阅读说明：本技术 一种海参皂甙的提取方法 (Method for extracting sea cucumber saponin ) 是由 梁志勇 王群 马迪 宋仁芳 于 2020-05-14 设计创作，主要内容包括：本发明公开了一种海参皂甙的提取方法,属于海洋生物技术领域。本发明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、乙醇沉淀和树脂吸附技术,研制开发出高体外抗氧化能力和抗癌功效的海参皂甙生物制品,其中混菌好氧发酵菌种主要包括酿酒酵母、枯草芽孢杆菌和冷解糖芽孢杆菌。所得海参皂甙具有较强的抗氧化能力和抗癌功能,光谱性强,可在保健、食品、医药等领域高效利用。该提取方法海参皂甙提取率高,能够实现海参加工废弃物的资源化再利用,便于工业化生产,市场前景广阔。(The invention discloses a method for extracting holothurian saponins, belonging to the technical field of marine organisms. The invention takes sea cucumber viscera which are wastes of deep processing of sea cucumber as raw materials, integrates the modern bioseparation technology, the biological enzymolysis technology, the mixed bacteria microbial fermentation technology, the ethanol precipitation and the resin adsorption technology, and develops a sea cucumber saponin biological product with high in-vitro oxidation resistance and anticancer efficacy, wherein the mixed bacteria aerobic fermentation strain mainly comprises saccharomyces cerevisiae, bacillus subtilis and bacillus saccharolyticus. The obtained sea cucumber saponin has strong oxidation resistance and anticancer function, strong spectrum, and can be used in fields of health promotion, food, medicine, etc. The extraction method has high extraction rate of holothurian saponin, can realize resource recycling of holothurian processing waste, is convenient for industrial production, and has wide market prospect.)

1. A method for extracting holothurian saponin 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% of lactobacillus 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 10000r/min to remove insoluble components;

(10) ethanol precipitation: adding ethanol into the centrifugate, and standing for more than 60 min;

(11) and (3) centrifugal concentration: centrifuging to remove precipitate, and concentrating under reduced pressure to obtain sea cucumber saponin extract.

2. The method for extracting holothurian saponins according to claim 1, characterized in that the complex enzyme preparation in step (4) is one or a mixture of more of protease, non-starch polysaccharidase and naringinase.

3. The method for extracting holothurian saponins according to claim 1, wherein the mass ratio of the protease, the non-starch polysaccharidase and the naringinase is 2: 1: 1.

4. the method for extracting sea cucumber saponins according to claim 1, wherein the compound microbial agent in the step (5) is a mixed microbial agent of saccharomyces cerevisiae, bacillus subtilis and bacillus psychrosaccharolyticus, and the mass ratio of the compound microbial agent to the bacillus psychrosaccharolyticus is 2: 1: 2.

5. the method for extracting holothurian saponins according to claim 1, wherein the ethanol concentration in the ethanol precipitation in the step (10) is 30-40%.

6. The method for extracting holothurian saponins according to claim 1, wherein the aeration rate in the fermentation tank during the aerobic fermentation in the step (5) is 0.2-0.3 m³/m³.min。

7. The method for extracting holothurian saponins according to claim 1, wherein the preparation method of the saccharomyces cerevisiae seed liquid comprises the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 10⁹cfu/mL。

8. The method for extracting sea cucumber saponins according to claim 1, wherein the preparation method of the seed liquid of bacillus subtilis and bacillus psychrosaccharolyticus comprises the following steps: under the aseptic condition, LB culture medium is adopted to culture for 24 hours under the condition of 28-32 ℃ to prepare liquid bacillus subtilis and bacillus psychrosaccharolyticus seed liquid, and the effective viable count of the seed liquid is required to be more than or equal to 10⁹cfu/mL。

9. The method for extracting holothurian saponins according to claim 1, wherein the holothurian saponins extract can be used for preparing holothurian saponins dry products by resin adsorption extraction, and the specific process steps are as follows: and (2) passing the sea cucumber saponin extracting solution through a macroporous adsorption resin column at the flow rate of 2-3 times of the column volume/h, then eluting with water at the same flow rate, then eluting the sea cucumber saponin components adsorbed on the macroporous adsorption resin with ethanol, collecting eluent, concentrating under reduced pressure to obtain an extract, and finally freeze-drying to obtain a sea cucumber saponin dry product.

10. The method for extracting holothurian saponins according to claim 9, wherein the concentration of the ethanol for elution is 60-70%, and the temperature in the process of reduced pressure concentration is controlled at 40-50 ℃.

Technical Field

The invention relates to a method for extracting sea cucumber saponin, in particular to a sea cucumber saponin biological product with high oxidation resistance and nutrition and health care effects prepared by taking sea cucumber viscera as raw materials through synergistic fermentation of microorganisms, 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.

In recent years, Stichopus japonicus has been researched and found that the main active component of Stichopus japonicus is holothurian saponin, which is a secondary metabolite of Stichopus japonicus, and the holothurian saponin has various pharmacological activities, such as anti-tumor effect, immunity improvement, neovascularization resistance, anticoagulation, fibrinolysis promotion, embolism formation inhibition, anti-inflammatory effect (such as arthritis) and anti-radiation activity, and HIV, hepatitis B virus, herpes simplex virus, etc., and has broad-spectrum antifungal effect. At present, many national research and development institutions are dedicated to research and development of products mainly containing holothurian saponins. Because the saponin in the processing result is mostly remained in the processing waste liquid, the research on recycling the processing waste to extract the holothurian saponin is less, and the serious waste and loss of resources are caused.

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 wastes, the application provides a sea cucumber saponin extraction method, integrates a modern bioseparation technology, a biological enzymolysis technology and a mixed bacteria microbial fermentation technology, develops and develops a sea cucumber saponin biological product with high in-vitro oxidation resistance, meets the application requirements of the fields of health care, medical treatment, food and the like on the sea cucumber saponin, and has the advantages of simple and high-efficiency fermentation process, strong oxidation resistance and free radical scavenging capacity of the obtained product, low production cost and easy industrial mass production.

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

a method for extracting holothurian saponin 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% of lactobacillus 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 10000r/min to remove insoluble components;

(10) ethanol precipitation: adding ethanol into the centrifugate, and standing for more than 60 min;

(11) and (3) centrifugal concentration: centrifuging to remove precipitate, and concentrating under reduced pressure to obtain sea cucumber saponin extract.

Preferably, the complex enzyme preparation in the step (4) is one or a mixture of more of protease, non-starch polysaccharidase and naringinase.

Preferably, the mass ratio of the protease, the non-starch polysaccharidase and the naringinase is 2: 1: 1.

preferably, the compound microbial agent in the step (5) is a mixed microbial agent of saccharomyces cerevisiae, bacillus subtilis and bacillus psychrosaccharolyticus, and the mass ratio of the compound microbial agent to the bacillus psychrosaccharolyticus is 2: 1: 2.

preferably, the ethanol concentration in the ethanol precipitation in the step (10) is 30-40%.

Preferably, the ventilation rate in the aerobic fermentation process in the step (5) is 0.2-0.3 m³/m³.min。

The preparation method of the saccharomyces cerevisiae seed liquid comprises the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 10⁹cfu/mL。

The preparation method of the bacillus subtilis and the bacillus psychrosaccharolyticus seed liquid comprises the following steps: under the aseptic condition, an LB culture medium is adopted at the temperature of 28-32 DEG CCulturing for 24h to obtain liquid Bacillus subtilis and Bacillus psychrosaccharolyticus seed solutions with effective viable count of 10 or more⁹cfu/mL。

Preferably, the holothurian extract can be used for preparing a holothurian saponin dry product by resin adsorption extraction, and the specific process steps are as follows: and (2) passing the sea cucumber saponin extracting solution through a macroporous adsorption resin column at the flow rate of 2-3 times of the column volume/h, then eluting with water at the same flow rate, then eluting the sea cucumber saponin components adsorbed on the macroporous adsorption resin with ethanol, collecting eluent, concentrating under reduced pressure to obtain an extract, and finally freeze-drying to obtain a sea cucumber saponin dry product.

Preferably, the concentration of the ethanol for elution is 60-70%, and the temperature in the reduced pressure concentration is controlled to be 40-50 ℃.

The invention provides an extraction method of sea cucumber saponin, the obtained sea cucumber saponin has stronger oxidation resistance, and the extract can be subjected to concentration, adsorption drying, alcohol precipitation, 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.

Compared with the prior art, the extraction method of the holothurian saponin provided by the invention has the following remarkable advantages:

(1) according to the method, sea cucumber viscera which are wastes of deep processing of sea cucumbers are used as raw materials, and a modern bioseparation technology, a biological enzymolysis technology and a mixed-bacterium microbial fermentation technology are integrated, so that the extraction rate and the antioxidant capacity of the sea cucumber saponin are remarkably improved; especially, although the naringinase has no obvious influence on improving the antioxidant capacity of the holothurian saponin extracting solution, the naringinase has obvious influence on improving the antioxidant capacity of the holothurian saponin extracting solution by the compound enzyme preparation;

(2) the application utilizes aerobic bacteria (saccharomyces cerevisiae, bacillus subtilis and bacillus psychrosaccharolyticus) to jointly ferment the viscera protein of the sea cucumber to extract the sea cucumber saponin, makes full use of the synergistic effect among microorganisms, and improves the extraction rate and the in-vitro antioxidant capacity of the sea cucumber saponin, wherein: the saccharomyces cerevisiae can generate various enzymes for degrading cell walls in the fermentation process, so that more holothurin is released from the cell walls, and the saccharomyces cerevisiae can generate the enzymes capable of releasing combined state phenolic acid, thereby being beneficial to improving the in-vitro antioxidant capacity of the holothurin; the bacillus subtilis can kill pathogenic bacteria in fermentation liquor in the high-temperature fermentation process, has strong capabilities of producing protease, protease and the like, and can degrade macromolecular starch and protein substances into microorganisms for utilization, so that the extraction rate of the holothurin is improved, but the influence on the antioxidant capacity of the holothurin is not obvious; the addition of the bacillus psychrosaccharolyticus can obviously improve the removing capability of holothurin-OH free radicals;

(3) the method adopts the lactobacillus to carry out anaerobic fermentation, so that the stability of the holothurian saponin is improved, and test results show that the temperature and the time have little influence on the inoxidizability of the holothurian saponin 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 present application, the activation method of saccharomyces cerevisiae, bacillus subtilis and bacillus psychrosaccharolyticus is as follows:

(1) the preparation method of the saccharomyces cerevisiae seed liquid comprises the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 10⁹cfu/mL；

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