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 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
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
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 Wort Preparation Yeast Propagation Fermentation
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
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
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
15°C with W34/70 Yeast Strain
Residual Gravity 93 h Post-Pitching
69 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 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), , 2012.

10. Lager strains differ in susceptibility to PYF (1)
W34/70 Yeast Strain
SMA 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)
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.

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
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
Suspended Yeast Cells
Residual Gravity
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,
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)
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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.
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Quain, D. E., W. G. Box, and E. F. Walton. An inexpensive and simple small-scale laboratory fermenter. Lab Pract , 34(84),1985.
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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.
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