apostolos g. panteloglou, katherine a. smart and david j. cook 23 january 2013 premature yeast...
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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
Yeast
-acetolactate
diacetyl
Green Beer
Non-enzymic oxidative decarboxylation
diacetyl
acetoin
O
O
O
OH
OH
OH
O
O
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 PYFPredictive 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 AssayIn-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 < 0.0001)
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.
15°C with W34/70 Yeast Strain Residual Gravity 93 h Post-Pitching
W34/70 Yeast Strain SMA Yeast Strain
40 hsignificant 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 Gravity Ethanol 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. 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.
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 Analysis ASBC FAN Method
Fermentation Profiles using our in-house PYF Assay
SMA 20 Million Cells 11°P Worts 15°C
Residual GravitySuspended 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 Utilization Maltotriose 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?
References
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