CN110894230A - Preparation method of acaudina molpadioides biological active peptide - Google Patents
Preparation method of acaudina molpadioides biological active peptide Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 54
- 241000145256 Acaudina molpadioides Species 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 34
- 229920001184 polypeptide Polymers 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 20
- 230000000813 microbial effect Effects 0.000 claims abstract description 13
- 230000000975 bioactive effect Effects 0.000 claims abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 8
- 230000004151 fermentation Effects 0.000 claims abstract description 8
- 230000015556 catabolic process Effects 0.000 claims abstract description 7
- 238000006731 degradation reaction Methods 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 7
- 238000004537 pulping Methods 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 239000012530 fluid Substances 0.000 claims abstract description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 16
- 108091005804 Peptidases Proteins 0.000 claims description 10
- 239000004365 Protease Substances 0.000 claims description 10
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 10
- 239000002002 slurry Substances 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 102000005600 Cathepsins Human genes 0.000 claims description 6
- 108010084457 Cathepsins Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 102000016943 Muramidase Human genes 0.000 claims description 6
- 108010014251 Muramidase Proteins 0.000 claims description 6
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 6
- 102000004142 Trypsin Human genes 0.000 claims description 6
- 108090000631 Trypsin Proteins 0.000 claims description 6
- 229940088598 enzyme Drugs 0.000 claims description 6
- 239000004325 lysozyme Substances 0.000 claims description 6
- 229960000274 lysozyme Drugs 0.000 claims description 6
- 235000010335 lysozyme Nutrition 0.000 claims description 6
- 239000008213 purified water Substances 0.000 claims description 6
- 239000012588 trypsin Substances 0.000 claims description 6
- 241000193749 Bacillus coagulans Species 0.000 claims description 5
- 229940054340 bacillus coagulans Drugs 0.000 claims description 5
- 241000965254 Apostichopus japonicus Species 0.000 claims description 4
- 241000194108 Bacillus licheniformis Species 0.000 claims description 4
- 244000063299 Bacillus subtilis Species 0.000 claims description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 4
- 241000186016 Bifidobacterium bifidum Species 0.000 claims description 4
- 244000017020 Ipomoea batatas Species 0.000 claims description 4
- 235000002678 Ipomoea batatas Nutrition 0.000 claims description 4
- 241000186660 Lactobacillus Species 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 4
- 150000004676 glycans Chemical class 0.000 claims description 4
- 229940039696 lactobacillus Drugs 0.000 claims description 4
- 229920001282 polysaccharide Polymers 0.000 claims description 4
- 239000005017 polysaccharide Substances 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
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- 238000003756 stirring Methods 0.000 claims description 2
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- 238000000926 separation method Methods 0.000 claims 1
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- 238000011160 research Methods 0.000 abstract description 6
- 102000008186 Collagen Human genes 0.000 abstract description 4
- 108010035532 Collagen Proteins 0.000 abstract description 4
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- 229920001436 collagen Polymers 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 239000006227 byproduct Substances 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 241000251511 Holothuroidea Species 0.000 description 5
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- 230000002255 enzymatic effect Effects 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- FJPHHBGPPJXISY-KBPBESRZSA-N (2s)-2-[[(2s)-2-[[2-[(2-aminoacetyl)amino]acetyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CNC(=O)CN)CC1=CC=C(O)C=C1 FJPHHBGPPJXISY-KBPBESRZSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 241000145248 Acaudina Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000258152 Caudinidae Species 0.000 description 1
- 241000258164 Molpadiida Species 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 244000187976 Spiraea japonica Species 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 235000011869 dried fruits Nutrition 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 108010065713 glycyl-glycyl-tyrosyl-arginine Proteins 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 229940115088 sea soft Drugs 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 235000011649 selenium Nutrition 0.000 description 1
- 229940091258 selenium supplement Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
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- 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]
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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
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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
-
- 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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of acaudina molpadioides bioactive peptide, which comprises the following steps: (1) pulping acaudina molpadioides; (2) domesticating microorganisms; (3) microbial fermentation degradation; (4) carrying out enzymolysis; (5) then centrifuging and taking supernatant fluid to obtain active peptide solution. The invention utilizes the acaudina molpadioides to carry out further comprehensive utilization, is beneficial to reducing pollution, improving the added value of byproducts and promoting the efficiency increase of cultivation and income. In the preparation process, on the basis of the previous research, the process steps are improved, the process time is shortened, the collagen in the acaudina molpadioides is changed into the polypeptide, the extraction rate of the polypeptide in the acaudina molpadioides is further improved, the content of the polypeptide with high biological activity and small molecular weight in the prepared polypeptide is greatly improved, and the short peptide with the molecular weight less than 1KDa accounts for 83.17 percent.
Description
Technical Field
The invention relates to a preparation method of acaudina molpadioides bioactive peptide, belonging to the technical field of biology.
Background
Dried fruit of Japanese meadow sweet (Haidi sweet potato)Acaudina molpadioides Semper) Commonly known as Heidi Guajaponi, Heidi eggplant, Xiangshen, etc. (from the order of Colocasiales)Molpadida) Jirimi (family of nojirimaceae)Caudinidae) Hai Di Gua (a) and (b)Acaudina) The length of the animals can reach 400mm at most, generally 100-200mm, and the animals are mainly distributed in soft mud at the bottom of the sea from the tidal zone to the water depth of 80 m in the east sea area.
The resources of the east China sea area are very rich, the current storage amount is about 106 t, the annual capture amount is about 3000 t, the sea cucumber is frequently eaten as low-value sea cucumber by folks, and because the sea cucumber is hard after being heated and lives in shallow sea soft mud, a large amount of harmful heavy metals such as lead, cadmium, mercury and the like are accumulated on the body surface, the sea cucumber resources are not fully developed and utilized. Researches show that the acaudina molpadioides are rich in nutritional ingredients, contain rich protein, polysaccharide, lower fat and amino acid, have extremely high contents of calcium, zinc and iron elements, and also contain selenium, vitamin A, vitamin B6, vitamin E and the like, wherein the content of crude protein of the acaudina molpadioides body wall accounts for 89.16% of the dry weight of the acaudina molpadioides, and is higher than that of the crude protein of stichopus japonicus, and the main ingredient of the protein is collagen with activity. In recent years, the acaudina molpadioides polypeptide is proved to have good biological activity as the same as the sea cucumber polypeptide, and the extraction of the acaudina molpadioides collagen peptide with antioxidant activity is a good way for improving the additional value and developing new products.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of acaudina molpadioides biological active peptide, and the invention perfects and improves the process of acaudina molpadioides biological active peptide on the basis of the prior art CN108410934A to prepare polypeptide with smaller molecular weight and stronger biological activity.
The invention provides application of bacillus in preparation of marine-source bioactive polypeptides. In particular to the application of the biological active polypeptide of the acaudina molpadioides.
Further, the bacillus, the bifidobacterium and the lactic acid bacteria are applied to the extraction of the biological active polypeptide of the acaudina molpadioides together.
Further, the bacillus of the invention is selected from one of bacillus licheniformis, bacillus subtilis and bacillus coagulans. Bacillus is a species of the genus Bacillus, gram-positive, and a species of aerobic bacteria. The strain is nontoxic, can secrete various enzymes such as protease, and if the strain can be applied to the extraction of biological polypeptide, each flow is well controlled, the extraction efficiency is greatly superior to that of a commercial complex enzyme. On the basis of the previous research, the invention further perfects the extraction process, shortens the process steps, improves the extraction efficiency and improves and perfects the strains with the synergistic effect. The research of the invention finds that if only bacillus is used, the fermentation efficiency is not optimal, and in the previous report, aspergillus oryzae, saccharomycetes and the like are used, and the invention is changed into the combination of bifidobacterium and lactic acid bacteria, so that the efficiency of fermenting and decomposing the small molecular polypeptide is higher.
By utilizing the research, the invention provides a preparation method of acaudina molpadioides bioactive peptide, which comprises the following steps:
(1) pulping acaudina molpadioides, and adding purified water in the pulping process, wherein the weight ratio of the purified water to the acaudina molpadioides is 3-5:1, so as to prepare acaudina molpadioides pulp.
(2) Domesticating microorganisms: inoculating Bacillus, Bacillus bifidus, and lactobacillus into the pulp of Japanese sea cucumber, and culturing for 1-2 days to obtain microorganism pulp.
(3) And (3) microbial fermentation degradation: with food grade NaHCO3Adjusting the pH value of the acaudina molpadioides pulp obtained in the step (1) to be alkalescent 7.1-7.5; adding the microbial slurry obtained in the step (2), wherein the weight ratio of the microbial slurry to the sweet potato slurry is 1-5: 100; fully and evenly stirred, and fermented for 5-10 h.
(4) Enzymolysis: continuously adding the compound protease, wherein the adding amount of the enzyme is 4000-.
(5) Then centrifuging and taking supernatant fluid to obtain active peptide solution.
Further, a step of removing sugar is added between the step (3) and the step (4), and the steps are as follows: and (4) adding ethanol after microbial fermentation and degradation, performing polysaccharide precipitation, taking supernate, and performing step (4) by using the supernate. After the sugar removal operation, the enzymolysis effect by using the compound enzyme is better.
The compound protease in the step (4) is trypsin, cathepsin and lysozyme, and the adding amount ratio of the compound protease is 3:2: 1. Further, the enzymolysis in step (4) comprises the following specific steps: adding lysozyme, and maintaining the temperature at 30-37 deg.C for 1-3 h; then adding trypsin and cathepsin, heating to 37-40 ℃ and maintaining for 7-9 h.
Preferably, in the process of the invention. The bacillus is selected from Bacillus licheniformis, Bacillus subtilis, and Bacillus coagulans.
Further, after the active peptide solution is obtained in the step (5), the invention uses an ultrafiltration membrane to separate polypeptides with different molecular weights.
The invention has the beneficial effects that:
the invention utilizes the acaudina molpadioides to carry out further comprehensive utilization, is beneficial to reducing pollution, improving the added value of byproducts and promoting the efficiency increase of cultivation and income.
In the preparation process, on the basis of the previous research, the process steps are improved, the process time is shortened, the collagen in the acaudina molpadioides is changed into the polypeptide, the extraction rate of the polypeptide in the acaudina molpadioides is further improved, the content of the polypeptide with high biological activity and small molecular weight in the prepared polypeptide is greatly improved, and the short peptide with the molecular weight less than 1KDa accounts for 83.17 percent.
Detailed Description
Example 1
The preparation method of the acaudina molpadioides bioactive peptide provided by the embodiment comprises the following steps:
(1) pulping the acaudina molpadioides, and adding purified water in the pulping process, wherein the weight ratio of the purified water to the acaudina molpadioides is 5:1, so as to prepare the acaudina molpadioides pulp.
(2) Domesticating microorganisms: inoculating Bacillus, Bacillus bifidus, and lactobacillus into the pulp of Japanese sea cucumber, and culturing for 1d to obtain microorganism pulp.
(3) And (3) microbial fermentation degradation: with food grade NaHCO3Adjusting the pH value of the acaudina molpadioides pulp obtained in the step (1) to be alkalescent 7.1; adding the microbial slurry obtained in the step (2), wherein the weight ratio of the microbial slurry to the sweet potato slurry is 1: 100; stirring thoroughly, and fermenting for 5 hr.
(4) Enzymolysis: continuously adding the compound protease, wherein the adding amount of the protease is 4000U/g, the enzymolysis time is 12h, and after the enzymolysis is finished, raising the temperature to inactivate the enzyme.
(5) Then centrifuging and taking supernatant fluid to obtain active peptide solution.
A step of removing sugar is added between the step (3) and the step (4), and the method specifically comprises the following steps: and (4) adding ethanol after microbial fermentation and degradation, performing polysaccharide precipitation, taking supernate, and performing step (4) by using the supernate.
The compound protease in the step (4) is trypsin, cathepsin and lysozyme, and the adding amount ratio of the compound protease is 3:2: 1. The enzymolysis in the step (4) comprises the following specific steps: adding lysozyme, and maintaining the temperature at 30 ℃ for 3 h; then adding trypsin and cathepsin, and heating to 37 ℃ for 9 h.
In the method of this embodiment. The bacillus is selected from bacillus coagulans.
Example 2 determination of the polypeptide content in the enzymatic supernatant
The determination of the polypeptide content in the enzymatic hydrolysate is carried out according to the literature (Luwei, the Tan nationality spectrum, Songmei protein hydrolysate determination method [ J ]. food science, 2005,26(7): 169-. Standing for 10min, centrifuging at 2000r/min for 10min, taking supernatant after centrifugation, determining OD value in a 540nm spectrophotometer, drawing a standard curve with the peptide concentration as abscissa and the OD value as ordinate, and calculating the polypeptide content in the sample by a regression equation.
Yield of polypeptide in enzymolysis liquid
By applying the method, the polypeptide content before and after enzymolysis of the enzymolysis liquid is detected, and the yield of the polypeptide is calculated as follows:
polypeptide yield (%) = (polypeptide content measured after enzymolysis-polypeptide content before enzymolysis)/total protein content
In the invention, the regression equation of the Gly-Gly-Tyr-Arg standard sample is y =1.7893x +0.1187 (R2 = 0.9997), the linear relation is good, and the method can be used for detecting the polypeptide in the solution.
The method of the embodiment is used for determining the polypeptide yield in the embodiment 1, and the yield can reach 86.41%.
Example 3 distribution of polypeptides of different molecular weights in enzymatic supernatants
The molecular weight of peptides is usually determined by classical gel column chromatography, but this method is cumbersome and has a long cycle. The molecular weight distribution of the small molecular weight peptides is analyzed rapidly and simply by GEL exclusion chromatography in HPLC using TSK-GEL G2000SWXL as analytical column.
The molecular weight of the acaudina molpadioides bioactive peptide prepared in example 1 is relatively close, the concentration ratio is relatively high, the molecular weight is below 3000Da, wherein the molecular weight is less than 1000Da, and the total content is 83.17%.
Claims (10)
1. Application of bacillus in preparing marine bioactive polypeptide.
2. Application of bacillus in acaudina molpadioides bioactive polypeptide.
3. Application of Bacillus, Bacillus bifidus, and lactobacillus in bioactive polypeptide of acaudina molpadioides is provided.
4. Use according to claims 1-3, wherein the Bacillus is selected from the group consisting of Bacillus licheniformis, Bacillus subtilis and Bacillus coagulans.
5. A preparation method of acaudina molpadioides bioactive peptide is characterized by comprising the following steps:
(1) pulping acaudina molpadioides, and adding purified water in the pulping process, wherein the weight ratio of the purified water to the acaudina molpadioides is 3-5:1, so as to prepare acaudina molpadioides pulp;
(2) domesticating microorganisms: inoculating Bacillus, Bacillus bifidus, and lactobacillus into the pulp of Japanese sea cucumber, and culturing for 1-2 days to obtain microorganism pulp;
(3) and (3) microbial fermentation degradation: with food grade NaHCO3Adjusting the pH value of the acaudina molpadioides pulp obtained in the step (1) to be alkalescent 7.1-7.5; adding the microbial slurry obtained in the step (2), wherein the weight ratio of the microbial slurry to the sweet potato slurry is 1-5: 100; fully and uniformly stirring, and fermenting for 5-10 h;
(4) enzymolysis: continuously adding the compound protease, wherein the enzyme addition amount is 4000-;
(5) then centrifuging and taking supernatant fluid to obtain active peptide solution.
6. The preparation method according to claim 5, wherein a step of removing sugar is added between the step (3) and the step (4), and specifically comprises the following steps: and (4) adding ethanol after microbial fermentation and degradation, performing polysaccharide precipitation, taking supernate, and performing step (4) by using the supernate.
7. The method according to claim 5, wherein the complex protease of step (4) is trypsin, cathepsin and lysozyme, and the ratio of the amounts added is 3:2: 1.
8. The preparation method according to claim 7, wherein the enzymolysis in step (4) comprises the following steps: adding lysozyme, and maintaining the temperature at 30-37 deg.C for 1-3 h; then adding trypsin and cathepsin, heating to 37-40 ℃ and maintaining for 7-9 h.
9. The method according to claim 5, wherein the Bacillus is selected from the group consisting of Bacillus licheniformis, Bacillus subtilis, and Bacillus coagulans.
10. The method of claim 5, wherein after the step (5) of obtaining the active peptide solution, the separation of polypeptides having different molecular weights is performed by using an ultrafiltration membrane.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911392520.0A CN110894230A (en) | 2019-12-30 | 2019-12-30 | Preparation method of acaudina molpadioides biological active peptide |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1475159A (en) * | 2002-08-15 | 2004-02-18 | 烟台芝罘岛海洋生物科技有限公司 | Fermentation type sea cucumbus nutrient product and its preparation method |
| CN104651435A (en) * | 2015-02-16 | 2015-05-27 | 江南大学 | Method for preparing antihypertensive peptides by fermenting marine organisms through probiotics |
| CN108410934A (en) * | 2018-03-19 | 2018-08-17 | 温州科技职业学院 | A kind of preparation method of acaudina molpadioidea collagen oligopeptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1475159A (en) * | 2002-08-15 | 2004-02-18 | 烟台芝罘岛海洋生物科技有限公司 | Fermentation type sea cucumbus nutrient product and its preparation method |
| CN104651435A (en) * | 2015-02-16 | 2015-05-27 | 江南大学 | Method for preparing antihypertensive peptides by fermenting marine organisms through probiotics |
| CN108410934A (en) * | 2018-03-19 | 2018-08-17 | 温州科技职业学院 | A kind of preparation method of acaudina molpadioidea collagen oligopeptide |
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