CN114634549B - Collagen tripeptide product and preparation method and application thereof - Google Patents
Collagen tripeptide product and preparation method and application thereof Download PDFInfo
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- CN114634549B CN114634549B CN202210367027.9A CN202210367027A CN114634549B CN 114634549 B CN114634549 B CN 114634549B CN 202210367027 A CN202210367027 A CN 202210367027A CN 114634549 B CN114634549 B CN 114634549B
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- collagen
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 162
- 102000008186 Collagen Human genes 0.000 title claims abstract description 162
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- 238000002360 preparation method Methods 0.000 title claims description 12
- 238000004519 manufacturing process Methods 0.000 title description 2
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- FKJQNJCQTKUBCD-XPUUQOCRSA-N Gly-Ala-His Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O FKJQNJCQTKUBCD-XPUUQOCRSA-N 0.000 description 1
- ITZOBNKQDZEOCE-NHCYSSNCSA-N Gly-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN ITZOBNKQDZEOCE-NHCYSSNCSA-N 0.000 description 1
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- HAMNKKUPIHEESI-UHFFFAOYSA-N aminoguanidine Chemical compound NNC(N)=N HAMNKKUPIHEESI-UHFFFAOYSA-N 0.000 description 1
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- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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, cold insoluble globulin [CIG]
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
Abstract
The application provides a collagen tripeptide product, which comprises at least one tripeptide with one end being basic amino acid, wherein 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 application 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.
Description
Technical Field
The application relates to a collagen tripeptide product, a preparation method of the collagen tripeptide product and application of the collagen tripeptide product in cosmetics.
Background
Collagen is an important component of extracellular matrix, and its content is about 1/3 of that of human protein, so that it can play an important role in maintaining the structure and function of human body. As people age, collagen is lost. Meanwhile, because collagen in the dermal extracellular matrix of the skin tissue has low reversibility, the collagen can more easily generate non-enzymatic reaction with metabolic molecules such as glucose and the like to generate saccharification end products (advanced glycationend products, AGE), such as glycosylated collagen and the like, and a series of adverse effects are generated on the tissue.
The collagen peptide is a micromolecular peptide product prepared by enzymolysis of macromolecular collagen. The research reports that the collagen peptide has the effects of improving skin moisture, reducing wrinkles, resisting aging and the like. Especially, the collagen tripeptide has small molecular weight and is easy to be absorbed and utilized by human bodies, so the collagen tripeptide has very good effect of deferring senility. The collagen tripeptide which is rich in basic amino acid has the anti-saccharification function and is beneficial to maintaining the normal functions of tissues. At present, collagen tripeptide products rich in basic amino acids are lacking in the market, and the anti-glycation effect of the collagen peptide is poor.
Disclosure of Invention
In order to fill the market gap, the application provides a collagen tripeptide product which contains abundant basic amino acids.
Accordingly, in one aspect, the present application provides a collagen tripeptide product, characterized in that the collagen tripeptide product comprises a collagen tripeptide; wherein the collagen tripeptide comprises a tripeptide with at least one end of basic amino acid, and the molecular formula of the tripeptide with at least one end of basic amino acid comprises Gly-X-Arg, gly-X-His and Gly-X-Lys, wherein X is amino acid; the content of the basic amino acid is not less than 10% based on the total weight of the collagen tripeptide; preferably, the basic amino acid content is 12-20%.
In another aspect, the present application provides a method for preparing a collagen tripeptide having at least one basic amino acid at one end, comprising the steps of:
1) Adding water into collagen, regulating the pH value of the collagen solution to be 5-8, and adding compound protease accounting for 0.5-5% of the weight of the collagen; carrying out enzymolysis for 2-6 hours at 45-60 ℃ to obtain enzymolysis liquid; wherein the complex protease comprises ficin and zingibain; preferably, the weight ratio of ficin to zingibain is 2-4:1;
2) Adjusting pH value of the enzymolysis liquid to 7-8.5, adding a starter which is 1-8% of the weight of the collagen, and fermenting for 2-4 hours at 30-42 ℃ to obtain fermentation liquid; the starter comprises Debaryomyces hansenii, pediococcus pentosaceus and Micrococcus variant; preferably, the weight ratio of the Debaryomyces hansenii, the Pediococcus pentosaceus and the Micrococcus variant is 1:1.8-2.2:1.8-2.2;
3) After solid-liquid separation of the fermentation broth, the filtrate was collected.
In a third aspect, the application provides the use of said collagen tripeptide product in cosmetics and functional food products.
The collagen tripeptide product rich in basic amino acids provided by the application contains a plurality of basic amino acids, and the content of the basic amino acids is high and is at least 10% of the total weight of the collagen tripeptide. Experiments prove that the anti-glycal effect of the collagen tripeptide product with high content of basic amino acid is very remarkable (37.2%), and is far higher than that of collagen tripeptide (8.2%) and collagen (5.5%).
Detailed Description
The application provides a collagen tripeptide product, which is characterized in that the collagen tripeptide product contains collagen tripeptide; wherein the collagen tripeptide comprises a tripeptide with at least one end of basic amino acid, and the molecular formula of the tripeptide with at least one end of basic amino acid comprises Gly-X-Arg, gly-X-His and Gly-X-Lys, wherein X is amino acid; the content of the basic amino acid is not less than 10% based on the total weight of the collagen tripeptide.
The amino acid represented by X may be any amino acid.
In a preferred embodiment, the amino acid represented by X is one or more of Pro, hyp, glu, ser, ala, phe, leu and Ile.
In order to obtain a better anti-glycation effect, the content of the basic amino acid is preferably 12-20%.
In order to obtain a better anti-glycation effect, preferably, the collagen tripeptide product contains 20-40 wt% of collagen tripeptide; preferably, the collagen tripeptide product contains 25-35 wt.% collagen tripeptide.
In order to facilitate easier absorption and utilization, a better anti-glycation effect is obtained, preferably the tripeptide having at least one basic amino acid at its end has an average molecular weight between 200 and 500 daltons.
In order to facilitate the acquisition of more basic amino acids, preferably, the preparation method of the collagen tripeptide product comprises the following steps:
1) Adding water into collagen, and adjusting the pH value of the collagen solution to 5-8, preferably 6-7; adding 0.5-5% of composite protease, preferably 1-4% of composite protease, more preferably 2-4% of composite protease by weight of collagen; carrying out enzymolysis for 2-6 hours, preferably 3-5 hours, at 45-60 ℃ to obtain an enzymolysis solution; wherein the compound protease comprises ficin and ginger protease, and the weight ratio of the ficin to the ginger protease is 2-4:1;
2) Adjusting pH of the enzymolysis liquid to 7-8.5, adding starter which is 1-8% of the weight of collagen, preferably 3-6%; fermenting at 30-42 deg.C for 2-4 hr to obtain fermentation liquor; the starter comprises Debaryomyces hansenii, pediococcus pentosaceus and Micrococcus mutans, wherein the weight ratio of the Pediococcus pentosaceus to the Micrococcus mutans is 1:1.8-2.2:1.8-2.2;
3) After solid-liquid separation of the fermentation broth, the filtrate was collected.
Preferably, in said step 2), said starter culture further comprises lactobacillus sake and/or staphylococcus botulinum, in order to obtain more basic amino acids.
Preferably, in the step 2), the starter further comprises lactobacillus sake and staphylococcus botulinum, and the debaryomyces hansenii, pediococcus pentosaceus, lactobacillus sake, and the mass ratio of micrococcus mutans and staphylococcus botulinum is 1:1.8-2.2:3.8-4.2:1.8-2.2:3.8-4.2.
The application also provides a method for preparing collagen tripeptide with at least one basic amino acid at one end, which is characterized by comprising the following steps:
1) Adding water into collagen, adjusting pH of the collagen solution to 5-8, adding compound protease with weight of collagen of 0.5-5%, preferably 1-4%, more preferably 3-4%; carrying out enzymolysis for 1-8 hours, preferably 2-6 hours, at 45-65deg.C, preferably 50-60deg.C; obtaining enzymolysis liquid; wherein the complex protease comprises ficin and zingibain; preferably, the weight ratio of ficin to zingibain is 2-4:1;
2) Adjusting pH of the enzymolysis liquid to 6.5-8.5, preferably pH 7-8, adding starter (1-8% by weight of collagen), preferably starter (2-7% by weight), and more preferably starter (3-6% by weight); at 25-45deg.C, preferably 30-40deg.C; fermenting for 1-6 hours, preferably 2-5 hours; obtaining fermentation liquor; the starter comprises Debaryomyces hansenii, pediococcus pentosaceus and Micrococcus variant; preferably, the weight ratio of the Debaryomyces hansenii, the Pediococcus pentosaceus and the Micrococcus variant is 1:1.5-2.5:1.5-2.5, preferably 1:1.8-2.2:1.8-2.2;
3) After solid-liquid separation of the fermentation broth, the filtrate was collected.
In a preferred embodiment, in said step 2), said fermenting agent further comprises lactobacillus sake and/or staphylococcus botulinum; preferably, the Debaryomyces hansenii, the Pediococcus pentosaceus, the Lactobacillus sake, the Micrococcus mutans and the Staphylococcus botulinum have a mass ratio of 1:1.5-2.5:3.5-4.5:1.5-2.5:3.5-4.5, preferably 1:1.8-2.2:3.8-4.2:1.8-2.2:3.8-4.2.
In order to remove the disturbance of impurities in the collagen, in a preferred embodiment, the method further comprises a step of refining the collagen before said step 1).
In a preferred embodiment, the clarifying is performed by adding a clarifying agent, stirring, and standing.
In a preferred embodiment, a clarifying agent is added to the collagen solution to adjust the pH of the collagen solution to 2-7, preferably the pH of the collagen solution to 3-6; after stirring uniformly, standing for 2-8 hours, preferably 3-6 hours, to obtain refined collagen.
The clarifying agent is not particularly limited, but in order to obtain a better impurity removing effect, in a preferred embodiment, the clarifying agent is egg white, sodium caseinate, okra polysaccharide; preferably, the weight ratio of the egg white, the sodium caseinate and the okra polysaccharide is 1:1-4:2-5, preferably 1:1-3:2.5-4.
In order to obtain better impurity removal effect, in a preferred embodiment, the clarifier is added in an amount of 0.5-3% by weight of collagen, preferably 1-2% by weight of collagen.
In order to obtain a better impurity removal effect, in a preferred embodiment, the clarifier has a pH of 3-5.
In order to obtain a better impurity removal effect, in the step 3), the flow rate of the homogeneous membrane filtration is 2-6cm/s, the operating voltage is 100-150V, the current is 50-100A, and the liquid with the outflow time of 30-60 minutes is collected.
The present application also provides a method for preparing an anti-glycation collagen tripeptide enriched in basic amino acids from a collagen-containing raw material, the method comprising the steps of:
step S1, preparing a crude extract of total protein: weighing raw materials, adding sodium hydroxide solution, soaking for 2-4 hours, cleaning for 2-3 times, adding dissolving water, homogenizing in a grinder to paste, and obtaining total protein crude extract;
step S2, preparing a crude collagen extract: adjusting the pH value of the total protein crude extract to 2-4, heating to 75-90 ℃, preserving heat and extracting gelatin for 3-8 hours to obtain a collagen crude extract;
step S3, refining the crude collagen extract: adding clarifier into the crude collagen extract, adjusting pH to 2-7, stirring, standing for 2-8 hr to obtain refined collagen;
step S4, preparation of collagen peptide: regulating pH of the collagen refined product to 5-8, and adding fructus fici extract and rhizoma Zingiberis recens extract for enzymolysis; carrying out enzymolysis for 2-6 hours at 45-60 ℃ to obtain enzymolysis liquid; regulating pH to 7-8.5, adding Debaryomyces hansenii, pediococcus pentosaceus, lactobacillus sake, micrococcus mutans, and Staphylococcus sarcose, fermenting at 30-40deg.C for 2-4 hr to obtain enzymolysis fermentation liquid;
step S5, preparation of collagen tripeptide rich in basic amino acid: filtering the enzymolysis fermentation liquor to remove impurities.
Preferably, the raw material in the step S1 is one of the skin, the scale or the bone of the animal, and the mass percentage of the sodium hydroxide solution is 0.6-1%.
The crude collagen extract prepared in the step S2 is not easily delaminated during refining, so that the yield is low. In order to improve the yield and promote layering, preferably, the clarifying agent in the step S3 is egg white, sodium caseinate and okra polysaccharide, wherein the mass ratio of the egg white to the sodium caseinate to the okra polysaccharide is 1:1-4:2-5, the mixture is mixed and stood for 2-6 hours, the adding amount of the clarifying agent is 0.5-3% of the mass of the crude collagen extract, and the pH value of the clarifying agent is 3-5.
Preferably, the compound protease in the step S4 is a combination of ficin and ginger protease, the mass ratio of the ficin to the ginger protease is 3-2:1, the addition amount of the compound protease is 0.5% -5% of the mass of the refined collagen, and the enzymolysis pH value is 6.5-8. The added starter is a combination of the Debaryomyces hansenii, pediococcus pentosaceus, lactobacillus sake, micrococcus mutans and staphylococcus sarcodactylis, and the weight ratio is 1-0.5:1:1.5-2.5:1:1.5-2.5, wherein the addition amount of the combined starter is 1-8% of the refined collagen, and the fermentation pH value is 7.0-8.5.
Preferably, the homogeneous membrane filtration flow rate in step 5 is 2-6cm/s, the operating voltage is 100-150V, the current is 50-100A, and the collagen tripeptide content of effluent liquid in different time periods is detected.
The application also provides application of the collagen tripeptide product in cosmetics and functional foods.
The cosmetic is helpful for delaying skin aging, and is especially beneficial for skin care of diabetes patients.
The collagen tripeptide product provided by the application can be used for functional foods, especially for acidic beverages, for example, the concentration of the collagen tripeptide solution in the beverage is 10 weight percent, and the pH value of the beverage is 2.5.
Example 1: collagen tripeptide rich in basic amino acid and preparation thereof
Step S1: weighing 100g of tilapia skin, cutting into small blocks, adding 2000mL of sodium hydroxide solution with mass fraction of 0.8%, soaking for 4 hours, cleaning for 3 times, adding softened water, homogenizing in a grinder to paste, and obtaining total protein crude extract;
step S2: adjusting the pH value of the total protein crude extract to 2.6, heating to 90 ℃, preserving heat and extracting gelatin for 5 hours to prepare a collagen crude extract;
step S3: 1g of egg white, 3g of sodium caseinate and 4g of okra polysaccharide are weighed, added into a collagen crude extract, the pH value is regulated to 3, stirred uniformly and then kept stand for 4 hours, and supernatant fluid is collected to obtain a collagen refined product;
step S4: regulating the pH value of the refined collagen to 7.5, adding 3g ficin and 1g ginger protease at 55 ℃ for enzymolysis for 6 hours to obtain an enzymolysis solution; the pH value of the enzymolysis liquid is adjusted to 8.0, the temperature is 37 ℃,1g of Debaryomyces hansenii, 1g of Pediococcus pentosaceus, 2g of lactobacillus sake, 1g of micrococcus mutans and 2g of staphylococcus botulinum are added, and the fermentation enzymolysis liquid is obtained after fermentation for 4 hours.
Step S5: removing impurities from the fermentation enzymolysis liquid by peak shifting filtration, filtering by a 1KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by nanofiltration, filtering by a homogeneous membrane at a flow rate of 5cm/s, an operating voltage of 120V and a current of 70A, intercepting the liquid for 30-60min, and spray-drying to obtain the collagen tripeptide product rich in alkaline amino acid.
Example 2: collagen tripeptide rich in basic amino acid and preparation thereof
Step S1: weighing 100g of tilapia skin, cutting into small blocks, adding 2000mL of sodium hydroxide solution with mass fraction of 0.8%, soaking for 4 hours, cleaning for 3 times, adding softened water, homogenizing in a grinder to paste, and obtaining total protein crude extract;
step S2: adjusting the pH value of the total protein crude extract to 2.6, heating to 90 ℃, preserving heat and extracting gelatin for 5h to prepare a collagen crude extract;
step S3: weighing 0.5g of egg white, 0.5g of sodium caseinate and 2g of okra polysaccharide, adding into the crude collagen extract, adjusting the pH value to 3, uniformly stirring, standing for 4 hours, and collecting supernatant to obtain a refined collagen product;
step S4: regulating the pH value of the refined collagen to 7.5, adding 3g ficin and 1g ginger protease at 55 ℃ for enzymolysis for 6 hours to obtain an enzymolysis solution; the pH value of the enzymolysis liquid is adjusted to 8.0, the temperature is 37 ℃, 0.1g of Debaryomyces hansenii, 0.2g of Pediococcus pentosaceus, 0.4g of lactobacillus sake, 0.2g of Micrococcus mutans and 0.4g of staphylococcus sarcodactylis are added, and fermentation is carried out for 4 hours, thus obtaining the fermentation enzymolysis liquid.
Step S5: removing impurities from the fermentation enzymolysis liquid by peak shifting filtration, filtering by a 1KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by nanofiltration, filtering by a homogeneous membrane at a flow rate of 6cm/s, operating voltage of 100V and current of 50A, intercepting the liquid for 30-60min, and spray-drying to obtain the collagen tripeptide product rich in alkaline amino acid.
Example 3: collagen tripeptide rich in basic amino acid and preparation thereof
A collagen tripeptide and a preparation method thereof are provided, wherein the collagen tripeptide is prepared by the following steps:
step S1: adding 10 g of commercially available collagen into 100g of water, and uniformly stirring;
step S2: adjusting the pH value of the collagen solution to 7.5 at 55 ℃, adding 2g ficin and 0.8g ginger protease, and performing enzymolysis for 6 hours to obtain an enzymolysis solution; the pH value of the enzymolysis liquid is adjusted to 8.0, the temperature is 37 ℃, 0.4g of Debaryomyces hansenii, 0.6g of Pediococcus pentosaceus and 0.4g of Micrococcus mutans are added, and the fermentation enzymolysis liquid is obtained after fermentation for 4 hours.
Step S5: removing impurities from the fermentation enzymolysis liquid by peak shifting filtration, filtering by a 1KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by nanofiltration, filtering by a homogeneous membrane at a flow rate of 4-6cm/s, operating at a voltage of 110V and a current of 60A, intercepting for 30-60min, and spray drying to obtain the collagen tripeptide product.
Example 4: collagen tripeptide rich in basic amino acid and preparation thereof
Step S1: weighing 100g of tilapia skin, cutting into small blocks, adding 2000mL of sodium hydroxide solution with mass fraction of 0.8%, soaking for 4 hours, cleaning for 3 times, adding softened water, homogenizing in a grinder to paste, and obtaining total protein crude extract;
step S2: adjusting the pH value of the total protein crude extract to 2.6, heating to 90 ℃, preserving heat and extracting gelatin for 5h to prepare a collagen crude extract;
step S3: 1g of egg white, 0.8g of sodium caseinate and 1g of okra polysaccharide are weighed, added into the crude collagen extract, the pH value is adjusted to 3, and the mixture is stirred uniformly and then kept stand for 4 hours. The crude extract liquid did not form a layer without supernatant.
Physical property detection of collagen tripeptide product
The collagen tripeptide products prepared in examples 1-3 and the commercially available collagen peptides were tested for their corresponding physical properties by the following procedure (provided by Beijing Cheng Meinuo Biotechnology Co., ltd.):
the content of tripeptide in the collagen tripeptide product is detected by HPLC-MS, and the result is shown in table 1; and tripeptide sequences, results are shown in tables 2-4).
HPLC-MS detection method: ZORBAXSB-C18 column (2.1 mm. Times.150 mm,5 μm); mobile phase a-water (0.1% tfa), B-acetonitrile (0.1% tfa); gradient elution is carried out for 0-7min, and B accounts for 5% -20%; 7-50min,20% -32% B;50-90min,32% -72% B; the sample injection amount is 50 mu L; the flow rate is 0.2mL min-1. Spraying voltage is 4.5kV; the capillary temperature is 300 ℃; nitrogen (N2) 253kPa; positive ion mode, primary mass spectrum scanning range m/z 300-1500, accurate mass number scanning (Zoom scan) and secondary mass spectrum (MS/MS) scanning are both data dependent scans (Data Dependent Scan); dynamic exclusion times 1; dynamic exclusion time 0.5min; the collision energy of the secondary mass spectrum is 35%.
The average molecular weight was measured according to the method of national standard GB22729 marine oligopeptide and the results are shown in Table 1.
The basic amino acid content was measured by an amino acid automatic analyzer (Saccharum model S433D), and the results are shown in Table 1.
TABLE 1
Table 2 shows the sequence of the collagen tripeptide containing basic amino acids in the collagen tripeptide product prepared in example 1
TABLE 2
Table 3 shows the sequence of the collagen tripeptide containing basic amino acids in the collagen tripeptide product prepared in example 2
TABLE 3 Table 3
| Sequence number | Collagen tripeptide sequences | Molecular weight |
| 1 | Gly-Pro-Arg | 328.36 |
| 2 | Gly-Hyp-Arg | 344.33 |
| 3 | Gly-Hyp-His | 325.29 |
| 4 | Gly-Hyp-Lys | 316.32 |
| 5 | Gly-Pro-His | 308.96 |
| 6 | Gly-Pro-Lys | 300.35 |
| 7 | Gly-Glu-Arg | 360.36 |
| 8 | Gly-Ser-Arg | 318.32 |
| 9 | Gly-Ala-His | 283.28 |
| 10 | Gly-Phe-Lys | 350.41 |
| 11 | Gly-Leu-His | 325.36 |
| 12 | Gly-Ile-Lys | 316.39 |
| 13 | Gly-Ala-Arg | 302.32 |
| 14 | Pro-Gly-Arg | 328.86 |
| 15 | Hyp-Gly-Arg | 344.33 |
Table 4 shows the sequence of the collagen tripeptide containing basic amino acids in the collagen tripeptide product prepared in example 3
| Sequence number | Collagen tripeptide sequences | Molecular weight |
| 1 | Gly-Hyp-Lys | 316.32 |
| 2 | Gly-Pro-Lys | 300.35 |
| 3 | Gly-Glu-Arg | 360.36 |
| 4 | Gly-Ser-Arg | 318.32 |
| 5 | Gly-Phe-Lys | 350.41 |
| 6 | Gly-Ala-Arg | 302.32 |
Anti-glycation capability assay
The collagen tripeptide products prepared in examples 1-3, as well as the commercially available collagen peptides, collagen tripeptides (available from Beijing Cheng Meinuo Biotechnology Co., ltd.) were tested for anti-glycation ability by the following two methods:
1. saccharification inhibition rate: 1mL of fructose solution (1.5 mol/L) is mixed with 1mL of collagen peptide component solution by adopting a BSA-fructose simulation reaction system, after incubation is carried out for 2 hours at 37 ℃, 1mL of 30mg/mL of BSA solution is added, the reactants are dissolved by using 50mmol/L of pH7.4 phosphate buffer (containing 0.1% sodium azide), the components of collagen peptide are replaced by aminoguanidine solution with the same mass concentration as a positive control group, the components of collagen peptide are replaced by phosphate buffer as a blank group, the fructose solution is replaced by phosphate buffer as a BSA and collagen peptide co-incubation group, after each sample is incubated for 6 days at 37 ℃ in a biochemical incubator, the fluorescence intensity of each sample is measured under the conditions of excitation wavelength of 370nm and emission wavelength of 440nm, and the inhibition rate R of collagen peptide on fluorescent AGEs is calculated by the following formula.
R/％＝(1-FA/FB)*100
Wherein: FA is the fluorescence intensity of each sample group; FB represents the fluorescence intensity of the blank group.
The test results are shown in Table 5.
2. The anti-glycation effect was evaluated in vivo using wild AB strain zebra fish as a study subject.
1. Detection material
1. Experimental animal
Zebra fish are all raised in 28 deg.c water (200 mg instant sea salt is added into 1L reverse osmosis water, the conductivity is about 500 mu S/cm, pH is about 7, and hardness is about 80mg/L CaCO) 3 ) The experimental animal use license number is provided by the breeding of the fish culture center of the company: SYXK (Zhe) 2012-0171, the feeding management meets the requirements of International AAALAC authentication (authentication number: 001458).
Wild type AB strain zebra fish is bred in a natural pairing mating breeding mode, and 300 tails are used.
2. Instrument, consumable and reagent
Ultraviolet phototherapy apparatus (KN-4006, xuzhou Keno medical instruments Co., ltd.); precision electronic balances (CP 214, OHAUS, america); multifunctional enzyme labelling apparatus (SPARK, tecan, switzerland); 6-well plates (Nest Biotech, china); 96-well plates (Nest Biotech, china); glycosylated hemoglobin ELISA detection kit (lot number 202004, taiwan jun organism, china).
2. Detection method
1. Evaluation of anti-glycation efficacy
Randomly selecting 210 tail 3dpf wild type AB strain zebra fish, equally dividing into 7 groups, placing the 7 groups in two 6-hole plates, and using 3mL of fish culture water for each hole, wherein each hole contains 30 tail zebra fish. The collagen tripeptide products prepared in examples 1-3, commercially available collagen peptides, and collagen tripeptides (available from Beijing Cheng Meinuo Biotechnology Co., ltd.) were each given a final concentration of 1250. Mu.g/mL for 2 hours; the normal control group and the model control group were each given the same volume of water. Then, other groups except the normal control group respectively adopt ultraviolet irradiation to cause the ultraviolet injury of zebra fish: and treating at 28 ℃ for 24 hours. After the ultraviolet irradiation is finished, the zebra fish is homogenized, and the glycosylation ELISA detection kit is used for reaction, and the anti-glycation efficacy of the collagen peptide is evaluated according to the statistical analysis result of the content of the glycation hemoglobin in the tissues. Statistical treatment results are expressed by mean+ -SE, and the calculation formula of the anti-glycation efficacy of the collagen peptide on the zebra fish is as follows:
statistical analysis with SPSS software, p <0.05 indicated significant differences.
TABLE 5
TABLE 6
As can be seen from tables 5 and 6 above: the anti-glycation effect of the collagen tripeptide product rich in the basic amino acid is far higher than that of the collagen tripeptide obtained by the commercial process, the glycation inhibition rate of the collagen tripeptide product rich in the basic amino acid is 53% at the minimum and 98% at the maximum, the glycation inhibition rate of the collagen tripeptide obtained by the commercial process is only 16%, and the glycation inhibition rate of the collagen tripeptide obtained by the commercial process is only 22%. The in vivo anti-glycation efficacy of the collagen tripeptide product rich in basic amino acids provided by the application is far higher than that of the commercially available collagen peptide and collagen tripeptide. In addition, the more the kinds of basic amino acids, the more favorable saccharification inhibition is.
Claims (14)
1. A collagen tripeptide product, wherein the collagen tripeptide product comprises a collagen tripeptide; wherein the collagen tripeptide comprises a tripeptide with at least one end of a basic amino acid, and the tripeptide with at least one end of a basic amino acid comprises a mixture of Gly-X1-Arg, gly-X2-His and Gly-X3-Lys, wherein X1, X2 and X3 are all amino acids; the content of the mixture of the basic amino acids Arg, his and Lys is not less than 10 percent based on the total weight of the collagen tripeptide; the amino acid represented by X1 is one of Pro, hyp, glu, ser and Ala; the amino acid represented by X2 is one of Hyp, pro, ala and Leu; the amino acid represented by X3 is one of Hyp, pro, phe and Ile.
2. The collagen tripeptide product according to claim 1, wherein the collagen tripeptide product comprises 20-40 wt.% collagen tripeptide.
3. The collagen tripeptide product according to claim 1, wherein said tripeptide with at least one basic amino acid at its end has an average molecular weight between 200 and 500 daltons.
4. The collagen tripeptide product according to claim 1, wherein the mixed content of basic amino acids Arg, his and Lys is 12-20%.
5. A method for preparing a collagen tripeptide having at least one basic amino acid at one end, the method comprising the steps of:
1) Adding water into collagen, regulating the pH value of the collagen solution to be 5-8, and adding compound protease accounting for 0.5-5% of the weight of the collagen; carrying out enzymolysis for 2-6 hours at 45-60 ℃ to obtain enzymolysis liquid; wherein the complex protease comprises ficin and zingibain; wherein the collagen is derived from the skin, scale or bone of an animal;
2) Adjusting pH value of the enzymolysis liquid to 7-8.5, adding a starter which is 1-8% of the weight of the collagen, and fermenting for 2-4 hours at 30-42 ℃ to obtain fermentation liquid; the starter comprises mixing of Debaryomyces hansenii, pediococcus pentosaceus, micrococcus mutans, lactobacillus sake and Staphylococcus sarcodactylis;
3) After solid-liquid separation of the fermentation broth, the filtrate was collected.
6. The method according to claim 5, wherein in the step 1), the collagen is derived from tilapia skin.
7. The method according to claim 5, wherein in the step 1), the weight ratio of ficin to ginger protease is 2-4:1.
8. The method according to claim 5, wherein in the step 2), the yeast debaryomyces hansenii, the pediococcus pentosaceus, the lactobacillus sake, the micrococcus mutans and the staphylococcus sarcoglucose are in a mass ratio of 1:1.8-2.2:3.8-4.2:1.8-2.2:3.8-4.2.
9. The method according to claim 5, further comprising the step of refining the collagen prior to said step 1).
10. The method according to claim 9, wherein the refining step comprises adding a clarifier to the collagen solution, adjusting the pH of the collagen solution to 2-7, stirring uniformly, and standing for 2-8 hours to obtain refined collagen.
11. The method of claim 10, wherein the clarifying agent is egg white, sodium caseinate, okra polysaccharide.
12. The method of claim 11, wherein the egg white, the sodium caseinate and the okra polysaccharide are present in a weight ratio of 1:1-4:2-5.
13. The method of claim 10, wherein the clarifying agent is added in an amount of 0.5-3% by weight of collagen and the pH of the clarifying agent is 3-5.
14. Use of a collagen tripeptide product according to any one of claims 1-4 for the preparation of a cosmetic or functional food.
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