CN113875909A - 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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- CN113875909A CN113875909A CN202111185393.4A CN202111185393A CN113875909A CN 113875909 A CN113875909 A CN 113875909A CN 202111185393 A CN202111185393 A CN 202111185393A CN 113875909 A CN113875909 A CN 113875909A
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- stichopus japonicus
- chinese herbal
- immunopotentiator
- bacillus licheniformis
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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 of feed: 40-60 mg/g of composite Chinese herbal medicine and 10 percent of bacillus licheniformis6～108cfu/g, ferrous gluconate 0.5-1.5 mg/g, and copper gluconate 0.2-0.8 mg/g. Weighing the Chinese herbal medicines, carrying out coarse grinding, then uniformly mixing the Chinese herbal medicines according to the weight percentage, and grinding by using an ultrafine grinder. Activating and resuspending the bacillus licheniformis, and adjusting the concentration of the bacterial liquid. Mixing the treated composite Chinese herbal medicine, bacillus licheniformis and oreAdding ferrous gluconate and copper gluconate into the feed of the stichopus japonicus according to a certain proportion, adding a small amount of seawater, stirring uniformly and feeding the stichopus japonicus. The components of the compound immunopotentiator act together and synergize to improve the disease resistance to comprehensively improve the nonspecific immunity function of the stichopus japonicus.
Description
Technical Field
The invention relates to a synergistic compound immunopotentiator for stichopus japonicus, in particular to an immunopotentiator prepared by matching Chinese herbal medicines, bacillus licheniformis, mineral elements of ferrous gluconate and copper gluconate, 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 and Stichopus, is rich in natural active substances such as protein, minerals, vitamins, etc., and has high nutritive and medicinal values. With the continuous expansion of the stichopus japonicus breeding scale and the non-standard operation under the intensive breeding mode, the problems of germplasm degradation, frequent disease, cost increase and the like occur in succession in the stichopus japonicus breeding process, and the healthy development of the stichopus japonicus breeding industry is seriously hindered. Stichopus japonicus belongs to invertebrate echinoderm, lacks specific immune system, and relies on natural immune mechanism to identify and eliminate foreign matters entering into organism. The quality of the stichopus japonicus feed directly influences the health of the cultivated stichopus japonicus and the quality of the cultivation environment, and the reasonably compatible feed additive can greatly improve the nonspecific immunity function of the stichopus japonicus by improving the intestinal health, improving the growth index and enhancing the disease resistance, thereby improving the cultivation benefit of the stichopus japonicus. Antibiotic drugs have great limitations in stichopus japonicus breeding due to problems of bacterial drug resistance, food and environmental safety and the like. At present, the immunopotentiator for stichopus japonicus in the market has poor quality and lacks of industrial specification and management. Therefore, the immune potentiator for the apostichopus japonicus, which is green, pollution-free and rich in nutrition, is imperative to be found.
CN200910017176.7 provides an antibacterial and immune double-effect compound Chinese herbal medicine for apostichopus japonicus skin rot syndrome, the medicine components are andrographis paniculata, dyers woad leaf, honeysuckle and ligusticum wallichii; the weight ratio of the medicines is 2: 1: 3: 2; the medicine is in powder form, and the granularity is 200 meshes. The invention relates to a special medicine for preventing and treating the skin rot syndrome of stichopus japonicus in an oral administration mode, which can be used in young and adult periods and has good prevention effect in the high-incidence period of the skin rot syndrome; the cure rate of the sick 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, is simple and convenient to use, can reduce or replace antibiotics, and is suitable for young ginseng cultivation and breeding production disease control.
CN200810012641.3 discloses a compound Chinese herbal medicine preparation for treating bacterial skin formation of stichopus japonicus, which comprises the following medicinal components in percentage by weight: 80-90% of dark plum fruit and 10-20% of Chinese gall. Decocting with water, and making into medicated bath or sprinkling in a whole pool for dissolving skin of Stichopus japonicus without residue in Stichopus japonicus; the addition amount is small, and the use is convenient; simple composition, stable source of the used raw materials, low price and simple production process.
CN201210206056.3 discloses a Chinese herbal medicine compound immunopotentiator for stichopus japonicus, which is characterized in that: comprises the following components: radix Codonopsis, Poria, fructus Schisandrae chinensis, herba Andrographitis, and flos Lonicerae; all the components of the stichopus japonicus Chinese herbal medicine composite immunopotentiator are Chinese medicines, the effects of sterilization, desinsectization and antivirus are strong, the stichopus japonicus compound immunopotentiator is non-toxic, free of side effect, free of pollution, low in cost and easy to prepare, the stichopus japonicus is echinoderm belonging to invertebrates, and the immune reaction mainly takes non-specific immunity as the main part.
At present, most of commercially available immunopotentiators for stichopus japonicus are prepared by adding probiotics preparations, Chinese herbal medicines or mineral elements into feed, and the preparation and application modes are single. Firstly, the active ingredients of the Chinese herbal medicines, such as starch, pectin, protease and the like, which are used in the prior art contain glucoside compounds, are difficult to absorb in the stichopus japonicus intestine, 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 the bacillus licheniformis needs a specific substrate to improve the survival rate of the bacillus licheniformis, and the intestinal environment of the stichopus japonicus is not suitable for the effective proliferation of the bacillus licheniformis, so that the bioavailability is low. Thirdly, the immunopotentiator used in the stichopus japonicus breeding must ensure no pollution, no residue, no drug resistance and no toxic and side effects, and the copper and iron inorganic salts 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 prepared by combining Chinese herbal medicines, bacillus licheniformis and mineral elements. The components of the compound immunopotentiator act together and synergize, and can comprehensively improve the nonspecific immunity function of the stichopus japonicus by improving the growth index, enhancing the activity of related enzymes, reducing the morbidity, improving the intestinal health and the like.
The technical solution of the invention is as follows:
a synergistic compound immunopotentiator for Stichopus japonicus is provided, wherein the additive concentration of compound Chinese herbal medicine, Bacillus licheniformis, ferrous gluconate, and copper gluconate in unit weight of feed is as follows: 40-60 mg/g of composite Chinese herbal medicine; bacillus licheniformis 106～108cfu/g; 0.5-1.5 mg/g of ferrous gluconate; 0.2-0.8 mg/g of copper gluconate.
Further, the preferable adding concentration of each component in the feed per unit weight is as follows: 50mg/g of compound Chinese herbal medicine; bacillus licheniformis 107cfu/g; ferrous gluconate 0.8 mg/g; copper gluconate 0.4 mg/g.
Furthermore, the composite immunopotentiator comprises the following Chinese herbal medicines in parts by mass: 15-25 parts of scutellaria baicalensis; 10-20 parts of phellodendron; 15-25 parts of angelica; 10-25 parts of rheum officinale; 5-10 parts of folium isatidis; 5-15 parts of liquorice; 15-25 parts of rhodiola rosea; 10-20 parts of angelica keiskei.
Further, the compound immunopotentiator comprises five or more of Chinese herbal medicines of radix scutellariae, cortex phellodendri, angelica, rhubarb, folium isatidis, liquorice, rhodiola rosea and angelica keiskei.
In order to achieve the above object, the present invention provides a method for preparing the above composite immunopotentiator. Wherein the preparation method comprises the following steps:
(1) weighing the Chinese herbal medicines according to the weight percentage, and respectively carrying out coarse grinding on the components by adopting a Chinese medicine grinder;
(2) sequentially putting various coarsely ground Chinese herbal medicine raw materials into a mixer, uniformly mixing, and putting into an ultrafine grinder for grinding for later use;
(3) activating and resuspending the bacillus licheniformis frozen in the ultra-low 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) after the bacillus licheniformis is cultured to a logarithmic growth phase, centrifuging and discarding supernatant, resuspending the bacterial solution by using sterile seawater, and adjusting the concentration of the bacterial solution by adopting a turbidimetric method for later use;
(6) the processed Chinese herbal medicines, the bacillus licheniformis, the mineral elements ferrous gluconate and copper gluconate are added into the stichopus japonicus feed in proportion and mixed evenly.
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 purpose, the invention also provides an application method of the composite immunopotentiator. The application method comprises the following steps:
(1) the mass of the stichopus japonicus monomer is 3-10 g, and the daily feed amount of the feed is 4-8% of the mass of the stichopus japonicus monomer. Adding the composite Chinese herbal medicine, the bacillus licheniformis, the ferrous gluconate and the copper gluconate into the feed in proportion, adding a small amount of seawater, uniformly stirring and feeding the stichopus japonicus.
(2) Feeding for 1 time every day, wherein the feeding time is 16: 00-18: 00 in the afternoon, air inflation is kept during the feeding period, water is changed periodically, the temperature of seawater is 15-20 ℃, the pH value is 7.8-8.1, and the salinity is 29-32. The dissolved oxygen is not lower than 5mg/L, and the ammonia nitrogen is not higher than 0.5 mg/L.
The technical scheme mechanism is as follows:
in the present invention, organic acids such as citric acid produced by Bacillus licheniformis are involved in the tricarboxylic acid cycle and ATP production and conversion. The reaction mechanism is shown in the following formula:
the organic acid can be respectively mixed with Fe in ferrous gluconate while improving the environment of digestive tract2+And Cu in copper gluconate2+The combination forms a complex product with high biological value and easy absorption.
According to the invention, the scutellaria baicalensis, the phellodendron amurense, the rheum officinale and the folium isatidis contain flavone, rhein and alkaloid substances, so that the growth of conditional pathogenic bacteria can be effectively inhibited, the capability of resisting virus and endotoxin infection of an organism is improved, and the barrier defense capability of the organism is enhanced. The angelica, the liquorice, the angelica keiskei and the rhodiola rosea are rich in various vitamins, mineral substances and good amino acids, can effectively participate in nutrient metabolism, promote nutrient digestion and the proliferation of intestinal probiotics, can effectively improve the activity and the antioxidant capacity of the enzyme related to the organism, and have the function of immunoregulation. The bacillus licheniformis is easy to colonize and grow in the organism, and the surface antigen or metabolite of the bacillus licheniformis acts as immunogen to continuously stimulate the immune defense system of the organism and enhance the nonspecific immunity of the organism. The bacillus licheniformis can produce various digestive enzymes and growth factors, promote the degradation of nutrient components and assist the nutrition metabolism of organisms. The bacillus licheniformis can inhibit the proliferation of partial pathogenic bacteria in the modes of nutrition competition, space competition and the like, and has the promotion and symbiosis effects on beneficial bacteria such as lactic acid bacteria and the like. Iron is a component of cytochrome systems and catalase and peroxidase, and plays an important role in respiration and biological oxidation processes. Iron deficiency affects protein synthesis and energy utilization, causing inorganic salt and vitamin disorders. Copper is an important component of metalloenzymes involved in energy metabolism and iron metabolism, and is an active factor constituting cytochrome oxidase, superoxide dismutase, catalase, etc., which improves the biological oxidation function and metabolic function of body tissue cells. Meanwhile, copper can promote the utilization of iron in the machine body.
Compared with the prior art, the invention has the following advantages and effects:
(1) the components of the composite immunopotentiator have a synergistic interaction effect. The composite Chinese herbal medicine, the bacillus licheniformis and the mineral elements are not simply and mechanically combined, but are combined through scientific selection and reasonable compatibility. All the components supplement 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 settling speed, low collapsibility and dissolubility when being added into feed, is favorable for improving the utilization rate of the feed, and is suitable for the ingestion habit of the apostichopus japonicus when being used for licking.
(2) The effective components of the Chinese herbal medicine contain glucoside compounds, are difficult to absorb in the intestines of the stichopus japonicus and have low bioavailability. Alpha-glycosidase and beta-glycosidase produced by the bacillus licheniformis in the metabolic process are the most important glycoside hydrolase in the Chinese herbal medicine metabolic reaction. The exogenous digestive enzymes can effectively activate the digestive enzyme activity of the stichopus japonicus, degrade cell walls of the Chinese herbal medicines, remove components such as starch, pectin and protease, decompose macromolecular substances in the Chinese herbal medicines into micromolecular substances such as active peptides and amino acids which are easy 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) Exogenous digestive enzyme generated by the bacillus licheniformis has multiple biological functions of oxidation, methylation, esterification, reduction and the like, can catalyze different substrates, and converts part of toxic components of the Chinese herbal medicines into pharmacologically active substances, thereby reducing the toxic and side effects of the Chinese herbal medicines. In turn, the Chinese herbal medicines can provide specific substrates for the growth of the bacillus licheniformis, and the survival rate of the bacillus licheniformis is improved. Moreover, the Chinese herbal medicine contains various nutrient components and bioactive components such as protein, trace elements, vitamins and the like, can provide nutrient substances for the growth, the propagation and the metabolism of the bacillus licheniformis, can increase the variety and the quantity of intestinal probiotics, inhibit the growth and the proliferation of harmful bacteria and maintain the micro-ecological balance of organisms.
(4) The ferrous gluconate and the copper gluconate are gluconic acid derivatives, belong to organic acid salts, and have the advantages of strong solubility, small toxic and side effects and high bioavailability. The ferrous gluconate and the copper gluconate are used as enzyme catalysis cofactors to directly participate in the maintenance and regulation of immunity and metabolism related enzyme activity in an organism, the pH value of an intestinal tract can be effectively reduced, an acidic environment which is more beneficial to the rapid proliferation of the bacillus licheniformis is created, the bioavailability is improved, and the absorption of the ferrous gluconate and the copper gluconate by the stichopus japonicus is effectively promoted.
(5) The compound Chinese herbal medicine, the bacillus licheniformis, the ferrous gluconate and the copper gluconate are jointly applied to the stichopus japonicus culture process, so that the immunity of the stichopus japonicus is effectively improved on the premise of ensuring no pollution, residue, drug resistance and toxic or side effect, and the method has important significance for improving the culture effect and increasing the economic benefit.
Drawings
FIG. 1 shows the effect of the compound immunopotentiator on the activity of acid phosphatase of Stichopus japonicus;
FIG. 2 shows the effect of the compound immunopotentiator on the activity of alkaline phosphatase of Stichopus japonicus;
FIG. 3 the effect of the compound immunopotentiator on the activity of superoxide dismutase of Apostichopus japonicus;
FIG. 4 the effect of the composite immunopotentiator on the lysozyme activity of Apostichopus japonicus;
FIG. 5 effect of composite immunopotentiator on Amylase Activity of Apostichopus japonicus;
FIG. 6 the effect of the composite immunopotentiator on the lipase activity of Apostichopus japonicus;
FIG. 7 shows the effect of the compound immunopotentiator on the abundance and diversity of the intestinal flora of Stichopus japonicus, in which FIG. A is the abundance index of the intestinal flora of Stichopus japonicus, and FIG. B is the diversity index of the intestinal flora of Stichopus japonicus;
FIG. 8 effect of composite immunopotentiators on cumulative mortality of challenge Stichopus japonicus;
FIG. 9 the effect of the compound immunopotentiator on the pathogenesis of challenge Stichopus japonicus;
FIG. 10 shows the inhibitory effect of the compound immunopotentiator on the challenge of Vibrio elatus.
Detailed Description
The invention is further illustrated by the following specific embodiments in conjunction with the accompanying drawings. The present invention includes, but is not limited to, the following embodiments. Modifications of the present invention in light of the prior art will be suggested without departing from the spirit and scope of the present invention.
The stichopus japonicus in the following examples is obtained from the breeding center of the research institute of marine aquatic science, liaison province, the mass of the stichopus japonicus monomer is 5.65 +/-0.74 g, and the stichopus japonicus monomer is temporarily cultured in four aquariums (100cm multiplied by 80cm multiplied by 75cm) with the same specification, and each aquarium has 50 heads. The aquariums are respectively numbered A, B, C, D. After temporarily culturing the stichopus japonicus for one week, adding a compound immunopotentiator.
Example 1:
weighing the following Chinese herbal medicines, wherein 15g of scutellaria baicalensis, 10g of phellodendron amurense, 15g of angelica, 10g of rheum officinale, 5g of folium isatidis, 5g of liquorice, 15g of rhodiola rosea and 10g of angelica keiskei. Respectively coarsely pulverizing the above components with a Chinese medicinal pulverizer to obtain 50 mesh powder, sequentially adding the coarsely pulverized Chinese medicinal materials into a mixer, mixing, and pulverizing with a micronizer to obtain 200 mesh powder. Activating and re-suspending Bacillus licheniformis frozen at-80 deg.C, adding activated Bacillus licheniformis suspension into MSB liquid culture medium at inoculation ratio of 5%, and performing shake culture at 28 deg.C and 140 rpm. After the bacillus licheniformis is cultured to the logarithmic growth phase, the supernatant is centrifuged and discarded, the bacterial solution is resuspended by sterile seawater, and the concentration of the bacterial solution is adjusted by a turbidimetric method. Adding the treated Chinese herbal medicines, the bacillus licheniformis, the ferrous gluconate and the copper gluconate into the stichopus japonicus feed in proportion, adding a small amount of seawater, and feeding the stichopus japonicus after uniformly stirring. The addition concentration of each component in the feed per unit weight is as follows: composite Chinese herbal medicine 40mg/g; bacillus licheniformis 106cfu/g; 0.5mg/g of ferrous gluconate; copper gluconate 0.2 mg/g.
Feeding the feed into the water tank A according to 4 percent of the mass of the stichopus japonicus. Sink D was used as a control, with no immunopotentiator added. The feeding experiment was continued for 30 days, 3 groups in total were established in parallel, and 3 replicates of each treatment were performed. Feeding for 1 time every day, wherein the feeding time is 16:30 in the afternoon, maintaining aeration during the feeding period, changing water every 2 days, the seawater temperature is 15 ℃, the pH value is 7.8, and the salinity is 29. The dissolved oxygen is not lower than 5mg/L, and the ammonia nitrogen is not higher than 0.5 mg/L.
Measuring the quality of the stichopus japonicus at the beginning and the end of the feeding experiment, and calculating the increase rate of the quality of the stichopus japonicus and the specific growth rate by referring to the following formulas:
mass gain ratio (WGR) of 100 × (W)t-W0)/W0
Specific Growth Rate (SGR) 100 × (LnW)t-LnW0)/t
Wherein W0And WtRespectively representing the initial body mass and the final body mass of the stichopus japonicus, wherein t is the experimental days.
Collecting the content of the stichopus japonicus intestinal tract by using a sterile centrifuge tube after the feeding experiment is finished for high-throughput sequencing of the intestinal flora. Randomly selecting 3 stichopus japonicus in each aquarium on 0 day, 10 days, 20 days and 30 days after the immunopotentiator is added, and dissecting the abdominal cavity to obtain body cavity liquid and intestinal tissue. Combining body cavity fluid of Stichopus japonicus, centrifuging at 4 deg.C and 3500r/min for 10min, and collecting supernatant for measuring activity of immunoenzyme. The intestinal tissues of Stichopus japonicus were pooled, washed with phosphate buffer (pH7.5), blotted with filter paper, and homogenized with 10-fold phosphate buffer. The homogenate was centrifuged at 3000r/min at 4 ℃ for 20min and the supernatant was used to determine the activity of the digestive enzyme. The activities of the immune enzyme and the digestive enzyme of the stichopus japonicus are measured by adopting a test box of Nanjing technology Limited. The activity of the acidic phosphatase and the activity of the alkaline phosphatase are determined by adopting a disodium phenylphosphate method, the activity of the superoxide dismutase is determined by adopting a WST-1 method, the activity of the lysozyme is determined by adopting a turbidimetric method, the activity of the amylase is determined by adopting an iodine-starch colorimetric method, the activity of the lipase is determined by adopting a methyl resorufin substrate method, and the specific determination steps are carried out according to the specification of the kit. The enzyme activity detection experiment is repeated for 3 times, and the enzyme activity is expressed in a specific activity form, and the unit is U/mg protein.
After the feeding experiment is finished for 30 days, the toxicity attacking experiment is carried out on the stichopus japonicus by vibrio splendidus. The semi-lethal concentration of vibrio splendidus on the stichopus japonicus is determined to be 2 x 10 by a preliminary test7cfu/mL. The challenge experiment is designed to be parallel to 3 groups, 25 stichopus japonicus is taken in each group, the injection dosage of vibrio splendidus is 0.1 ml/head, and the vibrio morbidity and cumulative mortality of the stichopus japonicus within 15 days after challenge are recorded. After the challenge experiment was completed, 3 stichopus japonicus was randomly taken from each group, and the intestinal tissue was obtained by dissection and homogenized with 30ml PBS buffer for 10 min. Diluting the intestinal tissue homogenate by 10 times with sterile physiological saline, coating 0.05ml of the diluent on a TCBS vibrio selective culture medium, performing inverted culture at 28 ℃, and counting the number of intestinal vibrios of the stichopus japonicus after 2 days.
Data were analyzed by One-way analysis of variance (One-way anova) using SPSS19.0 software and Duncan multiple comparisons expressed as mean ± standard deviation (X ± SD), with differences considered significant when P <0.05, expressed in different lower case letters. All the results of the study of the intestinal flora were visually analyzed using the "ggplot 2" package of the R-platform.
Example 2:
weighing the following Chinese herbal medicines, wherein 17g of scutellaria baicalensis, 13g of phellodendron amurense, 18g of angelica, 15g of rheum officinale, 7g of folium isatidis, 10g of liquorice, 17g of rhodiola rosea and 15g of angelica keiskei. Respectively coarsely pulverizing the above components with a Chinese medicinal pulverizer to obtain a pulverized particle size of 60 meshes, sequentially adding the coarsely pulverized Chinese medicinal materials into a mixer, mixing, and pulverizing with a superfine pulverizer to obtain a pulverized particle size of 300 meshes. Activating and re-suspending Bacillus licheniformis frozen at-80 deg.C, adding activated Bacillus licheniformis suspension into MSB liquid culture medium at 6%, and performing shake culture at 31 deg.C and 160 rpm. After the bacillus licheniformis is cultured to the logarithmic growth phase, the supernatant is centrifuged and discarded, the bacterial solution is resuspended by sterile seawater, and the concentration of the bacterial solution is adjusted by a turbidimetric method. Adding the treated Chinese herbal medicines, the bacillus licheniformis, the ferrous gluconate and the copper gluconate into the stichopus japonicus feed in proportion, adding a small amount of seawater, and feeding the stichopus japonicus after uniformly stirring. The addition concentration of each component in the feed per unit weight is as follows: 50mg/g of compound Chinese herbal medicine; lichenBacillus 108cfu/g; 1mg/g of ferrous gluconate; copper gluconate 0.4 mg/g.
Feeding the feed into the water tank B according to 5 percent of the mass of the stichopus japonicus. Sink D was used as a control, with no immunopotentiator added. The feeding experiment was continued for 30 days, 3 groups in total were established in parallel, and 3 replicates of each treatment were performed. Feeding for 1 time every day at 16:30 afternoon, charging air during feeding, changing water every 2 days, at seawater temperature of 18 deg.C, pH of 8.0, and salinity of 30. The dissolved oxygen is not lower than 5mg/L, and the ammonia nitrogen is not higher than 0.5 mg/L. The procedures of enzyme activity detection, challenge experiment and the like are the same as in example 1.
Example 3:
weighing the following Chinese herbal medicines, wherein 25g of scutellaria baicalensis, 20g of phellodendron amurense, 25g of angelica, 25g of rheum officinale, 10g of folium isatidis, 15g of liquorice, 25g of rhodiola rosea and 20g of angelica keiskei. Respectively coarsely pulverizing the above components with a Chinese medicinal pulverizer to obtain a pulverized particle size of 100 meshes, sequentially adding the coarsely pulverized Chinese medicinal materials into a mixer, mixing, and pulverizing with a superfine pulverizer to obtain a pulverized particle size of 400 meshes. Activating and re-suspending Bacillus licheniformis frozen at-80 deg.C, adding the activated Bacillus licheniformis suspension into MSB liquid culture medium at 8%, and performing shake culture at 35 deg.C and 180 rpm. After the bacillus licheniformis is cultured to the logarithmic growth phase, the supernatant is centrifuged and discarded, the bacterial solution is resuspended by sterile seawater, and the concentration of the bacterial solution is adjusted by a turbidimetric method. Adding the treated Chinese herbal medicines, the bacillus licheniformis, the ferrous gluconate and the copper gluconate into the stichopus japonicus feed in proportion, adding a small amount of seawater, and feeding the stichopus japonicus after uniformly stirring. The addition concentration of each component in the feed per unit weight is as follows: 60mg/g of compound Chinese herbal medicine; bacillus licheniformis 107cfu/g; 1.5mg/g of ferrous gluconate; copper gluconate 0.8 mg/g.
Feeding the feed into the water tank C according to 8 percent of the mass of the stichopus japonicus. Sink D was used as a control, with no immunopotentiator added. The feeding experiment was continued for 30 days, 3 groups in total were established in parallel, and 3 replicates of each treatment were performed. Feeding for 1 time every day, wherein the feeding time is 16:30 in the afternoon, maintaining aeration during the feeding period, changing water every 2 days, the seawater temperature is 20 ℃, the pH value is 8.1, and the salinity is 32. The dissolved oxygen is not lower than 5mg/L, and the ammonia nitrogen is not higher than 0.5 mg/L. The procedures of enzyme activity detection, challenge experiment and the like are the same as in example 1.
And (4) analyzing results: the effects of examples 1, 2 and 3 on the rate of mass increase and specific growth rate of stichopus japonicus are shown in table 1, and the rate of mass increase and specific growth rate of stichopus japonicus fed with the composite immunopotentiator is significantly higher than those of the control example, in which the rate of mass increase and specific growth rate of stichopus japonicus of example 3 are relatively highest. The compound immunopotentiator can enhance the absorption and utilization of the stichopus japonicus to nutrient substances and effectively promote the growth of the stichopus japonicus.
TABLE 1 Effect of Complex immunopotentiators on the growth Rate and specific growth Rate of Stichopus japonicus
Examples 1, 2 and 3 influence on the activities of immune enzymes and digestive enzymes of stichopus japonicus as shown in fig. 1 to 6, and among selected enzyme activity indexes, acid phosphatase and alkaline phosphatase are important hydrolases participating in immune defense activities in coelomic cells of stichopus japonicus. Superoxide dismutase can remove redundant free radicals in organism, block lipid peroxidation, and repair damaged cells. The lysozyme can destroy the cell wall of bacteria and eliminate pathogenic bacteria invading into the body. The amylase and the lipase can effectively reflect the absorption and utilization capacity of the stichopus japonicus to the nutrient substances. Before the compound immunopotentiator is used, the stichopus japonicus enzyme activity of each example is not significantly different from that of the control example. At 10 days, 20 days and 30 days of using the compound immunopotentiator, the activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, lysozyme and amylase and lipase in intestinal tracts in the body cavity liquid of the stichopus japonicus of each example is obviously higher than that of the control example, wherein the activating effect of the example 3 on the immunoenzyme and the digestive enzyme is better than that of the examples 1 and 2. The compound immunopotentiator is shown to be capable of remarkably improving the phosphatase response capability and the digestion function of the stichopus japonicus and effectively inhibiting oxidative damage.
Fig. 7 shows the effect of examples 1, 2 and 3 on the abundance and diversity of the intestinal flora of stichopus japonicus, and the abundance and diversity index of the intestinal flora of stichopus japonicus in each example are significantly higher than those in the control example. Compared with the control example, the stichopus japonicus intestinal flora composition in each example is more balanced, and the flora function is more stable. The influence of the composite immunopotentiator on the distribution of the intestinal flora of stichopus japonicus is shown in table 2, the proportion of oleophilic bacteria, colesevieria, dark brown bacillus, luminous bacteria and bacillus in each example is higher than that in the comparative example, wherein the oleophilic bacteria can degrade carcinogenic substances in petroleum sediments and can also decompose fatty acid residues in fish bodies. The coler veroni can produce extracellular active enzyme under low temperature condition, and reduce the risk of infection of host by pathogenic bacteria. The dark brown bacillus and the leucasmus belong to energy-converting organic nutritional bacteria, and have stronger intestinal adaptability and metabolic activity when being used as probiotics. The proportion of the bacillus in each embodiment is obviously higher than that of the control group, which indicates that the bacillus licheniformis in the compound immunopotentiator is successfully colonized in the intestinal tract of stichopus japonicus. The proportions of pathogenic bacteria such as actinobacillus, pseudoalteromonas and toxoplasma in the aquaculture are obviously lower than those in the comparative examples. The feed is added with the compound immunopotentiator, so that the intestinal flora structure of the stichopus japonicus can be effectively improved, the growth of intestinal beneficial bacteria is promoted, and the proliferation of pathogenic bacteria is inhibited.
TABLE 2 Effect of Complex immunopotentiators on the intestinal flora distribution of Stichopus japonicus
The effect of examples 1, 2 and 3 on the cumulative mortality of apostichopus japonicus was shown in fig. 8, where the first dead after challenge occurred in the control example, and the cumulative mortality of apostichopus japonicus in each example was significantly lower than that in the control example. The influence of examples 1, 2 and 3 on the onset of the attack-toxic Stichopus japonicus is shown in FIG. 9, and the control example showed different degrees of symptoms such as mouth swelling, shaking head, intestinal vomiting and skin formation from the Stichopus japonicus of each example. The superficial ulcer area of the stichopus japonicus in each example is smaller than that of the control example, and the response capability and the relative immune protection rate to external stimuli are higher than those of the control example. The inhibiting effect of the compound immunopotentiator on vibrio anguillarum is shown in figure 10, and the number of vibrio anguillarum in intestinal tracts of the stichopus japonicus in each example is obviously less than that in the control example. The compound immunopotentiator can inhibit the proliferation of vibrio splendidus and effectively improve the disease resistance of the stichopus japonicus.
By combining the test results, the components of the compound immunopotentiator act together and synergize synergistically, and the nonspecific immunity function of the stichopus japonicus can be comprehensively improved by improving the growth index, enhancing the activities of the immunoenzyme and the digestive enzyme, improving the intestinal health and improving the disease resistance.
The above embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the scope of the present invention, but should be construed as being limited only by the appended claims. Variations, modifications, substitutions, optimizations, etc. that may occur to those skilled in the art without departing from the spirit and principles of the invention are intended to be within the scope of the invention.
Claims (10)
1. The preparation method of the synergistic compound immunopotentiator for the stichopus japonicus is characterized in that the immunopotentiator contains the following compound Chinese herbal medicines, bacillus licheniformis, ferrous gluconate and copper gluconate, and the addition concentration of the compound Chinese herbal medicines in unit weight of feed is as follows: 40-60 mg/g of composite Chinese herbal medicine; bacillus licheniformis 106～108cfu/g; 0.5-1.5 mg/g of ferrous gluconate; 0.2-0.8 mg/g of copper gluconate.
2. The method for preparing the synergistic compound immunopotentiator for stichopus japonicus according to claim 1, wherein the additive concentrations of the compound Chinese herbal medicines, the bacillus licheniformis, the ferrous gluconate and the copper gluconate in the feed per unit weight are as follows: 50mg/g of compound Chinese herbal medicine; bacillus licheniformis 107cfu/g; ferrous gluconate 0.8 mg/g; copper gluconate 0.4 mg/g.
3. The method for preparing the synergistic compound immunopotentiator for the stichopus japonicus according to claim 1, wherein the immunopotentiator comprises the following compound Chinese herbal medicines by mass: 10-25 parts of scutellaria baicalensis; 10-20 parts of phellodendron; 15-25 parts of angelica; 10-25 parts of rheum officinale; 5-10 parts of folium isatidis; 5-15 parts of liquorice; 15-25 parts of rhodiola rosea; 10-20 parts of angelica keiskei.
4. The method of claim 3, wherein the immunopotentiator comprises five or more of the Chinese herbal medicines consisting of Scutellaria baicalensis, phellodendron amurense, Angelica sinensis, Rheum officinale, folium Isatidis, Glycyrrhiza uralensis, rhodiola rosea, and Angelica keiskei.
5. The method for preparing a synergistic compound immunopotentiator for apostichopus japonicus according to claim 1, comprising the steps of:
(1) weighing the Chinese herbal medicines according to the weight percentage, and respectively carrying out coarse grinding on the components by adopting a Chinese medicine grinder;
(2) sequentially putting various coarsely ground Chinese herbal medicine raw materials into a mixer, uniformly mixing, and putting into an ultrafine grinder for grinding for later use;
(3) activating and resuspending the bacillus licheniformis frozen in the ultra-low temperature refrigerator;
(4) adding the activated bacillus licheniformis suspension into an MSB liquid culture medium according to an inoculation proportion of 5-8%,
carrying out shaking culture at the temperature of 28-35 ℃ and at the rpm of 140-180;
(5) after the bacillus licheniformis is cultured to a logarithmic growth phase, centrifuging and discarding supernatant, resuspending the bacterial solution by using sterile seawater, and adjusting the concentration of the bacterial solution by adopting a turbidimetric method for later use;
(6) the processed Chinese herbal medicines, the bacillus licheniformis, the mineral elements ferrous gluconate and copper gluconate are added into the stichopus japonicus feed in proportion and mixed evenly.
6. The method for preparing the synergistic compound immunopotentiator for apostichopus japonicus according to claim 5, wherein in the step (1), the crushing particle size is 50-100 meshes; in the step (2), the crushing granularity is 200-400 meshes.
7. The application of the synergistic compound immunopotentiator for the stichopus japonicus is characterized by comprising the following steps:
(1) the mass of the stichopus japonicus monomer is 3-10 g, and the daily feed amount of the feed is 4-8% of the mass of the stichopus japonicus monomer. Adding the composite Chinese herbal medicine, the bacillus licheniformis, the ferrous gluconate and the copper gluconate into the feed in proportion, adding a small amount of seawater, uniformly stirring and feeding the stichopus japonicus.
(2) Feeding for 1 time every day, wherein the feeding time is 16: 00-18: 00 in the afternoon, air inflation is kept during the feeding period, water is changed periodically, the temperature of seawater is 15-20 ℃, the pH value is 7.8-8.1, and the salinity is 29-32. The dissolved oxygen is not lower than 5mg/L, and the ammonia nitrogen is not higher than 0.5 mg/L.
8. The use of the synergistic immunopotentiator for apostichopus japonicus as claimed in claim 7, wherein the immunopotentiator contains the following Chinese herbal medicines, bacillus licheniformis, ferrous gluconate, and copper gluconate added in a unit weight of feed: 40-60 mg/g of composite Chinese herbal medicine; bacillus licheniformis 106～108cfu/g; 0.5-1.5 mg/g of ferrous gluconate; 0.2-0.8 mg/g of copper gluconate.
9. The use of the synergistic immunopotentiator for apostichopus japonicus as claimed in claim 7, wherein the composition of the composite Chinese herbal medicine comprises the following components by weight: 10-25 parts of scutellaria baicalensis; 10-20 parts of phellodendron; 15-25 parts of angelica; 10-25 parts of rheum officinale; 5-10 parts of folium isatidis; 5-15 parts of liquorice; 15-25 parts of rhodiola rosea; 10-20 parts of angelica keiskei.
10. The method for preparing a synergistic immunopotentiator for Stichopus japonicus in accordance with claims 1-6, wherein the organic substance is generated by Bacillus licheniformisThe acid, which participates in the tricarboxylic acid cycle and ATP production and conversion, and Fe in ferrous gluconate2+And Cu in copper gluconate2+The combination forms a complex product with high biological value and easy absorption.
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