阅读说明：本技术 一种小分子牛骨髓肽及其提取方法 (Small molecule bovine bone marrow peptide and extraction method thereof ) 是由 于海峰 于 2020-06-28 设计创作，主要内容包括：本发明公开了一种小分子牛骨髓肽及其提取方法,所述提取方法包括如下步骤：将鲜骨进行破碎、清洗；对清洗后的鲜骨进行熬煮、过滤,收集熬煮液；将熬煮液进行离心去除脂肪,得到骨胶原蛋白溶液；调节骨胶原蛋白溶液浓度并添加复合酶解剂进行酶解,得到酶解液,其中,复合酶解剂由复配蛋白酶、骨蛋白水解酶和风味酶组成；对酶解液进行杀菌灭酶、固液分离、浓缩和干燥。本发明制备方法通过选用特定的复合酶解剂,通过一次水解,即可充分将骨胶原蛋白水解为小分子的多肽,提高了牛骨髓肽的提取率,同时还能够把多肽上含有的疏水氨基酸进行水解,能够改善苦味肽的口感,保障提取的牛骨髓肽具有优异的口感和风味。(The invention discloses a micromolecule bovine bone marrow peptide and an extraction method thereof, wherein the extraction method comprises the following steps: crushing and cleaning fresh bones; decocting the cleaned fresh bone, filtering, and collecting decoction; centrifuging the decoction to remove fat to obtain a bone collagen solution; adjusting the concentration of the ossein protein solution and adding a compound enzymolysis agent for enzymolysis to obtain enzymolysis liquid, wherein the compound enzymolysis agent consists of compound protease, bone protein hydrolase and flavor enzyme; and (4) sterilizing and inactivating the enzyme, performing solid-liquid separation, concentrating and drying on the enzymolysis liquid. The preparation method of the invention selects the specific compound zymolytic agent, can fully hydrolyze the ossein into micromolecular polypeptide through one-time hydrolysis, improves the extraction rate of the bovine bone marrow peptide, can hydrolyze hydrophobic amino acid contained in the polypeptide, can improve the taste of the bitter peptide, and ensures that the extracted bovine bone marrow peptide has excellent taste and flavor.)

1. A method for extracting small molecule bovine bone marrow peptide is characterized by comprising the following steps:

crushing and cleaning fresh bones;

decocting the cleaned fresh bone, filtering, and collecting decoction;

centrifuging the decoction to remove fat to obtain a bone collagen solution;

adjusting the concentration of the ossein protein solution and adding a compound enzymolysis agent for enzymolysis to obtain enzymolysis liquid, wherein the compound enzymolysis agent consists of compound protease, bone protein hydrolase and flavor enzyme;

and (4) sterilizing and inactivating the enzyme, performing solid-liquid separation, concentrating and drying on the enzymolysis liquid.

2. The extraction method according to claim 1, wherein the mass ratio of the compound protease to the bone proteolytic enzyme to the flavor enzyme in the compound enzymolysis agent is (1-3) to 1.

3. The extraction method according to claim 1 or 2, wherein the mass ratio of the compound protease, the bone protein hydrolase and the flavor enzyme in the compound enzymolysis agent is 2: 1.

4. The extraction method according to claim 1 or 2, wherein the bone proteolytic enzyme activity is (30-35) × 10⁴u/g, flavor enzyme activity of (3-6) × 10⁴u/g。

5. The extraction method according to claim 1 or 2, wherein each part of the compound protease is prepared by compounding 3 parts of bromelain, 2 parts of neutral protease and 3 parts of trypsin;

the pineapple proteinase activity is (20-30) × 10⁴u/g, neutral protease activity of (5-10) × 10⁴u/g, and the trypsin activity is 2000-4000 u/g.

6. The extraction method according to claim 1, wherein the concentration of the collagen solution is adjusted to 5-8 baume, and the mass ratio of the composite enzymolysis agent to the collagen solution is (1-2) to 100.

7. The extraction method according to claim 1, wherein the enzymatic hydrolysis comprises:

adjusting the pH value of the ossein protein solution to 6.0-6.2, and adding a compound enzymolysis agent at 54-56 ℃ for enzymolysis for 3-4 h.

8. The extraction method according to claim 1, wherein the steps of decocting and filtering the cleaned fresh bone and collecting the decoction comprise:

placing the cleaned fresh bone into clear water, and decocting and filtering for the first time to obtain a first decoction and decocted broken bone;

then, carrying out secondary boiling and filtering on the boiled broken bones and clear water to obtain secondary boiled liquid;

mixing the first decoction and the second decoction to obtain a decoction;

wherein: during the first time of boiling, the mass ratio of the fresh bones to the clear water is 1: 5-6, the boiling temperature is 110-120 ℃, and the boiling time is 2-3 h;

when decocting for the second time, the mass ratio of the decocted broken bones to the clear water is 1: (3-4), wherein the boiling temperature is 110-120 ℃, and the boiling time is 1-2 h.

9. The extraction method according to claim 1, wherein the filtration adopts a filter medium with a pore size of 150 meshes;

the aperture of the filter medium used for solid-liquid separation is 0.2-0.5 μm.

10. The small molecular bovine bone marrow peptide prepared by the extraction method according to any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of polypeptide preparation, in particular to a small-molecule bovine bone marrow peptide and an extraction method thereof.

Background

Bovine bone marrow peptide is a protein fragment obtained by enzymolysis of bovine bone marrow protein, can be directly absorbed by human body, and directly participates in life activities; the bovine bone marrow peptide has the beneficial effects of repairing damaged cells, stimulating immunoglobulin to generate antibodies, activating the activity of an intracellular lysozyme system and the like.

At present, the extraction method of bovine bone marrow peptide mainly comprises the steps of crushing, cooking, carrying out enzymolysis for multiple times, inactivating enzyme, filtering, concentrating and drying to obtain bovine bone marrow peptide; however, the bovine bone marrow peptide prepared by the existing method has the problems of low extraction rate, low content of the small molecular weight bovine bone marrow peptide and long production period caused by multiple times of enzymolysis.

In view of the above, the present invention is especially proposed!

Disclosure of Invention

The invention aims to provide a method for extracting small-molecule bovine bone marrow peptide, which can obviously improve the extraction rate of extracting bovine bone marrow peptide from bovine bones, only needs a complex enzyme preparation for enzymolysis once and has short production period.

The second purpose of the invention is to provide a small molecule bovine bone marrow peptide, which has high content of small molecule peptide and can be better absorbed by human body.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the first aspect of the embodiments of the present invention provides a method for extracting small molecule bovine bone marrow peptide, where the method includes the following steps:

crushing and cleaning fresh bones;

decocting the cleaned fresh bone, filtering, and collecting decoction;

centrifuging the decoction to remove fat to obtain a bone collagen solution;

adjusting the concentration of the ossein protein solution and adding a compound enzymolysis agent for enzymolysis to obtain enzymolysis liquid, wherein the compound enzymolysis agent consists of compound protease, bone protein hydrolase and flavor enzyme;

and (3) sterilizing and inactivating the enzyme of the enzymolysis liquid, performing solid-liquid separation, concentrating by using an ultrafiltration membrane and drying.

The preparation method can fully hydrolyze the ossein into micromolecular polypeptide by selecting the specific compound enzymolysis agent and performing primary hydrolysis, thereby improving the extraction rate of the bovine bone marrow peptide, simultaneously hydrolyzing hydrophobic amino acid contained in the polypeptide, improving the taste of the bitter peptide and ensuring that the extracted bovine bone marrow peptide has excellent taste and flavor.

Researches show that the main amino acids rather than polypeptides are contained in the bovine bone marrow peptide with the molecular weight below 180, so that the higher the content of the polypeptides with the molecular weight of more than 180 and close to the molecular weight of 180 is, the better the polypeptide content is, the technical scheme of the application can obviously improve the content of the polypeptides with the molecular weight of 180-500 in the extracted bovine bone marrow peptide by selecting the specific composite enzymolysis agent, and particularly can reach 41%, so that the bovine bone marrow peptide extracted by the application is easier to digest and absorb, and has a better utilization rate.

Preferably, the crushing is to crush fresh bones to 2-4 cm³(ii) a The cleaning is to clean the broken bones in a cleaning machine for 20-25 min and then soak the broken bones in clean water for 4-5 h.

Preferably, the mass ratio of the compound protease to the bone proteolytic enzyme to the flavor enzyme in the compound enzymolysis agent is (1-3) to 1; more preferably, the mass ratio of the compound protease, the bone proteolytic enzyme and the flavor enzyme in the compound zymolyte is 2: 1.

Preferably, the activity of the bone proteolytic enzyme is (30-35) × 10⁴u/g, flavor enzyme activity of (3-6) × 10⁴u/g。

Preferably, each part of the compound protease is prepared by compounding 3 parts of bromelain, 2 parts of neutral protease and 3 parts of trypsin, and more preferably, the activity of the bromelain is (20-30) × 10⁴u/g, neutral protease activity of (5-10) × 10⁴u/g, and the trypsin activity is 2000-4000 u/g.

Preferably, the enzymatic hydrolysis comprises:

adjusting the pH value of the ossein protein solution to 6.0-6.2, and adding a compound enzymolysis agent at 54-56 ℃ for enzymolysis for 3-4 h.

Through the specific limitation of enzymolysis conditions, the activity of each enzyme is regulated, the collagen is hydrolyzed into polypeptide by one-time hydrolysis under the combined action of different enzymes, and the amount and the size of the hydrolyzed polypeptide are ensured.

Preferably, the concentration of the collagen solution is adjusted to 5-8 Baume, and the mass ratio of the composite enzymolysis agent to the collagen solution is (1-2) to 100.

Preferably, the step of decocting and filtering the cleaned fresh bone and collecting the decoction comprises:

placing the cleaned fresh bone into clear water, and decocting and filtering for the first time to obtain a first decoction and decocted broken bone;

then, carrying out secondary boiling and filtering on the boiled broken bones and clear water to obtain secondary boiled liquid;

mixing the first decoction and the second decoction to obtain a decoction;

wherein: during the first time of boiling, the mass ratio of the fresh bones to the clear water is 1: 5-6, the boiling temperature is 110-120 ℃, and the boiling time is 2-3 h;

when decocting for the second time, the mass ratio of the decocted broken bones to the clear water is 1: (3-4), wherein the boiling temperature is 110-120 ℃, and the boiling time is 1-2 h.

Through the specific boiling process, the bovine bone marrow protein can be better extracted, and the extraction rate of the bovine bone marrow peptide can be better guaranteed.

Preferably, the filtration is carried out using a filter medium having a pore size of 150 mesh.

Preferably, the aperture of the filter medium used for solid-liquid separation is 0.2-0.5 μm.

The invention also provides a small molecular bovine bone marrow peptide prepared by the extraction method.

Compared with the prior art, the invention has the beneficial effects that at least:

the preparation method of the invention selects the specific compound zymolytic agent, can fully hydrolyze the ossein into micromolecular polypeptide through one-time hydrolysis, improves the extraction rate of the bovine bone marrow peptide, can hydrolyze hydrophobic amino acid contained in the polypeptide, can improve the taste of the bitter peptide, and ensures that the extracted bovine bone marrow peptide has excellent taste and flavor.

The bovine bone marrow peptide prepared by the invention has high content of polypeptide with molecular weight more than 180 and molecular weight close to 180, and the content of polypeptide with molecular weight between 180 and 500 in the bovine bone marrow peptide can reach 41%, so that the bovine bone marrow peptide extracted by the invention is easier to digest and absorb, and has better utilization rate.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

The following examples used the following starting materials:

the activity of the bone protein hydrolase is (30-35) × 10⁴u/g, purchased from bioscience, Inc., Yonghuadao, east;

flavor enzyme with activity of (3-6) × 10⁴u/g; purchased from biosciences, Inc., Yonghuadao, east;

each part of compound protease is formed by compounding 3 parts of bromelain, 2 parts of neutral protease and 3 parts of trypsin, wherein:

bromelain with activity of (20-30) × 10⁴u/g, purchased from Shanghai-derived leaf Biotechnology, Inc.;

neutral protease with activity of (5-10) × 10⁴u/g, purchased from bioscience, Inc., Yonghuadao, east;

trypsin: the activity is 2000-4000 u/g, and the product is purchased from Tonghengdao Biotechnology Co.