Low-yield high-grade alcohol high-yield ethyl lactate saccharomyces cerevisiae strain and construction method thereof

A strain of Saccharomyces cerevisiae and the technology of ethyl lactate, which is applied in the field of bioengineering, can solve the problems of low ability to synthesize ethyl lactate and high content of higher alcohols, and achieve the effect of overcoming uncoordinated flavors, excellent flavors, and broad market prospects

Inactive Publication Date: 2016-09-28
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem that Saccharomyces cerevisiae has a low ability to synthesize ethyl lactate and higher alcohol content in wine production, and provide a Saccharomyces cerevisiae strain with low production of higher alcohols and high production of ethyl lactate and its construction method

Method used

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  • Low-yield high-grade alcohol high-yield ethyl lactate saccharomyces cerevisiae strain and construction method thereof
  • Low-yield high-grade alcohol high-yield ethyl lactate saccharomyces cerevisiae strain and construction method thereof
  • Low-yield high-grade alcohol high-yield ethyl lactate saccharomyces cerevisiae strain and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of Saccharomyces cerevisiae strains with low production of higher alcohols and high production of ethyl lactate

[0033] The main construction process of the strain is as follows:

[0034] 1) Construction of pUC-BABPCK plasmid

[0035] Using pUC-19 as the base plasmid to construct the homologous recombination plasmid pUC-BABPCK, the construction process is as follows: figure 1 As shown (the strain construction method is basically the same), using Saccharomyces cerevisiae CICC32315 haploid α5 as a template, using primers BA-U (SEQ NO: 3) and BA-D (SEQ NO: 4) PCR amplification to obtain a 502bp upstream homology Arm BA and 501 bp downstream homology arm BB of BB-U (SEQ NO: 5) and BB-D (SEQ NO: 6). Using the pMD20-T-CALB-2 plasmid donated by South China University of Technology as a template, three target gene upstream primers Ci-U (SEQNO: 7), Cn-U (SEQNO: 8), Cα-U (SEQNO: 9 ) and downstream primers C-D (SEQ NO: 10) PCR amplification to obtain th...

Embodiment 2

[0049] Example 2: Fermentation experiment of sorghum raw material semi-solid liquor with INU strain containing inulinase signal peptide

[0050] 1) Fermentation process route:

[0051] Sorghum → soaking → liquefaction → saccharification → cooling → mixing koji, inoculation with lactic acid bacteria → cultivation for 12 hours → fermentation → distillation

[0052] 2) Process conditions: soaking conditions: 60-70°C, fully absorb water; cooking conditions: 115°C for about 30 minutes; liquefaction conditions: 85-90°C, high temperature resistant α-amylase, liquefaction for 60 minutes; saccharification conditions: 55-60°C , add glucoamylase, saccharification for 20min; fermentation conditions: 30°C, 7 days; wine steaming conditions: 100mL fermentation broth, add 100mL water, steam 100mL wine sample.

[0053] 3) Ingredients: sorghum flour: 78g; add water 200mL; high temperature resistant α-amylase: 25μL; glucoamylase: 45μL; acid protease: 1.2mL;

[0054] Carry out semi-solid Daqu l...

Embodiment 3

[0063] Example 3: Fermentation experiment of sorghum raw material semi-solid liquor with CalB self-signal peptide nsB strain

[0064] 1) Fermentation process route:

[0065] The fermentation process is consistent with 1) in Example 2.

[0066] 2) Process conditions: consistent with 2) in Example 2.

[0067] 3) batching: consistent with 3) in embodiment 2.

[0068]Carry out semi-solid Daqu liquor fermentation experiments on Saccharomyces cerevisiae starting strain α5 and selected strain α5CALBn according to the above-mentioned fermentation process; during the fermentation period, shake and weigh every 12 hours, and record the weight loss; after the fermentation is over, stop the cultivation and weigh; Residual sugar concentration, alcohol content and content of main aroma components. The comprehensive performance was characterized by fermentation capacity, residual sugar concentration and product production, and the results are shown in Table 4.

[0069] Table 4 Fermentatio...

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Abstract

The invention relates to a low-yield high-grade alcohol high-yield ethyl lactate saccharomyces cerevisiae strain and a construction method thereof. The method comprises acquisition of a target gene, construction of a chromosome integrated vector and fermented verification of the breeding bacterial strain. The selected bacterial strain performs heterogenous secrete expression on pseudonym antarctica lipase B, compared with parent strains, the fermentation performance of the constructed saccharomyces cerevisiae is not influenced, after fermentation by sorghum raw material semi-solid liquor for 6 days, ethyl lactate content of the breeding bacterial strain is increased by 57.4%-84.5%; the high-grade alcohol production amount is obviously reduced, isoamyl alcohol is reduced by 9.5%, isobutyl alcohol is reduced by about 50.0%, and active-amyl alcohol is reduced by about 44.0%. According to the breeding bacterial strain, the production amount of ethyl lactate is obviously increased, the high-grade alcohol production amount is obviously reduced, the problem of incompatibility of flavor of the wind can be solved at certain degree, and the method has wide application prospect in the wine-related filed.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to the breeding of industrial microorganisms, in particular to a Saccharomyces cerevisiae strain with low production of higher alcohol and high production of ethyl lactate and a construction method thereof. Background technique [0002] Trace flavor substances in liquor are the main factors affecting the quality of liquor, the content accounts for about 1%-2%, and esters account for 60%-90% of the trace components. Therefore, increasing the ester content of liquor is important for forming the typical flavor of liquor. is of great significance. A high content of ester can make the wine soft and harmonious, and improve the quality of drinking wine. Ethyl lactate is second only to ethyl acetate in the ester compounds of Fen-flavor liquor, and the content and ratio of ethyl lactate and ethyl acetate have a great influence on the flavor of liquor. For Laobaigan-flavored liquor, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12G3/02C12R1/865
CPCC12N9/1096C12G3/02C12N9/20C12Y206/01021
Inventor 张翠英肖冬光刘芳志刘鑫郭学武陈叶福杜丽平董健马立娟
Owner TIANJIN UNIV OF SCI & TECH
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