| Acetaldehyde is the volatile carbonyl compound with the highest content.Acetaldehyde in high concentration will bring unpleasant grass flavor to beer,shorten the shelf life of beer flavor and have adverse affect on human health.The acetaldehyde content in high-grade beer is below 3 mg?L-1.Yeast metabolism is the main source of acetaldehyde in beer,therefore the screening of brewing yeast with low acetaldehyde production is fundamental to control the acetaldehyde content in beer.In this thesis,the method of simultaneous determination of diethylacetal and acetaldehyde in beer was established and the low acetaldehyde production brewing yeast was obtained by ARTP mutation and high-throughput screening.Furthermore,the metabolic regulatory mechanism of acetaldehyde in the industrial brewing yeast was revealed through genome and transcriptome analysis and the key genes closely related to the metabolism of acetaldehyde were found.The main results are as follows:?1?A sensitive headspace gas chromatography method?HS-GC?was established for the simutaneous determination of diethylacetal and acetaldehyde content in beer.The limit of detection for diethylacetal and acetaldehyde analyses was both 0.005 mg·L-1 with relative standard deviation<5.5%.The recoveries of acetaldehyde and diethylacetal were 95-110%and 95-115%,respectively.The average contents of acetaldehyde and diethylacetal in 24commercial beer products were 11.83 and 4.36 mg·L-1,respectively.The Pearson correlation coefficient between diethylacetal and acetaldehyde was the highest?0.963?.The contents of diethylacetal and acetaldehyde reached the peak value after fermentation for 3 days and then decreased to a low value.In normal and forced aging storage,the diethylacetal content decreased and the acetaldehyde content increased gradually over time.When beer samples were stored in normal condition for 6 months,the diethylacetal content decreased by 25.0%and acetaldehyde content increased by 30.0%.This indicated acetaldehyde and ethanol would undergo reversible aldol condensation reaction under acidic condition.Therefore,it is necessary to detect both free-and bound-state acetaldehyde simultaneously during the process of beer fermentation and storage.?2?An optimized brewing yeast strain LAL-8a with lower acetaldehyde production was obtained using atmospheric and room temperature plasma?ARTP?mutagenesis technology and its growth performance was investigated and compared with M14 strain.The lag phase of strain M14 and LAL-8a were both short under 12°P wort.When the wort gravity was increased,the lag phase of strain M14 and LAL-8a were both extended.When the original gravity was 16°P and 20°P,the lag phase of LAL-8a strain was shorter than that of M14strain,which favored industrial production.The production of acetaldehyde in LAL-8a strain was stable in different generations.The acetaldehyde contents of LAL-8a strain in the triangle bottle and EBC tube fermentation were 6.00 mg·L-1 and 2.20 mg·L-1,respectively,which was70.00%and 85.00%lower than that of M14 strain.Meanwhile,the diethylacetal content in fermentation broth of LAL-8a strain was lower than that of M14 strain,which was 80%lower than that of the original strain at the end of the fermentation.In addition,the diacetyl,the ratio of esters to higher alcohols and total acid of LAL-8a strain were also reduced,which was beneficial to the coordination of beer flavor.Therefore,both the growth and fermentation performance of brewing yeast in high gravity wort was improved after ARTP mutagenesis.?3?The transcriptome of brewing yeasts was analyzed using RNA sequencing technology.Compared with the original M14 strain,the yeast will generate more mutagenesis in transcriptional level through ARTP mutation compared to the traditional UV mutation.9and 6 common differential genes were observed between strain D-A-14 and LAL-8a when they were fermented for 2 and 4 days.When fermentation for 6 days,the common differential gene number increased to 97.There was a close relationship between the increase of differential gene number during fermentation process and the final acetaldehyde content in the fermented liquid.There were 18,23 and 26 genes that had transcriptional changes in the glycolytic pathway at 2,4 and 6 days,respectively.These genes played important roles in glycogenesis,carbon metabolism,pyruvate metabolism,amino acid degradation and ethanol degradation,thus affecting the synthesis and reduction of acetaldehyde.In addition,the transcriptome sequencing data was verified by qRT-PCR and the results showed that the transcriptome sequencing results were highly reliable.The fermentation results of strains with gene over-expression indicated that gene ADH2 had a positive regulating effect on the conversion reaction of ethanol to acetaldehyde while its family genes ADH4 and ADH5 were involved in the conversion reaction of acetaldehyde to ethanol to a certain extent,making the acetaldehyde production of the corresponding strain M/adh4 and M/adh5 lower than that of the original strain.The acetaldehyde dehydrogenase genes ALD3 and ALD5 could catalyze the conversion of acetaldehyde to acetic acid and the acetaldehyde production of the corresponding over-expressed strains M/ald3 and M/ald5 was lower than that of the original strain.?4?To explain the regulation mechanism of acetaldehyde metabolism at the genetic level,the whole genome sequencing and repeat sequencing analysis of strain M14 and the mutant brewing yeasts were conducted.The results showed that the genome size of the industrial brewing yeast M14 was 22.84 M with the GC content of 38.98%and 9970 genes were annotated.The strain M14 was a hybrid of Saccharomyces cerevisiae×Saccharomyces uvarum,whose mitochondria were inherited from its parent S.uvarum.9 typical genes wer found in M14 strain.Mauve analysis showed a high degree of consistency between the gene sequence of M14 and its proximal strains.Mummer analysis showed a high degree of colinearity between M14 and Lager beer yeast S.pastorianus Weihenstephan 34/70.The repeat sequencing analysis of strain D-A-14 and LAL-8a was conducted with strain M14 as a reference genome and the number of SNPS were 41991 and 38080,respectively.The main mutation type in the two strains was base conversion,namely T:A>C,G and C:G>T:A.The number of Indel,copy number variation?CNV?and chromosome variation?SV?in the genome of strain D-A-14 and LAL-8a were 4152 and 4175,142 and 142,953 and 542,respectively.The level of acetaldehyde production is mainly related to the SNP mutation of acetoate decarboxylase gene PDC6 and acetaldehyde decarboxylase gene ALD3. |
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