WO2021142880A1 - 一种蛤蜊活性肽的生产方法 - Google Patents

一种蛤蜊活性肽的生产方法 Download PDF

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WO2021142880A1
WO2021142880A1 PCT/CN2020/076042 CN2020076042W WO2021142880A1 WO 2021142880 A1 WO2021142880 A1 WO 2021142880A1 CN 2020076042 W CN2020076042 W CN 2020076042W WO 2021142880 A1 WO2021142880 A1 WO 2021142880A1
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Prior art keywords
clam
protease
production method
water
meat
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PCT/CN2020/076042
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English (en)
French (fr)
Inventor
曹廷锋
樊芳
邹圣灿
冯郭君男
刘金丽
鲁秀
张曾亮
Original Assignee
美国琛蓝营养制品股份有限公司
青岛琛蓝医药科技发展有限公司
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Priority to US16/978,674 priority Critical patent/US20230118351A1/en
Priority to JP2022543690A priority patent/JP7422236B2/ja
Publication of WO2021142880A1 publication Critical patent/WO2021142880A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/10—Fish meal or powder; Granules, agglomerates or flakes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal 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
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • 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
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/40—Shell-fish
    • 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
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/50—Molluscs
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a production method of clam active peptides, in particular to a production method suitable for large-scale industrial production of clam active peptides.
  • Clams are one of the four largest cultured shellfish in my country, and they are rich in resources.
  • the clam has delicious meat, high protein, high vitamins, less fat, and contains more than ten kinds of amino acids and minerals necessary for the human body.
  • It is a bivalve sea product that is dual-purpose for food and medicine and has become a recognized high Nutritious, low-cost healthy food.
  • clams are mainly fresh and dried, and the problems of low processing level in the industry and fewer product types have become increasingly prominent.
  • the backward traditional processing technology can no longer meet human needs. Therefore, how to realize the deep processing and high-value utilization of clams and develop more and better functional clam products , Is a huge opportunity and severe challenge facing the clam industry.
  • the patent document "A method for extracting active peptides from clams” (China with application number 20161117416.8) uses two methods of flocculation-centrifugation-dextran gel column separation and enzymatic hydrolysis-dextran gel column separation to obtain polypeptides. The gel column separation process is difficult to apply to large-scale production.
  • the patent document "A method for extracting clam peptides” (China with the application number of 20161117416.8) uses enzymatic hydrolysis, which uses sodium hydroxide to adjust pH, enzymatic hydrolysis, extraction, enzyme inactivation, centrifugation, filtration adsorption, nanofiltration separation, concentration, and drying. , But the reaction process is complicated, the large-scale production cost is high, and sodium hydroxide and other chemicals are added in the middle.
  • the purpose of the present invention is to provide a production method suitable for large-scale industrial production of clam active peptides in view of the defects in the prior art.
  • a method for producing clam active peptides Fresh clam meat is washed with water, then water is added and homogenized with colloid to make clam meat slurry; clam meat slurry is hydrolyzed with water and compound protease, and after enzymatic hydrolysis, the enzyme is heated and inactivated; the enzymatic hydrolysis is collected by centrifugation The liquid is filtered through a microfiltration-ultrafiltration-nanofiltration membrane, and the enzymatic hydrolysate with a molecular weight of less than 2KDa is intercepted, and dried to obtain active clam peptides.
  • the present invention uses fresh clam meat as raw material, adopts composite enzyme-membrane coupling technology, and produces a clam activity that is pure in color, outstanding in taste, has blood pressure-lowering function and is easily absorbed by the human body through processing techniques such as enzymolysis, membrane separation and purification, and drying. Peptides to realize the high-value utilization of clam meat.
  • the composite protease in the production method is composed of neutral protease, alkaline protease, and flavor protease, and the addition ratio of each enzyme is 2:1:1 of neutral protease:alkaline protease:flavor protease.
  • the added amount of the composite protease in the production method is 0.1-0.3% of the mass of the clam pulp; continuous stirring during the enzymolysis process enables the composite protease to be fully utilized to ensure complete enzymolysis.
  • the weight ratio of clam meat to water during the enzymatic hydrolysis in the production method is 1:1 to 1:3.
  • the clam meat in the production method is red island clam meat.
  • the cleaning in the production method is deionized water cleaning.
  • deionized water is added to the production method; during the colloid grinding and homogenization process, the gap between the colloid grinding particles is controlled at 0-5 to ensure that the clam meat particles have a small and uniform particle size.
  • the enzymatic hydrolysis in the production method is enzymatic hydrolysis at 50-60°C for 4-6 hours under natural pH conditions.
  • the heat-killing enzyme in the production method is that the enzymatic hydrolysate is heated to 85° C. and kept for 10 minutes.
  • the centrifugation in the production method is to cool the enzymatic hydrolysate to below 40° C., filter through a 200-300 mesh filter, and then centrifuge at 16000 r/min.
  • the drying in the production method is that the filtrate after membrane filtration is sent to a drying tower for spray drying.
  • the spray drying temperature is 150-180°C, which can realize instantaneous drying into powder.
  • the industrial production method has mild conditions and is easy to control, and the obtained clam active peptide has pure flavor, small molecular weight, easy absorption, and higher quality. Therefore, the present invention also provides the clam active peptide prepared by the production method.
  • the present invention has at least one of the following beneficial effects:
  • the extraction of active peptides from clams generally has complicated processes, high production costs, long cycles, and is generally suitable for laboratory preparation, but not suitable for industrial production.
  • the production method of the present invention has simple process, mild conditions, short cycle, no inorganic or organic solvents, low energy consumption, high yield, and is more suitable for industrial production.
  • the prior art mostly uses single enzymes such as papain (endonuclease), neutral protease (endonuclease), and flavor protease (exonuclease) as enzyme preparations, and the protein recovery rate of the enzymatic hydrolysate prepared therefrom Low, the average relative molecular mass of clam active peptide is relatively large, the proportion of protein hydrolysate with relative molecular mass less than 1000U is small, and the peptide content is low.
  • the present invention adopts the composite enzyme-membrane coupling technology and adopts the composite protease to hydrolyze the protein in the clam meat.
  • the protein recovery rate of the clam enzymolysis solution is high (that is, the effective ingredients in the enzymolysis solution are high), reaching 90%, and the yield is
  • the proportion of protein hydrolysates with a relative molecular weight of less than 1000U is greater than 90%, and the content of polypeptides is greater than 80%.
  • the product quality qualification rate is high.
  • the enzymatic hydrolysate obtained by the compound protease hydrolysis of clam meat is then centrifuged-membrane filtration (microfiltration-ultrafiltration-nanofiltration)-drying technology.
  • the obtained enzymatic hydrolysate does not need to be decolorized, deodorized, and further purified and concentrated.
  • the process is simple, without the use of activated carbon, activated carbon fiber, etc. for decolorization and deodorization, and also avoids the generation of a large amount of solid waste, has high production efficiency and low cost, and is more suitable for industrial production.
  • the clam active peptides produced by the present invention are mainly tetra-hexapeptides, have high ACE inhibitory activity, the ACE inhibitory rate reaches 85%, and the blood pressure lowering functional activity is high.
  • the clam active peptide produced by the present invention has pure color, white-like appearance, no fishy smell, no other peculiar smell, pure color, excellent flavor, outstanding taste, high sensory evaluation, and is more popular with consumers.
  • the active peptides of clams have a small molecular weight and are easily absorbed by the human body.
  • they are rich in free amino acids, taurine, selenium and other nutrients, realizing the high-value utilization of clam meat.
  • Figure 1 shows the protein recovery rate of clam meat enzymatically hydrolyzed with different proteases in Example 1;
  • Figure 2 shows the proportion of the polypeptide content and relative molecular weight of the enzymolysis solution under different protease enzymolysis conditions in Example 1 that is less than 1000U;
  • Fig. 3 shows the chromatogram of the clam active peptide sample prepared in Example 2.
  • the invention discloses a production method of clam active peptides. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and product of the present invention have been described through the preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention. Invent technology.
  • a method for producing clam active peptides Fresh clam meat is washed with water, then water is added and homogenized with colloid to make clam meat slurry; clam meat slurry is hydrolyzed with water and compound protease, and after enzymatic hydrolysis, the enzyme is heated and inactivated; the enzymatic hydrolysis is collected by centrifugation The liquid is filtered through a microfiltration-ultrafiltration-nanofiltration membrane, and the enzymatic hydrolysate with a molecular weight of less than 2KDa is intercepted, and dried to obtain active clam peptides.
  • the clam meat in the production method is red island clam meat.
  • Fresh red island clam meat is used as the substrate for enzymolysis.
  • the source of clam meat is reliable and safe, with high nutritional value and extremely low content of harmful substances such as heavy metals.
  • the fresh clam meat is first washed with water for pre-treatment to clean the impurities on the surface of the clam meat.
  • Fresh clam meat itself is rich in free amino acids, vitamins, zinc, selenium and other nutrients. If the pre-treatment is not properly cleaned, a lot of nutrients will be lost.
  • the cleaning in the present invention is deionized water cleaning, followed by a little draining. Simply clean the surface of the clam meat with deionized water to remove impurities, and then slightly drain the cleaned clam meat.
  • the invention does not perform excessive washing and draining to dry, and can ensure that the moisture and nutrients in the clam meat are not lost.
  • the pretreatment process of the production method of the invention is simple, convenient and easy to operate.
  • the weight ratio of clam meat to water when the colloid is homogenized in the production method is 1:1.
  • the water is deionized water.
  • the gap between colloidal grinding particles is controlled at 0-5 to ensure that the clam meat particles are small and uniform, and the prepared clam meat slurry is fine and easy to be hydrolyzed and utilized by the compound protease. .
  • Endonuclease is a type of nuclease that can hydrolyze the phosphodiester bond from the middle of the protein molecule, thereby cutting the double-stranded protein; the exonuclease can only cut off from one end of the protein molecule.
  • the production method of the present invention uses a complex protease for enzymatic hydrolysis.
  • the composite protease is composed of neutral protease, alkaline protease, and flavor protease. It is a complex enzyme of a variety of endonucleases and exonucleases. It has many digestion sites and can completely digest the protein in clam meat.
  • Adopting the composite protease enzymolysis of the present invention can not only ensure the quality of the clam active peptide product, but also can greatly increase the yield of the product and increase economic benefits.
  • the addition ratio of each enzyme in the composite protease is 2:1:1 for neutral protease:alkaline protease:flavor protease.
  • the added amount of the composite protease in the enzymatic hydrolysis process in the production method is 0.1-0.3% of the mass of the clam pulp. In some embodiments, the added amount of the composite protease is 0.13% of the mass of the clam pulp. In some embodiments, the added amount of the composite protease is 0.2% of the mass of the clam pulp. In some embodiments, the added amount of the composite protease is 0.3% of the mass of the clam pulp.
  • the weight ratio of clam meat to water during the enzymatic hydrolysis in the production method is 1:1 to 1:3.
  • the water added during the enzymatic hydrolysis is deionized water.
  • the enzymatic hydrolysis in the production method is enzymatic hydrolysis at 50-60°C for 4-6 hours under natural pH conditions.
  • the enzymatic hydrolysis specifically refers to using the clam meat slurry obtained by homogenization as a substrate, sending the substrate to an enzymatic hydrolysis tank, adding deionized water, and raising the temperature in the enzymatic hydrolysis tank to 50-60°C.
  • Add compound protease enzymatically hydrolyze under natural pH conditions for 4-6h.
  • the natural pH is adopted in the enzymolysis process of the present invention, and there is no need to add chemical reagents such as hydrochloric acid or sodium hydroxide to adjust the pH of the clam pulp.
  • the process is simple, the operating conditions are mild, easy to control, no chemical reagents are added, and energy consumption is low. ,low cost.
  • the enzyme is heated to kill the enzyme after enzymolysis.
  • the heating to kill the enzyme means that the enzymatic hydrolysate is heated to 85°C and kept for 10 minutes.
  • the temperature and time of inactivation should not exceed the stated value, that is to ensure sufficient inactivation of the enzymatic hydrolysate, and avoid the Maillard reaction of the enzymatic hydrolysate itself at high temperature, so that the nutrients in the enzymatic hydrolysate are reduced and the color of the enzymatic hydrolysate is reduced. Deepening will eventually affect the quality of clam active peptides.
  • the enzyme hydrolysate is collected by centrifugation after the enzyme is inactivated.
  • the centrifugation involves cooling the enzymatic hydrolysate to below 40° C., filtering it through a 200-300 mesh filter, and then centrifuging at 16000 r/min.
  • the enzymatic hydrolysate after centrifugation is filtered through a membrane to intercept the enzymatic hydrolysate with a molecular weight of less than 2KDa.
  • the membrane filtration is specifically microfiltration-ultrafiltration-nanofiltration membrane filtration.
  • the drying in the production method is that the filtrate after membrane filtration is sent to a drying tower for spray drying.
  • the spray drying temperature is 150-180°C, which can realize instantaneous drying into powder.
  • the present invention adopts the enzymatic hydrolysate centrifugation-membrane filtration-spray drying process, which is simple, easy to operate, low cost, more suitable for industrial production, and the color of the clam active peptide product obtained by the process of the present invention Pure, good flavor, high quality, no need for decolorization and deodorization and further purification and concentration.
  • the production method of the invention has mild conditions and is easy to control, and the obtained clam active peptide has pure flavor, small molecular weight, mainly tetra-hexapeptide, and has the function of lowering blood pressure, is easy to absorb, and has higher quality. Therefore, the present invention also provides the clam active peptide prepared by the production method.
  • the present invention has at least one of the following beneficial effects:
  • the extraction of active peptides from clams generally has complicated processes, high production costs, long cycles, and is generally suitable for laboratory preparation, but not suitable for industrial production.
  • the production method of the present invention has simple process, mild conditions, short cycle, no inorganic or organic solvents, low energy consumption, high yield, and is more suitable for industrial production.
  • the prior art mostly uses single enzymes such as papain (endonuclease), neutral protease (endonuclease), and flavor protease (exonuclease) as enzyme preparations, and the protein recovery rate of the enzymatic hydrolysate prepared therefrom Low, the average relative molecular mass of clam active peptide is relatively large, the proportion of protein hydrolysate with relative molecular mass less than 1000U is small, and the peptide content is low.
  • the present invention adopts the composite enzyme-membrane coupling technology and adopts the composite protease to hydrolyze the protein in the clam meat.
  • the protein recovery rate of the clam enzymolysis solution is high (that is, the effective ingredients in the enzymolysis solution are high), reaching 90%, and the yield is
  • the proportion of protein hydrolysates with a relative molecular weight of less than 1000U is greater than 90%, and the content of polypeptides is greater than 80%.
  • the product quality qualification rate is high.
  • the enzymatic hydrolysate obtained by the compound protease hydrolysis of clam meat is then centrifuged-membrane filtration (microfiltration-ultrafiltration-nanofiltration)-drying technology.
  • the obtained enzymatic hydrolysate does not need to be decolorized, deodorized, and further purified and concentrated.
  • the process is simple, without the use of activated carbon, activated carbon fiber, etc. for decolorization and deodorization, and also avoids the generation of a large amount of solid waste, has high production efficiency and low cost, and is more suitable for industrial production.
  • the clam active peptides produced by the present invention are mainly tetra-hexapeptides, have high ACE inhibitory activity, the ACE inhibitory rate reaches 85%, and the blood pressure lowering functional activity is high.
  • the clam active peptide produced by the present invention has pure color, white-like appearance, no fishy smell, no other peculiar smell, pure color, excellent flavor, outstanding taste, high sensory evaluation, and is more popular with consumers.
  • the active peptides of clams have a small molecular weight and are easily absorbed by the human body.
  • they are rich in free amino acids, taurine, selenium and other nutrients, realizing the high-value utilization of clam meat.
  • the reagents involved in the embodiments of the present invention are all commercially available products, and all can be purchased through commercial channels.
  • the detection method of ACE inhibition rate is specifically to take 100 ⁇ L 5.0mmol/L hippuryl-histidyl-leucine (N-Hippuryl-His-Leu hydrate, HHL) solution and 30 ⁇ L clam peptide solution (ACEI) mix Put it in a 37°C water bath for 10 minutes, then add 10 ⁇ L 0.1U/mL ACE enzyme solution, mix well and continue the water bath reaction at 37°C for 30min, then add 250 ⁇ L 1mol/L HCl to the reaction system to stop the reaction, and then add 1.2mL frozen
  • a b the absorbance value of the reaction between ACE enzyme and HHL when the ACE inhibitor is not in the reaction;
  • Determination method of protein recovery rate According to the first method Kjeldahl method in GB 5009.5 "National Food Safety Standard Determination of Protein in Food", the protein content a1 in the raw clam meat and the protein in the clam active peptide powder are respectively determined Content b1. According to the method of the present invention to produce clam active peptides, record the feed amount A1 of fresh clam meat and the receipt amount B 1 of clam active peptide powder, and the protein recovery rate X of clam active peptide powder is calculated as:
  • Raw meat Red Island clam meat.
  • Test enzymes neutral protease, alkaline protease, papain, compound protease.
  • the enzymatic hydrolysis temperature is 50°C
  • the amount of enzyme added is 0.13% of the clam pulp
  • the pH is the natural pH value
  • the enzymatic hydrolysis is incubated for 6 hours, The enzyme was inactivated at 85°C for 10 min, centrifuged at 4000 r/min for 30 min, and the supernatant was taken.
  • the hydrolase is preferably a complex protease.
  • Example 2 Industrial production method of clam active peptide of the present invention
  • Raw meat Red Island clam meat.
  • Test enzyme compound protease.
  • the pH is the natural pH value; respectively heat the enzyme hydrolysis for 4 hours, inactivate the enzyme at 85°C for 10 minutes, centrifuge at 16000 r/min, take the supernatant for membrane filtration, and spray dry to obtain the active peptides of clams.
  • Example 3 Industrial production method of clam active peptide of the present invention
  • Raw meat Red Island clam meat.
  • Test enzyme compound protease.
  • the pH is the natural pH value; respectively heat the enzyme hydrolysis for 4 hours, inactivate the enzyme at 85°C for 10 minutes, centrifuge at 16000 r/min, take the supernatant for membrane filtration, and spray dry to obtain the active peptides of clams.
  • Example 4 Industrial production method of clam active peptide of the present invention
  • Raw meat Red Island clam meat.
  • Test enzyme compound protease.
  • Example 5 Industrial production method of clam active peptide of the present invention
  • Raw meat Red Island clam meat.
  • Test enzyme compound protease.
  • the pH is the natural pH value; respectively heat the enzyme hydrolysis for 6h, inactivate the enzyme at 85°C for 10min, centrifuge at 16000r/min, take the supernatant for membrane filtration, and spray dry to obtain the active peptide of clam.
  • the active peptides of clams prepared in each example were analyzed by liquid chromatography.
  • the results of the active peptides of clams prepared in Example 2 are shown in FIG. 3.
  • the detection data of the clam active peptide product obtained by the production method of the present invention meets the standard requirements and the quality is reliable.
  • the relative molecular weight of the clam active peptide product produced by the production method of the present invention is less than 1300 U, mainly small peptides such as tetrapeptide, pentapeptide, and hexapeptide, with small molecular weight and easy absorption.

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Abstract

公开了一种蛤蜊活性肽的制备方法。该方法为取新鲜的蛤蜊肉用水清洗，加水用胶体磨匀浆制成蛤蜊肉浆；蛤蜊肉浆加水和复合蛋白酶酶解，酶解后加热灭酶；离心收集酶解液，经过微滤-超滤-纳滤膜过滤，截取2KDa分子量以下的酶解液，干燥制得蛤蜊活性肽。该活性肽具有ACE抑制活性。

Description

一种蛤蜊活性肽的生产方法
本申请要求于2020年01月16日提交中国专利局、申请号为202010047257.8、发明名称为“一种蛤蜊活性肽的生产方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，具体涉及一种蛤蜊活性肽的生产方法，尤其是涉及一种适合于大规模工业化生产蛤蜊活性肽的生产方法。
背景技术
蛤蜊是我国四大养殖贝类之一，资源丰富。蛤蜊肉质鲜美，具有高蛋白、高维生素、少脂肪且含有人体必须的十多种氨基酸、矿物质的营养特点，它是一种食药两用的双壳贝海产品，已成为人们认可的高营养、低成本的健康食物。在国内蛤蜊主要以鲜食和干制为主，行业加工水平低和产品种类少等问题日益凸显。随着广大消费者对高质量海洋食品的需求提高，落后的传统加工技术已无法满足人类需求，因此，如何实现蛤蜊的精深加工和高值化利用，开发出更多更好的功能性蛤蜊产品，是蛤蜊产业面临的巨大机遇与严峻挑战。
随着海洋资源利用的不断深入，来源于生物酶解技术制备得到海洋生物的小肽，因其分子量小、生物效价高、生理活性好、稳定性好、安全易携带等优势而受到广泛关注。截止目前，国内市面已有海参肽、牡蛎肽、鲍鱼肽、胶原蛋白肽等物质大量销售，尚未见蛤蜊肽及相关蛤蜊提取物产品的销售。
从海洋生物中提取活性物质的研究早有报道。近代研究证明，海洋生物提取物具有提高免疫力、抗肿瘤、降血压、抗菌、抑制细胞微核形成、抗动脉粥样硬化等作用。因此，近年来，对于海洋生物活性肽的制备及其活性物质作用机理的研究成为热点。目前，利用酶解方 法从陆地生物蛋白及牡蛎、海参、海洋鱼等海洋生物蛋白中获取了大量的功能性活性肽，而对于酶解蛤蜊蛋白得到多肽的报道相对较少。现有酶解蛤蜊蛋白的报道中酶制剂的使用种类有胃蛋白酶、胰蛋白酶、木瓜蛋白酶、中性蛋白酶、碱性蛋白酶及动物水解蛋白酶等，而针对复合蛋白酶酶解红岛蛤蜊肉蛋白提取多肽的研究尚未见报道。
专利文献“一种蛤蜊活性肽的提取方法”(申请号为20161117416.8的中国)采用絮凝-离心-葡聚糖凝胶柱分离与酶法水解-葡聚糖凝胶柱分离两种方法得到多肽，凝胶柱分离工艺难以应用于规模化生产。专利文献“一种蛤蜊肽的提取方法”(申请号为20161117416.8的中国)使用酶解法，依次用氢氧化钠调pH、酶解、萃取、灭酶、离心、过滤吸附、纳滤分离浓缩、干燥，但反应过程复杂，大规模生产成本高且中间加入氢氧化钠等化学物质。
发明内容
有鉴于此，本发明的目的在于针对现有技术存在的缺陷，提供一种适合于大规模工业化生产蛤蜊活性肽的生产方法。
为实现本发明的目的，本发明采用如下技术方案：
一种蛤蜊活性肽的生产方法，取新鲜的蛤蜊肉用水清洗，加水用胶体磨匀浆制成蛤蜊肉浆；蛤蜊肉浆加水和复合蛋白酶酶解，酶解后加热灭酶；离心收集酶解液，经过微滤-超滤-纳滤膜过滤，截取2KDa分子量以下的酶解液，干燥制得蛤蜊活性肽。
本发明以新鲜蛤蜊肉为原料，采用复合酶-膜耦合技术，通过酶解、膜分离纯化、干燥等加工技术生产一种色泽纯正、口感突出，具有降血压功能活性且易于人体吸收的蛤蜊活性肽，以实现蛤蜊肉的高值化利用。
在本发明中，所述的生产方法中所述复合蛋白酶由中性蛋白酶、碱性蛋白酶、风味蛋白酶组成，各酶添加比例为中性蛋白酶:碱性蛋白酶:风味蛋白酶为2:1:1。
在本发明中，所述的生产方法中所述复合蛋白酶的加入量为蛤蜊肉浆质量的0.1～0.3％；所述酶解过程中连续搅拌，使复合蛋白酶被充分利用，确保酶解完全。
在本发明中，所述的生产方法中所述酶解时蛤蜊肉与水的重量比为1:1～1:3。
在本发明中，所述的生产方法中所述蛤蜊肉为红岛蛤蜊肉。
在本发明中，所述的生产方法中所述清洗为去离子水清洗。
在本发明中，所述的生产方法中所述匀浆为按蛤蜊肉：水＝1：1的重量比进行匀浆。
在本发明中，所述的生产方法中加入去离子水；所述胶体磨匀浆过程中，胶体磨颗粒间隙控制在0～5，确保蛤蜊肉粒粒度小且均一。
在本发明中，所述的生产方法中所述酶解为在自然pH条件下、50～60℃酶解4～6h。
在本发明中，所述的生产方法中所述加热灭酶为酶解液加热到85℃，保持10min。
在本发明中，所述的生产方法中所述离心为酶解液降温至40℃以下，经200～300目的滤网过滤，然后经16000r/min离心。
在本发明中，所述的生产方法中所述干燥为膜过滤后的滤液送入干燥塔中进行喷雾干燥。所述喷雾干燥温度为150～180℃，可实现瞬时干燥成粉。
上述工业化生产方法条件温和，易于控制，得到的蛤蜊活性肽风味纯正，分子量小，易于吸收，品质更高。因此本发明还提供了所述生产方法制得的蛤蜊活性肽。
与现有技术相比，本发明具有如下至少一种有益效果：
(1)现有技术在提取蛤蜊活性肽时普遍存在工艺复杂、生产成本高、周期长且通常适用于实验室内制备，不适于产业化生产。而本发明所述生产方法工艺简单，条件温和，周期短，不添加任何无机或有机溶剂，能耗低，产率高，更适合工业化生产。
(2)现有技术多采用如木瓜蛋白酶(内切酶)、中性蛋白酶(内切酶)、风味蛋白酶(外切酶)等单酶作为酶制剂，其制得的酶解液蛋白质回收率低，蛤蜊活性肽的平均相对分子质量较大，相对分子质量小于1000U的蛋白质水解物的占比小，多肽含量低。本发明采用复合酶-膜耦合技术，采用复合蛋白酶水解蛤蜊肉中的蛋白质，制得的蛤蜊酶解液的蛋白质回收率高(即酶解液中的有效成分高)，达到90％，产率高，且相对分子质量小于1000U的蛋白质水解物的占比大于90％，多肽含量大于80％，产品质量合格率高。进一步经复合蛋白酶酶解蛤蜊肉得到的酶解液，再经离心-膜过滤(微滤-超滤-纳滤)-干燥技术，制得的酶解液无需进行脱色脱腥与进一步的纯化浓缩等处理，工艺过程简单，同时无需使用活性炭、活性炭纤维等脱色脱腥，也避免了大量固废产生，生产效率高，成本低，更适合工业化生产。
(3)本发明生产的蛤蜊活性肽以四～六肽为主，具有较高的ACE抑制活性，ACE抑制率达到85％，降血压功能活性高。
(4)本发明生产的蛤蜊活性肽颜色纯正，呈类白色，无腥臭味，无其他异味，色泽纯正、风味优良、口感突出，感官评价高，更受消费者喜爱。且蛤蜊活性肽分子量小，易被人体吸收，另外含有丰富的游离氨基酸、牛磺酸以及硒等营养成分，真正实现蛤蜊肉的高值化利用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示实施例1不同蛋白酶酶解蛤蜊肉的蛋白质回收率；
图2示实施例1不同蛋白酶酶解条件下酶解液的多肽含量及相对分子质量小于1000U占比；
图3示实施例2制得的蛤蜊活性肽样品色谱图。
具体实施方式
本发明公开了一种蛤蜊活性肽的生产方法。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合，来实现和应用本发明技术。
为实现本发明的目的，本发明采用如下技术方案：
一种蛤蜊活性肽的生产方法，取新鲜的蛤蜊肉用水清洗，加水用胶体磨匀浆制成蛤蜊肉浆；蛤蜊肉浆加水和复合蛋白酶酶解，酶解后加热灭酶；离心收集酶解液，经过微滤-超滤-纳滤膜过滤，截取2KDa分子量以下的酶解液，干燥制得蛤蜊活性肽。
在一些实施方案中，所述的生产方法中所述蛤蜊肉为红岛蛤蜊肉。用新鲜红岛蛤蜊肉作为酶解底物，蛤蜊肉原料来源可靠、安全，营养价值高且重金属等有害物质含量极低。
在本发明中，所述的生产方法中新鲜的蛤蜊肉先用水清洗进行前处理，以清洗蛤蜊肉表面的杂质。新鲜蛤蜊肉本身含有丰富的游离氨基酸、维生素、锌、硒等营养成分，若前处理清洗不当，会导致大量的营养成分流失。本发明所述清洗为去离子水清洗，而后稍沥水。只用去离子水对蛤蜊肉表面进行简单清洗，可将杂质洗掉，然后将清洗后的蛤蜊肉轻微沥水即可。本发明不进行过度清洗与沥水晾干，可确保蛤蜊肉中的水分和营养成分不被流失。本发明所述生产方法前处理过程工艺简单、便捷，易操作。
在本发明中，所述的生产方法中所述胶体磨匀浆时蛤蜊肉与水的重量比为1：1。其中，在一些实施方案中，所述水为去离子水。
进一步的，在本发明中，所述胶体磨匀浆过程中，胶体磨颗粒间隙控制在0～5，确保蛤蜊肉粒粒度小且均一，制得的蛤蜊肉浆细腻，易被复合蛋白酶水解利用。
内切酶是一类能从蛋白质分子中间水解磷酸二酯键，从而切断双链蛋白质的核酸水解酶；外切酶只能从蛋白质分子的一端开始切断。本发明所述的生产方法以复合蛋白酶进行酶解。所述复合蛋白酶由中性蛋白酶、碱性蛋白酶、风味蛋白酶组成，其是多种内切酶与外切酶的复合酶，酶切位点较多，对蛤蜊肉中的蛋白质酶解比较彻底，可以提高蛤蜊肉中蛋白质的回收率，降低酶解液的平均相对分子质量，方便后面的膜过滤过程。采用本发明所述复合蛋白酶酶解既可以确保蛤蜊活性肽产品的品质，又可以大大提高产品的产率，增加经济效益。
在本发明中，所述复合蛋白酶中各酶添加比例为中性蛋白酶:碱性蛋白酶:风味蛋白酶为2:1:1。
在本发明中，所述的生产方法中酶解过程中所述复合蛋白酶的加入量为蛤蜊肉浆质量的0.1～0.3％。在一些实施方案中，所述复合蛋白酶的加入量为蛤蜊肉浆质量的0.13％。在一些实施方案中，所述复合蛋白酶的加入量为蛤蜊肉浆质量的0.2％。在一些实施方案中，所述复合蛋白酶的加入量为蛤蜊肉浆质量的0.3％。
在本发明中，所述的生产方法中所述酶解时蛤蜊肉与水的重量比为1:1～1:3。在一些实施方案中，所述酶解时添加的水为去离子水。
在本发明中，所述的生产方法中所述酶解为在自然pH条件下、50～60℃酶解4～6h。在一些实施方案中，所述酶解具体为将匀浆得到的蛤蜊肉浆为底物，将底物送至酶解罐中，加入去离子水，酶解罐中温度升至50～60℃，加入复合蛋白酶，在自然pH条件下酶解4～6h。本发明所述酶解过程中采用自然pH，无需加入盐酸或氢氧化钠等化学试剂对蛤蜊肉浆进行pH的调整，工艺简单，操作条件温和，易于控制，不添加任何化学试剂，能耗低，成本低。
进一步的，本发明所述酶解过程中进行连续搅拌，以使复合蛋白酶被充分利用，确保酶解完全。
本发明所述生产方法在酶解后进行加热灭酶。在本发明中，所述加热灭酶为酶解液加热到85℃，保持10min。灭活温度与时间不宜超 出所述的值，即确保酶解液灭活充分，又避免在高温下酶解液本身发生美拉德反应，使酶解液中的营养物质降低、酶解液颜色变深，最终影响蛤蜊活性肽的品质。
本发明所述生产方法在灭酶后离心收集酶解液。在一些实施方案中，所述离心为酶解液降温至40℃以下，经200～300目的滤网过滤，然后经16000r/min离心。
进一步的，离心后的酶解液经过膜过滤截取2KDa分子量以下的酶解液。所述膜过滤具体为微滤-超滤-纳滤膜过滤。
在本发明中，所述的生产方法中所述干燥为膜过滤后的滤液送入干燥塔中进行喷雾干燥。所述喷雾干燥温度为150～180℃，可实现瞬时干燥成粉。
相比于现有的技术，如离心-板框压滤-超滤-纳滤-负压浓缩-喷雾干燥、离心-超滤-减压浓缩-喷雾干燥、离心-树脂过滤吸附-纳滤-浓缩-喷雾干燥等工艺，本发明采用酶解液离心-膜过滤-喷雾干燥工艺，工艺简单，易操作，成本低，更适合工业化生产，且通过本发明的工艺方法得到的蛤蜊活性肽产品色泽纯正，风味较好，品质高，无需进行脱色脱腥与进一步的纯化浓缩等操作工艺。
本发明所述生产方法条件温和，易于控制，得到的蛤蜊活性肽风味纯正，分子量小，主要为四～六肽，且具有降血压功能，易于吸收，品质更高。因此本发明还提供了所述生产方法制得的蛤蜊活性肽。
与现有技术相比，本发明具有如下至少一种有益效果：
(1)现有技术在提取蛤蜊活性肽时普遍存在工艺复杂、生产成本高、周期长且通常适用于实验室内制备，不适于产业化生产。而本发明所述生产方法工艺简单，条件温和，周期短，不添加任何无机或有机溶剂，能耗低，产率高，更适合工业化生产。
(2)现有技术多采用如木瓜蛋白酶(内切酶)、中性蛋白酶(内切酶)、风味蛋白酶(外切酶)等单酶作为酶制剂，其制得的酶解液蛋白质回收率低，蛤蜊活性肽的平均相对分子质量较大，相对分子质 量小于1000U的蛋白质水解物的占比小，多肽含量低。本发明采用复合酶-膜耦合技术，采用复合蛋白酶水解蛤蜊肉中的蛋白质，制得的蛤蜊酶解液的蛋白质回收率高(即酶解液中的有效成分高)，达到90％，产率高，且相对分子质量小于1000U的蛋白质水解物的占比大于90％，多肽含量大于80％，产品质量合格率高。进一步经复合蛋白酶酶解蛤蜊肉得到的酶解液，再经离心-膜过滤(微滤-超滤-纳滤)-干燥技术，制得的酶解液无需进行脱色脱腥与进一步的纯化浓缩等处理，工艺过程简单，同时无需使用活性炭、活性炭纤维等脱色脱腥，也避免了大量固废产生，生产效率高，成本低，更适合工业化生产。
(3)本发明生产的蛤蜊活性肽以四～六肽为主，具有较高的ACE抑制活性，ACE抑制率达到85％，降血压功能活性高。
(4)本发明生产的蛤蜊活性肽颜色纯正，呈类白色，无腥臭味，无其他异味，色泽纯正、风味优良、口感突出，感官评价高，更受消费者喜爱。且蛤蜊活性肽分子量小，易被人体吸收，另外含有丰富的游离氨基酸、牛磺酸以及硒等营养成分，真正实现蛤蜊肉的高值化利用。
为了进一步理解本发明，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如无特殊说明，本发明实施例中所涉及的试剂均为市售产品，均可以通过商业渠道购买获得。
其中，ACE抑制率的检测方法具体为取100μL 5.0mmol/L马尿酰-组氨酰-亮氨酸(N-Hippuryl-His-Leu hydrate，HHL)溶液与30μL的蛤蜊肽液(ACEI)混合，置于37℃水浴10min，然后加入10μL 0.1U/mL的ACE酶液，混匀后于37℃继续水浴反应30min，随后向反应体系中加入250μL 1mol/L HCl终止反应，再加入1.2mL冷冻乙酸乙酯提取产生 的马尿酸，旋涡震荡混匀后以3500r/min离心5min，吸取1.0mL的乙酸乙酯层，于90℃烘箱中经1小时烘干、冷却后加入4mL蒸馏水充分溶解，漩涡混合后于228nm波长处测吸光值OD228。平行对照组除在反应前先加入250μL 1mol/L HCl以终止反应外，其他操作步骤均同实验组，重复测定3次，结果取平均值，具体操作步骤见表1：
表1 紫外分光光度计法测定ACE抑制率
Figure PCTCN2020076042-appb-000001
计算公式为：
Figure PCTCN2020076042-appb-000002
式中：A a—反应中ACE及其抑制剂都存在时，与HHL反应的吸光度值；A b—反应中不含ACE抑制剂时，ACE酶和HHL反应的吸光度值；A c—ACE和HHL空白反应的吸光度值。
蛋白质回收率的测定方法：依据GB 5009.5《食品安全国家标准 食品中蛋白质的测定》中的第一法凯氏定氮法，分别测定原料蛤蜊肉中的蛋白质含量a1与蛤蜊活性肽粉中的蛋白质含量b1。按照本发明方法生产蛤蜊活性肽，记录新鲜蛤蜊肉的投料量A1与蛤蜊活性肽粉的收料量B 1，蛤蜊活性肽粉的蛋白质回收率X计算公式为：
Figure PCTCN2020076042-appb-000003
实施例1：不同蛋白酶酶解效果比较
原料肉：红岛蛤蜊肉。
供试酶：中性蛋白酶、碱性蛋白酶、木瓜蛋白酶、复合蛋白酶。
酶解工艺流程：取冷冻的红岛蛤蜊肉，解冻后，按蛤蜊肉：水＝1:1的重量比加入去离子水进行匀浆；中性蛋白酶、碱性蛋白酶、木瓜蛋白酶、复合蛋白酶(配方为中性蛋白酶:碱性蛋白酶:风味蛋白酶＝2:1:1)的酶解温度为50℃，加酶量为蛤蜊肉浆的0.13％，pH为自然pH值；分别保温酶解6h，85℃灭酶10min，4000r/min离心30min，取上清液。
表2 酶解所得产品的检测结果
Figure PCTCN2020076042-appb-000004
由表2结果可见，筛选的4种蛋白水解酶中，复合蛋白酶水解的蛤蜊酶解液的蛋白质回收率、多肽含量、相对分子质量小于1000U的蛋白质水解物的占比均大于木瓜蛋白酶、中性蛋白酶、碱性蛋白酶，且复合蛋白酶水解的蛤蜊酶解液的平均相对分子质量最小。因此，水解酶优选复合蛋白酶。
实施例2：本发明所述蛤蜊活性肽的工业化生产方法
原料肉：红岛蛤蜊肉。
供试酶：复合蛋白酶。
酶解工艺流程：取冷冻的红岛蛤蜊肉，解冻后，按蛤蜊肉：水＝1:1的重量比加入去离子水进行匀浆，向蛤蜊肉浆中加入一定量的去离子水，使得最终蛤蜊肉：水＝1:2的重量比；复合蛋白酶(配方为中性蛋白酶:碱性蛋白酶:风味蛋白酶＝2:1:1)的酶解温度为50℃，加酶量为蛤蜊肉浆的0.13％，pH为自然pH值；分别保温酶解4h，85℃灭酶10min，16000r/min离心，取上清液进行膜过滤，喷雾干燥得蛤蜊活性肽。
实施例3：本发明所述蛤蜊活性肽的工业化生产方法
原料肉：红岛蛤蜊肉。
供试酶：复合蛋白酶。
酶解工艺流程：取冷冻的红岛蛤蜊肉，解冻后，按蛤蜊肉：水＝1:1的重量比加入去离子水进行匀浆，向蛤蜊肉浆中加入一定量的去离子水，使得最终蛤蜊肉：水＝1:3的重量比；复合蛋白酶(配方为中性蛋白酶:碱性蛋白酶:风味蛋白酶＝2:1:1)的酶解温度为50℃，加酶量为蛤蜊肉浆的0.13％，pH为自然pH值；分别保温酶解4h，85℃灭酶10min，16000r/min离心，取上清液进行膜过滤，喷雾干燥得蛤蜊活性肽。
实施例4：本发明所述蛤蜊活性肽的工业化生产方法
原料肉：红岛蛤蜊肉。
供试酶：复合蛋白酶。
酶解工艺流程：取冷冻的红岛蛤蜊肉，解冻后，按蛤蜊肉：水＝1:1的重量比加入去离子水进行匀浆，向蛤蜊肉浆中加入一定量的去离子水，使得最终蛤蜊肉：水＝1:2的重量比；复合蛋白酶(配方为中性蛋白酶:碱性蛋白酶:风味蛋白酶＝2:1:1)的酶解温度为60℃，加酶量为 蛤蜊肉浆的0.2％，pH为自然pH值；分别保温酶解4h，85℃灭酶10min，16000r/min离心，取上清液进行膜过滤，喷雾干燥得蛤蜊活性肽。
实施例5：本发明所述蛤蜊活性肽的工业化生产方法
原料肉：红岛蛤蜊肉。
供试酶：复合蛋白酶。
酶解工艺流程：取冷冻的红岛蛤蜊肉，解冻后，按蛤蜊肉：水＝1:1的重量比加入去离子水进行匀浆，向蛤蜊肉浆中加入一定量的去离子水，使得最终蛤蜊肉：水＝1:2的重量比；复合蛋白酶(配方为中性蛋白酶:碱性蛋白酶:风味蛋白酶＝2:1:1)的酶解温度为50℃，加酶量为蛤蜊肉浆的0.3％，pH为自然pH值；分别保温酶解6h，85℃灭酶10min，16000r/min离心，取上清液进行膜过滤，喷雾干燥得蛤蜊活性肽。
试验例
对各实施例制得的蛤蜊活性肽进行检测，结果见表3
对各实施例制得的蛤蜊活性肽进行液相色谱分析，其中实施例2制得的蛤蜊活性肽结果见图3。
表3蛤蜊活性肽检测结果
Figure PCTCN2020076042-appb-000005
Figure PCTCN2020076042-appb-000006
由表3结果可见，所述蛤蜊活性肽产品中相对分子质量≤2000U的蛋白质水解物占97％以上，相对分子质量＜1000U的蛋白质水解物均占90％以上，小分子肽含量较高，更容易被吸收；所述蛤蜊活性肽的重金属污染物指标和微生物指标均满足国家标准；所述蛤蜊活性肽ACE抑制率均大于85％，具有很好的ACE抑制活性，因此，本发明制得的蛤蜊活性肽降血压功能强。此外，所述蛤蜊活性肽产品色泽纯正，无腥臭味，无其他异味。
通过本发明所述生产方法得到的蛤蜊活性肽产品各项检测数据均符合标准要求，品质可靠。且本发明所述生产方法所生产的蛤蜊活性肽产品相对分子质量小于1300U，以四肽、五肽、六肽等小分肽为主，分子量小、易吸收。
以上所述，仅是对本发明的较佳实施例而已，并非是对本发明做其他形式的限制，应当指出，任何本技术领域的技术人员可能利用上述揭示的技术内容加以改进和润饰为等同变化的等效实施例。但是，凡是未脱离本发明方案内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化，仍属于本发明的保护范围。

Claims (10)

  1. 一种蛤蜊活性肽的生产方法，取新鲜的蛤蜊肉用水清洗，加水用胶体磨匀浆制成蛤蜊肉浆；蛤蜊肉浆加水和复合蛋白酶酶解，酶解后加热灭酶；离心收集酶解液，经过微滤-超滤-纳滤膜过滤，截取2KDa分子量以下的酶解液，干燥制得蛤蜊活性肽。
  2. 根据权利要求1所述的生产方法，所述复合蛋白酶由中性蛋白酶、碱性蛋白酶、风味蛋白酶组成，各酶添加比例为中性蛋白酶:碱性蛋白酶:风味蛋白酶为2:1:1。
  3. 根据权利要求1所述的生产方法，所述复合蛋白酶的加入量为蛤蜊肉浆质量的0.1～0.3％；所述酶解过程中连续搅拌，使复合蛋白酶被充分利用，确保酶解完全。
  4. 根据权利要求1所述的生产方法，所述酶解时蛤蜊肉与水的重量比为1:1～1:3。
  5. 根据权利要求1所述的生产方法，所述蛤蜊肉为红岛蛤蜊肉；所述清洗为去离子水清洗；所述匀浆为按蛤蜊肉：水＝1：1的重量比进行匀浆；加入去离子水；所述胶体磨匀浆过程中，胶体磨颗粒间隙控制在0～5，确保蛤蜊肉粒粒度小且均一。
  6. 根据权利要求1所述的生产方法，所述酶解为在自然pH条件下、50～60℃酶解4～6h。
  7. 根据权利要求1所述的生产方法，所述加热灭酶为酶解液加热到85℃，保持10min。
  8. 根据权利要求1所述的生产方法，所述离心为酶解液降温至40℃以下，经200～300目的滤网过滤，然后经16000r/min离心。
  9. 根据权利要求1所述的生产方法，所述干燥为膜过滤后的滤液送入干燥塔中进行喷雾干燥。
  10. 权利要求1-9所述的生产方法制得的蛤蜊活性肽。
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