microorganisms in the product. Therefore, lactic acid bacteria play an important role in the flavor and safety of the product. Traditional lactic acid-fermented foods are inseparable from the important role of lactic acid bacteria. In our daily life, there are many types of foods fermented by lactic acid bacteria, which can be divided into fermented vegetables, fermented seasonings, fermented sourdoughs, fermented dairy products, fermented meat products, etc. (Miao et al. 2015). Changes of microflora in fermented foods can be studied by transcriptomics techniques, which can systematically analyze the metabolic changes and responses of the entire microbial community. The following is an introduction to the application of transcriptomics from the perspective of different fermented foods.

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4.4.1  Fermented Vegetables The lactic acid bacteria-fermented vegetables are mainly sauerkraut, Sichuan kimchi, and kimchi. These fermented vegetables are not only crispy, delicious, and appetizing but also have the effect of promoting digestion and other special effects. For example, sauerkraut contains a large amount of cellulose, minerals, and organic compounds which are indispensable for human metabolism, among which are lactic acid, choline, acetylcholine, vitamin C, vitamin B12, and so on. Sauerkraut is not only unique in flavor but also has physiological functions such as regulating cholesterol, regulating blood balance, and preventing atherosclerosis. The main fermentation strain of sauerkraut is lactic acid bacteria. Studies have shown that in the long fermentation process, Leuconostoc is the dominant bacteria in the early fermentation of sauerkraut in Northeast China, and then the acid is produced by Lactobacillus acidophilus, Lactobacillus plantarum, and fermented Lactobacillus. The final fermentation process is mainly completed by Lactobacillus plantarum. It is clearly that the dominant bacterium in the early stage of sauerkraut fermentation is Leuconostoc and the dominant bacterium in the middle and late stages of fermentation is Phytophthora sinensis (Wu et al. 2014). The metagenomic analysis was used to study the distribution of microbial flora and the diversity of bacteria in Sichuan kimchi fermentation process. The study found that the microbial flora in Sichuan kimchi was mainly lactic acid bacteria. Weissella can reach 74.5% at the beginning of fermentation, while in the later stage, it is maintained at about 10%, and the dominant bacteria shit to Lactobacillus, and its content can reach 80%–85%. It is proved that in the fermentation process of Sichuan kimchi, Weissella is only a starter and the key bacterium in the fermentation process is Lactobacillus (Tong et al. 2015). Kimchi is a classical Korean fermented vegetable, with about 23 lactic acid bacteria involved in the fermentation of Korean kimchi (Nam et al. 2009; Jung et al. 2013). Jung et al. used RNA-Seq sequencing macrotranscriptome methods to study the structural micro-changes and genetic characteristics of microbial community and metabolic changes 29d fermentation of Korean kimchi. A total of 701,556 reading lengths were obtained by 454 sequencing from kimchi samples, with an average length of 438 bp. 16S rRNA sequencing analyses showed that the kimchi microbial community was mainly composed of three genera, namely, Leuconostoc, Lactobacillus, and Weissella (Jung et  al. 2011). The coverage of transcriptome sequencing data also showed that Leuconostoc mesenterica subspecies ATCC8293 and Lactobacillus sake 23K were highly expressed, suggesting the importance of these two strains in Korean kimchi. Leuconostoc mesenterica was the most active in the early stage of fermentation, while Lactobacillus sake and Weissella koreensis were more active in the late stage of fermentation (Bokulich et al. 2016). In addition, some bacteriophage DNA sequences were found, which proved that the strains in fermentation process had been contaminated by bacteriophages. In conclusion, the application of transcriptome to explore the evolution of microbial communities in fermented vegetables is of practical significance for the development of industrial fermented vegetables.

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4.4.2  Fermented Condiments The most common traditional fermentation condiments, such as brewing soy sauce, vinegar, tofu (fermented bean curd), soy sauce, etc., are rich in lactic acid bacteria in the fermentation process, which play an important role in its quality and taste. Lactic acid bacteria can ferment sugars to produce lactic acid, citric acid, and other organic acids, which may improve the flavor of condiments and soften the taste. At the same time, organic acids can esterify with ethanol produced by ethanol fermentation to produce esters and other flavor substances. Therefore, it is speculated that Lactobacillus may be related to the formation of organic acids and ester flavor substances and may contribute to the formation of flavor in fermented condiments. Secondly, Lactobacillus produces acid in the ethanol fermentation stage, which can produce synergistic bacteriostasis with ethanol to prevent contamination during the ethanol fermentation stage. The lactic acid bacteria in soy sauce are mainly Lactobacillus plantarum, Leuconostoc mesogenes, Lactobacillus brevis, Pediococcus lactis, and Tetracoccus (Zhang et al. 2014). The study of Lactobacillus in vinegar using macrogenomic method found that Lactobacillus, Staphylococcus, and Lactococcus were the main species of Lactobacillus (Wang et  al. 2016). Tetracoccus halophilis and salt-tolerant Lactobacillus such as Campylobacter and Lactobacillus casei are the main fermentation strains of tofu (Han et al. 2001). Cheonggukjang is a local delicacy in South Korea. It smells like a corpse, so it gets its name. This sauce is thick and tastes delicious. Transcriptomic studies found that the dominant bacteria in this food were Bacillus spp. and Lactobacillus spp. (Nam et al. 2012). Duan et al. used macrotranscriptome and 16S rRNA sequencing to explore the correlation between flavor formation and flora structure during shrimp paste fermentation (Duan et al. 2016). They found that Tetracoccus accounted for 95.1% of the total population. After searching the Nr database, 520 of 588 transcripts matched the transcripts of Tetracoccus halophilis. The citric acid cycle and oxidative phosphorylation of Tetracoccus halophilis were activated, but the lactate dehydrogenase gene was not expressed. It was proved that Tetracoccus halophilis mainly undergoes aerobic metabolism during the fermentation of shrimp paste. Amino acid metabolism, peptidase production, and gene expression of dipentene and pinene degradation pathway in Tetracoccus halophilis are also very active.

4.4.3  Fermented Sourdough The traditional sourdough dough is a flour product obtained by microbial fermentation of wet flour and is a fermenting strain which is necessary in the process of making steamed bread. The main fermentation bacteria in steamed bread fermentation process come from microorganisms in sourdough, which play a very important role in fermentation of pasta. That is to say, the fermentation process of sourdough is a process in which microorganisms decompose the protein and carbohydrate components in the

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dough and interact to produce flavor substances such as alcohol, phenol, aldehyde, ester, and so on. The fermentation process not only improves the texture of dough, forms unique flavor and taste, but also forms an acidic environment by microbial fermentation and prevents food spoilage caused by fungi or bacteria contamination. Sourdough is an extremely complex micro-ecological system. Studies have shown that there are no less than 50 kinds of lactic acid bacteria and about 20 kinds of yeasts, mainly Lactobacillus, Saccharomyces, and Candida in sourdough (De Vuyst and Neysens 2005). Zota et al. isolated 41 strains of lactic acid bacteria from the traditional sourdough “Cornetto” in Southern Italy, including Phytophthora, Leuconostoc, Lactobacillus plantarum, Lactobacillus curvatus, pentose Lactobacillus, and Weissella sinensis (Zotta et al. 2008). Metagenome analysis of Mexican fermented corn dough “pozol” found that it contains 14 species of bacteria, including Lactococcus lactis, Streptococcus suis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus alimentarius, Lactobacillus delbrueckii, Clostridium, etc. (Escalante et al. 2001). Macrotranscriptome analysis of traditional “fermentation starter” found that Lactobacillus plantarum and Lactobacillus fermentum are the most important lactic acid bacteria, followed by Pediococcus pentosaceus. Lactococcus lactis acts early in the fermentation of the entire flora ecosystem (Weckx et al. 2010). Wu and colleagues isolated and identified yeast and lactic acid bacteria from sourdough in the western part of Inner Mongolia, China. A total of 85 yeast strains and l08 lactic acid bacteria strains were isolated from 28 sourdough samples, including the following: 37 strains of Lactobacillus plantarum, 14 strains of Leuconostoc citreum, 10 strains of Weissella sinensis, 8 strains of Lactobacillus hominis, 7 strains of Weissella faecalis, and 7 strains of Lactobacillus helveticus (Wusiriguleng 2011).

4.4.4  Fermented Dairy Products Lactic acid bacteria-fermented dairy products are mainly divided into acidic fermented milk (yogurt, sour camel milk), alcoholic fermented milk (kumiss, posset), cheese, and sour cream. Dairy products fermented using lactic acid bacteria have the functions of improving the quality of dairy products, improving the flavor, enhancing the healthcare function, and extending the shelf life of products and are well received by people all over the world. Sun et  al. isolated and identified lactic acid bacteria in traditional sour camel milk produced by herders from Xinjiang and Mongolia, China (Sun et al. 2006). The lactic acid bacteria isolated from four parts of camel milk include four strains of Lactobacillus helveticus, two strains each of L. casei subsp. pseudoplantarum and Lactobacillus bulgaricus, and one strain each of Pediococcus lactis, Bacterium flexuosus, Pediococcus urinaeequi, and Enterococcus faecalis. The separation and identification of the kumiss fermenting bacteria were carried out with the horse milk wine from pastoral area in Xilin Gol, China, as the source of separation (Ma 2005). A total of 47 strains were isolated and belonged to 18 genera including Lactobacillus,

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Enterococcus, Leuconostoc, and Lactococcus. The leading strain of cheese fermentation is lactic acid bacteria, which decomposes lactose into lactic acid, thereby lowering the pH of the fermentation environment, inhibiting the growth and reproduction of spoilage bacteria to extend the preservation time of the cheese. Ma et al. isolated and identified lactic acid bacteria in cheese products from different pastoral areas in Xinjiang, China, and obtained 104 strains of lactic acid bacteria, of which 82 strains belonged to the genus Lactobacillus, 12 belonged to the genus Enterococcus, and 10 belonged to the genus Weissella. These lactic acid bacteria are Lactobacillus casei, Lactobacillus plantarum, Lactobacillus licheniformis, Lactobacillus militaris, Weissella sinensis, and Escherichia coli (Ma et al. 2011). Lactococcus lactis is widely used in the production of cheese. Lactococcus lactis faces different stress environments at every stage of cheese ripening. Cretenet et al. applied transcriptomics to study the expression of Lactococcus lactis in different processes of cheese making (Cretenet et al. 2011). The first step of cheese fermentation process is to produce lactic acid by Lactococcus lactis, and the pH is continuously lowered. Under acid stress, F0F1-ATPase acts on the elimination of cytoplasmic proton. Both the arginine deiminase and the citrate decarboxylase pathway are involved in pH homeostasis and energy production. At the 8 h time point of cheese fermentation, its pH was about 5.9. As showed by expression profiling, only three genes in the F0F1-ATPase system (seven in total) were overexpressed in the process of cheese fermentation. The genes atpG and atpD were overexpressed at 8 h time point of fermentation, and atpD and atpF were overexpressed at day 7 of fermentation. Citrate metabolism-related genes citC, citD, citE, and citF have transient overexpression at 8 h time point of fermentation. The transcriptomics analysis showed that the transcription of genes related to the F0F1-ATPase pathway did not change significantly after 8  h of Lactococcus lactis fermentation, which proved that the F0F1-ATPase pathway is not the acid stress pathway in Lactococcus lactis. The expression of arginine deiminase pathway activators ahrC, αrcB, and arcC1 increased, while the expression of arginine reverse transport system-related genes arcD1 and arcD2 decreased. The arginine deiminase pathway is involved in the pH stress response of Lactococcus lactis. Bisanz et al. is the first to use the RNA-Seq technique for transcriptomics analysis of two yogurts with different flavors of strawberry and vanilla and at different time points of fermentation (Bisanz et  al. 2014). They used the ABI SOLID4 sequencing platform to sequence mRNA, yielding 48,658,804 read reads and 37.2 time coverage per sample, but the sequence coverage of Lactococcus lactis is less than 39%. At the same time, they also compared the abundance of mRNA with high sequencing coverage in yogurt with different flavors and at different times. By clustering analysis of protein adjacent clusters (COG) of genes with RPKM values ≥200 and RPKM values ≥1000, the functions of the gene can be found in each microorganism. Genes functionally related to carbohydrate transport and metabolism, amino acid transport and metabolism protein translation, and amino acid transport and metabolism are all highly expressed. Statistics analysis showed that the high abundance genes in most microorganisms can be classified into the functional area of “transportation,” except Streptococcus thermophilus. Streptococcus

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thermophilus exhibits a high abundance in both carbohydrate transport and metabolic domains, including the beta-galactosidase gene annotated as “Lacz,” which primarily functions as a lactose-degrading function. There is also higher expression in Lactobacillus delbrueckii subsp. bulgaricus, but it is reversed in the Bifidobacterium animalis subsp. lactis. Analyzed using the Kyoto Gene and Genomic Encyclopedia (KEGG) pathway, genes involved in the carbon transport metabolic process and the glycolysis process are highly expressed in Streptococcus thermophilus, while the genes associated with energy production and transfer are highly expressed in Bifidobacterium lactate subspecies in animals. In addition, the KEGG analysis also showed that these highly expressed genes contain six F0F1-­ ATPase genes that play an important role in the tolerance of the bile salts and acids. Among the mRNA transcripts expressed from all microorganisms in different samples, the protein metabolism and specific ribosomal protein components are the most representative; further analysis revealed that prokaryotic bacteria can adapt to this fermentation environment. The research group from Inner Mongolia Agricultural University, China, also used RNA-Seq transcriptomics to compare the growth of Lactobacillus casei in milk and soymilk. When Lactobacillus casei Zhang was grown in cow’s milk, the expressions of 84 genes were significantly changed in the stable growth phase of pH 4.5 and the logarithmic growth phase of pH 5.2. Fifty-nine of them were significantly upregulated, and 40.5% of these genes are functionally associated with carbohydrate and energy metabolism. The major upregulated genes are associated with the PTC system and the pentose phosphate pathway. In the soymilk, there are 63 genes which had significant change in the expression at the stable growth period of pH 4.5 and at the logarithmic growth phase of pH 5.2, and expression of 162 genes changed significantly at the logarithmic growth phase of pH 5.2 and at the hysteretic growth phase of pH 6.4. Among them, 48.6% of the genes were upregulated in the logarithmic growth phase, 48.8% of the genes were upregulated in the stable growth phase, and all of them were related to the transport and metabolism of amino acids. The function of main upregulated genes is associated with proteolytic enzyme system (extracellular proteases, oligopeptide transport systems, and intracellular peptidases), amino acid (glutamic acid, lysine, and methionine), and nucleotide (purine and pyrimidine) metabolism. Particularly, the active expression of the genes related to proteolytic enzyme system makes Lactobacillus casei Zhang able to decompose soy protein in soymilk, providing sufficient amino acids and nucleotides for its growth, and may contribute to the better growth of Lactobacillus casei Zhang in soymilk than in cow’s milk (Wang 2012). Burenqiqige and colleagues studied the community structure and the expression of functional gene in sour horse milk samples from different fermentation periods (Burenqiqige 2014). Followed by transcriptomics metagenomics analyses, they adjusted and optimized the community structure and community function, which provides a theoretical basis for the safety, clinical application, and improvement of traditional fermentation of sour horse milk. The selected sour horse milk samples from the fermentation of six different stages were subjected to the metagenomic analyses of bacterial community structure succession during the fermentation pro-

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cess. Sour horse milk macrotranscriptome libraries were constructed from the samples of three different fermentation stages and subjected to RNA-Seq high-throughput sequencing to obtain functional gene expression during fermentation. The main results are as follows: a high-quality short sequence of 12.2 Gb from sour horse milk samples was obtained, and the number of Lactobacillus increased with the fermentation time and then decreased, while the number of Lactococcus has been increasing with the progress of fermentation. At the level of phylum, Proteobacteria and Firmicutes are the dominant bacteria in the acid horse milk, and genus Lactobacillus and the genus Lactococcus are the dominant genera. In this study, 64,410 mergeUnigenes were assembled, of which 5939 Unigenes could be directly linked to corresponding protein coding region (CDS) and sequence orientation, the remaining Unigenes were analyzed by software, and 2230 of them were predicted as the new protein coding sequence. The functional classification of merge-Unigene showed that the fermentation process was closely related to bacterial cell processing, catalytic ability, and metabolic process. The results of COG functional classification showed that the genes involved in amino acid transcription and metabolism play a key regulatory role at gene replication, recombination, and repair during the fermentation process of sour horse milk. According to the analysis of KEGG metabolic pathway, the metabolic pathway, the secondary metabolism of biosynthesis, and the microbial metabolic pathway under complex environment are the main signaling pathways in the fermentation process. The functional enrichment analysis of GO (differentially expressed genes) shows that the metabolic process, cell processing, and metabolic processing of organic substances are the main biological processes from the early to the middle stages of fermentation. During this period, the expressions of genes related to some molecular functions are relatively active, such as organic cyclic compound binding, heterocyclic compound binding, and small molecule compound binding. Gene related to biological processes, such as cell processing, cellular macromolecular metabolism, and single organism cell processing, plays a decisive role during the mid-to-end fermentation period. The expressions of genes associated with molecular structure activity, ribosome structure, and protein binding are very active as well at this period. KEGG-based enrichment analysis of differentially expressed gene pathways shows that the ribosome pathway is the dominant metabolic pathway in the early to middle stages of fermentation, while metabolic pathway of lipid metabolism, aminobenzoate degradation, steroid biosynthesis, mRNA monitoring, etc. play a major role in the middle to late stages of fermentation. The current yogurt on the market is dominantly fermented by the Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. These two strains stimulate each other’s growth by mutually utilizing their metabolites such as folic acid and carbon dioxide. By a combinatorial analysis of transcriptomics and related metabolite assays, Sieuwerts and colleagues found the relevance between the growth and metabolism of these two strains, mainly involving the metabolism of sputum, amino acids, and long-chain fatty acids (Sieuwerts et al. 2010). Formic acid, folic acid, and fatty acids are mainly produced by Streptococcus thermophiles, while the amino acids required for the growth of both strains are provided by protein hydrolysis activity of the Lactobacillus delbrueckii subsp. bulgaricus. However, observed

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from the expression levels of amino acid-related genes, the amino acids generated in the mixed system, such as sulfur-containing amino acids and branched-­chain amino acids, are not sufficient to support the growth for both strains. Genes related to iron absorption and epithelial production in Streptococcus thermophilus are also affected by the mixed system, and their expression levels are higher than those in the culture with single strain. These studies provide us a better understanding of the ecology and interaction mechanism of mixed culture of strains in fermented foods.

4.4.5  Fermented Meat Products Lactic acid bacteria-fermented meat products are more commonly found in China, such as cured meat, sour meat, sausage, bacon, plate duck, salted fish, etc. Appropriate pickling will give meat products a richer taste and good color and conducive to the meat preservation and storage. Lactic acid bacteria play a very important role in the fermentation of meat products, providing an acidic environment to prevent the growth of spoilage bacteria, promoting color development, inhibiting the formation of toxins, and enhancing the nutritional value (Pan 1997). Lactic acid bacteria are the dominant bacteria in fermented sausages and play a vital role in the fermentation, preservation, and flavor formation of sausages. Three kinds of lactobacilli were isolated from the fermented sour meat in the area of Dong minority, China, including Pediococcus pentosaceus, Lactobacillus delbrueckii, and Lactobacillus citrifolia (Zhang et al. 2008). Drosinos et al. isolated Lactobacillus plantar, Lactobacillus curvatus, and Lactobacillus sakei from Greek fermented sausages (Drosinos et al. 2005). Lactobacillus sakei strain 23K is a psychrophilic bacterium that is widely used in fermented meat products (Chaillou et al. 2005). In order to understand the characteristic gene expression of Lactobacillus sakei in the living environment of meat products, Xu et al. applied gene microarray transcriptome technology to study the transcriptional expression under different culture conditions, and the transcripts from a total of 551 genes were detected. Compared to the expression of the strain grown in the blank medium, the expressions of the genes involved in the hydrolysis of the peptide chain were upregulated in various degrees in the medium containing the muscle fibers and the sarcoplasm (Sun et al. 2015). The expression of the gene oppB and oppC, which is related to the amino acid and polypeptide transport systems, was upregulated. Except the glnA and meK genes, most of the genes involved in the metabolism of peptides, amino acids, and related molecules are overexpressed in media containing muscle fibers and sarcoplasm. The stress-­related genes were not induced to express in the medium containing muscle fiber. Lactic acid bacteria are involved in the fermentation of meat products, which is critical to the improvement of the taste of meat products, but a small number of lactic acid bacteria can cause the corruption of meat products. Lactococcus is a psychrophilic bacterium that is a spoilage organism causing meat products to decay during storage and produce oily and acidic odors during storage at low temperature.

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Studies have shown that the different growth environments of Lactococcus can cause great differences in degree of spoilage. By transcriptomic analysis of lactobacillus glucose metabolism at different time points, it was found that glucose was continuously consumed over time and gene expression of lactobacillus carbohydrate and glycerol metabolic pathways was upregulated gradually. At the same time, the expression of pyruvate metabolic pathway-related genes associated with the production of spoilage substances is abnormal. Through the transcriptomics approach, it was observed that Lactococcus can maintain its survival through the upregulation of related gene expression and the activation of metabolic pathway at the genetic level (Andreevskaya et al. 2015). Leuconostoc gelidum subsp. gasicomitatum is also a harmful microorganism that can cause the decay of meat products, often giving the meat a spoiled smell of butter. Gas chromatography-mass spectrometry (GCMS) combined with transcriptomics can be used to study the source of buttery taste of this strain under the growth conditions of different carbon sources (Jaaskelainen et al. 2015).

4.5  A  pplication of Transcriptomics in Study of Interactions Between LAB and Their Hosts The human body is a natural place where the microbial community can effectively reside and a large number of microorganisms parasitized to varying degrees in human body, including the mouth, digestive tract, respiratory tract, reproductive tract, and skin. Human and microbes are interdependent and maintain a dynamic balance. If this balance is broken, the human body may be faced with illness. For example, intestinal symbiotic microorganisms have a great influence on the physiological and pathological state of the host. Intestinal microbes can help the host to metabolize and absorb nutrients more efficiently and to resist the attack of foreign microorganisms or viruses. Comprehensive study of the relationship between human hosts and microorganisms will allow us to further understand the occurrence and treatment of human diseases (Hu et al. 2012). Although lactic acid bacteria have a small population in the human microbial community, most studies have shown that lactic acid bacteria play a positive role in the formation of human microbial communities and various functions of the human body.

4.5.1  Gut Environment Intestinal microbes provide the body with an important innate adaptation to the immune system and simultaneously regulate intestinal metabolism and immune balance. Probiotic lactic acid bacteria can regulate intestinal homeostasis and immune response. The transcriptomics study of living tissue suggested that the proto-oncogene BCL9, protein kinase ERK3, proto-oncogene JUN, and poly(ADP-­ribose)

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polymerase (PARP) 14 play an important role in the downstream signaling pathway in the immune response and can be signaled through interferon and transcriptional activation regulator IRF to regulate the production of cytokine Th1 (van Baarlen et  al. 2009). Van Baarlen et  al. used microarray transcriptomics to determine the gene expression of duodenal mucosa tissue after intervention with Lactobacillus plantarum and found that NF-κB regulatory pathway gene expression changes greatly, which is beneficial to human health by inducing human immune tolerance. Researchers applied hybrid microarray transcriptome to study the gene expression of Lactobacillus johnsonii in different locations in the intestine after oral supplementation of Lactobacillus johnsonii. The gene expression patterns of Lactobacillus johnsonii in different locations in the intestine are different. Alteration in gene expression of Lactobacillus johnsonii in the stomach was the most obvious, with 786 gene changes. Followed by the cecal and jejunal, there were 391 and 296 gene expression changes, respectively. Only 26 genes changed their expression in the colon. Among these expression-changed genes, besides the genes related to energy transport, most of them are related to sugar PTS transport, such the genes specifically expressed in the cecum, galactosamine PTS transport gene, and fructose, glucose, and fiber II sugar PTS transport-related genes in the jejunum. These genes display different expression patterns at different digestion sites (Denou et al. 2007). Marco et al. also studied the expression of functional genes by hybrid microarray transcriptomics after oral ingestion of Lactobacillus plantarum and found that Lactobacillus plantarum, which is clustered in the cecum, has a functional gene related to carbohydrate transport and metabolism (Marco et al. 2009). When oral supplementing Lactobacillus plantarum with different diets (Chinese or western style), it was found that the growth rate of Lactobacillus plantarum under western-­ style diet was lower and carbohydrate metabolism was altered. Kleerebezem and Vaughan compared the activity of bifidobacteria in infant and adult gut by macrotranscriptome sequencing (Kleerebezem and Vaughan 2009). The sequencing results showed that the expression of genes associated with oligosaccharide metabolism and vitamin-producing and trans-aldehyde enzyme had significant change. There is a very close correlation between gut microbes and human health. Prebiotics in the human ileum can selectively promote the growth of probiotics in the human gut to significantly improve intestinal flora balance. For example, both oligofructose and galactooligosaccharide have the effect of increasing the number of anaerobic bacteria in the human intestinal tract and can promote the growth and reproduction of bifidobacteria and lactic acid bacteria and inhibit the growth of pathogenic bacteria such as Enterobacter (Li 2012). Fructooligosaccharide is a commonly used prebiotic. Chen et al. used RNA-Seq transcriptomics sequencing technology to study the molecular mechanism of Lactobacillus plantarum ST-IIII using fructooligosaccharides by using glucose as control. Sequencing results showed that there were 363 gene expression changes, of which 324 genes were upregulated and 39 genes were downregulated. It was also found that two 75 kb and 4.5 kb gene clusters of Lactobacillus plantarum ST-III may be involved in the utilization of fructooligosaccharides. In order to more efficiently use fructooligosaccharides, the gene expression of fatty acid synthesis is downregulated to change the

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fatty acid composition and improve the fluidity of the cell membrane. By knockout experiments to remove the gene encoding the β-glucosidase (SacA) and PTS transport system (SacPTS1 and SacPTS2), it was found that fructooligosaccharides were transported to inside of cells by the PTS system and subsequently hydrolyzed to monosaccharides by SacA in Lactobacillus plantarum ST-III (Chen 2014). Probiotics promote intestinal mucosal repair and maintain intestinal balance. Probiotics are also often used in combination with antibiotics to treat intestinal infections. Lactobacillus has been used clinically to prevent and treat intestinal diseases in some infants. Kumar et al. used transcriptomics to understand the colonization of the strain in neonatal duodenum and ileum within 7 days after ingestion of L. rhamnosus LGG and Lactobacillus acidophilus (Kumar et al. 2014). Probiotic colonization in the gut and the benefits were determined by up- and downregulation of gene expression related to immune function, as well as metabolism of small molecular substances (such as vitamins and minerals) and macromolecular substances (such as carbohydrates, proteins, lipids, etc.). Compared with the level of transcription, the immune regulation and carbohydrate metabolism pathways are active when supplemented with LGG, while the energy and lipid metabolism are more active when supplemented with Lactobacillus acidophilus.

4.5.2  Oral Environment There are many microbial populations in the human oral environment, and it is an extremely complex micro-ecological system. The interaction between microbes in the mouth is closely related to the health condition of the human oral cavity. Microorganisms in the mouth have two living states, one is in a free state of the flowing saliva, and the other is on the surface of the organ, such as back of the tongue, cheek mucosa, dorsal tongue, crown plaque, and gingival sulcus. Excessive growth of pathogenic bacteria in the oral cavity or microorganisms that are unfavorable to the oral environment can cause problems such as dental caries, periodontal disease, and bad breath. Currently, probiotic therapy is gradually applied to the field of stomatology, and its advantages in the prevention and treatment of oral diseases are becoming more and more obvious. Lactic acid bacteria have a good preventive effect on dental caries and periodontal disease. Studies have shown that lactic acid bacteria isolated from the mouth of healthy people have the effect on inhibiting the activity of cariogenic bacteria and inhibiting the proliferation of periodontal bacteria. Yang screened 32 strains of Gram-positive bacilli from human saliva and plaque samples (Yang 2013). Two strains of lactic acid bacteria isolated from plaque samples are Lactobacillus fermentum Y29 and Lactobacillus brevis BBE-Y52. Lactobacillus brevis BBEY52 is a good oral probiotic, since it is able to synthesize hydrogen peroxide, and its acid production capability is also weaker than that of Lactobacillus salivarius and Streptococcus mutans, a dental caries pathogen. It was found that L. brevis BBE-­ Y52 has the ability to self-aggregate and co-aggregate with other oral microorgan-

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isms to form biofilm and adhere to oral epidermal cells, which is beneficial to its reproduction and survival in the oral environment. When L. brevis BBE-Y52 co-­ cultivated with human peripheral blood stimulated by S. mutans, the ratio of anti-­ inflammatory cytokine I-10 to pro-inflammatory cytokine IL-12p70 increased. This result suggests that L. brevis BBE-Y52 has the anti-inflammation activity in vitro and has potential for maintaining oral health as probiotic. A follow-up survey of children aged 1–6 found that children who had long-term intake of milk with R. rhamnosus had a lower probability of caries, probably due to the presence of R. rhamnosus in oral cavity inhibited the growth of caries pathogen S. mutans, reduced its amount, and prevented the occurrence of dental caries (Nase et al. 2001). The similar studies found that intake of Lactobacillus reuteri had the same control effect on dental caries (Caglar et al. 2006). Lactobacillus reuteri also has a certain effect on the treatment of periodontal disease. Krasse et al. found that the treatment with L. reuteri effectively reduced the clinical indicators of periodontal disease and reduced the deposition of bacterial plaque and relieved periodontal disease to a certain extent in a double-blind experiment on patients with moderate or higher gingivitis (Krasse et al. 2006). Hatakka et al. found that probiotics also play a role in the prevention and treatment of bad breath and oral candidiasis in the elderly (Hatakka et al. 2007).

4.5.3  Vaginal Environment The female reproductive tract is colonized with a large number of bacteria of different species, which plays a very important role in human health. Ling and colleagues studied the differences in vaginal microbial community diversity between bacterial vaginosis patients and healthy women from China by PCR-DGGE and 454 pyrosequencing. The study showed that there were far more bacterial strains in the vagina than in the healthy control group. Phylum Firmicutes accounts for the majority of vaginal flora in healthy women, of which the dominant flora includes lactic acid bacteria, mainly Lactobacillus, used to maintain the normal range of pH in the vagina and to inhibit the growth of potential pathogens. Micrococcus varians and Lactobacillus work together to maintain the health and balance of the vaginal micro-­ ecology. Lactobacillus inert is a dominant bacterium of the genus Lactobacillus in healthy women. It is considered to be a typical bacterium of the normal vaginal microbiota and a sensitive marker of vaginal microbial community changes. During the development of bacterial vaginal diseases, the copy number of inert Lactobacillus decreased by two to three orders of magnitude, even undetectable in pathogenic samples. In addition, other low-abundance lactobacilli, such as Lactobacillus curly and Lactobacillus jensenii, have been found (Ling 2012). Kohler et  al. found that mixed bacteria of Lactobacillus rhamnosus GR-I and Lactobacillus reuteri RC-14 completely inhibited the growth of pathogenic Candida albicans, which is prone to genital tract infections, and proposed the mechanism of interference exerted by probiotics. This result is important for studying how to

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maintain a healthy reproductive tract environment. Experiments have shown that the low pH environment caused by lactic acid plays an important role in inhibiting the growth of fungi. Finally, Candida albicans lost its metabolic activity and subsequently died. Transcriptomics was used to analyze the expression of stress-related genes in Candida albicans in this study. The genes involved in lactic acid utilization were effectively stimulated during the early stage and co-culture with lactic acid bacteria, such as cytochrome C oxidoreductase gene CYB2, lactic acid transport-­ related genes, stress-related genes, etc., and elucidated the fluconazole drug tolerance in the absence of lactic acid bacteria intervention (Kohler et al. 2012).

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Chapter 5

Proteomics of Lactic Acid Bacteria Yue Xiao, Yanjun Tong, and Wei Chen

5.1  Introduction to Proteomics Protein is the direct mediator of gene functions. The biological processes in protein level including dynamic modification, processing, transportation and localization, and structure formation cannot be predicted from gene content. The expression of mRNA cannot directly reflect the expression of the corresponding protein. Therefore, proteomics rather than genomics can provide direct evidence for the “true” occurrence of life. In the mid-1990s, proteomics research, as a newly merged discipline, initiated benefiting from the development on human genome project. The proteomes are of diversity and variability, wherein the compositions and abundances of protein pool are different in the different cells within the same organism. Meanwhile, the proteomes are also variable under different phases and conditions in the same cell. Therefore, proteomics can provide an effective means for research on complexity of protein functions during life process from dynamic and comprehensive perspectives.

5.1.1  Introduction to Proteomics 5.1.1.1  The Conception of Proteomics The concept of proteome was first proposed by Marc Wilkins, which refers to a whole set of proteins expressed and modified by a genome. Sometimes it also means a combination of proteins expressed by a cell at any given time and environment. Proteomics mainly focuses on studying the existence and activity of all proteins in cells. Proteome changes with tissues or even the environment. Proteomics mostly Y. Xiao · Y. Tong · W. Chen (*) Jiangnan University, Wuxi, China e-mail: [email protected]; [email protected] © Springer Nature Singapore Pte Ltd. and Science Press 2019 W. Chen (ed.), Lactic Acid Bacteria, https://doi.org/10.1007/978-981-13-7832-4_5

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concentrates on the dynamic description of gene regulation and thus quantitatively determines gene expression in protein level. Proteomics essentially refers to the study of protein characteristics at the large-scale level, including the expression levels of protein, posttranslational modifications, protein-protein interactions, etc., providing an overall and comprehensive understanding of the process of cell metabolism and disease occurrence at the protein level. 5.1.1.2  The Significance and Development Status of Proteomics With the implementation and advancement of the Human Genome Project, more than 150 genome projects (including the human genome project) have been published. Scientists are more concerned about the genome encoding the structure and function of proteins. The research of life science has entered the post-genome era. During the era, the main research objects of life sciences are functional genomics, including structural genomics and proteomics. Although the genomes of several species are now sequenced, the function of more than half of the genes in these genomes is currently unknown. Genomics, transcriptomics, and proteomics, respectively, study life activities from three levels of DNA, mRNA, and protein. From DNA to mRNA, and then to proteins, there are three levels of regulation, namely, transcriptional control, translational control, and posttranslational control. There is no one-to-one correlation between mRNA expression levels and protein levels, and transcriptional level regulation cannot wholly represent protein expression levels. In addition, complex posttranslational modifications of proteins, subcellular localization or migration of proteins, protein-protein interactions, etc. can hardly be judged from mRNA levels. Protein is the executor of physiological function and also directly shows life phenomenon. The study of protein structure and function will directly clarify how mechanism of life changes under different physiological or pathological conditions. In order to explore the existence and activity of proteins, such as posttranslational modifications, protein-protein interactions, and protein conformation, directly researching on proteins plays an important role. Therefore, using largescale, high-throughput and high-sensitivity proteomics techniques to study proteins at a global, dynamic, and network level by globally studying the expression profiles and functional maps of all proteins at different times and spaces contributes to a comprehensive and in-depth understanding of the complex activities of life. Proteomics research is developing rapidly; both theoretical basis and technical methods are constantly improved. In recent years, research techniques of proteomics have been applied to various fields of life science, such as cell biology and neurobiology and so on. In terms of research object, it covers the range of prokaryotic microorganisms, eukaryotic microorganisms, plants and animals, etc., and it involves various important biological phenomena, such as signal transduction, cell differentiation, and protein folding. In the future development, the research field of proteomics will be more extensive. In terms of proteomics research techniques, research methods of proteomics will have multiple technologies coexisting, each with its advantages and limitations, and it is difficult to form a comparatively consistent

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approach like genomics research. Except for developing new methods, it should put more emphasis on the integration and complementarity of the various methods to accommodate the different characteristics of different proteins. In addition, the intersection of proteomics and other disciplines will become increasingly significant and important. This crossover is the source of living water for new technologies and new methods, especially proteomics and other large-scale sciences (such as genomics, bioinformatics, etc.). The intersection of the omics biotechnology research methods presented by the system biology research model will become an exciting new frontier for the future life sciences.

5.1.2  Proteomics Classification 5.1.2.1  Comparative Proteomics Comparative proteomics is an important part of proteomics, which concerns on researching the identification of expression differences in protein level within the same biological system under different conditions or times, for example, the comparison between cells under normal state and harsh environmental stress, which is generally referred to as differential proteomics or comparative proteomics. By searching and screening differential protein components present in different protein expression profiles of different samples, it helps to study changes in protein components and reveal the progress and nature of cellular physiological states in different environments, which are also useful for researching the response of cells or tissues to external stress stimuli and cellular regulation mechanisms and qualitative, quantitative, and functional analysis of certain key proteins. In comparative proteomics, qualitative analysis was performed using the positional and density ratios of the colored spots in the twodimensional gel electrophoresis to analyze the differential expression of the proteins. 5.1.2.2  Structural Proteomics Structural proteomics focuses on studying proteins in an active conformation, depicting the three-dimensional structure of a protein or protein complex. The methods for determining the three-dimensional structure of proteins mainly include X-ray crystal diffraction pattern method, nuclear magnetic resonance method, and circular dichroism spectroscopy. 5.1.2.3  Functional Proteomics Functional proteomics is a broad term that includes many specific protein methods. The physiological functions of all proteins in the cell are determined by analyzing the interaction between proteins, the three-dimensional structure of the protein, the

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cellular localization of the protein, and the posttranslational modification of the protein. Functional proteomics focuses on the identification and classification of protein functions, protein interactions, and protein activities from a global perspective. The research methods of functional proteomics involve high-throughput multidimensional methods including molecular biology, biochemistry, and bioinformatics analysis. 5.1.2.4  Posttranslational Modification Proteomics Most of proteins in eukaryotes undergo posttranslational modification, and phosphorylation and glycosylation are the two major modifications of protein translation. All posttranslational modifications are accompanied by an increase or decrease in molecular weight. Therefore, mass spectrometry has become a rational tool for posttranslational modification proteomics identification and characterization. 5.1.2.5  Interaction Proteomics Interacting proteomics concerns on the interaction between genetic and physical proteins and the interaction between proteins and nucleic acids or small molecules. Analysis of protein interactions can not only provide functional information about the protein itself but also provide information about the role of proteins in metabolic pathways, regulatory networks, and complexes; research methods for interacting proteomics require different technology platforms to provide different information, which is closely linked to functional proteomics.

5.2  Technology of Proteomics Research 5.2.1  Protein Separation Technology 5.2.1.1  Two-Dimensional Gel Electrophoresis (2-DE) DE was put forward by Farrel in 1975. It can expand the complex protein mixture on two-dimensional planes based on the isoelectric point of protein (the first direction) and the relative molecular mass (the second direction). The gel is stained with Coomassie brilliant blue, silver, or fluorescent substance after electrophoresis, and then the electrophoresis images are analyzed using related software. Up to 10,000 proteins can be separated in a 2-DE. The protein spots are cut off from the gel, subsequently, hydrolyzed, digested, identified, and sequenced, which are usually manual and time-consuming. In addition, many factors make protein detection difficult: (a) low-copy-number proteins; (b) isoelectric focusing – the isoelectric point (pI) determined by 2-DE is 3  ~  10, some extremely acid or alkali protein cannot be

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detected; (c) protein molecular weight – the relative molecular mass range of proteins detected by 2-DE is 10  ~  200  kDa, extremely large (> 200  kDa) or small (< 10  kDa) protein cannot be separated; (d) proteins (including some important membrane proteins) which are difficult to dissolve in conventional separation. Although these factors limit the establishment of a good 2-DE method, 2-DE is still being used as a core technique for proteomics research. Analyze the increase or decrease of the intensity of protein spots in the gel to determine the differential expression of proteins in comparison with the protein 2-DE under normal growth environment and pressure conditions. The procedures of 2-DE generally include sample preparation, isoelectric focusing (the first direction), SDS polyacrylamide gel electrophoresis (SDS-PAGE) separation (the second direction), gel staining, image acquisition and analysis, protein identification, and so on. 5.2.1.2  Differential Gel Electrophoresis (DIGE) DIGE can separate multiple samples labeled by different fluorescence simultaneously in the same gel based on traditional 2-DE combined with multiple fluorometry. As different fluorescein-labeled samples have different excitation wavelengths, multiple samples can be separated and analyzed in the same gel. DIGE can be used to compare the distinction between different samples as well as avoid systematic errors effectively among different gels. There are certain limitations in the DIGE when selecting fluorochromes used for labeling protein sample. The isoelectric point of protein with fluorochromes should not be changed, and the molecular weight changes should be as small as possible after labeling. Furthermore, the molecular weight changes induced by different fluorochromes should be consistent. Cy2, Cy3, and Cy5 are the frequently used fluorochromes at present. 5.2.1.3  Isotope-Coded Affinity Tag (ICAT) ICAT is a new technique proposed by Gygi et al. (1999) for protein separation and analysis. Label sample proteins containing Cys with ICAT reagents selectively before hydrolyzed by protease. And then samples are identified by mass spectrometry after purified by affinity chromatography. According to the intensity level of a pair of peptide ions labeled by different ICAT reagents in the mass spectrogram, the relative abundance of the corresponding protein in the original sample can be quantitatively analyzed, and the difference of protein expression level between two samples can be compared accurately. The identification of protein can be achieved by determining peptides using tandem mass spectrometry. The key of ICAT is the application of ICAT reagents, which are generally composed of sulfhydryl-specific reaction groups, connecting parts (eight hydrogen or heavy hydrogen atoms), and biotin. And it is divided into light chain reagent and heavy chain reagent. ICAT separates and purifies proteins at the peptide level, which can solve the solubility problem of membrane proteins; furthermore, it can reduce

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the complexity of protein mixtures by selectively labeling peptide containing Cys. ICAT cannot be used for analyzing proteins without Cys, and ICAT reagent is a large modifier relative to the small peptide (the molecular weight of the ICAT reagent is approximately 0.5  kDa), which increases the complexity of protein analysis. 5.2.1.4  Isobaric Tags for Relative and Absolute Quantification (iTRAQ) iTRAQ was developed by Applied Biosystems, Incorporated, which is one of the most powerful methods for differential protein quantitative analysis with the highest flux and the smallest systematic error. At present, four (or eight) different isotopic coding reagents are used in iTRAQ to label the amino acid groups of polypeptides specifically and then analyze them by tandem mass spectrometry. The procedures of iTRAQ generally include extraction of proteins, elimination of proteins with high abundance, determination of protein concentration, reduction and alkylation, trypsin cleavage, labeling by iTRAQ reagent, and mixing of samples. The mixed samples were separated by online or offline liquid chromatography, detected by first-order mass spectrometry (MS) subsequently. The detected parent ions are dissociated by high-energy collision, and the obtained ions are tested by second-order MS. Comparing the relative molecular mass of different fragment ions obtained through the abovementioned detections with known databases can determine the corresponding protein precursors. The combination of iTRAQ and LC-MS/MS is widely used in quantitative study of proteomics at present. In the iTRAQ experiment, 2~8 samples can be labeled at the same time simply and efficiently. In addition, the iTRAQ reagent can label all enzymatic fragments in biological samples, including posttranslational modified fragments, without damaging some important structural information in proteins. 5.2.1.5  Multidimensional HPLC (MD-HPLC) MD-HPLC is a chromatographic technique of continuously using concentrated liquid chromatography to separate complex components to a greater degree. MD-HPLC is an effective method for the separation of complex samples, which belong to chromatography-­chromatography coupling technology. Two-dimensional liquid chromatography (2D-LC) is one of the most commonly used MD-HPLC. The separation and purification system of 2D-LC is composed of two tandem chromatographic columns with different separation mechanism and independent of each other. The samples are separated by one-dimensional ­chromatographic column and then purified by two-dimensional chromatographic column. As usual, 2D-LC separates and analyzes samples according to the differences of their molecular weight, isoelectric point, hydrophilicity, or special intermolecular forces. The common 2D-LC techniques include ion exchange chromatography-­ reversed-­ phase liquid chromatography (IEC-RPLC), affinity

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chromatography-­reversed-­phase liquid chromatography, molecular sieve chromatography-reversed-phase liquid chromatography, chromatofocusing-­reversed-­phase liquid chromatography, and so on.

5.2.2  Protein Identification Technology 5.2.2.1  Edman Degradation Edman degradation is a method to determine the primary structure of protein which mainly analyzes amino acid residue from the N-terminal of protein or polypeptide. The N-terminal amino acid residue is modified by phenylisothiocyanate; then the modified amino acid residue (phenylthiohydantoin amino acid) is cut from the polypeptide chain and identified by chromatography. The remaining polypeptide chain (one less residue) is recycled for the next cycle of degradation. In this way, each amino acid residue in the polypeptide chain can be identified by the analysis phenylthiohydantoin amino acids through an Edman and HPLP cycle. Edman degradation is generally divided into coupling, cutting, extraction, transformation, and identification. 5.2.2.2  Mass Spectrometry (MS) MS determines the type of protein by measuring the quality of the protein. Aston created the first velocity-focused MS in 1919, which became a milestone in the development of MS.  With the development of ion optics theory, MS has been improved and its application range has been expanded. The application of MS in the field of life science has formed a unique food MS technology. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS) are commonly used in protein identification. And MALDI-TOF-MS uses laser pulse to gasify and dry the samples in the crystal matrix so that the samples are charged, and the flight time of different samples with different nucleocytoplasmic ratio is different in the time-of-flight MS composed of accelerating electric field and magnetic field. We can obtain the spectral lines of different nucleocytoplasmic ratio by using the detection system, which form the peptide mass fingerprint. ESI-MS is often used for the separation and identification of complex proteins through ionizing the sample with high electric field.

5.2.3  Image Acquisition and Bioinformatics The application of imaging technology in proteomics enables the rapid development of proteomics study. Proteomics imaging can not only distinguish the characteristics and composition of proteome which is invisible to the naked eye but also

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analyze the differences between various proteome samples, making proteome analysis more intuitive. Proteomics imaging mainly includes 2-DE imaging, LC-MS imaging, molecular scanner imaging, and tissue MS imaging in vivo. In addition, a variety of analysis software such as PDQuest, ImageMaster 2D Elite, Biolmage Investigator can be used to collect and compare data of gel images.

5.2.4  Interaction Technology 5.2.4.1  Yeast Two-Hybrid System Yeast two-hybrid system was established by Fields and Song in 1989. It was used to study the transcriptional regulation of eukaryotic genes initially. During continuous improvement and development, it has become a simple and effective technique to study the interaction of proteins. More importantly, it is applied to the discovery of new unknown proteins interacting with known proteins. Yeast two-hybrid system is a highly sensitive technique for studying the relationship between proteins that carried out in the eukaryotic model yeast to study the interaction of proteins in cells. The weak and instantaneous interaction between proteins can also be detected sensitively by reporting gene expression products. Yeast two-hybrid system can be used to study the interaction between proteins encoded by either mammalian genome or higher plant genome. Therefore, yeast two-hybrid system is widely used in many research fields. 5.2.4.2  Co-immunoprecipitation Co-immunoprecipitation is based on the specific interaction of antigen-antibody, which is used to study the physiological interaction between proteins in intact cells. Many interactions among proteins in the cell non-denatured lysate are preserved. The basic principle of immunoprecipitation is perceived that the antibodies added to the lysate form specific immune complexes with the known antigens, and if there are proteins interacting with the antigens, they will be participating in this formation. Then the interaction between antigen and other proteins can be verified by SDS-PAGE, imprinting, or mass spectrometry. The object of study of co-immunoprecipitation is close to the physiological environment of organism, and it is carried out in the natural state of protein interaction, which can avoid the influence of human factors. However, co-immunoprecipitation also has some limitations, that is, the target protein needs to reach a certain concentration in order to form precipitation with the antibody, the technique is only suitable for the study of the target protein with high expression level, and its sensitivity is not as high as the affinity chromatography. In addition, co-immunoprecipitation can only detect soluble protein components and is not suitable for analyzing the interactions between insoluble macromolecular proteins.

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5.2.4.3  Protein Chip Protein chip, which is evolved from gene chip, is a high-throughput protein analysis technology, also known as protein array or protein microarray, and the research object of chip technology is protein. The principle of protein chip is as follows: firstly, the solid phase carrier is treated by special chemical method, and then the known molecules (antibodies, antigens, enzymes, cytokines) are immobilized on it; secondly, capture the proteins those can specifically bound to known molecules according to the biological characteristics of them; finally, analyze the proteins by laser scanning confocal microscope, atomic force microscope, mass spectrometry, or surface plasmon resonance detection techniques to high-throughput determine of protein-protein interactions or interactions between proteins and small molecules. At present, according to the application fields of protein chip, it can be divided into analytical protein chip and functional protein chip. Analytical protein chip is mainly used to identify proteins. Functional protein chip is mainly used to study the interaction between proteins and other molecules.

5.3  A  pplication of Proteomics in the Study of Lactic Acid Bacteria 5.3.1  A  pplication of Proteomics in Environmental Adaptation of Lactic Acid Bacteria The response of microorganisms to environmental changes is well documented (van de Guchte et al. 2002; de Angelis and Gobbetti 2004; zhao et al. 2014). Lactic acid bacteria can be affected by harsh environments in the gastrointestinal tract or in food processing. In the food industry, stable starter with good food processing adaptability is generally used. For probiotic strains, the highlight of the research is their adaptability to harsh environments in order to obtain better protection systems for strains against harsh environments. In the changeable environment of temperature, osmotic pressure, or oxidation level, proteomics can be used to analyze the response mechanism of lactic acid bacteria to harsh environments. In addition, genomics, transcriptomics, and metabolomics can be combined to provide new clues to reveal physiological changes in lactic acid bacteria under environmental stress. Lactobacillus can induce protein expression to change in stress response to external stress. The application of proteomics technology, through revealing the dynamic change of protein expression pattern from proteome level, helps to elucidate the molecular mechanism of the regulation of lactobacillus stress response.

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5.3.1.1  Heat Shock Response High temperature (usually refers to 40~65 °C) can remove the non-covalent interaction in the protein molecules or molecular and lead to its degeneration. Heat shock stress response is induced by a group of heat shock proteins (HSP), and heat shock proteins are not commonly used as stress response proteins. After Lactobacillus casei are heat-treated, the expression levels of chaperone proteins such as GroEL increased, and the rate of synthesis of total and secreted proteins also changed (Haddaji et al. 2015). Lactococcus lactis growing at high temperatures has at least a twofold increase in the expression of its chaperone protein GroESL, compared to low temperatures (Chen et al. 2015). DnaK, GroEL, and GroES as companion proteins can assist in the refolding of new proteins or denatured proteins (Guisbert and Morimoto 2013). The kinetics of heat shock protein expression is changed during heat shock stress reaction. During the initial phase of heat shock stress response, both DnaK and GroEL were induced to express immediately, while the remaining heat shock proteins were subsequently expressed. Proteomic analysis showed that Lactobacillus paracasei NFBC 338 overexpressed its chaperone protein GroEL in response to heat shock stress. In addition, studies have shown that overexpression of the chaperone protein GroES and GroEL in Lactococcus lactis and Lactobacillus paracasei transformants can increase the resistance of lactobacillus to solvents, especially butanol. This cross-protective effect may be associated with changes in cell membrane fluidity and membrane protein modification caused by solvent and thermal pressure (Desmond et al. 2004). Similar heat stress induced by heat shock proteins has also been reported in other lactobacilli (Fiocco et  al. 2007; Parente et al. 2010). Heat treatment was performed on Lactobacillus plantarum DPC2 at the middle stage of exponential growth and the stable stage, and then bidirectional gel electrophoresis was performed on the bacteria after heat treatment. The results revealed that there were 31 and 18 differentially expressed proteins in the strains with medium growth stage and 18 differentially expressed proteins in stable stage of heat treatment. And the researchers believe that the heat shock stress response of plant lactobacillus is a complex process, which involves the activity of chaperone proteins, ribosome stability, and temperature sensitivity (DE Angelis et  al. 2004). Similar protein modifications may occur under different stress conditions, suggesting that many proteins are involved in response to different environmental stresses (Mills et al. 2011). 5.3.1.2  Acid Shock Response An important feature of lactic acid bacteria is that saccharides can be converted to lactic acid by fermentation. Lactic acid can act as a preservative to inhibit the production of bacteria during fermentation and contribute to the development of the overall flavor and texture of the fermented product. Lactic acid bacteria have different response mechanisms in response to lactic acid stress during food processing

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and low pH environment in gastric juice. At present, the mechanism of resistance to acid stress in bacteria has been reported (Corcoran et al. 2008). A variety of lactic acid bacteria have been currently reported to protect against low pH.  Proteomic analysis of Lactobacillus brevis NCL912 under acid stress revealed 25 differential proteins, mainly pressure stress proteins, DNA repair-­ related proteins, protein synthesis-related proteins, and proteins involved in the glycolytic pathway, and 18 differential proteins were significantly upregulated, such as UspA, 50S ribosomal protein L10, and NADP-dependent glyceraldehyde-3-­ phosphate dehydrogenase (NADP-GAPDH). These differential proteins play an important role in the resistance of strains to acid stress (Huang et al. 2011). A study has recorded that changes in the protein profile of L. plantarum 423 when exposed to pH 2.5 by using a gel-free nanoLC-MS/MS proteomics approach; the proteins involved in the synthesis of biomacromolecules were significantly down-regulated; proteins involved in fatty acid synthesis like FabZ1, FabZ2, FabH2, FabG1, AccD2, AccA2, and AcpA2 were significantly downregulated, and the expression levels of Ddl, MurE1, and GlmU in peptidoglycan synthesis were also downregulated under acid stress. In the acid stress environment, the proteins involved in carbon metabolism also have certain changes. These altered carbon metabolism pathway proteins have certain effects on cell energy production and intracellular oxidation-reduction potential. In addition, the accumulation of alkaline substances in cells also has a certain effect on the environment against acid stress. Proteomic analysis of Lactobacillus casei Zhang strains in different H systems revealed 33 differential proteins in which proteins involved in their carbon source metabolism were significantly upregulated. These significantly upregulated proteins play an important role in the energy supply of Lactobacillus casei Zhang strain against acid stress (Wu et al. 2011). In addition, certain mechanisms can resist acid stress as well as other environmental stresses. By proteomic analysis of Lactobacillus reuteri under acid stress, it was found that the expression level of F0F1-ATP synthase was upregulated, while the protein involved in nucleotide and protein synthesis showed a downward trend, F0F1-ATP synthase can utilize both protons in the cellular environment to synthesize ATP and ATP hydrolysis to export protons out of the cell in order to protect against external acid stress (Koponen et al. 2012). Stress proteins that are resistant to low-pH environments are also involved in the protection of bile salts (Lee et al. 2008). Low pH and bile salts are two unfavorable conditions for lactic acid bacteria to pass through the gastrointestinal tract. Therefore, they can resist bile salt stress while resisting low pH. Proteomics studies of the response mechanism of Bifidobacterium longum to low-pH environments have revealed that low-pH environments had a certain effect on proteins involved in the glycolytic pathway such as alpha-1,4-glucosidase, glucose phosphate mutase, and UDP-glucose-4 isomerase, the expression levels of these enzymes were upregulated, and they were involved in the utilization of complex carbohydrates and the fructose-6-phosphate pathway. In addition, higher NH4+ concentrations in the cells can alleviate the intracellular low-pH environment. Therefore, Bifidobacterium longum responds to the acidic environment by altering the glycolysis flux and regulating its intracellular pH, whereas bile salt hydrolase

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exhibits a downward trend in acidic environments (Sánchez et al. 2007a). In addition, Jin et  al. (2012) studied the acid tolerance mechanism of Bifidobacterium longum using gene expression technology. The cell wall integrity and cell membrane permeability of Bifidobacterium longum in the acid environment were changed to prevent H+ from entering the cell. The acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus was studied by proteomics and transcriptomics (Fernandez et al. 2008), and it was found that many chaperone proteins were expressed during acid stress, such as GroES, GroEL, HrcA, GrpE, DnaK, DnaJ, ClpE, ClpP, and ClpL, and upregulation of chaperone expression can assist in the correct folding of intracellular proteins of Lactobacillus delbrueckii subsp. bulgaricus under acid stress or promote the hydrolysis of misfolded proteins. The expression of genes involved in fatty acid synthesis was upregulated, such as fabH, accC, and fabI; the expression of related genes involved in the synthesis of isoprenoid compounds was inhibited, such as mvaC and mvaS. Studies have shown that during acid stress process, Lactobacillus delbrueckii subsp. bulgaricus subspecies reconstituted pyruvate to fatty acids to alter cell membrane fluidity. However, during the acid stress of some lactic acid bacteria, there were no significant changes in pressure stress proteins such as GroEL, GroES, DnaK, and GrpE, which may be due to the fact that the lactic acid bacteria had a certain adaptability to low-pH environments (Koponen et al. 2012). At present, proteomics and transcriptomics studies on the acid adaptation of Lactobacillus delbrueckii subsp. bulgaricus found that the protein expression associated with the glycolytic pathway is upregulated to promote optimal use of carbohydrates and provide for cell growth and provide energy for strain growing. And it also revealed that the expression level of its transcriptional regulator Ldb0677 was also upregulated, and the change in its expression significantly increased the acid tolerance of the strain (Zhai et al. 2014). 5.3.1.3  Cold Shock Lactic acid bacteria are often in a low-temperature environment during the initial stages of production and storage or during the food processing stage. It is well known that low temperature causes a decrease in cell viability and damage to cell membranes (such as changes in cell membrane morphology and fluidity) and has a certain effect on replication, transcription, and translation (Passot et  al. 2012; Louesdon et al. 2015). The effect of low temperature on the bacteria also affects the technical performance of the bacteria, such as bacterial viability or acidification ability, and ultimately affects product quality. At present, research on the low-temperature tolerance of lactic acid bacteria is gradually carried out. It is reported that many bacteria increased their viability in a frozen environment by applying moderate or different pressures in advance (Mendoza et  al. 2014). Researchers used multidisciplinary approaches, including comparisons between proteomes. Studies have reported that moderate cooling temperature (26 °C) can improve the low-temperature tolerance of Lactobacillus acidophilus RD758 (Wang et al. 2005). Lactobacillus acidophilus RD758 cells will be

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obtained after cold treatment at 26 °C for 8 h optimal low-temperature tolerance. Under this treatment condition, the ratio of unsaturated fatty acids and saturated fatty acids increases and can induce the expression of ATP-dependent ClpP protein, as well as pyruvate kinase and glycoprotein endopeptidase. The ATP-dependent ClpP protein is required for cell growth under stress conditions and can participate in proteolysis due to misfolding or breakage due to cold stress. In an in-depth study, it has been revealed that the cryotolerance of L. bulgaricus CFL1 has been improved during the 30-min acidification at pH 5.15, which slightly minimized the acid shock after acidification (Streit et al. 2007). Further analysis of the proteomic analysis of the bacterial stress of Lactobacillus brevis in Bulgaria showed that 21 differential proteins were involved in energy metabolism, nucleotide and protein synthesis, and stress response. However, these differential proteins can be attributed to increased cold tolerance of the cells after acid stress, changes in saturated cyclic fatty acid concentrations, and decreased cell membrane fluidity (Streit et al. 2008). 5.3.1.4  Oxidative Stress Response Lactic acid bacteria produce certain oxidative stress reactions during fermentation, drying, storage, or oxidizing substances in the intestines or food. Reactive oxygen species such as hydrogen peroxide, superoxide anion, or hydroxyl radicals can react with lipids, proteins, or DNA, which leads to serious consequences for bacterial growth (Miyoshi et al. 2003; Dijkstra et al. 2014). When reactive oxygen species (ROS) exceeds a certain level, oxidative stress can activate the corresponding defense mechanism in bacteria. Arena et al. (2006) used proteomics to study the oxidative stress response of Streptococcus thermophilus. Two-dimensional gel electrophoresis, MALDI-TOF-MS analysis, and one-way gel electrophoresis-liquid chromatography-coupled electrospray ionization mass spectrometry were used to analyze differentially expressed proteins under different stresses. Studies have shown that hydrogen peroxide can promote the expression of general stress proteins, especially some proteins that can resist oxidative damage, such as NADH oxidase, manganese superoxide dismutase, iron-sulfur protein, glutathione reductase, Suf B, and Suf C, etc., and it was also found that the expression levels of related proteins involved in energy metabolism were reduced. 5.3.1.5  Osmotic Stress Response The substance which causes osmotic stress to lactic acid bacteria is generally a salt substance added during fermentation or food pickling, such as sodium chloride, potassium chloride etc., or a saccharide substance. Belfiore et al. (2013) performed proteomic analysis of Lactobacillus sakei CRL1756 strain under salt stress and found that the expression levels of 18 differential proteins were upregulated while the expression levels of some proteins involved in the glycolytic pathway (such as

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Fba, Pgk, Gpm5, and Tpi) were downregulated and the expression levels of stress proteins and proteins involved in nucleotide synthesis were upregulated. At the same time, it was pointed out that the addition of glycine-betaine could alleviate osmotic stress and promote the growth of cells. Proteomics studies have shown that many genes of Bifidobacterium longum were regulated by osmotic pressure, in which the expression of the chaperone protein was upregulated in heat stress and osmotic stress and the expression level of proteins involved in the metabolism of amino acid-related, nucleotide-related, and glycolysis-­ related was also upregulated (Sánchez et al. 2005). In addition, the expression of proteolytic enzymes of Lactococcus lactis and phospho-glucose transferase system (PTS system) was inhibited in osmotic stress, while in other stress responses, such as heat shock stress response or the acid stress response, the expression levels of these proteins were upregulated (Xie et al. 2004). The researchers used proteomics to analyze the stress response of Lactobacillus sakei to osmotic pressure at low temperatures and then constructed protein gene mutants with upregulated or downregulated expression (Marceau et al. 2004). The study found that at least six proteins were involved in the environmental stress response of Lactobacillus sakei in meat processing. The phosphofructokinase mutant had a reduced survival rate under low ambient temperature of 4 °C or 4% sodium chloride; likewise, methionine sulfoxide reductase A (MSRA), universal stress protein (USP family protein), and Alkaline shock proteins (ASP family proteins) were involved in the survival of Lactobacillus sakei at low temperatures. Moreover, functional genomics derived from the Lactobacillus acidophilus NCFM genome sketch sequence showed that cell division proteins (Cdp A) were involved in stress responses in various environments, such as the observed S-layer proteins and cell wall-associated proteins (Altermann et  al. 2004). In addition, Machado et al. (2004) found that the ratio of saturated/unsaturated fatty acids and cell membrane cyclopropane fatty acid concentrations in Lactobacillus casei ATCC 393 was changed in 1 mol/L sodium chloride. To overcome the limitations of proteomics in cell membrane and cell wall studies, it is necessary to use a variety of combinatorial methods to study responses under a variety of stress conditions. 5.3.1.6  Biliary Salt Stress Response Bacterial bile salt tolerance is an important feature of its survival as it passes through the gastrointestinal tract. Bile salts are secreted into the intestine during food digestion, and their main function is to emulsify and promote the absorption of fat. However, bile salts have a bactericidal effect by destroying the cell membrane structure of the cells, thereby studying the misfolding of proteins and the oxidative damage of DNA.  Hamon et  al. (2011) used comparative proteomics techniques to analyze the protein differences between Lactobacillus casei gallbladder-tolerant strains and bile salt-sensitive strains, and found that differential proteins are mainly involved in cell membrane-modifying proteins (e.g., NagA, RmlC), cytoprotective proteins, and detox proteins (e.g., ClpL and OpuA), as well as proteins involved in

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metabolism (e.g., Eno, Pky, Pta, etc.). In addition, studies have reported that biliary efflux can be promoted by regulating the membrane protein composition of the cell membrane to increase the bile salt tolerance of the strain (Elkins et al. 2001). Lactobacillus johnsonii PF01 is a strain currently reported to be more tolerant to bile salts. By proteomic analysis of Lactobacillus johnsonii PF01 strain exposed to 0.1%, 0.2%, and 0.3% bile salts, 215 differential proteins were found, of which 94 proteins were upregulated and 121 proteins are downregulated; these differential proteins were mainly involved in pressure stress proteins, cell division, transcription and translation, cell wall synthesis, carbohydrate metabolism, amino acid synthesis, etc. In addition, bile salt hydrolase can catalyze the early dissociation of bile salts to reduce the toxicity of bile salts. Under different concentrations of bile salt stress, the expression of bile salt hydrolase of Lactobacillus johnsonii PF01 was significantly upregulated (Lee et al. 2012). Proteomics method was used to study the difference of protein expression of Bifidobacterium longum under bile salt stress. Forty-four differential proteins were identified by mass spectrometry. And it was found that these differential proteins were involved in various aspects of bacterial metabolism by bioinformatics analysis. The response mechanism of bifidobacteria against bile salt stress was proposed: bile salt hydrolase, bile salt pumping transporter, and some membrane proteins, such as membrane proteins, which prevent bile salt invasion; general stress response, glucose metabolism, nucleotides. Bile salt adaptation mechanisms such as synthesis, amino acid synthesis, and transmembrane transport (An et al. 2014). In addition, bile salts had a certain effect on intracellular redox state-related proteins, and the result was associated with intracellular NADH and FAD levels (Sánchez et  al. 2007b). 5.3.1.7  High-Pressure Stress Response Nowadays, ultrahigh pressure is increasingly used as a new food preservation method due to its nonthermal properties and inhibition of harmful microorganisms and less impact on the sensory quality of food (Rastogi et al. 2007). Juices, oysters, and cooked meat products are often used for ultrahigh-pressure preservation. Ultrahigh pressure can affect deadly morphological, biochemical, and genetic changes in living cells (Wang Sui-lou et  al. 2006). The inactivation of bacteria caused by ultrahigh-pressure process is well documented. The effectiveness of ultra-high pressure is associated with not only the pressure level and duration but also the temperature, pH, osmotic pressure, and other factors in the ultra-high pressure process. Some natural pressure-tolerant microorganisms, such as those grown in deep-sea environments, can also grow at a pressure of 94 MPa (Abe and Horikoshi 2001). Hörmann et al. (2006) analyzed the proteomics of Lactobacillus sanfranciscensis DSM 20451T under pressure of 80 MPa and found 16 differential proteins, in which the differential protein similar to Clp protease was closely related to stress. Two-­dimensional gel electrophoresis analysis of low-temperature stress, high tem-

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perature stress, salt stress, acid stress, and starvation stress showed that the proteomics of high-pressure stress strains were similar to low-temperature stress or NaCl salt stress, and there were 11 identical differential proteins included. It is believed that there was no specific pressure stress response in the Lactobacillus sanfranciscensis DSM 20451T. After treatment at 400  MPa, the recovery of the morphology of two strains of Lactobacillus sakei, an Enterococcus and a Listeria monocytogenes strain, was strain dependent. Different strains produced different proteins to resist the stress of ultrahigh-pressure stress, showing different ultrahighpressure tolerance. Transcription factors, proteins involved in protein synthesis, and proteins involved in energy metabolism were induced to be expressed among the four species. However, the specificity of some stress proteins in two strains of Lactobacillus sakei was induced to be expressed. The differential proteins in Enterococcus were generally involved in energy metabolism, and the differential proteins of monocyte-­producing strains are mostly stress proteins and proteases. Listeria strains showed the strongest hyper-high-pressure sensitivity, while Enterococcus was the weakest in hyper-high pressure (Jofré et al. 2007). The study of the lactic acid bacteria proteome can provide an effective way to truly understand the life-operating mechanism of lactic acid bacteria, thereby improving the viability of probiotics under stress conditions and improving the process properties, which are beneficial to the better utilization of probiotics by lactic acid bacteria.

5.3.2  Application of Proteomics in Fermented Food Research 5.3.2.1  Sourdough There exists a typical complex food ecosystem in the sourdough, and the interaction of symbiotic Lactobacillus has an impact on species behavior and performance (Rul and Monnet 2015; Ventimiglia et al. 2015). The molecular mechanism of the interaction between lactic acid bacteria in sourdoughs was studied using proteomics techniques (di Cagno et  al. 2009; Shevchenko et  al. 2014). When Lactobacillus plantarum DC400 or Lactobacillus brevis CR13 were co-cultured to the late stationary phase, compared with co-culture growth of Lactobacillus plantarum DC400, Lactobacillus brevis CR13, or Lactobacillus reuteri A7, Lactobacillus sanfranciscensis CB1 has the largest number of dead or damaged cells in a solo medium (di Cagno et al. 2007). After two-dimensional gel electrophoresis analysis, the expression level of the induced expression protein of Lactobacillus brevis CB1 was significantly increased in the co-culture system with Lactobacillus plantarum DC400 or Lactobacillus brevis CR13. Nineteen overexpressed proteins had been identified, which were primarily involved in the glycolytic pathway and stress response. On the one hand, when Lactobacillus sanfranciscensis CB1 and Lactobacillus brevis CR13 were co-cultured, particularly with Lactobacillus plantarum DC400, dihydrofuranosyl-5-ethyl and 5-pentyl were considered to be signal molecules. On the

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other hand, when Lactobacillus plantarum DC400 was co-cultured with Lactobacillus sanfranciscensis DPPMA174 or with Lactobacillus reuteri A7, the cell viability was not affected (di Cagno et al. 2009). However, two-­dimensional gel electrophoresis analysis showed that the protein expression level under co-culture conditions was increased in the mid-log phase to the early stationary phase of Lactobacillus plantarum DC400. Induced expression of the polypeptide had been identified and was generally involved in energy metabolism, stress response, quorum sensing (adenosylmethionine synthase MetK), and elongation factor Tu (EFTu). When the Lactobacillus plantarum DC400 was co-cultured with other lactic acid bacteria, the self-inducing polypeptide Pln A can be expressed (di Cagno et al. 2010). When co-cultured with Lactobacillus plantarum DC400, it inhibited the survival rate of Lactobacillus sanfranciscensis DC400 and Pediococcus pentosaceus. Compared with the single culture of Lactobacillus sanfranciscensis, its proteomic group showed 58 differentially expressed proteins when co-cultured with Lactobacillus plantarum DC400, of which 47 proteins were upregulated and 11 proteins were downregulated. Similarly, proteomics techniques were used to study the physiological changes of Streptococcus thermophilus LMG18311  in milk fermentation (Herve-Jimenez et  al. 2008), and proteomics techniques were used to study the fermentation of yogurt, Streptococcus thermophilus, and bacterium Bulgarian subspecies co-culture (Herve-Jimenez et  al. 2009). During the milk fermentation process, Streptococcus thermophilus LMG18311 had two distinct growth stages, the second growth stage of which was significantly different from that of culture alone or coculture with Lactobacillus delbrueckii subsp. bulgaricus ATCC11842. When cultured alone, Streptococcus thermophilus LMG18311 had poor growth conditions, and when co-cultured with Lactobacillus delbrueckii subsp. bulgaricus. ATCC11842, it could overcome this unfavorable growth condition. The proteomic analysis of S. thermophilus LMG18311 cultured and co-cultured showed 27 differential proteins in two stages of growth, of which 13 proteins were downregulated and 14 proteins were upregulated. These differential proteins were mainly involved in the biosynthesis of amino acids, the metabolism of carbon and purine-pyrimidine, the stress regulator RR05, and other proteins of unknown function. Proteins involved in amino acid synthesis are p­ rimarily involved in the metabolism of cysteine and methionine. The conversion from homocysteine ​​to methionine and the proteins involved in the cysteine metabolic ​​ pathway were upregulated. Streptococcus thermophilus LMG18311, when cultured alone or co-cultured, induces the expression of amino acid and polypeptide transporters and sulfur-containing amino acids. When co-cultured with S. thermophilus LMG18311 and Lactobacillus brevisii subspecies, the sulfur-containing amino acid biosynthesis pathway was controlled by the interaction between Lactobacillus brevisii subsp. bulgaricus ATCC11842 and S. thermophilus LMG18311. In addition, at the later stage of the growth phase of S. thermophilus, the expression level of the stress regulator RR05 [is part of the twocomponent regulatory factor (2CRS)] was decreased, indicating that S. thermophilus is rapid or normal growth requires the presence of 2CRS.

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5.3.2.2  Fermented Dairy Products Proteomics technology can study the interaction between fermented dairy products and microbial proteins, such as in different cheeses, as well as different protein systems and complex microbial ecosystems. In addition, the main component of cheese comes from milk, including casein, whey protein, peptides, fats, minerals, and organic acids (Manso et al. 2005). The typical texture and flavor characteristics of cheese are mainly derived from protein hydrolysis. Proteomics technology can delve into the degradation of global casein to the hydrolysis of peptides and amino acids. Some researchers used high-performance liquid chromatography (HPLC) online and mass spectrometry to analyze the hydrolytic activity and specificity of different proteases (Gagnaire et  al. 2001). The initial requirement of proteomics technology was to isolate water-soluble proteins from insoluble cheese and then separate and purify them to reduce sample complexity (Gagnaire et al. 2004). For the proteomic analysis of Swiss cheese, the bacterial ecosystem of Swiss cheese is mainly Streptococcus thermophilus, Lactobacillus helveticus, Lactococcus lactis, and Streptococcus salivarius. Its protein component analysis identified 62 differential protein spots, and its differential proteins were mainly involved in hydrolysis, glycolysis pathway, stress response, and DNA and RNA repair and redox reactions. 5.3.2.3  A  pplication of Lactic Acid Bacteria to Produce Aromatic Substances Fermentation and drying can extend the shelf life and enhance the flavor and nutritional quality of the product. As the main starter, lactic acid bacteria can reduce the time of fermentation, improve the sensory properties of the product, and inhibit the growth of pathogenic bacteria and spoilage bacteria. What’s more, Lactobacillus is also regarded as beneficial microorganisms, which promotes the fermentation in milk-derived products. In terms of sensory properties, fermentation products such as cheese or sausage are ultimately acidified and dehydrated. The lactic acid bacteria carbohydrate fermentation products are mainly lactic acid, especially D-lactic acid. The lactic acid produced by fermentation reduces the fermentation pH to 5.0, resulting in protein coagulation and promoting the hardness of the product. At the same time, acidification can also reduce the water retention capacity of the fermented food and indirectly contributes to color development. Aromatic qualities such as taste, aroma, and scent are the most popular characteristics of food. In addition, organic acids produced during fermentation, such as lactic acid and acetic acid, can also inhibit pathogens and spoilage bacteria in food. The aromatic substances of the fermented products are mainly derived from the metabolism of raw materials such as sugars, proteins, and lipids (Steele et al. 2013). Aromatic substances are strain dependent, and initial culture conditions have a large effect on the final flavor characteristics of the product (Kieronczyk et  al. 2003).

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Food fermentation is usually carried out by mixed culture fermentation of strains of different species to enhance the flavor characteristics of the fermented food. The fermentation kinetics of the flora is critical to the fermentation performance. In early studies, the classical molecular biology method was used to analyze the changes of the flora in the mixed culture food fermentation. The availability of genomic sequences and advances in functional genomic technology have facilitated research between food microbes and food microbial metabolic activities. In this sense, Sieuwerts et al. (2008) used transcriptomics and proteomics to illustrate the interaction between microorganisms. Functional genomics can provide a basis for metabolomics research. From the entire genome sequence analysis, certain lactic acid bacteria can form aromatic precursors (Flahaut et al. 2013). In addition, in situ proteomics methods have been used in research and development in fermentation systems such as milk or cheese. Yvon et al. (2008) studied the enzymatic activity, proteomics, and metabolomics of aromatic substances in Lactococcus lactis subsp. lactis cell extracts during cheese fermentation. Proteomics is relatively less affected, and there were significant differences in bacterial metabolites produced after 7 days of acid stress and starvation stress. In addition, in mixed culture, proteomics methods are applied to identify proteins involved in cheese ripening. The proteins of Emmental cheese model were separated by two-dimensional gel electrophoresis and identified by MALDITOF-MS or de novo sequencing mass spectrometry.Among these differential proteins, about 20 are from Streptococcus thermophilus, 17 are from Lactobacillus helveticus, and 8 are from the genus Propionibacterium. Some of these differential proteins were involved in the sensory properties of cheese, such as PepN, PepE, PepO, and prolyl amino acid enzymes derived from Lactobacillus helveticus. These studies have shown that proteomics can identify proteins associated with the production of aromatic substances in fermented foods.

5.3.3  T  he Application of Proteomics in the Quorum Sensing of Lactic Acid Bacteria Biomes play an important role in the environment through their metabolic diversity and evolutionary capabilities. Bacteria have a highly complex mechanism for collecting, processing, and transforming environmental information. The diversity of the ecological niche depends on its ability to sense the external environment and adapt to the regulation of specific gene expression, generally including microbial synthesis, release detection, and hormonal small-molecule response. These small molecules participate in the process called “growth induction” (di Cagno et  al. 2011; Popat et  al. 2015). Among the Gram-negative strains, quorum sensing is mainly regulated by two-component LuxI/LuxR and acetyl homoserine lactone, and Gram-positive bacteria are characterized by a self-inducing polypeptide (AIP) as a specific community signal. It is speculated that bacteria can effectively transmit and

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receive signals, but the information of these signals is used for a certain purpose rather than for statistical purposes, which may explain that some organisms have multiple signaling systems. In the literature reports, language and behavior are often used to describe “group sensing” (von Bodman et al. 2008). The language or cross talk among bacteria or between bacteria and animal or plant hosts determines the outcome of the action (such as beneficial or pathogenic effects). Currently, research focuses on understanding and interpreting language signals between biomes (Castillo 2015). Given the large number of extracellular metabolites and the complexity of known quorum sensing signals, genomics and transcriptomics are the primary methods for studying quorum sensing mechanisms. Genomics provides the basis for comparative proteomics and functional proteomics. Proteomics shows the global nature of the proteins expressed in the genome and provides new and simple insights into bacterial behavior in quorum sensing phenomena. Proteomics can illuminate a large number of linguistic signals between biomes through regulatory mechanisms between different microbial populations. As a follow-up study of transcriptomics, proteomics can perform protein detection, cellular functional entities, and posttranslational modifications that cannot be predicted by mRNA expression analysis. In general, quorum sensing can be controlled through the operation of the posttranslational mechanism. Probiotics can confer a certain health benefit to the body when given a sufficient amount of probiotics (Bouchard et al. 2015), which must meet two conditions: one is the close interaction between the microorganism and the host, and the other is the microorganism has to have the ability to adapt to the environment. The mechanisms by which probiotics regulate health promotion are (1) inhibiting the growth of pathogens and repairing microbial balance through interactions between microorganisms, (2) enhancing the barrier function of epithelial cells, and (3) regulating immune responses (Bastani et  al. 2012; Bermudez-Brito et  al. 2012; Hemaiswarya et  al. 2013). Quorum sensing molecules show good biological activity and far exceed their transmission among bacteria, such as the acyl-homoserine lactones (AHL) synthesized by Pseudomonas aeruginosa, which has functions of ­antibacterial, pharmacological, and immunomodulatory activities (Diggle et  al. 2007). One of the important mechanisms is based on the two-component system, in which transmembrane domain of histidine protein kinase (HPK) receives signals and the bacteria react correspondingly by sensing the changes of the external environment through the two-component system. Therefore, the microflora density colonized in the intestinal tract has high density and diversity. Studies have speculated that the coordinated adaptation process of intestinal tract includes the competition and cooperation of nutrients and adhesion sites (Kaper and Sperandio 2005). Interactions between probiotics and human gut are mediated by proteins which were exposed to bacteria’s surface (Remus et al. 2013; Jensen et al. 2014). Therefore, these proteins are particularly important in assessing the prebiotic properties of certain bacteria. Surface layer proteins can be divided into covalent and non-covalent proteins. Covalent proteins include lipoproteins that bind to cell membranes through

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N-terminal LXXC sequences (lipid box sequences) as well as proteins that bind to S-layer through C-terminal PPXTG sequences. Non-covalent proteins include transmembrane proteins with specific C-terminal repeat sequences or glycine-­ tryptophan sequences that bind to lipophosphoric acid or phosphoric acid, as well as S-layer proteins that bind to cell wall matrix through specific SL domain polysaccharides. Due to the complexity of cell membrane and the different mechanisms of binding between proteins and membrane proteins, the related research is challenging. In terms of time, Bifidobacterium was first colonized in the intestinal tract. Bifidobacterium is considered to be the main bacteria that promote health and can regulate the immune response and maintain the integrity of the gastrointestinal barrier (Leahy et al. 2005; Amund et al. 2014). The factors involved in the interaction between Bifidobacterium are relatively unfamiliar. It is well known that S-Ribosylhomocysteinase (LuxS protein) plays a key role in the quorum sensing of Bifidobacterium longum NCC2705 (Yuan et al. 2007; Wei et al. 2014). Ruiz et al. studied the physiological characteristics of Bifidobacterium longum NCIMB8809 and Bifidobacterium brevis NCIMB8807 in co-culture conditions (2009). The physiological properties of bifidobacteria were analyzed by two-dimensional gel electrophoresis and mass spectrometry under co-culture conditions, it was found that 16 differential proteins belong to Bifidobacterium longum NCIMB8809 and Bifidobacterium breve NCIMB8807, and 10 of them were up-regulated by ribosomal protein expression (Ruiz et al. 2009). Among these 10 differential proteins, 5 are from Bifidobacterium longum and the remaining are from Bifidobacterium breve. Ribosomal proteins are essential for ribosome assembly and its stability and can sense changes in the external environment (Rutherford and Bassler 2012). The expression of transcriptional regulation factor clgR which regulates the expression of clpC gene and clpP operon was upregulated in Bifidobacterium breve (Lindner et  al. 2007; Milani et  al. 2016). Similarly, the expression of glycosyltransferase which involves peptidoglycan biosynthesis and cell division was upregulated (Mohammadi et al. 2007). Bifidobacterium brevis may respond to the inhibition of Bifidobacterium longum by enhancing cell wall biosynthesis. The expression of three different hydrolytic products of fructose-6-phosphotransferase was upregulated in Bifidobacterium longum. Strains perceive each other and regulate carbohydrate metabolism to increase their competitive power. Yuan et al. (2007) studied the proteomics of Bifidobacterium longum NCC2705 cultured in vitro and in vivo. The expression of 14 proteins was upregulated under the growth condition of imitating gastrointestinal tract, 4 of which were associated with membrane fluidity. All the proteins involved in adaptation of Bifidobacterium longum NCC2705  in gastrointestinal environment are mainly related to stress response and translation, for example, the extension factor Tu (EF-Tu) can act as an adhesion factor for Bifidobacterium and promote the attachment of strain. Bile salt hydrolase (BSH) can promote the interaction between bacteria and gastrointestinal tract, and more importantly, it can be used as the bacterial stress protein which can resist the harmful compounds of gastrointestinal tract. LuxS protein can change the

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membrane fluidity, and phosphorylated LuxS protein can act as the mechanism of interspecies signal interference by enterobacter. In the case of higher bacteria concentration and more species in human gastrointestinal tract, the concentration of signal factor Al-2 is also higher. Two different mechanisms have been proposed for Bifidobacterium longum NCC2705. The first mechanism is that when a large number of bacteria are present in the gastrointestinal tract, these signals can be used to identify the presence of Al-2. The second mechanism is to interfere with the signal transmission ability of other bacteria through distancing or destroying the Al-2 synthesized by other bacteria. The secretomics of Bifidobacterium longum NCIMB8809 have been reported, and its environmental perception information (Sánchez et  al. 2008) has been obtained. The secretomics study found 17 differential proteins, of which 14 proteins have been identified. The identified proteins include two solute binding protein ABC transporters: one is phosphate binding transporter, belonging to the ABC transport system, and the other is the cell wall synthase, which is involved in cell division-specific transpeptidase formed by the diaphragm, the cell wall hydrolase that invades related homologous proteins, and the protein that catalyzes cell wall turnover. Gastrointestinal mucosa adhesion is a common mechanism involving both small molecules and protein compounds in the process of adhesion (Bron et al. 2013; Gao et al. 2011; Glenting et al. 2013). Proteomics technique was used to study the cell aggregation process of Lactobacillus crispatus M247. Compared with the mutant strain without cell aggregation phenotype, the expression of elongation factor Tu was upregulated in wild strain, which indicated that Tu was involved in the adhesion process of strain (Siciliano et al. 2008). Lactobacillus reuteri RC-14 alters the virulence of Staphylococcus aureus by secreting intercellular signaling molecules (Laughton et al. 2006). In co-culture of two strains, Lactobacillus reuteri RC-14 can secrete one or more molecules to inhibit the expression of staphylococcal exotoxin. In addition, bacteria can transmit information with host by quorum sensing signals, which can be called cross-cutting intercellular signal transduction, and the signal transduction is almost analyzed by transcriptome technique (Wang et  al. 2008; Maeda et al. 2012; Jiang et al. 2013). At present, only the effects of Candida albicans and farnesol quorum sensing signaling molecules (Scheper et  al. 2008; Décanis et al. 2011) or Lactobacillus plantarum DC400 and pheromone PlnA (di Cagno et al. 2010) were studied by proteomics. In particular, PlnA can increase the viability of Caco-2/TC7 cells (human colon cancer cells). Caco-2 cells are usually used to simulate intestinal mucosa in vitro. Under the condition of culture, caco-2 cells have certain characteristics of morphology and intestinal cell function, mainly including tight intercellular connections and judging integrity of Caco-2 cells by measuring transepithelial electrical resistance (TEER). Compared to the negative test (isolated culture), PlnA significantly increased TEER levels (Yan et al. 2007). In addition, PlnA signaling molecules can block the damage of interferon γ to Caco-2/TC7 cells and eliminate the inhibition of cytokines.

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5.3.4  A  pplication of Proteomics in Energy Metabolism of Lactobacillus Lactobacillus originated about 3 billion years ago during the earth transition period from anaerobic to aerobic. During its evolution, it did not have the ability to biosynthesis heme (Carr et al. 2002). Porphyrin rings present in cytochrome, peroxidase, and catalase have respiration (including aerobic and anaerobic respiration) and detoxification of oxygen free radicals (Levering et al. 2012). Lactobacillus requires the addition of heme to obtain energy through respiration. The ATP obtained by fermentation is not enough to support the rapid growth of Lactobacillus, so it is necessary to change the strategy to obtain energy. There are three main energy production pathways in Lactobacillus: amino acid decarboxylation, malic acid decarboxylation, and arginine deaminase pathway (ADI pathway). These pathways not only provide energy but also play a role in pH regulation (Luo et al. 2012). Lactic acid fermentation can lead to lactic acid accumulation, decarboxylation of amino acid causes decrease of pH, and the ADI pathway can produce some ammonia. Different from strict anaerobic bacteria, Lactobacillus can tolerate oxygen with certain concentration (Brioukhanov and Netrusov 2007). Due to the lack of heme in Lactobacillus, the defense mechanism of Lactobacillus against oxygen is mainly based on the accumulation of metals: in fact, some Lactobacillus can accumulate large amounts of manganese (up to 25mmol/L), selenium, and zinc in cells. Manganese can act as an operating system similar to manganese superoxide dismutase, selenium can act as selenocysteine system to remove free radicals, and zinc can form oxygen free radical ion trap (de Angelis and Gobbetti 2004; Vandenplas et al. 2007; Hosseini Nezhad et al. 2015). These mechanisms make the anaerobic Lactobacillus have the oxygen tolerance, and Lactobacillus can be used as probiotics to reduce the metal ions in the gastrointestinal tract. Some heterozygous Lactobacillus can transform oxygen molecules into water through short electron transport chains (NADH-flavin-O2) (Brooijmans et al. 2007). In this system, acetic acid is generated by acetyl phosphate, and NADH is oxidized to form NAD+, which is accompanied by formation of ATP (Carr et al. 2002). Amino acid decarboxylation can be catalyzed by pyridoxal phosphate or pyruvyl-­ dependent decarboxylase (either soluble or membrane bound) (Fernández-Pérez et al. 2014; Alcántara et al. 2016). In addition to aspartic acid and glutamic acid, all amino acid can produce alkali compounds called biogenic amines. Although the decarboxylation of amino acids can reduce the acidity of the system, most of the amines (tyramine, histamine, and β-phenylethylamine) have different degree of pathogenicity to the central nervous system or blood vessels (Liu et al. 2013). In addition, putrescine and cadaverine will affect the sensory properties of food (Alvarez and Moreno-Arribas 2014). From different perspectives, the decarboxylation of glutamic acid and aspartic acid generates aminobutyric acid and β-alanine. The acidity of aminobutyric acid and β-alanine is lower than their precursors, but they are still acidic compounds and can increase the buffer capacity of pH slightly. As aminobutyric acid can regulate smooth muscle and central nervous system

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(Inoue et al. 2003), probiotic which can produce aminobutyric acid can be used for health products. The decarboxylation of acids and amino acids is accompanied by the electron reverse transport system (Wolken et al. 2006), which can produce PMF (PMF can synthesis ATP by F0F1-ATP synthase) and neutralize the intracellular acidic environment. In most Lactobacillus strains, the decarboxylase gene and the reverse transporter gene are located in the same operon (Capitani et al. 2003). Due to the acidified environment, some proteins can change their own decarboxylase conformation to obtain catalytic activity. Therefore, these pathways are complementary in lactic acid fermentation, and lactic acid accumulation can inhibit the decrease of pH. There is ADI pathway which can generate energy in bacteria especially in Lactobacillus (Brandsma et al. 2012). Arginine deaminase and arginine/ornithine transporter were the first hypothetical “reverse” urea cycle in which 1 mole of arginine was converted to form 1 mole of ornithine and 2 moles of NH3. However, activities of arginase and urease were not detected in arginine medium extracts. On the contrary, three different enzymes were obtained by isolation and purification: the arginine deaminase (ADI), ornithine transcarbamylase (OTC), and carbamate kinase (CK). In lactobacillus, these three enzymes are located in the same operator. 1 mol of arginine deimino group forms citrulline, and at the same time, it can produce 2 mol of NH3 and 1 mol of ATP. Citrulline is formed by carbamoylation to form ornithine. Malic acid decarboxylation reaction is accomplished by one-step catalysis of malolactic enzyme or through the catalysis of malolactic enzyme to form pyruvate intermediates, which are reduced to form lactic acid (Landete et al. 2013). Malolactic enzyme and malic enzyme both contain binding sites of Mn2+ and NAD+, and they are very similar in phylogenetic process. In industry, these decarboxylation reactions are called malolactic fermentation (MLF), which transforms dicarboxylic acid (malic acid) into monocarboxylic acid (lactic acid), which is more common in wine-­ making process and can increase the overall acidity of red wine. 5.3.4.1  Factors Affecting Alternative Energy Access The complex factors of Lactobacillus are different in each growth phase based on culture conditions, nutrient availability, strain characteristics, and auxotroph. According to the analysis of amines and proteomics, there was a positive correlation between the histamine accumulation and the excessive expression of histidine decarboxylase (HDC) in Lactobacillus in stable phase (Pessione et al. 2005), and a similar result was found in Lactococcus lactis NCDO2118 (Mazzoli et al. 2010). In the contrary, the expression of HDC gene in Lactobacillus hilgardii ISE 5211 in logarithmic growth phase was analyzed by Western blotting (Landete et al. 2006). Meanwhile, Mazzoli et al. researched HDC and ornithine decarboxylase in logarithmic growth phase, respectively (Mazzoli et al. 2009). PH, carbon source, ATP consumption, and nitrogen source can make certain effects to stable phase. In the whole fermentation process, there are different forms of mechanism, and different factors

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play an important role in different growth phase of bacteria. According to the literature, both the ADI and MLF pathways of the Lactobacillus sanguices and S. cerevisiae occur in the growth phase of the bacterial index (de Angelis et al. 2002). Carbohydrates can affect the decarboxylation pathway of amino acids and the output of Oenococcus oeni histamine in the absence of carbohydrates. It is reported that glucose does not affect the catalysis of HDC in Oenococcus oeni, and the biosynthesis controlled HDC operon was proposed (Coton et  al. 2010). In addition, Lactobacillus activates the HDC pathway during the stable phase when glucose is depleted, and glucose and fructose inhibit the expression of HDC operon in Lactobacillus hilgardii, Oenococcus oeni, and Pediococcus parvulus. Moreno-­ Arribas et al. reported that in the presence of fructose or fructose and L-malic acid, the accumulation of tyramine reached the maximum (Moreno-Arribas et al. 2003). The accumulation of tyramine reached the maximum during the exponential phase of Enterococcus faecalis DISAV1022 (Pessione et al. 2009). The effect of carbohydrates on the decarboxylation pathway of amino acids has not been fully elucidated. For amino acid decarboxylation, the ability of tyrosine decarboxylation of Lactobacillus brevis to form tyramine was increased tenfold and the activity of TDC in MRS medium was increased twofold (García-Ruiz et al. 2011). In the chemically defined medium (CDM) without tyrosine and phenylalanine, the tyramine production of Enterococcus faecalis DISAV1022 was only slightly increased, and the 2.5  mmol/L tyramine was obtained from CDM supplemented with 2.5  mmol/L tyrosine and 14 mmol/L phenylalanine (Pessione et al. 2009).The biosynthesis of tyrosine decarboxylase was found by studying the membrane proteomics of Enterococcus faecalis DISAV1022 strain. In Oenococcus oeni, the accumulation of cadaverine was accompanied by the increase of lysine, ornithine increased the accumulation of putrescine, and 100% of ornithine was converted to putrescine (Guerrini et al. 2002). It has been proven that histidine can induce histamine accumulation in Lactobacillus 30a and Lactobacillus hilgardii ISE5211. The increase of histamine was related to the biosynthesis of histidine in HDC operon, and the constitutive expression level of HDC was significantly increased. In addition, Landete et al. used Northern blotting to investigate Lactobacillus hilgardii, Oenococcus oeni, and Pediococcus parvulus and found that histidine had the same inductive effect on HDC operon (2007). In contrast, histidine can feedback inhibit the expression of HDC operon and the catalytic activity of HDC. The experimental study of CDM in Lactococcus lactis NCDO 2118 showed that the amount of biogenic amine-­ aminobutyric acid produced by glutamic acid decarboxylation was very small, which indicated there was no amino acid precursor. It is possible that glutamine is converted to glutamate in CDM, and glutamate can be decarboxylated to form aminobutyric acid (Mazzoli et al. 2010). Although the differential expression of glutamate decarboxylase has not been demonstrated by proteomics or transcriptomics techniques, the enhancement of aminobutyric acid biosynthesis can catalyze the regulation of glutamate decarboxylation. Therefore, when the constitutive expression level of the amino acid decarboxylase is low, or is completely induced to

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express, the precursor amino acid has a core role, which can activate the biosynthesis of the amino acid decarboxylase. In Lactococcus lactis, ADI enzyme and OTC enzyme are induced by arginine in the ADI pathway, while CK is constitutively expressed. In other dairy strains, such as Lactobacillus buchneri and Enterococcus faecalis and so on, the expression of ADI, OTC, and CK is induced by arginine, which is similar to the strain in wine. In contrast, ADI, OTC, and CK are all constitutive expressions in Oenococcus, and transcriptome analysis shows that arginine can enhance the transcription of ADI, OTC, and CK encoding genes (Tonon et al. 2001). In addition, it was found that the reverse transporter arcD involved in arginine/ornithine exchange was constitutively expressed through real-time quantitative PCR. Arginine can promote the formation of putrescine in Oenococcus oeni. Agmatine is formed through arginine decarboxylation, and then agmatine is converted to putrescine. Not all ornithine is used for reverse transport in the ADI pathway, and some of ornithine is decarboxylated to form putrescine. Arginine has a certain influence on the ADI pathway of Lactobacillus ft., and it was used in rye and wheat bread industry to produce bread by sourdough and Italian cake. The products of trypsin hydrolysates were analyzed by two-­ dimensional gel electrophoresis and peptide sequencing, and the 17mmol/L arginine could increase the expression of ADI, OTC, and CK by tenfold, fourfold, and twofold, respectively (DE Angelis et al. 2002). 5.3.4.2  Interrelationship of Energy Generation Pathways in Lactobacillus The effects of carbohydrate and acid compounds (including L-malic acid) on the decarboxylation of ADI/MLF and amino acids have been reported commonly, but the interactions between these production pathways have been studied rarely. Researchers have studied Lactobacillus that produce amines in foods to control the production of biogenic amines. Comparative proteomics is often chosen as the method for the determination of enzyme expression and can help to understand the complex networks of different metabolic pathways existing in bacterial biological systems. 1. Proteomics of the effect of amino acids on glucose metabolism At present, a large number of studies have focused on the regulation of carbohydrates to amino acid metabolism, while there are few reports on the effects of amino acids on glycolysis pathway (Embden-Meyerhof) and pentose phosphate pathway. Adding histidine and ornithine to the growth medium of Lactococcus lactis could inhibit the synthesis of Embden-Meyerhof pathway enzyme and glyceraldehyde 3-phosphate dehydrogenase II, compared with the control group, and the expression of both enzymes decreased by sevenfold (Pessione et al. 2005). The results indicated that glucose metabolism can be regulated by other energy production pathways. On the contrary, the research of Lactobacillus hilgardii ISE5211 by heterolactic fermentation in wine showed that there was no effect on the glucose metabolism during the growth phase under the condition of excessive histidine (Mazzoli et al. 2009; Lamberti et al. 2011). Transcriptomics and proteomic analysis

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of homofermentative Lactococcus lactis NCDO 2118 producing aminobutyric acid showed that glycolysis and glutamic acid decarboxylation were carried out at the same time, with or without glutamic acid (Oliveira et al. 2014). Comparison of proteomics and transcriptomics analysis showed that the expression of phosphoglycerate mutase, 6-phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase was upregulated, but the expression of pyruvate kinase, phosphoglycerate kinase, and fructose diphosphate aldolase was downregulated. Whether it is activating or inhibiting the glucose metabolism pathway, glutamic acid has a fine-tuning effect on the glucose metabolism pathway. On the contrary, Lactobacillus hilgardii, which has adapted to unfavorable growth conditions (low pH and high ethanol concentration), can grow in the pH range of 3.8–5.8 in wine. In addition, since Lactobacillus hilgardii is a kind of heterotypic lactic acid fermentation, the ethanol can make contribution to balance the production of lactic acid. In this case, the production of histamine can replenish energy demand through heterolactic fermentation (one molecule of ATP can be produced in each cycle) (Dias et al. 2015). In addition, in Lactococcus lactis and Lactobacillus, there is a unique photoregulation phenomenon. The maximum yield of biogenic amines was obtained in the stable growth phase, indicating that the amino acid decarboxylation reaction was rapid, and pH had no effect on the biosynthesis of biogenic amines (Mazzoli et al. 2010). When the main carbon sources (such as glucose) are depleted, ornithine, histidine, or glutamic acid decarboxylation reactions are performed to replenish the energy through the electron reverse transporter. It is worth mentioning that glutamate will be decarboxylated to aminobutyric acid which is still acid, causing only a little alkalinization. Thus, in these cases, amino acid decarboxylation seems to solve energy problems. Tyramine and β-phenylethylamine have a strong buffer capacity, and proteomics and metabonomics research can be used to further study the effects of pH on the production of biogenic amines. 2. Proteomics on the effects of amino acids on ADI pathway and amino acid decarboxylation The ADI pathway was first found in Enterococcus faecalis, and tyrosine and phenylalanine cannot regulate it (Barcelona-Andrés et  al. 2002). Compared with Lactococcus lactis, Lactobacillus hilgardii, and Oenococcus oeni, Enterococcus faecalis has the strongest acid resistance. Therefore, the ADI pathway and the amino acid decarboxylation reaction are carried out simultaneously to produce enough alkaline substances (ammonia, tyramine, β-phenylethylamine) to maintain the optimal growth pH and ensure efficient glycolysis pathways that provide sufficient energy. During the production process of aminobutyric acid by Lactococcus lactis NCDO 2118, there is a slight negative regulatory mechanism in the mRNA and protein levels (Mazzoli et al. 2010). In contrast, the biosynthesis level of aminobutyric acid increased in arginine-containing media, which may be due to the oxidative deamination of ornithine (the metabolite of arginine) to form glutamic semialdehyde which is easy to produce glutamate by oxidation. Therefore, the two energy metabolic pathways of arginine deamination and glutamate decarboxylation may be simultaneous, but there are specific regulatory effects. Considering the optimal growth pH of Lactococcus lactis, the transformation of glutamic acid into ami-

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nobutyric acid (which can produce a small amount of alkaline substances) may be more inclined to utilize ADI pathway to produce ammonia (Mazzoli et al. 2009). In Lactobacillus hilgardii ISE5211, histidine was not transformed into histamine by ADI pathway. The proteomics results showed that histidine could inhibit the expression of ADI enzyme. In this case, arginine in the medium inhibited the decarboxylation of histidine (Mazzoli et al. 2009). In addition, SDS-PAGE result showed that HDC was greatly affected by arginine. There was a competitive relation between ADI pathway and histidine decarboxylation in Lactobacillus hilgardii ISE5211. Comparative proteomics can provide some information under the condition of excessive amino acids regardless of the interaction between energy sources. The interaction between stress chaperone and protease has been reported gradually (Dougan et al. 2002). Chaperone can not only help the folding of new protein but also prevent the misfolding of protein and help the refolding of damaged proteins under stress conditions. In addition, chaperone can cooperate with protease to destroy the irreversible damage protein, and chaperone can obtain protease properties by activating proteolytic sites (Moliere and Turgay 2009). Therefore, it has been proven that stress response and proteolysis are closely related. In the study of Enterococcus faecalis DISAV1022 grown in tyrosine and phenylalanine, it was found that stress protein DnaJ and Gls24, protease, glutamyl aminopeptidase (Pep A), and some ABC transporters like glycine-betaine-carnitine-choline-binding protein were overexpressed. Glycine-betaine-carnitine-choline-binding protein is the first protective mechanism against osmotic pressure. Lactobacillus can respond to hypertonic or hypotonic stress by accumulating or releasing osmotic agents (betaine) (Konings 2006). In contrast, when Lactococcus lactis NCDO 2118 grew in the condition of glutamate excessively, the expression of glutamyl aminopeptidase, Clp P protease, and other stress proteins (such as superoxide dismutase and the Cts R) was downregulation detected by proteomics and transcriptomics analysis. And Cts R regulates the expression of Clp gene under stress conditions in Lactococcus lactis and Oenococcus oeni (Grandvalet et al. 2005). The regulation of productivity pathway on protein network in Lactobacillus (homolactic fermentation, heterolactic fermentation, lactic acid fermentation, amino acid decarboxylation, and arginine deamination pathway) is complex. At the same time, the cellular response of Lactobacillus that is exposed to different substrates is not only species-specific, but also strain-specific, and there is a certain hierarchical structure of energy utilization. Transcriptomics and proteomics techniques can provide information about energy metabolism pathways.

References Abe F, Horikoshi K (2001) The biotechnological potential of piezophiles. Trends Biotechnol 19(3):102–108 Alcántara C, Bäuerl C, Revillaguarinos A et  al (2016) Peptide and amino acid metabolism is controlled by an OmpR family -response regulator in lactobacillus casei. Mol Microbiol 1(100):25–41

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Chapter 6

Metabolomics of Lactic Acid Bacteria Wanqiang Wu and Nan Zhao

6.1  Overview of Metabolomics 6.1.1  Metabolomics Metabonomics/metabolomics, developed in the late 1990s, is a science that deals with the types, quantities, and changes of metabolites (molecular weight 75%), and the inhibition rate of some strains of Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus rhamnosus was even more than 90%. Fang et al. (2007) isolated a series of lactic acid bacteria strains from the feces of the elderly in Changshou Village, Bama, and then evaluated their inhibition on 4NQO gene toxicity by E. coli SOS chromogenic reaction. The results showed that the genotoxic clearance rates of Lactobacillus salivarius and Bifidobacterium were both above 80%, while the clearance rates of the commercial Lactobacillus casei strains and Lactobacillus rhamnosus strains were 73% and 9%, respectively. This model could effectivity distinguish and screen lactic acid bacteria with different anti-mutation characteristics. The authors also speculated that the relevant properties of lactic acid bacteria were directly or indirectly related to the metabolic activity of the strain. Zhaoyong et al. (2011) further studied the conditions that the requirement for L. salivarius to degradate 4NQO. He found that when the concentration of the strain cells reached 1011 cfu/ml, the obvious degradation was observed. And the degradation product of 4NQO can be detected when the concentration of the cells reaches 1013 cfu/ml. In addition to classical and general test methods such as the mutagenicity detection (Ames test), the SOS chromogenic reaction is a major method to evaluate the 4NQO genotoxicity inhibition ability of lactic acid bacteria. The reaction principle is mainly the exposure of E. coli PQ37 (genetically engineered bacteria) to the environment of mutagenic substances. Under such circumstances, the RecA protease could decompose the combined protein and activated the normal lac gene which was originally deleted, finally resulting in the positive correlation between galactosidase expression and mutagens. Then, the decomposition of o-nitrobenzene β-d-galactoside (ONPG) by galactosidase could produce yellow soluble substances. Therefore, quantitative determination can be performed (Quillardet and Hofnung 1985; Caldini et al. 2005). This experiment is a commonly used method for analyzing the ability of lactic acid bacteria to inhibit genotoxic effects of mutagenic chemicals due to its high s­ ensitivity

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and high specificity. Moreover, quantitative analysis of 4NQO content can be measured by high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), or combined techniques. Therefore, the degradation or adsorption ability of the strain could be concluded. For example, Verdenelli et al. (2010) co-cultured Lactobacillus rhamnosus IMC501 with 4NQO, and then the 4NQO degradation was discovered by GC/MS. Zhaoyong et al. (2011) demonstrated that intermediate product and final product of 4NQO degradation by lactic acid bacteria could be effectively detected by high-performance liquid chromatography. Its liquid mobile phase consists of water [0.1% trifluoroacetic acid (TFA) added] and methanol. The elution procedure was 0.01 min, 8% methanol; 3.0 min, 35% methanol; 4.0  min, 35% methanol; 15.0  min, 77% methanol; 20.0  min, 88% methanol; 21.0 min, stopped. Wang et al. (2008) also demonstrated that high-performance liquid chromatography can be an alternative to the SOS chromogenic reaction, with a correlation coefficient (R2) between the two methods up to 0.991. At the same time, the detection limit of liquid chromatography reaches 1.2 ng, which is more accurate and sensitive than the SOS chromogenic method. 7.2.1.2  N-Methyl-N′-Nitro-N-Nitrosoguanidine (MNNG) Experiment N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) is a nitrosamine chemical and a class of alkylating agents with strong carcinogenic and mutagenic functions. MNNG induces abnormal activation of the JNK/SAPK pathway in animals and human cells, leading to DNA damage and chromosomal aberrations (Isabelle et al. 2012; Zhang et al. 2014a). Both epidemiology and animal experiments have shown that MNNG can induce tumorigenesis in organs such as the digestive tract, liver, and kidney (Jing et  al. 2000; Manikandan et  al. 2011; Matsumura et  al. 2015). Similar to 4NQO, there have also been many researches on the anti-mutation properties of lactic acid bacteria by MNNG experiments. Park and Rhee (2001) isolated Lactobacillus plantarum KLAB21 from Korean kimchi. The whole cell, supernatant, and cell extract of this strain were found to have strong anti-mutation activity against MNNG.  Caldini et  al. (2005) tested the anti-mutagenic properties of 65 lactic acid bacteria strains and found that more lactic acid bacteria could restrain the genotoxicity caused by 4NQO, but only one strain of Lactobacillus acidophilus can inhibit the mutagenic activity of MNNG.  Ambalam et  al. (2011) found that Lactobacillus rhamnosus Lr231 isolated from human intestinal tract adsorbs MNNG and reduces its mutagenic activity. The adsorption process is related to teichoic acid and sulfhydryl groups on the cell surface, as well as involves hydrophobic interaction. Jinggang and Hong (1998) used MNNG to induce the Escherichia coli SOS reaction model and studied the anti-mutation characteristics of bifidobacteria. The results showed that Bifidobacterium whole bacteria, lipoteichoic acid, cell wall peptidoglycan, and culture supernatant have a certain degree of anti-mutation properties, in which lipoteichoic acid works best. They further found that Bifidobacterium can effectively inhibit DNA damage induced by MNNG in mouse intestinal mucosa by single-cell gel electrophoresis. The mechanism involved may

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be that the MNNG-active group is masked by binding to MNNG, resulting in decreased mutation activity (Jinggang et al. 1998). Similar to the 4NQO experiment, the MNNG experiment was used to evaluate the anti-mutation properties of lactic acid bacteria using common test methods such as Ames method, SOS color reaction, liquid chromatography, mass spectrometry, etc. (Caldini et al. 2005; Ambalam et al. 2011). In addition, single-cell gel electrophoresis, also known as comet assays, is often used to evaluate the results of MNNG experiments. The mechanism of action is mainly that DNA damage leads to an increase in fragmentation, looseness, and release of the DNA supercoiled structure, and a comet-like fluorescent head and tail can be obtained by ethidium bromide staining. Therefore, DNA damage and mutagenic effects of MNNG can be judged based on the tail length of the comet and the DNA content (Wollowski et al. 1999).

7.2.2  The In Vitro Adsorption Model According to the World Health Organization’s draft Global Food Safety Strategy, food safety issues are primarily related to microbiological and chemical hazards. The latter mainly includes natural toxic substances (such as microbial toxins) and environmental pollutants (such as heavy metals), which have become important safety hazard factors in food (Wei and Qixiao 2014). Lactic acid bacteria can effectively absorb chemical substances such as heavy metal ions, biological toxins, and pesticide residues due to their special surface structure. The adsorption mechanism may involve group bonding, ion exchange, physical deposition, membrane permeation, etc. on the surface of the cells and may also include active transport and endocytosis of the cells, and these modes of action are interrelated to form a complex adsorption mechanism. In order to look into the in vitro adsorption characteristics of lactic acid bacteria, a variety of mature adsorption models have been reported, which are described below. 7.2.2.1  Metal Ion Adsorption Harmful metal pollution has become a serious food safety and health problem. Take two common heavy metals such as lead and cadmium as examples. Lead is a harmful metal that has pernicious effects on the body. It has significant toxic effects on the liver, brain, kidney, and other organs. The ideal blood lead concentration should be zero. Cadmium has serious toxic effects on the bones, liver, kidneys, reproductive system, and blood system of the body. It is listed as the seventh toxic substance harmful to human health by the US Agency for Toxic Substances and Disease Registry and the National Environmental Protection Agency. It is classified as a class I carcinogen. Other toxic and harmful metals such as mercury, arsenic, aluminum, manganese, etc. may also cause significant harm to the body. At present, the problems of heavy metal pollution in foods are very common.

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In recent years, surveys have shown that the content of heavy metals in rice, fruits, and vegetables and aquatic products in developing countries including China is seriously increasing (Zhang et al. 2014b; Zhai et al. 2015). However, there is currently no very efficient and safe means for removing harmful metals from food. Lactic acid bacteria are considered to be food-safe microorganisms, and the special adsorption characteristics of some strains on metal ions make them have the potential to reduce the harmful metals in food. At present, many studies have used different evaluation models to investigate the adsorption characteristics of lactic acid bacteria and mainly consider the adsorption kinetics, thermodynamic properties, and adsorption stability of the strains. Bhakta et al. (2012) isolated 255 strains of lactic acid bacteria from environmental sludge samples. After screening, 26 strains were resistant to heavy metals such as lead and cadmium, and Lactobacillus reuteri Pb71-1 had the strongest removal capacity (59%) to lead. Lactobacillus reuteri Cd70-13 has the strongest adsorption capacity for cadmium (25%). In the first 24 h of adsorption, the adsorption capacity of the intracellular substance is near the cell membrane. At two times, at 48 h, the adsorption capacity of intracellular substances decreased, and the cell membrane adsorption capacity was greatly increased. The test conditions were as follows: initial lead concentration 6 mg/L, initial bacterial concentration 3 g/L (wet weight), culture temperature 37  °C, and culture time 2  h. Tian Fengwei et  al. screened Lactobacillus plantarum CCFM8661 with strong adsorption capacity for lead ions and used HNO3 (15  mmol/L and 1.5  mmol/L) and EDTA (1.0  mmol/L and 0.1 mmol/L) solutions, as well as ultrapure water. Desorption experiments were carried out on Lactobacillus plantarum CCFM8661 after the completion of adsorption of lead ions. The results showed that the adsorption of Pb2+ by CCFM8661 was stable and would not be easily desorbed. Through adsorption thermodynamic analysis, a series of solutions with different initial lead concentrations were selected for adsorption experiments, and equilibrium adsorption isotherms were used to balance the adsorption amount and equilibrium solution concentration, and different thermodynamic models were used to desorb the fitting. The results show that CCFM8661 is suitable for Langmuir model (assuming that the adsorbent surface is uniform and the adsorption energy is the same everywhere, and the adsorption process is monolayer adsorption. When the adsorbent surface is saturated with adsorbate, the adsorption amount reaches the maximum value, corresponding to the physical adsorption process). Both the Langmuir-Freundlich dual models have a good fit (Yin et al. 2016). A series of studies by Halttunen et  al. (2007, 2010) measured the adsorption capacity of ten strains of lactic acid bacteria including Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus casei, Bifidobacterium longum, and Bifidobacterium breve. Test conditions are as follows: initial cadmium concentration was 50 mg/L, initial pH was 6.0, initial bacterial concentration was 1 g/L (109 cells per ml), culture temperature was 22  °C, and culture time was 60  min. The results showed that although each strain had a certain adsorption capacity, the differences between the strains were significant and affected by the initial adsorption conditions. To further analyze the adsorption thermodynamic properties of the

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strain, the authors used different models to fit the existing adsorption isotherm data. According to the Langmuir model, the authors demonstrated that the theoretical monolayer maximum adsorption amount Qmax of Lactobacillus fermentum ME3 was 28.4 mg/g dry weight, Lactobacillus casei Qmax was 12.1 mg/g dry weight of bacteria, and Lactobacillus rhamnosus (the Qmax of Lactobacillus rhamnosus GG, LGG) was 13.2 mg/g dry weight of the cells. Qixiao (2015) screened a strain of Lactobacillus plantarum CCFM8610 with strong adsorption capacity for cadmium. Through adsorption thermodynamic analysis, it was found that the adsorption process of CCFM8610 has the best goodness of fit for the Langmuir-Freundlich model (R2 = 0.9928); the theoretical upper limit Qmax of the adsorption capacity of the strain is significantly higher than the commercial probiotic strain such as Lactobacillus rhamnosus LGG and Lactobacillus casei strains. Through adsorption kinetic analysis, it was found that the adsorption of the strain was a fast and efficient process, which accorded with the quasi-second-order kinetic equation, and reached 90% of the equilibrium adsorption amount at the time of 100 min. Further fitting using the Weber-Morris model revealed that the adsorption process can be divided into two phases, including the diffusion of cadmium ions from the solution onto the surface of the cells and the binding of cadmium to the surface active sites of the cells (Zhai et al. 2016). Ibrahim et al. (2006) found that the adsorption of cadmium by L. rhamnosus LC-705 could better fit the Langmuir model, which Qmax was 13.2 mg/g dry weight of the cells, and was influenced by the initial cadmium concentration, the initial bacterial concentration, the adsorption time and the initial pH. Through adsorption kinetic analysis, Teemu et al. (2008) found that the adsorption of cadmium by Lactobacillus fermentum ME3 and Bifidobacterium longum 46 was a rapid and efficient process and was associated with hydroxyl and phosphoryl groups on the surface of the cells. 7.2.2.2  Microbial Toxin Adsorption In general, microbial toxins refer to toxic chemicals produced by microorganisms during their growth and reproduction or under specific environmental conditions. The microbial toxins commonly found in people’s lives include aflatoxins, algal toxins, patulin, botulinum toxin, and cholera toxin. These toxins are difficult to remove once they contaminate food, posing a serious hazard to human food chain safety. According to the characteristics of different microbial toxins, the main methods for toxin removal include physical methods, chemical methods, and biodegradation methods. The biodegradation method does not use toxic and harmful chemical agents and does not affect the taste of food and the loss of nutrients in food. It is considered to be the best method of detoxification. The adsorption and subtraction functions of lactic acid bacteria on microbial toxins have been widely recognized. In different reports, there are some differences in the relevant adsorption models. Halttunen et al. (2010) studied the competence of a series of lactic acid bacteria to adsorb microcystins and aflatoxins. The test conditions were initial toxin

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c­ oncentration of 100 μg/L (microcystins) and 4 mg/L (aflatoxin), initial pH 7.0, initial bacterial concentration of 2 g/L (microcystins) and 109cfu/ml (aflatoxin), culture temperature of 22 °C (microcystins) and 37 °C (aflatoxin), and culture time of 24 h (micro-cytotoxin) and 60 min (aflatoxin). The results showed that Lactobacillus rhamnosus LC705 had the strongest adsorption capacity for microcystins, while Bifidobacterium breve E8/99 had the strongest removal ability for aflatoxins. Wang et al. (2010) examined the ability of three commercial lactic acid bacteria to reduce microcystins. The initial conditions were 7.0, the initial bacterial concentration was 109  cfu/L, the culture temperature was 37  °C, and the culture time was 24  h. Lactobacillus casei was found to have the best depletion ability, and the final removal rate was up to 43%. At the same time, the initial cell activity, cell concentration, and toxin concentration had an effect on the removal rate. After the addition of exogenous carbon source glucose, the efficiency of the strain to reduce algal toxin was significantly improved, reaching 92% after 24  h. Qian et  al. (2012a) screened a traditional fermented food to obtain a Lactobacillus casei BBE10-212 with high microcystin-reducing ability. The test conditions were as follows: initial toxin concentration of 1000 μg/L, initial pH of 7.0, initial bacteria concentration of 109 cfu/ml, culture temperature of 37 °C, and culture time of 24 h. The addition of 5% glucose increased the algal toxin removal rate from 19% to 52%. El-Nezami et al. (1998) studied the adsorption characteristics of aflatoxins B1 by five probiotics in vitro. The test conditions were as follows: the initial toxin concentration was 5–50 mg/ml, the initial pH was 7.3, and the initial bacterial content was 109–1010 cfu/ ml. The culture temperature was 4~37 °C, and the culture time was 4~72 h. The results showed that two strains of Lactobacillus rhamnosus (LGG and LC-705) had rapid and efficient subtraction ability to aflatoxin B1, and the removal rate was 80%. Fuchs et  al. (2008) used high-performance liquid chromatography (HPLC) to explore the degradation of patulin and ochratoxin by 30 strains of lactic acid bacteria. It was found that the patulin-degrading rate of Bifidobacterium animalis VM12 is more than 80%; furthermore, the degradation rate of ochratoxin by Lactobacillus acidophilus VM20 is above 95%. Subsequently, the cell experiments showed that these strains can effectively alleviate the genotoxic damage caused by mycotoxin to hepatocyte HepG2. Hatab et al. (2012) also detected the adsorption and removal of patulin by a series of lactic acid strains by HPLC. Testing condition are as follows: the initial conditions were 1 mg/L, the initial pH was 4.0, and the initial bacterial concentration was 1010 cfu/ml, the culture temperature was 37 °C, and the culture time was 24 h. The results showed that the toxin-reducing efficiency of the strain was closely related to the initial adsorption conditions and the activity of the strain. Bifidobacterium bifidum 6071 and Lactobacillus rhamnosus 6149 had the highest removal rate of patulin, which were 52.9% and 51.1%, respectively. El-Nezami et al. (2002a) used gas chromatography-mass spectrometry to study the ability of lactic acid bacteria to remove trichothecenes. Testing condition are as follows: the initial conditions were 20 mg/L, the initial pH was 7.3, and the initial bacterial concentration was 1010 cfu/ml, the culture temperature was 37 °C, and the culture time was 1 h. The results showed that the strain had specificity for the ability to remove

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toxins. Lactobacillus rhamnosus LGG could remove four of the seven toxins (removal rate varies from 18% to 93%), while Lactobacillus rhamnosus LC-705 could only remove two kinds (removal rate varies from 10% to 64%). El-Nezami’s research team used high-performance liquid chromatography to further study the removal mechanism of these food-grade lactic acid bacteria to corn ketene toxin. The results showed that more than 55% of the toxins were quickly adsorbed after contact with the strain, and the adsorption capacity and strain concentration were observed. The authors speculate that strains remove zearalenone more by adsorption (rather than physiological metabolism) (El-Nezami et al. 2002b).

7.2.3  In Vitro Antioxidant Model Oxidative stress and oxidative damage are important physiological and pathological processes for the development of metabolic and systemic diseases in the body. They have become the commonly used targets and research models in the development of drugs and functional foods. Numerous studies have shown that lactic acid bacteria may have the potential to alleviate oxidative stress caused by different factors, which have been confirmed by many in vitro and in vivo tests. The antioxidant abilities of lactic acid bacteria and its isolates in vitro, such as reducing activity, hydrogen peroxide tolerance, anti-lipid peroxidation, chelating properties of ferrous and copper ions, DPPH free radicals, and hydroxyl radical scavenging ability, are often used as an evaluation index to evaluate their antioxidant capacity. Next, we will briefly introduce several commonly used models for evaluating the in vitro antioxidant capacity of lactic acid bacteria. 7.2.3.1  DPPH Removal Model DPPH is 1,1-diphenyl-2-trinitrophenylhydrazine, alias 1,1-diphenyl-2-picryl (free radical), etc., which can be used for spectrophotometric determination of tocopherol and aliphatic sulfur. Alcohols, reducing substances, are also commonly used as polymerization inhibitors. Since 1958, the DPPH method has been widely used in the quantitative determination of antioxidant capacity such as food samples and biological samples. There is a single electron in the DPPH radical, which has a strong absorption at 517  nm, and the alcohol solution is a purple solution. After single electron pairing with the radical scavenger, its absorption gradually disappears, and the degree of fading of the solution is quantitatively related to the number of electrons received. Therefore, a rapid quantitative analysis of the scavenger removal ability of the radical scavenger can be realized by a spectrophotometer. A large number of experiments on the evaluation of in vitro antioxidant activity of lactic acid bacteria based on DPPH clearance showed that lactic acid bacteria have a strong ability to scavenge DPPH free radicals. Previous studies have ­evaluated the ability of selected 30 strains of lactic acid bacteria to scavenge DPPH

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free radicals and linoleic acid peroxidation and screened lactic acid bacteria L3 and L4 strains with strong antioxidant activity (Jiang-wei and Yu-sheng 2005; Jiang-wei et al. 2005). Gang et al. (2013) found that the DPPH clearance rate of extracellular secretions of lactic acid bacteria changed widely, with a minimum of 0.031% and a maximum of 98.153%. The clearest cell-free extract of lactic acid bacteria was La5 strain with a clearance rate of 98.153%. Wang Gang et al. (2013) determined the DPPH free radical scavenging of cell-free extracts and intact cell suspensions of different concentrations of lactic acid bacteria and found DPPH free radicals of CCFM1106, CCFM8661, CCFM1566, and ATCC53103 strains when the cell concentration was 1010 cfu/ml. The clearance rate is over 60%. The highest scavenging strain is CCFM1106, which is over 90%. Di et al. (2011) confirmed that cherry stem extracts fermented with Lactobacillus acidophilus have higher DPPH free radical scavenging ability. Zhan Jian-long et al. (2015) found that free radical scavenging rate of SR6 (Lactobacillus LAB26) was higher than 60% when detected by DPPH free radical scavenging experiments. They also studied the effects of fermentation conditions on the antioxidant activity of lactic acid bacteria. It is believed that nitrogen source, carbon source, and growth factor are the main effects of DPPH free radical scavenging rate. For SR6 strain, the degree of influence of the factors from large to small is carbon source>nitrogen source>lactose. 7.2.3.2  Hydroxyl Radical Scavenging Model Hydroxyl radical (·OH) is a free radical with great harm produced by human body in the process of metabolism. It can cause oxidative damage to sugars, amino acids, proteins, etc. in tissues, leading to cell necrosis and even cell mutation. The Fenton reaction is a phenomenon in which a H2O2 solution is mixed with a Fe2+ solution to produce a strong oxidation product. The mechanism is to generate a strong oxidizing OH in the reaction system. The reaction equation is H2O2 + Fe2 + →·OH + H2O· +Fe3+. At present, the Fenton reaction system has become a commonly used and effective method to evaluate the antioxidant capacity of substances. In addition to the Fenton reaction, there are many other methods that can be used to detect hydroxyl radicals: non-Fenton-type reactions of H2O2 under the action of catalysts, self-oxidation and reduction reactions of some thiol-containing organic compounds, and ultraviolet radiation of H2O2. Zhang Jiang-wei et al. (2005) found that the clearance rates of OH for L3 strains and L4 strains were relatively high, reaching 83.5% and 45.7%, respectively. Hu Xiao-li et al. (2009) established an in vitro model to simulate normal colon and iron-­ stimulated colonic environment and obtained three lactic acid bacteria with hydroxyl radical scavenging ability. The difference among strains was Lactobacillus rhamnosus> Lactobacillus paracasei> Lactobacillus, the highest clearance rate can reach about 60%, and the hydroxyl radical scavenging rate of strains in different environments is different. Wang Gang et al. (2013) used Fenton method and found that, when the cell concentration was 1010 cfu/ml, the clearance rate of ·OH of strains such as CCFM8661, CCFM1566, and ATCC53103 was above 60%.

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7.2.3.3  Anti-lipid Peroxidation Model Oxygen free radical reaction and lipid peroxidation play a very important role in human metabolism. Under normal conditions of coordination and dynamic balance, the two are responsible for maintaining many physiological, biochemical, and immune responses in the body. The lipid peroxidation products include malondialdehyde (MDA), 4-hydroxynonenal (HNE), and serum 8-isoprostane. The determination of MDA content can reflect the extent of lipid peroxidation damage to a certain extent and is recognized as a good indicator of lipid peroxidation. Lin and Yen (1999a) found that the inhibitory effects of cell extracts extracted from Lactobacillus acidophilus and Bifidobacterium longum on linoleic acid peroxidation, osteoblast membrane lipid peroxidation, and fluorescent tissue pigment accumulation, respectively, vary from 33% to 46%, 22% to 37%, and 20% to 39%. At the same time, the cell-free extracts of the two strains have a certain ability to scavenge the membrane lipid peroxidation product MDA. Lee et al. (2010) showed that the complete cell suspension (IC) and cell-free extract (CFE) of L. casei KCTC 3260 inhibited the linoleic acid peroxidation ability, and the inhibition rate reached 46.2% and 72.9%. In the experiment of studying the resistance to linoleic acid peroxidation, Zhang Jiang-wei et al. (2005) found that 27 strains of lactic acid bacteria inhibited the linoleic acid peroxidation reaction, the inhibition rate was at least 4.9%, and the highest was 75.2%. Liu Yang et al. (2012) found that the fermentation supernatants and intracellular extracts of Lactobacillus fermentum, Lactobacillus acidophilus, Lactococcus lactis, and Lactobacillus helveticus all showed certain anti-lipid peroxidation ability; among them, the lactobacillus fermentation supernatant and intracellular extract have the highest anti-lipid peroxidation ability. 7.2.3.4  Reduction Activity Determination Model There is a certain relationship between the reducing activity of organic compounds and their oxidation resistance. The reduction ability of the substance can be evaluated by the amount of Prussian blue (Fe4[Fe(CN)6]3) produced, and the magnitude of the reducing ability of the substance to be detected is evaluated by the absorbance at a wavelength of 700 nm. The larger the number of the A700nm, the stronger the reducing ability of the substance will be detected. Studies have shown that both the lactic acid bacteria L3 strain and the L4 strain detected reductive substances in acellular extracts (Jiang-wei et  al. 2005). The experimental result of Xi et al. (2010) showed that the all of the selected 35 LAB strains had reducing ability (A700nm = 0.001~3.169). Moreover, the extracellular secretion had a better reducing capacity. The strain La29 had the strongest reducing ability (A700nm = 3.168), while the La12 strain had the lowest reducing ability (A700nm = 0.029). The fermentation supernatants of the four lactic acid bacteria studied by Liu Yang et  al. (2012) showed certain reducing ability, among which Lactobacillus helveticus had the strongest reducing ability, while the intracellular extracts of all strains did not detect any reducing ability.

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7.2.3.5  Ferrous Ion Chelation Model Many researches have shown that translation metals including copper and iron can lead to the peroxidation of polyunsaturated fatty acids, which may cause further damage to biological membranes and finally result in severe harm in genetic level. Therefore, metal ion chelation ability is also an important indicator for the evaluation of antioxidant capacity of probiotics. Studies showed that lactic acid bacteria extracts have strong metal ion chelating ability. A series of previous studies on the antioxidant effects of lactic acid bacteria on intestinal mucosa found that Lactobacillus fermentum ME-3 increases antioxidant level by chelating metal ions and reducing lipid peroxidation (Lee et al. 2010; Kai et al. 2004). Studies have also shown that the antioxidant effect of Lactobacillus casei KCTC 3260 is achieved by chelation of Fe2+ and Cu2+. The chelating abilities were 10.6  mg/kg and 21.8  mg/kg, respectively. Lin and Yen’s (1999b, c) study showed that cell-free extract of 1010 live lactic acid bacteria can chelate Fe2+ with an amount of 2.5  ×  10–6  ~  72.7  ×  10–6. Hu Xiao-li et  al. (2009) established an in vitro model that mimics the normal colon and iron-stimulated colonic environment. The ability of the three lactic acid bacteria to scavenge hydroxyl radicals in a high-iron environment is consistent with their ability to inhibit proliferation of enterococci and chelate ferrous ions. This indicates that in a high-iron environment, the antibiotic ability of lactic acid bacteria was profoundly influenced by their ferrous ion chelating ability and inhibition for the proliferation of enterococci. 7.2.3.6  Hydrogen Peroxide Tolerance Model Chemical formula of hydrogen peroxide is H2O2 with a strong oxidizing property. Hydrogen peroxide can not only cause direct damage to cells or tissues but also indirectly participate in the oxidation process as a precursor of hydroxyl radicals (·OH). H2O2 stress is a widely used type of oxidative stress model. In general, lactic acid bacteria are less tolerant to H2O2, but some lactic acid bacteria strains are resistant to H2O2 and have potential antioxidant properties. Kullisaar et al. (2002) found that Lactobacillus fermentum E-3 and E-8 strains with antioxidant activities can survive 180 and 150  min when exposed to H2O2, while the survival time of Lactobacillus fermentum E-338-1-1 without antioxidant activity was 90  min. Lactobacillus have a variety of redox regulatory systems which are mainly composed of glutathione system, thioredoxin system, and “NADH oxidase/NADH peroxidase” system. Researchers investigated Lactobacillus plantarum and Lactobacillus brevis strains isolated from tomato and found that fermentation with Lactobacillus plantarum POM1 and POM35 can significantly improve ascorbic acid (ASC) and glutathione (GSH) content in tomato juice and also increase total antioxidant capacity (TTA) levels (Cagno et  al. 2009). Similarly, Kullisaar et  al. (2002) also concluded that most lactic acid bacteria could scavenge ·OH and H2O2 by producing superoxide dismutase and GSH.  Wang Gang et  al. (2013) used a three-layer MRS plate containing H2O2 to screen H2O2-tolerant strains, and 125

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strains were identified as lactic acid bacteria through Gram staining and contact enzyme reaction detection.

7.2.4  In Vitro Simulated Gastrointestinal Tolerance Model Probiotics exert their function mainly in the intestine. After oral ingestion, they need to experience various adverse factors to survive in the gastrointestinal tract, such as low pH caused by gastric acid, bile salts, and digestive enzymes. Tolerance of probiotics to the gastrointestinal tract is an important indicator for screening and evaluating beneficial bacteria. At present, commonly used digestive tract models include artificial gastric fluid (mainly hydrochloric acid and buffer) model and artificial intestinal fluid (mainly bile salts, digestive enzymes, and buffer) model. Besides, many complex factors in the complex upper gastrointestinal should also be considered, such as the buffering effect of food on gastric acid, gastrointestinal motility, the pH of the gastric juice as food intake changes, and the effects of digestive enzymes. The following part lists some frequently used models adopted to evaluate the tolerance of lactic acid bacteria to the gastrointestinal tract. 7.2.4.1  Mimical Gastric Fluid Model Many reports have indicated that lactic acid bacteria are resistant to low pH, while this ability varies among different lactic acid bacteria. According to Smith’s report (Smith 2010), the rabbits were fed with a bacterial consortium by drinking water with a concentration of 108 cfu/ml. After 24 h, the animals were slaughtered, and the pH of the stomach contents was 1.2. Only Lactobacillus survived, which indicates that Lactobacillus is more resistant to low pH environments than non-Lactobacillus and survives in the digestive tract of animals. Conway et al. (1987) found that most of Lactobacillus acidophilus survived after an incubation in pH 1.0 buffer for 0.5 h, but when duration time continued to 1 h, no live bacteria could be detectable. There were little effects on the survival of this strain when exposed to pH 3.0 and pH 5.0 buffers. Ruixia et al. (1996) studied the effects of bile salt and low pH environment on the activity of lactic acid bacteria. It was found that lactic acid bacteria had certain tolerance to low pH environment, but the number of live bacteria decreased with the decrease of pH. The results of Chen Lin et al. (2010) showed that several strains were well-tolerating in simulated gastric fluid (pH = 3) environment and the logarithmic decrease of live bacteria in 2–4 h was smaller than that of 0–2 h. Hao Qinghong et al. (2011) reported that the viable count of JM-11 strain increased by 12.16% after incubation for 2.5  h in pH  2.0 environment and the viable count increased by 32.97% after 3.5 h of culture. At pH 3.0, JM-11 was able to multiply, and its viable count increased by 7.39%, 7.95%, and 16.48% after an incubation of 1.5 h, 2.5 h, and 3.5 h, respectively.

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7.2.4.2  Mimical Intestinal Fluid Model Many lactic acid bacteria have been reported to be tolerant to bile salts. Chen Lin et al. (2010) found that many lactic acid bacteria strains could tolerate high concentrations of bile salts (0.3%) after a cultivation in a simulated intestinal fluid for 6 h. The viable count of most strains decreased with the prolongation of culture time. Hao Qinghong et al. (2011) studied the tolerance characteristics of chicken probiotic JM-11 and found that the survival rate of JM-11 strain reached 70.24% in 0.1% bile salt environment. As the concentration of bile salt increased, the survival rate of JM-11 strain decreased significantly, and 50% or even more bacteria could survive in 0.3% bile salt environment. Kanno et al. (2012) studied the bile salt tolerance of four Lactobacillus plantarum strains and one Leuconostoc mesogenes strain. These strains were found to grow in lactic acid MRS medium containing 3 g/L of bile. Zhang Dan-dan et al. (2014) showed that Lactobacillus helveticus has better tolerance to bile salts, and there are still 106 cfu/ml viable bacteria at 5 g/L bile salt. Meng Xiangchen et al. (2015) studied the tolerance of bile salts of two Lactobacillus plantarum strains. After an incubation in 0.3% bile salts for 8  h, no remarkable variation was observed in the viable counts, which were maintained at 108 cfu/ml. 7.2.4.3  In Vitro Gastrointestinal Biomimetic Simulation System Considering the gastrointestinal environment of the human body, the establishment of an in vitro gastrointestinal biomimetic simulation system can more reliably evaluate the tolerance of the strain to the gastrointestinal environment. Plenty of studies have been conducted to determine the digestive tract tolerance of probiotics using some simple gastrointestinal simulation systems. Hernández-Ledesma et al. (2004, 2007) simulated gastrointestinal digestion as follows: firstly, prepare 1 L buffer containing 500 mg/L peptide and 20 mg/g pepsin, and with pH 2.0; maintain this buffer at 37 °C for 1.5 h using the water bath. Secondly, adjust pH to 7.5, add pancreatin (40 mg/g), and continue the mixture to maintain at 37 °C for 150 min, and finally inactivate at 95 °C for 5 min. In vitro simulated gastrointestinal digestive test used by Zhang Yuanyuan et al. (2013) was as follows: 1.000 g of the sample is accurately weighed, 20 ml of the prepared artificial gastric fluid is added and shaken evenly and cultured at 37  °C for 1  h on a constant temperature shaking and then 80  ml artificial intestinal juice is added, cultured at 37 °C, 120 r/min for 2 h. Pipette 1 ml of the cultured suspension to be diluted in 9 ml of PBS buffer (dilution 10–3), and perform serial dilution according to the approximate viable cell count of the sample. Finally, the viable count was determined by the plate count method. On this basis, many researchers have designed an in vitro gastrointestinal biomimetic simulator to directly monitor the digestive process parameters and indicators through a computer to achieve continuous or semicontinuous simulation of the digestion process. Charteris et  al. (2002) studied the digestive tolerance of Lactobacillus and Bifidobacterium and found that Lactobacillus fermentum KLD was better tolerated

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in the simulated upper digestive tract environment, while others had a mortality rate greater than 90%. Mäkeläinen et al. (2009) studied the gastrointestinal tolerance of lactic acid bacteria in cheese by treating the cheese with a simulated upper digestive tract solution and then an artificial colon simulator (semicontinuous Enteromix® human colon simulator). The analysis results of the fermentation broth showed that the total number of Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus showed similar growth in the upper digestive tract and colon and the growth of other microorganisms was significantly influenced, indicating that the lactic acid bacteria can tolerate the gastrointestinal environment and thus successfully adhere to epithelial cells and colonize the intestine to exert a beneficial effect. Arroyo-LóPez et al. (2014) studied the in vitro dynamic gastrointestinal model and found that Lactobacillus rhamnosus LGG, Lactobacillus pentosus TOMC-LAB2, and Lactobacillus pentosus TOMC-LAB4 have strong gastrointestinal tolerance when compared to E. coli.

7.3  Cell and Ex Vivo Tissue Model Compared with animal and human experiments, cell and ex vivo tissue models are high efficiency, less consumable, and suitable to mimic host physiology. Therefore, they have become one of the most important and widely used models for studying lactic acid bacteria. Cell and ex vivo tissue models can be used as pre- or supplementary experiments for in vivo studies and with good stability and reproducibility. Besides, these models can be also used in studying in-depth mechanism.

7.3.1  Cytotoxic Model Cytotoxicity refers to the adverse effects of a physiological or functional disorder caused by a drug or biologically active substance. These harmful effects act on a cell, causing changes in its basic structure or physiological processes. And the following methods are mainly employed to study cytotoxic characteristics of lactic acid bacteria. 7.3.1.1  MTT Model 3-(4,5-Dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method is generally used to detect cell survival and growth, originally proposed by Mosmann, and later widely used in cytotoxicity evaluation. Succinate dehydrogenase in living cell mitochondria reduces yellow exogenous MTT to insoluble blue-­ purple crystals (formazan) and deposits them in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) dissolves blue-violet crystals in cells, and the number

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of viable cells can be detected by establishing a link between the amount of crystallization produced and the number of viable cells. The absorbance at a wavelength of 490  nm was obtained by enzyme-linked immunosorbent assay, which indirectly reflects the number of viable cells. The test process is simple, the degree of automation is high, the instrument is common and the structure is simple, and the relative quantity and relative vitality of the cells can be determined. Based on these principles, MTT colorimetry is generally adopted to study the effect of lactic acid bacteria on cell viability. Thirabunyanon et  al. (2009) obtained 54 lactic acid bacteria strains from fermented milk. In the MTT experiment, colon cancer cells were co-cultured with lactic acid bacteria and measured after 24 h of incubation. The results showed that Lactobacillus fermentum RM28 significantly inhibited the proliferation of colon cancer cells, indicating that this strain can be used as a potential functional food probiotic or products to relieve colon cancer. Chen et al. (2015) studied the biotransformation of aflatoxins in peanut meal by solid-state leavening agents, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. The aflatoxin and its derivatives in the untreated peanut meal and fermented peanut meal were extracted with a methanol-water mixed solution, and their cytotoxicity was analyzed by MTT assay in L929 cell line. The results showed that untreated peanut meal exhibited strong cell cytotoxicity while treated peanut meal had no obvious cytotoxicity, indicating the derivatives of aflatoxin formed in fermentation had low toxicity. All of these evidences suggest the feasibility of mycotoxin biotransformation by lactic acid bacteria. Zhang et al. (2015) studied the anticancer activity of malt extract fermented by Lactobacillus plantarum dy-1 and verified the effects of unfermented malt extract and fermented malt extract by Lactobacillus plantarum on cell proliferation. The results showed that unfermented malt extract could not inhibit the proliferation of HT-29 cell line, while fermented malt extract significantly inhibited the proliferation of HT-29 cell line, indicating that Lactobacillus plantarum fermented malt extract has antitumor effect. Tsai et  al. (2015) used MTT assay to determine the inhibition effect of the antibacterial peptide m2163 and m2386 produced by Lactobacillus casei ATCC 334 on the colon cancer cell line SW480. The results showed that compared to PBS-treated SW480, the antibacterial peptides m2163 and m2386 could inhibit its proliferation. They also compared the effects of the two antibacterial peptides on both cancer cells (BFTC 905, Caco-2, CHOK1) and normal cells (H184B5F5/M10). The results showed that the antimicrobial peptides m2163 and m2386 could selectively kill cancer cells but were not lethal to normal cells. Similarly, Dilna et al. (2015) studied the toxicity of Lactobacillus plantarum RJF4 on both cancer and normal cells. As a result, different doses of exopolysaccharide in Lactobacillus plantarum showed toxicity on MiaPaCa2 human pancreatic cancer cell lines, while no toxicity was observed in normal fibroblasts, suggesting a specific antiproliferative effect of extracellular polysaccharide of Lactobacillus plantarum on tumor cells.

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7.3.1.2  LDH Model Lactate dehydrogenase (LDH) is an important enzyme that catalyzes the redox reaction between lactic acid and pyruvic acid during glycolysis and gluconeogenesis. The cytoplasm of living cells in almost all tissues has LDH. Under normal circumstances, LDH cannot penetrate the cell membrane, but the destruction of cell membrane integrity caused by apoptosis or necrosis will cause LDH to be released extracellularly. Under the action of LDH, NAD+ is reduced to form NADH, which is then reduced to iodonitrotetrazolium (INT) by hydrogen donor phenazine dimethyl sulfate (PMS), and INT accepts H+ and is reduced to fuchsia formazan. The measurement was carried out by spectrophotometer, and the absorbance value was determined at a wavelength of 490 nm. The value was positively linearly correlated with the lactate dehydrogenase activity, that is, the activity of lactate dehydrogenase can be quantitatively measured by colorimetry. The cytotoxicity of endogenous and exogenous substances can be quantified by detecting the activity of LDH released from the cells into the culture solution. LDH release is considered to be an important indicator of cell membrane integrity and is commonly used in the detection of cytotoxicity. Borruel et al. (2002) studied the effects of probiotics on intestinal inflammation, co-cultured Lactobacillus casei DN-114001 and Lactobacillus bulgaricus LB10 with intestinal lymphocytes, and detected cell viability by LDH release method. The results showed that lactic acid bacteria had a certain regulatory effect on local inflammation. Yang Jingqiu (2009) obtained lactic acid bacteria with high antioxidant activity through experiments and studied its antioxidant capacity. The cell model was established after subculture of CT-26 in intestinal cancer cells. The antioxidant capacity of Lactobacillus acidophilus 874 was analyzed by LDH release method. The results indicated that the LDH in the culture supernatant of the intervention group was remarkably lower than that of the model group, indicating that this strain can protect cells from H2O2 oxidative damage. 7.3.1.3  Mitochondrial Membrane Potential Model Energy produced by respiration of mitochondria is stored in the inner membrane of this organelle in the form of electrochemical potential energy and causes an asymmetric distribution of proton and ions between inner and outer sides of the mitochondria membrane, which eventually forms a mitochondrial membrane potential (ΔΨm, MMP). MMP in the normal range is a prerequisite for ensuring normal physiological function of mitochondria, and its stability is conducive to maintaining cell stability and normal function. The destruction of the normal membrane potential of mitochondria occurs before the appearance of apoptosis in the nucleus and is considered to be one of the earliest events in the process of apoptosis cascade. Therefore, mitochondrial membrane potential is an important parameter that reflects the state of mitochondria in cells. Fluorescent probes commonly used in mitochondrial membrane potential models include JC-1, rhodamine 123 (Rh123), tetramethylrhodamine ethyl ester (TMRM), DioC6, etc. JC-1 is a novel cationic dye and a

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sensitive marker of mitochondrial membrane potential, which is commonly used in mitochondrial membrane potential detection. When the mitochondrial membrane potential is high, JC-1 will concentrate in the matrix to form J-aggregates; when the mitochondrial membrane potential is low, JC-1 cannot concentrate in the matrix, maintaining the monomer state. If the JC-1 monomer is excited by 488 nm/514 nm light, the monomer will emit green fluorescence with an absorption wavelength of 529 nm. If the J-aggregate is excited by 585 nm light, the aggregate will emit red fluorescence with an absorption wavelength of 590 nm. Therefore, changes in mitochondrial membrane potential can be measured indirectly. Rhodamine 123 is a cationic fluorescent dye that stains mitochondria in living cells and enters the mitochondrial matrix. The fluorescence intensity is reduced during the process. When apoptosis occurs, the mitochondrial membrane structure is destroyed, the mitochondrial membrane potential collapses, Rh123 in the membrane is released, and fluorescence is generated, changes in mitochondrial membrane potential and apoptosis can be detected by fluorescent signals. Rh123 is commonly used for the measurement of mitochondrial membrane potential and does not have any toxicity to cells. TMRM is a cationic fluorescent dye that penetrates the cell membrane and can fluoresce at a wavelength of around 580 nm with a 543 nm laser. TMRM has many advantages over other fluorescent probes. It only accumulates in the mitochondria because of changes in membrane potential, the toxicity is relatively small, and the cell organ binding rate is low, and it is suitable for quantitative analysis of mitochondrial membrane potential. Wang et al. (2014) established a model of HT-29 cells to study the induction of apoptosis by lactic acid bacteria. Using Rh123 as a fluorescent probe, the mitochondrial membrane potential of HT-29 cells was obtained by flow cytometry. The results showed that X12, the M5, and K14 strains have the ability to induce apoptosis in HT-29 cells, which is associated with cell wall extracts of the three strains. Ghoneum and Gimzewski (2014) studied the apoptotic effect of a new yoghurt cereal product on human multidrug resistance (MDR) myeloid leukemia cells in vitro and established a model for HL60/AR cells to explore apoptosis of the product, and using TMRM as a fluorescent probe, the mitochondrial membrane potential is detected by flow cytometry. The results indicate that the induction of apoptosis in this product is related to the activation of caspase 3, the decrease of Bcl-2 expression level, and the decrease of mitochondrial membrane potential polarization, so the yoghurt grain product can be used as a potential for MDR leukemia treatment method.

7.3.2  Intestinal Cell Adhesion Models Lactic acid bacteria can adhere to and colonize the intestine and form a biological barrier of the intestinal mucosa, regulate the immune response, repair the damaged gastrointestinal mucosa, and resist the interference of various harmful bacteria. The ability of lactic acid bacteria to adhere to the intestinal tract of the host is one of the

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important indicators for evaluating whether it is a probiotic. Intestinal cell adhesion is rapid, sensitive, and intuitive. At present, cell lines such as Caco-2 and HT-29 are mainly used as models. Tuomola and Salminen (1998) established an intestinal epithelial cell model with Caco-2 cancer cells in the study to study the adhesion characteristics of 12 different lactic acid bacteria. The study suggests that the probiotic effect of lactic acid bacteria is related to its adhesion ability, but it cannot be used as the only criterion for evaluation. Gopal et al. (2001) established a model of intestinal cell adhesion using HT-29, Caco-2, and HT29-MTX cells to study the adhesion characteristics and colonization characteristics of Lactobacillus rhamnosus DR20, Lactobacillus acidophilus HN017, and Bifidobacterium DR10 strains. The results showed that all the three strains had strong adhesion to intestinal cells. Tulini et al. (2013) isolated a bacteriocin-producing lactic acid bacterium FT259 from cheese and established a cell model with Caco-2 cells, which was found to have good intestinal adhesion and inhibit the growth of Listeria monocytogenes. Li-dong et  al. (2014) isolated a dominant strain HUCM 201 from the starter kefir and found that the strain has certain adhesion to Caco-2 cells. Chen Pei (2014) studied the adhesion of 18 strains of lactic acid bacteria to Caco-2 cell line. The results showed that the tested strains had certain adhesion to Caco-2 cell line, which was about 0.2–15.5 bacteria/cell. Among them, six strains of lactic acid bacteria have an adhesion capacity greater than eight bacteria/cell.

7.3.3  Immunomodulatory Model Lactic acid bacteria can regulate the body’s immune response, keep the body at a normal immune level, and play an important role in the body’s various probiotic functions. In recent years, the immunomodulatory function of lactic acid bacteria has been broadly concerned and studied. Lactic acid bacteria influence the secretion of cytokines. Zhu et al. (2011) studied the influence of three lactic acid bacteria strains derived from centenarians on activated macrophages. In their study, mouse macrophage cell line RAW264.7 was used, and then whole bacterial cells, bacterial cell walls, and cell-free extracts were co-cultured with macrophages. Finally, the production and phagocytic activity of NO and the production of IL-6 and TNF-α were tested. The results showed that these three strains could increase the activity of macrophages by increasing phagocytic activity and enhancing the expression of cytokines NO, IL-6, and TNF-α and could regulate immune activity. Ren Dayong (2013) established a Caco-2 cell inflammatory model to study the immunomodulatory effects of probiotic Lactobacillus. Lactic acid bacteria were co-cultured with inflammatory cells, and the results showed that Lactobacillus salivarius and Lactobacillus plantarum could downregulate the expression of cytokines such as IL-1α, IL-8, and NF-κB in Caco-2 cells, suggesting their potentials in resisting pathogenic infection and alleviating inflammatory reaction. Chen Pei (2014) established an inflammatory model of HT-29 cells to verify the regulation role of lactic acid bacteria on the mRNA expression and

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secretion of cytokine. Chen Pei (2014) established an inflammatory model using HT-29 cells, and compared to the control group, the expression levels of TNF-­α, IL-6, and IL-8 mRNA in L. casei CCFM0412 treatment group showed a significant downregulating trend, while the expression levels of IL-4 and IL-10 mRNA in the L. rhamnosus CCFM0528 and L. rhamnosus CCFM0528 treatment group showed a significant upregulating trend, indicating their immunomodulatory effects. Jiang et  al. (2016) studied the effects of Lactobacillus plantarum NDC 75017 on the expression of pro-inflammatory cytokines IL-1 and IL-6 and tumor necrosis factor by Caco-2 cells. The results showed that Lactobacillus plantarum NDC 75017 can regulate the immune processes associated with pro-inflammatory cytokines NF-κB and p38 MAPK and has the potential to act as an immunomodulator, an important type of functional foods. Rizzo et al. (2013) studied the effects of lactic acid bacteria on the immune response of Streptococcus pyogenes-infected human laryngeal epithelial cells Hep-2 and epithelial A549. The growth of S. pyogenes and the production of IL-17 and IL-23 were determined. The results showed that Lactobacillus plantarum inhibited the expression of TLR2/4 and the production of IL-17 and IL-23 and had immunomodulatory effects. Zhai Qixiao (2015) studied the lactic acid bacteria with the potential to reduce cadmium toxicity and found that lactic acid bacteria can reverse the increase of cytokines such as TNF-α, IL-1β, IL-6, and IL-8 in HT-29 cell line induced by cadmium exposure, indicating that probiotics can regulate intestinal immunity.

7.3.4  Oxidative Stress Model Oxidative stress is a series of adaptive responses caused by the imbalance between the prooxidant component and the antioxidant component in the body’s cells. Oxidative stress and its oxidative damage have become a basic pathological metabolic process in the development and progression of diseases in different systems. Cellular oxidative stress models are often used to evaluate the antioxidant capacity of lactic acid bacteria. Lin and Chang (2000) analyzed the antioxidant ability of both intact cells and cell-free extracts of lactic acid bacteria, respectively. The results showed that Lactobacillus acidophilus ATCC4356 and Bifidobacterium longum ATCC15708 strain can significantly reduce the cytotoxicity of 4-nitroquinoline-1-oxide (4NQO) on intestinal cells and inhibit the occurrence of lipid peroxidation in cell membrane differently. Yang Jingqiu (2009) studied the protection of oxidatively damaged CT-26 cell line by lactic acid bacteria and treated the CT-26 cell line with H2O2 to establish an oxidative damage model. The experimental results show that the lactic acid bacteria can increase the antioxidant capacity of cells and reduce the content of malondialdehyde, indicating that lactic acid bacteria may reduce the oxidative damage caused by hydrogen peroxide by scavenging free radicals. Li Sheng-yu et al. (2013)

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treated Caco-2 cell line with hydrogen peroxide for 12–48 h to establish oxidative damage model, and then studied the antioxidant effect of Lactobacillus plantarum C88. The results showed that Lactobacillus plantarum C88 showed strong antioxidant activity by increasing the free radical scavenging ability and antioxidant enzyme activity of Caco-2 cells. Achuthan et al. (2012) studied the antioxidant potential of Indian intestinal probiotics and the ability to increase the host cell’s antioxidant defense enzyme system under oxidative stress conditions. The results showed that Lactobacillus could significantly upregulate the expression of catalase, superoxide dismutase, and glutathione peroxidase in oxidative damage induced by H2O2  in HT-29 cell line, so lactic acid bacteria can be used as a potential intervention for diseases caused by oxidative stress. Lu Wei and Wei Ping (2013) established a model of chicken embryo fibroblasts infected with avian infectious bronchitis virus to analyze the activity of lactic acid bacteria on intracellular superoxide dismutase and glutathione peroxidase and analyzed the changes of malondialdehyde content. The results showed that the six strains of lactic acid bacteria significantly increased the total antioxidant capacity of the cells and decreased the content of malondialdehyde, which can reduce the oxidative damage caused by free radicals. Wu et  al. (2014) established an oxidative stress cell model with Caco-2 cell line to study the antioxidant capacity of lactic acid bacteria. The results showed that lactic acid bacteria can improve the ability of cells to scavenge superoxide anion free radicals and total antioxidant capacity. Zhai Qixiao (2015) studied the reduction of cadmium damage by lactic acid bacteria and found that Lactobacillus plantarum CCFM8610 can significantly reduce the content of reactive oxygen species (ROS) and malondialdehyde (MDA) due to cadmium exposure, which proves that the strain has the effect of relieving oxidative stress caused by cadmium exposure in cells.

7.4  Animal Model Functional evaluation based on animal models is an approach which uses animal model to investigate growth and metabolism and disease and homeostasis. Animal models follow three principles: it should have a similar regulation process with the human body, a similar behavioral appearance, as well as a similar response to drug treatment. Therefore, functional evaluation of probiotics using animal models is one of the most important links for the application of lactic acid bacteria. Even though living animal models used in functional evaluation are economically expensive and of long experimental period, relevant physiological and biochemical indicators measured in these experiments can directly reflect the beneficial effect of lactic acid bacteria on living animals. These studies can provide reference value for the improvement of feeding formula for farmed animals and also guide the prediction for the influence of lactic acid bacteria on the human physiology and pathology.

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7.4.1  Rat Model 7.4.1.1  Variety, Strain, and Biological Characteristics Rat (scientific name, Rattus norvegicus) is a mammalian, rodent, Muridae, Rattus animal. Rat strains are mainly divided into inbred lines and closed groups. Different varieties have different biological characteristics and medical applications. At present, there are more than 100 inbred lines in rats, among which are mainly ACI, F344/N, LEW, SHR, GH, BUF, and other lines. And common closed groups include Wistar, SD, and Long-Evans. Among them, ACI rats have a high incidence of spontaneous tumors such as pituitary tumors, testicular tumors, adrenal tumors, and various congenital renal abnormalities. F344 rats have a high incidence of breast tumors, pituitary tumors, testicular stromal cell tumors, and leukemia and are acceptable to various transplanted tumors. LEW rats can be the host of tumors and can be transplanted into many kinds of tumors; allergic encephalomyelitis, arthritis, and autoimmune complex hemoglobinosis can be induced by experimental allergic adjuvants. SHR rats have a high incidence of spontaneous hypertension and are often used in the screening of antihypertensive drugs. GH rats have hereditary hypertension combined with cardiovascular disease and are often adopted to study the hypertension and related cardiovascular diseases. Wistar rats are one of the most widely used rat breeds in the laboratory because of their docile character, strong reproductive capacity, and low tumor incidence. SD rats grow faster than Wistar rats and have strong disease resistance, which are one of the most commonly used rat strains in toxicology and nutrition research (Xinyou 1989; Lijun and Yunan 2012). 7.4.1.2  Applicable Functional Evaluation Model Rats are one of the most widely used animals in pharmacology, tumor models, nutritional metabolic disease models, hereditary disease models, neuroendocrine disease models, and infectious disease models. Common pharmacological studies include antihypertensive drugs, drugs for treating cardiovascular diseases, and drugs that affect neurotransmitter release. At the same time, in the toxicological study of drugs, rats are also often used to determine the maximum dose, metabolic changes, and accumulation characteristics of the drug. Rats can replicate a variety of tumor models and are among the most commonly used animals in cancer research. Rats are prone to liver cancer, and diethyl nitrosamine and diaminobiphenyl (DAB) are commonly used to replicate rat liver cancer model; rat esophageal cancer model is replicated by methylbenzyl nitrosamine. Because of their sensitivity to nutrient deficiencies, rats are often used to establish a variety of battalion-deficient disease models for the metabolism of vitamins, amino acids, calcium, and phosphorus. In recent years, rats have also been used in studies of alcoholism, atherosclerosis, malnutrition, and the like. In the introduction of rat breeds, it can be seen that

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some strains of rats have spontaneous genetic diseases, such as GH rats and SHR rats have a higher incidence of spontaneous hypertension. Such rats are often used to establish genetic disease models such as epilepsy, obesity, hypertension, and cataracts, making it easier to conduct in-depth studies of these diseases. Rats have a developed pituitary-adrenal system, which has a strong stress response to external stimuli. Experiments have been conducted to establish a rat model of stress gastric ulcer. In addition, endocrine experiments such as the adrenal gland, pituitary, and ovary can be performed by establishing a rat model of excised endocrine glands. It is also possible to establish a rat model of paratyphoid fever and bronchial pneumonia and then conduct an in-depth study on similar infectious diseases. At the same time, rats are often used in infectious disease models such as pasteurellosis, Staphylococcus infection (after hormone treatment), and Candida albicans. Rats are an important experimental animal model for the functional evaluation of lactic acid bacteria. Taking the diabetes model as an example, a rat model of type I diabetes was successfully established by inoculating rats with streptozotocin (STZ), and then the therapeutic effect of lactic acid bacteria on type I diabetes was explored (Yeo 2010) A rat type II diabetes model was established by feeding a high-sucrose-­ fat diet and injecting low-dose streptozotocin and explored the therapeutic benefits of lactic acid bacteria-fermented camel milk on type II diabetes (Manaer et  al. 2015). Specific methods are as follows: Male SPF Wistar rats weighing 160–200 g were fed with a high-sugar and high-fat diet (67% basal diet, 2.5% cholesterol, 0.5% cholate, 10% lard, 15% carbohydrate). After 6 weeks of feeding, the rats in the high-sugar and high-fat diet group were given STZ intraperitoneally at a dose of 30 mg/kg according to the body weight of the rats, while the normal diet group was injected with the same amount of citrate buffer. The model of type II diabetes was successfully established when the fasting blood glucose level of the mice was higher than 11.1  mmol/L within 1  week after injection. Furthermore, the hypoglycemic effect of lactic acid bacteria-fermented camel milk was investigated in groups. It was found that high-density fermentation of camel milk (6.97 × 108 lactic acid bacteria +2.2 × 106 yeast) had significant hypoglycemic effect on type II diabetic rats. He Qiuwen (2012) induced high-fat diet, injected low-dose STZ to obtain a rat model of type II diabetes, and further assessed the effect of Lactobacillus casei Zhang in preventing and treating type II diabetes in rats. The modeling method was similar to the above report, but the STZ dose was changed to 40 mg/kg. The model was successfully established when the fasting blood glucose was higher than 7 mmol/L after 1 week and the blood glucose was higher than 11 mmol/L 2 h after meal. Through perfusion of the probiotics, it was found that the probiotic L. casei Zhang had a good preventive and therapeutic effect on STZ-induced type II diabetes in rats. The glucose content in rats was significantly regulated, and the blood glucose tolerance was significantly improved. Rats are also the most frequently used animal models in hyperlipidemia experimental studies at home and abroad. In the modeling method, the high-fat feed method is widely used because of its low difficulty in operation. Zhang Dong et al. (2007) compared the differences in modeling results between different formulas by changing the formula of high-fat diet. Specific methods are as follows: 60 Wistar

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rats were randomly divided into 6 groups. In addition to the basic diet group, they were divided into five groups, which were intragastrically administered with different high-fat emulsions (formula 1 is lard, formula 2 is 20% lard + 10% cholesterol, formula 3 is 20% lard + 10% cholesterol + 2% No. 3 bile salt, formula 4 is 20% lard + 2% cholesterol + 0.2% propyl thiouracil, and formula 5 is 15% lard + 6% cholesterol + 2% biliary salt + 0.2% propyl thiouracil). These emulsions were administered at a dose of 10  mg/kg every day, and blood was taken through the eyeball every 10 days to measure the contents of total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein in the blood. The results showed that bile salt was combined with propylthiouracil, that is, formula 5 could get the ideal model (10 days) faster, and the stability of each index was higher. Based on the rat high-fat diet model, Banjoko et al. (2012) explored the probiotic effects of a mixture of various lactic acid bacteria (Lactobacillus acidophilus DSM 20242, Bifidobacterium DSM 20082, Lactobacillus helveticus CK 60) in regulating blood lipids. Usman and Hosono (2001) explored the lipid-lowering effect of the high-fat Wistar rat model by mixing lactic acid bacteria powder (Lactobacillus grisea SBT 0270) into high-fat diet. The results showed that total cholesterol and total bile acid were significantly decreased in the serum of rats in the probiotic powder treatment group. Lactic acid bacteria can significantly reduce the reabsorption of cholate and increase the discharge of sterols in feces. For alcoholic liver injury, Du et al. (2003) fed 20% ethanol in Wistar rats daily. After 8 weeks, the structure of rat hepatocyte membrane was destroyed, and liver function was damaged, which behaves consistently with alcoholic liver function damage in human. Thus, a rat model of chronic alcoholic liver injury was successfully established. Liu Keliang et  al. (2015) administered SD rats with a dose of 50% alcohol at 12 ml/kg. Compared with the control group, the liver cells in the model group were found to have severe diffuse steatosis, and more fat droplets were observed in the cells. Thus, a model of acute alcoholic liver injury was successfully created. Duan et al. (2002) gave lactic acid bacteria preparations before feeding Wistar rats with ethanol. Finally, it was found that lactic acid bacteria can effectively prevent severe macrovesicular lipid changes caused by ethanol, ensure the histological structure of the liver is stable, reduce the absorption of ethanol in the stomach, and reduce endotoxin translocation to prevent alcoholic liver damage. Qing and Wang (2008) explored the probiotic effects of lactic acid bacteria by establishing a rat model of steatohepatitis induced by ethanol. Specific methods are as follows: 25 Wistar rats weighing 180–200 g were adopted and split into three groups. The control group (five rats) were fed normally. The ethanol group (10) and the antialcoholic group (10) were fed with ethanol at a dose of 10 g/kg daily. The antialcoholic group was fed with a mixture of probiotics (containing Lactobacillus acidophilus 4 × 1010 cfu/ml and Bifidobacterium longum 2.5 × 107 cfu/ml) at a dose of 1.5 ml/100 g 30 min before feeding ethanol. The other groups were fed with the same dose of normal saline. After continuous feeding for 5 days, the blood, liver, and stomach were taken for sections, and the related enzyme activities (glutamic-­ oxaloacetic aminotransferase, alanine aminotransferase, alkaline phosphatase, etc.) were detected. The results showed that lactic acid bacteria can reduce the

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concentration of ethanol in the blood by accelerating the initial decomposition of ethanol in the stomach and liver. Compared with the ethanol group, lactic acid bacteria can effectively protect the liver tissue structure and significantly reduce the occurrence of alcoholic liver injury. In addition, rats fed with a high-cholesterol diet can exhibit the symptom of high cholesterol, the formation of which is similar to the human body, so it can be used for high cholesterol-related regulation studies. Kim et al. (2010) explored the effect of Lactobacillus-fermented soymilk on cholesterol content in rats by establishing a high-cholesterol rat model. Methods are as follows: 50 male SD rats weighing 240– 260 g were randomly divided into five groups. One group was fed with standard diet as control, and the other four groups were fed with high-fat diet containing 30% shortening oil. In addition, the other four groups were fed with normal saline, inhibitor (positive control), 25% fermented soymilk, and 50% fermented soymilk 200 μl, respectively. Daily intake and weight gain were recorded, and blood samples were collected from the heart for analysis after one night fasting. The results showed that fermented soybean milk could significantly reduce the LDL cholesterol content in the blood. Pan and Zhang (2005) established a high-cholesterol model by feeding rats with high-fat diet and evaluated the cholesterol-lowering and probiotic effects of yoghurt produced by the cholesterol-lowering Lactococcus lactis subspecies LQ-12. Forty-eight male SD rats weighing 180–220 g were divided into four groups on average. They were fed with basic diet, high-fat diet (besides 86.5% basic diet, 12% lard, 1% cholesterol, and 0.5% bovine bile salt were added), high-fat diet + sugary skimmed milk, and high-fat diet + yoghurt (made from the same amount of sugary skimmed milk as the former group). Weekly intake and weight changes were recorded, and the samples were tested at 14 days and 28 days, respectively. By measuring the cholesterol content in the liver and feces, it was found that the cholesterol content in the liver and feces of the yoghurt-feeding group decreased and the cholesterol content in the feces increased, indicating that the Lactobacillus not only promoted the excretion of excess cholesterol in vivo but also inhibited the cholesterol synthesis to some extent.

7.4.2  Mouse Model 7.4.2.1  Variety Strains and Biological Characteristics Mice are widely distributed all over the world. More than 1000 inbred lines and independent outbreeding groups have been formed after long-term artificial feeding and selective breeding. Mouse, as an experimental animal, has been the most frequently used and thoroughly studied mammalian experimental animal in the world since the seventeenth century. The strains of mice are mainly inbred lines, closed populations, and mutant lines. Among them, there are about 250 inbred lines and more than 350 mutant lines.

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About 44% of inbred strain A mice aged 6 months showed lupus erythematosus cells and antinuclear antibodies. It is deficient in complement C5 and is sensitive to X-ray irradiation. Compared with other inbred mice, BALB/c mice had the highest blood pressure and spontaneous hypertension and were susceptible to chronic pneumonia. CBA strain mice contain CBA/Br, CBA/Ca, CBA/J, CBA/St, CBA/H, and other subspecies. They have high blood pressure and are highly sensitive to vitamin K deficiency. AKR is deficient in complement C5, which is easy to induce immune tolerance. It has low reproductive rate in common environment but good reproduction in aseptic and non-specific pathogen (SPF) environment and short growth period. C57BL/6 mice are more sensitive to Mycobacterium tuberculosis and are easy to induce immune tolerance. C3H mice include C3H/Bi, C3H/He, C3H/HeJ, C3H/St, C3HeB/FeJ, C3H/DiSn, C3H/Sf, and other subfamilies. DBA/2 strain mice were sensitive to pertussis histamine susceptibility factor and resistant to plasmodium and typhus. The content of serum immunoglobulin in NZB strain mice was remarkably larger than that in other strain mice. The specific manifestation was that the content of IgM and IgG increased significantly, and the mice were susceptible to autoimmune hemolytic anemia. Several classical models of the mutant line include nude mice (hairless epidermis, T lymphocytes are damaged, and cellular immunity is weak due to thymic dysplasia), dwarf mice (infertile, congenital hypoperfusion of auxin and thyrotropin), diabetic mice (at 3–4 weeks of age, blood glucose increases significantly, abnormal fat masses can be found in subcutaneous tissue of the subaxillary and the groin), etc. The closed colony mice were KM mice, CFW mice, ICR mice, CFW mice, NIH mice, etc. 7.4.2.2  Applicable Functional Evaluation Model Mice have been used in many functional evaluation models because of their diversity and in-depth research. In immunological research, different types of immunodeficiency mice can be obtained by specific means, so as to better study the immune mechanism and other immune-related aspects. At the same time, mice are often used to produce monoclonal antibodies. In tumor models, mice are one of the most widely used animal models. Many strains of mice have higher incidence of spontaneous tumors, which are similar to human tumors in terms of genesis, and are often used for screening antineoplastic drugs and studying the related mechanisms. At the same time, mice are often induced to carcinogenesis or directly transplanted tumor cells for the study of tumor growth, metastasis, and treatment. Mice are often used to establish infectious disease models to study the pathogenesis and treatment of infectious diseases. Common infectious disease models include Salmonella model, Schistosomiasis japonicum model, leptospirosis model, poliomyelitis model, mold model, and so on. Mice are less resistant to many pathogens, so they are often used in the study of various pathogens. Common pathogenic infection models include influenza model, malaria model, encephalitis model, rabies model, and schistosomiasis infection model. Some strains of mice carry spontaneous genetic diseases, so they are often used as ideal laboratory animals to explore the pathogenesis, genetic

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characteristics, and treatment of related diseases. Common genetic disease models include melanosis model, albinism model, familial obesity model, hereditary anemia model, systemic lupus erythematosus model, and diabetes insipidus model (Xinyou 1989; Lijun and Yunan 2012). In the study of lactic acid bacteria, mice are commonly used animal models to evaluate immune regulation. Li Li et al. (2011) established an animal model of dust mite-sensitized and dust mite-stimulated female C57BL/6 mice and investigated the effect of oral Lactobacillus on the immunoregulatory function of spleen cells in mice with dust mite-induced allergic airway inflammation. The results showed that oral Lactobacillus could induce the proliferation of CD4+ regulatory T lymphocyte subsets in spleen cells of mice and reduce the content of Th1/Th2 cytokines by releasing IL-10, thus inhibiting the allergic airway inflammation induced by dust mites. Yuan et al. (2009) explored the relationship between the adhesion efficiency of lactobacillus to Peyer’s Patch and its immunomodulatory effect, the results showed that the adhesion of Lactobacillus increased the phagocytic activity of abdominal macrophages in OVA-sensitized mice and increased the content of fecal sIgA. Li et al. (2014) explored the immunomodulatory effect of Lactobacillus acidophilus NCFM exopolysaccharide on BALB/C mice. Short-term expression of interleukin-1 alpha (IL-1 alpha), chemokine C-C motif 2 (CCL2), tumor necrosis factor alpha (TNF-alpha), and penetrating hormone 3 (PTX3) was detected in the exopolysaccharide stimulation group. It has a certain inhibitory effect on colorectal adenocarcinoma. In addition, the organ coefficient, intestinal emptying index, and phagocyte index of mice stimulated by exopolysaccharide increased significantly. Cross et al. (2002) established antigen-sensitized mice to investigate the induction of T-cell factors Th1 and Th2 by Lactobacillus rhamnosus HN001. Specific steps are as follows: 6-week-old female BALB/c mice were fed with basic diet at 22 °C and 12-h light for 1 week. Thirty mice were randomly divided into two groups. The bacterial group was fed with 109 CFU live bacteria daily, 50 μg ovalbumin (OVA) with alum adjuvant was injected at 14 days and 21 days, and 20 μg OVA prepared by physiological salts were injected at 26 days and 27 days. Samples were taken at 28 days. In terms of antagonizing pathogens, Tsai et  al. (2011) explored the effect of mixed bacterial liquid and its additives on the ability of mice to resist Salmonella by feeding mice with different kinds of mixed lactic acid bacteria and mixed bacteria solution added skim milk. The results showed that different combinations of lactic acid bacteria could affect the anti-Salmonella infection ability of mice. Specific combinations often enhanced the anti-infection ability of mice liver and spleen. Chen et al. (2013) explored the probiotic effect of heat-lethal Lactobacillus on anti-­ Salmonella infection in mice by establishing a mouse model of Salmonella infection. The results showed that oral administration of the selected thermo-lethal Lactobacillus mixture could effectively reduce the risk of infection by Salmonella and infection-related inflammation in mice. The specific modeling steps were as follows: 20 male inbred BALB/c mice weighing 24–28 g were randomly fed with basic diet 1 week before the experiment, and then the mice were randomly divided into four groups. The mice were fed with sterile saline, mixed live bacteria (the

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density of each lactic acid bacteria was not less than 109 cfu/ml), mixed heat-killing bacteria 1 (100 °C 30 min sterilization, the density of each lactic acid bacteria was not less than 109 cfu/ml), and mixed thermo-lethal bacteria 2 (121 °C 15 min sterilization, the density of each lactic acid bacteria was not less than 109 cfu/ml). The dose was 200  μl/day. After 7  days of feeding, the mice were inoculated with 3.0  ×  107  cfu/ml of oral bacteria and 200 mu l of salmonella. The samples were taken from the mice on the third and sixth day after infection. Mouse models can also mimic many intestinal diseases, so as to study the regulation of lactic acid bacteria on intestinal health. Taking inflammatory bowel disease (IBD) as an example, according to different experimental needs, the establishment of inflammatory bowel disease model in mice can be divided into four methods: chemical-induced animal model (acetic acid, sodium glucan sulfate, trinitrobenzene sulfonic acid, etc.), genetic engineering animal model (gene knockout or transgenic), cell transplantation animal model, and spontaneous animal model (Yuefan and Nan 2011). Yao-Qing et al. (2011) established a mouse colitis model induced by DSS.  The inhibition effect of lactic acid bacteria on oxidative damage of colon mucosa was studied. The results showed that the selected lactic acid bacteria was able to inhibit the increase of free radical level in colon contents induced by DSS and increase the antioxidant capacity of colon. Cha et  al. (2014) established an inflammatory bowel disease model in mice induced by trinitrobenzene sulfonic acid (TNBS) and investigated the effect of lactic acid bacteria which inhibited the decomposition of mucopolysaccharide on inflammatory bowel disease. It was found that this kind of lactic acid bacteria could improve colon inflammation by inhibiting intestinal bacteria to degrade mucopolysaccharide and inhibit the expression of inflammatory cytokines. Cha et al. (2014) established an inflammatory bowel disease model in mice by injecting TNBS into the descending colon. The anti-­ inflammatory effects of the four Lactobacillus mixed bacteria preparations were investigated. It was found that the Lactobacillus preparations could effectively protect the colon from TNBS damage. Specific modeling steps are as follows: 6-week-­ old female ICR mice were anesthetized and injected 2% TNBS (formulated by 50% ethanol) 130 μl into the descending colon through the anus with a syringe. After 30 seconds of vertical posture, the mice were put back into the cage. Divided into bacteria group and control group, the bacteria group was fed with different lactic acid bacteria daily, and 2% TNBS was injected into colon 2 days later, and the control group was fed with normal saline, and 2% TNBS or 50% ethanol was injected into colon 2 days later. Three days after colonic injection, correlation analysis was performed (colon pathological section and related gene expression). In regulating intestinal flora, Wu Jing et al. (2013) used antibiotic interference to establish a mouse intestinal flora imbalance model and explored and proved the probiotic effect of Lactobacillus and Bifidobacterium preparation on intestinal flora imbalance mouse model. Yeom et al. (2015) established a mouse model of allergic dermatitis induced by trimethylamine (TMA). By feeding the model mice with Lactobacillus casei LCR35, it was observed that LCR35 could inhibit the development of allergic dermatitis by balancing intestinal flora. Millette et al. (2008) established a model of intestinal colonization against vancomycin enterococci. Through

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screening and obtaining lactic acid bacteria producing Streptococcus lactis peptide and fasciclin, the effect of reducing intestinal anti-vancomycin enterococci was explored. Specific steps are as follows: female CF-1 mice weighing 25–30 g were fed under SPF condition before modeling. The mice were subcutaneously injected with clindamycin at a dose of 1.4 mg per day for 5 days. Three days after the injection, an enterococcal colonization model of vancomycin-resistant Enterococcus was established by inoculating Enterococcus solution overnight cultured in brain heart infusion (BHI) broth at a dose of 250  μl (containing about 108  cfu live bacteria) through the stomach. Oral lactic acid bacteria test showed that oral lactic acid bacteria can effectively reduce the colonization of vancomycin-resistant Enterococcus in the intestine.

7.4.3  Piglet/Miniature Pig Model 7.4.3.1  Variety Strains and Biological Characteristics Currently, the breeds of pigs for biomedical research generally include common pig, Homer pig, Nebraska pig, Khan Foster pig, Hommel pig, Pitman-Moore pig, and Sinclair pig (because of the high cholesterol in the blood, scars in the arteries, a typical atheromatous lesion in the artery), Von Wenli Bligh pig (with congenital hemophilia for hemophilia study), Uktan pig (belonging to Mexico hairless pig, can be used to study diabetes), German Gottingen pigs, and Japan OMINI mini pig (cultured from small-scale black pigs in the northeast of China). The digestive system, cardiovascular system, nutritional needs, bone development, skin, and mineral metabolism of pigs are very similar to those of human beings, so they are often chosen as animal models for functional evaluation. The miniature pigs and piglets are more popular among researchers because of their low cost, short feeding cycle, and convenient feeding. The average life-span of miniature pigs is 16 years and the longest is 27 years. The weight of adult miniature pigs is about 30 kg (6-month-old), and that of miniature pigs is about 15 kg. Saliva contains more amylase. The stomach is a single chamber and can secrete a variety of digestive enzymes. Gallbladder bile concentration capacity is low. Cecum contains a large number of microorganisms and plays an important role in digestion. The piglet stage is a critical period for the growth and development of pigs and the highest requirement for feed and feeding management. The health of piglets is very important for the growth of pigs. At the same time, piglets have the characteristics of rapid growth and short experimental cycle, and in the key period of the establishment of intestinal flora and immune system, it is conducive to study the effects of lactic acid bacteria on the establishment of intestinal flora and immune system. In addition, the intestinal system and immune system of piglets are similar to that of human beings. The model of piglets can simulate the effect of Lactobacillus on infant health to some extent. This model can be used to study the probiotic effect of lactic acid bacteria on piglets, such as the growth performance model, the intestinal

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steady-state model, and so on, which can directly study the effects of lactic acid bacteria on the health of piglets and provide the basis for developing probiotic feed to promote healthy growth of pigs. 7.4.3.2  Applicable Functional Evaluation Model The functional evaluation of miniature pigs and piglets mainly includes cardiovascular disease, immune function, nutritional and metabolic diseases, genetic diseases, burns, cancer, and so on. It has the following characteristics: (1) The porcine coronary circulation is similar to humans in anatomy and hemodynamics. Eating high-cholesterol foods is as common as humans with typical lesions of atherosclerosis. The atherosclerosis of the arteries and coronary arteries and blood vessels of old pigs is similar to that of human, so pigs can be selected as the first choice for atherosclerosis research. (2) Since piglets can only get maternal antibodies through colostrum, there are very few γ-globulin and other immunoglobulins in the piglets during the first few weeks of birth, and the immune response of the serum is very low. There is no antibody in the body of aseptic pigs, and it can produce a good immune response by contacting certain antigens. Therefore, these characteristics can be used for immunological research. (3) The respiratory, urinary, and blood systems of piglets and young pigs are similar to those of newborns and can be used to study pediatric nutrition. (4) The tooth structure of miniature pig is similar to that of human beings. Caries and cariogenic food can produce the same caries as human beings. It is a good animal model for dental caries research. (5) Miniature pigs can also be used to study congenital diseases such as red eye disease and small eye disease, congenital lymphedema, and porphyria. (6) The skin structure of pig is similar to human, which can simulate the mechanism of humoral and metabolic changes of burn skin. Specially made pig skin can be used as a cover for human burns, which can shorten the healing time and reduce pain and infection. (7) American Sinclair miniature pig has spontaneous skin melanoma before and after birth. This melanoma has the same disease and transmission pattern as human melanoma. Tumor cell changes and clinical manifestations are very similar to those of human melanoma from benign to malignant. It is an ideal animal model for studying human melanoma (Xinyou 1989; Lijun and Yunan 2012) A large number of literatures reported that Lactobacillus feeding can effectively improve the growth performance of piglets. The inadequate digestive system, poor disease resistance, and unreasonable bacterial flora in the internal environment of newborn piglets often lead to diarrhea, which not only hinders the growth of piglets but also leads to the death of piglets and increases the economic losses in the process of breeding. Establishment of growth performance model mainly studied the effects of lactic acid bacteria on the health of piglets from the aspects of average daily gain and feed conversion rate of piglets. Jin Lingmei (2000) reported that microecological preparation at a dose of 0.2 g/one pig was administered to newborn piglets 1–2  h before lactation for 3 consecutive days, once every 7  days, and to 35 days after weaning. The results showed that microecological preparation could

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significantly improve the breeding rate and growth rate of piglets under the same feeding conditions. Ross et  al. (2010) found that feeding newborn piglets with Lactobacillus and Enterococcus faecium could increase feed conversion rate and daily gain of piglets. Estienne et al. (2005) also found that probiotics could increase average daily gain and feed conversion rate in pigs. Guo Yong et al. (2013) fed seven kinds of compound probiotics including Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus rhamnosus to newborn piglets and measured the body weight of newborn, 21-day-old, weaning age, and 70-day-old piglets. The results showed that the compound probiotics could effectively reduce weaning stress and increase the survival rate and average daily gain of piglets. Zhang Dongyan et al. (2011) used 64 weaned piglets (Duroc pig × Changbai pig × Dabai pig ternary hybrid), the average body weight (16.57 + 0.23) kg, and randomly divided into four groups, with four replicates in each group and four heads per replicate. The control group was fed with basal diet, and the experimental group was fed with basal diet supplemented with 0.25%, 0.50%, and 0.75% of Lactobacillus reuteri, respectively, with a cycle of 30 days. The results showed that the average daily gain of piglets with 0.75% lactobacillus was 20.07% (P