Saccharomyces cerevisiae capable of reducing biogenic amine in yellow rice wine and its construction method and application
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A technology of Saccharomyces cerevisiae and construction method, which is applied in the field of molecular biology and achieves the effect of wide application prospects
Active Publication Date: 2014-09-17
TIANJIN UNIV OF SCI & TECH
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[0011] The technical problem to be solved by the present invention is to provide a Saccharomyces cerevisiae genetically engineered bacterium that knocks out the PEP4 propeptide gene and the application of the strain in reducing the content of biogenic amines in rice wine, aiming at the common defects of biogenic amines in rice wine. Reduce the amount of free amino acids produced by proteins such as yeast autolyzed PrA secreted to extracellular decomposition bacteria in the late stage of rice wine fermentation, thereby reducing the production of biogenic amines
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Embodiment 1
[0063] According to the yeast genome data in Genebank and the integrated plasmid sequence, the primers in the following examples were designed.
[0064] Table 1. Primers used in this example
[0065]
[0066] The construction process of genetically engineered bacteria of the present invention is as follows:
[0067] 1) The upstream homology arm A segment of the homologous recombination of the purified PEP4 propeptide gene was single-digested with Hind III, and connected with the pUC19 plasmid after the same digestion to construct recombinant plasmid 1;
[0068] 2) performing a single enzyme digestion on the downstream homology arm B fragment of the purified PEP4 propeptide gene homologously recombined with Sac I, and connecting it with the recombinant plasmid 1 after the same digestion to construct recombinant plasmid 2;
[0069] 3), the KanMX gene fragment is single-digested with Kpn I, and connected with the recombinant plasmid 2 after the same digestion, to construct th...
Embodiment 2
[0076] Take one ring each of the genetically engineered bacteria obtained in Example 1 and the starting strain Saccharomyces cerevisiae RY1, inoculate them in 5 mL of wort medium, 30 ° C, 150 r / min, shake the flask for 12 hours and then transfer them to the same wort culture medium. In a 50mL Erlenmeyer flask, 30°C, 150r / min, continue to shake the flask for 24 hours, then inoculate 10% of the inoculum into the rice solid medium, and carry out the pre-fermentation experiment in a 30°C constant temperature incubator. After 5 days, adjust the constant temperature incubator After the temperature reached 30°C, the fermentation experiment was carried out for 15 days. After the fermentation, the supernatant was taken to pass through the membrane, and the liquid chromatography was ready for testing. At the same time, the fermentation broth was analyzed, including weight loss, residual sugar, alcohol content, and the content of important aroma substances. The results are shown in Table...
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Abstract
The invention discloses saccharomyces cerevisiae engineering bacteria capable of reducing biogenic amine in yellow rice wine and its construction method and application, and belongs to the technical field of molecular biology. Through knockout of a PEP4 propeptide gene of saccharomyces cerevisiae, the saccharomyces cerevisiae engineering bacteria having low protease A activity are obtained and can be used for fermentation production of yellow rice wine having low biogenic amine content. Under the premise of no influence on fermentation performances, a result of comparison between the converted offspring saccharomyces cerevisiae engineering bacteria and the parent bacteria shows that after simulative semi-solid state fermentation, main biogenic amine content is reduced, the reduction amounts of tyramine, cadaverine and histamine are maximum and compared with the original bacteria, the saccharomyces cerevisiae engineering bacteria have tyramine, cadaverine and histamine contents respectively reduced by 57.5%, 24.6% and 54.3%; and the screened engineering bacteria have no special requirements on fermentation equipment and conditions, are suitable for equipment and conditions of common wineries, have wide application prospects and can provide important guarantee for safe production of yellow rice wine.
Description
Technical field: [0001] The invention belongs to the technical field of molecular biology, and relates to a Saccharomyces cerevisiae genetically engineered bacterium capable of reducing the content of biogenic amines in rice wine fermentation and its application. Background technique: [0002] Biogenic amines are a class of basic nitrogen-containing compounds, which are mainly formed by the decarboxylation of free amino acids in microorganisms. Because of its potential toxicity and indication of production hygiene conditions, it has attracted widespread attention from consumers, manufacturers and researchers, and is one of the main objects of current food safety research. Among them, tyramine, cadaverine, and putrescine are the most widely present in various fermented foods, and their content is relatively high, which is more harmful. [0003] Generally speaking, there are three conditions for the production of biogenic amines by microorganisms: 1. There are free amino acid...
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