fermentation and food safety

Fermentation andFood Safety

Editors

Martin R.Adams, MSc, PhDReader in Food Microbiology

School of Biomedical and Life SciencesUniversity of Surrey

Guildford, United Kingdom

M. J. Robert Nout, PhDAssociate Professor

Laboratory of Food MicrobiologyWageningen University

Wageningen, The Netherlands

AN ASPEN PUBLICATIONAspen Publishers, Inc.

Gaithersburg, Maryland2001

The author has made every effort to ensure the accuracy of the information herein. However, appropriate infor-mation sources should be consulted, especially for new or unfamiliar procedures. It is the responsibility of everypractitioner to evaluate the appropriateness of a particular opinion in the context of actual clinical situations andwith due considerations to new developments. The author, editors, and the publisher cannot be held responsiblefor any typographical or other errors found in this book.

Aspen Publishers, Inc., is not affiliated with the American Society of Parenteral and Enteral Nutrition.

Library of Congress Cataloging-in-Publication Data

Fermentation and food safety/ Martin R. Adams and MJ. Robert Noutp. cm.

Includes bibliographical references and index.ISBN 0-8342-1843-7

1. Fermented foods. 2. Food industry and tradeSafety measures. I. Nout, M. J.Robert. II. Adams, Martin, R.

TP371.44.F472001660'.28448dc21

00-051087

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1 2 3 4 5

Contributors

Martin R. Adams, MSc, PhDReader in Food MicrobiologySchool of Biomedical and Life SciencesUniversity of SurreyGuildford, United Kingdom

A. AsanteScientistFood Safety UnitWorld Heath OrganizationGeneva, Switzerland

R.R. Beumer, PhDAssociate ProfessorLaboratory of Food MicrobiologyWageningen University Research CenterWageningen, The Netherlands

Leon Brimer, PhD, DScAssociate ProfessorDepartment of Pharmacology and

PathobiologyLaboratory of ToxicologyRoyal Veterinary and Agricultural

UniversityCopenhagen, Denmark

Michael J. Carter, BA, PhDReader in VirologySchool of Biomedical and Life SciencesUniversity of SurreyGuildford, United Kingdom

Wilhelm H. Hozapfel, PhDDirector and ProfessorInstitute of Biotechnology and Molecular

BiologyFederal Research Centre for NutritionKarlsruhe, Germany

Maurice O. Moss, PhD, ARCS, DICVisiting ReaderSchool of Biomedical and Life SciencesUniversity of SurreyGuildford, United Kingdom

Yasmine Motarjemi, PhDScientistFood Safety ProgrammeWorld Health OrganizationGeneva, Switzerland

M.J. Robert Nout, PhDAssociate ProfessorLaboratory of Food MicrobiologyWageningen UniversityWageningen, The Netherlands

Arthur C. Ouwehand, PhDSenior ScientistDepartment of Biochemistry and Food

ChemistryUniversity of TurkuTurku, Finland

Seppo J. Salminen, PhDProfessorDegree Programme on Health SciencesDepartment of Biochemistry and Food

ChemistryUniversity of TurkuTurku, Finland

M. Hortensia Silla-Santos, PhDSenior ScientistInstitute de Agroquimica y Tecnologia de

AlimentosConsejo Superior de Investigaciones

CientificasValencia, Spain

Mike Taylor, BVMS, PhD, MRCVS, CBiol,MIBiol

Head of Parasitology and EcotoxicologyVeterinary Laboratories AgencyNew Haw, United Kingdom

T. Verrips, PhDChief ScientistUnilever Research LaboratoryVlaardingen, The Netherlands

Atte J. von Wright, PhDProfessorInstitute of Applied BiotechnologyUniversity of KuopioKuopio, Finland

Foreword

In every part of the world, people wage a con-stant battle against food contamination and theresulting food-borne diseases and food wastage.Efforts to reduce the devastating consequencesof food contamination started long before writ-ten records. Besides cooking, smoking, andsimple sun drying, fermentation is one of theoldest technologies used for food preservation.Over the centuries, it has evolved and has beenrefined and diversified. Today, a large variety offoods are derived from this technology, which isused in households, small-scale food industries,and large-scale enterprises. Foods so producedform a major part of the human diet all over theworld but only a few people are aware of themultitude of fermented products and their im-portance in the human diet. In fact, all cultureshave in the course of their development learnedthe technique to preserve some of their foods byfermentation. However, the safety of fermentedfoods is a concern everywhere.

In the past, traditional fermentation technolo-gies were based on experiences accumulated byconsecutive generations of food producers, as aresult of trial and error. Only relatively recentlyhave science and technology started to contrib-ute to a better understanding of the underlyingprinciples of the fermentation process and of theessential requirements to ensure the safety aswell as nutritional and sensory quality of fer-mented foods. Since the days of Louis Pasteur,who pointed to the importance of hygiene in re-lation to fermentation, it is known that this tech-nology is easily influenced by various factors

during processing, and if not applied correctly,the safety and/or quality of the final product maybe jeopardized. As a matter of fact, causes andoutbreaks of food-borne illness have been tracedback to fermented food, in spite of the generalineffectiveness of food-borne disease surveil-lance programs in most countries.

Fermentation is also of economic importancein areas or for populations where preservationtechnologies such as cold storage (refrigeration)or hot-holding cannot be used for lack of re-sources or facilities. In such situations, fermen-tation may be considered an affordable technol-ogy, which if applied correctly results in thesafe preservation of foods, including comple-mentary foods for infants. Particularly in devel-oping countries, as a result of poor hygiene andincorrect application of fermentation, comple-mentary foods are often contaminated withpathogens and subsequently are a major cause ofinfant diarrhea and associated malnutrition.

Against this background, the World HealthOrganization (WHO), jointly with the Food andAgriculture Organization (FAO), organized in1995 a workshop to assess fermentation as ahousehold technology for improving foodsafety.1 This workshop was the first of its kind,highlighting the critical points in the fermenta-

!Fermentation: Assessment and Research. Reportof a Joint FAOAVHO Workshop, Pretoria, South Af-rica, 1 1-15 December 1995. WHO Consultations andWorkshops: WHO/FNU/FOS/96.1

tion process to ensure the safety of the resultingproducts, in line with the Hazard Analysis andCritical Control Point (HACCP) system. In away. this book is a result of this workshop. BothAspen Publishers and the two editors, MJ. Rob-ert Nout and Martin R. Adams, deserve applausefor this initiative and the unique approach theyhave adopted: the book focuses on food safety inall its aspects and is largely hazard based, whichhelps to identify those areas where knowledge islacking but needed for a satisfactory risk assess-ment to be made. This is in contrast to the exist-ing literature on fermented foods, which is gen-erally confined to descriptions of the product(s)

and the microbiology/biochemistry of their pro-duction.

In the interest of public health and food secu-rity, I wish this book a large and interested read-ership and for fermentation to result in safe, nu-tritionally adequate, and superbly tasting foodswith long shelf lives.

Fritz Kdfer stein, DVM9 PhDFormer Director, Programme of Food

Safety and Food Aid, WHO, Geneva;Current Distinguished Visiting Scientist,

Joint Institute for Food Safety and AppliedNutrition, Washington, DC

Preface

Fermented foods enjoy worldwide popularityas attractive, wholesome and nutritious compo-nents of our diet. They are produced on an enor-mous scale employing a huge variety of ingredi-ents and manufacturing techniques. Whethertraditional home-made foods, or high-tech prod-ucts derived from genetically modified organ-isms, the safety of the consumer remains of fore-most importance.

Fermented foods have always been generallyregarded as safe, but this reputation has been se-riously threatened in recent years by incidentssuch as outbreaks of illness caused by pathogensin soft cheeses and fermented meats, chroniccyanide intoxications from poorly processedcassava tubers, and mycotoxins in fermented ce-real foods. In addition, modern techniques of ge-netic engineering and biotechnology, which of-fer considerable opportunities in the area offermented food production, have also raisedsafety concerns among consumers. It is neces-sary therefore to have concrete guidelines on theconditions which lead to safe products and tohave a realistic view about what "guaranteedsafety" means. The massive impact of HACCPas a systematic approach to ensuring food safetyhas been widely apparent in recent years and thetechnique is as applicable to food fermentationas to any other food processing operation. Atpresent however, the literature on fermentedfoods has no focus on safety, but is mainly de-scriptive, concentrating on microorganisms re-sponsible for fermentation and on the biochemi-cal changes occurring in the food.

This book aims to integrate modern conceptsof safety assurance with the sometimes very tra-ditional environment of the production and dis-tribution of fermented foods. In particular, wehave taken a largely hazard-based approachrather than one centered on the different com-modities used. Introductory chapters aim to pro-vide a broad understanding of the nature of fer-mented foods, their production, distribution, anduse by consumers, and also discuss the generalfeatures of fermentation processes that contrib-ute to the product's overall safety and HACCP.For the bulk of the book, we have sought chap-ters which describe the principal individual haz-ards, both chemical and microbiological, and tryto provide some guidance on how these might becontrolled in food fermentations. These hazardsare discussed from the point of view of their se-verity and incidence, how they get into the food,which foods are specifically at risk, and what, ifany, are the conditions that remove or inactivatethese hazards. In many cases there is a dearth ofpublished material specifically on fermentedfoods, and contributors have used data obtainedin slightly different contexts to give some guid-ance. This exercise has proved useful in high-lighting where information is lacking and identi-fying areas where more research is desperatelyneeded.

It is hoped that the book will serve as a sourceof reference to support and help improve theproduction of safe fermented foods at all scales(household preparation up to large-scale indus-trial plants) and using all major food groups.

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Contents

Contributors ............................................................................................. vii

Foreword ................................................................................................. ix

Preface .................................................................................................... xi

1. Fermented Foods and Their Production ....................................... 1 Fermentation and Food Safety ....................................................................... 1 Food Fermentation Components .................................................................... 4 Diversity of Fermented Foods ......................................................................... 13 Process Unit Operations ................................................................................. 15 Process Conditions ......................................................................................... 17 Appendix 1-A: Flow Diagrams for Selected Food Fermentations .................. 20

2. Why Fermented Foods Can Be Safe ............................................. 39 Introduction ...................................................................................................... 39 Physical Processing ........................................................................................ 39 Microbial Activity .............................................................................................. 41 Overall Significance of Different Antimicrobial Factors .................................. 48

3. An Introduction to the Hazard Analysis and Critical Control Point (HACCP) System and Its Application to Fermented Foods ............................................................................ 53 Introduction ...................................................................................................... 53 Historical Development ................................................................................... 53 The Need for Change ...................................................................................... 54 What Is the HACCP System? ......................................................................... 55 Benefits of the HACCP System ...................................................................... 56 Areas of Application ........................................................................................ 56

iv Contents

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The HACCP System in Food Hygiene ............................................................ 57 Application of the HACCP Approach to Food Preparation ............................. 58 Application of the HACCP System to Gari ...................................................... 60 Appendix 3-A: HACCP Principles and Guidelines for Its Application ............. 67

4. Chemical Hazards and Their Control: Endogenous Compounds ..................................................................................... 71 Introduction ...................................................................................................... 71 Toxic and Antinutritional Glycosides in Food and Feed ................................. 71 Risks Associated with the Occurrence of Toxic Glycosides in Different

Commodities .......................................................................................... 77 Variation in Toxin Concentration among Varieties and Cultivars: The

Influence of Traditional Domestication and Modern Breeding .............. 83 Removal of Toxins through Processing .......................................................... 84 Conclusion ....................................................................................................... 88

5. Chemical Hazards and Their Control: Toxins .............................. 101 Microbial Toxins .............................................................................................. 101 Aflatoxins ......................................................................................................... 102 Ochratoxin A .................................................................................................... 105 Patulin .............................................................................................................. 107 Fusarium Toxins .............................................................................................. 108 Alternaria Toxins ............................................................................................. 111 Bacterial and Algal Toxins ............................................................................... 112

6. Toxic Nitrogen Compounds Produced during Processing: Biogenic Amines, Ethyl Carbamides, Nitrosamines .................... 119 Introduction ...................................................................................................... 119 Biogenic Amines .............................................................................................. 119 Ethyl Carbamides ............................................................................................ 126 Nitrosamines .................................................................................................... 129

7. Microbiological Hazards and Their Control: Bacteria ................. 141 Introduction ...................................................................................................... 141 Outbreaks Related to Fermented Products .................................................... 141 Bacterial Pathogens of Concern in Fermented Products ............................... 142

Contents v


Presence of Pathogens in Raw Materials ....................................................... 146 Effect of Fermentation Processes on the Survival of Bacterial

Pathogens .............................................................................................. 147 The Control of Microbial Hazards ................................................................... 148 HACCPs .......................................................................................................... 150 Validation of the Sausage Fermentation Process for the Control of

Pathogens .............................................................................................. 152

8. Microbiological Hazards and Their Control: Viruses ................... 159 Introduction ...................................................................................................... 159 Food-Borne Viruses ........................................................................................ 160 Potentially Food-Borne Viruses ...................................................................... 163 Assessment of Virus Risk ............................................................................... 168

9. Microbiological Hazards and Their Control: Parasites ................ 175 Introduction ...................................................................................................... 175 Nematodes ...................................................................................................... 175 Cestodes (Tapeworms) ................................................................................... 187 Trematodes (Flukes) ....................................................................................... 192 Protozoa .......................................................................................................... 200 Conclusion ....................................................................................................... 211

10. Biotechnology and Food Safety: Benefits of Genetic Modifications ................................................................................... 219 Introduction ...................................................................................................... 219 Supply Chain of Fermented Foods and Its Importance to Ensure Safe

Products ................................................................................................. 221 Two Sets of Examples of Safety Evaluation of Fermented Foods for

Which Recombinant DNA Is Used ......................................................... 229 Conclusion ....................................................................................................... 236

11. Safety Assessment of Probiotics and Starters ............................ 239 Safety Aspects for Probiotics and Biotechnology ........................................... 239 Safety of Current Probiotics ............................................................................ 242 Relevance of a Sound Taxonomic Basis ........................................................ 242 Transfer of Antibiotic Resistance .................................................................... 246

vi Contents


Genetically Modified Organisms ..................................................................... 246 Conclusion ....................................................................................................... 247

12. Practical Applications: Prospects and Pitfalls ............................. 253 Introduction ...................................................................................................... 253 Benefits of Lactic Acid Fermentation .............................................................. 254 Pitfalls of Fermentation .................................................................................... 255 Importance of Food Fermentation in Public Health ........................................ 257 Practical Intervention to Enhance Safety of Fermented Foods ...................... 259 Research Needs .............................................................................................. 271 Conclusion ....................................................................................................... 271

List of Sources ....................................................................................... 275

Index ....................................................................................................... 279

CHAPTER 1

Fermented Foods andTheir Production

MJ. Robert Nout

FERMENTATION AND FOOD SAFETYFermentation

Fermentation is one of the oldest methods offood processing. Bread, beer, wine, and cheeseoriginated long before Christ. Although modernfood technology has contributed to the present-day high standard of quality and hygiene of fer-mented foods, the principles of the age-old pro-cesses have hardly changed. In industrializedsocieties, a variety of fermented foods are verypopular with consumers because of their attractiveflavor and their nutritional value (Figure 1-1).

In tropical developing regions, fermentation isone of the main options for processing foods. Inthe absence of facilities for home refrigeration,freezing, or home canning, it serves as an afford-able and manageable technique for food preserva-tion. Fermentation can also increase the safety offoods by removing their natural toxic components,or by preventing the growth of disease-causingmicrobes. It imparts attractive flavor and nutritivevalue to many products. Fermentation is an attrac-tive technique because it is low cost and low tech-nology and it can be easily carried out at the house-hold level, often in combination with simplemethods such as salting, sun drying, or heating(e.g., boiling, steaming, frying).

Contrary to unwanted spoilage or toxin pro-duction, fermentation is regarded as a desirableeffect of microbial activity in foods. The mi-crobes that may be involved include molds

(mycelial fungi), yeasts (unicellular fungi), andbacteria. Examples of food fermentations andthe microbes responsible for the desired changeswill be presented in this chapter.

In general, the desirable effect of microbialactivity may be caused by its biochemical activ-ity. Microbial enzymes breaking down carbohy-drates, lipids, proteins, and other food compo-nents can improve food digestion in the humangastrointestinal tract and thus increase nutrientuptake. Several bacteria excrete B vitamins intofood. As a result of their growth and metabo-lism, substances of microbial origin are found inthe fermented food, including organic acids,alcohols, aldehydes, esters, and many others.These may have a profound effect on the qualityof the fermented product. For instance, lacticand acetic acids produced by lactic acid bacteria(LAB) have an inhibitory effect on spoilage bac-teria in sourdough bread and yogurt, and the pro-duction of ethanol and carbon dioxide deter-mines the acceptability of bread, beer, and wine(ethanol disappears from bread during the bak-ing process).

In addition to enzymes and metabolites, mi-crobial growth causes increased amounts of mi-crobial cell mass. This may be of nutritional andaromatic interest in yeast extract, for instance.The presence of living microbial cells such as innonpasteurized yogurt may well have advanta-geous effects on the intestinal microflora and,indirectly, on human health.

Nr.

123456789

10111213141516171819

Name of productquarkyogurtsauerkrautcultured milk ("karnemelk")treated black olivesgouda cheeseraw fermented sausagesyeast-leavened breadlager beerwhite winesherrylager beergruyere cheesepumpernickel breadmixed sourdough breadcamembert cheeseraw fermented sausagesoy saucetempeh

Major ingredient(s)cows' milkcows' milkwhite cabbagecows' milkolivescows' milkpork and/or beef meatwheat flourbarley, hopsgrapesgrapesbarley, hopscows' milkrye flourrye + wheat flourcows' milkpork and/or beef meatsoybeans and wheatsoybeans

Functional microorganismslactic acid bacteria (LAB)LABLABLABLABLABLAByeasts (Y)YYYYLAB + propionibacteriaLAB + YLAB + YLAB + moldsLAB + moldsMolds + LAB + YLAB + Molds + Y

Figure 1-1 Fermented foods representing different types of fermentations and raw materials

Food Safety

In its widest sense, the safety of food must beachieved through safe production, storage, andhandling in order to avoid food-borne illnessessuch as food intoxication, infectious diseases, or

other detrimental effects. In principle, such ill-nesses can be caused by agents of biological,chemical, or physical nature, as exemplified inTable 1-1.

In this book, the fermentation of food will beviewed in relation to these safety aspects. The

Table 1-1 Some Examples of Threats to Consumer Safety

Illness

Enteric infections

Liver cancerRespiratory failureHypertension

Cyanide intoxicationIntoxications

HypertensionAllergies

Injuries (cuts, perforations)

Specific Examples

Bacteria (Salmonella spp.)Viruses (Rotavirus)Parasites (Cryptosporidium spp.)Mycotoxin-producing fungi (Aspergillus flavus)Toxin-forming bacteria (Clostridium botulinum)Biogenic amine-forming bacteria (Enterobacteriaceae)Cyanogenic glycosides (linamarin) in cassava tubersPesticidesVeterinary drugsHeavy metalsBiogenic amines (e.g., in fish)PreservativesColorants

GlassMetal

Type of Causative Agents

Pathogenic microorganisms

Toxigenic microorganisms

PhytotoxinsEnvironmental contaminants

Toxic metabolitesFood additives and ingredients

Foreign matter

Nature

Biological

Chemical

Physical

book begins with a closer look at fermentation asa food processing technology. Next, the intrinsicsafety of fermented foods as well as the prin-ciples of the hazard analysis critical controlpoint (HACCP) system are discussed, followedby several examples of hazards that are potentialthreats to consumer safety.

The use of genetically modified ingredients ormicroorganisms in food fermentations, as wellas the use of microorganisms as probiotics infermented foods, are aspects that require a sys-tematic and critical assessment of their safety.

In the final synthesizing chapter, the HACCPapproach is used to illustrate and compare someof the critical processing steps that affect thesafety of fermented foods.

FOOD FERMENTATION COMPONENTS

Food IngredientsFood ingredients include the raw foods cho-

sen for fermentation which can be of plant oranimal origin. These foods contain a variety ofnutrients for the consumer but also some of thesenutrients will be required for the microbialgrowth and metabolism during the fermentationprocess. In order to enable microbial activity,sufficient water must be present. Consequently,water must be added in case the other ingredi-ents are too dry. For several reasons, such astaste or preservation, miscellaneous substancesmay be added to the ingredient mix. These willbe discussed in more detail below.

Food Groups Plant origin: Fermented foods of plant ori-

gin are derived from a variety of raw mate-rials of different chemical composition andbiophysical properties. Tuberous rootssuch as potatoes and cereals and tree cropslike breadfruit have a relatively high starchcontent. Legumes and oil seeds generallyhave a high protein content. Green veg-etables, carrots, beets, tomatoes, olives, cu-cumbers, okra, and forage crops for animalfeed silages have a high moisture content.Fruits contain high concentrations of re-ducing sugars.

Most fermentative preservations of veg-etables and cereals are due to the action of LAB,often in combination with yeasts. But other bac-teria, such as Bacillus spp., or mycelial fungi,such as Rhizopus and Aspergillus spp., areequally important in the fermentation of le-gumes and oil seeds.

Animal origin: Foods of animal origin thatare fermented are mainly milk, meat, fish,and seafood. These are all quite perishable,and fermentation has long been an effectivemethod for prolonging the shelf life ofthese valuable nutrients.

Milk originating from cows, buffaloes, sheep,and, occasionally, other animals has a highmoisture content; contains proteins, minerals,and vitamins; and has a neutral pH. A rapidacidification by lactic acid bacterial fermenta-tion to pH values of less than 4.5 strongly inhib-its the survival and growth of spoilage-causingor disease-associated bacteria. Milk containsample amounts of the fermentable carbohydratelactose, which is an essential ingredient to en-able this fermentation. Due to the reduced pH, aswell as bacterial glycocalix, the viscosity in-creases and various liquid fermented milk prod-ucts are obtained, ranging from fluid culturedmilk to stringy viscous Scandinavian milks,mixed sour-alcoholic fizzy fluids, and gel-typeproducts such as yogurt. Milk is also rather volu-minous; nomadic tribes developed methods tocurdle milk and separate the coagulated caseinfrom the residual liquid (whey). The coagulaterepresents only approximately 10% of the origi-nal milk volume. Fermentation by LAB, and oc-casional propionibacteria, yeasts, and molds, re-sults in a variety of cheeses.

Meat (cow, pork, goat, etc.) contains very lowlevels of fermentable carbohydrates, thus posinglimitations to lactic acid fermentation. Many ofthe fermented meats derive their long shelf lifefrom a combination of preservative effects.Acidification by LAB is usually boosted by add-ing sugar, and added salt, nitrate, or nitrite, aswell as some extent of dehydration, are majoraspects of the preservation of fermented sau-sages. It is interesting to note that most fer-

mented sausages have been prepared from rawmeat. In order to avoid the risk of raw meat-borne food infections by parasites, it is recom-mended to freeze the meat prior to processing.

Fish and seafood (e.g., shrimp) pose similarrestrictions to bacterial fermentation: They con-tain only low quantities of fermentable carbohy-drates. In practice, this has led to two majortypes of fish products preserved by fermenta-tion. The first is a mixture of fish and salt thatresults in liquid protein hydrolysate after severalmonths of fermentation. Halotolerant bacteriaand yeasts may play a role in flavor formation,but the high salt content is responsible for thepreservation. The second type of fish product isa mixture of fish, little salt, and cooked starchyfood (e.g., rice or cassava), the latter providingfermentable carbohydrates; these products arepreserved mainly by lactic acid fermentation.

Nutrients Required by Microorganisms Carbon and nitrogen: Most microorgan-

isms require some form of organic carbon.In natural raw materials, this is foundmainly in carbohydrates (e.g., monomers,dimers, etc., and a number of polymericfood-storage and structure-giving polysac-charides such as starch, pectins, hemicellu-lose, and cellulose). Molds and certain bac-teria (e.g., Bacillus spp.) are goodproducers of extracellular carbohydrate-de-grading enzymes that can release ferment-able mono- and oligomeric substances. Ac-counts of the response of yeasts to sourcesof carbon, nitrogen, phosphorus, and sulfurare available.34'41 Yeasts and LAB areknown to be poor converters of polysaccha-rides and pentoses. Although the presenceof chitin and acacia gum was shown to in-crease the rate of yeast growth and fermen-tation,31 these compounds were not me-tabolized as nutrients.

In addition to fermentable saccharides, othernutrients required for cell growth and metabo-lism include inorganic (e.g., NH4+) or organicsources of nitrogen (e.g., urea, amino acids, andpeptides, but rarely extracellular proteins),

phosporus (e.g., inorganic phosphate or phos-phate esters), and sulfur (e.g., sulfate, sulfite,methionine, glutathione). Fungi usually do notrequire additional nutrients in food fermenta-tion. Optimum carbon/nitrogen ratios for growthare 10-100. For use in industrial fermentations,it was shown that R. oryzae could grow wellwith only starch and nitrogen salts; the additionof vegetable juice further stimulated growth.37

Minerals: Iron, magnesium, potassium,sodium, and calcium are normally requiredfor cell growth.41 Of additional interest isthe effect of Ca2+ ions, which was reportedto increase the ethanol tolerance ofSaccha-romyces cerevisiae.24 Manganese plays animportant role in LAB; deficiencies maylead to fermentation failures.

Vitamins and other growth factors: Themost common growth factors for yeasts arebiotin, pantothenic acid, inositol, thiamine,nicotinic acid, and pyridoxine.41 Riboflavinand folic acid are synthesized by all yeasts,but are required by some bacteria. Fungalrequirements for additional vitamins in thefood environment seem to be negligible.On the contrary, they may contribute to thenutritive value of fermented foods by vita-min synthesis.

WaterWater is essential for microbial growth and

metabolism. The extent to which water is avail-able for biological metabolism is expressed aswater activity (Aw) or, occasionally, as water po-tential.15 Aw is the more commonly used termi-nology and is defined by the ratio of equilibriumwater vapor pressure of the food and of pure wa-ter, at a defined temperature such as 20 0C. Awranges from O to 1. Most microorganisms re-quire Aw to be greater than 0.70, with optimumgreater than 0.99.

Added Food IngredientsA variety of added ingredients affect the ac-

tivity of microorganisms and thus can be used toregulate the rate and extent of fermentation. Saltand sugar are well known for their antimicrobial

effect at high concentrations. Salt levels greaterthan 15% w/w and sugar concentrations greaterthan 25% w/w reduce the Aw considerably; inaddition, NaCl has a specific inhibitory influ-ence caused by Na+ ions. Herbs, spices, and soforth may contain inhibitory proteins, organicacids, essential oils, pigments, resins, phenoliccompounds, caffeine, and so on. On the otherhand, some spices are excellent sources of man-ganese, which is essential for LAB.

Microorganisms

Three groups of microorganisms are used infood fermentation, namely bacteria, yeasts, andmolds. Table 1-2 illustrates some prominentspecies of microorganisms, some of the food fer-mentations in which they are of importance, andtheir function.

How do microorganisms enter the fermenta-tion? Different scenarios of increasing complex-ity can be distinguished,25 as will be explained inthe following sections.

Natural Fermentation in Raw Substrate

Most raw foods of animal or plant origin con-tain a variety of microorganisms that arrived bychance or that have an ecological associationwith them, based on preharvest growing condi-tions. Food processing activities can also addcertain microorganisms to food. If food is al-lowed to ferment without prior heating, most ofthese microorganisms can multiply. However,their opportunities will be restricted by theirability to grow and compete in the food, and byexternal conditions. This usually results in a suc-cession of predominant microorganisms, finallystabilizing in a fermented product that contains amixed microbial population dominated by mi-croorganisms that are particularly suited to thephysicochemical conditions prevailing in the fi-nal product. This type of fermentation is exem-plified by sauerkraut, which is shredded andsalted cabbage that is fermented by LAB.11Drawbacks of natural fermentations are that theytake a relatively long time to complete, and theoutcome is always a surprise.

Use of Traditional Mixed StarterCultures in Raw or PreheatedSubstrateThe drawbacks of natural fermentation can be

reduced when a large quantity of microorgan-isms that occur in the final product are added.These may be expected to bring about a morerapid domination and a more predictable qualityof the fermented product. An example of thisapproach is the traditional fermentation of sour-dough, a mixture of cereal flour (wheat or rye)with water of dough consistency.12 A smallquantity of previously fermented sourdough ismixed into the new dough, and this practice canbe successfully carried out for many years toachieve dependable fermentations.

Traditional mixed-culture starters are also ap-plied in the fermentation of precooked ingredi-ents. Examples are the Indonesian fermentedsoybean food tempeh (also known as tempe\ 18as well as African alcoholic beverages such asGhanaian pito made from sorghum.9 Fortempeh, a mixed culture of molds (Rhizopus andMucor spp.) especially grown for this purposeon plant leaves (Hibiscus tileaceus) is used.27The fermentation starter for pito is an inocula-tion belt, a woven rope that is suspended in thefermenting beer and on which the predominatingyeasts (Saccharomyces spp.) are present as asediment. The fermentation of a next batch ofpito beer is started by immersing the inoculationbelt into the sugary liquid made from sorghum,which is a common cereal in tropical climates.

Use of Pure Cultures (Single or Mixed)in Preheated SubstrateWith increasing scale of production, more so-

phisticated technical facilities, and higher in-vestment and operational risks, the use of labo-ratory-selected and precultured starter culturesbecomes a necessity. Because equipment foraseptic processing at a large production scale isextremely expensive, common procedures in-clude pure culture maintenance and propagationunder aseptic conditions (e.g., sterile laboratoryand pilot fermentors). At production scale, thefood ingredient to be fermented is preheated in

Table 1-2 Selected Examples of Microorganisms and their Function in the Fermentation of Foods

Importance of Fermentation forProduct Characteristics

Contribute to flavor, shelf life, structure, andconsistency by the production of lactic acid,acetaldehyde, diacetyl, and polysaccharides

Produce ethanol, CO, and flavor

Form proteolytic and saccharolytic enzymes,enabling solubilization and flavor production

Fermented Foods

Yogurt

Alcoholic beverages(beers, wines)Soy sauce

Species

Lactic acid bacteria: Lactobacillus delbruckiissp. bulgaricus and Streptococcussalivarius ssp. thermophilus

Saccharomyces cerevisiae

Aspergillus sojae

Microbial Group

Bacteria

Yeasts

Molds

order to minimize the level of microbial contami-nation; subsequently, the pure culture starter(single or mixed cultures) is added and fermenta-tion takes place at the highest practicable and af-fordable level of hygienic protection.

Use of Pure Cultures in SterilizedSubstrateFor the propagation of starter cultures, or in

other situations requiring the absolute absenceof microbial contamination, food ingredients orotherwise suitable nutrients are sterilized andkept in sterile confinement. Pure cultures ofstarter microorganisms are added using aseptictechniques. Not only is this an expensive tech-nique, but it is also unnecessary in production-scale food fermentation. Moreover, severe heattreatments required to achieve sterility can harmheat-sensitive nutrients for both the microorgan-ism and the consumer.

Enzymes

The importance of enzymes in fermentationprocesses lies in their ability to degrade complexsubstrates. Some examples are in koji fermenta-

tion, where A. oryzae enzymes degrade starch,protein, and cell wall components, and intempeh production, where Rhizopus spp. en-zymes degrade cell wall components, protein,lipids, phytic acid, and oligosaccharides. Pro-teases produced by LAB are important degrad-ing proteins in fermented milk products. Someenzymes involved in the degradation of complexsubstrates include amylolytic, proteolytic, Ii-polytic, and cell wall degrading enzymes.

Amylolytic EnzymesStarch is composed of two polysaccharides,

amylose and amylopectin, both consisting ofglucose units only (Figure 1-2). Amylose is a lin-ear glycan in which the sugar residues are con-nected by a- 1,4 bonds; amylopectin is abranched glycan in which glucose residues in thebackbone and the side chains are a- 1,4 linked.The side chains are attached by a- 1,6 linkages.

Starch is degraded by several amylases work-ing simultaneously, a-amylases hydrolyzea- 1,4 glucosidic linkages. Iso-amylase is adebranching enzyme; it hydrolyzes a- 1,6 gluco-sidic linkages. Amyloglucosidase liberatessingle glucose units from the nonreducing end,

Figure 1-2 Amylopectin molecule and its degradation by several amylases. Adapted with permission fromUhlig, H. (1991). Enzyme arbeiten fur uns. Technische enzyme und ihre Anwendung. Miinchen, Wien: CarlHanser Verlag.43

Glucose Maltose

8-Amylase

Amyloglucosidase

a-Amylaseiso-amylase or

pullulanase

whereas (3-amylase liberates maltose units.These enzymes belong to the class of hydro-lases. In industrial-scale production of a-amy-lases, the enzyme is derived from the pancreaticgland of swine and cattle and from microbialcultures. Microorganisms that produce amylasesare B. subtilis and A. oryzae. B. licheniformisproduces a heat-stable amylase that can be usedat 100-1 10 0C. Gelatinization of starch by heat-ing enhances enzyme catalysis. Thus, the swol-len gelatinized starch substrate is degraded 300times faster by bacterial amylase and 105 timesfaster by fungal amylase than the native,unswollen, nongelatinized starch.

ProteasesProteases are classified according to their

source (e.g., animal, plant, microbial), their cata-lytic action (e.g., endopeptidase or exopeptidase),and the nature of the catalytic site. Endopeptidasesare the proteases that are used most commonly infood processing, but in some cases, they are ac-companied by exopeptidases. Endopeptidasescleave polypeptide chains at particularly suscep-tible peptide bonds distributed along the chain,whereas exopeptidases hydrolyze one amino acidfrom the end of the chain. The four major classesof endopeptidases are carboxyl proteases (i.e., as-partic protease), cysteine proteases, serine pro-teases, and metallo proteases (Table 1-3). As thenames imply, carboxyl, cysteine, and serine pro-teases have carboxyl, cysteine, and serine sidechains, respectively, as essential parts of the cata-lytic site. The carboxyl proteases generally havemaximum catalytic activity at acid pH. The serineproteases have maximum activity at alkaline pH;the closely related cysteine proteases usually showmaximum activity at more neutral pH values. Themetallo proteases contain an essential metal atom,usually Zn2+, and have an optimum activity at neu-tral pH. Ca2+ stabilizes these enzymes and strongchelating agents (such as ethylenediamine-tetraacetic acid, or EDTA) inhibit them.Enzymes Degrading Cell Wall

ComponentsIn order to understand how enzymes affect

plant cell walls, the structure of the walls first

must be reviewed. Microscopic investigationshave revealed that plant cell walls can be dividedinto three layers: the middle lamella, primarywall, and secondary wall (Figure 1-3). Themiddle lamella acts as an intercellular bindingsubstance and is mainly composed of pectin.Secondary cell walls contain less pectin but con-tain some lignin. Primary cell walls consist ofcellulose fibers called microfibrils embedded ina matrix of pectins, hemicelluloses, and proteins(Figures 1-3 and 1 )^. Pectin is the major bind-ing component of the cell wall, and its degrada-tion by pectolytic enzymes will cause fruit orvegetables to become soft. This is the first stepof enzymatic degradation of the cell wall.

Pectolytic enzymes: The basic structure ofpectins is a linear chain of oc-linked mol-ecules of pyranosyl D-galacturonic acid.Varying proportions of carboxylic groupscan be present as methyl esters. The esteri-fication is usually by methanol, in whichcase the pectin is said to be methylated.When less than 50% of the carboxyl groupsare methylated, the pectin is referred to aslow methoxyl pectin; when more than 50%of the carboxyl groups are methylated, thepectin is referred to as high methoxyl pec-tin. Pectin situated in the middle lamella isremoved from the cell wall relatively easilyand is most easily degraded by appropriateenzymes. In contrast, enzymes do not eas-ily degrade the pectin within primary andsecondary cell walls. Pectinases are definedand classified on the basis of their actiontoward the galacturonan part of the pectinmolecules. Two main groups are distin-guished, pectin esterases and pectindepolymerases.

Hemicellulases: Hemicelluloses arepolysaccharides that are extracted fromplant cell walls by strong alkali. They arecomposed of four major substances:arabinans, galactans, xyloglucans, and xy-lans. As an example, arabinans are de-graded by the enzymes, arabinanases.Arabinans are branched polysaccharideswith a backbone of a- 1,5 linked L-arabinose

Table 1-3 Overview of Proteases

Optimum StabilitypH Range

6.5-6.0

3.0-5.0

4.5-6.5

7.5-9.56.0-8.0

5.07.07.0-8.03.0-6.03.8-6.5

pH-Optimum

6.0-7.02.09.0

7.0-8.07.0-8.07.0-8.0

7.0-1 1 .06.0-9.0

3.0-4.05.5-7.56.0-9.53.5-4.5

5.0

Source

Stomach lining of calvesGastric lining of swine or bovinePancreas

Tropical melon tree (Carica papaya)Pineapple (fruit and stalk)Figs (Ficus carica)e.g., Bacillus subtilise.g., Bacillus thermoproteolyticusStreptomyces griseus

Aspergillus oryzaeAspergillus oryzaeAspergillus oryzaeMucor pusillusRhizopus chinensis

Type

Carboxyl proteaseCarboxyl protease

Cysteine proteaseCysteine proteaseCysteine protease

Serine proteaseMetallo protease

Carboxyl proteaseMetallo proteaseSerine proteaseCarboxyl proteaseCarboxyl protease

Name

Proteases of animal originChymosinPepsinPancreatic protease*Proteases of plant originPapainBromelainFicinBacterial proteasesAlkaline proteaseNeutral proteasePronaseFungal proteasesAcid proteaseNeutral proteaseAlkaline proteaseProteaseProtease

*A mixture of trypsin, chymotrypsin, and various peptidases with amylase and lipase as accompanying enzymes.

Primary wall

Middle lamella

Secondary wall

Figure 1-3 Schematic representation of the structure of plant cell walls. Reproduced with permission fromVoragen, A. G. J., Van den Broek, L. A. M. (1991). Fruit juices. Biotechnological Innovations in Food Process-ing, pp. 187-210. Edited by Biotol Team. Oxford, UK: Butterworth-Heinemann.44

units (Figure 1-5). To approximately everythird arabinose molecule, additional arabi-nose units are attached by a- 1,2- or a- 1,3linkages. This can produce complex struc-tures. There are two types of arabinanases:

arabinosidase (arabinofuranosidase) andendo-arabinanase. Arabinosidase can besubdivided into two forms, A and B. The Bform degrades branched arabinan to a lin-ear chain by splitting off terminal a- 1,3 or

Figure 14 Schematic representation of the primary cell wall. PGA and RG are part of pectin. Reproduced withpermission from Carpita, N. C., Gibeaut, D. M. (1993). Structural models of primary cell walls in floweringplants: consistency of molecular structure with the physical properties of the walls during growth. The PlantJournal*, 1-30.7

RG I witharabinogaiactanside-chains

PGA junctionzoneXyloglucan

cellulose fibril

Figure 1-5 The structure of arabinans. Adapted with permission from Beldman, G., Schols, H. A., Pitson, S. M.,Searle-van Leeuwen, M. J. F., Voragen, A. G. J. (1997). Arabinans and arabinan degrading enzymes. In Advancesin Macromolecular Carbohydrate Research. Vol. 1, pp. 1-64. Edited by RJ. Sturgeon. London: JAI Press.1

a- 1,2 linked side chains. At the same time,this enzyme slowly and sequentially breaksthe a- 1,5 linkages at the nonreducing endof linear arabinan. Endo-arabinanase hy-drolyzes linear arabinan in a random fash-ion, producing oligomers of shorterlengths. Arabinosidase A degrades thearabinan oligomers to monomers. Arabina-nases occur in some plants and microorgan-isms. Fungal arabinanases have an optimumpH of approximately 4.0; bacterialarabinanases have an optimum pH of ap-proximately 5 . 0-6 . 0 .

Cellulases: Cellulose is the best known ofall plant cell wall polysaccharides. It is par-ticularly abundant in secondary cell wallsand accounts for 20-30% of the total drymass. Cellulose is a linear chain of P- 1,4linked glucose units. In cellulose, these p-1,4 glucan chains aggregate by hydrogenbonds to rigid flat structures called fibrils(Figure 14). The degree of polymerizationcan be very high, up to 14,000 in secondarycell walls. The rigid structure makes cellu-lose very resistant to degradation by en-zymes. Cellulase (often called the cellulasecomplex) is a multienzyme complex sys-tem composed of several enzymes: endo-

glucanase, exo-glucanase, cellobiose hy-drolase, and cellobiase.

LipasesLipases degrade triglycerides. They only act

on an aqueous-lipid interface such as a micelle.Generally, the enzymes catalyze the hydrolysisof triglycerides to produce fatty acids and glyc-erol, but there are also specific enzymes thatcatalyze the hydrolysis of monoacylglycerides,phospholipids, and esters of sterols. Generally,the mode of action of lipases results in fatty ac-ids being preferentially hydrolyzed from 1- and 3-positions of triglycerides so as to leave 2-substi-tuted monoacylglycerides. Microbial lipases mayalso catalyze the hydrolysis in all three positions.The composition of the fatty acid (i.e., length,stereoconformation, and degree of saturation) af-fects the specificity and speed of the lipases.

PhytasesPhytases catalyze the hydrolytic removal of

phosphate groups from phytic acid (Figure 1-6),a substance that occurs widely in cereals and le-gumes and that is known to limit thebioavailability of macro- and micronutrients.The ability to degrade phytate occurs widelyamong molds (A.ficuum, R. oligosporus), yeasts

Figure 1-6 Structure of phytic acid

(Candida krusei, Schwanniomyces castelli), andLAB (Pediococcus pentosaceus, Lactobacillusagilis, Lb. confusus). Because the optimum pH ofmost phytases is approximately 5.5, the phytatedegradation can be less effective if a lactic acidfermentation has resulted in low pH values.

TannasesTannases catalyze the hydrolysis of polyphe-

nols (tannins). Fungal enzymes (e.g., from A.niger) are used in the food industry to degradetannins. Tannin removal results in reduced tur-bidity in tea-based beverages. It also benefits thenutrient bioavailability in tannin-containing fer-mented cereal products.2

DIVERSITY OF FERMENTED FOODS

There is a wide variety of fermented foodsworldwide. Various excellent reference bookson fermented foods are available,5'40'45 as aresource books on industrial aspects of food bio-technology, including food fermentation.23'33'39Mention was made already of the various foodingredients that can be fermented, as well as themicroorganisms and enzymes that are used infermentations.

In the strict sense, fermentation refers to aform of anaerobic energy metabolism. In thecontext of fermented foods, however, the micro-bial growth and metabolism can take place under

aerobic conditions as well. For example, moldfermentations require oxygen to facilitate theirgrowth and enzyme production.

An aspect of interest is the physical state un-der which the fermentation takes place. This canbe in the form of liquid in which the microorgan-isms are suspended while a relatively simplemixing device is used to ensure the homogeneityof microorganisms, substrates, and products. Inthese liquid fermentations, or LFs, the continu-ous phase is the liquid, and the control of tem-perature and levels of dissolved oxygen can beachieved with immersion coolers or heaters, andaeration. Quite a different situation occurs in aheap of cooked soybeans in which homogeneousgrowth of strictly aerobic molds is required.Whereas the particulate matter (i.e., soybeans)contains sufficient water to allow microbialgrowth, water is not the continuous phase; gas(i.e., air) is.This physical state is referred to assolid-state fermentation, or SSF. Because gas isa poor conductor of heat and mass, SSFs have atendency to develop gradients of temperatureand gas composition. Control of homogeneityrequires more complex measurement and mix-ing systems as compared to LF. In practice, in-termediate situations such as shredded veg-etables or particles such as olives, which arefermented while immersed in brine, can be en-countered. These can be considered as immer-sion LF because the continuous phase is liquidbut contains immersed particles. Obviously,these do not always behave like LF.

Table 1-4 illustrates selected examples offood fermentations representing different foodgroups, functional microorganisms and en-zymes, oxygen requirements, physical states,and present levels of industrialization. As men-tioned earlier, food ingredients of plant as wellas animal origin are used to prepare fermentedfoods. Of the functional microorganisms, LABplay the predominant role in the prolongation ofshelf life because of the antimicrobial effect oftheir acids. In addition, yeasts are often found asminority companions of LAB; they sometimescontribute to shelf life by scavenging residualassimilable carbon sources. This is the case, forexample, in fermented pickles. The major func-

Table 1-4 Examples of Food Fermentation Processes

References

30

16

26,28

3

10

36

42

8

Industrialization*

1-2

1-4

1-3

1-4

1-4

1-4

1-4

1-4

PhysicalState

Solid

Solid

Solid

Immersedin liquid

Liquid

Solid

Liquid

Solid

OxygenRequirement

Aerobic

Anaerobic

Aerobic

Anaerobic

Anaerobic

Anaerobic

Anaerobic

Aerobic

Enzymic Modificationsof The Product

Degradation of starchand cell walls

Degradation of starchand maltose

Degradation of protein,cell walls, and lipids

Degradation of lipids

Degradation ofproteins

Degradation ofproteins and lipids

Microorganisms andMajor Products*

Lactic acid bacteriaforming lactic andacetic acid

Lactic acid bacteria andyeasts forming lacticand acetic acid, andcarbon dioxide

Molds forming myceliumand enzymes

Lactic acid bacteriaforming lactic acid

Yeasts (and occasion-ally lactic acidbacteria) formingethanol and flavor

Micrococcaceae, lacticacid bacteria (andoccasionally molds)forming organic acidsand flavor andstabilizing meat color

Lactic acid bacteriaforming lactic acid,acetaldehyde, anddiacetyl and providingstructure

Lactic acid bacteria andmolds forming acidity,release enzymes forripening and flavordevelopment

Mode ofConsumption

Cooked paste

Bread

Fried snacks orcooked sidedish

Vegetable dishor relish

Alcoholicbeverage

Hearty foodingredient orsnack

Breakfast ordessert

Sandwichesand snacks

FermentedFood

Agbeli mawe

Sourdough

Tempeh

Sauerkraut

Wine

Raw driedsausages

Yogurt

Soft cheeses

Food GroupStarchy crops:

cassava

Cereals: wheatand rye

Legumes:soybean

Vegetables:cabbage

Fruits: grape

Meats: pork andbeef

Milk: cow,buffalo

Milk: cow,sheep, goat

*Mention is made only of functional groups of microorganisms.f1 , home-produced; 2, village style; 3, national market; 4, international market.

tion of yeasts in yeast-fermented products istheir copious production of carbon dioxide, suchas gas in bread and beer and ethanol in alcoholicbeverages. The functionality of molds is mainlyin their production of enzymes that degradepolymeric components such as cell wallpolysaccharides, proteins, and lipids. This canhave considerable consequences for the texture,flavor, and nutritional value of mold- fermentedfoods. Depending on the functional microorgan-isms that should be propagated, the incubation en-vironment (i.e., availability of oxygen for strictaerobes, anaerobic conditions when mold growthis undesirable) and physical state can be selectedand optimized. It is of interest to note that, in con-trast to predominantly LFs that are common in thepharmaceutical industry, a considerable numberof food products are fermented by SSF.

PROCESS UNIT OPERATIONS

The manufacture of fermented foods is orga-nized in a sequence of activities called unit op-erations.22 Irrespective of the scale at which theprocess takes place, the following types of unitoperations can be distinguished.

Physical Operations

TransportTransport is one of the most important unit

operations. It has the purpose of transferring in-gredients (i.e., transport of mass) to the desiredlocalities and/or equipment. Its aim is also to as-sist in heating and cooling (i.e., transport ofheat). There is a wide variety of materials andmethods that can be used for transport. Thechoice depends on the type of product, scale ofproduction, economic considerations, and localconditions.

Grading and/or SortingGrading and/or sorting have the purpose of

achieving homogeneity of size, color, maturity,hardness, and so forth. At the same time, itemsthat are spoiled, infected, or otherwise deterio-rated are removed. From a food safety point of

view, grading and sorting are important toolsthat can be used to optimize the quality of inputs.

Cleaning and/or WashingCleaning and/or washing are carried out to re-

move dirt, dust, insects, agricultural residues,and so forth. Depending on the type of ingredi-ents, dry or wet cleaning can be chosen. In cerealprocessing, for example, dry cleaning of wheatprior to flour milling is performed by a combina-tion of aspiration and sieving. But, in maize pro-cessing, maize kernels can be washed in waterprior to wet milling. Water is becoming everscarcer and expensive, causing increased inter-est in dry operations.

Physical SeparationsDehulling, peeling, trimming, and other sepa-

rations are aimed at obtaining the desired ana-tomical parts of plant or animal tissue while re-moving the undesired ones. Examples are theremoval of poorly digestible seedcoats fromsoybeans by dehulling, the removal of the cortexof the cassava root because of its bitter taste andpossible toxicity, and the trimming of fat fromred meat.

Moisture AdjustmentMoisture adjustment is required to achieve,

for example, the desired consistency and edibil-ity of dry seeds and grains. The need for a suffi-ciently high Aw for microbial metabolism wasmentioned earlier. Water can be added as an in-gredient and mixed; soaking or steeping in wateris also a common method to increase the mois-ture content of ingredients. Especially duringlonger periods of soaking at favorable tempera-tures, microbial activity can take place. Severalcereal fermentation processes combine soakingwith fermentation.

Size ReductionSize reduction of particulate matter is re-

quired to obtain meal or flour from seeds, to ob-tain pulp from tubers or fuits, or to prepare a ho-mogeneous slurry of meat and other ingredientsfor sausage making. Size reduction is performedby cutting, grinding, impact hammering, and so

forth using a wide variety of equipment that hasbeen developed to suit the processing of specificraw materials.

MixingMixing has the purpose of obtaining homoge-

neity of mass and heat. Mixing of mass is themost common type of mixing and is exemplifiedby the mixing of ingredients to obtain a homoge-neous product. Mixing at a small scale is rela-tively easy and can be carried out with simplekitchen utensils. The larger the scale of opera-tion, the more complicated mixing becomes.Mixing of dry components can be achieved us-ing specific mixing equipment such as tumblersor augur-type mixers, but it is also feasible tocombine mixing with other operations such asgrinding or transport. Mixing of liquids is oftenachieved in stirred tanks. Mixing of wet and dryingredients can be carried out in kneading ma-chines for doughs or in stirred tanks for less vis-cous suspensions.

Bioprocessing Operations

For microbial and enzymatic transformations,a first requirement is the presence of the requiredingredients, including the desired microorgan-ism^) and/or enzyme(s), the required substratesand cofactors, and sufficient water. Several ofthe unit operations mentioned above will be in-volved to fulfill these requirements.

In order to allow the transformations to takeplace, incubation under optimum conditions forthe correct period of time will be needed. In or-der to safeguard the constancy of these incuba-tion conditions, unit operations such as mixing,heating, cooling, and transport are needed to en-sure even distribution of mass and heat, compen-sate for heat losses or generated heat, and com-pensate for deficiencies or overproduction ofmass by additions or removal.

Thermal Processing Operations

Heating and cooling are both characterized asthe transport of heat. Heat treatments and cool-ing are of extreme importance in food process-

ing and thus merit specific attention. The pri-mary objective of heating is to render food palat-able. It causes the gelatinization of starch, dena-turation of protein, and softening of toughtissues, and transforms a number of flavors.

Heat treatments consist of a warming-upphase, a period of holding time, and a cooling-down phase. Temperatures exceeding 70 0Ccause enzyme inactivation and kill vegetativemicrobial cells. The combination of temperatureand time determines the lethal effect of heattreatments. In principle, the term pasteurizationcorresponds to mild heat that kills heat-sensitivevegetative cells of bacteria, yeasts, and molds.Sterilization signifies killing all living cells, in-cluding more heat-resistant spores of bacteriaand molds. In food processing practice, steriliza-tion is not always required to ensure long-termshelf life of foods. In this respect, the term com-mercial sterility indicates a situation wheresome heat-resistant spores may have survivedthe heat treatment, but the composition of thefood prevents their revival during storage.

In view of bioprocessing, the timing of heattreatments is of crucial importance. Fermenta-tion of ingredients without prior heat treatment(e.g., raw cereals, raw meat, etc.) has by defini-tion a mixed character: Microorganisms and en-zymes that were present in the ingredient willtake part in the fermentation, sometimes as soleactors and sometimes as an accompaniment toadded starter microorganisms. At larger scaleproduction, fermentations must be better definedand controlled, and selected or optimized startercultures must be used. In order to ensure opti-mum functioning and to remove potentially haz-ardous microorganisms, the ingredients can bepasteurized or sterilized prior to inoculation andfermentation. In such case, it should be realizedthat some heat-sensitive growth factors such asenzymes and vitamins may have to be replenishedin order to enable effective microbial metabolism.

Cooling in food fermentation can be used as aprocessing tool. Prior to inoculation, heat-treated ingredients must be cooled to inoculationtemperature. During fermentation, the excessiveproduction of metabolic heat must be removedby cooling in order to prevent fermentation fail-

ures. In certain products such as yogurt, the fer-mentation is halted by cooling in order to pre-vent excessive acid development. Not all fer-mented foods have a long shelf life. Refrigerateddistribution and storage contribute to shelf lifeand hygienic safety.

Organization in Flow Diagram

A flow diagram is a pictorial scheme illustrat-ing the sequence in which ingredients and unitoperations are combined and providing data re-garding processing conditions such as tempera-ture, time, mass, pH, and so forth. Figure 1-7 il-lustrates this principle.

Appendix 1 A provides flow diagrams for se-lected food fermentations. For each major food

group, two commodities have been selected.These flow diagrams are not intended to providerecipes or exclusive methods of preparing therespective products. Their purpose is to providean insight into the process conditions and tim-ing, as well as relevant environmental antimicro-bial conditions that are of importance in foodsafety.

PROCESS CONDITIONS

Appendix 1 A aims to provide concrete pa-rameter values (or ranges) affecting microbialgrowth, metabolism, survival, and death as wellas enzymatic activity/stability, that are relevantto food safety. These data can be useful in

cereal grains

grading, cleaning

grinding

flour

mixing, kneading

dough

fermentation 2-3 h, 25-30 0C

baking 20-30 min, 220-250 0C

bread

cooling to ambient temperature

packaging, distribution

consumption

water, salt, fat

baker's yeast

Ingredients, (intermediate) productsPhysical unit operations

Bioprocessing unit operations

Thermal unit operations

Figure 1-7 Principle of flow diagram (case of bread making)

HACCP approaches. Appendix 1 A providesflow diagrams and process conditions of rel-evance for the processing of starchy roots andtubers (cassava, Tables 1-A-l and l-A-2), ce-reals (barley, rye, and wheat, Tables l-A-3 andl-A-4), legumes (soybeans, Tables l-A-5 andl-A-6), vegetables (cabbage, Table l-A-7 andolives, Table l-A-8), fruits (grapes, Table 1-A-9 and palm sap, Table 1-A-l O), meat (pork,Tables 1-A-l 1 and l-A-12), fish (fresh water,Table l-A-13 and sea water, Table l-A-14),and milk (yogurt, Table 1-A-l 5 and cheese,Table 1-A-l 6). The icons explained in Figure1-7 are used throughout these flow diagrams toprovide a quick overview of the sequence of bio-and thermal unit operations. This is of specialimportance with regard to safety, as illustratedby the following cases.

REFERENCES

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2. Belitz, H. -D. & Grosch, W. (1987). Food Chemistry.New York: Springer- Verlag.

3. Buckenhuskes, H. J. (1993). Selection criteria for lacticacid bacteria to be used as starter cultures for variousfood commodities. FEMS Microbiol Rev 12(1-3), 253-272.

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6. Campbell-Platt, G. & Cook, P. E., eds. (1995). Fer-mented Meats. London: Blackie Academic & Profes-sional.

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8. Corroler, D., Mangin, I., Desmasures, N. & Gueguen,M. (1998). An ecological study of lactococci isolatedfrom raw milk in the Camembert cheese registered des-ignation of origin area. Appl Environ Microbiol 64(12),4729^735.

Case 1: Is the food heated at all, some-where during the process? This may be ofcrucial importance in view of viruses (referto Chapter 8) and parasites (Chapter 9).

Case 2: If heated, does this take place beforeor after fermentation? This will have seriousimplications in view of the activity of endog-enous enzymes, which may detoxify endog-enous toxic substances (Chapter 4), and inview of naturally occuring pathogens (Chap-ter 7), as well as the need for added safestarter cultures (Chapters 10 and 11).

Case 3: Is the fermented product usuallycooked prior to consumption (e.g., tempeh)or is it eaten uncooked (e.g., most yogurts)?Needless to say, cooking prior to consump-tion will provide an additional "safety net"to the consumer.

9. Demuyakor, B. & Ohta, Y. (1993). Characteristics ofsingle and mixed culture fermentation of pito beer. J SdFoodAgric 62(4), 401^08.

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14. Garrido Fernandez, A., Garcia, P. G. & Balbuena, M. B.(1995). Olive fermentations. In Biotechnology. Vol. 9,Enzymes, Biomass, Food and Feed, pp. 593627. Editedby H. J. Rehm, G. Reed, A. Puhler & P. Stadler.Weinheim, Germany: VCH Verlagsgesellschaft.

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APPENDIX 1-A

Flow Diagrams for SelectedFood Fermentations

Table 1-A-1 Food Group: Starchy Roots and Tubers Product Name: Gari (Fermented and Dried Cassava) Reference: 40Other Conditions of

Antimicrobial Relevance(Salt, pH, Preservatives, etc.)

from 65% moisture content toapprox 50% moisture content

organic acids (lactic, acetic)0.6-1 .2% w/w

initial pH 6.2final pH 4-4.5

dehydration to approx 8%moisture content

in polythene bags or hermetic tins

Thermal Data (Time at Temp)

12-96 hat 25-35 0C

15-20 min at 80-85-100 0C

Ingredients and Microorganisms

cassava (Manihot esculenta)

natural fermentation dominated byLactobacillus plantarum, Coryne-bacterium spp., Geotrichum candidum

Flow Diagram

cassava tuberswashpeel (remove cortex)grate to coarse pulpput in jute or

polypropylene bagpressurize by weight to

enable de-wateringferment

break lumpsgarify (toasting for

dehydration andstarch gelatinization)

sievepackage and storereconstitute with cold

water or milkconsume

Note: Factors contributing to shelf life: Dehydration to about 8% moisture content, combined with hermetic storage allows shelf life of several months.Other remarks: During this process, the grating facilitates the enzymatic degradation and detoxification of naturally occurring cyanide (mainly in the form of linamarin). HCN (ppm)

levels in cassava roots, peeled roots, grated peeled roots, fermented pulp, and finished gari were 306, 184, 104, 52, and 10, respectively.

Table 1-A-2 Food Group: Starchy Roots and Tubers Product Name: Tape Ketella (Fermented Cassava) Reference: 40Other Conditions of


ethanol to approx 2% v/vlactic acid 0. 1-0.3%final pH approx 5.0


1 0-30 min at 80-1 OO 0C

2-3 d at 25-30 0C


cassava (Manihot esculenta)

ragi tape (inoculum on rice powder)containing Amylomyces rouxii,Endomyces fibuligera, Endomycesburtonii, lactic acid bacteria

Flow Diagram

cassava tuberswashpeel (remove cortex)cut to 5-1 0 cm piecessteamcoolinoculate

ferment

consume

Note: Factors contributing to shelf life: Tape ketella has no shelf life. It is consumed as a snack or used occasionally as an ingredient in baked goods (cakes).

Table 1-A-3 Food Group: Cereals Product Name: Lager Beer Reference: 35

Other Conditions ofAntimicrobial Relevance

(Salt, pH, Preservatives, etc.)

generation of saccharolytic,proteolytlc, and other brewingenzymes; partial modification ofthe barley grain

hops have antimicrobial effects

continues


1-2 d at 10-20 0C with inter-mittent airing, until moisturecontent 45-50% w/w4-6 d at 15-20 0C

at 71 -92 0C to reducemoisture content to 4-5% w/w

infusion mashing (30 min-several h at 65 0C) ordecoction mashing (2-3 h atincreasing temperatures from35-10O0C)

1-3 hat 100 0C

to 2-5 0C


barley (Hordeum vulgare)

product: sweet wortbyproduct: spent grain

hops (Humulus lupulus)

product: wort

Flow Diagram

1. Malting:soaking

germination

kilning

2. Mashing:coarse millingmashing

filtration

3. Wort boiling:hops additionboilingfiltrationcoolingfiltration

Table 1-A-3 continued


(Salt, pH, Preservatives, etc.)Thermal Data (Time at Temp)

1-2 wks at 10-1 5 0C

1-4 wks at 2-6 0C

cooling to 0-2 0C

5 min at 60 0C holding temp


brewers' yeast (Saccharomyces cerevisiaeor S. uvarum) at approx 107 yeast cellsper ml wort

product: young ("green") beer

product: mature beer

Flow Diagram

4. Fermentation:pitching

primaryfermentationsedimentation

secondaryfermentation(lagering)filtration

5. Bottling

6. Pasteurizationconsume

Note: Factors contributing to shelf life: Lager beer has no shelf life unless it is kept anaerobic and refrigerated. Bottled lager beer derives its shelf life from pasteurization and theexclusion of air.

Table 1-A-4 Food Group: Cereals Product Name: Wheat Mixed Grain Sourdough Bread Reference: 38


(Salt, pH, Preservatives, etc.)

pH 4.6-4.7

0.5-0.7% lactic acid in bakedproduct


15-24 hat 23-31 0C

1-2 hat 26 0C

5 min

30-60 min at 30-40 0C35-40 min; oven temperature200-250 0Cto ambient temperature


rye flour 15 kgseed sour 0.3 kgwater 12.01yeasts (Candida krusei, Pichia saitoi,Saccharomyces cerevisiae, Torulopsisholmii) and lactic acid bacteria (Lacto-bacillus sanfranciscensis, Lb. plantarum,Lb. fermentum)seed sour 27 kgrye flour 15 kgwheat flour 70 kgbakers' yeast 2 kgsalt (NaCI) 2 kgwater 51 Isee above, but dominated by Saccharo-myces cerevisiae (added bakers' yeast)

Flow Diagram

mix ingredientsfor sourdough

fermentation ofsourdough

seed sourmixing andkneading ofbread doughingredients

fermentation ofbread doughdivide, make-upintermediateproofshape and tintin proofbake

coolconsume

Note: Factors contributing to shelf life: Packaging in paper or other bags permitting moisture migration.Other Remarks: Storage at 20 0C for several days, limited by staling.

Table 1-A-5 Food Group: Legumes Product Name: Tempe Kedele (Soybean Tempeh) Reference: 27Other Conditions of


pH soaking water decreased from6.5 to 4.1

ph beans 5.0-6.5

limited perforation allowsmycelium growth with very littlesporulationinitial pH 5.0;final pH 6.5-7.5NHs is formed due to proteolysis

Thermal Data (Time at Temp)6-24 h at 20-30 0C

20-40 min at 80-1 OO 0C

24-48 h at 25-30 0C externaltemp; internal temperaturemay become approx 1 0 0Chigher

few min in oil of 180 0C, or5-10 min in water or sauce of10O0C


soybeans (Glycine max), lactic acidbacteria (Lactobacillus plantarum), yeasts(Saccharomyces dairensis),Enterobacteriaceae

Rhizopus microsporus, R. Oligosporus, R.oryzae, Mucor indicusplant leaves (banana) or polyethylenesheets or boxes, with perforation holes

Flow Diagram

soak wholesoybeans in water

dehullcookcoolinoculate withmold starterpackage

ferment

harvest freshtempehslice or dicefry or stew

consume

Note: Factors contributing to shelf life: Fresh tempeh can be stored refrigerated or frozen.

Table 1-A-6 Food Group: Legumes Product Name: Koikuchi-Shoyu (Soy Sauce) Reference: 13Other Conditions of


NaCL approximately 18% (w/v)ethanol 2-3% (v/v), lactic acid1-2% (w/w), final pH 4.7-4.8

Thermal Data (Time at Temp)cooking: 40-45 min at 130 0Cor equivalent; roasting: fewmin at 170-1 80 0C

2-5 d at 25-30 0C

6-8 months

70-80 0C


soybeans (cooked, whole)wheat (roasted, crushed)koji starter containing Aspergillus sojae,Asp. oryzae

kojibrineTetragenococcus halophila,Zygosaccharomyces rouxii

Flow Diagram

mixing equal quantitiesof soybeans and wheat

inoculation

incubation to obtainmolded mass = kojimixing koji and brineto obtain moromi mashfermentation

pressing,residue used asanimal feed

obtain rawsoy saucepasteurizebottleconsume

Note: Factors contributing to shelf life: Combined effect of high salt concentration, mild acidity, and pasteurization.

Table 1-A-7 Food Group: Vegetables Product Name: Sauerkraut (Sour Fermented Cabbage) References: 1 1 , 29Other Conditions of


at least 1% w/w lactic acid shouldbe formed

4. canned sauerkraut

pasteurize 3 min at 74-820Chotfill in cans

continues


3-6 wks at 18-22 0C

3. fresh packaged long-lifesauerkrautadd preservative (0.1% w/wNa-benzoate)fill in glass or plastic


Brassica oleracea

byproduct: inner corebyproduct: outer leaves

add 1 .0-3.5 (typically 2.25)% w/w NaCI

using boards and weights or bags filledwith waternatural fermentation by microbialsuccession: Leuconostoc mesenteroides,Lactobacillus brevis, dominated byLactobacillus plantarum. Lactobacillusbavaricus occasionally added as a starterculture

2. fresh packaged sauerkraut

allow secondary fermentationto complete sugar depletionfill in plastic pouches

Flow Diagram

white cabbageharvest, transportgradingcoringtrimmingshredding to 1-2 mmthicksalting (andhomogenous mixing)load into fermentationtanks or vatscover with sheetheading (place weighton cabbage)fermentation

Postfermentation Options1 . fresh sauerkraut inbulk

refrigerate and sellshelf life 1 8-30 months

consume

refrigerate and sellshelf life 8-1 2 months

consume

refrigerate and sellshelf life> 2 months at < 7 0C; > 3months under CCk modifiedatmosphereconsume

Table 1-A-7 continued

refrigerate and sellshelf life 1-2 wks

consume

Note: Factors contributing to shelf life: The combination of acidity and depletion of fermentable sugars. In addition, exclusion of air, preservatives, pasteurization, or sterilization, asshown above.

Other remarks: Sauerkraut can be eaten without any heat treatment (e.g., in sandwiches), but it is also popular in cooked dishes with potatoes and meats.

Table 1-A-8 Food Group: Vegetables Product Name: Treated Black Olives in Brine References: 14, 29


(Salt, pH, Preservatives, etc.)fruits contain 0.5-1 .0% organicacids (citric, oxalic, malic), as wellas 1-3% phenolic compoundswith antimicrobial effect

pH to 5.8-7.9

final NaCI concentration approx1 .5% w/w

Thermal Data (Time at Temp)Ingredients and MicroorganismsOlea europaea sativa of various stages ofmaturation (yellowish green to purple)

5-7% w/v NaCI

natural fermentation; no starter added;lactic acid bacteria (dominated byLactobacillus plantarum) and yeasts(Pichia membranifaciens, Pichia vinii)1-2% w/v NaOH

purging with air; add iron salts to stabilizeblack color

2.5-5.0% w/v NaCI

add 1 .5% w/v NaCI brine

Flow Diagram

fresh olives

wash

brine

ferment

lye treatment

air oxidation(blackening)wash, neutralize

storage in brine

sort, sizecan

sterilizeconsume

Note: Factors contributing to shelf life: The combination of heat treatment and moderate salt and acidity. The antimicrobial effect of phenolic compounds occurring in fresh olives is notrelevant for the shelf life of the finished product because the mentioned substances have been degraded during the lye treatment.

Other remarks: Canned olives are often consumed without prior heating.

Table 1-A-9 Food Group: Fruits Product Name: Red Grape Wine References: 5, 10, 25


(Salt, pH, Preservatives, etc.)fermentation until fermentable

sugars less than 0.1%. Finalethanol 1 1-17% v/v. Acidity:volatile 0.1-0.15% w/v as aceticacid; total acidity 0.5-0.7% w/v astartaric acid

conversion of malic acid into lacticacid (typically 1 .5-3.5 g/l lacticacid)

fermentation and food safety

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