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Directional active peptide preparation method based on Caco-2 cell model

A technology of active peptides and cells, which is applied in the field of directional preparation of active peptides, can solve the problems of loss of activity, achieve wide application prospects, improve digestion resistance and easy absorption, and overcome the effects of digestive system damage or inability to be absorbed and utilized by the human body

Inactive Publication Date: 2020-04-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the identified food-derived bioactive peptides are degraded to some extent by the human digestive system, losing some or all of their activity

Method used

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  • Directional active peptide preparation method based on Caco-2 cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Preparation of rice protein hydrolyzate

[0024] Disperse the rice protein in water, the ratio of solid to liquid is 1:20, stir and mix well, adjust the pH value of the above rice protein dispersion to 8.0 with 1mol / L NaOH solution, add alkaline protease according to the ratio of 8000U / g, control the temperature as 50°C, enzymatic hydrolysis for 2 hours, after the end of the enzymatic hydrolysis, inactivate the enzyme in a 90°C water bath for 20 minutes, after cooling, centrifuge, take the supernatant and freeze-dry to obtain rice protein hydrolyzate 1.

[0025] Disperse the rice protein in water, the ratio of solid to liquid is 1:20, stir and mix well, adjust the pH value of the above rice protein dispersion to 7.5 with 1mol / L NaOH solution, add neutral protease according to the ratio of 7000U / g, control the temperature as 55°C, enzymatic hydrolysis for 2 hours, after the end of the enzymatic hydrolysis, inactivate the enzyme in a 90°C water bath for 20 m...

Embodiment 2

[0026] Example 2: In vitro simulation of gastrointestinal digestion

[0027] Prepare the rice protein hydrolyzate 1 in Example 1 into a 5% solution, add HCl solution to adjust the pH to 1.3, add pepsin, react at 37°C for 1 hour, and simulate gastric juice digestion; then digest the simulated gastric juice with NaOH Adjust the pH to 7.0, add trypsin, and react at 37°C for 2 hours to simulate intestinal juice digestion. After the in vitro simulated gastrointestinal digestion, the enzyme was inactivated in a water bath at 90°C for 20 minutes, centrifuged, and freeze-dried to obtain the anti-digestion active peptide mixture 1.

[0028] The rice protein hydrolyzate 2 in Example 1 was subjected to in vitro simulated gastrointestinal digestion by the same method, and the enzyme was inactivated, centrifuged, and freeze-dried to obtain the anti-digestion active peptide mixture 2.

Embodiment 3

[0029] Example 3: Caco-2 cell model simulates intestinal absorption

[0030] Caco-2 cells were inoculated into transwell chambers at a seeding density of 400,000 cells / well, the culture medium was changed three times a week, and cultured continuously for 2 weeks to make them grow into a complete cell layer. The active peptide mixture 1 obtained in Example 2 was made into a 1 mg / mL solution with PBS (pH 7.2) buffer, and 0.3 mL of the active peptide mixture solution prepared by PBS buffer was added to the upper chamber of the transwell chamber, and 1.2 mL of the active peptide mixture was added to the lower chamber. mL PBS buffer. After 2 hours of action, the substances in the lower chamber were collected and set aside.

[0031] The anti-digestion active peptide mixture 2 obtained in Example 2 was subjected to Caco-2 cell model to simulate intestinal absorption in the same way.

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Abstract

The invention discloses a directional active peptide preparation method based on a Caco-2 cell model, and belongs to the technical fields of deep processing of agricultural products and comprehensiveutilization of byproducts of agricultural products. Rice protein is used as a raw material, after protease treatment, by simulation of gastrointestinal digestion in vitro and simulation of a small intestine absorption process by the Caco-2 cell absorption model, an antioxidant peptide component resistant to digestion and easy to absorb is directionally and efficiently screened out, and then, gel chromatography and high performance liquid chromatography separation and purification are carried out to obtain a high-activity antioxidant peptide which has a clear amino acid sequence and is easy toabsorb and utilize by a human body. An efficient active peptide screening system is constructed based on simulation of gastrointestinal digestion in vitro and simulation of the small intestine absorption by the Caco-2 cell absorption model, the bioavailability of the antioxidant peptide of the rice protein is improved from the source, the problem of low bioavailability of the active peptide in vivo is solved to a certain extent, and the method has wide application prospects.

Description

technical field [0001] The invention relates to a method for directional preparation of active peptides based on a Caco-2 cell model, and belongs to the technical field of intensive processing of agricultural products and comprehensive utilization of by-products. Background technique [0002] Rice protein is a high-quality protein with high nutritional quality. It not only has a very reasonable amino acid composition, but also does not contain similar allergens, and is hypoallergenic. The biological value (BV) of rice protein is 77, and the protein utility ratio (PER) is 1.36-2.56, so the utilization rate of protein is higher than that of other grains. The use of rice protein to produce active peptides can not only overcome the defect that rice protein is insoluble in water, but also increase the added value, and has a good application prospect. [0003] Food-derived bioactive peptides have attracted more and more attention due to their good health-promoting effects and non...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/16
CPCC07K1/16C12P21/06
Inventor 王莉张新霞陈正行
Owner JIANGNAN UNIV
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