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Method for quantitative detection of microorganisms in mixed microbial fermentation process

A quantitative detection method and technology for mixing microorganisms, applied in the field of fermented food, can solve the problem of inability to count VBNC bacteria, and achieve the effects of optimizing PMA processing conditions, high sensitivity, and preventing false positive results.

Active Publication Date: 2018-09-28
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has the characteristics of accurate results, fast and efficient, and high sensitivity. It overcomes the shortcomings of the traditional viable counting method, such as long time-consuming, high operating intensity, and inability to count VBNC bacteria. Conditions are optimized on the basis of the existing PMA-qPCR

Method used

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  • Method for quantitative detection of microorganisms in mixed microbial fermentation process
  • Method for quantitative detection of microorganisms in mixed microbial fermentation process
  • Method for quantitative detection of microorganisms in mixed microbial fermentation process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The optimization of embodiment 1 propidium azide bromide (PMA) treatment condition

[0047] Experimental strains: Lactobacillus helveticus AF1-1, Lactobacillus plantarum AF1-9, Lactobacillus fermentum AF4-5, Acetobacter pasteurii CP-A11 (selected and preserved from vinegar unstrained spirits in this laboratory);

[0048] Preparation of propidium azide bromide mother liquor: take 1mg of PMA, add dimethyl sulfoxide to dissolve and quantify to 1mL, make the final concentration of the mother liquor 1mg / mL, and store in the dark at -20°C;

[0049] The mass fraction composition of vinegar fermented grain medium: bran 30%, rice husk 10%, glucose 2%, peptone 1%, beef extract 1%, yeast extract 0.5%, sodium acetate 0.5%, ammonium citrate 0.2%, Tween 80 0.1%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.058%, manganese sulfate 0.025%;

[0050] Preparation of live bacterial suspension: transfer Lactobacillus helveticus AF1-1, Lactobacillus plantarum AF1-9, Lactobacillu...

Embodiment 2

[0053] Example 2 Optimization of Sublethal Bacteria Restoration Solution Conditions

[0054] Components of the sub-lethal bacterial repair solution: reduced amount of broth medium (NB-) + foreign substances;

[0055] Reduced broth medium (NB-) consists of: peptone 1g / L, beef extract powder 0.3g / L, sodium chloride 0.5g / L;

[0056] Xenobiotics include: Tween 80, sodium pyruvate, catalase, MgCl 2 、Na 2 HPO 4 , MnCl 2 , FeCl 2 or moxifloxacin;

[0057] Resuspend the living bacteria (see Example 1 for operating conditions) with sterile PBS buffer, put it in a water bath at 75°C, and bathe in water for 5 minutes to obtain a damaged bacteria liquid containing injured bacteria, live bacteria and dead bacteria, 6000r / min centrifuged for 3min, then discard the supernatant, suspend the pellet with sterilized PBS buffer and mix well.

[0058] Take an appropriate amount of damaged bacteria liquid and centrifuge, remove the supernatant, dissolve the bacterial precipitate with an equ...

Embodiment 3

[0067] Embodiment 3 The comparison of repairing liquid of the present invention and contrast repairing liquid repairing effect

[0068] Preparation of live bacterial suspension: transfer Lactobacillus helveticus AF1-1, Lactobacillus plantarum AF1-9, Lactobacillus fermentum AF4-5, and Acetobacter pasteurianus CP-A11 stored in slant medium to shake flasks for culture , cultivate at a constant temperature at 37°C for about 12 hours, take 5 mL of uniform bacterial liquid in the vinegar fermented grain medium, and culture at a constant temperature at 37°C for about 12 hours, take 5 g of the sample into a 50 mL sterile centrifuge tube, add sterile PBS buffer to 50 mL, and resuspend Suspension, filter with sterile gauze, take the supernatant, centrifuge to collect the bacteria, and then resuspend twice with sterile PBS to clean the impurities in the vinegar unstrained spirits, collect the bacteria, and set aside;

[0069] Control repair solution: NB medium, LB medium, exogenous subst...

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Abstract

The invention relates to a method for quantitative detection of viable count of specific microorganisms in a vinegar solid fermentation process, and belongs to the field of fermented food. The methodhas the characteristics of accurate results, fast and efficient properties and high sensitivity, overcomes the shortcomings that consuming time is long, operating intensity is high and VBNC bacteria cannot be counted of a traditional viable counting method, and is optimized on the basis of existing PMA-qPCR. In particular, a repair solution is developed for repairing the membranes of sub-lethal bacteria cells and is suitable for repairing the cell membranes of bacteria with damaged cell membranes, false negative results caused by sub-lethal damage of a bacterium body is reduced, the accuracy of quantitative results is improved, and the purpose of quickly and accurately quantifying the number of live bacteria in the sample is achieved.

Description

Technical field: [0001] The invention relates to a method for quantitatively detecting the number of living bacteria of specific microorganisms in the solid-state fermentation process of vinegar, belonging to the field of fermented food. technical background: [0002] Traditional foods such as vinegar, soy sauce, bean paste, kimchi, rice wine, and white wine adopt an open mixed microbial fermentation process. During the fermentation process, the microbial composition is complex and diverse. The analysis of the microbial system during fermentation is particularly important, especially lactic acid bacteria and acetic acid bacteria, whose abundance accounts for more than 90% of the total bacterial community abundance. Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus fermentum and Acetobacter pasteurianus are important functional microorganisms, which play a leading role in the traditional food fermentation process. The metabolism of flavor substances such as bu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/06C12R1/225C12R1/25C12R1/02
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107
Inventor 郑宇王敏曹珊牟俊杨帅宋佳张强
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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