Handbook on Sourdough Biotechnology [2 ed.] 3031230833, 9783031230837

Table of contents :
Contents
1: History and Social Aspects of Sourdough
1.1 Sourdough: The Ferment of Life
1.2 History and Social Aspects of Sourdough in France
1.3 History and Social Aspects of Sourdough in Italy
1.4 History and Social Aspects of Sourdough in Germany
1.5 Global History
1.6 Recent Developments
References
2: Sourdough in a Regulatory Context
2.1 Sourdough as a Subject of Food Law
2.2 National Regulations
2.2.1 Austria
2.2.2 Germany
2.2.3 France
2.2.4 Spain
2.2.5 Czech Republic
2.2.6 Netherlands
2.2.7 Codes of Practice
2.3 Protected Geographical Indication (PGI) and Traditional Specialty Guaranteed (TSG)
References
3: Chemistry of Cereal Grains
3.1 Introductory Remarks
3.2 Grain Morphology and Chemical Composition
3.3 Carbohydrates
3.3.1 Starch
3.3.1.1 Changes in Starch Structure During Processing
3.3.1.2 Digestion of Starch as Affected by Structural Features
3.3.2 Nonstarch Polysaccharides (NSP)
3.3.2.1 Arabinoxylans
3.3.2.2 Other Non-starch Polysaccharides
3.4 Proteins
3.4.1 Storage Proteins
3.4.1.1 Wheat, Rye, Barley, and Oats Storage Proteins
3.4.1.2 Wheat Gluten
3.4.2 Storage Proteins of Maize, Millet, Sorghum, and Rice
3.4.3 Metabolic Proteins: Enzymes and Enzyme Inhibitors
3.5 Lipids
3.5.1 Lipid Composition
3.5.2 Effects of Lipids on the Baking Performance of Wheat Flour
3.6 Minor Constituents
3.6.1 Minerals
3.6.2 Vitamins
3.6.3 Bioactive Compounds or Phytochemicals
3.6.4 Antinutrients
Appendix
References
4: Technology of Sourdough Fermentation and Sourdough Application
4.1 Fermentation Schemes
4.1.1 Rye Processing
4.1.1.1 Industrial Nordic Rye Sourdoughs
4.1.1.2 Industrial Rye Sourdough Types
High Dry Matter Type 1 Sourdough (Firm Sourdough)
Low Dry Matter Industrial Sourdoughs (Liquid, Pumpable Sourdough)
Rye Sourdough Bread Making
4.1.2 Wheat Processing
4.2 Mother Dough
4.2.1 Development of Mother Dough by Spontaneous Fermentation
4.2.2 Cereal Based Sourdough Starters Obtained by Back-slopping
4.2.3 Dried Sourdoughs with Active Cultures
4.2.4 Freeze-Dried Cultures
4.3 Dried Sourdoughs
4.3.1 Ready-to-Use Industrial Sourdough
4.3.2 Spray-Dried and Drum-Dried Ready-to-Use Sourdoughs
4.4 The Future of Industrial Bread
References
5: Steamed Bread
5.1 Introduction
5.2 Types of Steamed Bread
5.2.1 Northern-Style Steamed Bread
5.2.2 Southern-Style Steamed Bread
5.2.3 Cantonese-Style Steamed Bread
5.2.4 Differences in Ingredients of the Three Types of Steamed Bread
5.2.5 Differences in Physical Properties
5.3 Process for Steamed Bread Preparation
5.3.1 Sourdough Procedure
5.3.2 Dough Preparation Procedures
5.3.3 Microbiology of Traditional Chinese Sourdoughs
5.3.4 Control of Proofing Conditions
5.3.5 Steaming
5.3.6 Cooling and Packing
5.3.7 Quick-Frozen Steamed Bread
5.4 Effect of Sourdough on Steamed Bread Quality
5.4.1 Texture
5.4.2 Volatile Compounds
5.4.3 Shelf Life
References
6: Taxonomy and Species Diversity of Sourdough Lactic Acid Bacteria
6.1 Taxonomy of Sourdough Lactic Acid Bacteria
6.1.1 Phylogenetic Position of Lactic Acid Bacteria
6.1.2 Classification of Lactobacillaceae
6.1.3 Occurrence and Identification of Lactobacillaceae in Sourdoughs
6.1.4 Lactobacillaceae Species First Isolated from Sourdough
6.1.5 Pediococcus, Leuconostoc, and Weissella as Subdominant Lactic Acid Bacteria Sourdough Species
6.2 Isolation of Sourdough Lactic Acid Bacteria
6.3 Identification of Sourdough Lactic Acid Bacteria
6.3.1 Culture-Dependent Approaches
6.3.1.1 Phenotypic Identification
6.3.1.2 Chemotaxonomic Identification
6.3.1.3 DNA Fingerprint-Based Molecular Identification
6.3.1.4 DNA Sequence-Based Molecular Identification
6.3.2 Culture-Independent Approaches
6.3.2.1 Oligonucleotide Probe/Primer-Based Identification
6.3.2.2 Whole-Community DNA Fingerprinting Identification
6.3.2.3 Metagenetic (PCR Amplicon)-Based Identification
6.3.2.4 Microarray-Based Metatranscriptomics
6.3.2.5 Sequencing-Based Metatranscriptomics
6.3.2.6 Metagenomics
6.3.2.7 Whole-Genome Sequencing
6.4 Meta-Analysis of the Species Diversity of Sourdough Lactic Acid Bacteria
6.5 Factors Influencing the Lactic Acid Bacterial Species Diversity of Sourdoughs
6.5.1 Influence of Geography
6.5.1.1 The Origin of Sourdough and the Sourdough-Specific Lactic Acid Bacterial Species Fructilactobacillus sanfranciscensis
6.5.1.2 The Origin of Sourdough Variation
6.5.1.3 Region-Specific Sourdoughs and Their Associated Microbiota
6.5.1.4 Influence of Cereals and Other Raw Materials
6.5.1.5 Influence of Technology
Sourdough Processing
Functional Starter Cultures
Fermentation Temperature
Acidity
Dough Yield
Oxygen Tension
Backslopping Parameters
Microbial Interactions
References
7: Taxonomy, Biodiversity, and Physiology of Sourdough Yeasts
7.1 Introduction
7.2 Yeast Taxonomy
7.2.1 Defining Yeast Taxa
7.2.2 Identifying Yeast Species in Sourdough
7.3 Yeasts Species Diversity
7.3.1 Yeast Species Detected in Sourdoughs
7.3.2 Geographical Distribution of Yeast Species Across Sourdoughs
7.4 Yeast Dispersion Across Environments
7.4.1 Dispersion Vectors
7.4.2 Origin of Sourdough Yeast Species
7.5 Abiotic Factors Influencing Yeast Species Diversity in Sourdoughs
7.5.1 Cereal Species
7.5.2 Bakery Practices
7.5.2.1 Scale of Production
7.5.2.2 Temperature
7.5.2.3 Hydration Rate
7.5.2.4 Time Between Back-Slopping and Re-feeding Practices
7.6 Yeasts Evolution in Sourdough
7.6.1 Intraspecific Diversity in Sourdough and the Selection of a New Starter
7.6.2 Domestication of S. cerevisiae for Bread-Making
7.6.3 Yeast–LAB and Yeast–Yeast Interactions
7.6.3.1 Co-occurrence and Facilitation
7.6.3.2 Metabolism
7.6.4 Yeast/Yeast Interactions
7.7 Physiology and Biochemistry of Sourdough Yeasts
7.7.1 The Yeast Nitrogen Metabolism
7.7.2 The Yeast Carbohydrate Metabolism
7.7.3 Stress Response in Sourdough Yeasts
7.7.3.1 Low Temperature
7.7.3.2 Acidity
7.7.3.3 Osmotic Stress
7.7.3.4 Membrane Lipids as Modulators of Stress Tolerance in Yeasts
7.7.4 Secondary Yeast Metabolites in Sourdough
7.8 Baker’s Yeast in the Bread-Making Industry
7.8.1 Baker’s Yeast Production
7.8.1.1 Raw Materials
7.8.1.2 Fermentation
7.8.2 General Characteristics of Fresh and Dry Baker’s Yeast
7.8.2.1 Fresh Baker’s Yeast
7.8.2.2 Dry Baker’s Yeast
References
8: Physiology and Biochemistry of Sourdough Lactic Acid Bacteria and Their impact on Bread Quality
8.1 Introduction
8.2 General Growth and Stress Parameters
8.3 Metabolism of Carbohydrates
8.3.1 Use of External Acceptors of Electrons
8.3.2 Metabolism of Organic Acids
8.3.3 Preferential and/or Simultaneous Use of Energy Sources
8.3.4 Metabolism of Oligosaccharides
8.4 Proteolysis and Catabolism of Free Amino Acids
8.4.1 Proteolysis
8.4.2 Amino Acid Metabolism
8.4.3 Synthesis of Kokumi-Active Peptides
8.5 Synthesis of Exopolysaccharides
8.5.1 EPS Biosynthesis and HoPS Structure
8.5.2 Ecological Function of HoPS Production and HoPS Formation in Dough
8.6 Antimicrobial Compounds from Sourdough Lactic Acid Bacteria
8.6.1 Antifungal Compounds from Sourdough Lactic Acid Bacteria
8.6.2 Antibacterial Compounds from Sourdough Lactic Acid Bacteria
8.7 Metabolism of Phenolic Compounds and Lipids
8.8 Metabolism of Sourdough Metacommunities
References
9: Sourdough: A Tool for Non-conventional Fermentations and to Recover Side Streams
9.1 Introduction
9.2 By-Products and Surplus from the Cereal Industry
9.2.1 Side-Streams Generation
9.2.2 Re-use and Valorization of Milling By-Products Through Fermentation
9.2.2.1 Wheat Bran
9.2.2.2 Wheat Germ
9.2.2.3 Rye Bran
9.2.2.4 Rice Germ and Bran
9.2.2.5 Milling By-Products from Other Cereals
9.3 Fermentation of the Cereal Industry Waste
9.3.1 Brewers’ Spent Grain
9.3.2 Surplus and Waste Bread
9.4 Fermentation of Legumes and Pseudo-cereals
9.4.1 Legumes
9.4.1.1 Nutritional Properties and Anti-nutritional Factors Issue
9.4.1.2 Sourdough Fermentation and Baked Goods Fortification
9.4.2 Pseudo-cereals
9.4.2.1 Nutritional and Functional Features
9.4.2.2 Potential of the Sourdough Fermentation Applied to Pseudo-cereals
References
10: Nutritional Aspects of Cereal Fermentation with Lactic Acid Bacteria and Yeast
10.1 Introduction
10.2 Effects on Cereal Biopolymers
10.2.1 Starch and Glycemic Index
10.2.2 Protein
10.2.3 Biogenic Peptides
10.2.4 Protein Digestibility
10.2.5 Dietary Fiber
10.2.6 Antinutritional Factors
10.3 Micronutrients
10.3.1 Vitamins
10.3.2 Minerals
10.3.3 Phytochemicals
10.4 Microbial Exopolysaccharides
10.5 Sourdough and Gut Microbiota
10.6 Future Prospects
References
11: Sourdough and Gluten-Free Products
11.1 Introduction: Gluten-Free Cereal Products
11.2 Sourdough Bread
11.3 Ecology of GF Fermentations and Development of GF Sourdough Starters
11.4 Proteolysis as a Tool to Improve the Baking Performances of GF Flours
11.5 Proteolysis for Reducing the Toxicity of Wheat Flour
11.6 Application of Legume-Based Flours for GF Bread
11.7 FODMAPs and GF Products
11.8 Exopolysaccharides: A Low-Cost Alternative to Hydrocolloids in GF Breads
11.9 Starch Hydrolysis for Delaying the Staling of GF Bread
11.10 Sourdough as a Natural Tool for Improving the Shelf Life of GF Bread
11.11 Sourdough Fermentation for Enhancing the Health Benefits of GF Bread
11.12 Germination and Sourdough Fermentation for the Development of GF Products
11.13 Conclusions
References
12: Sourdough and Cereal Beverages
12.1 Introduction
12.2 Boza
12.3 Togwa and Mahewu
12.4 Bushera
12.5 Gowé
12.6 Kwete
12.7 Malwa
12.8 Koko Sour Water
12.9 Pozol
12.10 Chicha
12.11 Kishk
12.12 Kvass
12.13 Sourish Shchi
12.14 Hulu-mur and Abreh
12.14.1 Abreh
12.15 Fermented Cereal Beverages with Live Microbiota
References
13: Perspectives
13.1 Microbial Ecology of Sourdough
13.2 Sourdough and Product Quality
13.3 Sourdough and Nutrition
13.4 Industrial and Artisanal Use of Sourdough
References
Index

Citation preview

Marco Gobbetti Michael Gänzle   Editors

Handbook on Sourdough Biotechnology Second Edition

Handbook on Sourdough Biotechnology

Marco Gobbetti  •  Michael Gänzle Editors

Handbook on Sourdough Biotechnology Second Edition

Editors Marco Gobbetti Faculty of Science and Technology Free University of Bolzano Bolzano, Italy

Michael Gänzle Department of Agricultural Food and Nutritional Science University of Alberta Edmonton, AB, Canada

ISBN 978-3-031-23083-7    ISBN 978-3-031-23084-4 (eBook) https://doi.org/10.1007/978-3-031-23084-4 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, 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 publisher, 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 publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

 1 History  and Social Aspects of Sourdough������������������������������������������������   1 Stefan Cappelle, Lacaze Guylaine, Michael Gänzle, and Marco Gobbetti  2 Sourdough  in a Regulatory Context��������������������������������������������������������  15 Markus J. Brandt  3 Chemistry  of Cereal Grains����������������������������������������������������������������������  25 Cristina M. Rosell and Peter Koehler  4 Technology  of Sourdough Fermentation and Sourdough Application����������������������������������������������������������������������  67 Markus J. Brandt, Jussi Loponen, and Stefan Cappelle  5 Steamed Bread�������������������������������������������������������������������������������������������  81 Bowen Yan and Dan Xu  6 Taxonomy  and Species Diversity of Sourdough Lactic Acid Bacteria����������������������������������������������������������������������������������  97 Luc De Vuyst, Víctor González-Alonso, Yohanes Raditya Wardhana, and Inés Pradal  7 Taxonomy,  Biodiversity, and Physiology of Sourdough Yeasts�������������� 161 Lucas von Gastrow, Andrea Gianotti, Pamela Vernocchi, Diana Isabella Serrazanetti, and Delphine Sicard  8 Physiology  and Biochemistry of Sourdough Lactic Acid Bacteria and Their impact on Bread Quality��������������������� 213 Michael Gänzle and Marco Gobbetti  9 Sourdough:  A Tool for Non-conventional Fermentations and to Recover Side Streams�������������������������������������������������������������������� 257 Erica Pontonio, Michela Verni, Marco Montemurro, and Carlo Giuseppe Rizzello 10 Nutritional  Aspects of Cereal Fermentation with Lactic Acid Bacteria and Yeast�������������������������������������������������������� 303 Kati Katina, Kaisa Poutanen, and Marco Gobbetti v

vi

Contents

11 Sourdough  and Gluten-Free Products ���������������������������������������������������� 325 Elke K. Arendt, Laila N. Shwaiki, and Emanuele Zannini 12 Sourdough  and Cereal Beverages������������������������������������������������������������ 351 Felicitas Pswarayi, Jussi Loponen, and Juhani Sibakov 13 Perspectives������������������������������������������������������������������������������������������������ 373 Michael Gänzle and Marco Gobbetti Index������������������������������������������������������������������������������������������������������������������  381

1

History and Social Aspects of Sourdough Stefan Cappelle, Lacaze Guylaine, Michael Gänzle, and Marco Gobbetti

1.1 Sourdough: The Ferment of Life The history of sourdough and related baked goods follows the entire arc of the development of human civilization, from the beginning of agriculture to the present. Sourdough bread and other sourdough baked goods made from cereals are examples of foods that summarize different types of knowledge, from agricultural practices and technological processes through to cultural heritage. Bread is closely linked to human subsistence in temperate climates and intimately connected to tradition, the practices of civil society, and religion. Christian prayer says, “Give us this day our daily bread” and the Gospels report that Jesus, breaking bread at the Last Supper, gave it to the Apostles to eat, saying, “This is my body given as a sacrifice for you.” Many languages also retain expressions that recall the close bond between life and bread: “to earn his bread” and “remove bread from his mouth” are just some of the most common idioms, not to mention the etymology of words in current use. For example “companio,” a word first used in late Latin or early French, is derived from cum panis, which means someone with whom you share your bread. In turn, “companio” was used in 2020 to name a genus of lactobacilli, Companilactobacillus, which frequently occurs in sourdoughs [1]. The word “lord” S. Cappelle · L. Guylaine Puratos Group, Dilbeek, Belgium e-mail: [email protected]; [email protected] M. Gänzle (*) Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada e-mail: [email protected] M. Gobbetti Faculty of Science and Technology, Free University of Bolzano, Bolzano, Italy e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. Gobbetti, M. Gänzle (eds.), Handbook on Sourdough Biotechnology, https://doi.org/10.1007/978-3-031-23084-4_1

1

2

S. Cappelle et al.

is derived from the Old English vocabulary hlaford, which translates as guardian of the bread [2]. The symbolic assimilation between bread and life is not just a template that has its heritage in the collective unconscious, but it is probably a precipitate of the history of culture and traditions. Throughout development of human civilization, (sourdough) bread was preferred over unleavened cereal products, supporting the hypothesis of a precise symbolism between the idea of elaborate and stylish, and that of sourdough. The transformation of raw cereals by fermentation and leavening was likely necessary to allow human societies to transition from a hunter-gatherer lifestyle to agricultural societies [3] but also produced an artifact in the sense of “made art.” Besides symbolism, sourdough bread has acquired a central social position over time. Bread, and especially sourdough bread, has become central in the diet of peasant societies. This suggests that the rural population empirically perceived sensory and nutritional transformations, which are also implemented through sourdough fermentation. In other words, the eating of bread, and especially of sourdough bread, was often a choice of civilization. Evidence of the production of leavened bread in the Fertile Crescent dates back more than 14,000 years. The knowledge of baking, along with brewing, thus likely pre-date human agriculture [4, 5]. The oldest leavened and acidified bread is over 5000 years old and was excavated in Switzerland [6]. The first documented production and consumption of sourdough bread can be traced back to the second millennium BC [7]. Egyptians employed bread fermentation to produce soft and light breads. Microscopic observations of yeast as well as measurements of the acidity of bread from early Egypt demonstrate that fermentation of bread dough involved yeasts and lactic acid bacteria [8, 9]. Eventually, the environmental contamination of dough was deliberately made by starting the fermentation with material from the previous fermentation process. Egyptians also made use of the foam of beer for bread making. At the same time, Egyptians also selected the best variety of wheat flour, adopted innovative tools for making bread, and used high-temperature ovens. The Jewish people learned the art of baking in Egypt. As the Bible says, the Jews fleeing from Egypt brought with them the dough before it was leavened. In Greece, bread was a food solely for home consumption in wealthy homes. Its preparation was reserved for women. Only in a later period, the literature mentioned the evidence of bakers, perhaps meeting in corporations, which prepared bread for retail sale. The use of sourdough was adopted from Egypt about 800 B.C. [8]. The Greek gastronomy had more than 70 varieties of breads, including sweet and savory, made with grains, and different preparation processes. The Greeks used to make votive offerings with flour, cereal grains, or toasted breads and cakes mixed with oil and wine. For instance, during the rites dedicated to Dionysus, the god of fertility, but also of euphoria and unbridled passion, the priestesses offered large loaves of bread. The step from the use of sacrificial bread to the use of curative bread was quick. Patients, who visited the temples dedicated to Asclepius, left bread, and, leaving the holy place, they received a part of it that had become imbued with the healing power attributed to the god [10, 11]. From antiquity to the most recent, the mystery of leavening was also unveiled from a scientific point of view. In 1857, Louis Pasteur definitively explained

1  History and Social Aspects of Sourdough

3

microbial leavening. The scientific research has also verified an assumption that the Greeks had already advanced: sourdough bread has greater nutritional value. Pliny the Elder wrote that it gave strength to the body. The history and social significance of the use of sourdough is further described for countries such as France, Italy, and Germany where this traditional biotechnology is widely used, and where its use is well documented.

1.2 History and Social Aspects of Sourdough in France The history of sourdough usage in France is linked to socio-cultural and socio-­ economic factors. There is little information about sourdough usage and bakery, in general, in France before the eighteenth century. Sourdough bread seemed to be introduced in Gaul by the Greeks living in Marseille in the fourth century BC. In 200 BC, the Gauls removed water from the bread recipe and replaced it with cervoise, a drink based on fermented cereal comparable to beer. They noticed that the cloudier the cervoise was, the more the dough was leavening. Thus, they started to use the foam of cervoise to leaven the bread dough. The bread obtained was particularly light. During the Middle Ages (400–1400 AC), bread making did not progress a lot and stayed a family activity. In the cities, the profession of baker appeared. The history of bread making in France is mainly linked to Parisian bakery because of the geographic localization of Paris. The regions with the biggest wheat production were near Paris, and Paris had the major importance in term of inhabitants. In that period, the production of bread was exclusively done by using sourdough fermentation, the only method known at that time. Further, using sourdough, thanks to its acidity, permitted baking without salt, an expensive and taxed (Gabelle) raw material, and obtaining breads for Middle Ages eating habits [12]. The seventeenth century marked a turning point in the history of French bakery. Until now, sourdough was used alone to ensure the dough fermentation even if in some French regions wine, vinegar, or rennet were added. Toward 1600 AC, French bakers again discovered the usage of brewer’s yeast for bread making. This yeast came from Picardie and Flanders in winter and from Paris breweries in summer. The breads obtained by this technique were named pain mollet because of the dough texture, which was softer than the bread produced until now (pain brie). Two French queens, Catherine de Medicis (Henri II’s wife) and Marie de Medicis (Henri IV’s wife) contributed to the success and development of these yeast fermented breads. In 1666, the use of brewer’s yeast was authorized for bread making but, after a lot of debate, in 1668, the use of brewers’ yeast was prohibited. Following the request of Louis XIV, the Faculty of Medicine of the Paris University studied the consequences of yeast usage on public health. According to the doctors, the yeast was harmful to human health because of its bitterness, coming from barley and rotting water. Despite this negative conclusion of the Faculty, the Parliament, by the decision of the 21st of March 1670, authorized the usage of brewer’s yeast for bread making in combination with sourdough. Besides the apparition of yeast in bread

4

S. Cappelle et al.

making, in that period, the eaten habits evolved to less acidic foods. Thus, back slopping techniques were adapted in order to reduce bread acidity [12]. The seventeenth century was also the period of the development of French philosophic and encyclopedic mind and, fortunately, bread making did not escape this movement. Two books detail the art of bread making and provide information of bread making techniques and knowledge at that period: “L’Art de la Boulangerie” [13] and “Le Parfait Boulanger” [14]. Already, we are learning that sourdough was obtained with a part of the leavened dough of the day. The volume of this dough piece is progressively increased by flour and water addition (back slopping) to have a sourdough ready to be used to ferment the dough. The originate piece of dough, called levain-chef, must be not too old and not too sour. The weight of the levain-­ chef is doubled or tripled by addition of water and flour leading to the levain de première. After, 6 or 7 h of fermentation, water and flour are added to give the levain de seconde, which is fermented 4 or 5 h. Again, water and flour are added. The dough obtained is called levain tout point and after 1 or 2 h of fermentation is added to the bread dough. This technique called travail sur 3 levains was recommended by Parmentier [14] who imputed the bad quality of Anjou bread to the bread making based on only one sourdough. Bread making based on 3 or 2 sourdoughs was mainly used in that period. In addition, it was understood that outside Paris, bread was mainly produced at home by women. It is interesting to notice that already in 1779, Malouin made the distinction between sourdough and artificial sourdough [13]. Artificial sourdough refers to sourdough obtained from a dough that may contain yeast. This distinction between sourdough and artificial sourdoughs remained in the nineteenth century. Until 1840, the yeast was always used in association with sourdough to initiate fermentation. At this date, an Austrian baker introduced in France a bread making based on yeast fermentation alone. This technique was called poolish. The bread obtained, called pain viennois, had a lot of success but this method stayed limited. In the middle of the nineteenth century, the bread making based on three stage sourdoughs disappeared progressively and was replaced by bread making on two stage sourdoughs. Indeed, the back slopping, necessary to maintain the fermentative activity of sourdoughs, imposed a hard working rhythm to the bakers. In 1872, the opening of the first factory of yeast on grain in France by Fould-Springer facilitated the development of bread making based on yeast to the detriment of sourdough bread making. This yeast was more active, more constant, with a nice flavor and most of all has a longer shelf life than brewer yeast. As a consequence, from 1885, bread making based on poolish fermentation was becoming more wide spread. Sourdough bread was, from that time on, called French bread. In 1910, a bill which prohibited night work and, in 1920, the reduction of working hours, implied modification within fermentation processes. Sourdough bread making regressed more and more in the cities when bread making on three stage sourdoughs totally disappeared even if, in 1914, the first fermentôlevain appeared. After the First World War, the use of yeast was extended from Paris to the province. Until the First World War, however, baker’s yeast was produced on cereal substrates and was invariably associated with lactic acid bacteria and thus a yeast-enriched

1  History and Social Aspects of Sourdough

5

sourdough. Yeast production on molasses since 1922 provided baker’s yeast in higher purity that also kept longer and was more easily distributed [9]. However, homemade loaves were still produced, even though they no longer existed in the cities, in the country until 1930 in the form of the levain chef, kept in stone jugs, and passed on from one family to another. The comeback of war in 1939 reduced the use of homemade sourdough bread again. In 1964, Raymond Calvel [15] wrote that “sourdough bread making does not exist anymore.” Indeed, baker’s yeast was systematically added to promote dough leavening, which permitted obtaining lighter breads. In addition, the use of baker’s yeast permitted to better manage bread quality and to reduce quality variations. Two sourdough bread making methods remained at this period. The first one was the method on two sourdoughs, mainly used West and South of Loire, and the second more commonly used method was on one sourdough with a high level of baker’s yeast. Between 1957 and 1960, the sensory qualities of bread decreased as the consequence of cost reduction. Fermentation time was reduced at its minimum. The sourdough bread was no longer produced. It was only during the 1980s that sourdough bread making gained popularity again thanks to the consumer request for authentic and tasty breads. Since 1990, the availability of starter cultures facilitated the re-introduction of sourdough in bread making processes. Indeed, these starters permit obtaining a levain tout-point with a single step and simplify the bread making process. A regulation issued on 13th September 1993 [16] defined sourdough and sourdough bread. According to article 4, sourdough is “dough made from wheat or rye, or just one of these, with water added and salt (optional), and which undergoes a naturally acidifying fermentation, whose purpose is to ensure that the dough will rise. The sourdough contains acidifying microbiota made up primarily of lactic bacteria and yeasts. Adding baker’s yeast (Saccharomyces cerevisiae) is allowed when the dough reaches its last phase of kneading, to a maximum amount of 0.2% relative to the weight of flour used up to this point.” This definition allowed dehydrating sourdough at the condition that microorganisms remained active (amounts of bacteria and yeast are indicated). Sourdough can also be obtained by addition of starter to flour and water. Article 3 of the same regulation declares that “Breads sold under the category of pain au levain must be made from a starter as defined by Article 4, just have a potential maximum pH of 4.3 and an acetic acid content of at least 900 parts per million.” The syndicat national des fabricants de produits intermédiaires pour boulangerie, patisserie et biscuiterie is working on a new definition of sourdough in order to be closer to the reality of sourdough bread.

1.3 History and Social Aspects of Sourdough in Italy The people in early Italy mainly cultivated barley, millet, emmer, and other grains, which were used for the preparation of non-fermented focacce and polenta. Emmer was not only used for making foods, but also performed as a vehicle of transmission of sacred rituals. At first, the Romans mainly consumed roasted or boiled cereals, seasoned with olive oil and combined with vegetables. After contact with the Greek

6

S. Cappelle et al.

civilization, Romans learned the process of baking and the technique of building bread ovens. Numa Pompilius sanctioned this gastronomic revolution with the introduction of the celebrations dedicated to Fornace, the ancient divinity who was the guardian for proper functioning of the bread oven. The Romans gave a great boost to both the improvement of the techniques of kneading and baking of leavened products, and regulated the manufacture and distribution by bakers (pistores). Cato the Elder described many varieties of bread in the De agri coltura (160 BC), which by then had already spread to Rome: the libum or votive bread; the placenta, a loaf of wheat flour, barley, and honey; the erneum, a kind of pandoro; and the mustaceus, a bread made with grape must. In the first century A.D., Pliny the Elder [17] refers to several alternative methods of dough leavening, including sourdough that was air-dried after 3  days of fermentation, the use of dried grapes as starter culture, and particularly the use of back-slopping of dough as most the common method to achieve dough leavening. Pliny the Elder specifically refers to sourdough in his indication that “it is an acid substance carrying out the fermentation.” According to the Pliny the Elder, it was generally acknowledged that “consumption of fermented bread improves health” [17]. After the triumph of classical baking, there was nothing novel in this field throughout the Middle Ages. Finding bread and flour in these centuries was difficult, due to involution of the agriculture and the rage of famine and epidemics. The bread was divided into two categories: the black bread, made from flours of different cereals, of little value and reserved to the most humble people, and the white bread, made from refined flour, more expensive and shown in the tables of the rich people. Special bread, which tradition has preserved to this day in different national or regional varieties, is the Brezel, originating from South Germany. It had the characteristic shape of knotted and dark red crust, which is generated by application of alkali prior to baking, and is sprinkled with coarse salt crystals. According to the legend, it was invented by a baker in Urach in South West Germany, who, to avoid job loss, was asked by the Duke of Württemberg to produce a bread that allows the sun to shine through three times. This special bread requires 2 days of working: the first to prepare the sourdough with wheat flour, and the second to mix it with water, flour, salt, lard, and malt. During the Renaissance, the practice to organize banquets in the courts of the lords led to the triumph of bread, which in various forms and preparations invariably supported the various dishes. In Venice for the Easter holidays, they used to prepare the “fugassa,” sweet bread made with sugar, eggs, and butter. In Tuscany, they used to prepare the “pane impepato” and in Milan, the “panettone.” Only toward the end of the 1600s was the use of yeast re-introduced for the distribution of luxury bread, which was salty and had added milk. In 1700, a very important innovation in the art of bread making was disseminated: the millstones in mills were replaced with a series of steel rollers. This allowed cheaper refining of flour. Also, pioneering mixers were set up. With the advances brought by the industrial revolution, bread was increasingly emerging as a staple food for workers. Rather than making the bread at home, people preferred to buy it from bakers. This change was criticized as distorting traditional values. At the same time, a health movement that originated in

1  History and Social Aspects of Sourdough

7

America started a battle against leavened bread, stating it was deleterious to health. Baker’s yeast was considered a toxic element, perhaps because it was derived from beer, while the sourdough gave a bad taste to the bread, which was remediated by the addition of potash, equally harmful. When Louis Pasteur discovered that microorganisms caused the fermentation, the concern over the toxicity of biological agents was amplified. Pasteur’s discovery eventually benefitted the supporters of the bread, as they stated that the use of selected yeast and related techniques was helpful in the manufacture of bread with a longer shelf life. The education of taste in different food cultures explains, however, the different relationship that has existed between the perception of the quality of bread and its level of acidity. During the First World War, so-called “military bread” was used in Europe, which was a 700 g loaf with a hard crust. It was initially distributed to soldiers and then also passed on to the civilian population. In the post-war period, thanks to the much-discussed Battle of Wheat, strongly supported by Mussolini, the production of wheat was plentiful and bread was brought to the table of the general population. The Second World War again resulted in an insufficient supply of bread. With the arrival of the American allies, the bread of liberation—a square white bread— became disseminated. Today, bread is regaining some importance. With a turnaround in the culinary habits of Westerners, bread made with unrefined flour, so-called black bread, is more widely consumed. A brief mention should be made, finally, of the various breads that are currently made with modern baking practices. Typical breads, with PDO (Denomination of Protected Origin) or PGI (Protected Geographical Indication) status, are the Altamura bread, the bread of Dittaino, the Coppia Ferrasese, the bread of Genzano, and the Cornetto of Matera. The manufacture of these breads is based on new processes, but still at an artisanal level [18].

1.4 History and Social Aspects of Sourdough in Germany Acidified and leavened bread has been produced in Central Europe (contemporary Austria, Germany, and Switzerland) for more than 5000 years. Leavened and acidified bread dating from 3600 was excavated near Bern, Switzerland [6]; comparable findings of bread or acidified flat bread were made in Austria (dating from 1800 BC) and Quedlinburg, Germany (dating from 800 BC) [19]. It remains unknown whether these breads represent temporary and local traditions or a permanent and widespread production of leavened and acidified bread; however, these archaeological findings indicate that the use of sourdough for production of leavened breads developed independently in Central Europe and the Mediterranean. Paralleling the use of leavening agents in France, sourdough was used as sole leavening agent in Germany until the use of brewer’s yeast became common in the fifteenth and sixteenth century [8, 19]. In many medieval monasteries, brewing and baking were carried out in the same facility to employ the heat of the baking ovens to dry the malt, and to use the spent brewer’s yeast to leaven the dough. The close connection between brewing and baking is also documented in the medieval gilds as

8

S. Cappelle et al.

bakers and brewers were often organized in the same gild. In many cities, bakers also enjoyed the right to brew beer [20]. Baker’s yeast has been produced for use as a leavening agent in baking since 1867 [8, 19, 21]. Baker’s yeast was initially produced with cereal substrates; the shortage of grains in Germany in the First World War forced the use of molasses as a substrate for baker’s yeast production [8]. The shift from cereal substrates to molasses for production of baker’s yeast in the first half of the twentieth century enabled the production of yeast biomass with minimal contamination by lactic acid bacteria. Although artisanal bread production relied on the use of sourdough as the main leavening agent until the twentieth century, the use of baker’s yeast gradually replaced sourdough as the leavening agent. Maurizio indicates in 1917 that baker’s yeast was the predominant leavening agent for white wheat bread, whereas whole grain and rye products continued to be leavened with sourdough [22]. In 1954, Neuman and Pelshenke refer to baker’s yeast as the main or sole leavening agent for wheat bread and as an alternative leavening agent in rye bread [23]. The industrial production of baker’s yeast to achieve leavening in straight dough processes was followed by the commercial production of sourdough starter cultures in Germany since 1910. The continued use of sourdough in Germany throughout the twentieth century relates to the use of rye flour in bread production. Rye flour requires acidification to achieve optimal bread quality. Acidification inhibits amylase activity and prevents starch degradation during baking; in addition, the solubilization of pentosans during sourdough fermentation improves the water binding and gas retention in the dough stage. Following the introduction of baker’s yeast as a leavening agent, the technological aim of sourdough fermentation in rye baking shifted from the use as a leavening agent to its use as an acidifying agent [21]. This use of sourdough for acidification of rye dough in Germany is paralleled in other European countries where rye bread has a major share in the bread market, including Sweden, Finland, the Baltic countries, Poland, and Russia. For example, the industrialization of bread production in the Soviet Union in the 1920s led to the development of fermentation equipment for the large scale and partially automated production of rye sourdough bread, a development that pre-dates the use of large scale and automated sourdough fermentation in other regions by many decades [24]. Chemical acidulants for the purpose of dough acidification became commercially available in the twentieth century as alternatives to sourdough fermentation; however, artisanal as well as industrial bakeries continued to use sourdough fermentation owing to the substantial difference in product quality. To differentiate between chemical and the more labor-intensive and expensive biological acidification, German food law provided a definition of sourdough as dough containing viable and metabolically active lactic acid bacteria, and defines sourdough bread as bread where acidity is exclusively derived from biological acidification. Sourdough is thus one of very few intermediates of food production that is regulated by legislation, and recognized by many consumers [8]. The consumer perception as well as the regulatory protection of the term “sourdough” in Germany and other European countries facilitated the recent renaissance of sourdough use in baking. In

1  History and Social Aspects of Sourdough

9

comparison, the term “sourdough” is not protected in the United States; here, the widespread labeling of chemically acidified bread as “sourdough bread” resulted in a widespread consumer perception of sourdough bread as highly acidic bread, and the use of alternative terminology to label bread produced with biological acidification.

1.5 Global History While this chapter focuses on the history of sourdough in a few European countries, it recognizes that sourdough is a fermentation process that originated in the Fertile Crescent and is used globally in temperate climates that support cultivation of wheat and rye—each of the regions and countries that have used sourdough have a comparable rich history of sourdough use (Fig.  12.1). Of note, the global diversity of sourdough baked goods is produced using comparable fermentation procedures and virtually identical fermentation organisms—fermentation protocols that achieve leavening without baker’s yeast apparently consistently select for a consortium of microbes that includes Kazachstania exigua (formerly Saccharomyces exiguus) or Kazachstania humilis (formerly Candida humilis or Candida milleri) and Fructilactobacillus sanfranciscensis (formerly Lactobacillus sanfranciscensis, L. sanfrancisco, and L. brevis subsp. lindneri) [1, 26]. Some historical aspects of the

Fig. 12.1  Overview on products that are produced worldwide with sourdough fermentation or with related fermentations to obtain cereal beverages or porridges. Countries are color-coded based on the preference for the types of bread or cereal product: Red, preference for wheat bread including sweet baked goods; red hatched, use or preference for rye bread; blue, preference for wheat flat breads; green, preference for wheat steamed breads; yellow, preference for beverages and porridges produced from maize, millet or sorghum; gray, no sourdough use or insufficient information. South and Central America are hatched as indigenous food fermentations use maize for fermentation while wheat bread is currently also consumed. The map was created with mapchart.net based on information in [25]

10

S. Cappelle et al.

use of sourdough for production of steamed bread in China is outlined in Chap. 5; sourdough is also part of the history of other regions of the world including North America. The use of sourdough as a leavening agent was essential whenever alternative means of dough leavening were not available. Examples include the Oregon Trail of 1848, the California gold rush of 1849, and the Klondike gold rush in the Yukon Territories, Canada, in 1898 and, more recently, the COVID-19 pandemic in March and April 2020, which resulted in a temporary shortage of baker’s yeast in many supermarkets and a resurgence of home-baking with sourdough [25]. During the 1849 gold rush, San Francisco was invaded by tens of thousands of men and women in the grip of gold fever. Following the gold rush, sourdough bread remained an element that distinguishes the local tradition until today. Some bakeries in San Francisco claim to use sourdough that has been propagated for more than 150 years. The use of sourdough during the Klondike gold rush in 1898 resulted in the use of “sourdough” to designate inhabitants of Alaska and the Yukon Territories to date. The Yukon definition of sourdough is “someone who has seen the Yukon River freeze and thaw,” i.e., a long-term resident of the area. The commercialization of dried sourdough with high titrable acidity constituted a compromise between economic bread production based on convenient use of baking improvers, and the use of sourdough fermentation for improved bread quality. These products were introduced commercially in the 1970s [21]. Their economic importance rapidly surpassed the importance of sourdough starter cultures. Dried or stabilized sourdoughs produced for acidification provided the conceptual template for the use of sourdough products as baking improvers in the past 30 years.

1.6 Recent Developments Recent developments include a revival of sourdough as the default process for bread production. Sourdough fermentation allows the production of bread with only two ingredients—flour and water—and this simplicity is a winning proposition on markets where “clean label” products are preferred by many customers. A recent systematic survey [27] showed that a relevant number of research articles (280), corresponding to almost the same number of products, dealt with the microbiological, biochemical, and/or technological features of typical/traditional sourdough baked goods, which spread in countries on all continents (Fig. 12.1). This analysis did not account for the publications that describe the fermentation of cereal beverages and porridges, which are preferentially consumed in Africa and South America (Fig. 12.1). The major part (246) characterized salty products (breads and substitutes), the remaining [24] dealt with sweet baked goods or both categories [10]. In Europe, Italy was the leading country with the characterization of more than 30 traditional varieties of salty and sweet sourdough baked goods. Emblematic reports [28, 29] showed the distinguishing compositional and functional features of sourdoughs used for making 19 typical breads and 18 sweet baked goods. Almost the same approach distinguished sourdoughs used for traditional French breads (baguettes), brioches, and rolls (e.g., [30]). Remaining in Europe, studies from

1  History and Social Aspects of Sourdough

11

Germany, Belgium, Scandinavia, and the Baltic area mainly deepened the sourdough rye bread tradition (e.g., [31]). In Asian countries, Iranian Barbari, Chinese steamed, and Indian Bhatura sourdough breads underwent investigation (e.g., [32]). In Africa, Egyptian Balady, Sudanese Kisra, and Ethiopian Injera sourdough breads were some of the most popular (e.g., [33]). The main sourdough products in South America were Mexican Tortillas, while industrial sourdough breads, rolls, crackers, and cookies attracted interest in the United States. Almost all the 280 articles concluded on the uniqueness of the microbial composition and functionality of each sourdough for every baked good. With an increasing trend, some recent research articles [16] dealt with the use of sourdough also for making pasta. The sourdough fermentation affected both sensory and rheology attributes. The proof of the irreplaceable sourdough potential comes from the extraordinary number of flours and agro-food by-products successfully fermented. Apart from the variable processing, the common purpose of these research articles was to exploit the sourdough potential for increasing the technological and nutritional attributes of conventional and non-conventional flours, and to recycle agro-food by-products. In detail, flours from 23 species/varieties of cereals, also using sprouted seeds, 10 pseudo-cereals, 19 varieties of legumes, and 25 miscellaneous vegetables were suitable for sourdough fermentation. While the research activity in the interval 1990–1999 mainly dealt with soft and durum wheat and rye, the most recent research articles enlarged the spectrum of cereal flours and mainly concerned legumes and pseudo-cereals, also used for gluten-free formulations, and other vegetable matrices. Numerous and heterogeneous agro-food by-products were recyclable by sourdough fermentation, almost all milling by-products and a diversity of other miscellaneous agricultural wastes. In practice, the sourdough fermentation was the irreplaceable technique for getting a consistent increase of the bran content in various baked good formulations [34]. At the same time, the sourdough fermentation had the potential to inhibit the lipase activity of the cereal germ, which allowed a prolonged shelf life and its use as a nutrient-rich ingredient in bread making formulas [35]. Sourdough fermentation also reduces the content of anti-nutritive components in wheat and rye, including phytate, the amylase-trypsin inhibitor, and fructans, which are thought to contribute to non-celiac wheat sensitivity and irritable bowel syndrome [3]. In addition, large scale and automated fermentation equipment is currently offered by many of the major suppliers of equipment to the baking industry, making the use of sourdough no longer a question of expensive versus cheap but of fixed cost for fermentation equipment and personnel versus variable cost for ingredients or additives. Sourdough fermentation is thus no longer confined to small scale, artisanal fermentation to achieve dough leavening and/or acidification but mainly carried out in industrial bakeries at a large scale matching the large-scale bread production, and in specialized ingredient companies for production of baking improvers specifically aiming to influence the storage life, as well as the sensory and nutritional quality of bread. In more than one way, contemporary baking is returning to its origin, to use yeasts and lactic acid bacteria to convert wheat, rye, and other cereals to nutritious and palatable products.

12

S. Cappelle et al.

References 1. Zheng J, Wittouck S, Salvetti E, Franz CMAP, Harris HMB, Mattarelli P, O’Toole PW, Pot B, Vandamme P, Walter J, Watanabe K, Wuyts S, Felis GE, Gänzle MG, Lebeer S (2020) A taxonomic note on the genus Lactobacillus: description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J System Evol Microbiol 70:2782–2858. https://doi.org/10.7939/ r3-­egnz-­m294 2. Mc Gee H (1989) Il cibo e la cucina. Scienza e cultura degli alimenti. Muzzio, Padova 3. Gänzle MG (2020) Food fermentations for improved digestibility of plant foods – an essential ex situ digestion step in agricultural societies? Curr Opin Food Sci 32:124–132 4. Arranz-Otaegui A, Gonzalez Carretero L, Ramsey MN, Fuller DQ, Richter T (2018) Archaeobotanical evidence reveals the origins of bread 14,400 years ago in northeastern Jordan. Proc Natl Acad Sci U S A 115:7925–7930 5. Hayden B, Canuel N, Shanse J (2013) What was brewing in the Natufian? An archaeological assessment of brewing technology in the epipaleolithic. J Archaeol Method Theory 20:102–150 6. Währen M (2000) Gesammelte Aufsätze und Studien zur Brot- und Gebäckkunde und – geschichte. In: Eiselen H (ed) . Deutsches Brotmuseum, Ulm 7. Adrrario C (2002) “Ta” Getreide und Brot im alten Ägypten. Deutsches Brotmuseum, Ulm 8. Samuel D (1999) A new look at old bread: ancient Egyptian baking. Archaeol Int 3:28–31. https://doi.org/10.5334/ai.3010 9. Brandt MJ (2005) Geschichte des Sauerteiges. In: Brandt MJ, Gänzle MG (eds) Handbuch Sauerteig, vol 2006, 6th edn. Behr’s Verlag, Hamburg, pp 1–5 10. Moiraghi C (2002) Breve storia del pane. Lions Club Milano Ambrosiano, Milano 11. Guidotti MC (2005) L’alimentazione nell’antico Egitto, in Cibi e sapèori nel Mondo antico. Sillabe, Livorno, pp 18–24 12. Roussel P, Chiron H (2002) Les pains français: évolution, qualité, production. Sciences et Technologie des Métiers de Bouche. Maé-Erti, Vezoul 13. Malouin PJ (1779) L’Art de la boulangerie ou La description de toutes les méthodes de pétrir, pour fabriquer les différentes sortes de pastes et de pains, 2nd edn, Paris 14. Parmentier AA (1778) Le parfait boulanger ou Traité complet sur la fabrication & le commerce du pain. Imprimerie royale, Paris 15. Calvel R (1964) Le pain et la panification. Que sais-je ? Presses Universitaires de France, Paris 16. Décret n°93-1074 du 13 septembre 1993 pris pour l’application de la loi du 1er août 1905 en ce qui concerne certaines catégories de pains 17. Pliny the Elder G (1972) Naturalis Historia, XVIII, 102-104, edition of Le Biniec H; Pline L’Ancien, Historie Naturelle, Livre XVIII, Societé D’Editions le Belles Lettres, Paris 18. Buonassisi V (1981) Storia del pane e del forno. SIDALM, Milano 19. Spicher G, Stephan H (1982) Handbuch Sauerteig, 1st edn. Behr’s Verlag, Hamburg 20. Krauß I (1994) Heute back’ ich, morgen brau’ ich. Eiselen Stiftung Ulm, Ulm 21. Brandt MJ (2007) Sourdough products for convenient use in baking. Food Microbiol 24:161–164 22. Maurizio A (1917) Die Nahrungsmittel aus Getreide. Parey, Berlin 23. Neumann MP, Pelshenke PF (1954) Brotgetreide und Brot, 5th edn. Parey, Berlin 24. Böcker G (2006) Grundsätze von Anlagen für Sauerteig. In: Brandt MJ, Gänzle MG (eds) Handbuch Sauerteig, 6th edn. Behr’s Verlag, Hamburg, pp 329–352 25. See e.g. https://trends.google.com/trends/ with the search terms “sourdough” and “COVID” (accessed in February 2021) 26. Kline L, Sigihara RF (1971) Microorganisms of the San Fransisco sour dough bread process. II. Isolation and characterization of undescribed bacterial species responsible for the souring activity. Appl Microbiol 21:459–465

1  History and Social Aspects of Sourdough

13

27. Arora K, Ameur H, Polo A, Di Cagno R, Rizzello CG, Gobbetti M (2021) Thirty years of knowledge on sourdough fermentation: a systematic review. Trends Food Sci Technol 108:71–83 28. Lattanzi A, Minervini F, Di Cagno R, Diviccaro A, Antonielli L, Cardinali G, Cappelle S, De Angelis M, Gobbetti M (2013) The lactic acid bacteria and yeast microbiota of eighteen sourdoughs used for the manufacture of traditional Italian sweet leavened baked goods. Int J Food Microbiol 163:71–79. https://doi.org/10.1016/j.ijfoodmicro.2013.02.010 29. Minervini F, Di Cagno R, Lattanzi A, De Angelis M, Antonielli L, Cardinali G, Cappelle S, Gobbetti M (2012) Lactic acid bacterium and yeast microbiotas of 19 sourdoughs used for traditional/typical Italian breads: Interactions between ingredients and microbial species diversity. Appl Environ Microbiol 78:1251–1264. https://doi.org/10.1128/AEM.07721-­11 30. Lhomme E, Lattanzi A, Dousset X, Minervini F, De Angelis M, Lacaze G, Onno B, Gobbetti M (2015) Lactic acid bacterium and yeast microbiotas of sixteen French traditional sourdoughs. Int J Food Microbiol 215:161–170. https://doi.org/10.1016/j.ijfoodmicro.2015.09.015 31. Ua-Arak T, Jakob F, Vogel RF (2017) Influence of levan-producing acetic acid bacteria on buckwheat-sourdough breads. Food Microbiol 65:95–104. https://doi.org/10.1016/j. fm.2017.02.002 32. Zhang G, Zhang W, Sadiq FA, Arbab SH, He G (2019) Microbiota succession and metabolite changes during the traditional sourdough fermentation of Chinese steamed bread. CyTA - J Food 17:172–179. https://doi.org/10.1080/19476337.2019.1569166 33. Baye K, Mouquet-Rivier C, Icard-Verni’ere C, Rochette I, Guyot JP (2013) Influence of flour blend composition on fermentation kinetics and phytate hydrolysis of sourdough used to make injera. Food Chem 138:430–436. https://doi.org/10.1016/j.foodchem.2012.10.075 34. Pontonio E, Dingeo C, Di Cagno R, Blandino M, Gobbetti M, Rizzello CG (2020) Brans from hull-less barley, emmer and pigmented wheat varieties: From by-products to bread nutritional improvers using selected lactic acid bacteria and xylanase. Int J Food Microbiol 313:108384. https://doi.org/10.1016/j.ijfoodmicro.2019.108384 35. Rizzello CG, Nionelli L, Coda R, De Angelis M, Gobbetti M (2010) Effect of sourdough fermentation on stabilisation, and chemical and nutritional characteristics of wheat germ. Food Chem 119:1079–1089. https://doi.org/10.1016/j.foodchem.2009.08.016

2

Sourdough in a Regulatory Context Markus J. Brandt

2.1 Sourdough as a Subject of Food Law The legal definition of the term “sourdough” primarily aims to enforce proper labeling and to protect consumers against misleading information. In general, sourdoughs can be defined as the fermentation product that is produced or formed from flour, water, lactic acid bacteria, and/or yeasts. It is used in the production of baked goods with the purpose of leavening and/or acidifying the dough and to create odor or taste. Sourdough fermentation is the oldest method for producing leavened baked goods. During fermentation the chemical composition of the flour is changed to varying degrees by the activity of the microorganisms, depending on the process conditions, time, and type of microorganisms. A descriptive characterization via chemical parameters is therefore difficult. The same is the case for viability of microbes at the end of fermentation, when the substrate is depleted or when the environmental conditions, e.g., pH, are too harsh, resulting in death of fermentation microbes. Sourdoughs can be produced separately in time and space from the bread production, if the sourdoughs are dried, frozen, or in other ways preserved. The use of sourdough varies in different countries, as do the flavor preferences (e.g., low acid in southern Europe, high in the north, use as leaving agent or baking improver). Thus, the appearance of sourdough has many faces. Therefore, it should be distinguished from products with similar technological objectives, such as other biological leavening agents (baker’s yeast) and chemical leavening agents (e.g., baking powder) on one hand, and on the other hand chemical acidifiers as additives (e.g., citric acid, lactic acid, or phosphates).

M. J. Brandt (*) Ernst Böcker GmbH & Co. KG, Minden, Germany e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. Gobbetti, M. Gänzle (eds.), Handbook on Sourdough Biotechnology, https://doi.org/10.1007/978-3-031-23084-4_2

15

16

M. J. Brandt

Sourdough is an intermediate product of bread production, having different meanings: (i) leavening agent for bread production; (ii) souring method for rye bread production; (iii) microbiologically active product used as starter culture; or (iv) the result of microbial fermentation [1]. According to Art. 9 (1) a) of Regulation EU 1169/2011 on the provision of food information to consumers [2] the labeling of the “name of the food” is mandatory for prepacked food. The “name of the food” can be a “legal name”: “legal name” means the name of a food prescribed in the Union provisions applicable to it or, in the absence of such Union provisions, the name provided for in the laws, regulations, and administrative provisions applicable in the Member State in which the food is sold, Art. 2 (2) n); a “customary name,” or, if both do not exist a “descriptive name” (Art 2 (2) o), p)). For the term “Sourdough bread” both “legal” and also “customary” names exist in several countries. “Sourdough bread” is predominantly leavened or exclusively acidified by sourdough. Other claims such as “with sourdough,” with “in-house sourdough,” “natural sourdough” or “leavened by sourdough” are to be treated in a differentiated manner and depend on the individual case. In the ingredient list, sourdough should be labeled as a compound ingredient, for example “rye sourdough (rye flour, water).” A first description of a customary use of sourdough can be found in the Codex Alimentarius Austriacus as early as 1912. The Codex is a collection of descriptions, designations, quality and composition parameters, basic production methods, procedures for examinations as well as assessment criteria of foodstuffs, which summarizes the current consumer expectations, the prevailing opinion of the responsible economy, and the current scientific findings. In Volume II of the first edition of the Codex Alimentarius Austriacus (1912), the following description for sourdough is found in its own Chap. 23 [3]: Sourdough is the name given to the flour set in fermentation by spontaneous infection, which is used in place of yeast to initiate dough fermentation in bakeries. Of the yeast species present in sourdough, Saccharomyces minor Engel is usually predominant. The sourdough itself is not an actual commodity good. As the name suggests, in addition to the multiplication of yeast, acid fermentation occurs in sourdough due to the presence of bacteria, producing acids, mainly lactic acid and acetic acid. The effect of sourdough, if the quantities of both leavening agents used in bakery are taken as a measure of their effect, is about six times less than that of yeast. A good sourdough should not be too acidic, because otherwise the proliferation of acid bacteria in the bread will make itself unpleasantly felt through an excessively sour taste. Under no circumstances should it smell or taste rotten, dull, or moldy. The little care required to maintain a healthy sourdough is disproportionate to the damage the baker suffers from using a spoiled sourdough, which is why complaints in this direction are very rare.

The Codex Alimentarius Austriacus was the model for the later worldwide Codex Alimentarius, today’s standard work of the Food and Agriculture Organization and the World Health Organization for international trade.

2  Sourdough in a Regulatory Context

17

2.2 National Regulations 2.2.1 Austria The Austrian codex was revised several times and is now in its fourth edition. It is prepared by a commission with representatives from governmental departments, food authorities, economic chambers, trade unions, and consumer organizations. Its legal base is in § 76 of the Food Safety and Consumer Protection Act (Lebensmittelsicherheits- und Verbraucherschutzgesetz). The Austrian Codex serves to promulgate designations, definitions, testing methods and assessment principles as well as guidelines for the production and marketing of goods. The current sourdough definition (revised 18.2.2010) can be found in chapter B18 (bakery products) of the Codex (Österreichisches Lebensmittelbuch) [4] under 2.1.1 Bread The doughs used to make bread are usually leavened with sourdough or baker’s yeast. Depending on the type of bread, other ingredients are used, such as table salt, spices and spice extracts, starch (including pregelatinised starch and soluble starch), pregelatinised flours, dough acidifiers, citric acid and tartaric acid as well as their salts, dairy products, sugar (sucrose) and types of sugar (dextrose, maltose, etc.), edible fats and oils as well as protein-rich substances of vegetable origin (e.g. wheat gluten, soy flour). Sourdough is a dough made from milled and peeled products, starter material [Anstellgut] and drinking water, sometimes with the addition of residual bread, mainly by lactic acid fermentation. Sourdough may be preserved by drying or thickening. The starter material [Anstellgut] is obtained by taking a ripe sourdough or by using a starter culture of acid-forming bacteria and/or sourdough yeasts (pure breeding [Reinzucht], pure culture [Reinkultur]) or, rarely, by spontaneous fermentation. If a starter culture of acid-forming bacteria and/or sourdough yeasts (pure breeding, pure culture) is used as the starter material [Anstellgut], the sourdough is referred to as “pure breeding sourdough” [Reinzuchtsauer (teig)]. A sourdough can only be called a “natural sourdough” if it was made with starter material [Anstellgut] obtained by taking from the mature sourdough. If the mature sourdough is originally a pure bred [Reinzuchtsauerteig] sourdough, then ripe dough [Anstellgut] must have been removed several times in succession for the designation “natural sourdough” to be permissible. If “natural sour” is mentioned in the presentation of a bread in addition to the name, only natural sourdough is used. Acidifiers are not added. If, in addition to the designation, the presentation of a bread refers to “sourdough”, it is predominantly acidified by sourdough. Only lactic acid and acetic acid are added as possible acidifiers.

Sourdough is also necessary for: 2.1.9.2 Country and Farmer’s Bread [Land- und Bauernbrot] Country [Landbrot] and farmer’s bread [Bauernbrot] are synonymous terms for breads that were baked in former times with the typical ingredients of the respective rural region. Today, these terms are associated with bread made from typical regional, mostly low extracted flour types, mostly as hearth bread, with a well-developed crust and a strong taste. The dough is mainly acidified with sourdough (over 50%). No additives other than acidifiers and L-ascorbic acid are used to treat wheat flours.

18

M. J. Brandt In accordance with the regional character, in the region produced dough pieces (deep-­ frozen dough pieces or half-baked breads), which are only baked at the point of sale, are not sold under these designations without indicating the origin; the origin of the dough pieces must be indicated in these cases.

“Pure bred sourdough” [Reinzuchtsauerteig] is a sourdough obtained by backslopping, which is also commercially available as starter culture. “Pure culture [Reinkultur] are microbial defined cultures. In Austria, the different methods to initiate sourdough fermentation are distinguished between, with a focus not on the activity of microorganisms, but more on the process and its result as e.g. dried sourdoughs are included.

2.2.2 Germany Similar to the Austrian system, a codex (Deutsches Lebensmittelbuch) for description of customary names of food in use was founded in 1958 and is now regulated in §15 and 16 of the German Food and Feed law book (Lebensmittel- und Futtermittelgesetzbuch). The Deutsches Lebensmittelbuch is a collection of guiding principles describing the production, composition, or other characteristics of foodstuffs that are of importance for the customary usage of the foodstuffs in trade. It is prepared by an equal manned commission from food authorities, science, food economy, and consumer organizations. A first description of sourdough was published in 1993 in the guiding principles for bread and small baked goods (Leitsätze für Brot und Kleingebäck). In 2021, it was revised [5]: 1.1.5 Sourdough Sourdough is a dough whose microorganisms (e.g. lactic acid bacteria, yeasts) from sourdough or sourdough starters are in an active state or can be reactivated. After the addition of cereal products and water, they are capable of continuous acid formation. Parts of a sourdough are used as starter material for new sourdoughs. The life activity of the microorganisms is only terminated by baking or cooking extrusion. The increase in acidity of the sourdough is based exclusively on its natural fermentation. Ingredients that influence the acidity (acidity level) (e.g. organic acids, dough acidifiers) are not used. It is possible to use sourdough bread in the sourdough production process. 2.1.5 Customary Use with Wholegrain If a bread or small baked good is named as wholemeal bread or roll, at least 90 per cent of the grain is wholemeal. If acid is added, at least two thirds of it comes from sourdough. 2.2 Special Characteristics: Ingredients The following minimum quantities shall be used or complied for ingredients which are expressed in the name or presentation of bread and small baked goods: 2.2.3.4 Sourdough The complete amount of added acid comes from sourdough. Reference is made to point 1.1.5 (e.g. sourdough bread or rolls). References to the co-use of sourdough are only customary if more than two thirds of the added acid come from sourdough. 2.3 Special Characteristics: Production Process 2.3.7 Pumpernickel

2  Sourdough in a Regulatory Context

19

Pumpernickel is made from at least 90% rye baking meal and/or wholemeal rye meal with baking times of at least 16 h at low temperatures (usually 100–120 °C). If pumpernickel is made from wholemeal coarse grain, at least two-thirds of the added acidity comes from sourdough. 2.4 Other Special Characteristics 2.4.5 Farmer’s Bread [Bauernbrot] Farmer’s breads are made with the addition of sourdough. They are hearth breads, usually round loaves of bread. A floured, cracked crust is characteristic. They have a rustic appearance along with a strong aroma in the crust and crumb. In farmer’s bread with a rye content of more than 20%, at least two thirds of the added acid comes from sourdough.

The two-thirds rule goes back to several court rulings on consumer deception regarding the use of dough acidifiers in country/farmer’s breads with sourdough in the 1980s.

2.2.3 France Decree n°93–1074 of 13 September 1993 with regard to certain categories of bread [6]: Article 3: Only breads defined in Articles 1 [pain maison] and 2 [pain de tradition francaise] with a maximum hydrogen potential (pH) of 4.3 and an endogenous acetic acid content of the crumb of at least 900 parts per million may be offered for sale or sold under a name bearing the additional wording “sourdough” [au levain]. Article 4: Sourdough is a dough composed of wheat and rye flour, or only one of these two ingredients, potable water, possibly with added salt, and subjected to a natural acidifying fermentation, the function of which is to ensure the rise of the dough. The sourdough contains an acidifying micro-flora consisting essentially of lactic acid bacteria and yeast. However, the addition of baker’s yeast (Saccharomyces cerevisiae) is allowed in the dough intended for the last phase of kneading, at a maximum dose of 0.2% of the weight of flour used. The leavening may be added to the dough at a maximum rate of 0.2% of the weight of the flour used at this stage. The leaven may be dehydrated provided that the dehydrated leaven contains a living flora of bacteria of the order of one billion and one to ten million yeasts per gram. After rehydration and, possibly, the addition of baker’s yeast (Saccharomyces cerevisiae) under the conditions laid down in the previous paragraph, it must be capable of ensuring the correct rising of the dough. The sourdough may be inoculated with micro-organisms authorised by order of the Minister for Agriculture and the Minister for Consumer Affairs, issued after consulting the Food Technology Commission set up by Decree No 89-530 of 28 July 1989 establishing the Food Technology Commission.

2.2.4 Spain In 2019, a sourdough definition was included in the quality standards for bread with Royal Decree 308/2019 in force from 1 July 2019 [7].

20

M. J. Brandt Article 8. Definition of cultured sourdough [masa madre de cultivo]. It is the active dough composed of wheat or other cereal flour, or mixtures thereof, and water, with or without added salt, subjected to a spontaneous acidifying fermentation whose function is to ensure the fermentation of the bread dough. The sourdough contains an acidifying microflora consisting essentially of lactic bacteria and wild yeasts. It may also be dehydrated if, after hydration, it contains a live flora of lactic acid bacteria and yeasts to ensure fermentation of the bread dough. Article 9. Definition of inactive sourdough [masa madre inactiva]. Sourdough in which the micro-organisms are in a physiologically inactive state, due to having undergone a drying, pasteurisation or equivalent treatment, but which retains organoleptic properties that improve the quality of the final products. Article 14. Voluntary food information. 4. Breads which incorporate baker’s yeast in their production process and ferment the dough, after kneading and before baking, at a temperature of more than 4 °C for at least eight hours, may bear the words “made with long fermentation” [elaborado con larga fermentación]. 5. Breads made by incorporating sourdough as defined in Article 8, in a proportion equal to or greater than 5% of the total weight of the flour of the final dough and without the addition of additives, may bear the words “made with sourdough” [elaborado con masa madre], provided that the following conditions are met: (a) The sourdough prepared to ensure fermentation of the bread dough, before incorporation into the bread dough, must have a pH of less than 4.2 and a total titratable acidity of more than 6, expressed as the milliliters of 0.1 M NaOH required to bring ten grams of sourdough to pH 8.5. (b) Bread dough before baking and bread after baking must have a pH of less than 4.8. The pH values indicated are solely the result of the acidifying biological action of the microflora present in the sourdough. Baker’s yeast may be incorporated, at the last stage of kneading, at a maximum rate of 0.2% by weight of the total flour used in the final dough.

2.2.5 Czech Republic Decree of 20 January 2020 on requirements for cereal mill products, pasta, bakery, and confectionery products and doughs [8], in force since 1 February 2020. (3) For the purposes of this Decree, the following shall mean (c) bread is a bakery product leavened with yeast or sourdough, or a combination thereof, in the form of a loaf, loaf or mold, weighing not less than 400 g, with the exception of sliced bread and non-traditional types of bread, which may be of a lower weight, (q) sourdough: a leavened semi-finished product of one or more cereal mill products, water and a leavening base, or, in the case of rye leaven or sourdough, a leavened semi-­ finished product of rye flour, water and a leavening base, the fermenting microorganisms of which are present in an active state and in the quantity necessary to sour the dough; mature leaven or rye leaven is capable of repeated multiplication, the leaven is produced without the use of additives or enzymes, the acid content of the leaven is due solely to fermentation, (r) stable sourdough: a stable fermented semi-finished product made from one or more grain mill products, water and a leavening base, modified in particular by drying or concentrating, for the acidification of which fermented vinegar or additives may be used, namely lactic acid or acetic acid in an amount not exceeding one-third of the total acidity of the stable ferment; the additives used shall be obtained exclusively by the fermentation process.

2  Sourdough in a Regulatory Context

21

From Annex 1 Table 9: bread of the ‘yeast type’ or ‘sourdough type’ [chléb “kvasového typu” nebo “kváskového typu”] is with stable sourdough sourdough bread [chléb “kvasový”, “kváskový”, “s kváskem” nebo “s kvasem”] is acidified exclusively with sourdough Traditional sourdough or Traditional sourdough bread [chléb “tradiční kvasový”, “tradiční kváskový”, “tradiční s kváskem” nebo “tradiční s kvasem”] is leavened and acidified exclusively with rye sourdough.

2.2.6 Netherlands From 1 July 2020, the Dutch Commodities act decree on flour and bread was expanded with a definition of sourdough [9]: In this decree the following terms shall have the following meanings h. (sour)dough [(zuur) desem]: a product that is the result of fermentation of a mixture on the basis of grain, water and naturally present microorganisms, whereby the micro-­ organisms are present in an active state or can be reactivated. Article 7d 1. The designation sourdough may only be used if: a. sourdough is used as the only leavening agent; and b. no more than 0.2% dry yeast or no more than 0.5% fresh yeast has been added to the flour component. 2. If a bread with fruit, nuts, seeds, and kernels (at least 30% of the total weight) is labeled with sourdough, the quantity of dry yeast may, contrary to paragraph 1(b), amount to a maximum of 0.5% dry yeast or a maximum of 1.2% fresh yeast of the flour component.

2.2.7 Codes of Practice Sourdoughs are used as leavening agent, for acidification and or general improvement of taste and flavor of breads. The expectations of sourdough or the fermentation results differ across Europe, likewise do the preferences for the bread quality. As shown above, there is no harmonized understanding of sourdough within the European Union, and consumers’ interest in sourdough is increasing. Therefore, a working group of FEDIMA (Federation of European Manufacturers and Suppliers of Ingredients to Bakery, Confectionary and Patisserie Industries) prepared a position paper on “Understanding sourdough: building a common ground” and published it in February 2022 [10]. There sourdough is described as: “Sourdough is a typical leavening agent with organoleptic properties in wheat bread production. Sourdough is a distinctive ingredient with its typical acidic characteristic in rye bread production. In all cases, a sourdough is a characteristic food ingredient obtained from flour of cereals or pseudo-cereals, fermented by microorganisms, mainly lactic acid bacteria and yeast without addition of acids to artificially adapt the acidity.” [10] Further explanations and agreements on labeling in the

22

M. J. Brandt

B2B and B2C context are mentioned in this paper. In the UK too, a code of practice was agreed between the relevant associations in the baking sector.

2.3 Protected Geographical Indication (PGI) and Traditional Specialty Guaranteed (TSG) Very detailed legal regulations regarding the use of sourdough are laid down in the specifications for breads registered with “protected geographical indication” (Regulation EU/1151/2012 [11]). For “Pan de Alfacar” [12] the sourdough has to be fermented in wooden bins or for “Pane di Matera” [13] the sourdough has to be started with incorporation of fruits. Other specifications are less detailed, e.g., for “Südtiroler Schüttelbrot” [14] a sourdough can be used for leavening, but if so, it has to be a natural in-house sourdough. The indication “traditional specialty guaranteed” (TSG, [11]) registered within the EU describes the traditional aspects of a specific food, such as the way the product is made or its composition, without being linked to a specific geographical area. There is only one bread with sourdough that has a TSG status: Salinātā rudzu rupjmaize [15], originating from Latvia. It is a rye bread prepared with a hot soaker and leavened without baker’s yeast.

References 1. Brandt MJ (2019) Industrial production of sourdoughs for the baking branch - an overview. Int J Food Microbiol 302:3–7 2. Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers. Official Journal of the European Union, L 304, 22 November 2011 3. Codex Alimentarius Austriacus, II. Band (1912), Verlag der K.K. Hof- und Staatsdruckerei, Vienna. p 341 4. Österreichisches Lebensmittelbuch, 4th edn, Kapitel/ B 18/Backerzeugnisse BMG-75210/0011-­II/B/7/2009 vom 18.2.2010 - https://www.verbrauchergesundheit.gv.at/ lebensmittel/buch/codex/B18_Backerzeugnisse.pdf?8bgbhq, accessed 20/03/2022 5. Neufassung der Leitsätze für Brot und Kleingebäck vom 01.04.2021, Bundesanzeiger AT 06.05.2021 B2, GMBl 29/2021, 654–659, https://www.deutsche-­lebensmittelbuch-­ kommission.de/fileadmin/Dokumente/leitsaetze_fuer_brot_und_kleingebaeck.pdf, accessed 20/03/2022 6. Décret n°93-1074 du 13 septembre 1993 pris pour l’application de la loi du 1er août 1905 en ce qui concerne certaines catégories de pains, https://www.legifrance.gouv.fr/loda/id/ JORFTEXT000000727617/, accessed 20/03/2022 7. Real Decreto 308/2019, de 26 de abril, por el que se aprueba la norma de calidad para el pan. Boletín oficial de estado 113 de 11 Mayo de 2019, 50168-50175, https://www.boe.es/ diario_boe/txt.php?id=BOE-­A-­2019-­6994, accessed 26/03/2022 8. Vyhláška č. 18/2020 Sb. Vyhláška o požadavcích na mlýnské obilné výrobky, těstoviny, pekařské výrobky a cukrářské výrobky a těsta, https://www.zakonyprolidi.cz/cs/2020-­18, accessed 26/03/2022 9. Warenwetbesluit meel en brood, Staatsblad van het Koninkrijk der Nederlanden. 14/2020. https://zoek.officielebekendmakingen.nl/stb-­2020-­14.html, accessed 26/03/2022

2  Sourdough in a Regulatory Context

23

10. FEDIMA (2022) Understanding Sourdough: building a common ground, https://www.fedima. org/images/2202_Fedima_Understanding_Sourdough_Paper.pdf, accessed 20/03/2022 11. Regulation (EU) No 1151/2012 of the European Parliament and of the Council of 21 November 2012 on quality schemes for agricultural products and foodstuffs, Official Journal of the European Union, L 343, 14 December 2012 12. Commission Implementing Regulation (EU) No 1112/2013 of 5 November 2013 entering a name in the register of protected designations of origin and protected geographical indications [Pan de Alfacar (PGI)], https://eur-­lex.europa.eu/legal-­content/EN/ALL/?uri=CELEX% 3A32013R1112, accessed 26/03/2022 13. Commission Regulation (EC) No 160/2008 of 21 February 2008 registering certain names in the Register of protected designations of origin and protected geographical indications (Pane di Matera (PGI), Tinca Gobba Dorata del Pianalto di Poirino (PDO)), https://eur-­lex.europa. eu/legal-­content/EN/ALL/?uri=CELEX%3A32008R0160, accessed 26/03/2022 14. Commission Implementing Regulation (EU) 2020/1084 of 17 July 2020 entering a name in the register of protected designations of origin and protected geographical indications ‘Südtiroler Schüttelbrot’/‘Schüttelbrot Alto Adige’ (PGI), https://eur-­lex.europa.eu/legal-­content/EN/ TXT/?uri=uriserv:OJ.L_.2020.239.01.0006.01.ENG&toc=OJ:L:2020:239:TOC, accessed 26/03/2022 15. Commission Implementing Regulation (EU) No 12/2014 of 8 January 2014 entering a name in the register of traditional specialities guaranteed [Salinātā rudzu rupjmaize (TSG)], https://eur-­ lex.europa.eu/legal-­content/EN/ALL/?uri=CELEX%3A32014R0012, accessed 26/03/2022

3

Chemistry of Cereal Grains Cristina M. Rosell and Peter Koehler

3.1 Introductory Remarks Cereals are the most important staple foods for humankind worldwide and represent the main constituent of animal feed. Cereals have been additionally used for energy production, for example, by fermentation yielding biogas or bioethanol. The major cereals are wheat, maize, rice, barley, sorghum, millet, oats, and rye. They are grown on nearly 60% of the cultivated land in the world. Wheat, maize, and rice take up the greatest part of the land cultivated by cereals and produce the largest quantities of cereal grains (Table  3.1) [1]. Botanically, cereals are grasses and belong to the monocot family Poaceae. Wheat, rye, and barley are closely related as members of the subfamily Pooideae and the tribus Triticeae. Oats are a distant relative of the Triticeae within the subfamily Pooideae, whereas rice, maize, sorghum, and millet show separate evolutionary lines. Cultivated wheat comprises five species: the hexaploid common (bread) wheat and spelt wheat (genome AABBDD), the tetraploid durum wheat and emmer (AABB), and the diploid einkorn (AA). Triticale and Tritordeum are man-made hybrids of durum wheat and rye (AABBRR) and durum wheat and barley (AABBH), respectively [2, 3]. Within each cereal species numerous varieties exist produced by conventional or very modern techniques of breeding in order to optimize agronomical, technological, and nutritional properties [4]. Additionally, climate change might have impacted on the chemical composition of C. M. Rosell (*) Institute of Agrochemistry and Food Technology (IATA-CSIC), Paterna, Spain Food and Human Nutritional Sciences Department, University of Manitoba, Winnipeg, Canada e-mail: [email protected]; [email protected] P. Koehler Biotask AG, Esslingen, Germany e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. Gobbetti, M. Gänzle (eds.), Handbook on Sourdough Biotechnology, https://doi.org/10.1007/978-3-031-23084-4_3

25

26

C. M. Rosell and P. Koehler

Table 3.1  Cereal production in 2018 [1] Species Maize Rice Wheat Barley Sorghum Millet Oats Triticale Rye

Cultivated area (million ha) 194 167 214 48 42 34 10 4 4

Grain production (million tons) 1147 782 [522]a 734 141 59 31 23 13 11

Referred to paddy rice and in brackets the rice milled equivalent

a

the cereals, since it has been demonstrated that CO2 levels have significant impact on some carbohydrate and lipid levels in wheat genetic lines, and a clear reduction in the protein level at elevated CO2 [5]. The farming of all cereals is, in principle, similar. They are annual plants and consequently, one planting yields one harvest. The demands on climate, however, are different. “Warm-season” cereals (maize, rice, sorghum, millet) are grown in tropical lowlands throughout the year and in temperate climates during the frostfree season. Rice is mainly grown in flooded fields, and sorghum and millet are adapted to arid conditions. “Cool-season” cereals (wheat, rye, barley, and oats) grow best in a moderate climate. Wheat, rye, and barley can be differentiated into winter or spring varieties. The winter type requires vernalization by low temperatures; it is sown in autumn and matures in early summer. Spring cereals are sensitive to frost temperatures and are sown in springtime and mature in midsummer; they require more irrigation and give lower yields than winter cereals.

3.2 Grain Morphology and Chemical Composition Cereals produce dry, one-seeded fruits, called the “kernel” or “grain,” in the form of a caryopsis, in which the fruit coat (pericarp) is strongly bound to the seed coat (testa). Grain size and weight vary widely from rather big maize grains (~350 mg) to small millet grains (~9 mg). The anatomy of cereal grains is fairly uniform: fruit and seed coats (bran) enclose the germ and the endosperm, with the aleurone layer at the inner site of the bran. The aleurone layer contains most of the minerals, vitamins, phenolic antioxidants, and lignans in the case of wheat [6]. In oats, barley, and rice the husk is fused together with the fruit coat and cannot be simply removed by threshing as can be done with common wheat and rye (naked cereals). Currently, there are digital tools to analyze the inner morphology of the grains and the changes of those during kernels development [7]. The chemical composition of cereal grains (moisture 11–14%) is characterized by the high content of carbohydrates (Table 3.2). Available carbohydrates, mainly starch deposited in the endosperm, amount to 56–74% and fiber, mainly located in

3  Chemistry of Cereal Grains

27

Table 3.2  Chemical composition of cereal grains (average values) [8, 9]

Moisture Protein (N × 6.25) Lipids Available carbohydrates Fiber Minerals

Wheat Rye (g/100 g) 12.6 13.6 11.3 9.4 1.8 1.7 59.4 60.3 13.2 1.7

13.1 1.9

Maize

Barley

Oats

Ricea

Sorghum

Millet

9.6 7.4 4.9 74.1

12.1 11.1 2.1 62.7

13.1 10.8 7.2 56.2

9.4 6.5 0.3 82.4

10.1 10.9 3.9 70.7

11.0 7.9 3.2 74.9

1.7 2.3

9.7 2.3

9.8 2.9

0.6 0.8

1.8 2.6

1.1 1.9

White (polished) rice

a

the bran, to 2–13%. The second important group of constituents is the proteins, which fall within an average range of about 8–11%. With the exception of oats (~7%), cereal lipids belong to the minor constituents (2–4%) along with minerals (1–3%). Antinutritional factors, vitamins and high-value proteins, besides other trace elements, are principally associated with the fruit coat and aleurone layer of the cereals grains. Therefore, variations on those tissues might have great impact on the chemical composition, and impact nutritional properties. Particularly, the relatively high content of B-vitamins is of nutritional relevance. With respect to structures and quantities of chemical constituents, notable differences exist between cereals and even between species and varieties within each cereal, which strongly affect the quality of the resulting foods. It should be emphasized that domestication of the cereals while increasing yields, has induced the so-called yield dilution phenomenon leading to high starch content and a reduction in the protein, vitamins, and minerals content [10]. In fact, protein and ash contents ranging from 22 to 28 g/100 g and 3.0 to 3.3 g/100 g, respectively, have been reported for diploid wild wheats [11]. Even a comparison of ancient versus modern wheat composition stressed the superior nutritional quality of ancient wheats, but the limited amount of intervention studies prevent a convincing conclusion [12]. In addition, very recent studies on in vitro digestibility of proteins carried out with a selection of ancient and modern wheats could not be associated with the year of introduction [13].

3.3 Carbohydrates Cereal grains contain 56–82% carbohydrates (Table  3.2); thus, this is by far the most abundant group of constituents. The major carbohydrate is starch (55–70%) followed by minor constituents such as arabinoxylans (1.5–8%), β-glucans (0.5–7%), sugars (~3%), cellulose (~2.5%), and fructans (traces–6.6%).

28

C. M. Rosell and P. Koehler

3.3.1 Starch Starch is the major storage carbohydrate of cereals and an important part of our nutrition. Because of its unique properties starch is important for the textural properties of many foods, in particular bread and other baked goods. In the endosperm, starch is present as intracellular granules of different sizes and shapes, depending on the cereal species. In contrast to most plant starches, wheat, rye, triticale, and barley starches usually have two granule populations differing in size. Small spherical B-type granules with an average size of 5 μm can be distinguished from large ellipsoid A-type granules with long axes around 22–36 μm [14]. The spatial distribution of starch granules within the endosperm is not homogeneous; in the case of rice, individual starch granules are principally located in the middle endosperm, elongated granules are distributed in the peripheral region adjacent to the subaleurone layer, and the interior hollow granules in the subaleurone layer [15]. Starch consists of two water-insoluble homoglucans, amylose and amylopectin. Cereal starches are typically composed of 25–28% amylose and 72–75% amylopectin [16]. Mutant genotypes may have an altered amylose/amylopectin ratio. “Waxy” cultivars have a very high amylopectin level (up to 100%), whereas “high amylose” or “amylostarch” cultivars may contain up to 70% amylose. This altered ratio of amylose/amylopectin affects the technological properties of these cultivars [17]. In wheat, the A-type granules contain higher total and apparent amylose content than B-type granules, and in the transition from wild type to waxy, a shift from B- to A-type granules is observed [18]. A-type granules exhibit higher pasting viscosities than B-type granules. Amylose consists almost exclusively of linear α-(1 → 4)-linked d-glucopyranosyl units. Parts of the molecules also have α-(1 → 6)-linkages providing slightly branched structures [16]. The degree of polymerization ranges from 500 to 6000 glucose units giving a molecular weight (MW) of 8 × 104 to 106. Amylopectin is responsible for the granular nature of starch. It contains 30,000–3,000,000 glucose units and, therefore, it has a considerably higher MW (107–109) than amylose. Amylopectin is a highly branched polysaccharide consisting of α-(1,4)-linked d-glucopyranosyl chains, which are interconnected via α-(1  →  4,6) branching points. The α-(1 → 4)-linked chains have variable length of 6 to more than 100 glucose units depending on the molecular site at which they are located. In fact, Bertoft et al. [19] proposed an amylopectin classification depending on its internal structure. Type 1 amylopectin contains mainly short chains but of a broad range of size distribution (A- and B1-chains). Type 4 amylopectin has a higher number of long chains with more than 36 glucose units (so-called B2- and B3 chains). Type 2 amylopectin contains a narrower distribution of short chains A- and B1 and more B2. Finally, type 3 amylopectin contains a considerably smaller number of the shortest internal B-chains (3–7 glucose units) [19]. Amylopectin has a tree-like structure, in which clusters of chains occur at regular intervals along the axis of the molecule [20]. Short A- and B1-chains of 12–15 glucose residues form clusters that have double-helical structures. The longer, less abundant B2-, B3-, and B4-chains

3  Chemistry of Cereal Grains

29

interconnect 2, 3, or 4 clusters, respectively. B2-chains contain approximately 35–40, B3-chains 70–80, and B4-chains up to more than 100 glucose residues. Under the polarization microscope, native starch granules are birefringent, indicating that ordered, partially crystalline structures are present in the granule. The combination of XRD, SAXS, SANS, and DSC has allowed understanding the structural architecture of the starch granules. The degree of crystallinity ranges from 20 to 40% [21] and is primarily caused by the structural features of amylopectin. In this structure, the amorphous and semi-crystalline layers are alternating into concentric growth rings [22]. The amorphous rings consist of amylose and amylopectin chains packed in an uneven organization, whereas the crystalline rings have a lamellar arrangement with alternate amorphous and crystalline regions located at repetitive distance of 9–10 nm [23]. The lamellae are formed by the side chains of the amylopectin interspersed with the amylose chains. It is thought that the macromolecules are oriented perpendicularly to the granule surface with the nonreducing ends of the molecules pointing to the surface. Crystalline regions contain amylopectin double helices of A- and B1-chains oriented in parallel fashion and possibly 18 nm-wide, left-handed superhelices formed from double helices. Amorphous regions represent the amylopectin branching sites, which may also contain a few amylose molecules. The lamellae are organized into larger spherical blocklets, which vary periodically in diameter between 20 and 500 nm [23]. The most recent model proposes that the amylopectin double helices may be structured as “building block backbone,” with small, branched units (building blocks) located along the long amylopectin chains, which are interlinked forming a long backbone. In this model, the amorphous lamellae consist of the backbone and most of the branches, and the short chains of the building blocks come perpendicularly into the crystalline lamellae [24]. This structural organization has a great impact on the starch properties.

3.3.1.1 Changes in Starch Structure During Processing Starch plays a fundamental role in cereal processing, principally in the fermentative process and those linked with thermal treatments. The structure of the native starch determines the extent and rate of hydrolysis by amylases [25], which in turn is fundamental to obtain fermentable sugars needed for processes involving fermentation. Yeast fermentation prefers glucose and fructose over maltose as a substrate, but the content of free fermentable sugars is too low for supporting the expansion of bread dough. Enzymatic hydrolysis of damaged starch by endogenous amylases provides the fermentable sugars to maintain yeast activity and dough expansion along the fermentation process. Damaged or gelatinized starch granules are more easily hydrolyzed by amylase enzymes, α-amylase and β-amylase, than intact granules, releasing dextrins or maltose, respectively. In many cereal manufacturing processes, flour and also starch is usually dispersed in water and finally heated. When heating is carried out below the onset temperature of gelatinization, this is referred to as annealing [26], and above that temperature, the gelatinization of the granule is induced involving swelling and finally disintegration [16]. Annealing starts at room temperature when starch granules are suspended under restricted water conditions (22%–60%). In those

30

C. M. Rosell and P. Koehler

conditions, the plasticizing effect of the water increases the mobility of β-glucan located in the amorphous lamella of the semicrystalline growth ring, modifying the starch structure to different extents. Starch swelling is affected by ways of processing besides the influence of genetic factors. Physical (hydrothermal treatment, high-­ pressure treatment), chemical, or enzymatic treatments of the starch granules modify the interaction between glucan chains, the spatial location of the free hydroxyl groups within the crystalline and amorphous regions, and the hydrophilic nature of the starch surface. Depending on water content, water distribution, and intensity of heat treatment, the molecular order of the starch granules can be completely transformed from the semicrystalline to an amorphous state. The mixing of starch in excess water at room temperature leads to a starch suspension. During mixing starch absorbs water up to 50% of its dry weight (1) because of physical immobilization of water in the void space between the granules, and (2) because of water uptake that promotes granule expansion, called swelling, which increases with temperature. This expansion includes the radial expansion of the granule and the tangential expansion in all directions; the latter more associated with the amylopectin component, whereas the amylose limits this tangential elongation [27]. Waxy starches swell more intensively than amylose rich starches because amylose retards swelling by retaining the integrity of the granule [28]. This is presumably related to the fact that waxy starches possess a higher number of short-­ chains and branch density [29]. First, the amorphous regions are hydrated, thereby increasing molecular mobility. This also affects the crystalline regions, in which the amylopectin double helices dissociate and the crystallites melt [30]. Nevertheless, different specific mechanisms have been proposed depending on the type of starches present, with predominance of short or long amylopectin branched chains. The type of the starch determines the predominance of radial or tangential elongations and the extension of the granule expansion [27]. These reactions are endothermic and irreversible. They are accompanied by the loss of birefringence, which can be observed under the polarization microscope. Endothermic melting of crystallites can also be followed by differential scanning calorimetry (DSC). Viscosity measurements, for example, in an amylograph, a rapid visco analyzer (RVA) or rapid force analyzer (RFA, Amylab), also allow monitoring the gelatinization process. Characteristic points are the onset temperature (T0; ca. 45 °C), which reflects the initiation of the process, as well as the peak (Tp; ca. 60 °C) and conclusion (Tc; ca. 75 °C) temperatures. For native starches, these temperatures are subject to change depending on the botanical source of the starch and the water content of the suspension [31]. The loss of molecular order and crystallinity during gelatinization is accompanied by further granule swelling due to increased water uptake and a limited starch solubilization. Mainly amylose is dissolved in water, which strongly increases the viscosity of the starch suspension. This phenomenon has been termed “amylose leaching,” and it is caused by a phase separation between amylose and amylopectin. During further heating beyond the conclusion temperature of gelatinization, swelling and leaching continue and a starch paste consisting of solubilized amylose and swollen, amorphous starch granules is formed. The shapes of the starch granules can still be observed unless shear force or higher temperatures are applied

3  Chemistry of Cereal Grains

31

[30, 32]. In general, type 1 amylopectin gelatinizes at lower temperature, whereas type 4 starches require higher temperatures to gelatinize [33]. Upon cooling with mixing, the apparent viscosity and hardness of a starch paste increases, whereas a starch gel is formed on cooling [31]. The second process is relevant in cereal baked goods. The changes that occur during cooling and storage of a starch paste have been summarized as “retrogradation” [34]. Generally, the amorphous system reassociates to a more ordered, partly crystalline state. Retrogradation processes can be divided into two subprocesses. The first is related to amylose and occurs in a time range of minutes to hours, the second is caused by amylopectin and takes place within hours or days. As amylose retrogradation proceeds, double helix formation increases and, finally, very stable crystalline structures are formed, which cannot be melted again by heating. Amylopectin retrogradation takes several hours or days and occurs in the granule remnants embedded in the initial amylose gel [35]. Therefore, amylose retrogradation is responsible for the initial hardness of a starch gel or bread, whereas amylopectin retrogradation determines the long-term gel structure, crystallinity, and hardness of a starch-containing food [36]. The amylopectin crystallites melt at ca. 60 °C and, therefore, aged bread can partly be “refreshed” by heating. This so-called “staling endotherm” can be measured by DSC to evaluate amylopectin retrogradation. Amylopectin retrogradation is strongly influenced by a number of conditions and substances, including pH, temperature, and the presence of low-molecular-weight compounds such as salts, sugars, and lipids [37, 38]. The interaction of amylose and lipids has been used as a strategy to modify the retrogradation behavior of baked products. The complexation of amylose with lipids reduces the gel rigidity after baking because those complexes aggregate in a more organized structure, although that effect is dependent on the fatty acids chain length [39].

3.3.1.2 Digestion of Starch as Affected by Structural Features Lately, the digestibility of starch has attracted much attention due to its association with the glycemic index and the incidence of diabetes and obesity. The selection of wheat and maize genotypes with the best performance for diabetic patients is an alternative for future breeding [40]. The tight structure of amylose, compared to amylopectin, results in lower postprandial levels of glycemic and insulinemic responses [10, 40]. In the case of waxy starches, their structure with more short and branched chains drives to slower enzymatic digestion in a single phase, which might be related to the difficulty of the digestive α-amylase to bind to the glucan chains [29]. With regard to digestion, starch is classified based on the hydrolysis rate into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) [41]. The RDS and SDS reflect the rate of in vivo starch digestion in the human small intestine, leading RDS to rapidly increase blood glucose and insulin levels, whereas SDS induces a more time-sustained blood glucose level. Starch from emmer wheat is SDS likely due to the complexity of the starch structure and high amylose content, or the high degree of crystallinity [42]. A very useful method for assessing the starch digestibility has been the in vitro method developed by Englyst

32

C. M. Rosell and P. Koehler

et al. [41], and the kinetic model of logarithm-of-slope (LOS) improved the first-­ order kinetic model and several relevant kinetic parameters were identified, including the rate constant (k) and the total starch digested (C∞) [43]. RS is the starch that escapes enzymatic digestion in the small intestine and goes directly to the colon for fermentation, acting as a substrate for the beneficial gut microbiota and releasing short-chain fatty acids [44]. Five different groups of RS have been defined. RS1 is physically entrapped starch, RS2 or native granular starch, RS3 is retrograded starch, RS4 refers to chemically modified starch, and RS5 corresponds to amylose-lipids complexes [45]. In the past decade, interest has focused on increasing the levels of RS owing to its association with hypoglycemic effect, prevention of colorectal cancer, lowering plasma cholesterol and triglyceride levels, leading to the recommendation that at least 14% of the total starch in starchy foods should be RS [46]. With the level of RS in mind, different transgenic cereals have been developed; for instance, high-amylose rice transgenic lines that have lower RDS and higher RS, likely related to their amylopectin structure containing high long-branch chain and short degree of branching [47]. Other alternatives for increasing the level of SDS and RS fractions involve the enzymatic, chemical, or physical treatments of the starches [44].

3.3.2 Nonstarch Polysaccharides (NSP) Polysaccharides other than starch are primarily constituents of the cell walls and are much more abundant in the outer than in the inner layers of the grains. Therefore, a higher extraction rate is associated with a higher content of NSP. This ingredient group is mainly composed of arabinoxylans (AX), fructans, β-glucans, glucomannan, and cellulose. In the particular case of wheat, outer layers or bran contain 17–33% arabinoxylans, 9–14% cellulose, 3–4% fructan, and 1–3% mixed β-glucans [48]. From a nutritional point of view, NSP are dietary fiber, which has been associated with positive health effects. For example, cereal dietary fiber has been related to a reduced risk of chronic life style diseases such as cardiovascular diseases, type II diabetes, and gastrointestinal cancer [49]. In addition, technological functionalities have been described for the arabinoxylans (AX) of wheat (reviewed by [50]) and rye. Within the EU FP6 HEALTHGRAIN program, the variability in the dietary fiber content of 26 wheat varieties was screened, showing that 80% of the total dietary fiber are NSP, and 18% accounted for water extractable compounds [51]. Nevertheless, the fiber functionality cannot be ascribed to the individual nonstarch polysaccharides; it is the combination of those constituents in the whole grain that lead to a slow fermentation in the colon, probably due to the structural complexity of the cell wall architecture [52].

3.3.2.1 Arabinoxylans AX are the major fraction (70%) of the dietary fiber in cereal grains. Different cereal species contain different amounts of AX. The highest contents are present in rye (6–8%), whereas wheat contains only 1.5–2% AX.  Wheat aleurone contains

3  Chemistry of Cereal Grains

33

20% of the total AX and 45% are located in the pericarp. On the basis of solubility, AX can be subdivided into a water-extractable (WEAX) and a water-unextractable fraction (WUAX). The former makes up 20–30% of total AX in wheat and 15–25% in rye. The solubility of the AX is dependent on the degree of arabinofuranose substitution, their distribution through the xylan backbone, molecular weight, and the aggregation of the unsubstituted AX molecules [53]. A substitution degree higher than 30% results in soluble AX, whereas solubility decreases with increasing interchain cross-links due to ferulic acid dimerization. In particular, WEAX have considerable functionality in breadmaking due to their viscosity in dough, although they have a very detrimental impact on the nutritive value for monogastric animals and in alcohol production. The molecular weight of the WEAX (200,000–300,000) is lower than that of WUAX. AX consist of linear β-(1,4)-d-xylopyranosyl-chains, which can be substituted at the O-2 and/or O-3-positions with α-l-arabinofuranose [53, 54]. A particular minor component of AX is ferulic or coumaric acid, which is bound to arabinose as an ester at the O-5 position and can be oxidized to dimers that cross-link AX chains [55]. AX of different cereals may vary substantially in content, substitutional pattern, and molecular weight [56]. WEAX mainly consist of two populations of alternating open and highly branched regions, which can be distinguished by their characteristic arabinose/xylose ratios, ranging between 0.3 and 1.1 depending on the specific structural region [57]. A high arabinose/xylose ratio is observed in the WUAX, which show a higher proportion of interchain cross-links by diferulates, preventing water solubility [58]. WUAX can be solubilized by mild alkaline treatment yielding structures that are comparable to those of WEAX. Solubilization is accompanied by de-esterification of ferulic acid and its dimers. The unique technological properties of AX are attributed to the WEAX that are able to absorb 15–20 times more water than their own weight and, thus, form highly viscous solutions, which may increase the gas holding capacity of wheat doughs via stabilization of the gas bubbles [54, 59]. In total, WEAX bind up to 25% of the added water in wheat doughs. Under oxidizing conditions, in particular under acidic pH, the so-called oxidative gelation [60] leads to AX gel formation probably due to di- and oligoferulic acid cross-links leading to higher loaf volume and softer crumbs [61, 62]. This is thought to be one major structure-forming reaction in rye sourdoughs. The structuring ability of the AX has even been confirmed in gluten free system models [63]. Apart from the impact on dough during mixing, the WEAX stabilize protein forms against thermal denaturation, and consequently, the gas retention during baking, thus the oven rise is delayed improving the crumb structure [54]. Conversely, WUAX are associated with a detrimental effect on breadmaking. Although they have high water-holding capacity and assist in water binding during dough mixing, they are considered to have a negative impact on wheat breadmaking due to possible water migration from gluten to AX and the physical barrier they form that interfere with the gluten network and, thus, destabilize the gas bubbles [54]. However, the baking performance can be affected by adding endoxylanases, which preferentially hydrolyze WUAX. This produces solubilized WUAX, which have techno-functional effects comparable to WEAX [54, 64–66].

34

C. M. Rosell and P. Koehler

AX and a small part of a water-soluble, highly branched arabinogalactan peptide [67] form the so-called pentosans. The former consists of β-(1,3) and β-(1,6) linked galactopyranose units with α-glycosidically bound arabinofuranose residues. The peptide is attached by 4-trans-hydroxyproline. Unlike AX, arabinogalactan peptides have no significant effects in cereal processing. From the nutritional point of view, apart from being considered the main constituents of the cereals dietary fiber, AX have an antiglycemic effect due to the inhibition of the α-glucosidase, although they do not affect the digestive amylase activity [68]. That inhibitory effect is dependent on the ferulic acid content, arabinose/xylose ratio, and the pattern of xylose distribution. The α-glucosidase affects the apparent maximum velocity and the Michaelis–Menten constant, suggesting an uncompetitive mechanism of inhibition.

3.3.2.2 Other Non-starch Polysaccharides Fructans have attracted more attention in the past decade due to their role as soluble fiber. Chemically, fructans consist of fructose linked via β-(2 → 1)- or β-(2 → 6)-glycosidic bonds giving different types of fructans. Among cereals, the amount of fructans is rather variable. Rye has the highest contents (3.3–6.6%), in contrast to wheat with values from 0.7 to 2.9% [69]. Maize and rice are considered free of fructans. Degradation of cereal fructans has been the focus of different studies because it has been suggested that their intake should be avoided by those suffering from irritable bowel syndrome. In many countries bread is the main source of fructans. Nevertheless, yeast or sourdough fermented breads have a significantly lower content of fructans than the cereal flour used as an ingredient. The extent of fructan degradation during breadmaking is dependent on the yeast strain present. Therefore, the selection of the right yeast strain might be a good strategy to reduce the content of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) in bread [70]. β-Glucans are also called lichenins and are present particularly in barley (3–7%), oats (2–6.6%), and rye (0.5–2.8%), whereas less than 2% β-glucans are found in other cereals. The chemical structure of these NSP is made up of linear d-glucose chains linked via mixed β-(1,3)- and β-(1,4)-glycosidic linkages. β-Glucans show a higher water solubility than AX (38–69% in barley, 65–90% in oats) and form viscous solutions, which in the case of barley may interfere in wort filtration during the production of beer. β-Glucans have been recognized for their health benefits, and health claims have been accepted in different legislations around the world. Wheat endosperm has low cellulose content (3%), and some traces of pectin and xyloglucan have been detected after removal of AX and β-(1,3; 1,4) glucans [71]. Very scarce information exists about the hemicellulosic polysaccharide mannan, although it has an important role on grain development. In the wheat endosperm, mannan is composed by linear chains of (1,4)-d-mannose residues that could be slightly acetylated [56].

3  Chemistry of Cereal Grains

35

3.4 Proteins The average protein content of cereal grains covers a relatively narrow range (8–11%, Table  3.2), variations, however, are quite noticeable. Wheat grains, for instance, may vary from less than 6% to more than 20%. The content depends on the genotype (cereal, species, variety) and the growing conditions (soil, climate, fertilization); amount and time of nitrogen fertilization are of particular importance. Proteins are distributed over the whole grain, their concentration within each compartment, however, is remarkably different. The germ and aleurone layer of wheat grains, for instance, contain more than 30% proteins, the starchy endosperm ~13%, and the bran ~7%. Regarding the different proportions of these compartments, most proteins of grains are located in the starchy endosperm, which is the source of white flours obtained by milling the grains and sieving. White flours are the most important products of grain milling. Therefore, the predominant part of the literature on cereal proteins deals with white flour proteins. The amino acid compositions of flour proteins from various cereals are shown in Table 3.3. Typical of all flours is the fact that glutamic acid almost entirely occurs in its amidated form as glutamine [72]. This amino acid generally predominates (15–31%), followed by proline in the case of wheat, rye, and barley (12–14%). Further major amino acids are leucine (7–14%) and alanine (4–11%). The nutritionally essential amino acids tryptophan (0.2–1.0%), methionine (1.3–2.9%), histidine (1.8–2.2%), and lysine (1.4–3.3%) are present only at very low levels, with lysine Table 3.3  Amino acid composition (mol%) of the total proteins of flours from various cereals [56] Amino acid Asxa Thr Ser Glxa Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp Amide group a

Wheat 4.2 3.2 6.6 31.1 12.6 6.1 4.3 1.8 4.9 1.4 3.8 6.8 2.3 3.8 1.8 1.8 2.8 0.7 31.0

Rye 6.9 4.0 6.4 23.6 12.2 7.0 6.0 1.6 5.5 1.3 3.6 6.6 2.2 3.9 1.9 3.1 3.7 0.5 24.4

Barley 4.9 3.8 6.0 24.8 14.3 6.0 5.1 1.5 6.1 1.6 3.7 6.8 2.7 4.3 1.8 2.6 3.3 0.7 26.1

Sum of Asp plus Asn and Glu plus Gln, respectively

Oats 8.1 3.9 6.6 19.5 6.2 8.2 6.7 2.6 6.2 1.7 4.0 7.6 2.8 4.4 2.0 3.3 5.4 0.8 19.2

Rice 8.8 4.1 6.8 15.4 5.2 7.8 8.1 1.6 6.7 2.6 4.2 8.1 3.8 4.1 2.2 3.3 6.4 0.8 15.7

Millet 7.7 4.5 6.6 17.1 7.5 5.7 11.2 1.2 6.7 2.9 3.9 9.6 2.7 4.0 2.1 2.5 3.1 1.0 22.8

Maize 5.9 3.7 6.4 17.7 10.8 4.9 11.2 1.6 5.0 1.8 3.6 14.1 3.1 4.0 2.2 1.4 2.4 0.2 19.8

36

C. M. Rosell and P. Koehler

being the limiting amino acid in most cereal species. Through breeding and genetic engineering, attempts are being made to improve the content of total proteins and of essential amino acids, and subsequently, modulating the amino acid score, particularly to obtain mutants high in lysine and methionine [73]. These approaches have been successful in the case of high-lysine barley and maize [74]. Traditionally, cereal flour proteins have been classified into four fractions (albumins, globulins, prolamins, and glutelins) according to their different solubility and based on the fractionation procedure of Osborne [72]. Albumins are soluble in water, while globulins are insoluble in pure water but soluble in dilute salt solutions. Prolamins are classically defined as cereal proteins soluble in aqueous alcohols, for example, 60–70% ethanol. Originally, glutelins were described as proteins that were insoluble in water, salt solution, aqueous alcohols, and soluble in dilute acids or bases. Later, it was shown that notable portions of glutelins are insoluble in dilute acids such as acetic acid, and that extraction with strong bases destroys the primary structure of proteins. Nowadays, complete solubility of glutelins is achieved by solvents containing a mixture of aqueous alcohols (e.g., 50% propanol), reducing agents (e.g., dithiothreitol), and disaggregating compounds (e.g., urea). Regarding their functions, most of the albumins and globulins are metabolic proteins, for example, enzymes or enzyme inhibitors (see Sect. 3.4.3). Oats are an exception because they contain considerable amounts of legume-like globulins such as 12S globulin [75]. Albumins and globulins are concentrated in the aleurone layer, bran, and germ, whereas their concentration in the starchy endosperm is relatively low. Predominantly, prolamins and glutelins are the storage proteins of cereal grains. Their only biological function is to supply the seedling with nitrogen and amino acids during germination. In particular, nitrogen is stored in the form of glutamine, and proline in the amino acid sequence leads to an effective, tight packing of the polypeptide chains. Prolamins and glutelins are located only in the starchy endosperm; in white flours, their proportions based on total proteins amount to 70–90%. In general, none of the Osborne fractions consists of a single protein, but of a complex mixture of different proteins. A small portion of proteins does not fall into any of the four solubility fractions. Together with starch, they remain in the insoluble residue after Osborne fractionation and mainly belong to the class of lipo (membrane) proteins. The prolamin fractions of the different cereals have been given trivial names: gliadin (wheat), secalin (rye), hordein (barley), avenin (oats), zein (maize), kafirin (millet, sorghum), and oryzin (rice). The glutelin fraction of wheat has been termed glutenin. Terms for the other glutelin fractions such as secalinin (rye), hordenin (barley), and zeanin (maize) are scarcely used today. Gliadin and glutenin fractions of wheat have been combined in the terms gluten or gluten proteins. The content of the Osborne fractions varies considerably and depends on genotype and growing conditions. Moreover, the results of Osborne fractionation are strongly influenced by experimental conditions, and the fractions obtained are not clear-cut. Therefore, data from the literature on the qualitative and quantitative composition of Osborne fractions is differing and, in parts, contradictory. On average, the smallest proportion of total protein is present in the globulin fraction, followed

3  Chemistry of Cereal Grains

37

by the albumin fraction. An exception is oat globulins amounting to more than 50% of total proteins. In most cereal flours, prolamins are the dominating fractions, oat prolamins, however, are minor protein components and rice flour is almost free of prolamins. Besides quantitative aspects, the Osborne procedure is useful for the preparation and characterization of flour proteins and the enrichment of different protein types.

3.4.1 Storage Proteins 3.4.1.1 Wheat, Rye, Barley, and Oats Storage Proteins Storage proteins (prolamins and glutelins) have been extensively investigated by the analysis of amino acid compositions, amino acid sequences, MW, and intra- and interchain disulfide linkages. The results indicated that, in accordance with phylogeny, the storage proteins of wheat, rye, and barley are closely related, whereas those of oats, in particular their glutelins, are structurally divergent. According to common structures, storage proteins have been classified into three groups by two different principles. Shewry and coworkers [75] defined all storage proteins as prolamins and grouped them into the high-molecular-weight (HMW), sulfur-poor (S-poor), and sulfur-rich (S-rich) prolamins based on differences in MW and sulfur (cysteine, methionine) content. To prevent confusion, however, the term “prolamin” is not used for all storage proteins in the present chapter, since classically the term prolamins comprises only the alcohol-soluble portions of storage proteins and does not include glutelins. Therefore, storage proteins have been classified according to related amino acid sequences and molecular masses into the following groups [76, 77]: (1) a HMW group; (2) a medium-molecular-weight (MMW) group; and (3) a LMW group. The proteins of these groups can be divided into different types on the basis of structural homologies (Table  3.4). Each type contains numerous closely related proteins; the small differences in their amino acid sequences can be traced back to substitutions, insertions, and deletions of single amino acids and short peptides. The nomenclature of storage protein types is rather confusing and inconsequential. On the one hand, prolamins have been termed according to their electrophoretic mobility in acid polyacrylamide gel electrophoresis (A-PAGE) with band regions designated as ω (lowest mobility), γ (medium mobility), and α/β (highest mobility). On the other hand, the nomenclature is based on their apparent sizes (after reduction of disulfide bonds) as indicated by sodium dodecyl sulfate (SDS-) PAGE; examples are HMW- and LMW-glutenin subunits (GS), HMW-secalins, D-, C-, and B-hordeins. Because of the different importance of HMW-GS for the bread-making quality of wheat, single subunits have been numbered according to their mobility on SDS-PAGE (original nos. 1–12), the genome (1A, 1B, or 1D), and the type (x or y); examples of nomenclature are HMW-GS 1Ax1, 1Bx7, and 1Dy10 [80]. The HMW group contains HMW-GS of wheat, HMW-secalins of rye, and D-hordeins of barley (Table 3.4); this type is missing in oats. HMW-GS and HMW-­ secalins can be subdivided into the x-type and the y-type. The proteins comprise

38

C. M. Rosell and P. Koehler

Table 3.4  Characterization of storage protein types from wheat, rye, barley, and oats [76–78] Partial amino acid composition (mol%)b Q P F +Y G L

V

Group/type HMW group HMW-GS x HMW-GS y HMW-secalin x HMW-secalin y D-hordein MMW group ω5-gliadin ω1,2-gliadin ω-secalin

Code

Repetitive unitb,c Residues statea (frequency)

Q6R2V1 Q52JL3 Q94IK6 Q94IL4 Q40054

815 637 760 716 686

a a a a a

QQPGQG (72×) QQPGQG (50×) QQPGQG (66×) QQPGQG (60×) QQPGQG (26×)

36 32 34 34 26

13 11 15 12 11

5.8 5.5 6.7 5.0 5.5

20 18 20 18 16

4.4 3.8 3.7 3.2 4.1

1.7 2.3 1.5 1.8 4.1

Q402I5 Q6DLC7 O04365

420 373 338

m m m

53 42 40

20 29 29

10.0 9.9 8.6

0.7 0.8 0.6

3.1 4.0 4.4

0.2 0.5 1.8

C-hordein

Q40055

328

m

(Q)QQQFP (65×) (QP)QQPFP (42×) (Q)QPQQPFP (32×) (Q)QPQQPFP (36×)

37

29

9.4

0.6

8.6

0.3

LMW group α/ß-gliadin

Q9M4M5

273

m

36

15

7.4

2.6

8.1

5.1

γ-gliadin

Q94G91

308

m

36

18

5.2

2.9

7.2

4.6

LMW-GS γ-40 k-secalind γ-75 k-secalin γ-hordein B-hordein avenin

Q52NZ4 – Q9FR41 P17990 P06470 Q09072

282 – 436 286 274 203

a m a m a m

QPQPFPPQQPYP (5×) (Q)QPQQPFP (15×) (Q)QQPPFS (11×) QPQQPFP QQPQQPFP (32×) QPQQPFP (15×) QQPFPQ (13×) PFVQQQQ (3×)

32 34 38 28 30 33

13 18 22 17 19 11

5.7 5.5 6.1 7.7 7.3 8.4

3.2 2.4 1.6 3.1 2.9 2.0

8.2 7.4 4.8 7.0 8.0 8.9

5.3 4.7 5.3 7.3 6.2 8.3

a aggregative, m monomeric One-letter-code for amino acids c Basic unit frequently modified by substitution, insertion, and deletion of single amino acid residues d Gellrich et al. [79] a

b

around 600–800 amino acid residues corresponding to MW of 70,000–90,000. The amino acid compositions are characterized by high contents of glutamine, glycine, and proline. The amino acid sequences can be separated into three structural domains: a nonrepetitive N-terminal domain A of ~100 residues, a repetitive central domain B of 400–700 residues, and a nonrepetitive C-terminal domain C with ~40 residues (Fig.  3.1) [79]. Domain B is dominated by repetitive sequences such as QQPGQG (one-letter-code for amino acids) as a backbone with inserted sequences like YYPTSL, QQG, and QPG with remarkable differences between x- and y-types (Table 3.5). Domains A and C have a more balanced amino acid composition and more amino acid residues with charged side chains. In the native state, the proteins of the HMW group are present as polymers aggregated through interchain disulfide bonds.

3  Chemistry of Cereal Grains

39

Fig. 3.1  Schematic structure and disulfide bonds of α/β-gliadins, γ-gliadins, LMW-, and HMW-GS (adapted from [78]). α α-gliadins, γ γ-gliadins, LMW LMW glutenin subunits, HMW HMW-glutenin subunits

The MMW group consists of the homologous ω1,2-gliadins of wheat, ω-secalins of rye, and C-hordeins of barley, including between 300 and 400 amino acid residues and MW around 40,000 (Table  3.4). Additionally, wheat contains unique ω5-gliadins with more than 400 residues and MW around 50,000. This group, likewise, is not present in oats. The proteins of the MMW group have extremely unbalanced amino acid compositions characterized by high contents of glutamine, proline, and phenylalanine, which together account for ~80% of total residues. Most sections of the amino acid sequences are composed of repetitive units such as QPQQPFP or QQQFP. This type of protein occurs as monomers and is readily soluble in aqueous alcohols and, partially, even in water. The LMW group consists of monomeric proteins, including α/β- and γ-gliadins of wheat, γ-40 k-secalins of rye, γ-hordeins of barley, and avenins of oats, and of aggregative proteins, including LMW-GS of wheat, γ-75  k-secalins of rye, and B-hordeins of barley (Table 3.4). They have around 300 amino acid residues and MW ranging from 28,000 to 35,000, besides γ-75 k-secalins (~430 residues, MW ~50,000) and avenins (~200 residues, MW ~23,000). The amino acid compositions of the LMW group proteins are characterized by a relatively high content of hydrophobic amino acids besides glutamine and proline. The amino acid sequences consist of four (α/β-gliadins five) different sequence sections (Fig. 3.1). The N-terminal section I is rich in glutamine, proline, and phenylalanine forming repetitive units such as QPQPFPPQQPY (α/β-gliadins), QQPQQPFP (γ-gliadins), QQPPFS (LMW-GS), or PFVQQQQ (avenins). Section I of γ-75 k-secalins is prolonged by around 130 residues as compared to γ-40 k-secalins and that of avenins is shortened

40

C. M. Rosell and P. Koehler

Table 3.5  Partial amino acid sequences of domain B of HMW-GS 1Dx2 (positions 93–338) and of HMW-GS 1Dy10 (positions 106–380) [78] Position 93 105 119 125 134 146 155 167 173 182 191 204 213 222 228 237 243 252 261 276 291 303 309 315 324 333 a

Sequencea YYPSVTSPQQVS YYPGQASPQRPGQG QQPGQG QQSGQGQQG YYP--TSPQQPGQW QQPEQGQPG YYP--TSPQQPGQL QQPAQG QQPGQGQQG QQPGQGQPG YYP-TSSQLQPGQL QQPAQGQQG QQPGQGQQG QQPGQG QQPGQGQQG QQPGQG QQPGQGQQG QQLGQGQQG YYP--TSLQQSGQGQPG YYP--TSLQQLGQGQSG YYP--TSPQQPGQG QQPGQL QQPAQG QQPGQGQQG QQPGQGQQG QQPGQG

Position 106 118 132 138 147 159 168 183 195 201 207 216 228 234 243 258 273 288 303 318 324 333 348 363 369 375

Sequencea YYPGVTSPRQGS YYPGQASPQQPGQG QQPGKW QEPGQGQQW YYP--TSLQQPGQG QQIGKGQQG YYP--TSLQQPGQGQQG YYP--TSLQHTGQR QQPVQG QQPEQG QQPGQWQQG YYP--TSPQQLGQG QQPRQW QQSGQGQQG HYP--TSLQQPGQGQQG HYL--ASQQQPGQGQQG HYP--ASQQQPGQGQQG HYP--ASQQQPGQGQQG HYP--ASQQEPGQGQQG QIPASQ QQPGQGQQG HYP--ASLQQPGQGQQG HYP--TSLQQLGQGQQT QQPGQK QQPGQG QQTGQG

One-letter-code for amino acids; − deletion

to around 40 residues. Section II is unique to α/β-gliadins and consists of a polyglutamine sequence (up to 18 Q-residues). Sections III, IV, and V possess more balanced amino acid compositions and most of the cysteine residues that form only intrachain disulfide bonds (monomeric proteins) or both intra- and interchain disulfide bonds (aggregative proteins). The comparison of the amino acid sequences demonstrates that sections III and V contain homologous sequences, whereas section IV is, in part, unique to each type (Table  3.6). γ-Type proteins (γ-gliadins, γ-40 k-secalins [81], γ-75 k-secalins, γ-hordeins) show the highest conformity; α/β-­gliadins, LMW-GS, and avenins have the lowest degree of homology within the LMW group. Most oat glutelins are globulin-like proteins and do not show any structural relationship with the HMW-, MMW-, and LMW-type proteins described above [82]. The reasons as to why they are not extractable with a salt solution are not yet clear. As mentioned above, the quantitative composition of storage protein is strongly dependent on both genotype and growing conditions. It even depends on the water

Type (a) Section III α/β-gliadin γ-gliadin LMW-GS γ-40 k-secalina γ-75 k-secalin γ-hordein B-hordein Avenin (b) Section IV α/β-gliadin γ-gliadin LMW-GS γ-40 k-secalina γ-75 k-secalin γ-hordein B-hordein Avenin (c) Section V α/β-gliadin

ILQQILQQQLIPCMDVVLQQHNIVHGRSQVLQQSTY-----QLLQELCCQHLWQIPEQSQCQAIHNVVHAIIL FIQPSLQQQLNPCKNILLQQCKPASLVSSL-WSIIWPQSDCQVMRQQCCQQLAQIPQQLQCAAIHSVVHSIIM IVQPSVLQQLNPCKVFLQQQCSPVAMPQRLARSQMWQQSRCHVMQQQCCQQLSQIPEQSRYDAIRAITYSIIL SIQLSLQQQLNPCKNVLLQQCSPVALVSSL-RSKIFPQSECQVMQQQCCQQLAQIPHHLQCAAIHSVVHAIIM SIQLSLQQQLNPCKNVLLQQCSPVALVSSL-RSKIFPQSECQVMQQQCCQQLAQIPQQLQCAAIHSVVHAIIM TIQLYLQQQLNPCKEFLLQQCRPVSLLSYI-WSKIVQQSSCRVMQQQCCLQLAQIPEQYKCTAIDSIVHAIFM YVHPSILQQLNPCKVFLQQQCSPVPVPQRIARSQMLQQSSCHVLQQQCCQQLPQIPEQFRHEAIRAIVYSIFL FLQPLLQQQLNPCKQFLVQQCSPVAAVPFL-RSQILRQAICQVTRQQCCRQLAQIPEQLRCPAIHSVVQSIIL

HQQQKQQQQPSSQVSFQQPLQQYPLGQGSFRPSQQN QQQQQQQQQQGMHIFLPLSQQQQVGQGSL QEQQQGFVQAQQQQPQQSGQGVSQSQQQSQQQLGQCSFQQPQQQLGQQPQQQQQQ QQEQREGVQILLPQSHQQLVGQGAL QQEQREGVQILLPQSHQQHVGQGAL QQGQRQGVQIVQQQPQPQQVGQCVL QEQPQQLVEGVSQPQQQLWPQQVGQCSFQQPQPQQVGQQQQ QQQQQQQQFIQPQLQQQVFQPQLQLQQQVFQPQLQQQVFQP

PQAQGSVQPQQLPQF-EEIRNLALQTLPAMCNVYIPPYCTI--APFGIFGTNYR

119–186 153–224 101–173 – 285–356 135–206 112–184 43–114

187–222 225–253 174–228 – 357–381 207–231 185–225 115–155

223–273

(continued)

Sequences

Positions

Table 3.6  Amino acid sequences of sections III, IV, and V of the LMW group [78]

3  Chemistry of Cereal Grains 41

a

Gellrich et al. [79]

Type γ-gliadin LMW-GS γ-40 k-secalina γ-75 k-secalin γ-hordein B-hordein Avenin

Positions 254–308 229–282 – 382–436 232–286 226–274 156–203

Table 3.6 (continued)

Sequences VQGQGIIQPQQPAQL-EAIRSLVLQTLPSMCNVYVPPECSIMRAPFASIVAGIGGQ VLQGTFLQPHQIAHL-EAVTSIALRTLPTMCSVNVPLYSATTSVPFAVGTGVSAY AQVQGIIQPQQLSQFNVGIVLQMLQNLPTMCNVYVPRQCPPSRRHLHAMSLVCGH AQVQGIIQPQQLSQL-EVVRSLVLQNLPTMCNVYVPRQCSTIQAPFASIVTGIVGH VQGQGVVQPQQLAQM-EAIRTLVLQSVPSMCNFNVPPNCSTIKAPFVGVVTGVGGQ VPQSAFLQPHQIAQL-EATTSIALRTLPMMCSVNVPLYRILRGVGPSVGV QLQQVFNQPQMQGQI-EGMRAFALQALPAMCDVYVPPQCPVATAPLGGF

42 C. M. Rosell and P. Koehler

3  Chemistry of Cereal Grains

43

Table 3.7  Relative proportions (%) of storage protein types of different wheat species, rye, and barley [85–87] Cereal

Common wheat Spelt wheat Durum wheat Emmer Einkorn Rye Barley

Variety

Rektor Schwabenkorn Biodur Unknown Unknown Halo Golden promise

Group HMW a 9.1 6.6 5.0 2.6 3.5 9.0 5.0

MMW m 10.4 10.4 6.7 10.8 12.8 17.6 35.8

LMW a 25.1 17.7 19.3 10.0 19.3 48.4 34.1

m/aa m 55.4 65.3 69.0 76.6 64.5 25.0 25.1

1.9 3.1 3.1 6.9 3.4 0.7 1.6

a aggregative, m monomeric

a

availability during the vegetative cycle and the grain formation [83]. In general, higher protein expression and gliadin occurs under drought stress, containing increased amounts of proline, glutamine, and phenylalanine. A high content of P and Q is characteristic of peptides associated with allergy and coeliac disease [84]. Nevertheless, some general statements can be made (Table 3.7) [85–87]: Proteins of the LMW group belong, by far, to the major components. Within this group, monomeric proteins (55–77% of total storage proteins) exceed aggregative proteins (10–25%) in the case of wheat species, whereas rye and barley are characterized by more aggregative (34–48%) than monomeric proteins (~25%). Proteins of the MMW and HMW groups belong to the minor components except ω-secalins (18%) and C-hordeins (36%) or are missing (oats). Within wheat species, significant differences can be observed. Common wheat is characterized by the highest values for aggregative proteins (HMW-, LMW-GS) and a low monomeric/aggregated (m/a) ratio, and the “ancient” wheat species emmer and einkorn by low proportions of HMW-GS and high ratios of monomeric to aggregated proteins.

3.4.1.2 Wheat Gluten Most information on the native storage (gluten) proteins is available for wheat because MW distribution (MWD) of gluten proteins has been recognized as one of the main determinants of the rheological properties of wheat dough. Native gluten proteins consist of monomeric α/β- and γ-gliadins with MW around 30,000 and monomeric ω5- and ω1,2-gliadins with MW between 40,000 and 55,000. They are alcohol-soluble and amount to ~50% of gluten proteins (Fig. 3.2). Besides monomers, the alcohol-soluble fraction contains oligomers with MW roughly ranging between 60,000 and 600,000. They are formed by modified gliadins with an odd number of cysteine residues and LMW-GS via interchain disulfide bonds and account for ~15% of gluten proteins [89]. Composition and quantity of the oligomeric fraction are strongly determined by the conditions of alcohol extraction, for example, by temperature and duration. The remaining proteins (~35%) are alcohol-­ insoluble, polymeric, and mainly composed of LMW-GS and HMW-GS linked by disulfide bonds. Their MW ranges approximately from 600,000 to more than 10

44

C. M. Rosell and P. Koehler

Fig. 3.2  Molecular weight distribution of native wheat storage (gluten) proteins (Modified from [88])

million. The largest polymers termed “glutenin macropolymer” (GMP) are insoluble in sodium dodecyl sulfate solutions and have MW well into the multimillions, indicating that they may belong to the largest proteins in nature [88]. Their amounts in flour (20–40 mg/g) are strongly correlated with dough strength and bread volume obtained when applying the straight dough process. Studies carried out with transgenic wheat lines associate wheat lines with overexpressed HMW-GS 1Ax1 with higher tenacity, extensibility, and deformation work (W); conversely, lines expressing 1Dx5 have lower extensibility and W and are thus unsuitable for breadmaking [90]. Rye storage proteins have a strongly different MWD as compared to wheat. Although rye shows higher proportions of aggregative to monomeric storage proteins than wheat (Table 3.7), the proportions of polymers is much lower (~23%) and the amount of GMP (~5 mg/g flour) strongly reduced [91, 92]. The deficiency of polymeric proteins is balanced by the higher proportion of oligomers (~30%), whereas the proportion of rye monomers (~47%) is similar to that of wheat. Obviously, rye storage proteins consist of many more chain terminations (e.g., γ-75 k-secalins, y-type HMW-secalins) and less chain extenders than wheat, which apparently prevents gluten formation during dough mixing. Information about the MWD of native barley and oat proteins is not yet available. Wheat is unique among cereals due to its ability to form a cohesive, viscoelastic dough, when flour is mixed with water. Wheat dough retains the gas produced during fermentation and this results in a leavened loaf of bread after baking. It is commonly accepted that gluten proteins (gliadins and glutenins) decisively account for the physical properties of wheat dough. Both protein fractions are important contributors to these properties, but their functions are divergent. Hydrated monomeric

3  Chemistry of Cereal Grains

45

and oligomeric proteins of the gliadin fraction have little elasticity and are less cohesive than glutenins; they contribute mainly to the viscosity and extensibility of dough. In contrast, hydrated polymeric glutenins are both cohesive and elastic, and are responsible for dough strength and elasticity. Thus, gluten is a “two-component glue,” in which gliadins can be considered as a “plasticizer” or “solvent” for glutenins [79]. A proper gliadin/glutenin ratio (1.4–2.1) is essential to give desirable dough and bread properties [93], although a more extended range has been described in recent studies (1.7–4.2) [94]. That ratio is much higher in ancient wheats, where it varies from 2.2 for spelt to 14 for einkorn [95]. Contents of gliadins, glutenins, and polymeric glutenin macropolymer are suitable for predicting wheat bread-­ making properties and particularly bread volume [93, 96]. Native gluten proteins are among the most complex protein networks in nature due to the presence of several hundred different protein components. Even small differences in the qualitative and quantitative protein composition determine the end-use quality of wheat varieties. Numerous studies demonstrated that the total amounts of gluten proteins (highly correlated with the protein content of flour), the ratio of gliadins to glutenins, the ratio of HMW-GS to LMW-GS, the amount of GMP, and the presence of specific HMG-GS determine dough and bread quality. Among chemical bonds, disulfide linkages play a key role in determining the structure and properties of gluten proteins, although it is rather difficult to directly relate molecular information on SH and SS-groups with dough rheology. Intrachain bonds stabilize the steric structure of both monomeric and aggregative proteins; large glutenin polymers consist of glutenin subunits connected via interchain disulfide bonds. The backbone of the polymers could be formed by x-type HMW-GS interconnected by disulfide bonds between N-terminals, C-terminals, and N-to-C-­ terminals. This is often referred to as head-to-head, tail-to-tail, and head-to-tail orientation [87]. The disulfide structure is not in a stable state, but undergoes a continuous change from the maturing grain to the end product (e.g., bread), and is chiefly influenced by redox reactions. These include (1) the oxidation of free SH groups to SS-linkages, which supports the formation of large aggregates, (2) the presence of SH-containing chain terminators (e.g., glutathione and gliadins with an odd number of cysteine), which stop polymerization, and (3) SH-SS interchange reactions, which affect the degree of polymerization of glutenins. Consequently, oxygen is known to be essential for optimal dough development and oxidizing agents, for example, dehydroascorbic acid (the oxidation product of ascorbic acid) [78] and glucose oxidase have been found to be useful as bread improvers or processing aids, respectively [97, 98]. Conversely, reducing agents such as cysteine and sodium metabisulfite or sourdough with heterofermentative lactobacilli [99] can be used to soften strong doughs, accompanied by decreased dough development time and resistance and increased extensibility. They are specifically in use as dough softeners for biscuits. The overall effect is to reduce the average MW of glutenin aggregates by SH/SS interchange. In addition to disulfide bonds, dityrosine and isopeptide bonds have been described as further covalent cross-links between gluten proteins. Compared with the concentration of disulfide bonds (~10  μmol per g flour) tyrosine-tyrosine

46

C. M. Rosell and P. Koehler

cross-­links (~0.7 nmol per g flour) appear to be only of marginal importance [100]. Interchain cross-links between lysine and glutamine residues (isopeptide bonds) are catalyzed by the enzyme transglutaminase (TG). Addition of TG to flour results in a decrease in the quantity of extractable gliadins and an increase of the glutenin fraction and the nonextractable fraction [101, 102]. The transglutaminase crosslinking results in an increase of the gluten yield and MWD of proteins due to glutamine-lysine linkages, although those could prevent the formation of disulfide bonds. It should be emphasized that the effect is enzyme dose-dependent and excessive activity can lead to glutamine deamidation and opposite effects on dough rheology [103]. The covalent structure of gluten proteins is complemented by noncovalent interactions, i.e., hydrogen bonds, ionic bonds, and hydrophobic bonds, which might compensate the role of the covalent bonds on the interactions of mesoscopic glutenin aggregates. Glutamine, predestinated for hydrogen bonds, is the most abundant amino acid in gluten proteins (Table  3.4) and chiefly responsible for the water-­ binding capacity of gluten. In fact, dry gluten absorbs about twice its own weight of water. Moreover, glutamine residues are involved in frequent protein-protein hydrogen bonds. Though the number of ionizable side chains is relatively low, ionic bonds are of importance for the interactions between gluten proteins. For example, salts such as NaCl are known to strengthen dough [104], obviously via ionic bonds with glutenins. Hydrophobic bonds can also contribute to the properties of gluten. Because the energy of hydrophobic bonds increases with increased temperature, this type of noncovalent bonds is particularly important for protein interactions during the oven phase. Through the use of different gluten modifying agents, including chemicals (glutathione, ascorbic acid, potassium bromate) and enzymes (glucose oxidase, transglutaminase), it has been possible to classify the different gluten polymer networks based on quantifying the level of lacunarity, branching rate, end-point rate, average protein length, and protein width [105]. The statistical analysis of those parameters allows describing five different protein networks with increasing level of lacunarity from type I (0–0.16) to type IV (>0.27), till type V composed of protein agglomerates with low viscosity and slight viscoelastic properties. Both covalent and noncovalent bonds determine the native steric structures (conformation) of gliadins and glutenins. Studies on the secondary structure have indicated that the repetitive sequences of gliadins and LMW-GS are characterized by β-turn conformation, whereas the nonrepetitive sections contain considerable proportions of α-helix and β-sheet structures [106]. The nonrepetitive sections of α/β-, γ-gliadins, and LMW-GS include intrachain disulfide bonds, which are concentrated in a relatively small area and form compact structures including two or three small rings and a large ring [107]. The nonrepetitive sections A and C of HMW-GS are dominated by α-helix and β-sheet structures, whereas the repetitive section B is characterized by regularly repeated β-turns [106]. They form a loose β-spiral similar to that of mammalian connective tissue elastin; β-spirals have been proposed to transfer elasticity to gluten. A range of models has been developed to explain the structure and functionality of glutenins. During common mixing, forces disrupt glutenin aggregates with a

3  Chemistry of Cereal Grains

47

simultaneous re-aggregation, yielding a double direction shift from sodium dodecyl sulfate insoluble proteins to soluble ones [108]. Re-aggregation of disulfide linked insoluble proteins is produced at even mild temperatures (35–45 °C) [108]. At some point during initial stages of baking, the heat induced aggregation overruns the mixing induced disruption, and even other protein fractions co-aggregate with the insoluble glutenins. Overall, it is a dynamic de- and re-structuring process in which water soluble, sodium dodecyl sulfate soluble and insoluble proteins are involved, until complete protein unfolding, in which SH/SS interchange, oxidation, and hydrophobic interactions are intertwined [108]. The most recent model is based on dough development time (ddt) and power input (Pwsp, J s−1 kg−1), in which only the insoluble glutenin polymer is correlated with the energy demand of flour (Ef) and specific critical power (Pwsp*) [109]. During mixing, gluten proteins assemble into microscopic lumps that progressively grow, reaching complete fibrillation at the ddt, involving disulfide cross-links and H-bonds interactions as “stress bearing” bonds (Fig.  3.3). When the Pwsp is lower than the flour specific critical power (Pwsp*) mixing results in continuous recovery and rupture of bonds that induce gluten stress-softening cycles but no fibrillation process occurs. Conversely, high Pwsp leads to the decrease of the content of SH-groups and accumulation of the oxidized side (disulfide bonds), and the mechanical stress applied to glutenin polymers can only be released by propagating along the polymer chain resulting in the alignment of the glutenin polymers and gluten fibrillation (Fig. 3.3). Gluten also affects the baking process due to its interactions with starch granules. Specifically, the gliadin/glutenin ratio determines the peak, through and final viscosities of starch pastes, which can be related to the increase in the gelatinization enthalpy observed when increasing the gliadin/glutenin ratio. The model proposed to explain the interaction between starch and gluten describes different scenarios depending on the amount of gliadins and glutenins [110]. In the absence of

Fig. 3.3  A model of dough development according to energy input [109] (Copyright permission)

48

C. M. Rosell and P. Koehler

glutenins, starch interacts with gliadins through hydrophobic forces that increase during heating until gliadin denaturation on the starch surface; with the water hydration of the amorphous regions and the amylose leaching, the dissolved chains form complexes with the denatured gliadin. When a balanced mixture of gliadins and glutenins exists, the gliadin denaturation preferentially occurs, and the amylosegliadin complexes are entrapped in the 3-D-glutenin network. In this case, the formation of a starch-gel network during starch retrogradation is hindered by the aggregated gliadin-glutenin and the amylose complexes. Conversely, in the absence of gliadins, when the covalently cross-linked glutenins reach the denaturation temperature, they are absorbed on the starch surface, resulting in a gel with lower viscosity than the one obtained by starch. In this case, during retrogradation, the complex of glutenin with amylose weaken the amylose and amylopectin arrangements [110]. Apart from the unique functional properties, gluten proteins can trigger hypersensitivity reactions associated with celiac disease, non-celiac gluten sensitivity, and wheat allergy [111]. In this context, the Codex Alimentarius defines gluten as the protein fraction present in wheat, rye, barley, and oats that is insoluble in water and 0.5 mol/l NaCl and cannot be tolerated by susceptible persons. In the regulatory scenario, prolamins represent 50% of the total gluten content, despite that the gliadin/glutenin ratio is variable, as was mentioned above, and it is dependent on the extraction and fractionation method used for quantification [94]. The repetitive amino acid sequences rich in glutamine and proline are involved in most of the wheat-derived hypersensitivities [111].

3.4.2 Storage Proteins of Maize, Millet, Sorghum, and Rice Overall, the storage proteins of maize, sorghum, millet, and rice are, in part, related and differ significantly from those of wheat, rye, barley, and oats. According to the amino acid composition, they contain less glutamine and proline and more hydrophobic amino acids such as leucine [61]. Maize storage proteins, called zeins, can be subgrouped into alcohol-soluble monomeric zeins and cross-linked zeins alcohol-­soluble only on heating or after reduction of disulfide bonds. With respect to different structures, zeins have been divided into four different subclasses [112]. α-Zeins are the major subclass (71–85% of total zeins), followed by γ- (10–20%), β- (1–5%), and δ-zeins (1–5%), respectively [113]. α-Zeins are monomeric proteins with an apparent MW of 19,000 and 22,000 determined by SDS-PAGE. Their amino acid sequences contain up to ten tandem repeats. Proteins of the other subclasses are cross-linked by disulfide bonds and their subunits have an apparent MW of 18,000 and 27,000 (γ-zein), 18,000 (β-zein), and 10,000 (δ-zein). In many ways, the storage proteins of sorghum and millet called kafirins are similar to zeins. Sorghum kafirins have also been subdivided into α, β-, γ-, and δ-subclasses based on solubility, MW, and structure [114]. α-Kafirins are monomeric proteins and represent the major subclass accounting for around 65–85% of total kafirins. Proteins of the other subclasses are highly cross-linked and

3  Chemistry of Cereal Grains

49

alcohol-­soluble only after reduction of disulfide bonds. On average, each of them accounts for less than 10% of total kafirins [115, 116]. Within the numerous millet species and varieties, the proteins of foxtail millet were studied in detail [117]. SDS-PAGE of unreduced kafirins revealed protein bands with an apparent MW ranging from 11,000 to 150,000. After the reduction of disulfide bonds, two major bands with MW of 11,000 (subunit A) and 16,000 (subunit B) were obtained. Unreduced proteins with higher MW were formed by cross-links of A and/or B subunits. The storage proteins of rice are characterized by the highly unbalanced ratio of prolamins to glutelins (~1:30). Both fractions show the lowest proline content (~5  mol%) among cereal storage proteins [61]. SDS-PAGE patterns of rice prolamins (oryzins) showed a major band with MW 17,000 and a minor band with MW 23,000 [106]. The apparent MW of glutelin subunits ranged from 20,000 to 38,000.

3.4.3 Metabolic Proteins: Enzymes and Enzyme Inhibitors Most proteins of the albumin and globulin fractions are metabolic proteins, mainly enzymes and enzyme inhibitors [118, 119]. Many of these proteins are located in the embryo and aleurone layer; others are distributed throughout the endosperm. They have nutritionally better amino acid compositions than storage proteins, particularly because of their higher lysine contents. Those enzymes that hydrolyze carbohydrates and proteins and, thereby provide the embryo with nutrients and energy during germination, are of most significant importance. From the technological point of view, enzymes exert an important role in fermentative processes by providing fermentable sugars but also affect the rheological properties of the doughs due to their action on carbohydrates and proteins. In this context, it should be highlighted that enzyme action depends on pH and temperature, with specific optima for each enzyme. For instance, in breadmaking processes, enzyme activities in straight doughs would be significantly different from those containing sourdough. The many carbohydrate-degrading enzymes include α-amylases, β-amylases, debranching enzymes, cellulases, β-glucanases, and glucosidases. Amylases are enzymes that hydrolyze the polysaccharides of starch granules. They can be classified as endohydrolases, which attack glucosidic bonds within the polysaccharide molecules, and exohydrolases, which attack glucosidic bonds at or near the end of the chains. The most important enzyme of the endohydrolase type is α-amylase [120]. The enzyme hydrolyzes α-(1 → 4)-glucosidic bonds of amylose and amylopectin and produces a mixture of dextrins together with smaller amounts of maltose and oligosaccharides; the pH-optimum is about 5 [121]. The other major amylase type is β-amylase, an exohydrolase, which hydrolyzes α-(1 → 4)-glucosidic bonds from the nonreducing ends of amylose and amylopectin to produce maltose. Its pH-­ optimum is similar to that of α-amylase. Both amylase types exist in multiple forms or isoenzymes with different chemical and physical properties. Neither α- nor β-amylase can break α-(1 → 6)-glucosidic bonds present in amylopectin. For this type of hydrolysis, debranching enzymes are present in cereal grains. Along with

50

C. M. Rosell and P. Koehler

α-glucosidases, they assist α- and β-amylases to yield a more complete conversion of starch to simple sugars and small dextrins. A number of other carbohydrate degrading enzymes exist; their amounts, however, are very low compared to amylases. Examples are α- and β-glucosidases, cellulases, and xylanases. Enzymes that hydrolyze proteins are called proteinases, proteases, or peptidases. They attack the peptide bond between amino acid residues and include both endoand exopeptidases. The latter are divided into carboxypeptidases, when acting from the carboxy terminal, and aminopeptidases, when acting from the amino terminal. The most important proteolytic enzymes are acidic peptidases. They exist in multiple forms having pH-optima between 4.2 and 5.5 and include both endo- and exotypes. On the basis of their catalytic mechanism, they can be classified as serine, metallo, aspartic, and serine peptidases. According to their biological function to provide the embryo with amino acids, their activity is highest during the germination of grains. Lipases are the most important enzymes that hydrolyze ester bonds. They attack triacylglycerols yielding mono- and diacylglycerols and free fatty acids. Lipase activity is important because free fatty acids are more susceptible to oxidative rancidity than fatty acids bound in triacylglycerols. The activity varies widely among cereals with oats and millet having the highest activity. Exogenous lipases are used to improve the baking performance of wheat flour. Phytase is an esterase that hydrolyzes phytic acid to inositol and free phosphoric acid. Even partial hydrolysis of phytic acid by phytase is desirable from a nutritional point of view because the strong complexation of cations such as zinc, calcium, and magnesium ions by phytic acid is significantly reduced. Lipoxygenase is present in high levels in the germ. It catalyzes the peroxidation of certain polyunsaturated fatty acids by molecular oxygen. Its typical substrate is linoleic acid containing isolated cis-configurated double bonds in the 9- and 12-­positions of the carbon chain. Polyphenoloxidases preferably occur in the outer layers of the grains. They catalyze the oxidation of phenols, such as catechol, pyrogallol, and gallic acid, to quinons by molecular oxygen. Peroxidase and catalase may be classified as hydroperoxidases catalyzing the oxidation of a number of aromatic amines and phenols, for example, ferulic acid in arabinoxylans, by hydrogen peroxide. Other oxidizing enzymes are ascorbic acid oxidase and glutathione dehydrogenase. Many investigators have isolated and characterized enzyme inhibitors from germ and endosperm [122–126]. Most important inhibitors are targeted on hydrolyzing enzymes to prevent the extensive degradation of starch and storage proteins during grain development and to defend plant tissues from animal (insect) or microbial enzymes. Predominant classes are amylase and protease inhibitors concentrated in the albumin/globulin fractions. Amylase inhibitors can be directed toward both cereal and noncereal amylases and protease inhibitors toward proteases from both cereals and animals. Some inhibitors appear to be bifunctional inhibiting amylases as well as proteases (amylase-trypsin inhibitors, ATI) and have been suggested to play a role in hypersensitivities toward cereals such as non-celiac-gluten/wheat-­ sensitivity (NCGS/NCWS) [127].

3  Chemistry of Cereal Grains

51

Cereal storage proteins contain many peptide sequences with biological activities, particularly in preventing chronic disease. Specifically, in wheat, barley, oat, and rice there is great occurrence of angiotensin-converting enzyme-inhibitor peptides, dipeptidyl peptidase-inhibitors, and peptides with antithrombotic, antioxidant, hypotensive, and opioid activities. Wheat and barley are the cereals with greatest biological activity, although in all cases, it is only possible to release those bioactive peptides from cereals through processes like enzymatic hydrolysis, fermentation, or germination [128].

3.5 Lipids 3.5.1 Lipid Composition Cereal lipids originate from membranes, organelles, and spherosomes and consist of different chemical structures. Depending on cereal species average lipid contents of 1.7–7% in the grains are present (Table 3.2). In particular, oats are rich in lipids (6–8%) in contrast to wheat and rye (1.7%). Lipids are mainly stored in the germ, to a smaller extent in the aleurone layer, and to the lesser extent in the endosperm. Nevertheless, the distribution pattern within the cereal is different depending on the grains [129]. In barley, rice, and sorghum, the lipids are concentrated in the germ and bran, whereas in oats, the oil is less concentrated in the bran fraction and the endosperm contains a significant amount of lipids. In wheat, the oil shows an intermediate distribution compared to the other cereals. Cereal lipids have similar fatty acid compositions, in which linoleic acid (C18:2) reaches contents of 36–62%, while oleic acid (C18:1) and palmitic acid (C16:0) make up 12–37% and 17–26%, respectively, and in minor proportions linolenic acid (C18:3) and stearic acid (C18:0) [129]. However, oat, rice, and sorghum have a higher proportion (32–37%) of oleic acid than wheat and barley (12–16%). The latter two have a higher proportion of linoleic acid (51–62%) than the others (36–45%). In durum wheat, oleic and palmitic acids have been identified as the predominant ones [130]. Although wheat lipids are only a minor constituent of the flour, they greatly impact the baking performance. By removing the outer layers of the kernels during pearling, the amount of lipids is decreased, but the important point is that this process changes the pattern of the lipids, reducing the unsaturated fatty acids (C18:1 and C18:3) and increasing the saturated ones (C16:0 and C18:0), whereas C18:1 is slightly modified [129]. Usually, lipids classes are defined based on their polarity. While triglycerides are the dominating lipid class in the germ and the aleurone layer, phospho- and glycolipids are present in the endosperm (Fig.  3.4). Depending on the extraction rate, wheat flour contains 0.5–3% lipids [131–133]. Extraction with a polar solvent at ambient temperature, i.e., water-saturated butanol, dissolves the nonstarch lipids that make up approximately 75% of the total flour lipids [134]. The residual 25% are the so-called starch lipids. The composition of the nonstarch lipids is given in Table  3.8. They contain about 60% nonpolar lipids, 24% glycolipids, and 15% phospholipids. By extraction with solvents of different polarities they can be further

52

C. M. Rosell and P. Koehler

Fig. 3.4  Polar lipids that affect the baking performance of wheat

Table 3.8  Composition of nonstarch lipids of wheat flour. Content (g/100 g) based on total lipid Nonstarch lipids: 1.70–1.95 g/100 g flour Polar Phospholipids Acylphosphatidyl ethanolamine Acyllysophosphatidyl ethanolamine Phosphatidyl ethanolamine/ phosphatidyl glycerol Phosphatidyl choline Phosphatidyl serine/phosphatidyl inosit Lysophosphatidyl ethanolamin Lysophosphatidyl glycerol Lysophosphatidyl choline Glycolipids Monogalactosyldiglycerides Monogalactosylmonoglycerides Digalactosyldiglycerides Digalactosylmonoglycerides

36–42 14–16 4.2–4.9 1.6–2.3 0.7–1.1 3.8–4.9 0.4–0.7 0.3–0.5 0.2–0.3 1.4–2.1 22–26 5.0–5.9 0.9–0.4 12.6–16.5 0.6–3.4

Nonpolar Sterol esters Triglycerides Diglycerides Esterified monogalactosyl-­ diglycerides/monoglycerides Esterified sterolglycerides

58–64 1.9–4.2 39.5–49.4 3.3–5.4 2.7–3.9 0.8–4.2

subdivided into a free (hexane extractable) and a bound fraction. The starch bound polar lipids have been related to the kernel hardness [135]. The nonpolar lipids are mainly present in the free lipid fraction, whereas glyco- and phospholipids are part of the bound fraction, in which they can be associated, for example, with proteins [136, 137]. The major glycolipid class is the digalactosyldiglycerides, followed by monogalactosyldiglyceride. Starch lipids are primarily composed of

3  Chemistry of Cereal Grains

53

lysophospholipids, which form inclusion complexes with amylose helices already in native starch [35].

3.5.2 Effects of Lipids on the Baking Performance of Wheat Flour Lipids are not uniformly distributed through the wheat kernel. The pericarp contains 5% of the total lipids, the aleurone 25–29%, the germ 30–36%, and the endosperm 35–45% [138]. The bran and germ lipids are mainly nonpolar and located within spherosomes [139]. In the starchy endosperm, there are starch lipids (40%) located in the starch granule that comprise mainly lysophospholipids, and nonstarch lipids (60%) that are nonpolar lipids mainly triacylglycerols. Starch lipids have been related to kernel hardness, specifically a negative relationship between the starch surface polar and nonpolar lipids and wheat hardness [140]. Specifically, endosperm hardness is positively correlated with the content of free glycolipids and negatively with the content of the non-polar fraction of starch surface lipids [140]. The outer layers of bran contain the highest level of free fatty acids and lipase activity. Therefore, bran obtained after the pearling process has lower lipid content and reduced lipase activity [139]. Only nonstarch lipids affect the rheological properties of wheat doughs. Interactions between starch lipids and starch are sufficiently strong so that this lipid fraction is not available before the starch gelatinizes. Studies with nonstarch lipids have shown that only the polar lipids have a positive effect on baking performance, whereas the nonpolar lipids have the opposite effect [113]. In particular, glycolipids have been shown to contribute to the high baking performance of wheat flour, namely galactolipids increase the stability of the gas bubbles by positioning on the air–water interface, preventing coalescence during the fermentation [141]. Phospholipids have a positive effect on loaf volume, acting as emulsifiers, which improve the baking performance of the dough, but they also interact with gluten proteins giving strength [142]. If the term “specific baking activity” would be defined, polar lipids would be found to affect the baking performance of wheat flour to a considerably greater extent than proteins. The addition of only 0.13% polar lipids would yield the same increase of loaf volume as a protein content increase of 1%. Polar lipids affect dough properties in many ways, i.e., the dough handling properties are improved and the gas-holding capacity during proofing is increased enabling a prolonged oven spring, increased loaf volume, better crumb resilience, and, in some cases, retardation of bread staling. The high baking activities of polar lipids, in particular of the glycolipids, might be explained by modes of action based on the formation of liquid films at the dough liquor/gas cell interface. Possible modes of action are the direct influence of the surfactants on the liquid film lamellae and gas cell interfaces through direct adsorption resulting in an increase of surface activity, as suggested by Sroan et al. [143, 144], as the secondary stabilizing mechanism in the so-called dual film theory. It suggests the presence of liquid lamellae, providing an independent mechanism of

54

C. M. Rosell and P. Koehler

gas cell stabilization. The effects of different surface active components may be explained by the type of monolayer that they form [145]. However, in particular, the positive effect of some polar lipids, such as acylated sterol glucosides and sterol glucosides, cannot be explained with this mode of directly stabilizing the liquid film lamellae. Here another mode of action could be the answer, for example, the indirect stabilization of the dough liquor/gas cell interface through this type of surfactant [145]. These polar lipid classes have a positive influence on the phase behavior of the endogenous lipids present in the dough liquor in that they lead to an increase in surface activity of the endogenous lipids and hence a better availability and accumulation at the liquid film lamellae/gas cell interface, thus increasing gas cell stabilization, and consequently the bread volume. Starch lipids significantly affect the pasting rate of the starch granule. Amylose is able to form helical inclusion complexes in particular with polar lipids, and this can occur in native (starch lipids; see below) as well as in gelatinized starch [145]. Inclusion complexes between amylose helices and polar lipids with one fatty acid residue are responsible for two effects. Complexes present in native starch (starch lipids) increase the temperature of gelatinization and, thus, prolong the time for pasting and the oven spring [18]. Inclusion complexes between amylose helices and polar lipids with one fatty acid residue may also form during and after the gelatinization process and are responsible for the anti-staling effect of some polar lipids, for example, monoglycerides. During gelatinization, amylose forms a left-handed single helix and the nonpolar moiety of the polar lipid is located in the central cavity. The inclusion complexes give rise to a V-type X-ray diffraction pattern. The presence of polar lipids strongly affects the retrogradation characteristics of the starch, because amylose-lipid complexes do not participate in the recrystallization process [146]. Complex formation is, however, strongly affected by the structure of the polar lipid [35]. For example, monoglycerides are more active than diglycerides and saturated fatty acids more active than unsaturated ones because inclusion complexes are preferably formed with linear hydrocarbon chains and with compounds having one fatty acid residue. In addition, lipids, in particular lysophospholipids (lysolecithin), are minor constituents of cereal starches in amounts of 0.8–1.2% [147, 148]. As so-called starch lipids, they are associated with amylose as well as with the outer branches of amylopectin [148]. These lipid complexes lead to a delay of the onset of gelatinization and affect the properties of the starch, especially in baking applications.

3.6 Minor Constituents 3.6.1 Minerals The mineral content of cereals ranges from ca. 1.0 to 2.5% (Table 3.2). The major portion of the minerals (>90%) is located in the outer layers of the grains, namely in the bran, the aleurone layer, and the germ that are removed during processing. The restoration of the minerals in the cereals flours has been of utmost importance in the

3  Chemistry of Cereal Grains

55

past decade, promoting the consumption of whole grains or the use of the aleurone as a bioavailable source of minerals [149]. Although wheat fortification is a voluntary or mandatory practice in many countries to provide the adequate amount of minerals in the diet, many minerals such as zinc are not added back. Therefore, the consumption of whole grains is recommended. The most abundant mineral is potassium followed by phosphorous and magnesium, although the absolute amounts vary from cereal to cereal (Table 3.9) [150].

3.6.2 Vitamins Cereals contain vitamins in concentrations ranging from below 1 to ca. 50 mg/kg, depending on the compound (Table 3.9). Thus, cereals are a good source of vitamins from the B-group, and, in industrial countries, they cover about 50–60% of the daily requirement of B-vitamins. Within the B vitamin group, cereals contain B1 (thiamine), B2 (riboflavin), B3 (niacin), B6 (pyridoxine), B9 (folate, folic acid or folacin), and B12 (cobalamin) [152]. The most important fat-soluble vitamins are vitamin E, comprising tocopherols (α, β, δ, and γ tocopherol) and tocotrienols (α, β, δ, and γ tocotrienols) [153]. Tocols are the major lipohilic secondary metabolites with antioxidant properties, besides the carotenoids [154]. Cereal tocopherols are concentrated in the germ fraction; conversely tocotrienols are present in the pericarp and the endosperm [155]. Wheat and barley are good sources of tocopherols and

Table 3.9  Mineral composition of cereal grains (average values) [150, 151] Wheat Calcium Magnesium Potassium Sodium Manganese Iron Copper Zinc Phosphorous

40 156 433 0.5 3.2 4.1 0.4 2.2 367

Vitamin B1 (thiamine) Vitamin B2 (riboflavin) Nicotinamide Panthothenic acid Vitamin B6 Folic acid Total tocopherols

4.6 0.9 51.0 12.0 2.7 0.9 41.0

Rye Maize (mg/100 g) 49 39 136 105 513 360 2.3 15 2.0 0.9 3.0 8 0.4 6 2.4 1.7 362 317 (mg/kg) 3.7 3.6 1.7 2.0 18.0 15.0 15.0 6.5 2.3 4.0 1.4 0.3 40.0 66.0

Barley

Oats

Rice

Sorghum

Millet

59 163 553 5.0 1.5 4.4 0.5 2.4 430

108 145 396 1.2 2.9 6.9 3.6 2.0 395

51 60 325 15 1.1 24 0.3 1 390

24 195 405 15 1.8 11 0.3 2 278

60 116 450 27 1.4 31 0.6 2 297

4.3 1.8 48.0 6.8 5.6 0.7 22.0

6.7 1.7 24.0 7.1 9.6 0.3 18.0

4.1 0.9 52.0 17.0 2.8 0.2 19.0

4.3 1.1 18.0 14.0 5.2 0.4 40.0

56

C. M. Rosell and P. Koehler

tocotrienols, which has been related with a reduction of the serum LDL cholesterol. In wheat, the tocol content varies with the variety; those with blue aleurone or purple pericarp contain higher tocols content than varieties with yellow or white grains [156]. Therefore, milling of cereals into white flour will remove most of the vitamins. Consequently, the use of whole grain products or products enriched in vitamin-containing tissues will be of nutritional benefit for the consumer.

3.6.3 Bioactive Compounds or Phytochemicals Cereal grains also contain important bioactive compounds, such as phenolic compounds that are the most abundant phytochemicals with antioxidant activity. Since the most important vitamins, minerals, and antioxidants are located in the bran, tangential abrasive dehulling, pearling, or debranning have been used to obtain brans enriched in bioactive compounds [156]. Polyphenol compounds include phenolic acids, flavonoids, and lignans. Alkylresorcinol is the major polyphenol present in whole grain wheats, whereas ferulic acid is the most important phenolic acid located in the bran, having antioxidant and anti-inflammatory effects. When comparing the amount of those in wheat, barley, rye, and oat, the highest value was found in barley > oat > wheat and rye [157]. Although the antioxidant activity greatly depends on the composition of phenolic compounds and their individual antioxidant activity, the presence of other compounds with antioxidant activities like phytosterols, saponins, phytoestrogens, carotenoids, tocopherols, and tocotrienols, also play a role. Within the low MW phenolics, quercetin is the most abundant in wheat germ [158]. Regarding carotenoids, their contents vary between 1.6 and 4.9 μg/g, and the most abundant one is lutein, followed by zeaxanthin. The role of the different cereals on oxidative stress and inflammation has been associated with their antioxidant activities, particularly in the case of oat, barley, rice, wheat, and rye [159]. In the case of oat, the antioxidant activity is related to avenanthramides (abundant polyphenol), tocopherols, tocotrienols, β-glucan, and phenolic compounds [159]. Rice also contains important antioxidant components, including phenolic compounds, γ-oryzanol, carotenoids (mainly lutein), tocopherols, and tocotrienols, which are much higher in red and black rice varieties. Particularly, the rice γ-oryzanol has shown cytotoxic effects on tumor cell lines, and that activity level is dependent on the synergistic effect with other bioactive compounds [160]. Additionally, bioactive compounds present in rice varieties have been described as effective antimicrobial agents, which opens the possibility of using rice bran as a natural preservative [160]. Sorghum has also been described as a good source of phenolic compounds, with flavonoids present in most of them and condensed tannins only in the pigmented varieties [161]. Those tannins are composed of flavan-3-ols of up to 8–10  units and have excellent antioxidant properties. Remarkable are sorghum anthocyanins, particularly 3-deoxyanthocyanins (luteolinidin and apigenidin) that could act as potent colorants at high pH.  In wheat, the aleurone is the kernel part with much higher antioxidant capacity due to its high content in ferulic acid [159]. Conversely, the outer layers of rye are responsible of

3  Chemistry of Cereal Grains

57

the antioxidant activity due to their high content in phenolic lipids like alkylresorcinols. Considering the health aspects of the bioactive compounds, some breeding programs are focused on varying the levels of total phenolics and carotenoids in wheats because the concentration of those components is linked to the grain genotype [162]. It has been confirmed that einkorn, emmer, and Khorasan wheat have a higher content of lutein than bread wheat, but not other bioactive compounds [152]. In addition, the contents of β-carotene and γ-tocotrienol decrease with the grain maturation; thus, immature rice grain was suggested as a good source of bioactive compounds [163]. Another approach to increase the level of bioactive components in cereal grains is the biological activation that occurs during grain germination, which is an emerging technique to produce health promoting compounds in cereals [164].

3.6.4 Antinutrients Cereals also contain compounds that reduce the nutritional value of essential nutrients such as starch, proteins, and minerals that are referred to as antinutritional factors. This group includes phytate, saponins, tannins, oxalates, and cyanogenic glycosides. Phytates or inositol hexakisphosphate are the chemical form in which phosphorous is present in cereals. Phosphorous bioavailability can be increased by its dephosphorylation using the phytase activity [165], but also selected lactic acid bacteria [166] and some bifidobacteria [167] can decrease the phytate content increasing the content of available phosphorous.

Appendix AX Arabinoxylans DSC Differential scanning calorimetry GMP Glutenin macropolymer GS Glutenin subunits HMW High-molecular-weight HPLC High-performance liquid chromatography LMW Low-molecular-weight m/a Monomeric/aggregated MMW Medium-molecular-weight MW Molecular weight MWD Molecular weight distribution NSP Nonstarch polysaccharides RDS Rapid digestible starch RS Resistant starch SANS Small angle neutron scattering SAXS Small-angle X-ray scattering SDS Slowly digestible starch

58

C. M. Rosell and P. Koehler

SDS-PAGE Sodium dodecyl sulfate Polyacrylamide gel electrophoresis TG Transglutaminase WEAX Water-extractable arabinoxylans WUAX Water-unextractable arabinoxylans XRD X-ray diffraction

References 1. Food and Agriculture Organization of the United Nations (2020) FAOSTAT Database. http:// faostat.fao.org/site/567/default.aspx#ancor. Accessed 18 November 2020 2. Jouve N, Diez N, Rodriguez M (1980) C-banding in 6x-triticale-x secale-cereale l-hybrid cytogenetics. Theor Appl Genet 57(2):75–79. https://doi.org/10.1007/bf00745033 3. Martin A, Alvarez JB, Martin LM, Barro F, Ballesteros J (1999) The development of tritordeum: a novel cereal for food processing. J Cereal Sci 30(2):85–95. https://doi.org/10.1006/ jcrs.1998.0235 4. Furbank RT, Sharwood R, Estavillo GM, Silva-Perez V, Condon AG (2020) Photons to food: genetic improvement of cereal crop photosynthesis. J Exp Bot 71(7):2226–2238. https://doi. org/10.1093/jxb/eraa077 5. Geng P, Sun JH, Chen P, Li YF, Peng B, Harnly JM et al (2020) A systematic approach to determine the impact of elevated CO2 levels on the chemical composition of wheat (Triticum aestivum). J Cereal Sci 95:103020. https://doi.org/10.1016/j.jcs.2020.103020 6. Brouns F, Hemery Y, Price R, Anson NM (2012) Wheat aleurone: separation, composition, health aspects, and potential food use. Crit Rev Food Sci Nutr 52(6):553–568. https://doi. org/10.1080/10408398.2011.589540 7. Zhao LX, Cai CH, Wei CX (2020) An image processing method for investigating the morphology of cereal endosperm cells. Biotech Histochem 95(4):249–261. https://doi.org/10.108 0/10520295.2019.1677940 8. Belitz H-D, Grosch W (2009) P S. Cereals and cereal products. In: Belitz H-D, Grosch W, Schieberle P (eds) Food chemistry, 4th edn. Springer, Berlin, pp 670–675 9. Souci SW, Fachmann WHK (2008) Deutsche Forschungsanstalt für Lebensmittelchemie (ed) Food composition and nutrition tables. Deutsche Forschungsanstalt für Lebensmittelchemie. MedPharm Scientific Publishers, Stuttgart 10. Arzani A, Ashraf M (2017) Cultivated ancient wheats (Triticum spp.): a potential source of health-beneficial food products. Compr Rev Food Sci Food Saf 16(3):477–488. https://doi. org/10.1111/1541-­4337.12262 11. Brandolini A, Hidalgo A, Gabriele S, Heun M (2015) Chemical composition of wild and feral diploid wheats and their bearing on domesticated wheats. J Cereal Sci 63:122–127. https:// doi.org/10.1016/j.jcs.2015.03.005 12. Dinu M, Whittaker A, Pagliai G, Benedettelli S, Sofi F (2018) Ancient wheat species and human health: Biochemical and clinical implications. J Nutr Biochem 52:1–9. https://doi. org/10.1016/j.jnutbio.2017.09.001 13. Gulati P, Brahma S, Graybosch RA, Chen YH, Rose DJ (2020) In vitro digestibility of proteins from historical and modern wheat cultivars. J Sci Food Agric 100(6):2579–2584. https:// doi.org/10.1002/jsfa.10283 14. Jane JL, Kasemsuwan T, Leas S, Zobel H, Robyt JF (1994) Anthology of starch granule morphology by scanning electron-microscopy. Starch-Starke 46(4):121–129. https://doi. org/10.1002/star.19940460402 15. Cai CH, Huang J, Zhao LX, Liu QQ, Zhang CQ, Wei CX (2014) Heterogeneous structure and spatial distribution in endosperm of high-amylose rice starch granules with different morphologies. J Agric Food Chem 62(41):10143–10152. https://doi.org/10.1021/jf502341q

3  Chemistry of Cereal Grains

59

16. Tester RF, Karkalas J, Qi X (2004) Starch - composition, fine structure and architecture. J Cereal Sci 39(2):151–165. https://doi.org/10.1016/j.jcs.2003.12.001 17. Van Hung P, Maeda T, Fujita M, Morita N (2007) Dough properties and breadmaking qualities of whole waxy wheat flour and effects of additional enzymes. J Sci Food Agric 87(13):2538–2543. https://doi.org/10.1002/jsfa.3025 18. Geera BP, Nelson JE, Souza E, Huber KC (2006) Composition and properties of A- and B-type starch granules of wild-type, partial waxy, and waxy soft wheat. Cereal Chem 83(5):551–557. https://doi.org/10.1094/cc-­83-­0551 19. Bertoft E, Piyachomkwan K, Chatakanonda P, Sriroth K (2008) Internal unit chain composition in amylopectins. Carbohydr Polym 74(3):527–543. https://doi.org/10.1016/j. carbpol.2008.04.011 20. Hejazi M, Fettke J, Paris O, Steup M (2009) The two plastidial starch-related dikinases sequentially phosphorylate glucosyl residues at the surface of both the a- and b-type allomorphs of crystallized maltodextrins but the mode of action differs. Plant Physiol 150(2):962–976. https://doi.org/10.1104/pp.109.138750 21. Jenkins JPJ, Cameron RE, Donald AM (1993) A universal feature in the structure of starch granules from different botanical sources. Starch-Starke 45(12):417–420 22. Gallant DJ, Bouchet B, Baldwin PM (1997) Microscopy of starch: evidence of a new level of granule organization. Carbohydr Polym 32(3–4):177–191. https://doi.org/10.1016/ s0144-­8617(97)00008-­8 23. Blazek J, Gilbert EP (2011) Application of small-angle X-ray and neutron scattering techniques to the characterisation of starch structure: A review. Carbohydr Polym 85(2):281–293. https://doi.org/10.1016/j.carbpol.2011.02.041 24. Bertoft E (2013) On the building block and backbone concepts of amylopectin structure. Cereal Chem 90(4):294–311. https://doi.org/10.1094/cchem-­01-­13-­0004-­fi 25. Qi X, Tester RF (2016) Effect of composition and structure of native starch granules on their susceptibility to hydrolysis by amylase enzymes. Starch-Starke 68(9–10):811–815. https:// doi.org/10.1002/star.201600063 26. Jayakody L, Hoover R (2008) Effect of annealing on the molecular structure and physicochemical properties of starches from different botanical origins - a review. Carbohydr Polym 74(3):691–703. https://doi.org/10.1016/j.carbpol.2008.04.032 27. Vamadevan V, Bertoft E (2020) Observations on the impact of amylopectin and amylose structure on the swelling of starch granules. Food Hydrocoll 103:105663. https://doi. org/10.1016/j.foodhyd.2020.105663 28. Hsieh CF, Liu WC, Whaley JK, Shi YC (2019) Structure and functional properties of waxy starches. Food Hydrocoll 94:238–254. https://doi.org/10.1016/j.foodhyd.2019.03.026 29. Guo L (2018) In vitro amylase hydrolysis of amylopectins from cereal starches based on molecular structure of amylopectins. Food Hydrocoll 77:238–247. https://doi.org/10.1016/j. foodhyd.2017.09.039 30. Waigh TA, Gidley MJ, Komanshek BU, Donald AM (2000) The phase transformations in starch during gelatinisation: a liquid crystalline approach. Carbohydr Res 328(2):165–176. https://doi.org/10.1016/s0008-­6215(00)00098-­7 31. Garzon R, Rosell CM (2021) Rapid assessment of starch pasting using a rapid force analyzer. Cereal Chem 98:305–314. https://doi.org/10.1002/cche.10365 32. Miles MJ, Morris VJ, Orford PD, Ring SG (1985) The roles of amylose and amylopectin in the gelation and retrogradation of starch. Carbohydr Res 135(2):271–281. https://doi. org/10.1016/s0008-­6215(00)90778-­x 33. Vamadevan V, Blennow A, Buleon A, Goldstein A, Bertoft E (2018) Distinct properties and structures among b-crystalline starch granules. Starch-Starke 70(3–4):1700240. https://doi. org/10.1002/star.201700240 34. Hug-Iten S, Escher F, Conde-Petit B (2003) Staling of bread: role of amylose and amylopectin and influence of starch-degrading enzymes. Cereal Chem 80(6):654–661

60

C. M. Rosell and P. Koehler

35. Morrison WR, Law RV, Snape CE (1993) Evidence for inclusion complexes of lipids with V-amylose in maize, rice and oat starches. J Cereal Sci 18(2):107–110. https://doi. org/10.1006/jcrs.1993.1039 36. Gray JA, BeMiller JN (2003) Bread staling: molecular basis and control. Compr Rev Food Sci Food Saf 2(1):1–21. https://doi.org/10.1111/j.1541-­4337.2003.tb00011.x 37. Barcenas M, Haros M, Rosell C (2003) An approach to studying the effect of different bread improvers on the staling of pre-baked frozen bread. Eur Food Res Technol 218(1):56–61. https://doi.org/10.1007/s00217-­003-­0816-­y 38. Rojas JA, Rosell CM, Benedito de Barber C (2001) Role of maltodextrins in the staling of starch gels. Eur Food Res Technol 212(3):364–368 39. Blazek J, Gilbert EP, Copeland L (2011) Effects of monoglycerides on pasting properties of wheat starch after repeated heating and cooling. J Cereal Sci 54(1):151–159. https://doi. org/10.1016/j.jcs.2011.02.014 40. Aziz MS, Nawaz R, Haider N, Rehman ZU, Aamir AUH, Imran M (2019) Starch composition, antioxidant potential, and glycemic indices of various varieties of Triticum aesitivum L. and Zea mays L. available in Pakistan. J Food Biochem 43(8):e12943. https://doi. org/10.1111/jfbc.12943 41. Englyst HN, Kingman SM, Cummings JH (1992) Classification and measurement of nutritionally important starch fractions. Eur J Clin Nutr 46:S33–S50 42. Dhanavath S, Rao U (2017) Nutritional and nutraceutical properties of triticum dicoccum wheat and its health benefits: an overview. J Food Sci 82(10):2243–2250. https://doi. org/10.1111/1750-­3841.13844 43. Butterworth PJ, Warren FJ, Grassby T, Patel H, Ellis PR (2012) Analysis of starch amylolysis using plots for first-order kinetics. Carbohydr Polym 87(3):2189–2197. https://doi. org/10.1016/j.carbpol.2011.10.048 44. Magallanes-Cruz PA, Flores-Silva PC, Bello-Perez LA (2017) Starch structure influences its digestibility: A review. J Food Sci 82(9):2016–2023. https://doi. org/10.1111/1750-­3841.13809 45. Dupuis JH, Liu Q, Yada RY (2014) Methodologies for increasing the resistant starch content of food starches: A review. Compr Rev Food Sci Food Saf 13(6):1219–1234. https://doi. org/10.1111/1541-­4337.12104 46. EFSA (2011) Panel on Dietetic Products, Nutrition and Allergies (NDA). Scientific Opinion on the substantiation of health claims related to resistant starch and reduction of post - prandial glycaemic responses (ID 681),“digestive health benefits”(ID 682) and “favours a normal colon metabolism”(ID 783) pursuant to Article 13 (1) of Regulation (EC) No 1924/2006. EFSA Journal 9(4):2024 47. Huang J, Shang ZQ, Man JM, Liu QQ, Zhu CJ, Wei CX (2015) Comparison of molecular structures and functional properties of high-amylose starches from rice transgenic line and commercial maize. Food Hydrocoll 46:172–179. https://doi.org/10.1016/j.foodhyd.2014.12.019 48. Hemdane S, Jacobs PJ, Dornez E, Verspreet J, Delcour JA, Courtin CM (2016) Wheat (Triticum aestivum L.) Bran in bread making: a critical review. Compr Rev Food Sci Food Saf 15(1):28–42. https://doi.org/10.1111/1541-­4337.12176 49. Anderson JW, Baird P, Davis RH Jr, Ferreri S, Knudtson M, Koraym A et al (2009) Health benefits of dietary fiber. Nutr Rev 67(4):188–205 50. Goesaert H, Brijs K, Veraverbeke WS, Courtin CM, Gebruers K, Delcour JA (2005) Wheat flour constituents: how they impact bread quality, and how to impact their functionality. Trends Food Sci Technol 16(1–3):12–30. https://doi.org/10.1016/j.tifs.2004.02.011 51. Gebruers K, Dornez E, Bedo Z, Rakszegi M, Fras A, Boros D et  al (2010) Environment and genotype effects on the content of dietary fiber and its components in wheat in the HEALTHGRAIN diversity screen. J Agric Food Chem 58(17):9353–9361. https://doi. org/10.1021/jf100447g 52. Lu SY, Flanagan BM, Williams BA, Mikkelsen D, Gidley MJ (2020) Cell wall architecture as well as chemical composition determines fermentation of wheat cell walls by a faecal inoculum. Food Hydrocoll 107:105858. https://doi.org/10.1016/j.foodhyd.2020.105858

3  Chemistry of Cereal Grains

61

53. Kiszonas AM, Fuerst EP, Morris CF (2013) Wheat arabinoxylan structure provides insight into function. Cereal Chem 90(4):387–395. https://doi.org/10.1094/cchem-­02-­13-­0025-­fi 54. Courtin CM, Delcour JA (2002) Arabinoxylans and endoxylanases in wheat flour bread-­ making. J Cereal Sci 35(3):225–243. https://doi.org/10.1006/jcrs.2001.0433 55. Freeman J, Lovegrove A, Wilkinson MD, Saulnier L, Shewry PR, Mitchell RAC (2016) Effect of suppression of arabinoxylan synthetic genes in wheat endosperm on chain length of ­ arabinoxylan and extract viscosity. Plant Biotechnol J 14(1):109–116. https://doi. org/10.1111/pbi.12361 56. Verhertbruggen Y, Falourd X, Sterner M, Guillon F, Girousse C, Foucat L et  al (2019) Challenging the putative structure of mannan in wheat (Triticum aestivum) endosperm. Carbohydr Polym 224:9. https://doi.org/10.1016/j.carbpol.2019.115063 57. Izydorczyk MS, Biliaderis CG (1994) Studies on the structure of wheat-endosperm arabinoxylans. Carbohydr Polym 24(1):61–71. https://doi.org/10.1016/0144-­8617(94)90118-­x 58. Courtin CM, Gelders GG, Delcour JA (2001) Use of two endoxylanases with different substrate selectivity for understanding arabinoxylan functionality in wheat flour breadmaking. Cereal Chem 78(5):564–571. https://doi.org/10.1094/cchem.2001.78.5.564 59. Biliaderis CG, Izydorczyk MS, Rattan O (1995) Effect of arabinoxylans on bread-making quality of wheat flours. Food Chem 53(2):165–171. https://doi. org/10.1016/0308-­8146(95)90783-­4 60. Hoseney RC, Faubion JM (1981) A mechanism for the oxidative gelation of wheat-flour water-soluble pentosans. Cereal Chem 58(5):421–424 61. Cleemput G, Booij C, Hessing M, Gruppen H, Delcour JA (1997) Solubilisation and changes in molecular weight distribution of arabinoxylans and protein in wheat flours during bread-­ making, and the effects of endogenous arabinoxylan hydrolysing enzymes. J Cereal Sci 26(1):55–66. https://doi.org/10.1006/jcrs.1996.0099 62. Labat E, Morel MH, Rouau X (2000) Effects of laccase and ferulic acid on wheat flour doughs. Cereal Chem 77(6):823–828. https://doi.org/10.1094/cchem.2000.77.6.823 63. Espinosa-Ramirez J, Garzon R, Serna-Saldivar SO, Rosell CM (2020) Exploring the potential of arabinoxylan as structuring agent in model systems for gluten-free yeast-leavened breads. J Cereal Sci 95:103080. https://doi.org/10.1016/j.jcs.2020.103080 64. Driss D, Bhiri F, Siela M, Bessess S, Chaabouni S, Ghorbel R (2013) Improvement of breadmaking quality by xylanase GH11 from Penicillium occitanis Pol6. J Texture Stud 44(1):75–84. https://doi.org/10.1111/j.1745-­4603.2012.00367.x 65. Lebesi DM, Tzia C (2012) Use of endoxylanase treated cereal brans for development of dietary fiber enriched cakes. Innov Food Sci Emerg Technol 13:207–214. https://doi. org/10.1016/j.ifset.2011.08.001 66. Butt MS, Tahir-Nadeem M, Ahmad Z, Sultan MT (2008) Xylanases and their applications in baking industry. Food Technol Biotechnol 46(1):22–31 67. Delcour JA, Van Win H, Grobet PJ (1999) Distribution and structural variation of arabinoxylans in common wheat mill streams. J Agric Food Chem 47(1):271–275. https://doi. org/10.1021/jf9805294 68. Malunga LN, Izydorczyk M, Beta T (2017) Antiglycemic effect of water extractable arabinoxylan from wheat aleurone and bran. J Nutr Metabol 2017. https://doi. org/10.1155/2017/5784759 69. Verspreet J, Dornez E, Van den Ende W, Delcour JA, Courtin CM (2015) Cereal grain fructans: structure, variability and potential health effects. Trends Food Sci Technol 43(1):32–42 70. Laurent J, Timmermans E, Struyf N, Verstrepen KJ, Courtin CM (2020) Variability in yeast invertase activity determines the extent of fructan hydrolysis during wheat dough fermentation and final FODMAP levels in bread. Int J Food Microbiol 326:108648 71. Chateigner-Boutin AL, Bouchet B, Alvarado C, Bakan B, Guillon F (2014) The wheat grain contains pectic domains exhibiting specific spatial and development-associated distribution. PLoS One 9(2):e89620. https://doi.org/10.1371/journal.pone.0089620 72. Osborne TB, Harris IF (1906) The chemistry of the protein bodies of the wheat kernel. Part II. Preparation of the proteins in quantity for hydrolysis. Am J Physiol 17(3):223–230

62

C. M. Rosell and P. Koehler

73. Shewry PR (2007) Improving the protein content and composition of cereal grain. J Cereal Sci 46(3):239–250. https://doi.org/10.1016/j.jcs.2007.06.006 74. Hospodarenko HM, Karpenko VP, Liubych VV, Novikov VV (2018) Characterization of amino acid content of grain of new wheat varieties and lines. Agric Sci Pract 5(3):12–18. https://doi.org/10.15407/agrisp5.03.012 75. Shewry PR, Tatham AS (1990) The prolamin storage proteins of cereal seeds - structure and evolution. Biochem J 267(1):1–12 76. Wieser H, Koehler P (2008) The biochemical basis of celiac disease. Cereal Chem 85(1):1–13. https://doi.org/10.1094/cchem-­85-­1-­0001 77. Payne PI, Nightingale MA, Krattiger AF, Holt LM (1987) The relationship between hmw glutenin subunit composition and the bread-making quality of british-grown wheat-varieties. J Sci Food Agric 40(1):51–65. https://doi.org/10.1002/jsfa.2740400108 78. Grosch W, Wieser H (1999) Redox reactions in wheat dough as affected by ascorbic acid. J Cereal Sci 29(1):1–16. https://doi.org/10.1006/jcrs.1998.0218 79. Gellrich C, Schieberle P, Wieser H (2005) Studies of partial amino acid sequences of gamma-­40k secalins of rye. Cereal Chem 82(5):541–545. https://doi.org/10.1094/cc-­82-­0541 80. http://pir.georgetown.edu DUPKT 81. Robert LS, Nozzolillo C, Altosaar I (1985) Characterization of oat (Avena-sativa L) residual proteins. Cereal Chem 62(4):276–279 82. Wieser H (2000) Comparative investigations of gluten proteins from different wheat species I. Qualitative and quantitative composition of gluten protein types. Eur Food Res Technol 211(4):262–268. https://doi.org/10.1007/s002170000165 83. Brzozowski B, Stasiewicz K (2017) Effects of water stress on the composition and immunoreactive properties of gliadins from two wheat cultivars: Nawra and Tonacja. J Sci Food Agric 97(4):1134–1142. https://doi.org/10.1002/jsfa.7839 84. Tuckova L, Sanchez D, Tlaskalova-Hogenova H, Panzner P (2012) Wheat-dependent exercise-induced anaphylaxis: pros and cons of recombinant omega-5gliadin and glutenins, or their epitope peptides, in diagnosis. Clin Exp Allergy 42(8):1146–1149. https://doi. org/10.1111/j.1365-­2222.2012.04050.x 85. Gellrich C, Schieberle P, Weiser H (2003) Biochemical characterization and quantification of the storage protein (secalin) types in rye flour. Cereal Chem 80(1):102–109. https://doi. org/10.1094/cchem.2003.80.1.102 86. Lange M, Vincze E, Wieser H, Schjoerring JK, Holm PB (2007) Suppression of C-hordein synthesis in barley by antisense constructs results in a more balanced amino acid composition. J Agric Food Chem 55(15):6074–6081. https://doi.org/10.1021/jf0709505 87. Lutz E, Wieser H, Koehler P (2012) Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation. J Agric Food Chem 60(14):3708–3716. https://doi.org/10.1021/jf204973u 88. Weegels PL, Hamer RJ, Schofield JD (1996) Functional properties of wheat glutenin. J Cereal Sci 23(1):1–18. https://doi.org/10.1006/jcrs.1996.0001 89. Schmid M, Wieser H, Koehler P (2017) Disulphide structure of high-molecular-weight (HMW-) gliadins as affected by terminators. J Cereal Sci 78:66–74. https://doi.org/10.1016/j. jcs.2016.12.007 90. Barro F, Barcelo P, Lazzeri PA, Shewry PR, Ballesteros J, Martin A (2003) Functional properties of flours from field grown transgenic wheat lines expressing the HMW glutenin subunit 1Ax1 and 1Dx5 genes. Mol Breed 12(3):223–229. https://doi.org/10.1023/a:1026367214120 91. Wieser H, Seilmeier W, Kieffer R, Altpeter F (2005) Flour protein composition and functional properties of transgenic rye lines expressing HMW subunit genes of wheat. Cereal Chem 82(5):594–600. https://doi.org/10.1094/cc-­82-­0594 92. Altpeter F, Popelka JC, Wieser H (2004) Stable expression of 1Dx5 and 1Dy10 high-­ molecular-­weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutelin fraction in rye flour. Plant Mol Biol 54(6):783–792. https://doi.org/10.1007/ s11103-­004-­0122-­5

3  Chemistry of Cereal Grains

63

93. Thanhaeuser SM, Wieser H, Koehler P (2014) Correlation of quality parameters with the baking performance of wheat flours. Cereal Chem 91(4):333–341. https://doi.org/10.1094/ cchem-­09-­13-­0194-­cesi 94. Hajas L, Scherf KA, Torok K, Bugyi Z, Schall E, Poms RE et al (2018) Variation in protein composition among wheat (Triticum aestivum L.) cultivars to identify cultivars suitable as reference material for wheat gluten analysis. Food Chem 267:387–394. https://doi. org/10.1016/j.foodchem.2017.05.005 95. Wieser H, Mueller KJ, Koehler P (2009) Studies on the protein composition and baking quality of einkorn lines. Eur Food Res Technol 229(3):523–532. https://doi.org/10.1007/ s00217-­009-­1081-­5 96. Geisslitz S, Wieser H, Scherf KA, Koehler P (2018) Gluten protein composition and aggregation properties as predictors for bread volume of common wheat, spelt, durum wheat, emmer and einkorn. J Cereal Sci 83:204–212. https://doi.org/10.1016/j.jcs.2018.08.012 97. Abedi E, Pourmohammadi K (2020) The effect of redox agents on conformation and structure characterization of gluten protein: an extensive review. Food Sci Nutr 8(12):6301–6319. https://doi.org/10.1002/fsn3.1937 98. Bonet A, Rosell CM, Caballero PA, Gomez M, Perez-Munuera I, Lluch MA (2006) Glucose oxidase effect on dough rheology and bread quality: a study from macroscopic to molecular level. Food Chem 99(2):408–415 99. Tang KX, Zhao CJ, Gänzle MG (2017) Effect of glutathione on the taste and texture of type I sourdough bread. J Agric Food Chem 65(21):4321–4328 100. Hanft F, Koehler P (2005) Quantitation of dityrosine in wheat flour and dough by liquid chromatography-tandem mass spectrometry. J Agric Food Chem 53(7):2418–2423. https:// doi.org/10.1021/jf048005t 101. Bauer N, Koehler P, Wieser H, Schieberle P (2003) Studies on effects of microbial transglutaminase on gluten proteins of wheat. I. Biochemical analysis. Cereal Chem 80(6):781–786. https://doi.org/10.1094/cchem.2003.80.6.781 102. Rosell CM, Wang J, Aja S, Bean S, Lookhart G (2003) Wheat flour proteins as affected by transglutaminase and glucose oxidase. Cereal Chem 80(1):52–55 103. Amiri A, Shahedi M, Kadivar M (2017) Structural properties of gluten modified by ascorbic acid and transglutaminase. Int J Food Prop 20:1588–1599. https://doi.org/10.1080/1094291 2.2017.1349141 104. Avramenko NA, Hopkins EJ, Hucl P, Scanlon MG, Nickerson MT (2020) Role of NaCl level on the handling and water mobility in dough prepared from four wheat cultivars. J Texture Stud 51(5):766–778. https://doi.org/10.1111/jtxs.12525 105. Lucas I, Becker T, Jekle M (2018) Gluten polymer networks. A microstructural classification in complex systems. Polymers 10(6):617. https://doi.org/10.3390/polym10060617 106. Tatham AS, Miflin BJ, Shewry PR (1985) The beta-turn conformation in wheat gluten proteins - relationship to gluten elasticity. Cereal Chem 62(5):405–412 107. Muller S, Wieser H (1997) The location of disulphide bonds in monomeric gamma-type gliadins. J Cereal Sci 26(2):169–176. https://doi.org/10.1006/s0733-­5210(97)90100-­4 108. Rosell CM, Altamirano-Fortoul R, Don C, Dubat A (2013) Thermomechanically induced protein aggregation and starch structural changes in wheat flour dough. Cereal Chem 90(2):89–100. https://doi.org/10.1094/cchem-­05-­12-­0056-­r 109. Baudouin F, Nogueira TL, van der Mijnsbrugge A, Frederix S, Redl A, Morel MH (2020) Mechanochemical activation of gluten network development during dough mixing. J Food Eng 283:110035. https://doi.org/10.1016/j.jfoodeng.2020.110035 110. Li MF, Yue QH, Liu C, Zheng XL, Hong J, Li LM et al (2020) Effect of gliadin/glutenin ratio on pasting, thermal, and structural properties of wheat starch. J Cereal Sci 93:102973. https:// doi.org/10.1016/j.jcs.2020.102973 111. Scherf KA, Koehler P, Wieser H (2016) Gluten and wheat sensitivities - an overview. J Cereal Sci 67:2–11. https://doi.org/10.1016/j.jcs.2015.07.008 112. Esen A (1987) A proposed nomenclature for the alcohol-soluble proteins (zeins) of maize (Zea-mays-L). J Cereal Sci 5(2):117–128. https://doi.org/10.1016/s0733-­5210(87)80015-­2

64

C. M. Rosell and P. Koehler

113. Wilson CM (1991) Multiple zeins from maize endosperms characterized by reversed-­ phase high-performance liquid-chromatography. Plant Physiol 95(3):777–786. https://doi. org/10.1104/pp.95.3.777 114. Shull JM, Watterson JJ, Kirleis AW (1991) Proposed nomenclature for the alcohol-soluble proteins (kafirins) of sorghum-bicolor (L Moench) based on molecular-weight, solubility, and structure. J Agric Food Chem 39(1):83–87. https://doi.org/10.1021/jf00001a015 115. Hamaker BR, Mohamed AA, Habben JE, Huang CP, Larkins BA (1995) Efficient procedure for extracting maize and sorghum kernel proteins reveals higher prolamin contents than the conventional method. Cereal Chem 72(6):583–588 116. Watterson JJ, Shull JM, Kirleis AW (1993) Quantitation of alpha-karafins, beta-karafins, and gamma-kafirins in vitreous and opaque endosperm of sorghum-bicolor. Cereal Chem 70(4):452–457 117. Danno G, Natake M (1980) Isolation of foxtail millet proteins and their subunit structure. Agric Biol Chem 44(4):913–918. https://doi.org/10.1080/00021369.1980.10864039 118. Filova V, Valentova O, Daussant J, Kas J (1992) Some methods for isolation and assays of enzymes occurring in cereals and legumes. Food Rev Intl 8(4):559–572. https://doi. org/10.1080/87559129209540952 119. Guzman-Ortiz FA, Castro-Rosas J, Gomez-Aldapa CA, Mora-Escobedo R, Rojas-Leon A, Rodriguez-Marin ML et al (2019) Enzyme activity during germination of different cereals: a review. Food Rev Intl 35(3):177–200. https://doi.org/10.1080/87559129.2018.1514623 120. Muralikrishna G, Nirmala M (2005) Cereal alpha-amylases - an overview. Carbohydr Polym 60(2):163–173. https://doi.org/10.1016/j.carbpol.2004.12.002 121. Rosell C, Haros M, Escriva C, de Barber C (2001) Experimental approach to optimize the use of alpha-amylases in breadmaking. J Agric Food Chem 49(6):2973–2977 122. Gong LX, Feng DN, Wang TX, Ren YQ, Liu YL, Wang J (2020) Inhibitors of alpha-amylase and alpha-glucosidase: potential linkage for whole cereal foods on prevention of hyperglycemia. Food Sci Nutr 8(12):6320–6337. https://doi.org/10.1002/fsn3.1987 123. Reig-Otero Y, Manes J, Manyes L (2018) Amylase-trypsin inhibitors in wheat and other cereals as potential activators of the effects of nonceliac gluten sensitivity. J Med Food 21(3):207–214. https://doi.org/10.1089/jmf.2017.0018 124. Victor Z, Kahn M, Tenzer S, Schild H, Schuppan D (2014) Cereal alpha-amylase/trypsin inhibitors (atis) as nutritionals triggers of innate immunity: bioactivity in various foods. Gastroenterology 146(5):S348–S3S9 125. Goesaert H, Elliott G, Kroon PA, Gebruers K, Courtin CM, Robben J et al (2004) Occurrence of proteinaceous endoxylanase inhibitors in cereals. Biochim Biophy Acta Proteins Proteom 1696(2):193–202. https://doi.org/10.1016/j.bbapap.2003.08.015 126. Goesaert H, Gebruers K, Brijs K, Courtin CM, Delcour JA (2003) TAXI type endoxylanase inhibitors in different cereals. J Agric Food Chem 51(13):3770–3775. https://doi.org/10.1021/ jf0262155 127. Junker Y, Zeissig S, Kim SJ, Barisani D, Wieser H, Leffler DA et al (2012) Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4. J Exp Med 209(13):2395–2408. https://doi.org/10.1084/jem.20102660 128. Cavazos A, de Mejia EG (2013) Identification of bioactive peptides from cereal storage proteins and their potential role in prevention of chronic diseases. Compr Rev Food Sci Food Saf 12(4):364–380. https://doi.org/10.1111/1541-­4337.12017 129. Liu KS (2011) Comparison of lipid content and fatty acid composition and their distribution within seeds of 5 small grain species. J Food Sci 76(2):C334–CC42. https://doi. org/10.1111/j.1750-­3841.2010.02038.x 130. Guven M, Kara HH (2016) Some chemical and physical properties, fatty acid composition and bioactive compounds of wheat germ oils extracted from different wheat cultivars. J Agric Sci Tarim Bili Dergisi 22(3):433–443 131. Morrison WR, Mann DL, Soon W, Coventry AM (1975) Selective extraction and quantitative-­ analysis of non-starch and starch lipids from wheat-flour. J Sci Food Agric 26(4):507–521. https://doi.org/10.1002/jsfa.2740260416

3  Chemistry of Cereal Grains

65

132. Przybylski R, Eskin NAM (1994) 2 simplified approaches to the analysis of cereal lipids. Food Chem 51(2):231–235. https://doi.org/10.1016/0308-­8146(94)90263-­1 133. Hammann S, Korf A, Bull ID, Hayen H, Cramp LJE (2019) Lipid profiling and analytical discrimination of seven cereals using high temperature gas chromatography coupled to high resolution quadrupole time-of-flight mass spectrometry. Food Chem 282:27–35. https://doi. org/10.1016/j.foodchem.2018.12.109 134. Azudin MN, Morrison WR (1986) Nonstarch lipids and starch lipids in milled rice. J Cereal Sci 4(1):23–31. https://doi.org/10.1016/s0733-­5210(86)80004-­2 135. Finnie SM, Jeannotte R, Morris CF, Faubion JM (2010) Variation in polar lipid composition among near-isogenic wheat lines possessing different puroindoline haplotypes. J Cereal Sci 51(1):66–72. https://doi.org/10.1016/j.jcs.2009.09.006 136. McCann TH, Small DM, Batey IL, Wrigley CW, Day L (2009) Protein-lipid interactions in gluten elucidated using acetic-acid fractionation. Food Chem 115(1):105–112. https://doi. org/10.1016/j.foodchem.2008.11.070 137. Bojsen K, Poulsen CH, Soe JB. Method of improving dough and bread quality. 2003 138. Hargin KD, Morrison WR (1980) The distribution of acyl lipids in the germ, aleurone, starch and non-starch endosperm of 4 wheat-varieties. J Sci Food Agric 31(9):877–888. https://doi. org/10.1002/jsfa.2740310904 139. De Brier N, Delcour JA (2017) Pearling affects the lipid content and composition and lipase activity levels of wheat (Triticum aestivum L.) roller milling fractions. Cereal Chem 94(3):588–593. https://doi.org/10.1094/cchem-­09-­16-­0242-­r 140. Konopka I, Rotkiewicz D, Tanska M (2005) Wheat endosperm hardness. Part II. Relationships to content and composition of flour lipids. Eur Food Res Technol 220(1):20–24. https://doi. org/10.1007/s00217-­004-­1038-­7 141. Salt LJ, Gonzalez-Thuillier I, Chope G, Penson S, Tosi P, Haslam RP et al (2018) Intrinsic wheat lipid composition effects the interfacial and foaming properties of dough liquor. Food Hydrocoll 75:211–222. https://doi.org/10.1016/j.foodhyd.2017.08.020 142. Min B, Gonzalez-Thuillier I, Powers SJ, Wilde P, Shewry PR, Haslam RP (2017) Effects of cultivar and nitrogen nutrition on the of wheat flour lipid composition. J Agric Food Chem 65(26):5427–5434. https://doi.org/10.1021/acs.jafc.7b01437 143. Sroan BS, Bean SR, MacRitchie F (2009) Mechanism of gas cell stabilization in bread making. I. The primary gluten-starch matrix. J Cereal Sci 49(1):32–40. https://doi.org/10.1016/j. jcs.2008.07.003 144. Sroan BS, MacRitchie F (2009) Mechanism of gas cell stabilization in breadmaking. II. The secondary liquid lamellae. J Cereal Sci 49(1):41–46. https://doi.org/10.1016/j.jcs.2008.07.004 145. Selmair PL, Koehler P (2009) Molecular structure and baking performance of individual glycolipid classes from lecithins. J Agric Food Chem 57(12):5597–5609. https://doi. org/10.1021/jf9008982 146. Selmair PL, Koehler P (2008) Baking performance of synthetic glycolipids in comparison to commercial surfactants. J Agric Food Chem 56(15):6691–6700. https://doi.org/10.1021/ jf800692b 147. Liu L, Guo Q, He ZH, Xia XC, Waters DLE, Raymond CA et al (2017) Genotypic variation in wheat flour lysophospholipids. Molecules 22(6):909. https://doi.org/10.3390/ molecules22060909 148. Liu L, Tong C, Bao JS, Waters DLE, Rose TJ, King GJ (2014) Determination of starch lysophospholipids in rice using liquid chromatography-mass spectrometry (LC-MS). J Agric Food Chem 62(28):6600–6607. https://doi.org/10.1021/jf500585j 149. Aslam MF, Ellis PR, Berry SE, Latunde-Dada GO, Sharp PA (2018) Enhancing mineral bioavailability from cereals: Current strategies and future perspectives. Nutr Bull 43(2):184–188. https://doi.org/10.1111/nbu.12324 150. Adeyeye A, Ajewole K (1992) Chemical-composition and fatty-acid profiles of cereals in nigeria. Food Chem 44(1):41–44. https://doi.org/10.1016/0308-­8146(92)90255-­z

66

C. M. Rosell and P. Koehler

151. Rodehutscord M, Ruckert C, Maurer HP, Schenkel H, Schipprack W, Knudsen KEB et al (2016) Variation in chemical composition and physical characteristics of cereal grains from different genotypes. Arch Anim Nutr 70(2):87–107. https://doi.org/10.1080/1745039x.2015.1133111 152. Shewry PR, Hey S (2015) Do “ancient” wheat species differ from modern bread wheat in their contents of bioactive components? J Cereal Sci 65:236–243. https://doi.org/10.1016/j. jcs.2015.07.014 153. Lachman J, Hejtmankova A, Orsak M, Popov M, Martinek P (2018) Tocotrienols and tocopherols in colored-grain wheat, tritordeum and barley. Food Chem 240:725–735. https://doi. org/10.1016/j.foodchem.2017.07.123 154. Atanasova-Penichon V, Barreau C, Richard-Forget F (2016) Antioxidant secondary metabolites in cereals: potential involvement in resistance to fusarium and mycotoxin accumulation. Front Microbiol 7:566. https://doi.org/10.3389/fmicb.2016.00566 155. Falk J, Krahnstover A, van der Kooij TAW, Schlensog M, Krupinska K (2004) Tocopherol and tocotrienol accumulation during development of caryopses from barley (Hordeum vulgare L.). Phytochemistry 65(22):2977–2985. https://doi.org/10.1016/j.phytochem.2004.08.047 156. Chen YF, Dunford NT, Goad C (2013) Evaluation of wheat bran obtained by tangential abrasive dehulling device. Food Bioprocess Technol 6(7):1655–1663. https://doi.org/10.1007/ s11947-­012-­0809-­6 157. Zielinski H, Kozlowska H (2000) Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions. J Agric Food Chem 48(6):2008–2016. https://doi.org/10.1021/jf990619o 158. de Vasconcelos M, Bennett R, Castro C, Cardoso P, Saavedra MJ, Rosa EA (2013) Study of composition, stabilization and processing of wheat germ and maize industrial by-products. Ind Crop Prod 42:292–298. https://doi.org/10.1016/j.indcrop.2012.06.007 159. Lee YM, Han SI, Song BC, Yeum KJ (2015) Bioactives in commonly consumed cereal grains: implications for oxidative stress and inflammation. J Med Food 18(11):1179–1186. https://doi.org/10.1089/jmf.2014.3394 160. Castanho A, Lageiro M, Calhelha RC, Ferreira I, Sokovic M, Cunha LM et  al (2019) Exploiting the bioactive properties of -oryzanol from bran of different exotic rice varieties. Food Funct 10(5):2382–2389. https://doi.org/10.1039/c8fo02596g 161. Dykes L, Rooney LW (2006) Sorghum and millet phenols and antioxidants. J Cereal Sci 44(3):236–251 162. Ziegler JU, Schweiggert RM, Carle R (2015) A method for the simultaneous extraction and quantitation of lipophilic antioxidants in Triticum sp by HPLC-DAD/FLD-MSn. J Food Compos Anal 39:94–102. https://doi.org/10.1016/j.jfca.2014.11.011 163. Ji CM, Shin JA, Cho JW, Lee KT (2013) Nutritional evaluation of immature grains in two Korean rice cultivars during maturation. Food Sci Biotechnol 22(4):903–908. https://doi. org/10.1007/s10068-­013-­0162-­1 164. Singh A, Sharma S (2017) Bioactive components and functional properties of biologically activated cereal grains: a bibliographic review. Crit Rev Food Sci Nutr 57(14):3051–3071. https://doi.org/10.1080/10408398.2015.1085828 165. Rosell CM, Santos E, Sanz Penella JM, Haros M (2009) Wholemeal wheat bread: a comparison of different breadmaking processes and fungal phytase addition. J Cereal Sci 50(2):272–277. https://doi.org/10.1016/j.jcs.2009.06.007 166. Palacios MC, Haros M, Sanz Y, Rosell CM (2008) Selection of lactic acid bacteria with high phytate degrading activity for application in whole wheat breadmaking. LWT Food Sci Technol 41(1):82–92 167. Palacios MC, Sanz Y, Haros M, Rosell CM (2006) Application of Bifidobacterium strains to the breadmaking process. Process Biochem 41(12):2434–2440

4

Technology of Sourdough Fermentation and Sourdough Application Markus J. Brandt, Jussi Loponen, and Stefan Cappelle

Sourdough has been produced for millennia in different ways and at different scale and will continue to be for many years to come. Sourdough fermentation technology ranges from small bowls used by artisanal bakers to large, stirred fermentation vessels used by industrial bakeries. Application of sourdough on production of baked goods is not limited to wheat and rye breads, it is used for pizza, viennoise pastry, and sweet baked goods like Panettone and also improves the quality of gluten free baked goods. This chapter aims is to summarize the principles of applied sourdough fermentation technology and sourdough products.

4.1 Fermentation Schemes Based on experience, countless sourdough schemes have been developed and established over the centuries, usually resulting in good quality breads and baked goods. New ones are added, or existing ones are adapted in response to changing milling qualities or working time arrangements, so that in the following only the predominant schemes will be presented. Most of the management schemes are quite traditional and are described in the older literature [1, 2] where the designations of the different sourdough stages Grundsauer (basic sour) and Vollsauer (full sour), which M. J. Brandt (*) Ernst Böcker GmbH & Co. KG, Minden, Germany e-mail: [email protected] J. Loponen Oy Karl Fazer Ab, Vantaa, Finland e-mail: [email protected] S. Cappelle Puratos Group, Groot-Bijgaarden, Belgium e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. Gobbetti, M. Gänzle (eds.), Handbook on Sourdough Biotechnology, https://doi.org/10.1007/978-3-031-23084-4_4

67

68

M. J. Brandt et al.

are still common today in the German-speaking countries, can be found. In the processing of rye, wheat, spelt, and other cereal flours, a basic distinction can be made between single-stage and multi-stage leavening. In the case of single-stage leavening, the sourdough is ripened until it reaches the stationary phase. These doughs change their composition slowly; therefore, they have a high processing tolerance and are used in both craft and industry. As a rule, baker’s yeast must be added to the bread dough to achieve sufficient leavening activity. Sufficient leavening activity can also be achieved in single-stage schemes but this is done at the expense of processing tolerance and results in long proof times. In multistage processing, the lactic acid bacteria and yeasts are kept in an active state by frequent back-slopping, i.e., the organisms are at the exponential phase of growth for much of the fermentation scheme. These sourdoughs should be processed to the point to provide sufficient leavening power for baking without the addition of baker’s yeast. The main difference between artisanal and industrial processing relates to the dough yields. Sourdough fermentation at a larger scale requires liquid sourdoughs to allow transport via pumps.

4.1.1 Rye Processing For the propagation of rye sourdough, several fermentation schemes were developed (Table 4.1) [3]. These schemes all have advantages and disadvantages regarding workload, scheduling, and bread quality. All those fermentation processes are based on two main schemes, the one-stage or multistage procedures. Multistage fermentation (Fig. 4.1) has the advantage that the sourdough microorganisms are maintained in a highly metabolically active state. Usually, these sourdoughs achieve higher concentrations of acetic acid in relation to lactic acid as in one-stage processes. Nevertheless, the working tolerances of these sourdoughs are quite short, and small deviations in the production process may lead to a major impact on sourdough quality and final bread. Because of the lower microbial activity compared to multistage sourdoughs, the one-stage fermentation with fermentation times of 15–24 h are generally easier to handle. Usually, the amount of sourdough starter (mother-dough) used in the recipes is smaller than in multistage processes. These one-stage sourdoughs (uncooled) have a longer process tolerance. The ratio of lactic to acetic acids in one-stage fermented sourdoughs is normally 80:20 to 70:30 (w/w) in contrast to the ratios that are usually found in multistage fermentations (ca. 70:30 to 60:40, w/w). This can be explained by the higher total titratable acidity (tta) and the fact that acetic acid is mainly formed at the beginning of the sourdough fermentations when fructose is available.

4.1.1.1 Industrial Nordic Rye Sourdoughs Industrial sourdough bread making is often based on back-slopping of sourdough, which can give some unique features for the end product, the delicious rye sourdough bread. Back-slopping has advantages for the bakeries using those sourdoughs. Back-slopping is cost effective, and it allows the maintenance of the unique

4  Technology of Sourdough Fermentation and Sourdough Application

69

Table 4.1  Examples for fermentation protocols for sourdoughs

Three-stage scheme

Mother Rye Mother dough (Anfrischsauer) 25–26 °C 5–8 h

1. Stage

2. Stage

Bread dough

Grundsauer 23–28 °C 8–12 h

Vollsauer 28–31 °C 3 h

About 50% of flour fermented 32 °C 1.5–2.5 h ca. 40% of flour fermented Baker’s yeast necessary 50–70% of flour fermented Baker’s yeast essential 30–40% of flour fermented

One stage scheme

One stage sour; 30 to 24 °C decreasing 15–24 h

Berlin short sour

Short sour 35 °C, 3 h

Salted sourdough

San-Francisco-­ Sourdough-­ Bread

20% Anstellsauer

Wheat Mother sponge

2% salt (flour base) 30–35 °C to 20–25 °C 18–24 h, up to 3 days storage 23–27 °C 7–8 h

Pugliese

Madre

Refreshed two times (rinfresco) 18–22 °C, 2 h

Panettone

Madre (from second girata)

Refreshed three times (girata) 18–22 °C Each 4–6 h

Livieto naturale 18–22 °C, 2 h Impasto bianco Salt, butter, sugar 18–22 ° C, 6–8 h

15–20% of flour fermented 29–33 °C 6–8 h Biga 18–22 °C, 2 h

Impasto giallo (Salt, butter, sugar, eggs, raisins) 30 °C, 6–7 h

microbial composition and special characteristics of a sourdough. The sourdough microbes and their metabolism are adapted to the particular sourdough conditions and the sourdough metabolism remains relatively stable. Empirical experience indicates that the sourdough microbiota can undergo changes overtime in strain and even in species level, but despite this, the overall sourdough phenotype remains practically the same. This means that the metabolism occurring in the sourdough would not change even if the microbial composition changes; however, the long-­ term functional stability of industrial sourdoughs is not very well documented in the scientific literature. Keeping the sourdough phenotype unchanged mainly requires that the sourdough temperature, fermentation time, agitation or mixing, formulation (e.g., starter %, dough yield), and raw materials (enzyme activity and ash content) remain the same. The sourdough conditions, especially the fermentation temperature, play a more important role with regard to the microbial composition of sourdough than the raw

70

M. J. Brandt et al.

1st refreshment (Anfrischsauer)

24°C - 26°C 6h

Flour Water Mother (Anstellgut)

2nd refreshment (Grundsauer)

23°C - 27°C 8-10 h

Flour Water 1st refreshment (Anfrischsauer)

Full sour (Vollsauer) Flour Water 2nd refreshment (Grundsauer)

28°C - 32°C 3h

Bread dough Flour Water Full sour (Vollsauer) Salt (Optional addition of baker’s yeast) Fig. 4.1  Three-stage fermentation scheme for rye bread

material or formulation. A point in case is that of the well-known San Francisco sourdough, which is prepared using white wheat flour and produced in bakeries located in San Francisco, California. The San Francisco sourdoughs practically always harbor Fructilactobacillus sanfranciscensis and Kazachstania humilis as the dominant lactobacilli and yeast, respectively. When Finnish rye sourdough is produced in Lahti, Finland, using wholegrain rye flour, different dough yield and in industrial scale process, the microbial composition turns out to be identical, with Fl. sanfranciscensis and K. humilis as the dominant sourdough microbes. The

4  Technology of Sourdough Fermentation and Sourdough Application

71

adaptation and dominance of these two sourdough specific microorganisms requires basically just that the temperature is in Type 1 sourdough temperature range, i.e., around 20–30 °C and frequent and long-term back-slopping in a bakery environment. There is of course a good explanation for the dominance of these sourdough microbes, i.e., the symbiosis they have as K. humilis is unable to utilize maltose, which Fl. sanfranciscensis utilizes while releasing one glucose moiety for use by K. humilis. On the other hand, Fl. sanfranciscensis produces acetic acid, which is well tolerated by K. humilis. In rye-bread processing or in rye containing mixed breads, the use of sourdough is essential. Historically, the main reason that necessitated the use of sourdough in rye baking was to inhibit amylase activity. In rye, the endogeneous amylase activity, which is active during gelatinization (49–56 °C) of starch, needs to be reduced by a drop in pH. Otherwise, the α-amylase activity would weaken the crumb structure in such a way that the bread crumb could rip from the crust during the baking process. In fact, the endogenous enzyme activity is important for good quality sourdough as it releases nutrients for sourdough microbes’ carbohydrate metabolism (maltose liberated by amylases) and nitrogen metabolism (peptides liberated by proteases of rye). In the end, these metabolisms are important for the sourdough’s contribution to sensory properties such as sourness and formation of flavor compounds. Although sprouting in the field is not a problem anymore with the actual rye varieties grown [4], and current rye crops usually do not suffer from pre-harvest sprouting and rye flour can actually be too low in amylase enzyme activities. This means that the use of sourdough would not be obligatory from this point of view. Nevertheless, rye breads do not have an acceptable crumb elasticity without sourdough. The continued need for use of sourdough relates to the activity of cereal arabinoxylanases that solubilize water insoluble arabinoxylans during sourdough fermentation. This improves dough hydration, increases bread volume and crumb elasticity. In addition, the main function of sourdough nowadays is to produce good sensory properties to the bread, i.e., mild sour taste and aromatic bread flavor, and this is also dependent on the activity of rye enzymes. Endogenous rye enzymes are located predominantly in the outer layers of the kernel. Kernels of modern rye varieties are much higher in starchy endosperm, which means that the proportion of outer layers from the whole kernel are lower than in traditional varieties. This also affects the sourdough baking quality of rye as the enzyme activities are lower due to the smaller proportion of outer layers. Because of the low enzyme activity, it is sometimes necessary to boost it by added enzyme sources that can be enzyme preparations or bread improvers containing, e.g., amylases or grain-derived materials with higher enzyme activity such as rye bran or enzyme-active malts. The bigger proportion of starchy endosperm and smaller proportion of kernel outer layers in modern rye varieties also means that there is less ash in rye flours and therefore less buffering capacity. Buffering capacity is another important feature in industrial sourdough processes, as there is usually a certain level of acidity that is targeted. The amount of total acid is usually measured as total titratable acidity (TTA). If the desired TTA level cannot be reached, it is possible to increase the

72

M. J. Brandt et al.

buffering capacity of sourdough by adding grain fractions with higher ash contents, e.g., bran, and thereby to increase the total acidity.

4.1.1.2 Industrial Rye Sourdough Types  igh Dry Matter Type 1 Sourdough (Firm Sourdough) H Industrial firm rye sourdoughs have low dough yield (~180). They are referred to as Type 1 sourdoughs and fermented in ambient bakery temperature at ~25–30  °C often in dough mixer bowls. Firm sourdoughs cannot be pumped due to the high consistency. Usually, the fermentation time is short (less than 10 h) and the amount of seed sourdough is high. The seed sourdough is ripe sourdough originating from a previous batch and works as the sourdough starter. The sourdough microbiota typically consists of Fl. sanfranciscensis and K. humilis along with some additional lactic acid bacteria. The sourdough yeast and, to a lesser extent, heterofermentative lactobacilli contribute to the leavening of the dough [5], and the process can be modified to also work without added baker’s yeast. L ow Dry Matter Industrial Sourdoughs (Liquid, Pumpable Sourdough) Industrial liquid sourdoughs are pumpable and can be differentiated based on their fermentation temperature; those fermented around ~30 °C are here referred to as Type 1 liquid sourdoughs and those fermented at around ~40 °C or above are here referred to as Type 2 liquid sourdoughs. For both types, the fermentation time is usually between 10 and 20 h but can be extended to several days. The amount of seed sourdough is below 10%. The targeted TTA values are 10–20 for Type 1 liquid sourdoughs and 25–30 for Type 2 liquid sourdoughs. It is worth noting that the TTA values can be strongly influenced by the selection of the raw material used for sourdough. The fermentation temperature practically determines the microbial compositions of liquid sourdoughs. Type 1 liquid sourdoughs, fermented at ~30 °C, contain typical Type 1 sourdough microbiota, i.e., sourdough yeast, often K. humilis, and very often Fl. sanfranciscensis as a dominant lactic acid bacteria along with 1–2 other dominant lactobacilli strains. As in Type 1 firm sourdoughs, the sourdough yeast in Type 1 liquid sourdoughs contributes to the leavening of the dough and the bread making is possible with less or without added baker’s yeast. Bread making without added baker’s yeast requires a careful process and longer proofing times. The Type 2 liquid sourdoughs are fermented at higher temperature around ~40 °C or above. Dominant lactobacilli include Lactobacillus species, including Lactobacillus amylovorus and Lactobacillus crispatus, and Limosilactobacillus species, including Limosilactobacillus frumenti, Limosilactobacillus pontis and Limosilactobacillus secaliphilus. Due to the higher fermentation temperature, there is no yeast present and the acidification is usually more intensive. There is some notable difference usually in the flavor properties of the two liquid sourdough types.

4  Technology of Sourdough Fermentation and Sourdough Application

73

 ye Sourdough Bread Making R Sourdough bread making is a relatively simple process. The ripe sourdough and other ingredients are mixed into a fairly soft dough. The dough is then allowed to rest, then molded into the desired shape, proofed, and baked in the oven. It is worth noting that the sourdough metabolism continues during the process and the flavor formation occurs during baking.

4.1.2 Wheat Processing In wheat processing, the generation of flavor compounds is the main reason for using sourdough. Rye processing uses one-stage fermentation schemes with the addition of baker’s yeast and multiple stage schemes with the aim of achieving leavening by sourdough. In addition, sponge doughs started with baker’s yeast are also very common. In one-stage processes, the sourdough is usually fermented between 23 and 30 °C for 12–16 h [6]. It is added to the bread dough in 5–20% of flour depending on the desired total titratable acidity of the bread. The consumer preference for acidity depends on the region. Whole-grain or coarse bread have higher acidity. One-­ stage processes are the standard process for industrial production of wheat breads with sourdough. If a leavening activity is desired in combination with a low acidity, a multiple stage process must be used. The growth of yeast is less affected at lower temperature when compared to growth of lactic acid bacteria [5, 7]. Therefore, these fermentation schemes all work between 16 and 22 °C with 2 to 3 refreshments per day. Examples are listed in Table 4.1. It is important to use a mother dough with a microbiota that fits the process; for multistage processes aiming for leavening, the mother dough should have a higher count of yeasts. A preferment, also termed sponge dough or poolish is started by addition of baker’s yeast. Sponge doughs are used only in wheat baking and these doughs are not back-slopped. The main aim of their use is to improve the flavor of baked goods; for sweet doughs, providing time for adaption of the yeast to the high osmotic pressure is an additional aim. Fermentation times are from 0.5 to 24 h depending on the product. In preferments with long fermentation times, the amount of yeast is lower (0.1–0.2% on flour base) and the dough is fermented at lower temperatures, 22–25 °C [3]. In some countries, it is usual to supplement a sourdough with such amounts of baker’s yeast. Preferments with shorter fermentation times of 2–3 h are usually made with higher amounts of yeast (1–2% on flour base). As baker’s yeast also contains significant amounts of lactic acid bacteria [8], longer fermented preferments result in a sourdough (Type 0) with high cell counts of lactic acid bacteria and a pH of less than 4.5.

74

M. J. Brandt et al.

4.2 Mother Dough As mother dough can be derived by spontaneous fermentation of flour, often with addition of plant material or even manure at the first fermentation step, followed by continuous and long-term back-slopping, or from a defined microbial culture. The substrate flour has a relatively high microbial load of ca. 106 cfu/g, consisting mainly of plant-associated Enterobacteriaceae, bacilli and lactic acid bacteria. Flour microorganisms cannot be eliminated by heating because this would also gelatinize the starch and denature proteins. Microbial cultures to start a sourdough must be very competitive. The microorganisms should be well adapted to their substrate (cereals, pseudo-cereals, legumes, etc.) as different chemical composition may affect competitiveness of strains. In general, there are no major differences between fermentation of wheat or rye flours but gluten free sourdoughs are not known to harbor F. sanfranciscensis. Table 4.2 lists some microorganisms occurring in commercial sourdough starters.

4.2.1 Development of Mother Dough by Spontaneous Fermentation As soon as water is added to flour, a spontaneous fermentation starts. Usually, fermentation is initiated by plant-associated Enterobacteriaceae [11, 12], but lactic acid bacteria, including Leuconostoc and Lactococcus species, also grow and lower the pH. Refreshing, i.e., mixing flour, water, and part of the previous dough marks the end of spontaneous fermentation and a natural selection of lactic acid bacteria and yeast starts. With each refreshment, the initial pH is somewhat lower as in the stage before (Fig. 4.2), which favors growth of yeasts and lactic acid bacteria [13]. Generally, flour, the bakery equipment, the human skin, or any added non-cereal material, for example, onions, raisins, honey, or yoghurt [14], introduce enough lactic acid bacteria to make the mother dough suitable for baking after 3–5 refreshments. Usually, it takes much more refreshments to obtain a stable microbiota with good sensory and technological properties. The fermentation scheme of a Table 4.2  Lactic acid bacteria and yeasts used in commercial starter preparationsa Frozen/ freeze-dried/ spray-dried preparations Cereal-based preparations

a

Lactic acid bacteria Levilactobacillus brevis, Lactiplantibacillus plantarum, Fructilactobacillus sanfranciscensis, Lacticaseibacillus casei, Lactobacillus delbrueckii, Limosilactobacillus fermentum, Pediococcus pentosaceus, P. acidilactici F. sanfranciscensis, Limosilactobacillus pontis, Lactobacillus helveticus, Lv. brevis, Companilactobacillus paralimentarius, Lp. plantarum, Lm. fermentum, Lentilactobacillus buchneri, C. crustorum, Furfurilactobacillus rossiae, Weissella confusa, Leuconostoc lactis

References [9, 10] and unpublished observations of the authors

Yeasts Saccharomyces cerevisiae, Torulaspora delbrueckii Kazachstania humilis, S. cerevisiae, S. pastorianus

4  Technology of Sourdough Fermentation and Sourdough Application

75

Fig. 4.2  Development of initial pH (black) and pH after 16 h fermentation (gray) in a spontaneous started sourdough based on spelt wholegrain flour (28 °C, dough yield 200)

spontaneously obtained sourdough should follow the planned scheme for bread production as temperature and amount of mother are the main determining factors to maintain a stable microbiota.

4.2.2 Cereal Based Sourdough Starters Obtained by Back-slopping Originally developed from spontaneous fermentation and back-slopped further— sometimes over decades—active sourdough starters are sold as liquid or stiff dough. As microbial activity decreases over time, these preparations have a shelf life of 4 weeks to 6 months. Usually, the microbial composition of these so-called undefined cultures are very well known and undergo only minor changes over time [15]. As this kind of starters are kept in sourdough in an active state, they are characterized by short lag-phases in sourdough and usually contain both lactic acid bacteria and yeasts.

4.2.3 Dried Sourdoughs with Active Cultures Sourdoughs can be dried at lower temperatures in a way that resistant sourdough microorganisms survive (type III-sourdoughs). Although these dried sourdoughs can be used to initiate a new fermentation, they are usually used for direct bread production while fulfilling labeling regulations that require a minimum viable cell count in some countries.

76

M. J. Brandt et al.

4.2.4 Freeze-Dried Cultures Lactic acid bacteria and yeasts from sourdough can generally be grown as pure cultures in specific culture broth. Biomass production in sterilized aseptic fermenters at optimized conditions yields high cell counts. At the end of the fermentation, the biomass is collected by centrifugation, washed, and freeze dried to remove water while preserving culture viability. Freeze drying remains the optimal way to obtain a viable preparation of dried bacteria. The powders are highly concentrated and can contain up to 1012CFU/g after freeze drying but declines during storage. In order to create more complex starter cultures for sourdough, different dried strains are blended together. The addition of starter cultures allows controlling exactly the type of bacteria and yeast, as well as the amount that is being added to each batch of sourdough.

4.3 Dried Sourdoughs 4.3.1 Ready-to-Use Industrial Sourdough Another category of sourdough products that are industrially produced is a wide range of ready-to-use sourdoughs. The fermentation step for bakers can be considered as outsourced because the sourdough is fermented by the supplier for use in the bakeries. Such an operation requires extra care and extra steps in the sourdough process. While a sourdough being freshly produced in a bakery can be added immediately into the final dough, a ready-to-use sourdough that is produced by a supplier and shipped to the bakery requires a much longer shelf life and a better stability to accommodate shipment and supply management. Therefore, some crucial adaptations to the process and ingredients can be implemented to safeguard the safety and quality of ready-to-use sourdoughs. The blending system is often an external mixing unit to prevent flour dust from accumulating on the ceiling of the fermenter room. When these flour residues in the production environment absorb humidity, molds or undesired bacteria can easily grow and cause quality issues for the sourdough. In a fully automated fermentation system, many will choose to work with a starter culture instead of a perpetual refreshment of an undefined mother dough. The use of starter cultures allows cleaning and sanitation of the production equipment after each batch. The use of starter cultures also has the advantage of improved control of the fermentation. Every batch of sourdough is inoculated with a known quantity of well-characterized lactic acid bacteria and yeasts. Initially, lactic acid bacteria will grow fast and outcompete the natural microbial population present in the flour. Rapid acidification inhibits plant-associated bacteria including Enterobacteriaceae, bacilli, and many of the plant-associated lactic acid bacteria, preventing these organisms from influencing the fermentation and flavor profile of the sourdough. At the end of the fermentation, the sourdough is cooled to 4 °C until it is used for the final bread manufacturing. Because these production facilities are highly automated, the sourdoughs are quite

4  Technology of Sourdough Fermentation and Sourdough Application

77

liquid with a dough yield of more than 200. At the end of the fermentation, they can be pumped to the required packaging. The shelf life of a fresh sourdough is quite limited, even at 4 °C because the fermentation continues slowly. First, the yeasts will suffer from the highly acidic environment, so the dough’s leavening capacity is reduced quite quickly. This will still result in a very flavorful sourdough, but commercial yeast has to be applied in the final dough to achieve leavening of the dough. Dependent on the desired length of the final dough fermentation, a baker will add 0.2% to 2% yeast in addition to the sourdough. After a certain period of storage of the sourdough, the lactic acid bacteria will go into a dormant phase and finally loose viability. The objective of producing RTU sourdough is providing the bakery industry with a broad variety of sourdoughs to be used to give more taste to a specific bread. There is an almost unlimited potential of developing different sourdoughs. The sourdough will be defined mainly through the fermentation step but additionally, the way a sourdough is stabilized (e.g., drying) can contribute to the flavor profile of a sourdough. During the fermentation, the choice of the flour will define the main characteristics of the sourdough. Mostly rye and wheat are used, but other types of flour such as kamut, spelt, sorghum, or any other type of cereal can be fermented with lactic acid bacteria and yeasts. Different cereals ferment better or worse, depending on the release of free sugar and soluble nitrogen by endogenous enzymes in the flour. Lactic acid bacteria have very high nutritional requirements and need minerals and trace elements. In addition, the high ash content in the flour will buffer the pH drop during the acidification and allow the lactic acid bacteria to continue their growth longer. Growth of lactic acid bacteria in sourdough is usually limited by the pH rather than the concentration of organic acids because the intracellular acidification inhibits metabolism at a certain extracellular pH. Therefore, whole meal flours will result in sourdoughs with much higher acidity compared to its white flour counterparts.

4.3.2 Spray-Dried and Drum-Dried Ready-to-Use Sourdoughs The short shelf life of a fresh ready-to-use sourdough limits the geographical area of distribution. Inspired by the dairy industry, where fresh milk is dried to milk powder to achieve a long shelf life, the same technologies were applied to sourdough. Spray drying is most commonly used to produce dried sourdoughs. The liquid sourdough, with typical DY of around 300, will be pulverized in the chamber of a large spray dryer. The entrance air temperature reaches temperatures of around 200 °C while the outlet is set at around 90 °C. Water evaporates after the sourdough is atomized in little droplets. As long as the water evaporates, evaporation cooling maintains the core of the sourdough droplet at around 70 °C, preventing Maillard reactions. This results in a very fruity powder that is collected at the bottom of the dryer and separated from the air by cyclones.

78

M. J. Brandt et al.

Since there is no Maillard reaction, a wheat-based sourdough will result in a white powder, similar to its raw material used. When using rye flour as substrate, the color will be more beige due to the acidification of the rye flour. A powdered sourdough is very stable and can easily be kept for 1  year at ambient temperature. Almost all the micro-organisms are killed during the spray drying process. A second drying technology used to turn a liquid sourdough into a powder is drum drying. The liquid sourdough will be spread directly on a steel drum, heated at 200 °C. Due to the direct contact with the drum, water will evaporate immediately and Maillard reactions will start to occur as soon as all the water has evaporated. The temperature of the drum and the speed at which the drum is turning will determine how much Maillard reactions occur. The slower it rotates, the darker and more roasted the sourdough will be. This allows a wide range of taste profiles. Obviously, in both spray dried and drum dried, sourdough powder are all lactic acid bacteria and yeasts deactivated. The use of this kind of sourdough is purely for its organoleptic properties which, different from other products, transfers roasty Maillard flavors into the crumb. Many bakers use bakery mixes to produce specialty breads. Powdered sourdoughs are often used in these blends to optimize the taste of the obtained breads.

4.4 The Future of Industrial Bread In rye baking, sourdough continued to be used throughout the twentieth century because it achieved two technological aims, inhibition of rye amylases and activation of rye amyloxylanases. Even if baking with contemporary rye varieties are less dependent on these aims, sourdough fermentation remains essential to achieve the typical taste profiles that are expected by consumers. In the wheat bread application, most traditional sourdoughs disappeared in favor of commercial baker’s yeast. The short and consistent process obtained with commercial baker’s yeast allowed the industrialization of bread production and the time-­ consuming sourdough fermentation was abandoned. Despite the advantages of straight dough processes in bread production, many traditional sourdoughs have been maintained by baker’s families. Puratos started collecting many different traditional sourdoughs in the Puratos sourdough library. It allows studying the vast microbial diversity to be found in sourdoughs around the world and preserves them for the future (Fig. 4.3). For several years now, there is a revival of sourdough in many bakery applications. Sourdough research has revealed a lot of benefits of using sourdough during bread making. Industrial ready-to-use sourdough has also seen an important evolution as new insights appear. The sole benefit of industrial ready to use sourdough is no longer only flavor. In recent years, new legislation was introduced, for example in Spain, that defines the use of the term sourdough. In Spain, the fermentation microbiota is

4  Technology of Sourdough Fermentation and Sourdough Application

79

Fig. 4.3  The Puratos sourdough library in Sankt-Vith, Belgium

not only required to be viable but also to originate from a traditional fermentation rather than from defined starter cultures. Industry adapts and improves the processes in order to maintain viability of fermentation microbes in ready to use sourdough and refreshment schemes for mother doughs. Another important challenge is the leavening power of the sourdough, which is more difficult to control than acidification and the total titrable activity. Leavening with sourdough as the sole leavening agent is rarely, if ever, mastered at large industrial scales. Improved control of fermentation time and storage/shipment conditions to enable use of ready to use sourdoughs as the sole leavening agent will offer an alternative to the use of baker’s yeast, while providing more taste and texture to the bread. Last but not least, sourdough has a huge potential to improve the nutritional properties of bread. Up to 15% of the world population suffers from some kind of intolerance to wheat and encounters problems digesting wheat bread made with the fast modern bread-making processes based on baker’s yeast, often used in conjunction with high energy mixing and short proofing times. The use of sourdough allows lactic acid bacteria to pre-digest parts of the flour to make them better digestible for humans. Our intestinal track contains trillions of bacteria and the use of sourdough “extends” the digestive track and toxic compounds are degraded. The selection of specific lactic acid bacteria with high enzymatic activity will help to speed up the breakdown of many compounds in the flour and help digestion. A healthy gut counts for a good immune system and a good mood. The future of sourdough is secured. Sourdough can be made in many ways, at home, in the bakery but also in specialized industrial productions. Sourdough is back and will stay because of the organoleptic and nutritional benefits it brings to bread.

80

M. J. Brandt et al.

References 1. Wahl GG (1829) Die Kunst Brod und andere Gebäcke zu backen oder Anweisung wie man gutes, gesundes und wohlschmeckendes Brod u.s.w. bäckt. Serig, Leipzig 2. Von Wagner JR (1860) Theorie und Praxis der Gewerbe- Hand und Lehrbuch der Technologie, vol Band 3. Otto Wigand, Leipzig 3. Neumann H, Stephan H, Brümmer JM (2006) Roggen als Rohstoff und Technik der Roggensauerteigführung. In: Brandt MJ, Gänzle MG (eds) Handbuch Sauerteig, 6th edn. Behr’s Verlag, Hamburg, pp 285–327 4. Brandt MJ (2014) Quality improvement and fermentation control in dough fermentations. In: Holzapfel W (ed) Advances in fermented food and beverages - improving quality, technologies and health benefits. Woodhead Publishing, Cambridge, pp 391–407 5. Brandt MJ, Hammes WP, Gänzle MG (2004) Effects of process parameters on growth and metabolism of Lactobacillus sanfranciscensis and Candida humilis during rye sourdough fermentation. Eur Food Res Int 218:333–338 6. Seiffert M (2006) Technik der Weizenvor- und Sauerteig-Führungen in Deutschland und Europa. In: Brandt MJ, Gänzle MG (eds) Handbuch Sauerteig, 6th edn. Behr’s Verlag, Hamburg, pp 285–327 7. Gänzle MG, Ehmann M, HAmmes WP (1998) Modelling of growth of Lactobacillus sanfranciscensis and Candida milleri in response to process parameters of the sourdough fermentation. Appl Environ Microbiol 64:2616–2623 8. Reale A, Di Renzo T, Succi M, Tremonte P, Coppola R, Sorrentino E (2013) Microbiological and fermentative properties of Baker’s yeast starter used in breadmaking. J Food Sci 78:1224–1231 9. Hammes WP, Vogel RF (2007) Sauerteig. In: Holzapfel W (ed) Mikrobiologie der Lebensmittel – Lebensmittel pflanzlicher Herkunft Behr’s Verlag, Hamburg, pp 285–314 10. Poitrenaud B (2003) Commercial starters in France. In: Kulp K, Lorenz K (eds) Handbook of dough fermentations. Marcel Dekker, New York, pp 197–230 11. Ercolini D, Pontonio E, De Filipis F, Minervin F, La Storia A, Gobbetti M, Di Cagno R (2013) Microbial ecology dynamics during rye and wheat sourdough preparation. Appl Environ Microbiol 79:7827–7836 12. Hochstrasser RE, Ehret A, Geiges O, Schmidt-Lorenz W (1993) Mikrobiologie der Brotteigherstellung. II.  Mikrobiologische Untersuchungen an unterschiedlich hergestellten Spontansauerteigen aus Weizenmehl. Mitt Gebiete Lebensm Hyg 84:356–381 13. Dinardo FR, Minervini F, De Angelis M, Gobbetti M, Gänzle MG (2019) Dynamics of Enterobacteriaceae and lactobacilli in model sourdoughs are driven by pH and concentrations of sucroe and ferulic acid. LWT 114:108394 14. Ripari V, Gänzle MG, Berardi E (2016) Evolution of sourdough microbiota in spontaneous sourdoughs started with different plant materials. Int J Food Microbiol 232:35–42 15. Ehrmann MA, Behr J, Böcker G, Vogel RF (2011) The genome of L. sanfranciscensis after 18 years of continuous propagation. In: 10th Symposium of Lactic Acid Bacteria, Egmont an Zee, Abstract book, A005

5

Steamed Bread Bowen Yan and Dan Xu

5.1 Introduction Steamed bread, also known as Chinese Mantou or Mo, is a staple food in China [1]. Steamed bread is a traditional fermented food that is formulated with cereal flour, mainly wheat flour, water and a leavening agent, and processed by steaming [2]. It accounts for 40% of the wheat consumption in China [3, 4] and is also popular in surrounding countries [5]. The earliest history of Chinese people eating steamed bread can be traced back to the Warring States Period, 475–221 BCE. As described in the work “Book of Qi” written by Zixian Xiao, “mian qi bing,” a type of bread with soft texture produced from fermented cereal flour, was used for sacrificial ceremonies [6]. The original fermentation method of steamed bread was “rice wine fermentation” [2], and nowadays, Chinese people generally use sourdough in combination with the addition of alkali to neutralize the acids produced during fermentation to make steamed bread [7]. Although baker’s yeast has been widely used in recent decades, the use of sourdough remains preferred because of the improved flavor associated with the use of sourdough [4]. According to the differences in ingredients and product properties, Huang and Quail defined three typical styles of steamed bread in East and Southeast Asia, including northern-style, southern-style, and Cantonese-style steamed bread [8]. This chapter will systematically introduce the types of Chinese steamed bread and their differences, the sourdough and dough production process, and the effects of sourdough on steamed bread quality (Fig. 5.1).

B. Yan (*) · D. Xu School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People’s Republic of China © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. Gobbetti, M. Gänzle (eds.), Handbook on Sourdough Biotechnology, https://doi.org/10.1007/978-3-031-23084-4_5

81

82

B. Yan and D. Xu

Fig. 5.1  Some types of steamed bread

5.2 Types of Steamed Bread 5.2.1 Northern-Style Steamed Bread Traditional northern-style steamed bread is very popular as a staple food in northern China, and due to the process complexity and low cost, traditional northern-style steamed bread is still made at home, especially in the countryside [4]. Qiangmian mantou and Gaozhuang mantou are the typical representation of traditional northern-­style steamed bread, which should have good chewing properties and a natural wheat smell. Sourdough is generally used for northern-style steamed bread making in order to improve the specific texture quality [9, 10]. As a consequence of urbanization, “machine-made” northern-style steamed bread has become predominant in northern China. Compared to the traditional northern-style steamed bread, industrially produced steamed bread has a less firm texture but retains some of the chewy characteristic because of the addition of an extra 2–3% water to the dough. There is a trend toward softer northern-style steamed bread in the cities of northern China [11].

5  Steamed Bread

83

5.2.2 Southern-Style Steamed Bread Because of the warm and rainy climate, rice-based food rather than wheat-based steamed bread is the staple food in southern China. Compared with the northern-­ style steamed bread, southern-style steamed bread with soft and a bit chewy texture is preferred as a popular breakfast food in southern China [12]. Most of the southern-­ style steamed bread is manufactured in factories due to its one step fermentation process, resulting in a rapid increase in the market share.

5.2.3 Cantonese-Style Steamed Bread According to the local dietary habits of the Guangdong region, Cantonese-style steamed bread has developed into one of the typical styles in China. The quality properties of Cantonese-style steamed bread is similar to that of southern-style, but it is very soft and less cohesive [2]. Western influences resulted in Cantonese-style steamed bread with a sweet taste and small size, which is preferred as a dessert in the Guangdong and Fujian provinces as well as Hong Kong and other Southeast Asian countries. In recent years, Cantonese-style steamed bread has become the most common type of frozen packaged steamed bread in Chinese supermarkets, and frozen packaged products are also exported to other Asian countries.

5.2.4 Differences in Ingredients of the Three Types of Steamed Bread The ingredients of steamed bread are mainly flour, water, and yeast or sourdough. Salt is not necessary for steamed bread making, but the addition of alkali is common to neutralize the acidity of dough with sourdough or long-term spontaneous fermentation [7]. Different from northern- and southern-style steamed breads, recipes for Cantonese-style steamed bread may contain up to 25% sugar and 10% fat. In addition, milk powder or fresh milk is also used to improve the flavor of products, except for the traditional northern-style. The flour quality is the key factor for various styles of steamed bread. For the northern-style steamed bread, the optimum level of water addition to flour is around 60–63%, while around 58–60% of water is added to southern-style steamed bread [8, 13]. Cantonese-style steamed bread with or without fat requires the addition of 58–60% and 54–57% of water, respectively [14]. The wet gluten content of flour for northern-style steamed bread is at least 28%, while flour less than 28% wet gluten is used for southern-style and Cantonese-style steamed bread. Dough strength as a flour quality factor is more important than the protein content, and hard wheat is more suitable than soft wheat for northern-style steamed bread [13]. The appropriate protein content for the firm and very firm types of steamed bread should be 10.5–12.5% and 10.7–13.5%, respectively [15]. However, a protein content of around 10% is suitable for southern-style steamed bread and a protein content of

84

B. Yan and D. Xu

12% resulted in a poor appearance of products. Compared to northern style breads, the protein content had a stronger effect on the specific volume of southern-style steamed bread. Similarly, protein content also plays an important role for the quality of Cantonese-style steamed bread. The total quality score of Cantonese-style steamed bread rapidly increased with an increasing protein content from 7.8 to 10.5%, and medium dough strength is better for Cantonese-style steamed bread making [12].

5.2.5 Differences in Physical Properties The basic physical properties of steamed bread are specific volume, spread ratio, skin smoothness, as well as color and texture; these quality differences also define the differences between northern-style, southern-style, and Cantonese-style steamed bread. The structure of northern-style steamed bread is dense with a firm and cohesive eating quality. In contrast, the southern-style steamed bread is soft and a bit chewy. Cantonese-style steamed bread has a specific eating quality, which is sweet, very soft and not cohesive. The national standard for the specific volume of steamed bread is no less than 1.7. Specifically, the volume of the traditional and non-­ traditional northern-style steamed bread is 2.0 and 2.5, respectively. The softer texture of southern-style and Cantonese-style steamed bread relates to a larger specific volume, which is around 3.0 and 2.6–3.4, respectively [16]. The spread ratio is defined as the ratio of width/height, which is only applicable for the well-rounded products. Steamed breads with a northern-style shape have a spread ratio of 1.2–1.5 while a spread ratio of 1.4–1.6 is typical for southern-style or Cantonese-style steamed bread [16].

5.3 Process for Steamed Bread Preparation 5.3.1 Sourdough Procedure • Traditional Sourdough (Laomian) Laomian, also known as Mianfei (flour fertilizer), Mianzhong (flour seed), and Jiaomian, among others, is the traditional sourdough starter for making steamed bread [1]. The method of making Laomian varies from region to region in China, and it is based on similar principles as sourdoughs used for bread making. The traditional method to make Laomian is as follows: To make the steamed bread dough, flour, water, and leavening agent are mixed and fermented for a period of time. A part of the dough is not used for steaming but set aside in a bowl or wrapped in lotus or sweet potato leaves for future use as a starter. Laomian can also be dried for long-term storage. In some areas, Laomian is mixed with wheat bran or corn flour, then fully fermented to obtain the granulated dough, which is also called Jiaozi [5]. High temperature and exposure to sunlight should be

5  Steamed Bread

85

avoided during drying and the water content of Laomian is 15–20%, so as to keep its microbial activity and fermenting power. Laomian has the advantages of long storage time, low cost, and a good flavor and taste of the final products [1]. The shelf life of Laomian is related to its water content. Generally, Laomian can be stored for more than 1 month below 25 °C, but if it is completely air-dried, the time can be extended to more than half a year. The composition of microorganisms in Laomian can be variable depending on the fermentation parameters, and the metabolic activities of the dominant microorganisms improve the flavor and texture of steamed bread [17]. However, the fermentation process with Laomian still has some drawbacks. First, the traditional production process is extensive, and the culture conditions largely depend on the experience of the producer but may result in unstable composition of the microbiota. In addition, the production efficiency is lower compared with baker’s yeast, due to its lower fermenting power and longer making process. • Jiaozi Jiaozi as a leavening agent is a traditional starter that is widely used in northern areas of China for steamed bread making [5]. Jiaozi is prepared from corn, rice, or millet flour and inoculated with jiuqu. Jiuqu is a saccharification culture that is prepared for fermentation of grain liquors and vinegars; its microbiota is dominated by plant-­associated Enterobacteriaceae, bacilli, lactic acid bacteria, as well as yeasts and fungi. The propagation of Jiaozi generally relies on more than three refreshments within 24 h and lasts for several days [18]. After propagation, it is naturally dried in order to reduce the moisture content for long-term storage and to keep strains in a viable state (Fig. 5.2). Depending on the different preparation and propagation steps, Jiaozi had a higher pH and lower TTA compared to the type I sourdough, and the moisture content values were also significantly different [18, 19]. Jiaozi has been applied for hundreds of years and the preparation of Jiaozi is based on empirical knowledge and relies on spontaneous fermentation. Therefore, the product quality and stability are often variable due to the

Fig. 5.2  The preparation processing of type I sourdough and Jiaozi, respectively

86

B. Yan and D. Xu

uncontrolled–sterile fermentation conditions as well as the occasional change of environmental conditions, such as temperature and water content of fermentation. However, traditional Jiaozi is still applied in steamed bread making due to the unique flavor and taste of the product attained. Jiuqu is used for the preparation of Jiaozi, and it enriches Jiaozi with more enzymes (i.e., proteases), which have pH-­optima between 4.2 and 5.5 [20]. The acidification by LAB during Jiaozi fermentation favors the activation of proteinases to be responsible for the production of more amino acids, which serve as flavor precursor [21]. Furthermore, pre-treatment of Jiaozi is required before steamed bread making, which is triturating or crushing and soaking in warm water until there are no particles in order to activate the leavening activity of Jiaozi [22].

5.3.2 Dough Preparation Procedures The difference of steamed bread production technology mainly lies in the difference of leavening agent and fermentation method, and fermentation is the most critical link in the dough preparation procedure, which directly affects the quality of steamed bread. There are three major and two minor dough preparation methods for steamed bread, as shown in Fig. 5.3 [1, 2]. Some of the methods are suitable for industrial production, while others are only suitable for manual operation in small workshops.

Fig. 5.3  Dough preparation procedures for steamed bread

5  Steamed Bread

87

• One-Step Procedure The one-step procedure is a rapid fermentation method, in which all ingredients are put into a dough mixer at one time, and directly mixed, divided, molded, proofed, and steamed [23]. This method takes the least time and has a short production cycle, and is suitable for industrial production with improved production efficiency. However, this process also has many disadvantages, the yeast usage is higher than other procedures and the products are less preferred by consumers for lack of flavor [24]. • Two-Step Procedure The two-step procedure is similar to the sponge and dough procedure for making bread, in which the dough is mixed twice [25]. First, 60–80% of the flour and all the baker’s yeast were mixed into a “sponge,” and after fermentation, the remaining ingredients were added and mixed to form the dough. The other ingredients include fat, sugar, salt, and alkali solution, depending on the style of steamed bread. This method is improved from the traditional dough procedure and is compatible with the advantages of traditional and modern technology. The steamed bread produced with the two-step process has larger volume, smoother appearance, better texture and flavor, less yeast requirement, and lower rate of substandard products compared to steamed bread made by the one-step process [24]. • Sourdough Procedure Laomian is used as the main leavening agent in this method, and a small amount of other starter cultures (baker’s yeast or Jiaozi as supplement) are sometimes added to improve the gas production ability. The dough is made by mixing 5–15% (flour basis) of sourdough, 33% of flour, and half of the water (37–42% water in total) in the first dough mixing stage, and then fermented under ambient conditions 25–30  °C) for around 10  h. After the overnight fermentation, the remaining ingredients and an appropriate amount of alkali needed to neutralize the acidity of the dough are remixed, and the final dough is obtained and carried in the subsequent processing. • Liquid Ferment Procedure Strictly speaking, this procedure should belong to the liquid sourdough procedure. The liquid sourdough is a mixture of flour, water, and a large number of microorganisms, which is obtained after a long time of fermentation under proper stirring condition [26, 27]. Compared with the sourdough (with Laomian as starter) procedure, the ferment condition of the liquid ferment procedure is easy to control and suitable for industrial mass production. • Fermented Glutinous Rice Procedure Rice wine, also known as jiuniang, is a sweet wine produced from steamed glutinous rice and fermented with jiuqu (starter culture containing yeasts or molds) for about 2 days. This production also can be used as an ingredient for fermenting steamed bread dough [7]. This procedure provides a low cost of leavening agent and unique flavor in steamed bread. However, it is difficult to control fermentation conditions due to its wide variety of microorganisms, and it is also not suitable for commercial production [2].

88

B. Yan and D. Xu

5.3.3 Microbiology of Traditional Chinese Sourdoughs Laomian is a complex biological ecosystem, which has a microbial composition similar to sourdough used for making bread. Different types of traditional sourdough (Laomian) are widely used and distributed in China, and their microbial composition also varies between regions. According to the current reports of sourdoughs, Saccharomyces cerevisiae, Kazachstania humilis, and Fructilactobacillus sanfranciscensis were the most common species [19, 28, 29]. In addition, Lactobacillus pontis and Lactobacillus plantarum were also reported as the dominant species [29, 30]. Of the other genera, Wickerhamomyces, Lactobacillus, Pediococcus, and Leuconostoc were identified to dominate the traditional Chinese sourdoughs [31–33], and Pichia membranifaciens was first demonstrated to exist in Chinese sourdough by Liu et al. [17]. Different from Laomian, Jiaozi is usually fermented with jiuqu as starter and requires various preparation and propagation steps other than the one required for type I sourdough (Fig. 5.2). Jiuqu starter is used for fermentation of Chinese liquors and vinegars making and includes daqu, xiaoqu, and fuqu [34–36]. The addition of jiuqu not only determines the microbial diversity of Jiaozi, but also significantly contributes to the flavor characteristic providing fungal and microbial enzymes, particularly proteases and amylases [37–39]. To ensure proper consistency of Jiaozi starters and to improve the quality of steamed bread, various studies have explored the effect of varying raw materials and ecological parameters on the microflora of Jiaozi [40–42]. The predominant lactic acid bacteria generally have remarkable resistance toward dryness and include W. cibaria, P. pentosaceus, and Lp. plantarum [18, 19]. Moreover, W. cibaria has been reported as the dominant LAB species in daqu and xiaoqu [43], which is the main reason for the high abundance of Weissella in Jiaozi. High-throughput sequencing and culture-dependent approaches of fungal community diversity reveal that S. cerevisiae and W. anomalus are the predominant species in most of Jiaozi from Henan province [5]. It is worth noting that Aspergillus and Penicillium were present in a very low abundance in Jiaozi samples, which probably came from the starter culture of xiaoqu. Jiuniang is fermented with jiuqu, which has a strong saccharification effect on glutinous rice. The microbial composition of this kind of jiuqu is relatively clear. The filamentous fungi genera, e.g., Rhizopus, Mucor, Monilia, and Aspergillus were frequently detected in jiuqu [44, 45]. For the yeast strains, besides the typical ethanol-­producing yeast Saccharomyces cerevisiae, non-Saccharomyces yeasts like Pichia kudriavzevii and Saccharomycopsis fibuligera were found in jiuqu samples [46, 47]. During fermentation, the filamentous fungi and yeast release variety of enzymes (alpha and beta amylase, glucoamylase and protease) to convert starch and protein present in rice, which results in improved digestibility of rice.

5.3.4 Control of Proofing Conditions Proofing is a key step in the production of steamed bread, and the correct proofing conditions are a prerequisite for maintaining the sensory and flavor quality of

5  Steamed Bread

89

products. During proofing, the dough expands due to the carbon dioxide produced by yeast aerobic respiration, and air bubbles are formed continuously in the dough, which provides an internal spongy structure of steamed bread [48]. At the same time, microbial metabolites also accumulate during the proofing stage and generate a series of chemical changes to improve the food flavor [49]. For proofing conditions, it is essential that temperature, humidity, and proofing time are all carefully controlled. Details as follows: • Proofing Temperature Proofing temperature is mainly determined by yeast fermentation temperature. The optimum temperature for yeast is 30 °C, while its gas production capacity is the strongest at 38 °C. To prevent the dough collapse caused by long proofing time, 38–40 °C is usually used as the optimum proofing temperature [2]. Low temperatures prolong the proofing time due to poor gas production by yeast, causing a flattened appearance of products. High temperatures accelerate yeast fermentation, and too short proofing time is more likely to result in overproofing, which may lead to surface cracks and bubbles of steamed bread. • Proofing Humidity Humidity is the most critical parameter in the steamed bread proofing process. The relative humidity required in the proofer is preferably in the range of 70–90%, and usually not less than 60% [2]. Low humidity will cause surface dryness and hinder the expansion of the dough, resulting in a rough appearance and surface cracking of products. Distinct from bread proofing, if the humidity is too high (over 95%), bubbles will be produced on the steamed bread surface, inducing a dull color and sticky finished skin. • Proofing Time The proofing time is dependent on the specific product and production process. Dough underproofed will result in products with a small volume and poor internal structure. And overproofed makes products taste sour or firm, and have an uneven, dull and rough skin, and an open and rough cell structure with large honeycomb holes. Normally, proofing time of the products prepared using the one-step dough procedure is in the range of 50–80 min, while a shorter time is needed for products manufactured by the two-step procedure. Proofing time can be accomplished within this desired range by adjusting yeast addition, dough temperature, and water level.

5.3.5 Steaming Steaming, one of the key steps in steamed bread production, directly affects the quality. The main functions of steaming are heating, sterilizing, and transition state, whose essence is to transform the semi-solid plastic dough into solid elastic steamed bread. During steaming, conduction and convection are the main heat transfer modes. Moreover, a series of physical, chemical, and biochemical changes have been attributed to the steaming process. The dough volume expands rapidly, the

90

B. Yan and D. Xu

protein molecules are denatured to form a gluten protein network with a stable structure, and the starch is gelatinized to replace the protein network as a major contributor to product texture. Furthermore, changes in moisture and formation of skin are significantly different from bread, which has an important impact on the steamed bread quality [50]. Therefore, effective control of steaming conditions, such as steaming pressure and steaming time, is crucial for high-quality products production. • Control of Steaming Conditions In specific practice, the steaming pressure and time should be properly acted in line with the degree, type and size of the dough proofing. Steaming cabinets or chambers are usually used in the industrial manufacture of steamed products, and the entering vapor pressure cannot be lower than 0.01 MPa or higher than 0.06 MPa. Excessive steam pressure makes cooking faster and may hinder the expansion of dough, leading to a smaller food volume or skin blisters, dimples, and jelly spots on the surface of steamed bread. With lower pressure, heat exchange is insufficient and longer steaming time is required, which may result in overproofed doughs and bring about collapse and shrinkage. As for the steaming time, the larger the steam quantity in the steaming cabinet and the smaller the product volume, the shorter the steaming time. Generally, 26–27 min steaming time is sufficient for 135 g dough pieces at a pressure of 0.02–0.04 MPa. • Changes in Moisture During steaming, the moisture content of steamed bread shows an upward trend, contrary to the water loss in the bread baking process. In the initial stage of steaming, due to the temperature difference between the dough surface and the steam, the water vapor quickly condenses on the dough skin and forms a layer of mist droplets. When the surface temperature of steamed bread exceeds the dew point, condensation ceases and condensation and evaporation are in a dynamic balance in the later stage of steaming. Moreover, a pressure difference is established between the inner and outer layers of the dough during steaming; the steam and the area formed by condensation are continuously transferred to the steamed bread core. On completion of steaming, only tightly bound water and weak bound water exist in steamed bread, and the moisture distribution becomes more even [51]. • Formation of Skin After steaming, a thin white skin is formed on the surface of steamed products [52], which is very elastic and leathery, differing from the browning crust of bread. Owing to gelatinization of starch after heating, a dense epidermis film is established on the surface. A proper steaming process ensures superior circulation and even distribution of steam, to avoid blistering and scalding on the products skin.

5  Steamed Bread

91

5.3.6 Cooling and Packing To facilitate large-scale production and sales, steamed bread must be cooled and then packaged, and the temperature should be low enough to prevent condensation forming on the products or package [2]. Generally, cooling to 50–60 °C is the ideal packaging temperature for steamed bread, so that it will not lose too much internal moisture and maintains its softness. Packaging at the appropriate temperature also prevents condensate water in the package. Natural cooling is the most commonly used cooling method, and hot steamed bread is usually placed in a cooling room and cooled for 20–30  min. The cooling rate depends on the temperature difference between the products and room temperature, relative humidity, air convection, size and spacing of products. Because of the difficulty of controlling temperature and humidity in the natural cooling process, low humidity may lead to rapid skin dehydration, cracking and firmer products texture. High humidity in the cooling room prevents excessive water loss, thereby obtaining the products with smooth surface and a soft texture. Thus, environmental humidity should be increased as much as possible during cooling of steamed bread. To increase the cooling rate, moist cold air convection can be used. Steamed products are packed either to prevent skin dehydration, shrinkage, and delay retrogradation or to protect them from pollution and damage in the process of freezing, storage, transportation, and sales. There are various inert materials with good moisture and gas barrier properties for steamed bread packing, such as nitrocellulose film, polyethylene, and polypropylene. Polyethylene bags are a popular choice, which may have a couple of small holes for steam evaporation to prevent condensation. In addition, to reduce the quality deterioration of steamed bread and meet the trend of environmental protection, some advanced packaging systems, such as modified atmosphere packaging [53], active packaging, intelligent packaging, nanopackaging, biodegradable and edible films and coatings, have also been applied to steamed bread packing [54].

5.3.7 Quick-Frozen Steamed Bread Quick-frozen steamed bread is quick-frozen after steaming and kept at −18 °C or lower. Quick-freezing is a freezing process that ensures formation of small ice crystals; the thermal center temperature reaches −18 °C and below, which extends the shelf life and enables large-scale production and remote sales of products. During freezing, residence at temperatures of +10 to −6 °C must be minimized to avoid rapid retrogradation of starch. The freezing units that commonly achieve the best freezing effect are individual quick freezing units, plate freezing units, tunnel freezing units, and spiral freezing units. The freezing time of steamed bread is usually 1–1.5 h, depending on the freezing rate. Quick-frozen steamed bread products are split into quick-frozen dough and quick-frozen finished products.

92

B. Yan and D. Xu

• Quick-Frozen Dough Products Frozen dough technology is a new food processing technology that is increasingly being produced worldwide in recent years and widely used in bakery food production [55]. The frozen dough products have the advantages of a centralized manufacturing and distribution process as well as quality standardization. Its application is also a development trend of steamed bread production [56]. Larger steamed bread factories produce the frozen dough by blending, molding, fermenting, and quick-freezing, and deliver them to various markets, supermarkets, and retail stores by cold chain. These consumers can get fresh steamed bread with a consistent quality only by thawing, proofing, and steaming. • Quick-Frozen Finished Products Quick-frozen steamed bread is made from cooked steamed bread by quick-­ freezing, which can be frozen for a long time and eaten after appropriate reheating. It can be reheated by steaming, microwave or baking, and the specific reheating conditions are according to the manufacturer’s packaging instructions. Since the production conditions of such products are easy to control, convenient for storage and transportation, and simple for pre-processing before eating, most manufacturers choose to produce quick-frozen finished products at present.

5.4 Effect of Sourdough on Steamed Bread Quality 5.4.1 Texture The texture is an important eating quality item of steamed bread. Compared with baking, steaming of bread leads to the formation of an outer elastic skin rather than a dry crust, higher moisture content, but similar specific volume and crumb hardness [57]. Although the heat transfer is different between baking and steaming, the principle of improving the texture of steamed bread by using sourdough is similar to that of bread. The yeasts and lactic acid bacteria (LAB) in sourdough affect the microstructure of dough not only by producing exopolysaccharides, but also by producing CO2 and lowering the pH [58, 59].

5.4.2 Volatile Compounds With the increasing use of commercial dry yeast, the production efficiency of steamed bread has been greatly improved. However, the flavor of steamed bread produced with sourdough is irreplaceable by commercial yeast. It is reported that 2-pentylfuran (wheat-like, fruity), ethyl acetate (fruity), ethyl lactate (floral, caramel-­like) hexyl acetate (fruity), and benzaldehyde (almond) are the key aroma compounds in sourdough-based steamed breads as compared to the baker’s yeast steamed bread [60, 61]. Besides, alkali addition neutralizes the acids and inhibits the production of aroma-negative compounds, such as butanoic acid, 1-octen-3-ol, hexanal, and heptanal, and further improves the overall aroma profile [7].

5  Steamed Bread

93

5.4.3 Shelf Life Compared with bread, steamed bread has a higher moisture content, higher staling rate, and more amylopectin crystallites, which lead to the spoilage and unacceptable changes in texture, appearance, or flavor and reduce the shelf life of steamed bread [62]. The addition of sourdough can improve the steamed bread quality, especially the EPS produced by lactic acid bacteria can enhance its antifirming property, and the mechanism is similar to that in sourdough bread [63]. EPS can act as hydrocolloids with anti-staling property and the functionality of sourdough containing EPS is higher in steamed bread than in bread [58].

References 1. Liu C (2019) Production techniques of steamed products, 3rd edn. Chemical Industry Publisher, Beijing 2. Huang S, Miskelly D (2018) Steamed bread - a review of manufacturing, flour quality requirements, and quality evaluation. Cereal Chem 96(1):8–22 3. Zhang G, Wu T, Sadiq F, Yang H, Liu T, Ruan H, He G (2016) A study revealing the key aroma compounds of steamed bread made by Chinese traditional sourdough. J Zhejiang Univ Sci B 17(10):787–797 4. Kim Y, Huang W, Zhu H, Rayas-Duarte P (2009) Spontaneous sourdough processing of Chinese Northern-style steamed breads and their volatile compounds. Food Chem 114(2):685–692 5. Li Z, Li H, Bian K (2016) Microbiological characterization of traditional dough fermentation starter (Jiaozi) for steamed bread making by culture-dependent and culture-independent methods. Int J Food Microbiol 234:9–14 6. Xiao Z (2005) Book of Qi edn. Jilin People’s Publishing House, Jilin 7. Sun X, Liu C, Wang Y (2020) Influence of Na2CO3 on the quality of dough with rice wine sourdough and steamed bread. Int J Food Sci Technol 55(5):2261–2270 8. Huang S, Quail K (1996) Flour quality guidelines for southern style Chinese steamed bread. In: Wrigley CW (ed) Cereals 96 proceedings of the 46th Australian cereal chemistry conference. Cereal Chemistry Division, Royal Australian Chemical Institute, pp 315–318 9. Wu C, Liu R, Huang W, Rayas-Duarte P, Wang F, Yao Y (2012) Effect of sourdough fermentation on the quality of Chinese Northern-style steamed breads. J Cereal Sci 56(2):127–133 10. Yan B, Yang H, Wu Y, Lian H, Zhang H, Chen W, Fan D, Zhao J (2020) Quality enhancement mechanism of alkali-free Chinese northern steamed bread by sourdough acidification. Molecules 25(3):726 11. Huang S (2005) Current styles of steamed bread in China. In: Blanchard CL, Truong H, Allen HM, Blakeney AB, O’Brien L (eds) Proceedings of the 55th Australian cereal chemistry conference. Royal Australian Chemical Institute, pp 197–200 12. Huang S, Quail K, Moss R (1998) The optimization of a laboratory processing procedure for southern-style Chinese steamed bread. Int J Food Sci Technol 33(4):345–357 13. Huang S, Yun S-H, Quail K, Moss R (1996) Establishment of flour quality guidelines for northern style Chinese steamed bread. J Cereal Sci 24(2):179–185 14. Rubenthaler G, Huang M, Pomeranz Y (1990) Steamed bread. I. Chinese steamed bread formulation and interactions. Cereal Chem 67(5):471–475 15. Su D (2005) Studies on classification and quality evaluation of staple Chinese steamed bread. China Agricultural University, Beijing

94

B. Yan and D. Xu

16. Huang S (2014) Steamed bread. In: Zhou W, Hui YH, Leyn ID, Pagani MA, Rosell CM, Selman JD, Therdthai N (eds) Bakery products science and technology, 2nd edn. Wiley, New York, NY, pp 539–562 17. Liu T, Yang L, Sadiq FA, Yang H et  al (2018) Predominant yeasts in Chinese traditional sourdough and their influence on aroma formation in Chinese steamed bread. Food Chem 242:404–411 18. Li H, Li Z, Qu J, Wang J (2017) Bacterial diversity in traditional Jiaozi and sourdough revealed by high-throughput sequencing of 16S rRNA amplicons. LWT-Food Sci Technol 81:319–325 19. Yan B, Sadiq FA, Cai Y, Fan D, Chen W, Zhang H, Zhao J (2019) Microbial diversity in traditional type I sourdough and jiaozi and its influence on volatiles in Chinese steamed bread. LWT-Food Sci Technol 101:764–773 20. Spicher G, Nierle W (1988) Proteolytic activity of sourdough bacteria. Appl Microbiol Biotechnol 28(4–5):487–492 21. Wang Y, Zhao J, Xu F, Wu X, Hu W, Chang Y, Zhang L, Chen J, Liu C (2020) GC-MS, GC-O and OAV analyses of key aroma compounds in Jiaozi Steamed Bread. Grain Oil Sci Technol 3(1):9–17 22. Liang Y, Liu C, Li Z, Zhang J, Huang S (2012) Preliminary study on effect of jiaozi mixed with yeast on industrialized steamed bread. Sci Technol Food Ind 9:114–119 23. He S, Liu C, Zuo J (2004) Study on the technics of one-time-fermentation-method steamed-­ bread making procession. Grain Processing 29(4):47–52 24. Han J, Wang Y, Liu C (2016) Study on different fermentation methods and characteristics of steamed bread. Grain Sci Technol Econ 41(2):60–62 25. Liu C, Li H, Fan Y, Bian K (2017) Change of water distribution of steamed bread with different fermentation methods. Food Ind 38(12):39–42 26. Liu C, Zhang H, Li Z (2014) Effect of batter fermentation conditions on quality of steamed bread. Sci Technol Cereals Oils Foods 1:63–67 27. Fan Y, Liu C, Chen L, Qu L (2015) Effect of Jiaozi paste fermentation condition on quality of steamed bread. Food Ind 36(9):12–15 28. Zhang G, Zhang W, Sadiq F, Sakandar H, He G (2019) Microbiota succession and metabolite changes during the traditional sourdough fermentation of Chinese steamed bread. Cyta J Food 17(1):172–179 29. Wang X, Zhu X, Bi Y, Zhao R, Wenqiao Y (2020) Dynamics of microbial community and changes of metabolites during production of type Ι sourdough steamed bread made by retarded sponge-dough method. Food Chem 330:127316 30. Zhang J, Liu W, Sun Z, Bao Q, Wang F, Yu J, Chen W, Zhang H (2011) Diversity of lactic acid bacteria and yeasts in traditional sourdoughs collected from western region in Inner Mongolia of China. Food Control 22(5):767–774 31. Zhao Zheng M, Taihua HS (2019) Microbial characterization of five Chinese traditional sourdoughs by high-throughput sequencing and their impact on the quality of potato steamed bread. Food Chem 274:710–717 32. Liu X, Zhou M, Chen J, Luo Y, Ye F, Jiao S, Hu X, Zhang J, Xin L (2018) Bacterial diversity in traditional sourdough from different regions in China. LWT Food Sci Technol 96:251–259 33. Suo B, Nie W, Wang Y, Ma J, Zhilu A (2020) Microbial diversity of fermented dough and volatile compounds in steamed bread prepared with traditional Chinese starters. LWT- Food Sci Technol 126:109350 34. Du H, Wang X, Zhang Y, Xu Y (2019) Exploring the impacts of raw materials and environments on the microbiota in Chinese Daqu starter. Int J Food Microbiol 297:32–40 35. Sakandar HA, Hussain R, Farid Khan Q, Zhang H (2020) Functional microbiota in Chinese traditional Baijiu and Mijiu Qu (starters): a review. Food Res Int 138:109830 36. Zheng X, Han B (2016) Baijiu (白酒), Chinese liquor: history, classification and manufacture. J Ethnic Foods 3(1):19–25 37. Xu D, Zhang H, Xi J, Jin Y, Chen Y, Guo L, Jin Z, Xu X (2020) Improving bread aroma using low-temperature sourdough fermentation. Food Biosci 37:100704

5  Steamed Bread

95

38. Zheng X, Yan Z, Han B, Zwietering MH, Samson RA, Boekhout T, Robert Nout MJ (2012) Complex microbiota of a Chinese “Fen” liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods. Food Microbiol 31(2):293–300 39. Xu D, Yin Y, Ali B, Zhang Y, Guo L, Xu X (2019) Isolation of yeast strains from Chinese liquor Daqu and its use in the wheat sourdough bread making. Food Biosci 31:100443 40. Changhong L, Yanhua C, Zhijian L, Hongmei L (2012) Effect of ratio of yeast to Jiaozi on quality of Chinese steamed bread. Procedia Environ Sci 12:1203–1207 41. Zhijian L, Haifeng L, Kedan S, Mingyu C (2019) Performance of non-Saccharomyces yeasts isolated from Jiaozi in dough fermentation and steamed bread making. LWT-Food Sci Technol 111:46–54 42. Shiwei W, Haifeng L, Zhijian L, Yanmei S, Jinshui W, Meng L (2018) Fitness of Jiaozi starter for steamed bread production using a two-stage procedure. Food Sci Nutr 6(6):1394–1401 43. Jin G, Zhu Y, Xu Y (2017) Mystery behind Chinese liquor fermentation. Trends Food Sci Technol 63:18–28 44. Cai H, Zhang T, Shen L, Peng P, Dai J, Zhang Q, Luo J, Mao K, Cai C, J M (2019) Isolation and identification of fungal and bacterial strains from Chinese sweet rice wine starters. Food Res Dev 40(24):204–210 45. Wang W, Wang X, Li Z, Ma Y, D L (2008) Molecular identification and sequence analysis of 18S rDNA gene of mold in sweet rice wine starter. Liquor-Making Sci Technol 011:33–35 46. Yao S, Zhang Y, Qin Z, Xu Q, Ma L, Chen L, W S (2013) Diversity of yeast in sweet fermented glutinous rice. China Brewing 32(7):61–64 47. Sakandar HA, Hussain R, Khan QF, Zhang H (2020) Functional microbiota in Chinese traditional Baijiu and Mijiu Qu (starters): a review. Food Res Int 138(B):109830 48. Yue Q, Liu C, Li L, Zheng X, Bian K (2019) Effects of fermentation on the rheological characteristics of dough and the quality of steamed bread. J Food Process Preserv 43(10):e14115 49. Liu T, Li Y, Sadiq FA, Yang H, Gu J, Yuan L, Yk L, He G (2018) Predominant yeasts in Chinese traditional sourdough and their influence on aroma formation in Chinese steamed bread. Food Chem 242:404–411 50. Huang S, Miskelly D (2016) Chapter 7 - Processing - making products. In: Huang S, Miskelly D (eds) Steamed breads. Woodhead Publishing, pp 81–99 51. Sha K, Qian P, Wang L, Lu Z, Li L (2007) Effect of storage time on the physicochemical and sensory properties of Man-tou (Chinese steamed bread). Int J Food Eng 3(3) 52. Huang S, Moss R, Resmini P, Gallant DJ (1991) Light-microsocpy observations on the mechanisim of dough development in Chinese steamed bread production. Food Struct 10(4):289–293 53. Wang X, Ma Z, Li X, Liu L, Yin X, Zhang K, Liu Y, Hu X (2018) Food additives and technologies used in Chinese traditional staple foods. Chem Biol Technol Agric 5(1):1 54. Pasqualone A (2019) Chapter 17 - Bread packaging: features and functions. In: Preedy VR, Watson RR (eds) Flour and breads and their fortification in health and disease prevention, 2nd edn. Academic Press, New York, NY, pp 211–222 55. Ke Y, Wang Y, Ding W, Leng Y, Lv Q, Yang H, Wang X, Ding B (2020) Effects of inulin on protein in frozen dough during frozen storage. Food Funct 11(9):7775–7783 56. Huang S, Miskelly D (2016) Chapter 8  - Frozen steamed products and frozen doughs. In: Huang S, Miskelly D (eds) Steamed breads. Woodhead Publishing, Sawston, pp 101–107 57. Ananingsih VK, Sim EYL, Chen X, Zhou W (2014) Heat transfer during steaming of bread. Int J Food Eng 10(4):613–623 58. Xu D, Hu Y, Wu F, Jin Y, Gänzle MG (2020) Comparison of the Functionality of Exopolysaccharides Produced by Sourdough Lactic Acid Bacteria in Bread and Steamed Bread. J Agric Food Chem 68:8907–8914 59. Tang X, Liu R, Huang W, Zhang B, Wu Y (2018) Impact of in situ formed exopolysaccharides on dough performance and quality of Chinese steamed bread. LWT-Food Sci Technol 96:549–525 60. Xi J, Xu D, Wu F, Jin Z, Yin Y, Xu X (2020) The aroma compounds of Chinese steamed bread fermented with sourdough and instant dry yeast. Food Biosci 38:100775

96

B. Yan and D. Xu

61. Yan B, Sadiq FA, Cai Y, Fan D, Zhang H, Zhao J, Chen W (2019) Identification of key aroma compounds in type I sourdough-based Chinese steamed bread: application of untargeted metabolomics analysis. Int J Mol Sci 20(4):818 62. Zhu F (2016) Staling of Chinese steamed bread: quantification and control. Trends Food Sci Technol 55:118–127 63. Chen D, Wang J, Jia F, Zhang C (2018) Effects of sourdough addition on the quality and shelf life of chinese steamed bread. Grain Oil Sci Technol 1(2):85–90

6

Taxonomy and Species Diversity of Sourdough Lactic Acid Bacteria Luc De Vuyst, Víctor González-Alonso, Yohanes Raditya Wardhana, and Inés Pradal

6.1 Taxonomy of Sourdough Lactic Acid Bacteria 6.1.1 Phylogenetic Position of Lactic Acid Bacteria LAB comprise a heterogeneous group of cocci-shaped or rod-shaped, Gram-­ positive, catalase-negative, non-sporulating, strictly fermentative, and facultatively anaerobic (aerotolerant) bacteria that produce lactic acid as the main end-metabolite of carbohydrate metabolism and that play an important role in the safety, organoleptic, health-promoting, and technological aspects of various fermented food products [1–6]. Phylogenetically, LAB belong to the phylum of the Firmicutes, class Bacilli, order Lactobacillales. Those found in sourdough belong to families such as the Lactobacillaceae [including the former Leuconostocaceae and, hence, covering in particular the genera Fructobacillus, Lactobacillus, Lactobacillus-related genera (see below), Leuconostoc, Oenococcus, Paralactobacillus, Pediococcus, and Weissella], Enterococcaceae (in particular the genera Enterococcus and Tetragenococcus), and Streptococcaceae (in particular the genera Lactococcus and Streptococcus). With respect to sourdough ecosystems, lactobacilli, lactobacilli-­ related genera, leuconostocs, pediococci, and weissellas occur mostly [7–16]. Enterococci, lactococci, and streptococci are the minor genera occurring in sourdoughs [9, 17–19].

L. De Vuyst (*) · V. González-Alonso · Y. R. Wardhana · I. Pradal Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, Belgium e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 M. Gobbetti, M. Gänzle (eds.), Handbook on Sourdough Biotechnology, https://doi.org/10.1007/978-3-031-23084-4_6

97

98

L. De Vuyst et al.

6.1.2 Classification of Lactobacillaceae Initially, the classification of LAB was based on morphological and physiological characteristics [20]. At a later stage, also chemotaxonomical properties, such as cellular fatty acid and cell wall compositions, were included. However, in LAB as well as in many other bacterial groups, phenotypic characters are often limited in their taxonomic usefulness for discrimination of closely related species and suffer from poor interlaboratory exchangeability. As a result, differentiation of LAB solely based on phenotypic traits is generally only considered reliable at genus level. The introduction of DNA-based techniques, such as genomic guanine plus cytosine content (mol%  G  +  C), DNA–DNA hybridization, sequencing of ribosomal RNA (rRNA) and housekeeping genes, and whole-genome sequence analysis brought significant changes to LAB taxonomy [21–24]. Especially the use of rRNA gene sequences as evolutionary chronometers allowed the elucidation of phylogenetic relationships between LAB species. As a result, the comparison of 16S rRNA gene sequences with such sequences available in public online databases became a standard approach for the identification of unknown LAB isolates. However, the low evolutionary rate of ribosomal genes compromised differentiation between LAB species exhibiting identical or nearly identical 16S rRNA gene sequences [25–28]. Alternatively, the use of multiple housekeeping genes, encoding essential cellular functions, was proposed for sequence-based identification of LAB.  For instance, classification of Lactobacillaceae species based on sequence analysis of the housekeeping genes pheS (encoding the phenylalanyl-tRNA synthase) and rpoA (encoding the DNA-dependent RNA polymerase alpha-subunit) proved to be highly congruent with 16S rRNA gene phylogeny [29–31]. Finally, whole-genome sequencing, which has been increasingly applied during the past two decades, and the application of average nucleotide identity (ANI) values of genes shared between bacterial genomes have revolutionized the approach to delineate new bacterial species [32, 33]. Indeed, today, ANI (95% within a range of 94–96%) is the only accepted criterion for delineation of new species and core genome phylogeny is a powerful tool to interrogate the evolutionary and phylogenetic relationships between different LAB. As is the case for many fermented food ecosystems, Lactobacillaceae are by far the most frequently recovered LAB from sourdough ecosystems [2, 5–16, 34–37]. Furthermore, in sourdoughs, at least 73 former Lactobacillus species, among 111 different LAB species, representing a high gamma-diversity, have been identified (Table  6.1). Taking into account that until March 2020 at least 260 species were present in the Lactobacillus genus, which includes the genus Pediococcus as an integral part, and primarily based on its complex taxonomy, the genus Lactobacillus has been reclassified into 25 genera [24]. Whereas a clear distinction can be made between homofermentative and heterofermentative Lactobacillus-related genera, homolactate and heterolactate fermentation is conserved at the family level in other LAB genera [24, 38]. These Lactobacillus-related genera include the emended genus Lactobacillus (homofermentative), the emended genus Paralactobacillus (homofermentative), and 23 novel genera (hence defined in 2020), among which the homofermentative Amylolactobacillus, Companilactobacillus, Schleiferilactobacillus, Lacticaseibacillus, Latilactobacillus, Loigolactobacillus,

Table 6.1  Homofermentative and heterofermentative lactic acid bacteria (LAB) species found in mature sourdoughs ranked according to their frequency (the number between brackets indicates the number of times the species was found in a total of 1364 sourdoughs consulted in the literature)

a

Homofermentative LAB species (62 species)

Heterofermentative LAB species (49 species)

Lactiplantibacillus plantarum (579) Companilactobacillus paralimentarius (220) Pediococcus pentosaceus (137) Pediococcus parvulus (131) Latilactobacillus sakei (95) Lacticaseibacillus paracasei (71) Latilactobacillus curvatus (61) Lacticaseibacillus casei (45) Lactiplantibacillus pentosus (45) Lactococcus lactis (45) Companilactobacillus alimentarius (40) Companilactobacillus farciminis (38) Loigolactobacillus coryniformis (37) Lactiplantibacillus paraplantarum (26) Pediococcus damnosus (26) Lactiplantibacillus xiangfangensis (22) Schleiferilactobacillus harbinensis (21) Lactobacillus delbrueckii (20) Lactobacillus helveticus (20) Enterococcus faecium (16) Lactobacillus acidophilus (13) Pediococcus acidilactici (13) Companilactobacillus crustorum (12) Latilactobacillus graminis (11) Lacticaseibacillus rhamnosus (9) Pediococcus ethanolidurans (9) Companilactobacillus nantensis (8) Companilactobacillus mindensis (7) Companilactobacillus kimchii (6) Lactobacillus acetotolerans (6) Lactobacillus gallinarum (6) Enterococcus durans (5) Enterococcus hirae (5) Lactobacillus amylovorus (5) Lactococcus garvieae (5) Ligilactobacillus salivarius (5) Enterococcus mundtii (4) Enterococcus faecalis (3) Lactobacillus crispatus (3) Lacticaseibacillus pantheris (2) Lacticaseibacillus zeae (2) Ligilactobacillus agilis (2) Amylolactobacillus amylophilus (1) Carnobacterium divergens (1) Companilactobacillus heilongjiangensis (1) Enterococcus cecorum (1) Enterococcus pseudoavium (1) Enterococcus raffinosus (1) Lacticaseibacillus saniviri (1) Lacticaseibacillus songhuajiangensis (1)a Lactobacillus amylolyticus (1) Lactobacillus johnsonii (1) Lactobacillus ultunensis (1) Liquorilactobacillus uvarum (1) Pediococcus argentinicus (1) Pediococcus cerevisiae (1) Pediococcus inopinatus (1) Schleiferilactobacillus perolens (1) Streptococcus constellatus (1) Streptococcus equinus (1) Streptococcus thermophilus (1) Tetragenococcus halophilus (1)

Fructilactobacillus sanfranciscensis (508) Levilactobacillus brevis (494) Furfurilactobacillus rossiae (111) Leuconostoc mesenteroides (84) Weissella cibaria (82) Leuconostoc citreum (77) Leuconostoc lactis (74) Limosilactobacillus fermentum (73) Weissella confusa (56) Limosilactobacillus pontis (30) Levilactobacillus senmaizukei (29) Levilactobacillus parabrevis (25) Leuconostoc pseudomesenteroides (24) Lentilactobacillus kefiri (21) Levilactobacillus spicheri (20) Levilactobacillus hammesii (19) Fructilactobacillus fructivorans (16) Lentilactobacillus diolivorans (16) Levilactobacillus zymae (14) Weissella paramesenteroides (14) Weissella viridescens (13) Levilactobacillus acidifarinae (10) Lentilactobacillus hilgardii (9) Limosilactobacillus frumenti (9) Limosilactobacillus panis (9) Lentilactobacillus buchneri (8) Leuconostoc holzapfelii (8) Lentilactobacillus parabuchneri (7) Leuconostoc gelidum (6) Levilactobacillus namurensis (6) Limosilactobacillus reuteri (6) Leuconostoc kimchii (4) Levilactobacillus koreensis (4) Secundilactobacillus odoratitofui (4) Secundilactobacillus malefermentans (3) Lentilactobacillus kisonensis (2) Leuconostoc fallax (2) Limosilactobacillus mucosae (2) Limosilactobacillus vaginalis (2) Weissella fabalis (2) Fructilactobacillus lindneri (1) Fructobacillus durionis (1) Fructobacillus fructosus (1) Furfurilactobacillus siliginis (1)a Lentilactobacillus farraginis (1) Lentilactobacillus otakiensis (1) Limosilactobacillus secaliphilus (1) Paucilactobacillus vaccinostercus (1) Secundilactobacillus collinoides (1)

Corresponding sourdough not characterized in detail

100

L. De Vuyst et al.

Liquorilactobacillus, Ligilactobacillus, and Lactiplantibacillus, and the heterofermentative Furfurilactobacillus, Paucilactobacillus, Limosilactobacillus, Secundilactobacillus, Levilactobacillus, Fructilactobacillus, and Lentilactobacillus harbor at least one sourdough LAB species. To this end, a polyphasic approach based on (conserved) pairwise average amino acid identity (AAI), core-gene average amino acid identity (cAAI), core genome phylogeny, clade-specific signature genes, physiological traits, and ecological criteria, has been applied [24]. Yet, before this reclassification came into practice, several of the proposed species names were already synonymized. Also, different phylogenetic clades (phylogroups) were proposed before, such as the Lactobacillus delbrueckii group (now Lactobacillus strictu sensu), the Lactobacillus alimentarius group (now Companilactobacillus), the Lactobacillus perolens group (now Schleiferilactobacillus), the Lactobacillus casei group (now Lacticaseibacillus), the Lactobacillus sakei group (now Latilactobacillus), the Lactobacillus plantarum group (now Lactiplantibacillus), the Lactobacillus rossiae group (now Furfurilactobacillus), the Lactobacillus reuteri group (now Limosilactobacillus), the Lactobacillus collinoides group (now Secundilactobacillus), the Lactobacillus brevis group (now Levilactobacillus), and the Lactobacillus buchneri group (now Lentilactobacillus), taking into account shared ecological and metabolic properties of the species involved but still phylogenetically and physiologically diverse [24, 38–41]. Former reclassifications of importance for the LAB species diversity in sourdoughs further include the transfer of some Leuconostoc species to the genus Fructobacillus, namely Leuconostoc durionis (now Fructobacillus durionis) and Leuconostoc fructosus (now Fructobacillus fructosus) [42].

6.1.3 Occurrence and Identification of Lactobacillaceae in Sourdoughs As a result of natural contamination through the flour, the environment, or by deliberate introduction via ingredients other than flour, a wide taxonomic range of LAB species has been found in sourdoughs [7–15, 37, 43]. In sourdough environments, LAB live in association with yeasts and are generally considered to contribute most to the process of acidification during flour fermentation, whereas yeasts are primarily responsible for dough leavening through carbon dioxide formation [7–9, 44–47]. Both microbial groups contribute to sourdough flavor formation during flour fermentation. Although homofermentative LAB species have also been isolated from sourdoughs, heterofermentative LAB species have the best potential and competitiveness to survive and grow in this particular matrix [7–9, 11, 15, 16, 48, 49]. The LAB species diversity associated with sourdoughs has been reviewed by several authors in the past two decades [7–9, 14–16, 37, 43, 48–51]. An updated overview of the homofermentative and heterofermentative LAB species found in mature sourdoughs up to now is compiled in Table 6.1. Given the taxonomic complexity of the Lactobacillaceae, accurate identification of unknown Lactobacillus and Lactobacillus-related sourdough isolates is not easy

6  Taxonomy and Species Diversity of Sourdough Lactic Acid Bacteria

101

and requires specific expertise, for example, the use of methods that offer sufficient taxonomic resolution as well as the correct interpretation of the identification results, for instance, by comparison with complete and up-to-date databases. In most of the older studies, however, identification of Lactobacillaceae mainly or even exclusively relied on phenotypic approaches with limited taxonomic resolution at species level. Therefore, it is safe to assume that some of the Lactobacillus(related) species previously reported in sourdough matrices and environments may have been incorrectly identified at the species or even at the genus level. A typical example is the taxonomic situation in the former L. plantarum group (now the Lactiplantibacillus genus), for which discrimination between the ubiquitous sourdough bacterium Lactiplantibacillus plantarum (previously Lactobacillus plantarum) and the phylogenetically highly related Lactiplantibacillus paraplantarum (previously Lactobacillus paraplantarum) and Lactiplantibacillus pentosus (previously Lactobacillus pentosus) may be problematic when identification methods with insufficient taxonomic resolution are used. In this regard, Torriani and co-­ workers [26] were among the first to suggest that sequences of housekeeping genes, such as recA (encoding a protein essential for repair and maintenance of DNA), rather than 16S rRNA gene sequences, should be recommended to distinguish between members of this phylogenetically tight species group (now genus). Likewise, several sourdough isolates initially assigned to Companilactobacillus alimentarius (previously Lactobacillus alimentarius) may in fact belong to the later described and closely related Companilactobacillus paralimentarius (previously Lactobacillus paralimentarius) due to former phenotypic misidentification [52, 53]. Also in this case, molecular fingerprint- and sequence-based methods are required to differentiate between both species [53]. Similarly, primers targeting the 16S/23S rRNA intergenic spacer region are used to discriminate species within the Latilactobacillus genus (previously the Lactobacillus sakei group) [54]. In Furfurilactobacillus rossiae (previously Lactobacillus rossiae), a remarkable intraspecific heterogeneity, leading to the identification of several subspecific clusters based on pheS gene sequencing, may complicate unambiguous identification of such strains [55]. A similar intraspecific heterogeneity occurs among Fructilactobacillus sanfranciscensis strains [56–59]. Finally, nomenclatural issues may also be a cause for taxonomic confusion. Corrections of originally misspelled specific epithets, such as “Lactobacillus sanfrancisco” [60] (later Lactobacillus sanfranciscensis and now F. sanfranciscensis) and “Lactobacillus rossii” [61] (later L. rossiae and now Ff. rossiae), and the synonymization of species, such as the recognition of Lactobacillus cellobiosus as a later synonym of Limosilactobacillus fermentum (previously Lactobacillus fermentum) [62], Lactobacillus suntoryeus as a later synonym of Lactobacillus helveticus [63], Lactobacillus homohiochii as a later synonym of Fructilactobacillus fructivorans (previously Lactobacillus fructivorans) [64], and Leuconostoc argentinum as a later synonym of Leuconostoc lactis [65] can take a while to be introduced in subsequent taxonomic and general literature. At present, a website hosted by two universities (www.lactobacillus.ualberta. ca and www.lactobacillus.uantwerpen.be) provides guidance on the current taxonomy of all lactobacilli.

102

L. De Vuyst et al.

6.1.4  Lactobacillaceae Species First Isolated from Sourdough Triggered by the introduction of molecular DNA-based taxonomic methods in sourdough microbiology and the growing number of 16S rRNA gene sequences as well as whole-genome sequences in public databases, many Lactobacillaceae species have been described since 2000 that were first isolated from a sourdough environment. It concerns Limosilactobacillus frumenti (previously Lactobacillus frumenti) [66], Companilactobacillus mindensis (previously Lactobacillus mindensis) [67], Levilactobacillus spicheri (previously Lactobacillus spicheri) [68], Levilactobacillus acidifarinae (previously Lactobacillus acidifarinae) [69], Levilactobacillus zymae (previously Lactobacillus zymae) [69], Levilactobacillus hammesii (previously Lactobacillus hammesii) [70], Ff. rossiae (previously L. rossiae) [61], Furfurilactobacillus siliginis (previously Lactobacillus siliginis) [71], Companilactobacillus nantensis (previously Lactobacillus nantensis) [72], Limosilactobacillus secaliphilus (previously Lactobacillus secaliphilus) [73], Companilactobacillus crustorum (previously Lactobacillus crustorum) [74], Levilactobacillus namurensis (previously Lactobacillus namurensis) [75], and Lacticaseibacillus songhuajiangensis (previously Lactobacillus songhuajiangensis) [76]. However, some of these species have only been reported in sourdough ecosystems once or seldom and are, hence, rarely isolated from this type of fermented food matrix, or are represented by only a few strains. Therefore, it is not clear which of these species are really typical for sourdough environments and, if so, what their geographical distribution is. In fact, only a few Lactobacillaceae species, such as the heterofermentative F. sanfranciscensis (insect-adapted lifestyle) and the homofermentative C. paralimentarius (lifestyle associated with other Lactobacillaceae), seem to be optimally adapted to the sourdough ecosystem and are rarely isolated from other sources [7–9, 15, 16]. Other heterofermentative LAB species, such as Levilactobacillus brevis (environmental or plant-associated lifestyle), and homofermentative LAB species, such as Lp. plantarum (nomadic lifestyle), are also frequently isolated from sourdoughs, but have also been found in many other food and non-food environments [7–9, 16, 38, 77]. Furthermore, Lactobacillus and Limosilactobacillus species have an intestinal lifestyle [16, 40].

6.1.5  Pediococcus, Leuconostoc, and Weissella as Subdominant Lactic Acid Bacteria Sourdough Species Although the LAB microbiota of sourdoughs is clearly characterized by lactobacilli and related genera, other less predominant or subdominant LAB species may also be found, including members of the genera Fructobacillus, Leuconostoc, Weissella, Pediococcus, Lactococcus, Enterococcus, and Streptococcus (Table 6.1). Of these, specific species of Pediococcus, Leuconostoc, and Weissella are particularly well adapted to survive and grow in plant-derived materials [48, 78]. The taxonomy of those three genera of the Lactobacillaceae family is much less complex than that of Lactobacillus and Lactobacillus-related genera, and their presence in sourdoughs is

6  Taxonomy and Species Diversity of Sourdough Lactic Acid Bacteria

103

restricted to only a few species. Within the homofermentative pediococci, the species Pediococcus pentosaceus and Pediococcus parvulus are most commonly found in sourdoughs (Table 6.1). Also Pediococcus damnosus and Pediococcus acidilactici occur, but less frequently. Differentiation between biochemically and phylogenetically related pediococcal species, such as P. acidilactici and P. pentosaceus, can be achieved by fingerprinting methods such as ribotyping [79, 80], amplified ribosomal DNA restriction analysis (e.g., 16S rDNA-ARDRA) [81], randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) [82, 83], and by sequence analyses of the 16S rRNA gene, the ribosomal intergenic spacer region (displaying high polymorphism), and the heat-shock protein 60 gene [84]. Concerning the heterofermentative genus Leuconostoc, the majority of sourdough isolates so far identified belong to Leuconostoc mesenteroides, Leuconostoc citreum, and Leuconostoc lactis (Table 6.1). In the species Lu. mesenteroides, further taxonomic distinction is made at subspecies level between Lu. mesenteroides subsp. mesenteroides, Lu. mesenteroides subsp. dextranicum, and Lu. mesenteroides subsp. cremoris. These three subspecies can be separated by RAPD-PCR fingerprinting [85]. Also Leuconostoc pseudomesenteroides occurs, but less frequently. Fructobacilli, closely related to leuconostocs, occur only sporadically (Table 6.1). Weissellas are heterofermentative LAB, of which a number of species produce dextran, as some leuconostoc species do [86–88]. In sourdoughs, the dextran-producing species Weissella cibaria and Weissella confusa are most frequently found [89–91]. Both species are positioned together on one of the four phylogenetic branches in this genus based on 16S rRNA gene sequence analysis [92], but can be differentiated using restriction analysis of the amplified 16S rDNA [93], RAPD-PCR [94], and a PCR assay targeting the 16S–23S rRNA intergenic spacer region [95].

6.2 Isolation of Sourdough Lactic Acid Bacteria Isolation of LAB from sourdough environments is challenging for three main reasons. First, sourdoughs are complex ecosystems not only in terms of their microbial composition but also in terms of the microbial interactive effects that occur among types of sourdough production processes and ingredients. The utilization of soluble carbohydrates by LAB and, thus, their energy yield are greatly influenced by the associated yeasts and vary according to the type of carbohydrates [96–98]. However, as many media for selective isolation of LAB incorporate yeast-inhibiting agents, such as cycloheximide, pimaricin, and amphotericin B, the trophic interaction between LAB and yeasts is in these cases disturbed, which may affect the recovery potential of LAB species/strains that strongly rely on this association. Second, sourdough production through backslopping (continuous propagation) is a dynamic process, in which fast-acidifying LAB species initially dominate the ecosystem and are then gradually replaced by typical sourdough LAB species that are more acid-­ tolerant and largely contribute to the organoleptic and textural properties of the end-product [7–9, 13, 17, 99–101]. Depending on whether the early subdominant LAB species and/or the final dominant LAB species are the target of the isolation

104

L. De Vuyst et al.

approach, it may thus be necessary to include multiple samples taken at different time points during the backslopping process. Finally, the LAB communities in sourdoughs may consist of metabolically very diverse groups, including homofermentative and heterofermentative LAB species, yeasts, and acetic acid bacteria [7–9, 15, 16, 43, 44, 48, 49, 102, 103]. As some of these LAB species have specific growth requirements in terms of the incubation medium and conditions (e.g., auxotrophies, temperature, pH, atmosphere, time, etc.), it seems inevitable that different medium formulations and/or sets of incubation parameters, so-called culturomics, are required to cover the entire metabolic LAB spectrum present in a sourdough sample. Initially, sourdough LAB were mostly isolated on de Man-Rogosa-Sharpe (MRS) medium [104], which is the general medium used for the isolation and enumeration of Lactobacillaceae from fermented food products. M17 medium is often used for the isolation and enumeration of Streptococcaceae [105]. MRS medium contains glucose as the main carbohydrate source. Triggered by growing insights into the species diversity of sourdough-associated LAB, several more specialized media were developed for the selective isolation of typical sourdough LAB species. For the specific detection of F. sanfranciscensis, Kline and Sugihara [60] proposed the Sour Dough Bacteria (SDB) medium that contains maltose as the carbohydrate source in addition to freshly prepared yeast extract (FYE) to further enhance its growth under anaerobic incubation conditions. The SDB medium is still frequently used for the isolation of LAB from sourdough [18, 78, 95, 100, 106–137]. In addition, FYE is often used as a supplement for other isolation media, for instance (modified) MRS medium [95, 109, 110, 113, 114, 116, 120, 121, 123–125, 132, 135, 138]. The Sanfrancisco medium was developed for the isolation and description of Limosilactobacillus pontis and C. mindensis [67, 139] and further used for general sourdough LAB isolations [78, 118, 128, 140–142]. This medium contains three flour carbohydrates (maltose, fructose, and glucose), FYE, cysteine, and rye or wheat bran, and anaerobic incubation conditions are applied. In parallel to the design of new media, several authors have also described variations of the original MRS medium formulation for the general isolation of sourdough LAB. Vogel and co-workers [139] further proposed a modified MRS medium with higher pH value (6.3) and anaerobic incubation conditions, whereas the MRS-5 medium [143] contains the three major carbohydrates present in flour (i.e., maltose, fructose, and glucose) in addition to cysteine and a mixture of six vitamins, has a lower pH value (5.8), and is suggested to be incubated under anaerobic conditions. In subsequent studies, the MRS-5 medium and slightly modified versions thereof have been repeatedly used for the enumeration and isolation of LAB during sourdough productions based on different types of flour and made under laboratory or bakery conditions and from mature sourdoughs, either under aerobic or anaerobic incubation conditions [68, 99, 118, 123, 129, 132, 144–163]. Also, it has been successfully used for the isolation of several novel Lactobacillaceae species from sourdough, such as Lv. spicheri [68], Lv. namurensis [75], and C. crustorum [74]. From these descriptions, it thus appears that the use of a modified MRS formulation with a lowered pH (