CN112225796A - Sheep skin collagen peptide with DPP-IV inhibitory activity and preparation method thereof - Google Patents
Sheep skin collagen peptide with DPP-IV inhibitory activity and preparation method thereof Download PDFInfo
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- CN112225796A CN112225796A CN202011086514.5A CN202011086514A CN112225796A CN 112225796 A CN112225796 A CN 112225796A CN 202011086514 A CN202011086514 A CN 202011086514A CN 112225796 A CN112225796 A CN 112225796A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Mycology (AREA)
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Abstract
The invention discloses a sheep skin collagen peptide with DPP-IV inhibitory activity and a preparation method thereof, wherein the amino acid sequence of the sheep skin collagen peptide is Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val. The invention provides a collagen peptide with DPP-IV inhibitory activity and a preparation method thereof, which not only realizes the comprehensive utilization of sheepskin and makes full use of the edible quality and the nutritional value of sheepskin, but also avoids the waste of resources and the pollution to the environment, and has great economic and social benefits.
Description
Technical Field
The invention relates to the technical field of comprehensive utilization of sheepskin resources, in particular to a preparation method of a DPP-IV (dipeptidyl peptidase IV) inhibiting collagen peptide.
Background
Diabetes is a metabolic disease characterized by hyperglycemia, and is a serious long-term disease. It is estimated that 400 million people die worldwide in 2017, which is one of ten major causes of adult death. Among them, type ii diabetes is the most common form of disease, accounting for approximately 90-95% of all cases diagnosed with diabetes. Type II diabetes has various etiologies, and GLP-1/DPP-IV inhibitors have become a novel means for treating type II diabetes. Glucagon-like peptide 1(GLP-1) stimulates rapid release of insulin in vivo, stimulates insulin biosynthesis, inhibits beta cell apoptosis, and promotes beta cell differentiation. GLP-1 is inactivated by the rapid cleavage by DPP-IV, therefore, DPP-IV inhibitors which inhibit DPP-IV activity and thus do not break down GLP-1 have been one of the leading targets for the treatment of type II diabetes. The DPP-4 inhibitors which are currently studied most intensively and have been applied clinically are Sitagliptin (Sitagliptin), Vildagliptin (Vildagliptin) and Saxagliptin (Saxagliptin). However, although the chemically synthesized medicines have the effect of relieving type II diabetes, certain toxic and side effects exist. Collagen peptide with DPP-IV inhibitory activity derived from natural protein draws attention to people due to the advantages of high edible safety, no toxic or side effect and the like.
In the leather industry of China, the whole sheepskin is mainly used for preparing high-grade leather, and the broken sheepskin cannot be used for preparing the leather and is discarded, so that resources are seriously wasted, and even the environment is polluted. However, the sheepskin contains a large amount of collagen, not only has good eating quality such as soft mouthfeel and easy digestion, but also contains various beneficial nutrient substances for human body, and has a plurality of functional properties. The collagen has good biocompatibility and high absorption and utilization rate of human bodies, and the reported collagen peptide has good bioactivity, so the collagen is an ideal protein source. In the prior art, a process for preparing collagen peptide with DPP-IV inhibitory activity by taking sheepskin as a raw material is rarely reported. The collagen is extracted from the sheepskin and the collagen peptide is prepared, so that the comprehensive utilization rate of the sheepskin can be improved, and the economic benefit and the social benefit of the sheepskin can be improved.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or other problems associated with existing DPP-IV inhibitory collagen peptides.
Therefore, one of the purposes of the invention is to overcome the defects of the existing collagen peptide products with DPP-IV inhibitory activity and provide a sheepskin collagen peptide with DPP-IV inhibitory activity.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: an ovine skin collagen peptide having DPP-IV inhibitory activity comprising: the amino acid sequence of the sheep skin collagen peptide is Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val.
The invention also aims to provide a preparation method of the sheep skin collagen peptide with DPP-IV inhibitory activity.
To solve the above technical problem, according to an aspect of the present invention, the present invention provides the following technical solutions: a preparation method of sheep skin collagen peptide with DPP-IV inhibitory activity comprises the following steps:
pretreatment of sheepskin: physically degreasing unhaired sheep skin, cleaning, shearing into sheep skin grains of 1 × 1cm, then deliming, degreasing and deproteinizing, and cleaning the sheep skin grains again after the treatment is finished;
extracting sheep skin collagen: soaking the sheepskin grains obtained through pretreatment in an alkali solution, collecting the sheepskin grains after soaking, performing decalcification and hydrogen peroxide treatment, washing the sheepskin grains with clear water, and performing pyrolysis treatment to obtain a solution, namely a sheepskin collagen solution;
enzymatic hydrolysis of collagen: adding Alcalase alkaline protease, neutral protease and flavourzyme into the pH value of the obtained sheep skin collagen solution for hydrolysis, inactivating enzymes at high temperature after the hydrolysis is finished, and collecting supernatant for later use;
preparation of collagen peptide: and clarifying the supernatant of the sheep skin collagen peptide subjected to enzymolysis, carrying out DPP-IV inhibitory activity detection on the enzymolysis liquid, concentrating the solution, and freeze-drying to obtain the sheep skin collagen peptide with DPP-IV inhibitory activity.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the pretreatment of the sheepskin comprises the following steps:
soaking and cleaning raw sheep skin: rinsing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking and maintaining water change in the soaking process, shearing off excessive fat, then cleaning under stirring and maintaining water change in the cleaning process,
soaking and deliming: adding the cleaned sheepskin into an ammonium sulfate solution, stirring and soaking at normal temperature, and keeping the solution changed. Washing with deionized water and adjusting the pH to 7.5 after deashing;
removing foreign proteins: adding the delimed sheepskin into salt water, stirring and soaking at normal temperature, replacing the solution once, and washing with water after soaking;
secondary deliming: soaking, adding the cleaned sheepskin grain skin blocks into a soda solution for soaking and stirring, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with ethanol, soaking for degreasing, filtering to remove the ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into a NaCl solution, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with the distilled water for later use.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the extraction of the sheep skin collagen comprises the following steps:
soaking in alkali liquor: and (3) adding the degreased sheepskin particles into a sodium hydroxide solution for soaking at room temperature, stirring for 3 times every day, and replacing alkali liquor with the same concentration and volume once every day.
Decalcification treatment: and soaking the alkali-treated sheep skin particles in a sodium sulfate solution, and standing at room temperature for 24 hours. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking the decalcified sheep skin grains in hydrogen peroxide solution for 24 h. The hydrogen peroxide solution is replaced after a period of time. Subsequently, the sheepskin particles were washed clean with water.
And (3) pyrolysis treatment: soaking cleaned sheepskin particles into water, boiling glue, and raising the temperature for a period of time.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the enzymatic hydrolysis of collagen comprises the following steps:
adjusting pH and temperature to optimum pH and temperature of protease, adding protease, and performing enzymolysis for sufficient time. Heating the enzymolysis liquid obtained after enzymolysis for enzyme deactivation.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the preparation of the collagen peptide comprises the following steps:
clarifying and filtering: taking diatomite or active carbon as an adsorbent, performing suction filtration by using a sand core funnel (three layers of quantitative filter paper), centrifuging filtrate at a high rotating speed by using a centrifuge, and removing upper-layer grease and lower-layer precipitate to obtain a clarified solution;
concentrating and drying: rotating and evaporating the clarified solution, and concentrating by more than half of the volume; and freeze-drying the concentrated solution to obtain the collagen peptide.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the protease in the enzyme hydrolysis is Alcalase alkaline protease, neutral protease and flavourzyme.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the dosage of the protease in the enzymatic hydrolysis is 7200-16000U.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the protease in the enzymatic hydrolysis is Alcalase alkaline protease, and the dosage of the protease is 9600U.
As a preferred embodiment of the method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity of the present invention, wherein: the concentration of the alkali in the alkali liquor soaking is 0.5 mol/L.
The invention provides a collagen peptide with DPP-IV inhibitory activity, which not only realizes the comprehensive utilization of sheepskin and makes full use of the edible quality and the nutritive value of sheepskin, but also avoids the waste of resources and the pollution to the environment, has great economic and social benefits, and simultaneously has the following advantages:
(1) the sheep skin collagen peptide has high DPP-IV (dipeptidyl peptidase IV) inhibition activity and good gastrointestinal stability, the DPP-IV inhibition activity of the collagen peptide (10ug/ml) is 9.528%, the DPP-IV inhibition activity retention rate after gastrointestinal digestive tract digestion is 5.866%, and the high DPP-IV inhibition activity is still maintained. Therefore, the sheep skin collagen peptide with DPP-IV inhibitory activity has a good application prospect when being added into health food as a functional factor;
(2) the method combines chemical hydrolysis and enzyme hydrolysis, the whole preparation process is simple and easy to control, the enzymolysis efficiency is high, and the obtained collagen peptide with DPP-IV inhibitory activity is safe and has no side effect;
(3) the raw material adopted by the invention is the leather processing waste, namely the sheepskin, thereby effectively improving the resource utilization rate and avoiding the environmental pollution.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is an HPLC-MS/MS spectrum of sheep skin collagen peptide having DPP-IV inhibitory activity in the present invention;
FIG. 2 is a graph showing the effect of the sheep skin collagen peptide having DPP-IV inhibitory activity on DPP-IV in the present invention;
FIG. 3 is a graph showing the effect of gastrointestinal digestive enzymes on the activity of the sheep skin collagen peptide having DPP-IV inhibitory activity in the present invention.
FIG. 4 is a comparison of the inhibitory activity of the collagen peptides prepared by different protease hydrolysis and non-enzymatic hydrolysis on DPP-IV
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.5M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheepskin collagen solution to 8.5 and the temperature to 55 ℃, adding Alcalase alkaline protease with the enzyme dosage of 9600U for heat preservation and enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 2
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.25M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheepskin collagen solution to 8.5 and the temperature to 55 ℃, adding Alcalase alkaline protease with the enzyme dosage of 9600U for heat preservation and enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 3
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.75M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheepskin collagen solution to 8.5 and the temperature to 55 ℃, adding Alcalase alkaline protease with the enzyme dosage of 9600U for heat preservation and enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 4
The protein content of the sheepskin collagen solution obtained by the pyrolysis treatment in the examples 1-3 is determined according to the formula:
the protein extraction rate was calculated and the protein extraction rate obtained is shown in table 1:
TABLE 1 protein extraction yield in examples 1-3
| Examples | Protein extraction ratio/%) |
| Example 1 | 39.12882983 |
| Example 2 | 31.74603175 |
| Example 3 | 24.36323367 |
As can be seen from Table 1, the protein extraction rate in example 1 is the highest, and when the concentration of the alkali liquor in the alkali liquor soaking process is increased or decreased from 0.5M (mol/L), the protein extraction rate is decreased, and the concentration of the alkali liquor in the alkali liquor soaking process is selected to be 0.5M (mol/L).
Example 5
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.5M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheepskin collagen solution to 8.5 and the temperature to 55 ℃, adding Alcalase alkaline protease with the enzyme dosage of 7200U, preserving the temperature for enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation is finished;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 6
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.5M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheep skin collagen solution to 8.5 and the temperature to 55 ℃, adding Alcalase alkaline protease with the enzyme dosage of 12000U for heat preservation and enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 7
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.5M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheep skin collagen solution to 8.5 and the temperature to 55 ℃, adding 16000U of Alcalase alkaline protease for heat preservation and enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation is finished;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 8
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.5M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheep skin collagen solution to 8.5 and the temperature to 55 ℃, adding neutral protease with the enzyme dosage of 7200U, 9600U, 12000U and 16000U respectively, performing heat preservation and enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping the temperature for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation is finished;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 9
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.5M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
enzymolysis: adjusting the pH value of the obtained sheepskin collagen solution to 8.5 and the temperature to 55 ℃, adding flavourzyme with the enzyme dosage of 72000U, 9600U, 12000U and 16000U, carrying out heat preservation and enzymolysis for 5 hours, heating the enzymolysis liquid obtained after enzymolysis to 90 ℃, keeping for 15min for enzyme deactivation, and collecting the supernatant for later use after the enzyme deactivation is finished;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Comparative example 1
Soaking and cleaning raw sheep skin: washing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking in clear water at 25 ℃ for 3 days, and changing water every 6 hours until the sheep skin is soft. Cutting sheepskin into small pieces of 1 × 1cm, and cutting off excessive fat; adding tap water, and cleaning for 3h under stirring, and changing water every 1 h; adding deionized water, cleaning for 3h, changing water every 1h, stirring at a rotation speed of 300r/min, and cleaning until the conductivity of the aqueous solution is less than 450 mu;
soaking and deliming: taking the stirred solution, adding 3% ammonium sulfate solution at a material-liquid ratio of 1:3, stirring and soaking for 3h at normal temperature, changing the solution once every 1h, washing with deionized water after deashing, and measuring the pH once every 1h until the pH is 7.5;
removing foreign proteins: taking the solution with the adjusted pH, adding 1% saline solution according to the ratio of the materials to the solution of 1:5, stirring and soaking for 6h at normal temperature, changing the solution every 2h, and washing with distilled water at 30 ℃ for 3 times after soaking;
secondary deliming: adding the washed leather blocks into 5% soda solution to soak for 18h in an environment of 6 ℃, stirring once every 2h, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with 60% ethanol in a material-liquid ratio of 1:5 in an environment at 6 ℃, soaking and degreasing for 20 hours, filtering to remove ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into 10% NaCl solution in a material-liquid ratio of 1:5, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with distilled water for 3 times for later use;
soaking in alkali liquor: adding the degreased sheep skin particles into a 0.5M (mol/L) sodium hydroxide solution according to a feed-liquid ratio of 1:10, soaking for 24h at room temperature, stirring for 3 times a day, and changing the alkali liquor with the same concentration and volume once a day;
decalcification treatment: soaking the alkali-treated sheep skin particles in a sodium sulfate solution with the concentration of 0.75mol/L at the material-to-liquid ratio of 1:10, and standing at room temperature for 24 h. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking sheepskin grains in 2mol/L hydrogen peroxide solution with 10 times volume, standing at 6 ℃ for 24h, replacing the hydrogen peroxide solution every 6h, and then washing the sheepskin grains with water with 10 times volume for 3 times;
and (3) pyrolysis treatment: soaking sheepskin particles into deionized water, controlling the substrate concentration to be about 5% (w/v), boiling glue, and carrying out pyrolysis treatment at 90 ℃ for 5 hours to obtain a sheepskin collagen solution;
clarifying and filtering: taking 1% diatomite or activated carbon as adsorbent, vacuum filtering with sand core funnel (three-layer quantitative filter paper), centrifuging the filtrate at 10000rpm for 20min with centrifuge, removing upper layer oil and lower layer precipitate to obtain clarified solution;
concentrating and drying: carrying out rotary evaporation on the clarified solution, setting the rotary evaporation temperature to be 60 ℃, the rotating speed to be 45rpm, the vacuum degree to be below 0.9, and concentrating the volume by more than half; and freeze-drying the concentrated solution to obtain the collagen peptide.
Example 10
The supernatant obtained in the examples 5 to 10 after the enzymolysis is subjected to the determination of the peptide content (percentage) with the molecular weight distribution of <1kDa, and the peptide content (percentage) with the molecular weight distribution of <1kDa is calculated according to the following formula:
the data for the peptide content with a molecular weight distribution <1kDa of the supernatants obtained in examples 5-10 are shown in Table 2:
TABLE 2 data of collagen peptide having molecular weight distribution <1kDa in examples 5 to 10
As can be seen from table 2, when the dosage of Alcalase alkaline protease is 9600U, the peptide with the molecular weight distribution of <1kDa is the highest, the optimal dosage of Alcalase alkaline protease is 9600U, when neutral protease or flavourzyme is used in the enzymolysis, the optimal dosage of Alcalase alkaline protease is 12000U, and it can be seen that when Alcalase alkaline protease is used in the enzymolysis, the effect of increasing the peptide content with the molecular weight distribution of <1kDa is significant, and the preferred protease type used in the enzymolysis treatment is Alcalase alkaline protease.
The content of the peptide with the molecular weight distribution of less than 1kDa in the comparative example 1 is measured, the measurement result is 1.46%, and compared with the content of the collagen peptide with the molecular weight distribution of less than 1kDa in the examples 5-10, the enzyme digestion process has great improvement effect on the content of the collagen peptide with the molecular weight distribution of less than 1kDa, namely the content of the collagen peptide with the DPP-IV inhibitory activity in the sheep skin.
As shown in FIG. 1, the collagen peptide prepared in example 1 has DPP-IV inhibitory activity.
According to fig. 2, the collagen peptide with DPP-IV inhibitory activity prepared in the present invention has the structure Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val, and binds to the DPP-IV active site to inhibit the DPP-IV activity, thereby achieving the effects of inhibiting the rapid release and synthesis of insulin without decomposition of glucagon-like peptide 1(GLP-1), inhibiting the withering of β cells, and promoting the differentiation of β cells.
The inhibitory activity of the collagen peptide on DPP-IV before and after digestion is calculated to obtain a graph shown in figure 3, and the calculation formula of the inhibitory activity (DI) of DPP-IV is as follows: the formula for DPP-IV inhibitory activity (DI) is:
according to the calculated results, as shown in FIG. 3, the inhibitory activity of the collagen peptide (10ug/ml) on DPP-IV is 9.528%, the DPP-IV inhibitory activity retention rate after gastrointestinal tract digestion is 5.866%, and high DPP-IV inhibitory activity is still maintained. The formula of the intake concentration and the DPP-IV inhibition rate after digestion is as follows: y 0.4816ln (x) +4.7997, for a certain amount of consumers, the collagen peptide prepared by the present invention can be ingested in an appropriate amount according to the above formula to achieve an economical and good inhibitory effect on DPP-IV.
As shown in FIG. 4, the results of comparison of the DPP-IV inhibitory activities of the collagen peptides obtained by performing enzymatic hydrolysis with the different enzymes of examples 1 to 3 and 5 to 9 with those of the collagen peptide obtained in comparative example 1 show that, as shown in FIG. 4, the inhibitory activity of the collagen peptide after Alcalase alkaline protease treatment is higher than that of the collagen peptide after other protease treatment or the collagen peptide without enzymatic hydrolysis treatment.
The invention prepares the collagen peptide with DPP-IV inhibitory activity by taking the sheepskin as a raw material, combines the chemical hydrolysis method and the enzymatic hydrolysis method, and is scientific and reasonable, and the process is easy to operate. The obtained collagen peptide is safe, has no side effect, has higher DPP-IV inhibitory activity, and still keeps higher DPP-IV inhibitory activity after being digested by gastrointestinal digestive tract; in addition, the raw material adopted by the method for preparing the collagen peptide is leather processing waste sheepskin, so that the environmental pollution is effectively avoided, the resource utilization rate is improved, and the method has higher practical value and commercial value.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A sheep skin collagen peptide having DPP-IV inhibitory activity, characterized by: the amino acid sequence of the sheep skin collagen peptide is Gly-Pro-Ala-Gly-Pro-Ile-Gly-Pro-Val.
2. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 1, wherein: the method comprises the following steps:
pretreatment of sheepskin: physically degreasing unhaired sheep skin, cleaning, shearing into sheep skin grains of 1 × 1cm, then deliming, degreasing and deproteinizing, and cleaning the sheep skin grains again after the treatment is finished;
extracting sheep skin collagen: soaking the sheepskin grains obtained through pretreatment in an alkali solution, collecting the sheepskin grains after soaking, performing decalcification and hydrogen peroxide treatment, washing the sheepskin grains with clear water, and performing pyrolysis treatment to obtain a solution, namely a sheepskin collagen solution;
enzymatic hydrolysis of collagen: adding Alcalase alkaline protease, neutral protease and flavourzyme into the pH value of the obtained sheep skin collagen solution for hydrolysis, inactivating enzymes at high temperature after the hydrolysis is finished, and collecting supernatant for later use;
preparation of collagen peptide: and clarifying the supernatant of the sheep skin collagen peptide subjected to enzymolysis, carrying out DPP-IV inhibitory activity detection on the enzymolysis liquid, concentrating the solution, and freeze-drying to obtain the sheep skin collagen peptide with DPP-IV inhibitory activity.
3. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 2, wherein: the pretreatment of the sheepskin comprises the following steps:
soaking and cleaning raw sheep skin: rinsing the dehaired sheep skin with clear water until the skin material water is free of sand, soaking and maintaining water change in the soaking process, shearing off excessive fat, then cleaning under stirring and maintaining water change in the cleaning process,
soaking and deliming: adding the cleaned sheepskin into an ammonium sulfate solution, stirring and soaking at normal temperature, and keeping the solution changed. Washing with deionized water and adjusting the pH to 7.5 after deashing;
removing foreign proteins: adding the delimed sheepskin into salt water, stirring and soaking at normal temperature, replacing the solution once, and washing with water after soaking;
secondary deliming: soaking, adding the cleaned sheepskin grain skin blocks into a soda solution for soaking and stirring, and finally draining and washing for 3 times;
degreasing: mixing the cleaned sheepskin grains with ethanol, soaking for degreasing, filtering to remove the ethanol, cleaning the sheepskin grains with distilled water, adding the sheepskin grains into a NaCl solution, soaking for 24 hours, filtering, and cleaning the obtained sheepskin grains with the distilled water for later use.
4. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 2, wherein: the extraction of the sheep skin collagen comprises the following steps:
soaking in alkali liquor: and (3) adding the degreased sheepskin particles into a sodium hydroxide solution for soaking at room temperature, stirring for 3 times every day, and replacing alkali liquor with the same concentration and volume once every day.
Decalcification treatment: and soaking the alkali-treated sheep skin particles in a sodium sulfate solution, and standing at room temperature for 24 hours. Washing the sheepskin particles with deionized water at room temperature until the pH of the solution is 7.5;
hydrogen peroxide treatment: soaking the decalcified sheep skin grains in hydrogen peroxide solution for 24 h. The hydrogen peroxide solution is replaced after a period of time. Subsequently, the sheepskin particles were washed clean with water.
And (3) pyrolysis treatment: soaking cleaned sheepskin particles into water, boiling glue, and raising the temperature for a period of time.
5. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 2, wherein: the enzymatic hydrolysis of the collagen comprises the following steps:
adjusting pH and temperature to optimum pH and temperature of protease, adding protease, and performing enzymolysis for sufficient time. Heating the enzymolysis liquid obtained after enzymolysis for enzyme deactivation.
6. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 2, wherein: the preparation of the collagen peptide comprises the following steps:
clarifying and filtering: taking diatomite or active carbon as an adsorbent, performing suction filtration by using a sand core funnel (three layers of quantitative filter paper), centrifuging filtrate at a high rotating speed by using a centrifuge, and removing upper-layer grease and lower-layer precipitate to obtain a clarified solution;
concentrating and drying: rotating and evaporating the clarified solution, and concentrating by more than half of the volume; and freeze-drying the concentrated solution to obtain the collagen peptide.
7. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 5, wherein: the protease in the enzyme hydrolysis is Alcalase alkaline protease, neutral protease and flavourzyme.
8. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 5, wherein: the dosage of the protease in the enzymatic hydrolysis is 7200-16000U.
9. The method for preparing the collagen peptide of sheepskin having DPP-IV inhibitory activity according to any one of claims 5, 7 and 8, wherein: the protease in the enzymatic hydrolysis is Alcalase alkaline protease, and the dosage of the protease is 9600U.
10. The method for preparing the collagen peptide of sheep skin having DPP-IV inhibitory activity according to claim 4, wherein: the concentration of alkali in the alkali liquor soaking is 0.5 mol/L.
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