CN113875909B - Preparation method of synergistic composite immunopotentiator for stichopus japonicus - Google Patents
Preparation method of synergistic composite immunopotentiator for stichopus japonicus Download PDFInfo
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- CN113875909B CN113875909B CN202111185393.4A CN202111185393A CN113875909B CN 113875909 B CN113875909 B CN 113875909B CN 202111185393 A CN202111185393 A CN 202111185393A CN 113875909 B CN113875909 B CN 113875909B
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- stichopus japonicus
- chinese herbal
- bacillus licheniformis
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- gluconate
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a preparation method of a synergistic compound immunopotentiator for stichopus japonicus, which comprises the following components in unit weight: 40-60 mg/g of compound Chinese herbal medicine, 10 6～108 cfu/g of bacillus licheniformis, 0.5-1.5 mg/g of ferrous gluconate and 0.2-0.8 mg/g of copper gluconate. Weighing the Chinese herbal medicines, performing coarse grinding, uniformly mixing the Chinese herbal medicines according to the weight percentage, and grinding by using an ultrafine grinder. Activating and re-suspending bacillus licheniformis, and adjusting the concentration of bacterial liquid. The processed composite Chinese herbal medicine, bacillus licheniformis and mineral element ferrous gluconate and copper gluconate are added into stichopus japonicus feed according to a proportion, a small amount of seawater is added, and the stichopus japonicus is fed after being uniformly stirred. The components of the composite immunopotentiator act together and synergistically, and the disease resistance is improved to comprehensively improve the nonspecific immunity of the stichopus japonicus.
Description
Technical Field
The invention relates to a synergistic composite immunopotentiator for stichopus japonicus, in particular to a immunopotentiator prepared from Chinese herbal medicines, bacillus licheniformis and mineral elements of ferrous gluconate and copper gluconate, and a preparation method and a use technology thereof, and belongs to the technical field of echinoderm immunology.
Background
Stichopus japonicus (Apostichopus japonicus) belongs to Echinodermata, stichopus japonicus class, is rich in various natural active substances such as protein, mineral, vitamins, etc., and has high nutritive and medicinal values. Along with the continuous expansion of the stichopus japonicus culture scale and the irregular operation in the intensive culture mode, the problems of germplasm degradation, disease frequency, cost increase and the like appear in succession in the stichopus japonicus culture process, and the healthy development of the stichopus japonicus culture industry is seriously hindered. Apostichopus japonicus belongs to invertebrate echinoderm, lacks specific immune system, and mainly relies on natural immune mechanism to identify and remove foreign matters entering organism. The quality of the stichopus japonicus feed directly influences the health of the stichopus japonicus and the quality of the breeding environment, and the reasonably-compatible feed additive can greatly improve the nonspecific immunity of the stichopus japonicus by improving the health of intestinal tracts, the growth index and the disease resistance, thereby improving the breeding benefit of the stichopus japonicus. Antibiotics have great limitations in stichopus japonicus cultivation due to the problems of bacterial drug resistance, food and environmental safety and the like. The immunopotentiator for stichopus japonicus in the market at present has good quality and is lack of industry specification and management. Therefore, it is imperative to find an immunopotentiator for stichopus japonicus, which is green and pollution-free and has rich nutrition.
CN200910017176.7 provides an antibacterial immune double-effect compound Chinese herbal medicine for treating stichopus japonicus beancurd skin syndrome, wherein the medicine components are common andrographis herb, dyers woad leaf, honeysuckle and szechuan lovage rhizome; the weight ratio of the medicines is 2:1:3:2; the medicine is in powder form with granularity of 200 meshes. The invention is a special drug for preventing and treating the stichopus japonicus putrescence Pi Zeng syndrome in an oral way, can be used in both young ginseng stage and adult ginseng stage, and has good preventing effect in the high-frequency period of the stichopus japonicus putrescence syndrome; the cure rate of the stichopus japonicus in 2-3 weeks reaches 80-90%, and the survival rate reaches more than 85%. The compound Chinese herbal medicine has the effects of immunity and disease resistance, has no toxic or side effect, has simple and convenient use method, can reduce or replace antibiotics, and is suitable for disease control of young ginseng cultivation and cultivation production.
CN200810012641.3 discloses a compound Chinese herbal medicine preparation for treating bacterial skin-dissolving disease of stichopus japonicus, which comprises the following pharmaceutical components in percentage by weight: 80-90% of dark plum and 10-20% of Chinese gall. The water is decocted and then is used for bath or full pool sprinkling of the stichopus japonicus, and no residue is left in the stichopus japonicus body; the addition amount is small, and the use is convenient; the composition is simple, the sources of the used raw materials are stable, the price is low, and the production process is simple.
CN201210206056.3 discloses a stichopus japonicus Chinese herbal medicine composite immunopotentiator, which is characterized in that: comprises the following components: radix Codonopsis, poria, fructus Schisandrae chinensis, herba Andrographitis, and flos Lonicerae; the Chinese herbal medicine composite immunopotentiator for stichopus japonicus has the advantages of being prepared from traditional Chinese medicines, strong in sterilization, insect expelling and virus resisting effects, non-toxic, free of side effects, pollution-free, low in cost and easy to prepare, being used as echinoderm, belonging to invertebrates, and mainly taking nonspecific immunity as a main component.
At present, most of commercial immunopotentiators for stichopus japonicus are prepared by adding probiotic preparations, chinese herbal medicines or mineral elements into feeds singly and have single preparation and application modes. Firstly, the active ingredients of the Chinese herbal medicines such as starch, pectin, protease and the like in the prior art contain glucoside compounds, are difficult to absorb in the intestines of stichopus japonicus, and have low bioavailability. Moreover, the dissolution rate of active substances of macromolecular substances in the Chinese herbal medicine is low, which is not beneficial to the absorption of organisms. And the toxic components of part of the Chinese herbal medicines also directly influence the cultivation effect of the stichopus japonicus. Secondly, the growth of bacillus licheniformis needs a specific substrate to improve the survival rate, and the environment in the intestinal canal of stichopus japonicus is not suitable for the effective proliferation of bacillus licheniformis, so that the bioavailability is low. And thirdly, the use of the immunopotentiator in stichopus japonicus culture must ensure no pollution, no residue, no drug resistance and no toxic or side effect, and the inorganic salts of copper and iron of mineral elements have biological toxicity and have certain negative effects on the growth index and the immunocompetence of the stichopus japonicus.
Disclosure of Invention
The invention aims to make up the defects of the prior art and provides a compound immunopotentiator for stichopus japonicus, which is used by combining Chinese herbal medicines, bacillus licheniformis and mineral elements. The components of the composite immunopotentiator act together and synergistically, and the nonspecific immunity of the stichopus japonicus can be comprehensively improved by improving the growth index, enhancing the activity of related enzymes, reducing the morbidity, improving the intestinal health and the like.
The technical scheme of the invention is as follows:
A synergistic composite immunopotentiator for stichopus japonicus, wherein the additive concentration of composite Chinese herbal medicine, bacillus licheniformis, ferrous gluconate and copper gluconate in the feed per unit weight is as follows: 40-60 mg/g of compound Chinese herbal medicine; bacillus licheniformis 10 6～108 cfu/g; 0.5-1.5 mg/g of ferrous gluconate; copper gluconate 0.2-0.8 mg/g.
Further, the preferable additive concentration of each component in the feed per unit weight is: compounding 50mg/g of Chinese herbal medicine; bacillus licheniformis 10 7 cfu/g; ferrous gluconate 0.8mg/g; copper gluconate 0.4mg/g.
Further, the composite immunopotentiator comprises the following Chinese herbal medicines in percentage by mass: 15-25 parts of radix scutellariae; 10-20 parts of phellodendron; 15-25 parts of Chinese angelica; 10-25 parts of rheum officinale; 5-10 parts of dyers woad leaf; 5-15 parts of liquorice; 15-25 parts of rhodiola rosea; 10-20 parts of Angelica keiskei.
Further, the compound immunopotentiator epidemic comprises five or more of Chinese herbal medicines including radix Scutellariae, cortex Phellodendri, radix Angelicae sinensis, radix et rhizoma Rhei, folium Isatidis, glycyrrhrizae radix, radix Rhodiolae, and Angelica keiskei.
In order to achieve the aim of the invention, the invention provides a preparation method of the composite immunopotentiator. The preparation method comprises the following steps:
(1) Weighing the Chinese herbal medicines according to the weight percentage, and coarsely crushing the components by adopting a traditional Chinese medicine crusher;
(2) Sequentially adding the coarsely crushed various Chinese herbal medicine raw materials into a mixer, uniformly mixing, and putting into an ultrafine grinder for grinding for later use;
(3) Activating and re-suspending the bacillus licheniformis frozen in the ultralow temperature refrigerator;
(4) Adding the activated bacillus licheniformis suspension into an MSB liquid culture medium according to an inoculation proportion of 5-8%, and carrying out shaking culture at 28-35 ℃ and 140-180 rpm;
(5) Culturing Bacillus licheniformis to logarithmic phase, centrifuging, removing supernatant, re-suspending the bacterial liquid with sterile seawater, and adjusting bacterial liquid concentration by turbidimetry;
(6) The processed Chinese herbal medicine, bacillus licheniformis and mineral element ferrous gluconate and copper gluconate are added into stichopus japonicus feed according to a proportion, and are uniformly mixed.
In the step (1), the crushing granularity is 50-100 meshes. In the step (2), the crushing granularity is 200-400 meshes.
In order to achieve the above object, the present invention further provides a method for using the composite immunopotentiator. The application method comprises the following steps:
(1) The weight of the stichopus japonicus monomer is 3-10 g, and the daily feed dosage is 4-8% of the weight of the stichopus japonicus body. The compound Chinese herbal medicine, bacillus licheniformis and ferrous gluconate as well as copper gluconate are added into the feed according to a proportion, a small amount of seawater is added, and the stichopus japonicus is fed after being uniformly stirred.
(2) Feeding for 1 time every day, wherein the feeding time is 16:00-18:00 pm, aeration is kept during the feeding period, water is changed periodically, the temperature of seawater is 15-20 ℃, the pH is 7.8-8.1, and the salinity is 29-32. Dissolved oxygen is not lower than 5mg/L, and ammonia nitrogen is not higher than 0.5mg/L.
The technical scheme mechanism is as follows:
In the present invention, organic acids produced by Bacillus licheniformis, such as citric acid, are involved in tricarboxylic acid cycle and in the generation and conversion of ATP. The reaction mechanism is shown in the following formula:
the organic acid can be respectively combined with Fe 2+ in ferrous gluconate and Cu 2+ in copper gluconate to form a complex product with high biological value, which is easy to absorb, while improving the environment of the digestive tract.
In the invention, the baikal skullcap root, the amur corktree bark, the rhubarb and the dyers woad leaf contain flavone, rhein and alkaloid substances, which can effectively inhibit the growth of conditional pathogenic bacteria, improve the capability of resisting virus and endotoxin infection of organisms and strengthen the barrier defensive capability of the organisms. The angelica sinensis, the liquorice, the Angelica keiskei and the rhodiola rosea are rich in various vitamins, minerals and good amino acids, can effectively participate in nutrient metabolism, promote nutrient digestion and proliferation of intestinal probiotics, can effectively improve the relevant enzyme activity and antioxidant capacity of organisms, and has an immunoregulation function. Bacillus licheniformis is easy to grow in vivo, and its surface antigen or metabolite acts as immunogen to stimulate the immune defense system of body and strengthen the nonspecific immunity of body. Bacillus licheniformis can produce various digestive enzymes and growth factors, promote nutrient degradation and assist organism nutrient metabolism. Bacillus licheniformis can inhibit the proliferation of partial pathogenic bacteria by nutrition competition, space competition and other modes, and has promoting and symbiotic effects on beneficial bacteria such as lactobacillus. Iron is a component of the cytochrome system and catalase, peroxidases, and plays an important role in respiration and biological oxidation processes. Iron deficiency affects protein synthesis and energy utilization, and causes inorganic salts and vitamin disorders. Copper is an important component of metalloenzymes related to energy metabolism and iron metabolism, is an active factor constituting cytochrome oxidase, superoxide dismutase, catalase and the like, and can improve biological oxidation function and metabolic function of organism tissue cells. Meanwhile, copper can promote the utilization of iron in the body.
Compared with the prior art, the invention has the following advantages and effects:
(1) The components of the composite immunopotentiator have synergistic effect. The composite Chinese herbal medicine, bacillus licheniformis and mineral elements applied by the invention are not simply and mechanically combined, but are combined by scientific selection and reasonable compatibility. The components complement each other and promote each other, and have obvious synergistic effect. The compound immunopotentiator has no mildew, deterioration, putrefaction and other phenomena, has good adsorptivity and stability, high sedimentation speed and lower collapsibility and solvent loss when added into feed, is favorable for improving the utilization rate of the feed, and is suitable for the ingestion habit of benthonic licking of stichopus japonicus.
(2) The effective components of the Chinese herbal medicine contain glucoside compounds, are difficult to absorb in the intestines of stichopus japonicus, and have low bioavailability. Whereas the alpha-glycosidases and beta-glycosidases produced by Bacillus licheniformis during metabolism are the most important glycoside hydrolases in the metabolic reaction of Chinese herbal medicine. The exogenous digestive enzymes can effectively activate the digestive enzyme activity of the stichopus japonicus, degrade the cell walls of the Chinese herbal medicines, remove starch, pectin, protease and other components, decompose macromolecular substances in the Chinese herbal medicines into active peptide, amino acid and other small molecular substances which are easier to absorb, improve the dissolution rate of bioactive substances, release the active ingredients of the Chinese herbal medicines to the maximum extent, and facilitate the absorption of organisms.
(3) The exogenous digestive enzyme produced by the bacillus licheniformis has various biological functions such as oxidation, methylation, esterification, reduction and the like, and can catalyze different substrates to convert toxic components of part of Chinese herbal medicines into pharmacological active substances, so that the toxic and side effects of the Chinese herbal medicines are reduced. In turn, the Chinese herbal medicine can provide a specific substrate for the bacillus licheniformis to grow, and the survival rate of the bacillus licheniformis is improved. In addition, the Chinese herbal medicine contains various nutritional ingredients such as protein, trace elements, vitamins and the like and bioactive ingredients, can provide nutrients for the growth and the reproduction and the metabolism of bacillus licheniformis, can increase the types and the quantity of intestinal probiotics, inhibit the growth and the reproduction of harmful bacteria and maintain the microecological balance of organisms.
(4) Ferrous gluconate and copper gluconate are derivatives of gluconic acid, belong to organic acid salts, and have strong solubility, small toxic and side effects and high bioavailability. Ferrous gluconate and copper gluconate serve as cofactors for enzyme catalysis to directly participate in maintenance and regulation of enzyme activities related to immunity and metabolism in an organism, so that the pH value of an intestinal tract can be effectively reduced, an acidic environment which is more conducive to rapid proliferation of bacillus licheniformis is created, the bioavailability is improved, and absorption of ferrous gluconate and copper gluconate by stichopus japonicus is effectively promoted.
(5) The invention combines the compound Chinese herbal medicine, bacillus licheniformis, ferrous gluconate and copper gluconate to be applied to the stichopus japonicus cultivation process, effectively improves the immune function of the stichopus japonicus on the premise of ensuring no pollution, no residue, no drug resistance and no toxic or side effect, and has important significance for improving the cultivation effect and increasing the economic benefit.
Drawings
FIG. 1 effect of composite immunopotentiator on acid phosphatase activity of Apostichopus japonicus;
FIG. 2 effect of composite immunopotentiator on alkaline phosphatase activity of Apostichopus japonicus;
FIG. 3 effect of composite immunopotentiator on superoxide dismutase activity of Apostichopus japonicus;
FIG. 4 effect of composite immunopotentiator on stichopus japonicus lysozyme activity;
FIG. 5 effect of composite immunopotentiator on stichopus japonicus amylase activity;
FIG. 6 effect of composite immunopotentiator on stichopus japonicus lipase activity;
FIG. 7 effects of a composite immunopotentiator on the richness and diversity of the intestinal flora of stichopus japonicus, wherein FIG. A is an index of richness of the intestinal flora of stichopus japonicus, and FIG. B is an index of diversity of the intestinal flora of stichopus japonicus;
FIG. 8 effect of composite immunopotentiator on cumulative mortality of offending stichopus japonicus;
FIG. 9 effect of composite immunopotentiator on the onset of the offending stichopus japonicus;
FIG. 10 inhibition of the virulent vibrio splendidus by composite immunopotentiator.
Detailed Description
The invention is further illustrated by the following specific examples in conjunction with the accompanying drawings. The present invention includes, but is not limited to, the following embodiments. Changes may be made in the present invention in accordance with the prior art without departing from the spirit of the invention and still fall within the scope of the invention.
In the following examples, stichopus japonicus was obtained from the center for breeding by the national institute of marine aquatic science, liaoning, and single stichopus japonicus was cultured in four aquariums (100 cm. Times.80 cm. Times.75 cm) of the same specifications, each 50 heads. The aquariums are respectively numbered A, B, C, D. After the stichopus japonicus is temporarily raised for one week, a compound immunopotentiator is added.
Example 1:
Weighing the following Chinese herbal medicines, wherein the weight of the Chinese herbal medicines is 15g of baical skullcap root, 10g of amur corktree bark, 15g of Chinese angelica, 10g of rhubarb, 5g of dyers woad leaf, 5g of liquoric root, 15g of rhodiola rosea and 10g of Angelica keiskei. Coarse pulverizing the above materials with a traditional Chinese medicine pulverizer to obtain powder with particle size of 50 mesh, sequentially adding the coarse pulverized Chinese medicinal materials into a mixer, mixing, and pulverizing with an ultrafine pulverizer to obtain powder with particle size of 200 mesh. The bacillus licheniformis frozen at the temperature of minus 80 ℃ is activated and resuspended, and the activated bacillus licheniformis bacterial suspension is added into MSB liquid culture medium according to the inoculation proportion of 5 percent, and is cultured at the temperature of 28 ℃ and 140rpm in a shaking way. Culturing Bacillus licheniformis to logarithmic phase, centrifuging, removing supernatant, re-suspending the bacterial liquid with sterile seawater, and adjusting bacterial liquid concentration by turbidimetry. The processed Chinese herbal medicine and bacillus licheniformis and ferrous gluconate and copper gluconate are added into stichopus japonicus feed according to a proportion, a small amount of seawater is added, and the stichopus japonicus is fed after being uniformly stirred. The addition concentration of each component in the feed per unit weight is as follows: 40mg/g of compound Chinese herbal medicine; bacillus licheniformis 10 6 cfu/g; ferrous gluconate 0.5mg/g; copper gluconate 0.2mg/g.
Feeding into a water tank A according to 4% of the stichopus japonicus body mass. Sink D was used as a control, without immunopotentiator added. The feeding experiment was continued for 30 days with 3 groups of replicates, 3 replicates per treatment. Feeding for 1 time every day, wherein the feeding time is 16:30 pm, aeration is kept during feeding, water is changed every 2 days, the seawater temperature is 15 ℃, the pH is 7.8, and the salinity is 29. Dissolved oxygen is not lower than 5mg/L, and ammonia nitrogen is not higher than 0.5mg/L.
The stichopus japonicus mass was measured at the beginning and end of the feeding experiment, and the rate of increase in stichopus japonicus mass and specific growth rate were calculated with reference to the following formula:
mass increase rate (WGR) =100× (W t-W0)/W0
Specific Growth Rate (SGR) =100× (LnW t-LnW0)/t
Wherein W 0 and W t represent initial and final body masses of Stichopus japonicus, respectively, and t is the number of days of experiment.
The intestinal contents of stichopus japonicus were collected with a sterile centrifuge tube for high throughput sequencing of the intestinal flora after the end of the feeding experiment. 3 stichopus japonicus are randomly selected in each aquarium on the 0 th day, the 10 th day, the 20 th day and the 30 th day after the immunopotentiator is added, and the abdominal cavity is dissected to obtain body cavity fluid and intestinal tissues. Mixing the body cavity liquids of the stichopus japonicus, centrifuging at 4deg.C and 3500r/min for 10min, and collecting supernatant for measuring immune enzyme activity. The intestinal tissues of Apostichopus japonicus were pooled, washed with phosphate buffer (pH 7.5), blotted dry with filter paper, and homogenized with 10-fold phosphate buffer. The homogenate was centrifuged at 3000r/min for 20min at 4℃and the supernatant was used to determine the digestive enzyme activity. The immune enzyme and digestive enzyme activities of stichopus japonicus are measured by using a test box of Nanjing established technology limited company. The acid phosphatase activity and the alkaline phosphatase activity are measured by adopting a disodium phosphate method, the superoxide dismutase activity is measured by adopting a WST-1 method, the lysozyme activity is measured by adopting a turbidimetry method, the amylase activity is measured by adopting an iodine-starch colorimetric method, the lipase activity is measured by adopting a methyl resorufin substrate method, and the specific measuring steps are carried out according to a kit instruction book. The enzyme activity detection experiment is repeated for 3 times, and the enzyme activity is expressed in terms of specific activity and the unit is U/mg protein.
After the 30-day feeding experiment was completed, the stichopus japonicus was subjected to a toxicity test with vibrio splendidus. The semi-lethal concentration of Vibrio splendidus to stichopus japonicus was determined to be 2X 10 7 cfu/mL by a preliminary experiment. The toxicity attack experiment is carried out by setting 3 groups in parallel, taking 25 stichopus japonicus per group, the injection dosage of vibrio splendidus is 0.1 ml/head, and recording the vibrio morbidity and the cumulative mortality of the stichopus japonicus within 15 days after toxicity attack. After the toxicity attack experiment is finished, 3 stichopus japonicus selenka are randomly taken from each group, intestinal tissues are obtained through dissection, and the intestinal tissues are homogenized for 10min by using 30mLPBS buffer solution. Diluting intestinal tissue homogenate by 10 times with sterile physiological saline, spreading 0.05ml of the diluted solution on TCBS vibrio selective culture medium, culturing at 28deg.C in an inverted manner, and counting the number of intestinal vibrio of stichopus japonicus after 2 days.
Data were analyzed by SPSS19.0 software for One-way analysis of variance (One-wayANOVA) and Duncan multiple comparisons, with results expressed as mean.+ -. Standard deviation (X.+ -. SD), differences were considered significant when P <0.05, expressed in different lower case letters. All studies of intestinal microbiota were visually analyzed using the "ggplot2" package of the R platform.
Example 2:
Weighing the following Chinese herbal medicines, wherein the weight of the Chinese herbal medicines is 17g of baical skullcap root, 13g of amur corktree bark, 18g of Chinese angelica, 15g of rhubarb, 7g of dyers woad leaf, 10g of liquoric root, 17g of rhodiola rosea and 15g of Angelica keiskei. Coarse pulverizing the above materials with a pulverizer with granularity of 60 mesh, sequentially adding the coarse pulverized Chinese herbal materials into a mixer, mixing, and pulverizing with superfine pulverizer with granularity of 300 mesh. The bacillus licheniformis frozen at the temperature of minus 80 ℃ is activated and resuspended, and the activated bacillus licheniformis bacterial suspension is added into MSB liquid culture medium according to the inoculation proportion of 6 percent, and is cultured at the temperature of 31 ℃ and 160rpm in a shaking way. Culturing Bacillus licheniformis to logarithmic phase, centrifuging, removing supernatant, re-suspending the bacterial liquid with sterile seawater, and adjusting bacterial liquid concentration by turbidimetry. The processed Chinese herbal medicine and bacillus licheniformis and ferrous gluconate and copper gluconate are added into stichopus japonicus feed according to a proportion, a small amount of seawater is added, and the stichopus japonicus is fed after being uniformly stirred. The addition concentration of each component in the feed per unit weight is as follows: compounding 50mg/g of Chinese herbal medicine; bacillus licheniformis 10 8 cfu/g; ferrous gluconate 1mg/g; copper gluconate 0.4mg/g.
Feeding feed to the water tank B according to 5% of the stichopus japonicus body mass. Sink D was used as a control, without immunopotentiator added. The feeding experiment was continued for 30 days with 3 groups of replicates, 3 replicates per treatment. Feeding for 1 time every day, wherein the feeding time is 16:30 pm, aeration is kept during feeding, water is changed every 2 days, the seawater temperature is 18 ℃, the pH is 8.0, and the salinity is 30. Dissolved oxygen is not lower than 5mg/L, and ammonia nitrogen is not higher than 0.5mg/L. The enzyme activity detection, toxicity attack experiment and the like are the same as those of the example 1.
Example 3:
Weighing the following Chinese herbal medicines, wherein the weight of the Chinese herbal medicines is 25g of baical skullcap root, 20g of amur corktree bark, 25g of Chinese angelica, 25g of rhubarb, 10g of dyers woad leaf, 15g of liquoric root, 25g of rhodiola rosea and 20g of Angelica keiskei. Coarse pulverizing the above materials with a pulverizer with granularity of 100 mesh, sequentially adding the coarse pulverized Chinese herbal materials into a mixer, mixing, and pulverizing with superfine pulverizer with granularity of 400 mesh. The bacillus licheniformis frozen at the temperature of minus 80 ℃ is activated and resuspended, and the activated bacillus licheniformis bacterial suspension is added into MSB liquid culture medium according to the inoculation proportion of 8 percent, and is cultured at the temperature of 35 ℃ and 180rpm in a shaking way. Culturing Bacillus licheniformis to logarithmic phase, centrifuging, removing supernatant, re-suspending the bacterial liquid with sterile seawater, and adjusting bacterial liquid concentration by turbidimetry. The processed Chinese herbal medicine and bacillus licheniformis and ferrous gluconate and copper gluconate are added into stichopus japonicus feed according to a proportion, a small amount of seawater is added, and the stichopus japonicus is fed after being uniformly stirred. The addition concentration of each component in the feed per unit weight is as follows: compounding 60mg/g of Chinese herbal medicine; bacillus licheniformis 10 7 cfu/g; ferrous gluconate 1.5mg/g; copper gluconate 0.8mg/g.
Feeding into a water tank C according to 8% of the stichopus japonicus body mass. Sink D was used as a control, without immunopotentiator added. The feeding experiment was continued for 30 days with 3 groups of replicates, 3 replicates per treatment. Feeding for 1 time every day, wherein the feeding time is 16:30 pm, aeration is kept during feeding, water is changed every 2 days, the seawater temperature is 20 ℃, the pH is 8.1, and the salinity is 32. Dissolved oxygen is not lower than 5mg/L, and ammonia nitrogen is not higher than 0.5mg/L. The enzyme activity detection, toxicity attack experiment and the like are the same as those of the example 1.
Analysis of results: the effects of examples 1,2 and 3 on the rate of increase in the quality and the specific growth rate of stichopus japonicus are shown in table 1, and the rate of increase in the quality and the specific growth rate of stichopus japonicus added with the composite immunopotentiator in the feed are significantly higher than those of the control, wherein the rate of increase in the quality and the specific growth rate of stichopus japonicus of example 3 are relatively highest. The compound immunopotentiator can strengthen the absorption and utilization of the stichopus japonicus on nutrient substances, and effectively promote the growth of the stichopus japonicus.
TABLE 1 Effect of composite immunopotentiators on the rate of increase in Apostichopus japonicus mass and specific growth rate
Examples 1,2 and 3 influence on the activities of the immune and digestive enzymes of stichopus japonicus as shown in fig. 1 to 6, acid phosphatase and alkaline phosphatase are important hydrolases for the cellular internal parameters of the body cavity of stichopus japonicus and the immune defense activities among the selected indexes of enzyme activities. Superoxide dismutase can remove redundant 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. The amylase and the lipase can effectively reflect the absorption and utilization capacity of the stichopus japonicus on nutrient substances. The stichopus japonicus enzyme activity of each example was not significantly different from that of the control example before the use of the composite immunopotentiator. The activities of acid phosphatase, alkaline phosphatase, superoxide dismutase, lysozyme and amylase and lipase in the intestinal tract of the stichopus japonicus body cavity liquid of each example were significantly higher than those of the control example at 10 days, 20 days and 30 days when the composite immunopotentiator was used, wherein the activation effect of the immune enzyme and digestive enzyme of example 3 was superior to those of examples 1 and 2. The compound immunopotentiator can obviously improve the phosphatase response capability and digestion function of stichopus japonicus and effectively inhibit oxidative damage.
The effects of examples 1, 2 and 3 on the intestinal flora richness and diversity of stichopus japonicus are shown in fig. 7, and the intestinal flora richness and diversity index of stichopus japonicus in each example is significantly higher than that of the control example. Compared with the control example, the intestinal flora composition of the stichopus japonicus in each example is more balanced, and the flora function is more stable. The influence of the composite immunopotentiator on the intestinal flora distribution of stichopus japonicus is shown in Table 2, and the proportion of oleaginous bacteria, col Wei Ershi bacteria, dark brown bacillus, armillariella and bacillus in each example is higher than that in the comparative example, wherein the oleaginous bacteria can degrade the carcinogenic substances in petroleum sediments and can also decompose fatty acid residues in fish carcasses. The Col Wei Ershi bacteria can produce extracellular active enzyme under low temperature condition, so as to reduce the risk of infection of host by pathogenic bacteria. The dark brown bacillus and the armillariella belong to chemolithotrophic bacteria, and have stronger intestinal adaptability and metabolic activity when being used as probiotics. The proportion of bacillus in each example is significantly higher than that of the control group, which indicates that bacillus licheniformis in the composite immunopotentiator has successfully colonized the intestinal tract of stichopus japonicus. The proportion of pathogenic bacteria such as the photo-bacillus, the pseudoalteromonas and the toxobacter which are common in aquaculture in each embodiment is obviously lower than that of the control embodiment. The compound immunopotentiator is added into the feed, so that the intestinal flora structure of the stichopus japonicus can be effectively improved, the growth of beneficial intestinal bacteria is promoted, and the proliferation of pathogenic bacteria is inhibited.
TABLE 2 Effect of composite immunopotentiator on intestinal flora distribution in Apostichopus japonicus
The effects of examples 1, 2, and 3 on the cumulative mortality rate of the offending stichopus japonicus are shown in fig. 8, and the first dead individuals appear in the control after the offending, and the cumulative mortality rate of the stichopus japonicus in each example is significantly lower than that in the control. The effects of examples 1, 2 and 3 on the disease conditions of the offensive stichopus japonicus are shown in fig. 9, and the stichopus japonicus of the comparative example and each example show symptoms such as mouth swelling, head shaking, intestinal vomiting, skin peeling and the like in different degrees. The area of the epidermal ulcer of the stichopus japonicus in each embodiment is smaller than that of the control, and the response capability and the relative immune protection rate to external stimulus are higher than those of the control. The inhibition effect of the composite immunopotentiator on the vibrio splendidus of the stichopus japonicus is shown in figure 10, and the number of the vibrio splendidus in the intestinal tract of the stichopus japonicus is obviously smaller than that of the control example. The compound immunopotentiator can inhibit the proliferation of vibrio splendidus and effectively improve the disease resistance of stichopus japonicus.
By combining the test results, the components of the composite immunopotentiator act together and synergistically, and the nonspecific immunity of the stichopus japonicus can be comprehensively improved by improving the growth index, enhancing the activities of immune enzymes and digestive enzymes, improving the intestinal health and improving the disease resistance.
The above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention as defined in the claims. Variations, modifications, substitutions, and optimizations, etc., which do not depart from the spirit and principle of the present invention, are included in the protection scope of the present invention.
Claims (6)
1. The preparation method of the synergistic composite immunopotentiator for stichopus japonicus is characterized in that the immunopotentiator is compounded with Chinese herbal medicines, bacillus licheniformis, ferrous gluconate and copper gluconate in the feed with the following additive concentrations in unit weight: 40-60mg/g of compound Chinese herbal medicine; bacillus licheniformis 10 6-108 cfu/g; 0.5-1.5mg/g of ferrous gluconate; copper gluconate 0.2-0.8mg/g; the organic acid generated by the bacillus licheniformis participates in the cycle of tricarboxylic acid and the generation and conversion of ATP, and is combined with Fe 2+ in ferrous gluconate and Cu 2+ in copper gluconate to form a complex product with high biological value, wherein the complex product is easy to absorb; the organic acid can be respectively combined with Fe 2+ in ferrous gluconate and Cu 2+ in copper gluconate to form a complex product with high biological value, which is easy to absorb, while improving the environment of the digestive tract; ferrous gluconate and copper gluconate serve as cofactors for enzyme catalysis to directly participate in maintenance and regulation of immunity and metabolism related enzyme activities in an organism, reduce the pH value of intestinal tracts, and create an acidic environment which is more conducive to rapid proliferation of bacillus licheniformis; wherein, the immunopotentiator is compounded with Chinese herbal medicines according to mass fraction as follows: 10-25 parts of radix scutellariae; 10-20 parts of phellodendron; 15-25 parts of Chinese angelica; 10-25 parts of rheum officinale; 5-10 parts of dyers woad leaf; 5-15 parts of liquorice; 15-25 parts of rhodiola rosea; 10-20 parts of Angelica keiskei; the preparation method comprises the following steps:
(1) Weighing the Chinese herbal medicines according to the weight percentage, and coarsely crushing the components by adopting a traditional Chinese medicine crusher;
(2) Sequentially adding the coarsely crushed various Chinese herbal medicine raw materials into a mixer, uniformly mixing, and putting into an ultrafine grinder for grinding for later use;
(3) Activating and re-suspending the bacillus licheniformis frozen in the ultralow temperature refrigerator;
(4) Adding the activated bacillus licheniformis bacterial suspension into MSB liquid culture medium according to an inoculation proportion of 5-8%, and carrying out shaking culture at 28-35 ℃ and 140-180 rpm;
(5) Culturing Bacillus licheniformis to logarithmic phase, centrifuging, removing supernatant, re-suspending the bacterial liquid with sterile seawater, and adjusting bacterial liquid concentration by turbidimetry;
(6) The processed Chinese herbal medicine, bacillus licheniformis and mineral element ferrous gluconate and copper gluconate are added into stichopus japonicus feed according to a proportion, and are uniformly mixed.
2. The preparation method of the synergistic compound immunopotentiator for stichopus japonicus according to claim 1, wherein the compound Chinese herbal medicine, bacillus licheniformis, ferrous gluconate and copper gluconate are added into the feed in unit weight at the following concentration: compounding 50mg/g of Chinese herbal medicine; bacillus licheniformis 10 7 cfu/g; ferrous gluconate 0.8mg/g; copper gluconate 0.4mg/g.
3. The method for preparing a synergistic composite immunopotentiator for stichopus japonicus according to any one of claims 1 to 2, wherein in the step (1), the pulverization particle size is 50 to 100 mesh; in the step (2), the crushing granularity is 200-400 meshes.
4. The use of a synergistic composite immunopotentiator for stichopus japonicus prepared by the preparation method according to any one of claims 1 to 3, characterized by comprising the steps of:
(1) The weight of the stichopus japonicus monomer is 3-10g, and the daily feed dosage is 4-8% of the weight of the stichopus japonicus body; adding the composite Chinese herbal medicine, bacillus licheniformis, ferrous gluconate and copper gluconate into the feed according to a proportion, adding a small amount of seawater, uniformly stirring, and feeding stichopus japonicus;
(2) Feeding for 1 time every day, wherein the feeding time is 16:00-18:00 pm, aeration is kept during the feeding period, water is changed periodically, the temperature of seawater is 15-20 ℃, the pH is 7.8-8.1, the salinity is 29-32, the dissolved oxygen is not lower than 5mg/L, and the ammonia nitrogen is not higher than 0.5mg/L.
5. The application of the synergistic compound immunopotentiator for stichopus japonicus of claim 4, which is characterized in that the additive concentration of the compound Chinese herbal medicine, bacillus licheniformis, ferrous gluconate and copper gluconate in the feed per unit weight is as follows: 40-60mg/g of compound Chinese herbal medicine; bacillus licheniformis 10 6-108 cfu/g; 0.5-1.5mg/g of ferrous gluconate; copper gluconate 0.2-0.8mg/g.
6. The application of the synergistic compound immunopotentiator for stichopus japonicus according to claim 4, wherein the compound Chinese herbal medicine comprises the following components in parts by mass: 10-25 parts of radix scutellariae; 10-20 parts of phellodendron; 15-25 parts of Chinese angelica; 10-25 parts of rheum officinale; 5-10 parts of dyers woad leaf; 5-15 parts of liquorice; 15-25 parts of rhodiola rosea; 10-20 parts of Angelica keiskei.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105326898A (en) * | 2015-10-20 | 2016-02-17 | 辽宁省海洋水产科学研究院 | Compound immunopotentiator for apostichopus japonicus |
| WO2020177236A1 (en) * | 2019-03-04 | 2020-09-10 | 山东省海洋生物研究院 | Method for promoting growth and coloration of apostichopus japonicus selenta |
| CN111728104A (en) * | 2020-07-24 | 2020-10-02 | 山东省海洋资源与环境研究院 | Micro-ecological type special feed for stichopus japonicus and preparation method |
| CN111789054A (en) * | 2020-07-29 | 2020-10-20 | 辽宁省海洋水产科学研究院 | Method for applying bacillus liquid to stichopus japonicus seedling culture |
-
2021
- 2021-10-12 CN CN202111185393.4A patent/CN113875909B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105326898A (en) * | 2015-10-20 | 2016-02-17 | 辽宁省海洋水产科学研究院 | Compound immunopotentiator for apostichopus japonicus |
| WO2020177236A1 (en) * | 2019-03-04 | 2020-09-10 | 山东省海洋生物研究院 | Method for promoting growth and coloration of apostichopus japonicus selenta |
| CN111728104A (en) * | 2020-07-24 | 2020-10-02 | 山东省海洋资源与环境研究院 | Micro-ecological type special feed for stichopus japonicus and preparation method |
| CN111789054A (en) * | 2020-07-29 | 2020-10-20 | 辽宁省海洋水产科学研究院 | Method for applying bacillus liquid to stichopus japonicus seedling culture |
Non-Patent Citations (9)
| Title |
|---|
| Effects of Bacillus spp. on growth, gut microbiota compositions and immunity of sea cucumber (Apostichopus japonicus);Lu, Zhen et.al;《AQUACULTURE RESEARCH》;20210831;第3978-3990页 * |
| 余元勋等.《中国医学分子微量元素学》.安徽科学技术出版社第一版,2009,第86页. * |
| 吕选忠.《元素生物学》.中国科学技术大学出版社第一版,2011,第434-435页. * |
| 吕选忠等.《元素生物学》.中国科学技术大学出版社第一版,2011,第418页. * |
| 复合免疫增强剂对刺参生长和非特异性免疫酶活性的影响;张宇鹏等;《大连海洋大学学报》;第178-183页 * |
| 张力等.《动物营养与饲料》.中国农业大学出版社第1版,2007,第162页. * |
| 李树伟.《生物化学》.北京邮电大学出版社第1版,2012,第58页. * |
| 石若夫.《应用微生物技术》.北京航空航天大学出版社第1版,2020,第176页. * |
| 董三白等.《食物相克与饮食宜忌 修订版》.中国轻工业出版社第1版,2007,第321页. * |
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