High Throughput Screening Of Industrial Brewing Yeast With Low Acetaldehyde Production

Aldehydes are important off-flavor compounds in beer,among which acetaldehyde is the highest concentrations substance of volatile aldehydes.A pungent smell of grass is brought by high levels of acetaldehyde and the quality of beer has declined dramatically.For mature beer with high quality,the acetaldehyde concentrations usually range from 2 to 10 mg?L^-1.Acetaldehyde is produced by metabolism of yeast during the consuming with wort.Therefore,it is urgent to obtain a brewing strain with the lower concentration of acetaldehyde.In this study,industrial Lager brewing yeast was modified by traditional mutagenic breeding,adaptive evolution and high throughput screening which combined with traditional breeding methods and directed evolution strategies to reduce the production of acetaldehyde of the mutant strain during beer brewing.The main contents are as follows:Traditional breeding methods for industrial brewing yeast with low acetaldehyde production.In this study,the Lager industrial brewing yeast was modified by atmospheric and room temperature plasma(ARTP)and Co⁶⁰?ray mutagenesis,two simple and practicable techniques,to construct the mutant library.Among Schiff's reagent,acetaldehyde and disulfiram,the latter two reagents were selected as screening inhibitors for industrial brewing yeast with low acetaldehyde production.The resistant medium plate and domestication culture medium were established and optimized for screening industrial brewing yeast with low acetaldehyde production.Furthermore,the mutant strains were coated on YPD plate with acetaldehyde at the concentration of 2.8 g?L^-1and basic carbon source medium plate with ethanol(10 g?L^-1)as the sole carbon source and containing disulfiram(0.3 mg?L^-1)to screen the dominant strains.Subsequently,the screened dominant strains were inoculated into YPD medium with acetaldehyde at the concentration of 3.2 g?L^-1and basic carbon source liquid medium with ethanol(10 g?L^-1)and disulfiram(2.5 mg?L^-1)for long-term domestication.Finally,166 strains were obtained following the establishment of the screening methods of"multiple rounds of superimposed ARTP mutation-plate screening-liquid domestication"and"combination of single Co⁶⁰?mutagenesis and four rounds of liquid domestication",associated with continuous increase of the concentration of inhibitors in the screening plate and domestication medium.Our study for the first time constructed a high-throughput method for the detection of acetaldehyde content in beer.A comparison was carried out focusing on the chromogenic effect of various chromogenic reagents combined with acetaldehyde.Corresponding results revealed that the detection limit and linear range of Schiff's reagent and sodium nitroferrocyanide reagent could not meet the requirement of acetaldehyde content in beer.Accordingly,a high-throughput method for the determination of the content of acetaldehyde in beer was established and optimized based on spectrophotometry for the first time with3-methyl-2-benzothiazolinone hydrozone(MBTH)as the chromogenic reagent.The proposed method is characterized by low detection limit and wide linear range.In the subsequently re-screening experiment,the mutant strains obtained in the preliminary screening were cultured in the basic carbon source medium containing 5 g?L^-1ethanol and the acetaldehyde solution containing 100 mg?L^-1ethanol,respectively.Afterwards,the absorbance value was detected at the wavelength of 610 nm to detect the acetaldehyde production and metabolism of the mutant strain within a certain period of time.Eventually,six re-screened strains with relatively low acetaldehyde synthesis and relatively high metabolism were obtained for the validation of beer fermentation simulation at a shake flask level.The acetaldehyde production,growth capability,genetic stability and fermentability of the mutant strain were determined by the simulated fermentation of beer through the flask.The results showed that the acetaldehyde production of all the mutant strains(except Lager-4)compared with the original strain were decreased by more than 55%.Thereinto,the acetaldehyde production of Lager-5 finished beer was 15.74 mg?L^-1,lower than the original strain 63.42%.Besides,there were no obvious differences in the main aroma components between the mutant strain and the original strain.The ratio of higher alcohols to esters of the mutant strain was significantly reduced,which is good for the aroma of beer.Meanwhile,the mutant strain had good growth capability and grew faster than the original strain.In addition to,the mutant strain had a stable acetaldehyde production after continuous fermentation for 5generations.As generalized above-mentioned,the Lager-5 could basically sufficed the requirements of the fermentation with some industrial application.The results of this study will provide a theoretical basis for further developing industrial brewer's yeast and industrial production of high-quality beer.