阅读说明：本技术 一种提高海参补肾壮阳功效的方法 (Method for improving kidney-tonifying and yang-strengthening effects of sea cucumber ) 是由 王群 马迪 于文静 贾盼盼 于 2019-10-10 设计创作，主要内容包括：本发明公开了一种提高海参补肾壮阳功效的方法,属于海洋生物技术领域。以海参深加工废弃物海参内脏为原料,集成现代生物分离技术、生物酶解技术、混菌微生物发酵技术,研制开发出显著补肾壮阳功效的海参多肽生物制品,其中混菌好氧发酵菌种主要包括富硒酿酒酵母、枯草芽孢杆菌和地衣芽孢杆菌。所得海参多肽具有较强的抗氧化能力,提取液可根据应用途径做浓缩、吸附干燥、醇析或树脂纯化等处理,满足在保健、食品、医药等领域的高效利用。该工艺技术简单高效、所得产品抗氧化能力和补肾壮阳功效强,产品稳定性强,生产成本低,易于工业化规模生产。(The invention discloses a method for improving kidney-tonifying and yang-strengthening effects of sea cucumbers, and belongs to the technical field of marine organisms. The sea cucumber polypeptide biological product with the obvious kidney tonifying and yang strengthening effects is developed by taking sea cucumber deep processing waste sea cucumber viscera as raw materials and integrating the modern biological separation technology, the biological enzymolysis technology and the mixed bacteria microbial fermentation technology, wherein the mixed bacteria aerobic fermentation strain mainly comprises selenium-enriched saccharomyces cerevisiae, bacillus subtilis and bacillus licheniformis. The obtained sea cucumber polypeptide has strong oxidation resistance, and the extract can be subjected to concentration, adsorption drying, alcohol precipitation or resin purification and other treatments according to application approaches, so that the high-efficiency utilization in the fields of health care, food, medicine and the like is met. The process is simple and efficient, the obtained product has strong oxidation resistance, strong kidney-tonifying and yang-strengthening effects, strong product stability and low production cost, and is easy for industrial mass production.)

1. A method for improving the kidney-tonifying and yang-strengthening effects of sea cucumbers is characterized by comprising the following steps:

(1) cleaning: cleaning the processed fresh sea cucumber viscera with tap water, cleaning with natural mineral water or purified water, and draining;

(2) homogenizing: adding the sea cucumber viscera obtained in the step (1) into a high-speed homogenizer, adding natural mineral water or purified water, and performing high-speed homogenization until all the sea cucumber viscera can pass through a round sieve with the aperture of 2 mm;

(3) and (3) sterilization: sterilizing the homogenized internal organs of the sea cucumber by a high-pressure steamer, and cooling for later use;

(4) compound enzymolysis: adding a complex enzyme preparation with the mass ratio of 2% into the sterilized slurry, and stirring and performing enzymolysis for 30min at the temperature of 40-60 ℃;

(5) aerobic fermentation: adding a compound microbial agent with the mass ratio of 3% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and carrying out aerobic fermentation for 8h at the temperature of 28-35 ℃;

(6) anaerobic fermentation: after the aerobic fermentation is finished, 2% bifidobacterium bifidum powder is added, and anaerobic fermentation is carried out for 12 hours at the temperature of 28-35 ℃;

(7) and (3) filtering: performing coarse filtration to remove fermentation thalli;

(8) enzyme deactivation: inactivating enzyme at 100 deg.C for 10 min;

(9) centrifuging: centrifuging at 3000r/min to obtain supernatant as sea cucumber polypeptide extractive solution.

2. The method for improving kidney-tonifying and yang-strengthening effects of sea cucumbers according to claim 1, wherein 5 mass percent of alginate oligosaccharides of sea cucumbers viscera is added in the homogenization process of the step (2).

3. The method as claimed in claim 2, wherein the molecular weight of the alginate oligosaccharide is 3000-5000 Da.

4. The method for improving the kidney-tonifying and yang-strengthening effects of the sea cucumber according to claim 1, wherein the compound protease in the step (4) is a combination of trypsin, flavourzyme and papain, and the mass ratio of the flavourzyme to the papain is 2:2: 1.

5. The method for improving kidney-tonifying and yang-strengthening effects of sea cucumbers according to claim 1, wherein the compound microbial agent in the step (5) is a mixed microbial agent of selenium-enriched saccharomyces cerevisiae, bacillus subtilis and bacillus licheniformis, and the mass ratio of the compound microbial agent to the bacillus subtilis to the bacillus licheniformis is 2: 3: 1.

6. the method for improving kidney-tonifying and yang-strengthening effects of sea cucumber according to claim 1, wherein the aerobic fermentation process in the step (5) is performedThe ventilation rate is 0.2-0.3 m when the fermentation is carried out in a fermentation tank³/m³.min。

7. The method for improving the kidney-tonifying and yang-strengthening effects of the sea cucumber according to claim 1, wherein the preparation method of the selenium-enriched saccharomyces cerevisiae seed solution comprises the following steps: under the aseptic operation condition, picking two-ring strains from a yeast YDP inclined plane by using an inoculating ring, and inoculating a saccharomyces cerevisiae activation culture medium, wherein the glucose concentration of the culture medium is 5 percent, and the yeast extract concentration is 1 percent; culturing at 28-32 deg.C for 24 hr, adding 10g/L sodium selenite into culture medium respectively at 6 hr, 12 hr and 18 hr to prepare yeast seed liquid with effective viable count of more than 10⁹cfu/mL。

8. The method for improving kidney-tonifying and yang-strengthening effects of sea cucumbers according to claim 1, wherein the preparation method of the bacillus subtilis and bacillus licheniformis seed solution comprises the following steps: under the aseptic condition, LB culture medium is adopted to culture for 24 hours at the temperature of 28-32 ℃ to prepare liquid bacillus subtilis and bacillus licheniformis seed liquid, and the effective viable count of the seed liquid is required to be more than or equal to 10⁹cfu/mL。

9. A sea cucumber polypeptide biological product prepared by the process of claims 1-8.

10. The sea cucumber polypeptide biological product of claim 9, which is applied to health care, food and medicine.

Technical Field

The invention relates to a method for improving kidney-tonifying and yang-strengthening effects of sea cucumbers, in particular to a sea cucumber polypeptide biological product which is prepared by taking sea cucumber viscera as raw materials and performing microbial synergistic fermentation and has remarkable kidney-tonifying and yang-strengthening effects, belonging to the technical field of marine organisms.

Background

Sea cucumber is an important marine invertebrate, has tender meat quality and rich nutrition, is a typical high-protein low-fat food, has extremely high nutritional value and medicinal value, is a dietary therapy product for patients with hypertension, coronary heart disease, hepatitis and the like and the old, can enhance the immunity and hematopoietic function of organisms, and can inhibit the growth and transfer of various moulds and human cancer cells and the like. As early as one thousand years ago, China has a habit of eating sea cucumbers, and the sea cucumbers are regarded as precious nourishments and are listed as the head of 'eight delicacies in sea'. The 'herbal medicine records that the sea cucumber has the efficacy of' sweet, salty, warm, kidney tonifying, essence boosting, yang strengthening and impotence treating 'in the' herbal medicine compendium supplement, records 'sea cucumber sexual warmth supplement, foot enemy ginseng is named as sea cucumber for reasons'; sweet and salty in taste, tonifying kidney channel, replenishing essence, eliminating phlegm, arresting urination, tonifying yang, treating flaccidity, and killing sore and worm.

At present, the main edible part of the sea cucumber is the sea cucumber body wall, so the research on the sea cucumber is focused on the active substances on the sea cucumber body wall. The sea cucumber viscera which are byproducts in the sea cucumber processing are mostly discarded, the utilization rate is low, the research on the sea cucumber viscera at home and abroad is less, and the developed related products are very little. However, the viscera of the sea cucumber not only contain chemical components similar to the body wall, but also are rich in taurine, arginine, phospholipid, unsaturated fatty acid, various high-activity trace elements, pigments, active enzymes and functional bacteria, and the edible value and the medicinal value of the viscera of the sea cucumber are higher than those of the body wall of the sea cucumber within a certain range. At present, the lack and neglect of the knowledge of the active substances in the internal organs of the sea cucumbers cause serious waste and loss of resources.

Disclosure of Invention

In order to overcome the defects of the prior art and meet the requirements of high-efficiency extraction and high-value utilization of sea cucumber processing waste, the application provides a method for improving the kidney-tonifying and yang-strengthening effects of the sea cucumber, integrates a modern bioseparation technology, a biological enzymolysis technology and a mixed bacteria microbial fermentation technology, develops and develops a sea cucumber polypeptide biological product with remarkable kidney-tonifying and yang-strengthening effects, meets the application requirements of fields such as health care, medical treatment, food and the like on the sea cucumber polypeptide, has a simple and high-efficiency fermentation process, has remarkable kidney-tonifying and yang-strengthening health-care effects, is low in production cost, and is easy for industrial mass production.

The invention realizes the technical effects through the following technical scheme:

a method for improving the kidney-tonifying and yang-strengthening efficacy of sea cucumber is characterized in that the preparation method comprises the following steps:

(1) cleaning: cleaning the processed fresh sea cucumber viscera with tap water, cleaning with natural mineral water or purified water, and draining;

(2) homogenizing: adding the sea cucumber viscera obtained in the step (1) into a high-speed homogenizer, adding natural mineral water or purified water, and performing high-speed homogenization until all the sea cucumber viscera can pass through a round sieve with the aperture of 2 mm;

(3) and (3) sterilization: sterilizing the homogenized internal organs of the sea cucumber by a high-pressure steamer, and cooling for later use;

(4) compound enzymolysis: adding a complex enzyme preparation with the mass ratio of 2% into the sterilized slurry, and stirring and performing enzymolysis for 30min at the temperature of 40-60 ℃;

(5) aerobic fermentation: adding a compound microbial agent with the mass ratio of 3% of the enzymolysis liquid into the enzymolysis liquid after enzymolysis, and carrying out aerobic fermentation for 8h at the temperature of 28-35 ℃;

(6) anaerobic fermentation: after the aerobic fermentation is finished, 2% bifidobacterium bifidum powder is added, and anaerobic fermentation is carried out for 12 hours at the temperature of 28-35 ℃;

(7) and (3) filtering: performing coarse filtration to remove fermentation thalli;

(8) enzyme deactivation: inactivating enzyme at 100 deg.C for 10 min;

(9) centrifuging: centrifuging at 3000r/min to obtain supernatant as sea cucumber polypeptide extractive solution.

Preferably, the brown algae oligosaccharide with the mass ratio of 5 percent of the viscera of the sea cucumber is added in the process of homogenizing the viscera of the sea cucumber.

Further preferably, the molecular weight of the brown alginate oligosaccharide is 3000-5000 Da.

The compound protease in the step (4) is a combination of trypsin, flavourzyme and papain, and the mass ratio of the flavourzyme to the papain is 2:2: 1.

The compound microbial agent in the step (5) is a mixed microbial agent of selenium-rich saccharomyces cerevisiae, bacillus subtilis and bacillus licheniformis, and the mass ratio of the compound microbial agent to the bacillus subtilis to the bacillus licheniformis is 2: 3: 1.

the ventilation rate is 0.2-0.3 m when the aerobic fermentation process in the step (5) is carried out in a fermentation tank³/m³.min。

The preparation method of the selenium-rich saccharomyces cerevisiae seed liquid comprises the following steps: under aseptic operation conditions, two-ring strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, and a saccharomyces cerevisiae activation culture medium is inoculated, wherein the glucose concentration of the culture medium is 5 percent, and the yeast isThe concentration of the paste is 1%; culturing at 28-32 deg.C for 24 hr, adding 10g/L sodium selenite into culture medium respectively at 6 hr, 12 hr and 18 hr to prepare yeast seed liquid with effective viable count of more than 10⁹cfu/mL。

The preparation method of the bacillus subtilis and bacillus licheniformis seed solution comprises the following steps: under the aseptic condition, LB culture medium is adopted to culture for 24 hours at the temperature of 28-32 ℃ to prepare liquid bacillus subtilis and bacillus licheniformis seed liquid, and the effective viable count of the seed liquid is required to be more than or equal to 10⁹cfu/mL。

The invention provides a method for improving kidney-tonifying and yang-strengthening effects of sea cucumber, the obtained sea cucumber polypeptide has strong kidney-tonifying and yang-strengthening effects, and the extract can be subjected to concentration, adsorption drying, alcohol precipitation or resin purification and other treatments according to application ways, so that the efficient utilization in the fields of health care, food, medicine and the like is met.

The invention provides a method for improving the kidney-tonifying and yang-strengthening efficacy of sea cucumber, which has the following remarkable advantages compared with the prior art:

(1) the sea cucumber visceral protein is used as a raw material, and the modern bioseparation technology, the biological enzymolysis technology and the mixed bacteria microbial fermentation technology are integrated, so that the oxidation resistance of the sea cucumber visceral protein is remarkably improved; more importantly, the application creatively discovers that the kidney tonifying and yang strengthening effects of the sea cucumber polypeptide prepared by the enzymolysis and fermentation method can be obviously improved by adding the brown algae oligosaccharide in a certain proportion into the sea cucumber viscera dissolving solution;

(2) the application utilizes aerobic bacteria (selenium-enriched saccharomyces cerevisiae, bacillus subtilis and bacillus licheniformis) to jointly ferment the viscera proteins of the sea cucumber to extract the sea cucumber polypeptide, makes full use of the synergistic effect among microorganisms, improves the extraction rate of the sea cucumber polypeptide and the health care effects of tonifying kidney and strengthening yang, wherein: the selenium-rich saccharomyces cerevisiae can generate various enzymes for degrading cell walls in the fermentation process, so that more functional active substances such as polypeptide and the like are released from the cell walls, and the yeast is an enzyme capable of releasing combined state phenolic acid, so that the kidney-tonifying and yang-strengthening effects of the sea cucumber polypeptide can be improved; the bacillus subtilis can kill pathogenic bacteria in fermentation liquor in the high-temperature fermentation process, has strong abilities of producing amylase, protease and the like, and can degrade macromolecular starch and protein substances into microorganisms for utilization, thereby improving the oxidation resistance and the nutritional and health-care effects of active peptide; the addition of the bacillus licheniformis can obviously improve the stability of the sea cucumber polypeptide and the health-care effects of tonifying kidney and strengthening yang;

(3) the method adopts bifidobacterium bifidum to carry out anaerobic fermentation, improves the stability of the sea cucumber polypeptide, and test results show that the temperature and the time have little influence on the inoxidizability of the sea cucumber visceral hydrolyzed peptide within the range of 20-80 ℃, the inoxidizability begins to reduce along with the increase of the temperature and the time when the temperature exceeds 100 ℃, and the stability is higher than the technical level reported at present;

(4) by adopting liquid fermentation, the growth rate of the thalli is improved, the fermentation time is shortened, the production efficiency is improved, the fermentation process conditions are mild, the production cost is low, the large-scale production is easy, and the method plays an important role in improving the industrial economic benefit and large-scale popularization and application.

Detailed Description

In the application, the activation method of the selenium-rich saccharomyces cerevisiae, the bacillus subtilis and the bacillus licheniformis comprises the following steps:

(1) the preparation method of the selenium-rich saccharomyces cerevisiae seed liquid comprises the following steps: under the aseptic operation condition, picking two-ring strains from a yeast YDP inclined plane by using an inoculating ring, and inoculating a saccharomyces cerevisiae activation culture medium, wherein the glucose concentration of the culture medium is 5 percent, and the yeast extract concentration is 1 percent; culturing at 28-32 deg.C for 24 hr, adding 10g/L sodium selenite into culture medium respectively at 6 hr, 12 hr and 18 hr to prepare yeast seed liquid with effective viable count of more than 10⁹cfu/mL；

(2) The preparation method of the bacillus subtilis and bacillus licheniformis seed solution comprises the following steps: under the aseptic condition, LB culture medium is adopted to culture for 24 hours at the temperature of 28-32 ℃ to prepare liquid bacillus subtilis and bacillus licheniformis seed liquid, and the effective viable count of the seed liquid is required to be more than or equal to 10⁹cfu/mL。