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Wine Saccharomyces cerevisiae strain with high glycerol yield and low acetic acid yield and construction method thereof

A Saccharomyces cerevisiae strain and Saccharomyces cerevisiae technology, applied in the field of Saccharomyces cerevisiae strains and constructions for wine with high glycerin and low acetic acid production, to achieve the effects of reducing acetic acid production, high sugar and alcohol tolerance, and increasing glycerin production

Active Publication Date: 2017-09-19
梁恒宇 +1
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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 the acetic acid content of Saccharomyces cerevisiae is too high and the glycerin content is low during the alcoholic fermentation of wine, and a gene knockout Saccharomyces cerevisiae strain and construction method for wine with high glycerol and low acetic acid production are provided

Method used

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  • Wine Saccharomyces cerevisiae strain with high glycerol yield and low acetic acid yield and construction method thereof
  • Wine Saccharomyces cerevisiae strain with high glycerol yield and low acetic acid yield and construction method thereof
  • Wine Saccharomyces cerevisiae strain with high glycerol yield and low acetic acid yield and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] [Example 1] Isolation, purification and mating type identification of Saccharomyces cerevisiae NJZCC17 (CGMCC No.3284) and NJZSC5 (CGMCC No.3732) haploid strains

[0064] Two strains of NJZCC17 and NJZSC5 preserved in the laboratory were cultured on YEPD agar medium at 28°C for 48 hours, and activated twice continuously. Pick a single colony of the activated strain and transfer it to the international universal spore-forming medium (McClary medium) to induce sporulation, culture at 28°C for 7 days, observe the formation of ascospores under a microscope, and use a hemocytometer to count The total number of yeast and ascospores were calculated, and the sporulation rate was calculated. After culturing for 7 days, the sporulation rate of the two parental strains was observed under a microscope and both were above 70%.

[0065] Sporulation rate (%) = ascospore number / total cell number.

[0066] Take a clean glass slide, add a little distilled water dropwise, take a little ...

Embodiment 2

[0071] [Example 2] Knockout of Saccharomyces cerevisiae haploid strain ALD6 gene

[0072] Utilize Plasimid Mini Kit I (Omega, USA) plasmid extraction kit to contain pUG6 plasmid (such as Figure 5 ) DH5α Escherichia coli for plasmid extraction, and its concentration was diluted to 20-50ng / μl, and stored in a -20°C refrigerator for later use as template DNA. According to the sequence of the ALD6 gene (Genbank number: 856044) in the Yeast genome, design a pair of primers for constructing a knockout plasmid, and the 54bp upstream primer is 5'-AACATCAAGAAACATCTTTAACATACACAAACACAT CTTCGTACGCTGCAG GTC-3' and 62bp downstream primer 5'-TTTGTGTATATGACGGAAAGAAATGCAGGTTGGTACATTA GCATAGGCCACTAG TGGATCTG -3', synthesized by Shanghai Bioengineering Technology Service Co., Ltd. (the sequence underlined at the bottom is the complementary primer sequence to the pUG6 plasmid). The PCR amplification conditions are: 94°C×4min, one cycle; 94°C×30s, 62°C×30s, 72°C×1min, 30 cycles; 72°C×10mi...

Embodiment 3

[0075] [Example 3] Knockout of ALD6 gene knockout strain G418 selection marker gene

[0076] Utilize Plasimid Mini Kit I (Omega, USA) plasmid extraction kit to contain pSH65 (such as Figure 8 ) (GAL1-cre, phleomycin resistance) plasmid DH5α Escherichia coli for plasmid extraction, and the method in Example 2 to 17A24-ald6△-KanMX and 5125-ald6△-KanMX two gene knockout strains were transformed again, and used The YEPD medium containing 100 μg / ml phleomycin was used for selection. After the transformants grew out, they were induced and cultured in YEPG liquid medium containing galactose for 5 hours. The presence of galactose induced the expression of Cre recombinase on the pSH65 plasmid, thereby mediating the integration into the adjacent haploid yeast genome. Knockout of the KanMX (G418 resistance) gene between loxP sites. Then spread on the YEPD solid culture plate, after obtaining a single colony, copy it to the YEPD solid plate containing 200 μg / ml G418, and screen the str...

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Abstract

The invention discloses a wine Saccharomyces cerevisiae strain with high glycerol yield and low acetic acid yield and a construction method thereof and belongs to the technical field of industrial microorganisms. Saccharomyces cerevisiae NJZCC17 and NJZSC5 are used as original strains subjected to haploid separation, ALD6 gene in haploids is knocked out separately to obtain genetically-modified strains, the genetically-modified strains 17A24-ald6 Delta (MATa) and 5125-ald6 Delta (MAT alpha) are subjected to diploid hybridization fusion to obtain diploid hybrid strain NJZ17A5-ald6 Delta. The wine Saccharomyces cerevisiae strain has the advantages that acetic yield during alcohol fermentation is lowered, and more carbon metabolic fluxes are diverted to generate and accumulate glycerol; the yield of ethyl acetate is lowered; the yields of isoamyl alcohol and 2-phenylethyl alcohol are increased. Therefore, a novel method for lowering acetic acid yield and increasing glycerol yield is provided for wine making industry.

Description

technical field [0001] The invention belongs to the technical field of industrial microbes, and in particular relates to a high-yield glycerol-low acetic acid-yielding Saccharomyces cerevisiae strain for wine and a construction method. Background technique [0002] With the significant improvement of my country's comprehensive national strength and people's living standards, the consumption of wine has increased year by year, and the market has expanded year by year. Under normal circumstances, the volatile acid in wine is between 0.4 and 0.6g / L, and should not exceed 1.1g / L. The change of its content can be regarded as a "barometer" of the health status of wine making and storage. Excessive volatile acid content will seriously affect the sensory quality of wine, causing unpleasant burning sensation and pungent smell when tasting wine, and producing pungent sour taste when sniffing, covering up other aromas of wine. The main volatile organic acid in wine is acetic acid, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/04C12N1/19C12G1/022C12P7/20C12P7/22C12P7/04C12R1/865
CPCC12G1/0203C12N15/04C12N15/81C12P7/04C12P7/20C12P7/22
Inventor 梁恒宇徐农王凯李遥力
Owner 梁恒宇
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