CN115553402A - Biological polypeptide preparation capable of improving nonspecific immunity function of haliotis discus hannai and application thereof - Google Patents
Biological polypeptide preparation capable of improving nonspecific immunity function of haliotis discus hannai and application thereof Download PDFInfo
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- CN115553402A CN115553402A CN202211312357.4A CN202211312357A CN115553402A CN 115553402 A CN115553402 A CN 115553402A CN 202211312357 A CN202211312357 A CN 202211312357A CN 115553402 A CN115553402 A CN 115553402A
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- haliotis discus
- discus hannai
- bacillus subtilis
- biological polypeptide
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- 241000143510 Haliotis discus hannai Species 0.000 title claims abstract description 62
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 56
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 56
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 230000036039 immunity Effects 0.000 title claims abstract description 19
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 28
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 28
- 239000000843 powder Substances 0.000 claims abstract description 17
- 229920002774 Maltodextrin Polymers 0.000 claims abstract description 8
- 239000005913 Maltodextrin Substances 0.000 claims abstract description 8
- 229940035034 maltodextrin Drugs 0.000 claims abstract description 8
- 239000005018 casein Substances 0.000 claims abstract description 5
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 235000021240 caseins Nutrition 0.000 claims abstract description 5
- 238000000855 fermentation Methods 0.000 claims description 36
- 230000004151 fermentation Effects 0.000 claims description 36
- 210000000601 blood cell Anatomy 0.000 claims description 17
- 206010057249 Phagocytosis Diseases 0.000 claims description 14
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000001694 spray drying Methods 0.000 claims description 6
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims description 5
- 230000008020 evaporation Effects 0.000 claims description 5
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- 229920002261 Corn starch Polymers 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
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- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 239000008120 corn starch Substances 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
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- 230000000844 anti-bacterial effect Effects 0.000 abstract description 4
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- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 8
- 229960000274 lysozyme Drugs 0.000 description 8
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- 239000004325 lysozyme Substances 0.000 description 8
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 7
- 102000013563 Acid Phosphatase Human genes 0.000 description 7
- 108010051457 Acid Phosphatase Proteins 0.000 description 7
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- 206010059866 Drug resistance Diseases 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
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- 244000144974 aquaculture Species 0.000 description 2
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- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
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- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 241001489139 Haliotis discus Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
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- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
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- 230000009471 action Effects 0.000 description 1
- 230000003260 anti-sepsis Effects 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
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Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- 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
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- 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
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- 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
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- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention relates to a biological polypeptide preparation capable of improving the nonspecific immunity function of haliotis discus hannai and application thereof, belonging to the technical field of feeds. The biological polypeptide preparation comprises 75-80 parts of active polypeptide powder, 10-15 parts of maltodextrin, 1-5 parts of casein and 5-6 parts of bacillus subtilis by weight, can improve the nonspecific immunity function of the haliotis discus hannai, improve the antibacterial capability of the haliotis discus hannai, promote the growth and development of the haliotis discus hannai and reduce the death rate of the haliotis discus hannai, and is green and environment-friendly.
Description
Technical Field
The invention belongs to the technical field of feeds, and particularly relates to a biological polypeptide preparation capable of improving the nonspecific immunity function of haliotis discus hannai and application thereof.
Background
The haliotis discus hannai is a rare marine shellfish, which is named as abalone, haliotis discus hannai, abalone, purple abalone and the like, and has high edible and medicinal values. Abalone culture is an important component of aquaculture industry, the culture scale is enlarged year by year, but the modern abalone culture industry ubiquitous has the problems of unreasonable plant layout, high-density culture, discharge of culture sewage into sea areas and the like, pathogenic microorganisms breed in a large amount in culture water, as abalone only has a nonspecific immune system, namely a congenital immune system, the resistance is relatively low, as long as the stress environmental condition or the change of culture management occurs, the immunity of abalone obviously decreases, pathogenic microorganisms reach the level of pathogenic value, diseases can be developed, and huge economic loss is caused to abalone culture. Especially in the alternate season of spring summer and autumn, as the weather changes infrequently, the meteorological factors such as air temperature, air pressure and rainfall change violently, so that the abalones are easy to be sick, and especially young abalones and adult abalones with weak constitution are easy to die.
At present, antibiotic medicines are commonly used in aquaculture production to prevent and treat abalone diseases, but the medicines cannot fundamentally solve the problem that abalone is low in immunity and easy to cause pathological changes and die, medicine residues can also influence the quality of abalone, environmental pollution is generated, and excessive use of antibiotics can cause drug resistance, so that the using effect of antibiotics is reduced, and hidden troubles are left for subsequent abalone culture.
Disclosure of Invention
The invention aims to solve the technical problems that a biological polypeptide preparation capable of improving the nonspecific immunity function of the haliotis discus hannai is provided, the nonspecific immunity function of the haliotis discus hannai is improved, the antibacterial capability of the haliotis discus hannai is improved, the growth and development of the haliotis discus hannai are promoted, the death rate of the haliotis discus hannai is reduced, and the biological polypeptide preparation is green and environment-friendly; the invention also provides application of the biological polypeptide preparation in haliotis discus hannai cultivation.
The biological polypeptide preparation comprises, by weight, 75-80 parts of active polypeptide powder, 10-15 parts of maltodextrin, 1-5 parts of casein and 5-6 parts of bacillus subtilis.
The viable count of the bacillus subtilis is 1.5 multiplied by 10 6 -1.7×10 6 CFU/g。
The active polypeptide powder mainly comprises the active polypeptide substances which are generated by a ribosome synthesis mechanism in the fermentation and metabolism process of bacillus subtilis and secreted to the outside of cells and have the functions of immunoregulation, bacteriostasis, antisepsis, intestinal regulation and the like, wherein N-terminal amino acid forms a beta-sheet structure, C-terminal amino acid forms firm alpha-helix, and a disulfide bond with a stable structure exists in each of the beta-sheet layer and the alpha-helix. The preparation method comprises the following steps:
(1) And (3) amplification culture: carrying out amplification culture on the bacillus subtilis seed solution until logarithmic phase to obtain bacillus subtilis fermentation seed solution, wherein the preferred concentration of the bacillus subtilis seed solution is 8.2 multiplied by 10 8 CFU/mL, the concentration of the bacillus subtilis fermentation seed liquid after the enlarged culture is 1.0 multiplied by 10 9 -1.1×10 9 CFU/mL, wherein the culture medium for the expanded culture is LB liquid culture medium;
(2) Fermentation culture: inoculating the seed liquid to fermentation culture medium, and fermenting to obtain Bacillus subtilis fermentation liquid with viable count of 3.6 × 10 or more 9 CFU/mL；
Fermentation culture process parameters: the inoculation amount is 10 percent of the volume of the fermentation medium, the rotating speed is 100r/min, the temperature is 37 ℃, the ventilation is 1000L/min, the air pressure is 0.03MPa, and the culture time is 40h;
the fermentation culture medium comprises the following components: 4g/L of peptone, 2g/L of glucose, 5g/L of sodium chloride, 6g/L of corn flour, 5g/L of yeast powder and 1.5g/L of magnesium sulfate;
(3) And (3) evaporation and concentration: centrifuging the bacillus subtilis fermentation liquor, evaporating and concentrating the supernatant to 1/10 of the volume of the fermentation liquor after centrifugation, setting the centrifugal rotation speed to be 7100r/min, and setting the evaporation temperature to be 80 ℃;
(4) Spray drying: adding maltodextrin or corn starch with the volume of 15% of the fermentation liquor into the concentrated solution, uniformly mixing, and performing spray drying to obtain active polypeptide powder, wherein the air inlet temperature of a dryer is 180 ℃, and the air outlet temperature is 70-80 ℃;
preferably, a spray dryer is used, the air inlet and the spray nozzle of the dryer are simultaneously opened, the material enters an atomized state from the spray nozzle, and after the material is dried by hot air, the formed active polypeptide powder is discharged from the air outlet.
It should be noted that in the fermentation culture process, real-time monitoring is needed to perform defoaming control according to the conditions in the tank; before the amplification culture and the fermentation culture, equipment and a culture medium are required to be disinfected at the temperature of 121 ℃ for 30min, and then the culture is carried out after cooling.
The biological polypeptide preparation is applied to haliotis discus hannai cultivation, and is preferably applied to improving the nonspecific immune function of haliotis discus hannai.
The application comprises the following steps: the application of the compound in improving the number of blood cells and the phagocytosis rate of Haliotis discus hannai, the application in improving the respiratory outbreak vitality of Haliotis discus hannai and the application in promoting the Haliotis discus hannai to resist vibrio parahaemolyticus.
The application method is that the adding amount of the biological polypeptide preparation accounts for 0.2-1.5% of the weight of the feed.
Compared with the prior art, the invention has the following beneficial effects:
1. the biological polypeptide preparation can improve the nonspecific immunity function of abalone, increase the number of blood cells in the organism of haliotis discus hannai, enhance the phagocytosis of external pathogenic microorganisms through the action of acid phosphatase and lysozyme in the blood cells, improve the respiratory burst activity of haliotis discus hannai, and finally achieve the remarkable effect of improving the nonspecific immunity function of haliotis discus hannai.
2. The biological polypeptide preparation can improve the resistance of haliotis discus hannai to vibrio parahaemolyticus and reduce the death rate.
3. The biological polypeptide preparation can obviously promote the growth of the haliotis discus hannai, obviously improve three important production indexes of specific growth rate, specific weight gain rate and specific meat-shell ratio, and create favorable conditions for large-scale cultivation of the haliotis discus hannai.
4. The biological polypeptide preparation is green and environment-friendly, has no problems of drug residue and drug resistance, and has no pollution to the environment.
Detailed Description
The present invention will be further described with reference to the following examples.
It should be noted that: in actual practice, the temperature control allowed a fluctuating temperature difference of 2 ℃ and all the starting materials used in the examples were commercially available, except where otherwise specified.
Example 1
The biological polypeptide preparation comprises 80 parts of active polypeptide powder, 12 parts of maltodextrin, 3 parts of casein and 5 parts of bacillus subtilis according to parts by weight;
wherein the viable count of Bacillus subtilis is 1.7 × 10 6 CFU/g；
The molecular weight of the active polypeptide powder is 4.4KD, and the preparation method comprises the following steps:
(1) And (3) amplification culture: carrying out amplification culture on the bacillus subtilis seed solution until logarithmic phase to obtain bacillus subtilis fermentation seed solution, wherein the preferred concentration of the bacillus subtilis seed solution is 8.2 multiplied by 10 8 CFU/mL, the concentration of the bacillus subtilis fermentation seed liquid after the amplification culture is 1.09 multiplied by 10 9 CFU/mL, wherein the culture medium for the expanded culture is LB liquid culture medium;
(2) Fermentation culture: inoculating the seed liquid to fermentation culture medium, and fermenting to obtain Bacillus subtilis fermentation liquid with viable count of 3.9 × 10 9 CFU/mL；
Fermentation culture process parameters: the inoculation amount is 10 percent of the volume of the fermentation medium, the rotating speed is 100r/min, the temperature is 37 ℃, the ventilation is 1000L/min, the air pressure is 0.03MPa, and the culture time is 40h;
the fermentation culture medium comprises the following components: 4g/L of peptone, 2g/L of glucose, 5g/L of sodium chloride, 6g/L of corn flour, 5g/L of yeast powder and 1.5g/L of magnesium sulfate;
(3) And (3) evaporation and concentration: centrifuging the Bacillus subtilis fermentation liquid, evaporating and concentrating the supernatant to 1/10 of the volume of the fermentation liquid, setting the centrifugal rotation speed to be 7100r/min, and setting the evaporation temperature to be 80 ℃;
(4) And (3) spray drying: adding maltodextrin or corn starch 15% of the volume of the fermentation liquor into the concentrated solution, uniformly mixing, drying by using a spray dryer, simultaneously opening an air inlet and a spray nozzle of the dryer, allowing the material to enter an atomization state from the spray nozzle, drying by hot air, discharging the formed active polypeptide powder from the air outlet, and setting the air inlet temperature of the dryer to be 180 ℃ and the air outlet temperature to be 70 ℃.
Example 2
The biological polypeptide preparation comprises 75 parts of active polypeptide powder, 10 parts of maltodextrin, 5 parts of casein and 6 parts of bacillus subtilis by weight;
wherein the viable count of Bacillus subtilis is 1.5 × 10 6 CFU/g；
The molecular weight of the active polypeptide powder is 4.4KD, and the preparation method is the same as that of the active polypeptide powder in example 1.
Example 3
The biological polypeptide preparation disclosed by the invention is applied to haliotis discus hannai cultivation, the non-specific immune function of the haliotis discus hannai is improved, the biological polypeptide preparation prepared in the embodiment 1 is added into haliotis discus hannai feed, and a cultivation test is carried out.
1. Design of experiments
The cultivation test is carried out in Qingdao Marster biotechnology limited (Jiaodong base), an indoor water circulation system is adopted, and the water environment is as follows: the water temperature is 20-25 ℃, the salinity is 31-33 per mill, the pH value is 7.5-8.0, and the dissolved oxygen is more than 6.8mg/L. The test is provided with 4 groups, namely a control group, a test group a, a test group b and a test group c, wherein each group comprises 3 culture ponds, the control group is fed with basic feed, the test group is fed with feed added with biological polypeptide preparation, and the formula of the feed of each group is shown in table 1.
The initial weight of the haliotis discus hannai before cultivation is 2.06 +/-0.01 g, the shell length is 25.42 +/-0.18 mm, the haliotis discus hannai is fed after being saturated at 17 pm every day in the cultivation process, the haliotis discus hannai attachment plate is brushed at 8% in the next morning, the residual ear is absorbed, the water pollution is avoided, and the cultivation lasts 115 days.
TABLE 1 feed formula table for haliotis discus hannai
2. Test results
And after the culture test is finished, measuring and counting various growth indexes of the haliotis discus hannai, and collecting samples for detection.
(1) The influence of the biological polypeptide preparation on the growth index of haliotis discus hannai
And measuring and counting growth indexes of each group of haliotis discus hannai, including survival rate, daily increase of shells, weight gain rate, specific growth rate, meat-shell ratio and meat yield. Wherein the shell length measuring instrument of the haliotis discus hannai is a digital vernier caliper, the accuracy is 0.01mm, the haliotis discus hannai is anesthetized by using a 5% edible vinegar water solution before measurement, and the shell length measuring method is to use the vernier caliper to measure the lengths of the front and rear edges of the shells; the body weight measurement is carried out after the haliotis discus hannai is taken out and placed on dry gauze for 10 s. The growth index data are shown in table 2.
TABLE 2 Haliotis discus hannai growth index data sheet
Note: a, b, c indicate significant differences between groups (P < 0.05).
According to the growth index data in table 2, the biological polypeptide preparation has a promotion effect on the growth of haliotis discus hannai, when the addition amount of the biological polypeptide is 0.5%, the terminal weight, shell day increase, weight gain rate and specific growth rate of the haliotis discus are all significantly higher than those of a control group (P < 0.05), the meat-shell ratio is the highest, and the survival rate is more than 97%.
(2) The influence of the biological polypeptide preparation on the nonspecific immune function of haliotis discus hannai
The detection indexes include: the number of blood cells of the haliotis discus hannai, the activity of blood cell acid phosphatase and lysozyme, the phagocytosis rate of blood cells, the respiratory burst activity of the haliotis discus hannai and the detection results are shown in table 3.
TABLE 3 detection data table for nonspecific immunologic function index of haliotis discus hannai
As can be seen from Table 2, compared with the control group, the number of blood cells, the activity of acid phosphatase and lysozyme, the phagocytosis rate of blood cells and the respiratory burst activity of Haliotis discus hannai fed with the biological polypeptide preparation are all significantly improved, especially when the addition amount of the biological polypeptide is 0.5%, the number of blood cells of Haliotis discus hannai is increased by 40% compared with the control group, the phagocytosis rate of blood cells is increased by 58.84%, the respiratory burst activity is increased by 184.6%, and the activity of acid phosphatase and lysozyme are respectively as high as 17.31 +/-0.38U/100 ml and 4.41 +/-0.40U/ml.
The shellfish does not have immune globulin and lymphoid cells, the immune function of the shellfish depends only on nonspecific immunity, and in most shellfish, the phagocytosis performed by granulocytes in blood cells is the main defense line of shellfish against external invasion, and plays an important role in immune epidemic prevention of shellfish. After the granulocytes have completed phagocytosis of foreign bodies, they are processed in two ways: firstly, a phagosome is fused with a lysosome, and the content of the phagosome is digested and degraded by hydrolase in the lysosome; secondly, a large amount of active oxygen free radicals (superoxide anions, hydrogen peroxide, singlet oxygen and hydroxyl free radicals) are generated by respiratory burst accompanied by phagocytosis, and the swallowed microorganisms are killed.
The blood cells play an important role in the nonspecific immunity of the shellfish, so the number of the blood cells can be increased to enhance the nonspecific immunity function of the shellfish. The acid phosphatase and the lysozyme are also important components in the nonspecific immune system of the shellfish and have dual functions of decomposition and defense, and the acid phosphatase is a marker enzyme of lysosomes and can destroy or eliminate foreign matters invading into the body; lysozyme can dissolve the cell wall of bacteria, and after a phagosome is fused with a lysosome, the content of the lysozyme can be digested and degraded by lysozyme and acid phosphatase in the lysosome. Phagocytosis of blood cells is the main defense line of shellfish against external invasion, so the non-specific immunity of Haliotis discus hannai is enhanced after the phagocytosis rate is increased. The respiratory burst activity is positively correlated with the phagocytosis rate of blood cells, and the respiratory burst activity accompanied with the phagocytosis is improved by promoting the phagocytosis of the blood cells, so that the effect of promoting the nonspecific immunity of Haliotis discus hannai.
(3) The influence of the biological polypeptide preparation on the antibacterial capacity of Haliotis discus hannai
After the culture test is finished, 100 Haliotis discus hannai Hance are randomly extracted from each group for challenge test, pathogenic bacteria used for challenge is vibrio parahaemolyticus, which is provided by aquatic virus and immunity laboratory of China ocean university, and bacterial liquid concentration is 10 after pre-test screening 8 cfu/mL, the period of counteracting the toxic substance is 10 days, the temperature of the seawater is 24 ℃, the accumulated mortality of each group of Haliotis discus hannai is counted after the test is finished, and the result is shown in Table 3.
TABLE 3 cumulative mortality of Haliotis discus hannai in challenge test
| Item | Control group | Test group a | Test group b | Test group c |
| Cumulative mortality | 36％ | 13％ | 22％ | 26％ |
As can be seen from Table 3, after the toxicity attack test, the cumulative mortality of the test group fed with the biological polypeptide preparation of the invention is significantly lower than that of the control group, and the maximum reduction amplitude reaches 63.9%, which indicates that the biological polypeptide preparation of the invention can enhance the antibacterial ability of Haliotis discus hannai, enhance the resistance ability to vibrio parahaemolyticus, and reduce the mortality of Haliotis discus hannai.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A biological polypeptide preparation capable of improving the nonspecific immunity function of haliotis discus hannai is characterized in that: the composition comprises, by weight, 75-80 parts of active polypeptide powder, 10-15 parts of maltodextrin, 1-5 parts of casein and 5-6 parts of bacillus subtilis.
2. The biological polypeptide formulation of claim 1, wherein: the viable count of Bacillus subtilis is 1.5 × 10 6 -1.7×10 6 CFU/g。
3. The biological polypeptide formulation of claim 1, wherein: the active polypeptide powder mainly contains polypeptide substances which are generated and secreted to the outside of cells through a ribosome synthesis mechanism in the fermentation and metabolism process of bacillus subtilis.
4. The biological polypeptide preparation of claim 3, wherein: the preparation method comprises the following steps:
(1) And (3) amplification culture: carrying out amplification culture on the bacillus subtilis seed liquid to logarithmic phase to obtain bacillus subtilis fermentation seed liquid;
(2) Fermentation culture: inoculating the Bacillus subtilis fermentation seed liquid into a fermentation culture medium for fermentation culture to obtain a Bacillus subtilis fermentation liquid with a viable count of not less than3.6×10 9 CFU/mL；
(3) And (3) evaporation and concentration: centrifuging the bacillus subtilis fermentation liquor, and evaporating and concentrating the supernatant to 1/10 of the volume of the fermentation liquor after centrifugation;
(4) Spray drying: adding maltodextrin or corn starch with the volume of 15% of the fermentation liquor into the concentrated solution, uniformly mixing, and carrying out spray drying to obtain the active polypeptide powder.
5. The biological polypeptide preparation of claim 4, wherein: the fermentation culture medium comprises the following components: 4g/L of peptone, 2g/L of glucose, 5g/L of sodium chloride, 6g/L of corn flour, 5g/L of yeast powder and 1.5g/L of magnesium sulfate.
6. The biological polypeptide formulation of claim 4, wherein: the air inlet temperature of the spray drying is 180 ℃, and the air outlet temperature is 70-80 ℃.
7. Use of a biological polypeptide formulation according to claim 1, wherein: is applied to the culture of haliotis discus hannai.
8. Use of a biological polypeptide preparation according to claim 7, wherein: is applied to improve the nonspecific immunity function of the haliotis discus hannai.
9. Use of a biological polypeptide formulation according to claim 8, wherein: the application comprises the following steps: the application of the compound in improving the number of blood cells and the phagocytosis rate of Haliotis discus hannai, the application in improving the respiratory outbreak vitality of Haliotis discus hannai and the application in promoting the Haliotis discus hannai to resist vibrio parahaemolyticus.
10. Use of a biological polypeptide formulation according to any one of claims 7 to 9, wherein: the application method is that the adding amount of the biological polypeptide preparation accounts for 0.2-1.5% of the weight of the feed.
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