Construction method and application of oenococcus oeni engineering bacteria

A technology of Oenococcus oeni and its construction method, applied in the construction field of Oenococcus oeni engineering bacteria, which can solve the problems of low cell transformation rate, inability to use antibiotic resistance genes, inability to apply Oenococcus oeni, etc.

Active Publication Date: 2020-02-28
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, since Oenococcus oeni is directly used in food, there are currently strict restrictions on the genetic modification of edible fungi for food, especially antibiotic resistance genes cannot be used for marking, and the existing conventional markers from edible fungi cannot be applied to wine. Oenococcus, as a result, there is currently no relevant report on genetic modification of Oenococcus. In addition, the low transformation rate of Oenococcus competent cells is also the main reason restricting its transformation

Method used

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  • Construction method and application of oenococcus oeni engineering bacteria
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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] A construction method of Oenococcus oeni (Oenococcus oeni) engineering bacteria, the steps are as follows:

[0083] (1) Extract the genome of Lactobacillus plantarum according to the method of bacterial genome DNA extraction kit, add 50 μL ddH 2 O, stored at -20°C;

[0084] (2) The lipase gene of Lactobacillus plantarum on GenBank, the following primers were designed

[0085] F:5'-ATGCAAGTTATTAAGCAAAAAATTAAC-3'SEQ ID NO.1

[0086] R: 5'-CTAACGATTATCAGCTAGCCATTCAAG-3'SEQ ID NO.2

[0087] (3) the step (2) lipase gene primer is used as a template in the step (1) plant Lactobacillus genome, and the lipase gene lipase fragment is amplified by PCR;

[0088] The step PCR amplification system is as follows, total system 50 μ l:

[0089]

[0090] The PCR amplification procedure is as follows:

[0091] Pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 55°C for 30s, extension at 68°C for 1min, 35 cycles; final extension at 68°C for 10min;

[00...

Embodiment 2

[0123] Application of the above-mentioned Oenococcus oeni engineering bacteria in the production of wine of embodiment 2

[0124] With the bacterial classification that the present invention constructs, through adding the SO of 10mg / L 2 , 10% (volume percent) absolute ethanol and acidic tomato medium (ATB medium) of 20U / mL concentration Nisin for 3-5d acclimatization; then add 5% mass percent to the grape juice, and cultivate at 20°C for 5d Expanding the culture; then adding 1-3% mass percentage to the wine fermentation broth that has been fermented by yeast in the early stage for post-ripening, and adding food additive Nisin at a concentration of 10-20U / mL.

Embodiment 3

[0126] For the determination of ethyl acetate, butyl acetate, isoamyl acetate and ethyl hexanoate in wine, refer to the QBT4850-2015 standard.

[0127] By adding different Oenococcus oeni, the content of the above esters was measured, and the test results were as follows:

[0128]

[0129] Result analysis

[0130] Through the analysis of the above test results, it can be concluded that the Oenococcus oeni engineering bacteria have a certain degree of degradation effect on the above esters, especially for ethyl acetate, whose content has been reduced by more than 80%.

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Abstract

The invention relates to a construction method and application of oenococcus oeni engineering bacteria. The construction method comprises the following steps: (1) preparing lipase gene lipase; (2) preparing homologous arm genes with lipase genes; (3) preparing plasmid pMG36e-lipase; (4) preparing nisl fragments; (5) preparing a linear fragment without containing an erythromycin sequence; (6) preparing an expression vector pMG36n-lipase; and (7) transforming the expression vector pMG36n-lipase into oenococcus oeni competent cells to obtain the oenococcus oeni engineering bacteria. The oenococcus oeni engineering bacteria with a function of degrading esters obtained by the invention can be applied to production of wine, so that adverse effects on taste, aroma and the like of the wine causedby excessive esters in the current wine brewing process are avoided, the preservation time of the wine is shortened, and the production period of the wine reaching the same taste is shortened.

Description

technical field [0001] The invention relates to a construction method and application of Oenococcus oeni engineering bacteria, belonging to the technical field of microbial engineering. Background technique [0002] Oenococcus oeni (Oenococcus oeni) is a kind of lactic acid bacteria, which is a very important strain in addition to yeast in the wine production process. Its role is mainly in the post-ripening of wine, that is, in the later stage of wine brewing, using the special function of Oenococcus oeni to degrade a large amount of malic acid produced in the fermentation into lactic acid and CO 2 , so as to enhance the wine's taste and reduce its acidity, this unique fermentation process is called malolactic fermentation (MalolacticFermentation MLF). Oenococcus and yeast are therefore the only species allowed to be added to wine production in various countries. [0003] Chinese patent document CN104845811A (application number 201510237503.5) discloses a rice wine brewing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/74C12N1/21C12G1/022C12R1/01
CPCC12N9/20C12N15/74C12G1/0203C12Y301/01003
Inventor 何熹韩宁侯冬冬赵新节
Owner QILU UNIV OF TECH
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