Importance of yeast foralcoholic fermentationMustafa. H. VohraResearch AssociateDepartment of Alcohol TechnologyVasantdada Sugar Institute
Yeast Yeast is generally defined as unicellular fungi, which in a stageof their life cycle occurs as a single cell reproducing commonlyby budding or less frequently by fission. Occurrence: Widely distributed in nature, soil, marine, organicmatter, fruits. Isolation: Soil of wine yards, fruits, insects, distilleryenvironment. Generally in distilleries Saccharomyces cerevisiae andSchizosaccharomyces pombe are used. Pyscrophilic – Below 15oC Mesophilic- 20-35oC Thermophilic- above 45oC Taxonomy: 50 Genera, 500 Species and 1500 Strains.
Morphology- (shapes) Vegetative cells are spherical, global & oval –Depending on age & cultural conditions- Elongatedspecial shapes Some cells form pseudomycellium under stress conditions.(Size) depends on age/cultural conditions Width- 1 to 9µm Length- 2- 10 µm (20-50 µm) Spherical cells- 4-6 µm much larger than bacteria (µm)
Required characteristic of better yeast foralcoholic fermentation High alcohol tolerance (above 12%). High ethanol yield and productivity. Tolerance to inhibitors. Minimum nutrient supplementation High sugar tolerance. High temperature tolerance (above 400C). Flocculating type. Less by products formation during the fermentation. Broad substrate utilization range.
Saccharomyces cerevisiae “Sacchoaromyces” is Greek for “sugar mold” and“cerevisiae” is Latin for “for beer”. Used since ancient times in baking and brewing. Significant eukaryotic model organism. First isolated from the skin of grapes. Flocs and settles well, thus it is easy to filter out.– Flocculation- process of coming out of solutionand clumping together.One such yeast…….
Yeast cell structure Cell wall: Surrounded by a rigid envelope, which comprised ofcarbohydrate (glucan, mannan) and glycosylated protein(phosphomanno-protein). Determines the shape of the cell. Provides a barrier againsthigh osmotic pressure in cytoplasm and generally maintains theintegrity of the cell. The cell wall is a complex extra cellular organelle, capable ofdynamic changes in response to changes in external conditionsand to different stages of the life cycles. Capsular material: Produced extracellularly. Cytoplasmic membrane: present between cell wall &cytoplasm. Function: Selective transport of low molecular weightcompounds.
Cytoplasm: Ground matrix- Cell structures are suspended.Consists of polyphosphates, glycolytic & hydrolytic, pentosecycle enzymes, glycogen, trehalose, ribosomes. Nucleus: Nucleolus- 90% DNA, RNA, Polyphosphatecompounds. Number of chromosomes vary. Nuclear membrane: pair of unit membrane with pores. (85nm). Cell division: Nucleus divides. Function: Genetic and hereditary control. Endoplasmic reticulum: Double membrane system.Ribosomes are present which are responsible for proteinsynthesis.
Vacuoles: Spherical, more transparent, vary in size,site of storage and hydrolysis In active cells more vacuoles are present & in oldcells single large vacuoles are present surrounded byunit membrane. Contain ribonucleases, proteases, esterases, freeamino acids, polyphosphates. Function: Autolysis. Mitochondria: Powerhouse of cell, site of oxidativereactions. Contains DNA, RNA, RNA-Polymerase, Enzymes ofTCA & ETC. Lipid globules: Contain lipids, no membrane.
Yeast Life Cycle : AsexualNew daughters must grow before initiatingtheir first cell cycle
Yeast Life Cycle: SexualHaploid CellsMating PairBudding ZygoteDiploid Cellaa/a
Yeast NutritionMacronutrients: Building blocks needed fornew cell material.Micronutrients: Catalysts needed to facilitatebiochemical reactions.
Categories of Yeast Nitrogen SourcesCompound may be used as that compound forbiosynthesis.Compound may be converted to relatedcompounds for biosynthesis.Compound may be degraded with release ofnitrogen.
Yeast Nitrogen SourcesDegradation may depend upon availability ofother components: vitamins and oxygen.Utilization impacted by other environmentalfactors such as pH.Inorganic nitrogen like most ammonium saltare used.Organic nitrogen like Amino acids & urea areused.
MicronutrientsMinerals and Trace Elements: Mg, Ca, Mn, K, Zn,Fe, Cu.The requirement of minerals are is very low but theyare essential for the growth.Vitamins: act as catalytic functions.Some yeast can synthesize their own vitamins whilesome have multiple requirements like biotin,pantothenic acid, folic acid, niacin, p-amino benzoicacid, inositol, thiamin, riboflavin, pyridoxine, etc.All these vitamins are present in molasses.
Most of the fermentation is conductedby stationary phase cellsStationary phase:1. rate of growth = rate of death2. quiescent, no growth, no death
How Does Ethanol Inhibit Yeast? Displaces water of hydration changing the propertiesof protein-lipid interactions. Denatures proteins. Disrupts protein active sites. Allows increased passage of protons from themedium into the cell leading to acidification of thecytoplasm. Removal of protons requires expenditure of energy.
Cultural conditions for yeastAs the yeast is facultative organism, for growthaerobic conditions are required.MGYP medium:Malt extract- 0.3%Glucose- 1.0%Yeast extract- 0.3%Peptone- 0.5%D/W (make up volume) 100 mlPhysical parameters:Temperature- 32.50CpH- 4.50RPM- 180
Yeast Growth/metabolism Yeast can either produce energy by fermentation oroxidative phosphorylation (ox/phos). Yeast greatly prefer fermentation over ox/phos andwill not start ox/phos until all the sugars areconverted to ethanol. Ethanol is a good source of energy and in thepresence of oxygen yeast use it up as well. Fermentation also produces 2 CO2 molecules permolecule of sugar-- as a by product. =)
MetabolismSpecial flavors and aromas of beers arise from minor biochemical reactionsMajor Reaction: Glucose to Carbon Dioxide and Ethanol
Fundamentals of yeast growth(facultative veg. Reproduction – td = 2h)Aerobic AnaerobicGlycolytic (EMP) pathway(cytoplasm)EMP-TCA-oxidativephosphorylationEtoH is final e- acceptor O2 is final e- acceptorPartial degradation Complete degradation2 ATP/ glucose 36 ATP/ glucoseWeak cell membrane Cell membrane with ufa &sterols (nicotinic acid)fermentation EtoH tolerance, respiration
A- Lag phase dx/dt = 0 Synthesis & degradation of cellular material Adaptation to new environment Process design-Avoid lag phase Composition of growth medium kept sameB- Accelerating phase dx/dt = µx ----------- (1)(µ relevant to medium & strain)µ = 0 to µmaxC- Exponential phase dx/dt = µmax ------------(2) Reproduction & multiplication at max. rate Necessary nutrient in excess amount No hidden limitation by incomplete mixing Absence of inhibitors( SO2, Sulphites, org.acids, cupric ion,ethanol, DCO2
On integration, ln x-ln xo= µmax . t ------------------------(3)Plot of ln x Vs t , slope = µmax (experiment)When eq (3) is expressed in formln (x/xo) = µmax . t ------------------------ (4)The fundamental eq. For batch growthx = xo e(µmax . t ) ------------------------(5)When x=2xo, t = td = doubling timetd = ln 2/ µmax = 0.693/ µmax --------------- (6)D-Decelarating phase: again dx/dt = µt Concentration of one of the nutrient is low. Accumulation of inhibitory compounds.
E-Stationary phase Substrate is exhausted No growth Beginning of starvation, degradation of cellular material Later- cell lysis – nutrients For other cell- few cells still divide So keep your culture in exponential phase & reduce down time.Oxygen solubility In liquid culture O2 is obtained by yeast from dissolved state Aeration – blowers/ compressors Solubility of O2 in DW at 20oC = 9.2 mg/L. Decreases withincreasing temp & increasing solute conc. In its life cycle- yeast cell divides on an average 24 times (bud scars,chitin) Below 1PPM DO – yeast cannot multiply for move than 4-5generations.Therefore, keep low sugar & high aeration rate
Importance of Yeast in BrewingFermentation Flavors What are some of the brewing fermentationflavor compounds? What influences their production? Sensory evaluation of some of these flavorcompounds.
Control of Fermentation FlavorsImportant Factors Yeast– Strain– Pitching rate and consistency– Viability (autolytic “yeasty” flavours)– Contaminants Wort– Gravity, pH, dissolved oxygen, contaminants, yeast foods Fermentation– Design/geometry, temperature, pressure
Ester production by yeast Over 90 distinct esters in beer - floweryand fruity flavors and aromas. Desirable at low concentrations, butundesirable at high concentrations. Important esters: Isoamyl acetate, ethylacetate, isobutyl acetate, ethyl caproate,2-phenyl acetate.
Glycerol Production by YeastGlycerol is quantitatively one of the mostimportant products of yeast fermentationand contributes to the viscosity and “body”of beer (and wine).
Higher alcohols Increased by– high fermentation temp– mixing duringfermentation– low concentration ofamino-acids in wort– intensive aeration– high gravity Reduced by– high pitching rate– cold pitching temperatureand fermentation– pressure duringfermentation– avoid oxygen after pitching– enough amino-acids 80 % of higher alcohols are built during fermentation.They can´t be influenced during maturation.
Yeast AutolysisFlavors and Aromas Gives meat-like flavors and aromas, harshbitternessYeast viability significantly decreases.Yeast releases proteases.pH increases. Increased by:– Length of time beer sits on yeast.– Temperature of yeast. Reduced by:– Timely remove settled yeast.
Sulfur Aromas Increased by:– Low wort oxygenation.– Restricted yeast growth.– Slow fermentation.– Poor venting of fermenter gas.– Time on yeast.– Short storage time.
Products of Fermentation• Ethanol and CO2 are the main products offermentation.• Glycerol is sometimes produced and it cansmoothen the taste and impart viscosity.• Higher-alcohol esters and aldehydes can beproduced as flavor compounds.
Unpleasant By-products• Succinic and acetic acid production causeswine defects.• Malic acid occurs naturally in some grapes andcan give wine a very sour taste (it is used toproduce SweetTarts candy).• Malolactic fermentation using Oenococcusoeni can decrease malic acid content thuslowering acidity of the wine.