Method with function of reducing accumulation of ethyl carbamate in rice wine fermentation

A technology of urethane and amino acid, applied in the field of microbial genetics and molecular biology, can solve problems such as accumulation of high-concentration urea

Inactive Publication Date: 2016-01-27
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Regulation of the NCR effect leads to the accumu

Method used

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  • Method with function of reducing accumulation of ethyl carbamate in rice wine fermentation
  • Method with function of reducing accumulation of ethyl carbamate in rice wine fermentation
  • Method with function of reducing accumulation of ethyl carbamate in rice wine fermentation

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Embodiment 1

[0034] Construction of Gln3p and Gat1p nuclear localization regulatory sequence deletion and truncated expression vector: primers were designed using the genome of Saccharomyces cerevisiae CEN. site; the reverse primer adds a SacI restriction endonuclease site) (see Table 1), PCR amplification to obtain truncated GLN3 and GAT1. The amplified gene fragment and the pYX212 plasmid were digested with SalI and SacI restriction enzymes respectively, and then ligated with DNA ligase to obtain pYX212-Gln3p 1-653 and pYX212-Gat1p 1-375 . The resulting plasmid was transformed into E.coliJM109 strain and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. It is verified that the plasmid with the correct gene sequence is used for the next step of the experimental operation. Construction of complete expression vectors of Gln3p and Gat1p (experimental control): primers were designed to amplify complete GLN3 and GAT1 (see Table 1). Construct pYX212-Gln3p and pYX212-Gat1p in ...

Embodiment 2

[0043] Example 2: Application

[0044] Referring to the rice wine production process of Zhejiang Guyue Longshan Company, the fermentation process of rice wine was simulated in the laboratory. The specific steps are as follows: take 100g of rice, soak it at room temperature for 2-3 days; after regular pressure cooking, add 17.4g of wheat koji (13.5g of raw wheat koji, 3.4g of cooked wheat koji), 170g of water, and the activated Saccharomyces cerevisiae The bacterial strain was inoculated into the culture medium according to the inoculation amount of 10% (v / v); the shake flask was added with a fermentation plug and left to ferment at 30°C; weighed every day, when the CO 2 Stop the pre-fermentation when the weight loss is less than 2g; place the shake flask at 15°C to continue the static fermentation, and finish the post-fermentation after 15 days, and take the fermentation liquid to test the content of urethane, urea, amino acids and flavor substances.

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Abstract

The invention belongs to the field of microbial genetics and molecular biology and discloses a method with a function of reducing accumulation of ethyl carbamate in rice wine fermentation. Genetically engineered bacteria modified by nitrogen catabolite repression regulation factors are adopted for rice wine fermentation, and engineered saccharomyces cerevisiae eliminates nuclear localization regulatory sequences of the regulation factors Gln3p and Gat1p and mutates phosphorylation sites of the nuclear localization sequences. In a simulated rice wine fermentation system, compared with rice wine with wild strains for fermentation, rice wine fermented with the genetically engineered bacteria has the advantages that contents of carbamide and ethyl carbamate in the rice wine are decreased by 63% and 72% respectively, the content of the ethyl carbamate in the rice wine is decreased to about 55.53 microgram/L, and contents of major nutrient substances and characteristic flavor substances are less in difference. Therefore, the method has a huge potential of application to rice wine production.

Description

technical field [0001] The invention relates to a method capable of reducing the accumulation of ethyl carbamate in rice wine fermentation, and belongs to the fields of microbial genetics and molecular biology. Background technique [0002] Urethane is considered a potential carcinogen to humans, and was classified as a Class 2B carcinogenic compound by the International Agency for Research on Cancer (IARC) in 1974. Since then, researchers have discovered that urethane can also directly induce human liver cancer, which further prompted IARC to raise the carcinogenic level of urethane from 2B to 2A (same level as formaldehyde) in 2007. The ethyl carbamate in rice wine has become an important hidden danger that endangers the health of consumers and affects its market competitiveness. [0003] In sake and rice wine, urea produced by the metabolism of Saccharomyces cerevisiae is the most important precursor of ethyl carbamate. During the fermentation process, urea in yeast cel...

Claims

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Application Information

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IPC IPC(8): C12G3/02C12N1/19C12N15/81C12R1/865
Inventor 周景文陈坚堵国成吕永坤赵鑫锐
Owner JIANGNAN UNIV
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