阅读说明：本技术 一种具有促益生菌生长和抗氧化作用的牛骨蛋白糖肽及其制备方法与应用 (Bovine bone protein glycopeptide with probiotic growth promoting and oxidation resisting effects and preparation method and application thereof ) 是由 张恒 罗永康 刘怀高 于 2021-02-19 设计创作，主要内容包括：本发明涉及动物食品加工技术领域,具体涉及一种具有促益生菌生长和抗氧化作用的牛骨蛋白糖肽及其制备方法与应用。本发明提供的牛骨蛋白糖肽制备方法,包括如下步骤：以牛骨为原料,将经预处理的原料经两步酶解制得酶解物；所述两步酶解中,第一步酶解使用的复合蛋白酶为碱性蛋白酶、中性蛋白酶、胰蛋白酶,第二步酶解使用的复合蛋白酶为风味蛋白酶、木瓜蛋白酶。本发明所述的牛骨胶原蛋白糖肽是牛骨胶原蛋白肽与低聚果糖的糖基化反应产物,且含有特定的氨基酸多肽序列,具有促进益生菌生长和抗氧化的功能。(The invention relates to the technical field of animal food processing, in particular to bovine bone protein glycopeptide with probiotic growth promoting and anti-oxidation effects and a preparation method and application thereof. The preparation method of the bovine bone protein glycopeptide provided by the invention comprises the following steps: taking ox bone as a raw material, and performing two-step enzymolysis on the pretreated raw material to prepare an zymolyte; in the two-step enzymolysis, the compound protease used in the first step of enzymolysis is alkaline protease, neutral protease and trypsin, and the compound protease used in the second step of enzymolysis is flavourzyme and papain. The bovine bone collagen glycopeptide is a glycosylation reaction product of bovine bone collagen peptide and fructo-oligosaccharide, contains a specific amino acid polypeptide sequence, and has the functions of promoting the growth of probiotics and resisting oxidation.)

1. The preparation method of the bovine bone protein glycopeptide is characterized by comprising the following steps: taking ox bones as a raw material, and performing two-step enzymolysis on the pretreated raw material to prepare an enzymolysis product; and performing glycosylation reaction on the enzymolysis product after ultrafiltration and purification and fructo-oligosaccharide.

2. The preparation method according to claim 1, wherein in the two steps of enzymolysis, the alkaline protease in the compound protease used in the first step of enzymolysis is: neutral protease: trypsin is (1-2): 1: 1; and (3) performing enzymolysis on the compound protease used in the second step: the papain is (1-2): 1.

3. the preparation method according to claim 2, wherein the bone protein content of the bovine bone protein fluid obtained by the pretreatment is 11-13%; the compound protease used in the first step of enzymolysis accounts for 0.5 to 0.9 percent of the bone protein; the compound protease used in the second step of enzymolysis accounts for 0.1-0.3% of the bone protein.

4. The preparation method according to claim 3, wherein the conditions of the first step of enzymolysis are that the reaction is carried out for 0.5-2h at 50-60 ℃; the second step enzymolysis is carried out at 50-60 deg.C for 0.2-1h, and then the temperature is kept at 95-100 deg.C for 10-20 min.

5. The method of manufacturing according to claim 4, wherein the pre-treatment comprises:

adding water into the pulverized Os bovis Seu Bubali, treating at 125 deg.C for 90-150min at 100-.

6. The process according to any one of claims 1 to 5, wherein the ultrafiltration purification of the product of the two steps of enzymatic hydrolysis comprises the following steps:

and (3) performing ultrafiltration by using a nano-grade ceramic membrane with the pore diameter of 6000 daltons and 3000 daltons to separate a protein peptide solution with the molecular weight of less than 3000.

7. A bovine bone protein glycopeptide, which is produced by the production method according to any one of claims 1 to 6; the bovine bone protein glycopeptide is a glycosylation reaction product of bovine bone collagen peptide and fructo-oligosaccharide.

8. The bovine bone protein glycopeptide of claim 7, comprising more than 50% of the small peptides Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala.

9. Use of the bovine bone protein glycopeptide according to any one of claims 1-6 or 7-8 for the preparation of a pharmaceutical product, a food product or a food additive.

10. A pharmaceutical, food or food additive comprising the bovine osteocalcin glycopeptide of any one of claims 7 to 8.

Technical Field

The invention relates to the technical field of animal food processing, in particular to a bovine bone protein glycopeptide with functions of promoting growth of probiotics and resisting oxidation and a preparation method of the bovine bone protein glycopeptide.

Background

Probiotics are a class of microorganisms that are of great significance to human health. The common probiotics include Bifidobacterium, Lactobacillus, lactococcus, and Streptococcus thermophilus. The probiotics have the functions of promoting the organism to absorb nutrient substances, improving the organism immunity, improving the organism oxidation resistance level, inhibiting intestinal inflammation and the like, have important effects on the human health, and how to efficiently promote the growth of the probiotics becomes a problem to be solved. At present, many studies report that functional oligosaccharides can selectively promote proliferation of lactic acid bacteria, and common functional oligosaccharides include soybean oligosaccharide, fructo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide and the like.

In recent years, with the intensive research on human health and the progress of techniques for separating, purifying and identifying substances, the physiological functions of polypeptides between amino acid monomers and macromolecular proteins are gaining increasing scientific attention. According to the raw material source of the peptide, the exogenous bioactive peptide can be divided into animal-derived bioactive peptide, plant-derived bioactive peptide, microbial bioactive peptide and the like. Among various sources of the bioactive peptide, the animal bone protein resource is rich, and the difference between the physiological structure and the physiological process and other materials is large, so that the bioactive peptide becomes a treasure house of the exogenous bioactive peptide resource. At present, a series of bioactive peptides with physiological functions of resisting oxidation, reducing blood pressure, reducing blood sugar, resisting tumors and the like are found in the bone protein bioactive peptides. However, no related patent and research reports exist about the effect of the bone protein peptide on the growth of the probiotics.

Disclosure of Invention

The invention aims to provide the bovine bone protein glycopeptide with the functions of promoting the growth of probiotics and resisting oxidation and the preparation method of the bovine bone protein glycopeptide.

The invention aims to develop bovine bone protein glycopeptide which has the functions of promoting the growth of probiotics and resisting oxidation. Aiming at the characteristics of the raw material characteristics, the components and the like of the bovine bone, the process for producing the bovine bone protein glycopeptide with the functions of promoting the growth of probiotics and resisting oxidation by taking the bovine bone and fructo-oligosaccharide as raw materials is developed. Polypeptide products of protein in the bovine bone after enzymolysis are quite complex in composition, and comprise a large number of polypeptides with unknown sequences and unknown functions, and active peptides with specific functions may comprise a plurality of peptides with different amino acid compositions and different molecular weights, so that the peptides are difficult to distinguish through certain common characteristics. These all bring great difficulty in the development of bovine bone protein peptides having specific functions.

The bovine bone protein peptide in the prior art does not have the function of promoting the growth of probiotics, and the bovine bone protein peptide with better functions of resisting oxidation and promoting probiotics is less. In addition, in the prior art, acid and alkali are needed to be added to adjust the pH value in the enzymolysis process, which not only increases the complexity and cost of the process, but also may affect the functional characteristics of the product and increase the ash content of the product.

In the enzymolysis development process in the prior art, although the peptide with smaller molecular weight can be obtained, the obtained peptide does not have the function of promoting probiotics at all, and some methods need to rely on acid and alkali to repeatedly adjust pH. In a plurality of methods, the invention determines a two-step enzymolysis method which is suitable for bovine bones and does not depend on acid, alkali or pH, and the specific bovine bone collagen peptide obtained by enzymolysis is subjected to glycosylation reaction with fructo-oligosaccharide, so that the bovine bone collagen glycopeptide with better functions of resisting oxidation and promoting the growth of probiotics can be obtained.

In a first aspect, the present invention provides a method for preparing bovine bone protein glycopeptide having probiotic growth promoting and antioxidant functions, comprising the steps of: taking ox bones as a raw material, and performing two-step enzymolysis on the pretreated raw material to prepare an enzymolysis product; and performing glycosylation reaction on the enzymolysis product after ultrafiltration and purification and fructo-oligosaccharide.

In the preparation method, in the two-step enzymolysis, the alkaline protease in the compound protease used in the first step enzymolysis comprises the following steps: neutral protease: trypsin is (1-2): 1: 1; in the compound protease used in the second step of enzymolysis, flavourzyme: the papain is (1-2): 1.

in the preparation method provided by the invention, the bovine bone protein content in the bovine bone protein liquid obtained by pretreatment is 11-13%; the compound protease used in the first step of enzymolysis accounts for 0.5 to 0.9 percent of the bovine bone protein; the compound protease used in the second step of enzymolysis accounts for 0.1-0.3% of the bovine bone protein.

In the preparation method provided by the invention, the condition of the first step of enzymolysis is that the reaction is carried out for 0.5 to 2 hours at the temperature of between 50 and 60 ℃; the second step enzymolysis is carried out at 50-60 deg.C for 0.2-1h, and then the temperature is kept at 95-100 deg.C for 10-20 min.

In the preparation method provided by the invention, the pretreatment comprises the following steps: adding water into the crushed bovine bone, treating at the temperature of 100-. The specific frequency ultrasonic wave is combined with the ultrahigh pressure technology for processing, so that the tissue structure of the bone protein can be changed, and the obtained bovine bone protein liquid has higher bone protein content.

In the preparation method provided by the invention, the product obtained after the two steps of enzymolysis is subjected to ultrafiltration purification, and the preparation method comprises the following steps:

ultrafiltering with nanometer ceramic membrane with pore size of 6000 daltons and 3000 daltons, and separating protein peptide solution with molecular weight less than 3000 daltons;

separating the protein peptide liquid with the molecular weight less than 3000 by SephadexG-15 gel, detecting at 280nm and collecting the 2 nd elution peak; and (3) carrying out RP-HPLC reversed-phase high performance liquid chromatography on the collected protein peptide liquid, and collecting the protein peptide liquid when the time is 9-12 minutes.

In the preparation method provided by the invention, the mass ratio of the protein peptide solution obtained by RP-HPLC reversed-phase high performance liquid chromatography to fructo-oligosaccharide is adjusted to (8-10): 1, reacting at 80-90 ℃ for 30-60min, concentrating, and freeze-drying to obtain the bovine bone protein glycopeptide product.

The main components of the protein peptide solution obtained by RP-HPLC reversed-phase high performance liquid chromatography are measured by LC-MS/MS, and the detection result shows that the content of the peptide with the amino acid sequence of Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala is 50-55%.

The invention aims at the problems that the bone resources in China are rich and the bone protein resources are not fully utilized at present, according to the characteristics of bone protein, the bovine bone is taken as a raw material, the high-pressure, high-frequency ultrasonic wave, high-static pressure water and the like of the bovine bone are pretreated, multiple protein complex enzymes are utilized for step-by-step enzymolysis under the condition of not adding any acid and alkali, and the bovine bone protein glycopeptide with the functions of promoting the growth of probiotics and resisting oxidation is developed by the membrane separation, gel separation and reversed-phase HPLC separation technologies, so that a set of bovine bone protein glycopeptide with specific functions is established.

Specifically, the preparation method of the bovine bone protein glycopeptide with the functions of promoting the growth of probiotics and resisting oxidation comprises the following steps:

(1) taking bovine bones as a raw material, crushing the raw material into particles with the particle size of 8-15mm, then adding water with the weight of 1-3 times of the weight of the bone particles, treating at the temperature of 100 ℃ and 125 ℃ for 90-150 minutes, and removing fat and bone residues to obtain bovine bone protein liquid;

(2) adjusting the bovine bone protein liquid obtained in the step (1) to 70-80 ℃, and after carrying out ultrasonic treatment for 20-30 minutes by using 110-130kH, carrying out treatment for 20-30 minutes by using 400-500Mpa to change the tissue structure of the bone protein;

(3) regulating the protein content in the bovine bone protein liquid obtained in the step (2) to 11-13%, and adding compound protease accounting for 0.6-1.1% of the weight of the protein to perform two-step enzymolysis; alkaline protease in the complex enzyme used in the first step of enzymolysis: neutral protease: trypsin is (1-2): 1: 1, carrying out enzymolysis for 0.5-2.0h at 50-60 ℃; and (3) performing enzymolysis on the compound protease used in the second step: the papain is (1-2): 1, carrying out enzymolysis at 50-60 ℃ for 0.2-1h, then preserving the temperature at 95-100 ℃ for 10-20 min, cooling to room temperature, filtering and separating through a plate frame, and collecting a separation solution;

(4) performing two-step ultrafiltration treatment on the separation liquid obtained in the step (3), performing ultrafiltration by using a nano-grade ceramic membrane with the aperture of 6000 daltons and 3000 daltons, and separating the bovine bone protein peptide liquid with the molecular weight of less than 3000;

(5) taking bovine bone protein peptide liquid with the molecular weight less than 3000, carrying out Sephadex G-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and collecting the 2 nd elution peak; concentrating, freezing and drying the collected bovine bone protein peptide liquid to obtain bovine bone protein peptide;

(6) performing RP-HPLC reversed-phase high performance liquid chromatography on the bovine bone protein peptide liquid obtained by gel separation and collection, and collecting the protein peptide solution for 9-12 minutes, wherein the separation condition of the reversed-phase HPLC is that 5-90% acetonitrile solution is used as eluent; adjusting the mass ratio of the protein peptide solution obtained by RP-HPLC reversed-phase high performance liquid chromatography to fructo-oligosaccharide to (8-10): 1, reacting at 80-90 ℃ for 30-60min, concentrating, and freeze-drying to obtain the bovine bone protein glycopeptide product.

In a second aspect, the present invention provides a bovine bone protein glycopeptide prepared by the above preparation method; the bovine bone protein glycopeptide is a glycosylation reaction product of bovine bone collagen peptide and fructo-oligosaccharide.

The bovine bone protein glycopeptide provided by the invention contains more than 50% of small peptides shown by Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala.

The bovine bone protein glycopeptide provided by the invention is a product obtained by glycosylation reaction of protein peptide containing Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala and fructo-oligosaccharide; test and detection prove that the bovine bone protein glycopeptide has the effects of promoting the growth of probiotics and resisting oxidation, wherein the probiotics are lactobacillus acidophilus NCFM and/or bifidobacterium animalis Bb-12.

According to the understanding of the skilled person, the present invention also claims the above-mentioned preparation method or the above-mentioned application of the bovine bone protein glycopeptide in the preparation of a pharmaceutical product, a food product or a food additive. The invention also claims a food, a medicine or a food additive containing the bovine bone protein glycopeptide.

The invention has the beneficial effects that:

(1) the novel bovine bone protein glycopeptide developed by utilizing the bovine bone and the fructo-oligosaccharide has no acid or alkali added for adjusting the pH in the whole processing process, and the product keeps better functional characteristics;

(2) the bovine bone protein glycopeptide product developed by the invention has better functions of promoting the growth of probiotics and resisting oxidation;

(3) the bovine bone protein liquid treated by combining specific frequency ultrasonic waves with an ultrahigh pressure technology obviously improves the sensitivity of bone protein to enzyme, the protein content in the enzymolysis liquid is more than 11%, and the use amount of the enzyme is reduced;

(4) the invention adopts a plurality of food-grade compound proteases (alkaline protease, neutral protease, trypsin, flavourzyme and papain), bovine bone protein peptide obtained by moderate enzymolysis under mild conditions reacts with fructo-oligosaccharide under mild conditions, no additive is added, and the developed product is safe;

(5) the product developed by the invention has good flavor and color and can be widely applied to special foods and nutritional foods;

(6) the product developed by the invention has a clear peptide composition, wherein the content of the peptides of Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala is more than 50 percent.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Unless otherwise specified, test materials, reagents, instruments and the like used in the examples of the present invention are commercially available; all technical measures in the examples of the present invention are conventional measures well known to those skilled in the art, unless otherwise specified.

The enzyme activity unit of the protease used in the embodiment of the invention is as follows:

alkaline protease: 500000-110000U/mL; neutral protease: 300000 and 650000U/mL; trypsin: 400000 and 1000000U/mL; flavor protease: 150000-250000U/mL; papain: 500000 and 1350000U/mL.

Example 1 an osteocalcin glycopeptide having probiotic growth promoting and antioxidant effects and a method for preparing the same

The embodiment provides a bovine bone protein glycopeptide with probiotic growth promoting and antioxidant effects, and the preparation method comprises the following steps:

(1) selecting frozen ox bone, cleaning with clean water meeting drinking water sanitary standard, pulverizing into granules with particle diameter of 8-15mm with a pulverizer, adding water 2.0 times of bone weight, processing at 125 deg.C for 90 min to obtain ox bone slurry, removing upper fat to obtain defatted ox bone slurry, removing bone residue with a centrifugal separator to obtain ox bone protein liquid;

(2) adjusting the temperature of the bovine bone protein liquid obtained in the step (1) to 80 ℃, treating the bovine bone protein liquid for 20 minutes by ultrasonic waves (the frequency is 110kH) by using an ultrasonic generator, and treating the bovine bone protein liquid for 20 minutes by using high hydrostatic pressure (500Mpa) to change the tissue structure of bone protein;

(3) adjusting the protein content in the bone protein liquid to 13%, adding compound protease according to the weight percentage of 1.0% of the weight of the protein in the bone protein liquid for step-by-step enzymolysis, wherein in the first step, the compound protease (composed of alkaline protease, neutral protease and trypsin in a mass ratio of 2: 1: 1) with the weight of the protein of 0.8% is subjected to enzymolysis reaction for 2.0 hours at the temperature of 50 ℃; the second step is that compound protease (flavourzyme and papain with the mass ratio of 1: 1) with the weight of protein of 0.2 percent is subjected to enzymolysis reaction for 0.5h at the temperature of 55 ℃; preserving the heat at 98 ℃ for 10 minutes, cooling to room temperature, filtering and separating through a plate frame, and collecting a separation solution;

(4) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 6000 daltons, separating proteins and polypeptides with the molecular weight of less than 6000 daltons, and separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons;

(5) taking protein peptide liquid with molecular weight less than 3000, and then carrying out Sephadex G-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, and freeze-drying to obtain bone protein peptide;

separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, wherein the reversed-phase HPLC separation condition is to take a peptide solution collected by 9-12 minutes by taking a 5-90% acetonitrile solution as an eluent;

the content of peptides with amino acid sequences of Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala, which are determined by LC-MS/MS for the main components of the peptide solution, is 51.5 percent;

(6) adjusting the mass ratio of the peptide solution obtained by RP-HPLC reversed-phase high performance liquid chromatography in the step (5) to fructo-oligosaccharide to be 8: 1, reacting at 90 ℃ for 30min, and concentrating, freezing and drying to obtain the bovine bone protein glycopeptide product.

Example 2 bovine bone protein glycopeptide having probiotic growth promoting and antioxidant effects and preparation method thereof

The embodiment provides a bovine bone protein glycopeptide with probiotic growth promoting and antioxidant effects, and the preparation method comprises the following steps:

(1) selecting frozen ox bone, cleaning with clean water meeting drinking water sanitary standard, pulverizing into granules with particle diameter of 8-15mm with a pulverizer, adding water 1.0 times of bone weight, treating at 115 deg.C for 150min to obtain ox bone slurry, removing upper fat to obtain defatted ox bone slurry, and removing bone residue with centrifugal separation stage to obtain ox bone protein liquid;

(2) adjusting the temperature of the bovine bone protein liquid obtained in the step (1) to 70 ℃, treating the bovine bone protein liquid for 30 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 120kH), and treating the bovine bone protein liquid for 30 minutes by using high hydrostatic pressure (500Mpa), so as to change the tissue structure of the bovine bone protein;

(3) adjusting the protein content in the bone protein liquid to 11%, adding compound protease according to the weight percentage of 1.1% of the weight of the protein in the bone protein liquid for step-by-step enzymolysis, wherein in the first step, the compound protease (composed of alkaline protease, neutral protease and trypsin in a mass ratio of 2: 1: 1) with the weight of the protein is 0.8%, and carrying out enzymolysis reaction for 2.0h at the temperature of 50 ℃; the second step is that compound protease (flavourzyme and papain with the mass ratio of 2: 1) with the weight of protein of 0.3 percent is subjected to enzymolysis reaction for 1.0 hour at the temperature of 50 ℃; preserving the heat at 95 ℃ for 20 minutes, cooling to room temperature, filtering and separating through a plate frame, and collecting a separation solution;

(4) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 6000 daltons, separating proteins and polypeptides with the molecular weight of less than 6000 daltons, and separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons;

(5) taking protein peptide liquid with molecular weight less than 3000, and then carrying out Sephadex G-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, and freeze-drying to obtain bone protein peptide;

separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, wherein the reversed-phase HPLC separation condition is to take a peptide solution collected by 9-12 minutes by taking a 5-90% acetonitrile solution as an eluent; the content of peptides with amino acid sequences of Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala, which are 52.7 percent by LC-MS/MS measurement of the main components of the peptide solution;

(6) adjusting the mass ratio of the peptide solution obtained by RP-HPLC reversed-phase high performance liquid chromatography in the step (5) to fructo-oligosaccharide to be 10: 1, reacting at 80 ℃ for 60min, concentrating, and freeze-drying to obtain the bovine bone protein glycopeptide.

Example 3 an osteocalcin glycopeptide having probiotic growth promoting and antioxidant effects and a method of making the same

The embodiment provides a bovine bone protein glycopeptide with probiotic growth promoting and antioxidant effects, and the preparation method comprises the following steps:

(1) selecting fresh or frozen ox bone, cleaning with clean water meeting drinking water sanitary standard, pulverizing into granules with particle diameter of 8-15mm with a pulverizer, adding water 3.0 times of bone weight, treating at 105 deg.C for 150min to obtain ox bone slurry, removing upper fat to obtain defatted ox bone slurry, and removing bone residue by centrifugal separation to obtain ox bone protein liquid;

(2) adjusting the temperature of the bovine bone protein liquid obtained in the step (1) to 75 ℃, treating the bovine bone protein liquid for 25 minutes by using an ultrasonic generator through ultrasonic waves (the frequency is 120kH), and treating the bovine bone protein liquid for 25 minutes by using high hydrostatic pressure (450Mpa), so as to change the tissue structure of bone protein;

(3) regulating the protein content in the bone protein liquid to be 12%, adding compound protease according to the weight percentage of 0.6% of the weight of the protein in the bone protein liquid for carrying out stepwise enzymolysis, wherein in the first step, the compound protease (composed of alkaline protease, neutral protease and trypsin in a mass ratio of 1: 1: 1) with the weight of the protein of 0.5% is subjected to enzymolysis reaction for 1.0h at the temperature of 55 ℃; the second step is that compound protease (the mass ratio of flavourzyme to papain is 1: 1) with the weight of protein of 0.1 percent is subjected to enzymolysis reaction for 0.5h at the temperature of 60 ℃; preserving the heat at 95 ℃ for 15 minutes, cooling to room temperature, filtering and separating through a plate frame, and collecting a separation solution;

(4) treating the separation liquid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using a ceramic membrane with the aperture of 6000 daltons, separating proteins and polypeptides with the molecular weight of less than 6000 daltons, and separating protein peptides with the molecular weight of less than 3000 daltons by using a membrane with the aperture of 3000 daltons;

(5) taking protein peptide liquid with molecular weight less than 3000, and then carrying out Sephadex G-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 2 nd elution peak is collected; concentrating, and freeze-drying to obtain bone protein peptide;

separating the protein peptide liquid for 1 time by RP-HPLC reversed-phase high performance liquid chromatography, wherein the reversed-phase HPLC separation condition is to take a 5-90% acetonitrile solution as an eluent and take a peptide solution collected by 9-12 minutes;

the content of peptides with amino acid sequences of Gly-Pro-Ala-Gly-Ala-Asp-Gly-Ala and Hyp-Gly-Val-Ala-Pro-Gly-Pro-Ala, which are 53.1 percent by determining the main components of the peptide solution through LC-MS/MS;

(6) adjusting the mass ratio of the peptide solution obtained by RP-HPLC reversed-phase high performance liquid chromatography in the step (5) to fructo-oligosaccharide to 9: 1, reacting at 85 ℃ for 60min, concentrating, and freeze-drying to obtain the bovine bone protein glycopeptide.

Experimental example 1 measurement test of ability of bovine bone protein glycopeptide to promote growth of probiotic

Test samples: samples 1, 2 and 3 are bovine osteocalcin glycopeptides prepared in example 1, example 2 and example 3, step (6), respectively; samples 4, 5 and 6 are the products of example 1, step (3), (4) and (5), respectively, after freeze-drying.

The probiotic growth promotion assay was performed as follows:

inoculating activated 2 probiotic strains (Lactobacillus acidophilus NCFM and Bifidobacterium animalis Bb-12) to corresponding liquid culture medium (control group is pure culture medium, experiment 1 group, 2 group, 3 group, 4 group, 5 group and 6 group are respectively added with 5% of samples 1, 2, 3, 4, 5 and 6) in the corresponding culture medium, culturing the culture medium inoculated with the bacterial liquid at 37 ℃ under anaerobic condition, culturing for 6, 12, 18 and 24h, respectively taking 200 mu L of bacterial liquid in a 96-well plate, measuring the absorbance of the bacterial liquid at 600nm by using a microplate reader, and measuring the OD600 value. Meanwhile, 0.5mL of bacterial liquid cultured for 24h is taken to be subjected to gradient dilution in 4.5mL of sterile physiological saline, 100 mu L of bacterial suspension with proper dilution gradient is taken to be coated in a solid culture medium, the coated flat plate is placed under anaerobic conditions and cultured at 37 ℃ for 36h, and the total number of colonies is taken out and calculated.

TABLE 1 Lactobacillus acidophilus NCFM growth test results of bovine bone protein glycopeptide of the present invention

TABLE 2 growth test results of Bifidobacterium (Bb-12), an actuator of the bovine bone protein glycopeptide of the present invention

Experimental example 2 measurement test of antioxidant Activity of bovine bone protein glycopeptide of the present invention

Test samples: samples 1, 2 and 3 are protein peptide products prepared in step (6) of example 1, example 2 and example 3, respectively; samples 4, 5 and 6 are the products prepared by freeze-drying in step (3), (4) and (5) of example 1, respectively; samples 7, 8 are synthetic pure peptide products.

The antioxidant activity was measured as follows:

(1) ability to scavenge DPPH free radicals: taking 1.5mL of 0.2mg/mL sample, adding 1.5mL of 99.5% ethanol and 0.675mL of 0.02% DPPH ethanol solution, mixing, oscillating, mixing uniformly, carrying out water bath at room temperature in a dark place for 30min, and detecting the light absorption value of the system at 517 nm. The lower the light absorption value, the stronger the DPPH free radical scavenging ability of the system. The blank control is to change 1.5mL of sample solution to 1.5mL of deionized water.

DPPH radical scavenging capacity ═ ((blank absorbance-sample absorbance)/blank absorbance) × 100;

(2) and (3) reduction force determination: a0.2 mg/mL sample (1 mL) was added with 2.5mL of 0.2M phosphate buffer (pH 6.6) and 2.5mL of 1% (mass fraction) potassium ferricyanide solution, mixed well, and then heated in a water bath at 50 ℃ for 20 min. Taking out and rapidly cooling, adding 2.5mL of 10% (mass fraction) trichloroacetic acid (TCA) solution, mixing uniformly, and then centrifuging at 3000g for 10 min. Taking 2.5mL of supernatant, adding 2.5mL of deionized water and 0.5mL of 1% (mass fraction) ferric trichloride solution, mixing well, reacting at room temperature for 10min, and measuring absorbance at 700nm wavelength. The reducing power can be expressed as the absorbance at 700 nm.

TABLE 3 antioxidant Activity test results of the bovine bone protein glycopeptide of the present invention

The results in Table 3 show that the bovine bone protein glycopeptide of the invention has better oxidation resistance, and under the condition of 0.2mg/mL, the bovine bone protein glycopeptide has the DPPH free radical scavenging capacity of more than 91 percent and the reducing power of more than 0.90, and is a better oxidation resistance substance.

As can be seen from tables 1 and 2, the bovine bone protein glycopeptide can effectively promote the growth of probiotics, and the developed bovine bone protein glycopeptide has a remarkable promotion effect on the growth of the probiotics compared with a control group. Table 3 shows that the bovine bone protein glycopeptide developed by the invention has obvious effect of promoting the growth of probiotics and also has obvious antioxidant effect.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.