Yeasts in foods and beverages: impact on product quality and safety Graham H Fleet

Yeasts in foods and beverages: impact on product quality and safety Graham H Fleet The role of yeasts in food and beverage production extends beyond the well-known bread, beer and wine fermentations. Molecular analytical technologies have led to a major revision of yeast taxonomy, and have facilitated the ecological study of yeasts in many other products. The mechanisms by which yeasts grow in these ecosystems and impact on product quality can now be studied at the level of gene expression. Their growth and metabolic activities are moderated by a network of strain and species interactions, including interactions with bacteria and other fungi. Some yeasts have been developed as agents for the biocontrol of food spoilage fungi, and others are being considered as novel probiotic organisms. The association of yeasts with opportunistic infections and other adverse responses in humans raises new issues in the field of food safety. Addresses School of Chemical Sciences and Engineering, The University of New South Wales, Sydney, New South Wales, Australia Corresponding author: Fleet, Graham H (g.fleet@unsw.edu.au) This review comes from a themed issue on Food biotechnology Edited by Christophe Lacroix and Beat Mollet Available online 1st February 2007 0958-1669/$ see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2007.01.010 Introduction The impact of yeasts on the production, quality and safety of foods and beverages is intimately linked to their ecology and biological activities. Recent advances in understanding the taxonomy, ecology, physiology, biochemistry and molecular biology of yeasts have stimulated increased interest in their presence and significance in foods and beverages. This has led to a deeper understanding of their roles in the fermentation of established products, such as bread, beer and wine, and greater awareness of their roles in the fermentation processes associated with many other products. As the food industry develops new products and processes, yeasts present new challenges for their control and exploitation. Food safety and the linkage between diet and health are issues of major concern to the modern consumer, and yeasts have emerging consequences in this context. On the positive side, there is increasing interest in using yeasts as novel probiotic and biocontrol agents, and for the nutrient fortification of foods. On the negative side, food-associated yeasts could be an under-estimated source of infections and other adverse health responses in humans. Two books, entirely devoted to the occurrence and significance of yeasts in foods and beverages, have recently been published [1,2 ] and another includes several chapters on food spoilage yeasts [3]. These publications demonstrate the expanding academic and industrial interest in the field. This article reviews recent developments in understanding the ecology and biology of yeasts in foods and beverages and discusses how these impact on product quality and safety. New analytical tools The ability to isolate, enumerate and identify yeasts to genus, species and strain levels is fundamental to understanding their occurrence and significance in foods and beverages. Although cultural procedures remain basic to these needs, molecular methods are making the study of yeast ecology much more attractive and convenient than ever before [4,5]. Yeast taxonomy and species identification Whereas the identification of new yeast isolates once required the laborious completion of 80 to 100 morphological, biochemical and physiological analyses, this task is now quickly achieved by DNA sequencing. The DNA sequences of the genes encoding the D1/D2 domain of the large (26S) subunit of ribosomal RNA are known for all yeast species, and the sequence of the ITS1-ITS2 region of rrna, as well as other genes, is known for many. These sequence phylogenetic data have led to a complete revision of yeast taxonomy, and the description of many new genera and species [6 ]. Although sequencing of ribosomal genes is now the accepted method for yeast identification, restriction fragment length polymorphism (RFLP) analysis of the ITS1-ITS2 region is a less expensive, faster alternative, and databases containing the results of such analyses have been established for food yeasts [5]. Nucleic acid probes and real-time PCR detection methods have been described for some species, such as Saccharomyces cerevisiae, Brettanomyces bruxellensis and Zygosaccharomyces bailii [4,5,7], and a novel probe-flow cytometric assay has been reported for various Candida species [8]. Strain differentiation The distinctive character of many breads, beers and wines can be linked to particular strains of S. cerevisiae used in www.sciencedirect.com

Yeasts in foods and beverages Fleet 171 the fermentation [9]. Consequently, differentiation of yeasts at the subspecies level is an important requirement. Molecular methods developed for this purpose include pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and PCR-based methods such as random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), RFLP, and profiling of microsatellite DNA. A simpler, faster method is based on RFLP analysis of mitochondrial DNA, where no PCR amplification of DNA is required [4,5,10]. These methods are not only useful for quality assurance typing of yeast starter cultures and spoilage species, but they have been used to reveal the ecological complexity of the yeast flora associated with many food and beverage fermentations. For example, it is now known that the fermentation of wine, cheese, meat sausages and other products not only involves the successional contributions from many different species of yeast, but successional growth of numerous strains within each species also occurs [11,12 ]. Culture-independent analysis Most branches of microbial ecology now accept that viable but non-culturable species occur in many habitats, including foods and beverages. Detection of these organisms requires extraction and analysis of the habitat DNA. One approach that is finding increasing application is PCR in conjunction with denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE). Total DNA is extracted from the food, and yeast DNA is specifically amplified using PCR and primers targeting regions of rdna. The yeast DNA is then resolved into amplicons for individual species by DGGE or TGGE. These amplicons are extracted from the gel and their species identity determined through sequence analysis. PCR-DGGE/TGGE has been applied to analyse the yeast communities associated with grapes, wine, sourdough, cocoa bean, coffee bean and meat sausage fermentations [4,5,13,14,15]. There is good agreement in the results obtained by cultural and PCR-DGGE/TGGE methods, although in some cases species that were not identified by agar culture were recovered by PCR-DGGE suggesting the presence of non-culturable flora. However, the reverse also occurs, where PCR-DGGE has not detected yeasts that were isolated by culture. Many factors affect the performance of PCR-DGGE/TGGE analyses and further research is required to understand and optimize the assay conditions [4,13 ]. Molecular understanding of the yeast response As yeasts grow in foods and beverages, they utilize carbon and nitrogen substrates and generate a vast array of volatile and non-volatile metabolites that determine the chemosensory properties of the product and its appeal to the consumer. Some yeasts produce extracellular proteases, lipases, amylases and pectinases that also impact on product flavour and texture. The biochemistry of these reactions and their linkage to product quality are generally well known [16 ]. Now, genomic studies using sequence, DNA array, and proteomic analyses enable the linkage of these responses to the expression and regulation of individual genes [17 ]. Only a few such studies have been performed with food and beverage yeasts, and these have yielded interesting new insights. For example, during wine and beer fermentations, S. cerevisiae exhibits sequential expression and regulation of many genes associated with carbon, nitrogen and sulfur metabolism, as well as other genes required to tolerate stresses such as high sugar concentration, low ph, ethanol and nutrient deficiency [17,18,19]. Genomic analyses also give molecular explanations of the remarkable tolerance of some yeasts to the extremes of high salt and sugar contents in some foods (e.g. Debaryomyces hansenii in cheese brines, Zygosaccharomyces rouxii in sugar syrups and fruit juice concentrates), and to organic acid preservatives in other foods (e.g. Z. bailii in salad dressings and soft drinks) [20 ]. Beyond brewing, baking and wine yeasts Although research on the contribution of S. cerevisiae to beer, bread and wine fermentations continues to be a focus, there is expanding interest in the role of yeasts in other products [12 ]. It is now well recognized that yeasts make important contributions to the process of cheese maturation, where various strains of D. hansenii, Yarrowia lipolytica, Kluyveromyces marxianus and S. cerevisiae frequently grow to high populations. They contribute to the development of cheese flavour and texture through proteolysis, lipolysis, utilization of lactic acid, fermentation of lactose and autolysis 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 style sausages and country cured hams [15,22]. Many breads, especially sour dough varieties, are still produced by traditional fermentation processes where no commercial strains of baker s yeast are added. Although indigenous strains of S. cerevisiae are prominent in many of these fermentations, other yeasts are significant and include Saccharomyces exiguus, Candida milleri, Candida humilis, Candida krusei (Issatchenkia orientalis), Pichia anomala, Pichia membranifaciens and Y. lipolyitica. These yeasts grow in cooperation with lactic acid bacteria, giving distinctive flavours to the final product [23]. High-value cash crops such as cocoa beans and coffee beans also undergo processes that involve the action of yeasts [24]. Coca beans must be fermented to generate the precursors of chocolate flavour, and various species of Saccharomyces, Hanseniaspora, Candida, Issatchenkia and Pichia contribute to the process [14,25]. Coffee beans are processed to remove pulp and other mucilaginous www.sciencedirect.com

172 Food biotechnology materials that surround the seeds, and species of Candida, Saccharomyces, Kluyveromyces, Saccharomycopsis, Hanseniaspora, Pichia and Arxula have been associated with these fermentations [26]. A vast array of traditional fermented foods and beverages are produced in African, Asian and South American countries from raw materials such as maize, wheat, cassava, rice, soy beans and fruit. Fermentation is essential in contributing to the quality, safety and nutritional value of these products. Aspects of their microbial ecology are just starting to emerge, and demonstrate important contributions from numerous yeast species [27,28 ]. Collectively, the ecological studies of yeasts in products other than beer, bread and wine are providing the knowledge base for developing a new generation of yeast starter cultures, beyond S. cerevisiae. Microbial interactions and biocontrol Yeasts rarely occur in food and beverage ecosystems as single cultures. Exceptions occur in highly processed products where spoilage outbreaks by single, welladapted species are known: for example, Z. rouxii in high sugar products [29]. Generally, most habitats are comprised of a mixture of yeasts, bacteria, filamentous fungi and their viruses, and product quality is determined by the interactive growth and metabolic activity of the total microflora. Even within yeasts themselves, there can be significant species and strain interactions that impact on the population dynamics of the ecosystem. The diversity and complexity of these microbial interactions is just beginning to emerge [11,30,31]. A network of yeast yeast interactions occurs in most ecosystems, and is observed in fermentations of wine, cheese, meat, and cocoa beans. These interactions manifest themselves as the successive growth and death of different yeast species and strains within each species, as the fermentation progresses. The mechanisms underlying these ecological shifts are numerous. Explanations include the different rates of nutrient transport and uptake by the different species and strains, their sensitivities to metabolic end products (e.g. ethanol), and responses to killer toxins [11]. Cell cell interactions might also occur through the production of quorum sensing molecules [32 ] and unexplained spatial phenomena [33 ]. Defining the metabolic outcomes of these interactions and their impact on product quality remains a greater challenge, as demonstrated by the interactive effects of S. cerevisiae and Saccharomyces bayanus strains on the chemical composition and flavour of wines [34]. Interactions between yeast and bacteria are often seen as the inhibitory effects of yeasts on bacteria through ethanol production; however, the relationships are much broader than this. The death and autolysis of yeast cells releases vitamins and other nutrients that stimulate the growth of important flavour-enhancing bacteria, such as the malolactic bacteria in wine fermentations [11,31], staphylolcocci, micrococci and brevibacteria during cheese maturation [21], and lactic acid bacteria during sour dough fermentations [23]. Ethanol, produced by yeasts during cocoa bean fermentations, stimulates the growth of acetic acid bacteria that oxidize the ethanol to acetic acid. This acid is essential for killing the cocoa beans (seeds) and triggering endogenous bean metabolism that generates the precursors of chocolate flavour [24,25]. Some yeasts utilize the organic acids that occur in cheeses, fruit products and salad dressings, causing an increase in product ph and growth of spoilage and pathogenic bacteria [30]. Some bacteria are antagonistic towards yeasts. Excessive growth of lactic acid bacteria and acetic acid bacteria on grapes produces acetic acid and other substances that inhibit the growth of yeasts in grape juice, causing stuck or sluggish wine fermentations and loss of process efficiency [11,31]. Interactions between yeast and fungi have not been widely studied, except in the context of biocontrol. Fungal growth on wine grapes produces substances that inhibit the growth of yeasts during grape juice fermentation [11]. By contrast, some yeasts improve the growth of Penicillium spp. during the maturation of cheeses [35]. Several species within the genera Candida, Pichia, Metschnikowia, Cryptococcus and Pseudozyma have strong antifungal properties mediated through the production of lytic enzymes, toxic proteins, toxic fatty acids and ethyl acetate, and have potential for the biocontrol of fungi. Commercial preparations of some species are now available for the pre- and post-harvest control of fruit, vegetable and grain spoilage fungi [36,37]. Yeasts and food safety As part of daily life, humans consume large populations of yeasts without adverse impact on their health. Unlike bacteria and viruses, yeasts are rarely associated with outbreaks of foodborne gastroenteritis, intoxications or other infections. Nevertheless, caution is needed, and further research on this topic is required [38 ]. Significant lay literature connects the dietary intake of yeasts with a range of gastrointestinal, respiratory, skin, migraine and even psychiatric disorders. Overgrowth of yeasts in the gastrointestinal tract might contribute to the development of these disorders, but immune reactions to yeast cell wall polysaccharides and responses to yeastproduced amines and sulfur dioxide could also occur. The connection between yeast, the human response and food is largely based on dietary observations. If foods suspected to contain yeasts or their products are removed from the diet, the adverse responses disappear, but return when such foods are reintroduced [38,39]. www.sciencedirect.com

Yeasts in foods and beverages Fleet 173 Yeasts are not aggressive, infectious organisms, but some species such as Candida albicans and Cryptococcus neoformans are opportunistic pathogens that cause a range of mucocutaneous, cutaneous, respiratory, central nervous system and organ infections, as well as general fungemia [40]. Individuals with weakened health and immune systems are at greatest risk, and include cancer, AIDS and hospitalized patients, and those undergoing treatment with immunosuppressive drugs, broad spectrum antibiotics and radio- chemotherapies. The greater frequency of such individuals in the community has led to increased reporting of yeast infections. Moreover, an increasing number of yeast species has been implicated, including many found in foods (e.g. S. cerevisiae, C. krusei, C. famata, P. anomola, Rhodotorula spp. [38,41]. Infections caused by S. cerevisiae are notable because of its extensive use in the food industry, and infections with this yeast have been reported in immunocompetent individuals [42,43]. It is thought that hospitalized patients become exposed to high levels of yeasts through the biofilms they form on catheters and other invasive devices, and that these yeasts probably originate from the hands of hospital workers and the foods brought into the hospital environment [38 ]. More research is needed to establish stronger linkages between the role of foods in contributing to yeast infections. Information is needed on the survival and growth of yeasts throughout the gastrointestinal system, the potential for yeasts to translocate from the gastrointestinal tract to the blood system, and the general occurrence of yeasts in the hospital and health care environments. The circumstances whereby a nonpathogenic yeast, such as S. cerevisiae, becomes pathogenic also require investigation. Probiotic and other health benefits Probiotics are viable microorganisms that are beneficial to consumers when ingested in appropriate quantities. Although certain species of lactic acid bacteria are prominent as probiotic organisms, there is increasing interest in yeasts as probiotics [38,44,45]. S. cerevisiae var boulardii has been used for many years as an oral biotherapeutic agent for treating a range of diarrheal disorders. This species colonizes the intestinal tract where, in a probiotic function, it combats diarrhoea-causing bacteria [44,46]. Food carrier systems for this yeast need to be developed for its commercial application as a probiotic, but technical obstacles have been encountered. When incorporated into some products, it caused gassy, ethanolic spoilage and off-flavours [47,48]. Of greater concern, are reports of fungemia infections caused by S. boulardii [42,43]. Other yeasts mentioned as potential probiotics include D. hansenii, Kluy. marxianus, Y. lipolytica, I. orientalis, P. farinosa and P. anomala, but further research is required [38 ]. Yeasts are increasingly used as probiotics in the livestock and aquaculture industries [38 ]. Yeast products, principally derived from S. cerevisiae, have been used for many years as ingredients and additives in food processing. These products include flavourants, enzymes, antioxidants, vitamins, colourants and polysaccharides [49,50]. Three points are worthy of mention. First, many of these products are prepared from yeast cells after they have been processed by autolysis. Despite its commercial significance, molecular understanding of yeast autolysis is still very limited and more research is needed to optimize this process [51,52]. Second, most products are derived from S. cerevisiae. The yield and range of products could be increased by screening for their presence in other yeast species and strains, as demonstrated for the vitamin folic acid [53], cell wall polysaccharides [54] and autolysates [55]. Finally, there remains undiscovered bioactivity and functionality in yeast products. Whereas the glucan polysaccharides from the walls of S. cerevisiae were originally valued for their water-binding and rheological functionalities, it is now recognized that they can stimulate the immune system, lower serum cholesterol, exhibit antitumour activity, and adsorb substances such as mycotoxins [38,49]. Conclusions Advances in molecular technologies have provided new analytical tools for studying the diversity and biological activities of yeasts associated with food and beverage production, although more research is still required on the ecology and activities of yeasts in products other than beer, bread and wine. The interactions between yeasts and the ecosystems in which they occur provide another area for future study; yeasts form interactions with other species and strains, along with bacteria, other fungi, protozoans and their viruses, but as yet these relationships remain poorly described and understood. Interest in the public health significance of yeasts in foods and beverages is also increasing, in both positive and negative contexts. Again, we are likely to see future developments in this regard. Update Debaryomyces hansenii is one of the most significant yeasts in food and beverage production, and this is highlighted in a recent review of its phylogeny, ecology, physiology, molecular biology and its biotechnological potential [56]. As mentioned in the conclusion, yeast interactions between themselves and with other organisms have implications for food quality and safety, and further research is needed on these topics. Aspects of yeast cell interactions have been considered in a recent review that discusses their underlying molecular mechanisms, how they impact on growth and survival and how they affect pathogenicity [57]. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: 1. of special interest of outstanding interest Boekhout T, Robert V (Eds): Yeasts in Food. Beneficial and Detrimental Aspects. Behr s Verlag; 2003. www.sciencedirect.com

174 Food biotechnology Comprenhensive discussions of yeasts in foods and beverages an emphasis is placed on commodities. 2. Querol A, Fleet GH (Eds): Yeasts in Food and Beverages. Springer; 2006. Comprehensive discussions of yeasts in foods and beverages emphasis on ecology and biology of yeasts. 3. Blackburn C (Ed): Food Spoilage Microorganisms. CRC Press; 2006. 4. Beh AL, Fleet GH, Prakitchaiwattana C, Heard GM: Evaluation of molecular methods for the analyses of yeasts in foods and beverages. In Advances in Food Mycology. Edited by Hocking AD, Pitt JT, Samson RA, Thrane U. Springer; 2006:69-106. Reviews and lists recent literature on molecular methods used for the analysis of yeasts in foods and beverages. 5. Fernandez-Espinar JT, Martorell P, de Llanos R, Querol A: Molecular methods to identify and characterise yeasts in foods and beverages. In Yeasts in Food and Beverages. Edited by Querol A, Fleet GH. Springer; 2006:55-82. 6. Kurtzman CP, Fell JW: Yeast systematics and phylogeny implications of molecular identification methods for studies in ecology. In Biodiversity and Ecophysiology of Yeasts. Edited by Rosa CA, Peter G. Springer; 2006:11-30. Outlines the most recent changes to yeast classification and taxonomy, based on DNA sequencing and phylogenetic analyses. 7. Rawsthorne H, Phister T: A real-time PCR assay for the enumeration and detection of Zygosaccharomyces bailii from wine and fruit juices. Int J Food Microbiol 2006, 112:1-7. 8. Page BT, Kurtzman CP: Rapid identification of Candida species and other clinically important yeast species by flow cytometry. Appl Environ Microbiol 2005, 43:4507-4514. 9. Fleet GH: Saccharomyces and related genera.in Food Spoilage Microorganisms. Edited by Blackbrun C. CRC Press; 2006:306-335. 10. Schuller D, Valero E, Dequin S, Caseal M: Survey of molecular methods for the typing of wine yeast strains. FEMS Microbiol Lett 2004, 231:19-26. 11. Fleet GH: Yeast interactions and wine flavour. Int J Food Microbiol 2003, 86:11-22. 12. Romano P, Capece A, Jespersen L: Taxonomic and ecological diversity of food and beverage yeasts. In Yeasts in Food and Beverages. Edited by Querol A, Fleet GH. Springer; 2006:13-53. Good overview of diversity and roles of yeasts in fermented foods and beverages. 13. Prakitchaiwattana J, Fleet GH, Heard GM: Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes. FEMS Yeast Res 2004, 4:865-877. Provides a critical discussion of merits and limitations of the use of DGGE for analysing yeasts in foods and beverages. 14. Nielsen DS, Hanholt S, Tano-Debrah K, Jespersen L: Yeast populations associated with Ghanaian cocoa fermentation analysed using denaturing gradient gel electrophoresis (DGGE). Yeast 2005, 22:271-284. 15. Cocolin L, Urso R, Rantsiou K, Cantoni C, Comi G: Dynamics and characterisation of yeasts during natural fermentation of Italian sausages. FEMS Yeast Res 2006, 6:692. 16. Swiegers JH, Bartowsky EJ, Henschke PA, Pretorius IS: Yeast and bacterial modulation of wine aroma and flavour. Aust J Grape Wine Res 2005, 11:139-173. Comprehensive, well illustrated review of the biochemical production of flavour and aroma compounds by microorganisms in foods and beverages. 17. Bond U, Blomerg A: Principles and applications of genomics and proteomics in the analysis of industrial yeast strains. In Yeasts in Food and Beverages. Edited by Querol A, Fleet GH. Springer; 2006:173-213. Good, basic introduction to yeast genomics and its applications in food and beverage fermentations. 18. Varela C, Cardenas J, Melo F, Agosin E: Quantitative analysis of wine yeast gene expression profiles under winemaking conditions. Yeast 2005, 22:369-383. 19. Brejning J, Arneborg N, Jespersen L: Identification of genes and proteins induced during the lag and early exponential phase of lager brewing yeasts. J Appl Microbiol 2005, 98:261. 20. Tanghe A, Prior B, Thevelein JM: Yeast responses to stress. In Biodiversity and Ecophysiology of Yeasts. Edited by Rosa CA, Peter G. Springer; 2006:175-195. Good, general review of the biology and practical significance of the stress responses in yeasts. 21. Addis E, Fleet GH, Cox JMC, Kolak D, Leung T: The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses. Int J Food Microbiol 2001, 69:25-36. 22. Samelis J, Sofos JN: Yeasts in meat and meat products. In Yeasts in Food Beneficial and Detrimental Aspects. Edited by Boekhout T, Robert V. Behr s Verlag; 2003:239-265. 23. De Vuyst LD, Neysens P: The sourdough microflora: biodiversity and metabolic interactions. Trends Food Sci Technol 2005, 16:43-56. 24. Schwan R, Wheals AE: Mixed microbial fermentations of chocolate and coffee. In Yeasts in Food Beneficial and Deterimental Aspects. Edited by Boekhout T, Robert V. Behr s-verlag; 2003:429-449. 25. Ardhana M, Fleet GH: The microbial ecology of cocoa bean fermentations in Indonesia. Int J Food Microbiol 2003, 86:87-99. 26. Masoud W, Cesar LB, Jespersen L, Jakobsen M: Yeasts involved in fermentation of Coffee arabica in East Africa, determined by genotyping and by direct denaturing gradient gel electrophoresis (DGGE). Yeast 2004, 21:549-556. 27. Aidoo KE, Nout MJR, Sarkar PK: Occurrence and function of yeasts in Asian indigenous fermented foods. FEMS Yeast Res 2006, 6:30-39. 28. Nout MJR: Traditional fermented products from Africa, Latin Amercia and Asia. In Yeasts in Food Beneficial and Detrimental Aspects. Edited by Boekhout T, Robert V. Behr s-verlag; 2003:451-473. Demonstrates the diversity and significance of yeasts in many products little known to western consumers. 29. Stratford M: Food and beverage spoilage yeasts. In Yeasts in Food and Beverages. Edited by Querol A, Fleet GH. Springer; 2006:335-380. 30. Viljoen B: Yeast ecological interactions. Yeast-yeast, yeastbacteria, yeast-fungi interactions and yeasts as biocontrol agents. In Yeasts in Food and Beverages. Edited by Querol A, Fleet GH. Springer; 2006:83-110. 31. Alexandre H, Costello PJ, Remize F, Guzzo J, Guilloux-Benatier M: Saccharomyces cerevisiae Oenococcus oeni interactions in wine: current knowledge and perspectives. Int J Food Micorbiol 2004, 93:141-154. 32. Hogan DA: Quorum sensing: alcohols in a social situation. Curr Biol 2006, 16:R457-R458. Novel discussion of the concept of quorum sensing and its mechanisms in yeast biology. 33. Arneborg N, Siegumfeldt H, Andersen GH, Nissen P, Daria VR, Rodrigo PJ, Gluckstad J: Interactive optical trapping shows that confinement is a determinant of growth in a mixed yeast culture. FEMS Microbiol Lett 2005, 245:155-159. Novel use of laser optical technology to demonstrate that spatial phenomena might affect yeast cell cell interactions. 34. Howell K, Cozzolino D, Bartowsky E, Fleet GH, Henschke PA: Metabolic profiling as a tool for revealing Saccharomyces interactions during wine making. FEMS Yeast Res 2006, 9:91-101. 35. Hansen TK, van der Tempel T, Cantor MD, Jakobsen M: Saccharomyces cerevesiae as a starter culture in mycelia. Int J Food Microbiol 2001, 69:101-111. 36. Fleet GH: Yeasts in fruit and fruit products.in Yeasts in Food Beneficial and Detrimental Aspects. Edited by Boekhout T, Robert V. Behr s-verlag; 2003:267-287. www.sciencedirect.com

Yeasts in foods and beverages Fleet 175 37. Passoth V, Fredlund E, Druvefors UA, Schnurer J: Biotechnology, physiology and genetics of the yeast Pichia anomala. FEMS Yeast Res 2006, 6:3-13. 38. Fleet GH, Balia R: The public health and probiotic signficance of yeasts in foods and beverages. In Yeasts in Food and Beverages. Edited by Querol A, Fleet GH. Springer; 2006:381-398. First major review of positive and negative public health issues relating to yeasts in foods and beverages. 39. Eaton TK: Moulds, yeasts, ascospores, basidiospores, algae and lichens: toxic and allergic reactions. J Nutrit Environ Med 2004, 14:187-201. 40. Hazen KC, Howell SA: Candida, Cryptococcus and other yeasts of medical importance. In Manual of Clinical Microbiology 8 th edition. Edited by Murray PR. American Society for Microbiology; 2003:1693-1711. 41. Hobson RP: The global epidemiology of invasive Candida infections is the tide turning? J Hosp Infect 2003, 55:159-168. 42. Enache-Angoulvant A, Hennequin C: Invasive Saccharomyces infections: a comprehensive review. Clin Inf Dis 2005, 41:1559-1568. A thorough review and discussion of human infections caused by S. cerevisiae an industrial yeast not normally considered to be a risk to human health. 43. de Llanos R, Querol A, Peman J, Gobernado M, Fernandez-Espinar MT: Food and probiotic strains from the Saccharomyces cerevisiae species as a possible origin of human systemic infections. Int J Food Microbiol 2006, 110:286-290. 44. van der Aa Kuhle A, Skovgaard K, Jespersen L: In vitro screening of probiotic properties of Saccharomyces cerevisae var boulardii and foodborne Saccharomyces cerevisiae strains. Int J Food Microbiol 2005, 101:29-40. Provides a good discussion of issues related to the use of yeasts as probiotic organisms. 45. Sullivan A, Nord CE: The place of human probiotics in human intestinal infections. Int J Antimicrob Agents 2003, 20:313-319. 46. Czervoka D, Rampal P: Experimental effects of Saccharomyces boulardii on diarrheal pathogens. Microbes Infect 2002, 4:733-739. 47. Lourens-Hattingh A, Viljoen BC: Growth and survival of probiotic yeast in dairy products. Food Res Int 2001, 34:791-796. 48. Heenan CN, Adams MC, Hosken RW, Fleet GH: Survival and sensory acceptability of probiotic microorganisms in a non-fermented frozen, vegetarian dessert. Lebensm Wiss Technol 2004, 37:461-466. 49. Dawson KA: Not just bread or beer: new applications for yeast and yeast products in human health and nutrition. In Nutritional Biotechnology in the Feed and Food Industry. Edited by Lyons TP, Jaques FA. Nottingham University Press; 2002:225-232. 50. Abbas CA: Production of antioxidants, aromas, colours, flavours and vitamins by yeasts. In Yeasts in Food and Beverages. Edited by Querol A, Fleet GH. Springer; 2006:285-334. 51. Zhao J, Fleet GH: Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides. J Ind Microbiol Biotechnol 2005, 32:415-423. 52. Alexandre H, Guilloux-Benatier M: Yeast autolysis in sparkling wine a review. Aust J Grape Wine Res 2006, 12:119-217. 53. Hjortmo S, Patring J, Jastrebova J, Andlid T: Inherent biodiversity of folate content and composition in yeasts. Trends Food Sci Technol 2005, 16:311-316. 54. Nguyen TH, Fleet GH, Rogers PL: Composition of the cell wall of several yeast species. Appl Microbiol Biotechnol 1998, 50:206-212. 55. Lukondeh T, Ashbolt NJ, Rogers PL: Evaluation of Kluyveromyces marxianus as a source of yeast autolysates. J Ind Microbiol Biotechnol 2003, 30:52-56. 56. Breuer U, Harms H: Debaryomyces hansenii an extremophilic yeast with biotechnological potential. Yeast 2006, 23:415-437. 57. Palkova Z, Vachova L: Life within a community: benefit to yeast long term survival. FEMS Microbiol Rev 2006, 30:806-824. www.sciencedirect.com