Petite Mutations and their Impact of Beer Flavours. Maria Josey and Alex Speers ICBD, Heriot Watt University IBD Asia Pacific Meeting March 2016

Transcription

1 Petite Mutations and their Impact of Beer Flavours Maria Josey and Alex Speers ICBD, Heriot Watt University IBD Asia Pacific Meeting March 2016

2 Table of Contents

3 What Are They? No or reduced mitochondrial function Spontaneous genetic mutation Also called respiratory deficient (or RD) cells Figure 1. TTC Overlay Technique with Petite Mutations Noted with an Arrow

4 Petites in the Industry Exist in frequencies of: 0.1% to 0.5% 1% to 4% Strain Dependent Higher if yeast is abused

5 Previous Research: The RD mutant produced considerably higher amounts of isobutanol and isoamyl alcohol Ernandes, 1993 Isoamyl alcohol has a pear drop flavour Isobutanol has a solvent-like aroma

6 Method for Fermentation (ASBC Yeast-14) End of Fermentation Flavour Analysis Ethidium bromide to produce 3.66, 5.10, 8.67, and 10.77% RD s measured by TTC Overlay method Throughout Fermentation Density and Absorbance Measurements

7 Mini-fermentation Assay Originated for flocculation determination Use 12.6 o P wort plus 4% glucose & 3 reps, Turbidity/density measurements taken 10 x 21 o C over 75 h, Model sugar consumption and yeast in suspension, Standard method ASBC Yeast-14 7

8 Absorbance at 600nm Mini-fermentation Assay Tilted Gaussian fit [1] Key to assay is the analysis of both change in o P/SG and A 600 These non-linear curves A P σ F(t)= Rt + Ae - 1 ætè ç 2 2 ö ø can be fit with Excel and statistically compared with Prism µ A Amplitude µ Mean σ Standard Deviation R Rotation (slope term) Height of peak, P = Rt+A Time (hr)

9 Absorbance (600nm) Yeast in Suspension Trends For All Four Fermentations Absorbance (600nm) % % 8.39% 10.77% Time (hr) Figure 2. Yeast in suspension trends with fermentations containing varying levels of petites No significant (p>0.05) difference for yeast in suspension characteristics as the petite mutations present in the fermentation increased

10 Method for Fermentation (ASBC Yeast-14) End of Fermentation Flavour Analysis Throughout Fermentation Density and Absorbance Measurements

11 Flavours Analysed Significant means the flavour compounds present in the beer changed as the percentage of petites changed No significant difference in all higher alcohols analysed and acetone Table 1. Results showing if there was a significant change in flavour compound levels post beer fermentation as the percentage of petites present increased up to 11% Flavour Compound Acetaldehyde Butanedione Ethyl acetate Isobutyl acetate Ethyl butyrate Pentanedione Ethyl Octanoate Iso amyl acetate Ethyl hexanoate 3-Methyl butanol 2-Methyl butanol Propan-1-ol Isobutanol Acetone Significant (p>0.05) No No No No No

12 Acetaldehyde (mg/l) Acetaldehyde Levels Post Fermentation R 2 = p> Linear increase in acetaldehyde level post fermentation with increasing levels of petite mutations Same trends found with other eight flavour compounds % Petite Mutations Figure 4. Acetaldehyde levels in the beer post fermentation as the percentage of mutated cells increased in the population

13 Flavours Analysed Flavours outlined showed a significant change in flavour levels as the petite mutations in the pitching yeast changed. Table 1. Results showing if there was a significant change in flavour compound levels post beer fermentation as the percentage of petites present increased up to 11% Flavour Compound Acetaldehyde Butanedione Ethyl acetate Isobutyl acetate Ethyl butyrate Pentanedione Ethyl Octanoate Iso amyl acetate Ethyl hexanoate 3-Methyl butanol 2-Methyl butanol Propan-1-ol Isobutanol Acetone Significant (p>0.05) No No No No No

14 Does increase in flavour compounds make noticeable differences? Acetaldehyde Isoamyl acetate Flavour Threshold 5mg/L Flavour Threshold 1.1mg/L From experimental values, a 10% increase in petites would result in a 1.44mg/L increase in acetaldehyde 10% increase in petites would result in 0.58mg/L increase in isoamyl acetate

15 Does increase in flavour compounds make noticeable differences? Ethyl Acetate Flavour threshold - 5mg/L Butanedione Flavour threshold 0.01mg/L With 10% increase in petites, 5.6 mg/l increase of ethyl acetate 10% increase resulted in 0.05mg/L increase in butanedione Possible Index Compounds?

16 Ethyl octanoate (mg/l) Conclusions from Petite Mutation Experiment At low levels of petites, flocculation characteristics didn t change Nine of the flavour compounds analysed increased with increasing petite mutations in the population % Petite Mutations

17 Conclusions continued Higher alcohols or acetone did not change with increasing levels of petites Based on results, it s likely ethyl acetate and butanedione will be the first noticeable flavour changes in beer if a high number of petites are present

18 Thank you for listening, Questions? Acknowledgements IBD Ph.D. Studentship M Josey Alltech ICBD donation

19 References American Society of Brewing Chemists. Methods of Analysis, 2012, 12 th ed, Yeast-14 Miniature Fermentation Assay. The Society, St. Paul, MN. Ernandes, J.R., et al., Respiratory Deficiency in Brewing Yeast Strains Effects on Fermentation, Flocculation, and Beer Flavor Components. Journal of the American Society of Brewing Chemists, (1): p Lawrence, S.J., et al., The Relationship Between Yeast Cell Age, Fermenter Cone Environment, and Petite Mutant Formation in Lager Fermentations. Journal of the American Society of Brewing Chemists, (2): p Morrison, K.B. and A. Suggett. Yeast handling, petite mutants, and lager flavour. in European Brewery Convention London, UK: IRL Press Limited. Pires, E.J., et al., Yeast: the soul of beer's aroma - a review of flavour-active esters and higher alcohols produced by the brewing yeast. Applied Microbiology and Biotechnology, : p Quilter, M.G., et al., The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae. Journal of the Institute of Brewing, (1): p Slonimski, P.P., G. Perrodin, and J.H. Croft, Ethidium bromide induced mutation of yeast mitochondria: Complete transformation of cells into respiratory deficient non-chromosomal petites. Biochemical and Biophysical Research Communications, (3): p

20 Propan-1-ol (mg/l) Propan-1-ol Levels Post Fermentation Increasing levels of petite mutations showed no significant (p>0.05) in propan-1-ol Same trends found with other higher alcohols and acetone % Petites 5% Petites 8% Petites 11% Petites Figure 3. Propan-1-ol (mg/l) in beer fermented with varying levels of petite mutated yeasts

21 Iso amyl acetate (mg/l) Ethyl hexanoate (mg/l) Ethyl octanoate (mg/l) Flavour compounds present in beer increase as the percentage of mutated cells increase R 2 =0.844 p> R 2 = p> R 2 = p> % Petite Mutations % Petite Mutations % Petite Mutations

22 Higher Alcohol Formation Fig. 1 Taken from Pires, Teixeira et al The Ehrlich pathway and the main genes involved in the synthesis of enzymes catalyzing each reaction. The reversible transamination reaction uses different BAT-encrypted enzymes while BAT2 catalyses the transfer of the amino group from the amino acid to α-ketoglutarate (AKG), BAT1 is usually required on the reverse transamination for amino acid biosynthesis (Pires, Teixeira et al. 2014)

23 Acetate Esters and MCFA Ethyl Ester Formation Taken from Pires, Teixeira et al Fig. 3 A scheme of the chemical reactions involving the biosynthesis of acetate esters (a) and medium-chain fatty acid ethyl esters (b). The main genes involved in each reaction are presented above the reaction arrows (Pires, Teixeira et al. 2014)