CN114634549A - 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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- CN114634549A CN114634549A CN202210367027.9A CN202210367027A CN114634549A CN 114634549 A CN114634549 A CN 114634549A CN 202210367027 A CN202210367027 A CN 202210367027A CN 114634549 A CN114634549 A CN 114634549A
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- collagen
- tripeptide
- amino acid
- basic amino
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- 108091005804 Peptidases Proteins 0.000 claims abstract description 21
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- 241000235036 Debaryomyces hansenii Species 0.000 claims abstract description 20
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- 238000000855 fermentation Methods 0.000 claims abstract description 19
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- DHDOADIPGZTAHT-YUMQZZPRSA-N Gly-Glu-Arg Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DHDOADIPGZTAHT-YUMQZZPRSA-N 0.000 description 2
- YLEIWGJJBFBFHC-KBPBESRZSA-N Gly-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 YLEIWGJJBFBFHC-KBPBESRZSA-N 0.000 description 2
- GAAHQHNCMIAYEX-UWVGGRQHSA-N Gly-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN GAAHQHNCMIAYEX-UWVGGRQHSA-N 0.000 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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- JYPCXBJRLBHWME-IUCAKERBSA-N Gly-Pro-Arg Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JYPCXBJRLBHWME-IUCAKERBSA-N 0.000 description 1
- ZZJVYSAQQMDIRD-UWVGGRQHSA-N Gly-Pro-His Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O ZZJVYSAQQMDIRD-UWVGGRQHSA-N 0.000 description 1
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- HAMNKKUPIHEESI-UHFFFAOYSA-N aminoguanidine Chemical group 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
- 108010025801 glycyl-prolyl-arginine Proteins 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 invention provides a collagen tripeptide product, which contains tripeptide with at least one end being basic amino acid, the molecular formula of the tripeptide with the basic amino acid comprises Gly-X-Arg, Gly-X-His and Gly-X-Lys, wherein X is the 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 composite protease accounting for 0.5-5% of the weight of the collagen; enzymolysis is carried out for 2-6 hours at 45-60 ℃; the compound protease comprises ficin and ginger protease; adjusting pH of the enzymolysis solution to 7-8.5, adding leaven 1-8% of collagen weight, and fermenting at 30-42 deg.C for 2-4 hr; the leavening agent comprises Debaryomyces hansenii, Pediococcus pentosaceus and Micrococcus varians; and (4) carrying out solid-liquid separation on the fermentation liquor, and collecting filtrate.
Description
Technical Field
The invention 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, has about 1/3 of human protein content, and plays an important role in maintaining the structure and function of the organism. As people age, collagen is lost. Meanwhile, because the collagen in the dermal extracellular matrix of the skin tissue has low reversibility, the collagen is easier to generate non-enzymatic reaction with glucose and other metabolic molecules to generate advanced glycation end products (AGEs), such as glycosylated collagen, and the like, and a series of adverse effects are generated on the tissue.
The collagen peptide is a small molecular peptide product prepared by enzymolysis of macromolecular collagen. According to research reports, the collagen peptide has the effects of improving skin moisture, reducing wrinkles, resisting aging and the like. Especially, the collagen tripeptide is easy to be absorbed and utilized by human body due to small molecular weight, so the collagen tripeptide has very good effect of delaying senility. The collagen tripeptide rich in basic amino acid has the function of resisting saccharification, and is beneficial to maintaining the normal function of tissues. At present, collagen tripeptide products rich in basic amino acids are lacked in the market, and the anti-glycation effect of the collagen peptide is poor.
Disclosure of Invention
In order to fill up the market blank, the invention provides a collagen tripeptide product which contains abundant basic amino acids.
Accordingly, in one aspect, the present invention provides a collagen tripeptide product, characterized in that the collagen tripeptide product comprises collagen tripeptides; wherein the collagen tripeptide comprises tripeptide with at least one basic amino acid end, and the molecular formula of the tripeptide with at least one basic amino acid end 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 percent based on the total weight of the collagen tripeptide; preferably, the content of the basic amino acid is 12 to 20%.
In another aspect, the present invention provides a method for preparing a collagen tripeptide with at least one basic amino acid at one end, comprising the steps of:
1) adding water into collagen, adjusting pH of the collagen solution to 5-8, and adding compound protease 0.5-5% of the weight of the collagen; carrying out enzymolysis for 2-6 hours at the temperature of 45-60 ℃ to obtain an enzymolysis liquid; wherein the complex protease comprises ficin and zingiberin; preferably, the weight ratio of ficin to zingiberin is 2-4: 1;
2) adjusting pH of the enzymolysis solution to 7-8.5, adding a leaven 1-8% of the weight of the collagen, and fermenting at 30-42 deg.C for 2-4 hr to obtain a fermentation solution; the leavening agent comprises Debaryomyces hansenii, Pediococcus pentosaceus and Micrococcus varians; preferably, the weight ratio of debaryomyces hansenii, pediococcus pentosaceus and micrococcus varians is 1: 1.8-2.2: 1.8-2.2;
3) and (4) carrying out solid-liquid separation on the fermentation liquor, and collecting filtrate.
In a third aspect, the present invention provides the use of the collagen tripeptide product in cosmetics and functional foods.
The collagen tripeptide product rich in basic amino acid contains a plurality of basic amino acids, and the content of the basic amino acids is high and at least accounts for 10 percent of the total weight of the collagen tripeptide. Experiments in the application prove that the collagen tripeptide product with high content of basic amino acid has a very significant (37.2%) sugar-resistant effect which is far higher than the sugar-resistant effects of the collagen tripeptide (8.2%) and the collagen (5.5%).
Detailed Description
The invention provides a collagen tripeptide product, which is characterized by comprising collagen tripeptide; wherein the collagen tripeptide comprises tripeptide with at least one basic amino acid end, and the molecular formula of the tripeptide with at least one basic amino acid end 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% by weight 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 to 20%.
In order to obtain 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, and to obtain better anti-glycation effect, it is preferred that the tripeptide with at least one basic amino acid end has an average molecular weight between 200 and 500 daltons.
In order to facilitate the availability of more basic amino acids, preferably, the method for preparing the collagen tripeptide product comprises the following steps:
1) adding water into collagen to adjust pH of the collagen solution to 5-8, preferably pH 6-7; adding 0.5-5% of compound protease, preferably 1-4% of compound protease, more preferably 2-4% of compound protease based on the weight of collagen; performing enzymolysis at 45-60 deg.C for 2-6 hr, preferably for 3-5 hr to obtain enzymolysis solution; wherein the compound protease comprises ficin and zingiberin, and the weight ratio of the ficin to the zingiberin is 2-4: 1;
2) adjusting pH of the enzymolysis solution to 7-8.5, adding 1-8% of fermenting agent (preferably 3-6%) of collagen; fermenting at 30-42 deg.C for 2-4 hr to obtain fermentation liquid; the leavening agent comprises Debaryomyces hansenii, Pediococcus pentosaceus and Micrococcus varians, and the weight ratio of the Debaryomyces hansenii, the Pediococcus pentosaceus and the Micrococcus varians is 1: 1.8-2.2: 1.8-2.2;
3) and (4) carrying out solid-liquid separation on the fermentation liquor, and collecting filtrate.
Preferably, in the step 2), the leavening agent further comprises lactobacillus sake and/or staphylococcus carnosus, so as to obtain more basic amino acids.
Preferably, in the step 2), the leavening agent further comprises lactobacillus sake and staphylococcus carnosus, and the mass ratio of the debaryomyces hansenii, the pediococcus pentosaceus, the lactobacillus sake, the micrococcus mutans and the staphylococcus carnosus is 1: 1.8-2.2: 3.8-4.2:1.8-2.2: 3.8-4.2.
The present invention also provides a method for preparing a collagen tripeptide with at least one basic amino acid at one end, comprising the steps of:
1) adding water into collagen, adjusting pH of collagen solution to 5-8, adding 0.5-5% of compound protease, preferably 1-4% of compound protease, more preferably 3-4% of compound protease by weight of collagen; performing enzymolysis at 45-65 deg.C, preferably 50-60 deg.C for 1-8 hr, preferably 2-6 hr; obtaining enzymolysis liquid; wherein the complex protease comprises ficin and zingiberin; preferably, the weight ratio of ficin to zingiberin is 2-4: 1;
2) adjusting pH of the enzymolysis solution to 6.5-8.5, preferably pH of 7-8, adding 1-8% of fermenting agent, preferably 2-7% of fermenting agent, more preferably 3-6% of fermenting agent based on collagen weight; at 25-45 deg.C, preferably 30-40 deg.C; fermenting for 1-6 hours, preferably fermenting for 2-5 hours; obtaining fermentation liquor; the leavening agent comprises Debaryomyces hansenii, Pediococcus pentosaceus and Micrococcus varians; preferably, the weight ratio of debaryomyces hansenii, pediococcus pentosaceus and micrococcus varians is 1: 1.5-2.5:1.5-2.5, preferably 1: 1.8-2.2: 1.8-2.2;
3) and (4) carrying out solid-liquid separation on the fermentation liquor, and collecting filtrate.
In a preferred embodiment, in the step 2), the leavening agent further comprises lactobacillus sake and/or staphylococcus carnosus; preferably, the mass ratio of the debaryomyces hansenii, the pediococcus pentosaceus, the lactobacillus sake, the micrococcus varians and the staphylococcus carnosus is 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 interference 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 clarification is carried out by adding a clarifying agent, stirring and standing.
In a preferred embodiment, the clarifying agent is added to the collagen solution to adjust the pH of the collagen solution to 2-7, preferably 3-6; stirring, standing for 2-8 hr, preferably 3-6 hr to obtain refined collagen.
The clarifying agent is not particularly limited, but in order to obtain a better impurity removal 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, and preferably 1:1-3: 2.5-4.
In order to obtain a better decontamination effect, in a preferred embodiment, the clarifying agent is added in an amount of 0.5-3% by weight of the collagen, preferably 1-2% by weight of the collagen.
In order to obtain a better stripping effect, in a preferred embodiment, the pH of the clarifying agent is 3-5.
In order to obtain 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 flowing out for 30-60 minutes is collected.
The present invention also provides a method for preparing an anti-glycation collagen tripeptide rich in basic amino acids from a collagen-containing raw material, the method comprising the steps of:
step S1 preparation of crude total protein extract: weighing raw materials, adding sodium hydroxide solution, soaking for 2-4 hr, cleaning for 2-3 times, adding water, and homogenizing in a grinder to obtain total protein crude extract;
step S2 preparation of collagen crude extract: adjusting pH of the crude extract to 2-4, heating to 75-90 deg.C, keeping the temperature, and extracting gel for 3-8 hr to obtain crude extract of collagen;
step S3 refining of collagen crude extract: adding clarifier into the crude collagen extract, adjusting pH to 2-7, stirring, standing for 2-8 hr to obtain refined collagen extract;
step S4 preparation of collagen peptide: adjusting pH of the refined collagen to 5-8, adding fructus fici extract and rhizoma Zingiberis recens extract, and performing enzymolysis; carrying out enzymolysis for 2-6 hours at the temperature of 45-60 ℃ to obtain an enzymolysis liquid; adjusting pH to 7-8.5, adding Debaryomyces hansenii, Pediococcus pentosaceus, Lactobacillus sake, Micrococcus mutans, and Staphylococcus carnosus, and fermenting at 30-40 deg.C for 2-4 hr to obtain enzymolysis fermentation broth;
step S5 preparation of collagen tripeptide rich in basic amino acids: filtering the enzymolysis fermentation liquid to remove impurities.
Preferably, the raw material in step S1 is one of skin, scale or bone of animal, and the sodium hydroxide solution is 0.6-1% by mass.
The crude collagen extract prepared in step S2 is not easily separated into layers during purification, resulting in a low yield. In order to improve the yield and promote the layering, preferably, the clarifying agent in the step S3 is egg white, sodium caseinate and okra polysaccharide, the mass ratio of the egg white, the sodium caseinate and the okra polysaccharide is 1:1-4:2-5, the mixture is mixed and kept still 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 described in step S4 is a combination of ficin and zingiberin, the mass ratio of the two enzymes is 3-2:1, the amount of the combined enzyme is 0.5-5% of the mass of the collagen refined product, and the enzymolysis pH value is 6.5-8. The added leaven is the combination of Debaryomyces hansenii, Pediococcus pentosaceus, Lactobacillus sake, Micrococcus mutans and Staphylococcus carnosus, and the weight ratio is 1-0.5: 1: 1.5-2.5: 1:1.5-2.5, the addition amount of the combined leaven is 1-8% of the mass of the collagen refined substance, and the fermentation pH value is 7.0-8.5.
Preferably, the flow rate of the step 5 homogeneous membrane filtration is 2-6cm/s, the operating voltage is 100-.
The invention also provides application of the collagen tripeptide product in cosmetics and functional foods.
The cosmetic is effective in delaying skin aging, and is especially suitable for skin care of diabetes patients.
The collagen tripeptide product provided by the invention can be used for functional food, 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 pieces, adding 2000mL of sodium hydroxide solution with the mass fraction of 0.8%, soaking for 4 hours, cleaning for 3 times, adding softened water, and homogenizing in a grinder to obtain a paste to obtain a total protein crude extract;
step S2: adjusting the pH value of the total protein crude extract to 2.6, heating to 90 ℃, and carrying out heat preservation and gel extraction for 5 hours to prepare a collagen crude extract;
step S3: weighing 1g of egg white, 3g of sodium caseinate and 4g of okra polysaccharide, adding the egg white, 3g of sodium caseinate and 4g of okra polysaccharide into the collagen crude extract, adjusting the pH value to 3, uniformly stirring, standing for 4 hours, and collecting supernatant to obtain a collagen refined extract;
step S4: adjusting pH of the refined collagen to 7.5, adjusting temperature to 55 deg.C, adding ficin 3g and ginger protease 1g, and performing enzymolysis for 6 hr to obtain enzymolysis solution; adjusting the pH value of the enzymolysis liquid to 8.0, adjusting the temperature to 37 ℃, adding 1g of Debaryomyces hansenii, 1g of Pediococcus pentosaceus, 2g of Lactobacillus sake, 1g of Micrococcus mutans and 2g of Staphylococcus carnosus combination, and fermenting for 4 hours to obtain the fermentation enzymolysis liquid.
Step S5: filtering the fermentation enzymolysis liquid by adopting peak staggering filtration to remove impurities, then filtering by adopting a 1KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by adopting nanofiltration, filtering the liquid with the flow rate of 5cm/s, the operating voltage of 120V and the current of 70A for 30-60min by adopting a homogeneous membrane, and performing spray drying to obtain the collagen tripeptide product rich in the basic amino acid.
Example 2: collagen tripeptide rich in basic amino acid and preparation thereof
Step S1: weighing 100g of tilapia skin, cutting into small pieces, adding 2000mL of sodium hydroxide solution with the mass fraction of 0.8%, soaking for 4 hours, cleaning for 3 times, adding softened water, and homogenizing in a grinder to obtain a paste to obtain a total protein crude extract;
step S2: adjusting the pH value of the crude extract of the total protein to 2.6, heating to 90 ℃, and performing heat preservation and gel extraction for 5 hours to prepare a crude extract of the collagen;
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 4h, and collecting supernatant to obtain a refined collagen extract;
step S4: adjusting pH of the refined collagen to 7.5, adjusting temperature to 55 deg.C, adding ficin 3g and ginger protease 1g, and performing enzymolysis for 6 hr to obtain enzymolysis solution; adjusting the pH value of the enzymolysis liquid to 8.0, adjusting the temperature to 37 ℃, adding 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 carnosus combination, and fermenting for 4 hours to obtain the fermentation enzymolysis liquid.
Step S5: filtering the fermented enzymolysis liquid by adopting peak staggering to remove impurities, then filtering by adopting a 1KD ultrafiltration membrane, removing inorganic salts and water-soluble free amino acids by adopting nanofiltration, filtering the liquid with the flow rate of 6cm/s, the operating voltage of 100V and the current of 50A for 30-60min by adopting a homogeneous membrane, and performing spray drying to obtain a collagen tripeptide product rich in basic amino acids.
Example 3: collagen tripeptide rich in basic amino acid and preparation thereof
A collagen tripeptide and a preparation method thereof are 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, adjusting the temperature to 55 ℃, adding 2g of ficin and 0.8g of ginger protease, and carrying out enzymolysis for 6h to obtain an enzymolysis solution; adjusting pH of the enzymolysis solution to 8.0, heating to 37 deg.C, adding 0.4g of Debaryomyces hansenii, 0.6g of Pediococcus pentosaceus and 0.4g of Micrococcus varians, and fermenting for 4 hr to obtain fermentation enzymolysis solution.
Step S5: filtering the fermented enzymolysis liquid by adopting peak staggering filtration to remove impurities, filtering by adopting a 1KD ultrafiltration membrane, removing inorganic salt and water-soluble free amino acid by adopting nanofiltration, filtering the liquid at the flow rate of 4-6cm/s, the operating voltage of 110V and the current of 60A for 30-60min by adopting a homogeneous membrane, and performing 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 pieces, adding 2000mL of sodium hydroxide solution with the mass fraction of 0.8%, soaking for 4 hours, cleaning for 3 times, adding softened water, and homogenizing in a grinder to obtain a paste to obtain a total protein crude extract;
step S2: adjusting the pH value of the crude extract of the total protein to 2.6, heating to 90 ℃, and performing heat preservation and gel extraction for 5 hours to prepare a crude extract of the collagen;
step S3: weighing 1g of egg white, 0.8g of sodium caseinate and 1g of okra polysaccharide, adding into the crude collagen extract, adjusting the pH value to 3, uniformly stirring, and standing for 4 h. The crude extract liquid failed to form a layer, and no supernatant.
Detection of physical properties of collagen tripeptide product
The collagen tripeptide products prepared in examples 1 to 3 and the commercially available collagen peptide, collagen tripeptide (provided by shenmei noy biotechnology limited, beijing) were tested for their respective physical properties by the following methods:
detecting and detecting the content of tripeptide in a collagen tripeptide product by HPLC-MS, wherein the result is shown in Table 1; and tripeptide sequences, as 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 for 0-7min, 5% -20% B; 7-50min, 20% -32% B; 50-90min, 32% -72% B; the sample volume is 50 mu L; the flow rate was 0.2mL min-1. The spraying voltage is 4.5 kV; the temperature of the capillary tube is 300 ℃; nitrogen (N2)253 kPa; the positive ion mode, the primary mass spectrum scanning range m/z 300-1500, the accurate mass number scanning (Zoom Scan) and the secondary mass spectrum (MS/MS) scanning are both Data Dependent scanning (Data Dependent Scan); dynamic exclusion times 1; dynamic elimination time is 0.5 min; secondary mass collision energy 35%.
The average molecular weight was determined according to the method of national standard GB22729 marine oligopeptide, and the results are shown in Table 1.
The content of basic amino acids was measured using an automatic amino acid analyzer (Secalm model S433D), and the results are shown in Table 1.
TABLE 1
Table 2 shows the tripeptide sequence of collagen containing basic amino acids in the tripeptide product of collagen prepared in example 1
TABLE 2
Table 3 shows the tripeptide sequence of collagen containing basic amino acids in the tripeptide product of collagen prepared in example 2
TABLE 3
| Serial 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 tripeptide sequence of collagen containing basic amino acids in the tripeptide product of collagen prepared in example 3
| Serial number | Collagen tripeptide sequence | 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 ability assay
The collagen tripeptide products prepared in examples 1 to 3 and the commercially available collagen peptide, collagen tripeptide (provided by shengmenor biotechnology limited, beijing) were tested for their anti-glycation ability by the following two methods:
1. glycation inhibition ratio: adopting a BSA-fructose simulated reaction system, mixing 1mL of fructose solution (1.5mol/L) with 1mL of collagen peptide component solution, incubating at 37 ℃ for 2 hours, adding 1mL of 30mg/mL of BSA solution, dissolving the above reactants with 50mmol/LpH7.4 phosphate buffer solution (containing 0.1% sodium azide), substituting aminoguanidine solution with same mass concentration for collagen peptide solution as positive control group, substituting phosphate buffer solution for collagen peptide solution as blank group, substituting phosphate buffer solution for fructose solution as BSA and collagen peptide co-incubation group, incubating each sample in biochemical incubator at 37 deg.C for 6d, the fluorescence intensity of each sample was measured at an excitation wavelength of 370nm and an emission wavelength of 440nm, and the inhibition rate R of the collagen peptide on the formation of fluorescent AGEs was calculated by the following formula.
R/％＝(1-FA/FB)*100
In the formula: FA is the fluorescence intensity of each sample set; FB represents fluorescence intensity of blank.
The results are shown in Table 5.
2. The wild AB strain zebra fish is taken as a research object to evaluate the anti-saccharification effect in vivo.
Firstly, detecting material
1. Laboratory animal
The zebra fish are all raised in water for fish culture at 28 deg.C (water quality: 200mg of instant sea salt per 1L of reverse osmosis water, conductivity about 500 μ S/cm, pH about 7, and hardness about 80mg/L CaCO)3) The fish culture center of the company breeds and provides the fish culture, and the license number for experimental animals is as follows: SYXK (Zhe) 2012-0171, and the feeding management meets the requirements of the international AAALAC certification (certification number: 001458).
Wild type AB strain zebra fish, carried out in a natural mating breeding mode, 300 tails in total.
2. Instruments, consumables and reagents
Ultraviolet light therapy apparatus (KN-4006, Xuzhou Kenuo medical instruments equipment Co., Ltd.); precision electronic balances (CP214, OHAUS, America); multifunctional microplate readers (SPARK, Tecan, Switzerland); 6-well plates (Nest Biotech, China); 96-well plates (Nest Biotech, China); glycated hemoglobin ELISA test kit (lot No. 202004, taiwan force jun organism, China).
Second, detection method
1. Evaluation of anti-glycation efficacy
Randomly selecting 210 tail 3dpf wild type AB strain zebra fish, averagely dividing the zebra fish into 7 groups, placing the 7 groups in two 6-hole plates, wherein each hole is provided with 30 tail zebra fish, and each hole is provided with 3mL of fish culture water. The experimental groups were treated with the collagen tripeptide product prepared in examples 1-3, a commercially available collagen peptide, and a collagen tripeptide (provided by shengmeno biotechnology limited, beijing) at a final concentration of 1250 μ g/mL for 2 hours, respectively; the normal control group and the model control group were given the same volume of water, respectively. Then, except for a normal control group, the other groups respectively adopt ultraviolet irradiation to cause the ultraviolet damage of the zebra fish: treating at 28 deg.C for 24 h. And (3) homogenizing the zebra fish after the ultraviolet irradiation is finished, reacting by using a glycosylation ELISA detection kit, and evaluating the anti-glycation effect of the collagen peptide according to the result of statistical analysis of the content of the glycated hemoglobin in the tissue. The statistical treatment results are expressed by mean ± SE, and the anti-glycation efficacy of the collagen peptide on the zebra fish is calculated according to the following formula:
statistical analysis was performed with SPSS software and p <0.05 indicated significant differences.
TABLE 5
TABLE 6
As can be seen from tables 5 and 6 above: the collagen tripeptide product rich in basic amino acid provided by the invention has the anti-glycation effect far higher than that of commercially available collagen peptide and collagen tripeptide, the glycation inhibition rate of the collagen tripeptide product rich in basic amino acid provided by the invention is 53% at least and 98% at most, while the glycation inhibition rate of the commercially available collagen peptide is only 16% and the glycation inhibition rate of the commercially available collagen tripeptide is only 22%. The collagen tripeptide product rich in basic amino acids provided by the application has in vivo anti-glycation efficacy far higher than that of commercially available collagen peptides and collagen tripeptides. Further, the more the basic amino acid species are, the more favorable the glycation inhibition is.
Claims (10)
1. A collagen tripeptide product, comprising a collagen tripeptide product; wherein the collagen tripeptide comprises tripeptide with at least one basic amino acid end, and the molecular formula of the tripeptide with at least one basic amino acid end 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 percent based on the total weight of the collagen tripeptide; preferably, the content of the basic amino acid is 12-20%; preferably, the amino acid represented by X is one or more of Pro, Hyp, Glu, Ser, Ala, Phe, Leu and Ile.
2. The collagen tripeptide product according to claim 1, comprising 20-40 wt% collagen tripeptide.
3. The collagen tripeptide product according to claim 1, wherein the tripeptide with at least one basic amino acid end has an average molecular weight between 200 and 500 daltons.
4. The collagen tripeptide product according to any one of claims 1 to 3, wherein the preparation method comprises the following steps:
1) adding water into collagen, adjusting pH of the collagen solution to 5-8, and adding compound protease 0.5-5% of the weight of the collagen; carrying out enzymolysis for 2-6 hours at the temperature of 45-60 ℃ to obtain an enzymolysis liquid; wherein the compound protease comprises ficin and zingiberin, and the weight ratio of the ficin to the zingiberin is 2-4: 1;
2) adjusting pH of the enzymolysis solution to 7-8.5, adding a leaven 1-8% of the weight of the collagen, and fermenting at 30-42 deg.C for 2-4 hr to obtain a fermentation solution; the leavening agent comprises Debaryomyces hansenii, Pediococcus pentosaceus and Micrococcus varians, and the weight ratio of the Debaryomyces hansenii, the Pediococcus pentosaceus and the Micrococcus varians is 1: 1.8-2.2: 1.8-2.2;
3) and (4) performing solid-liquid separation on the fermentation liquor, and collecting filtrate.
5. The collagen tripeptide product according to claim 4, characterized in that in step 2) the starter culture further comprises Lactobacillus sake and/or Staphylococcus carnosus.
6. The collagen tripeptide product according to claim 5, wherein in the step 2), the fermentation agent further comprises lactobacillus sake and staphylococcus carnosus, and the mass ratio of the debaryomyces hansenii, the pediococcus pentosaceus, the lactobacillus sake, the micrococcus mutans and the staphylococcus carnosus is 1: 1.8-2.2: 3.8-4.2:1.8-2.2: 3.8-4.2.
7. A method for preparing a collagen tripeptide with at least one basic amino acid at one end, comprising the steps of:
1) adding water into collagen, adjusting pH of the collagen solution to 5-8, and adding compound protease 0.5-5% of the weight of the collagen; carrying out enzymolysis for 2-6 hours at the temperature of 45-60 ℃ to obtain enzymolysis liquid; wherein the complex protease comprises ficin and zingiberin; preferably, the weight ratio of ficin to zingiberin is 2-4: 1;
2) adjusting pH of the enzymolysis solution to 7-8.5, adding a leaven 1-8% of the weight of the collagen, and fermenting at 30-42 deg.C for 2-4 hr to obtain a fermentation solution; the leavening agent comprises Debaryomyces hansenii, Pediococcus pentosaceus and Micrococcus varians; preferably, the weight ratio of debaryomyces hansenii, pediococcus pentosaceus and micrococcus varians is 1: 1.8-2.2: 1.8-2.2;
3) and (4) carrying out solid-liquid separation on the fermentation liquor, and collecting filtrate.
8. The method according to claim 7, wherein in the step 2), the leavening agent further comprises lactobacillus sake and/or staphylococcus carnosus; preferably, the mass ratio of the debaryomyces hansenii, the pediococcus pentosaceus, the lactobacillus sake, the micrococcus varians and the staphylococcus carnosus is 1: 1.8-2.2: 3.8-4.2:1.8-2.2: 3.8-4.2.
9. The method according to claim 7, further comprising a step of refining the collagen before the step 1); preferably, the clarifying agent is added into the collagen solution, the pH value of the collagen solution is adjusted to 2-7, and the refined collagen is obtained after the collagen solution is stirred uniformly and then stands for 2-8 hours; preferably, the clarifying agent is egg white, sodium caseinate and okra polysaccharide; preferably, the weight ratio of the egg white to the sodium caseinate to the okra polysaccharide is 1:1-4: 2-5; preferably, the amount of the clarifying agent added is 0.5-3% by weight of the collagen, and the pH value of the clarifying agent is 3-5.
10. Use of the collagen tripeptide product according to any one of claims 1 to 6 in cosmetics and functional foods.
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