阅读说明：本技术 一种硫酸软骨素超滤废液制备胶原蛋白肽的方法 (Method for preparing collagen peptide from chondroitin sulfate ultrafiltration waste liquid ) 是由 王斌 尤加宇 赵贵吉 王咏梅 毛节元 贾玉红 王洪英 魏茂军 于 2021-04-08 设计创作，主要内容包括：本发明提供了一种硫酸软骨素联产高纯度胶原蛋白肽的方法：原料中加入蛋白酶在水中进行酶解,调节pH,加热灭活酶,过滤得酶解液I；酶解液I过截留分子量为10kDa的膜进行超滤,获得浓缩液I和流出液I；浓缩液I分离纯化后制备硫酸软骨素；流出液I经纳滤膜浓缩,获得浓缩液II；浓缩液II以强阴离子树脂吸附；树脂滤除后添加蛋白酶酶解,加热灭活酶,过滤得酶解液II；酶解液II调节pH后添加活性炭吸附,然后过滤,获得滤液；将滤液灭菌、过滤、干燥获得胶原蛋白肽。本发明的制备工艺,过程简单、易于操作。本发明从制备硫酸软骨素的超滤废液中制备胶原蛋白肽,不仅降低排污压力,还能够变废为宝,可为提升企业的综合效益。(The invention provides a method for co-producing high-purity collagen peptide by chondroitin sulfate, which comprises the following steps: adding protease into raw materials, performing enzymolysis in water, adjusting pH, heating to inactivate enzyme, and filtering to obtain enzymolysis solution I; ultrafiltering the enzymolysis solution I through a membrane with the molecular weight cutoff of 10kDa to obtain a concentrated solution I and an effluent I; separating and purifying the concentrated solution I to prepare chondroitin sulfate; concentrating the effluent I by a nanofiltration membrane to obtain a concentrated solution II; adsorbing the concentrated solution II by strong anion resin; filtering with resin, adding protease for enzymolysis, heating to inactivate enzyme, and filtering to obtain enzymolysis solution II; adjusting the pH of the enzymolysis liquid II, adding activated carbon for adsorption, and filtering to obtain a filtrate; sterilizing the filtrate, filtering and drying to obtain the collagen peptide. The preparation process is simple and easy to operate. According to the invention, the collagen peptide is prepared from the ultrafiltration waste liquid for preparing the chondroitin sulfate, so that the pollution discharge pressure is reduced, waste can be changed into valuable, and the comprehensive benefit of an enterprise can be improved.)

1. A method for co-producing high-purity collagen peptide by chondroitin sulfate is characterized by comprising the following steps:

(1) adding protease into raw materials, performing enzymolysis in water, adjusting pH, heating to inactivate enzyme, and filtering to obtain enzymolysis solution I;

(2) ultrafiltering the enzymolysis solution I through a membrane with the molecular weight cutoff of 10kDa to obtain a concentrated solution I and an effluent I;

(3) separating and purifying the concentrated solution I to prepare chondroitin sulfate; concentrating the effluent I by a nanofiltration membrane to obtain a concentrated solution II and an effluent II;

(4) adjusting the pH of the concentrated solution II, and adsorbing by strong anion resin;

(5) filtering with resin, adjusting pH, heating, adding protease for enzymolysis, heating to inactivate enzyme, and filtering to obtain enzymolysis solution II;

(6) adjusting the pH of the enzymolysis liquid II, adding activated carbon for adsorption, and filtering to obtain a filtrate;

(7) sterilizing the filtrate, filtering and drying to obtain the collagen peptide.

2. The method according to claim 1, wherein in the step (1), the mass-to-volume ratio of the raw material to the water is 1: 5-10;

in the step (1), the adding amount of the protease is 0.5-1.5% of the mass of the cartilage;

in the step (1), the enzymolysis temperature is 45-50 ℃, the enzymolysis time is 2-4h, and the enzymolysis pH is 8-9;

in the steps (1) and (5), the pH is 4.2-5.0, and the temperature for inactivating the protease is 75-85 ℃;

in the step (5), the pH value is 8-9; the enzymolysis temperature is 45-50 ℃.

3. The method according to claim 1, wherein in step (1), the protease is subtilisin; in the step (5), the protease is alkaline protease.

4. The method according to claim 1, wherein in step (5), the protease is a mixture of subtilisin and trypsin; the addition amount of subtilisin is 0.1-0.3 wt% of protein, and the addition amount of trypsin is 0.2-0.5 wt% of protein.

5. The method according to claim 1, wherein in step (5), the method further comprises the step of adding a protease activator before the temperature rise; the protease activator is selected from at least one of soluble calcium salt, soluble zinc salt or soluble magnesium salt; the adding amount of the soluble calcium salt is 10-20% of the mass of the protease, and the adding amount of the soluble zinc salt or the soluble magnesium salt is 20-30% of the mass of the protease;

before the step (1), the method also comprises a raw material heat treatment step: heating cartilage in water at 90-100 deg.C for 2-4 hr.

6. The method of claim 1, wherein in step (2), the ultrafiltration endpoint is the OD of effluent I₂₈₀Not more than 0.6; maintaining the volume of the ultrafiltration to be 1/4-1/6 of the volume of the enzymolysis liquid I;

in the step (3), the molecular weight cut-off of the nanofiltration membrane is 200-500 daltons; the nanofiltration conditions are as follows: the pressure is 4.2-5.5bar, and the solution temperature is 50-60 ℃; the conductivity of effluent II was 5-12 ms/cm.

7. The method according to claim 1, wherein in step (4), the pH is 6.8 to 7.2; the volume ratio of the strong anion resin to the concentrated solution II is 2-5: 100.

8. The method of claim 7, wherein the strong anion resin is selected from the group consisting of FPA98 resin, D201 resin, and D202 resin.

9. The method according to claim 1, wherein in step (6), the pH is 5.0-6.0; the mass volume ratio of the active carbon to the concentrated solution II is 1-2%; the adsorption time is 1.5-2 h.

10. The method according to claim 1, wherein in step (7), the sterilization is autoclaving; the temperature is 110-; the drying is selected from spray drying or freeze drying.

Technical Field

The invention belongs to the field of biological medicine raw material preparation and sewage treatment, and relates to a method for preparing collagen peptide from chondroitin sulfate ultrafiltration waste liquid.

Background

Chondroitin Sulfate (Chondroitin Sulfate) is acidic mucopolysaccharide extracted from animal cartilage tissue, has molecular weight of 10000-50000 Da, and can be widely used for treating cardiovascular and cerebrovascular diseases, arthralgia, arthritis, eye drop, etc. The current mainstream technology is chondroitin sulfate produced by adopting an enzymolysis ultrafiltration technology. In the process, when the pig nasal middle bone is taken as a raw material for extracting the chondroitin, 1000 kilograms of the pig nasal middle bone raw material contains 350 kilograms of the chondroitin, 450 kilograms of collagen and 200 kilograms of other organic matters and inorganic matters; when the marine fish bone is used as a raw material for extracting the chondroitin, 1000 kilograms of the fish bone raw material contains 280 kilograms of the chondroitin, 480 kilograms of the collagen and 240 kilograms of other organic matters and inorganic matters. Therefore, a large amount of collagen is treated as production sewage through ultrafiltration after enzymolysis, and the collagen is a nitrogenous macromolecular organic matter, so that the ammonia nitrogen and COD values are very high, and great treatment pressure is brought to a sewage treatment department.

Collagen is a main structural protein widely existing in extracellular spaces of various connective tissues in animals, and is also a functional protein with the largest content and the widest distribution in mammals. Collagen has good biocompatibility, biodegradability and bioactivity, and is widely applied to the fields of health-care foods, medicines, cosmetics and the like. Therefore, in order to improve the utilization rate of raw materials and reduce the difficulty of wastewater treatment, there are a plurality of patent documents relating to the extraction of collagen from chondroitin sulfate ultrafiltration waste liquid:

the publication number is CN108264582A discloses a new process for preparing chondroitin sulfate, which mainly adopts the technical scheme that a certain amount of alcohol ketone polar solvent is added into an enzymolysis system, and a homogenizing agent is introduced. In a method for producing chondroitin sulfate and co-producing hydrolyzed collagen by using fish cartilage, publication No. CN103320486A discloses a method for producing chondroitin sulfate and co-producing hydrolyzed collagen by using fish cartilage, which comprises the following steps: firstly, preprocessing; secondly, enzymolysis; thirdly, heat treatment/filtration concentration; fourthly, resin adsorption; fifthly, refining the chondroitin sulfate; sixthly, recovering the protein. The invention adopts a complex enzyme hydrolysis technology to realize synchronous hydrolysis of chondroitin sulfate and collagen, and the protein content of the hydrolyzed collagen is about 92 percent. In a method for extracting chondroitin sulfate and collagen from animal cartilage, publication No. CN109232772A discloses a method for extracting chondroitin sulfate and collagen from animal cartilage, which comprises a chondroitin sulfate extraction step and a collagen extraction step; carrying out ultrafiltration filtration on the cartilage leaching liquor to obtain a concentrated solution and a permeate, wherein the concentrated solution is used for extracting chondroitin sulfate; the permeate is used for extracting peptone or albumen powder; realizes the purpose of simultaneously extracting chondroitin sulfate and collagen in animal cartilage.

The collagen produced by the method has larger molecular weight, is not easy to absorb and utilize, and has polysaccharide residue, and the prepared collagen powder is easy to absorb moisture and has poor dissolving effect. Collagen can be classified into: 100-1000 amino acids, 30-50 amino acids and 3 amino acids. The absorption rate of macromolecular collagen is very low, allergic rejection reaction is easily caused, collagen peptide can be absorbed by human body, but the absorption and use effect is not good enough, the collagen peptide can be directly absorbed and utilized by human body, under the condition of equal intake, the absorption rate of the collagen peptide is 6-7 times of that of the collagen peptide, and no allergic reaction exists, because the collagen peptide is the minimum unit of collagen, the structure of the collagen peptide can be expressed as (GLY-X-Y), the collagen peptide is a high-purity tripeptide with glycine at the N end, and the tripeptide can be directly absorbed by small intestine and can be conveyed to the required part of the body through blood. At present, the collagen peptide is mainly prepared by taking fish skin or fish scales as raw materials through multi-stage enzymolysis, for example, CN1032431444B adopts the fish skin as the raw material for three-purpose compound enzymolysis, then collagenase extracted by self is used for enzymolysis, and then the collagen peptide is prepared by centrifugation, fishy smell removal and decoloration, concentration and spray drying; CN101870995B takes fish skin as raw material, and collagen peptide is prepared by alkali treatment, heat treatment and three times of enzymolysis; CN109897101B takes fish scales as raw materials, and collagen peptide is prepared by alkali treatment, heat treatment, high-efficiency compound enzymolysis, ceramic membrane filtration, high-temperature high-pressure treatment, concentration and spray drying. The fish scales and the fish skins have high content of fat components, a large amount of acid-base treatment is needed in the preparation process, and when acid-base acts on part of amino acids in collagen, harmful substances are generated, so that more proper preparation raw materials are very necessary to select. The cartilage has low fat content, and acid-base treatment is not needed, but the price of the cartilage is more expensive compared with that of fish skin and fish scales, and the joint production of collagen peptide while preparing chondroitin sulfate is an ideal scheme for reducing the manufacturing cost.

At present, no literature report exists on the production of collagen peptide while chondroitin sulfate is prepared, and the prepared collagen peptide has high protein content, good solubility and obvious whitening effect while the high-purity chondroitin sulfate is prepared. Meanwhile, the pressure of sewage treatment is greatly reduced, the original waste components are reasonably utilized, and the comprehensive benefit is improved.

Disclosure of Invention

The invention provides a method for preparing high-purity collagen peptide from chondroitin sulfate ultrafiltration waste liquid.

In order to achieve the purpose, the invention adopts the following technical scheme.

A method for co-producing high-purity collagen peptide by chondroitin sulfate comprises the following steps:

(1) adding protease into raw materials, performing enzymolysis in water, adjusting pH, heating to inactivate enzyme, and filtering to obtain enzymolysis solution I;

(2) ultrafiltering the enzymolysis solution I through a membrane with the molecular weight cutoff of 10kDa to obtain a concentrated solution I and an effluent I;

(3) separating and purifying the concentrated solution I to prepare chondroitin sulfate; concentrating the effluent I by a nanofiltration membrane to obtain a concentrated solution II and an effluent II;

(4) adjusting the pH of the concentrated solution II, and adsorbing by strong anion resin;

(5) filtering with resin, adjusting pH, heating, adding protease for enzymolysis, heating to inactivate enzyme, and filtering to obtain enzymolysis solution II;

(6) adjusting the pH of the enzymolysis liquid II, adding activated carbon for adsorption, and filtering to obtain a filtrate;

(7) sterilizing the filtrate, filtering and drying to obtain the collagen peptide.

Before the step (1), the method also comprises a raw material heat treatment step: heating cartilage in water at 90-100 deg.C for 2-4 hr.

In the step (1), the mass volume ratio of the raw materials to the water is 1: 5-10.

In the step (1), the adding amount of the protease is 0.5-1.5% of the mass of the cartilage. The protease is subtilisin.

In the step (1), the enzymolysis temperature is 45-50 ℃, the enzymolysis time is 2-4h, and the enzymolysis pH is 8-9.

In the step (1), the pH is 4.2-5.0, and the temperature for inactivating the protease is 75-85 ℃.

In the step (2), the endpoint of the ultrafiltration is the OD of the effluent I₂₈₀Not greater than 0.6.

In the step (2), the volume of the enzymolysis solution is maintained to be 1/4-1/6 of the volume of the enzymolysis solution I through ultrafiltration.

In the step (3), the molecular weight cut-off of the nanofiltration membrane is 200-500 daltons.

In the step (3), the nanofiltration conditions are as follows: the pressure is 4.2-5.5bar, and the solution temperature is 50-60 deg.C.

In step (3), the conductivity of effluent II was 5-12 ms/cm.

In the step (4), the pH is 6.8-7.2.

In the step (4), the volume ratio of the strong anion resin to the concentrated solution II is 2-5: 100. The strong anion resin is selected from FPA98 resin, D201 resin or D202 resin.

In the step (5), the pH is 8-9. The enzymolysis temperature is 45-50 ℃.

In the step (5), the protease is alkaline protease; preferably a mixture of subtilisin and trypsin. More preferably, subtilisin is added in an amount of 0.1 to 0.3% by weight of the protein and trypsin is added in an amount of 0.2 to 0.5% by weight of the protein.

In the step (5), a step of adding a protease activator is further included before the temperature is raised. The protease activator is selected from at least one of soluble calcium salt, soluble zinc salt or soluble magnesium salt, such as calcium chloride, zinc chloride, and magnesium chloride. The adding amount of the soluble calcium salt is 10-20% of the mass of the protease, and the adding amount of the soluble zinc salt or the soluble magnesium salt is 20-30% of the mass of the protease.

In the step (5), the pH of the inactivated enzyme is 4.2-5.0, and the heating temperature is 75-85 ℃.

In the step (6), the pH is 5.0 to 6.0.

In the step (6), the mass volume ratio of the activated carbon to the concentrated solution II is 1-2%.

In the step (6), the adsorption time is 1.5-2 h.

In the step (7), the sterilization is high-pressure steam sterilization; preferably, the temperature is 110-130 ℃, the pressure is 0.1-0.3Mpa, and the time is 10-20 min.

In the step (7), the drying is selected from spray drying or freeze drying.

The invention has the following advantages:

the preparation process is simple and easy to operate. By controlling the types of enzymes and the enzymolysis conditions, the collagen in the chondroitin sulfate waste liquid can be efficiently recovered, the pollution discharge pressure is reduced, and the utilization rate of raw materials is improved. The preparation method of the invention firstly carries out enzymolysis after heat treatment of cartilage and then carries out ultrafiltration to prepare concentrated solution and filtrate. Collecting ultrafiltration filtrate by nanofiltration, removing small molecular substances such as amino acid residues, oligosaccharides, salts and the like in the nanofiltration process, adsorbing the concentrated solution by resin to remove anionic protein and polysaccharide impurities, adding composite protease and cationic enzymolysis reinforcing agent for secondary enzymolysis, inactivating the protease, adding active carbon for adsorption, removing pigments and fishy smell substances in the product, then carrying out high-temperature and high-pressure treatment, and finally inactivating, spraying and drying to obtain collagen peptide powder, wherein the prepared collagen peptide powder has a molecular weight distribution diagram showing that most of the collagen peptide powder is 200 plus 500 daltons, and the average molecular weight of the collagen peptide powder is 280 daltons. The measurement by the lowry method shows that the protein content in the collagen exceeds 99.5 percent, and the collagen hardly contains harmful microorganisms such as bacteria and the like through high-temperature sterilization in the production process, and the indexes of heavy metal and burning residues meet the requirements of national medicines and health-care foods, and can be used in the fields of health-care foods, medicines, cosmetics and the like. According to the invention, the collagen peptide is prepared from the ultrafiltration waste liquid for preparing the chondroitin sulfate, so that the pollution discharge pressure is reduced, waste can be changed into valuable, and the comprehensive benefit of an enterprise can be improved.

Detailed Description

The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.

Example 1 preparation of collagen peptide from Ultrafiltration waste solution of chondroitin sulfate derived from pig cartilage

(1) Adding 7000L of water into a 10-ton reaction tank, adding 1000kg of pig trachea, throat and cartilage, starting stirring, heating steam to 90 ℃, keeping the temperature for 2h, cooling to 50 ℃, adjusting the pH to 8.5 by using 20% sodium hydroxide until the enzymolysis is finished, adding 5kg of Novoxil Alcalase 3.0T protease for enzymolysis for 4h, adjusting the pH to 4.5 by using 15% hydrochloric acid, heating to 85 ℃ for 30min to inactivate the enzyme, standing and settling for 2h, and filtering the feed liquid to obtain 7200L of enzymolysis liquid I;

(2) transferring the enzymolysis solution I to an ultrafiltration tank, starting the ultrafiltration machine, performing ultrafiltration with an ultrafiltration membrane with cut-off molecular weight of 10kDa, and continuously adding 2% sodium chloride solution to maintain ultrafiltration volume of about 2400L (1/4 of enzymolysis solution I) until OD of effluent₂₈₀Not more than 0.6, obtainingAbout 1500L of concentrate I and about 90000L of effluent I;

(3) carrying out primary precipitation, oxidation decoloration, secondary precipitation and the like on the concentrated solution I to prepare 297.3kg of chondroitin sulfate with the purity of 99.5 percent; concentrating the effluent I at 60 deg.C under 4.2bar with a nanofiltration membrane of 200Da until the conductivity of the effluent is 12ms/cm to obtain about 1800L of concentrated solution II and effluent II;

(4) adjusting pH of the concentrated solution II to 7.0, adding 50L of FPA98 strong anion resin, stirring, and adsorbing for 1 h;

(5) filtering the feed liquid, determining protein content in the solution, adjusting pH to 8.5, adding 0.2kg of calcium chloride and 0.3kg of zinc chloride, heating to 50 ℃, adding 0.5kg of Novoxin liquid protease Esperase and 0.7kg of trypsin (produced by Biochemical products, Inc. of Deyang, Sichuan province) for enzymolysis for 1h, adjusting pH to 4.5, heating at 85 ℃ for 30min to inactivate enzyme, standing for settling for 2h, and filtering to obtain about 1750L of enzymolysis liquid II;

(6) adjusting the pH value of the enzymolysis liquid II to 6.0, adding about 30kg of active carbon to the mass volume ratio of 1.5%, adsorbing for 1.5h, and filtering to obtain a filtrate;

(7) sterilizing the filtrate at 121 deg.C under 0.1Mpa with high temperature steam for 20min, cooling to room temperature, filtering, and spray drying the obtained solution to obtain 157.6kg collagen peptide.

Example 2 preparation of collagen peptide from Ultrafiltration waste solution of chondroitin sulfate derived from shark bone

(1) Adding about 10000L of water into a 15-ton reaction tank, adding 1000kg of shark cartilage, starting stirring, heating by steam to 100 ℃, preserving heat for 2h, cooling to 45 ℃, adjusting the pH to 8.0 by using 20% sodium hydroxide until enzymolysis is finished, adding 15kg of Novoxil Alcalase 3.0T protease for enzymolysis for 2h, adjusting the pH to 4.2 by using 15% hydrochloric acid, heating to 80 ℃ for 30min to inactivate the enzyme, standing and settling for 2h, and filtering feed liquid to obtain about 10400L of enzymolysis liquid I;

(2) transferring the enzymolysis solution I to an ultrafiltration tank, starting the ultrafiltration machine, performing ultrafiltration with ultrafiltration membrane with cut-off molecular weight of 10kDa, and continuously adding 2% sodium chloride solution to maintain ultrafiltration volume of about 2000L (1/5 of enzymolysis solution I volume) until OD of effluent₂₈₀Not more than 0.6, about 1500L of concentrate I and about 94000L of effluent I are obtained;

(3) performing primary precipitation, oxidation decoloration, secondary precipitation and the like on the concentrated solution I to prepare 265.2kg of chondroitin sulfate with the purity of 99.7 percent; concentrating the effluent I at 50 deg.C under 5.5bar with a nanofiltration membrane of 300Da until the conductivity of the effluent is about 5ms/cm to obtain about 1500L of concentrated solution II and effluent II;

(4) adjusting the pH value of the concentrated solution II to 6.8, adding 100L of D201 strong anion resin, stirring and adsorbing for 1.5 h;

(5) filtering the feed liquid, measuring the protein content in the solution, adjusting the pH value to 9.0, adding 0.1kg of calcium chloride and 0.3kg of magnesium chloride, heating to 45 ℃, adding 0.6kg of Novoxil liquid protease Esperase and 0.8kg of trypsin (produced by Biochemical products, Inc. of Deyang, Sichuan province) for enzymolysis for 1h, adjusting the pH value to 4.2, heating to 80 ℃ for 30min to inactivate the enzyme, standing and settling for 2h, and filtering to obtain about 1500L of enzymolysis liquid II;

(6) adjusting the pH value of the enzymolysis liquid II to 5.0, adding about 24kg of active carbon to the mass-volume ratio of 1.5%, adsorbing for 1.5h, and filtering to obtain a filtrate;

(7) sterilizing the filtrate at 121 deg.C under 0.1Mpa with high temperature steam for 20min, cooling to room temperature, filtering, and spray drying the obtained solution to obtain collagen peptide 162.9 kg.

Example 3 preparation of collagen peptide from bovine bone-derived chondroitin sulfate Ultrafiltration waste liquid

(1) Adding 8500L of water into a 10-ton reaction tank, adding 1000kg of bovine crescent bone, starting stirring, heating to 95 ℃ with steam, keeping the temperature for 2h, cooling to 47.8 ℃, adjusting the pH to 9 with 20% sodium hydroxide until enzymolysis is finished, adding 12kg of Novoxil Alcalase 3.0T protease for enzymolysis for 2h, adjusting the pH to 5 with 15% hydrochloric acid, heating to 85 ℃ for 30min to inactivate the enzyme, standing and settling for 2h, and filtering the feed liquid to obtain 9000L of enzymolysis liquid I;

(2) transferring the enzymolysis solution I to an ultrafiltration tank, starting the ultrafiltration machine, performing ultrafiltration with ultrafiltration membrane with cut-off molecular weight of 10kDa, and continuously adding 2% sodium chloride solution to maintain ultrafiltration volume of about 1500L (1/6 of enzymolysis solution I volume) until OD of effluent₂₈₀No greater than 0.6, yielding about 1500L of concentrate I and about 105000L of effluent I;

(3) performing primary precipitation, oxidation decoloration, secondary precipitation and the like on the concentrated solution I to prepare 205.6kg of chondroitin sulfate with the purity of 95.8 percent; concentrating the effluent I at 54 deg.C under 5bar with a 200Da nanofiltration membrane until the conductivity of the effluent is 7ms/cm to obtain about 1500L of concentrated solution II and effluent II;

(4) adjusting the pH value of the concentrated solution II to 7.2, adding 90L of D202 strong anion resin, stirring and adsorbing for 1 h;

(5) filtering the feed liquid, measuring the protein content in the solution, adjusting the pH value to 8.3, adding 0.3kg of calcium chloride and 0.5kg of zinc chloride, heating to 47.8 ℃, adding 0.5kg of subtilisin and 0.8kg of trypsin (produced by Biochemical products, Inc. of Deyang, Sichuan province) for enzymolysis for 1h, adjusting the pH value to 5, heating at 85 ℃ for 30min to inactivate enzyme, standing for settling for 2h, and filtering to obtain 1450L of enzymolysis liquid II;

(6) adjusting the pH value of the enzymolysis liquid II to 5.5, adding about 35kg of active carbon to a mass volume ratio of 2%, adsorbing for 1.5h, and filtering to obtain a filtrate;

(7) sterilizing the filtrate at 121 deg.C under 0.1Mpa with high temperature steam for 20min, cooling to room temperature, filtering, and spray drying the obtained solution to obtain 190.7kg collagen peptide.

Example 4 detection of physicochemical Properties of collagen peptide

The molecular weight distributions of the collagen peptides prepared in examples 1 to 3 were measured by a size exclusion high performance liquid chromatography (SEC-HPLC) method, and a calibration curve was prepared using glycylethylacetylamino acid (Mr 189.1), glycine-hydroxyproline-serine (Mr 276.3), glycine-tyrosine-arginine (Mr 451.2), human angiotensin ii (Mr 1045.5), tryptase (Mr 6511), cytochrome C (Mr 12355), and bovine immunoglobulin (Mr 15000) as relative molecular mass standards, and the results are shown in table 1:

table 1 examples 1-3 preparation of collagen peptide molecular weight distribution

。

The collagen peptides prepared in examples 1 to 3 were tested for appearance, odor, loss on drying, pH of 1% solution, light transmittance of 1% solution (620 nm), protein content, ash content, sulfur dioxide, peroxide, heavy metals, total number of colonies, pathogenic bacteria, etc., respectively, and the results are shown in table 2:

TABLE 2 examples 1-3 preparation of collagen peptide samples physicochemical Properties

As can be seen from the data in tables 1 and 2, the protein content of the collagen peptides prepared by the examples 1-3 is above 95%, and the content of oligopeptides with molecular weights of 280 Da-450 Da is about 70%, which indicates that the content of functional peptides in the prepared products is high, and the content of harmful impurities and pathogenic bacteria meets the standard.

Comparative example 1

Chondroitin sulfate and collagen peptide were prepared from the same starting material by the method of example 1, except that trypsin was used instead of novacin Alcalase 3.0T protease in step (1), and the addition ratio was 0.9% by weight of cartilage.

Comparative example 2

Chondroitin sulfate and collagen peptide were prepared from the same starting material by the method of example 1, except that papain was used in place of novacin Alcalase 3.0T protease in step (1), and the addition ratio was 1.5% of the weight of cartilage.

Comparative example 3

Chondroitin sulfate and collagen peptide were prepared from the same starting material by the method of example 1, except that in step (1), novacin Alcalase 3.0T protease was replaced with flavourzyme, and the addition ratio was 2.1% by weight of cartilage.

Comparative example 4

Chondroitin sulfate and collagen peptide were prepared from the same raw materials by the method of example 1, except that papain was used instead of the novacin liquid protease in step (5), and the addition ratio was 0.5% by weight of the protein.

Comparative example 5

Chondroitin sulfate and collagen peptide were prepared from the same raw materials using the method of example 1, except that papain was used instead of trypsin in step (5), and the addition ratio was 0.5% by weight of the protein.

Comparative example 6

Chondroitin sulfate and collagen peptide were prepared from the same raw material using the method of example 1, except that in step (5), flavourzyme was used instead of trypsin, and the addition ratio was 0.5% by weight of the protein.

Comparative example 7

Chondroitin sulfate and collagen peptide were prepared from the same raw material using the method of example 1, except that in step (5), flavourzyme was used instead of norretin liquid protease, and the addition ratio was 0.5% by weight of the protein.

Comparative example 8

Chondroitin sulfate and collagen peptide were prepared from the same raw material using the method of example 1, except that the nanofiltration of step (3) used a 600Da cut-off membrane.

Comparative example 9

Chondroitin sulfate and collagen peptide were prepared from the same raw material using the method of example 1, except that no anion resin adsorption was used and the filtering operations in step (4) and step (5) were omitted.

Example 5 detection of physicochemical Properties of collagen peptides

In the above comparative examples, the addition ratio of the used alternative protease is close to the optimal ratio of the enzyme, and the exploration process is not detailed, mainly comparing the product differences caused by the difference of the types of the used proteases and the process steps, including chondroitin yield, chondroitin heteroprotein, collagen peptide yield, 280Da oligopeptide content, storage stability and the like. The results are shown in Table 3:

TABLE 3 yield and quality of collagen peptide obtained by different processes

The comparison results in the table show that after the key control links in the process are changed, the process can generate great changes compared with the examples, including increase of chondroitin sulfate heteroprotein, low yield of the prepared collagen peptide, less proportion of 280-plus-450 Da oligopeptide, easy moisture absorption and unfavorable storage, and the like. Therefore, the reasonable technology and relevant parameters for preparing the collagen peptide from the chondroitin sulfate ultrafiltration waste liquid are mastered, the prepared collagen peptide has high yield and high active ingredient ratio, and the product is easy to store for a long time.

Application example 1 measurement of biological Activity of collagen peptide

The collagen peptide has various biological activity effects in the molecular weight region of 200-500Da, and the molecular weight of the collagen peptide prepared by the invention is more than 80% below 500 Da. In order to evaluate the collagen peptides prepared in examples 1 to 3 for efficacy, the results of verifying the efficacy in whitening, moisturizing, etc. are shown in table 4:

table 4 examples 1-3 collagen peptide preparation efficacy test (n = 3)

According to the results in table 4, the inhibition rate of the 2% solution of the collagen peptide sample prepared in the example on tyrosinase exceeds 30%, and the collagen peptide sample has a good whitening effect. For the moisturizing effect, the product prepared in the embodiment also shows good body surface and in-vitro moisturizing effect, and can effectively prevent water loss.

The collagen peptides prepared in the comparative example were formulated into 2% solutions for tyrosinase inhibition assay, the results of which are shown in table 5:

TABLE 5 average inhibition of tyrosinase (n = 3) for the collagen peptide solutions prepared in comparative examples 1-9

As can be seen from the results in Table 5, the products prepared in the comparative examples had tyrosinase inhibition rates of 18% to 30%, which were lower than those in the examples.