阅读说明：本技术 一种牛骨胶原蛋白肽及其生产方法 (Bovine bone collagen peptide and production method thereof ) 是由 王朝辉 于 2021-02-02 设计创作，主要内容包括：本发明属于胶原蛋白肽领域,一种牛骨胶原蛋白肽及其生产方法,生产方法包括以下步骤：(1)原料前处理；(2)原料纯化；(3)乙酸溶液震摇浸提；(4)一次酶解；(5)二次酶解；(6)活性碳过滤；(7)无机纳滤陶瓷膜过滤；(8)浓缩；(9)喷雾干燥。本发明充分利用牛类加工副产物牛骨制备胶原蛋白肽,成本低廉,胶原提取率大于85％；制备的牦牛骨胶原蛋白寡肽为白色或淡黄色粉末,纯度大于90％,分子量小于1000Da,无异味；本发明将牛骨制备成骨粉后,利用NaOH溶液、正己烷溶液和乙二胺四乙酸二钠盐浓缩溶液纯化可以将牛骨泥中的非胶原物质、脂肪和钙质基本分离去除,制备的活性肽产品不粘结,基本不含脂肪、胆固醇等杂质。(The invention belongs to the field of collagen peptide, and relates to bovine bone collagen peptide and a production method thereof, wherein the production method comprises the following steps: (1) pretreating raw materials; (2) purifying raw materials; (3) shaking and leaching the acetic acid solution; (4) carrying out primary enzymolysis; (5) carrying out secondary enzymolysis; (6) filtering with activated carbon; (7) filtering with inorganic nanofiltration ceramic membrane; (8) concentrating; (9) and (5) spray drying. The invention fully utilizes the byproducts of the processing of cattle, namely the ox bone, to prepare the collagen peptide, has low cost, and the extraction rate of the collagen is more than 85 percent; the prepared yak bone collagen oligopeptide is white or faint yellow powder, the purity is more than 90%, the molecular weight is less than 1000Da, and no peculiar smell exists; according to the invention, after the ox bone is prepared into bone powder, the non-collagen substance, fat and calcium in the ox bone paste can be basically separated and removed by using NaOH solution, n-hexane solution and ethylenediaminetetraacetic acid disodium salt concentrated solution for purification, and the prepared active peptide product is not sticky and basically does not contain impurities such as fat, cholesterol and the like.)

1. The production method of the bovine bone collagen peptide is characterized by comprising the following steps:

(1) pretreatment of raw materials: removing impurities on bones of the ox bones, crushing the ox bones into bone blocks of 2-3 cm by using a bone crusher, cleaning the crushed ox bones, and crushing the ox bones into 50 meshes;

(2) raw material purification: placing the crushed bovine bone meal into 0.1mol/L NaOH solution with the mass being 18-20 times of that of the crushed bovine bone meal, shaking for 1-3 hours at the temperature of 3-5 ℃ to remove non-collagen substances, filtering, repeatedly washing a bone sample with distilled water to be neutral, draining, placing the bone sample into n-hexane solution with the concentration of 10% according to the material-to-liquid ratio of 1: 6-8, shaking for degreasing for 1-3 hours at the temperature of 3-5 ℃ to remove fat, filtering, repeatedly washing the bone sample with distilled water to be neutral, draining, adding the bone sample into 0.25mol/L disodium ethylenediamine tetraacetate concentrated solution according to the material-to-liquid ratio of 1: 8-10, shaking for decalcification for 1-3 hours at the temperature of 3-5 ℃ to remove calcium, and freeze-drying to obtain purified bovine bone meal;

(3) placing the purified bovine bone meal into 0.5mol/L acetic acid solution according to the material-to-liquid ratio of 1: 8-10, extracting for 10-20h under shaking at the temperature of 3-5 ℃, centrifuging and collecting supernatant to obtain bovine bone collagen solution;

(4) primary enzymolysis: adjusting the pH value of the bovine bone collagen solution to 7-9, and adding bone collagenase to carry out ultrasonic-assisted enzymolysis, wherein the primary enzymolysis temperature is 50-55 ℃, the primary enzymolysis time is 1-3 h, and the addition amount of the bone collagenase is 0.1-0.3% of the mass of the bovine bone collagen; after enzymolysis, heating to 80-85 ℃, keeping the temperature for 10-20 min, and inactivating enzyme to obtain primary enzymolysis liquid;

(5) secondary enzymolysis: adjusting the pH value of the primary enzymolysis liquid to 5-7, adding a secondary compound enzyme for ultrasonic-assisted enzymolysis, wherein the secondary compound enzyme adopts pepsin, trypsin, papain and flavourzyme, and obtaining a secondary enzymolysis liquid after the secondary enzymolysis reaction is finished; carrying out microwave enzyme deactivation treatment on the secondary enzymatic hydrolysate, and obtaining enzyme-deactivated enzymatic hydrolysate after the microwave enzyme deactivation is finished;

(6) filtering with activated carbon: adding activated carbon into the enzyme-deactivated enzymolysis liquid, wherein the adding amount of the activated carbon is 2-4% of the mass of the enzyme-deactivated enzymolysis liquid, heating the enzyme-deactivated enzymolysis liquid to 80-100 ℃, and preserving the heat for 5-10 min; then cooling to 70-80 ℃, separating by diatomite, decoloring and removing fishy smell to obtain enzymatic hydrolysate;

(7) filtering with an inorganic nanofiltration ceramic membrane: filtering the enzymatic hydrolysate filtered by the activated carbon by using an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da to obtain a permeate which is a high-purity bovine bone collagen peptide solution;

(8) concentration: controlling the material temperature to be 55-65 ℃, concentrating the material fed into the concentrator in each batch for no more than 30 minutes under the vacuum degree of 0.06-0.08 Mpa until the Baume degree is 20-22 Be, and temporarily storing the concentrated solution in a concentrated solution storage tank;

(9) spray drying: the material temperature is 45-50 ℃, the Baume degree is 20-22 Bee, the air inlet temperature of the spraying tower is adjusted to be 180-190 ℃, the air outlet temperature is 85-95 ℃, and the materials are dried into powder.

2. The method for producing bovine bone collagen peptide according to claim 1, wherein the filter core of the inorganic nanofiltration ceramic membrane in the step (7) is made of any one of silicon, aluminum, magnesium, silicon-aluminum and silicon-magnesium.

3. Bovine bone collagen peptide produced by the method of any one of claims 1 to 2.

Technical Field

The invention belongs to the field of collagen peptides, and particularly relates to bovine bone collagen peptide and a production method thereof.

Background

The collagen is rich in amino acids such as glycine, proline, hydroxyproline and the like required by human bodies. Collagen is a high molecular functional protein, which is the main component of skin and accounts for 80% of the proportion of the dermis layer of the skin, and forms a fine elastic net in the skin, firmly locks water and supports the skin. The collagen is a spiral fibrous protein formed by twisting three peptide chains, is also the most abundant protein in human body, is widely distributed in connective tissues, skin, bones, visceral intercellular substances, muscle cavities, ligaments, sclera and other parts, approximately accounts for more than 30 percent of the total protein of the human body, is rich in collagen characteristic amino acids such as proline, hydroxyproline and the like required by the human body, and is an important component in human body cells, particularly skin extracellular matrix. Collagen is a macromolecular protein, the molecular weight of which is above 300,000Da, and cannot be directly absorbed by human bodies.

Collagen peptide is a product of collagen hydrolysis, and is a substance between amino acids and macromolecular protein, 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. Peptides are precise fragments of proteins whose molecules are only nanometer in size. Modern researches show that compared with protein, the peptide is easier to digest and absorb, can rapidly provide energy for organisms, has the characteristics of no protein denaturation, low allergy, good water solubility and the like, and has multiple bioactive functions. The bovine bone collagen peptide obtained by enzymolysis of collagen peptide extracted from bovine bone has special functional effects in increasing bone density, preventing and treating osteoarthritis and osteoporosis, inhibiting activity of angiotensin converting enzyme, inhibiting activity of platelet aggregation, resisting oxidation activity and resisting tumor activity.

Chinese invention patent application (CN201510556199.0) discloses a preparation method of bovine bone collagen peptide, which relates to a preparation method of protein peptide. The invention mainly aims to solve the problems of low extraction rate, easy browning, high energy consumption and long production period of the existing production process for preparing the bovine bone collagen peptide by adopting two crushing, two re-cooking, high-temperature cooking and ultrafiltration membrane concentration technologies in sequence on bovine bones and combining a specific complex enzyme preparation. The method comprises the following steps: firstly, screening; secondly, crushing for the first time; thirdly, secondary crushing; fourthly, cleaning; fifthly, high-temperature extraction; sixthly, re-boiling for the second time; seventhly, filtering and separating; eighthly, liquid-liquid separation; ninth, enzymolysis; tenth, sterilizing and inactivating enzyme; eleven, solid-liquid separation; twelfth, concentrating by using an ultrafiltration membrane, thirteen, and spray drying; fourteen, packaging to obtain the bovine bone collagen peptide.

Chinese patent application (CN201710823158.2) discloses a method for extracting bovine bone collagen peptide. Comprises the following steps of firstly, breaking bone; secondly, feeding and cleaning; thirdly, feeding, cooking and discharging soup; fourthly, hydrolysis and filtration; fifthly, concentration; sixthly, drying and spraying powder. The extraction rate of the bovine bone collagen peptide obtained by the method is not enough, and the small molecular active polypeptide is not enough.

Disclosure of Invention

The invention aims to provide the bovine bone collagen peptide which is simple and safe, easy to operate, low in cost and high in purity of the produced product and the production method thereof, so that byproducts of bovine processing are fully utilized, waste is turned into wealth, and the bovine bone collagen peptide with wide application prospect is developed.

The technical scheme for solving the technical problems is as follows:

a method for producing bovine bone collagen peptide comprises the following steps:

(1) pretreatment of raw materials: removing impurities on bones of the ox bones, crushing the ox bones into bone blocks of 2-3 cm by using a bone crusher, cleaning the crushed ox bones, and crushing the ox bones into 50 meshes;

(2) raw material purification: placing the crushed bovine bone meal into 0.1mol/L NaOH solution with the mass being 18-20 times of that of the crushed bovine bone meal, shaking for 1-3 hours at the temperature of 3-5 ℃ to remove non-collagen substances, filtering, repeatedly washing a bone sample with distilled water to be neutral, draining, placing the bone sample into n-hexane solution with the concentration of 10% according to the material-to-liquid ratio of 1: 6-8, shaking for degreasing for 1-3 hours at the temperature of 3-5 ℃ to remove fat, filtering, repeatedly washing the bone sample with distilled water to be neutral, draining, adding the bone sample into 0.25mol/L disodium ethylenediamine tetraacetate concentrated solution according to the material-to-liquid ratio of 1: 8-10, shaking for decalcification for 1-3 hours at the temperature of 3-5 ℃ to remove calcium, and freeze-drying to obtain purified bovine bone meal;

(3) placing the purified bovine bone meal into 0.5mol/L acetic acid solution according to the material-to-liquid ratio of 1: 8-10, extracting for 10-20h under shaking at the temperature of 3-5 ℃, centrifuging and collecting supernatant to obtain bovine bone collagen solution;

(4) primary enzymolysis: adjusting the pH value of the bovine bone collagen solution to 7-9, and adding bone collagenase to carry out ultrasonic-assisted enzymolysis, wherein the primary enzymolysis temperature is 50-55 ℃, the primary enzymolysis time is 1-3 h, and the addition amount of the bone collagenase is 0.1-0.3% of the mass of the bovine bone collagen; after enzymolysis, heating to 80-85 ℃, keeping the temperature for 10-20 min, and inactivating enzyme to obtain primary enzymolysis liquid;

(5) secondary enzymolysis: adjusting the pH value of the primary enzymolysis liquid to 5-7, adding a secondary compound enzyme for ultrasonic-assisted enzymolysis, wherein the secondary compound enzyme adopts pepsin, trypsin, papain and flavourzyme, and obtaining a secondary enzymolysis liquid after the secondary enzymolysis reaction is finished; carrying out microwave enzyme deactivation treatment on the secondary enzymatic hydrolysate, and obtaining enzyme-deactivated enzymatic hydrolysate after the microwave enzyme deactivation is finished;

(6) filtering with activated carbon: adding activated carbon into the enzyme-deactivated enzymolysis liquid, wherein the adding amount of the activated carbon is 2-4% of the mass of the enzyme-deactivated enzymolysis liquid, heating the enzyme-deactivated enzymolysis liquid to 80-100 ℃, and preserving the heat for 5-10 min; then cooling to 70-80 ℃, separating by diatomite, decoloring and removing fishy smell to obtain enzymatic hydrolysate;

(7) filtering with an inorganic nanofiltration ceramic membrane: filtering the enzymatic hydrolysate filtered by the activated carbon by using an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da to obtain a permeate which is a high-purity bovine bone collagen peptide solution;

(8) concentration: controlling the material temperature to be 55-65 ℃, concentrating the material fed into the concentrator in each batch for no more than 30 minutes under the vacuum degree of 0.06-0.08 Mpa until the Baume degree is 20-22 Be, and temporarily storing the concentrated solution in a concentrated solution storage tank;

(9) spray drying: the material temperature is 45-50 ℃, the Baume degree is 20-22 Bee, the air inlet temperature of the spraying tower is adjusted to be 180-190 ℃, the air outlet temperature is 85-95 ℃, and the materials are dried into powder.

Further, the filter element of the inorganic nanofiltration ceramic membrane in the step (7) is made of any one of siliceous material, aluminous material, magnesian material, silicoaluminophosphate material and silicomagnesian material. After enzymolysis, a technical means of filtering by using an inorganic nanofiltration ceramic membrane is adopted to remove substances which are not easy to dissolve and sugar components which are easy to absorb moisture, so that the water re-solubility of the finally obtained collagen peptide finished product is improved, and the finished product is not easy to absorb moisture and agglomerate in the storage process. Compared with an organic nanofiltration membrane, the inorganic nanofiltration ceramic membrane has the advantages of organic solvent resistance, high temperature resistance, acid and alkali resistance, high mechanical strength, large flux and long service life. The method only needs to adopt a nanofiltration membrane for treatment once, obviously simplifies the process steps compared with the prior art which needs to adopt an ultrafiltration membrane to intercept macromolecular substances and adopt a nanofiltration membrane for desalination and concentration, and has different membrane treatment effects.

Further, the method produces the obtained bovine bone collagen peptide.

Compared with the prior art, the invention has the beneficial effects that: (1) the invention fully utilizes the byproducts of the processing of cattle, namely the ox bone, to prepare the collagen peptide, has low cost, and the extraction rate of the collagen is more than 85 percent; the prepared yak bone collagen oligopeptide is white or faint yellow powder, the purity is more than 90%, the molecular weight is less than 1000Da, and no peculiar smell exists;

(2) after the bovine bone is prepared into bone powder, the non-collagen substances, fat and calcium in the bovine bone paste can be basically separated and removed by purifying NaOH solution, normal hexane solution and ethylene diamine tetraacetic acid disodium salt concentrated solution, and the prepared active peptide product is not sticky and basically does not contain impurities such as fat, cholesterol and the like;

(3) the bovine bone collagen peptide is filtered and purified by adopting secondary enzymolysis in cooperation with activated carbon and an inorganic nanofiltration ceramic membrane, so that the impurity removal and concentration effects are good, and the method is suitable for large-scale industrial production.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1

A method for producing bovine bone collagen peptide comprises the following steps:

(1) pretreatment of raw materials: removing impurities on bones of the ox bones, crushing the ox bones into bone blocks of 2cm by using a bone crusher, cleaning the crushed ox bones, and crushing the ox bones into 50 meshes;

(2) raw material purification: placing the crushed bovine bone powder into 0.1mol/L NaOH solution with the mass being 18 times of that of the crushed bovine bone powder, shaking for 3 hours at the temperature of 3 ℃ to remove non-collagen substances, filtering, repeatedly washing a bone sample to be neutral by using distilled water, draining, placing the bone sample into n-hexane solution with the concentration of 10% according to the material-liquid ratio of 1:6, shaking for degreasing for 3 hours at the temperature of 3 ℃, filtering, repeatedly washing the bone sample to be neutral by using distilled water, draining, adding the bone sample into 0.25mol/L disodium ethylenediamine tetraacetate concentrated solution according to the material-liquid ratio of 1:8, shaking for decalcification for 3 hours at the temperature of 3 ℃, removing calcium, and freeze-drying to obtain purified bovine bone powder;

(3) placing the purified bovine bone meal into 0.5mol/L acetic acid solution according to the material-liquid ratio of 1:8, shaking and leaching for 20h under the condition of the temperature of 3, centrifuging and collecting supernatant to obtain bovine bone collagen solution;

(4) primary enzymolysis: adjusting the pH value of the bovine bone collagen solution to 7, and adding bone collagenase to carry out ultrasonic-assisted enzymolysis, wherein the primary enzymolysis temperature is 50 ℃, the primary enzymolysis time is 3 hours, and the adding amount of the bone collagenase is 0.1 percent of the mass of the bovine bone collagen; heating to 80 deg.C after enzymolysis, maintaining the temperature for 10min to inactivate enzyme to obtain primary enzymolysis liquid;

(5) secondary enzymolysis: adjusting the pH value of the primary enzymolysis liquid to 5, adding a secondary compound enzyme for ultrasonic-assisted enzymolysis, wherein the secondary compound enzyme adopts pepsin, trypsin, papain and flavourzyme, and obtaining a secondary enzymolysis liquid after the secondary enzymolysis reaction is finished; carrying out microwave enzyme deactivation treatment on the secondary enzymatic hydrolysate, and obtaining enzyme-deactivated enzymatic hydrolysate after the microwave enzyme deactivation is finished;

(6) filtering with activated carbon: adding active carbon into the enzyme-deactivated enzymolysis liquid, wherein the adding amount of the active carbon is 2% of the mass of the enzyme-deactivated enzymolysis liquid, heating the enzyme-deactivated enzymolysis liquid to 80 ℃, and preserving the heat for 5 min; then cooling to 70 ℃, separating by diatomite, decoloring and removing fishy smell to obtain enzymatic hydrolysate;

(7) filtering with an inorganic nanofiltration ceramic membrane: filtering the enzymatic hydrolysate filtered by the activated carbon by using an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da to obtain a permeate which is a high-purity bovine bone collagen peptide solution;

(8) concentration: controlling the material temperature at 55 ℃, concentrating the material fed into the concentrator in each batch for no more than 30 minutes under the vacuum degree of 0.06MPa until the Baume degree is 20 Be, and temporarily storing the concentrated solution in a concentrated solution storage tank;

(9) spray drying: the material temperature is 45 ℃, the baume degree is 20 Be, the air inlet temperature of the spraying tower is adjusted to be 180 ℃, the air outlet temperature is 85 ℃, and the materials are dried into powder.

The filter element of the inorganic nanofiltration ceramic membrane in the step (7) is made of any one of siliceous material, aluminum material, magnesium material, silicon-aluminum material and silicon-magnesium material.

Example 2

A method for producing bovine bone collagen peptide comprises the following steps:

(1) pretreatment of raw materials: removing impurities on bones of the ox bones, crushing the ox bones into bone blocks of 2cm by using a bone crusher, cleaning the crushed ox bones, and crushing the ox bones into 50 meshes;

(2) raw material purification: placing the crushed bovine bone powder into 0.1mol/L NaOH solution with the mass 20 times of the crushed bovine bone powder, shaking for 3 hours at the temperature of 4 ℃ to remove non-collagen substances, filtering, repeatedly washing a bone sample to be neutral by using distilled water, draining, placing the bone sample into n-hexane solution with the concentration of 10% according to the material-liquid ratio of 1:7, shaking for degreasing for 3 hours at the temperature of 4 ℃, filtering, repeatedly washing the bone sample to be neutral by using distilled water, draining, adding the bone sample into 0.25mol/L disodium ethylenediamine tetraacetic acid concentrated solution according to the material-liquid ratio of 1:8, shaking for decalcification for 3 hours at the temperature of 4 ℃, removing calcium, and freeze-drying to obtain purified bovine bone powder;

(3) placing the purified bovine bone meal into 0.5mol/L acetic acid solution according to the material-liquid ratio of 1:8, extracting for 20 hours at the temperature of 4 ℃ by shaking, centrifuging and collecting supernatant to obtain bovine bone collagen solution;

(4) primary enzymolysis: adjusting the pH value of the bovine bone collagen solution to 8, and adding bone collagenase to carry out ultrasonic-assisted enzymolysis, wherein the primary enzymolysis temperature is 53 ℃, the primary enzymolysis time is 3 hours, and the adding amount of the bone collagenase is 0.1 percent of the mass of the bovine bone collagen; heating to 83 deg.C after enzymolysis, maintaining the temperature for 20min to inactivate enzyme to obtain primary enzymolysis liquid;

(5) secondary enzymolysis: adjusting the pH value of the primary enzymolysis liquid to 6, adding a secondary compound enzyme for ultrasonic-assisted enzymolysis, wherein the secondary compound enzyme adopts pepsin, trypsin, papain and flavourzyme, and obtaining a secondary enzymolysis liquid after the secondary enzymolysis reaction is finished; carrying out microwave enzyme deactivation treatment on the secondary enzymatic hydrolysate, and obtaining enzyme-deactivated enzymatic hydrolysate after the microwave enzyme deactivation is finished;

(6) filtering with activated carbon: adding active carbon into the enzyme-deactivated enzymolysis liquid, wherein the adding amount of the active carbon is 3% of the mass of the enzyme-deactivated enzymolysis liquid, heating the enzyme-deactivated enzymolysis liquid to 90 ℃, and preserving the heat for 10 min; then cooling to 80 ℃, separating by diatomite, decoloring and removing fishy smell to obtain enzymatic hydrolysate;

(7) filtering with an inorganic nanofiltration ceramic membrane: filtering the enzymatic hydrolysate filtered by the activated carbon by using an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da to obtain a permeate which is a high-purity bovine bone collagen peptide solution;

(8) concentration: controlling the material temperature at 60 ℃, concentrating the material fed into the concentrator in each batch for no more than 30 minutes under the vacuum degree of 0.07Mpa until the baume degree is 21 Be, and temporarily storing the concentrated solution in a concentrated solution storage tank;

(9) spray drying: the material temperature is 47 ℃, the baume degree is 21 Bee, the air inlet temperature of the spraying tower is adjusted to be 185 ℃, the air outlet temperature is adjusted to be 90 ℃, and the materials are dried into powder.

The filter element of the inorganic nanofiltration ceramic membrane in the step (7) is made of any one of siliceous material, aluminum material, magnesium material, silicon-aluminum material and silicon-magnesium material.

Example 3

A method for producing bovine bone collagen peptide comprises the following steps:

(1) pretreatment of raw materials: removing impurities on bones of the ox bones, crushing the ox bones into bone blocks of 2cm by using a bone crusher, cleaning the crushed ox bones, and crushing the ox bones into 50 meshes;

(2) raw material purification: placing the crushed bovine bone powder into 0.1mol/L NaOH solution with the mass 20 times of the crushed bovine bone powder, shaking for 3 hours at the temperature of 5 ℃ to remove non-collagen substances, filtering, repeatedly washing a bone sample to be neutral by using distilled water, draining, placing the bone sample into n-hexane solution with the concentration of 10% according to the material-liquid ratio of 1:8, shaking for degreasing for 3 hours at the temperature of 5 ℃ to remove fat, filtering, repeatedly washing the bone sample to be neutral by using distilled water, draining, adding the bone sample into 0.25mol/L disodium ethylenediamine tetraacetate concentrated solution according to the material-liquid ratio of 1: 10, shaking for decalcification for 3 hours at the temperature of 5 ℃, removing calcium, and freeze-drying to obtain purified bovine bone powder;

(3) placing the purified bovine bone meal into 0.5mol/L acetic acid solution according to the material-liquid ratio of 1: 10, extracting for 20 hours at the temperature of 5 ℃ by shaking, centrifuging and collecting supernatant to obtain bovine bone collagen solution;

(4) primary enzymolysis: adjusting the pH value of the bovine bone collagen solution to 9, and adding bone collagenase to carry out ultrasonic-assisted enzymolysis, wherein the primary enzymolysis temperature is 55 ℃, the primary enzymolysis time is 3 hours, and the adding amount of the bone collagenase is 0.3 percent of the mass of the bovine bone collagen; heating to 85 deg.C after enzymolysis, maintaining the temperature for 20min to inactivate enzyme to obtain primary enzymolysis solution;

(5) secondary enzymolysis: adjusting the pH value of the primary enzymolysis liquid to 7, adding a secondary compound enzyme for ultrasonic-assisted enzymolysis, wherein the secondary compound enzyme adopts pepsin, trypsin, papain and flavourzyme, and obtaining a secondary enzymolysis liquid after the secondary enzymolysis reaction is finished; carrying out microwave enzyme deactivation treatment on the secondary enzymatic hydrolysate, and obtaining enzyme-deactivated enzymatic hydrolysate after the microwave enzyme deactivation is finished;

(6) filtering with activated carbon: adding activated carbon into the enzyme-deactivated enzymolysis liquid, wherein the adding amount of the activated carbon is 4% of the mass of the enzyme-deactivated enzymolysis liquid, heating the enzyme-deactivated enzymolysis liquid to 100 ℃, and preserving the heat for 10 min; then cooling to 80 ℃, separating by diatomite, decoloring and removing fishy smell to obtain enzymatic hydrolysate;

(7) filtering with an inorganic nanofiltration ceramic membrane: filtering the enzymatic hydrolysate filtered by the activated carbon by using an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 800Da to obtain a permeate which is a high-purity bovine bone collagen peptide solution;

(8) concentration: controlling the material temperature at 65 ℃, concentrating the material fed into the concentrator in each batch for no more than 30 minutes under the vacuum degree of 0.08Mpa until the baume degree is 22 Be, and temporarily storing the concentrated solution in a concentrated solution storage tank;

(9) spray drying: the material temperature is 50 ℃, the baume degree is 22 Bee, the air inlet temperature of the spraying tower is adjusted to be 190 ℃, the air outlet temperature is 95 ℃, and the materials are dried into powder.

The filter element of the inorganic nanofiltration ceramic membrane in the step (7) is made of any one of siliceous material, aluminum material, magnesium material, silicon-aluminum material and silicon-magnesium material.

(1) And (3) detecting the enzymolysis effect of the high-efficiency compound enzyme preparation:

in examples 1 to 3, after the bovine bone is subjected to raw material pretreatment and oil-water separation, enzymolysis is performed by using a high-efficiency compound enzyme preparation under the conditions of no pH value adjustment and no temperature control, and in order to evaluate the enzymolysis effect, after the enzymolysis is finished, the method in appendix A of national standard GB/T22729-:

TABLE 1

The results in Table 1 show that the enzymolysis products of examples 1 to 3 have small and concentrated molecular weight, low free amino acid content (less than 200Da of less than 5%), a collagen peptide ratio of less than 1000Da of molecular weight of 100%, and a collagen peptide ratio of 200 to 1000Da of more than 95%. Therefore, the combination technical means of specific twice enzymolysis and high-efficiency complex enzyme enzymolysis provided by the invention can obviously improve the enzymolysis efficiency of the enzymolysis collagen.

(2) Detecting the water re-solubility of the finished product of the bovine bone collagen peptide and the light transmittance of the bovine bone collagen peptide solution:

and (3) respectively pouring 8g of the finished bovine bone collagen peptide powder obtained in the examples 1-3 into different cups, mixing with 300mL of warm boiled water at about 60 ℃, dissolving completely within 1s to obtain a bovine bone collagen peptide solution, and observing the solution by naked eyes to be clear and transparent without any precipitate. The light transmittance of the bovine bone collagen peptide solutions was measured at 620nm using an ultraviolet spectrophotometer, and the results are shown in the following table 4:

TABLE 2

	Example 1	Example 2	Example 3
				Transmittance of item (%)	96	94	95

From the results in table 2, it can be seen that the method adopts the technical means of inorganic nanofiltration ceramic membrane filtration to remove the substances which are not easy to dissolve, and then adopts the technical means of high-temperature and high-pressure treatment of peptide solution to further improve the solubility of the finished product, so that the obtained finished product of bovine bone collagen peptide is extremely easy to dissolve in water and has excellent water re-solubility, the dissolved solution after dissolving in water is clear and transparent, has no any precipitate and has high light transmittance, and when a consumer soaks the product, the product does not need to be stirred, is convenient to eat and has good eating experience.

(3) Shelf life test of the finished bovine bone collagen peptide product:

8g of the bovine bone collagen peptide finished product powder obtained in the examples 1-3 is respectively poured into a packaging bag, placed on an indoor display rack in an open mode at room temperature, and observed by naked eyes to obtain the caking time of the finished product, and the results are shown in the following table 3:

TABLE 3

	Example 1	Example 2	Example 3
				Time (day) when the item caked	56	54	53

Bovine bone collagen peptide produced by the prior art is easy to absorb moisture and agglomerate in the storage process, and the sale of products is influenced. The results in table 3 show that the method of the present invention using the inorganic nanofiltration ceramic membrane filtration technology can remove the sugar components that are easy to absorb moisture, so that the finished product is not easy to absorb moisture and agglomerate during storage, and is easy to store, which is a technical effect that the prior art cannot achieve.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.