CN108251482B

CN108251482B - Production method of three-enzyme one-fermentation chick embryo peptide

Info

Publication number: CN108251482B
Application number: CN201810167938.0A
Authority: CN
Inventors: 不公告发明人
Original assignee: Anhui Precision Medical Industry Research Institute Co ltd
Current assignee: Anhui Precision Medical Industry Research Institute Co ltd
Priority date: 2018-02-28
Filing date: 2018-02-28
Publication date: 2021-01-22
Anticipated expiration: 2038-02-28
Also published as: CN108251482A

Abstract

The production method of the three-enzyme one-fermentation chick embryo peptide provided by the invention firstly decomposes starch and the like through salivary amylase enzymolysis so as to avoid influencing the enzymolysis of pepsin and trypsin on protein and the like in chick embryos and improve the peptide yield. Then the enzymolysis temperature, pH environment, enzymolysis time and the like of pepsin and trypsin are controlled, so that the enzymolysis efficiency is improved, and the molecular weight of the peptide is ensured to be smaller. After 3 times of enzymolysis, streptomycete fermentation is carried out, and the small peptides are further decomposed, so that the molecular weight of the prepared chick embryo peptides is smaller and the chick embryo peptides are easier to be absorbed by people. Then the obtained product active peptide has high yield, the loss amount is less than 1 percent, and the relative molecular mass is less than 2KD and the small molecular peptide is more than 91 percent after separation, ultrafiltration, concentration, redissolution and chromatographic column purification. Compared with the prior art, the method adopts a bionic enzymolysis method, and the chicken embryo is completely enzymolyzed through three times of enzymolysis and one time of fermentation, so that the obtained chicken embryo peptide molecules are smaller, the yield is higher, the absorption by a human body is facilitated, and the effect of the chicken embryo peptide molecules is exerted.

Description

Production method of three-enzyme one-fermentation chick embryo peptide

Technical Field

The invention relates to a production method of chick embryo peptide, in particular to a production method of three-enzyme one-fermentation chick embryo peptide.

Background

The amino group of one amino acid can be condensed with the carboxyl group of another amino acid to form a peptide, and the amide group formed is referred to as a peptide bond in protein chemistry. The molecule of amino acid is the smallest, the molecule of protein is the largest, two or more amino acids are dehydrated and condensed to form a plurality of peptide bonds to form a peptide, and a plurality of peptides are folded in multiple stages to form a protein molecule.

The chicken embryo is the embryo of chicken, and is edible egg without shell breaking after fertilized egg is hatched for a certain number of days. Research has shown that the nutrient components contained in the chick embryo include proteins, polypeptides, amino acids, carbohydrates (glucose, fructose, galactose, arabinose, furanose, etc.), lipids, fatty acids, etc. In addition, the chick embryo also comprises anti-infection components and bioactive substances. The anti-infective component comprises globulin, interferon, fetal protein, lysozyme and phosphodiesterase; the bioactive substances include sialic acid, catecholamine, insulin-like growth factor-I, insulin, kininase, histaminase, choline, erythropoietin, deoxyribonuclease, ribonuclease, etc. Compared with the unhatched eggs, the content of nutrient components, particularly small molecular proteins or polypeptides, in the chick embryos is richer, and the content of fat, cholesterol and the like which are not ideal for human bodies is greatly reduced.

Disclosure of Invention

The invention aims to provide a production method of three-enzyme one-fermentation chick embryo peptide, which sequentially utilizes salivary amylase, pepsin and trypsin for enzymolysis, and finally utilizes streptomycete for fermentation, and the whole process is subjected to bionic enzymolysis, so that the obtained chick embryo peptide has high activity, high total peptide content and high safety.

The invention provides a production method of three-enzyme one-fermentation chick embryo peptide, which comprises the following steps:

1) taking out the chick embryo incubated for 8-15 days, crushing, adding water, homogenizing, adjusting the pH value to 6.5-7.5, adding salivary amylase, and carrying out enzymolysis reaction at the temperature of 37-37.5 ℃;

2) then adjusting pH to 1.5-2.0, adding pepsin, and carrying out enzymolysis reaction at 37-42 deg.C;

3) then adjusting the pH value to 7.5-8.0, adding trypsin, and carrying out enzymolysis reaction at the temperature of 37-42 ℃;

4) adding streptomyces into the system prepared in the step 3), fermenting, centrifuging, separating, and ultrafiltering the filtrate to obtain filtrate, and vacuum drying the filtrate;

5) and 4) adding water to dissolve the dried product, purifying by using a chromatographic column, concentrating under reduced pressure, and drying to obtain the chick embryo peptide.

The step 1) of adding water for homogenizing refers to adding water with the mass 1-3 times of that of the chick embryos.

The adding amount of the salivary amylase in the step 1) is 0.6-5.5% of the mass of the chick embryo.

Carrying out the enzymolysis reaction in the step 1) for 1-2 h;

the adding amount of the pepsin in the step 2) is 0.3-3.5% of the mass of the chick embryos.

Carrying out the enzymolysis reaction in the step 2) for 2-4 h;

the adding amount of the trypsin in the step 3) is 0.3-3% of the mass of the chick embryo.

Carrying out the enzymolysis reaction in the step 3) for 3-5 h;

the addition amount of the streptomyces in the step 4) is 0.05-1% of the mass of the chick embryos.

The fermentation condition in the step 4) is fermentation at 30-37 ℃ for 20-30 h.

The centrifugation in the step 4) refers to centrifugation for 15-20min under the conditions of 8000-10000 r/min.

The ultrafiltration in the step 4) is carried out by using a hollow fiber membrane with the interception relative molecular mass of 3 KD.

The step 5) of adding water for dissolving refers to adding 3-5 times of water for dissolving.

And 5) purifying the chromatographic column in the step 5) by using DA-201C type macroporous resin column chromatography, eluting with water, eluting with ethanol, and collecting the eluate.

The invention firstly carries out enzymolysis by salivary amylase, and firstly decomposes starch and the like so as to avoid influencing the enzymolysis of pepsin and trypsin on proteins and the like in chicken embryos, thereby improving the peptide yield. Then the enzymolysis temperature, pH environment, enzymolysis time and the like of pepsin and trypsin are controlled, so that the enzymolysis efficiency is improved, and the molecular weight of the peptide is ensured to be smaller. After 3 times of enzymolysis, streptomycete fermentation is carried out, and the small peptides are further decomposed, so that the prepared chick embryo peptides have smaller molecular weight and higher solubility and are easier to be absorbed by people. Then the obtained product active peptide has high yield, the loss amount is less than 1 percent, and the relative molecular mass is less than 2KD and the small molecular peptide is more than 91 percent after separation, ultrafiltration, concentration, redissolution and chromatographic column purification.

Compared with the prior art, the invention adopts a bionic enzymolysis method, sequentially utilizes salivary amylase, pepsin and trypsin for enzymolysis, and finally utilizes streptomycete for fermentation. Through three times of enzymolysis and one time of fermentation, the chicken embryo is completely enzymolyzed, and the obtained chicken embryo peptide molecules are smaller, the yield is more, and the chicken embryo peptide molecules are more easily absorbed by human bodies and play the role of the chicken embryo peptide molecules.

Detailed Description

Example 1

A production method of three-enzyme one-fermentation chick embryo peptide comprises the following steps:

1) taking out the chick embryo which is hatched for 10 days, crushing the chick embryo, adding 2 times of water for homogenizing, adjusting the pH value to 7, adding salivary amylase accounting for 1 percent of the mass of the chick embryo, and carrying out enzymolysis reaction for 2 hours at the temperature of 37.5 ℃;

2) then adjusting the pH value to 1.5, adding pepsin accounting for 0.5 percent of the mass of the chick embryo, and carrying out enzymolysis reaction for 4 hours at the temperature of 38 ℃;

3) then adjusting the pH value to 7.5, adding trypsin with the mass of 0.5 percent of that of the chick embryo, and carrying out enzymolysis reaction for 5 hours at the temperature of 38 ℃;

4) adding streptomyces with the mass of 0.08 percent of that of the chick embryo into the system prepared in the step 3), fermenting for 25h at 33 ℃, centrifuging for 15min under the condition of 10000r/min, ultrafiltering the separated filtrate by using a hollow fiber membrane with the intercepted relative molecular mass of 3KD, and drying the obtained filtrate in vacuum;

5) and 4) adding 3 times of water into the dried product to dissolve, performing DA-201C type macroporous resin column chromatography, eluting with water, eluting with ethanol, collecting eluate, concentrating under reduced pressure, and drying to obtain the chick embryo peptide.

The prepared small peptide is measured by HPLC, and the chick embryo peptide prepared by the application can be obtained, wherein the relative molecular mass of the chick embryo peptide is less than 92.3% of that of the small peptide with 2 KD.

Example 2

1) taking out the chick embryo which is hatched for 12 days, crushing the chick embryo, adding 1.5 times of water for homogenizing, adjusting the pH value to 7, adding salivary amylase accounting for 2 percent of the mass of the chick embryo, and carrying out enzymolysis reaction for 1.5 hours at the temperature of 37.5 ℃;

2) then adjusting the pH value to 2, adding pepsin accounting for 2 percent of the mass of the chick embryo, and carrying out enzymolysis reaction for 3.5 hours at the temperature of 37.5 ℃;

3) then adjusting the pH value to 8, adding trypsin accounting for 1.5 percent of the mass of the chick embryo, and carrying out enzymolysis reaction for 4 hours at the temperature of 37.5 ℃;

4) adding streptomyces with the mass of 0.2 percent of that of the chick embryo into the system prepared in the step 3), fermenting at 35 ℃ for 22h, centrifuging for 20min under the condition of 8000r/min, ultrafiltering the separated filtrate by using a hollow fiber membrane with the intercepted relative molecular mass of 3KD, and drying the obtained filtrate in vacuum;

5) and 4) adding 4 times of water into the dried product to dissolve, performing DA-201C type macroporous resin column chromatography, eluting with water, eluting with ethanol, collecting eluate, concentrating under reduced pressure, and drying to obtain the chick embryo peptide.

The chicken embryo peptide prepared by the method can be obtained by measuring the prepared small peptide by HPLC, and the relative molecular mass of the chicken embryo peptide is less than 91.8% of that of the small peptide with 2 KD.

Claims

1. A method for producing a tri-enzyme mono-fermented chick embryo peptide, comprising the steps of:

1) taking out the chick embryo which is hatched for 8-15 days, crushing, adding 2 times of water for homogenizing, adjusting the pH value to 6.5-7.5, adding salivary amylase accounting for 1% of the mass of the chick embryo, and carrying out enzymolysis reaction for 2 hours at the temperature of 37.5 ℃;

2) then adjusting the pH value to 1.5-2.0, adding pepsin with the mass of 0.5% of that of the chick embryo, and carrying out enzymolysis reaction for 4 hours at the temperature of 38 ℃;

3) then adjusting the pH value to 7.5-8.0, adding trypsin with the mass of 0.5% of that of the chick embryo, and carrying out enzymolysis reaction for 5 hours at the temperature of 38 ℃;

4) adding streptomyces with the mass of 0.08 percent of that of the chick embryo into the system prepared in the step 3), fermenting for 25h at 33 ℃, centrifuging, ultrafiltering the separated filtrate by using a hollow fiber membrane with the intercepted relative molecular mass of 3KD, and drying the obtained filtrate in vacuum;

5) and 4) adding water to dissolve the dried product, performing DA-201C type macroporous resin column chromatography, eluting with water, eluting with ethanol, collecting the eluent, concentrating under reduced pressure, and drying to obtain the chick embryo peptide.

2. A method for producing a tri-enzyme mono-fermented chick embryo peptide, comprising the steps of:

1) taking out the chick embryo which is hatched for 8-15 days, crushing, adding 1.5 times of water for homogenizing, adjusting the pH value to 6.5-7.5, adding salivary amylase accounting for 2% of the mass of the chick embryo, and carrying out enzymolysis reaction for 1.5h at the temperature of 37.5 ℃;

2) then adjusting the pH value to 1.5-2.0, adding pepsin accounting for 2% of the mass of the chick embryo, and carrying out enzymolysis reaction for 3.5 hours at the temperature of 37.5 ℃;

3) then adjusting the pH value to 7.5-8.0, adding trypsin accounting for 1.5% of the mass of the chick embryo, and carrying out enzymolysis reaction for 4 hours at the temperature of 37.5 ℃;

4) adding streptomyces with the mass of 0.2 percent of that of the chick embryo into the system prepared in the step 3), fermenting for 22h at 35 ℃, centrifuging, ultrafiltering the separated filtrate by using a hollow fiber membrane with the intercepted relative molecular mass of 3KD, and drying the obtained filtrate in vacuum;