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Microporous plate for quantitatively detecting pantothenic acids with microbiological method, kit thereof and preparation method thereof

A technology of microporous plate and quantitative method, applied in the field of microporous plate, can solve the problems of strain loss, wrong result, high detection background, etc., and achieve the effect of reducing the loss of strain and improving accuracy

Active Publication Date: 2017-05-17
BEIJING ZHONGJIAN BAOTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are many defects in the kit coated with Lactobacillus plantarum. The existing method freezes the microbial suspension in the microwell plate at -80°C, and then freezes the microbial mass in the microwell plate under vacuum.
Some microorganisms mutate or recombine to adapt to unfavorable environments, so that after adding water or samples, some microorganisms can still grow in the absence of vitamins, resulting in high detection background and, in individual cases, erroneous results
In addition, existing methods using freeze-drying under vacuum also result in a loss of at least about 23% of the strain

Method used

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  • Microporous plate for quantitatively detecting pantothenic acids with microbiological method, kit thereof and preparation method thereof
  • Microporous plate for quantitatively detecting pantothenic acids with microbiological method, kit thereof and preparation method thereof
  • Microporous plate for quantitatively detecting pantothenic acids with microbiological method, kit thereof and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] 1. Microplate Preparation

[0054] (1) Activated Lactobacillus plantarum ATCC 8014

[0055] Add 11g of skimmed milk powder to 89ml of distilled water, mix well, dispense into test tubes in units of 10ml, and sterilize at 115°C for 15 minutes. This solution is a liquid medium.

[0056] Add 0.3g of powdered Lactobacillus plantarum ATCC 8014 strain into 10ml of culture medium, culture at 34°C until curdling, and store in a refrigerator at 0°C.

[0057] Add 0.3ml of the cultured bacteria in the above step to 10ml of the culture medium, and cultivate at 38°C until the milk curds.

[0058] Repeat the previous step several times until the curd time is stable, indicating that the bacteria have been activated.

[0059] (2) Preparation of test bacteria solution

[0060] Inoculate the activated Lactobacillus plantarum ATCC 8014 strain into the Lactobacillus broth medium, culture at 36°C±1°C for 24h, centrifuge at 2100 rpm for 2min, stop the cultivation, and discard the supernat...

Embodiment 2

[0074] 1. Microplate Preparation

[0075] Lactobacillus plantarum ATCC 8014 was used as the coated Lactobacillus plantarum strain, and the steps were as follows: Inoculate the activated bacterial strain (the activation treatment method is the same as in Example 1) into the Lactobacillus broth medium, cultivate it at 38°C ± 1°C for 18h, and incubate at 2000 Centrifuge at rpm for 3 min to stop the culture and discard the supernatant. Add 190mM sucrose and 9mM CaCl 2 Mix 7ml of protective agent mixture, centrifuge for 2min, discard the supernatant, add 7ml of protective agent, and mix evenly. Centrifuge as before and discard the supernatant. Add 7ml of protective agent and mix well. Aspirate 2ml of the bacterial suspension into 10ml of protective agent, mix well to make the test bacterial liquid.

[0076] Add 6 μl of the bacterial solution to each well of the microwell plate, and heat the bacterial solution slightly under the low vacuum environment of 25mTorr, 50mTorr, 500mTo...

experiment example 1

[0088] This experimental example is to study the precision of the kit of the present invention.

[0089] Use 3 batches of kits (kits prepared in Example 1) to detect the National Institute of Standards and Technology milk powder reference substance 1849E (pantothenic acid concentration is 6.82mg / 100g), do parallel experiments, the results are shown in Table 1. 1 dilution of milk powder reference substance 1849E was tested per batch (final concentration within the range of the standard curve).

[0090] Table 1

[0091]

[0092]

[0093] The coefficient of variation (1.80%) for determining the concentration of milk powder reference substance is very small, indicating high precision. The variation in the raw results of the standards among the 3 batches was less than 10%.

[0094] Use 3 batches of kits (kits prepared in Example 2) to detect milk powder reference substance 1849E [concentration target value is 6.82mg / 100g], do parallel experiments, the results are shown in T...

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Abstract

The invention provides a microporous plate for quantitatively detecting pantothenic acids with a microbiological method. The microporous plate is prepared through the following steps: carrying out inoculation treatment on activated lactobacillus plantarum strains, and then adding a mycose / saccharose and calcium chloride mixed liquor to be prepared into a test bacteria solution; then adding the test bacteria solution into each pore of the microporous plate, heating the test bacteria solution in a low vacuum environment, and finally carrying out drying treatment. The invention also provides a kit containing the microporous plate and a preparation method of the kit. By adding the mycose / saccharose and calcium chloride mixed liquor protective agent in the preparation and improving the drying method for the bacteria solution, the strain loss in the kit is reduced, and the accuracy of pantothenic acid detection results is improved. Through viable counting, the quantity of active strains stored in lactobacillus plantarum dry foam in the prepared microporous plate exceeds 92% of the total quantity of strains in the added bacteria solution (at least about 23% of strains are lost in an ordinary freeze-drying method).

Description

technical field [0001] The invention relates to a microporous plate for the quantitative detection of pantothenic acid by a microbial method, a kit and a preparation method thereof, and belongs to the field of microbial detection. Background technique [0002] At present, the detection methods for pantothenic acid at home and abroad are relatively complicated, generally including microbial method, high performance liquid phase method and enzyme-linked immunosorbent method. Among them, the lower limit of HPLC detection is much higher than that of microbial method, and the accuracy is better, but it requires expensive precision instruments and reagents, and the sample processing process is relatively complicated, so it is difficult to popularize and apply in production; in addition, enzyme-linked immunoassay detection The result is quite different from the actual value. The microbial method can sensitively detect the biologically active pantothenic acid, which is the biggest ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12R1/225
CPCC12Q1/02
Inventor 刘龙飞高天垒卜庆婧
Owner BEIJING ZHONGJIAN BAOTAI BIOTECH
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