03-31250128 Improving Beer Flavour Stability
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Transcript 03-31250128 Improving Beer Flavour Stability
BIO301
Improving Beer Flavour
Stability
About the Author
Executive Summary
Background
Paper 1
Paper 2
Comparison
Critical Review
Personal Comments
References
Kristen Wolter
31250128
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About the Author
Background
My name is Kristen Wolter and I am currently in my third and final year of
study, majoring in Biotechnology and Molecular Biology with a minor in
Forensic Biology.
This semester I am studying Biochemistry 2 and Industrial Bioprocessing and
Bioremediation.
I chose to do this review on improving the flavour of beer as our course
does not cover a lot of detail about brewing and the two papers I have
selected also have a genetics aspect to them as well which I find interesting.
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Background
Executive Summary
Comparison of two papers exploring the effects of altered characteristics of
yeast on the stability of beer flavour.
Both papers utilise UV mutation to create mutant strains of Saccharomyces
cerevisiae, a common yeast used in beer brewing.
Both papers achieved their aim and both mutant strains were proved to be
genetically stable allowing commercial use.
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Paper 1
Background to Papers
The flavour of beer often has a lot to do with the fermentation conditions used. For example, the production of ale is generally
done by top-fermentation at 15-200C, while the production of lager is by bottom fermentation at 5-140C (Sicard et al, 2011). As
the following two articles demonstrate, by altering by-product formation of the yeasts during fermentation the flavour stability
of beer can be improved.
Paper 1:
Chen, Y., Yang, X., Zhang, S., Wang, X., Guo, C., Guo, X and Xiao, D. (2012). “Development of Saccharomyces cerevisiae Producing
Higher Levels of Sulfur Dioxide and Glutathione to Improve Beer Flavour Stability”. Applied Biochemistry and Biotechnology. 166: 402413.
Background: The level of acetaldehyde concentration in beer is a large concern in the beer industry as too much creates a pungent
aroma whilst an appropriate amount creates a desirable green apple aroma. The amount of acetaldehyde also affects beer staling,
and thus reducing the amount of acetaldehyde should improve the shelf life of beer. In this experiment this was achieved by
modifying a strain of industrial brewers yeast with 4-Methylpyrazole (4-MP) which is a competitive inhibitor of alcohol
dehydrogenase 2 which is responsible for the conversion of ethanol back to acetaldehyde.
Paper 2:
Shen, N., Wang, J., Yin, H., Liu, C., Li, Y and Li, Q. (2013). “Development of Industrial Brewing Yeast with Low Acetaldehyde Production
and Improved Flavour Stability”. Applied Biochemistry and Biotechnology. 169: 1016-1025.
Background: Sulphur compounds such as SO2, H2S and glutathione (GSH) play a role in the stability of beer flavour. While SO2 and
GSH both have antiaging effects, H2S is an unwanted product due to its unpleasant smell. Thus, by increasing levels of antiaging
compounds the stability of the beer flavour should increase, while decreasing the amount of H 2S will assist the aroma of the beer as
not only will there be reduced H2S but also a reduced number of subsequently formed compounds with undesirable components. In
this paper, this was achieved by performing mutagenesis on a strain of Saccharomyces cerevisiae to produce a mutant with increased
production of GSH and SO2 and a lowered conversion of SO2 to H2S.
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Paper 1 Cont.
Paper 1: Shen et al
Development of Industrial Brewing Yeast with Low Acetaldehyde Production and Improved Flavour
Stability
Aim: To create a mutant yeast strain which produces lowered amounts of acetaldehyde to improve flavour stability.
Methods:
Table of strains used in the
experiment.
Mutagenesis: Mutagenesis of yeast strains was completed using ultraviolet mutation with 4-MP as a selection marker. Mutant strains
were chosen using Schiff reagent (which turns purple in the presence of acetaldehyde).
Fermentation and Pilot-Scale Brewing: Yeast cultures were inoculated in wort culture for 48 hours and then transferred to a shaking
flask for a further 48 hours. The cultures were then transferred to a European Brewery Convention tube for pilot-scale brewing. The
main fermentation was carried out for 6 days followed by a post fermentation of 7 days.
Genetic Stability: The mutant strain was bred for 15 generations with the 1st, 4th, 12th and 15th generation strains processed for
conical flask fermentation to determine genetic stability of the mutant strain.
Analytical Methods: The acetaldehyde concentration from the final beer was measure using headspace gas chromatography. The
preservative qualities of the beer were determined by measuring the thiobarbituric acid (TBA) value and the resistant staling value
(RSV). The ethanol concentration was measured using the Alcolyzer Plus Beer machine WBA-505B.
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Paper 2
Paper 1 Cont.
Results and Conclusions:
Acetaldehyde concentration was shown to be lower in the mutant
strain (MA12) compared to the wild type (MI4) over all days of
fermentation. Due to a decreased level of acetaldehyde, the MA12
strain also showed higher ethanol quantities than the MI4. Also, all
generations of the mutant strain were successful in genetic stability
tests indicating safe use in the food and beverage industry. At the
end of fermentation several parameters associated with beer quality
were tested as indicated below, with one of the main off-flavour
compounds (diacetyl) showing a decrease in the mutant strain.
Importantly, the lowered acetaldehyde levels led to a decrease in the
TBA value (which reflects the amount of staling compounds) and
thus an increase in the RSV value (a higher value indicates longer
flavour freshness). Furthermore, the mutant strain displayed similar
results to the wild type in parameters associated with aroma (ethyl
acetate, isoamyl acetate, N-propanol, isobutanol and isoamylol).
Measurement of acetaldehyde production of the two strains
over the fermentation period.
It was found that not only did the mutant strain
produce less acetaldehyde, thus increasing flavour
stability, but the UV mutagenesis did not affect any
other brewing properties, nor was the strain
genetically unstable. Thus, the new strain is a
promising application prospect.
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Paper 2: Chen et al
Paper 2 Cont.
Development of Saccharomyces cerevisiae Producing Higher Levels of Sulfur Dioxide and Glutathione to Improve
Beer Flavour Stability
Aim: To create a mutant strain of S. cerevisiae which produces higher content of GSH and SO2 to improve the stability of beer flavour.
Methods:
The strains used in the experiment were and industrial brewing yeast strain S5 (Saccharomyces cerevisiae ), M8 (mutant of S5) and
MV16 (mutant of M8).
Mutagenesis: Mutagenesis of yeast strains were performed via ultraviolet mutation using lead acetate and different sulphur sources
plates to determine mutagenic candidates.
Analytical Methods: SO2 concentrations were measured using iodometric titration, while H2S was measured spectrophotometrically.
GSH content was determined using 2-nitrobenzoic acid. Preservative qualities of the beer were determined by measuring the TBA
and RSV values. Alcohol concentration was measured using a beer analyzer.
Beer Fermentation: Seed culture was incubated in 50ml of wort for 24 hours. Following this flask culture experiments were
performed containing medium and seed culture for eight days.
Genetic Stability: The mutant strain was bred for 20 generations after which generations 1, 5, 10, 15 and 20 were processed for SO 2,
GSH and H2S production to determine stability.
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Results and Conclusions:
Paper 2 Cont.
Comparison
The strain M8 was shown to have an increased SO2 production of 30.8% and a decreased production of H2S of 82.1% and thus was
chosen for further mutagenesis by UV to produce MV16 which showed increased GSH.
MV16 showed and increase over the wild type
for both SO2 and GSH and also showed a
decrease in H2S. Also, the MV16 showed no
adverse affects to the mutagenesis in regards to
fermentation ability. An important aspect of this
was the levels of acetaldehyde did not increase
and the other aroma affecting compounds also
stayed similar to the wild type as shown below.
Also, genetic stability tests were carried out and
the mutant strains were proven to be genetically
stable.
To determine the degree of beer staling the TBA and RSV
values were measured. Both M8 and MV16 displayed a
lower TBA value compared to the wild type and a higher
RSV value indicating an increase in beer flavour stability in
the mutant strains. Thus, by increasing the antiaging
compounds the beer flavour stability did increase. As
genetic stability tests were passed, the mutant would be
useable for the beer industry.
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Critical Review
Comparison
Both papers essentially attempt to do the same thing: improve beer flavour stability. They differ however, in the method of
which they attempt to achieve this. Or, more simply put, they alter different characteristics of the yeast.
Paper 1
Paper 2
Aim
Paper 1 focuses on acetaldehyde
production to improve beer flavour
stability.
Paper 2 focuses on sulphur
containing compounds to improve
beer flavour stability.
Yeast
Industrial brewers yeast.
Industrial brewers yeast.
Mutagenesis
UV mutation using 4-MP as a
selection marker.
UV mutation using lead acetate and
different sulphur sources as
selection markers.
Fermentation
Inoculation in wort medium followed
by pilot-scale brewing.
Inoculation in wort medium.
Analytical Methods
Gas Chromatography, TBA and RSV
measurements, Alcolyzer Beer Plus
machine, GS/MS
Iodometric titration, TBA and RSV
measurements, beer analyzer, GC
Both papers reported results not only their specific component tested (i.e. acetaldehyde), but also provided results of other
aroma compounds involved in beer (e.g. ethyl acetate). Thus, it could be shown whether the mutagenesis affected any other
components in the flavour of beer which may make it unsuitable for commercial use. Likewise, both papers provided genetic
stability testing results, which indicated that both mutant strains are useable in the beer industry.
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Personal Comments
Critical Review
Both papers set out to create a mutant strain of yeast which contained an altered property which could be used to increase the
beer flavour stability. In both cases this was achieved, and both papers demonstrated that their mutant strains were genetically
stable and thus safe for commercial use. Furthermore, both papers indicated that the mutagenesis had no adverse affects on
any other fermenting properties of the yeast or the production of compounds associated with the aroma of beer. However,
neither paper (with the exception of paper 1 which explains diacetyl) explains whether we want a higher or a lower
concentration of these compounds. So while it is great that they show that there is no major difference between their mutant
strains and the wild type, the information is not as helpful as it could be as we don’t know how much of these compounds we
would like in beer.
Overall, the presentation of Paper 1 is good. It presents diagrams which are easy to understand and represent the results of
their data that are relevant to the papers aim. Furthermore, each section of the paper is succinctly written but still provides
enough detail for someone to replicate the experiment. However, the paper does not explicitly say that the strain of yeast is
Saccharomyces cerevisiae, rather you are meant to assume that this is the strain used by a diagram of acetaldehyde metabolism
in this yeast.
The presentation of Paper 2 is perhaps not as good as Paper 1. With the experiment performing two mutagenesis’ to get to
their desired strain of yeast there are a lot of diagrams presented which are not as relevant to the overall aim of the paper. As
such there is a bit of an information overload of aspects of the experiment which could have been left out of the report, or
perhaps placed in an appendix. Although, each section of the paper is well explained and the experiment would be able to be
replicated.
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References
Personal Comments
Although I don’t personally drink beer, with the size of the industry it is not surprising that there is research into improving
aspects of beer flavour and stability. From the view point of the industry, any leg up companies can gain by improving their beer
flavour and shelf life, will ultimately be a huge economical gain. So although the taste of beer doesn’t have any solutions to
environmental problems such as ozone depletion which many people would think is far more important to be spending money
on research on, the demand of beer is not going away any time soon. Thus, it could be argued that we do need this kind of
research. And to all those who do drink beer, I am sure that they would not be complaining about any research to improve the
taste!
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References
Chen, Y., Yang, X., Zhang, S., Wang, X., Guo, C., Guo, X and Xiao, D. (2012).
“Development of Saccharomyces cerevisiae Producing Higher Levels of
Sulfur Dioxide and Glutathione to Improve Beer Flavour Stability”. Applied
Biochemistry and Biotechnology. 166: 402-413.
Shen, N., Wang, J., Yin, H., Liu, C., Li, Y and Li, Q. (2013). “Development of
Industrial Brewing Yeast with Low Acetaldehyde Production and Improved
Flavour Stability”. Applied Biochemistry and Biotechnology. 169: 1016-1025.
Sicard, D and Legras, J. (2011). “Bread, beer and wine: Yeast domestication
in the Saccharomyces sensu stricto complex”. Competes Rendus Biologies.
334: 229-236.
Glossary
Wort: the liquid extracted from the mashing process during the brewing of beer or whisky. It
contains the sugars that are fermented by yeast to produce alcohol.
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Iodometric Titration: A method of volumetric chemical analysis where the appearance or
disappearance of iodine indicates the end point.
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