Method for quickly distinguishing lactic acid bacterium strains

A technology of lactic acid bacteria and strains, applied in the field of microbiology, can solve the problems of high price, cumbersome operation of distinguishing lactic acid bacteria strains, unstable test results, etc.

Inactive Publication Date: 2016-05-11
BRIGHT DAIRY & FOOD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved in the present invention is to overcome the time-consuming 16SrDNA sequencing method for identifying strains in the prior art and the existing lactic acid bacteria strain identification kit API50 kit for distinguishing lactic acid bacteria strains is cumbersome, expensive, and unstable in detection results technical problems, a method for quickly distinguishing lactic acid bacteria strains is provided

Method used

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  • Method for quickly distinguishing lactic acid bacterium strains
  • Method for quickly distinguishing lactic acid bacterium strains
  • Method for quickly distinguishing lactic acid bacterium strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1 distinguishes Lactobacillus bulgaricus (Lacobacillus bulgaricus) and Lactobacillus helveticus (Lactobacillus bulgaricus)

[0030] Streak Lactobacillus bulgaricus and Lactobacillus helveticus on the MRS solid medium, pick a single colony and inoculate the two strains in the MRS liquid medium, culture at 37°C for 48 hours, collect the supernatant by centrifugation, and dilute the sample 25 times Use D-LDH and L-LDH to measure the ratio of D-lactic acid and L-lactic acid, and the specific detection reaction is carried out according to the system in Table 2 below:

[0031] Table 2 Reaction system for lactic acid determination

[0032]

[0033] The glycylglycine buffer is 0.5mol / L, pH8.9 glycylglycine buffer. Add D-LDH or L-LDH to react for 4 minutes, read the absorbance value (A2) after the reaction, calculate the change value of absorbance in the blank and sample (A2-A1), and subtract the change value of the absorbance of the sample from the absorbance val...

Embodiment 2

[0040] The distinction of embodiment 2 plant lactobacillus (Lacobacillus plantarum) and lactobacillus casei (Lacobacillus casei)

[0041] Streak culture of Lactobacillus plantarum and Lactobacillus casei on MRS solid medium, select a single clone in MRS liquid medium, culture at 37°C for 36 hours, centrifuge to take supernatant, dilute 20 times and use as shown in Table 3 below The system measures the concentration of D-lactic acid and L-lactic acid:

[0042] Table 3 Reaction system for lactic acid determination

[0043]

[0044]The glycylglycine buffer is 0.5mol / L, pH8.9 glycylglycine buffer. Add D-LDH / L-LDH to react for 6 minutes. After the reaction, read the absorbance value (A2), calculate the change value of absorbance in the blank and sample (A2-A1), and subtract the change value of the absorbance of the sample from the absorbance value of the blank The change value of , that is, ΔA D-乳酸 and ΔA L-乳酸 ,

[0045] Calculate the concentration of D-lactic acid and L-la...

Embodiment 3

[0050] The distinction of embodiment 3 Lactobacillus plantarum and Lactobacillus casei

[0051] Streak culture of Lactobacillus plantarum and Lactobacillus casei on MRS solid medium, select a single clone in MRS liquid medium, culture at 37°C for 36 hours, centrifuge to take supernatant, dilute 20 times and use as shown in Table 3 below The system measures the concentration of D-lactic acid and L-lactic acid:

[0052] Table 3 Reaction system for lactic acid determination

[0053]

[0054] The glycylglycine buffer is 0.5mol / L, pH8.9 glycylglycine buffer. Add D-LDH / L-LDH and react for 5 minutes. After the reaction, read the absorbance value (A2), calculate the change value of absorbance in the blank and sample (A2-A1), and subtract the change value of the absorbance of the sample from the absorbance value of the blank The change value of , that is, ΔA D-乳酸 and ΔA L-乳酸 ,

[0055] Calculate the concentration of D-lactic acid and L-lactic acid by the following calculation f...

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Abstract

The invention provides a method for quickly distinguishing lactic acid bacterium strains. The method includes the steps that firstly, a culture solution of lactic acid bacterium strains to be detected is centrifuged, and supernate is taken and diluted by 20-25 folds; secondly, the supernate, water, a glycylglycine buffer solution, NAD+ and D-GPT are mixed for 2-4 min before D or L-lactic dehydrogenase is added according to the table 1, a light-absorbing value A1 is read, then D-or L-LDA is added for reacting for 4-6 min, and a light-absorbing value A2 is read; thirdly, a light-absorbing value change value (A2-A1) of samples is subtracted by a contrasted light-absorbing value change value (A2-A1) to obtain deltaAD-lactic acid or deltaAL-lactic acid, the deltaAD-lactic acid or deltaAL-lactic acid is substituted into the following formula to calculate the concentration of D-lactic acid or L-lactic acid of the lactic acid bacterium strains to be detected, and please see the formula in the description. The lactic acid bacterium strains to be detected are distinguished according to the concentration ratio of D-lactic acid and L-lactic acid of the lactic acid bacterium strains to be detected.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a method for quickly distinguishing lactic acid bacteria strains. Background technique [0002] Lactic acid is a naturally occurring organic acid. Since its molecule contains an asymmetric carbon atom, it has two configurations, D- and L-. D-lactic acid is right-handed; L-lactic acid is left-handed; when D-lactic acid and L-lactic acid are mixed in equal proportions, it is racemic DL-lactic acid. [0003] Generally, lactic acid bacteria can synthesize D-type, L-type or DL-type lactic acid, depending on the species. For lactic acid bacteria with similar colony morphology, it is difficult to distinguish during the cultivation process, which increases the uncertainty of bacterial contamination. The common method of strain identification is generally to select a single clone for culture, extract the genome, amplify the 16SrDNA sequence, then perform sequencing, and perform ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/52C12Q1/32C12Q1/04
CPCC12Q1/52C12Q1/04C12Q1/32
Inventor 黄艳娜李楠游春苹刘振民
Owner BRIGHT DAIRY & FOOD CO LTD
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