A biologically active preparation for promoting the synthesis of sea cucumber saponins

Abstract

The present application relates to the field of bio-agricultural technology, and in particular to a bioactive preparation and animal feed that promotes the synthesis of sea cucumber saponins. The components of the bioactive preparation of the present application respond to each other and synergize to enhance the effect, which can significantly activate the expression of key enzyme genes that regulate the synthesis of sea cucumber saponins, and effectively promote the synthesis and accumulation of sea cucumber saponins; at the same time, it can also comprehensively enhance the non-specific immune function of sea cucumbers by enhancing the activity of immune enzymes and improving disease resistance, and has the advantages of clear ingredients, small dosage, and green environmental protection.

Description

Technical Field

[0001] The present application relates to the field of biological agricultural technology, and in particular to a bioactive preparation for promoting the synthesis of sea cucumber saponins and an animal feed thereof. Background Art

[0002] Sea cucumber belongs to the phylum Echinodermata and class Holothuria. It is rich in proteins, minerals, vitamins and other natural active substances, and has extremely high nutritional and medicinal value. Sea cucumber saponins are the main secondary metabolites of sea cucumbers to resist natural enemies. They are a class of glycoside compounds mainly composed of aglycones and sugar chains. They have physiological effects such as improving immunity, anti-tumor, anti-aging, anti-coagulation, and preventing hyperlipidemia and arteriosclerosis. It has important development value and application prospects in food and medicine. The content of saponin monomers in sea cucumbers is low, and the content of other secondary metabolites produced by complex metabolic processes is high. Therefore, it is imperative to develop a bioactive preparation with clear ingredients, green and pollution-free, which can promote the synthesis of sea cucumber saponins.

[0003] Polysaccharides are macromolecules formed by free monosaccharide groups connected by glycosidic bonds. Compared with other types of biopolymers, polysaccharides can carry a large amount of biological information. Polysaccharide structures are complex and diverse, and have rich biological effects, such as anti-tumor, anti-viral, anti-oxidation, anti-coagulation, hypolipidemic and immune regulation. Polysaccharides are widely available. In plants, polysaccharide components include starch, cellulose, fructan, hemicellulose, gum, mucilage and mucosa. Plant polysaccharides have the characteristics of no toxic side effects and no residue, and can mediate physiological activities such as animal cell differentiation, proliferation, and signal transduction. Unlike plant polysaccharides, fungal polysaccharides are active polysaccharides isolated from fungal fruiting bodies and have a helical stereo configuration. Most fungal polysaccharides can activate immune cells, activate the complement system, promote cytokine production, and can regulate the biological immune system through various pathways. In aquaculture, plant polysaccharides and fungal polysaccharides as feed additives or bioactive substances can promote growth, enhance immunity, regulate the structure of intestinal flora, and improve disease resistance.

[0004] However, there is no report or disclosure in the prior art about the rational combination of plant polysaccharides and fungal polysaccharides to improve the synthesis and accumulation of sea cucumber saponins. There is also no report or disclosure about using psyllium husk powder, melatonin, and octyl glucoside as additional components of a composite polysaccharide extract to promote the synthesis of sea cucumber saponins. In view of this, the present application is hereby proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present application studies a bioactive preparation that promotes the synthesis of sea cucumber saponins. The bioactive preparation has clear ingredients, is green and environmentally friendly, and has low cost. More importantly, its functional components synergize to effectively promote the synthesis and accumulation of sea cucumber saponins, and can significantly enhance the nonspecific immune function of sea cucumbers, making it suitable for large-scale production and promotion.

[0006] Specifically, this application provides the following technical solutions:

[0007] The present application first provides a bioactive preparation for promoting the synthesis of sea cucumber saponins, wherein the bioactive preparation comprises a complex polysaccharide extract, psyllium husk powder, melatonin and octyl glucoside.

[0008] Furthermore, the composite polysaccharide extract includes plant polysaccharide extract and fungal polysaccharide extract;

[0009] Preferably, the plant polysaccharide extract is ginkgo polysaccharide; the plant polysaccharide extract is mulberry linterus polysaccharide, versicolor polysaccharide and Flammulina velutipes polysaccharide.

[0010] Furthermore, the mass concentrations of the polysaccharide extracts are: 2-3% of ginkgo polysaccharide, 1-1.5% of mulberry linterus polysaccharide, 1-1.5% of versicolor polysaccharide and 1-1.5% of Flammulina velutipes polysaccharide.

[0011] Furthermore, the mass concentrations of the psyllium husk powder, melatonin and octyl glucoside are 0.3-0.6% of psyllium husk powder, 0.02-0.04% of melatonin and 0.5-1.5% of octyl glucoside.

[0012] Furthermore, the optimal concentrations of the components of the bioactive preparation are as follows: 2.5% ginkgo polysaccharide, 1.5% mulberry linterus polysaccharide, 1.2% versicolor polysaccharide, 1.4% Flammulina velutipes polysaccharide, 0.5% psyllium husk powder, 0.03% melatonin, and 1% octyl glucoside.

[0013] Furthermore, the preparation of the polysaccharide extract includes: cleaning ginkgo, mulberry linterus, versicolor and enoki mushroom respectively, drying in the shade through ventilation, and then crushing; soaking in 75% ethanol solution, filtering to remove impurities, and then concentrating under reduced pressure and spray drying to obtain the polysaccharide extract.

[0014] Furthermore, the preparation of the polysaccharide extract specifically includes: washing clean and fresh ginkgo, mulberry, versicolor, and enoki mushrooms with normal color with distilled water, ventilating and drying them in the shade, and then crushing them with a homogenizer to a particle size of 100 meshes. Adding 75% ethanol solution (solid-liquid ratio of 1g / 25ml), extracting for 1h under the action of ultrasound with a working frequency of 60KHZ and a power of 100W, filtering with sterile gauze to remove impurities, and decompressing and concentrating the extract, spray drying, to obtain a polysaccharide extract.

[0015] Furthermore, the plant polysaccharide ginkgo polysaccharide and the fungal polysaccharides mulberry linterus polysaccharide, versicolor polysaccharide, and enoki mushroom polysaccharide in the bioactive preparation have a synergistic effect; the plant polysaccharide has a diverse structure and complex composition, mainly comprising starch, cellulose, fructan, hemicellulose, gum, mucilage, and adhesive; the high molecular weight polymer of fungal polysaccharide separated from the fungal fruiting body has a spiral three-dimensional structure; after the fungal polysaccharide and the plant polysaccharide are compounded, polymerization occurs in the structure, the polymer chains are combined into aggregates, and the polysaccharide polymers are formed through orderly combination and distribution of the residue bonds between the polysaccharides. ; It can work synergistically on multiple pathways and targets to activate the expression of key enzyme genes that regulate the synthesis of sea cucumber saponins, and effectively promote the synthesis and accumulation of sea cucumber saponins; melatonin participates in regulating the body's growth, development and stress response by regulating signal transduction pathways, indirectly affecting the gene expression and enzyme activity related to saponin synthesis, and has a positive effect on the metabolic pathways related to saponin synthesis; octyl glucoside can bind to proteins to form a stable complex, which is beneficial to promote the folding and stabilization of proteins, making it easier to release substances in cells, and helping to promote the synthesis and accumulation of saponins.

[0016] The present application also provides the use of the bioactive preparation in promoting the synthesis of sea cucumber saponins.

[0017] The present application also provides a sea cucumber imitation feed, which contains the aforementioned bioactive preparation.

[0018] The present application also provides a method for preparing the sea cucumber imitation feed, which comprises adding a composite polysaccharide extract, psyllium husk powder, melatonin, and octyl glucoside to the sea cucumber imitation feed in proportion, and adding a small amount of seawater and stirring evenly.

[0019] The present application also provides a method for promoting the synthesis of sea cucumber saponins, comprising: adding the aforementioned bioactive agent to sea cucumber feed, adding a small amount of seawater, stirring evenly, and then spraying it into the sea cucumber breeding water body with a water temperature of 12-30°C.

[0020] Advantages of this application:

[0021] 1) This application is the first to scientifically match plant polysaccharides Ginkgo polysaccharides with fungal polysaccharides (Phellinus igniarius polysaccharides, Versicolor polysaccharides, Flammulina velutipes polysaccharides) and use them together to promote the synthesis and accumulation of sea cucumber saponins. Compared with polysaccharides from a single source, plant polysaccharides and fungal polysaccharides interact and respond to each other, synergizing. The two form new polysaccharide polymers through polymerization, which can synergize in multiple pathways and targets, activate the expression of key enzyme genes that regulate the synthesis of sea cucumber saponins, effectively promote the synthesis and accumulation of sea cucumber saponins, and have higher biological efficacy.

[0022] 2) Melatonin participates in regulating the growth and development of the body and stress response by regulating signal transduction pathways, indirectly affecting gene expression and enzyme activity related to saponin synthesis, and having a positive effect on metabolic pathways related to saponin synthesis. TritonX-100 is a non-ionic surfactant that can destroy cell membrane structure, activate the activity of phospholipases and proteases, and improve the synthesis efficiency of saponins; however, Triton X-100 has certain chemical toxicity to cells. High concentrations or long-term use will affect the normal structure and function of cells and will also cause harm to the breeding ecological environment. As a green alternative to Triton X-100, octyl glucoside comes from natural plants and is synthesized from natural raw materials glucose and fatty alcohols. It has excellent compatibility, biodegradability and low irritation. Octyl glucoside can bind to proteins to form a stable complex, which is conducive to promoting protein folding and stabilization, making it easier to release substances in cells, and helping to promote the synthesis and accumulation of saponins. At the same time, octyl glucoside can improve the physical properties of feed and can effectively improve the dispersion and uniformity of feed.

[0023] 3) Psyllium husk powder is rich in dietary fiber, which can promote intestinal peristalsis of sea cucumbers, help digestion and excretion, and reduce the occurrence of intestinal diseases. Moreover, most polysaccharides are soluble in water or lose a lot of water, while psyllium husk powder contains a lot of soluble fiber, which is easy to form gel material, has thickening and bonding effects, can increase the sedimentation rate of feed, reduce the disintegration rate and dissolution rate of polysaccharides in feed, and is suitable for the feeding habits of sea cucumbers that lick the bottom.

[0024] 4) The bioactive preparation of the present application can comprehensively enhance the non-specific immune function of sea cucumbers by enhancing the activity of immune enzymes and improving disease resistance.

[0025] 5) The overall components of the bioactive preparation of this application have the advantages of interactive response and synergistic synergy. At the same time, the preparation has clear ingredients, low cost, simple preparation and convenient storage. It has the characteristics of no pollution, no drug resistance, no toxic side effects, no aquatic product safety risks and environmental safety issues, and is suitable for large-scale production and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Schematic diagram of the effect of the bioactive preparation of the present application on the expression of key enzyme genes for the synthesis of sea cucumber saponins;

[0028] Figure 2 Schematic diagram of the effect of the bioactive preparation of the present application on the content of sea cucumber saponins;

[0029] Figure 3 Schematic diagram of the effect of the bioactive preparation of the present application on the activity of acid phosphatase of sea cucumber;

[0030] Figure 4 Schematic diagram of the effect of the bioactive preparation of the present application on the activity of alkaline phosphatase of sea cucumber;

[0031] Figure 5 Schematic diagram of the effect of the bioactive preparation of the present application on the activity of superoxide dismutase of sea cucumber;

[0032] Figure 6 Schematic diagram of the effect of the bioactive preparation of the present application on the activity of sea cucumber lysozyme;

[0033] Figure 7 Schematic diagram of the effect of the bioactive preparation of the present application on the cumulative mortality of poison-challenged sea cucumbers;

[0034] Figure 8 Schematic diagram of the inhibitory effect of the bioactive preparation of the present application on Vibrio splendens, a pathogenic bacteria of Apostichopus japonicus. DETAILED DESCRIPTION

[0035] The embodiments of the present application will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be considered to limit the scope of the present application. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0036] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0037] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0038] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0039] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0040] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0041] The core technology of this application is described in detail:

[0042] A bioactive preparation for promoting the synthesis of sea cucumber saponins. The functional components of the bioactive preparation include complex polysaccharide extract, psyllium husk powder, melatonin and octyl glucoside.

[0043] In some embodiments, the complex polysaccharide extract includes a plant polysaccharide extract and a fungal polysaccharide extract;

[0044] In some preferred embodiments, the plant polysaccharide extract is ginkgo polysaccharide; the plant polysaccharide extract is mulberry linterus polysaccharide, versicolor polysaccharide and Flammulina velutipes polysaccharide.

[0045] It can be summarized through some embodiments of the present application that the mass concentrations of the polysaccharide extracts described in the present application can be: 2-3% of ginkgo polysaccharide, 1-1.5% of mulberry linterus polysaccharide, 1-1.5% of versicolor polysaccharide and 1-1.5% of Flammulina velutipes polysaccharide.

[0046] It can be summarized through some embodiments of the present application that the mass concentrations of psyllium husk powder, melatonin and octyl glucoside described in the present application can be 0.3-0.6% of psyllium husk powder, 0.02-0.04% of melatonin and 0.5-1.5% of octyl glucoside respectively.

[0047] In the above concentration range, they all show advantages over existing bioactive preparations. In addition, in some specific embodiments of the present application, the optimal concentrations of the components of the bioactive preparation are: 2.5% ginkgo polysaccharide, 1.5% mulberry linterus polysaccharide, 1.2% versicolor polysaccharide, 1.4% enokitake polysaccharide, 0.5% psyllium husk powder, 0.03% melatonin, 1% octyl glucoside, and the best effect is shown under this ratio.

[0048] Without limitation, the preparation of the polysaccharide extract described in the present application may include the following basic steps: clean Ginkgo, Coriolus linteus, Coriolus versicolor and Flammulina velutipes respectively, pulverize after ventilation and shade drying; soak in 75% ethanol solution, filter to remove impurities, and then reduce pressure concentration and spray drying to obtain a polysaccharide extract.

[0049] In some specific embodiments, the preparation of the polysaccharide extract includes: washing clean and fresh ginkgo, mulberry, versicolor, and enoki mushrooms with normal color with distilled water, drying in the shade, and crushing with a homogenizer to a particle size of 100 mesh. Adding 75% ethanol solution (solid-liquid ratio of 1g / 25ml), extracting for 1h under the action of ultrasound with a working frequency of 60KHZ and a power of 100W, filtering with sterile gauze to remove impurities, decompressing and concentrating the extract, and spray drying to obtain a polysaccharide extract.

[0050] It can be understood that when used, different polysaccharide extracts, psyllium husk powder, melatonin, and octyl glucoside are added to the sea cucumber feed in proportion, a small amount of seawater is added and stirred evenly, and then sprinkled into the sea cucumber breeding water.

[0051] The plant polysaccharides (gingko polysaccharides) and fungal polysaccharides (mulberry linterus polysaccharides, versicolor polysaccharides, and velutipes polysaccharides) in the bioactive preparation of the present application have a synergistic effect. The structure of plant polysaccharides is relatively diverse and the composition is complex, mainly including starch, cellulose, fructan, hemicellulose, gum, mucilage and mucus, etc. Fungal polysaccharides are high molecular weight polymers separated from fungal fruiting bodies, with a spiral three-dimensional structure. After compounding fungal polysaccharides with plant polysaccharides, polymerization will occur in the structure, and the polymer chains will be combined into aggregates, and polysaccharide polymers will be formed through orderly combination and distribution of residue bonds between polysaccharides. It can work synergistically on multiple pathways and targets, activate the expression of key enzyme genes that regulate the synthesis of sea cucumber saponins, and effectively promote the synthesis and accumulation of sea cucumber saponins. Melatonin participates in regulating the growth and development of the body and the stress response by regulating the signal transduction pathway, indirectly affecting the gene expression and enzyme activity related to saponin synthesis, and has a positive effect on the metabolic pathways related to saponin synthesis. Octyl glucoside can bind to proteins to form stable complexes, which is beneficial to promoting the folding and stabilization of proteins, making it easier to release substances in cells, and helping to promote the synthesis and accumulation of saponins.

[0052] The implementation scheme of the present application is described in detail below in conjunction with the examples. The present application includes but is not limited to the following implementation schemes. Any changes made to the present application based on the prior art that do not deviate from the essence of the present application still fall within the scope of protection of the present application.

[0053] Example

[0054] Example 1 Preparation of biologically active preparations

[0055] The mass percentages of the components of the bioactive preparations added to the sea cucumber feed are: 2% ginkgo polysaccharide, 1.5% mulberry linterus polysaccharide, 1% versicolor polysaccharide, 1.3% Flammulina velutipes polysaccharide, 0.3% psyllium husk powder, 0.04% melatonin, and 1% octyl glucoside.

[0056] The preparation method of the polysaccharide extract is as follows: clean and fresh ginkgo, mulberry, versicolor and enoki mushrooms with normal color are cleaned with distilled water respectively, ventilated and dried in the shade, and then crushed with a homogenizer to a particle size of 100 meshes. Add 75% ethanol solution (solid-liquid ratio is 1g / 25ml), extract for 1 hour under the action of ultrasound with a working frequency of 60KHZ and a power of 100W, filter with sterile gauze to remove impurities, reduce the pressure and concentrate the extract, and spray dry to obtain the polysaccharide extract.

[0057] Different polysaccharide extracts, psyllium husk powder, melatonin, and octyl glucoside are added to the imitation sea cucumber feed in proportion, and a small amount of seawater is added and stirred evenly before being sprinkled into the sea cucumber breeding water. The bioactive agent is suitable for long-term use during the breeding cycle, with a suitable water temperature of 12-30℃ and is used once a day.

[0058] Plant polysaccharides (ginkgo polysaccharides) and fungal polysaccharides (mulberry linterus polysaccharides, versicolor polysaccharides, and enokitake polysaccharides) in bioactive preparations have synergistic effects. Plant polysaccharides have diverse structures and complex components, mainly including starch, cellulose, fructan, hemicellulose, gum, mucilage, and colloids. Fungal polysaccharides are high molecular weight polymers isolated from fungal fruiting bodies, with a spiral three-dimensional structure. When fungal polysaccharides are compounded with plant polysaccharides, polymerization occurs in the structure, and the polymer chains are combined into aggregates, and polysaccharide polymers are formed through orderly combination and distribution of residue bonds between polysaccharides. They can work synergistically in multiple pathways and targets, activate the expression of key enzyme genes that regulate the synthesis of sea cucumber saponins, and effectively promote the synthesis and accumulation of sea cucumber saponins. Melatonin participates in the regulation of the body's growth and development and stress response by regulating signal transduction pathways, indirectly affecting the gene expression and enzyme activity related to saponin synthesis, and has a positive effect on the metabolic pathways related to saponin synthesis. Octyl glucoside can bind to proteins to form stable complexes, which is beneficial to promoting the folding and stabilization of proteins, making it easier to release substances in cells, and helping to promote the synthesis and accumulation of saponins.

[0059] Example 2 Preparation of biologically active preparation 2

[0060] The preparation process and administration method of the bioactive preparation in Example 2 are basically the same as those in Example 1. The difference is that the mass percentages of the components of the bioactive preparation added to the sea cucumber feed are: 3% ginkgo polysaccharide, 1% mulberry linterus polysaccharide, 1.5% versicolor polysaccharide, 1.5% Flammulina velutipes polysaccharide, 0.6% psyllium husk powder, 0.03% melatonin, and 0.5% octyl glucoside.

[0061] Example 3 Preparation of biologically active preparations

[0062] The preparation process and administration method of the bioactive preparation in Example 3 are basically the same as those in Example 1. The difference is that the mass percentages of the components of the bioactive preparation added to the sea cucumber feed are: 2.5% of ginkgo polysaccharide, 1.3% of mulberry linterus polysaccharide, 1.2% of versicolor polysaccharide, 1% of Flammulina velutipes polysaccharide, 0.4% of psyllium husk powder, 0.02% of melatonin, and 1.5% of octyl glucoside.

[0063] Specific experiments:

[0064] The imitation sea cucumbers in each embodiment were taken from the breeding center of Liaoning Marine Fisheries Science Research Institute. The imitation sea cucumbers had a single mass of 7.27±0.84g and were temporarily kept in six aquariums of the same specifications, with 50 heads in each box. The aquariums were numbered A, B, C, D, E, and F. After the imitation sea cucumbers were temporarily kept for one week, the bioactive agent was added, and the feed was fed to each aquarium at 5% of the body mass of the imitation sea cucumbers. Aquarium A was used as a control example, and no bioactive agent was applied. Aquarium B was used as a plant polysaccharide comparison example, and only plant polysaccharide extract (3% ginkgo polysaccharide) and 0.6% psyllium husk powder, 0.04% melatonin, and 1.5% octyl glucoside were added. Aquarium C was used as a fungal polysaccharide control, and only fungal polysaccharide extracts (1.5% mulberry linterus polysaccharide, 1.5% versicolor polysaccharide, 1.5% enokitake polysaccharide) and 0.6% psyllium husk powder, 0.04% melatonin, and 1.5% octyl glucoside were added. Aquariums D, E, and F were groups of Examples 1, 2, and 3, respectively. Each treatment group was administered with a bioactive agent once a day, and the control group was not administered with a bioactive agent. Other breeding management was the same as that of each treatment group. The experiment was set up in 3 parallel groups, and each treatment was repeated 3 times. During the feeding period, aeration was maintained, and the water was changed once every 2 days. The seawater temperature was 16-20°C, pH 8.1-8.3, and salinity 30. Dissolved oxygen was not less than 5 mg / L, and ammonia nitrogen was not higher than 0.5 mg / L. The experiment lasted for 30 days. Five imitation sea cucumbers were randomly selected from each aquarium on the 0th, 10th, 20th, and 30th days after the addition of the bioactive agent, and the abdominal cavity was opened to obtain the body cavity fluid. The coelomic fluid of the imitation sea cucumber was combined, centrifuged at 4°C and 4000r / min for 10min, and the supernatant was used to determine the immunoenzyme activity. The test kit of Nanjing Jiancheng Technology Co., Ltd. was used to determine the immunoenzyme activity of the sea cucumber, and the specific determination steps were carried out according to the instructions of the kit. The enzyme activity detection test was repeated 3 times, and the enzyme activity was expressed in the form of specific activity, with the unit of U / mg protein. The coelomic fluid of the imitation sea cucumber was taken at each sampling, and the effect of biologically active agents on the expression level of key enzyme genes (MVD, PMK, FPS, IDI) for the synthesis of imitation sea cucumber saponins was detected by the method of fluorescent real-time quantitative polymerase chain reaction (SYBR GREEN I Real-TimePolymerase Chain Reaction). The imitation sea cucumber saponins were extracted by water-alcohol extraction. The sea cucumber is homogenized, the homogenate is repeatedly boiled in water, filtered and separated, the filtrates are combined and recovered under reduced pressure to obtain an extract, 70% by volume ethanol is added, stirred evenly, and the precipitate is discarded after standing to obtain a supernatant, and 95% by volume ethanol is finally added to the supernatant, a white precipitate is collected, the precipitate is washed with benzene and dissolved in chloroform, the insoluble matter is separated and removed, and the supernatant is recovered under reduced pressure to obtain crude sea cucumber saponins.

[0065] After the 30-day feeding experiment, the sea cucumbers were challenged with Vibrio splendens. The semi-lethal concentration of Vibrio splendens to the sea cucumbers was determined to be 2×10 7cfu / mL. The challenge test was set up in three parallel groups, and the injection dose of Vibrio splendens was 0.1 ml / head. The incidence of Vibrio and the cumulative mortality of the sea cucumbers within 15 days after the challenge were recorded. After the challenge test, 5 sea cucumbers were randomly selected from each group, and the intestinal tissue was dissected to obtain the intestinal tissue, which was homogenized with 30 mL of PBS buffer for 10 minutes. The intestinal tissue homogenate was diluted 10 times with sterile saline, and 0.05 ml of the dilution was spread on TCBS Vibrio selective culture medium, inverted and cultured at 28°C, and the number of Vibrio in the sea cucumber intestine was counted after 2 days. The Vibrio splendens (MCCC1A04096) used was purchased from the Marine Microbial Culture Collection Management Center.

[0066] Experimental Results

[0067] The effects of the bioactive preparation of the present application on the gene expression of key enzymes in the synthesis of sea cucumber saponins are as follows: Figure 1 As shown. It can be seen that, except for the control group, the expression of the key enzyme genes for the synthesis of sea cucumber saponins in each treatment group was enhanced. Compared with the plant polysaccharide control and the fungal polysaccharide control, the expression levels of the key enzyme genes for the synthesis of sea cucumber saponins in Examples 1, 2, and 3 were significantly higher. Among them, the gene expression levels of FPS and IDI were higher in Example 1, the PMK gene expression level was the highest in Example 2, and the MVD gene expression level was the highest in Example 3.

[0068] The effects of the bioactive preparation of the present application on the content of sea cucumber saponins are as follows: Figure 2 As shown. It can be seen that, except for the control group, the content of sea cucumber saponin in each treatment group gradually increased as the experiment progressed. The content of sea cucumber saponin in the plant polysaccharide comparison and the fungal polysaccharide comparison was significantly lower than that in each example, and the content of sea cucumber saponin in Example 2 was slightly higher than that in Examples 1 and 3.

[0069] The effects of Examples 1, 2 and 3 on the activity of the immune enzymes of the sea cucumber are shown in Figure 3-6As shown, among the selected enzyme activity indicators, acid phosphatase and alkaline phosphatase are important hydrolases involved in immune defense activities in the coelomocytes of the sea cucumber. Superoxide dismutase can remove excess free radicals in the body, block lipid peroxidation, and repair damaged cells. Lysozyme can destroy the cell wall of bacteria and eliminate pathogenic bacteria invading the body. Before using the biologically active preparation, there was no significant difference in the activity of the sea cucumber enzymes in each embodiment compared with the control example. As the experiment proceeded, the acid phosphatase, alkaline phosphatase, superoxide dismutase, and lysozyme activities in the coelomic fluid of the sea cucumber in each treatment group were significantly higher than those in the control example. Compared with the plant polysaccharide control example and the fungal polysaccharide control example, the immune enzyme activity of the sea cucumber in Examples 1, 2, and 3 is higher. Among them, Example 1 has a better activation effect on acid phosphatase and alkaline phosphatase than Example 2 and Example 3, and the superoxide dismutase activity of Example 2 is the highest, and the lysozyme activity of Example 3 is higher than that of Example 1 and Example 2. Vibrio splendens can cause pathogenic effects on aquatic animals by secreting hemolysins, enterotoxins, etc. It can infect shellfish, shrimps and fish in a short period of time and cause mass deaths.

[0070] Effects of bioactive agents on the cumulative mortality of poison-challenged sea cucumbers Figure 7 As shown. The first dead individual after Vibrio splendidus infection appeared in the control example, while the cumulative mortality of the imitation sea cucumber in each treatment group was significantly lower than that in the control example. Among them, the cumulative mortality of the imitation sea cucumber in the plant polysaccharide comparison and the fungal polysaccharide comparison was higher than that in each embodiment, and the cumulative mortality of the imitation sea cucumber in embodiment 3 was lower than that in embodiments 1 and 2.

[0071] The inhibitory effect of bioactive agents on Vibrio splendens Figure 8 As shown. The control example has the largest number of Vibrio, while the number of brilliant Vibrio in the sea cucumber intestinal tract in each example is less than the plant polysaccharide control example and the fungal polysaccharide control example. Among them, the number of brilliant Vibrio in the sea cucumber intestinal tract in Example 3 is the smallest, and the inhibitory effect of the bioactive preparation on brilliant Vibrio is higher than that in other examples.

[0072] Based on the above results, it can be seen that the components in the active preparation established by this organism can respond to each other and synergize, which can significantly activate the expression of key enzyme genes that regulate the synthesis of sea cucumber saponins, and effectively promote the synthesis and accumulation of sea cucumber saponins. At the same time, this biological active preparation can also comprehensively enhance the non-specific immune function of sea cucumbers by enhancing the activity of immune enzymes and improving disease resistance.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A bioactive agent for promoting the synthesis of sea cucumber saponins, characterized in that: The bioactive preparation comprises a composite polysaccharide extract, psyllium husk powder, melatonin and octyl glucoside; the composite polysaccharide extract comprises a plant polysaccharide extract and a fungal polysaccharide extract; the plant polysaccharide extract is ginkgo polysaccharide; the fungal polysaccharide extract is mulberry linterus polysaccharide, versicolor polysaccharide and enokitake polysaccharide; the mass concentrations of the composite polysaccharide extract are: 2-3% ginkgo polysaccharide, 1-1.5% mulberry linterus polysaccharide, 1-1.5% versicolor polysaccharide and 1-1.5% enokitake polysaccharide; the mass concentrations of the psyllium husk powder, melatonin and octyl glucoside are: 0.3-0.6% psyllium husk powder, 0.02-0.04% melatonin and 0.5-1.5% octyl glucoside.

2. The biologically active agent according to claim 1, characterized in that The preparation of the polysaccharide extract comprises: cleaning ginkgo, mulberry linterus, versicolor versicolor and enoki mushroom respectively, drying in the shade through ventilation, and then crushing; soaking in 75% ethanol solution, filtering to remove impurities, and then concentrating under reduced pressure and spray drying to obtain the polysaccharide extract.

3. Use of the bioactive preparation according to any one of claims 1 to 2 in promoting the synthesis of sea cucumber saponins.

4. A sea cucumber imitation feed, characterized in that: The sea cucumber imitation feed comprises the bioactive preparation according to any one of claims 1-2.

5. The method for preparing the imitation sea cucumber feed according to claim 4, characterized in that: The method comprises adding the compound polysaccharide extract, psyllium husk powder, melatonin and octyl glucoside to the sea cucumber feed in proportion, and adding a small amount of seawater to stir evenly.

6. A method for promoting the synthesis of sea cucumber saponins, characterized in that: The method comprises: adding the bioactive agent according to any one of claims 1 to 2 to the sea cucumber feed, adding a small amount of seawater, stirring evenly, and then spraying into the sea cucumber breeding water body with a water temperature of 12-30°C.

Images