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<ul><li><p>Yeasts in foods and beverages: impact on product qualityand safetyGraham H Fleet</p><p>The role of yeasts in food and beverage production extends</p><p>beyond the well-known bread, beer and wine fermentations.</p><p>Molecular analytical technologies have led to a major revision of</p><p>yeast taxonomy, and have facilitated the ecological study of</p><p>yeasts inmanyotherproducts. The mechanismsbywhichyeasts</p><p>grow in these ecosystems and impact on product quality can</p><p>now be studied at the level of gene expression. Their growth and</p><p>metabolic activities are moderated by a network of strain and</p><p>species interactions, including interactions with bacteria and</p><p>other fungi. Some yeasts have been developed as agents for the</p><p>biocontrol of food spoilage fungi, and others are being</p><p>considered as novel probiotic organisms. The association of</p><p>yeasts with opportunistic infections and other adverse</p><p>responses in humans raises new issues in the field of food safety.</p><p>AddressesSchool of Chemical Sciences and Engineering, The University of</p><p>New South Wales, Sydney, New South Wales, Australia</p><p>Corresponding author: Fleet, Graham H (</p><p>Current Opinion in Biotechnology 2007, 18:170175</p><p>This review comes from a themed issue on</p><p>Food biotechnology</p><p>Edited by Christophe Lacroix and Beat Mollet</p><p>Available online 1st February 2007</p><p>0958-1669/$ see front matter</p><p># 2007 Elsevier Ltd. All rights reserved.</p><p>DOI 10.1016/j.copbio.2007.01.010</p><p>IntroductionThe impact of yeasts on the production, quality and safetyof foods and beverages is intimately linked to theirecology and biological activities. Recent advances inunderstanding the taxonomy, ecology, physiology, bio-chemistry and molecular biology of yeasts have stimu-lated increased interest in their presence and significancein foods and beverages. This has led to a deeper under-standing of their roles in the fermentation of establishedproducts, such as bread, beer and wine, and greaterawareness of their roles in the fermentation processesassociated with many other products. As the food industrydevelops new products and processes, yeasts present newchallenges for their control and exploitation. Food safetyand the linkage between diet and health are issues ofmajor concern to the modern consumer, and yeasts haveemerging consequences in this context. On the positiveside, there is increasing interest in using yeasts as novel</p><p>Current Opinion in Biotechnology 2007, 18:170175</p><p>probiotic and biocontrol agents, and for the nutrient for-tification of foods. On the negative side, food-associatedyeasts could be an under-estimated source of infectionsand other adverse health responses in humans.</p><p>Two books, entirely devoted to the occurrence andsignificance of yeasts in foods and beverages, haverecently been published [1,2] and another includesseveral chapters on food spoilage yeasts [3]. These pub-lications demonstrate the expanding academic and indus-trial interest in the field. This article reviews recentdevelopments in understanding the ecology and biologyof yeasts in foods and beverages and discusses how theseimpact on product quality and safety.</p><p>New analytical toolsThe ability to isolate, enumerate and identify yeasts togenus, species and strain levels is fundamental to under-standing their occurrence and significance in foods andbeverages. Although cultural procedures remain basic tothese needs, molecular methods are making the study ofyeast ecology much more attractive and convenient thanever before [4,5].</p><p>Yeast taxonomy and species identification</p><p>Whereas the identification of new yeast isolates oncerequired the laborious completion of 80 to 100 morpho-logical, biochemical and physiological analyses, this task isnow quickly achieved by DNA sequencing. The DNAsequences of the genes encoding the D1/D2 domain of thelarge (26S) subunit of ribosomal RNA are known for allyeast species, and the sequence of the ITS1-ITS2 region ofrRNA, as well as other genes, is known for many. Thesesequencephylogenetic data have led to a complete revi-sion of yeast taxonomy, and the description of many newgenera and species [6]. Although sequencing of ribosomalgenes is now the accepted method for yeast identification,restriction fragment length polymorphism (RFLP) analysisof the ITS1-ITS2 region is a less expensive, faster alterna-tive, and databases containing the results of such analyseshave been established for food yeasts [5].</p><p>Nucleic acid probes and real-time PCR detection methodshave been described for some species, such as Saccharo-myces cerevisiae, Brettanomyces bruxellensis and Zygosacchar-omyces bailii [4,5,7], and a novel probe-flow cytometricassay has been reported for various Candida species [8].</p><p>Strain differentiation</p><p>The distinctive character of many breads, beers and winescan be linked to particular strains of S. cerevisiae used in</p><p></p><p></p></li><li><p>Yeasts in foods and beverages Fleet 171</p><p>the fermentation [9]. Consequently, differentiation ofyeasts at the subspecies level is an important require-ment. Molecular methods developed for this purposeinclude pulsed-field gel electrophoresis (PFGE) of chro-mosomal DNA and PCR-based methods such as randomamplification of polymorphic DNA (RAPD), amplifiedfragment length polymorphism (AFLP), RFLP, and pro-filing of microsatellite DNA. A simpler, faster method isbased on RFLP analysis of mitochondrial DNA, where noPCR amplification of DNA is required [4,5,10]. Thesemethods are not only useful for quality assurance typingof yeast starter cultures and spoilage species, but theyhave been used to reveal the ecological complexity of theyeast flora associated with many food and beverage fer-mentations. For example, it is now known that thefermentation of wine, cheese, meat sausages and otherproducts not only involves the successional contributionsfrom many different species of yeast, but successionalgrowth of numerous strains within each species alsooccurs [11,12].</p><p>Culture-independent analysis</p><p>Most branches of microbial ecology now accept that viablebut non-culturable species occur in many habitats, includ-ing foods and beverages. Detection of these organismsrequires extraction and analysis of the habitat DNA. Oneapproach that is finding increasing application is PCR inconjunction with denaturing gradient gel electrophoresis(DGGE) or temperature gradient gel electrophoresis(TGGE). Total DNA is extracted from the food, and yeastDNA is specifically amplified using PCR and primerstargeting regions of rDNA. The yeast DNA is thenresolved into amplicons for individual species by DGGEor TGGE. These amplicons are extracted from the gel andtheir species identity determined through sequenceanalysis. PCR-DGGE/TGGE has been applied to analysethe yeast communities associated with grapes, wine, sour-dough, cocoa bean, coffee bean and meat sausage fermen-tations [4,5,13,14,15]. There is good agreement in theresults obtained by cultural and PCR-DGGE/TGGEmethods, although in some cases species that were notidentified by agar culture were recovered by PCR-DGGE suggesting the presence of non-culturable flora. How-ever, the reverse also occurs, where PCR-DGGE has notdetected yeasts that were isolated by culture. Many factorsaffect the performance of PCR-DGGE/TGGE analysesand further research is required to understand and optimizethe assay conditions [4,13].</p><p>Molecular understanding of the yeastresponseAs yeasts grow in foods and beverages, they utilize carbonand nitrogen substrates and generate a vast array ofvolatile and non-volatile metabolites that determinethe chemosensory properties of the product and its appealto the consumer. Some yeasts produce extracellular pro-teases, lipases, amylases and pectinases that also impact</p><p></p><p>on product flavour and texture. The biochemistry of thesereactions and their linkage to product quality are gener-ally well known [16]. Now, genomic studies usingsequence, DNA array, and proteomic analyses enablethe linkage of these responses to the expression andregulation of individual genes [17]. Only a few suchstudies have been performed with food and beverageyeasts, and these have yielded interesting new insights.For example, during wine and beer fermentations,S. cerevisiae exhibits sequential expression and regulationof many genes associated with carbon, nitrogen and sulfurmetabolism, as well as other genes required to toleratestresses such as high sugar concentration, low pH, ethanoland nutrient deficiency [17,18,19]. Genomic analysesalso give molecular explanations of the remarkabletolerance of some yeasts to the extremes of high salt andsugar contents in some foods (e.g. Debaryomyces hansenii incheese brines, Zygosaccharomyces rouxii in sugar syrups andfruit juice concentrates), and to organic acid preservativesin other foods (e.g. Z. bailii in salad dressings and softdrinks) [20].</p><p>Beyond brewing, baking and wine yeastsAlthough research on the contribution of S. cerevisiae tobeer, bread and wine fermentations continues to be afocus, there is expanding interest in the role of yeasts inother products [12].</p><p>It is now well recognized that yeasts make importantcontributions to the process of cheese maturation, wherevarious strains of D. hansenii, Yarrowia lipolytica, Kluyver-omyces marxianus and S. cerevisiae frequently grow to highpopulations. They contribute to the development ofcheese flavour and texture through proteolysis, lipolysis,utilization of lactic acid, fermentation of lactose and auto-lysis of their biomass [21]. In a similar way, D. hansenii,Y. lipolytica and various Candida species affect flavour,texture and colour development in fermented salami stylesausages and country cured hams [15,22]. Many breads,especially sour dough varieties, are still produced bytraditional fermentation processes where no commercialstrains of bakers yeast are added. Although indigenousstrains of S. cerevisiae are prominent in many of thesefermentations, other yeasts are significant and includeSaccharomyces exiguus, Candida milleri, Candida humilis,Candida krusei (Issatchenkia orientalis), Pichia anomala, Pichiamembranifaciens and Y. lipolyitica. These yeasts grow incooperation with lactic acid bacteria, giving distinctiveflavours to the final product [23].</p><p>High-value cash crops such as cocoa beans and coffeebeans also undergo processes that involve the action ofyeasts [24]. Coca beans must be fermented to generatethe precursors of chocolate flavour, and various species ofSaccharomyces, Hanseniaspora, Candida, Issatchenkia andPichia contribute to the process [14,25]. Coffee beansare processed to remove pulp and other mucilaginous</p><p>Current Opinion in Biotechnology 2007, 18:170175</p></li><li><p>172 Food biotechnology</p><p>materials that surround the seeds, and species of Candida,Saccharomyces, Kluyveromyces, Saccharomycopsis, Hansenias-pora, Pichia and Arxula have been associated with thesefermentations [26]. A vast array of traditional fermentedfoods and beverages are produced in African, Asian andSouth American countries from raw materials such asmaize, wheat, cassava, rice, soy beans and fruit. Fermen-tation is essential in contributing to the quality, safety andnutritional value of these products. Aspects of theirmicrobial ecology are just starting to emerge, and demon-strate important contributions from numerous yeastspecies [27,28].</p><p>Collectively, the ecological studies of yeasts in productsother than beer, bread and wine are providing the knowl-edge base for developing a new generation of yeast startercultures, beyond S. cerevisiae.</p><p>Microbial interactions and biocontrolYeasts rarely occur in food and beverage ecosystems assingle cultures. Exceptions occur in highly processedproducts where spoilage outbreaks by single, well-adapted species are known: for example, Z. rouxii in highsugar products [29].</p><p>Generally, most habitats are comprised of a mixture ofyeasts, bacteria, filamentous fungi and their viruses, andproduct quality is determined by the interactive growthand metabolic activity of the total microflora. Even withinyeasts themselves, there can be significant species andstrain interactions that impact on the population dynamicsof the ecosystem. The diversity and complexity of thesemicrobial interactions is just beginning to emerge[11,30,31].</p><p>A network of yeastyeast interactions occurs in mostecosystems, and is observed in fermentations of wine,cheese, meat, and cocoa beans. These interactions mani-fest themselves as the successive growth and death ofdifferent yeast species and strains within each species, asthe fermentation progresses. The mechanisms under-lying these ecological shifts are numerous. Explanationsinclude the different rates of nutrient transport anduptake by the different species and strains, their sensi-tivities to metabolic end products (e.g. ethanol), andresponses to killer toxins [11]. Cellcell interactionsmight also occur through the production of quorum sen-sing molecules [32] and unexplained spatial phenomena[33]. Defining the metabolic outcomes of these inter-actions and their impact on product quality remains agreater challenge, as demonstrated by the interactiveeffects of S. cerevisiae and Saccharomyces bayanus strainson the chemical composition and flavour of wines [34].</p><p>Interactions between yeast and bacteria are often seen asthe inhibitory effects of yeasts on bacteria through etha-nol production; however, the relationships are much</p><p>Current Opinion in Biotechnology 2007, 18:170175</p><p>broader than this. The death and autolysis of yeast cellsreleases vitamins and other nutrients that stimulate thegrowth of important flavour-enhancing bacteria, such asthe malolactic bacteria in wine fermentations [11,31],staphylolcocci, micrococci and brevibacteria duringcheese maturation [21], and lactic acid bacteria duringsour dough fermentations [23]. Ethanol, produced byyeasts during cocoa bean fermentations, stimulates thegrowth of acetic acid bacteria that oxidize the ethanol toacetic acid. This acid is essential for killing the cocoabeans (seeds) and triggering endogenous bean metab-olism that generates the precursors of chocolate flavour[24,25]. Some yeasts utilize the organic acids that occur incheeses, fruit products and salad dressings, causing anincrease in product pH and growth of spoilage and patho-genic bacteria [30]. Some bacteria are antagonistictowards yeasts. Excessive growth of lactic acid bacteriaand acetic acid bacteria on grapes produces acetic acid andother substances that inhibit the growth of yeasts in grapejuice, causing stuck or sluggish wine fermentations andloss of process efficiency [11,31].</p><p>Interactions between yeast and fungi have not beenwidely studied, except in the context of biocontrol. Fun-gal growth on wine grapes produces substances thatinhibit the growth of yeasts during grape juice fermenta-tion [11]. By contrast, some yeasts improve the growth ofPenicillium spp. during the maturation of cheeses [35].Several species within the genera Candida, Pichia, Metsch-nikowia, C...</p></li></ul>