阅读说明：本技术 一种海地瓜生物活性肽的制备方法 (Preparation method of acaudina molpadioides biological active peptide ) 是由 徐仰丽 苏来金 杨会成 胡世伟 于 2019-12-30 设计创作，主要内容包括：本发明公开了一种海地瓜生物活性肽的制备方法,包括下列步骤：(1)将海地瓜打浆；(2)微生物驯养；(3)微生物发酵降解；(4)酶解；(5)然后离心取上清液,得活性肽溶液。本发明利用海地瓜开展进一步的综合利用,有助于减少污染,提升副产物附加值,促进养殖增收增效。本发明在制备工艺中,在此前的研究基础上,改进工艺步骤,缩短工艺时间,将海地瓜中的胶原蛋白变成多肽,进一步提高海地瓜中多肽的提取率,制备的多肽中生物活性更高的小分子量的多肽含量大大提高,分子量小于1KDa的短肽占比为83.17%。(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.)

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.

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