Presentation on theme: "Apostolos G. Panteloglou, Katherine A. Smart and David J"— Presentation transcript:
1 Premature Yeast Flocculation (PYF) – its causes, nature and significance Apostolos G. Panteloglou, Katherine A. Smart and David J. Cook January 2013
2 Contents What is Premature Yeast Flocculation (PYF)? Development of an assay to predict the PYF status of maltsThe importance of yeast strain on the severity of PYFInvestigating the “antimicrobial peptide hypothesis”Conclusions
3 What is Premature Yeast Flocculation (PYF)? Early or abnormally heavy onset of flocculationlow suspended cell countsincomplete fermentation of sugars to alcoholSporadic problem in brewery fermentationsregion and harvest conditions dependentInduced by factor(s) which have been shown to originate from malt (van Nierop et al., 2004)
4 Why PYF is Important? Incomplete conversion of sugars to alcohol Flavour abnormalities (e.g. diacetyl)Potential issues with the re-use of the yeastRequirement to blendSignificant financial and logistical problemsThe uptake of diacetyl by yeast: an important aspect of flavour maturation in lager beers
5 What causes PYF?Two main theories - each originate with fungal infection of barley/maltFungi secretes enzymes which degrade husk materialsGenerates soluble high molecular weight polysaccharides (HMWP)HMWP form bridges between flocculent yeast cells, increasing floc size1. The bridging polysaccharide theoryBarley responds to stress of fungal infection with production of anti-microbial peptides (AP)AP survive brewhouse processing and have ‘anti-yeast’ activity due to impairment of membrane function2. The anti-microbial peptide theoryAxcell et al. (2000) ; Axcell, (2003) ; van Nierop et al., (2004 & 2006)
6 Predictive tests for PYF Standard malt analysis is unable to predict PYFLaboratory-scale fermentation assays are the most widely adoptedFermentation tests take several days to be completedMonitor gravity and measure suspended yeast cellsSome rapid and micro-scale tests have been developed
7 In-house PYF Assay Wort Preparation Yeast Propagation Fermentation Barley milling (80 g)↓Mashing → 63°C (60 min)72°C (1°C/min) for 25 min76°C (1°C/min) for 5 minCoolingFiltration → Recycle first 100 mLSparging (100 mL mash water at 63°C)Gravity Adjustment (11°P)Autoclave (30 min at 121°C)StorageFull loop cells↓10 mL YPD25°C (1 day)100 mL YPD25°C (3 days)Spinning50% w/w yeast slurryCell countingYeast cells (20 million cells/mL)↓200 mL 11°P wort (+ 4% w/v glucose)AerationPanteloglou, A. G., Box, W. G., Smart, K. A., and Cook, D. J. Optimization of a small-scale fermentation test to predict the premature yeast flocculation potential of malts. J. Inst. Brew, 116(4), 413–420, 2010.
8 Typical test results for PYF+ve & PYF-ve fermentations 15°C with W34/70 Yeast StrainResidual Gravity 93 h Post-Pitching69 h statistical significant differences (P < )
9 Residual Gravity 93 h Post-Pitching The effect of re-pitching PYF-ve wort with yeast from a previous PYF+ve fermentation15°C with W34/70 Yeast StrainResidual Gravity 93 h Post-PitchingPanteloglou, A.G., Smart, K.A., and Cook, D.J. Malt-induced premature yeast flocculation: current perspectives. Industrial Microbiology & Biotechnology, 39(6), , 2012.
10 Lager strains differ in susceptibility to PYF (1) W34/70 Yeast StrainSMA Yeast Strain40 hsignificant differences (P < 0.01)Panteloglou, A. G., Box, W. G., Smart, K. A., and Cook, D. J. Optimization of a small-scale fermentation test to predict the premature yeast flocculation potential of malts. J. Inst. Brew, 116(4), 413–420, 2010.
11 Lager strains differ in susceptibility to PYF (2) Fermentation ProfilesResidual GravityEthanol YieldPanteloglou, A.G., Smart, K.A., and Cook, D.J. PYF from the perspective of brewing yeast: impacts of nutrient uptake and yeast fermentation characteristics. In: Proceedings of the American Society of Brewing Chemists, Sanibel Island, Florida, Oral Presentation O17, 2011.
12 Lager strains differ in susceptibility to PYF (3) Fermentation Profiles using different PYF+ve Worts and the ‘Industrial’ Lager YeastPanteloglou, A.G., Smart, K.A., and Cook, D.J. The importance of wort nutrients and yeast strain on the incidence of premature yeast flocculation. In preparation.
13 INVESTIGATING THE “ANTIMICROBIAL PEPTIDE HYPOTHESIS” ‘MINI-FV’ EXPERIMENTS Quain, D.E., Box , W.G., and Walton, E.FPanteloglou, A.G., Smart, K.A., and Cook, D.J. The effect of premature yeast flocculation factor(s) on and mechanical agitation on fermentation rate, yeast physiology and nutrient uptake. In preparation.
14 ‘Mini-FV’ experiments (100 mL) Designed to investigate:-The effects of PYF factor(s) on yeast physiological characteristics- Nutrient uptake in PYF+ve and PYF-ve fermentation mediaSamples: Industry sourced PYF+ve and PYF-ve malts- Matched in terms of barley variety, harvest year and region of production33 PYF+ve and 33 PYF-ve fermentations were conducted simultaneously, enabling destructive time-point sampling-15°P worts; SMA yeast pitched at 20 million cells/mL-Continuous stirring (280 rpm) at 15°C for 7 days
15 Measurements taken during the mini FV experiments Cell Density and Budding IndexGravity and EthanolFree Amino NitrogenFermentable Sugars (sucrose, fructose, glucose, maltose, maltotriose)
16 Worts Fermentable Sugars & Free Amino Nitrogen Composition HPLC AnalysisASBC FAN MethodPYF+ve and PYF-ve malts were matched in terms of barley variety, harvest year and region of production
17 Fermentation Profiles using our in-house PYF Assay SMA 20 Million Cells 11°P Worts 15°CSuspended Yeast CellsResidual GravityEthanol Yield
18 Impact of PYF Factor(s) on Yeast’s Physiological Characteristics Cell Density and Budding Index
19 Impact of PYF Factor(s) on Yeast’s Physiological Characteristics Viable Cells
20 Impact of PYF factor(s) on fermentation progression Gravity and Ethanol Content
21 Impact of PYF factor(s) on FAN utilisationFree Amino Nitrogen Utilization
22 Impact of PYF Factor(s) on Sugar Uptake Glucose UtilizationFructose Utilization
23 Impact of PYF Factor(s) on Sugar Uptake (2) Maltose UtilizationMaltotriose Utilization
24 ConclusionsYeast strain is a factor in the severity of PYF which is presented in a breweryLager yeasts have different degrees of susceptibility even to the same PYF factor(s)The more flocculent yeast strain SMA exhibited a higher degree of susceptibility than the less flocculent yeast strain W34/70The fermentation performance for a PYF+ve wort could be improved by using a less flocculent yeast which is less sensitive to PYF
25 Conclusions (2): Mini-FV experiments Many markers of fermentation performance and nutrient uptake were ‘normal’ in the PYF+ve fermentationsIn this experiment the PYF factor(s) did not appear to unduly influence nutrient uptake, inhibit yeast growth or cause the death of the cellsPYF+ve sample probably represented the ‘bridging polysaccharide’ type of PYF rather than the ‘antimicrobial peptide’
26 AcknowledgmentsThis research was financed by the UK Home Grown Cereal Authority (HGCA) and the University of NottinghamAdditional thanks to my colleagues in The University of Nottingham Brewing Science Group for their help, support and advice
28 ReferencesAmerican Society of Brewing Chemists. Wort-12 Free Amino Nitrogen (International Method). Methods of Analysis, 8th ed., The Society: St. Paul, MN,Axcell, B., van Nierop, S., and Vundla, W. Malt induced premature yeast flocculation. Tech. Q. Master Brew. Assoc. Am, 37(4), , 2000.Axcell, B.C. Impact of wort composition on flocculation. In: Brewing Yeast Fermentation Performance, Smart. K. (Ed). Blackwell Science, Oxford, , 2003.Jibiki, M., Sasaki, K., Kaganami, N., and Kawatsura, K. (2006). Application of a newly developed method for estimating the premature yeast flocculation potential of malt samples. J. Am. Soc. Brew. Chem., 2006, 64,Koizumi, H. Barley malt polysaccharides inducing premature yeast flocculation and their possible mechanisms. J. Am. Soc. Brew. Chem, 66(3), , 2008.Koizumi, H., Kato, Y., and Ogawa, T. Barley malt polysaccharides inducing premature yeast flocculation and their possible mechanism. J. Am. Soc. Brew. Chem, 67(3), , 2009.Lake, J. C. and Speers, A. R. A discussion on malt-induced premature yeast flocculation. Tech. Q. Master Brew. Assoc. Am., 45(3), , 2008.
29 References (2)Okada, T., Yoshizumi, H., and Terashima, Y. A . Lethal Toxic Substance for Brewing Yeast in Wheat and Barley. Part I. Assay of Toxicity on Various Grains, and Sensitivity of Various Yeast Strains. Agr. Biol. Chem, 34(7), , 1970.Okada, T. and Yoshizumi, H. A Lethal Toxic Substance for Brewing Yeast in Wheat and Barley. Part II. Isolation and Some Properties of Toxic Principle. Agr. Biol. Chem, 34(7), , 1970.Panteloglou, A. G., Box, W. G, Smart, K. A., and Cook, D. J. Optimization of a small-scale fermentation test to predict the premature yeast flocculation potential of malts. J. Inst. Brew, 116(4), , 2010.Quain, D. E., W. G. Box, and E. F. Walton. An inexpensive and simple small-scale laboratory fermenter. Lab Pract , 34(84),1985.Smart, K. A. Optimizing lager fermentations, IBD Symposium. Cork, 2008.Stratford, M. Yeast Flocculation: A new Perspective. Advances in Microbial Physiology, 33, 1-72, 1992.van Nierop, S. N. E., Rautenbach, M., Axcell, B.C., Cantrell, I.C. The impact of microorganisms on barley and malt quality—a review. J. Am. Soc. Brew. Chem, 64, 69–78, 2006.van Nierop, S., Cameron-Clarke, A. and Axcell, B. C. Enzymatic generation of factors from malt responsible for premature yeast flocculation. J. Am. Soc. Brew. Chem, 62, , 2004.Verstrepen, K. J., Derdelinckx, G., Verachtert, H., and Delvaux, F. R. Yeast Flocculation: what Brewers should know. Appl. Microbiol. Biotechnol, 61, , 2003.