Natural and Bio-Based Antimicrobials for Food Applications 9780841233058, 0841233055, 9780841232976

Table of contents :
Content: Preface .............................................................................................................................. ix1. Natural and Bio-based Antimicrobials: A Review ............................................... 1Xuetong Fan, Helen Ngo, and Changqing Wu2. Natural Food Antimicrobials: Recent Trends in Their Use, Limitations, andOpportunities for Their Applications in Food Preservation .............................. 25Thomas Matthew Taylor3. Plant-Based Antimicrobials for Clean and Green Approaches to FoodSafety ....................................................................................................................... 45Sadhana Ravishankar4. Organic Acids, Detergents, and Their Combination for Inactivation ofFoodborne Pathogens and Spoilage Microorganisms ......................................... 63Dong Chen and Tong Zhao5. Improving the Microbial Food Safety of Fresh Fruits and Vegetables withAqueous and Vaporous Essential Oils .................................................................. 87Juan Yun, Changqing Wu, Xihong Li, and Xuetong Fan6. Antimicrobial Activities of Olive Leaf Extract and Its Potential Use in FoodIndustry ................................................................................................................. 119Yanhong Liu, Lindsay C. McKeever, Yujuan Suo, Tony Z. Jin, and Nasir S. A. Malik7. Control of Foodborne Pathogens by Hops Beta Acids in Food Systems ......... 133Cangliang Shen8. The Use of Natural Antimicrobials Combined with Nonthermal TreatmentsTo Control Human Pathogens ............................................................................. 149Behnoush Maherani, Samia Ayari, and Monique Lacroix9. Packaging Methods To Effectively Deliver Natural Antimicrobials onFood ....................................................................................................................... 171Tony Z. Jin, Mingming Guo, and Wenxuan Chen10. Antimicrobial Potential of Sophorolipids for Anti-Acne, Anti-Dental Caries,Hide Preservation, and Food Safety Applications ............................................ 193Richard D. Ashby, Daniel K. Y. Solaiman, Xuetong Fan, and Modesto Olanya11. New Classes of Antimicrobials: Poly-Phenolic Branched-Chain FattyAcids ...................................................................................................................... 209Helen Ngo, Karen Wagner, Alberto Nunez, Jianwei Zhang, Xuetong Fan, andRobert A. Moreau12. Evaluation of Toxicity and Endocrine Disruption Potential of the Naturaland Bio?Based Antimicrobials ............................................................................ 223Changqing Wu and Ying PengEditors>
' Biographies .................................................................................................... 243IndexesAuthor Index ................................................................................................................ 247Subject Index ................................................................................................................ 249

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Natural and Bio-Based Antimicrobials for Food Applications

ACS SYMPOSIUM SERIES 1287

Natural and Bio-Based Antimicrobials for Food Applications Xuetong Fan, Editor Eastern Regional Research Center U.S. Department of Agriculture, Agricultural Research Service Wyndmoor, Pennsylvania

Helen Ngo, Editor Eastern Regional Research Center U.S. Department of Agriculture, Agricultural Research Service Wyndmoor, Pennsylvania

Changqing Wu, Editor Department of Animal and Food Sciences University of Delaware Newark, Delaware

Sponsored by the ACS Division of Agricultural and Food Chemistry, Inc.

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Library of Congress Cataloging-in-Publication Data Names: Fan, Xuetong, editor. | Ngo, Helen, editor. | Wu, Changqing. 1975editor. Title: Natural and bio-based antimicrobials for food applications / Xuetong Fan, editor ; Helen Ngo, editor ; Changqing Wu, editor. Description: Washington, DC : American Chemical Society, [2018] | Series: ACS symposium series ; 1287 | Includes bibliographical references and index. Identifiers: LCCN 2018028810 (print) | LCCN 2018042117 (ebook) | ISBN 9780841232976 (ebook) | ISBN 9780841233058 Subjects: LCSH: Antibiotics in food preservation. | Anti-infective agents. Classification: LCC TP371.2 (ebook) | LCC TP371.2 N365 2018 (print) | DDC 664/.028--dc23 LC record available at https://lccn.loc.gov/2018028810

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2018 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

Contents Preface .............................................................................................................................. ix 1.

Natural and Bio-based Antimicrobials: A Review ............................................... 1 Xuetong Fan, Helen Ngo, and Changqing Wu

2.

Natural Food Antimicrobials: Recent Trends in Their Use, Limitations, and Opportunities for Their Applications in Food Preservation .............................. 25 Thomas Matthew Taylor

3.

Plant-Based Antimicrobials for Clean and Green Approaches to Food Safety ....................................................................................................................... 45 Sadhana Ravishankar

4.

Organic Acids, Detergents, and Their Combination for Inactivation of Foodborne Pathogens and Spoilage Microorganisms ......................................... 63 Dong Chen and Tong Zhao

5.

Improving the Microbial Food Safety of Fresh Fruits and Vegetables with Aqueous and Vaporous Essential Oils .................................................................. 87 Juan Yun, Changqing Wu, Xihong Li, and Xuetong Fan

6.

Antimicrobial Activities of Olive Leaf Extract and Its Potential Use in Food Industry ................................................................................................................. 119 Yanhong Liu, Lindsay C. McKeever, Yujuan Suo, Tony Z. Jin, and Nasir S. A. Malik

7.

Control of Foodborne Pathogens by Hops Beta Acids in Food Systems ......... 133 Cangliang Shen

8.

The Use of Natural Antimicrobials Combined with Nonthermal Treatments To Control Human Pathogens ............................................................................. 149 Behnoush Maherani, Samia Ayari, and Monique Lacroix

9.

Packaging Methods To Effectively Deliver Natural Antimicrobials on Food ....................................................................................................................... 171 Tony Z. Jin, Mingming Guo, and Wenxuan Chen

10. Antimicrobial Potential of Sophorolipids for Anti-Acne, Anti-Dental Caries, Hide Preservation, and Food Safety Applications ............................................ 193 Richard D. Ashby, Daniel K. Y. Solaiman, Xuetong Fan, and Modesto Olanya

vii

11. New Classes of Antimicrobials: Poly-Phenolic Branched-Chain Fatty Acids ...................................................................................................................... 209 Helen Ngo, Karen Wagner, Alberto Nuñez, Jianwei Zhang, Xuetong Fan, and Robert A. Moreau 12. Evaluation of Toxicity and Endocrine Disruption Potential of the Natural and Bio‐‐Based Antimicrobials ............................................................................ 223 Changqing Wu and Ying Peng Editors’ Biographies .................................................................................................... 243

Indexes Author Index ................................................................................................................ 247 Subject Index ................................................................................................................ 249

viii

Preface It is estimated that foodborne microorganisms in the United States cause 48 million sicknesses, 128,000 hospitalizations and 3,000 deaths each year. Food spoilage due to decay-causing microorganisms is also an issue in both developing and developed countries with 30-40% annual loss of fruits and vegetables. Synthesized antimicrobials (preservatives) are commonly used by the food industry to enhance microbial safety and increase shelf-life. However, food and agricultural industries are experiencing a lack of potent antimicrobial agents to secure the safety and maintain the quality of food products. Some synthetic preservatives may produce harmful by-products and damage the environment. There is also increasing incidence of antibiotics-resistant pathogens which has drawn great concern from the scientific communities and public health professionals. Therefore, there is increasing interest in the use of natural antimicrobials to enhance microbial safety, reduce spoilage and extend the shelf life of food. Natural antimicrobials are from plants, microorganisms and animals. They cover a wide variety of compounds including phenolics, terpenes, bacteriocins, peptides, proteins, natural polymers, fatty acids (lipids), and organic acids. Another trend in recent years is the rapid development of sustainable and value-added agricultural bio-products. Production of bio-products from renewable and sometime agricultural wastes would increase sustainability and reduce costs of natural products such as antimicrobials. Unlike natural antimicrobials which are directly extracted or purified from naturally occurring biological products, biobased antimicrobials are made using chemical reactions from agricultural products without the use of petrochemicals as precursors. The use of natural or bio-based antimicrobials over synthetic chemicals has several potential advantages. These include the potential for a smaller chance of microorganisms to develop resistance, improve biodegradability performance, minimize impact on environment, and a possible increased efficacy in inactivating microorganisms. In 2016, we organized a symposium on natural antimicrobials at the 252nd American Chemical Society National Meeting in Philadelphia, PA. The symposium covered the latest research on development and application of natural and bio-based natural antimicrobials. This book composes of the presentations made at this symposium with the majority of the speakers contributing to this book. In addition, two researchers were invited to contribute chapters. The book is divided into sections with different topics. Overall reviews of well-known natural or bio-based antimicrobials are first presented (Chapters 1, 2, 3). This book also has a heavy emphasis on antimicrobials of plant sources and their applications in foods and animal health (Chapters 4, 5, 6, 7, 8). A number of ix

chapters (Chapters 1, 2, 9) address the combinations of natural antimicrobials with non-thermal processing technologies to achieve additive and synergistic effects. The use of natural antimicrobials in packaging and coating is discussed in chapter 10, as well as in Chapters 2 and 9. In addition, both well-studied and novel biobased antimicrobials are discussed (Chapters 4, 11, 12). Furthermore, the needs for toxicological evaluations of natural and bio-based antimicrobials are presented and protocols are recommended (Chapter 13). Moreover, there is discussion in many of the chapters on the modes of action, mechanisms, and industrial aspects of applying natural or bio-based antimicrobials. Although it is difficult to cover all aspects of this ongoing active research in one book, this book on natural and bio-based antimicrobials serves the purpose of providing valuable information to the industry, academics and researchers who are searching for alternatives to synthetic compounds. In addition, we hope that the book will stimulate further research and promote the commercial applications of natural and bio-based antimicrobials. We are grateful to the authors for their outstanding effort in preparing their book chapters within a short time frame.

Xuetong Fan Lead Scientist/ Research Food Technologist USDA, ARS, Eastern Regional Research Center 600 E. Mermaid Lane Wyndmoor, PA 19038, United States

Helen Ngo Research Chemist USDA, ARS, Eastern Regional Research Center 600 E. Mermaid Lane Wyndmoor, PA 19038, United States

Changqing Wu Associate Professor and Food Toxicologist 044 Townsend Hall 531 S. College Avenue Department of Animal and Food Sciences University of Delaware Newark, DE 19716, United States

x

Chapter 1

Natural and Bio-based Antimicrobials: A Review Xuetong Fan,*,1 Helen Ngo,1 and Changqing Wu2 1USDA,

ARS, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038, United States 2Department of Animal and Food Sciences, University of Delaware, Newark, Delaware 19716, United States *E-mail: [email protected].

Natural antimicrobials, from plants, microorganisms and animals, cover a wide variety of compounds including phenolics, terpenes, bacteriocins, peptides, enzymes/proteins, natural polymers, fatty acids (lipids), organic acids and mixtures of bioactive compounds such as essential oils and plant extracts. Bio-based antimicrobials are modified, or synthesized from chemicals obtained from biological sources. This review discusses major types of natural and bio-based antimicrobials, inactivation mechanisms against microorganisms, structure-effect relationship, considerations for commercial application and future research needs.

Chemical preservatives are often used to extend shelf-life and reduce spoilage of various foods. However, consumers are increasingly becoming concerned with the safety of synthetic food additives and preservatives. As a result, “real food” without artificial ingredients or synthetic preservatives, are gaining popularity in recent years. The so-called “clean label movement” demands food products containing natural, familiar and easy-to-recognize and understand ingredients (1). Another trend in recent years is the rapid development of sustainable and value-added agricultural bio-products. Production of bio-products from renewable and agricultural wastes would increase sustainability and liberate us from our dependence on fossil fuel. A bio-based product derived from renewable © 2018 American Chemical Society

resources should have characteristics of biodegradability, commercial viability and environmental acceptability. Additionally, there has been a dramatic rise in the number of antibioticresistant pathogens in recent decades, stimulating interests to identify and develop antimicrobials/antibiotics that are active against multi-resistant bacteria. Natural and bio-based antibacterial agents are being developed and are considered to circumvent the increasing resistance of some pathogens, with a hypothesis that bacteria have less potential to develop resistance towards natural antimicrobials (2, 3). This review describes some well-known types of natural antimicrobials from plants, animals and microorganisms, along with inactivation mechanisms of selected antimicrobials, and the relationship between chemical structure and antimicrobial activity. Furthermore, bio-based antimicrobials are defined and addressed. In addition, considerations for commercial application of natural antimicrobials and future research needs are discussed.

Types of Natural Antimicrobials To be considered a “natural antimicrobial,” it is generally understood that this type of compound must be naturally occurring and be extracted from natural sources using simple methods. Natural antimicrobials are generally from animals, microorganisms, and plants. Some examples of natural antimicrobials from animal sources (e.g. milk, eggs, and crustaceans) are lysozyme, lactoferrin, lactoperoxidase, chitosan, megainin, pleurocidin, curvacin A, spheniscin and free fatty acids (4). Most of the antimicrobials of animal origins are enzymes/proteins and peptides. The lactoperoxidase system (LPS), consisting of lactoperoxidase, thiocyanate (SCN−) and hydrogen peroxide (H2O2), is an antimicrobial system that occurs naturally in milk and other secretions such as in saliva and tears. Well-known natural antimicrobials for microbial sources are nisin, natamycin, diplococcin, acidophilin, and pediocins, most of which are cationic, amphiphilic, and membrane-permeabilizing peptides (bacteriocins) which are produced by Gram-positive bacteria. Nisin and natamycin have been used commercially for various purposes. Nisin is one of the bacteriocins mostly applied by the food industry, and used in cheese, liquid eggs, and sauces. It exhibits a wide-spectrum antimicrobial action against Gram-positive bacteria such as Listeria monocytogenes. Natural antimicrobials can be obtained via a fermentation process and can be applied as partially purified or purified concentrates. Reuterin (β-hydroxypropionaldehyde) is an antimicrobial compound produced by some strains of Lactobacillus reuteri during the anaerobic fermentation of glycerol and has antimicrobial activity towards a broad spectrum of foodborne pathogens and spoilage organisms. Also, competitive-exclusion microorganisms and bacteriophages have been investigated and some are commercially applied (5, 6). Plants provide a wide range of antimicrobials. Some plant tissues, such as herbs and spices, are well known for their antimicrobial activities and have been used for centuries in foods to add flavors and fragrances. Agricultural and 2

food industries (particularly of fruits and vegetables) generate huge amounts of waste annually. Utilization of the waste and other low-value agricultural and food by-products for the recovery of bioactive compounds, including natural antimicrobials, is promising from food safety and human health perspectives (7). Antimicrobial compounds can be extracted from various parts of plants such as seeds, peels, pulps, and husks. By-products of fruit and vegetable processing could be good sources of organic acids, and phenolics, although the cost would be higher than corresponding synthetic compounds. Many plant-derived compounds are phenolics or their oxygen-substituted derivatives. Some examples of phenolics include thymol in thyme, vanillin in vanilla, benzoic acid from cranberries, cinnamic acid and eugenol in cinnamon, eugenol in cloves, terpene in sage and rosemary and many phytophenols in spices. Phenolic compounds have been shown to have antimicrobial activity against human pathogens and spoilage microorganisms (8, 9). Saleem et al. (10) surveyed natural metabolites with antimicrobial activity (with minimum inhibitory concentrations (MICs) in the range of 0.02–10 mg/mL) in the period of 2000–2008 literature. It is shown that phenolic compounds were the largest group (47) followed by quinones (19) and alkaloids (14) among 145 compounds examined. Possible toxic effects of quinones and alkaloids need to be thoroughly examined before being further considered for food applications. Most natural antimicrobials from plants are secondary metabolites and play important roles in the biochemistry and physiology of plants. These natural compounds are also vital in the defense of plants against invading pathogens, including bacteria, fungi, and viruses. Many compounds are biosynthesized in plants from shikimate pathway (for example, phenylpropanoids and phenolics) (11) and mevalonate and methylerythritol phosphate pathways (such as terpenes and terpenoids) (12). Some common antimicrobials such as sorbic acid and benzonic acid used can be found in nature, and have been widely used in food industry for spoilage reduction and shelf-life extension. However, the ones widely used by the food industry are primarily created through chemical synthesis due to economical reason (13). Essential oils are extracted or purified from various plant parts, such as leaves, flowers, seeds, balks, bulbs, and rhizomes. Certain essential oils stand out as better antibacterials than others for food applications. For example, coriander, clove, oregano and thyme oils were found to be effective at levels of 5–20 μl/g in inhibiting L. monocytogenes, Aeromonas hydrophila and autochthonous spoilage flora in meat products, sometimes causing a marked initial reduction in the number of recoverable cells (14). Essential oils are very complex natural mixtures which can contain ca. 20–60 components at quite different concentrations (15). They are characterized by two or three major components at fairly high concentrations (20–70%) compared to other components present in trace amounts. Generally, these major components determine the biological properties of the essential oils. The components of essential oils include three groups of distinct biosynthetical origins: shikimate, mevalonate, and methylerythritol phosphate pathway. Terpenes and terpenoids are synthesized in plants via mevalonate and/or methylerythritol phosphate pathways while other aromatic and aliphatic 3

constituents are from shikimate. All essential oil compounds are characterized by low molecular weight. Organic acids are naturally occurring compounds found in all living organisms, most notably from fruits, such as citric acid from citrus fruit, and malic acid from apples. The amounts of acids can be 1% in some immature fruits (16). These acids may play a significant role in defending the fruit against invading insects and microorganisms. Even though organic acids are found naturally, commercial production of organic acids from fruits and vegetables would be costly, making them not economically feasible. Therefore, most organic acids are either chemically synthesized or produced through a fermentation process. For example, industrial processes for the production of acetic acid are dominated by methanol carbonylation and the oxidation of hydrocarbons such as acetaldehyde and ethylene (17). Almost the entire quantity of citric acid is produced by microorganisms, mainly through submerged fermentation of starchor sucrose-based media, using the filamentous fungus Aspergillus niger (18). Other organic acids such as lactic acids can also be obtained from microbial fermentations. U.S. Federal regulatory agencies have not developed regulations on the definition of “natural,” although the FDA recently requested comments on use of the term “natural” on food labeling (19). “Natural flavor” and “natural flavoring” has been described by FDA. “The term natural flavor or natural flavoring means the essential oil, oleoresin, essence or extractive, protein hydrolysate, distillate, or any product of roasting, heating or enzymolysis, which contains the flavoring constituents derived from a spice, fruit or fruit juice, vegetable or vegetable juice, edible yeast, herb, bark, bud, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products, or fermentation products” (20). In another description, the FDA considered the term “natural” to mean that nothing artificial or synthetic (including all color additives regardless of source) has been included in, or has been added to, a food that would not normally be expected to be in that food. Interestingly, thymol, carvocrol and formic acid which are natural compounds are on the FDA synthetic flavoring substances and adjuvants list under Title 21, Sec. 172.515 19. Many organic acids and some flavor compounds are listed as GRAS which could be either natural or synthetic compounds (21).

Bio-Based Antimicrobials Presently, there is no established definition for bio-based antimicrobials. In this book, bio-based antimicrobials are defined as non-natural antimicrobial compounds that have been modified or synthesized from materials obtained from biological sources. The precursors of bio-based antimicrobials are generally regarded safe as they are from food and agricultural by-products. Bio-based antimicrobials may be synthesized from natural and renewable resources (e.g. phenolic fatty acids from phenolic compounds and fatty acids, thiamine dilauryl sulfate from vitamin B1, or lauryl alginate from arginine, Figure 1). These types of antimicrobials have advantages in terms of sustainability and renewability over 4

other compounds synthesized from petrochemicals. However, regardless of these advantages, these compounds are synthesized and do not exist in nature, and therefore, according to current regulations, they cannot be claimed as “natural” or clean labeled ingredients.

Figure 1. Examples of bio-based antimicrobials.

There are cases that compounds extracted from natural sources should be considered as “bio-based” antimicrobials. To extract natural compounds, solvents, acids, alkalines, and/or heat are often involved. The harsh conditions can potentially alter the structure of their natural forms. For example, when chitin isolated from shells of shrimp and other crustaceans is treated with an alkaline solution, chitosan is produced due to alkaline-induced partial deacetylation of chitin. Unlike chitin, chitosan is soluble in aqueous acidic media, which has a broader application. However, because chitosan is structurally different from its natural form (chitin), chitosan is a “pseudonatural” polymer (22). These types of compounds may be defined as bio-based antimicrobials. While it may be acceptable for natural compounds to be extracted using physical, enzymatic or microbiological processing, the use of genetically engineered microorganisms (GMOs) as a source of natural compounds is debatable. Most consumers think that “natural” should mean no GMOs (23) even though the genetic engineering process does not necessarily change the nature of natural compounds in the GMOs. For example, citric acid is commonly produced by a fungus, A. niger. The fungus may have been genetically modified to increase the yield of citric acid. Claiming antimicrobials from these types of genetic engineering as natural may be reasonable as they are not structurally altered. A term “natural-equivalent” has been used to promote the use of compounds that have the same exact composition and isomeric structure as the natural sources. However, there are instances that compounds are modified from the natural forms via genetic engineering to increase efficacy and stability. For example, 5

some antimicrobial peptides (AMPs) are genetically modified and the resulting structures of compounds are slightly different from their natural forms. In other instances, plants (such as tobaccos) are genetically modified (via recombinant DNA) to produce bacteriocins while non-transgenic plants do not naturally produce the specific bacteriocins. Thus, all these compounds can be considered bio-based antimicrobials.

Structure-Activity Relationship It is well-known that variations in the structure of compounds and chemical composition of the extracts result in differences in their antimicrobial activities (24, 25) (Figure 2). Comparing the antimicrobial activity of natural compounds with similar structural characteristics may be used to elucidate the importance of certain functional groups. Furthermore, comprehensive understanding of structure-activity is essential to guide the rational design and discovery of new and better antimicrobials. A key characteristic of antimicrobials is amphiphilicity. An amphiphilic antimicrobial has both hydrophilic and lipophilic properties with the ability to pass through cell membranes and be soluble in the aqueous phase. Hsiao and Siebert (26) applied the principal components analysis (PCA) method to analyze properties that could be related to antimicrobial activity of 17 acids commonly found in food. Among 11 properties analyzed, polar groups (PC1), number of double bonds (PC2), molecular size (PC3), and solubility in nonpolar solvents (PC4) were found related to antimicrobial activities. Furthermore, regression models of organic acid antimicrobial activity as a function of acid principal properties were developed by Nakai and Siebert (27) and successfully predicted MICs reported by Hsiao and Siebert (26). Both PC1 and PC4 represented polarities of a molecule, suggesting that polarity may have a great impact on the antimicrobial activity of acids. It is well known that molecular solubility is closely related to its polarity: the higher polarity of the molecule, the better its solubility in polar solvents (28). Therefore, better solubility of a compound may render the compound easier access to bacteria cells. In addition, molecular volume, interatomic distance and electric properties are prevailing structure factors that affect antimicrobial activity as these factors determine the configuration of molecules and interaction with membranes and cellular organelles (29). The functional groups (hydroxyl, oxygenated, double bound) of essential oil compounds affect their antimicrobial activity on multiple levels. They play a role in the polarity, solubility, hydrogen bonding capacity and pKa of the essential oil compounds, which all might influence the antimicrobial activity. Griffin et al. (30) found that the water solubility was the most important factor for determining in vitro antimicrobial activity of terpenoids after H-bonding capacity. The results also suggest that the essential oil compounds should be dissolved in the polar aqueous phase to be active against microorganisms. Kalemba and Kunicka (31) noted that among essential oils, phenols had the highest antimicrobial activity, followed by aldehydes, ketones, alcohols, ethers and hydrocarbons. 6

Figure 2. Chemical structures of selected natural antimicrobials.

Phenolic compounds possess great structural variations and are one of the most diverse groups of secondary metabolites. The hydroxyl (OH) group is most important for the antimicrobial activity of phenolic compounds (32). For example, phenols such as thymol and carvacrol, showed higher antibacterial 7

activity compared to hydrocarbon monoterpenes (33). The substitution of the OH group with an amino group reduces the antimicrobial activity. Compounds were more active with OH than with OCH3 or with COOH group (34). In addition, increasing the number of OH group on the benzene ring increased antimicrobial activity (34). The OH groups in phenolic compounds, acting as proton exchangers, are thought to interact with the cell membrane of bacteria, resulting in the destabilization of the cytoplasmic membrane and reduced pH gradient across the cytoplasmic membrane, which ultimately lead to the leakage of cellular components and cell death (35–38). The position of the OH group also influences the antimicrobial effectiveness of phenolic compounds. For example, thymol and carvacrol, having a similar structure, were different in antimicrobial effectiveness in agar medium (14). This difference has been attributed to the OH group located at the meta position in thymol compared to the ortho position in carvacrol. In addition, the length of the saturated side chain are influencing factors for the antimicrobial activity of phenolic compounds. The number of double bonds in phenolics and other compounds are also important in relation to antimicrobial effectiveness (39). For example, among citronellol, geraniol and nerol, citronellol was found to be less effective due to the presence of only one double bond whereas geraniol and nerol, with two double bonds, showed higher antimicrobial activity against tested bacteria (B. cereus, E. coli, S. aureus) and yeast (Candida albicans) (35). Antimicrobial activity of fatty acids is mainly a result of the undissociated molecule, similar to organic acids. For free fatty acids and monoglycerides, their antimicrobial activity depends on many factors such as the length of the acyl chain and the presence, number, position, and orientation of double bonds in the chain (40). Among all naturally existing fatty acids, the most active short chain saturated fatty acids is lauric acid (C12), the most active monounsaturated fatty acids is palmitoleic acid (C16:1), and the most active polyunsaturated fatty acid is linoleic acid (C18:2). The position and number of double bonds are important too, for example, the cis-form is more active than trans form. The activity of monoglycerides is strongly affected by the type of glycerol head group (40). For esterified fatty acids, single or multiple hydroxyl groups are necessary for antimicrobial activity. For instance, fatty acids esterified to a monohydric alcohol are inactive. Esterification to polyols increases activity. A class of novel antimicrobial phenolic branched-chain fatty acids was synthesized (41). When fatty acids were esterified to polyols, the antimicrobial activity of resulting phenolic branched-chain fatty acids increased compared with corresponding fatty acids (41). Interestingly, esterification of phenolic branched-chain fatty acids to phenolic fatty acid methyl esters and replacement of hydroxyl group in the phenolics with silyl group eliminated the antimicrobial activity of the compounds, suggesting that the carboxylic group in the fatty acid moiety and the hydroxyl group on the phenollic are important for the antimicrobial efficacy of the compounds against L. innocua (41).

8

Mechanisms of Action Although various mechanisms and theories have been proposed for many natural and bio-based antimicrobials, no uniform mechanism can be used to explain their antimicrobial properties. Considering the large variation both in compositions and chemical structures of natural antimicrobials, it is not a surprise that the exact mechanism of action is not well defined for most natural antimicrobials. As a result, the mechanisms of many natural and bio-based antimicrobials remain unknown. It is known that nisin and other bacteriocins are more effective against Gram-positive bacteria than Gram-negative bacteria (42). The resistance of Gram-negative bacteria to bacteriocins (and perhaps other natural antimicrobials) is explained by the presence of a protective outer membrane. However the activity of nisin against Gram-negative bacteria can be enhanced by combining with other membrane disrupting agents such as ethylenediaminetetracetic acid (EDTA). Many bacteriocins are AMPs that inhibit other closely related species of bacteria (4). Some bacteriocins such as nisin, pediocin and reuterin are the by-products of lactic acid bacteria fermentation. They are active against Gram-positive vegetative bacterial cells by a process of binding, insertion, aggregation, and pore formation in the cell membrane of bacteria, causing leakage of cellular molecules and cell death (43, 44). Although they vary in length, amino acid composition and secondary structure, AMPs have a distinct amphipathic conformation with a cationic charge and a significant proportion of hydrophobic residues. The cationic charge facilitate their binding to microbial cell membrane which is negatively charged. The hydrophobicity promotes interactions with the fatty acyl chains of phospholipids in the cell membrane (45). Based on secondary structures, AMPs can be classified into groups: α-helical peptides, β-sheet peptides and extended peptides (46). Extended AMPs, which do not fold into regular secondary structure elements, often contain high proportions of certain amino acids, such as histidine, arginine, glycine or tryptophan. Even though multiple mechanisms other than targeting cell membrane have been proposed, the interaction of AMPs with membrane and membrane components of microorganisms is probably a pre-requisite for the antimicrobial activities. Once peptides bind on the membrane and reach a certain threshold concentration, the peptides can create pores and channels in the membrane. Several mechanisms of interaction between peptides and the bacterial cell membrane have been proposed, including aggregate or toroidal pore, barrel-stave and carpet models (Figure 3) (48, 49). The aggregate or toroidal model involves the formation of an array of AMPs inside the bacterial lipid membrane in the same orientation as phospholipids. The barrel-starve model has aggregates of antimicrobial peptides which insert into the membrane bilayer so that the hydrophobic peptide regions align with the lipid core region and the hydrophilic peptide regions form the interior region of the pore (48). In the carpet model, the peptides disrupt the membrane by orienting AMPs parallel to the surface of the lipid bilayer and forming an extensive layer or carpet. The mechanism of nisin antimicrobial activity is well studied (50). Nisin, which has a net positive charge, attaches to the negatively charged cell membrane 9

surface. The hydrophobic N-terminal of nisin initially forms a complex with Lipid II, a precursor molecule in the synthesis of peptidoglycan (components of bacterial cell walls). The nisin-lipid II complex is then incorporated into the cytoplasmic membrane and forming pores (the barrel-starve model), and allows the efflux of essential cellular contents (Figure 4). The leakage of cytoplasm contents and/or cell lysis results in cell death.

Figure 3. Common models of AMPs against microorganisms. The interaction of AMPs with bacterial cell membranes: (A) Barrel-stave pore model; (B) toroidal pore model; (C) carpet model. The AMPs cause channels to form within the membranes, causing leakage of cytoplasmic content. Adapted with permission from ref. (47). Copyright 2009 Feinstein Institute for Medical Research. These models are established mainly based on in vitro studies using lipid vesicles and may not fully explain the interaction of AMPs with the complex bacterial cell membranes (52). Recent studies demonstrate that AMPs also achieve their antimicrobial activity by disrupting various key cellular processes such as DNA and protein synthesis, protein folding, enzymatic activity and cell wall synthesis (53). It is believed that AMPs should not cause widespread resistance due to their preferential attack on the cell membrane (54). It is difficult to correlate antimicrobial activity with specific structure characteristics of AMPs. However, it is generally agreed that the two main features required for their antimicrobial activity are cationic charge and induced amphipathic conformation (55). 10

Figure 4. A structure of nisin A (top) and mode of nisin action (bottom). After nisin reaches the bacterial plasma membrane, it binds to Lipid II via two of its amino-terminal rings, and forms a pore by orientating eight nisin molecules with four Lipid II. Adapted with permission from ref. (51). Copyright 2006 Springer Nature.

It has been shown that AMPs attach to the outer leaflet of the membrane and induce formation of blebs protruding from the microbial surface (56). In many instances, cellular damage lags substantially behind the time required for antimicrobial killing, indicating that some of the ultrastructural damage is a symptom of AMPs. It has been found that thiamine dilauryl sulfate (a vitamin B1 derivative) and sophorolipids induced similar blebs on the surface of Listeria cells (57) (Figure 5). However, it is unclear whether the appearance of the blebs is artificial, or an indication of loss in bacterial viability. Organic acids in their undissociated form (the protonated form) are lipophilic and can diffuse across the bacterial cell membrane. Once inside the cells, organic acids, encounter a near-neutral pH environment and dissociate into the free protons and acid anion (13). It is proposed that the microbial membrane is impermeable to protons, requiring proton motive force to actively efflux protons in order to maintain pH homeostasis in the cellular interior environment (58). However, continuous and large influx of organic acids dissipates the proton potential, depletes the endogenous energy sources and results in lowering the pH of cytoplasm. Low pH interrupts the normal cellular function and proton motive force of bacteria and jeopardizes the survival of most microorganisms. In addition, organic acids denature protein, and inhibit membrane transport leading to starvation of cells. Because organic acids exert their effects in the undissociated form, the dissociation constant (pKa) of the acids should be considered when 11

applying the acids in foods (59). The pKa is the pH at which 50% of the total acid is dissociated. The pKa values of almost all organic acids are below 6 (Table 1). Therefore, organic acids are mostly effective at low pHs (below 6), limiting their use in juices or other low pH foods. However, some organic acids in salt form such as sodium diacetate or sodium lactate have antimicrobial activity. The acidification of the cell cytoplasm by organic acids can not completely explain the effects of salts of organic acids (60). It is believed that the antimicrobial action of the compounds may be due to other actions such as chelating properties, dissipation of the transmembrane proton potential, interference with microbial metabolism, anion toxicity and reducing water activity of food products (61).

Figure 5. Representative SEM Images of Listeria cells treated with 20% ethanol (A) and 1% TDS dissolved in 20% ethanol (B) for 2 h. Magnifications were 50,000 x. It has been proposed that the antimicrobial action of essential oils is attributed to their ability to penetrate through bacterial cell membranes, and consequently, inhibit activity and functional properties of the cell, such as cellular energy (ATP) generation system, and disrupt the proton motive force (14, 62–64). In addition, essential oils and phenolic compounds may disrupt the cell membrane and cause leakage of the internal content of the cell (62). The disrupted permeability of the cytoplasmic membrane can result in cell death. Bio-based antimicrobials such as sophorolipids and thiamine dilauryl sulfate (57, 65) also exert their effects through interactions with the membrane, by separating cell membranes for Gram-negative bacteria and formation of blebs (Figure 5) on the surface of Gram-positive bacteria. It has also been suggested that thiamine dilauryl sulfate penetrates past the membrane into the cytoplasm to target other essential cellular functions (66). Lactoferrin, an iron-binding glycoprotein extracted from milk, possesses antimicrobial activity against a wide range of bacteria and viruses (24, 67). Lactoferrin has been approved in the United States and applied as an antimicrobial in a variety of meat products (68). In terms of antimicrobial action, lactoferrin may limit microbial access to nutrients via iron chelation, which produces an iron 12

deficient medium. Another possibility is that lactoferrin destabilizes the outer membrane of Gram-negative bacteria by releasing lipopolysaccharides from the membrane surface and increased membrane permeability. Hydrolytic proteins, such as lysozymes and chitinases, degrade the key structural components of the cell wall of bacteria and fungi. Like antimicrobial peptides, lysozyme is more effective against Gram-positive bacteria whose cell wall consists of peptidoglycan, which makes it susceptible to the activity of lysozyme (69). Gram-negative bacteria are generally resistant to lysozyme due to their lipopolysaccharidic layer of outer membrane which acts as a physical barrier. However, the susceptibility of Gram-negative bacteria to lysozyme can be increased by the use of detergents and chelators (EDTA) as membrane disrupting agents.

Table 1. Chemical Properties of Selected Organic Acids Name

Chemical formula

Molecular weight

Solubility (g/L) (temperature)

pKa1

Acetic acid

C2H4O2

60

Miscible

4.76

Adipic acid

C6H10O4

146

24 (25°C)

4.43

Caprylic acid

C8H16O2

144

0.68 (20°C)

4.89

Citric acid

C6H8O7

192

1478 (20°C)

3.14

Formic acid

CH2O2

46

Miscible

3.77

Fumaric acid

C4H4O4

116

4.9 (20°C)

3.03

Lactic acid

C3H6O3

90

Miscible

3.86

Levulinic acid

C5H8O3

116

675 (20°C)

4.59

Malic acid

C4H6O5

134

558 (20°C)

3.4

Propionic acid

C3H6O2

74

Miscible

4.87

Sodium diacetate

NaC4H7O4

142

570 (20°C)

4.75

Succinic acid

C4H6O4

118

58 (20°C)

4.16

5.61

Tartaric acid

C4H6O6

150

206 (20°C)

2.98

4.34

13

pKa1

pKa3

4.77

6.39

4.44

5.11

Chitosan is obtained from deacetylation of chitin by exposing chitin to strong NaOH solutions or to the enzyme chitinase. Chitosan exhibits strong antimicrobial effects against a variety of pathogenic and spoilage organisms (70). Structurally, chitin (poly-N-acetylglucosamine) resembles cellulose, except that the hydroxyl (–OH) group at the C-2 atom is substituted with an acetylated amino group (–NH–CO–CH3). The exact mechanism(s) of the antimicrobial activities of chitosan are unknown (71). However, it is has been proposed that the polycationic chitosan binds to negatively charged components on the cell surface via electrostatic interactions, altering transport and/or membrane permeability (72). Chitosan at 0.025% caused extensive cell surface alterations and covered the outer cell membrane with vesicular structures, thus explaining the loss of the bacterial barrier function (73). A number of mechanisms for the antimicrobial activity of fatty acids have been proposed (74) including stimulating oxygen uptake, inhibition of the membrane-located transport of amino acids into the cell, uncoupling energy systems and induced leakage of amino acids from cells. The effect of fatty acids and the type of inhibition produced by fatty acids depend on concentrations. At high concentrations, the effects are irreversible and bactericidal while fatty acids are bacteriostatic or have no effect at low concentrations. Because fatty acids and their esters have several modes of action that are nonspecific, the development of resistance to these compounds has not been reported. Monolaurin (a monoglyceride ester of fatty acids and a GRAS emulsifier), has been commercially used. It is believed that monoglyceride esters of fatty acids destabilize membranes by increasing membrane fluidity. Similar to essential oils, the sensitivity of cells to fatty acid esters depends on the lipid composition, fluidity, and curvature of the membrane (40). Although a number of mechanisms have been proposed for natural and bio-based antimicrobials, including interaction with proteins, enzymes, and membrane function, it seems that the most common mechanism is the action on cell membrane of microorganisms, and consequent cellular leakage. It is unclear whether the cellular leakage is the cause of cell inactivation (loss of viability) by natural antimicrobials or the symptoms as a result of other mechanisms. Often, when studying inactivation of microorganisms by natural and bio-based antimicrobials, too high concentrations and excessive treatment times are used (M. Davidson, personnel communication). It is possible that damage to cell membrane is a consequence of other mechanisms. For example, thymol and carvacrol interfere with ergosterol biosynthesis of Candida leading to changes in membrane fluidity and disruption of normal membrane activity (75). Cinnamaldehyde was found to inhibit cell wall synthesis enzymes: β-(1,3)-glucan synthase and chitin synthase 1 (76). Most natural antimicrobials are more effective against Gram-positive bacteria which have a one layer membrane and thick cell walls consisting of peptidoglycan and teichoic acid. The cell wall may play an important role in the sensitivity of Gram-positive bacteria to some natural antimicrobials.

14

Combinations with Other Antimicrobial Measures No single antimicrobial can control all types of bacteria, yeasts and molds in all food matrices. Often natural and bio-based antimicrobials alone are not effective enough or have too intense negative effect on food properties, such as smell and taste. As a result, the use of natural and bio-based antimicrobials may not be sufficient to meet the demands in microbiological safety, sensory quality, and retention of nutritional values. Thus, it is often desirable to use them in combination either with other natural antimicrobials or with physical preservation processes, such as high pressure processing (HPP) and packaging to achieve synergistic or additive effects against microorganisms, while the damage to the sensory and nutritional parameters of the food is kept to the minimum. Techathuvanan et al. (77) compared the efficacy of several commercial natural antimicrobials including white mustard essential oil (WMEO), citrus flavonoid and acid blend (CFAB), olive extract (OE), Nisaplin (a compound containing nisin), and lauric arginate (LAE) individually and in combination against foodborne pathogens and spoilage microorganisms. When WMEO was combined with other antimicrobials, the effects were usually additive except for WMEO plus Nisaplin and WMEO+OE, which had synergistic activity against L. monocytogenes and Salmonella Enteritidis, respectively. For WMEO+LAE+CFAB, additive antimicrobial effects were noted against all strains tested except Staphylococcus aureus, where a synergistic effect occurred. These findings suggest that these commercial natural antimicrobials can be combined to enhance food safety by inhibiting foodborne pathogens and extending product shelf life (77). Natural antimicrobials and plant extracts have also been used in combination with antibiotics to combat (overcome) the multi-resistance of pathogens (78–81). For example, essential oils containing carvacrol, cinnamaldehyde, cinnamic acid, eugenol and thymol, when combined with antibiotics, had a synergistic effect against human pathogens (82). It is proposed that antibiotics and the essential oil components may act synergistically by affecting multiple targets, by physicochemical interactions and by inhibiting antibacterial-resistance mechanisms (82). Natural antimicrobials have been combined with non-thermal processing technology such as high pressure processing (HPP). It has been shown that HPP inactivates vegetative microbial cells and, hence, produces microbiologically safe and stable products. As a non-thermal processing technology, HPP retains the nutritional and sensory characteristics of the fresh product better than the traditional thermal processing. While low molecular weight molecules like aroma compounds, vitamins, and minerals are less affected by HPP because of their very low compressibility of covalent bonds, large molecules such as proteins and starch, can change their native structure in response to HPP (83). Furthermore, the effects of HPP on many enzymes are limited. For example, enzymes such as polyphenol oxidase (PPO), peroxidase (POD), and pectin methylesterase (PME) are highly tolerant to HPP and are, at most, partially inactivated under conditions used by the industry (84). The residual activity of these enzymes would have impact on the quality (such as flavor and color) and shelf-life of foods. In terms of inactivating microorganisms, sub lethal 15

high-pressure injuries only inactivates a fraction of the microbial population; and certain bacterial strains and spore-forming bacteria are baroresistent. These injured and survival microorganisms can recover and multiply, posing a risk to the safety and preservation of foods. In order to overcome the limitations of HPP, the technology has been combined with natural antimicrobials, such as plant-origin antimicrobial agents (essential oils, oleoresins and vegetal extracts), acids from natural fermentation (vinegars) and their salts, and animal/microbial-origin compounds (antimicrobial peptides, active lipids, chitosan, lactoperoxidase systems, lysozyme, bacteriocins), competitive flora and bacteriophages (85). In many of these cases, natural antimicrobials act as additional hurdles or exert synergistic effects on spoilage and pathogenic microorganisms (85). Masschalck et al. (86) tested HPP inactivation of eight bacteria (two Escherichia coli strains, two Salmonella strains, two Shigella strains, Pseudomonas fluorescens and Staphylococcus aureus) in the presence of bovine lactoferrin (500 μg/mL), pepsin hydrolysate of lactoferrin (500 μg/mL), lactoferricin (20 μg/mL), and nisin (100 IU/mL). None of these compounds, at the indicated dosage, were bactericidal when applied at atmospheric pressure, except nisin, which caused a low level of inactivation of the bacteria. Under high pressure, lactoferrin, lactoferrin hydrolysate and lactoferricin displayed bactericidal activity against some of the test bacteria. The bactericidal efficiency and spectrum of nisin were also enhanced under high pressure. The authors proposed that pressure promoted uptake of these antimicrobial proteins and peptides in Gram-negative bacteria. Sanz-Puig et al. (87) demonstrated that the HPP treatment (200 MPa, 2 min) caused only one log reduction of Salmonella Typhimurium, but when combined with cauliflower or mandarin by-product infusions, S. Typhimurium was reduced by 5 log in 6 h at 37 °C. By-products of citrus species (mandarin, orange, and lemon) have phenolic compounds and essential oils with antimicrobial properties while by-products from Brassicaceae species contain compounds such as glucosinolates, flavonoids and polyphenols. It appears that a synergistic antimicrobial effect against S. Typhimurium took place when HHP treatment was combined with cauliflower or mandarin by-product infusion. Montiel et al. (88) combined reuterin and lactoperoxidase system with HPP to inactivate L. monocytogenes in cooked ham. They found that HPP at 450 MPa for 5 min only achieved 0.8 log reductions of L. monocytogenes, and the small difference between the HPP samples and control disappeared during 35 d storage at 4 and 10 °C. When reuterin or lactoperoxidase were applied in combination with HPP there was a synergistic antimicrobial effect against L. monocytogenes, avoiding the recovery observed with individual treatments. Furthermore, the combined treatments reduced levels of S. Enteritidis and E. coli O157:H7 below the detection limit ( 5.0 log10-cycles (31). Sohaib et al. (33) recently reviewed reports of PEF application for poultry and red meat products, indicating limited utility of the treatment due to concerns over composition of fresh meat products and low voltage impacts against microbes. The utility of PEF and other such treatments may be enhanced in fresh and further processed meats and poultry products by the inclusion of one or more natural antimicrobial agents (34, 35). Irradiation processing has been repeatedly evaluated for its antimicrobial impacts on foodborne spoilage and pathogenic microbes across multiple food types, and in many research studies natural antimicrobials have been incorporated in order to reduce the applied dosage(s). Irradiation has both primary and secondary effects against microorganisms, but one that has been reported, especially at higher doses, is the negative impacts of oxy- and hydroxy-radical formation on product quality attributes (color, odor, etc.) (35). Hence, using an antimicrobial that may also provide some antioxidant functionality (such as some plant-derived antimicrobials) can assist in preserving product quality by reducing the necessary dose applied to the food and protecting against free radicals formation. Research into the combined use of antimicrobials and irradiation has indicated desirable antimicrobial impacts of the hurdle processing on both plant-derived and animal-derived foods for multiple human pathogens and spoilage microorganisms. Over et al. (36) reported the combination of tartaric acid and 1.0 or 2.0 kGy electron beam processing on vacuum-packed chicken breast meat produced approximately 2.0 or 6.0 log10 CFU/g reductions in L. monocytogenes counts, respectively, during post-process storage. Use of natural antimicrobial-containing extracts, however, was not reported to impact pathogen reductions by authors. Kim et al. (37) reported the counts of aerobic bacteria and coliforms were reduced to non-detectable on irradiated pork loins when irradiation application was paired with 2% lactic, citric, or acetic acid, though secondary oxidation products were not reduced by antimicrobial treatments. Reductions in L. monocytogenes on fully cooked frankfurters were enhanced when 3,000 or 6,000 IU of the bacteriocin pediocin were applied in combination with 1.2 to 3.5 kGy irradiation processes (38). Lacroix et al. (39) applied Gamma irradiation to different foods inoculated with gram negative and positive pathogens. Salmonella Typhimurium was highly sensitive to irradiation in MAP and oxygen-permeable packaging films, although modified atmosphere packaging (MAP) use produced steeper reduction curves versus those obtained for beef not MAP-packaged. Additionally, combined application of carvacrol and 1.2 kGy treatment to cells of Bacillus cereus reduced the intracelluar ATP content by more than half versus cells treated by 1.2 kGy alone (39). Additionally, a variety of studies have been published detailing the usefulness of irradiation in combination with natural antimicrobial preservatives (applied directly or via edible coatings) for decontaminating bacterial and fungal pathogens from fresh vegetables, fruits and cereal grains (40–43). The combined application of irradiation (ionizing, 31

non-ionizing) with natural antimicrobials, similar to the combining of natural antimicrobials with other forms of processing, can facilitate greater application of irradiation processing in food stabilization and safety protection, assisting food sustainability and reduced waste of foods.

Enhancement of Natural Antimicrobial Efficacy or Delivery to Foodborne Microorganisms through Encapsulation or Incorporation into Edible Films/Coatings Encapsulation of Antimicrobials for Delivery to Foods The encapsulation of natural antimicrobials has been reported to facilitate increased transport of the antimicrobial agent(s) into the food system, with enhanced interaction with targeted microorganisms. Encapsulation systems vary in their complexity, stability, composition, and payload release properties. Examples of encapsulation technologies for food applications include micelles and liposomes, solid-lipid nanoparticles, protein- or modified polysaccharide-derived polymeric nanoparticles, repeating sub-unit co-polymer micelles or nano-capsules, multi-layer micro- and nano-capsules, and many others. Encapsulated antimicrobials, furthermore, may be added to the food through incorporation into the food formulation, adhered to packaging material, sprayed onto food surfaces or contact food through washing solutions. Taylor et al. (44) previously reviewed the applications for liposomes comprised of phospholipids and other amphipathic lipids for entrapment of differing compounds for agricultural and food applications, including food antimicrobials. These authors identified liposomes as thermodynamically self-assembled systems capable of high encapsulation capacity with good delivery into aqueous systems, including many foods, although encapsulate structures are not highly stable during long-term storage. Nonetheless, many studies and reviews have reported the utility of liposomal structures to encapsulate and deliver differing antimicrobial compounds to foodborne pathogenic bacteria in model systems and differing foods (45–52). Schmidt et al. (45) and da Silva Malheiros et al. (53) reported the antimicrobial efficacy of liposome-entrapped nisin, an antibacterial peptide produced by the gram positive bacterium Lactococcus lactis, indicating inhibition of L. monocytogenes was achieved in reduced fat milks stored at refrigeration following liposome addition. Encapsulation can assist the protection of encapsulated compounds from other food components that would otherwise interfere with the antimicrobial’s inhibition of contaminating microbes (54, 55). Antimicrobial-bearing micelles are nano-structured molecules comprised of amphiphilic surfactants that form around hydrophobic compounds that are aqueously dispersed. Like liposomes, their formation is thermodynamically favored when hydrophobic compounds are aqueously dispersed, and like liposomes they’ve been repeatedly studied for their utility in delivering antimicrobials to foodborne pathogenic microbes for liquid and solid food systems. Micelles and/or micellar structures can be formed from lipid-type surfactants, or from polymers that possess amphipathicity, allowing the encapsulation of 32

hydrophobic or amphipathic payloads/antimicrobials and their application into food systems. Gaysinsky et al. (56–59) reported the plant EOCs eugenol and carvacrol, encapsulated in surfactant micelles, exhibited inhibitory activity against the bacterial pathogens E. coli O157:H7 and L. monocytogenes in fluid medium and in fluid milk systems, in some instances producing greater reductions in pathogen counts versus controls than non-encapsulated EOC. Pérez-Conesa et al. (60, 61) applied plant EOC-loaded micelles to reduce the numbers of pathogens in biofilms, an innovative technology for food plant sanitary condition maintenance. Biofilms of L. monocytogenes Scott A and E. coli O157:H7 were reported to have pathogen counts reduced by >4.0 log10-cycles in one trial following 20 minutes of exposure to carvacrol-loaded micelles (60). Asker et al. (62) reported on the formation of mixed micelles containing the antimicrobial surfactant lauric arginate ester (LAE) with negatively-charged polysaccharides and the non-ionic surfactant tween 20, indicating that mixed micelles exhibited greater stability during storage. Similar results were obtained in subsequent research wherein lecithin micelles encapsulating the plant EOC thymol were stabilized against size change by inclusion of differing bio-polymers (e.g., gelatin, gum arabic) (63, 64). Xue et al. (65) recently reported that lecithin/gelatin mixed micelle nanoparticles encapsulating the plant EOC thymol were capable of reducing E. coli O157:H7 and L. monocytogenes counts by 4.5 to 5.0 log10 CFU/ml in skim milk stored at 21°C for 48 hr. Pathogen counts were reduced to non-detectable levels in 2% reduced fat milk and in cantaloupe juice products stored similarly to skim milk. Multiple log10 reductions in L. monocytogenes, S. aureus, and E. coli counts were recently also reported when pathogens were exposed to clove oil-containing micelles (66). Solid lipid nanoparticles (SLNs), containing 12.0 μg/ml limonene, were recently reported to exert inhibitory effects towards S. aureus in liquid medium, achieving between 3.0 and 4.0 log10 CFU/ml reductions in the pathogen’s numbers (67). Of particular interest regarding nano-encapsulation of antimicrobials for food safety protection has been a recent series of studies describing the application of antimicrobial micelles and micelle-like nanoparticles for pathogen decontamination on solid food surfaces. Zhang et al. (68) reported the formulation and characterization of micellar structures for produce decontamination from pathogenic bacteria. Ruengvisesh et al. (69) reported the minimum inhibitory concentrations (MICs), shearing properties, and antimicrobial potential against Salmonella Saintpaul and E. coli O157:H7 inoculated onto spinach leaf surfaces. Maximal reductions in pathogens were observed when spinach leaves were submerged in washing fluid containing LAE-containing micelles loaded with the plant EOC eugenol. Nevertheless, application of plant-derived EOC micelles to beef trimmings failed to reduce numbers of O157 and non-O157 Shiga toxin-producing E. coli (STEC) during post-treatment grinding and refrigerated storage (70, 71). Yegin et al. (72) developed nanoparticles comprised of the co-polymer Pluronic® F-127 and loaded with the rose EOC geraniol to decontaminate spinach from bacterial pathogens. MICs of nanoparticle-entrapped geraniol against E. coli O157:H7 and Salmonella were at least half those of unencapsulated geraniol, and pathogens on spinach leaves were reduced by 1.5 to 4.0 log10 CFU/cm2 depending on whether nanoparticles were applied 33

via spray or immersion for up to 5 minutes. Other researchers have reported on decontamination of foods using other non-micelle types of encapsulate structures. Gomes et al. (73, 74) reported the decontamination of leafy vegetables from bacterial pathogens following application of essential oils of cinnamon and other spice plants within β-cyclodextrins and the co-polymer poly (DL-lactide-co-glycolide) (PLGA) encapsulates, in one instance also detailing the capacity of nanoparticles to enhance spinach decontamination achieved through electron-beam ionizing irradiation from pathogens. D10 values (the required absorbed dose of radiative energy necessary to produce a 90% reduction in the count of a targeted microorganism) for Salmonella isolates inoculated onto baby spinach leaves were reduced from 0.19 kGy for the control by 0.05-0.08 kGy by application of encapsulates bearing either garlic extract or eugenol (74). Hill et al. (75, 76) later reported similar antimicrobial activities of nanoparticles bearing either eugenol, cinnamic aldehyde, or cinnamon bark oil against L. monocytogenes and Salmonella Typhimurium on spinach leaves, expanding understanding of the antimicrobial range of these technologies.

Natural Antimicrobials Incorporated into Edible Films, Coatings, and Food Packaging Materials for Food Preservation. As with natural antimicrobial delivery into foods via encapsulates, the incorporation of antimicrobials into edible films, coatings, or into food packaging materials (i.e. active packaging) represents an opportunity for enhancing the overall stability of food by applying a long-term selective pressure against cross-contaminating microorganisms in the food. In some instances, researchers have integrated nanoparticles or nano-encapsulate into the formulation of a film or coating for antimicrobial delivery onto a food surface (77, 78). Such technologies also present challenge to food processors, especially concerning the cost of applying such technologies into food processing systems and customers’ willingness to absorb the additional costs for purchasing foods bearing such systems. Malhotra et al. (79) recently discussed various applications of antimicrobial packaging appearing in the literature, discussing their observations of discrepancies between research data and opportunities for industry application. Authors discussed differences in release kinetics of film-loaded antimicrobials in research trials versus in food processing environments, whereby significantly higher loads of antimicrobials might be required during industrial processing versus laboratory trials for microbial inhibition. As with encapsulation of antimicrobials, researchers should consider laboratory-controlled experiments as indicating possible usefulness of antimicrobial-bearing films rather than as confirmatory for food safety protection during commercial food processing. Nevertheless, antimicrobial-bearing coatings and packaging polymers have been reported to be effective for pathogen inhibition and spoilage delay on various foods, and may be in the future very useful in preventing consumer exposure to microbial pathogens on fresh and further processed meat and poultry products, as well as minimally processed produce items (80, 81). 34

Consumer Demands for Clean-Label Foods Bearing Fewer Traditional and Synthetic Ingredients without Compromise on Product Quality and Safety As discussed above, there has been in recent years a pronounced trend in consumers of desiring foods bearing fewer traditional and/or synthetic preservatives. This demand for “clean label” foods requires a selection of foods possessing physico-chemical, organoleptic, and microbiological properties (including shelf stability) not differing from other foods formulated with traditional preservatives and ingredients. This has facilitated their commercialization in absence of any regulatory definition of clean label in the U.S. (82, 83) Formulating foods according to a clean label target presents food industry members and food safety specialists with, simultaneously, both opportunity and challenges for food safety protection and spoilage prevention as it relates to antimicrobials selection and use. Abbaspourad et al. (82), in a recent paper in a food industry professional serial, described fundamental challenges with clean label formulation stemming from the replacement of synthetic ingredients, including preservatives, with natural ingredients, including: • • •

Potential for reduced activity in the natural food additives, due to lack of potency in natural extracts, or heterogeneity in its formula/composition; Reduced stability of natural extracts and ingredients as compared to traditional/synthetic compounds, and; Additions to food cost derived from costs of ingredient extraction, processing, and refinement/purification (82).

Taylor (84) recently discussed other possible challenges and concerns in addition to those listed above related to clean label foods and natural antimicrobials use. The opportunity to produce a broader range of food products is an attractive outcome, potentially even a necessary one for food industry members to retain customer loyalty. Nonetheless, food industry members must consider and conduct trials during product development to guard against: •

•

•

Undesirable interactions of the natural antimicrobial with other food constituents, leading to loss of either food physico-chemical and/or organoleptic properties; Reduced apparent activity of the natural antimicrobial against microbes in the food based on reduced potency, activity based on antimicrobial mechanisms, or partitioning of the antimicrobial away from the microbes (related to previous concern); A need to add a higher content of the natural antimicrobial when replacing a synthetic or traditional preservative to gain the same antimicrobial activity demands that leads to a change in the food’s organoleptic properties (e.g., plant-derived antimicrobials with aromatic properties rendering a food less acceptable at concentrations giving effective antimicrobial activity).

35

•

A reduction in consumer understanding of food composition or consumer misunderstanding as to a product’s manufacture, as can be observed currently in the U.S. by the marketing of cured meat and poultry products as uncured when a natural source of nitrate/nitrite is used during product formulation, as compared to sodium nitrate and/or nitrite.

Abbaspourad et al. (82), in response to the concerns identified regarding clean label foods development, recommended a system-type approach to food companies developing new clean label foods to protect against product quality or safety loss through the replacement of synthetic antimicrobials with natural antimicrobials (82). Technologies such as antimicrobial encapsulation, combining use of a natural antimicrobial with other forms of processing, and other food safety-protecting systems not discussed here (e.g., use of hazard analysis critical control point [HACCP], sanitation, etc.) can aid the successful formulation of clean label food products to respond to consumer desires for such products (85, 86). For example, the delivery of plant-derived antimicrobials into a food product (e.g., vegetable juice requiring a 5.0 log10 CFU/ml reduction in a non-sporulating pathogen for pasteurization completion) via an encapsulate could be integrated into either a HPP or PEF-type processing system, or a thermal pasteurization system, allowing for a reduced-intensity physical process while protecting against pathogen re-growth or product spoilage. Additionally, the use of differing biopreservative technologies, most notably pathogen-antagonizing bacterial microorganisms, antimicrobial-containing fermentates, and bacteriophages, represent a strategy for food safety protection during post-processing food handling (87–89).

Natural Antimicrobials Utilization and the Changing Food Safety Regulatory Landscape in the U.S. With the passage of the FDA FSMA and its implementing rules, particularly those pertaining to Current Good Manufacturing Practices, Hazard Analysis, and Food Safety Preventive Controls for Human Food (90) and Standards for the Growing, Harvesting, Packing and Holding of Produce for Human Consumption (i.e. Produce Safety Rule) (91), the U.S. landscape for natural antimicrobials usage in food safety protection has changed. Key provisions of 21 CFR §117.135, 155, and 160 include the development for preventive controls, including process preventive controls, as well as a requirement for process preventive controls to be validated for efficacy. These requirements are similar to those in place for other FDA-covered food products subject to HACCP (seafoods, juices) (32, 92), or U.S. Department of Agriculture-regulated foods (meat, poultry, egg products, catfish) subject to HACCP (93). Procedures for validation of an antimicrobial have been detailed in multiple published studies, and multiple sets of guidelines for design and execution of antimicrobial interventions, including the use of chemical antimicrobials are published to assist food industry members with validating the utility of a HACCP or Food Safety plan (94–96). 36

In addition to the implementation of Food Safety Preventive Controls, regulations impacting the fresh produce industry are likely to open new doors to natural antimicrobial research and commercial applications, in order to reduce potential for pathogen transmission via minimally processed, ready-to-eat (RTE) produce to consumers. Painter et al. (97) reported fresh produce was the leading vehicle for onset of human foodborne disease cases in instances where food vehicles were identified in conjunction with sporadic and outbreak cases of human foodborne disease. While specific mandates for antimicrobials application during pre- or post-harvest produce handling incorporating antimicrobials are not elaborated in the final rule (91), produce growers covered by the final rule and its requirements may determine the use of a natural antimicrobial to be useful in produce decontamination and food safety hazard reduction. Natural antimicrobials lend themselves well to such activities, given the likely needs of growers to both reduce hazard transmission while also protecting natural/minimal processing claims for harvested commodities. Researchers have described opportunities for evaluating the utility of natural antimicrobials alongside other food processing technologies from the perspective of performance objectives and food safety objectives (FSOs) development. Performance objectives and FSOs can be useful in describing, following completion of a quantitative risk assessment for a food safety hazard, procedures to reduce the risk of hazard transmission and human disease throughout the food chain. David et al. (98) described processes to evaluate the performance of an antimicrobial agent for the purpose of developing a performance objective during product manufacture and handling. Performance objectives have the capacity to assist food industry members with identifying an achievable food safety standard within the processing environment, as well as quantifying the efficacy of hazard reducing interventions that assist in achieving the performance objective.

What Does the Future Hold for Natural Antimicrobials Usage? Given popular estimates of a global population of 9 billion or more by the year 2050, and the consequent needs for a food supply for all that is safe and wholesome, the need for antimicrobials usage will continue, if not grow, in years to come. Over the past decade, many new natural and traditional antimicrobial technologies have been introduced into the U.S. food industries from various sources; many of these have been natural antimicrobials, including biopreservatives (e.g., bacteriophages). The advent and growth of the organic and natural food industry sectors, and the explosion of interest in clean label food products in many consumers, indicates the demand for natural antimicrobials will continue to grow as food product developers work to meet consumer demands without compromising product safety and shelf-life. Nevertheless, while natural antimicrobials, due to their higher costs versus traditional antimicrobials, are unlikely to replace all use of traditional antimicrobials, it is an exciting time to be involved in the identification, characterization, application and verification of new and existing natural antimicrobials to protect foods. 37

Summary Natural food antimicrobials are a very important form of food preservation technology, typically regulated as food additives requiring some form of declaration of their presence in foods on product package labeling. Multiple factors influence the utility of an antimicrobial in a food. These include factors related to the chemistry of the food product itself, the processing of the food and its post-process handling, the microbiota that are targeted by the antimicrobial, and the antimicrobial agent itself. In the U.S. and around the world today there is great interest in natural antimicrobials to replace or supplement the use of traditional antimicrobials in order to produce foods bearing a clean label. Multiple strategies for enhancing the efficacy of natural antimicrobials, or to overcome their inherent limitations to use, have been explored. These have included using antimicrobials alongside other food safety hurdles, encapsulation of the antimicrobial compound in various manners, including encapsulated antimicrobial additives, films, coatings, and active packaging systems. Natural antimicrobials open doors to clean labels for foods not bearing synthetic ingredients, but replacing traditional antimicrobials is not a simple task and much work to validate no loss of product safety, wholesomeness, or loss of consumer acceptance is required. The ongoing use of food safety regulations, namely HACCP, and the initiation of food safety preventive controls and the FDA FSMA, drive processors’ interest in antimicrobials use. Developing understanding of how antimicrobial preservatives can be integrated into performance and food safety objectives design and application may yet aid greater food safety protection efficiency in years to come. Natural antimicrobials are still need of much research to yield greater understanding of which antimicrobials are of greatest utility in differing foods, critical parameters for their harvest or extraction to yield greatest antimicrobial activity, and how to successfully match them to foods for the best possible consumer acceptance and microbiological safety and quality protection.

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Chapter 3

Plant-Based Antimicrobials for Clean and Green Approaches to Food Safety Sadhana Ravishankar* School of Animal and Comparative Biomedical Sciences, University of Arizona, 1117 E. Lowell Street, Tucson, Arizona 85721, United States *E-mail: [email protected]. Phone: 520-626-1499.

Foodborne illnesses due to the consumption of contaminated food are on the rise in recent years. Since contamination can occur anywhere during the production chain, appropriate control measures are needed to reduce the microbial load post-harvest. Current consumers prefer natural ingredients and processes over chemical or synthetic additives, since they are aware of the health risks that could arise from using synthetic ingredients. Current consumers also want healthy and safe foods with good taste, freshness, nutrition, as well as ease of use. Hence, to meet consumer preferences, the food industry is seeking natural options in food production. To maintain good taste, freshness, nutrition and sensory attributes, multiple hurdle approach may be a good option for the industry. In multiple hurdle approach, several interventions are combined to produce safe food with good nutrition, freshness and other sensory attributes. Plant-based compounds are potential candidates to include in the multiple hurdle approach, since these are well known for their antimicrobial and other health promoting properties. Many of these compounds are Generally Recognized as Safe (GRAS), are considered natural, food-compatible and hence, have the potential to be acceptable by the consumers. Plant-based compounds could be considered a viable option in organic production, since the organic food industries are limited in what they can use, based on the requirements of the USDA-National Organic Program. Research has shown © 2018 American Chemical Society

that some plant-based compounds have potent antimicrobial activity in vitro and in/on foods. In this chapter, the potential for application of plant-based antimicrobials in multiple hurdle approach will be discussed. The antimicrobial activity of these compounds in vitro will be described. The application of plant-based antimicrobials for the post-harvest decontamination of fresh produce with emphasis on organic production will be covered. The use of these compounds to improve the microbial and toxicological safety of meat and poultry products will be explained. A unique way of applying some plant-based antimicrobials in foods via edible films will also be discussed. Plant-based compounds have the potential to decontaminate foods and improve their microbial safety, impart unique flavors and health-promoting properties to foods, reduce potentially carcinogenic compounds formed in grilled meats, and therefore, can help improve public health.

Introduction Food safety is an issue concerning public health since every year there are about 48 million people that get sick, about 128,000 people get hospitalized and about 3000 people die due to consuming contaminated food (1). The sources of food contamination are varied and can occur anywhere during the production and processing of food. Hence, farm to fork food safety is important. Since it is difficult to control the environmental factors leading to contamination during the production of food, stringent food safety measures are needed post-harvest. Control measures post-harvest are even more important for foods that are consumed raw such as salad vegetables or other ready-to-eat food products that do not undergo any lethal step prior to consumption. Present day consumers are aware of the health issues arising due to the consumption of synthetic or chemical additives present in foods. Hence, current consumers prefer all natural ingredients in foods and also prefer natural processing of their food products. Current health conscious consumers want safe foods with a fresh taste, appealing color and texture, good nutrition and convenient packaging. The food industry is seeking appropriate measures to meet the preferences of consumers and produce safe food with minimal effects on the sensorial attributes of food. One such measure is the multiple hurdle approach in which food is treated with more than one intervention. Natural food-compatible compounds derived from plants with strong antimicrobial properties can be potential candidates for a multi hurdle approach. Plant-based compounds are well known for their effectiveness against a number of microorganisms. Various forms of plant compounds that have been studied for their antimicrobial activity include essential oils, their active components, plant extracts and powders, and spices and herbs. Essential oils, their active components and plant extracts have demonstrated antiviral, 46

antimycotic, antiparasitic, insecticidal, antitoxigenic, and antibacterial properties (2–4). Examples of essential oils and their active components include oregano oil and carvacrol, cinnamon oil and cinnamaldehyde, clove bud oil and eugenol, lemongrass oil and citral, etc. Essential oils contain aromatic compounds and they can be obtained from various parts of the plant including leaves, bark, buds, flowers, fruits, seeds, and roots. Due to the presence of aromatic compounds, essential oils can add unique flavors to foods. Essential oils have wide applications in foods and cosmetic products. They are very effective against microorganisms even in small quantities. Due to their pungency, the amount that can be added to foods is limited. Plant extracts and powders can be obtained from various parts of the plants and depending on the flavor characteristics of the plant parts, the aromas and flavors of the extracts and powders can vary. For examples, olive extract made from olive juice can have the olive flavor, while apple extract made from apple skins may not have any flavor. Essential oils and plant extracts can contain over 60 different components with specific compounds comprising up to 85-90% of the oil or extract whereas others are only present in very small amounts (2, 4). This major component is called the active component. Examples of compounds found in essential oils and extracts include phenols, polyphenols, terpenoids, flavonoids, flavones, flavonols, tannins, quinones, coumarins, alkaloids, lectins, and polypeptides (5). Plant extracts and spices are usually added in higher quantities than essential oils, since they may not affect the sensory properties of foods. Various parts of the plants can be used as spices, and they generally have characteristics similar to essential oils, since spices contain aromatic compounds. Spices are usually added in small quantities to foods. Examples of spices include cinnamon bark, clove bud, cardamom, allspice, cumin, etc. Herbs constitute the leaves and stems of a plant and generally contain aromatic compounds as well. Examples of herbs include thyme, rosemary, mint, basil, sage, etc. Plant compounds are well known for their health benefits and medicinal values (4). For example, cinnamon is involved in reducing blood sugar, and turmeric has anti-inflammatory properties. Compounds found in olive extracts such as 4-hydroxytyrosol are known to have antioxidant, anticancer, anti-cholesterol, anti-aging, antidiabetic, and antimicrobial properties as well as protect against bone loss, heart disease, oxidative injury of kidney cells, and suppress oxidative stress (6). Many plant compounds are generally recognized as safe (GRAS) by the US Food and Drug Administration (7). Due to these benefits, plant compounds should be welcomed and easily acceptable as a “green technology” by consumers. Even though plant compounds have numerous advantages over synthetic/chemical additives, one major concern faced by the food industry could be the pungency of some compounds affecting the sensory attributes of foods. To overcome this concern, a combination application may be a viable option, where an aromatic essential oil or an active component of essential oil can be combined with a non-aromatic plant extract, both at lower concentrations. Synergistic or additive antimicrobial activities are possible from combination applications, which will be discussed later in this chapter.

47

Multiple Hurdle Approach The multiple hurdle approach embraces the concept of combining more than one treatment to achieve better effectiveness against microbes without adversely affecting the sensory attributes of food. A physical intervention can be combined with a chemical and/or a natural intervention or a chemical/natural intervention can be combined with another chemical/natural intervention to improve food safety. For instance, thermal treatment could be combined with a chemical preservative and/or a plant antimicrobial to inactivate microorganisms of concern in a food product. The intensity or concentration of each intervention can be significantly reduced, thereby maintaining the sensory quality of food. Since the mechanism of action of one intervention may differ from another, multiple hurdle approach may help achieve better inactivation by targeting various components of a microbial cell. Often times synergistic or additive effects are demonstrated when interventions are combined. The addition of carvacrol, the active component of oregano essential oil, to heated ground beef patties enhanced the thermal inactivation of Escherichia coli O157:H7 (8). Additional reductions of 2.5, 3.3 and 5.0 logs were obtained with combination treatments in comparison to heat alone, at 65, 70 and 80°C, respectively (8). When spinach leaves inoculated with Salmonella enterica serovar Typhimurium were electrostatically sprayed with a combination of 3% grape seed extract and 2% malic acid, reductions of 2.6-3.3 log CFU/g in the bacterial populations were observed after 14 days of storage, and the combination treatment had higher reductions compared to when the 3% grape seed extract was sprayed individually (9). Lv et al. (10) found that the combinations of oregano-basil, oregano-bergamot, basil-bergamot, and oregano-perilla essential oils were synergistic against Staphylococcus aureus; combinations of oregano with basil or bergamot had additive effects on Bacillus subtilis; and combinations of oregano with basil or perilla had additive effects on E. coli and yeast. These authors concluded that essential oil combinations inhibited bacterial growth at lower concentrations than was needed when they were used individually. Rada et al. (11) investigated the efficacy of 0.1% cinnamon or oregano essential oils in combination with 3% olive extract against Salmonella enterica serovar Newport on organic romaine and iceberg lettuces and baby and mature spinach. These investigators found that these combination treatments induced reductions in Salmonella population of up to 3.5-4 logs and 3-4.4 logs CFU/g on organic baby spinach and romaine lettuce, respectively, which were better than the reductions obtained when individual treatments were used.

Effectiveness of Plant Antimicrobials in Vitro Essential oils and their active components have been evaluated for their antimicrobial activity in vitro against numerous microorganisms. Lemongrass oil was effective against Candida albicans, E. coli, S. Typhimurium, Serratia marcescens, Staphylococcus aureus and other potential pathogens in vitro (12–15). Twenty seven essential oils out of a total of 96 and 12 of their active 48

components out of a total of 23 evaluated were found to be effective against the predominant foodborne pathogens including Salmonella, E. coli O157:H7, Listeria monocytogenes and Campylobacter jejuni (13). Elgayyar et al. (16) evaluated the antimicrobial activity of anise, angelica, basil, carrot, celery, cardamom, coriander, dill weed, fennel, oregano, parsley, and rosemary essential oils against L. monocytogenes, S. aureus, E. coli O157:H7, Yersinia enterocolitica, Pseudomonas aeruginosa, Lactobacillus plantarum, Aspergillus niger, and Geotrichum rhodotorula. Oregano, basil, and coriander essential oils showed strong inhibitory activity against both the bacteria and fungi, while anise essential oil was only effective against the fungi (16). Lemon, orange, and bergamot essential oils and their active components were found to be effective against C. jejuni, E. coli O157:H7, L. monocytogenes, B. cereus, and S. aureus in vitro, and among the compounds tested, bergamot, citral, and linalool showed the greatest activity (17). Shan et al. (3) evaluated 46 different extracts from spices and medicinal herbs for antimicrobial activity against Bacillus cereus, L. monocytogenes, S. aureus, E. coli, and S. Anatum. Twelve out of the 46 extracts including shiliupo, oregano, cinnamon, clove, diyu, huzhang, and cassia with a high phenol content showed antimicrobial activity against all five test organisms, with S. aureus being the most sensitive and E. coli being the most resistant (3). Cinnamaldehyde, the active component of cinnamon oil and carvacrol, at 0.1% or higher concentrations, showed rapid antimicrobial activity against both antibiotic-resistant and non-resistant C. jejuni strains, in phosphate buffered saline (18). Carvacrol and cinnamaldehyde were effective against antibiotic resistant and non-resistant Salmonella serotypes in 0.1% phosphate buffered saline (19). Several researchers have investigated the effectiveness of plant extracts and powders against numerous microorganisms in vitro. Commercial green tea leaf and rosemary powders were effective against B. cereus, S. aureus and L. monocytogenes in laboratory media (20). Olive extracts are made from the leaf, pomace or fresh juice of olives and contain a number of components that have shown antimicrobial activity. Olive leaf extracts exhibited antimicrobial activity against P. aeruginosa, K. pneumoniae as well as fungi including Candida albicans and Cryptococcus neoformans (21). The active components of olive leaf extract including hydroxytyrosol and oleuropein were effective against S. Typhi, Vibrio parahaemolyticus, V. cholera, and V. alginolyticus. The efficacy of olive leaf extract was evaluated against 122 microorganisms and it was found to be effective against C. jejuni and S. aureus including strains of methicillin resistant S. aureus (MRSA) (22). Friedman et al. (23) evaluated olive pomace and olive juice freeze-dried extracts against E. coli O157:H7, S. enterica, L. monocytogenes, and S. aureus and found them to be effective. The active component hydroxytyrosol was effective against all four pathogens, while another active component oleuropein was only effective against S. aureus and S. enterica (23). The olive juice freeze-dried extract was more effective against S. aureus and L. monocytogenes at the same concentrations as the pure hydroxytyrosol, suggesting the presence of other components in the extract that could have contributed to the increased activity (23). 49

Grape seed is an industrial waste byproduct and can contain a number of antimicrobial components such as phenolic compounds and proanthocyanidins (24, 25). Grapeseed extract was effective against S. aureus including MRSA strains (23, 26) and the efficacy in general, was better against Gram positives than Gram negatives (24). In another investigation, grapeseed extract was effective against both Gram positives and Gram negatives including B. cereus, B. coagulans, B. subtilis, S. aureus, E. coli, and P. aeruginosa; however, the concentrations required to inhibit Gram negatives were higher (1250-1500 ppm) than those required to inhibit Gram positives (850-1000 ppm) (27). The survival of E. coli O157:H7, S. enterica and L. monocytogenes in roselle (hibiscus) calyx aqueous (RCA) or roselle leaf aqueous (RLA) extracts over 72 h at 4, 8 and 25 °C was investigated (28). No E. coli O157:H7 and Salmonella survivors were detected in RCA or RLA at 24 h and all temperatures. The following reductions in L. monocytogenes populations were observed: 5 and 3 logs in RCA and RLA, respectively, at 24 h and all temperatures; by 4–6 logs at 4 °C and 8 °C; and to undetectable levels at 25 °C, at 48 h (28). Roselle extracts were effective against S. aureus, Bacillus stearothermophilus, Micrococcus luteus, Serratia marcescens, Clostridium sporogenes, E. coli, K. pneumoniae, B. cereus, and P. fluorescens in laboratory media (29) and against MRSA, K. pneumoniae, P. aeruginosa, and Acinetobacter baumanii (30). Plant compounds have been found to be effective against the enterotoxins produced by foodborne pathogens. One of the active components of olive extract, 4-hydroxytyrosol was effective in inactivating the enterotoxin A produced by S. aureus (6). Another active component of olive extract, oleuropein was found effective in inhibiting S. aureus enterotoxin B production (31, 32). Apple extracts containing polyphenolic compounds reduced the biological activity of S. aureus enterotoxin A (33).

Applications of Plant Antimicrobials in Produce The consumption of fresh produce has increased in recent years. The source of many outbreaks involving fresh produce has been identified as the pre-harvest environment and hence, stringent post-harvest measures are needed to decontaminate fresh produce. The main application of plant-based antimicrobials for produce could be in the wash water to decontaminate fresh produce. These compounds could serve as a good alternative to the currently used chemical-based sanitizers such as chlorine for the following reasons: a) plant compounds are effective against pathogenic microorganisms, thereby, increasing the microbial safety of produce; b) these compounds are also active against spoilage organisms and can thus, help increase produce shelf-life; c) they are GRAS-listed compounds and can be considered clean-label technology that may be acceptable by the consumers and the food industry; d) their efficacy is not affected in the presence of organic matter; e) they are more environmentally friendly, since they are biodegradable, and unlike chlorine, do not form carcinogenic compounds upon reacting with organic matter; f) plant antimicrobials have residual activity, which could help improve microbiological safety and shelf-life during transportation 50

and storage in the retail and consumer homes; g) they are effective at cold and ambient temperatures, thereby, are energy efficient; h) these compounds can enable recycling of wash water without reduction in their efficacy; and i) can provide health benefits to the consumers and unique flavors to foods. Some essential oils such as oregano oil are already added to salad dressings to improve flavor. Due to these benefits, plant antimicrobial application in produce has been the focus of research by many investigators. Essential oils and their active components could be potential candidates for application in the wash water to decontaminate produce. Yossa et al. (34) evaluated the effects of a 1 min treatment of 1000 ppm cinnamaldehyde against E. coli O157:H7 and a Salmonella cocktail on baby spinach. E. coli O157:H7 was not detected on the spinach leaves, whereas, there was a recovery of 1.66 log CFU/g of Salmonella after 14 days (34). Carvacrol and cinnamaldehyde at 1% were effective against antibiotic resistant S. Newport on celery (19). Oregano oil at 75 ppm and myrtle oil at 1000 ppm caused reductions of up to about 2.0 log CFU/g in S. Typhimurium population on whole tomatoes and iceberg lettuce, with a 20 min exposure (35, 36). This low reduction could be attributed to the very low concentration of oregano oil used, and also the essential oils were suspended in distilled water, which could have resulted in a low solubility of these hydrophobic compounds. Carvacrol and cinnamaldehyde in vapor phase were effective against Salmonella and E. coli O157:H7 on sliced and whole tomatoes (37). Carvacrol, cinnamaldehyde and thymol exhibited antimicrobial activity against B. cereus strains in carrot broth (38). The organic produce industry is limited in its options for adding sanitizers in the wash water to decontaminate organic produce, since they have to follow guidelines of the USDA-National Organic Program. Plant antimicrobials may be a viable option for the organic produce industry, since these compounds have shown effectiveness on organic produce. The antimicrobial activity of olive and apple powder extracts and hibiscus aqueous extract against S. Newport on organic iceberg lettuce, romaine lettuce, baby spinach and mature spinach stored at 4°C for 3 days was investigated (39). The antimicrobial activities were concentrationand storage time- dependant, with olive, apple and hibiscus extracts showing 2-3, 1-2 and 1 log reductions, respectively, while hydrogen peroxide (control) showed 1 log reduction (39). The efficacy of hibiscus tea, grapeseed and green tea extracts against antibiotic resistant S. Newport on organic romaine and iceberg lettuces and bunched mature and packaged baby spinaches was investigated (40). Compared to the water control, the concentration-dependent reductions in Salmonella populations by hibiscus tea, grapeseed and green tea extracts ranged between 0.7-2.3 log CFU/g, 0.4-2.0 log CFU/g, and 0.2-1.6 log CFU/g, respectively (40). The antimicrobial activities of lemongrass essential oil against S. N ewport were investigated on organic leafy greens stored for 3 days at 4 and 8°C, and romaine and iceberg lettuces, and mature and baby spinach samples showed between 0.6–1.5 log, 0.5–4.3 log, 0.5–2.5 log and 0.5–2.2 log CFU/g reductions in S. Newport population by day 3, respectively (41). The antimicrobial activity of oregano oil against S. Newport on organic leafy greens was investigated and reductions of up to 5 logs (no survivors detected) were obtained at both 4 and 8°C 51

(42). Cinnamon oil treatments of organic iceberg lettuce, romaine lettuce, baby spinach and mature spinach showed concentration-, storage time- and exposure time-dependant activities against S. Newport (43). The efficacy of essential oils and their active components has been investigated in fruits and fruit-based products. Citron essential oil was effective against S. Enteritidis, E. coli, and L. monocytogenes in fruit-based salads (44). Duan and Zhao (45) evaluated the effectiveness of lemongrass oil against E. coli O157:H7 and S. Enteritidis in strawberry juice. Citral and geraniol, the main active components present in lemongrass oil were effective against S. enterica and E. coli O157:H7 in apple juice (46). Roselle aqueous extract exhibited antimicrobial activity against L. monocytogenes, E. coli O157:H7, S. aureus, and B. cereus in apple juice (47). Plant-based compounds have been found to be effective in suppressing growth of spoilage microorganisms and hence, can enhance the produce shelf-life. Tea tree and clove essential oils exhibited antimicrobial effects against background microflora including bacteria, yeasts and molds on romaine lettuce leaves (48). Plant extracts such as olive, apple and hibiscus extracts were effective against background microflora of organic iceberg and romaine lettuce and baby and mature spinach (39). Cinnamon oil stunted the hyphal growth of Colletotrichum coccodes, Cladosportium herbarum, Aspergillus niger, Botrytis cinerea, and Rhizopus stolonifer as well as reduced the spore production (49). Basil essential oil was effective against spoilage microorganisms such as Aeromonas hydrophila and P. fluorescens on lettuce (50). Combining plant antimicrobials to reduce any adverse effects on the sensory properties of produce has been the subject of interest by many researchers. Since the combination treatments can affect multiple targets in a bacterial cell, the effectiveness may be better than that of individual treatments. Schneider et al. (51), evaluated a combination of clove bud oil (0.1%) and olive extract (5%) as well as individual treatments against S. Newport on organic romaine and iceberg lettuces and baby and mature spinaches. Reductions from individual treatments ranged from 0.40-2.5 log CFU/g and were less effective than the combination treatments that yielded reductions ranging from 0.87-3.6 log CFU/g in Salmonella population on organic leafy greens (51). Lv et al. (10) investigated combinations of various essential oils and showed that combinations of oregano-basil, oregano-bergamot, basil-bergamot, and oregano-perilla essential oils were synergistic against S. aureus; combinations of oregano with basil or bergamot had additive effects on B. subtilis; and combinations of oregano with basil or perilla had additive effects on E. coli and yeast. Additive, synergistic, and antagonistic activities have been studied by a number of researchers using various combinations of essential oils, their active components and plant extracts. Delaquis et al. (52) combined extracts of cilantro, dill, eucalyptus, and coriander essential oils and demonstrated additive, synergistic, or antagonistic activity when mixed in varying combinations. Al-Bayati (53) showed additive effects with combinations of phytochemicals against P. aeruginosa, which was found to be resistant to all of the test compounds when they were used individually. A combination of cinnamaldehyde and eugenol inhibited growth of Bacillus, Staphylococcus, Micrococcus, and Enterobacter 52

species for over 30 days, whereas their individual applications at the same concentrations did not inhibit growth (54). The extracts of clove, pomegranate, thyme, and jambolan were effective against P. aeruginosa and synergistic activity was exhibited when lower concentrations of the extracts were combined (55).

Applications of Plant Antimicrobials in Muscle Foods Muscle foods are complex matrices containing sugars, proteins, fats, vitamins, and minerals which could affect the activity of plant antimicrobials. Hence, it is difficult to achieve microbial inactivation in these matrices. However, plant antimicrobials have been found to be effective against pathogens in meat products. The application of plant antimicrobials in muscle foods is usually in the form of ingredients which are cooked prior to consumption. Hence, it is important that the antimicrobials withstand the heating process and are still active during storage after cooking. The influence of heat (70°C for 5 min) and subsequent cold storage (4°C up to 7 days) on the effectiveness of oregano and cinnamon essential oils and olive and apple extracts against multidrug resistant S. enterica serovar Typhimurium DT104 in ground pork was investigated (56). The plant compounds were stable during cooking, and heating did not affect their antimicrobial activity during storage. Cinnamon oil and olive extract were effective against S. Typhimurium resulting in 1.3 and 3 log CFU/g reductions, respectively, after 7 days of storage and their activity was concentration- and storage time-dependent (56). Hoque et al. (57) also found that exposure to 100°C for 30 min did not affect the antimicrobial activity of cinnamon and clove oils against L. monocytogenes, S. aureus, and S. Enteritidis. The application of 4% oregano and cinnamon bark extracts (as curing juice) into raw pork resulted in 2- and 3-log reductions in S. enterica populations, respectively, during storage at 20°C up to 9 days (58). Grape seed, pine bark and rosemary extracts at 1% concentration caused about 1-log reduction in populations of S. Typhimurium, L. monocytogenes, and E. coli O157:H7 in raw ground beef after 9 days of storage (59). Carvacrol and cinnamldehyde were effective against antibiotic resistant S. Newport on oysters during storage at 4 °C up to 3 days, exhibiting a reduction of about 2 and 5 logs CFU/g, respectively (19). Lemongrass oil inhibited the growth of foodborne pathogens in minced meat products (60). Roselle aqueous extract demonstrated antimicrobial activity against L. monocytogenes, E. coli O157:H7, S. aureus, and B. cereus in ground beef (47). The antimicrobial effects of garlic, ginger, carrot and turmeric pastes against E. coli O157:H7 in laboratory buffer and ground beef were investigated and it was found that the antimicrobial activity was reduced in ground beef (in comparison to laboratory buffer), with only commercial ginger paste showing 1-2 log CFU/g reduction after 3 days of storage (61). Garlic, ginger and turmeric pastes inhibited the growth of multi-drug resistant S. Typhimurium DT 104 in heat-treated ground beef stored at 4°C for 10 days (62). Plant antimicrobials have been shown to have antioxidative activity and because of this property, they can be applied in meats for their dual benefitsantimicrobial and anti-carcinogenic activities. Ground beef products such as 53

hamburger patties can be contaminated with E. coli O157:H7. Undercooked hamburger patties have been implicated in outbreaks of E. coli O157:H7 in which there were fatalities of young children. The regulatory agencies mandate that ground beef products be heated to an internal temperature of 71.1°C at the cold spot to inactivate E. coli O157:H7. While the patties are being heated/grilled to reach 71.1°C at the cold spot, the periphery of the patty can reach temperatures as high as 200°C. At such high temperatures, potentially carcinogenic compounds, such as heterocyclic amines can be formed. Epidemiological studies have shown a good correlation between consumption of well done meats containing heterocyclic amines and incidence of cancer in humans (63, 64). The dual benefits of carvacrol were evaluated in heated ground beef patties inoculated with E. coli O157:H7, and the active component was able to simultaneously inactivate the pathogen (2.5-5 log CFU/g reduction) and reduce the formation of heterocyclic amines (58-78% reduction in the three major amines) (8). Olive extract at 5% and lemongrass oil at 1% reduced the E. coli O157:H7 population to below detection limits and olive extract reduced the two predominant heterocyclic amines formed in grilled red meats, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by 79.5% and 84.3%, respectively, in heated ground beef patties (65). Apple extract at 5% and clove bud oil at 1% reduced the E. coli O157:H7 population by 1.6 log CFU/g, respectively, and apple extract reduced the heterocyclic amines, MeIQx and PhIP by 76.1% and 82.1%, respectively, in heated ground beef patties, while onion powder reduced PhIP by 94.3% (65). Olive and apple extracts, clovebud oil and onion powder exhibited concentration-dependent effects against E. coli O157:H7 and heterocyclic amines- MeIQx and PhIP (66). Plant antimicrobial addition in hamburger patties can help improve the microbial safety of these products and benefit human health. The use of less heat to cook the meat will also save energy (8).

Application of Plant Antimicrobials via Edible Films A novel way of applying plant-based antimicrobials to food products is through incorporating them in edible films. Edible films are thin films that can be made using various plant parts such as fruits (apple), vegetables (carrot), leaves (spinach), and flowers (hibiscus). The plant material is pureed and essential oils are added. These along with other ingredients are homogenized and cast into thin films that are dried and cut into any desired shape and size. Edible films can also be made from other natural substrates such as algin, chitosan, casein or whey proteins. Edible films can either be used as wrappings on food products such as meats or added as ingredients in packages such as salad bags. Edible films can impart the following benefits to the food products: reducing the microbial load present in foods; preventing cross-contamination from the environment; preventing browning in some fruits and vegetables such as apples and potatoes; preventing lipid oxidation in foods containing fats; preventing loss of moisture from foods; preventing the food from outside odors; preventing loss of volatile flavors from foods (67); and imparting of unique flavors to 54

foods from the essential oils in the films. Edible films and coatings can serve as carriers for various food additives, including antimicrobials. The incorporation of antimicrobial compounds into edible films or coatings provides a novel way to control foodborne pathogen contamination (68). The effectiveness of antimicrobial edible films against foodborne pathogens in vitro and on foods has been investigated by many researchers. Edible appleand tomato-based films containing plant essential oils (oregano, lemongrass, and cinnamon) and their active components (carvacrol, citral, and cinnamaldehyde) reduced the populations of E. coli O157:H7, S. enterica, and C. jejuni in vitro (69–72). Oregano and cinnamon oil containing edible films were effective against S. enterica on agar, both by direct contact with the bacteria and indirectly by vapors emanating from the films (73, 74). Apple-alginate based edible coatings containing lemongrass (1.0-1.5%) and oregano (0.5%) essential oils exhibited strong antimicrobial activity against L. innocua, reducing the bacterial population to below detection limits within 7 days (70). Whey protein films containing 2% oregano oil were effective against E. coli O157:H7, S. aureus, S. Enteritidis, L. monocytogenes, and Lactobacillus plantarum (75). Chitosan and alginate-based films containing garlic oil showed antimicrobial activity against E. coli, S. aureus, S. Typhimurium, L. monocytogenes, and B. cereus (76, 77). Films made of partially hydrolyzed sago starch and alginate mixture containing lemongrass oil inhibited E. coli O157:H7 (14). Apple, carrot and hibiscus-based edible films containing three concentrations (0.5, 1.5 and 3%) of carvacrol or cinnamaldehyde were added as ingredients in salad bags containing organic leafy greens, and their antimicrobial effectiveness against S. Newport during storage at 4°C for 7 days was evaluated (78). All 3 types of 3% carvacrol films reduced the Salmonella population by 5 log CFU/g at day 0 and 1.5% carvacrol films reduced Salmonella by 1-4 log CFU/g at day 7, while the films with 3% cinnamaldehyde showed 0.5-3 log reductions on different leafy greens at day 7 (78). Alginate films containing cinnamon, clove, and lemongrass oils or their active components cinnamaldehyde, eugenol, and citral reduced E. coli O157:H7 population by 4 logs on fresh-cut Fuji apples (79). An alginate-based edible coating containing essential oils, their active components and malic acid significantly reduced S. Enteritidis population in fresh-cut melon (80). Adding 1.5-2.0% chitosan to the methyl cellulose coating on fresh-cut cantaloupe reduced the mesophilic aerobes, psychrotrophs, lactic acid bacteria, and total coliforms by 3-4 log CFU/g (81). Apple-based edible films containing 3 concentrations of carvacrol and cinnamaldehyde wrapped on chicken breast and ham and stored for 72 hours at 4 and 23°C exhibited concentration- and storage time- dependent activities against S. Enteritidis, E. coli O157:H7 and L. monocytogenes (82). Apple, carrot and hibiscus-based films containing 3 concentrations of carvacrol and cinnamaldehyde wrapped on ham and bologna and stored at 4°C for up to 7 days were effective against L. monocytogenes with a maximum of 3 logs reduction (83). In general carvacrol containing films were more effective than those containing cinnamaldehyde; inactivation by apple films was greater than that by carrot or hibiscus films; and films were more effective on ham than on bologna (83). Apple-based edible films containing 3 concentrations of carvacrol 55

and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken breast stored at 4 or 23°C for 72 hours (84). Cinnamaldehyde containing films were more effective (causing reductions below the detection limit) than those with carvacrol and their activity was better at 23 than at 4°C (84). Antimicrobial edible films can be used as wrappings on meat and poultry products to reduce contamination by foodborne pathogens.

Mechanisms of Action of Plant-based Antimicrobials Plant-based antimicrobials have multiple active components that could contribute to the antimicrobial activity and hence, it is difficult to pinpoint one type of mechanism for any specific type of an antimicrobial (85, 86). Thus, one antimicrobial compound may have several targets of action in a bacterial cell. Also, not all mechanisms observed may be separate targets; some may occur as a consequence of another target or mechanism (87). In general, it is believed that the polyphenolic compounds present in many plant antimicrobials could be membrane active compounds disrupting the cell membrane and eventually causing cell lysis (88, 89). Essential oils are lipophilic compounds that can enter microbial cells, disrupt the membrane and/or permeabilize it (90). Membrane permeabilization is usually followed by the loss of ions and the reduction of potential, the collapse of proton pump and the depletion of ATP pool (91). The mechnisms of action have been investigated for a few active components of essential oils. Leakage of phosphate ions was observed in case of S. aureus and P. aeruginosa cells that were treated with carvacrol (92) and adenosine triphosphate release was observed following disruption of E. coli cell membranes by carvacrol (93). Being a lipophilic compound, carvacrol dissolved in the phospholipid bilayer of cell membrane and caused expansion and destabilization of the membrane, thereby, resulting in an increase in membrane fluidity and passive permeability (94, 95). Cinnamaldehyde and eugenol have been shown to interfere with the activity of enzymes. In Enterobacter aerogenes, cinnamaldehyde interfered with the action of amino acid decarboxylases (96) and inhibited the separation of B. cereus cells after cell division (97). Studies have shown that eugenol can inhibit ATPase, amylase, protease, and histidine decarboxylase (96, 98, 99). Essential oils with aldehydes or phenols as major components (examples; cinnamaldehyde, citral, carvacrol, eugenol, or thymol) are usually the most effective against microorganisms (90), followed by those containing terpene alcohols (100), and then those containing esters (examples; β-myrcene, α-thujone, or geranyl acetate) (101, 102).

Conclusions Farm to fork food safety is important in food production, and since it is difficult to control pre-harvest contamination, appropriate post-harvest control measures are necessary. Plant-based antimicrobials are potential candidates 56

for application in a multiple hurdle approach to enhance the microbiological safety of foods. These compounds are safe, natural, food-compatible and have added benefits in comparsion to chemical additives. They are well suited for the organic industry which has limited options for control measures. Plant-based antimicrobials have demonstrated efficacy against a number of foodborne pathogens and spoilage microorganisms in vitro and in model food systems, and hence, have the potential to be applied to foods to improve their microbiological safety and shelf-life. These antimicrobials also have antioxidative properties and can be used for dual benefits to inactivate foodborne pathogens and reduce the formation of carcinogenic heterocyclic amines in grilled meat products. Plant antimicrobials can be used in the wash water for washing fresh produce as a clean label technology that can allow for the recycling of wash water. These compounds can be added as ingredients in meat products, since they are stable under cooking conditions and can inactivate pathogens of concern in meat products. Antimicrobial edible films can help reduce surface contamination of meat and poultry products and inactivate pathogens in bagged salads. Future research will aid in commercial applications of these compounds by the food industry.

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Chapter 4

Organic Acids, Detergents, and Their Combination for Inactivation of Foodborne Pathogens and Spoilage Microorganisms Dong Chen1 and Tong Zhao*,2 1College

of Food Science, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400715, China 2Center for Food Safety, College of Agricultural and Environmental Sciences, University of Georgia, 1109 Experiment Street, Griffin, Georgia 30223, United States *E-mail: [email protected].

Organic acids have been broadly used by meat and poultry industries as a spray treatment of meat carcasses for reduction of foodborne pathogens. Their benefits, efficacy, and problems were evaluated by different studies. The combination of levulinic acid and sodium dodecyl sulfate (SDS) has recently received attention for its antimicrobial efficacy against several bacteria and viruses in food matrices or on multiple food and non-food surfaces. Both ingredients are an FDA approved flavoring substance and multipurpose food additive, respectively. Synergism between levulinic acid and SDS was observed, and the assumed mechanism of action was presented. The antimicrobial efficacy of levulinic acid and SDS remained high even when organic materials are present. The other features, including foamability and readily solubility, extend its potential applications to decontamination of hard-to-reach surfaces and control of foodborne pathogens on food contact surfaces.

© 2018 American Chemical Society

Inactivation of Foodborne Pathogens with Organic Acids Organic acids, because of their low pH, have been documented inhibitory or lethal on various foodborne pathogens. Among them, acetic acid, citric acid, octanoic acid, lactic acid, levulinic acid, propionic acid, and phenyllactic acid are revealed possessing bactericidal effects. Lactic acid is often the acid of first choice because it is odorless compared with other acids (1). The general explanation of bactericidal mechanism is the reduction of medium pH resulting in a decrease of the cytoplasmic pH of microbes by ionization of undissociated acid molecules. In order to maintain intracellular pH close to neutrality, bacterial cells have to pump out protons by hydrolyzing ATP, which depletes energy causing their death eventually (2–4). Results from a recall of ground beef containing Escherichia coli O157:H7 indicate that the contamination level was low and the selective enrichment was necessary for its isolation (5). Our investigation on the largest outbreak of E. coli O157:H7 linked to consumption of ground beef revealed that the median most probable number of the pathogen was 1.5 cells/g (range, 0.05) change before and after the hot surface spray treatment (7). Recent studies on the efficacy of acid-based intervention for reduction of Shiga toxin-producing E. coli (STEC), especially for E. coli O157:H7, in meat processing facilities have revealed that these bacteria have unique acid-tolerance characteristics, and have been associated with various acidic foods, including apple cider (8), sausages (9), and tomato ketchup (10). Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) 64

considers E. coli O157:H7 and six non-O157 serotypes of STEC, including O26, O45, O103, O111, O121, and O145 strains, as adulterants in both raw ground and whole muscle cuts (11).

Reduction of Detergents on Foodborne Pathogens Based on their chemical structure, detergents can be classified into three species, including 1) anionic detergents with a structure of alkylbenzenesulfonate, 2) cationic detergents with a structure of quaternary ammonium, and 3) non-ionic and zwitterionic detergents with a structure of the uncharged hydrophilic group. The main purpose of their application in the food industry is for cleaning of food facilities with low inhibitory effect on foodborne pathogens. In 1961, the United States Public Health Service recommended that personnel need to wash their hands with soap and water for 1-2 minutes before and after client contact. The result indicated that the antibacterial soaps can remove 65 to 85% bacteria from human skin (12). Food handlers, including those who deliver and serve foods, should follow this recommendation to reduce potential cross-contamination. A recent study indicates that some bacterial strains, such as Pseudomonas aeruginosa, may become detergent-resistant, which leads to their survival even in the presence of high concentration of soaps (13).

Combination of Levulinic Acid plus Sodium Dodecyl Sulfate Background Information One chemical treatment that in recent years has shown considerable promise as an antimicrobial intervention is a combination of levulinic acid and sodium dodecyl sulfate (SDS). Levulinic acid (4-oxopentanoic acid, CH3C(O)CH2CH2CO2H, molecular mass: 116.12 Da) is a 5-carbon organic acid which has been designated as Generally Recognized as Safe (GRAS, 21 CFR, 172.515) by FDA for direct addition to food products as a flavoring agent (14). It can be produced at a low cost yet in a high yield from renewable feedstocks (15). Addition of sodium levulinate in ready-to-eat (RTE) meats was effective at inhibiting growth of spoilage bacteria and Listeria monocytogenes without a noticeable different flavor (16, 17). S. Enteritidis populations in pure culture held at 21°C were reduced by 3.4 log CFU/ml when exposed to 0.3% levulinic acid for 30 minutes (18). SDS, an anionic surfactant, is designated by FDA as a multipurpose food additive (21 CFR 172.822). It is approved for use in a variety of foods, including egg whites, fruit juices, vegetable oils, and gelatin as a whipping or wetting agent, and also widely used in household products such as toothpastes, shampoos, shaving creams, and bubble baths (18). Various microbial isolates have been individually evaluated for their sensitivity to levulinic acid with SDS. Our results reveal that all bacterial isolates tested are killed instantly within 10-15 seconds of contact with levulinic acid with SDS while retaining the quality of treated products. The microorganisms 65

tested include but not limited to 1) yeasts and molds, such as Candida magnoliae, Debaryomyces hansenii, Geotrichum candidum, Saccharomyces cerevisiae, and Zygosaccharomyces bailii; 2) viruses, such as norovirus; 3) bacterial spores, such as Bacillus anthracis spores; 4) bacteria, such as Aerococcus viridans, B. anthracis, Campylobacter jejuni, Citrobacter freundii, E. coli O26:H11, O111:NM and O157:H7, L. monocytogenes, Klebsiella oxytoca and K. pneumoniae, S. Enteritidis, S. Medegridis, S. Montevideo, and S. Typhimurium, Staphylococcus aureus, Streptococcus gordonii, S. mitis, S. mutans and S. oralis, Vibrio cholerae, Yersinia pestis and Y. pseudotuberculosis; 5) plant pathogens, such as Acidovorax avenae subsp. citrulli, Erwinia carotovora, Xanthomonas campestris, Xanthomonas axonopodis, and Pantoea ananatis. Unfortunately, this novel disinfectant cannot be used to inactivate foodborne parasites (19, 20). Cryptosporidium and microsporidia are not inactivated when treated for various periods of time with 2% levulinic acid and 1% SDS or 3% levulinic acid and 2% SDS at 20°C (20).

Inactivation on Yeasts and Molds The pure culture of five isolates of yeasts, including Saccharomyces cerevisiae, Debaryomyces hansenii, Candida magnoliae, Zygosaccharomyces bailii, and Geotrichum candidum, was individually determined at 21°C for its sensitivity to the solution containing levulinic acid plus SDS at different concentrations. Results revealed that the inactivation of levulinic acid plus SDS on yeasts was concentration- and species-dependent. As shown in Table 1, the killing effect of 0.5% levulinic acid plus 0.05% SDS is weak, and a longer contact time (>10 minutes) was required for a significant (P < 0.05) reduction. Some of them, especially Z. bailii, require high concentrations of 2% levulinic acid plus 1% SDS and long contact time (20 minutes) to reach a reduction of >5 log CFU/ml. The pure culture of four mold isolates, including Mucor hiemalis, Penicillium pubeses, P. expansum, and Paecylomyces variotri, was individually determined at 21°C for its sensitivity to levulinic acid plus SDS at different concentrations. As shown in Table 2, the killing effect on molds was various and species-dependent. Generally, the combination with higher concentrations would achieve a greater reduction. Some of them (e.g. M. hiemalis) was more resistant, suggesting that other factors, like heating, could be considered if mold contamination is the sole concern.

Virucidal Efficacy Virucidal efficacy of levulinic acid plus SDS was tested, with such that 5 % levulinic acid plus 2 % SDS inactivated murine norovirus (MNV-1) surrogate by 2.5 1og PFU/ml, and stainless steel-inoculated MNV-1 by >1.50 log PFU/ml, after 1 min of exposure (21). Lower concentrations of 0.5% levulinic acid plus 0.5% SDS induced 2.7-, 1.4-, and 2.4-log PFU/ml reductions for hepatitis A virus 66

(HAV), MNV-1, and MS2 bacteriophage, respectively, after 2 min of exposure (22). The virucidal efficacy of this combination was not significantly affected by the presence of organic matters (up to 10%), indicating this combination could be effective for reducing infectious virus in a clinical matrix, where stool or vomit, which can protect viruses from inactivation by a sanitizer, often exists (21). High concentrations of 5% levulinic acid plus 2% SDS was also determined to be virucidal for influenza A H3N2 virus (23). Thus, this novel disinfectant may be an alternative sanitizer that can be used as a part of an egg decontamination strategy and as a tool to mitigate human and avian influenza transmission by eggs.

Inactivation on Bacterial Spores Isolates of B. cereus, B. subtilis, B. circulans and Alicyclobacilli acidoterrestris were individually induced to form spores by standard procedures. These spores were treated by heat at 65°C for 30 minutes for inactivation of germinated bacteria. Results revealed that the killing effect of this formulation (levulinic acid plus SDS) on Bacillus species was variable, even among the same species. The killing effect is very high on spores induced from isolates of B. subtilis (ATCC #82), even at low concentrations of 0.5% levulinic acid plus 0.05% SDS. All tested spores (>7 log CFU/ml) were instantly inactivated (12-15 seconds of processing time) at 21°C. However, the killing effect at 21°C on spores induced from isolates of B. subtilis (ATCC #31028) was very weak, even at high concentrations of 20% levulinic acid plus 3% SDS for an extended contact time (120 minutes). Similar results were demonstrated for spores induced from isolates of B. circulans (#47-10), B. cereus (ATCC #10987), and A. acidoterrestris (#SAC, #OS-CAJ, and #N-110). Inactivation of spores by 3% levulinic acid plus 2% SDS with the treatment of raised temperatures at 62, 70, and 80°C for different contact times was also determined. Results revealed that the treatment of 3% levulinic acid plus 2% SDS at 62°C instantly killed all the tested spores induced from different Bacillus species, but the spores induced from isolates of A. acidoterrestris species were more resistant to the same treatment. The killing effect was directly related to the temperature. At 80°C, all tested spores even induced from isolates of A. acidoterrestris were inactivated within 1 min. These results indicate that the treatment composed of levulinic acid plus SDS is a powerful disinfection agent for inactivation of spores when it is used with heating.

67

Table 1. Effect of Levulinic Acid Plus Sodium Dodecyl Sulfate (SDS) at Different Concentrations at 21°C on Various Yeast Species Yeasta

Saccharomyces cerevisiae

68

Debaryomyces hansenii

Candida magnoliae

Treatments

Yeast counts (log CFU/ml) at minute: 0b

1

2

5

10

20

30

60

0.1 M PBS (control)

5.2

5.3

5.5

5.3

5.3

5.2

5.3

5.3

2.0% levulinic acid

5.4

5.3

5.5

5.4

5.3

5.3

5.5

5.0

1.0% SDS

2.7

2.4

2.6

2.3

2.8

2.7

2.3

2.4

0.5% levulinic acid plus 0.05% SDS

4.9

4.5

3.9

3.2

2.7

1.7

1.3