construction of HD34-1 strain and method for making alcohol-free beer with HD34-1 strain as ferment strain

A technology for fermenting strains and alcohol-free beer, which is applied in the field of fermenting strains for brewing alcohol-free beer and the construction of genetically engineered strains, and can solve problems such as difficulty in wide-scale application, poor stability, and susceptibility to miscellaneous bacteria.

Inactive Publication Date: 2005-03-16
HEILONGJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these existing methods have more or less corresponding problems and disadvantages in technology: 1. Special yeast controls fermentation: because the mash contains a large amount of unfermented maltose, it has a very strong sweet taste, and during the fermentation process Due to the low-alcohol and high-sugar mash environment, the microbial control requirements of the fermented mash are very strict. It must be ensured that there is only one kind of yeast in the mash during the fermentation process, otherwise it will lead to the failure of the fermentation; 2. Interrupted fermentation: due to fermentation Incomplete, its stability is not as good as traditional fermented beer; 3. Low temperature controlled fermentation: In addition to high residual sugar

Method used

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  • construction of HD34-1 strain and method for making alcohol-free beer with HD34-1 strain as ferment strain
  • construction of HD34-1 strain and method for making alcohol-free beer with HD34-1 strain as ferment strain

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specific Embodiment approach 1

[0005] Specific embodiment one: the genetic engineering bacterial strain HD34-1 of present embodiment is constructed like this: a, design 5 ' end, 3 ' end primers according to the ADH gene sequence registered on GenBank, at 5 ' end primers and 3 ' end primers Recognition sites for Sac I and BamH I were added GAGCTC and GGATCC b, using the PCR method to amplify the ADH gene with the genomic DNA of Bacillus subtilis as a template; c, linking the obtained product with the expression vector pYC6 / CT; d, transforming the constructed recombinant DNA into Saccharomyces cerevisiae HD34 using the LiAc transformation method; e. Preliminary screening was performed on the obtained transformants by the isoenzyme method, and ADH qualitative detection was performed on the strains after the primary screening, and a bacterial strain HD34-1 with high ADH activity was obtained.

specific Embodiment approach 2

[0006] Specific embodiment two: the genetically engineered bacterial strain HD34-1 of the present embodiment is constructed like this:

[0007] a. Design 5′ and 3′ primers according to the ADH gene sequence registered on GenBank, and add Sac I and BamH I recognition sites to the 5′ and 3′ primers respectively GAGCTC and GGATCC :

[0008] Bacillus subtilis ADH (Alcohol Dehydrogenase) gene sequence registered on GenBank NC 000964. for:

[0009] ttat

[0010] 3182941 accgttttag gcgtaaggtt aaagcagcgt ttgtagatcc agttgtatgc tttttcattt

[0011] 3183001 aaatctctag ggtttccgaa ggtttgcgga tctttcattg cttctttaga caagcgttcg

[0012] 3183061 atcatatcag gtgatactcc ctgctcttct aaagtcggca cttctaaatc ttcgaccaga

[0013] 3183121 tcatacatcc aattgacaga tgctttcgca gcttcttctg ttgtcatctt gcttgtatca

[0014] 3183181 ataccgaacg ctttcgcaat acgcgcaaac ttctcaggat agcccttcca gttgtattcc

[0015] 3183241 atgacagggc ccatcat...

specific Embodiment approach 3

[0126] Specific embodiment three: The non-alcoholic beer of this embodiment is realized in the following way: a. Use wort culture medium to activate HD34-1 on a solid slope; b. Transfer the activated strain to 500ml at a temperature of 20-40°C In the wort liquid culture medium, shake the flask for 46-50 hours, the temperature is 20-40 °C, and the rotation speed is 120 rpm; c. Cool the expanded bacterial suspension to 3-5 °C, press 2-5 °C 4% inoculum amount inoculates the expansion strain into the fermenter containing wort; d, then oxygenates the fermenter for 2 to 3 minutes, and ferments at 10 to 14°C; e, measures the sugar content in days, Stop fermentation when the sugar content drops to 5°BX; f, ​​cool down to 3-5°C, leave the tank for a week to obtain non-alcoholic beer.

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Abstract

The invention relates to a method of engineered bacterium HD34-1 construction and alcohol free beer brewing using engineered bacterium HD34-1 as zymogen. HD34-1 is constructed as follows: a.designing 5' and 3' primer; b. amplifying ADH gene; c. connecting with expression vector pYC6/CT; d. transforming Saccharomyces cerevisiae HD34; e. screening transformer to obatain HD34-1 strain. Alcohol free beer brewing method using HD34-1 as zymogen is as follows : a. activating HD34-1; b. switching strain to medium and shaking culture; c. inoculating strain to be amplified in malt juice containing fermenter; d. aerating and fermenting e.determing the sugar degree, and stopping fermentation when sugar degree decrease to 5 DEG BX; f. lowering the temperature. The inventive strain can catalyze positive reaction of primary alcohol with aldehyde, lower ethanol yield and simplify the process.

Description

Technical field: [0001] The invention relates to a method for constructing a genetically engineered strain and a method for brewing non-alcoholic beer, in particular to a method for constructing a genetically engineered strain and a method for brewing non-alcoholic beer using the strain as a fermentation strain. Background technique: [0002] Alcohol dehydrogenase (E.C.1.1.1.1, referred to as ADH) is a broadly specific zinc-containing metalloenzyme (Drewkw et al., 1988), which can use NAD as a coenzyme to catalyze the reversible reaction between primary alcohols and aldehydes. It is an important fermentation enzyme in the beer fermentation process, and its positive reaction catalytic activity is of great significance for reducing the ethanol content in beer. Therefore, this enzyme is a key enzyme for the production of low-alcohol or no-alcohol beer by microbial fermentation. Alcohol-free beer refers to beer with no alcohol or very little alcohol content. Like ordinary beer,...

Claims

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Application Information

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IPC IPC(8): C12C12/00C12N1/19C12N15/53C12N15/81
Inventor 平文祥葛菁萍宋刚楼庄伟贾树彪吴国峰赵辉周东坡
Owner HEILONGJIANG UNIV
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