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Kit for rapidly detecting activity of probiotics and detection method thereof

A detection method and active reagent technology, which is applied in the rapid detection of probiotic activity kit and its detection field, can solve the problems of inaccurate detection and death of probiotics, and achieve the effect of improving efficiency and high accuracy

Pending Publication Date: 2021-11-19
湖南省中医药研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, MTT is toxic, and it is easy to cause the death of probiotics during centrifugation and other operations, which to a certain extent caused inaccurate detection.

Method used

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  • Kit for rapidly detecting activity of probiotics and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The present invention is as follows to the detection method step of the Lactobacillus plantarum sample of market purchase:

[0032] S1: Take 1ml of the Lactobacillus plantarum sample purchased from the market in a sterile centrifuge tube, add Polygonatum polysaccharide extract at a volume ratio of 1:1 (probiotics: Polygonatum polysaccharide), centrifuge at 8000rpm for 10min, and discard supernatant;

[0033] S2: In a sterile environment, add 4ml of PBS buffer (phosphate buffered saline solution) to the bacterial solution of S1 to resuspend the bacterial pellet, vortex and shake until the sterile block;

[0034] S3: Add 0.5ml of MTT staining solution to the mixture of S2 and mix well;

[0035] S4: Put the mixed solution of S3 in a light-proof 37°C incubator to react for 2h, then centrifuge at 12000rpm for 5min, and discard the supernatant;

[0036] S5: Add 3ml of dimethyl sulfoxide to the reaction product after centrifugation in S4, vortex and oscillate to a sterile bl...

Embodiment 2

[0040] The present invention is as follows to the Lactobacillus bulgaricus sample detection method step of market purchase:

[0041] S1: Take 1ml of the bacterial solution of Lactobacillus bulgaricus sample purchased from the market in a sterile centrifuge tube, add Polygonatum polysaccharide extract at a volume ratio of 1:0.5 (probiotics: Polygonatum polysaccharide), centrifuge at 10000rpm for 10min, and discard supernatant;

[0042] S2: In a sterile environment, add 4ml of PBS buffer to the bacterial solution of S1 to resuspend the bacterial pellet, vortex and shake until the sterile block;

[0043] S3: Add 1ml of MTT staining solution to the mixture of S2 and mix well;

[0044] S4: Put the mixture of S3 in a dark incubator at 37°C to react for 2 hours, then centrifuge at 10,000 rpm for 5 minutes, and discard the supernatant;

[0045] S5: Add 1ml of dimethyl sulfoxide to the reaction product after centrifugation in S4, vortex and oscillate to a sterile block, place at room...

Embodiment 3

[0049] The present invention is to the Lactobacillus johnsonii sample detection method step of market purchase as follows:

[0050] S1: Take 1ml of the bacterial solution of Lactobacillus johnsonii sample purchased from the market in a sterile centrifuge tube, add the Polygonatum polysaccharide extract at a volume ratio of 1:1 (probiotics: Polygonatum polysaccharide), centrifuge at 10000rpm for 10min, discard remove the supernatant;

[0051] S2: In a sterile environment, add 5ml of PBS buffer to the bacterial solution of S1 to resuspend the bacterial pellet, vortex and shake until the sterile block;

[0052] S3: Add 2ml of MTT staining solution to the mixture of S2 and mix well;

[0053] S4: Place the mixture of S3 in a dark incubator at 37°C to react for 4 hours, then centrifuge at 10,000 rpm for 5 minutes, and discard the supernatant;

[0054] S5: Add 5ml of dimethyl sulfoxide to the centrifuged reaction product of S4, vortex and oscillate to a sterile block, place in the ...

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Abstract

The invention discloses a kit for rapidly detecting activity of probiotics and a detection method thereof. The kit comprises a PBS buffer solution, a rhizoma polygonati polysaccharide extracting solution, an MTT staining solution and dimethyl sulfoxide. The specific detection method comprises the following steps of S1, putting 1-5ml of probiotic liquid into a sterile centrifugal tube, adding a polygonatum sibiricum polysaccharide extracting solution according to a volume ratio of 1:(0.5-1), centrifuging, and discarding supernate; S2, 4-5 ml of a PBS buffer solution being added into the bacterial liquid obtained in the S1, thallus precipitation being resuspended, and vortex oscillation being performed till a sterile block is formed; S3, adding 0.5-2ml of MTT staining solution into the mixed solution in the S2, and uniformly mixing; S4, placing the mixed solution in S3 in a dark incubator at 37 DEG C to react for 0.5-4 hours, then centrifuging for 4-6 minutes, and discarding supernate; S5, adding 1-5ml of dimethyl sulfoxide into a reaction product centrifuged in the step S4, performing vortex oscillation to obtain a sterile block, placing the sterile block in a dark place at room temperature, performing centrifugation, and discarding a precipitate; and S6, putting the supernate in the step S5 into an ultraviolet spectrophotometer, and measuring a light absorption value of the supernate. According to the method, determination accuracy is improved, and the detection speed is high.

Description

technical field [0001] The invention relates to the technical field of probiotic activity detection, in particular to a kit for rapidly detecting the activity of probiotics and a detection method thereof. Background technique [0002] In recent years, it has been found that the thiazolium blue (MTT) colorimetric method has the characteristics of simplicity, rapidity, sensitivity, and stability, and has begun to be applied to the detection of live bacteria in probiotics. However, MTT is toxic, and it is easy to cause the death of probiotics during centrifugation and other operations, which to a certain extent caused inaccurate detection. Therefore, it is of great significance to invent a fast, efficient and accurate probiotic detection kit. Contents of the invention [0003] Based on the technical problems existing in the background technology, the present invention proposes a kit for rapidly detecting probiotic activity and a detection method thereof, which improves the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 金剑钟灿谢珍妮张水寒
Owner 湖南省中医药研究院
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