CN111733117B - Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof - Google Patents

Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof Download PDF

Info

Publication number
CN111733117B
CN111733117B CN202010824328.0A CN202010824328A CN111733117B CN 111733117 B CN111733117 B CN 111733117B CN 202010824328 A CN202010824328 A CN 202010824328A CN 111733117 B CN111733117 B CN 111733117B
Authority
CN
China
Prior art keywords
antibacterial peptide
fermentation
liquid
solid
tank
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010824328.0A
Other languages
Chinese (zh)
Other versions
CN111733117A (en
Inventor
彭健
方素云
胡晓珂
闵军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhongke Marine Microbial Industry Technology Research Institute Shandong Co ltd
Yantai Institute of Coastal Zone Research of CAS
Original Assignee
Zhongke Marine Microbial Industry Technology Research Institute Shandong Co ltd
Yantai Institute of Coastal Zone Research of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhongke Marine Microbial Industry Technology Research Institute Shandong Co ltd, Yantai Institute of Coastal Zone Research of CAS filed Critical Zhongke Marine Microbial Industry Technology Research Institute Shandong Co ltd
Priority to CN202010824328.0A priority Critical patent/CN111733117B/en
Publication of CN111733117A publication Critical patent/CN111733117A/en
Application granted granted Critical
Publication of CN111733117B publication Critical patent/CN111733117B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • 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
    • A23K20/195—Antibiotics
    • 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
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • 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
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/50—Treatment of water, waste water, or sewage by addition or application of a germicide or by oligodynamic treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • 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
    • 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
    • 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Animal Husbandry (AREA)
  • Birds (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hydrology & Water Resources (AREA)
  • Water Supply & Treatment (AREA)
  • Environmental & Geological Engineering (AREA)
  • Insects & Arthropods (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a marine bacillus for producing antibacterial peptide and a fermentation method and application thereof, wherein the marine bacillus is bacillus subtilis M252 obtained by separating and purifying bottom mud samples collected from a certain culture pond in a new area of south China sea in Weihai city, has obvious inhibition effects on vibrio alginolyticus, staphylococcus aureus, aeromonas hydrophila and escherichia coli, and has a preservation number of CCTCC NO: m2020057, the bacillus subtilis M252 can prepare three antibacterial peptide products of solid-liquid powder in different use states through liquid fermentation and solid fermentation, the fermentation products are diversified and can be applied to different fields of aquaculture, animal feed, crop fertilizer and the like, in addition, the components of a culture medium used by the bacillus subtilis M252 in the fermentation process are all derived from raw materials with lower cost, the fermentation cost is reduced while the fermentation efficiency is ensured, and the bacillus subtilis M252 is more suitable for industrial production and application.

Description

Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof
Technical Field
The invention relates to a strain, a fermentation method and application thereof, in particular to a marine bacillus for producing antibacterial peptide, a fermentation method and application thereof, and belongs to the technical field of microorganisms.
Background
Antibiotics have been the most important weapon held by humans in the fight against pathogenic bacteria. However, with the use of antibiotics in medical treatment, feed additives and the like, many pathogenic bacteria have evolved into multiple mechanisms to survive in many antibiotics, and antibiotic resistance also becomes one of the most serious challenges facing the present society. The problems of drug resistance and drug residue are increasingly serious, and the health of human beings is influenced. Therefore, the search for alternatives to antibiotics is pressing.
Screening for chemical substances that are effective against pathogens has been a focus of research in the field of life sciences. Antibacterial peptides, also called host defense peptides, are usually small molecular polypeptides consisting of 7-100 amino acids, are important components of the natural immune defense system of organisms, are the first line of defense of various organisms against invading pathogens, and have attracted extensive attention as a potential candidate which can be extracted from natural sources and used for resisting antibiotic-resistant bacteria. The antibacterial peptide has broad-spectrum antibacterial, antiviral, antifungal, antiprotozoal, even antitumor cell activities, and has the activities of promoting body tissue healing and regulating in vivo immune system.
Using Bacillus subtilis (B), (B)Bacillus subtilis) Bacillus amyloliquefaciens (A) and (B)Bacillus amyloliquefaciens) Most of the bacilli represented by the bacillus subtilis have the advantages of rapid growth, simple nutritional requirement and strong stress resistance, and can generate polypeptide antibiotics and enzymes with bacteriolysis. As a non-pathogenic bacterium widely existing in nature, most of the bacillus is harmless to human and livestock and does not cause environmental pollution. Therefore, the bacillus and the metabolite of the antibacterial peptide thereof are the natural biological agent which is the most safe, effective and has the most application prospect for controlling the pathogenic bacteria diseases.
In the prior art, the application of producing antibacterial peptide by using Bacillus terrigenus is common. The production cost and the effect are comprehensively considered, and the application of directly immobilizing the antibacterial peptide-producing microorganisms and metabolites thereof into a microbial agent is increasing. The application of various antibacterial peptides in marine aquatic product culture can improve the production performance, digestive enzyme activity, antioxidation and immunologic functions of aquatic animals, but the particularity of the seawater environment needs to be considered when the microbial agent is applied. Therefore, the method is a key link in the production of aquaculture and livestock breeding feeds and is a source for controlling the food quality safety.
Disclosure of Invention
The invention aims to: the method comprises the steps of screening a marine microorganism for producing the antibacterial peptide by considering the specificity of a marine environment in marine aquatic product culture, providing a fermentation method of the marine microorganism for producing the antibacterial peptide, and simultaneously providing an application method of a microbial agent prepared by fermenting the marine microorganism for producing the antibacterial peptide.
In order to achieve the above object, the present invention adopts the following technical solutions:
a marine bacillus for producing antibacterial peptide is characterized in that the marine bacillus is a bacillus subtilis M252 obtained by separating and purifying a bottom sediment sample collected from a certain culture pond in a new area of south China sea in Weihai city, has obvious inhibition effects on vibrio alginolyticus, staphylococcus aureus, aeromonas hydrophila and escherichia coli, and has a preservation number of CCTCC NO: m2020057, the preservation date is 2020, 03 and 24 days, the preservation unit is China center for type culture Collection, and the preservation address is Wuhan, China.
The first fermentation method of the marine bacillus for producing the antibacterial peptide is characterized by comprising the following steps of:
step 1: purifying bacillus subtilis M252, and then inoculating the purified bacillus subtilis M252 into a first-stage seed tank, wherein the formula of a culture medium in the first-stage seed tank is as follows: 10g/L of peptone, 5g/L of yeast extract and 1g/L of sodium chloride, preparing with purified seawater, and culturing at 35 ℃ and 200rpm for 40h to obtain first-stage seed liquid;
step 2: the first-stage seed liquid is inoculated into a second-stage seed tank, and the formula of a culture medium in the second-stage seed tank is as follows: 40g/L glucose, 10g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate and 1g/L calcium chloride, preparing by purified seawater, culturing at 35 ℃ and 300rpm for 20h to obtain secondary seed liquid;
and step 3: and (3) inoculating the secondary seed liquid into a solid fermentation tank, wherein the formula of a culture medium in the solid fermentation tank is as follows: 5g/kg of sodium chloride, 2g/kg of monopotassium phosphate, 2g/kg of ammonium sulfate, the balance of bran, rice bran or soybean meal, the water content of which is controlled at 20-40%, the humidity of which is controlled at 30%, culturing for 56h at 35 ℃ and 8rpm, and then carrying out low-temperature air blast drying to obtain the antibacterial peptide solid.
The application of the antibacterial peptide solid preparation obtained by the first fermentation method is characterized in that the antibacterial peptide solid preparation is used as a feed additive.
The second fermentation method of the marine bacillus for producing the antibacterial peptide is characterized by comprising the following steps of:
step 1: purifying bacillus subtilis M252, and then inoculating the purified bacillus subtilis M252 into a first-stage seed tank, wherein the formula of a culture medium in the first-stage seed tank is as follows: 10g/L of peptone, 5g/L of yeast extract and 1g/L of sodium chloride, preparing with purified seawater, and culturing at 35 ℃ and 200rpm for 40h to obtain first-stage seed liquid;
step 2: the first-stage seed liquid is inoculated into a second-stage seed tank, and the formula of a culture medium in the second-stage seed tank is as follows: 40g/L glucose, 10g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate and 1g/L calcium chloride, preparing by purified seawater, culturing at 35 ℃ and 300rpm for 20h to obtain secondary seed liquid;
and step 3: inoculating the secondary seed liquid into a liquid fermentation tank, wherein the formula of a culture medium in the liquid fermentation tank is as follows: 1g/L of ammonium chloride, 3g/L of sodium acetate, 0.2g/L of magnesium chloride, 0.2g/L of calcium chloride, 0.6g/L of monopotassium phosphate, 1g/L of sodium chloride, 2g/L of yeast extract, 2g/L of glucose and purified seawater are prepared, the set temperature is 35 ℃, the rotating speed is 400rpm, the ventilation quantity is 0.75vvm and the DO value is 25 percent, the pH value of the fermentation liquor is controlled to be 7-8, a defoaming agent is manually added, the fermentation is carried out for 56 hours to obtain the fermentation liquor, the fermentation liquor is centrifuged at 4 ℃ and 4000rpm, the obtained supernatant is concentrated by a rotary evaporator by 10 times, and thalli and precipitates are removed from the concentrated liquor through a 0.22 mu m filter membrane to obtain the antimicrobial peptide liquid;
or, inoculating the secondary seed liquid into a solid fermentation tank, wherein the formula of the culture medium in the solid fermentation tank is as follows: 5g/kg of sodium chloride, 2g/kg of monopotassium phosphate, 2g/kg of ammonium sulfate, the balance of bran, rice bran or soybean meal, the water content of which is controlled at 20-40%, the humidity of which is controlled at 30%, culturing at 35 ℃ and 8rpm for 56h, then carrying out low-temperature air blast drying to obtain an antibacterial peptide solid, and then adding the antibacterial peptide solid into the antibacterial peptide solid according to the weight ratio of 10: 1, adding sterile purified water, leaching for 2 hours at 180rpm, centrifugally concentrating the leaching liquor, and sterilizing by a 0.22-micron filter membrane to obtain the antibacterial peptide liquid.
The application of the antibacterial peptide liquid obtained by the second fermentation method is characterized in that the antibacterial peptide liquid is used as a functional fertilizer for culturing prawns and aquatic fishes and is directly applied to a culture pond.
The third fermentation method of the marine bacillus for producing the antibacterial peptide is characterized by comprising the following steps of:
step 1: purifying bacillus subtilis M252, and then inoculating the purified bacillus subtilis M252 into a first-stage seed tank, wherein the formula of a culture medium in the first-stage seed tank is as follows: 10g/L of peptone, 5g/L of yeast extract and 1g/L of sodium chloride, preparing with purified seawater, and culturing at 35 ℃ and 200rpm for 40h to obtain first-stage seed liquid;
step 2: the first-stage seed liquid is inoculated into a second-stage seed tank, and the formula of a culture medium in the second-stage seed tank is as follows: 40g/L glucose, 10g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate and 1g/L calcium chloride, preparing by purified seawater, culturing at 35 ℃ and 300rpm for 20h to obtain secondary seed liquid;
and step 3: inoculating the secondary seed liquid into a liquid fermentation tank, wherein the formula of a culture medium in the liquid fermentation tank is as follows: 1g/L of ammonium chloride, 3g/L of sodium acetate, 0.2g/L of magnesium chloride, 0.2g/L of calcium chloride, 0.6g/L of monopotassium phosphate, 1g/L of sodium chloride, 2g/L of yeast extract, 2g/L of glucose and purified seawater are prepared, the set temperature is 35 ℃, the rotating speed is 400rpm, the ventilation quantity is 0.75vvm and the DO value is 25 percent, the pH value of the fermentation liquor is controlled to be 7-8, a defoaming agent is manually added, the fermentation is carried out for 56 hours to obtain the fermentation liquor, the fermentation liquor is centrifuged at 4 ℃ and 4000rpm, the obtained supernatant is concentrated by a rotary evaporator by 10 times, and thalli and precipitates are removed from the concentrated liquor through a 0.22 mu m filter membrane to obtain the antimicrobial peptide liquid; or, inoculating the secondary seed liquid into a solid fermentation tank, wherein the formula of the culture medium in the solid fermentation tank is as follows: 5g/kg of sodium chloride, 2g/kg of monopotassium phosphate, 2g/kg of ammonium sulfate, the balance of bran, rice bran or soybean meal, the water content of which is controlled at 20-40%, the humidity of which is controlled at 30%, culturing at 35 ℃ and 8rpm for 56h, then carrying out low-temperature air blast drying to obtain an antibacterial peptide solid, and then adding the antibacterial peptide solid into the antibacterial peptide solid according to the weight ratio of 10: 1, adding sterile purified water, leaching for 2 hours at 180rpm, centrifugally concentrating the leaching solution, and sterilizing by a 0.22-micron filter membrane to obtain an antibacterial peptide liquid;
and 4, step 4: adjusting the pH value of the antibacterial peptide liquid to 2.0, standing at 4 ℃ for 12h, centrifuging at 4 ℃ and 10000rpm to obtain a precipitate, dissolving the precipitate with 90% ethanol, uniformly stirring, adjusting the pH value of the solution to 7.0, standing at room temperature for 3h, centrifuging at 4 ℃ and 10000rpm to obtain an extracting solution, concentrating the extracting solution with a rotary evaporator, and freeze-drying the concentrated solution to obtain the antibacterial peptide powder.
The application of the antibacterial peptide powder obtained by the fermentation of the third fermentation method is characterized in that the antibacterial peptide powder is used as a functional fertilizer for holothurian culture, and is prepared into a solution to be applied to an aquaculture pond.
The invention has the advantages that:
1. the bacillus subtilis M252 obtained by screening is separated from bottom mud of an aquaculture pond, has obvious inhibition effects on vibrio alginolyticus, staphylococcus aureus, aeromonas hydrophila and escherichia coli, is easy to culture and high in propagation speed, and belongs to dominant strains during mixed culture of the strains;
2. the antibacterial peptide products prepared by fermenting the bacillus subtilis M252 obtained by screening have obvious effect of inhibiting pathogenic bacteria such as vibrio alginolyticus, staphylococcus aureus, aeromonas hydrophila, escherichia coli and the like;
3. each antibacterial peptide product prepared by fermenting the bacillus subtilis M252 obtained by screening comprises three different using states of solid (antibacterial peptide solid agent B1) liquid (antibacterial peptide liquid agent A2 and antibacterial peptide liquid agent B2) powder (antibacterial peptide powder agent A3 and antibacterial peptide powder agent B3), and the fermented product is diversified and can be applied to different fields of aquaculture, animal feed, crop fertilizer and the like;
4. the culture medium components used in the fermentation process of the bacillus subtilis M252 obtained by screening are all derived from raw materials with lower cost, the fermentation efficiency is ensured, the fermentation cost is reduced, and the method is more suitable for industrial production and application.
Drawings
FIG. 1 is a correlation graph of the culture period and the bacteriostatic activity of Bacillus subtilis M252;
FIG. 2 is a graph showing the change in relative activity and action temperature of antibacterial peptide produced by Bacillus subtilis M252;
FIG. 3 is a graph showing the relative activity of antimicrobial peptide produced by Bacillus subtilis M252 versus pH of action.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
First, separation, purification and identification of bacillus subtilis M252
1. Separation and purification of bacillus subtilis M252
The bacillus subtilis M252 obtained by screening is obtained by separating and purifying a sediment sample collected from a certain culture pond in a new area of south China sea in Weihai city, and the separation and purification processes are as follows:
(1) taking 10g of bottom mud sample collected from a certain culture pond in a new area of south China sea in Weihai city, and adding the bottom mud sample into 100ml of NaCl sterile solution with the concentration of 0.85 percent to prepare turbid liquid.
(2) And (2) performing gradient dilution on the prepared turbid solution, and then coating 100 mu l of the turbid solution diluted by 10000 times onto a peptone culture medium in a plate coating manner, wherein the peptone culture medium has the formula: 10g/L peptone, 5g/L yeast extract, 15g/L agar, 1g/L sodium chloride, prepared with purified seawater, and this step totally coated with 3 plates, after which 3 plates coated with the turbid solution were placed in an incubator at 37 ℃ and cultured upside down for 24 hours, at which time approximately 60-90 single colonies grew on each plate.
(3) Randomly picking 3 single colonies from each plate to a new peptone culture medium, purifying the single colonies by adopting a plate streaking mode, placing the 9 plates in a 37 ℃ incubator for inverted culture for 24 hours after streaking is finished, growing about 50 single colonies on each plate, and finally randomly storing a single colony which is round in shape, irregular in edge, wrinkled on the surface, opaque and yellowish in color, wherein the single colony is the single colony finally screened by the invention, and correspondingly, the strain in the single colony is the strain finally screened by the invention.
2. Identification of Bacillus subtilis M252
(1) Observing colony morphology
The strain obtained by the final screening of the invention is coated on a peptone medium in a plate coating manner, and is subjected to inverted culture in an incubator at 37 ℃ for 24 hours, wherein about 100 single colonies grow on the plate, and the colonies are round, irregular in edge, wrinkled on the surface, opaque and yellowish in color.
(2) Gel electrophoresis detection
The strain obtained by the final screening of the invention is inoculated into LB liquid culture medium and cultured for 12h at 37 ℃ and 150rpm to obtain bacterial liquid.
The DNA of the bacteria in the bacterial suspension was extracted with a bacterial genomic DNA extraction kit, and then sequence amplification was carried out using the extracted DNA as a template, 27F (sequence AGA GTT TGA TCC TGG CTCAG) as an upstream primer, and ITS1 (sequence TCCGTAGGTGAACCTGCGG) as a downstream primer. The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 1 min, annealing at 56 ℃ for 1 min, extension at 72 ℃ for 1 min, 31 cycles; 5 min at 72 ℃.
After the amplification is finished, the amplification product is detected by 1% (w/v) agarose gel electrophoresis. The detection result shows that: the PCR amplification of the universal primer is successful.
(3) 16S rRNA identification
The strain obtained by the final screening of the invention is sent to Beijing Odoku Dingsheng Biotechnology limited company for 16S rRNA sequence determination, and the 16S rRNA sequence of the strain is compared with the homologous sequence in Gen BankSearching, and comparing the searching results to display: the strain and bacillus subtilis (B)Bacillussubtilis) The similarity reaches 100%.
Finally, the strain finally obtained by screening is identified as bacillus subtilis M252.
Second, determination method of bacteriostatic effect
And (3) taking vibrio alginolyticus, staphylococcus aureus, aeromonas hydrophila and escherichia coli as indicator bacteria, and determining the antibacterial effect of the solution to be detected by an Oxford cup plate diffusion method.
Manufacturing a bacteriostatic board: respectively adding vibrio alginolyticus, staphylococcus aureus, aeromonas hydrophila and escherichia coli into the molten LB solid culture medium, wherein the adding concentration is 1% (v/v), then respectively pouring the mixture into different culture dishes, and cooling to obtain the corresponding antibacterial plates.
After the bacteriostatic plates are manufactured, 4 Oxford cups are placed on each bacteriostatic plate, wherein 1 Oxford cup is used as a blank control group, the other 3 Oxford cups are used as parallel experiment groups, 100 mu l of sterile water is added into the Oxford cups in the blank control group, 100 mu l of solution to be tested is added into each Oxford cup in the parallel experiment groups respectively, the solution to be tested is cultured for 24 hours at the normal temperature of 35 ℃, and then the diameter (mm) of a bacteriostatic circle on each bacteriostatic plate is measured and recorded.
Thirdly, researching liquid fermentation bacteriostasis of the bacillus subtilis M252
After purifying the bacillus subtilis M252, inoculating the purified bacillus subtilis M252 into a first-stage seed tank according to the inoculation amount of 5%, wherein the formula of a culture medium in the first-stage seed tank is as follows: 10g/L of peptone, 5g/L of yeast extract and 1g/L of sodium chloride, preparing with purified seawater, and culturing at 35 ℃ and 200rpm for 40h to obtain first-stage seed liquid; then inoculating the first-stage seed liquid into a second-stage seed tank according to the inoculation amount of 5%, wherein the formula of a culture medium in the second-stage seed tank is as follows: 40g/L glucose, 10g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate and 1g/L calcium chloride, preparing by purified seawater, culturing at 35 ℃ and 300rpm for 20h to obtain secondary seed liquid; then inoculating the secondary seed liquid into a liquid fermentation tank according to the inoculation amount of 5%, wherein the formula of a culture medium in the liquid fermentation tank is as follows: 1g/L of ammonium chloride, 3g/L of sodium acetate, 0.2g/L of magnesium chloride, 0.2g/L of calcium chloride, 0.6g/L of monopotassium phosphate, 1g/L of sodium chloride, 2g/L of yeast extract and 2g/L of glucose are prepared by purified seawater, the set temperature is 35 ℃, the rotating speed is 500rpm, the ventilation volume is 0.75vvm, the DO value is 25 percent, the pH value of the fermentation liquor is controlled to be 7-8, an antifoaming agent is manually added, and the fermentation liquor is fermented for 70 hours to obtain the fermentation liquor A1.
During the whole fermentation process, we sampled periodically and measured the OD of the fermentation broth600nmAnd the diameter of the inhibition zone (taking vibrio alginolyticus as a representative of indicator bacteria), and correlating the culture period and the bacteriostasis of the bacillus subtilis M252, finally obtaining a correlation diagram of the culture period and the bacteriostasis of the bacillus subtilis M252 shown in figure 1, and as can be seen from figure 1:
(1) the bacillus subtilis M252 can still continuously generate and accumulate the antibacterial peptide in the vigorous growth period, and the antibacterial peptide accumulation is more, so that the antibacterial effect is more obvious;
(2) at 56h of fermentation, the diameter of the inhibition zone of the fermentation broth is maximized (i.e. antibacterial peptide activity is maximized) and then decreased, and the optimal fermentation time is 56 h.
Then, an appropriate amount of the fermentation liquid A1 was centrifuged at 4000rpm at 4 ℃ for 30min, the obtained supernatant was concentrated 10 times by a rotary evaporator, and the concentrated solution was passed through a 0.22 μm filter to remove the cells and the precipitate, thereby obtaining an antimicrobial peptide solution A2.
The antibacterial effect of the antibacterial peptide liquid A2 is detected, and the specific result is detailed in Table 1.
TABLE 1 bacteriostatic effect of Bacillus subtilis M252 by liquid fermentation
Figure DEST_PATH_IMAGE001
As can be seen from Table 1, the antimicrobial peptide liquid A2 prepared by liquid fermentation of Bacillus subtilis M252 has obvious inhibitory effects on Vibrio alginolyticus, Staphylococcus aureus, Aeromonas hydrophila and Escherichia coli, wherein the inhibitory effects on the marine pathogenic bacteria Vibrio alginolyticus are the most obvious, and the inhibitory effects on Escherichia coli are relatively weak, which may be caused by the fact that the Bacillus subtilis M252 is from the sea and the antimicrobial peptides generated by metabolism have higher action specificity on the marine microorganisms.
Fourthly, researching solid state fermentation bacteriostasis of bacillus subtilis M252
Purifying bacillus subtilis M252, inoculating the purified bacillus subtilis M252 into a first-stage seed tank according to the inoculation amount of 5%, culturing the bacillus subtilis M252 in the first-stage seed tank for 40 hours at 35 ℃ and 200rpm to obtain a first-stage seed solution; then, inoculating the primary seed solution into a secondary seed tank according to the inoculation amount of 5%, culturing the secondary seed tank with the culture medium in the same way as the primary seed tank at 35 ℃ and 300rpm for 20 hours to obtain secondary seed solution; then inoculating the second-stage seed liquid into a solid fermentation tank according to the inoculation amount of 5%, wherein the formula of a culture medium in the solid fermentation tank is as follows: 5g/kg of sodium chloride, 2g/kg of monopotassium phosphate, 2g/kg of ammonium sulfate, the balance of bran, rice bran or bean pulp (the bran is adopted in the embodiment), the water content is controlled to be 20-40%, the humidity is controlled to be 30%, the materials are cultured at 35 ℃ and 8rpm for 64h, and the materials are subjected to low-temperature blast drying to obtain an antibacterial peptide fixing agent B1; then, taking a proper amount of the antibacterial peptide solid agent B1 according to the weight ratio of 10: 1, adding sterile purified water, leaching for 2 hours at 180rpm, centrifugally concentrating the leaching liquor, and sterilizing by a 0.22 mu m filter membrane to finally obtain the antibacterial peptide liquid B2.
The antibacterial effects of the antibacterial peptide solid B1 and the antibacterial peptide liquid B2 are detected, and the specific results are shown in Table 2.
TABLE 2 antibacterial effect of antibacterial peptide solid B1 and antibacterial peptide liquid B2
Figure 451548DEST_PATH_IMAGE002
As can be seen from Table 2, the antibacterial peptide solid B1 and the antibacterial peptide liquid B2 prepared by solid state fermentation of Bacillus subtilis M252 all have obvious inhibitory effects on Vibrio alginolyticus, Staphylococcus aureus, Aeromonas hydrophila and Escherichia coli, but compared with the antibacterial peptide liquid A2, the inhibitory effects of the antibacterial peptide solid B1 and the antibacterial peptide liquid B2 are relatively weaker, which is probably caused by different culture media used when the antibacterial peptide is produced by fermentation of Bacillus subtilis M252, and the liquid state fermentation culture medium has more abundant material components than the solid state fermentation culture medium and is more beneficial to the fermentation metabolic activity of microorganisms.
Fifthly, preparing powdery antibacterial peptide (namely antibacterial peptide powder) and researching the bacteriostatic activity of the powdery antibacterial peptide
Taking a proper amount of the antibacterial peptide liquid A2 and the antibacterial peptide liquid B2, respectively adjusting the pH value to 2.0 by using 3mol/L HCl, standing for 12h at 4 ℃, centrifuging for 20min at 10000rpm to obtain precipitates, dissolving the obtained precipitates by using 90% ethanol, uniformly stirring, adjusting the pH value to 7.0 by using 3mol/L NaOH, standing for 3h at room temperature, centrifuging for 10min at 4 ℃ and 10000rpm to obtain an extracting solution, concentrating the extracting solution by using a rotary evaporator, and freeze-drying the concentrated solution to obtain antibacterial peptide powder A3 and antibacterial peptide powder B3.
Sterile water is respectively added into the antibacterial peptide powder A3 and the antibacterial peptide powder B3 to prepare antibacterial peptide solutions with the concentration of 25% (w/v), and then the antibacterial effects of the two antibacterial peptide solutions are detected, and the specific results are shown in Table 3.
TABLE 3 bacteriostatic results of antibacterial peptide powder A3 and antibacterial peptide powder B3
Figure DEST_PATH_IMAGE003
As can be seen from table 3, compared with the antimicrobial peptide liquid a2 and the antimicrobial peptide liquid B2, the inhibitory effect of the antimicrobial peptide powder A3 and the antimicrobial peptide powder B3 on the indicator bacteria is more significant, which indicates that the antimicrobial peptides prepared from the antimicrobial peptide liquid a2 and the antimicrobial peptide liquid B2 by the acid precipitation method indicate that the active ingredients generated after the fermentation of bacillus subtilis M252 are polypeptide substances, and the polypeptide substances have significant inhibitory effects on the indicator bacteria.
Method for determining activity of antibacterial peptide
The antibacterial peptide is a basic polypeptide substance with antibacterial activity, and is a peptide with antibacterial capability consisting of amino acid residues. The definition of the biological enzyme is a high-efficiency catalyst with biological specificity, the biological enzyme is a protein, and the protein is composed of polypeptides. It is currently believed that the bacteriostatic mechanism of antimicrobial peptides is that they act on cell membranes and form ion channels across the membrane, destroying the integrity of the membrane, causing leakage of the cell contents, thereby killing the cells. Therefore, the invention analyzes the activity characteristics of the antibacterial peptide produced by the bacillus subtilis M252 compared with the method for measuring the biological lysozyme.
And (3) carrying out antibacterial peptide activity determination on the antibacterial peptide solid agent, the antibacterial peptide liquid agent and the antibacterial peptide powder by using a turbidimetric method. And (3) respectively determining the activities of the antibacterial peptide solid agent, the antibacterial peptide liquid agent and the antibacterial peptide powder according to the turbidity change of the bacterial suspension by using the micrococcus muralis as a substrate.
1. Preparation of a suspension of a muralytic Micrococcus
Inoculating Micrococcus muralis in LB liquid culture medium, culturing at 30 deg.C and 160rpm to exponential phase, centrifuging at 5000rpm for 10min to collect thallus, washing with sterile water, and adjusting to Micrococcus muralis suspension A with 0.1mol/L phosphate buffer450=1.0。
2. Respectively preparing antibacterial peptide solid preparation, antibacterial peptide liquid preparation and antibacterial peptide powder preparation into solutions with appropriate concentrations
The antibacterial peptide solid preparation is dissolved by using sterile water to prepare an antibacterial peptide solution with the concentration of 40% (w/v).
The antimicrobial peptide solution was diluted with sterile water to prepare a 30% (v/v) antimicrobial peptide solution.
The antibacterial peptide powder is dissolved by using sterile water to prepare an antibacterial peptide solution with the concentration of 20% (w/v).
3. Determination of activity of antibacterial peptide solid, antibacterial peptide liquid and antibacterial peptide powder by turbidimetry
Placing the wall-dissolving microspherical bacteria suspension and the antibacterial peptide solution corresponding to the antibacterial peptide solid agent, the antibacterial peptide liquid agent and the antibacterial peptide powder agent in a water bath kettle at 25 ℃ for 20min, preparing a plurality of colorimetric tubes, adding 3mL of wall-dissolving microspherical bacteria suspension into each colorimetric tube, and measuring A of each colorimetric tube at the moment450Adding 0.2mL of antibacterial peptide solution corresponding to antibacterial peptide solid agent, antibacterial peptide liquid agent and antibacterial peptide powder agent into each colorimetric tube, shaking up rapidly, and recording A of each colorimetric tube every 30s450The value is obtained.
Definition of activity units: at 25 ℃ and pH 7, A450The value decreases by 0.001 every 60s, i.e. 1 activity unit, thus obtaining the antibodyThe formula for calculating the activity of the bacteriocin is as follows:
(1) calculation formula of activity of antibacterial peptide liquid
Antimicrobial peptide Activity Unit = [ A ] per 1mL450（0s）- A450(60 s) ]. times.1000/ml sample.
(2) Calculation formula of activity of antibacterial peptide solid agent and antibacterial peptide powder
Activity unit of antibacterial peptide per 1mg = [ A450（0s）- A450(60 s) ]. times.1000 in mg/sample.
Seventhly, verifying the fermentation effect and fermentation performance of the bacillus subtilis M252
Example 1: liquid state fermentation
Purifying bacillus subtilis M252, inoculating the purified bacillus subtilis M252 into a first-stage seed tank according to the inoculation amount of 5%, culturing the bacillus subtilis M252 in the first-stage seed tank for 40 hours at 35 ℃ and 200rpm in the same manner as the culture medium in the first-stage seed tank to obtain first-stage seed liquid; then inoculating the primary seed liquid into a secondary seed tank according to the inoculation amount of 5%, wherein the culture medium in the secondary seed tank is the same as the above, the dosage of the culture medium is 5L, and the culture is carried out at 35 ℃ and 300rpm for 20h to obtain a secondary seed liquid; then inoculating the secondary seed liquid into a liquid fermentation tank according to the inoculation amount of 5%, wherein the culture medium in the liquid fermentation tank is the same as the above, the dosage of the culture medium is 50L, the set temperature is 35 ℃, the rotating speed is 400rpm, the ventilation rate is 0.75vvm, the DO value is 25%, the pH value of the fermentation liquid is controlled to be 7-8, the antifoaming agent is manually added, and the fermentation is carried out for 56h, so that the fermentation liquid A1 is obtained’(ii) a Finally, taking a proper amount of fermentation liquor A1’Centrifuging at 4 deg.C and 4000rpm for 30min, concentrating the obtained supernatant with rotary evaporator by 10 times, and filtering the concentrated solution with 0.22 μm filter membrane to remove thallus and precipitate to obtain antibacterial peptide liquid A2’0.5L of the above-mentioned antibacterial peptide liquid A2 was collected’Adjusting pH to 2.0 with 3mol/L HCl, standing at 4 deg.C for 12h, centrifuging at 4 deg.C and 10000rpm for 20min to obtain precipitate, dissolving the precipitate with 90% ethanol, stirring, adjusting pH to 7.0 with 3mol/L NaOH, standing at room temperature for 3h, centrifuging at 4 deg.C and 10000rpm for 10min to obtain extractive solution, concentrating the extractive solution with rotary evaporator, and freeze drying the concentrated solution to obtain antibacterial peptide powder A3’。
Example 2: solid state fermentation
Purifying bacillus subtilis M252, inoculating the purified bacillus subtilis M252 into a first-stage seed tank according to the inoculation amount of 5%, culturing the bacillus subtilis M252 in the first-stage seed tank for 40 hours at 35 ℃ and 200rpm in the same manner as the culture medium in the first-stage seed tank to obtain first-stage seed liquid; then inoculating the primary seed liquid into a secondary seed tank according to the inoculation amount of 5%, wherein the culture medium in the secondary seed tank is the same as the above, the dosage of the culture medium is 5L, and the culture is carried out at 35 ℃ and 300rpm for 20h to obtain a secondary seed liquid; then inoculating the second-stage seed liquid into a solid fermentation tank according to the inoculation amount of 5%, culturing the second-stage seed liquid in the solid fermentation tank with the culture medium in the same manner as the solid fermentation tank, controlling the using amount of the culture medium to be 50L, controlling the humidity to be 30%, culturing the second-stage seed liquid at 35 ℃ and 8rpm for 56h, and then drying the second-stage seed liquid by low-temperature blast to obtain the antibacterial peptide solid agent B1’(ii) a Finally, 1kg of the antibacterial peptide solid preparation B1 was taken’According to the weight ratio of 10: 1 adding sterile purified water, leaching at 180rpm for 2h, centrifuging and concentrating the leaching solution, sterilizing with 0.22 μm filter membrane to obtain antibacterial peptide liquid B2’(ii) a Collecting 0.5L of the above antibacterial peptide liquid B2’Adjusting pH to 2.0 with 3mol/L HCl, standing at 4 deg.C for 12h, centrifuging at 4 deg.C and 10000rpm for 20min to obtain precipitate, dissolving the precipitate with 90% ethanol, stirring, adjusting pH to 7.0 with 3mol/L NaOH, standing at room temperature for 3h, centrifuging at 4 deg.C and 10000rpm for 10min to obtain extractive solution, concentrating the extractive solution with rotary evaporator, and freeze drying the concentrated solution to obtain antibacterial peptide powder B3’。
Respectively detecting antibacterial peptide solid reagent B1’Antimicrobial peptide liquid A2’Antimicrobial peptide liquid B2’Antibacterial peptide powder A3’And antibacterial peptide powder B3’Concentration (polypeptide content using coomassie brilliant blue) and viability. The results are shown in tables 4, 5 and 6.
TABLE 4 concentration and Activity of antibacterial peptide solid B1
Figure 588131DEST_PATH_IMAGE004
TABLE 5 concentration and Activity of antimicrobial peptide liquid A2' and antimicrobial peptide liquid B2
Figure DEST_PATH_IMAGE005
TABLE 6 concentration and viability of antimicrobial peptide powder A3' and antimicrobial peptide powder B3
Figure 725852DEST_PATH_IMAGE006
As shown in tables 4, 5 and 6, bacillus subtilis M252 can not only produce high-concentration and high-activity antimicrobial peptides by fermentation, but also has significant stability in fermentation performance and activity of the produced antimicrobial peptides.
Eighthly, the optimal action temperature and the optimal action pH of the antibacterial peptide solid agent, the antibacterial peptide liquid agent and the antibacterial peptide powder agent
1. Optimum action temperature of antibacterial peptide solid, antibacterial peptide liquid and antibacterial peptide powder
Measuring antibacterial peptide solid B1 at pH 7 at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C and 50 deg.C respectively’Antimicrobial peptide liquid A2’Antimicrobial peptide liquid B2’Antibacterial peptide powder A3’And antibacterial peptide powder B3’Then calculating the relative activity (i.e. specific activity, i.e. the number of enzyme activity units per mg or ml of enzyme protein), and then plotting the variation curve between the relative activity of the antibacterial peptide and the temperature, wherein the obtained variation curve is shown in fig. 2.
As shown in FIG. 2, the antibacterial peptide solid preparation B1’The optimum action temperature of (2) is 35 ℃, and the antibacterial peptide liquid agent A2’And antimicrobial peptide liquid B2’The optimum action temperature of the antibacterial peptide powder A3 is 30 DEG C’And antibacterial peptide powder B3’The optimum temperature for the reaction is 35 ℃.
2. Optimum action pH of antibacterial peptide solid, antibacterial peptide liquid and antibacterial peptide powder
Antimicrobial peptide solid reagent B1 was assayed at 35 deg.C and different pH values (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5), respectively’Antimicrobial peptide liquid A2’Antimicrobial peptide liquid B2’Antibacterial peptide powder A3’And antibacterial peptide powderB3’Then calculating the relative activity, and then drawing a change curve between the relative activity of the antimicrobial peptide and the pH value, wherein the obtained change curve is shown in figure 3.
As can be seen from FIG. 3, the antibacterial peptide solid preparation B1’Has an optimum pH of 8.0, and antibacterial peptide liquid A2’And antimicrobial peptide liquid B2’Has an optimum action pH of 7.5-8.0, and antibacterial peptide powder A3’And antibacterial peptide powder B3’The optimum pH for the action of (2) was 8.0.
Antibacterial peptide solid preparation, antibacterial peptide liquid preparation and antibacterial peptide powder preparation, and application method and effect thereof
1. Application method and application effect of antibacterial peptide solid preparation
The antibacterial peptide solid preparation is used as a feed additive, and the use method comprises the following steps:
mixing antibacterial peptide B1’Adding 40% (w/w) of the additive into broiler feed in a chicken farm for feeding and applying, and feeding No. 1, No. 2 and No. 3 chicken sheds with antibacterial peptide solid agent B1 according to the culture period of 7 weeks of broilers’The feed of (4) chicken coop was fed with normal feed as a control.
After the 7-week breeding period is finished, the change condition of the average weight of the broilers in each chicken shed is shown in table 7.
TABLE 7 average weight change of broiler chickens in each shed
Figure 938789DEST_PATH_IMAGE008
From table 7, it is known that when the antibacterial peptide solid agent is used as a feed additive to feed broilers, the growth rate of the broilers can be remarkably increased, and the slaughtering rate of the broilers can be effectively increased.
2. Method for using antibacterial peptide liquid and using effect
The antibacterial peptide liquid is used as a pond culture functional fertilizer for prawns, aquatic fishes and the like, and the use method comprises the following steps:
mixing antibacterial peptide solution A2’And antimicrobial peptide liquid B2’Directly throwing the mixture into a No. 1 prawn culture pond and a No. 2 prawn culture pond respectively according to the dosage of 1 kg/mu,no. 3 prawn culture pond is used as a control without adding antibacterial peptide liquid. The weight change of the prawns in each aquaculture pond is shown in table 8.
TABLE 8 weight change of prawns in aquaculture ponds
Figure 161960DEST_PATH_IMAGE010
From table 8, it is known that the antibacterial peptide liquid can significantly increase the growth rate of the prawns when applied as a functional fertilizer during the cultivation of the prawns.
3. Application method and application effect of antibacterial peptide powder
The antibacterial peptide powder is used as a functional fertilizer for holothurian culture, and the use method comprises the following steps:
mixing antibacterial peptide powder A3’And antibacterial peptide powder B3’Dissolving with purified water to obtain antibacterial peptide solution with concentration of 20% (w/v), respectively recording as antibacterial peptide solution A3’’And antimicrobial peptide solution B3’’Then the antibacterial peptide solution is applied to the sea cucumber aquaculture pond according to the dosage of 1kg of antibacterial peptide solution per 5 mu.
Selecting 5 20 mu holothurian culture ponds in the same area, respectively recording the ponds as a pond 1, a pond 2, a pond 3, a pond 4 and a pond 5, and respectively applying 4kg of antibacterial peptide solution A3 to the pond 1 and the pond 2’’4kg of antimicrobial peptide solution B3 was administered to each of the ponds 3 and 4’’The remaining pond 5 was not dosed with any microbial agent (control), after which the antimicrobial peptide solution A3 was added every 7d’’And antimicrobial peptide solution B3’’Once, 2kg of the microbial inoculum is applied each time, 6w of the microbial inoculum is continuously applied, and no microbial inoculum is applied to a control group all the time.
The weight change of the sea cucumbers in each aquaculture pond is observed and recorded every 7 days, and the specific results are detailed in a table 9.
TABLE 9 weight change of Stichopus japonicus in each aquaculture pond
Figure 367814DEST_PATH_IMAGE012
After 6 weeks, the water quality in each aquaculture pond was observed, and the specific results are detailed in table 10.
TABLE 10 Water quality in various aquaculture ponds
Pond 1 Pond 2 Pond 3 Pond 4 Pond 5
Before application Good taste Good taste Good taste Good taste Good taste
After 6 weeks Is preferably used Is preferably used Is preferably used Is preferably used Is poor
From tables 9 and 10, it is known that the antimicrobial peptide powder not only can significantly increase the growth rate of sea cucumbers, but also can effectively improve the water quality in the culture pond in the process of culturing the sea cucumbers.
Ten, preservation of Bacillus subtilis M252
The use effects of the antibacterial peptide solid agent, the antibacterial peptide liquid agent and the antibacterial peptide powder agent prepared by utilizing the bacillus subtilis M252 are provided, the bacillus subtilis M252 obtained by screening has very high practical application value in the aspects of poultry cultivation and aquaculture, so the bacillus subtilis M252 is subjected to strain preservation, and the preservation information is as follows:
the preservation unit is China center for type culture Collection, the preservation date is 2020, 03 and 24 days, and the preservation number is CCTCC NO: m2020057, the preservation address is Wuhan in China.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.

Claims (7)

1. A marine bacillus for producing antibacterial peptide is characterized in that the marine bacillus is a bacillus subtilis M252 obtained by separating and purifying a bottom sediment sample collected from a certain culture pond in a new area of south China sea in Weihai city, has obvious inhibition effects on vibrio alginolyticus, staphylococcus aureus, aeromonas hydrophila and escherichia coli, and has a preservation number of CCTCC NO: m2020057, the preservation date is 2020, 03 and 24 days, the preservation unit is China center for type culture Collection, and the preservation address is Wuhan, China.
2. The method for fermenting Bacillus marinus producing antibacterial peptide according to claim 1, comprising the steps of:
step 1: purifying bacillus subtilis M252, and then inoculating the purified bacillus subtilis M252 into a first-stage seed tank, wherein the formula of a culture medium in the first-stage seed tank is as follows: 10g/L of peptone, 5g/L of yeast extract and 1g/L of sodium chloride, preparing with purified seawater, and culturing at 35 ℃ and 200rpm for 40h to obtain first-stage seed liquid;
step 2: the first-stage seed liquid is inoculated into a second-stage seed tank, and the formula of a culture medium in the second-stage seed tank is as follows: 40g/L glucose, 10g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate and 1g/L calcium chloride, preparing by purified seawater, culturing at 35 ℃ and 300rpm for 20h to obtain secondary seed liquid;
and step 3: and (3) inoculating the secondary seed liquid into a solid fermentation tank, wherein the formula of a culture medium in the solid fermentation tank is as follows: 5g/kg of sodium chloride, 2g/kg of monopotassium phosphate, 2g/kg of ammonium sulfate, the balance of bran, rice bran or soybean meal, the water content of which is controlled at 20-40%, the humidity of which is controlled at 30%, culturing for 56h at 35 ℃ and 8rpm, and then carrying out low-temperature air blast drying to obtain the antibacterial peptide solid.
3. Use of the antibacterial peptide solid obtained by fermentation according to the fermentation method of claim 2 as a feed additive.
4. The method for fermenting Bacillus marinus producing antibacterial peptide according to claim 1, comprising the steps of:
step 1: purifying bacillus subtilis M252, and then inoculating the purified bacillus subtilis M252 into a first-stage seed tank, wherein the formula of a culture medium in the first-stage seed tank is as follows: 10g/L of peptone, 5g/L of yeast extract and 1g/L of sodium chloride, preparing with purified seawater, and culturing at 35 ℃ and 200rpm for 40h to obtain first-stage seed liquid;
step 2: the first-stage seed liquid is inoculated into a second-stage seed tank, and the formula of a culture medium in the second-stage seed tank is as follows: 40g/L glucose, 10g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate and 1g/L calcium chloride, preparing by purified seawater, culturing at 35 ℃ and 300rpm for 20h to obtain secondary seed liquid;
and step 3: inoculating the secondary seed liquid into a liquid fermentation tank, wherein the formula of a culture medium in the liquid fermentation tank is as follows: 1g/L of ammonium chloride, 3g/L of sodium acetate, 0.2g/L of magnesium chloride, 0.2g/L of calcium chloride, 0.6g/L of monopotassium phosphate, 1g/L of sodium chloride, 2g/L of yeast extract, 2g/L of glucose and purified seawater are prepared, the set temperature is 35 ℃, the rotating speed is 400rpm, the ventilation quantity is 0.75vvm and the DO value is 25 percent, the pH value of the fermentation liquor is controlled to be 7-8, a defoaming agent is manually added, the fermentation is carried out for 56 hours to obtain the fermentation liquor, the fermentation liquor is centrifuged at 4 ℃ and 4000rpm, the obtained supernatant is concentrated by a rotary evaporator by 10 times, and thalli and precipitates are removed from the concentrated liquor through a 0.22 mu m filter membrane to obtain the antimicrobial peptide liquid;
or, inoculating the secondary seed liquid into a solid fermentation tank, wherein the formula of the culture medium in the solid fermentation tank is as follows: 5g/kg of sodium chloride, 2g/kg of monopotassium phosphate, 2g/kg of ammonium sulfate, the balance of bran, rice bran or soybean meal, the water content of which is controlled at 20-40%, the humidity of which is controlled at 30%, culturing at 35 ℃ and 8rpm for 56h, then carrying out low-temperature air blast drying to obtain an antibacterial peptide solid, and then adding the antibacterial peptide solid into the antibacterial peptide solid according to the weight ratio of 10: 1, adding sterile purified water, leaching for 2 hours at 180rpm, centrifugally concentrating the leaching liquor, and sterilizing by a 0.22-micron filter membrane to obtain the antibacterial peptide liquid.
5. The application of the antibacterial peptide liquid obtained by fermentation according to the fermentation method of claim 4 is characterized in that the antibacterial peptide liquid is used as a functional fertilizer for prawn and aquatic fish culture and is directly applied to a culture pond.
6. The method for fermenting Bacillus marinus producing antibacterial peptide according to claim 1, comprising the steps of:
step 1: purifying bacillus subtilis M252, and then inoculating the purified bacillus subtilis M252 into a first-stage seed tank, wherein the formula of a culture medium in the first-stage seed tank is as follows: 10g/L of peptone, 5g/L of yeast extract and 1g/L of sodium chloride, preparing with purified seawater, and culturing at 35 ℃ and 200rpm for 40h to obtain first-stage seed liquid;
step 2: the first-stage seed liquid is inoculated into a second-stage seed tank, and the formula of a culture medium in the second-stage seed tank is as follows: 40g/L glucose, 10g/L yeast extract, 5g/L corn steep liquor, 1g/L ammonium sulfate, 0.5g/L magnesium sulfate, 1.5g/L potassium dihydrogen phosphate and 1g/L calcium chloride, preparing by purified seawater, culturing at 35 ℃ and 300rpm for 20h to obtain secondary seed liquid;
and step 3: inoculating the secondary seed liquid into a liquid fermentation tank, wherein the formula of a culture medium in the liquid fermentation tank is as follows: 1g/L of ammonium chloride, 3g/L of sodium acetate, 0.2g/L of magnesium chloride, 0.2g/L of calcium chloride, 0.6g/L of monopotassium phosphate, 1g/L of sodium chloride, 2g/L of yeast extract, 2g/L of glucose and purified seawater are prepared, the set temperature is 35 ℃, the rotating speed is 400rpm, the ventilation quantity is 0.75vvm and the DO value is 25 percent, the pH value of the fermentation liquor is controlled to be 7-8, a defoaming agent is manually added, the fermentation is carried out for 56 hours to obtain the fermentation liquor, the fermentation liquor is centrifuged at 4 ℃ and 4000rpm, the obtained supernatant is concentrated by a rotary evaporator by 10 times, and thalli and precipitates are removed from the concentrated liquor through a 0.22 mu m filter membrane to obtain the antimicrobial peptide liquid;
or, inoculating the secondary seed liquid into a solid fermentation tank, wherein the formula of the culture medium in the solid fermentation tank is as follows: 5g/kg of sodium chloride, 2g/kg of monopotassium phosphate, 2g/kg of ammonium sulfate, the balance of bran, rice bran or soybean meal, the water content of which is controlled at 20-40%, the humidity of which is controlled at 30%, culturing at 35 ℃ and 8rpm for 56h, then carrying out low-temperature air blast drying to obtain an antibacterial peptide solid, and then adding the antibacterial peptide solid into the antibacterial peptide solid according to the weight ratio of 10: 1, adding sterile purified water, leaching for 2 hours at 180rpm, centrifugally concentrating the leaching solution, and sterilizing by a 0.22-micron filter membrane to obtain an antibacterial peptide liquid;
and 4, step 4: adjusting the pH value of the antibacterial peptide liquid to 2.0, standing at 4 ℃ for 12h, centrifuging at 4 ℃ and 10000rpm to obtain a precipitate, dissolving the precipitate with 90% ethanol, uniformly stirring, adjusting the pH value of the solution to 7.0, standing at room temperature for 3h, centrifuging at 4 ℃ and 10000rpm to obtain an extracting solution, concentrating the extracting solution with a rotary evaporator, and freeze-drying the concentrated solution to obtain the antibacterial peptide powder.
7. The use of the antibacterial peptide powder fermented by the fermentation method according to claim 6 is characterized in that the antibacterial peptide powder is used as a functional fertilizer for holothurian culture, and is prepared into a solution and then applied to an aquaculture pond.
CN202010824328.0A 2020-08-17 2020-08-17 Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof Active CN111733117B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010824328.0A CN111733117B (en) 2020-08-17 2020-08-17 Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010824328.0A CN111733117B (en) 2020-08-17 2020-08-17 Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof

Publications (2)

Publication Number Publication Date
CN111733117A CN111733117A (en) 2020-10-02
CN111733117B true CN111733117B (en) 2020-12-08

Family

ID=72658511

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010824328.0A Active CN111733117B (en) 2020-08-17 2020-08-17 Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof

Country Status (1)

Country Link
CN (1) CN111733117B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481267A (en) * 2021-07-05 2021-10-08 武汉华扬天乐动物保健品有限公司 Antibacterial peptide fermentation process
CN114766597B (en) * 2022-03-24 2024-04-05 焦作市佰役安生物工程有限公司 Method for preparing antibacterial peptide feed additive through duplex mixed fermentation

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999048912A1 (en) * 1998-03-25 1999-09-30 Korea Advanced Institute Of Science And Technology A novel antimicrobial peptide isolated from parasilurus asotus and its uses
CN103881936A (en) * 2012-12-20 2014-06-25 青岛中仁药业有限公司 Preparation method of solid-state microecological preparation for bacillus natto and bacillus subtilis culture
CN104946565A (en) * 2015-06-29 2015-09-30 浙江惠嘉生物科技有限公司 Solid-state continuous fermentation method of Bacillus subtilis
CN106434508A (en) * 2016-10-11 2017-02-22 上海邦成生物工程有限公司 Process for producing feed antimicrobial peptides by bacillus subtilis and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999048912A1 (en) * 1998-03-25 1999-09-30 Korea Advanced Institute Of Science And Technology A novel antimicrobial peptide isolated from parasilurus asotus and its uses
CN103881936A (en) * 2012-12-20 2014-06-25 青岛中仁药业有限公司 Preparation method of solid-state microecological preparation for bacillus natto and bacillus subtilis culture
CN104946565A (en) * 2015-06-29 2015-09-30 浙江惠嘉生物科技有限公司 Solid-state continuous fermentation method of Bacillus subtilis
CN106434508A (en) * 2016-10-11 2017-02-22 上海邦成生物工程有限公司 Process for producing feed antimicrobial peptides by bacillus subtilis and application thereof

Also Published As

Publication number Publication date
CN111733117A (en) 2020-10-02

Similar Documents

Publication Publication Date Title
CN112358999B (en) Lactobacillus reuteri and application thereof
CN106282072B (en) Compound lactobacillus microecological preparation and preparation method and application thereof
CN108251335B (en) Enterococcus faecalis HKF7 with lactic acid activity and screening culture method and application thereof
CN108504601A (en) One plant of Pediococcus acidilactici HEW-AP27 and its application
CN105176874B (en) Bacillus coagulans FM603 and its application
CN105175518B (en) The bacteriocin and preparation method thereof that bacillus coagulans FM603 is generated
CN112574924B (en) Bacillus subtilis strain, microecological preparation and application thereof
CN114085789B (en) Pediococcus pentosaceus MA.WTPQJ01 and application thereof
CN111733117B (en) Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof
CN113040390B (en) Probiotic salt-tolerant lactobacillus johnsonii and application thereof in preventing and treating pathogenic bacteria in livestock and poultry aquiculture
CN106148231B (en) One plant of enterococcus faecalis Y17 and its screening and culturing and application
CN102517227B (en) Enterococcus faecalis and applications and feed additive and leavening agent thereof
CN111269865B (en) Brevibacillus laterosporus strain S62-9 and application thereof
CN115505546B (en) Salt-tolerant bacillus for inhibiting aeromonas hydrophila and application thereof
CN111996139B (en) Composite microecological preparation for improving production benefit of meat calves and application thereof
CN104774781B (en) One bacillus subtilis DCU and application thereof
CN113502243B (en) Lactobacillus plantarum GBW-LP001 capable of highly producing lactic acid and antibacterial agent alternative thereof and application
CN107937301B (en) Bacillus amyloliquefaciens and application thereof in aquaculture
CN104726359B (en) One bacillus pumilus BP and application thereof
CN105104712B (en) A kind of additive for microbe feedstuff and preparation method thereof
CN113604387B (en) Salt-tolerant and high-temperature-resistant lactobacillus reuteri and application thereof in prevention and treatment of pathogenic bacteria in livestock and poultry aquaculture
CN112592851B (en) Lactobacillus acidophilus with broad-spectrum antagonistic effect on aquatic pathogenic bacteria and application thereof
CN114806944A (en) Lactobacillus plantarum LP11, fermentation liquor thereof, preparation method and application
CN108048341B (en) Lactic acid bacteria and application thereof
CN117965340B (en) Bacillus subtilis for preventing and treating nocardia and regulating blood sugar and blood fat and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant