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  1. BIO301 Improving Beer Flavour Stability • About the Author • Executive Summary • Background • Paper 1 • Paper 2 • Comparison • Critical Review • Personal Comments • References Kristen Wolter 31250128

  2. Home Background About the Author • 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.

  3. Home 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.

  4. Home 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 (Sicardet 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 cerevisiaeProducing Higher Levels of Sulfur Dioxide and Glutathione to Improve Beer Flavour Stability”. Applied Biochemistry and Biotechnology. 166: 402-413. 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 H2S 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 cerevisiaeto produce a mutant with increased production of GSH and SO2 and a lowered conversion of SO2 to H2S.

  5. Home Paper 1 Cont. Paper 1: Shenet alDevelopment 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: 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. Table of strains used in the experiment.

  6. Home Paper 2 Paper 1 Cont. 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). Results and Conclusions: 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.

  7. Home Paper 2 Cont. Paper 2: Chen et alDevelopment of Saccharomyces cerevisiaeProducing Higher Levels of Sulfur Dioxide and Glutathione to Improve Beer Flavour Stability Aim: To create a mutant strain of S. cerevisiaewhich 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 SO2, GSH andH2S production to determine stability.

  8. Home Comparison Paper 2 Cont. 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. • Results and Conclusions: 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. 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.

  9. Home 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. • 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.

  10. Home 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.

  11. Home 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!

  12. Home References • Chen, Y., Yang, X., Zhang, S., Wang, X., Guo, C., Guo, X and Xiao, D. (2012). “Development of Saccharomyces cerevisiaeProducing 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 sensustrictocomplex”. Competes RendusBiologies. 334: 229-236.

  13. 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. Back • Iodometric Titration: A method of volumetric chemical analysis where the appearance or disappearance of iodine indicates the end point. Back