A kind of immunoactive peptide in fermented milk and its enrichment method and application

An immunoactive peptide and fermented milk technology, applied in the field of bioengineering, can solve the problems of various active peptides and low concentration, and achieve the effects of good immune promotion, high safety and mild operating conditions.

Active Publication Date: 2021-07-09
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complexity of microbial enzyme systems, the types of active peptides formed are diverse and the concentration is very low, how to enrich and accumulate peptides with specific functions has become an urgent problem to be solved

Method used

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  • A kind of immunoactive peptide in fermented milk and its enrichment method and application
  • A kind of immunoactive peptide in fermented milk and its enrichment method and application
  • A kind of immunoactive peptide in fermented milk and its enrichment method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 active peptide BP 1 Separation and purification preparation

[0039] (1) Activation of bacterial strains: inoculate Streptococcus thermophilus JIM 8232 into M17 liquid medium with 4% inoculum, and inoculate Lactobacillus bulgaricus ATCC 11842 into MRS broth medium with 4% inoculum, cultivate at 37°C for 18- 24h respectively to get the activated strains. After the cultivation, centrifuge at 8000rpm / min at 4°C for 5min to discard the culture solution, mix the activated strains, resuspend the bacteria solution with pre-sterilized skim milk medium with a mass concentration of 12%, and resuspend the bacteria solution at 8000rpm / min at 4°C for 4 The cells were collected by centrifugation at ℃ for 5 min, and washed three times.

[0040] (2) Skim milk fermentation: Inoculate the activated Streptococcus thermophilus ATCC 11842 and Lactobacillus bulgaricus JIM 8232 bacteria solution into the skim milk medium at a 5% (1:1, v / v) inoculum, and ferment at 42°C After 6...

Embodiment 2

[0051] Embodiment 2 active peptide BP 1 Optimization of fermentation conditions

[0052] (1) Active peptide BP 1 Quantitative determination of concentration: accurately weigh the active peptide finished product BP synthesized in Example 1 1 10mg, dissolved in ultrapure water, and prepared as a 1mg / mL mother solution. Dilute the mother liquor into 0.05, 0.1, 0.2, 0.4, 0.6, 0.8mg / mL working solutions in turn, pass through a 0.45μm filter membrane and inject into the liquid phase sampling bottle, and use liquid chromatography to measure the active peptide concentration and peak area The dose relationship between. The liquid phase program is: 0-30min, 95%-70%A, 5%-30%B; 30-45min, 70%-50%A, 30%-50%B; 45-50min, 50%-95% A, 50%-5% B. Mobile phase A and B are ultrapure water and acetonitrile containing 0.1% formic acid respectively, the injection volume is 20 μL, and the liquid phase column is: Waters Sunfire C 18 Column (25 cm x 4.6 mm x 5 μm).

[0053] The result is as Figu...

Embodiment 3

[0063] Embodiment 3 active peptide BP 1 Effects on the secretion of RAW.264.7 cytokines in macrophages

[0064] (1) Macrophage culture

[0065] Take out a macrophage RAW264.7 cell (purchased from the Cell Bank of the Chinese Academy of Sciences) from the liquid nitrogen irrigation. Fetal bovine serum cultured in DMEM medium. Add the lysed cells to a 15mL centrifuge tube containing 5mL of medium, centrifuge at 1000rpm / min for 3min, discard the supernatant, resuspend the cells in 2mL of complete medium, mix well, inoculate into a culture bottle, and place the culture bottle in an incubator Cultivate, CO 2 The concentration is 5%, and the temperature is 37°C. The cells were grown to a density of about 90%, the supernatant was discarded, and an appropriate volume of 0.25% trypsin was added to digest the cells. After the cells became round, a complete medium was added to terminate the reaction. Pipette the cells in the cell culture flask, collect the mixture into a 15mL test t...

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Abstract

The invention discloses an immunoactive peptide BP in fermented milk 1 , the active peptide BP 1 The amino acid sequence of NQFLPYPYYAKPA is NQFLPYPYYAKPA, and the active peptide BP1 can significantly promote the secretion of cytokines TNF-α and IL-6, and has a good immune-promoting effect. The invention also discloses a method for enriching the active polypeptide. The invention uses the active peptide BP 1 content as an index, the parameters of milk fermentation were optimized by response surface methodology, and finally the active peptide BP was obtained 1 The content reaches 0.213 mg / mL, which is significantly better than the conventional fermentation technology. This method can be used to increase the content of immunoactive peptides in fermented milk, provide process guidance for further exploration of extracting bioactive peptides from fermented milk, and provide guidance for the development of dairy products with immune regulation function and health care products are of great significance.

Description

[0001] Technical solutions [0002] The invention belongs to the field of bioengineering, and in particular relates to an immunoactive peptide in fermented milk and its enrichment method and application. Background technique [0003] Bioactive peptides are a class of specific protein fragments that have a positive impact on the function or state of the body, and ultimately have a benign effect on the health of the body. The intake of bioactive peptides can have a positive impact on the body's cardiovascular system, digestive system, immune system and nervous system. These specific bioactive peptides reduce the risk of suffering from clinical diseases, promote the body's autoimmunity and improve the health status of the human body, so they have received widespread attention in the field of functional food research. [0004] Milk protein mainly includes casein, whey protein and a small amount of lactoferrin and other protein components, and is the most important source of bioac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12P21/02C07K1/34C07K1/14A61K38/10A61P37/00
CPCA61K38/00A61P37/00C07K14/4732C12P21/02
Inventor 杨振泉俞慧芬饶胜其高璐李熠
Owner YANGZHOU UNIV
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