CN116574171A - Production process and application of in-situ liquid collagen peptide for industrial production - Google Patents
Production process and application of in-situ liquid collagen peptide for industrial production Download PDFInfo
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- CN116574171A CN116574171A CN202310465272.8A CN202310465272A CN116574171A CN 116574171 A CN116574171 A CN 116574171A CN 202310465272 A CN202310465272 A CN 202310465272A CN 116574171 A CN116574171 A CN 116574171A
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- collagen peptide
- enzymolysis
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- fish scales
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 97
- 102000008186 Collagen Human genes 0.000 title claims abstract description 90
- 108010035532 Collagen Proteins 0.000 title claims abstract description 90
- 229920001436 collagen Polymers 0.000 title claims abstract description 90
- 239000007788 liquid Substances 0.000 title claims abstract description 61
- 238000004519 manufacturing process Methods 0.000 title abstract description 6
- 238000011065 in-situ storage Methods 0.000 title abstract description 4
- 238000009776 industrial production Methods 0.000 title description 4
- 241000251468 Actinopterygii Species 0.000 claims abstract description 32
- 108091005658 Basic proteases Proteins 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 108090000526 Papain Proteins 0.000 claims abstract description 20
- 239000004365 Protease Substances 0.000 claims abstract description 20
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- 238000000034 method Methods 0.000 claims description 23
- 230000000694 effects Effects 0.000 claims description 18
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 10
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- 238000001179 sorption measurement Methods 0.000 claims description 5
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- 150000003254 radicals Chemical class 0.000 description 3
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- GMGWMIJIGUYNAY-UHFFFAOYSA-N MeIQ Chemical compound CC1=CC2=NC=CC=C2C2=C1N(C)C(N)=N2 GMGWMIJIGUYNAY-UHFFFAOYSA-N 0.000 description 2
- DVCCCQNKIYNAKB-UHFFFAOYSA-N MeIQx Chemical compound C12=NC(C)=CN=C2C=CC2=C1N=C(N)N2C DVCCCQNKIYNAKB-UHFFFAOYSA-N 0.000 description 2
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- 230000002000 scavenging effect Effects 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- YHVQIDWAIRCSOQ-UHFFFAOYSA-N 1-nitrotetrazol-2-ium chloride Chemical compound [Cl-].[O-][N+](=O)N1C=[NH+]N=N1 YHVQIDWAIRCSOQ-UHFFFAOYSA-N 0.000 description 1
- LAZSIJHHPMHKQI-UHFFFAOYSA-N 3,4,8-Trimethyl-1H,2H,3H-imidazo[4,5-F]quinoxalin-2-imine Chemical compound C12=NC(C)=CN=C2C=C(C)C2=C1N=C(N)N2C LAZSIJHHPMHKQI-UHFFFAOYSA-N 0.000 description 1
- VQWMRFGOLQDJSV-UHFFFAOYSA-N 3,7,8-trimethylimidazo[4,5-f]quinolin-2-amine Chemical compound C1=C2N=C(C)C(C)=CC2=C2N=C(N)N(C)C2=C1 VQWMRFGOLQDJSV-UHFFFAOYSA-N 0.000 description 1
- CFEPPLAUXZCOJL-UHFFFAOYSA-N 3,8-dimethylimidazo[4,5-f]quinolin-2-amine Chemical compound C12=CC(C)=CN=C2C=CC2=C1N=C(N)N2C CFEPPLAUXZCOJL-UHFFFAOYSA-N 0.000 description 1
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241001597062 Channa argus Species 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 241000252230 Ctenopharyngodon idella Species 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- 206010013911 Dysgeusia Diseases 0.000 description 1
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- 208000001132 Osteoporosis Diseases 0.000 description 1
- UQVKZNNCIHJZLS-UHFFFAOYSA-N PhIP Chemical compound C1=C2N(C)C(N)=NC2=NC=C1C1=CC=CC=C1 UQVKZNNCIHJZLS-UHFFFAOYSA-N 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 241000276707 Tilapia Species 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000004227 calcium gluconate Substances 0.000 description 1
- 229960004494 calcium gluconate Drugs 0.000 description 1
- 235000013927 calcium gluconate Nutrition 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
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- 239000013068 control sample Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
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- 108010007119 flavourzyme Proteins 0.000 description 1
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- 239000003205 fragrance Substances 0.000 description 1
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- 239000013505 freshwater Substances 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 230000007358 intestinal barrier function Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/66—Proteins
-
- 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Mycology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The application provides a production process and application for industrially producing an in-situ liquid collagen peptide, and relates to the technical field of food biology. The production process comprises heating fish scales, swelling, performing enzymolysis to obtain enzymolysis liquid under the action of alkaline protease and neutral papain, inactivating enzyme, extracting, adsorbing, and sterilizing to obtain liquid collagen peptide. The application can efficiently carry out enzymolysis on the macromolecular collagen of the fish scales into the collagen peptide with high peptide segment proportion and molecular weight within the range of 250-500Da, has good water supplementing and antioxidant effects, good taste of the collagen peptide, higher stability, no heterocyclic amine and other substances, and is safe and healthy. The collagen peptide is applied to the preparation of collagen peptide drinks and is deeply favored by consumers.
Description
Technical Field
The application belongs to the technical field of food biology, and particularly relates to a production process and application of an industrial production in-situ liquid collagen peptide.
Background
The collagen peptide is a product which is produced by taking fresh animal tissues (including skin, bones, tendons, scales and the like) rich in collagen as raw materials, extracting, hydrolyzing and refining and has the relative molecular mass of less than 10000 Da. The collagen peptide has the effects of beautifying and protecting skin, resisting hypertension, protecting gastric mucosa, preventing and inhibiting arthritis, osteoporosis and the like. Collagen beverage prepared by taking collagen peptide as raw material is more toughed by young women.
The preparation method of the collagen peptide mainly comprises an enzymatic hydrolysis method, a chemical method, a microbial fermentation method and a genetic method. The chemical method is mainly used in laboratory pilot scale research, and the research cost is high; the fermentation time of the microbial fermentation method is longer, the requirements on fermentation conditions are more severe, and the fermentation product is influenced by various fermentation factors, so that the prepared fermentation product is nonuniform, more and more mixed in components; in the genetic engineering method, the expression efficiency of the gene is low, and safety problems are easily caused by expression errors. The main characteristic of collagen peptide is high content of small molecules, and if the molecular weight is high, the collagen peptide is difficult to digest and absorb. And enzymatic hydrolysis can hydrolyze collagen into numerous small fragments of low molecular weight collagen peptides with a higher probability of crossing the intestinal barrier, resulting in increased biological activity and reduced antigenicity.
The Chinese patent No. 102533913B discloses a method for preparing antioxidant active peptide by hydrolyzing fish scale collagen with co-immobilized double enzymes, which comprises the steps of firstly, carrying out soaking pretreatment on fish scales by using a crosslinking method to immobilize double enzymes, removing fish silver, pigment substances and soluble impurity proteins, carrying out directional hydrolysis on the fish scale proteins by using the co-immobilized enzymes to obtain fish scale proteolysis solution, decoloring and deodorizing, and carrying out post-treatment such as ultrafiltration separation, vacuum concentration, freeze drying and the like to obtain the active peptide. The method has high enzymolysis efficiency, the enzyme can be used for multiple times, and the enzymolysis product is easy to purify; provides a new way for the effective utilization of the freshwater fish wastes; and the active peptide product can be used in the fields of medicines, foods, cosmetics and the like.
The Chinese patent No. 114045323A discloses a method for preparing collagen peptide powder from fish skin and fish scales by a multistage enzymolysis process, which comprises the following steps: cleaning and crushing fish skin and scale to prepare raw material slurry, adding sodium hydroxide, and performing ultrasonic treatment to obtain primary hydrolysis feed liquid; adding alkaline protease to obtain primary biochemical degradation feed liquid; adding alkaline protease to obtain secondary biochemical degradation feed liquid; adding collagenase to obtain three-stage biochemical degradation feed liquid; adding neutral protease and flavourzyme for reaction to obtain four-level biochemical degradation feed liquid; adding papain, cystine, calcium chloride solution and calcium gluconate to obtain five-stage biochemical degradation feed liquid; filtering to obtain collagen peptide glue solution; adding activated carbon powder for decolorization, centrifuging, and lyophilizing to obtain collagen peptide powder. The application adopts a multistage enzymolysis process, adopts ultrasonic wave for assistance after deodorization treatment, improves enzyme activity, has high hydrolysis degree and high utilization rate, and can thoroughly hydrolyze, and the prepared collagen peptide powder has good smell and high yield.
In order to reduce bacterial contamination and to extend shelf life, collagen peptide raw materials are often present in powder form obtained by spray drying collagen stock solutions. The collagen peptide powder is produced through spray drying, which is to be treated by hot air at about 180 ℃, and the high temperature treatment can lead the collagen peptide to generate heterocyclic amine with a certain dosage, thus forming hidden trouble for health and safety of consumers. In order to save the cost, manufacturers often use the collagen peptide powder with a shelf life fast enough to prepare the liquid collagen peptide, active ingredients in the collagen peptide drink drunk by consumers are lost in a large amount, the taste is changed, and the safety and health index is reduced. In addition, the enzyme hydrolysis collagen has high requirement on the action of the enzyme, and the collagen cannot be hydrolyzed into small molecular collagen peptide fully by selecting unsuitable enzyme and enzymolysis conditions, so that the water supplementing and antioxidation capabilities are low.
In view of the above, the application provides a preparation method of liquid collagen peptide, which can efficiently carry out enzymolysis on fish scale collagen to obtain collagen peptide with high peptide segment proportion of 250-500, has good water supplementing and antioxidant effects, good taste of the collagen peptide, high stability, no heterocyclic amine and other substances, and is safe and healthy.
Disclosure of Invention
Aiming at the problems existing in the prior art, the application provides a production process and application for producing the liquid collagen peptide in industrial production. The application can efficiently carry out enzymolysis on the fish scale collagen into the collagen peptide with high peptide segment proportion within the range of 250-500Da, has good water supplementing and antioxidant effects, good taste of the collagen peptide, higher stability, no heterocyclic amine and other substances, and is safe and healthy. The collagen peptide is applied to the preparation of collagen peptide drinks and is deeply favored by consumers.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
in one aspect, the present application provides a method for preparing a liquid collagen peptide, comprising the steps of:
(1) Moistening and expanding: mixing fish scales with water, heating and stirring to obtain pretreatment liquid;
(2) Enzymolysis: cooling the pretreatment liquid to 45-60 ℃, adding sodium hydroxide to adjust the pH to 6.8-7.4, adding alkaline protease and neutral papain for enzymolysis for 2-6h to obtain enzymolysis liquid; the weight ratio of the alkaline protease to the fish scales is 0.2-0.7 percent to 1; the weight ratio of the neutral papain to the fish scales is 0.25-0.7 percent to 1;
(3) And (3) enzyme deactivation and extraction: heating the enzymolysis liquid to 70-95 ℃ and preserving heat for 20-50min to obtain an enzymolysis protein peptide solution;
(4) Adsorption: cooling the enzymolysis protein peptide solution to 45-55 ℃, adding active carbon, stirring for 15-25min, filtering, and sterilizing to obtain the liquid collagen peptide.
Preferably, in the step (1), the solid-to-liquid ratio of the fish scales to the water is 1:4-8,g/mL.
Further preferably, the solid-to-liquid ratio of the fish scales to water is 1:5-7,g/mL.
Preferably, in step (1), the heating and stirring are specifically: heating to 80-90 deg.C, and stirring at 65-80rpm for 1-3 hr.
Further preferably, the heating and stirring are specifically: heating to 85-88 deg.C, and stirring at 70rpm for 2 hr.
Preferably, in step (2), the temperature of the cooling is 50 ℃.
Preferably, in step (2), the concentration of sodium hydroxide is 2-8%.
Preferably, in step (2), the weight ratio of alkaline protease to neutral papain is 1-2.6:1.
Further preferably, the weight ratio of alkaline protease to neutral papain is 2.5:1.
Preferably, in the step (2), the weight ratio of the alkaline protease to the fish scales is 0.6-0.65% to 1.
Further preferably, the weight ratio of alkaline protease to fish scale is 0.64%:1.
Preferably, in the step (2), the weight ratio of the neutral papain to the fish scales is 0.2-0.3 percent to 1.
Further preferably, the weight ratio of the neutral papain to the fish scales is 0.26 percent to 1.
Preferably, in the step (2), the enzyme activity of the alkaline protease and the neutral papain is 15-25 ten thousand U/g.
Further preferably, the enzyme activity of the alkaline protease and the neutral papain is 20 ten thousand U/g.
Preferably, in the step (3), the enzyme-inactivating extraction is specifically: and heating the enzymolysis liquid to 80-90 ℃ and preserving heat for 40-50min to obtain an enzymolysis protein peptide solution.
Further preferably, the enzyme-inactivating extraction is specifically: and heating the enzymolysis liquid to 90 ℃ and preserving heat for 45min to obtain an enzymolysis protein peptide solution.
Preferably, in the step (4), the enzymatic protein peptide solution is cooled to 50 ℃.
Preferably, in the step (4), the addition amount of the activated carbon is 2-10% of the mass of the fish scales.
Further preferably, the addition amount of the activated carbon is 6-8% of the mass of the fish scales.
Preferably, in the step (4), the filtering device is a ceramic membrane filtering device and a nanofiltration membrane device.
Further preferably, the pore diameter of the ceramic membrane filtration device is 200nm, and the pressure is 0.4MPa; the aperture of the nanofiltration membrane equipment is 200D.
Preferably, in the step (4), the sterilization treatment is specifically performed at a sterilization temperature of 80-98 ℃ for 20-50min.
Further preferably, the sterilization treatment is specifically sterilization at 95 ℃ for 30min.
In another aspect, the present application provides a liquid collagen peptide prepared by the above preparation method.
Finally, the application provides application of the liquid collagen peptide prepared by the preparation method in preparation of collagen peptide drinks.
Compared with the prior art, the application has the following beneficial effects:
1. the application provides a preparation method of liquid collagen peptide, which comprises the steps of swelling, enzymolysis, enzyme deactivation extraction, adsorption and the like, wherein in the enzymolysis step, alkaline protease and neutral papain are added at one time under alkaline conditions, so that the enzymolysis process is simplified, and the liquid collagen peptide obtained by enzymolysis has high peptide segment proportion with the molecular weight of 250-500Da and good moisturizing and antioxidation effects.
2. In the preparation method of the liquid collagen peptide, the concentration spraying step is not needed, and the liquid collagen peptide has better taste and no peculiar smell flavor through enzymolysis and adsorption under specific conditions.
3. The liquid collagen peptide has good stability after high-temperature sterilization, is not easy to dye bacteria, does not contain substances such as heterocyclic amine, and is safe and healthy.
Drawings
FIG. 1 is a diagram showing the dissolution of a liquid collagen peptide prepared according to the present application and a commercially available collagen peptide powder.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the application and are not intended to limit the application in any way. The following is merely exemplary of the scope of the application as claimed and many variations and modifications of the application will be apparent to those skilled in the art in light of the disclosure, which are intended to be within the scope of the application as claimed.
The application is further illustrated by means of the following specific examples. The various chemical reagents used in the examples of the present application were obtained by conventional commercial means unless otherwise specified.
In the following examples, the scales are decalcified and decolored pretreated scales derived from tilapia scales, grass carp scales and snakehead scales. The manufacturer of alkaline protease and papain is Denmark (China) Limited company, nanning east Henghua channel biotechnology Limited liability company, shandong Long Kete enzyme preparation Limited company, norwestine (China) investment Limited company.
The raw materials and the reality of the application, products of different manufacturers and products of different models have no obvious influence on the effect.
Example 1
(1) Moistening and expanding: mixing 1kg of fish scales with 6kg of distilled water, heating the water to 87 ℃, and stirring for 2 hours under the condition of a stirring rotating speed of 70rpm to obtain a pretreatment liquid;
(2) Enzymolysis: cooling the pretreatment liquid to 50 ℃ at room temperature, adding 5% NaOH to adjust the pH to 7.1, adding 4.9g of alkaline protease and 4.1g of neutral papain, and stirring and carrying out enzymolysis for 5 hours at 50 ℃ to obtain an enzymolysis liquid;
(3) And (3) enzyme deactivation and extraction: heating the enzymolysis liquid to 90 ℃ and preserving heat for 45min to obtain enzymolysis protein peptide liquid;
(4) Adsorption: cooling the protein peptide liquid to 50 ℃, adding 70g of active carbon, stirring for 20min, sequentially passing through a ceramic membrane filter device with the aperture of 200nm and the pressure of 0.4MPa and a nanofiltration membrane device with the aperture of 200D, and sterilizing at 95 ℃ for 30min to obtain the liquid collagen peptide.
Examples 2 to 4
Examples 2-4 differ from example 1 in that in step (2) the weight ratio of alkaline protease to neutral papain is different, the specific ratios are as follows:
| alkaline protease, g | Neutral protease, g | Alkaline protease: neutral protease | |
| Example 1 | 4.9 | 4.1 | 1.2:1 |
| Example 2 | 6 | 3 | 2:1 |
| Example 3 | 4.5 | 4.5 | 1:1 |
| Example 4 | 6.4 | 2.6 | 2.5:1 |
The remaining steps and parameters were the same as in example 1.
Example 5
Unlike example 4, in step (2), the temperature of the enzymolysis was 60 ℃. The remaining steps and parameters were the same as in example 4.
Example 6
Unlike example 4, in step (2), the pH was 6.8. The remaining steps and parameters were the same as in example 4.
Examples 7 to 9
Unlike example 4, in step (2), the time for the enzymolysis is different, and the specific enzymolysis time is as follows:
| enzymolysis time, h | |
| Examples4 | 5h |
| Example 7 | 2h |
| Example 8 | 4h |
| Example 9 | 6h |
The remaining steps and parameters were the same as in example 4.
Comparative examples 1 to 2
Unlike example 4, in step (2), the weight ratio of alkaline protease to neutral papain is different, and the specific ratio is as follows:
| alkaline protease, g | Neutral protease, g | Alkaline protease: neutral protease | |
| Comparative example 1 | 2.2 | 6.6 | 1:2.7 |
| Comparative example 2 | 4.1 | 4.9 | 1:1.2 |
The remaining steps and parameters were the same as in example 4.
Comparative examples 3 to 4
Unlike example 4, in step (2), the time for the enzymolysis is different, and the specific enzymolysis time is as follows:
| enzymolysis time, h | |
| Comparative example 3 | 7h |
| Comparative example 4 | 8h |
The remaining steps and parameters were the same as in example 4.
Comparative example 5
Unlike example 4, in step (2), the temperature of the enzymolysis was 40 ℃. The remaining steps and parameters were the same as in example 4.
Comparative example 6
Unlike example 4, in step (2), the pH was 8.0. The remaining steps and parameters were the same as in example 4.
Comparative example 7
Unlike example 4, in step (2), the enzyme used was replaced with neutral protease which was obtained by fermentation and extraction of Bacillus subtilis and had an enzyme activity of 20U/g.
The remaining steps and parameters were the same as in example 4.
Test 1 collagen peptide molecular weight determination
The liquid collagen peptides obtained in examples and comparative examples were subjected to peptide molecular weight measurement.
The measuring method comprises the following steps: the distribution of the peptide molecular weight was detected according to GB/T22492 method, with a detection wavelength of 220nm. The measurement results of the proportions (%) of the respective molecular weight ranges are shown in Table 1.
TABLE 1
The molecular weight of the liquid collagen peptide is in the optimal molecular weight range of 245-500, the stable collagen tripeptide structure in the peptide range is easy to be absorbed by human body, the absorption rate is high, the liquid collagen peptide has good antioxidant and moisturizing effects, collagen is effectively synthesized by collagen fibroblasts in the dermis of the skin, and the higher the collagen tripeptide content is, the better the effect is.
As can be seen from Table 1, in examples 1 to 4 and comparative examples 1 to 2, the ratio of the molecular weight of 245 to 500 was the highest in examples 2 and 4, and the ratio of comparative examples 1 to 2 was the lowest in examples 1 and 3 times. Wherein example 4 is about 2.2 times that of comparative examples 1-2. It can be seen that the ratio of alkaline protease to neutral protease has a significant influence on the molecular weight.
As is clear from the results of examples 7 to 9 and comparative examples 3 to 4, the enzymolysis time also has an important effect on the ratio of molecular weights, and although the enzymolysis time is long and the content of small molecules is high, in examples 7 to 9 and comparative examples 3 to 4, a part of small molecule peptides have no water supplementing and antioxidation effects, and therefore, the effect is not as good as the longer the enzymolysis time is.
Test 2 Water make-up Effect test
1. Test object: a total of 320 female subjects aged 30-50 years, and skin moisture less than or equal to 12%.
2. Sample: there were 16 total groups of samples, examples 1-9 and comparative examples 1-7, respectively.
3. Instrument: grease moisture tester (German CK SD 27)
4. The test method comprises the following steps: 320 subjects were randomized into 16 groups of 20 persons each using a double-blind randomized group, inter-group and self-two control design. Each group took 1 bottle (50 ml) of samples of the corresponding examples 1-9 and comparative examples 1-7 1 time a day. The observation was continued for 35 days. The 16 subjects during the trial had to take other skin moisture retaining items and eat normally.
5. Skin moisture index determination
The skin between the forehead was tested for moisture. The measurement environment is carried out in a test room with wide space environment, good ventilation condition, stable temperature, humidity and the like. The measured part is cleaned by dipping a clean cotton ball in distilled water in a quiet state, the water content is measured 15min after the cleaning, and the measuring work before and after the test is operated by the same instrument and the same person.
6. Statistics of test data
Results are expressed as mean ± standard deviation, paired t-test for self-comparison before and after ingestion of the test, and paired t-test for inter-group comparison.
7. Results and analysis
The skin moisture change is shown in Table 2. Skin moisture changes before and after the test were compared,%, mean ± standard deviation.
TABLE 2
Note that: comparison before and after the experiment, through t-test, P <0.01 and P <0.05; example 1 and comparative example 1 were compared with example 4, respectively, and P <0.01 was represented by t-test # s; example 1 was compared with comparative example 1, and after t-test, & indicates P <0.01.
(1) Example 1, example 2, example 4, example 5, example 8 showed very significant differences in skin moisture after and before test (P < 0.01), example 3, example 6, example 7 and example 9 showed significant differences in skin moisture after and before test (P < 0.05), and comparative examples 1-7 showed no significant differences in skin moisture after and before test, indicating that the group samples of example 19 had significant skin moisture increasing effects, and the comparative examples 1-7 showed insignificant skin moisture increasing effects and possibly longer intake times.
(2) The comparative moisture delta t-test analysis of example 2 and example 4, both have no significance, indicating that the samples of example 2 and example 4 have no difference in the effect of increasing skin moisture, and the effect is the same; the comparative moisture delta t-test analysis of example 1 and example 3, without significant relationship, demonstrates that the two groups of samples have the same effect of moisturizing the skin.
(3) Example 4 was significantly correlated (P < 0.01) as compared to the moisture delta t-test analysis of example 1 and comparative example 1, indicating that the samples of example 4 were significantly better in moisturizing skin than example 1 and comparative example 1; example 1 moisture delta was compared to the comparative example 1 moisture delta t-test analysis and the significance correlation (P < 0.01) indicated that the sample of example 1 was significantly different from comparative example 1 in increasing skin moisture, and example 1 was superior to comparative example 1. Indicating that the ratio of alkaline protease and the ratio of neutral protease have an influence on the effect of water replenishment.
Test 3 liquid collagen peptide antioxidant test
The liquid collagen peptides prepared in examples 1 to 9 and comparative examples 1 to 7 were subjected to an antioxidant test.
1. Hydroxyl radical scavenging test
The method comprises the following steps: mixing 1.0mL of 1, 10-phenanthroline (1.865 mmol/L) and 2.0mL of sample solution in a test tube with a stopper, and adding 1.0mL of LFASO 4 . 7H 2 0 (1.865 mmol/L), finally 1.0. 1.0mLH is added 2 O 2 The reaction was started from the solution (0.03%, v/v), the test tube was placed in a 37℃water bath for 60min, the absorbance of the reaction mixture was measured at 536nm, distilled water was used as a negative control instead of the sample solution, and distilled water was used as a blank control instead of hydrogen peroxide.
The scavenging activity of the sample on OH radicals was calculated as follows:
OH radical clearance (%) = (D-D) 0 /(D 0 -D 1 )×100％；
Wherein: d is the absorbance of the sample, D 1 Absorbance as negative control, D 0 The average of five measurements for absorbance of the blank control is shown in table 3.
2、O 2 - Determination of radical scavenging Activity
1mL of the sample solution was placed in a test tube, 1mL of nitrotetrazolium chloride (NBT, 2.52 mmol/L) and 1mL of nicotinamide adenine dinucleotide (624 mmol/L) were added, 1mL of phenazine methosulfate solution (PMS, 120. Mu. Mol/L) was added to initiate the reaction, absorbance at 560nm of the product was measured after 5min of the reaction at 25℃while distilled water was used as a blank control sample solution for O 2 - The scavenging activity of the free radicals was calculated as follows:
O 2 - radical clearance (%) = (C 0 -C)/C 0 ×100％；
Wherein: c (C) 0 The absorbance of the blank control, the absorbance of the sample, and the average value of the five measurements was taken, and the detection results are shown in table 3.
3. DPPH free radical scavenging test method: 2.5mL of the sample solution was placed in a test tube, 2.5mL of DPPH solution (0.1 mmoL) dissolved in 95% ethanol was added, the mixture was vigorously shaken for 10s, then left to react at room temperature for 30min, after the reaction was completed, the absorbance of the reaction mixture was measured at 517nm, and 2.5mL of distilled water was used as a blank control instead of the sample solution.
The DPPH free radical scavenging activity of the sample solution is calculated as follows:
DPPH radical clearance (%) = (B) 0 -B)/B 0 ×100％；
Wherein: b (B) 0 The absorbance of the blank control, the absorbance of the sample, and the average value of the five measurements was taken, and the detection results are shown in table 3.
TABLE 3 Table 3
Note that: compared with example 4, P <0.01 and P <0.05 are represented by t-test; comparative examples 1-7 compared to example 1, t-test, # represents P <0.01, # represents P <0.05
Examples 1-3 and comparative examples 1-2 are compared to the data of example 4, where the hydroxyl radical and superoxide anion removal rates are significantly different from example 2 compared to example 4, the balance being significantly different, whereas the DPPH radical removal rate is not different from example 4, the balance being significantly different, indicating that the antioxidant activity is more sensitive to the ratio of alkaline protease to neutral protease.
Example 4 has no significant difference from example 4 in the DPPH radical elimination rate of example 8 alone, and has a significant difference from example 4 in the hydroxyl radical elimination rate of example 8, and the balance is the difference from the significant difference, as compared with examples 7 to 9; comparative examples 3-4 all have very significant differences compared to example 1, indicating that the enzymatic hydrolysis time has an effect on the antioxidant activity, optimally 5h.
Test 4 test-taking taste measurement
The liquid collagen peptide obtained in example 4 was evaluated for taste as a commercially available collagen peptide powder.
1. Assessment method
The evaluation group consists of 10 people, 5 men and 5 women, the ages are 25-35, the group personnel numbers 1-10 are healthy, no bad taste exists, the color, the fragrance and the taste are better resolved and the sensitivity is higher, the evaluation is carried out at room temperature, and the mouth is rinsed with warm water before each sensory evaluation to keep the mouth fresh. The experiment is composed of 3 factors of smell, taste and bitter taste, and the evaluation of each factor is rated according to the corresponding scores of three grades of excellent, medium and poor.
2. Sample preparation
(1) Sample 1: the sample produced in example 4, 50ml each, contained 5g collagen peptide;
(2) Sample 2: commercially available collagen peptide powder (model: J018, manufacturer: asahi biological technology Co., ltd.) was prepared by adding water to a volume of 50ml per 5g of collagen peptide.
3. Sensory evaluation reference criteria are specifically shown in table 4.
TABLE 4 Table 4
4. Sensory evaluation results
The specific sensory evaluation results are shown in table 5:
TABLE 5
From the above results, the two samples showed no significant difference in smell, taste, and bitterness, indicating that the collagen peptide liquid was acceptable in sense.
FIG. 1 is a drawing showing a liquid collagen peptide prepared in example 4 of the present application dissolved in a commercially available collagen peptide powder, wherein test tube (1) is model J018 collagen peptide of Shanxi Long Bei Biotechnology Co., ltd; (2) the test tube number is the liquid collagen peptide prepared in the example 4; (3) a state diagram of a model J018 collagen peptide powder of Shanxi Long Bei biotechnology Co., ltd after dissolution and sterilization at 90 ℃ for 30 min; (4) the test tube number is self-made collagen peptide with the sterilization condition of 90-30 min in example 4; (5) a state diagram of the model J018 collagen peptide powder of Shanxi Long Bei biotechnology Co., ltd, with pH adjusted to 4.0 after dissolution; (6) the test tube No. is a state diagram of the liquid collagen peptide prepared in example 4 with pH adjusted to 4.0. As can be seen from FIG. 1, the liquid collagen peptide prepared by the method has no precipitation and is stable in state.
Test 5 heterocyclic amine assay
The liquid collagen peptide obtained in example 4 and the collagen peptide of Shandong Long Bei Biotechnology Co., ltd., lot C-YL-220820 were subjected to detection of heterocyclic amine, and the heterocyclic amine was detected with reference to standard GB 5009.243-2016.
The detection results are shown in Table 6:
TABLE 6
| Detecting items | Example 4 | Long Bei collagen peptide |
| 2-amino-3, 4-dimethylimidazo [4.5-f]Quinoline (MeIQ) | Not detected<0.1ug/kg) | 0.73ug/kg |
| 2-amino-3, 8-dimethylimidazo [4,5-f]Quinoline (MeIQx) | Not detected<0.3ug/kg) | Not detected<0.3pg/kg) |
| 2-amino-3, 4, 8-trimethylimidazo [45-f](4,8-DiMeIQx) | Not detected<0.2ug/kg) | Not detected<0.2pg/kg) |
| 2-amino-3, 7, 8-trimethylimidazo [4,5-f]Quinoline (7, 8-DiMeIQx) | Not detected<0.2pg/kg) | Not detected<0.2pg/kg) |
| 2-amino-1-methyl-6-phenyl-imidazo [4,5-b]Pyridine (PhIP) | Not detected<0.1ug/kg) | 0.35ug/kg |
Finally, it should be noted that the above description is only for illustrating the technical solution of the present application, and not for limiting the scope of the present application, and that the simple modification and equivalent substitution of the technical solution of the present application can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present application.
Claims (10)
1. A method for preparing a liquid collagen peptide, comprising the steps of:
(1) Moistening and expanding: mixing fish scales with water, heating and stirring to obtain pretreatment liquid;
(2) Enzymolysis: cooling the pretreatment liquid to 45-60 ℃, adding sodium hydroxide to adjust the pH to 6.8-7.4, adding alkaline protease and neutral papain for enzymolysis for 2-6h to obtain enzymolysis liquid; the weight ratio of the alkaline protease to the fish scales is 0.2-0.7%:1, a step of; the weight ratio of the neutral papain to the fish scales is 0.25-0.7 percent to 1;
(3) And (3) enzyme deactivation and extraction: heating the enzymolysis liquid to 70-95 ℃ and preserving heat for 20-50min to obtain an enzymolysis protein peptide solution;
(4) Adsorption: cooling the enzymolysis protein peptide solution to 45-55 ℃, adding active carbon, stirring for 15-25min, filtering, and sterilizing to obtain the liquid collagen peptide.
2. The preparation method according to claim 1, wherein in the step (1), the solid-to-liquid ratio of the fish scales to water is 1:4-8,g/mL; the heating and stirring steps are as follows: heating to 80-90 deg.C, and stirring at 65-80rpm for 1-3 hr.
3. The method according to claim 1, wherein in the step (2), the weight ratio of the alkaline protease to the neutral papain is 1-2.6:1.
4. The preparation method according to claim 1, wherein in the step (2), the weight ratio of the alkaline protease to the fish scales is 0.6-0.65%:1; the weight ratio of the neutral papain to the fish scales is 0.2-0.3 percent to 1.
5. The preparation method according to claim 1, wherein in the step (2), the enzyme activities of the alkaline protease and the neutral papain are 15 ten thousand to 25 ten thousand U/g.
6. The preparation method according to claim 1, wherein in the step (3), the enzyme-inactivating extraction is specifically: and heating the enzymolysis liquid to 80-90 ℃ and preserving heat for 40-50min to obtain an enzymolysis protein peptide solution.
7. The preparation method according to claim 1, wherein in the step (4), the addition amount of the activated carbon is 2-10% of the mass of the fish scales.
8. The method according to claim 1, wherein in the step (4), the filtration device is a ceramic membrane filtration device and a nanofiltration membrane device; the aperture of the ceramic membrane filtering equipment is 200nm, and the pressure is 0.4MPa; the aperture of the nanofiltration membrane equipment is 200D.
9. The method according to claim 1, wherein in the step (4), the sterilization treatment is specifically performed at a sterilization temperature of 80 to 98 ℃ for 20 to 50 minutes.
10. Use of the liquid collagen peptide prepared by the preparation method of any one of claims 1 to 9 in the preparation of collagen peptide beverage.
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