CN102952838A - Preparation method of immune-enhancing mussel enzymatic hydrolytic polypeptide and preparation method of corresponding tablet thereof - Google Patents
Preparation method of immune-enhancing mussel enzymatic hydrolytic polypeptide and preparation method of corresponding tablet thereof Download PDFInfo
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Abstract
A preparation method of immune-enhancing mussel enzymatic hydrolytic polypeptide and corresponding tablets comprises the following steps: mixing a raw material of mussel dry powder with a buffer according to a weight volume ratio of 1:20-1:25, homogenizing the mixture, adjusting the pH value to 9.5-10 to obtain a mixed solution; heating the mixed solution to 50-55 DEG C, preheating for 10-15 min, respectively adding an alkaline protease and a neutral protease with a weight being 1.0%-1.5% of the raw material, performing enzymatic hydrolysis at 50-55 DEG C for 2-4 hours; performing enzyme deactivation of the enzymatic hydrolytic product to obtain an enzymatic hydrolytic solution, then orderly performing desalination, concentration, and spray drying of the enzymatic hydrolytic solution to obtain the immune-enhancing enzymatic hydrolytic polypeptide. The invention also discloses a related immune-enhancing mussel polypeptide tablet. The preparation method of the invention is simple; the obtained immune-enhancing polypeptide and its tablet have high activity; the raw material resource is abundant; and the preparation method has strong industrial implementation potential.
Description
Technical field
What the present invention relates to is a kind of method that is prepared immunostimulant mussel enzymolysis polypeptide and respective tablets by mollusk enzymolysis such as mussels.
Background technology
Mussel belongs to Mollusca, lamellibranchiata, mussel order, Mytilidae, mussel belongs to, the marine products bivalve shellfishs, be commonly called as blue or green mouthful, Hai Hong, have the title of " marine egg ", its protein contains 8 kinds of indispensable amino acids such as the α-amino-isovaleric acid, leucine of human body needs, and content is far above egg, chicken, duck, fish, shrimp and meat.
The good protein groups of mussel becomes the mussel deep development provides assurance, extracts treatment rheumatic arthritis and osteoarticular medicine such as New Zealand from mussel, and Russia produces a kind of anti-atherosclerotic food with mussel specially.But China mussel substantially rests on band shell marketing fresh, is processed into various frozen product, fried food product and mussel wet goods aspect, and the development and use of intensive processing product are few.Therefore, with other large sea-food Comparatively speaking, the processing and utilization rate of mussel is extremely low, has caused the significant wastage of high nutrition mussel resource, thereby has limited the sustained and rapid development of China's mussel industry.Therefore, Efficient Development utilizes the mussel protein resource to have wide market outlook and important social effect.
In recent years, the Some Domestic scholar is studied the preparation technology of mussel enzymolysis polypeptide.As Chinese patent CN102020709A " a kind of Mytilus coruscus antibacterial peptide and preparation method and purposes " namely studied from Mytilus crassitesta Lischke prepare the method for antibacterial peptide and feed as the application of additive; Chinese patent CN 101991840A " preparation method and the application of anti-prostate cancer mussel enzymatic hydrolyzed extract " has namely studied method and the application thereof that utilizes Alcalase to prepare anti-prostate cancer mussel enzymatic hydrolyzed extract; Chinese patent CN 101991840A " the enzymolysis mussel protein prepares the method for blood pressure lowering peptide " has namely studied a kind of method of utilizing Sumizyme MP enzymolysis mussel protein to prepare blood pressure lowering peptide.The polypeptide that above method utilizes the enzymolysis process preparation to have given efficacy, but because single enzyme enzymolysis relative efficiency is lower, thereby the polypeptide yield with given efficacy is extremely low, is difficult to reach the industrialization demand.In addition, Chinese patent CN 101991840A " preparation method of mussel active polypeptide " has namely studied and has utilized the vacuum ultrasonic reactor to prepare the method for mussel active polypeptide under a kind of acidic conditions, although it is simple that the method has technique, be suitable for the advantage of suitability for industrialized production, but the gained enzymolysis product is except containing polypeptide, still containing the many kinds of substances such as polysaccharide, is the mixture of Multiple components, and the gained prepared product can not be directly used in the high purposes of purity requirement.
The applicant finds that at retrieving China is take mussel as raw material, the technical study of utilizing zymolysis technique to prepare the immuno-potentiation polypeptide is in the blank stage, for practicable enzymolysis process report not still, and have no especially report as preparation technology and the prescription that raw material prepares the immunity enhancement type polypeptide piece.
Summary of the invention
First technical problem to be solved by this invention provides a kind of preparation method of immunostimulant mussel enzymolysis polypeptide, take mussel as raw material, and aboundresources, simultaneously preparation technology's mucin peptide purity simple, that make is high and have a higher activity.
First technical problem to be solved by this invention provides a kind of preparation method of mussel polypeptide piece, be prepared take the mussel protein mucin peptide as raw material, the tablet that makes can be used as the elderly and valetudinarian's daily health caring product, also can be used as the medical food of postoperative patient.
The present invention solves the technical scheme that above-mentioned first technical problem adopts: a kind of preparation method of immunostimulant mussel enzymolysis polypeptide is characterized in that may further comprise the steps:
1) with mussel dry powder as raw material, with raw material and pH be 9.5～10.5 glycine-NaOH damping fluid according to the weightmeasurement ratio of 1g: 20ml～1g: 25ml mix, homogenate, then regulate pH value to 9.5～10, get mixed solution;
2) mixeding liquid temperature is risen to 50～55 ℃ and stir preheating 15～20 minutes, add respectively Sumizyme MP and neutral protease by 1.0%～1.5% of raw materials quality, hydrolysis temperature is 50～55 ℃, enzymolysis time 2～4h;
3) enzymolysis product of gained is processed through the enzyme that goes out first, is got enzymolysis solution, again with enzymolysis solution successively through desalination, concentrated and spraying drying, obtain immunostimulant mussel enzymolysis polypeptide.
As preferably, described step 1) mussel in is Mytilus edulis, Mytilus crassitesta Lischke or Perna uiridis (Linnaeus).
The pH of the damping fluid as preferably, described step 1) is 10.
The enzyme activity of the Sumizyme MP preferred again, described step 2) 〉=1.9 * 10
4U/g, the enzyme activity of neutral protease 〉=4.9 * 10
4U/g/g.
At last, described step 3) enzyme that goes out in is treated to: with step 2) enzymolysis product of gained is warming up to 95～100 ℃, and keep 10～15min in this temperature, and be cooled to again 0～10 ℃ to stop enzyme reaction, then centrifugal, get enzymolysis solution;
Described step 3) desalination in, concentrated and spray-dired detailed process are: enzymolysis solution is made the solution of concentration 10mg/ml～20mg/ml, carried out the macroporous resin desalination; Enzymolysis solution after the desalination carries out vacuum concentration under≤40 ℃ low temperature, the proportion that obtains concentrated enzymolysis solution is 1.05～1.15g/cm
3Concentrated enzymolysis solution is to carry out spraying drying under 160～200 ℃ carrying out temperature, and temperature out is 65～110 ℃.
The mussel enzymolysis polypeptide that obtains shows through the SEC-HPLC test: the enzymolysis product molecular-weight average is 385.9kDa, and (80～2500u) content are 90.89% to molecular weight.
The present invention solves the technical scheme that above-mentioned second technical problem adopt: a kind of preparation method of immunostimulant mussel enzymolysis polypeptide tablet is characterized in that may further comprise the steps:
1) with mussel dry powder as raw material, adopt above-mentioned zymolysis technique to prepare immunostimulant mussel enzymolysis polypeptide;
2) enzymolysis polypeptide and the mixing diluents that makes is even, cross 60～80 mesh sieves, add an amount of ethanol, mixture is made softwood, cross 20～24 mesh sieves, the granulation shape, again air seasoning under 50～60 ℃ of conditions after 18～22 mesh sieves, obtains the film-making particle;
3) add an amount of disintegrating agent and lubricant in gained film-making particle, mix, be pressed into tablet at tabletting machine at last, film coating namely gets immunity enhancement type mussel polypeptide piece.
As preferably, described thinner is microcrystalline cellulose and starch, and described enzymolysis polypeptide and microcrystalline cellulose and starch mix according to mass ratio 1: 0.8～1: 0.3～0.6.
Preferred again, described disintegrating agent is dry starch, and described lubricant is talcum powder; The amount of used dry starch is 2.5%～3.5% of film-making particle weight, and used talcous amount is 1%～2% of film-making particle weight.
Compared with prior art, the invention has the advantages that:
1, craft science of the present invention is reasonable, simple to operate, select Sumizyme MP and neutral protease as the enzymolysis enzyme, merge simultaneously the means such as macroporous resin desalination and spraying drying is prepared by biologic enzymolysis method, enzymolysis process is easily monitored, mucin peptide is farthest discharged, improve the utilization ratio of raw material, the mucin peptide that makes simultaneously and tablet thereof have higher activity;
2, the raw material that adopts of the present invention is mussel, aboundresources, and cost is lower, and added value that simultaneously can the Effective Raise mussel has great importance to the Sustainable development of mussel industry;
3, the prepared mucin peptide of the present invention is that mussel protein makes through enzymic hydrolysis, have no side effect safely,, and immuno-potentiation is remarkable, the elderly, valetudinarian and postoperative patient had good health-care effect, mucin peptide can be used as medicine, protective foods, food, foodstuff additive etc., mucin peptide tablet of the present invention can be used as the elderly and valetudinarian's daily health caring product, also can be used as the medical food of postoperative patient.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
A kind of preparation technology and method for preparing tablet thereof thereof of immunostimulant mussel enzymolysis polypeptide, preparation technology's flow process is as follows: mussel dry powder → enzymolysis → macroporous resin desalination → spraying drying → mucin peptide → tablet.
Concrete steps are:
1, take by weighing mussel degreasing dry powder 5.0g, add pH and be 10 glycine-NaOH damping fluid 125mL, homogeneous 5min in tissue mashing machine is 9.5 with 1mol/L HCl with this miscellany adjust pH, gets mixed liquid.
2, mixeding liquid temperature is risen to 55 ℃ and stir preheating, and under whipped state, be incubated 20min; Then add 0.075g Sumizyme MP (enzyme activity 〉=1.9 * 10
4U/g) and 0.050g neutral protease (enzyme activity 〉=4.9 * 10
4U/g) start enzyme digestion reaction, hydrolysis temperature is 53～55 ℃, continuously stirring, and temperature and the pH constant (temperature is 53～55 ℃, and pH is 9.5) of maintenance reaction system, enzymolysis time is 3h.Get enzymolysis product.
3, with step 2) enzymolysis product of gained processes through the enzyme that goes out first, gets enzymolysis solution, and described enzymolysis solution through macroporous resin desalination, concentrated and spraying drying, gets mucin peptide successively.Concrete experiment is:
1. enzyme goes out: with the enzymolysis product enzyme 10min that goes out in 95 ℃ of water-baths, then be cooled to 10 ℃; Thereby the termination enzyme reaction, then centrifugal 15min under 5000r/min gets supernatant liquor as enzymolysis solution.
2. D101 macroporous resin desalination: it is 10mg/ml solution that enzymolysis solution is made mass concentration, join D101 macroporous resin chromatography column, to wash except salt and impurity in the enzymolysis solution, then zymolyte desorb from the D101 macroporous resin is got off with the flow velocity of 2.0BV/h with 70% ethanol, low temperature (≤40 ℃) vacuum concentration gets the enzymolysis solution concentrated solution.
3. spraying drying: enzymolysis solution proportion is 1.1g/cm during spraying drying
3, to carry out temperature and should be 175～180 ℃, temperature out should be 75～80 ℃.
The mussel enzymolysis polypeptide of gained shows through the SEC-HPLC test: the enzymolysis product molecular-weight average is 385.9kDa, and (80～2500u) content are 90.89% to molecular weight.
The mussel enzymolysis polypeptide that makes is done the immuno-potentiation experiment (on the impact of mouse lymphocyte multiplication capacity, on the impact of mice serum hemolysin and the impact of mouse carbon being cleaned up ability) of mouse body, measuring method is with reference to Xu Zenglai, Wang Qiong, Zhao Meng etc. " Study immune regulation of Huai Chinese yam polysaccharose " [being recorded in " the precious traditional Chinese medical science traditional Chinese medicines of generation ", the 5th phase in 2007 (the 18th volume): 1040-041 page or leaf]
Experimental result shows: the mussel enzymolysis polypeptide can significantly strengthen the mouse lymphocyte multiplication capacity, promote mouse antibodies to generate, strengthen mouse carbon cleans up ability, that is the mussel enzymolysis polypeptide all has certain enhancement to humoral immunization, cellular immune function, the non-specific immune function of mouse.
Table 1 mussel enzymolysis polypeptide is on the impact (n=3) of mouse lymphocyte multiplication capacity
Table 2 mussel enzymolysis polypeptide is on the impact (n=3) of mice serum hemolysin
Table 3 mussel enzymolysis polypeptide is cleaned up the impact (n=3) of ability on mouse carbon
4, enzymolysis polypeptide and microcrystalline cellulose and starch were mixed with 1: 0.9: 0.5, cross 60 mesh sieves, add an amount of ethanol, mixture is made softwood, cross 22 mesh sieves, the granulation shape, again air seasoning under 55 ℃ of conditions after 20 mesh sieves, obtains the film-making particle.
5, the film-making particle is added weight and account for the starch of film-making particle 3.0% and 1.5% talcum powder, mix, be pressed into tablet at tabletting machine, film coating namely gets immunity enhancement type mussel polypeptide piece.
Mucin peptide of the present invention can be used as medicine, protective foods, food, foodstuff additive etc., and craft science is reasonable, and is simple to operate, has stronger industrial implementation.Mucin peptide tablet of the present invention can be used as the elderly and valetudinarian's daily health caring product, also can be used as the medical food of postoperative patient.
The actual usage of the mucin peptide sheet that adopts the inventive method preparation and get is: oral, and every day 3 times, each 4～6.
At last, still should be noted, what more than enumerate only is a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (9)
1. the preparation method of an immunostimulant mussel enzymolysis polypeptide is characterized in that may further comprise the steps:
1) with mussel dry powder as raw material, with raw material and pH be 9.5～10.5 glycine-NaOH damping fluid according to the weightmeasurement ratio of 1g: 20ml～1g: 25ml mix, homogenate, then regulate pH value to 9.5～10, get mixed solution;
2) mixeding liquid temperature is risen to 50～55 ℃ and stir preheating 15～20 minutes, add respectively Sumizyme MP and neutral protease by 1.0%～1.5% of raw materials quality, hydrolysis temperature is 50～55 ℃, enzymolysis time 2～4h;
3) enzymolysis product of gained is processed through the enzyme that goes out first, is got enzymolysis solution, again with enzymolysis solution successively through desalination, concentrated and spraying drying, obtain immunostimulant mussel enzymolysis polypeptide.
2. preparation method according to claim 1 is characterized in that described step 1) in mussel be Mytilus edulis, Mytilus crassitesta Lischke or Perna uiridis (Linnaeus).
3. preparation method according to claim 1 is characterized in that described step 1) in the pH of damping fluid be 10.
4. preparation method according to claim 1 is characterized in that described step 2) in the enzyme activity 〉=1.9 * 10 of Sumizyme MP
4U/g, the enzyme activity of neutral protease 〉=4.9 * 10
4U/g/g.
5. preparation method according to claim 1, it is characterized in that described step 3) in the enzyme that goes out be treated to: with step 2) enzymolysis product of gained is warming up to 95～100 ℃, and keeps 10～15min in this temperature, is cooled to 0～10 ℃ to stop enzyme reaction again, then centrifugal, get enzymolysis solution;
Described step 3) desalination in, concentrated and spray-dired detailed process are: enzymolysis solution is made the solution of concentration 10mg/ml～20mg/ml, carried out the macroporous resin desalination; Enzymolysis solution after the desalination carries out vacuum concentration under≤40 ℃ low temperature, the proportion that obtains concentrated enzymolysis solution is 1.05～1.15g/cm
3Concentrated enzymolysis solution is to carry out spraying drying under 160～200 ℃ carrying out temperature, and temperature out is 65～110 ℃.
6. preparation method according to claim 1 is characterized in that described step 3) in the enzymolysis product molecular-weight average of mussel enzymolysis polypeptide of gained be 385.9Da, the content of molecular weight 80～2500Da is 90.89%.
7. the preparation method of an immunostimulant mussel enzymolysis polypeptide tablet is characterized in that may further comprise the steps:
1) with mussel dry powder as raw material, adopt that arbitrary zymolysis technique method prepares immunostimulant mussel enzymolysis polypeptide in the right 1～5;
2) enzymolysis polypeptide and the mixing diluents that makes is even, cross 60～80 mesh sieves, add an amount of ethanol, mixture is made softwood, cross 20～24 mesh sieves, the granulation shape, again air seasoning under 50～60 ℃ of conditions after 18～22 mesh sieves, obtains the film-making particle;
3) add an amount of disintegrating agent and lubricant in gained film-making particle, mix, be pressed into tablet at tabletting machine at last, film coating namely gets immunity enhancement type mussel polypeptide piece.
8. preparation method according to claim 7 is characterized in that described thinner is microcrystalline cellulose and starch, and described enzymolysis polypeptide and microcrystalline cellulose and starch mix according to mass ratio 1: 0.8～1: 0.3～0.6.
9. preparation method according to claim 7 is characterized in that described disintegrating agent is dry starch, and described lubricant is talcum powder; The amount of used dry starch is 2.5%～3.5% of film-making particle weight, and used talcous amount is 1%～2% of film-making particle weight.
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| CN107573405B (en) * | 2017-08-09 | 2020-08-28 | 山东欣悦肽生物技术有限公司 | Mussel polypeptide extract and preparation method thereof |
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| CN115246871A (en) * | 2021-12-27 | 2022-10-28 | 浙江海洋大学 | Preparation method of mytilus coruscus immunocompetence hexapeptide |
| CN115246871B (en) * | 2021-12-27 | 2024-02-27 | 浙江海洋大学 | Preparation method of thick-shell mussel immunocompetent hexapeptide |
| CN114287630A (en) * | 2021-12-30 | 2022-04-08 | 湖北鑫祥顺生物工程有限公司 | Immunoregulation preparation and preparation method thereof |
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Effective date of registration: 20230531 Address after: Room 1869, 18th Floor, Block B, Cross border E-commerce Building, Huguang Road, Shushan District, Hefei City, Anhui Province, 230031 Patentee after: Hefei little hedgehog Information Technology Co.,Ltd. Address before: No. 127 Datong Road, Zhujiajian Street, Putuo District, Zhoushan City, Zhejiang Province Patentee before: ZHEJIANG OCEAN University Effective date of registration: 20230531 Address after: 405-2, 4th Floor, Building 2, Zhongmeng Science and Technology Park, No. 5 Binhe Road, Nanning City, Guangxi Zhuang Autonomous Region, 530000 Patentee after: Guangxi Zhongbao Zerui Biotechnology Co.,Ltd. Address before: Room 1869, 18th Floor, Block B, Cross border E-commerce Building, Huguang Road, Shushan District, Hefei City, Anhui Province, 230031 Patentee before: Hefei little hedgehog Information Technology Co.,Ltd. |
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