Immunoactive peptide in fermented milk as well as enrichment method and application thereof

A technology of immunoactive peptides and fermented milk, which is applied in the field of bioengineering, can solve the problems of complex types of active peptides and low concentration, and achieve good immune promotion effects, high safety, and low cost effects

Active Publication Date: 2018-10-09
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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  • Immunoactive peptide in fermented milk as well as enrichment method and application thereof
  • Immunoactive peptide in fermented milk as well as enrichment method and application thereof
  • Immunoactive peptide in fermented milk as well as enrichment method and application thereof

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 BP1 in fermented milk. The amino acid sequence of the active peptide BP1 is NQFLPYPYYAKPA. The active peptide BP1 can obviously promote secretion of cell factors TNF-alpha and IL-6, and has a good immunological enhancement effect. The invention further discloses an enrichment method of the active peptide. Milk fermentation parameters are optimized through a response surface method by taking the content of the active peptide BP1 as an index, so that the content of the finally obtained active peptide BP1 is 0.213 mg / mL, which is obviously higher than that of a conventional fermentation technology. The method can be used for increasing the content of the immunoactive peptide in the fermented milk; a process instruction is provided for further exploration of extraction of a biological active peptide in the fermented milk; the immunoactive peptide BP1 in the fermented milk has important significance for developing dairy products and health products which have immune modulating functions.

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 Applications(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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