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?

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