CN110870470A - Preparation method of feed for enhancing immunity of stichopus japonicus and application of feed for enhancing immunity of stichopus japonicus in stichopus japonicus culture - Google Patents
Preparation method of feed for enhancing immunity of stichopus japonicus and application of feed for enhancing immunity of stichopus japonicus in stichopus japonicus culture Download PDFInfo
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- CN110870470A CN110870470A CN201811015206.6A CN201811015206A CN110870470A CN 110870470 A CN110870470 A CN 110870470A CN 201811015206 A CN201811015206 A CN 201811015206A CN 110870470 A CN110870470 A CN 110870470A
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- stichopus japonicus
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- 238000002386 leaching Methods 0.000 claims abstract description 45
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- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 claims description 2
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- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/30—Culture of aquatic animals of sponges, sea urchins or sea cucumbers
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/22—Animal feeding-stuffs from material of animal origin from fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Marine Sciences & Fisheries (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Insects & Arthropods (AREA)
- Birds (AREA)
- Biodiversity & Conservation Biology (AREA)
- Inorganic Chemistry (AREA)
- Biomedical Technology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a preparation method of a feed for enhancing the immunity of stichopus japonicus and application of the feed for enhancing the immunity of stichopus japonicus in stichopus japonicus breeding. The feed prepared by the feed preparation method for enhancing the immunity of the stichopus japonicus and the breeding method can improve the organism immune response of the stichopus japonicus, enhance the disease resistance of the stichopus japonicus to a certain extent and promote the growth of the stichopus japonicus. The preparation method of the feed for enhancing the immunity of the stichopus japonicus comprises the following steps: crushing the basic raw materials, adding additives for granulation, and drying and then finishing granules; the preparation method of the additive motherwort extract in the step 2 comprises the following steps: carrying out ultrasonic leaching on motherwort, and purifying by using an alumina column; the preparation method of the additive rhizoma cibotii extract comprises the following steps: ultrasonic leaching rhizoma Cibotii, and purifying with resin column; the feed is used as bait, the initial bait feeding amount is 5% of the weight of the sea cucumbers, the feeding is properly adjusted according to the ingestion condition of each box of the sea cucumbers every day to achieve the purpose of satiation feeding, the feeding is carried out for 1 time every day, the feeding time is 17:00, the residual bait and excrement are sucked off on the next day by 8:00, and fresh seawater is supplemented.
Description
Technical Field
The invention belongs to the technical field of agricultural cultivation, and particularly relates to a preparation method of a feed for enhancing the immunity of stichopus japonicus and application of the feed for enhancing the immunity of stichopus japonicus in stichopus japonicus cultivation.
Background
Stichopus japonicus (Apostichopus japonicus), also known as Apostichopus japonicus, is customarily called Stichopus japonicus, belongs to Echinodermata, Stichopus, tenodactyles, Apostichopus, and has high economic and medicinal health-care values. With the improvement of living standard and the enhancement of health care consciousness of people, the demand of stichopus japonicus is continuously increased, thereby causing the over fishing of stichopus japonicus and leading the natural resources of stichopus japonicus to be endangered to be exhausted. In order to solve the contradiction between the demand and the resource, the artificial stichopus japonicus breeding technology is produced and rapidly developed, and becomes another large breeding variety of Chinese prawns and scallops. However, large-scale disordered breeding of stichopus japonicus induces stichopus japonicus disease breeding, and seriously restricts the health and sustainable development of the industry. Excessive abuse of chemicals and antibiotics brings environmental pollution, causes food safety hidden danger, and destroys internal stability and autoimmune reaction of the stichopus japonicus. Therefore, it is imperative to find safe, effective and healthy immunopotentiators.
Stichopus echinoderm, like other invertebrates, lacks a specific immune system, and the key defense mechanism is regulated by cellular immune response and acellular humoral response, and relies on the natural immune mechanism to recognize and eliminate foreign bodies entering the animal body, or to change the foreign bodies into harmless substances, and to recover wounds, etc. Improving the activity of body cavity cells and having important significance for enhancing the immune response of the stichopus japonicus.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of a feed for enhancing the immunity of stichopus japonicus and application of the feed for enhancing the immunity of stichopus japonicus in stichopus japonicus culture. The feed prepared by the feed preparation method for enhancing the immunity of the stichopus japonicus and the breeding method can improve the organism immune response of the stichopus japonicus, enhance the disease resistance of the stichopus japonicus to a certain extent and promote the growth of the stichopus japonicus.
The invention is realized by the following technical scheme:
a preparation method of a feed for enhancing the immunity of stichopus japonicus comprises the following steps: crushing the basic raw materials, adding additives for granulation, and drying and then finishing granules;
step 1, crushing basic raw materials: the basic raw materials comprise the following raw materials in parts by weight, wherein the gulfweed comprises the following components in parts by weight: 25-35 parts of zostera marina: 25-35 parts of sea mud: 10-20 parts of kelp powder: 8-15 parts of soybean meal: 5-10 parts of fish meal: 5-10 parts of crushed basic raw materials, wherein the granularity of the crushed basic raw materials is less than 100 meshes;
and 2, adding additives for granulation: adding an additive and water into the basic raw material obtained after the step 1 is finished, wherein the addition amount of the additive is 0.5-2% of the weight of the basic raw material, and the additive is at least one of a motherwort extract and a east Asian tree fern rhizome extract; the adding amount of the water is 25-50% of the weight of the base raw material; uniformly mixing to prepare particles with the diameter of 0.5-1.5 mm;
step 3, drying and granulating: and (3) drying the granules obtained after the step (2) is finished, and grading to obtain the feed for enhancing the immunity of the stichopus japonicus, wherein the particle size of the feed is 40-80 meshes.
In the technical scheme, the content of crude protein in the product feed is 18-22%, and the content of crude fat is 2.5-3.5%.
In the technical scheme, in the step 2, the uniformly mixed raw materials are extruded into particles with the diameter of 0.8-1 mm and the length of 1-1.2 mm by using a double-screw extruder.
In the technical scheme, a blast drier is adopted for drying in the step 3, the drying temperature is 40-50 ℃, and the drying time is 20-30 min.
In the above technical solution, the preparation method of the motherwort extract in step 2 comprises: carrying out ultrasonic leaching on motherwort, and purifying by using an alumina column;
ultrasonic leaching of motherwort: adding a first solvent into motherwort, wherein the adding amount of the first solvent is 8-12 times of the weight of the motherwort, leaching in an ultrasonic emitter, wherein the ultrasonic frequency is 100-120 KHz, the leaching time is 30-50 min, and filtering after leaching is finished to obtain filtrate, namely motherwort leachate;
purifying by an alumina column: concentrating the motherwort leach liquor to a weight which is 0.8-2 times of the weight of the motherwort, adsorbing the concentrated motherwort leach liquor by using an alumina column, eluting the alumina column with saturated adsorption by using water with a weight which is 15-25 times of the weight of the alumina column, and evaporating and drying the eluate in a water bath to obtain a solid dry product which is the motherwort extract.
In the above technical scheme, the first solvent is water, a 1 wt% hydrochloric acid solution, a 95 wt% ethanol solution or a 0.1 wt% hydrochloric acid ethanol solution.
In the technical scheme, the ultrasonic leaching process is carried out twice, namely the first solvent is added into the filter cake obtained after the first filtration again, the adding amount of the first solvent is 8-12 times of the weight of the motherwort, the motherwort is leached in an ultrasonic emitter, the ultrasonic frequency is 100-120 KHz, the leaching time is 30-50 min, and the filtrate obtained after the completion of leaching and the filtrate obtained after the first filtration are combined to obtain a motherwort leaching solution;
in the above technical solution, the preparation method of the cibotium barometz extract in the step 2 comprises: ultrasonic leaching rhizoma Cibotii, and purifying with resin column;
ultrasonic leaching of rhizoma cibotii: adding a second solvent into the rhizoma cibotii powder, wherein the adding amount of the second solvent is 5-15 times of the weight of the rhizoma cibotii powder, leaching in an ultrasonic emitter, the ultrasonic frequency is 100-120 KHz, the leaching time is 30-50 min, filtering is carried out after leaching is finished, filtrate is evaporated to dryness, water is added into residue after evaporation to dryness to dissolve the residue again, the adding amount of the water is 8-12 times of the weight of the rhizoma cibotii powder, and filtering is carried out again to obtain filtrate which is rhizoma cibotii leaching liquid;
purifying by using a resin column: putting the rhizoma cibotii leachate into a macroporous adsorption resin bed layer for adsorption, wherein the volume ratio of the rhizoma cibotii leachate to the macroporous adsorption resin bed layer is 4-6: 1, the adsorption time is 60-100 min, after adsorption is finished, the resin saturated in adsorption is firstly subjected to water elution and impurity removal, the volume ratio of water for elution and impurity removal to the macroporous adsorption resin bed layer is 2.5-3.5: 1, the elution and impurity removal time is 2.5-3.5 h, after elution and impurity removal, 10-20 wt% of ethanol aqueous solution is adopted for elution on the macroporous adsorption resin bed layer, the ethanol aqueous solution elution process is not less than two times, the volume ratio of the ethanol aqueous solution for elution to the macroporous adsorption resin bed layer is 3-5: 1, and the obtained eluent is combined to be used as the rhizoma cibotii extract.
In the above technical scheme, the second solvent is acetone, n-butanol or ethanol, and the macroporous adsorption resin is D101 type macroporous adsorption resin.
The feed is used as bait, the initial bait feeding amount is 5% of the weight of the sea cucumbers, the feeding is properly adjusted according to the ingestion condition of each box of the sea cucumbers every day to achieve satiation feeding, the feeding is carried out for 1 time every day, the feeding time is 17:00, residual bait and excrement are absorbed in 8:00 days next time, fresh seawater is supplemented, the feeding period is continuously aerated, the water temperature is controlled to be (17 +/-0.5) DEG C, the salinity is 27-29, the pH is 7.8-8.2, the dissolved oxygen is not lower than 5mg/L, the ammonia nitrogen is not higher than 0.5mg/L, and the nitrite nitrogen is not higher than 0.1 mg/L.
The invention has the advantages and beneficial effects that:
the feed is added with the extracts of motherwort and east Asian tree fern rhizome, which has certain promotion effect on the growth of the stichopus japonicus, can improve the immune response of the organism and enhance the disease resistance of the stichopus japonicus to a certain extent.
Detailed Description
In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.
The body cavity cell of echinoderm is the effector cell which participates in immune reaction, and it reacts to the stimulus to generate phagocytosis and participate in the immune function of body. When phagocytosis of coelomic cells is performed, active oxygen free radicals with killing effect such as O can be induced by bacteria or bacterial cell wall components2 -(ii) a Nitric oxide produced by NOS can inhibit the metabolism of nucleic acid and protein of bacterial virus, and has correlation with the immunity of the organism. SOD is an important antioxidant enzyme in the body, can remove redundant oxygen free radicals in the body and eliminate the harm of the SOD to the body. Therefore, NOS, SOD is also regarded as an important nonspecific immune index reflecting the immune system of Apostichopus japonicus.
Measurement of phagocytic Activity 100. mu.L of coelomic cell suspension was added to a 96-well plate, and adhered to the wall for 30min, and the supernatant was discarded. Adding 100 μ L0.001M neutral red solution, phagocytizing for 30min, washing out uncophagocytized neutral red with physiological saline, adding 100 μ L cell lysate (glacial acetic acid: absolute ethanol ═ 1:1, v/v), lysing for 20min, and measuring absorbance at 540nm with microplate reader. Phagocytic activity was expressed as absorbance per 106 individual luminal cells under experimental conditions.
Coelomic cell O2 -Measurement of yield (COP) 200. mu.L of coelomic cell suspension was added to a 96-well plate, then an equal volume of 2mg/mL NBT (dissolved in 2% NaCl in Tris-HCl buffer, pH 7.6) was added, the reaction was carried out at room temperature in the dark for 1 hour, centrifugation was carried out at 300r/min for 10min, the supernatant was removed, washing was carried out with isotonic buffer for 3 times, 200. mu.L methanol was added, centrifugation was carried out at 750r/min for 10min after immobilization for 10min, the supernatant was removed and washed with 50% methanol for 3 times, and finally the supernatant was removed by centrifugation and then dried at room temperature. After drying, 240. mu.L of LKOH (2mol/L) and 280. mu.L of DMSO were added to dissolve well. And measuring the light absorption value of the solution at the wavelength of 630nm in a microplate reader.
Determination of superoxide dismutase (SOD) and Nitric Oxide Synthase (NOS) Activity in CLS the SOD activity unit is defined as that the corresponding SOD amount is 1 enzyme activity unit (U) when the SOD inhibition rate reaches 50% in 1mL of reaction solution per milligram of protein, and the SOD activity is expressed as U/mg of protein; the unit of NOS activity in CLS is defined as 1 unit of enzyme activity (U) producing 1nmol of NO per minute per mg of protein in the sample, and the NOS activity is expressed as U/mg protein. The activity was measured using Nanjing kit.
Example one
A preparation method of a feed for enhancing the immunity of stichopus japonicus comprises the following steps: crushing the basic raw materials, adding additives for granulation, and drying and then finishing granules;
step 1, crushing of basic raw materials: the basic raw materials comprise the following raw materials in parts by weight, wherein the gulfweed comprises the following components in parts by weight: 30 parts of zostera marina: 30 parts, sea mud: 15 parts of kelp powder: 10 parts of soybean meal: 8 parts, fish meal: 7 parts, crushing the basic raw material, wherein the granularity of the crushed basic raw material is less than 100 meshes;
and 2, adding additives for granulation: adding a motherwort extract and water into the basic raw material obtained after the step 1, wherein the addition amount of the motherwort extract is respectively 0.5%, 1% and 2% of the weight of the basic raw material, and the motherwort extract is sequentially numbered as feed I, II and III; the adding amount of the water is 25-50% of the weight of the base raw material; after being uniformly mixed, a double-screw extruder (F-26 (II) university of south China reason worker) is adopted to extrude the uniformly mixed raw materials into particles with the diameter of 1mm and the length of 1-1.2 mm;
step 3, finishing after drying: and (3) drying the granules obtained after the step (2) in a blast dryer at 44 ℃, crushing the dried feed, sieving, taking the intermediate granules of 40-80 meshes as a product feed, and sealing and storing.
And finally, the content of crude protein in the feed product is 18-22%, and the content of crude fat is 2.5-3.5%.
According to the feed for cultivating the stichopus japonicus, the feed is used as the bait, the initial feeding amount is 5% of the weight of the stichopus japonicus, the feeding is properly adjusted according to the ingestion condition of each box of the stichopus japonicus every day to achieve the purpose of satiation feeding, the feeding is carried out for 1 time every day, the feeding time is 17:00, residual bait and excrement are absorbed in 8:00 days next time, fresh seawater is supplemented, the air is continuously inflated during the feeding period, the water temperature is controlled to be (17 +/-0.5) DEG C, the salinity is 27-29, the pH is 7.8-8.2, the dissolved oxygen is not lower than 5mg/L, the ammonia nitrogen is not higher than 0.5mg/L, and the nitrite nitrogen is not higher than 0.1 mg.
For judging whether the stichopus japonicus reaches the satiation feeding, the method comprises the following steps: performing microscopic examination on the feed before and after feeding, performing microscopic examination after feeding for half an hour, and considering that the feeding amount is appropriate if the color of the feed in the stomach is obvious and the color of the feed still can be seen in the stomach before feeding next time; if the stomach of the stichopus japonicus is empty before the next feeding, the feeding amount needs to be increased; if the water tank contains residual bait, the feeding is considered to be excessive.
The preparation method of the feed prepared from the basic raw materials of the control group feed is basically the same as that of the feed prepared by the method, and the method for cultivating the stichopus japonicus is completely the same without adding the additives of the motherwort extract and the cibotium barometz extract.
After 8 weeks of cultivation, the cultivated stichopus japonicus is starved for 24h, and then each repetition is counted and weighed. Sampling and determining the immune index. Dissecting Stichopus japonicus, and collecting body cavity fluid. Absorbing anticoagulant [0.068mol/L Tris-HCl, 0.02mol/L EDTA, 0.48% NaCl, 0.019mol/L KCl, pH 7.6,]until the volume ratio of the anticoagulant to the coelomic fluid is 1:1, and one part of the obtained anticoagulant coelomic fluid is directly used for counting coelomic cells and measuring phagocytic activity; then centrifuged for 10min (3000r/min, 4 ℃), the supernatant discarded and the cell pellet of the body cavity was quickly washed with frozen isotonic buffer [0.001mol/L EDTA, 0.53mol/L NaCl,0.01mol/L Tris-HCl, pH 7.6]Adjusted to 2X 106cells/mL. Applying 600 μ L coelomic cell suspension to sea cucumber coelomic cell O2 -Yield determination, the remaining samples were quickly stored in liquid nitrogen and stored in a-80 ℃ freezer for additional analysis. Coelomic cell samples were thawed at 4 ℃ and 0.1mmol/L PMSF was added, homogenized with ultrasound for 6s (output 22kHz, 0 ℃) and centrifuged for 10min (3000r/min, 4 ℃). The obtained supernatant is the supernatant of coelomic cell disruption product (CLS), and is immediately used for measuring various immunity indexes.
The indexes are shown in table 1.
TABLE 1 Effect of adding herba Leonuri extract on Stichopus japonicus Immunity: (
n＝10)
Example two
A preparation method of a feed for enhancing the immunity of stichopus japonicus comprises the following steps: pulverizing the basic raw materials, adding additives, granulating, drying, and grading;
step 1, crushing of basic raw materials: the basic raw materials comprise the following raw materials in parts by weight, wherein the gulfweed comprises the following components in parts by weight: 30 parts of zostera marina: 30 parts, sea mud: 15 parts of kelp powder: 10 parts of soybean meal: 8 parts, fish meal: 7, crushing and grinding the basic raw material, wherein the granularity of the ground basic raw material is less than 100 meshes;
step 2, adding additives for granulation: adding a rhizoma cibotii extract and water into the basic raw material obtained after the step 1 is finished, wherein the adding amount of the rhizoma cibotii extract is respectively 0.5 percent, 1 percent and 2 percent of the weight of the basic raw material, and the rhizoma cibotii extract is numbered as IV, V and VI feeds in sequence; the adding amount of the water is 25-50% of the weight of the base raw material; after being uniformly mixed, a double-screw extruder (F-26 (II) university of south China reason worker) is adopted to extrude the uniformly mixed raw materials into particles with the diameter of 1mm and the length of 1-1.2 mm;
step 3, drying, crushing and screening: and (3) drying the granules obtained after the step (2) in a blast dryer at 44 ℃, crushing the dried feed, sieving, taking the intermediate granules of 40-80 meshes as a product feed, and sealing and storing.
And finally, the content of crude protein in the feed product is 18-22%, and the content of crude fat is 2.5-3.5%.
According to the feed for cultivating the stichopus japonicus, the feed is used as the bait, the initial feeding amount is 5% of the weight of the stichopus japonicus, the feeding is properly adjusted according to the ingestion condition of each box of the stichopus japonicus every day to achieve the purpose of satiation feeding, the feeding is carried out for 1 time every day, the feeding time is 17:00, residual bait and excrement are absorbed in 8:00 days next time, fresh seawater is supplemented, the air is continuously inflated during the feeding period, the water temperature is controlled to be (17 +/-0.5) DEG C, the salinity is 27-29, the pH is 7.8-8.2, the dissolved oxygen is not lower than 5mg/L, the ammonia nitrogen is not higher than 0.5mg/L, and the nitrite nitrogen is not higher than 0.1 mg.
The preparation method of the feed prepared from the basic raw materials of the control group feed is basically the same as that of the feed prepared by the method, and the method for cultivating the stichopus japonicus is completely the same without adding the additives of the motherwort extract and the cibotium barometz extract.
After 8 weeks of cultivation, the cultivated stichopus japonicus is starved for 24h, and then each repetition is counted and weighed. Sampling and determining the immune index. Dissecting Stichopus japonicus, and collecting body cavity fluid. Absorbing anticoagulant [0.068mol/LTris-HCl, 0.02mol/L EDTA, 0.48% NaCl, 0.019mol/LKCl, pH 7.6]Until the volume ratio of the anticoagulant to the coelomic fluid is 1:1, and one part of the obtained anticoagulant coelomic fluid is directly used for counting coelomic cells and measuring phagocytic activity; then centrifuged for 10min (3000r/min, 4 ℃), the supernatant discarded and the cell pellet of the body cavity was quickly washed with frozen isotonic buffer [0.001mol/L EDTA, 0.53mol/L NaCl,0.01mol/L Tris-HCl, pH 7.6]Adjusted to 2X 106cells/mL. Applying 600 μ L coelomic cell suspension to sea cucumber coelomic cell O2 -Yield determination, the remaining samples were quickly stored in liquid nitrogen and stored in a-80 ℃ freezer for additional analysis. Coelomic cell samples were thawed at 4 ℃ and 0.1mmol/L PMSF was added, homogenized with ultrasound for 6s (output 22kHz, 0 ℃) and centrifuged for 10min (3000r/min, 4 ℃). The obtained supernatant is the supernatant of coelomic cell disruption product (CLS), and is immediately used for measuring various immunity indexes.
The indexes are shown in Table 2.
TABLE 2 Effect of rhizoma Cibotii extract on Stichopus japonicus Immunity: (
n＝10)
EXAMPLE III
A preparation method of a feed for enhancing the immunity of stichopus japonicus comprises the following steps: pulverizing the basic raw materials, adding additives, granulating, drying, and grading;
step 1, crushing of basic raw materials: the basic raw materials comprise the following raw materials in parts by weight, wherein the gulfweed comprises the following components in parts by weight: 30 parts of zostera marina: 30 parts, sea mud: 15 parts of kelp powder: 10 parts of soybean meal: 8 parts, fish meal: 7, crushing and grinding the basic raw material, wherein the granularity of the ground basic raw material is less than 100 meshes;
step 2, adding additives for granulation: adding an additive and water into the basic raw material obtained after the step 1, wherein the addition amount of the additive is respectively 0.5%, 1% and 2% of the weight of the basic raw material, and the additives are numbered as VII, VIII and IX feeds in sequence; the additive is composed of a motherwort extract and a cibotium barometz extract according to a weight ratio of 1:1, and the adding amount of water is 25-50% of the weight of the basic raw material; after being uniformly mixed, a double-screw extruder (F-26 (II) university of south China reason worker) is adopted to extrude the uniformly mixed raw materials into particles with the diameter of 1mm and the length of 1-1.2 mm;
step 3, drying, crushing and screening: and (3) drying the granules obtained after the step (2) in a blast dryer at 44 ℃, crushing the dried feed, sieving, taking the intermediate granules of 40-80 meshes as a product feed, and sealing and storing.
And finally, the content of crude protein in the feed product is 18-22%, and the content of crude fat is 2.5-3.5%.
According to the feed for cultivating the stichopus japonicus, the feed is used as the bait, the initial feeding amount is 5% of the weight of the stichopus japonicus, the feeding is properly adjusted according to the ingestion condition of each box of the stichopus japonicus every day to achieve the purpose of satiation feeding, the feeding is carried out for 1 time every day, the feeding time is 17:00, residual bait and excrement are absorbed in 8:00 days next time, fresh seawater is supplemented, the air is continuously inflated during the feeding period, the water temperature is controlled to be (17 +/-0.5) DEG C, the salinity is 27-29, the pH is 7.8-8.2, the dissolved oxygen is not lower than 5mg/L, the ammonia nitrogen is not higher than 0.5mg/L, and the nitrite nitrogen is not higher than 0.1 mg.
The preparation method of the feed prepared from the basic raw materials of the control group feed is basically the same as that of the feed prepared by the method, and the method for cultivating the stichopus japonicus is completely the same without adding the additives of the motherwort extract and the cibotium barometz extract.
After 8 weeks of cultivation, the cultivated stichopus japonicus is starved for 24h, and then each repetition is counted and weighed. Sampling and determining the immune index. Dissecting Stichopus japonicus, and collecting body cavity fluid. Suction anticoagulant [0.068mol/L Tris-HCl，0.02mol/L EDTA，0.48％NaCl，0.019mol/L KCl，pH＝7.6]Until the volume ratio of the anticoagulant to the coelomic fluid is 1:1, and one part of the obtained anticoagulant coelomic fluid is directly used for counting coelomic cells and measuring phagocytic activity; then centrifuged for 10min (3000r/min, 4 ℃), the supernatant discarded and the cell pellet of the body cavity was quickly washed with frozen isotonic buffer [0.001mol/L EDTA, 0.53mol/L NaCl,0.01mol/L Tris-HCl, pH 7.6]Adjusted to 2X 106cells/mL. Applying 600 μ L coelomic cell suspension to sea cucumber coelomic cell O2 -Yield determination, the remaining samples were quickly stored in liquid nitrogen and stored in a-80 ℃ freezer for additional analysis. Coelomic cell samples were thawed at 4 ℃ and 0.1mmol/L PMSF was added, homogenized with ultrasound for 6s (output 22kHz, 0 ℃) and centrifuged for 10min (3000r/min, 4 ℃). The obtained supernatant is the supernatant of coelomic cell disruption product (CLS), and is immediately used for measuring various immunity indexes.
The indexes are shown in Table 3.
TABLE 3 influence of herba Leonuri extract and rhizoma Cibotii extract added at a ratio of 1:1 on Stichopus japonicus immunity: (
n＝10)
Note: in tables 1, 2 and 3, a is less than 0.05 compared with the control group, and b is less than 0.01 compared with the control group.
The addition of the motherwort and rhizoma cibotii extracts in the feed has no influence on the survival rate of the stichopus japonicus, and each treatment group is 100%. The addition of herba Leonuri and rhizoma Cibotii extract can improve active oxygen yield (COP), superoxide dismutase (SOD), Nitric Oxide Synthase (NOS), and phagocytosis activity of body cavity cells of Stichopus japonicus in different degrees, and is dose dependent. Wherein the coelomic cell active oxygen yield, SOD, NOS and phagocytic activity of 1.0% and 2.0% of the added group stichopus japonicus are obviously higher than those of a control group and 0.5% of the added group (P is less than 0.05); under the condition of not increasing the adding proportion, the two are added simultaneously according to the proportion of 1:1, and each index has obvious difference (P is less than 0.01) with the control group. The comparison of the comprehensive results shows that: (1) the feed is added with the extracts of motherwort and east Asian tree fern rhizome, so that the yield of stichopus japonicus culture can be improved, and meanwhile, the nonspecific immunity and disease resistance of the stichopus japonicus can be improved; (2) in the research, the motherwort and the cibotium barometz extracts are fed during the whole-period culture period, so that the immune fatigue or other side effects cannot be caused.
Example four
A method for preparing rhizoma Cibotii extract comprises: ultrasonic leaching rhizoma Cibotii, and purifying with resin column;
ultrasonic leaching of rhizoma cibotii: adding 50ml of 60% acetone into 5.0g of rhizoma cibotii powder, leaching in an ultrasonic emitter, wherein the ultrasonic frequency is 100-120 KHz, the leaching time is 30min, filtering after leaching is finished, evaporating filtrate to dryness, adding 50ml of water into residue after evaporation to dryness, dissolving the residue again, and filtering again to obtain filtrate which is rhizoma cibotii leachate (containing crude drug content of 0.1 g/ml);
purifying by using a resin column: putting the rhizoma Cibotii leachate into a D101 type macroporous adsorbent resin bed layer for adsorption, wherein the volume ratio of the rhizoma Cibotii leachate to the D101 type macroporous adsorbent resin bed layer is 6:1, the adsorption time is 90min, after adsorption is completed, eluting saturated resin with water to remove impurities, the volume ratio of the water for eluting and removing impurities to the D101 type macroporous adsorption resin bed layer is 3:1, the elution impurity removal time is 3h, after the elution impurity removal is finished, 10 wt% of ethanol water solution is firstly used for elution, the ratio of the volume of 10 wt% ethanol water solution to the volume of the D101 type macroporous adsorption resin bed layer is 4:1, then 20 wt% ethanol water solution is adopted for elution, the volume ratio of the 20 wt% ethanol water solution used for elution to the D101 type macroporous adsorption resin bed layer is 4:1, and the eluates obtained by two times of elution are combined to be used as the cibotium barometz extract. The rhizoma Cibotii extract contains rhizoma Cibotii total tannin 69.5g per 100 g.
Example 5
A preparation method of herba Leonuri extract comprises: carrying out ultrasonic leaching on motherwort, and purifying by using an alumina column;
ultrasonic leaching of motherwort: adding 10 times of water by weight of motherwort into motherwort, leaching in an ultrasonic emitter, wherein the ultrasonic frequency is 110KHz, the leaching time is 40min, performing primary filtration after leaching to obtain primary filtrate, adding 10 times of water by weight of motherwort into a filter cake obtained after primary filtration, leaching in the ultrasonic emitter, wherein the ultrasonic frequency is 110KHz, the leaching time is 40min, performing secondary filtration after leaching, and combining the obtained secondary filtrate and the primary filtrate to obtain motherwort leachate;
purifying by an alumina column: concentrating the motherwort leach liquor to a weight 1 time of that of the motherwort, adsorbing the concentrated motherwort leach liquor by using an alumina column (neutral, 100-200 meshes, and the weight is the same as that of the motherwort leach liquor), eluting the alumina column with saturated adsorption by using water with the weight 20 times of that of the alumina column, and evaporating and drying the eluate in a water bath to obtain a solid dry product which is the motherwort extract. Each 100g of the herba Leonuri extract contains herba Leonuri total alkaloids 70.3 g.
Moreover, relational terms such as "first" and "second," and the like, may be used solely to distinguish one element from another element having the same name, without necessarily requiring or implying any actual such relationship or order between such elements.
The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
Claims (10)
1. The preparation method of the feed for enhancing the immunity of the stichopus japonicus is characterized by comprising the following steps: crushing the basic raw materials, adding additives for granulation, and drying and then finishing granules;
step 1, crushing basic raw materials: the basic raw materials comprise the following raw materials in parts by weight, wherein the gulfweed comprises the following components in parts by weight: 25-35 parts of zostera marina: 25-35 parts of sea mud: 10-20 parts of kelp powder: 8-15 parts of soybean meal: 5-10 parts of fish meal: 5-10 parts of crushed basic raw materials, wherein the granularity of the crushed basic raw materials is less than 100 meshes;
and 2, adding additives for granulation: adding an additive and water into the basic raw material obtained after the step 1 is finished, wherein the addition amount of the additive is 0.5-2% of the weight of the basic raw material, and the additive is at least one of a motherwort extract and a east Asian tree fern rhizome extract; the adding amount of the water is 25-50% of the weight of the base raw material; uniformly mixing to prepare particles with the diameter of 0.5-1.5 mm;
step 3, drying and granulating: and (3) drying the granules obtained after the step (2) is finished, and grading to obtain the feed for enhancing the immunity of the stichopus japonicus, wherein the particle size of the feed is 40-80 meshes.
2. The preparation method of the feed for enhancing the immunity of the stichopus japonicus according to claim 1, wherein the feed for enhancing the immunity of the stichopus japonicus contains 18-22% of crude protein and 2.5-3.5% of crude fat.
3. The preparation method of the feed for enhancing immunity of stichopus japonicus according to claim 1, wherein in the step 2, the uniformly mixed raw materials are extruded into granules with the diameter of 0.8-1 mm and the length of 1-1.2 mm by a double-screw extruder.
4. The preparation method of the feed for enhancing immunity of stichopus japonicus according to claim 1, wherein the drying in the step 3 is performed by using a blast dryer, the drying temperature is 40-50 ℃, and the drying time is 20-30 min.
5. The method for preparing the feed for enhancing the immunity of the stichopus japonicus according to claim 1, wherein the motherwort extract in the step 2 is prepared by: carrying out ultrasonic leaching on motherwort, and purifying by using an alumina column;
ultrasonic leaching of motherwort: adding a first solvent into motherwort, wherein the adding amount of the first solvent is 8-12 times of the weight of the motherwort, leaching in an ultrasonic emitter, wherein the ultrasonic frequency is 100-120 KHz, the leaching time is 30-50 min, and filtering after leaching is finished to obtain filtrate, namely motherwort leachate;
purifying by an alumina column: concentrating the motherwort leach liquor to a weight which is 0.8-2 times of the weight of the motherwort, adsorbing the concentrated motherwort leach liquor by using an alumina column, eluting the alumina column with saturated adsorption by using water with a weight which is 15-25 times of the weight of the alumina column, and evaporating and drying the eluate in a water bath to obtain a solid dry product which is the motherwort extract.
6. The method for preparing the feed for enhancing the immunity of the stichopus japonicus according to claim 5, wherein the first solvent is water, a 1 wt% hydrochloric acid solution, a 95 wt% ethanol solution or a 0.1 wt% hydrochloric acid ethanol solution.
7. The method for preparing the feed for enhancing immunity of stichopus japonicus according to claim 5, wherein the ultrasonic leaching process is performed twice, namely, the first solvent is added into the filter cake obtained after the first filtering, the adding amount of the first solvent is 8-12 times of the weight of the leonurus japonicus, the leonurus japonicus is leached in an ultrasonic emitter, the ultrasonic frequency is 100-120 KHz, the leaching time is 30-50 min, and the filtrate obtained by filtering after the leaching is completed and the filtrate obtained by filtering in the first filtering are combined to obtain the leonurus japonicus leaching solution.
8. The preparation method of the feed for enhancing the immunity of the stichopus japonicus according to claim 1, wherein the preparation method of the cibotium barometz extract in the step 2 comprises the following steps: ultrasonic leaching rhizoma Cibotii, and purifying with resin column;
ultrasonic leaching of rhizoma cibotii: adding a second solvent into the rhizoma cibotii powder, wherein the adding amount of the second solvent is 5-15 times of the weight of the rhizoma cibotii powder, leaching in an ultrasonic emitter, the ultrasonic frequency is 100-120 KHz, the leaching time is 30-50 min, filtering is carried out after leaching is finished, filtrate is evaporated to dryness, water is added into residue after evaporation to dryness to dissolve the residue again, the adding amount of the water is 8-12 times of the weight of the rhizoma cibotii powder, and filtering is carried out again to obtain filtrate which is rhizoma cibotii leaching liquid;
purifying by using a resin column: putting the rhizoma cibotii leachate into a macroporous adsorption resin bed layer for adsorption, wherein the volume ratio of the rhizoma cibotii leachate to the macroporous adsorption resin bed layer is 4-6: 1, the adsorption time is 60-100 min, after adsorption is finished, the resin saturated in adsorption is firstly subjected to water elution and impurity removal, the volume ratio of water for elution and impurity removal to the macroporous adsorption resin bed layer is 2.5-3.5: 1, the elution and impurity removal time is 2.5-3.5 h, after elution and impurity removal, 10-20 wt% of ethanol aqueous solution is adopted for elution on the macroporous adsorption resin bed layer, the ethanol aqueous solution elution process is not less than two times, the volume ratio of the ethanol aqueous solution for elution to the macroporous adsorption resin bed layer is 3-5: 1, and the obtained eluent is combined to be used as the rhizoma cibotii extract.
9. The method for preparing the feed for enhancing the immunity of the stichopus japonicus according to claim 8, wherein the second solvent is acetone, n-butanol or ethanol, and the macroporous adsorption resin is D101 type macroporous adsorption resin.
10. The application of the feed for enhancing the immunity of the stichopus japonicus, which is prepared by the preparation method of the feed for enhancing the immunity of the stichopus japonicus according to any one of claims 1 to 9, in stichopus japonicus cultivation is characterized in that the feed is used as a bait, the initial bait feeding amount is 5% of the weight of the stichopus japonicus, the bait feeding is adjusted to be full according to the ingestion condition of each box of the stichopus japonicus every day, the bait feeding is carried out for 1 time every day, the feeding time is 17:00, the next day is 8:00, residual bait and excrement are sucked, fresh seawater is supplemented, the feeding period is continuously aerated, the water temperature is controlled to be 17 +/-0.5 ℃, the salinity is 27-29, the pH is 7.8-8.2, the dissolved oxygen is not lower than 5mg/L, the ammonia nitrogen is not higher than 0.5mg/L, and.
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|---|---|---|---|---|
| CN112956612A (en) * | 2021-04-12 | 2021-06-15 | 山东省海洋生物研究院 | Feed for improving disease resistance of stichopus japonicus and preparation method and application thereof |
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