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Detection method of germs in traditional zymotic soybean paste

A detection method and fermented soybean technology, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve the problem of inability to accurately detect bacteria, etc. The effect of ensuring accuracy and reducing analytical bias

Inactive Publication Date: 2010-01-06
HEILONGJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the current PCR-DGGE technology cannot accurately detect bacteria in traditional fermented soybean paste, and provide a detection method for bacteria in traditional fermented soybean paste

Method used

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  • Detection method of germs in traditional zymotic soybean paste
  • Detection method of germs in traditional zymotic soybean paste
  • Detection method of germs in traditional zymotic soybean paste

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specific Embodiment approach 1

[0023] Specific embodiment one: the detection method of bacteria in the traditional fermented soybean paste of the present embodiment is carried out according to the following steps: one, traditional fermented soybean paste sample bacterial total genome DNA extraction:

[0024] a. Pretreatment of traditional fermented soybean paste samples: Suspend 10 g of traditional fermented soybean paste samples with 50 mL of phosphate buffer solution with a pH value of 7.0 and a concentration of 0.1 mol / L, then add glass beads and vibrate for 5 minutes with a shaker, and then shake at 200 r Centrifuge for 5 min under the condition of 0.1 mol / L to collect the supernatant, then use the pH value of 7.0 and the concentration of 0.1 mol / L phosphate buffer to suspend, centrifuge and repeat 3 times, wherein each centrifugation speed is 200r / min , the centrifugation time is 5min, and then the centrifuged supernatant is collected and centrifuged at a speed of 9000r / min for 12min to collect the cent...

specific Embodiment approach 2

[0044] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is: in step 3, the sample volume is 5.0 μL each time, and the PCR amplification product obtained in 2.5 μL step 2 is mixed with 2.5 μL neutral loading solution composition. Other steps and parameters are the same as those in Embodiment 1.

[0045] In step 3 of this embodiment, the PCR amplification product obtained in step 2 is directly used without purification of the PCR amplification product.

specific Embodiment approach 3

[0046] Specific embodiment three: the difference between this embodiment and specific embodiment two is: 10 mL of neutral sample solution is composed of 2 g sucrose, 100 μ L of Tris-HCl with a concentration of 1 mol / L and a pH value of 7.8, and 20 μ L of Tris-HCl with a concentration of 0.5 mol / L L, EDTA with a pH value of 8.0, 10 mg of bromophenol blue and the remainder of deionized water. Other steps and parameters are the same as those in Embodiment 2.

[0047] In this embodiment, the neutral sample solution is stored in an environment of 4°C.

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Abstract

The invention relates to a detection method of germs in soybean paste, in particular to a detection method of germs in traditional zymotic soybean paste, solving the problem that the prior PCR-DGGE technology can not accurately detect the germs in the traditional zymotic soybean paste. The detection method comprises the following steps: (1) the DNA extraction of a germ common gene group of a traditional zymotic soybean paste sample; (2) a V3 section of 16S rDNA of PCR amplified germs; and (3) the PCR-DGGE of the germs. The method of the invention thoroughly clear up other contents of the traditional zymotic soybean paste except microbial cells while avoiding the loss of the species and the amount of microorganism whenever possible, and the whole detection process does not change the DNA content, reduces the final analysis bias and ensures the precision of a detection result.

Description

technical field [0001] The invention relates to a detection method for bacteria in soybean paste. Background technique [0002] There are many types of bacteria in traditional fermented soybean paste. These bacteria are the key to the traditional fermentation of soybean paste, directly affect the fermentation speed and product flavor of soybean paste, and even determine the success or failure of fermentation. In the past, the detection of bacteria in traditional fermented soybean paste was mainly based on the results of morphological identification, while the non-culturable microorganisms and the very small number of microorganisms in soybean paste were often unable to be detected, resulting in inaccurate detection results. [0003] With the development of molecular biology, the use of PCR-DGGE technology has been able to detect uncultivable microorganisms and extremely small microorganisms in environmental samples. However, the composition of traditional fermented soybean ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/447C12R1/44C12R1/24C12R1/125C12R1/07C12R1/01
Inventor 葛菁萍蔡柏岩平文祥宋刚赵丹高冬妮凌宏志陈丽
Owner HEILONGJIANG UNIV
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