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Screening method for alpha-amylase inhibitor producing bacteria

A technology for producing amylase inhibitors and bacteria, which is applied in the fields of biochemical equipment and methods, and the measurement/inspection of microorganisms. The relationship between activity and high-throughput identification of microplate reader can not be solved, so as to achieve the effect of reducing sample processing, high-throughput identification and reliable results

Active Publication Date: 2014-07-02
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 has a greater influence on the measurement interval of 0.4-0.7, and it is difficult to determine the relationship between inhibitor concentration and 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 identification using a microplate reader.
At present, there is no efficient, sensitive, and rapid identification method for microbial-derived 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
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Effect test

Embodiment 1

[0036] (1) Preparation of culture medium:

[0037] Preparation of plate culture medium and inclined surface culture medium: The composition of the medium is as follows (g / L): sucrose 25g / l, peptone 2g / l, tyrosine 1g / l, K 2 HPO 4 ·3H 2 O0.5g / l, KCl0.5g / l, MgSO 4 ·7H 2 O0.5g / l, FeSO 4 ·7H 2 O0.1g / 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 O0.5g, KCl0.5g, MgSO 4 ·7H 2 O0.5g, FeSO 4 ·7H 2 O0.1g, and stir until completely dissolved, then make up to 1000ml.

[0038] Pour each 5ml into a 20ml test tube, sterilize at 121°C for 20min, place the inclined plane, and cool to obtain the inclined plane medium; at the same time, cool the sterilized medium to 60°C under aseptic conditions Pour into a sterilized petri dish and cool to obtain a petri dish culture medium. Fermentation medium preparation: Add 80g of maltose, 20g ...

Embodiment 2

[0047] (1) Preparation of culture medium:

[0048] Preparation of slant medium, plate medium, seed medium, and fermentation medium: slant medium and plate medium are medium with the same composition, and are prepared as follows: add 20g agar strips to 1000mL of water, and stir while heating until Dissolve completely, then add 30g sucrose, 2g peptone, 1g tyrosine, K 2 HPO 4 ·3H 2 O1g, KCl0.5g, MgSO 4 ·7H 2 O0.5g, FeSO 4 ·7H 2 O0.1g, and stir until completely dissolved, then add water to 1000ml, adjust the pH to 7.0. Pour each 5ml into a 20ml test tube, sterilize at 121°C for 20min, place the inclined plane, and cool to obtain the inclined plane medium; at the same time, cool the sterilized medium to 60°C under aseptic conditions Pour it into a sterilized petri dish and cool to obtain a petri dish culture medium;

[0049] Fermentation medium: add 40g maltose, 40g glucose, 15g soybean meal powder, 5g calcium carbonate, 5g glycerol, 5g sodium glutamate in 1000ml water, adjust the pH t...

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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 title of the invention of the original application: α-amylase inhibitor producing bacteria and its screening method, application date: December 25, 2010. (1) Technical field [0002] The present invention relates to a method for identifying bacteria producing alpha-amylase inhibitors. (2) Background technology [0003] Diabetes is a major multiple disease, which is widespread worldwide. Due to the huge change in lifestyle and the lack of people’s awareness of health care, there are currently 92.4 million diabetic patients in my country, including 50.2 million men and 42.2 million women, surpassing India and becoming the country with the most diabetes patients in the world, with an incidence rate (9.7%). ) Is second only to the United States (11%), of which type II diabetes patients account for 93.7%. Currently commonly used drugs for the treatment of type II diabetes include: biguanide...

Claims

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Application Information

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Patent Type & Authority Patents(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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