cheese flavor and the genomics of lactic acid bacteria€¦ · century, that the door to...

Cheese Flavor and the Genomicsof Lactic Acid BacteriaGenomics and molecular biology are valuable in helping to define howthese bacteria contribute to the flavor and texture of cheeses

Jeffery R. Broadbent and James L. Steele

Cheese—milk’s leap toward immortality-Clifton Paul Fadiman

Humans place great value on technol-ogies to improve the keeping quali-ties of foods, and one of the mostancient of these practices depends onlactic acid bacteria (LAB) to ferment

milk. Because these bacteria are constituents ofraw milk, cheese and other fermented milk foodshave likely been part of the diet since humans firstcollected milk and held it in crude containers.Over the centuries, these “accidental” fermenta-tions were controlled and molded into the morethan 1,000 unique cheeses, yogurts, and fer-mented milks that are available today.

Because fermented dairy foods developed be-fore the emergence of microbiology as a science,manufacturing processes for all varieties longrelied upon naturally occurring LAB to acidifymilk. It was not until discovery of the lactic acidfermentation by Pasteur in 1857, and develop-ment of pure LAB starter cultures later thatcentury, that the door to industrialized cheeseand milk fermentations opened. Since then, pro-duction of fermented milk and especially cheesehave undergone dramatic, sustained growth. Inthe United States alone, for example, cheeseproduction increased more than 200% in thelast quarter century, and total worldwide pro-duction now runs approximately 13 milliontons per year.

To sustain such growth and productivity, thedairy industry has evolved into a leader instarter microbiology and fermentation technol-ogy. Decades of experience have proved thatlarge-scale production of uniform, high-quality

cheese is facilitated by the use of thoroughlycharacterized starter bacteria. Thus, eventhough some traditional cheese fermentationsrely on the natural souring of raw milk, the greatmajority of industrialized processes use startercultures. Since future growth and economic vi-tality of the cheese industry depends on startercultures with known, predictable, and stablecharacteristics, fundamental understanding ofLAB genetics and physiology holds enormousvalue globally.

Genome Studies in Dairy

Lactic Acid Bacteria

LAB are a relatively heterogeneous group ofgram-positive cocci, coccobacilli, and bacillithat inhabit a broad range of ecological niches,yet share several defining characteristics, includ-ing: (i) low (�55 mol%) G � C content; (ii) highacid tolerance; (iii) non-spore forming; (iv) nu-tritionally fastidious; (v) aerotolerant but notaerobic; (vi) unable to synthesize porphyrins;and (vii) strictly fermentative metabolism withlactic acid as the major metabolic end product.Included within this group are several species ofLactobacillus, Lactococcus, Leuconostoc, andStreptococcus that serve as starter cultures forthe commercial manufacture of cheese and fer-mented milks.

Genetics research in “food-grade” LAB beganabout 35 years ago, during which period fourbasic types of genetic elements were character-ized in dairy LAB: plasmid DNA, transposableelements, bacteriophages, and complete chro-mosomes. Representatives from all four of thesegenetic elements affect milk fermentation. How-ever, detailed knowledge of LAB chromosome

Jeffery R. Broad-bent is a professorof food science inthe Department ofNutrition and FoodSciences and West-ern Dairy Center,Utah State Univer-sity, Logan, andJames L. Steele isa professor of foodscience in the De-partment of FoodScience, Universityof Wisconsin,Madison.

Volume 71, Number 3, 2005 / ASM News Y 121

structure and organization is of particular valuebecause genes for all the essential housekeeping,catabolic, and biosynthetic activities of the cellare located in the chromosome.

As with many microorganisms, efforts tocharacterize LAB chromosomes began in ear-nest with the advent of pulsed field gel electro-phoresis (PFGE) technology during the early1980s. Researchers quickly learned that LABhave a single, relatively small (1.8 to 3.4 Mbp),circular chromosome, and that genome size andorganization differ among individual speciesand strains. Although PFGE is still useful inchromosome studies, the most exciting and in-novative research in this realm of microbiologyis now fueled by genomic nucleotide sequenceanalysis.

Genome sequence information for the first ofseveral industrially important LAB starter spe-cies appeared in 2001, when Sorokin and co-

workers released the genomic DNA sequencefor Lactococcus lactis IL1403 (Table 1). Ge-nome sequence information for several otherimportant dairy LAB is also now available, andadditional sequencing projects are under wayand, indeed, sequence information is being gath-ered for more than one strain of a particularspecies (Table 1). The latter development couldprovide insight to the molecular basis for com-mercially significant strain-dependent proper-ties, such as the ability to produce specific fla-vors, propensity for autolysis, acidificationrates, and cell vitality in frozen or lyophilizedstarter concentrates, which are commonly en-countered in dairy LAB.

Because of their economic relevance, many ofthese sequences are being mined for intellectualproperty and are not yet available to the generalscientific community. Nonetheless, nucleotidesequence data is publicly available for more than

Table 1. Genome sequencing projects for dairy-related lactic acid bacteria and other species

Species Strain

Genomesize(MBp) Project sponsora Public

Lactobacillus acidophilus ATCC700396 2.0 Dairy Management, Inc. and Rhodia, Inc. (U.S.) NoL. brevis ATCC 367 2.0 JGI-LABGCb (U.S.) YesL. casei ATCC 334 2.9 JGI-LABGC (U.S.) YesL. casei BL23 2.6 INRA (France) NoL. delbrueckii subsp. bulgaricus ATCCBAA-365 2.3 JGI-LABGC (U.S.) YesL. delbrueckii subsp. bulgaricus ATCC11842 2.3 INRA and Genoscope (France) NoL. delbrueckii subsp. bulgaricus DN-100107 2.1 Danone Vitapole (France) NoL.gasseri ATCC 33323 2.0 JGI-LABGC (U.S.) YesL. helveticus CNRZ32 2.4 Dairy Management, Inc. and Chr. Hansen, Inc.(U.S.) NoL.helveticus DPC 4571 NRc Teagasc and University College, Cork (Ireland) NoL. johnsonii NCC533 2.0 Nestle (Switzerland) YesL. plantarum WCFS1 3.3 Wageningen Centre for Food Sciences (Netherlands) YesL. rhamnosus HN001 2.4 Fonterra Research Center (New Zealand) NoLactococcus lactis subsp.

cremorisSK11 2.3 JGI-LABGC (U.S.) Yes

L.lactis subsp. cremoris MG1363 2.6 Univ. Groningen (Ne); INRA (France) NoL. lactis subsp. lactis IL1403 2.3 INRA and Genoscope (France) YesLeuconostoc mesenteroides ATCC 8293 2.0 JGI-LABGC (U.S.) YesPediococcus pentosaceus ATCC 25745 2.0 JGI-LABGC (U.S.) YesStreptococcus thermophilus LMD-9 1.8 JGI-LABGC (U.S.) YesS. thermophilus LMG18311 1.9 Univ. Catholique de Louvain (Belgium) NoS. thermophilus CNRZ1066 1.8 INRA (France) NoBifidobacterium longum NCC2705 2.3 Nestle (Switzerland) YesB. longum DJ010A 2.1 JGI-LABGC (U.S.) YesB. breve NCIMB8807 2.4 University College, Cork (Ireland) NoBrevibacterium linens ATCC9174 3.0 JGI-LABGC (U.S.) YesPropionibacterium freundenreichii ATCC6207 2.6 DSM Food Specialties (Netherlands) No

a As of 1 January 2005.b JGI-LABGC, Department of Energy Joint Genome Institute and Lactic Acid Bacteria Genomics Consortiumc NR, not reported.

122 Y ASM News / Volume 71, Number 3, 2005

Broadbent Finds Pleasure in Studying Lactobacilli, Watching RaptorsJeffery Broadbent finds pleasurestudying and teaching about bac-teria that, instead of being harm-ful, do much good. In particular,lactic acid bacteria (LAB) providea central service worldwide to thefermentation and bioprocessingindustries—helping, for instance,to produce 13 million tons ofcheese annually. The many foodsproduced with these bacteria “re-main steeped in artisan traditionand thus provide fertile opportu-nity to researchers like myselfwho are intrigued by microbialecology and physiology in com-plex environments,” he says.

“How many other foods do weknowingly consume that containmillions of live bacteria?” Broad-bent continues. “One of the mostbasic goals of science is to teach usto view seemingly common ob-jects with renewed curiosity andappreciation. I hope the next timepeople find themselves enjoying agood piece of cheese, they pauseto savor the microbiological mar-vel it represents.”

Broadbent, 43, is professor ofdairy microbiology in the depart-ment of nutrition and food sci-ences at Utah State University inLogan City, where he focuses onthis diverse group of gram-posi-tive cocci, coccobacilli, and ba-cilli. “The future of LAB researchis bright with promise; with ge-nome sequences and molecular bi-ology tools now available for sev-eral key species, opportunities toinvestigate LAB evolution, genet-ics, physiology, and metabolismhave never been greater,” he says.

His interest in science, how-ever, did not originate withcheese-making bacteria, but in-stead flourished from a “lifelongenchantment with wild fauna and

flora, particularly birds,” he says.“Some of my earliest memoriesare rooted in these experiences—catching frogs in the Utah moun-tains with my grandfather, beingspellbound as a young boy by thedazzling cacophony of color andsound in cage-loads of exoticbirds at an open air market.”

A native of Utah, Broadbentmoved with his family as a youngchild to places that further fueledthese interests. For example, hisfamily spent five years in SaoPaulo, Brazil, “where my fascina-tion with birds and other wildlifebecame indelibly imprinted intomy psyche, and also where Ilearned to feed my curiositythrough a fairly voracious readinghabit,” he says.

By the time Broadbent beganhigh school, in Tempe, Ariz., hehad blossomed into a competentamateur ornithologist, and wasparticularly interested in birds ofprey. Determined then to pursue acareer in wildlife biology, he be-came involved in several researchprojects in his biology and zool-ogy courses, and eventually spentthe summer before his senior yearas a volunteer ornithologist doingfield research on a peregrine fal-con project in the Gila NationalForest of New Mexico.

Broadbent enrolled at UtahState University in the fall of1979, but his undergraduate stud-ies were disrupted several timesdue to financial problems, keep-ing him from completing hisbachelor of sciences degree until1987. By then, his coursework fo-cus had shifted from wildlife re-search to microbial biotechnol-ogy, a field he found equallyfascinating.

“After graduation, I had the re-

markable good fortune of landinga research tech position in the lab-oratory of Dr. Jeffrey Kondo, aworld-renowned geneticist in thelactic acid bacteria community,”Broadbent says. “Jeff’s thoroughand articulate research opened myeyes to the elegance of lactic acidbacteria and introduced me to afield that, to this day, is brightwith opportunity.”

He became a graduate fellowand, while still under Kondo’s su-pervision, received his Ph.D. innutrition and food sciences in1992. Later, he accepted a posi-tion on the faculty at Utah State.

While Broadbent’s researchand academic interests shiftedfrom wildlife to lactic acid bacte-ria, he continues to pursue thoseearly passions. He and his wife—ahigh school special educationteacher—and their two daugh-ters, 12 and 11, maintain “whatsometimes feels like a small zoo”in their home, he says. It includesa falcon, four dogs, a cat, a hedge-hog, fish, a loft of homing pi-geons, and three egg-laying chick-ens. “As you might expect,leaving town for a family vacationrequires some planning,” he says.

Broadbent also is a practicingfalconer, having engaged in thesport since high school. With hisfalcon and two of the dogs, bothEnglish Setters, he pursues ducks,pheasant, grouse, and partridge.“I am still enchanted by raptors,and falconry provides the connec-tion to wildlife I have soughtthroughout my life,” he says.

Marlene Cimons

Marlene Cimons is a freelance writerwho lives in Bethesda, Md.


half of the sequenced LAB strains (Table 1), and10 of the 14 publicly accessible sequences werecontributed as part of a joint venture betweenthe Department of Energy Joint Genome Insti-tute and the U.S.-based Lactic Acid BacteriaGenomics Consortium (LABGC). The LABGCmission is to advance academic and industrialresearch on LAB through release of genome se-quence information for microorganisms promi-nently associated with the fermented foods indus-try. (For additional information on the LABGCeffort, see http://wineserver.ucdavis.edu/people/Faculty/mills/LABGC/lab.htm).

Cheese Flavor Basics: Add

Lactic Acid Bacteria

Converting bland and rubbery fresh curds into adelicious mature cheese is a complex and dy-namic process whose intricacies are dictated bythe type and composition of milk being used, thecultures and enzymes that are added, and thespecific manufacturing and ripening regimensthat are applied. Many cheese types are stored atlow temperature for months or years to attaintheir characteristic flavor and body attributes.During storage, the microorganisms and en-zymes that are trapped in the cheese matrix acton carbohydrates, citrate, proteins, and lipids ina manner that is heavily influenced by the curdmicroenvironment and which ultimately yieldsdistinct types of cheeses.

Although a link between LAB and cheeseflavor was first postulated more than 100years ago, complexities in microbiology, en-zymology, and cheese microenvironmentsconfounded early efforts to establish a defin-itive role for these bacteria affecting flavors.However, in the late 1950s, Elisabeth Sharpeand coworkers at the Institute for Food Re-search in Shinfield, England, developed tech-nologies to manufacture cheeses aseptically,enabling researchers to prove LAB are essen-tial for flavor development in Cheddar andother cheeses.

LAB that contribute to this process mayinclude deliberately added starters, adjunctbacteria (select strains that intensify or accel-erate flavor development), and adventitiousspecies, called nonstarter lactic acid bacteria(NSLAB), that enter curd from the process-ing environment. While many different LABspecies may affect cheese flavor, research inthis area mainly focuses on Lactococcus lac-

tis, which serves as the starter bacterium forCheddar, Gouda, and many other cheeses, andon dairy-related species of Lactobacillus (Fig.1). Interest in lactobacilli such as Lactobacillushelveticus and Lactobacillus delbrueckii subsp.bulgaricus stems from their widespread use asboth starter and adjunct cultures, and becauseNSLAB populations are almost always domi-nated by facultatively heterofermentative Lacto-bacillus sp. such as Lactobacillus casei.

Because of the role of LAB in developingflavor, efforts to define its biochemical basis incheese focus on the physiology of these micro-organisms. The numbers of starter bacteriacommonly exceed 109 CFU per gram of cheesewhen ripening begins (Fig. 2), but the microen-vironment of ripening cheese is harsh. For in-stance, it is typified by an absence of residuallactose, high levels of NaCl, low pH, and lowtemperature. Those conditions extract a toll onstarter viability, and, typically, a sizable fractionof the starter cells undergo autolysis, which re-leases intracellular enzymes and other cellularcomponents into the cheese matrix where they,too, can influence ripening.

Meanwhile, NSLAB populations, whose ini-tial numbers are typically below 102 CFU/g,begin to grow and eventually plateau at celldensities of 107-109 CFU/g after 3–9 months ofaging (Fig. 2). Depending on the species that isused and whether a particular strain can grow in

TABLE 2. Components of the Lactobacillus helveticus CNRZ32proteolytic enzyme system isolated before and after genomesequence determination

Genes isolated prior tosequencing project(1990–2001)

New genes from genome annotation(2001–2004)

Proteinases:prtH prtH2 plus 9 additional proteasesEndopeptidases:pepE, pepO, pepO2 pepE2, pepF, pepO3, plus 2

glycoprotein endopeptidasesAminopeptidases:pepC, pepN, pepX pepC2 plus 7 additional

aminopeptidasesDi-Tripeptidases:pepD, pepI, pepQ, pepR pepD2, pepD3, pepD4, pepQ2, pepT1,

and pepT2Oligo- and di-tripeptide transport

systems:None oppA,oppA2, oppB-D, oppF, and dtpA,

dtpA2, and dtpTMultiple amino acid transporters


ripening cheese, populations of adjunct bacteriamay mirror those of the starter or NSLAB frac-tions.

Key Modes of Microbial Action

in Cheese Ripening

Starter, adjunct, and NSLAB collectively influ-ence flavor development through several basicmechanisms that include fermenting lactose,converting milk proteins (primarily caseins) intopeptides and free amino acids, and breakingdown citrate, lipids, esters, and amino acids intovolatile aroma compounds.

Fermenting lactose into L-lactic acid is a pri-mary function of any starter culture in cheesemanufacture. Acid productivity is critical forcontrolling cheese quality because the culturedetermines the final pH and mineral content ofthe curd, which affects the protein structure andamount of residual coagulant in the curd, and,

thus, texture and flavor properties. Lactate itselfis also a component of cheese flavor and inSwiss-type cheeses serves as a key nutrient forpropionibacteria. They convert it into propionicacid, which is another important flavor compo-nent, and carbon dioxide, which gives the cheeseits “eyes.” If starter bacteria rapidly depleteresidual milk sugar in the curd, they can help toprevent its use as a substrate for undesirableadventitious bacteria, such as heterofermenta-tive Lactobacillus brevis, that can produce seri-ous flavor and texture defects.

Proteolysis and its secondary reactions alsoplay a major role in bacterially ripened cheeses,making casein hydrolysis and its relationship toflavor development an area of intense researchinterest for decades. The hydrolysis of intactcaseins is almost exclusively catalyzed by thecoagulant and endogenous milk proteinases(e.g., plasmin), while LAB proteinases and pep-

F I G U R E 1

Colored scanning electron micrographs of representative cheese starter (A-C) and nonstarter (D-F) lactic acid bacteria (LAB). Species showninclude Lactobacillus helveticus (A), Lb. delbrueckii subsp. bulgaricus (B), Lactococcus lactis (C), Lb. casei (D), Pediococcus pentosaceus (E),and Lb. brevis (F). Images provided by B. McManus and J. Broadbent.


tidases are responsible for producing water-sol-uble peptides and free amino acids.

Together, primary and secondary proteolysisof caseins influences cheese flavor in at leastthree significant ways. First, casein networkbreakdown softens cheese texture, which facili-tates the release of flavor compounds when thecheese is consumed. Second, some of the low-molecular-weight peptides produced in these re-actions directly affect flavor, but this conse-quence is generally negative since these peptidesimpart bitterness. Third, the free amino acidsthat are liberated can also directly affect flavor.For instance, glutamate and aspartate residuesenhance flavors.

More commonly, released amino acids areprecursors for a broad range of potent aromacompounds. These reactions are of particularinterest because a growing body of evidenceindicates that LAB’s converting of free aminoacids into aroma compounds is the rate-limitingstep in the development of mature cheese aro-mas. The products of amino acid catabolism,which may arise via decarboxylation, deamina-tion, transamination, desulfuration, or sidechain removal, can impart desirable or undesir-able flavor attributes.

Much of the research on amino acid catabo-lism by LAB has been directed toward the fatesof aromatic, sulfur-containing, and branched-chain classes of amino acids because of their key

role in aroma. For example, convert-ing methionine into volatile sulfurcompounds such as methanethiol,hydrogen sulfide, dimethyl sulfide,and dimethyl trisulfide is thought tocontribute desirable “sulfur” flavorsto many cheese types, whereasbreaking down leucine is the likelysource of a desirable nutty flavornote in Cheddar cheese. In contrast,breaking down aromatic amino ac-ids contributes several undesirable“off-flavors” to cheese, including de-rivatives such as indole, skatole,[para]-cresol, and phenyl acetalde-hyde.

Free fatty acids formed by lipaseor esterase activity on milk fat alsodirectly affect cheese flavor, and canhave further effects by serving as pre-cursors for esters and other flavor

compounds. Moreover, esterases and lipasescatalyze the hydrolysis or synthesis of esters,depending on cheese water activity and levels ofother available fatty acids and alcohols. En-zymes involved in these reactions may comefrom rennet pastes, from milk itself, and fromstarter and nonstarter LAB.

It is well established, for example, that pregas-tric lipases and esterases from ruminants areresponsible for the sharp, fatty acid-based fla-vors that characterize some Italian cheeses. Incheeses such as Parmesan that do not use pre-gastric lipases and esterases, however, flavornotes associated with lipolysis are probably dueto indigenous milk enzymes and microbial en-zymes. Most LAB lack lipolytic activity andhave very low esterolytic activity, but in cheesewith long ripening times these cells can generateenough free fatty acids and esters to impactflavor.

Finally, LAB use citrate to produce succinateor diacetyl. Succinate, a compound withmonosodium glutamate-like flavor-enhancingproperties, can be isolated from several cheesevarieties, and sensory studies suggest it contrib-utes savory flavor to Swiss-type cheese and to afull, aged flavor in Cheddar. In Swiss and othercheeses where Propionibacterium freudenreichiisubsp. shermanii attain high numbers, succinateproduction is attributed to aspartic acid catabo-lism by the propionibacteria. In Cheddar and

F I G U R E 2

0

Log

10 C

FU

/g c

hees

e

10

Ripening time

8

6

4

2

Non-starter lactics

Lactococcal starterLactococcal starter

Microbiology of ripening Cheddar cheese.


other varieties, however, NSLAB produce succi-nate from citrate via the reductive tricarboxylicacid pathway.

The other important citrate-derived flavorcomponent, diacetyl, imparts a “buttery” notewhose importance in butter, buttermilk, andsome cheese types has been recognized for dec-ades. Diacetyl is formed by oxidative decompo-sition of �-acetolactate, an intermediate in thepathways for pyruvate metabolism and aminoacid biosynthesis. In recent years, detailedknowledge of citrate metabolism and diacetylproduction has yielded effective strategies forengineering L. lactis strains to enhance diacetylproduction.

Genomics Will Propel Further

Advancements

Though great progress has been made towardunderstanding LAB physiology and the pro-cesses that drive cheese flavor development,much remains to be learned about these reac-tions. Currently, significant research advancesdepend on recombinant DNA technology. Thecomplexity of the peptidase enzyme system inLAB, for example, confounded earlier efforts toestablish the role of individual enzymes in caseinhydrolysis and cheese ripening. However, toolsfor constructing isogenic strains that differ in theactivity of only single peptidases now are pro-viding researchers with an effective approach todetermine how individual enzymes contribute tocell growth and cheese properties.

By combining molecular tools with genomics,researchers in industry and academia are creat-ing even greater opportunities to investigate themeans by which LAB act within and respond tocheese and milk microenvironments. Hence, re-search to better define the relationship betweenLAB physiology and flavor development should,whenever possible, focus on strains that: (i) pos-sess established flavor-producing capabilities;(ii) are amenable to genetic manipulation; and(iii) are analyzed at the genome sequence level.

One such candidate strain for forthcomingstudy is Lactobacillus helveticus CNRZ32, acommercial cheese flavor adjunct that can re-duce bitterness and intensify flavor develop-ment. We recently assembled a draft (fourfoldcoverage) genome sequence for CNRZ32, andare currently using that sequence to investigate

mechanisms by which this strain affects cheeseflavor.

For example, because proteolysis plays such acritical part in cheese ripening, one of us (Steeleand his collaborators) spent more than a decadecloning and characterizing CNRZ32 genes thatencode proteolytic enzymes (Table 1), develop-ing gene transfer systems, and constructing aseries of single and multiple deletion mutantslacking functional genes for many of those en-zymes. Despite such concerted efforts, initialannotation of the genome sequence revealed alarge number of additional genes in CNRZ32whose products are predicted to contribute tothe proteolytic enzyme system of this bacterium.

From our perspective, such data underscoreboth the power of genome sequence informationfor applied bacteriology, and the challenges onemust face in interpreting and applying that in-formation. Although sequencing efforts ex-panded the genetic database for the CNRZ32proteolytic enzyme system by about fivefold,efforts to confirm and characterize all the newgene assignments will require more time andresources. Nonetheless, functional analysis ofthe newly discovered endopeptidase genes hasalready identified enzymes with important rolesin the hydrolysis of bitter peptides in cheese.

Functional genomics is also being used toinvestigate pathways for amino acid biosynthe-sis and catabolism in Lactobacillus helveticusCNRZ32. In cheese, converting amino acidsinto volatile cheese flavor compounds may oc-cur directly or through interactions amongstarter, adjunct, and NSLAB components. Somestrains can independently convert amino acidsinto aroma compounds, while others may pro-duce or degrade only one or more metabolicintermediates.

The basis for this phenomenon has not beendetermined, but all LAB are auxotrophic for oneor more amino acids, and the primary mecha-nism for amino acid breakdown by LAB in-volves the reversible action of enzymes involvedin biosynthetic pathways. Thus, much of theinterplay that occurs between LAB in aminoacid catabolism probably reflects the nature ofamino acid auxotrophies among the differentbacteria in cheese. Since the primary sequencesof most enzymes involved in these reactions arerelatively well-conserved, access to genome se-quence information should dramatically en-hance our ability to predict—and test—how in-


dividual organisms contribute to amino acidcatabolism in cheese.

Dairy technologists and microbiologists haveidentified many of the fundamental mechanismsby which LAB affect flavor, and this knowledgeis facilitating industry efforts to accelerate and

intensify flavors. There is still a great deal to belearned, however, and the combined strengthsof genomics and molecular biology tools arecertain to play a leading role in research todefine the molecular dynamics of LAB in pro-ducing fine cheeses.

SUGGESTED READING

Beresford, T. P., N. A. Fitzsimons, N. L. Brennan, and T. M. Cogan. 2001. Recent advances in cheese microbiology. Int. DairyJ. 11:259–274.Broadbent, J. R. 2001. Genetics of lactic acid bacteria, pp. 243–299. In J. L. Steele and E. H. Marth (eds.), Applied DairyMicrobiology, 2nd ed. Marcel Dekker, Inc., New York.Davidson, B. E., N. Kordias, M. Dobos, and A. J. Hillier. 1996. Genomic organization in lactic acid bacteria. Antonie vanLeeuwenhoek 70:161–183.Klaenhammer, T., E. Altermann, F. Arigoni, A. Bolotin, F. Breidt, J. Broadbent, R. Cano, S. Chaillou, J. Deutscher, M.Gasson, M. van de Guchte, J. Guzzo, T. Hawkins, P. Hols, R. Hutkins, M. Kleerebezem, J. Kok, O. Kuipers, M. Lubbers, E.Maguin, L. McKay, D. Mills, A. Nauta, R. Overbeek, H. Pel, D. Pridmore, M. Saier, D. van Sinderen, A. Sorokin, J. Steele, D.O’Sullivan, W. de Vos, B. Weimer, M. Zagorec, and R. Siezen. 2002. Discovering lactic acid bacteria by genomics. Antonievan Leeuwenhoek 82:29–58.Olson, N. F. 1990. The impact of lactic acid bacteria in cheese flavor. FEMS Microbiol. Rev. 87:131–148.Yvon, M., and L. Rijnen. 2001. Cheese flavour formation by amino acid catabolism. Int. Dairy J. 11:185–202.


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