microbiology and technology of fermented foods 2006 - hutkins

fer-Fermentation became an even more spread practice during the Roman Empire, as wide-Fermented Foods and Human History Fermented foods were very likely among the first foods consumed by

Microbiology and Technology of

Fermented Foods

Robert W Hutkins

Microbiology and Technology of

Fermented Foods

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Microbiology and Technology of

Fermented Foods

Robert W Hutkins

• Biofilms in the Food Environment (Hans P Blaschek, Hua Wang, and Meredith E Agle)

• Food Carbohydrate Chemistry (Ronald E Wrolstad)

• Food Irradiation Research and Technology (Christopher H Sommers and Xuetong Fan)

• High Pressure Processing of Foods (Christopher J Doona, C Patrick Dunne, and Florence

E Feeherry)

• Hydrocolloids in Food Processing (Thomas R Laaman)

• Multivariate and Probabilistic Analyses of Sensory Science Problems (Jean-Francois

Meullenet, Hildegarde Heymann, and Rui Xiong)

• Nondestructive Testing of Food Quality (Joseph Irudayaraj and Christoph Reh)

• Preharvest and Postharvest Food Safety: Contemporary Issues and Future Directions

(Ross C Beier, Suresh D Pillai, and Timothy D Phillips, Editors; Richard L Ziprin, sociate Editor)

As-• Regulation of Functional Foods and Nutraceuticals: A Global Perspective (Clare M.

Hasler)

• Sensory and Consumer Research in Food Product Development (Howard R Moskowitz,

Jacqueline H Beckley, and Anna V.A Resurreccion)

• Thermal Processing of Foods: Control and Automation (K.P Sandeep)

• Water Activity in Foods: Fundamentals and Applications (Gustavo V Barbosa-Canovas,

Anthony J Fontana Jr., Shelly J Schmidt, and Theodore P Labuza)

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First edition, 2006

Library of Congress Cataloging-in-Publication Data

Hutkins, Robert W (Robert Wayne)

Microbiology and technology of fermented foods / Robert W Hutkins.

—1st ed.

p cm.

Includes bibliographical references and index.

ISBN-13: 978-0-8138-0018-9 (alk paper)

ISBN-10: 0-8138-0018-8 (alk paper)

1 Fermented foods—Textbooks 2 Fermented foods—Microbiology—Textbooks I Title.

TP371.44.H88 2006

The last digit is the print number: 9 8 7 6 5 4 3 2 1

In organizing this book, I have followedthe basic outline of the course I teach,Microbiology of Fermented Foods Stu-dents in this course, and hopefully readers

of this text, are expected to have had a sic course in microbiology, at minimum, aswell as courses in food microbiology andfood science An overview of microorgan-isms involved in food fermentations, theirphysiological and metabolic properties,and how they are used as starter cultureprovides a foundation for the succeedingchapters Nine chapters are devoted to themajor fermented foods produced aroundthe world, for which I have presented bothmicrobiological and technological featuresfor the manufacture of these products Iconfess that some subjects were consid-ered, but then not included, those beingthe indigenous fermented foods and thenatural fermentations that occur duringprocessing of various “non-fermented”foods, such as cocoa beans and coffeebeans These topics are thoroughly cov-ered in the above mentioned texts.One of my goals was to provide a histor-ical context for how the manufacture offermented foods evolved, while at the sametime emphasizing the most current sci-ence To help accomplish this goal I haveincluded separate entries, called “Boxes,”that describe, in some detail, current topicsthat pertain to the chapter subjects Some

ba-of these boxes are highly technical,whereas others simply provide sidebar in-formation on topics somewhat apart frommicrobiology or fermentation Hopefully,the reader will find them interesting and apleasant distraction from the normal text

This project started out innocently

enough, with the simple goal of providing

a resource to students interested in the

mi-crobiology of fermented foods Since

1988, when I first developed a course in

fermentation microbiology at the

Univer-sity of Nebraska, there has not been a

suit-able student text on this subject that I

could recommend to my students

Peder-son’s Microbiology of Food

Fermenta-tionshad last been published in 1979 and

Fermented Foods,by A.H Rose, was

pub-lished in 1982 Brian Wood’s two volume

Microbiology of Fermented Foods,

pub-lished in 1998 (revised from an earlier

1985 edition), is an excellent resource and

is considered to be one of the most

thor-ough texts on fermented foods, but it and

other handbooks are generally beyond the

scientific scope (and budget) of most

stu-dents in a one-semester-long course

Fi-nally, there are many excellent resources

devoted to specific fermented foods The

recently published (2004) Cheese

Chem-istry, Physics and Microbiology(edited by

Fox, McSweeney, Cogan, and Guinee) is an

outstanding reference text, as are

Jack-son’s Wine Science Principles, Practice,

and Perceptions and Steinkraus’

Industri-alization of Indigenous Fermented Foods.

However, their coverage is limited to only

those particular foods

I hope this effort achieves the dual

pur-poses for which it is intended, namely to

be used as a text book for a college course

in fermentation microbiology and as a

gen-eral reference on fermented food

microbi-ology for researchers in academia,

indus-try, and government

ix

Finally, in an effort to make the text

eas-ier to read, I made a conscious decision to

write the narrative portion of the book

with minimal point-by-point referencing

Each chapter includes a bibliography fromwhich most source materials were ob-tained The box entries, however, are fullyreferenced

Shoaf, Jennifer Huebner, Jun Goh, JohnRupnow, and the SMB group.The editorialstaff at Blackwell Press, especially MarkBarrett and Dede Pederson, have been in-credibly patient, for which I am very ap-preciative

I thank my wife, Charla, and my kids,Anna and Jacob, for being such goodsports during the course of this project.Atleast now you know why I was busier thanusual these past two years

Finally, I would not be in a position ofwriting an acknowledgment section, muchless this entire text, were it not for mygraduate mentors, Robert Marshall, LarryMcKay, Howard Morris, and Eva Kashket.Role models are hard to find, and I was for-tunate to have had four My greatest inspi-ration for writing this book, however, hasbeen the many students, past and present,that have made teaching courses and con-ducting research on fermented foods mi-crobiology such a joy and privilege

I am grateful to the many colleagues who

reviewed chapters and provided me with

excellent suggestions and comments Any

questionable or inaccurate statements,

however, are due solely to the author (and

please let me know).To each of the

follow-ing reviewers, I thank you again: Andy

Benson, Larry Beuchat, Lloyd Bullerman,

Rich Chapin, Mark Daeschel, Lisa Durso,

Joe Frank, Nancy Irelan, Mark Johnson,

Jake Knickerbocker, David Mills, Dennis

Romero, Mary Ellen Sanders, Uwe Sauer,

Randy Wehling, and Bart Weimer

For the generous use of electron

micro-graphs, photos, and other written

materi-als used in this text, I thank Kristin Ahrens,

Andreia Bianchini, Jeff Broadbent, Lloyd

Bullerman, Rich Chapin (Empyrean Ales),

Lisa Durso, Sylvain Moineau, Raffaele de

Nigris, John Rupnow, Albane de Vaux, Bart

Weimer, Jiujiang Yu, and Zhijie Yang

For their encouragement and support

during the course of this project, special

thanks are offered to Jim Hruska, Kari

xi

Microbiology and Technology of

Fermented Foods

Introduction

“When our souls are happy, they talk about food.”

Charles Simic, poet

than the raw materials from which they weremade Despite the “discovery” that fermentedfoods tasted good and were well preserved, itmust have taken many years for humans to fig-ure out how to control or influence conditions

to consistently produce fermented food ucts It is remarkable that the means for produc-ing so many fermented foods evolved indepen-dently on every continent and on an entirelyempirical basis.Although there must have beencountless failures and disappointments, small

prod-“industries,” skilled in the art of making mented foods, would eventually develop Aslong ago as 3000 to 4000 B.C.E., for example,bread and beer were already being mass pro-duced by Egyptian bakeries and Babylonianbreweries Likewise, it is clear from the histori-cal record that the rise of civilizations aroundthe Mediterranean and throughout the MiddleEast and Europe coincided with the productionand consumption of wine and other fermentedfood and beverage products (Box 1–1) It isnoteworthy that the fermented foods con-sumed in China, Japan, and the Far East werevastly different from those in the Middle East;yet, it is now apparent that the fermentationalso evolved and became established aroundthe same time

fer-Fermentation became an even more spread practice during the Roman Empire, as

wide-Fermented Foods and Human History

Fermented foods were very likely among the

first foods consumed by human beings.This was

not because early humans had actually planned

on or had intended to make a particular

fer-mented food, but rather because fermentation

was simply the inevitable outcome that resulted

when raw food materials were left in an

other-wise unpreserved state.When, for example,

sev-eral thousands of years ago, milk was collected

from a domesticated cow, goat, or camel, it was

either consumed within a few hours or else it

would sour and curdle, turning into something

we might today call buttermilk.A third

possibil-ity, that the milk would become spoiled and

pu-trid, must have also occurred on many

occa-sions Likewise, the juice of grapes and other

fruits would remain sweet for only a few days

before it too would be transformed into a

pleas-ant, intoxicating wine-like drink Undoubtedly,

these products provided more than mere

suste-nance; they were also probably well enjoyed for

aesthetic or organoleptic reasons Importantly, it

must have been recognized and appreciated

early on that however imperfect the soured

milk, cheese, wine, and other fermented foods

may have been (at least compared to modern

versions), they all were less perishable and were

usually (but not always) safer to eat and drink

3

new raw materials and technologies were

adopted from conquered lands and spread

throughout the empire Fermented foods also

were important for distant armies and navies,

due to their increased storage stability Beer

and wine, for example, were often preferred

over water (no surprise there), because the

lat-ter was often polluted with fecal malat-terial or

other foreign material During this era, the

means to conduct trade had developed, and

cheese and wine, as well as wheat for

bread-making, became available around the

Mediter-ranean, Europe, and the British Isles

Although manufacturing guilds for breadhad existed even during the Egyptian empire,

by the Middle Ages, the manufacture of manyfermented foods, including bread, beer, andcheese, had become the province of craftsmenand organized guilds The guild structure in-volved apprenticeships and training; oncelearned, these skills were often passed on tothe next generation For some products, partic-ularly beer, these craftsmen were actuallymonks operating out of monasteries andchurches, a tradition that lasted for hundreds

of years Hence, many of the technologies and

Box 1–1. Where and When Did Fermentations Get Started? Answers from Biomolecular Archaeologists

Although the very first fermentations were certainly inadvertent, it is just as certain that humanbeings eventually learned how to intentionally produce fermented foods When, where, andhow this discovery occurred have been elusive questions, since written records do not exist.However, other forms of archaeological evidence do indeed exist and have made it possible tonot only establish the historical and geographical origins of many of these fermentations, butalso to describe some of the techniques likely used to produce these products

For the most part, investigations into the origins of food fermentations have focused on holic fermentations, namely wine and beer, and have been led primarily by a research group atthe University of Pennsylvania Museum of Archaeology and Anthropology’s Museum AppliedScience Center for Archaeology (http://masca.museum.upenn.edu) These “biomolecular ar-chaeologists” depend not so much on written or other traditional types of physical evidence(which are mostly absent), but rather on the chemical and molecular “records”obtained from ar-tifacts discovered around the world (McGovern et al., 2004)

alco-Specifically, they have extracted residues still present in the ancient clay pottery jars and vesselsfound in excavated archaeological sites (mainly from the Near East and China) Because these ves-sels are generally porous, any organic material was adsorbed and trapped within the vessel pores

In a dehydrated state, this material was protected against microbial or chemical decomposition.Carbon dating is used to establish the approximate age of these vessels, and then various analyti-cal procedures (including gas chromatography-mass spectroscopy, Fourier transform infraredspectrometry, and other techniques) are used to identify the chemical constituents

The analyses have revealed the presence of several marker compounds, in particular, tartaricacid, which is present in high concentrations in grapes (but is generally absent elsewhere), andtherefore is ordinarily present in wine, as well (Guash-Jané et al., 2004; McGovern, 2003) Based

on these studies (and others on “grape archaeology”), it would appear that wine had been duced in the Near East regions around present-day Turkey, Egypt, and Iran as long ago as the Neolithic Period (8500 to 4000 B.C.E.)

pro-Recent molecular archaeological analyses have revealed additional findings In 2004, it was ported that another organic marker chemical, syringic acid (which is derived from malvidin, apigment found in red wines), was present in Egyptian pottery vessels.This was not a real sur-prise, because the vessels were labeled as wine jars and even indicated the year, source, andvintner.What made this finding especially interesting, however, was that one of the vessels hadoriginally been discovered in the tomb of King Tutankhamun (King Tut, the “boy king”).Thus,not only does it now appear that King Tut preferred red wine, but that when he died (at aboutage 17), he was, by today’s standards, not even of drinking age

re-Introduction 5

manufacturing practices employed even today

were developed by monks Eventually,

produc-tion of these products became more

priva-tized, although often under some form of state

control (which allowed for taxation)

From the Neolithic Period to the Middle Ages

to the current era, fermented foods have been

among the most important foods consumed by

humans (Figure 1–1) A good argument can be

made that the popularity of fermented foods

and the subsequent development of

technolo-gies for their production directly contributed to

the cultural and social evolution of human

his-tory Consider, after all, how integral fermented

foods are to the diets and cuisines of nearly all

civilizations or how many fermented foods and

beverages are consumed as part of religious

cus-toms, rites, and rituals (Box 1–2)

Fermented Foods:

From Art to Science

It is difficult for the twenty-first century reader

to imagine that fermented foods, whose facture relies on the intricate and often subtleparticipation of microorganisms, could havebeen produced without even the slightest no-tion that living organisms were actually in-volved The early manufacturers of fermentedfoods and beverages obviously could not haveappreciated the actual science involved intheir production, since it was only in the last

manu-150 to 200 years that microorganisms and zymes were “discovered.” In fact, up until themiddle of the nineteenth century, much of thescientific community still believed in the con-cept of spontaneous generation The very act

en-of fermentation was a subject for philosophers

As noted above, the origins of wine making in the Near East can be reliably traced to about

5400 B.C.E.The McGovern Molecular Archaeology Lab group has also ventured to China in aneffort to establish when fermented beverages were first produced and consumed (McGovern etal., 2004).As described in Chapter 12,Asian wines are made using cereal-derived starch ratherthan grapes Rice is the main cereal used Other components, particularly honey and herbs,were apparently added in ancient times

As had been done previously, the investigators analyzed material extracted from Neolithic(ca 7000 B.C.E.) pottery vessels In this case, the specific biomarkers would not necessarily bethe same as for wine made from grapes, but rather would be expected to reflect the differentstarting materials Indeed, the analyses revealed the presence of rice, honey, and herbal con-stituents, but also grapes (tartaric acid) Although domesticated grape vines were not intro-duced into China until about 200 B.C.E., wild grapes could have been added to the wine (as asource of yeast) Another explanation is that the tartaric acid had been derived from other na-tive fruits and flowers.Additional analyses of “proto-historic” (ca 1900 to 700 B.C.E.) vessels in-dicate that these later wines were cereal-based (using rice and millet) Thus, it now appearsclear that fermented beverage technology in China began around the same time as in the NearEast, and that the very nature of the fermentation evolved over several millennia

References

Guasch-Jané, M.R., M Ibern-Gómez, C.Andrés-Lacueva, O Jáuregui, and R.M Lamuela-Raventós 2004 uid chromatography with mass spectrometry in tandem mode applied for the identification of wine markers in residues from ancient Egyptian vessels.Anal Chem 76:1672–1677.

Liq-McGovern, P.E 2003 Ancient Wine:The Search for the Origins of Viniculture Princeton University Press Princeton, New Jersey.

McGovern, P.E., J Zhang, J.Tang, Z Zhang, G.R Hall, R.A Moreau,A Nuñez, E.D Butrym, M.P Richards, S.Wang, G Cheng, Z Zhao, and C.Wang 2004 Fermented beverages of pre- and proto-historic China Proc Nat.Acad Sci 101:17593–17598.

C.-Box 1–1. Where and When Did Fermentations Get Started? Answers from Biomolecular Archaeologists (Continued)

and alchemists, not biologists Although the

Dutch scientist Antonie van Leeuwenhoek had

observed microorganisms in his rather crude

microscope in 1675, the connection between

Leeuwenhoek’s “animalcules” and their

biologi-cal or fermentative activities was only slowly

realized It was not until later in the next

cen-tury that scientists began to address the tion of how fermentation occurs

ques-Initially it was chemists who began to studythe scientific basis for fermentation In the late1700s and early 1800s, the chemists Lavoisierand Gay-Lussac independently described theoverall equations for the alcoholic fermenta-

About 14 million bushels

of grain per year must

be imported into Rome to

feed the inhabitants

Wine grapes reintroduced into Alsatia, France by Roman emperor Probus (after being displaced for 200 years by wheat)

618

Kumyss, an ethanol-containing fermented mare's milk product

1202 277

16

Neanderthal man

becomes Homo sapiens

As glaciers retreat in the North, wild grains begin to grow in the Near East.

48 11,000

B.C.E.

A brewery, later named

as Löwenbräu in 1552,

opens in Bavaria

Sherry, a type of fortified

wine, is produced by the

wine house, Valdespino,

at Jerez de la Frontera, Spain.

1630

Soy sauce introduced in Japan by a company that will become Kikkoman

The Gekkeikan Sake Co.

begins producing sake in Kyoto, Japan Today, it

is the world's largest producer.

1637

White Mission grapes are introduced into southern California

1697 1430

1383

The French Benedictine monk, Dom Pierre Pérignon, develops techniques for retaining carbon dioxide in wine, leading to the development of Champagne.

1698

Henry Holt and Co., New York, and other sources.

Introduction 7

tion Improvements in microscopy led Kützing,

Schwann, and others to observe the presence of

yeast cells in fermenting liquids, including beer

and wine These observations led Schwann to

propose in 1837 (as recounted by Barnett,

2003) that “it is very probable that, by means of

the development of the fungus, fermentation is

started.” The suggestion that yeasts were ally responsible for fermentation was not widelyaccepted, however; and instead it was argued byhis contemporaries (namely Berzelius, Liebig,and Wưhler) that fermentation was caused byaerobic chemical reactions and that yeasts wereinert and had nothing to do with fermentative

Moët and Chandon, now the world's

largest Champagne producer, begins

operations in Épernay France

Thomas Jefferson, seeking to develop viniculture in the Virginia colony, plants grape vines at Monticello; later he will try to grow olives Both efforts are unsuccessful.

Captain Cook, by feeding sauerkraut to his crewman, is awarded a medal by the Royal Society for conquering scurvy.

1743 1773 1775

Frederick Accum, a professor at the Surrey

Institution flees to Berlin in order to escape

industry wrath for publishing "A Treatise on

Adulteration of Food and Culinary Poisons".

This publication revealed the identities of

those producers of wine, beer, bread,

vinegar, cheese, pickles, and other products

that were deliberately adulterated.

Port au Salut cheese is

invented by Trappist Monks

in Touraine, France

Charles Heidsick produces his first Champagne in Reims, France

Emmenthaler cheese introduced to America

2,000 bakeries operate in the U.S., but more than 90%

of all bread consumed is baked at home Wheat consumption in the U.S is over 200 pounds per person per year (in 2004, it is less than 150) Sour dough bread becomes

a staple among gold prospectors in California

The first modern chemistry

text book, Traité

élementaire de chime

(Elements of Chemistry),

by Lavoisier, is published.

90% of Americans are engaged

in farming and food production.

Today, only about 2% are.

Although reportedly around since the 12th century, Camembert cheese is "re-invented".

The round little box that permits transport of the fragile cheese is invented 100 years later.

1790

Nicole-Barbe Cliquot invents the

“remuage” technique, used to remove yeast sediment from champagne.

1796

processes.The debate over the role of

microor-ganisms in fermentation was brought to an

un-equivocal conclusion by another chemist, Louis

Pasteur, who wrote in 1857 that “fermentation,

far from being a lifeless phenomenon, is a living

process” which “correlates with the

develop-ment of cells and plants which I have

pre-pared and studied in an isolated and pure state”

(Schwartz, 2001) In other words, fermentationcould only occur when microorganisms werepresent.The corollary was also true—that whenfermentation was observed, growth of the mi-croorganisms occurred

In a series of now famous publications, teur described details on lactic and ethanolicfermentations, including those relevant to milk

Pas-1857 1873 1864 1877

Brick cheese, a milder version of Limburger invented

in Wisconsin

The H.J Heinz Co.

introduces White Vinegar and Apple Cider Vinegar

Louis Pasteur, at age

32, shows that bacteria

are responsible for the

lactic acid fermentation

in milk

Heineken beer, made using a customized yeast strain, is produced

in Amsterdam

First varietal grape vines

are planted in California,

symbolizing the beginning

of the California wine

industry

The first pizzeria opens in the U.S in New York City

Lister isolates Lactococcus

lactis from milk

1880 1895

1907 1916 1950 1964

Bacteriophage against lactic acid bacteria identified by New Zealand researchers

Kraft introduces process cheese; Velveeta arrives

in 1928, Singles in 1947, and Cheese Whiz in 1953

Russian microbiologist

Ellie Metchnikoff isolates

Lactobacillus from a

fermented milk product;

his findings are published

in "The Prolongation of

Life"

Yoplait yogurt introduced

by SODIAAL, a French dairy cooperative

Dannon Milk Products Inc.

introduces yogurt in New York

1942

Miller Brewing Co.

Introduces Miller Lite

Plasmids in lactic acid

1974

Lactococcus lactis becomes

the first lactic acid bacterium

to have its genome sequenced

2006

Introduction 9

fermentations, beer, and wine He also identified

the organism that causes the acetic acid (i.e.,

vinegar) fermentation and that was responsible

for wine spoilage.The behavior of yeasts during

aerobic and anaerobic growth also led to

impor-tant discoveries in microbial physiology (e.g.,

the aptly named Pasteur effect, which accounts

for the inhibitory effect of oxygen on glycolytic

metabolism) Ultimately, the recognition that

fer-mentation (and spoilage) was caused by

mi-croorganisms led Pasteur to begin working on

other microbial problems, in particular,

infec-tious diseases Future studies on fermentationswould be left to other scientists who had em-braced this new field of microbiology

Once the scientific basis of fermentationwas established, efforts soon began to identifyand cultivate microorganisms capable of per-forming specific fermentations Breweries such

as the Carlsberg Brewery in Copenhagen andthe Anheuser-Busch brewery in St Louis wereamong the first to begin using pure yeaststrains, based on the techniques and recom-mendations of Pasteur, Lister, and others By the

Box 1–2. Fermented foods and the Bible.

The importance of fermented foods and beverages to the cultural history of human societies isevident from many references in early written records Of course, the Bible (Old and New Tes-taments) and other religious tracts are replete with such references (see below) Fermentedfoods, however, also serve a major role in ancient Eastern and Western mythologies

The writers , who had no scientific explanation for the unique sensory and often intoxicatingproperties of fermented foods, described them as “gifts of the gods.” In Greek mythology, for ex-ample, Dionysus was the god of wine (Bacchus, according to Roman mythology).The Iliad andthe Odyssey, classic poems written by the Greek poet Homer in about 1150 B.C.E., also containnumerous references to wine, cheese, and bread Korean and Japanese mythology also refers tothe gods that provided miso and other Asian fermented foods (Chapter 12)

Fermented foods and the Bible

From the Genesis story of Eve and the apple, to the dietary laws described in the books of Exodusand Leviticus, food serves a major metaphoric and thematic role throughout the Old Testament.Fermented foods, in particular, are frequently mentioned in biblical passages, indicating that thesefoods must have already been well known to those cultures and civilizations that lived during thetime at which the bible was written

In Genesis (9:20), for example, one of the first actions taken by Noah after the flood watershad receded was to plant a vineyard In the very next line, it is revealed that Noah drank enoughwine to become drunk (and naked), leading to the first, but certainly not last, episode in whichdrunkenness and nakedness occur Later in Genesis (18:8), Abraham receives three strangers(presumably angels), to whom he offers various refreshments, including “curds.”

Perhaps the most relevant reference to fermentation in the Bible is the Passover story.As scribed in Exodus (12:39), once Moses had secured the freedom of the Hebrew slaves, theywere “thrust out of Egypt, and could not tarry.”Thus, the dough could not rise or become leav-ened, and was baked instead in its “unleavened” state.This product, called matzoh, is still eatentoday by people of the Jewish faith to symbolically commemorate the Hebrew exodus

de-Ritual consumption of other fermented foods is also prescribed in Judaism Every Sabbath, forexample, the egg bread, Challah, is to be eaten, and grapes or wine is to be drunk, preceded byappropriate blessings of praise

Fermented foods are also featured prominently in the New Testament.At the wedding in Cana( John 2:1–11), Jesus’ first miracle is to turn water into wine Later ( John 6:1–14), another mira-cle is performed when five loaves of bread (and two fish) are able to feed 5,000 men.The Sacra-ment of Holy Communion (described by Jesus during the Last Supper) is represented by breadand wine

early 1900s, cultures for butter and other dairy

products had also become available The dairy

industry was soon to become the largest user

of commercial cultures, and many specialized

culture supply “houses” began selling not only

cultures, but also enzymes, colors, and other

products necessary for the manufacture of

cheese and cultured milk products (Chapter 3)

Although many cheese factories continued to

propagate their own cultures throughout the

first half of the century, as factory size and

prod-uct throughput increased, the use of dairy

starter cultures eventually became

common-place Likewise, cultures for bread, wine, beer,

and fermented meats have also become the

norm for industries producing those products

The Modern Fermented

Foods Industry

The fermented foods industry, like all other

seg-ments of the food processing industry, has

changed dramatically in the past fifty years

Cer-tainly, the average size of a typical production

facility has increased several-fold, as has the rate

at which raw materials are converted to

fin-ished product (i.e., throughput) Although

small, traditional-style facilities still exist, as is

evident by the many microbreweries, small

wineries, and artisanal-style bakery and cheese

manufacturing operations, the fermented foods

industry is dominated by producers with large

production capacity

Not only has the size of the industry

changed, but so has the fundamental manner

in which fermented foods are produced (Table1–1) For example, up until the past forty or soyears, most cheese manufacturers used raw,manufacturing (or Grade B) milk, whereas pas-teurized Grade A milk, meeting higher qualitystandards, is now more commonly used Manu-facturing tanks or vats are now usually en-closed and are constructed from stainless steel

or other materials that facilitate cleaning andeven sterilization treatments In fact, modernfacilities are designed from the outset with anemphasis on sanitation requirements, so thatexposure to air-borne microorganisms andcross-contamination is minimized

Many of the unit operations are mechanizedand automated, and, other than requiring a fewkeystrokes from a control panel, the manufac-ture of fermented foods involves minimal hu-man contact Fermented food production isnow, more than ever before, subject to time andscheduling demands In the so-called “old days,”

if the fermentation was slow or sluggish, it ply meant that the workers (who were probablyfamily members) would be late for supper, andlittle else In a modern production operation, aslow fermentation may mean that the workershave to stay beyond their shift (requiring thatthey be paid overtime), and in many cases, itcould also affect the entire production sched-ule, since the production vat could not beturned over and refilled as quickly as needed.Al-though traditional manufacturing practices maynot have always yielded consistent products, lotsizes were small and economic losses due to anoccasional misstep were not likely to be too se-

Small scale (craft industry) Large scale (in factories)

Non-sterile medium Pasteurized or heat-treated medium

Insensitive to time Time-sensitive

Significant exposure to contaminants Minimal exposure to contaminants

Varying quality Consistent quality

Safety a minor concern Safety a major concern

Introduction 11

rious Besides, for every inferior cask of wine or

wheel of cheese, there may have been an

equally superior lot that compensated for the

one that turned out badly Even the absolute

worst case scenario—a food poisoning

out-break as a result of an improperly manufactured

product—would have been limited in scope

due to the small production volume and narrow

distribution range

Such an attitude, today, however, is simply

beyond consideration A day’s worth of

prod-uct may well be worth tens, if not hundreds of

thousands of dollars, and there is no way a

pro-ducer could tolerate such losses, even on a

spo-radic basis Food safety, in particular, has

be-come an international priority, and there is

generally zero tolerance for pathogens or other

hazards in fermented foods Quality assurance

programs now exist throughout the industry,

which strive to produce safe and consistent

products In essence, the fermented foods

in-dustry has evolved from a mostly art- or

craft-based practice to one that relies on modern

science and technology Obviously, the issues

discussed above—safety, sanitation, quality, and

consistency—apply to all processed foods, and

not just fermented foods However, the

fer-mented foods industry is unique in one major

respect—it is the only food processing

indus-try in which product success depends on the

growth and activity of microorganisms.The

im-plications of this are highly significant

Microorganisms used to initiate

fermenta-tions are, unlike other “ingredients,” not easily

standardized, since their biochemical activity

and even their concentration (number of cells

per unit volume) may fluctuate from lot to lot

Although custom-made starter cultures that are

indeed standardized for cell number and

activ-ity are readily available, many industrial

fer-mentations still rely, by necessity, on the

pres-ence of naturally-occurring microflora, whose

composition and biological activities are often

subject to considerable variation In addition,

microorganisms are often exposed to a variety

of inhibitory chemical and biological agents in

the food or environment that can compromise

their viability and activity Finally, the culture

organisms are often the main means by whichspoilage and pathogenic microorganisms arecontrolled in fermented foods If they fail toperform in an effective and timely manner,the finished product will then be subject tospoilage or worse Thus, the challenge con-fronting the fermented foods industry is tomanufacture products whose very production

is subject to inherent variability yet satisfy themodern era demands of consistency, quality,line-speed, and safety

Properties of Fermented Foods

As noted in the previous discussion, fermentedfoods were among the first “processed” foodsproduced and consumed by humans Theirpopularity more than 5,000 years ago was due

to many of the same reasons why they tinue to be popular today (Table 1–2)

con-Preservation

The preservation aspect of fermented foodswas obviously important thousands of yearsago, when few other preservation techniquesexisted A raw food material such as milk ormeat had to be eaten immediately or it wouldsoon spoil Although salting or smoking could

be used for some products, fermentation musthave been an attractive alternative, due toother desirable features Preservation was un-doubtedly one of the main reasons why fer-mented foods became such an integral part ofhuman diet However, even today, preservation,

or to use modern parlance, shelf-life (or tended shelf-life), is still an important feature

ex-of fermented foods For example, specializedcultures that contain organisms that produce

Enhanced preservation Enhanced nutritional value Enhanced functionality Enhanced organoleptic properties Uniqueness

Increased economic value

specific antimicrobial agents in the food are

now available, providing an extra margin of

safety and longer shelf-life in those foods

Nutrition

The nutritional value of fermented foods has

long been recognized, even though the

scien-tific bases for many of the nutritional claims

have only recently been studied Strong

evi-dence that fermentation enhances nutritional

value now exists for several fermented

prod-ucts, especially yogurt and wine Fluid milk is

not regularly consumed in most of the world

because most people are unable to produce

the enzyme ␤-galactosidase, which is

neces-sary for digestion of lactose, the sugar found

naturally in milk Individuals deficient in

␤-galactosidase production are said to be lactose

intolerant, and when they consume milk,

mild-to-severe intestinal distress may occur This

condition is most common among Asian and

African populations, although many adult

Cau-casians may also be lactose intolerant

Many studies have revealed, however, that

lactose-intolerant subjects can consume yogurt

without any untoward symptoms and can

therefore obtain the nutritional benefits (e.g.,

calcium, high quality protein, and B vitamins)

contained in milk In addition, it has been

sug-gested that there may be health benefits of

yo-gurt consumption that extend beyond these

macronutrients Specifically, the

microorgan-isms that perform the actual yogurt

fermenta-tion, or that are added as dietary adjuncts, are

now thought to contribute to gastrointestinal

health, and perhaps even broader overall

well-being (Chapter 4)

Similarly, there is now compelling evidence

that wine also contains components that

con-tribute to enhanced health (Chapter 10)

Spe-cific chemicals, including several different

types of phenolic compounds, have been

iden-tified and shown to have anti-oxidant activities

that may reduce the risk of heart disease and

cancer.That wine (and other fermented foods)

are widely consumed in Mediterranean

coun-tries where mortality rates are low has led to

the suggestion that a “Mediterranean diet” may

be good for human health

Functionality

Most fermented foods are quite different, interms of their functionality, from the raw, start-ing materials Cheese, for example, is obviouslyfunctionally different from milk However, func-tional enhancement is perhaps nowhere moreevident than in bread and beer When humansfirst collected wheat flour some 10,000 yearsago, there was little they could do with it, otherthan to make simple flat breads However, oncepeople learned how to achieve a leaveneddough via fermentation, the functionality ofwheat flour became limitless Likewise, barleywas another grain that was widespread and haduse in breadmaking, but which also had limitedfunctionality prior to the advent of fermenta-tion Given that barley is the main ingredient(other than water) in beer manufacture, couldthere be a better example of enhanced func-tionality due to fermentation?

Organoleptic

Simply stated, fermented foods taste cally different than the starting materials Indi-viduals that do not particularly care for Lim-burger cheese or fermented fish sauce mightargue that those differences are for the worse,but there is little argument that fermentedfoods have aroma, flavor, and appearance at-tributes that are quite unlike the raw materialsfrom which they were made.And for those in-dividuals who partake of and appreciate Lim-burger cheese, the sensory characteristics be-tween the cheese and the milk make all thedifference in the world

dramati-Uniqueness

With few exceptions (see below), there is noway to make fermented foods without fermen-tation Beer, wine, aged cheese, salami, andsauerkraut simply cannot be produced anyother way For many fermented products, the

Introduction 13

very procedures used for their manufacture are

unique and require strict adherence For

exam-ple, Parmesan cheese must be made in a

de-fined region of Italy, according to traditional

and established procedures, and then aged

un-der specified conditions Any deviation results

in forfeiture of the name Parmesan For those

“fermented” foods made without fermentation

(which includes certain fresh cheeses,

sau-sages, and even soy sauce), their manufacture

generally involves direct addition of acids and/

or enzymes to simulate the activities normally

performed by fermentative microorganisms

These products (which the purist might be

in-clined to dismiss from further discussion) lack

the flavor and overall organoleptic properties

of their traditional fermented counterparts

Economic value

Fermented foods were the original members of

the value-added category Milk is milk, but add

some culture and manipulate the mixture just

right, age it for a time, and the result may be a

fine cheese that fetches a price well above the

combined costs of the raw materials, labor, and

other expenses Grapes are grapes, but if grown,

harvested, and crushed in a particular

environ-ment and at under precise conditions, and the

juice is allowed to ferment and mature in an

op-timized manner, some professor may well pay

up to $6 or $7 (or more!) for a bottle of the

fin-ished product.Truly, the economic value of

fer-mented foods, especially ferfer-mented grapes, can

reach extraordinary heights (apart from the

pro-fessor market) As noted in Chapter 10, some

wines have been sold for more than $20,000

per bottle Even some specialty vinegars

(Chap-ter 11) sell for more than $1,000 per li(Chap-ter It

should be noted that not all fermented foods

command such a high dollar value In truth, the

fermented foods market is just as competitive

and manufacturers are under the same market

pressures as other segments of the food

indus-try Fermented foods are generally made from

in-expensive commodities (e.g., wheat, milk, meat,

etc.) and most products have very modest profit

margins (some products,such as “current”or

un-aged cheese, are sold on commodity markets,with very tight margins).There is a well-knownjoke about the wine business that applies toother products as well, and that summarizes thechallenge in making fermented foods:“How doyou make a million dollars in the wine business?Easy, first you start with two million dollars.” Fi-nally, on a industry-wide basis, fermented foodsmay have a significant economic impact on a re-gion, state or country In California, for example,the wine industry was reported to contributemore than $40 billion to the economy in 2004(according to a Wine Institute report; www.wineinstitute.org) A similar analysis of the U.S.beer industry (www.beerinstitute.org) reported

an overall annual impact of more than $140 lion to the U.S economy

bil-Fermented Foods in the Twenty-first Century

For 10,000 years, humans have consumed mented foods As noted above, originally andthroughout human history, fermentation pro-vided a means for producing safe and well-preserved foods Even today, fermented foodsare still among the most popular type of foodconsumed No wonder that about one-third ofall foods consumed are fermented In the UnitedStates, beer is the most widely consumed fer-mented food product, followed by bread,cheese, wine, and yogurt (Table 1–3) Global sta-tistics are not available, but it can be estimatedthat alcoholic products head the list of mostpopular fermented foods in most of the world

fer-In Asia, soy sauce production and consumptionranks at or near the top Collectively, sales of fer-mented foods on a global basis exceeds a trilliondollars, with an even greater overall economicimpact

Although fermented foods have been part ofthe human diet for thousands of years, as theworld becomes more multicultural and cuisinesand cultures continue to mix, it is likely that fer-mented foods will assume an even more impor-tant dietary and nutritional role Foods such askimchi (from Korea), miso (from Japan), and ke-

fir (from Eastern Europe) are fast becoming part

of the Western cuisine Certainly, the desirable

flavor and sensory attributes of traditional, as

well as new-generation fermented foods, will

drive much of the interest in these foods

Consumption of these products also will

likely be increased as the potential beneficial

ef-fects of fermented foods on human health

be-come better established, scientifically and

clini-cally.As noted above, compelling evidence now

exists to indicate that red wine may reduce the

risk of heart disease and that live bacteria

pre-sent in cultured milk products may positively

in-fluence gastrointestinal health.Armed now with

extensive genetic information on the

micro-organisms involved in food fermentations that

has only become available in the last century,

it is now possible for researchers to

custom-produce fermented foods with not only specific

flavor and other functional characteristics, butthat also impart nutritional properties that ben-efit consumers

References

Barnett, J.A 2003 Beginnings of microbiology and biochemistry: the contribution of yeast research Microbiol 149:557–567.

Bulloch,W 1960.The History of Bacteriology Oxford University Press, London.

Cantrell, P.A 2000 Beer and ale In K.F Kiple, K.C.

Ornelas (ed) Cambridge World History of Food,

p 619–625 Cambridge University Press, bridge, United Kingdom

Cam-Steinkraus, K.H 2002 Fermentations in world food processing Comp Rev Food Sci Technol 1: 23–32.

a Sources: 2001–2004 data from USDA,WHO, and industry organizations

b Per person, per year

c Not available

Microorganisms and Metabolism

“We can readily see that fermentations occupy a special place in the series of

chem-ical and physchem-ical phenomena What gives to fermentations certain exceptional

characters, of which we are only now beginning to suspect the causes, is the mode

of life in the minute plants designated under the generic name of ferments, a mode

of life which is essentially different from that of other vegetables, and from which

result phenomena equally exceptional throughout the whole range of the

chem-istry of living beings.”

From The Physiological Theory of Fermentation by Louis Pasteur, 1879

popular in Indonesia, is made by inoculating

soybeans with the fungal organism Rhizopus oligosporus The manufacturing process lendsitself, however, to chance contamination withother microorganisms, including bacteria thatsynthesize Vitamin B12, making tempeh a goodsource of a nutrient that might otherwise be ab-sent in the diet of individuals who consume thisproduct

A Primer on Microbial Classification

For many readers, keeping track of the manygenus, species, and subspecies names assigned

to the organisms used in fermented foods can be

a challenging task However, knowing which ganisms are used in specific fermented foods israther essential (to put it mildly) to understand-ing the metabolic basis for how microbial fer-mentations occur.Therefore, prior to describingthe different groups of microorganisms involved

or-in food fermentations, it is first necessary to view the very nature of microbial taxonomy(also referred to as systematics) and how micro-biologists go about classifying, naming, and iden-tifying microorganisms

re-Although this might seem to be a thanklesstask, it is, after all, part of human nature to sort

or order things; hence, the goal of taxonomy is

When one considers the wide variety of

fer-mented food products consumed around the

world, it is not surprising that their

manufac-ture requires a diverse array of microorganisms

Although lactic acid-producing bacteria and

ethanol-producing yeasts are certainly the most

frequently used organisms in fermented foods,

there are many other bacteria, yeast, and fungi

that contribute essential flavor, texture,

appear-ance, and other functional properties to the

finished foods In most cases, more than one

organism or group of organisms is involved in

the fermentation

For example, in the manufacture of

Swiss-type cheeses, thermophilic lactic acid bacteria

from two different genera are required to

fer-ment lactose, produce lactic acid, and acidify

the cheese to pH 5.2, a task that takes about

eighteen hours Weeks later, another organism,

Propionibacterium freudenreichii subsp

sher-mani, begins to grow in the cheese, producing

other organic acids, along with the carbon

diox-ide that eventually forms the holes or eyes that

are characteristic of Swiss cheese

Even for those fermented foods in which

only a single organism is responsible for

per-forming the fermentation, other organisms may

still play inadvertent but important supporting

roles Thus, tempeh, a fermented food product

15

to achieve some sense of order among the

mi-crobial world Specifically, taxonomy provides

a logical basis for: (1) classifying or arranging

organisms into related groups or taxa; (2)

es-tablishing rules of nomenclature so that those

organisms can be assigned names on a rational

basis; (3) identifying organisms based on the

accepted classification scheme and

nomencla-ture rules; and (4) understanding evolutionary

relationships of species, one to another

As will be noted later in this and successive

chapters, rules for classification are not

per-manently fixed, but rather can be amended

and re-defined in response to new, more

dis-criminating methods For the most part, these

new classification methods are based on

mo-lecular composition and genetic properties,

which can also be used to determine

phyloge-netic or evolutionary relationships between

related organisms

The three domains of life

According to modern taxonomy, life on thisplanet can be grouped into three branches or

domains—the Eukarya, the Bacteria, and the Archaea(Figure 2–1).This organization for clas-sifying all living organisms was proposed andestablished in the 1980s by Carl Woese and isbased on the relatedness of specific 16S rRNAsequences using a technique called oligonu-cleotide cataloging.This three-branch tree of lifedisplaced the classical taxonomy that had rec-ognized only two groups, eukaryotes and pro-karyotes, and that was based primarily on mor-phology and biochemical attributes All of themicroorganisms relevant to fermented foods(and food microbiology, in general) belong to ei-

ther the Eukarya or Bacteria The Archaea,

while interesting for a number of reasons, sists of organisms that generally live and grow in

con-Figure 2–1. Phylogenetic tree of life (based on 16S rRNA sequences) Courtesy of the Joint Genome tute (U.S Department of Energy).

Insti-Microorganisms and Metabolism 17

extreme environments (e.g., very high

tempera-ture, very low pH, very high salt), but rarely are

they associated with foods

Classification of organisms as eukaryotic

or prokaryotic is based on a variety of

char-acteristics (Bergey’s Manual lists more than

50 cytological, chemical, metabolic,

molecu-lar, and reproductive properties), but the

traditional distinguishing feature is the

pres-ence of a nuclear membrane in eukaryotes

Included within the Eukarya domain are

ani-mals, plants, protists, and fungi.The latter are

represented by the Kingdom Fungi, which

in-cludes both yeasts and molds

Fungi

The Kingdom Fungi is very large, containing

many species, and although the exact number

is unknown, it is thought to possibly number as

many as 1.5 million Only about 10% of these

have been observed and described In the

cur-rent system of taxonomy (2004), four major

groups or phyla are recognized among the true

fungi or Eumycota.These are Chytridiomycota,

Zygomycota , Ascomycota, and Basidiomycota.

The Chytridiomycota are primitive fungi

and may be a link to ancestral fungi They are

considered true fungi based upon metabolic

patterns, the presence of chitin in their cell

walls, and small subunit rDNA sequence

com-parisons with other fungi.The chytrids, as they

are sometimes called, live in wet soil, fresh

wa-ter and marine environments, and are not

com-mon in foods, nor are they involved in food

fer-mentations They may be unicellular or may

form simple branching chains of cells or

primi-tive hyphae, which are coenocytic, that is,

without cross walls

The Zygomycota group, sometimes called

zygomycetes, also produces coenocytic hyphae

and reproduce sexually by fusion of two cells

known as gametangia that form a

zygospo-rangium containing zygospores.This group also

produces asexual sporangiospores, stolons, and

rhizoids Sporangiospores are produced in a

sac-like structure on an aerial stalk that extends

into the air from the point of attachment to the

substrate by the rhizoid The rhizoids are cialized root-like hyphae that anchor the mold

spe-to its substrate, secrete enzymes, and absorb trients.The stolons are rapidly growing hyphaethat run over the surface of the substrate from

nu-one rhizoid to another The Zygomycota may

contain as many as 900 species, but those portant in foods and food fermentations are

im-found primarily in the Rhizopus and Mucor

genera

The Ascomycota is a very large phylum of

fungi, containing as many as half of all fungalspecies The ascomycetes (members of the

Ascomycota) have septate mycelia, with crosswalls or septa, and produce sexual spores,called ascospores within a structure known as

an ascus (plural asci) The ascomycetes alsoproduce asexual reproductive states, where re-production occurs by production of asexualspores known as conidia, which are sporesborne on the end of special aerial fertile hy-phae (conidiophores) Conidia are producedfree, often in chains and not in any enclosingstructure.The asexual state or stage is referred

to as the anamorphic state and the fungus iscalled an anamorph This state is sometimescalled the “imperfect” stage or state of the fun-gus The sexual state (ascogenous) is referred

to as the teleomorphic state, and the cospore-producing fungus as a teleomorph.The sexual stage is also referred as the “per-fect” stage or state of the fungus

as-Another group of fungi that are related tothe ascomycetes, but which are not given phy-lum status, are the deuteromycetes, sometimesreferred to as Deuteromycota This is a group

of fungi that are similar to members of the comycetes, but which have no observed sexualstage in their life cycle They are anamorphsand are sometimes (in older literature) referred

as-as the “imperfects” or Fungi Imperfecti

The zygomycetes, ascomycetes, and teromycetes are fungi that are also calledmolds, or micro fungi, because of their smallsize and because they include the fungalspecies important in foods and food fermenta-tions One group of ascomycetes, also known

deu-as hemideu-ascomycetes because they do not

produce a separate ascoma, are primarily

sin-gle celled organisms known as yeasts Yeasts

may produce ascospores within the yeast cell,

but they also reproduce asexually by a process

known as budding, and are very important in

foods and food fermentations

The Basidiomycota includes certain plant

pathogens, such as rusts and smuts, and macro

fungi such as mushrooms, puffballs, and bracket

fungi that grow on trees and fallen logs, as well

as ectomycorrhizal fungi that are associated

with certain plant roots.Another group of fungi

that form the most common type of

mycor-rhizal associations with plant roots are the

Glo-males, sometimes referred to as a fifth phylum,

the Glomeromycota.

Bacteria

Within the Bacteria domain, there has been

less consensus among taxonomists on how

to organize bacteria into higher taxa (e.g.,

Kingdom, Sub-Kingdom, etc.).The Ninth

Edi-tion of Bergey’s Manual of Determinative

Bacteriology (published in 1994) described

three major categories of bacteria and one of

archaeobacteria, and then further divided the

bacteria categories into thirty different

de-scriptive groups According to the most

cur-rent taxonomy (Garrity, et al., 2004), the

Bacteria are now divided into twenty-four

different phyla Nearly all of the bacteria

im-portant in food fermentations, including

lac-tic acid bacteria, belong to a single phylum,

the firmicutes Beyond the phyla, bacteria can

be further divided into classes (and

sub-classes), orders (and sub-orders), families, and

genera Other details on their taxonomic

po-sitions will be described below

Nomenclature

Like all living organisms, microorganisms are

named according to Latinized binomial

no-menclature, meaning they are assigned two

names, a genus and a species By convention,

both the genus and species names are

itali-cized, but only the genus is capitalized.Thus,the name of the common food yeast is writ-

ten as Saccharomyces cerevisae For some

or-ganisms, a trinomial system is applied to cate a subspecies epithet, as is the case for

indi-the dairy organism Lactococcus lactis subsp lactis

Microorganisms are named according to therules established by the appropriate governingbody For bacteria and fungi, the InternationalCommittee on Systematic Bacteriology and theInternational Association for Plant Taxonomy,respectively, define the nomenclature rules.These rules are then published in the respec-tive “codes,” the International Code of Nomen-clature of Bacteria and the International Codefor Botanical Nomenclature In addition, there

is a “running list,” called the List of BacterialNames with Standing in Nomenclature (www.bacterio.cict.fr), that provides updated bacter-ial nomenclature To be included in these listsand considered as “valid,” a name must havebeen published in the scientific literature, alongwith a detailed description and relevant sup-porting data

In some cases, a validly named organismmay be referred to by another name, indicated

as a synonym In other instances, the name of

an organism may have been replaced by a newname, in which case the original name is indi-cated as a basonym In situations where a namewas “unofficially” assigned to an organism anddoes not appear on the list, that name is con-sidered to be illegitimate and its use should bediscontinued However, even if the name as-signed to a particular organism is supported by

a valid publication, this does not mean that thename is or must be accepted by the scientificcommunity

Although names are indeed based on cial rules (where each taxon has a valid name),the utility of a given classification scheme, onwhich a given organism is named, is left up tothe scientists who use it That is, a researchermay propose that a given organism be assigned

offi-a “new” noffi-ame, offi-and hoffi-ave the supporting dence published in a valid journal, but othermicrobiologists are entitled to disagree with

evi-Microorganisms and Metabolism 19

the taxonomy and reject the proposed

classifi-cation

It is relevant to raise these issues, because

many of the organisms used in food

fermenta-tions have either undergone nomenclature

re-visions or have been reclassified into new taxa

For example, the official name of the dairy

or-ganism mentioned above, L lactis subsp lactis

was originally Bacterium lactis (the first

or-ganism isolated in pure culture and named by

Joseph Lister in 1873) It was renamed

Strepto-coccus lactisin 1909, which is how this

organ-ism was known for more than 70 years (and

which still shows up occasionally in “current”

texts), before the new genus, Lactococcus, was

adopted In other cases, a name was proposed,

then rescinded (see below) There are also

in-stances of organisms that had been assigned

“unofficial” names (see above), and through

frequent use, had acquired some level of

valid-ity, however undeserved A good example of

the latter situation was for Lactobacillus

sporo-genes, an organism that is properly classified as

Bacillus subtilis.Yeast nomenclature, although

under the authority of the International Code

for Botanical Nomenclature, is also subject to

taxonomical challenges and changes in

classifi-cation (see below)

Microbial taxonomy and

methods of analysis

If microbiology began with Pasteur in the

mid-dle of the nineteenth century, then for the next

120 years, microbial classification was based

primarily on phenotypic characteristics

Al-though many of these traits remain useful as

di-agnostic tools, by far, the most powerful means

of classifying microorganisms is now via

mo-lecular techniques

Originally, routine tests were based on

nu-cleic acid composition (mol% G  C) and

DNA-DNA and DNA-RNA hybridization.The

lat-ter has long been considered the gold standard

for defining a species, in that organisms

shar-ing high DNA homology (usually greater than

70%) are regarded as members of the same

species However, more recently, the nucleic

acid sequence of the 16S rRNA region has come the most common way to distinguish between organisms, to show relatedness, andultimately to classify an organism to the genus

be-or species level

These sequences can be easily determinedfrom the corresponding DNA after PCR ampli-fication By comparing the sequence from agiven organism to those obtained from refer-ence organisms in the 16S rRNA database, it ispossible to obtain highly significant matches or

to infer relationships if the sequence is unique

In addition, DNA probes, based on genus- orspecies-specific 16S rRNA sequences from aparticular reference organism, are also usefulfor identifying strains in mixed populations.Other techniques that essentially provide amolecular fingerprint and that can be used todistinguish between strains of the same speciesinclude restriction fragment length polymor-phism (RFLP), randomly amplified polymorphicDNA (RAPD), pulsed field gel electrophoresis(PFGE), and DNA microarray technology Finally,

it should be noted that nucleic acid-based ods for classification, whether based on 16SrRNA sequences, DNA-DNA hybridization val-ues, or even whole genome analyses, are notnecessarily the be-all, end-all of microbial taxon-omy Rather, a more holistic or integrative taxon-omy, referred to as polyphasic taxonomy, hasbeen widely adopted and used to classify manydifferent groups of bacteria Polyphasic taxon-omy is based on genotypic as well as pheno-typic and phylogenetic information and is con-sidered to represent a “consensus approach” forbacterial taxonomy

meth-Bacteria Used in the Manufacture

of Fermented Foods

Despite the diversity of bacteria involved rectly or indirectly in the manufacture of fer-mented foods, all are currently classified in one

di-of three phyla, the Proteobacteria, Firmicutes, and the Actinobacteria.Within the Firmicutes

are the lactic acid bacteria, a cluster of positive bacteria that are the main organismsused in the manufacture of fermented foods

Gram-This phylum also includes the genera Bacillus

and Brevibacterium that contain species used

in the manufacture of just a few selected

fer-mented foods

The Proteobacteria contains Gram negative

bacteria that are involved in the vinegar

fermen-tation, as well as in spoilage of wine and other

alcoholic products The Actinobacteria

con-tains only a few genera relevant to fermented

foods manufacture, and only in a rather indirect

manner These include Bifidobacteriim,

Kocu-ria , Staphylococcus, and Micrococcus In fact,

Bifidobacterium do not actually serve a

func-tional role in fermented foods; rather they are

added for nutritional purposes (see below)

While species of Kocuria and the

Staphylo-coccus /Micrococcus group are used in

fer-mented foods, they are used for only one

prod-uct, fermented meats, and for only one purpose,

to impart the desired flavor and color.It is worth

emphasizing that fermented foods may contain

many other microorganisms, whose presence

occurs as a result of inadvertent contamination

However, the section below describes only

those bacteria whose contribution to

fer-mented foods manufacture is known

The Lactic Acid Bacteria

From the outset, it is important to recognize

that the very term “lactic acid bacteria” has no

official status in taxonomy and that it is really

just a general term of convenience used to

de-scribe a group of functionally and genetically

related bacteria Still, the term carries rather

significant meaning among microbiologists

and others who study food fermentations, and,

therefore, will be used freely in this text

Ac-cordingly, the lactic acid bacteria are generally

defined as a cluster of lactic acid-producing,

low %GC, non-spore-forming, Gram-positive

rods and cocci that share many biochemical,

physiological, and genetic properties (Table

2–1).They are distinguished from other Gram

positive bacteria that also produce lactic acid

(e.g., Bacillus, Listeria, and Bifidobacterium)

by virtue of numerous phenotypic and

geno-typic differences

In addition to the traits described above,other important properties also characterizethe lactic acid bacteria, but only in a generalsense, given that exceptions occasionally exist.Most lactic acid bacteria are catalase-negative,acid-tolerant, aerotolerant, facultative anaer-obes In terms of their carbon and energyneeds, they are classified as heterotrophicchemoorganotrophs, meaning that they re-quire pre-formed organic carbon both as asource of carbon and energy

Until recently, it was thought that all lacticacid bacteria lack cytochrome or electrontransport proteins and, therefore, could not de-rive energy via respiratory activity This view,however true for the majority of lactic acidbacteria, has been revised, based on recentfindings that indicate some species may indeedrespire, provided the medium contains thenecessary nutrients (Box 2–1) Still, substratelevel phosphorylation reactions that occurduring fermentative pathways (see below) arethe primary means by which ATP is obtained.The lactic acid bacteria as a group are oftendescribed as being fastidious with complex nu-tritional requirements, and indeed, there arespecies that will grow only in nutrient-rich,well-fortified media under optimized condi-tions However, there are also species of lacticacid bacteria that are quite versatile with re-spect to the growth environment and thatgrow reasonably well even when the nutrientcontent is less than ideal Furthermore, somelactic acid bacteria are actually known for theirability to grow in inhospitable environments,including those that often exist in fermentedfoods This is reflected by the diverse habitats

Table 2.1. Common characteristics of lactic acid bacteria

Gram positive Fermentative Catalase negative Facultative anaerobes Non-sporeforming Low mol% G  C Non-motile Acid-tolerant

Microorganisms and Metabolism 21 Box 2–1. Lactic acid bacteria learn new tricks

Look up “lactic acid bacteria” in any older (or even relatively recent) text, and in the section onphysiological characteristics, it will likely be stated that these bacteria “lack cytochromes andheme-linked electron transport proteins” and are, therefore,“unable to grow via respiration.” Infact, this description was considered to be dogma for generations of microbiology students andresearchers who studied these bacteria, despite the occasional report that suggested otherwise(Ritchey and Seeley, 1976; Sijpesteijn, 1970) In the past few years, however, it has become ap-parent that this section on physiology of lactic acid bacteria will have to be re-written, becausebiochemical and genetic evidence, reported by researchers in France, now supports the exis-

tence of an intact and functional respiratory pathway in Lactococcus lactis and perhaps other

lactic acid bacteria (Duwat et al., 2001; Gaudu et al., 2002)

Respiratory metabolism

Respiration is the major means by which aerobic microorganisms obtain energy During ration, electrons generated during carbon metabolism (e.g., citric acid cycle) are transported orcarried across the cytoplasmic membrane via a series of electron carrier proteins These pro-teins are arranged such that the flow of electrons (usually in the form of NADH+) is toward in-creasing oxidation-reduction potentials Along the way, protons are translocated across themembrane, effectively converting an oxidation-reduction potential into a proton electrochemi-cal potential.This proton potential or proton motive force (PMF) can drive transport, operateflagella motors, or perform other energy-requiring reactions within the membrane It can also

respi-be used to make ATP directly via the ATP synthase.This reaction (called oxidative tion) provides aerobes with the bulk of their ATP and is the coupling step between the respira-tory electron transport system and ATP formation

phosphoryla-Respiration in lactic acid bacteria

For respiration to occur in lactococci, several requirements must be met First, the relevantgenes encoding for electron transport proteins must be present, and then the functional pro-teins must be made Based on physiological and genetic data, these genes are present and the

respiratory pathway is intact in a wild-type strain of Lactococcus lactis (Vido et al., 2004) In this

and presumably other strains of lactococci, this pathway is comprised of several proteins, cluding dehydrogenases, menaquinones, and cytochromes In particular, cytochrome oxidase

in-(encoded by cydAB) serves as the terminal oxidase and is essential (Gaudu et al., 2002)

Miss-ing, however, is a system for making porphyrin groups.The latter is combined with iron to makeheme, which is required for activity of cytochrome proteins, as well as the enzyme catalase.Thus, heme (or a heme precursor) must be added to the medium for respiration to occur Res-piratory growth follows a fermentative period and occurs primarily during the later stages ofgrowth Interestingly, expression of the heme transport system is subject to negative regulation,mediated by the catabolite control protein CcpA (Gaudu et al., 2003) Recall that the activity ofthis regulator is high during rapid growth on glucose and that genes for other catabolic path-ways are repressed Thus, it appears that CcpA might be the metabolic conduit between fer-mentation and respiration

Importantly, it was observed that when lactococci were grown under respiring conditions(i.e., aerobic, with heme added), not only did cells perform respiratory metabolism, they alsogrew better (Duwat et al., 2001) That is, they grew for a longer time and reached higher celldensities compared to non-respiring cells Moreover, during prolonged incubation at 4°C, respir-ing cells remained viable for a longer time, in part because the pH remained high (near 6.0),since less lactic acid was produced via fermentation.These findings are of considerable indus-trial importance, given that one of the main goals of the starter culture industry (Chapter 3)

(continued)

occupied by lactic acid bacteria, which include

not only plant material, milk, and meat, but also

salt brines, low pH foods, and ethanolic

envi-ronments

Perhaps the most relevant properties of

lac-tic acid bacteria are those related to nutrient

metabolism Specifically, the main reason why

lactic acid bacteria are used in fermented foods

is due to their ability to metabolize sugars and

make lactic and other acid end-products Two

fermentative pathways exist In the

homofer-mentative pathway, more than 90% of the sugar

substrate is converted exclusively to lactic acid

In contrast, the heterofermentative pathway

re-sults in about 50% lactic acid, with the balance

as acetic acid, ethanol, and carbon dioxide

Lac-tic acid bacteria possess one or the other of

these two pathways (i.e., they are obligate homofermentative or obligate heterofermenta-tive), although there are some species that havethe metabolic wherewithal to perform both(facultative homofermentative) These path-ways will be described in detail later in thischapter

The genera of lactic acid bacteria

According to current taxonomy, the lactic acidbacteria group consists of twelve genera (Table

2–2) All are in the phylum Firmicutes, Order, Lactobacillales Based on 16S rRNA sequenc-ing and other molecular techniques, the lacticacid bacteria can be grouped into a broad phy-logenetic cluster, positioned not far from other

is to maximize cell biomass while maintaining cell viability Thus, the respiration story has certainly captured the attention of the starter culture industry (Pedersen et al., 2005) Finally,since lactococci do not have an intact citric acid cycle, the electrons that feed the respiratorychain cannot be supplied from the citric acid cycle-derived NADH pool (Vido et al., 2004) In-stead, they must be generated though other pathways

These recent findings on lactococci have not yet been extended to other lactic acid bacteria

However, based on several sequenced genomes, it appears that cyd genes may be present in other lactococci as well as some oenococci and leuconostocs (but are absent in Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgaricus) If, and under what circum-

stances these bacteria actually perform respiration, is not known.As had initially been reportedthirty years ago, and re-confirmed by these more recent studies, molar growth and ATP yieldsare higher during respiratory growth.Thus, it may well be that the respiratory pathway providesthese bacteria an alternative and efficient life-style choice, were they to find themselves backout in nature rather than in the confines of food environments (Duwat et al., 2001)

References

Duwat, P., S Sourice, B Cesselin, G Lamberet, K.Vido, P Gaudu,Y Le Loir, F.Violet, P Loubière, and A Gruss.

2001 Respiration capacity of the fermenting bacterium Lactococcus lactis and its positive effects on

growth and survival J Bacteriol 183:4509–4516.

Gaudu, P., G Lamberet, S Poncet, and A Gruss 2003 CcpA regulation of aerobic and respiration growth in

Lactococcus lactis Mol Microbiol 50:183–192.

Gaudu, P., K.Vido, B Cesselin, S Kulakauskas, J.Tremblay, L Rezạki, G Lamberet, S Sourice, P Duwat, and A.

Gruss 2002 Respiration capacity and consequences in Lactococcus lactis.Antonie van Leeuwenhoek

82:263–269.

Pedersen, M.B., S.L Iversen, K.I Sørensen, and E Johansen 2005.The long and winding road from the search laboratory to industrial applications of lactic acid bacteria FEMS Microbiol Rev 29:611–624 Ritchey,T.W., and H.W Seely 1976 Distribution of cytochrome-like respiration in streptococci J Gen Mi- crobiol 93:195–203.

re-Sijpesteijn, A.K 1970 Induction of cytochrome formation and stimulation of oxidative dissimilation by

hemin in Streptococcus lactis and Leuconostoc mesenteroides.Antonie Van Leeuwenhoek 36:335–348.

Box 2–1. Lactic acid bacteria learn new tricks (Continued)

Microorganisms and Metabolism 23

low G  C Gram positive bacteria (Figure 2–2)

Five sub-clusters are evident from this tree,

including: (1) a Streptococcus-Lactococcus

branch (Family Streptococcaceae), (2) a

Lacto-bacillus branch (Family Lactobacillaceae), (3)

a separate Lactobacillus-Pediococcus branch

(Family Lactobacillaceae); (4) an

Oenococcus-Leuconostoc-Weisella branch (Family

Leu-conostocaceae ), and (5) a

Carnobacterium-

Aerococcus-Enterococcus-Tetragenococcus-Vagococcus branch (Families

Carnobacteri-aceae, Aerococcaceae , and Enterococcaceae).

It is worth noting that this phylogeny is not

entirely consistent with regard to the

morpho-logical and physiomorpho-logical characteristics of

these bacteria For example, Lactobacillus and

Pediococcusare in the same sub-cluster, yet the

former are rods and include

heterofermenta-tive species, whereas the latter are

homofer-menting cocci Likewise, Carnobacterium are

obligate heterofermentative rods, and coccus and Vagococcus are homofermentative

Entero-cocci

Seven of the twelve genera of lactic acid

bacteria, Lactobacillus, Lactococcus, ostoc , Oenococcus, Pediococcus, Streptococcus, and Tetragenococcus, are used directly in food fermentations Although Enterococcus sp are

Leucon-often found in fermented foods (e.g., cheese,sausage, fermented vegetables), except for afew occasions, they are not added directly Infact, their presence is often undesirable, inpart, because they are sometimes used as indi-cators of fecal contamination and also becausesome strains may harbor mobile antibiotic-resistance genes

Importantly, some strains of Enterococcus

are capable of causing infections in humans

Likewise, Carnobacterium are also

undesir-able, mainly because they are considered as

Lactobacillus delbrueckii

Staphylococcus aureus Bacillus subtilis

Listeria monocytogenes

Tetragenococcus halophilus

Lactobacillus casei Lactobacillus brevis Pediococcus pentosaceus Lactobacillus plantarum Lactobacillus gasseri Lactobacillus johnsonii

Streptococcus pneumoniae

Streptococcus thermophilus Clostridium botulinum

0.1

(un-rooted) was generated using the neighbor-joining method.

Microbiology and

Table 2.2. Genera of lactic acid bacteria and their properties 1,2

Cell Fermentation Growth at: Growth in NaClat: Growth at pH: Lactic acid

1 Adapted from Axelsson, 2004

2 Refers to the general properties of the genus; some exceptions may exist

3Species of Lactobacillus may be homofermentative, heterofermentative, or both

4 This phenotype is variable, depending on the species

5 Some species produce D-, L-, or a mixture of D- and L-lactic acid.

Microorganisms and Metabolism 25

spoilage organisms in fermented meat

prod-ucts Finally, species of Aerococcus,

Vagococ-cus , and Weisella are not widely found in

foods, and their overall significance in food is

unclear In the section to follow, the general

properties, habitats, and practical

considera-tions of the genera that are relevant to food

fer-mentations will be described; descriptions of

the important species will also be given

Infor-mation on the genetics of these bacteria, based

on genome sequencing and functional

geno-mics, will be presented later in this chapter

Lactococcus

The genus Lactococcus consists of five

phylo-genetically-distinct species: Lactococcus lactis,

Lactococcus garviae , Lactococcus piscium,

Lactococcus plantarum , and Lactococcus

raf-finolactis(Figure 2–3).They are all non-motile,

obligately homofermentative, facultative

anaer-obes, with an optimum growth temperature

near 30°C They have a distinctive microscopic

morphology, usually appearing as cocci in

pairs or short chains

One species in particular L lactis, is among

the most important of all lactic acid bacteria

(and perhaps one of the most important

organ-isms involved in food fermentations, period)

This is because L lactis is the “work horse” of

the dairy products industry—it is used as astarter culture for most of the hard cheesesand many of the cultured dairy products pro-duced around the world There are actually

three L lactis subspecies: L lactis subsp lactis, Lactococcus lactis subsp cremoris, and Lacto- coccus lactis subsp hordinae Only L lactis subsp lactis and L lactis subsp cremoris, how- ever, are used as starter cultures; L lactis subsp hordinaehas no relevance in fermented food

manufacture.Another variant of L lactis subsp lactis , formerly named L lactis subsp diaceti- lactis (or Lactococcus lactis subsp lactis bio- var diacetylactis), is distinguished based on its

ability to metabolize citrate.This species is notincluded in the current List of Bacterial Names,

and instead is encompassed within L lactis subsp lactis (see below) However, Lacto- coccus lactis subsp diacetilactis is listed in Bergey’s Taxonomical Outline of the Prokary- otes(2004 version)

Plant material has long been considered to

be the “original” habitat of both L lactis subsp lactis and L lactis subsp cremoris The sugges-

tion, however, that milk is now their “new”habitat is supported by several observations

Lactococcus raffinolactis

Lactococcus plantarum Lactococcus piscium Lactococcus garvieae Lactococcus lactis subsp cremoris Lactococcus lactis subsp lactis Lactococcus lactis subsp hordniae

0.01

sequence analysis.