CN116769861B - Collagen tripeptide with high GPH content and preparation method thereof - Google Patents
Collagen tripeptide with high GPH content and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a collagen tripeptide with high GPH content and a preparation method thereof. The preparation method comprises the following steps: the fish skin is taken as a raw material, mixed with water, and subjected to enzymolysis treatment by adding a certain amount of compound protease, and then subjected to enzyme deactivation, centrifugation, concentration and freeze drying to obtain the propeptide tripeptide. The collagen tripeptide is prepared by the complex enzyme hydrolysis method, no food additive is needed in the process, and the collagen tripeptide has the advantages of simple preparation process, low equipment requirement, low cost, green and safety and the like. The collagen tripeptide prepared by the invention has the characteristic of high GPH content, wherein the glycine GPH content can reach 3.6%.
Description
Technical Field
The invention relates to the technical field of processing of propeptide tripeptides, in particular to a collagen tripeptide and a preparation method thereof, wherein the GPH content of the collagen tripeptide is high.
Background
In daily life, many people consider that foods such as pig trotters and chicken feet contain abundant collagen, so that it is considered that the skin of the pig trotters, chicken feet and the like which are frequently eaten and supplement collagen is good, but the skin is not actually so. The practice proves that the molecular weight of the collagen in the foods such as pig trotters, chicken feet and the like is very large, the collagen is not easy to be absorbed by human bodies, and the collagen really needed by skin is the collagen consisting of 3 amino acid molecules, namely 'collagen tripeptide'.
The collagen tripeptide is a small peptide which is obtained by hydrolyzing collagen and consists of three amino acids, and has various biological activities and higher application value; the collagen tripeptide hydrolyzed by the collagen has the functions of moisturizing, nourishing, brightening skin, tightening, preventing wrinkles and the like, and is mainly applied to the fields of cosmetics industry and food industry.
With the rapid development of the age, women are defined with a plurality of new roles, life and work, so that the women always have fatigue and anxiety states, and the skin also enters a sub-health state with time. Technological advances have made it possible for people to continue the beauty. Collagen peptide and collagen tripeptide are also applied to medical industry at present, and are used for building anti-aging products which are most suitable for eastern face and bone phase, so that better experience is brought to consumers.
On the market, a plurality of products are marked with collagen-rich collagen tripeptide, and have the effects of whitening, resisting aging and compacting. Collagen has important physiological functions and biological activities as an important structural protein in a human body, and is widely applied in the medical field; however, collagen is a macromolecular compound and needs to be metabolized into small-molecule short peptides in vivo to exert its effect. Meanwhile, the manually extracted collagen contains certain immunogenicity, and the potential risk of application of the collagen is increased. Collagen tripeptide (collagen tripeptide, CTP) is the smallest molecular peptide in collagen metabolites, consists of 3 amino acids, has the advantages of small relative molecular mass, high purity, no antigenicity, hypoallergy and the like, becomes a research hotspot in various industries at present, and has a trend of replacing collagen in the application field.
Patent CN114805550a filed by shandong hengxin biotechnology limited discloses a method for producing collagen tripeptide from cow leather, wherein the collagen tripeptide is prepared by the following steps: pretreating, denaturing, exciting, inactivating enzyme, centrifuging, collecting supernatant, decolorizing with K15 activated carbon column, and spray drying to obtain collagen tripeptide. The method can thoroughly hydrolyze the collagen, and the enzymolysis product has small and concentrated molecular weight, high collagen tripeptide yield, high hydroxyproline content, high total nitrogen content, low ash content, fine finished product and good water solubility, and is beneficial to large-scale production. However, the method has complex procedures and high production cost.
Patent CN115449535a filed by sea Nanhua, collagen technologies and companies, discloses a collagen tripeptide of bone origin and a preparation method thereof, wherein the collagen tripeptide is prepared by the following method: steaming and boiling the beef bones for 1-2 hours, removing meat, fascia and grease attached to the beef bones, and then soaking the beef bones in 0.1-0.2 mol/L sodium hydroxide solution for 2-4 hours to obtain clean beef bones. And then 25-35% of compound protease (mass percent), 25-35% of flavourzyme, 25-35% of alkaline protease, 5-10% of animal proteolytic enzyme and 2-5% of enzyme activator are used for enzymolysis, and finally the bone remote collagen tripeptide is obtained. Although the method adopts compound enzyme to carry out enzymolysis on the collagen, the collagen peptide with small molecular weight and high activity can be obtained. However, this technique has the disadvantages that: a large amount of alkali liquor is needed in the experiment, so that waste liquor is difficult to treat, the enzyme dosage is large, and the production cost is high.
Patent CN115669822a filed by sea Nanhua collagen research science and technology company discloses a fruit-flavored beverage rich in collagen tripeptide and a processing technology thereof, wherein the fruit-flavored beverage comprises the following components: corn silk juice, fresh orange juice, fruit particles, collagen tripeptide, vitamin C, sucralose, citric acid, sodium citrate, potassium sorbate, edible essence, xylitol, suspending agent and purified water. The collagen tripeptide fruity beverage prepared by adopting the collagen tripeptide fruity beverage rich in collagen and the processing technology thereof has rich nutrition, has the effect of beautifying, and simultaneously has better mouthfeel and better stability. The invention discloses a production method of collagen tripeptide for skin repair, which is characterized in that fish skin is used as a raw material, and the collagen tripeptide for skin repair with the effects of moisturizing, whitening, moisturizing and the like is prepared through water milling, filtering, enzymolysis and the like, and filter cloth is embedded in an annular cavity area of a double-layer filter cylinder to form a multi-V-shaped sequentially distributed shape, so that the assembled multi-stage radioactive filter cylinder can be used for the filtering operation in the whole process of preparing the collagen tripeptide, single multiple filtering of a solution is realized, the purity of a collagen tripeptide product is greatly improved, the complicated operation of multiple filtering in the prior art is reduced, and the production efficiency is effectively improved. Patent CN114634549A of Beijing Cheng Meinuo biotechnology limited company discloses a collagen tripeptide product and a preparation method and application thereof, wherein the collagen tripeptide comprises at least one tripeptide with one end being basic amino acid, and the molecular formula of the tripeptide of the basic amino acid comprises Gly-X-Arg, gly-X-His and Gly-X-Lys, wherein X is amino acid; the content of basic amino acid is not less than 10% based on the total weight of collagen tripeptide. The invention also provides a method for preparing collagen tripeptide, which comprises the steps of adding water into collagen, wherein the pH value of the solution is 5-8, and adding compound protease accounting for 0.5-5% of the weight of the collagen; enzymolysis at 45-60deg.C for 2-6 hr; the compound protease comprises ficin and zingibain; adjusting pH value of the enzymolysis liquid to 7-8.5, adding starter accounting for 1-8% of the weight of collagen, and fermenting at 30-42 ℃ for 2-4 hours; the starter comprises Hansenula debaryomyces, pediococcus pentosaceus and Micrococcus variant; and (3) carrying out solid-liquid separation on the fermentation liquor, and collecting filtrate. However, the process of preparing the collagen tripeptide by the disclosed invention is complex, multiple filtration is needed, the pH value is regulated for multiple times, and the content of glycine GPH in the prepared collagen tripeptide is low.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a collagen tripeptide and a preparation method thereof, wherein the glycine GPH content in the collagen tripeptide prepared by a simple process is up to 3.6%.
The collagen tripeptide with high glycine GPH content is prepared by mainly taking fish skin as a raw material, adding endoprotease, alkaline protease 37071 and papain for hydrolysis according to a certain mass ratio, controlling enzymolysis treatment and the like, and has the advantages of simple process, low cost, no need of adding any food additive, simple preparation process, low equipment requirement, low cost, greenness, safety and the like.
The object of the invention is achieved by at least one of the following technical solutions.
A preparation method of collagen tripeptide with high GPH content comprises the following steps:
Stirring and mixing the fish skin and water, heating to 100-110 ℃ and keeping for 30-40 min, and after collagen in the fish skin is depolymerized, mixing the fish skin according to the mass ratio: complex protease = 100:0.5 to 100:2, adding compound protease, and hydrolyzing to obtain fish skin collagen hydrolysate; inactivating enzyme, centrifuging, concentrating, and freeze drying to obtain collagen tripeptide.
Further, the mass ratio of the fish skin to the water is 1:1 to 1:9.
Further, the added complex proteases include endoprotease proline, alkaline protease 37071 and papain.
Further, the mass ratio of the endoprotease proline, the alkaline protease 37071 and the papain is 1:1:1.
Further, the temperature of the hydrolysis treatment is 50-65 ℃, and the time of the hydrolysis treatment is 6-12 h.
Further, the enzyme deactivation condition is that the enzyme is heated for 30-60 min at 85-95 ℃.
Further, the rotational speed of the centrifugation is 4000 r/min-8000 r/min, and the centrifugation time is 10 min-15 min.
Further, the freeze drying temperature is-40 ℃ to-60 ℃, and the freeze drying time is 24h to 48h.
The invention takes fish skin as raw material, depolymerizes collagen by high temperature, and adds a certain amount of compound protease (endoprotease proline, alkaline protease 37071 and papain) to carry out hydrolysis reaction on the collagen under a certain condition, thus forming the peptide tripeptide. It has a high glycine GPH content and a high CTP content.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The invention uses fish skin as raw material of collagen, adds a certain amount of proline endoprotease, alkaline protease 37071 and papain to carry out enzymolysis reaction under a certain condition, thus forming the propeptide tripeptide.
(2) The collagen tripeptide is prepared by the complex enzyme hydrolysis method, no food additive is needed in the process, and the collagen tripeptide has the advantages of simple preparation process, low equipment requirement, low cost, green and safety and the like.
(3) The collagen tripeptide prepared by the invention has the characteristic of high GPH content, wherein the glycine GPH content can reach 3.6%.
Detailed Description
The following is a further description and illustration of specific embodiments of the invention in connection with examples. Embodiments of the present invention are not limited thereto. It should be noted that the following is performed under conventional conditions or conditions recommended by the manufacturer, unless specific conditions are noted. The raw materials, reagents, etc. used, which are not noted to the manufacturer, are conventional products commercially available.
Comparative example 1
Mixing 500g of tilapia skin and 500g of water, stirring, heating to 100 ℃ and maintaining for 30min, adding 1.2% of proline endoprotease (6 g based on the weight of the substrate tilapia skin) after the tilapia skin collagen is depolymerized, stirring and hydrolyzing, hydrolyzing for 8h at 50 ℃ to obtain tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 85 ℃, and maintaining for 30min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residue, concentrating, and freeze drying at-40deg.C for 24 hr to obtain comparative example 1-collagen tripeptide No. 1.
Example 1
Mixing 500g of tilapia skin and 500g of water, stirring, heating to 100 ℃ and keeping for 30min, adding 1.2% of compound protease (6 g based on the weight of the tilapia skin as a substrate) after the tilapia skin collagen is depolymerized, wherein the mass of proline endoprotease is 2g, the mass of alkaline protease 37071 is 2g, and the mass of papain is 2g, stirring and hydrolyzing, hydrolyzing at 50 ℃ for 8h, and obtaining tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 85 ℃, and maintaining for 30min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residue, concentrating, and freeze-drying at-40deg.C for 24 hr to obtain the final product of example 1-collagen tripeptide No. 1.
Comparative example 2
Mixing 100g of tilapia skin with 500g of water, stirring, heating to 110 ℃ and keeping for 40min, adding 1.2% papain (1.2 g based on the weight of the tilapia skin substrate) after the tilapia skin collagen is depolymerized, stirring and hydrolyzing, hydrolyzing at 60 ℃ for 12h, and obtaining tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 95 ℃ and maintaining for 40min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residue, concentrating, and freeze drying at-60deg.C for 48 hr to obtain comparative example 2-collagen tripeptide No. 2
Example 2
Mixing 100g of tilapia skin with 500g of water, stirring, heating to 110 ℃ and keeping for 40min, adding 1.2% of compound protease (1.2 g based on the weight of the tilapia skin serving as a substrate) after the collagen of the tilapia skin is depolymerized, wherein the mass of proline endoprotease is 0.4g, the mass of alkaline protease 37071 g is 0.4g, and the mass of papain is 0.4g, hydrolyzing at 60 ℃ for 12h, and obtaining tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 95 ℃ and maintaining for 40min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residue, concentrating, and freeze drying at-60deg.C for 48 hr to obtain example 2-collagen tripeptide No. 2.
Comparative example 3
Mixing 500g of tilapia skin and 1500g of water, stirring, heating to 105 ℃ and keeping for 35min, adding 1.5% alkaline protease 37071 (7.5 g based on the weight of the substrate tilapia skin) after the tilapia skin collagen is depolymerized, stirring and hydrolyzing, hydrolyzing for 10h at 55 ℃ to obtain tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 90 ℃ and keeping for 35min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residues, concentrating, and freeze drying at-50deg.C for 36 hr to obtain comparative example 3-collagen tripeptide No. 3.
Example 3
Mixing 500g of tilapia skin and 1500g of water, stirring, heating to 105 ℃ and keeping for 35min, adding 1.5% of compound protease (7.5 g based on the weight of the tilapia skin as a substrate) after the tilapia skin collagen is depolymerized, wherein the mass of proline endoprotease is 2.5g, the mass of alkaline protease 37071 g is 2.5g, and the mass of papain is 2.5g, stirring and hydrolyzing, hydrolyzing for 10h at 55 ℃ to obtain tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 90 ℃ and keeping for 35min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residue, concentrating, and freeze drying at-50deg.C for 36 hr to obtain the final product of example 3-collagen tripeptide No. 3.
Comparative example 4
Mixing 500g of tilapia skin and 1500g of water, stirring, heating to 105 ℃ and keeping for 35min, adding 2% of alkaline protease 37071 (10 g based on the weight of the substrate tilapia skin) after the collagen of the tilapia skin is depolymerized, stirring and hydrolyzing, hydrolyzing for 10h at 55 ℃ to obtain tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 90 ℃ and keeping for 35min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residue, concentrating, and freeze drying at-50deg.C for 36 hr to obtain comparative example 4-collagen tripeptide No. 4.
Example 4
Mixing 500g of tilapia skin and 1500g of water, stirring, heating to 105 ℃ and keeping for 35min, adding 2% of compound protease (10 g based on the weight of the tilapia skin as a substrate) after the collagen of the tilapia skin is depolymerized, wherein the mass of endoprotease proline is 3.33g, the mass of alkaline protease 37071 g and the mass of papain is 3.33g, stirring and hydrolyzing, hydrolyzing at 55 ℃ for 10h, and obtaining tilapia skin collagen hydrolysate after the hydrolysis is completed; heating to 90 ℃ and keeping for 35min to inactivate enzyme; centrifuging at 8000r/min for 20min to remove residue, concentrating, and freeze drying at-50deg.C for 36 hr to obtain the final product of example 4-collagen tripeptide No. 4.
Effect verification
The related detection and evaluation method related to the effect of the invention comprises the following steps:
1. method for detecting glycine- (GPH) content in collagen tripeptide
The GPH content of the single enzyme hydrolysates collagen tripeptides 1,2, 3, 4 of comparative examples 1-4 was determined by reverse phase chromatography using the combined hydrolysates collagen tripeptides 1,2, 3, 4 of examples 1-4 endoprolinase + alkaline protease 37071+ papain. The chromatographic conditions were set as follows:
chromatographic column: ZORBAXSB-Aq,46X250mm 5m; mobile phase: 0.1% trifluoroacetic acid solution; detection wavelength: 220nm; flow rate: 1mL/min; column incubator: 50"C; sample injection volume: 10uL, analysis time: 15min.
And calculating according to the established standard curve to obtain the glycine- (GPH) ratio in the sample.
2. Method for detecting content of Collagen Tripeptide (CTP) in collagen tripeptide
CTP content of collagen tripeptides No. 1, no. 2, no. 3, and No. 4, which are single enzyme hydrolysates of comparative examples 1 to 4, was determined by high performance size exclusion chromatography using the combination of proline endoprotease + alkaline protease 37071+ papain. The chromatographic conditions were set as follows:
Chromatographic column: superdex' 30Increate10/300 GL and a protective column are added; detection wavelength: 214nm; flow rate: 0.3mL/min; sample injection volume: 10pL; analysis time: 75min.
And calculating according to the established standard curve to obtain the relative content of the Collagen Tripeptide (CTP) in the sample.
TABLE 1 Glycine- (GPH) content in collagen tripeptides
| GPH content (%) | |
| Comparative example 1-collagen tripeptide No. 1 | 1.8 |
| EXAMPLE 1 collagen tripeptide No. 1 | 2.5 |
| Comparative example 2-collagen tripeptide No. 2 | 1.2 |
| EXAMPLE 2 collagen tripeptide No. 2 | 2.8 |
| Comparative example 3-collagen tripeptide No. 3 | 1.4 |
| EXAMPLE 3 collagen tripeptide No. 3 | 3.0 |
| Comparative example 4-collagen tripeptide No. 4 | 1.6 |
| EXAMPLE 4 collagen tripeptide No. 4 | 3.6 |
As can be seen from table 1, the Glycine (GPH) content in the collagen tripeptides of examples 1,2, 3 and4 was significantly increased, since the Glycine (GPH) content in the collagen tripeptides was increased from 2.5% to 3.6% in the range of 0 to 2% of the enzyme addition amount (based on the weight of the substrate fish skin) under the same fish skin to water mass ratio, enzymatic hydrolysis temperature and time. In addition, the Glycine (GPH) content of the collagen tripeptides of examples 1 to 4 was improved to some extent as compared with comparative examples 1 to 4. The effect of increasing Glycine (GPH) content in collagen tripeptide by using composite protease is better than that by using single protease.
TABLE 2 Collagen Tripeptide (CTP) content
| CTP content (%) | |
| Comparative example 1-collagen tripeptide No. 1 | 15 |
| EXAMPLE 1 collagen tripeptide No. 1 | 26 |
| Comparative example 2-collagen tripeptide No. 2 | 13 |
| EXAMPLE 2 collagen tripeptide No. 2 | 25 |
| Comparative example 3-collagen tripeptide No. 3 | 14 |
| EXAMPLE 3 collagen tripeptide No. 3 | 28 |
| Comparative example 4-collagen tripeptide No. 4 | 18 |
| EXAMPLE 4 collagen tripeptide No. 4 | 30 |
As can be seen from table 2, examples 1,2, 3 and 4 all showed a certain increase in the content of Collagen Tripeptide (CTP) compared with comparative examples 1,2, 3 and 4, indicating that the effect of increasing the content of Collagen Tripeptide (CTP) by using the complex protease was better than that by using the single protease. The Collagen Tripeptide (CTP) content may reach 30%.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structural changes made by the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (7)
1. The preparation method of the collagen tripeptide with high GPH content is characterized by comprising the following steps:
Stirring and mixing the fish skin and water, heating to 100-110 ℃ and keeping for 30-40 min, and after collagen in the fish skin is depolymerized, mixing the fish skin according to the mass ratio: complex protease = 100:0.5 to 100:2, adding compound protease, and hydrolyzing to obtain fish skin collagen hydrolysate; inactivating enzyme of the hydrolysate, centrifuging, concentrating, and freeze drying to obtain collagen tripeptide;
The added compound protease comprises endoprotease proline, alkaline protease 37071 and papain; the mass ratio of the endoprotease proline to the alkaline protease 37071 to the papain is 1:1:1, a step of;
the temperature of the hydrolysis treatment is 50-65 ℃, and the time of the hydrolysis treatment is 6-12 h.
2. The method for preparing collagen tripeptide with high GPH content according to claim 1, wherein the mass ratio of the fish skin to the water is 1:1 to 1:9.
3. The method for preparing collagen tripeptide with high GPH content according to claim 1, wherein the condition for inactivating the enzyme is heating at 85-95 ℃ for 30-60 min.
4. The method for preparing collagen tripeptide with high GPH content according to claim 1, wherein the rotational speed of the centrifugation is 4000 r/min-8000 r/min and the time of the centrifugation is 10 min-15 min.
5. The method for preparing collagen tripeptide with high GPH content according to claim 1, wherein the freeze-drying temperature is-40 ℃ to-60 ℃ and the freeze-drying time is 24h to 48h.
6. The collagen tripeptide prepared by the method according to any one of claims 1 to 5.
7. The collagen tripeptide according to claim 6, wherein the collagen tripeptide has a glycine GPH content of up to 3.6%.
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