Food fermentations by yeasts and filamentous fungi
advertisement
Food fermentations by yeasts and filamentous fungi Prof. Anna Maráz Department of Microbiology and Biotechnology Faculty of Food Science Corvinus University of Budapest ERASMUS IP Maribor 2011 Position of yeasts and moulds (filamentous microfungi) in fungal taxonomy Fungi (Eumycota) Yeasts ~ 800 species In food: ~ 50 species Chytridiomycetes Zygomycetes Ascomycetes Basidiomycetes Deuteromycetes (Fungi imperfecti) Moulds ~ 80.000 species In food: ~ 100 species Main differences between yeasts and filamentous fungi (moulds) Characteristics Yeasts Moulds Vegetative cells Unicellular (budding and pseudomycelia) Mycelia Growth rate (generation time) 1-2 hours 6-48 hours Metabolism Facultative aerobic/anaerobic Aerobic Lytic enzymes (hydrolases) Few Many Toxin production No Yes Dimorfic fungi: ability for budding and pseudomycelia or true mycelia Yeasts - - Cell morphology: ellipsoidal, cylindrical, lemon-shaped, filamentous (with pseudohyphae or septate hyphae) Metabolism: respiration and in some cases ethanolic fermentation Saccharomyces cerevisiae Budding Budscars Saccharomyces cerevisae Fission yeasts e.g. Schizosaccharomyces pombe Pseudomycelium formation e.g. Candida albicans Ethanolic fermentation Piruvatdecarboxylase Glycolysis Alcoholdehydrogenase Glucose 2 pyruvate 2 acetaldehide 2 etanol C6H12O6 CH3-CO-COOH CH3-CHO CH3-CH2-OH 2 CO2 2 NADH2 2 NAD C6H12O6 = 2C2H5OH + 2CO2 180 g glucose 92 g ethanol + 88 g (44,8 normal liter) CO2 End products of alcoholic fermentation and pathways connected to it Formation of higher alcohols during the synthesis of amino acids Tradition of food fermentations • Ancient tribes: milk clotting, spontaneous fermentation of beverages • Sumerians B.C. 3800: beer from cereals • Ancient Egypt: beer from cereals, dough for leavening bread • • Greek and Roman civilizations: culture of wine, beer (Dionysos, Bacchus) Middle ages: beer, wine, cheese production in monasteries Recognition of microorganisms: - Louis Pasteur: diseases of wine and beer caused by microbes - Christian Hansen: use of pure cultures of yeast for beer fermentation 19. century: development of biochemistry: - Fermentation in cell free extracts - Recognition of enzymes and intermediers of fermentation - The glycolytic sequence: Embden-Meyerhof pathway 20th century: • Exploitation of microorganisms in the fermentation and processing of foods: - Bread, alcoholic beverages, dairy products - Meat products, vegetables • Development of starter cultures • Industrial fermentations - Natural antimicrobial substances - Pharmaceuticals, fine chemicals, enzymes 21st century: • Modern biotechnology • Molecular biology and genetic engineering Role of microorganisms in the production of alcoholic beverages (examples) ________________________________________________________ Yeasts Sacch. cerevisiae fermentation of wine, ale-beer Sacch. pastorianus fermentation of lager beer Hanseniaspora / Kloeckera spontaneous fermentation of wine Schizosacch. pombe fermentation of rum Moulds Aspergillus oryzae koji preparation of sake Bacteria Oenococcus oenos malo-lactic fermentation of wine Enterobacteria fermentation of lambic beer Acetic acid bacteria flavour development in rum Zymomonas mobilis fermentation of pulque ________________________________________________________ Major alcoholic beverages – summary of production Beer Raw ingradients Barley, adjuncts (rice, wheat, maize, etc.) Whyisky Wine Spirits, liqueurs Barley (Malt whisky Barley, wheat (Grain whisky) Grapes Barley, maize, molasses, grapes, whey, etc. Pre-treatment Malting, meshing Malting, meshing Crushing, macerati on Variable, dependent on substrate Boiling Yes (hops) No No No Fermentation S. cerevisae S. pastorianus S. cerevisae S. cerevisae S. bayanus S. cerevisae K. marxianus (whey) Yeast recycling Yes No No No Destillation No Yes No Yes Maturation Yes: weeks Yes: years Yes: months, years Varies Final alcohol content (%, v/v) 3-6 40-45 8-14 35-45 Graeme, 1998 BREWING History of brewing in pictures Brewery in the XVIth century Fermentation in open container Modern brewery Fermentation in tanks The brewing process Malting - Barley soaked for 5-7 days to germinate and activate amylases; then dried, crushed Mashing - Malt mixed with water, adjuncts added, heated to hydrolyse starch, then at 74oC enzymes inactivated, the wort settled, filtered Wort boiling- Hops extract solubilized, microbes inactivated Fermentation - Brewer’s yeast added, 5-15 oC for 1-2 weeks Aging - Young (‘green’) beer stored, lagered at 0oC for weeks to months; yeast settled Finishing - Filtering, carbonation, sterilization, packaging Process steps Main biochemical conversions _____________________________________________________________________________________ Water → Water, adjuncts → Spent grains Hops ← → Spent hops Yeast Yeast ← → ← Barley grains ↓ Malting ----- Development of enzymes (amylase, protease) ↓ Kilning ----- Stop respiration in germinating barley ↓ Milling ↓ Mashing ----- Enzymatic hydrolysis of barley ↓ Wort separation ↓ Wort boiling ↓ Wort separation, cooling ↓ Fermentation ----- Production of ethanol, CO2, other metabolites (eg. diacetyl) ↓ Beer clarification ↓ Maturation, lagering ----- Final flavor development ↓ Filtration ↓ Packaging ↓ Pasteurization Flow chart of brewing Typical changes in beer fermentation Traditional use in brewing Pitching yeast: Saccharomyces cerevisiae – lager beer Sacharomyces pastorianus – ale beer Advantages: converting carbohydrates to ethanol, wort to beer, producing flavors, reducing contamination Novel use before steeping: Control of mycotoxin producing fusaria by Debaryomyces hansenii Lambic beer (gueuze) spontaneous, natural fermentation Brettanomyces, Candida, etc, lactics, enterobacters Weiβbier: top fermenting yeasts Wine fermentation Traditional use in wine making Wine yeasts: Saccharomyces cerevisiae Saccharomyces bayanus – low temperature Saccharomyces uvarum – Botryotized grape Advantages: rapid fermentation, ethanol tolerance, absence of off-flavours ‘Wild’ yeasts are always present: Hanseniaspora / Kloeckera start fermentation Candida, Pichia, Metschnikowia contribute to some degree in developing the ‘bouque’ Novel trends: use of mixed starters Outline of wine fermentation White wines Red wines ___________________________________________________ Grapes Grapes Stemming, crushing Stemming, crushing Settling, pressing Maceration with skin Yeast fermentation Yeast fermentation Malo-lactic fermentation Clarification Maturation, fining Filtration Bottling Growth of yeast species during the fermentation of wine o S. cerevisiae • Hanseniaspora / Kloeckera ■ Candida spp Other alcoholic beverages Special types of wine - Champagne and sparkling wines - Sherry - Sake - Cider Spirits (distilled alcoholic beverages) - Whisky, rum, vodka, gin, tequila, etc Indigenous fermented foods Regions Orient Indian African American Products Soy Vegetables Mixed Fish The indigenous fermented foods constitute a group of foods that are produced in homes, villages, and small cottage industries Some indigenous food fermentations such as soy sauce (shoyu), Japanese miso, Indonesian tempe, Indonesian tape ke ten, Japanese sake, Indian idli and dosai, and fish and shrimp sauces and pastes are commercialized at large scale Types of indigenous foods 1. Basic type of fermentations made by koji, in which microorganisms are grown on a cereal-grain or legume substrate to produce a crude enzyme concentrate, koji, that can be used to hydrolyse components in particular fermentation (e.g. soy sauce, miso, tempe, oncom, tape, and rice wines such as sake); 2. Fermentations involving proteolysis of vegetable proteins by microbial enzymes in the presence of salt and/or acid with production of amino acid and peptide mixtures with a meatlike flavour (e.g. soy sauce, Indonesian miso) 3. Fermentations involving enzymic hydrolysis of fish and shrimp or other marine animals in the presence of relatively high salt concentrations to produce meatflavoured sauces and pastes Types of indigenous foods (cont.) 4. Fermentations producing a meat-like texture in a cerealgrain-legume substrate by means of fungal mycelium that knits the particles together (e.g. Indonesian and Malaysian tempe, Indonesian oncom) 5. Fermentation in which organic acids are major products (this category includes Korean kimchi, African ogi, idli, dosai, tape, and also tempe, in which acidification occurs during the initial soaking of soybeans) 6. Fermentations in which ethanol is a major product (e.g. rice wines, palm toddies, sugar cane wines, agave pulque, pito beer, and tape ketan) Examples of indigenous fermented beverages Genetic improvement of yeast starters – brewing yeasts - Introduction of glucoamylase activity to produce low alcoholic beer - Elimination of phenolic off-flavor - Reduction of vicinal diketone formation - Introduction of killer activity / killer resistance (also in wine yeast) Examples of genetically modified brewer’s yeasts Transformed gene(s) Effect STA2 S.cer.var. diastaticus Glucoamylase - A.awamori dextrin fermenting, α-amylase & glucoamylase higher ethanol yield - Schwan. occidentalis FLO1 S.cerevisiae Acetolactate decarboxylase MET25 sulfuhydrase better flocculation reduced production diacetyl reduced H2S production References Meaden et al.1985 Cole et al.1988 Dohmen et al.1990 Lancashire et al.1989 Watari et al.1991 Fuji et al.1990 Shimizu et al.1989 Omura et al.1995 Genetic improvement of yeast starters – wine yeasts - Reduction of fusel alcohol production - Reduction of H2S production - Introducing flocculation and reducing of foaming - Increasing yield of glycerol Examples of genetically modofied wine yeasts Transformed gene(s) Effects Pectate lyase Gonzales-Candelas et al. 1995 ML fermentation Volschenk et al.1997 filtering,clarification malolactic genes (Lb.lactis) GDP1 S. cerevisiae References sensory quality Michnick et al.1997 K1 killer toxin S. cer. competitive advantage Boone et al. 1990 Resveratrol synthase (poplar) resveratrol & coenzyme-A ligase (grape) production Becker et al. 2003 Approval of GM food yeasts Baker’s yeast, maltose derepressed 1989 Brewer’s yeast, expressing STA gene 1993 Sake yeast, flavour enhanced Wine yeast, malolactic fermentation 2002 2003 Effects of wine consumption on the death risk from different deseases Risk of death depending wine consumption Halálozási kockázat aon borfogyasztás függvényében (12 3400034000 persons, between (12 years, éves vizsgálat, főre,males 40-60 éves francia40-60, férfiak,Nancy Nancy) rákos Cancerbetegség egyéb Other okú betegségek Cardio-vasculare desase cardiovasculáris betegségek halálozási kockázat, bázis =1 Relative risk of death 1,8 1,6 1,4 1,2 1 0,8 0,6 0 1-2 pohár 2-3 pohár 3-5 pohár bor naponta Glass pohár of wine / day 5-7 pohár 7-12 pohár > 12 a rákos betegségek emésztőszervi és fül-orrgégészeti jellegűek Negative effects of wine consumption • Ethanol – Toxic effects for central nervous system, liver, stomach etc. – Moderate consumption (linked to meal) is not harmful • Females: 1,5 – 2 dl wine /day • Males:2-3 dl wine /day – „French paradox” • Methanol – high toxicity: consumption of 8-10 g causes serious visual disturbances (lethal dose: 40-100 g) – methanol content of Hungarian wines: 0.02 - 0.3 g/l Brakedown of methanol in human body CH3 – OH Xantin-oxidáz-kataláz (XOK) CH2 = O B12 vitamine HCOOH Formic acid – high toxicity! Negative effects of wine consumption (cont.) • sulphur dioxide – relatively low toxicity for humans (in solution) – allergic reaction in some consumers (particularly asthmatics): wheezing, flushing, tingling etc. – its role in headache is possible but not fully proven • biogenic amines (except serotonin) – headache (red-wine induced headache symptom) – allergic reactions – decomposition of amines in human body by monoamine-oxidase enzyme is inhibited by ethanol! The main biogenic amines of wines MYCOTOXINS ? The only known toxin that my be present in wine is Ochratoxin-A (OTA) 0,7 OTA production of some mould on sterile grape musts 0,6 (pH 3,2; 20 oC, 2 weeks) 0,4 0,3 0,2 0,1 0 Kontroll Botrytis Penicillium Asp. ochraceus 0,7 0,6 0,5 0,3 0,2 0,1 Chardonnay Sauvignon Irsai Olivér 0 Aszú wine (after fermentation) OTA concentrations measured in different wines 0,4 Aszú base (before fermentation) OA ug/l OA ug/l 0,5 Positive effects of wine consumption Correlation between the yearly wine consumption and the death rate (/1000 persons) of cardio-vasculare desases Death / 100 persons „French paradox” Wine consumption Log L/person/year Positive effects of wine consumption (cont.) • Resveratrol – Chemical nature: stylbene (phenolic compound) – Origin: phytoalexyne, produced by the plant (grape) in stress situation, e.g. as a response to fungal infection – Effects: Increase of the plasma level of high-density lipoprotein (H.D.L.), maintenance of low level of “bad” cholesterol (low density lipoprotein: L.D.L) prevention of arteriosclerosis and coronary heart disease. – Level in wines: 0 - 8 mg/l (red wines contain more than white wines) Isomers of resveratrol Transresveratrol Cisresveratrol R=sugar 3,4,5trihidroxisztilbén3-ß-mono-Dglükozid Procyanidines: condensed katechines and leucoanthocyanines Procianidindimer Maintenance of good-cholesterol level in blood Procianidintetramer Positive effects of wine consumption (cont.) • Minerals, organic acids – positive nutritional effects • Serotonin (biogenic amine): – antidepressive effect • Wine is poor vitamin source! Conclusion A glass of wine a day keeps the doctor away! Thank you for your attention!
