CN114807277A - Method for preparing sea cucumber peptide by double enzymes step by step - Google Patents
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- CN114807277A CN114807277A CN202210379719.5A CN202210379719A CN114807277A CN 114807277 A CN114807277 A CN 114807277A CN 202210379719 A CN202210379719 A CN 202210379719A CN 114807277 A CN114807277 A CN 114807277A
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- 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
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- 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
Abstract
The invention belongs to the technical field of marine organisms, and discloses a method for preparing sea cucumber peptide by double enzymes step by step, which comprises the following steps: (1) cleaning viscera of sea cucumber, and grinding into thick liquid; (2) uniformly mixing the sea cucumber viscera protoplasm with deionized water, heating until denaturation, cooling, and performing enzymolysis step by using double enzymes; (3) after the enzymolysis is finished, adding active carbon to remove the odor, then adjusting the pH value to 6.0-7.0, and heating to inactivate the enzyme; (4) filtering with inorganic ceramic membrane; (5) separating sea cucumber oligopeptide by adopting a nano-scale membrane separation technology; (6) freeze drying; (7) and (4) preparing particles. The invention has the advantages of thorough enzymolysis, high extraction rate, high oligopeptide content, more than 90 percent of oligopeptide content in the product components, and the peptide molecular weight distribution result: peptides above 1000Da are less than 10%, and small molecule peptides within 1000Da are more than 90%; the invention changes the waste sea cucumber viscera of the sea cucumber processing factory into all water-soluble active ingredients through a biological double-enzyme stepwise enzymolysis technology, the oligopeptide content of the product is high, and the aim of changing waste into valuables is fulfilled.
Description
Technical Field
The invention belongs to the technical field of marine biotechnology, and particularly relates to a method for preparing sea cucumber peptide by double enzymes step by step.
Background
Sea Cucumber (Sea Cucumber), an animal of the class Holothuria of the phylum Echinodermata, lives from seaside to 8000 meters, usually lives on the Sea floor with a rather low water temperature, usually takes the impurities in the filtered sand as food, and is called as "Qingdao in the Sea". Sea cucumber is distinguished among various mountain delicacies and sea tastes as eight delicacies, and people in China thought that sea cucumber is not only a delicacy but also a nourishing health-care product hundreds of years ago. In Ming Dynasty, the book "five zang ancient sacrificial offerings" has a record about sea cucumber. The book Bencao gang mu Shi Yi compiled by Qing dynasty Zhao academic Ming has the following description: the sea cucumber is warm in nature and sufficient for the enemy ginseng, so the sea cucumber is named; sweet and salty in taste, tonifying kidney channel, benefiting essence, eliminating phlegm, controlling urination, strengthening yang, treating flaccidity, and killing sore and worm. The medicinal value of the sea cucumber is more thoroughly recorded in the medicine identification and the medicine property examination. The sea cucumber not only contains more than 50 nutrient components needed by human bodies, such as amino acids, vitamins, chemical elements and the like, but also contains various bioactive substances, such as acidic mucopolysaccharide, saponin, collagen and the like, and modern pharmacological research shows that the sea cucumber has various active components and has various pharmacological actions, such as tumor resistance, immunity regulation, oxidation resistance and the like.
The bioactive peptide has various human body physiological regulation functions, has the effects of improving immunity, regulating hormone, resisting oxidation, bacteria and viruses, reducing blood pressure, reducing blood fat and the like, can meet the all-round requirement of delaying senility, has high edible safety and easy digestion and absorption, and is the hottest research topic and the functional factor with great development prospect in the current international food industry. Meanwhile, researches show that the active peptide has good compatibility and stability in cosmetics, and the small molecular active polypeptide can relieve wrinkles, make the skin smoother and lighten color spots.
The viscera of the sea cucumber contains a plurality of active ingredients such as protein, polysaccharide, saponin and the like, and has a plurality of efficacies of tumor resistance, inflammation resistance, blood coagulation resistance and the like; however, most of the viscera of the sea cucumber are discarded as wastes of sea cucumber processing at present, so that the resources are greatly wasted; the invention takes the viscera of the sea cucumber as the raw material, utilizes the technology of preparing the sea cucumber peptide by double enzymes step by step, has thorough enzymolysis and high extraction rate, realizes changing waste into valuable and greatly increases the additional value of sea cucumber processing. Has very important significance for the deep development of the sea cucumber.
Disclosure of Invention
The invention aims to solve the main problems that most of the viscera of the sea cucumber are discarded as wastes for processing the sea cucumber, so that the resources are greatly wasted; the technology for preparing the sea cucumber peptide by using double enzymes step by step has thorough enzymolysis and high extraction rate, changes waste into valuable and greatly increases the additional value of sea cucumber processing.
The invention is implemented by the following technical scheme: a method for preparing sea cucumber peptide by double enzymes step by step comprises the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 90-100 ℃ for 10-15 min to moderately denature sea cucumber protein, cooling, and then adding soluble protease step by step for enzymolysis, wherein the amount of trypsin added in the first step is 20000U-30000U/g, the enzymolysis condition is pH7.5-9.0, the temperature is 30-40 ℃, and the time is 2-5 h; secondly, adding 10-50LAPU (leucine aminopeptidase unit)/g of flavourzyme, wherein the enzymolysis condition is that the pH value is 6.0-7.0, the temperature is 30-40 ℃, and the time is 3-6 hours;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(5) filtering the sea cucumber peptide clear filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(6) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide;
(7) and (3) putting the nanofiltration permeate of the sea cucumber peptide into a freeze drying device for freeze drying, controlling the temperature to be below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide.
Further, in the step (2), the two enzymes are trypsin and compound flavor protease respectively, wherein the compound flavor protease is a product with the specification of 1000LAPU/g of Novitin.
Further, in the step (4), the filter element of the inorganic ceramic membrane is made of an alkali pottery clay material.
Further, in the step (5), a roll-type nanofiltration membrane made of 100Da polyamide material is adopted, and a nanofiltration system works
The parameters are: the operating pressure was 8bar, the feed temperature was 45 ℃ and the feed PH = 6.
Fresh sea cucumber viscera is used as a raw material, the protein content of the sea cucumber viscera reaches 50-60%, and the sea cucumber viscera also contains various active substances such as sea cucumber polysaccharide, sea cucumber saponin and the like, is rich in vitamins and trace elements, has low fat content, basically does not contain cholesterol, and has very important significance for developing deep-processed products of the sea cucumber viscera.
The invention has the following remarkable beneficial effects:
(1) complete enzymolysis, high extraction rate, high oligopeptide content, high peptide content in product components up to more than 80 percent, and the result of peptide molecular weight distribution: peptides above 1.1000Da are less than 10%, and small molecule peptides within 1000Da are more than 90%; the internal organs of the sea cucumber are changed into all water-soluble active ingredients through a double-enzyme stepwise enzymolysis technology through the double-enzyme stepwise enzymolysis technology, so that the absorption is complete and rapid, the oligopeptide content of the product is high, and the absorption and the utilization of the active ingredients of the sea cucumber are facilitated;
(2) removing odor: after the taste of the sea cucumber peptide is removed by the active carbon, the sea cucumber peptide is more acceptable in taste, and the filtering efficiency and the quality are improved;
(3) after enzymolysis, a technical means of filtering by using an inorganic nanofiltration ceramic membrane is adopted to remove substances which are not easy to dissolve and sugar components which are easy to absorb moisture, so that the water re-solubility of the finally obtained sea cucumber peptide finished product is improved, and a good foundation is laid for the next operation unit;
(4) the product has less fishy smell, is safe and easy to absorb, and has good health care and health preserving effects.
Detailed Description
In order that the invention may be readily understood, the invention will now be further described with reference to specific embodiments thereof.
Example 1
A method for preparing sea cucumber peptide by double enzymes step by step comprises the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 95 ℃ for 15min to moderately denature sea cucumber protein, cooling, and then adding soluble protease step by step for enzymolysis, wherein the amount of trypsin added in the first step is 30000U/g, the enzymolysis condition is pH9.0, the temperature is 35 ℃, and the time is 5 hours; secondly, adding flavourzyme with the amount of 50LAPU (leucine aminopeptidase unit)/g, and carrying out enzymolysis under the conditions of pH6.5, the temperature of 40 ℃ and the time of 6 hours;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(4) filtering the sea cucumber peptide clear filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(5) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide;
(6) freeze-drying the nanofiltration permeate of the sea cucumber peptide in a freeze-drying device at the temperature of below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze-drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide;
example 2
A method for preparing sea cucumber peptide by double enzymes step by step comprises the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 100 ℃ for 15min to moderately denature sea cucumber protein, cooling, and then adding soluble protease step by step for enzymolysis, wherein the amount of the added trypsin in the first step is 20000U/g, the enzymolysis condition is pH9.0, the temperature is 35 ℃, and the time is 5 h; secondly, adding 20LAPU (leucine aminopeptidase unit)/g of flavourzyme, wherein the enzymolysis condition is pH6.5.0, the temperature is 40 ℃, and the time is 6 hours;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(4) filtering the sea cucumber peptide clear filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(5) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide;
(6) freeze-drying the nanofiltration permeate of the sea cucumber peptide in a freeze-drying device at the temperature of below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze-drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide;
example 3
A method for preparing sea cucumber peptide by double enzymes step by step comprises the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 95 ℃ for 10-15 min to moderately denature sea cucumber protein, cooling, and then adding soluble protease step by step for enzymolysis, wherein the amount of trypsin added in the first step is 30000U/g, the enzymolysis condition is pH9.0, the temperature is 35 ℃, and the time is 3 hours; secondly, adding flavourzyme with the amount of 50LAPU (leucine aminopeptidase unit)/g, and carrying out enzymolysis under the conditions of pH6.5, the temperature of 40 ℃ and the time of 4 hours;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(4) filtering the sea cucumber peptide clear filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(5) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide;
(6) freeze-drying the nanofiltration permeate of the sea cucumber peptide in a freeze-drying device at the temperature of below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze-drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide;
comparative example 1
A method for preparing sea cucumber peptide by double enzymes step by step comprises the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 95 ℃ for 15min to moderately denature sea cucumber protein, cooling, adding soluble protease step by step for enzymolysis, adding 30000U/g of trypsin, wherein the enzymolysis condition is pH9.0, the temperature is 35 ℃, and the time is 5 h;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(4) filtering the sea cucumber peptide clear filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(5) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide;
(6) freeze-drying the nanofiltration permeate of the sea cucumber peptide in a freeze-drying device at the temperature of below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze-drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide;
comparative example 2
A method for preparing sea cucumber peptide by double enzymes step by step comprises the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 95 ℃ for 15min to moderately denature sea cucumber protein, cooling, adding soluble protease step by step for enzymolysis, adding flavourzyme with the amount of 50LAPU (leucine aminopeptidase unit)/g, wherein the enzymolysis condition is pH6.5, the temperature is 40 ℃, and the time is 6 h;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(4) filtering the sea cucumber peptide clear filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(5) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide;
(6) freeze-drying the nanofiltration permeate of the sea cucumber peptide in a freeze-drying device at the temperature of below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze-drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide;
comparative example 3
A method for preparing sea cucumber peptide by double enzymes step by step comprises the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 95 ℃ for 15min to moderately denature sea cucumber protein, cooling, and then adding soluble protease step by step for enzymolysis, wherein the amount of the added papain is 30000U/g, the enzymolysis condition is pH6.5, the temperature is 37 ℃, and the time is 5 h;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(4) filtering the sea cucumber peptide clear filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(5) and (3) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide.
(6) Freeze-drying the nanofiltration permeate of the sea cucumber peptide in a freeze-drying device at the temperature of below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze-drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide;
and (3) detecting the enzymolysis effect of two enzymes:
in examples 1 to 3 and comparative examples 1 to 3, after the internal organs of the sea cucumber are subjected to pretreatment and heating treatment of the raw materials, enzymolysis is performed by using two steps of double enzymes, in order to evaluate the enzymolysis effect, after the enzymolysis is finished, the detection of the enzymolysis products is performed by using the method in appendix A of national standard GB/T22729-:
table 1 shows the molecular weight distribution ratio (%)
The results in table 1 show that the enzymolysis products in examples 1 to 3 have small and concentrated molecular weights, the enzymolysis products have low free amino acid content (the proportion of less than 200Da is less than 5%), the proportion of the sea cucumber peptide with the molecular weight of less than 1000Da reaches 100%, the proportion of the sea cucumber peptide between 200Da and 1000Da reaches more than 95.5%, and the molecular weights of the enzymolysis products are obviously better than those of comparative examples 1 to 3. Therefore, the method can obviously improve the enzymolysis efficiency of the sea viscera holothurian protein by adopting the two-enzyme two-step technical means provided by the invention, and the enzymolysis is thorough.
The technical contents of the present invention are further illustrated by the examples only for the convenience of the reader, but the embodiments of the present invention are not limited thereto, and any technical extension or re-creation based on the present invention is protected by the present invention. The protection scope of the invention is subject to the claims.
Claims (4)
1. A method for preparing sea cucumber peptide by double enzymes step by step is characterized by comprising the following steps:
(1) cleaning fresh sea cucumber viscera, cutting into small pieces, and grinding to obtain sea cucumber viscera raw pulp;
(2) 1, mixing the sea cucumber viscera protoplasm with deionized water: 1, uniformly mixing, heating to 90-100 ℃ for 10-15 min to moderately denature sea cucumber protein, cooling, and then adding soluble protease step by step for enzymolysis, wherein the amount of trypsin added in the first step is 20000U-30000U/g, the enzymolysis condition is pH7.5-9.0, the temperature is 30-40 ℃, and the time is 2-5 h; secondly, adding 10-50LAPU (leucine aminopeptidase unit)/g of flavourzyme, wherein the enzymolysis condition is that the pH value is 6.0-7.0, the temperature is 30-40 ℃, and the time is 3-6 hours;
(3) after the enzymolysis is finished, adding activated carbon accounting for 17% of the weight of dry matters for deodorization, then adjusting the pH value to 6.0-7.0, heating, inactivating enzymes, sterilizing, cooling to 68-72 ℃, and standing for 4 hours;
(4) filtering the clear sea cucumber peptide filtrate by adopting a 0.5 micron inorganic nanofiltration ceramic membrane to obtain a permeate as a clear sea cucumber peptide solution;
(5) introducing the sea cucumber peptide ceramic membrane permeate into a nanofiltration system to separate sea cucumber oligopeptide;
(6) freeze-drying the nanofiltration permeate of the sea cucumber peptide in a freeze-drying device at the temperature of below 0 ℃, removing water from ice crystals to steam in a vacuum state, and freeze-drying for 20-40 hours to control the water content to be 3-5% to prepare the flaky sea cucumber peptide;
(7) preparing particles: and crushing the obtained flaky sea cucumber peptide into powder with the fineness of 100-200 meshes in a low-temperature state by using a freezing crusher, and preparing the powder sea cucumber peptide into granules with the granularity of 1-2 mm by using a granulator to obtain the sea cucumber peptide powder.
2. The method for preparing sea cucumber peptide by two enzymes step by step according to claim 1, wherein in the step (2), the two enzymes are trypsin and flavourzyme, respectively, wherein the flavourzyme adopts the product with the specification of 1000LAPU/g of Novoxil.
3. The method for preparing sea cucumber peptides by two enzymes step by step according to claim 1, wherein in the step (4), the filter element of the inorganic ceramic membrane is made of an alkali pottery clay material.
4. The method for preparing sea cucumber peptide by using double enzymes step by step according to claim 1, wherein in the step (5), a 100Da polyamide roll type nanofiltration membrane is adopted, and the operating parameters of a nanofiltration system are as follows: the operating pressure was 8bar, the feed temperature was 45 ℃ and the feed PH = 6.
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