Multiple polymerase chain reaction (PCR) technology for simultaneously detecting four functional microbes in vinegar culture
A functional microorganism and microorganism technology, applied in the field of multiple PCR technology, can solve the problems of poor process controllability, large flavor difference, product quality batch difference, etc.
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Embodiment 1
[0032] Embodiment 1: Extraction of the total DNA of the microbial community of vinegar fermented grains.
[0033] 1. Sample collection: collect vinegar unstrained spirits samples at different time points during the vinegar fermentation process. If the DNA cannot be extracted in time after sample collection, it should be frozen immediately.
[0034] 2. Extraction method of microbial total DNA in vinegar fermented grains: Weigh 2 g of vinegar fermented grains, add liquid nitrogen in a mortar and grind thoroughly, transfer to a 50 mL centrifuge tube. Add 6 mL of DNA extraction buffer and 100 μL of lysozyme (50 mg / mL) to the centrifuge tube, and shake at 37 °C for 30 min on a shaker at 225 rpm. Add 1.5 mL of 10% SDS to the centrifuge tube, bathe in water at 65°C for 2 h, invert gently every 15-20 min, and centrifuge at 6000×g for 10 min at room temperature. Collect the supernatant; extract once with an equal volume of chloroform-isoamyl alcohol (24:1, V / V), precipitate with 0.6 t...
Embodiment 2
[0035] Example 2: Analysis of the structure of the microbial community of vinegar fermented grains by PCR-DGGE technology.
[0036] 1. PCR amplification
[0037] Using the above-mentioned extracted vinegar fermented grain community genomic DNA as a template, PCR amplification was carried out with primers P1 and P2 of the bacterial 16S rDNA V3 region, and the target fragment was about 196 bp, which corresponded to E. coli 16S rDNA 341 to 534, the primer sequence is: P1: 5'-ATT ACC GCG GCT GCT GG-3'; P2: 5'-[CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G]CC TAC GGG AGG CAG CAG-3'; PCR products were checked by 1.0% agarose gel electrophoresis, and the DNA concentration was determined by DyNA QuantTM 200 concentration instrument.
[0038] 2. DGGE analysis
[0039]DGGE was analyzed by electrophoresis using Bio-Rad Dcode system. The electrophoresis conditions were: 8% polyacrylamide gel, 30%-50% denaturing gradient, and the DNA loading amount was 200 ng. Electrophoresis b...
Embodiment 3
[0044] Example 3: Screening of functional microorganisms in the microbial community of fermented grains of vinegar.
[0045] 1. Isolation and purification of microorganisms in vinegar fermented grains: Take 5 g of vinegar fermented grains samples and put them into a 250 mL Erlenmeyer flask mixed with glass beads and 45 mL of sterile water, vibrate on a shaker at 37°C at 120 rpm for 30 min, and then take bacteria 1 mL of the suspension was appropriately diluted with sterile saline, spread on GYC plate, MRS plate and broth medium plate respectively, and cultured at 37°C for 48-72 h; There were colonies with large transparent circles on the GYC plate, and a total of 60-80 strains were obtained, which were further purified and preserved.
[0046] 2. Enzyme digestion and classification of purified strains: 16S rDNA amplification was carried out on the above strains, and the amplified products were digested with restriction endonucleases Hinf Ⅰ and Csp6 Ⅰ, separated by electrophores...
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