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Premature Yeast Flocculation (PYF) – its causes, nature and significance

Premature Yeast Flocculation (PYF) – its causes, nature and significance. Apostolos G. Panteloglou, Katherine A. Smart and David J. Cook 23 January 2013. Contents.

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Premature Yeast Flocculation (PYF) – its causes, nature and significance

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  1. Premature Yeast Flocculation (PYF) – its causes, nature and significance Apostolos G. Panteloglou, Katherine A. Smart and David J. Cook 23 January 2013

  2. Contents • What is Premature Yeast Flocculation (PYF)? • Development of an assay to predict the PYF status of malts • The importance of yeast strain on the severity of PYF • Investigating the “antimicrobial peptide hypothesis” • Conclusions

  3. What is Premature Yeast Flocculation (PYF)? • Early or abnormally heavy onset of flocculation • low suspended cell counts • incomplete fermentation of sugars to alcohol • Sporadic problem in brewery fermentations • region and harvest conditions dependent • Induced 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 yeast • Requirement to blend • Significant financial and logistical problems The 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/malt 2. The anti-microbial peptide theory 1. The bridging polysaccharide theory Fungi secretes enzymes which degrade husk materials Barley responds to stress of fungal infection with production of anti-microbial peptides (AP) Generates soluble high molecular weight polysaccharides (HMWP) AP survive brewhouse processing and have ‘anti-yeast’ activity due to impairment of membrane function HMWP form bridges between flocculent yeast cells, increasing floc size Axcell et al. (2000) ; Axcell, (2003) ; van Nierop et al., (2004 & 2006)

  6. Predictive tests for PYF • Standard malt analysis is unable to predict PYF • Laboratory-scale fermentation assays are the most widely adopted • Fermentation tests take several days to be completed • Monitor gravity and measure suspended yeast cells • Some rapid and micro-scale tests have been developed

  7. In-house PYF Assay Yeast Propagation Fermentation Wort Preparation Full loop cells ↓ 10 mL YPD ↓ 25°C (1 day) ↓ 100 mL YPD ↓ 25°C (3 days) ↓ Spinning ↓ 50% w/w yeast slurry ↓ Cell counting Barley milling (80 g) ↓ Mashing → 63°C (60 min) 72°C (1°C/min) for 25 min 76°C (1°C/min) for 5 min ↓ Cooling ↓ Filtration → Recycle first 100 mL ↓ Sparging (100 mL mash water at 63°C) ↓ Gravity Adjustment (11°P) ↓ Autoclave (30 min at 121°C) ↓ Storage Yeast cells (20 million cells/mL) ↓ 200 mL 11°P wort (+ 4% w/v glucose) ↓ Aeration ↓ 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.

  8. Typical test results for PYF+ve & PYF-ve fermentations Residual Gravity 93 h Post-Pitching 15°C with W34/70 Yeast Strain 69 h statistical significant differences (P < 0.0001)

  9. The effect of re-pitching PYF-ve wort with yeast from a previous PYF+ve fermentation 15°C with W34/70 Yeast Strain Residual Gravity 93 h Post-Pitching Panteloglou, A.G., Smart, K.A., and Cook, D.J. Malt-induced premature yeast flocculation: current perspectives. Industrial Microbiology & Biotechnology, 39(6), 813-822, 2012.

  10. Lager strains differ in susceptibility to PYF (1) SMA Yeast Strain W34/70 Yeast Strain 40 h significant 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) Ethanol Yield Fermentation Profiles Residual Gravity Panteloglou, 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 Yeast Panteloglou, 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.F. 1985. Panteloglou, 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 media • Samples: Industry sourced PYF+ve and PYF-ve malts • - Matched in terms of barley variety, harvest year and region of production • 33 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 Index • Gravity and Ethanol • Free Amino Nitrogen • Fermentable Sugars (sucrose, fructose, glucose, maltose, maltotriose)

  16. Worts Fermentable Sugars & Free Amino Nitrogen Composition HPLC Analysis ASBC FAN Method PYF+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°C Residual Gravity Suspended Yeast Cells Ethanol 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 utilisation Free Amino Nitrogen Utilization

  22. Impact of PYF Factor(s) on Sugar Uptake Glucose Utilization Fructose Utilization

  23. Impact of PYF Factor(s) on Sugar Uptake (2) Maltose Utilization Maltotriose Utilization

  24. Conclusions • Yeast strain is a factor in the severity of PYF which is presented in a brewery • Lager 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 W34/70 • The 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 fermentations • In this experiment the PYF factor(s) did not appear to unduly influence nutrient uptake, inhibit yeast growth or cause the death of the cells • PYF+ve sample probably represented the ‘bridging polysaccharide’ type of PYF rather than the ‘antimicrobial peptide’

  26. Acknowledgments This research was financed by the UK Home Grown Cereal Authority (HGCA) and the University of Nottingham Additional thanks to my colleagues in The University of Nottingham Brewing Science Group for their help, support and advice

  27. Thank You For Your Attention! Any Questions?

  28. References • American Society of Brewing Chemists. Wort-12 Free Amino Nitrogen (International Method). Methods of Analysis, 8th ed., The Society: St. Paul, MN, 1992. . • Axcell, B., van Nierop, S., and Vundla, W. Malt induced premature yeast flocculation. Tech. Q. Master Brew. Assoc. Am, 37(4), 501-504, 2000. • Axcell, B.C. Impact of wort composition on flocculation. In: Brewing Yeast Fermentation Performance, Smart. K. (Ed). Blackwell Science, Oxford, 120-128, 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, 79-85. • Koizumi, H. Barley malt polysaccharides inducing premature yeast flocculation and their possible mechanisms. J. Am. Soc. Brew.Chem, 66(3), 137-142, 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), 129-134, 2009. • 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), 129-134, 2009. • Lake, J. C. and Speers, A. R. A discussion on malt-induced premature yeast flocculation. Tech. Q. Master Brew. Assoc. Am., 45(3), 253-262, 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), 1084-1088, 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), 1089-1094, 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), 413-420, 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, 108-116, 2004. • Verstrepen, K. J., Derdelinckx, G., Verachtert, H., and Delvaux, F. R. Yeast Flocculation: what Brewers should know. Appl. Microbiol. Biotechnol, 61, 197-205, 2003.

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