CN115553402B - Biological polypeptide preparation capable of improving nonspecific immunity function of Haliotis discus hand-Mazz and application thereof - Google Patents
Biological polypeptide preparation capable of improving nonspecific immunity function of Haliotis discus hand-Mazz and application thereof Download PDFInfo
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- CN115553402B CN115553402B CN202211312357.4A CN202211312357A CN115553402B CN 115553402 B CN115553402 B CN 115553402B CN 202211312357 A CN202211312357 A CN 202211312357A CN 115553402 B CN115553402 B CN 115553402B
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 51
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 51
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 241001489139 Haliotis discus Species 0.000 title claims abstract description 30
- 230000036039 immunity Effects 0.000 title claims abstract description 21
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 32
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 32
- 239000000843 powder Substances 0.000 claims abstract description 18
- 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
- 230000001737 promoting effect Effects 0.000 claims abstract description 7
- 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 42
- 230000004151 fermentation Effects 0.000 claims description 42
- 241000143510 Haliotis discus hannai Species 0.000 claims description 24
- 230000000694 effects Effects 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 19
- 210000000601 blood cell Anatomy 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 9
- 230000019254 respiratory burst Effects 0.000 claims description 9
- 238000001704 evaporation Methods 0.000 claims description 8
- 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
- 230000000242 pagocytic effect Effects 0.000 claims description 5
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims description 4
- 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 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
- 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
- 235000012054 meals 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
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- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 2
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- 206010057249 Phagocytosis Diseases 0.000 description 9
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- 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
- 102000016943 Muramidase Human genes 0.000 description 7
- 108010014251 Muramidase Proteins 0.000 description 7
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 7
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
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- 238000009423 ventilation Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 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
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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
- A23K20/142—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
-
- 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
-
- 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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- Genetics & Genomics (AREA)
- Biochemistry (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to a biological polypeptide preparation capable of improving the nonspecific immunity function of Haliotis discus hand, and application thereof, and belongs to the technical field of feeds. The biological polypeptide preparation disclosed by the invention 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, and can be used for improving the nonspecific immunity of haliotis discus, improving the antibacterial capability of haliotis discus, promoting the growth and development of haliotis discus, reducing the death rate of haliotis discus, and is 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 makinoica and application thereof.
Background
The Haliotis discus majoris is a precious marine shellfish, and the aliases are abalone, haliotis discus majoris and the like, and has high edible and medicinal values. Abalone culture is an important component of the aquaculture industry, the culture scale expands year by year, but the modern abalone culture industry generally has the problems that the layout of a culture farm is unreasonable, the culture sewage is discharged into a sea area and the like, pathogenic microorganisms are greatly propagated in a culture water body, the resistance is relatively low as the abalone has a nonspecific immune system, namely an innate immune system, and the abalone has obviously reduced immunity as long as the stress environmental condition or culture management changes, the pathogenic microorganisms reach the level of a pathogenic value, and the disease can be exploded, so that huge economic loss is caused to abalone culture. Especially in the season alternation period of spring, summer and autumn, weather elements such as air temperature, air pressure, precipitation and the like are extremely changed due to unusual weather changes, so that abalones are easy to be ill, and particularly young abalones and adult abalones with weaker physique are easy to die.
At present, antibiotics are often used for preventing and treating abalone diseases in the breeding production, but the problems of easy lesions and death due to low abalone immunity cannot be fundamentally solved by using the antibiotics, the abalone quality is also affected by the drug residues, the environmental pollution is generated, the drug resistance can be caused by excessive use of the antibiotics, the use effect of the antibiotics is reduced, and hidden danger is left for subsequent abalone breeding.
Disclosure of Invention
The invention aims to solve the technical problem of providing the biological polypeptide preparation capable of improving the nonspecific immunity of the Haliotis discus, improving the antibacterial capability of the Haliotis discus, promoting the growth and development of the Haliotis discus, reducing the death rate of the Haliotis discus, and being environment-friendly; the invention also provides application of the biological polypeptide preparation in culture of haliotis discus hannai.
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 1.5X10 6 -1.7×10 6 CFU/g。
The active polypeptide powder mainly comprises polypeptide substances which are produced by a ribosome synthesis mechanism in the fermentation and metabolism process of bacillus subtilis and secreted outside cells and have the effects of immunoregulation, bacteriostasis, corrosion prevention, intestinal tract regulation and the like, wherein N-terminal amino acid forms a beta-lamellar structure, C-terminal amino acid forms a firm alpha-helix, and disulfide bonds with a stable structure are respectively arranged on the beta-lamellar structure and the alpha-helix. The preparation method comprises the following steps:
(1) And (3) performing expansion culture: performing amplification culture on the bacillus subtilis seed liquid to a logarithmic phase to obtain bacillus subtilis fermentation seed liquid, wherein the concentration of the bacillus subtilis seed liquid is preferably 8.2X10 8 CFU/mL, the concentration of the bacillus subtilis fermentation seed liquid after the amplification culture is 1.0X10% 9 -1.1×10 9 CFU/mL, the culture medium for the enlarged culture is LB liquid culture medium;
(2) Fermentation culture: inoculating the bacillus subtilis fermentation seed liquid into a fermentation medium for fermentation culture to obtain bacillus subtilis fermentation liquid, wherein the viable count of the bacillus subtilis fermentation liquid is more than or equal to 3.6X10 9 CFU/mL；
Fermentation culture process parameters: the inoculation amount is 10% 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 medium comprises the following components: 4g/L of peptone, 2g/L of glucose, 5g/L of sodium chloride, 6g/L of corn meal, 5g/L of yeast powder and 1.5g/L of magnesium sulfate;
(3) And (3) evaporating and concentrating: centrifuging bacillus subtilis fermentation liquor, evaporating and concentrating supernatant to 1/10 of the volume of the fermentation liquor after centrifuging, setting the centrifugal speed to 7100r/min and the evaporating temperature to 80 ℃;
(4) Spray drying: adding maltodextrin or corn starch accounting for 15% of the volume of the fermentation liquor into the concentrated liquor, 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, an air inlet and a spray opening of the dryer are simultaneously opened, materials enter an atomized state from the spray opening, and active polypeptide powder formed after hot air drying is discharged from an air outlet.
It should be noted that, in the fermentation culture process, defoaming control is required to be performed in real time according to the conditions in the tank; the equipment and the culture medium are sterilized before the expansion culture and the fermentation culture, the temperature is 121 ℃ and the time is 30min, and the culture is carried out after cooling.
The biological polypeptide preparation provided by the invention is applied to culture of haliotis discus, and is preferably applied to improving the nonspecific immunity of haliotis discus.
The application comprises: the application of the method in improving the number of blood cells and phagocytic rate of the haliotis discus hannai, the application in improving the respiratory burst activity of the haliotis discus hannai and the application in promoting the haliotis discus hannai to resist vibrio parahaemolyticus.
The application method is that the addition 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 of abalone, increase the number of blood cells in the body of the 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 the Haliotis discus hannai, and finally achieve the remarkable effect of improving the nonspecific immunity of the Haliotis discus hannai.
2. The biological polypeptide preparation can improve the resistance of the haliotis discus to vibrio parahaemolyticus and reduce the death rate.
3. The biological polypeptide preparation provided by the invention can obviously promote the growth of the haliotis discus hannai, obviously improve three important production indexes of specific growth rate, weight gain rate and meat-shell ratio, and create favorable conditions for large-scale culture 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 invention is further described below with reference to examples.
It should be noted that: in practice, the temperature control allows a fluctuating temperature difference of 2℃and all the raw materials used in the examples are commercially available, except for the specific descriptions.
Example 1
The biological polypeptide preparation comprises, by weight, 80 parts of active polypeptide powder, 12 parts of maltodextrin, 3 parts of casein and 5 parts of bacillus subtilis;
wherein the viable count of the bacillus subtilis is 1.7X10 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) performing expansion culture: performing amplification culture on the bacillus subtilis seed liquid to a logarithmic phase to obtain bacillus subtilis fermentation seed liquid, wherein the concentration of the bacillus subtilis seed liquid is preferably 8.2X10 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, the culture medium for the enlarged culture is LB liquid culture medium;
(2) Fermentation culture: inoculating the bacillus subtilis fermentation seed liquid into a fermentation medium for fermentation culture to obtain bacillus subtilis fermentation liquid with viable count of 3.9X10 9 CFU/mL；
Fermentation culture process parameters: the inoculation amount is 10% 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 medium comprises the following components: 4g/L of peptone, 2g/L of glucose, 5g/L of sodium chloride, 6g/L of corn meal, 5g/L of yeast powder and 1.5g/L of magnesium sulfate;
(3) And (3) evaporating and concentrating: centrifuging bacillus subtilis fermentation liquor, evaporating and concentrating supernatant to 1/10 of the volume of the fermentation liquor after centrifuging, setting the centrifugal speed to 7100r/min and the evaporating temperature to 80 ℃;
(4) Spray drying: and adding maltodextrin or corn starch accounting for 15% of the volume of the fermentation liquor into the concentrated liquor, uniformly mixing, drying by using a spray dryer, simultaneously opening an air inlet and an air spraying opening of the dryer, enabling materials to enter an atomized state from the air spraying opening, discharging formed active polypeptide powder from an air outlet after hot air drying, 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 in parts by weight;
wherein the viable count of the bacillus subtilis is 1.5X10 6 CFU/g；
The molecular weight of the active polypeptide powder was 4.4KD, and the preparation method thereof was the same as that of the active polypeptide powder of example 1.
Example 3
The biological polypeptide preparation disclosed by the invention is applied to culture of haliotis discus hannai, the nonspecific immunity 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 culture test is carried out.
1. Test design
The cultivation test is carried out in Qingdao Maston biotechnology Co., ltd (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 is 7.5-8.0, and the dissolved oxygen is more than 6.8mg/L. The test set comprises 4 groups, namely a control group, 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 preparations, and the feed formulas of the groups are shown in table 1.
Before cultivation, the corrugated plate Bao Chuchong is 2.06+/-0.01 g, the shell length is 25.42+/-0.18 mm, the corrugated plate is fed by feeding 17:00 pm every day in the cultivation process, the corrugated plate abalone attaching plate is brushed 8:00 the next day earlier, the residual lugs are sucked and removed, water pollution is avoided, and cultivation is carried out for 115 days.
Table 1 table of feed formulation for haliotis discus
2. Test results
After the cultivation test is finished, measuring and counting all growth indexes of the Haliotis discus hannai, and collecting samples for detection.
(1) Influence of the biological polypeptide preparation of the invention on the growth index of Haliotis discus
The growth indexes of the abalone groups are measured and counted, wherein the growth indexes comprise survival rate, daily shell growth, weight gain rate, specific growth rate, meat-shell ratio and meat yield. The measuring instrument for the shell length of the haliotis discus hannai, which is a digital vernier caliper with the accuracy of 0.01mm, is used for anaesthetizing the haliotis discus hannai with 5% edible vinegar aqueous solution before measurement, and the shell length measuring method is to use the vernier caliper to measure the front and rear edge lengths of the shells; the weight measurement is carried out after taking out the Haliotis discus majoris and placing the Haliotis discus majoris on dry gauze for 10 s. Growth index data are shown in table 2.
TABLE 2 data sheet of growth index of Haliotis discus
Note that: a, b, c represent significant differences between groups (P < 0.05).
According to the growth index data in Table 2, the biological polypeptide preparation has an effect of promoting the growth of the Haliotis discus hand, when the addition amount of the biological polypeptide is 0.5%, the powder weight, the daily shell growth, the weight gain rate and the specific growth rate of the Haliotis discus hand are all obviously higher than those of a control group (P < 0.05), the meat-shell ratio level is the highest, and the survival rate is more than 97%.
(2) Effect of the biological polypeptide preparation of the invention on the nonspecific immunity function of Haliotis discus
The detection indexes comprise: the number of blood cells, the acid phosphatase and lysozyme activities of the blood cells, the phagocytosis rate of the blood cells and the respiratory burst activity of the Haliotis discus hannai, and the detection results are shown in Table 3.
TABLE 3 data sheet for detecting nonspecific immune function index of Haliotis discus hannai
As can be seen from Table 2, compared with the control group, the biological polypeptide preparation of the corrugated plate Bao Siwei has obviously improved blood cell number, blood cell acid phosphatase and lysozyme activity, blood cell phagocytic rate and respiratory burst activity, especially when the biological polypeptide addition amount is 0.5%, the blood cell number of the corrugated plate abalone is increased by 40% compared with the control group, the blood cell phagocytic rate is increased by 58.84%, the respiratory burst activity is increased by 184.6%, and the acid phosphatase activity and the lysozyme activity are respectively up to 17.31+/-0.38U/100 ml and 4.41+/-0.40U/ml.
Shellfish has no immunoglobulin and lymphocyte, the immune function only depends on nonspecific immunity, and in most shellfish, phagocytosis by granulocytes in blood cells is a main defense line of shellfish against external invasion, and plays an important role in immunization and epidemic prevention of shellfish. After granulocytes complete phagocytosis of foreign bodies, they are treated by two ways: firstly, the phagosome and the lysosome are fused, and the content of the phagosome is digested and degraded by hydrolytic enzymes in the lysosome; secondly, a large amount of active oxygen radicals (superoxide anions, hydrogen peroxide, singlet oxygen, hydroxyl radicals) are generated by respiratory burst accompanied by phagocytosis, and the ingested microorganisms are killed.
Blood cells play an important role in nonspecific immunity of shellfish, so that the nonspecific immunity of shellfish can be enhanced after the number of blood cells is increased. Acid phosphatase and lysozyme are also important components in the nonspecific immune system of shellfish, play a dual role in decomposition and defense, and the acid phosphatase is a marker enzyme of lysosomes and can destroy or remove foreign matters invading the organism; lysozyme can lyse the cell wall of bacteria, and when phagosome and lysosome are fused, the content can be digested and degraded by lysozyme and acid phosphatase in lysosomes. The phagocytosis of blood cells is a main defense line of shellfish against external invasion, so that the nonspecific immunity of the Haliotis discus hannai can be enhanced after the phagocytosis rate is improved. Respiratory burst activity is positively related to the phagocytic rate of blood cells, and the effect of promoting nonspecific immunity of Haliotis discus hannai is achieved by promoting phagocytosis of blood cells and improving respiratory burst activity accompanied with phagocytosis.
(3) Effect of the biological polypeptide preparation of the invention on the antibacterial ability of Haliotis discus
After the cultivation test is finished, 100 abalones are randomly extracted from each group to carry out a toxicity attack test, and pathogenic bacteria used for toxicity attack are secondaryVibrio haemolyticus provided by aquatic virus and immunity laboratory of China university, and has bacterial liquid concentration of 10 8 cfu/mL, the toxin attacking period is 10 days, the seawater temperature is 24 ℃, the cumulative mortality of the abalones in each group is counted after the test is finished, and the result is shown in Table 3.
TABLE 3 cumulative mortality statistics for Haliotis discus hannai in toxicity test
| Project | Control group | Test group a | Test group b | Test group c |
| Cumulative mortality rate | 36％ | 13％ | 22％ | 26％ |
As can be seen from Table 3, after the toxicity attack test, the cumulative death rate of the test group fed with the biological polypeptide preparation of the invention is obviously lower than that of the control group, and the maximum amplitude reduction reaches 63.9%, which indicates that the biological polypeptide preparation of the invention can enhance the antibacterial capability of the haliotis discus, enhance the resistance to vibrio parahaemolyticus and reduce the death rate of the haliotis discus.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A biological polypeptide preparation capable of improving the nonspecific immunity function of haliotis discus, which is characterized in that: 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;
the viable count of the bacillus subtilis is 1.5X10 6 -1.7×10 6 CFU/g；
The active polypeptide powder is mainly effective components of polypeptide substances which are produced and secreted outside cells through a ribosome synthesis mechanism in the fermentation and metabolism process of bacillus subtilis, wherein N-terminal amino acid forms a beta-lamellar structure, C-terminal amino acid forms firm alpha-helix, and disulfide bonds with a stable structure are respectively arranged on the beta-lamellar structure and the alpha-helix, and the preparation method comprises the following steps:
(1) And (3) performing expansion culture: performing expansion culture on the bacillus subtilis seed liquid to a logarithmic phase to obtain bacillus subtilis fermentation seed liquid;
(2) Fermentation culture: inoculating the bacillus subtilis fermentation seed liquid into a fermentation medium for fermentation culture to obtain bacillus subtilis fermentation liquid, wherein the viable count of the bacillus subtilis fermentation liquid is more than or equal to 3.6X10 9 CFU/mL; the fermentation medium comprises the following components: 4g/L of peptone, 2g/L of glucose, 5g/L of sodium chloride, 6g/L of corn meal, 5g/L of yeast powder and 1.5g/L of magnesium sulfate;
(3) And (3) evaporating and concentrating: centrifuging the bacillus subtilis fermentation liquor, and evaporating and concentrating supernatant after centrifuging to 1/10 of the volume of the fermentation liquor;
(4) Spray drying: and adding maltodextrin or corn starch accounting for 15% of the volume of the fermentation liquor into the concentrated solution, uniformly mixing, and performing spray drying to obtain the active polypeptide powder.
2. The biologic polypeptide formulation of claim 1, wherein: the air inlet temperature of spray drying is 180 ℃ and the air outlet temperature is 70-80 ℃.
3. The use of a biologic polypeptide preparation according to claim 1, wherein: is applied to the culture of haliotis discus hannai.
4. The use of a biologic polypeptide preparation according to claim 3, wherein: is applied to improving the nonspecific immunity of the haliotis discus.
5. The use of a biologic polypeptide preparation according to claim 4, wherein: the application comprises: the application of the method in improving the number of blood cells and phagocytic rate of the haliotis discus hannai, the application in improving the respiratory burst activity of the haliotis discus hannai and the application in promoting the haliotis discus hannai to resist vibrio parahaemolyticus.
6. The use of a biologic polypeptide preparation according to any one of claims 3-5, characterized in that: the application method is that the addition amount of the biological polypeptide preparation accounts for 0.2-1.5% of the weight of the feed.
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