Screening method for alpha-amylase inhibitor producing bacteria

A technology of amylase inhibitors and screening methods, which is applied in the fields of biochemical equipment and methods, measurement/inspection of microorganisms, etc., and can solve the problems of reporting the screening methods of microbial-sourced α-amylase inhibitors and difficult determination of inhibitor concentration and enzyme activity Problems such as the relationship between high-throughput screening and the inability to perform high-throughput screening can achieve the effects of reducing sample processing, improving detection efficiency, and high-throughput screening

Active Publication Date: 2013-01-09
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in this reaction, the influence of soluble starch and α-amylase on the background Abs546 value of the measurement system is close to 0.2, and the background influence is relatively large for the measurement interval 0.4-0.7, and it is difficult to determine the relationship between the inhibitor concentration and the enzyme activity
At the same time, the color development of the reaction requires a boiling water bath, which cannot be carried out on a 96-well plate, and cannot be used for high-throughput screening using a microplate reader.
At present, there is no efficient, sensitive, and rapid screening method for microbial-sourced α-amylase inhibitors reported.

Method used

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  • Screening method for alpha-amylase inhibitor producing bacteria
  • Screening method for alpha-amylase inhibitor producing bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Preparation of medium:

[0037] Preparation of plate medium and slant medium: the medium components are as follows (g / L): sucrose 25g / l, peptone 2g / l, tyrosine 1g / l, K 2 HPO 4 ·3H 2 O 0.5g / l, KCl 0.5g / l, MgSO 4 ·7H 2 O 0.5g / l, FeSO 4 ·7H 2 O 0.1g / l, agar 20g / l, solvent is water, initial pH 7.0; when preparing, add 20g agar strips to 1000mL water, stir while heating until completely dissolved, then add 25g sucrose, 2g peptone, 1g tyrosine Acid, K 2 HPO 4 ·3H 2 O 0.5g, KCl 0.5g, MgSO 4 ·7H 2 O 0.5g, FeSO 4 ·7H 2 O 0.1g, and stir until completely dissolved, then add water to 1000ml.

[0038] Pour 5ml into a test tube with a volume of 20ml, sterilize at 121°C for 20min, place a slant, and cool to obtain a slant medium; at the same time, cool the sterilized medium to 60°C, Pour it into a sterilized plate and cool down to obtain a medium plate. Fermentation medium preparation: Add 80g of maltose, 20g of glucose, 10g of soybean cake powder, 5g of calcium car...

Embodiment 2

[0047] (1) Preparation of medium:

[0048] Preparation of slant medium, plate medium, seed medium, and fermentation medium: slant medium and plate medium are mediums with the same composition, and are prepared as follows: add 20g agar strips to 1000mL water, and stir while heating until Completely dissolve, then add 30g of sucrose, 2g of peptone, 1g of tyrosine, K 2 HPO 4 .3H 2 O 1g, KCl 0.5g, MgSO 4 .7H 2 O 0.5g, FeSO 4 .7H 2O 0.1g, and stir until completely dissolved, then add water to 1000ml, adjust pH to 7.0. Pour 5ml into a test tube with a volume of 20ml, sterilize at 121°C for 20min, place a slant, and cool to obtain a slant medium; at the same time, cool the sterilized medium to 60°C, Pour into a sterilized petri dish and cool down to obtain a dish culture medium;

[0049] Fermentation medium: Add 40g of maltose, 40g of glucose, 15g of soybean cake powder, 5g of calcium carbonate, 5g of glycerin, and 5g of sodium glutamate in 1000ml of water to adjust the pH to...

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Abstract

The invention discloses a screening method for alpha-amylase inhibitor producing bacteria, which comprises the following steps of: successively inoculating a bacterial strain into a plate culture medium, a slope culture medium and a fermentation culture medium to be cultured; collecting to obtain fermentation liquor; enabling the fermentation liquor to be centrifuged; diluting supernate with water by 10-100 times to obtain a sample to be tested; respectively adding 30mu l of 0.1U / ml of salivary amylase, 40mu l of 5mmol / l of Gal-G2-alpha-CNP (C-type natriuretic peptide), 90mu l of 50mmol / l of disodium hydrogen phosphate-citrate buffer solution with the pH (potential of hydrogen) being 6.0 and 40mu l of sample to be tested into a 96 plate hole; a control group is characterized in that buffer solution of the equal volume is used for replacing the sample to be tested; Abs405 is detected on a 405nm position by a microplate reader; and a digit is read once in every five seconds until Abs405 is not increased; the sample hole of Abs405, which is lowered by above 30% relatively to the control group, is marked as a positive bacterial strain; and the positive bacterial strain is stored. The screening method provided by the invention is simple and effectively and has a high sensitivity, a great quantity of samples can be screened in a short time, and the detection efficiency is improved.

Description

[0001] This application is a divisional application of patent application No. 2010106059123. The original application's invention name is: α-amylase inhibitor producing bacteria and its screening method, and the application date is December 25, 2010. (1) Technical field [0002] The invention relates to a method for screening alpha-amylase inhibitor producing bacteria. (2) Background technology [0003] Diabetes is a major multiple disease that is prevalent worldwide. Due to the huge change in lifestyle and the lack of people's awareness of health care, there are currently 92.4 million diabetics in my country, including 50.2 million males and 42.2 million females, surpassing India and becoming the country with the largest number of diabetics in the world. The incidence rate (9.7%) ) is second only to the United States (11%), of which type II diabetes patients accounted for 93.7%. Currently commonly used drugs for the treatment of type II diabetes include: biguanides that inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/40C12Q1/04
Inventor 郑裕国王远山冯志华薛亚平王亚军沈寅初
Owner ZHEJIANG UNIV OF TECH
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