Presentation on theme: "Apostolos G. Panteloglou, Katherine A. Smart and David J. Cook 23 January 2013 Premature Yeast Flocculation (PYF) – its causes, nature and significance."— Presentation transcript:
Apostolos G. Panteloglou, Katherine A. Smart and David J. Cook 23 January 2013 Premature Yeast Flocculation (PYF) – its causes, nature and significance
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
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) What is Premature Yeast Flocculation (PYF)?
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
Two main theories - each originate with fungal infection of barley/malt Axcell et al. (2000) ; Axcell, (2003) ; van Nierop et al., (2004 & 2006) What causes PYF? Fungi secretes enzymes which degrade husk materials Generates soluble high molecular weight polysaccharides (HMWP) HMWP form bridges between flocculent yeast cells, increasing floc size 1. The bridging polysaccharide theory Barley 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 function 2. The anti-microbial peptide theory
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
In-house PYF Assay 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 Wort Preparation Yeast cells (20 million cells/mL) ↓ 200 mL 11°P wort (+ 4% w/v glucose) ↓ Aeration ↓ Yeast Propagation Fermentation 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 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.
15°C with W34/70 Yeast Strain 69 h statistical significant differences (P < ) Residual Gravity 93 h Post-Pitching
The effect of re-pitching PYF-ve wort with yeast from a previous PYF+ve fermentation Panteloglou, A.G., Smart, K.A., and Cook, D.J. Malt-induced premature yeast flocculation: current perspectives. Industrial Microbiology & Biotechnology, 39(6), , °C with W34/70 Yeast StrainResidual Gravity 93 h Post-Pitching
W34/70 Yeast StrainSMA Yeast Strain 40 h significant differences (P < 0.01) Lager strains differ in susceptibility to PYF (1) 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.
Lager strains differ in susceptibility to PYF (2) Fermentation Profiles Residual GravityEthanol Yield 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.
Lager strains differ in susceptibility to PYF (3) 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. Fermentation Profiles using different PYF+ve Worts and the ‘Industrial’ Lager Yeast
INVESTIGATING THE “ANTIMICROBIAL PEPTIDE HYPOTHESIS” ‘MINI-FV’ EXPERIMENTS Quain, D.E., Box, W.G., and Walton, E.F 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.
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 ‘Mini-FV’ experiments (100 mL)
Cell Density and Budding Index Gravity and Ethanol Free Amino Nitrogen Fermentable Sugars (sucrose, fructose, glucose, maltose, maltotriose) Measurements taken during the mini FV experiments
Worts Fermentable Sugars & Free Amino Nitrogen Composition PYF+ve and PYF-ve malts were matched in terms of barley variety, harvest year and region of production HPLC AnalysisASBC FAN Method
Fermentation Profiles using our in-house PYF Assay SMA 20 Million Cells 11°P Worts 15°C Residual Gravity Suspended Yeast Cells Ethanol Yield
Impact of PYF Factor(s) on Yeast’s Physiological Characteristics Cell Density and Budding Index
Impact of PYF Factor(s) on Yeast’s Physiological Characteristics Viable Cells
Impact of PYF factor(s) on fermentation progression Gravity and Ethanol Content
Impact of PYF factor(s) on FAN utilisation Free Amino Nitrogen Utilization
Impact of PYF Factor(s) on Sugar Uptake Fructose UtilizationGlucose Utilization
Maltose UtilizationMaltotriose Utilization Impact of PYF Factor(s) on Sugar Uptake (2)
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
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’
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 Acknowledgments
Thank You For Your Attention! Any Questions?
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