CN114921385A - Bacillus subtilis and application thereof in feed addition and antibiotic-free culture - Google Patents
Bacillus subtilis and application thereof in feed addition and antibiotic-free culture Download PDFInfo
- Publication number
- CN114921385A CN114921385A CN202210681316.6A CN202210681316A CN114921385A CN 114921385 A CN114921385 A CN 114921385A CN 202210681316 A CN202210681316 A CN 202210681316A CN 114921385 A CN114921385 A CN 114921385A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- application
- feed
- preparation
- antibiotic
- 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.)
- Granted
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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Polymers & Plastics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Physiology (AREA)
- Molecular Biology (AREA)
- Animal Husbandry (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Hydrology & Water Resources (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention belongs to the field of agricultural microorganisms, and relates to bacillus subtilis and application thereof in feed addition and antibiotic-free culture. The Bacillus subtilis is Bacillus subtilis RX05, is preserved in China center for type culture Collection with a preservation date of 2022 years, 6 months and 15 days, and has a preservation number of: CCTCC NO: M2022887, and the preservation address is as follows: wuhan university, Wuhan, China zip code 430072. The bacillus subtilis provided by the invention has the characteristics of high colony growth speed, large spore production amount, high environmental adaptability, multiple enzyme production types, strong antibiotic substance production capacity and the like, can improve the utilization rate of feed, has the functions of broad-spectrum antibiosis and mycotoxin degradation, can perform environmental deodorization and water body purifying agent waste treatment, and is wide in application.
Description
Technical Field
The invention belongs to the field of agricultural microorganisms, and relates to bacillus subtilis and application thereof in feed addition and antibiotic-free culture.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The antibiotic-free feed is divided into two aspects from the feed nutrition perspective: the feed is a broad-sense non-antibiotic feed, and the feed does not contain antigens, anti-nutritional factors and antibiotics; secondly, the non-antibiotic feed in the narrow sense refers to the feed without any preventive growth-promoting antibiotics. Antibiotic substitution mainly comprises antibacterial peptide, enzyme preparation, Chinese herbal medicine extract, probiotics and the like. Among several antibiotic alternatives, probiotics are considered to be the most promising one. Bacillus is a beneficial group of microorganisms that can maintain intestinal microecological system balance. According to the research of the inventor, the probiotic bacillus for feeding shows strong effects on the aspects of improving the immunity of organisms, promoting digestion, improving the utilization rate and reproductive capacity of feed, purifying the environment and the like by maintaining the colony balance of the digestive tract, so that the bacillus capable of serving as an antibiotic substitute is required to be provided.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the bacillus subtilis and the application thereof in feed addition and nonreactive culture.
In order to achieve the purpose, the technical scheme of the invention is as follows:
on one hand, the Bacillus subtilis is Bacillus subtilis RX05, is preserved in China Center for Type Culture Collection (CCTCC) with a preservation date of 2022 years, 6 months and 15 days, and has a preservation number of: CCTCC NO: M2022887, and the preservation address is as follows: wuhan, Wuhan university, zip code 430072.
In another aspect, a Bacillus subtilis preparation comprises the Bacillus subtilis, a fermentation product of the Bacillus subtilis, and/or a metabolite of the Bacillus subtilis.
In a third aspect, any one of the following applications of the bacillus subtilis and/or the bacillus subtilis microbial inoculum is provided;
a. the application in feed addition;
b. the application in spore production;
c. the application of replacing antibiotics in non-antibiotic breeding;
d. the application in the preparation of pathogenic bacteria inhibitor;
e. the application in preparing mycotoxin degrading agent;
f. the application in treating feces odor;
g. the application in treating breeding wastewater;
h. application in the treatment of breeding waste.
The Bacillus subtilis RX05 provided by the invention has good heat resistance, and bacterial colonies can normally grow at 60 ℃; although not growing at 70 ℃, RX05 still can keep vitality after 3 days of treatment and can grow normally when transferred to 30 ℃.
The Bacillus subtilis RX05 provided by the invention has strong salt tolerance, and RX05 can keep normal growth under the treatment of 5% NaCl.
The Bacillus subtilis RX05 provided by the invention has strong acid and alkali resistance, and can still keep normal growth at about pH 3 and pH 10.
The Bacillus subtilis RX05 provided by the invention is used as a feed additive, and has the main function that the Bacillus subtilis RX05 can form endospores, can resist high temperature, extrusion, acid and bile salt, and can ensure the quality of feed in the feed processing and storage processes.
The Bacillus subtilis RX05 provided by the invention can improve the use efficiency of feed and promote the absorption and utilization of nutrient substances by organisms, and researches show that RX05 can secrete various enzymes such as amylase, protease, cellulase and the like, promote the efficient decomposition of nutrient components such as starch, protein and the like in the feed, promote the absorption and utilization of nutrient substances by the organisms and improve the growth performance of livestock and poultry.
The Bacillus subtilis RX05 provided by the invention has an inhibiting effect on various pathogenic microorganisms, and the antibacterial spectrum of the Bacillus subtilis RX05 comprises staphylococcus aureus, escherichia coli, salmonella typhimurium, salmonella choleraesuis, Bacillus thuringiensis, septicemia pleuropneumoniae, klebsiella pneumoniae and the like, so that RX05 can effectively replace antibiotics and promote nonreactive breeding of livestock and poultry; it is also possible to produce an antibiotic substance (pathogen inhibitor) by using itself or a fermentation product, a metabolite, or the like.
The Bacillus subtilis RX05 provided by the invention can degrade mycotoxin, and researches show that RX05 especially has strong degradation capability on vomitoxin and aflatoxin, wherein the degradation rate of RX05 on vomitoxin in feed reaches 76.85%, and the degradation rate on aflatoxin in feed reaches 67.53%.
The Bacillus subtilis RX05 provided by the invention has strong capability of producing anti-spore, and researches show that after 3 days of culture, the spore yield is 31.5 multiplied by 10 6 cfu/mL, yield up to 77.1%.
The Bacillus subtilis RX05 provided by the invention can enhance the metabolic activity of microorganisms in the digestive tract, reduce the emission of nitrogen, reduce the generation of odor, utilize ammonia nitrogen in excrement, reduce the odor in livestock and poultry breeding houses, and provide a good growth environment for livestock and poultry.
The Bacillus subtilis RX05 provided by the invention can decompose residual baits and organic matters in water, remove the content of ammoniacal nitrogen and nitrous nitrogen in sewage, and inhibit the propagation of germs in water.
The Bacillus subtilis RX05 provided by the invention can secrete chitinase, keratinase and other biomass degrading enzymes, can effectively degrade animal stumps, rotten feathers, lignocellulose waste and the like generated in the livestock and poultry breeding process, and realizes resource utilization and harmless treatment of waste.
The invention has the beneficial effects that: the Bacillus subtilis RX05 provided by the invention has the characteristics of high colony growth speed, large spore production quantity, high environmental adaptability, multiple enzyme production types, strong capability of producing antibiotic substances and the like, can be used as a safe, reliable and pollution-free microecological preparation adding strain, can effectively improve the quality and the use efficiency of feed, reduce odor pollution and water body pollution in livestock and poultry cultivation, and utilize cultivation wastes as resources, and has good application prospects in the aspects of improving the production environment and the growth performance of livestock and poultry.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a diagram of the whole genome sequencing result of RX05 provided by the embodiment of the present invention;
FIG. 2 is a colony morphology of RX05 provided in accordance with an embodiment of the present invention;
fig. 3 is a salt tolerance diagram of RX05 according to an embodiment of the present invention;
fig. 4 is a heat resistance diagram of RX05 according to an embodiment of the present invention;
fig. 5 is a diagram illustrating acid and alkali resistance of RX05 according to an embodiment of the present invention;
fig. 6 is a characteristic diagram of digestive enzymes (protease, amylase, cellulase) of RX05 according to the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The invention provides bacillus subtilis and application thereof in feed addition and nonreactive culture.
The invention provides a typical implementation mode of Bacillus subtilis, which is Bacillus subtilis RX05 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2022 years, 6 months and 15 days, and the preservation numbers are as follows: CCTCC NO, M2022887, preservation address is as follows: wuhan, Wuhan university, zip code 430072. The bacterial strain is separated from saline-alkali wetland sediment of a natural protection area in the yellow river delta country, RX05 bacterial strain has completed whole genome sequencing, the genome of the bacterial strain is a circular genome, the size is 4063959bp, the GC content is 43.84%, and 4,217 coding genes are totally contained.
RX05 has the identifying characteristics of Bacillus subtilis. The expression is as follows: the bacterial colony grows rapidly on a nutrient gravy culture medium, is a circular bacterial colony with the size of 1-2mm, and has neat edges, light yellow color and non-transparency; the surface is rough and dark, the texture is dry, the surface is rough, the edge is irregular, the front surface is grey white or light, the shape is flat, the picking is easy, and no pigment is produced; gram-positive staining, rod-like, little chain formation and uniform staining; the flagella grow sideways and can move; endospores were 0.8 × (1.5-1.8) μm, and the surface coloration of free spores was weak.
In another embodiment of the present invention, a bacillus subtilis microbial inoculum is provided, which comprises the bacillus subtilis, a fermentation product of the bacillus subtilis and/or a metabolite of the bacillus subtilis.
In a third embodiment of the present invention, there is provided any one of the following applications of the above-mentioned bacillus subtilis and/or bacillus subtilis microbial agent;
a. the application in feed addition;
b. the application in producing spores;
c. the application of replacing antibiotics in non-antibiotic breeding;
d. the application in the preparation of pathogenic bacteria inhibitor;
e. the application in preparing mycotoxin degrading agent;
f. the application in treating feces odor;
g. the application in treating breeding wastewater;
h. application in the treatment of breeding waste.
More specifically, in the application of feed addition, Bacillus subtilis RX05 is mainly used as a feed additive to improve the use efficiency of the feed and promote the absorption and utilization of nutrients by organisms. Therefore, the invention provides a livestock and poultry feed, which comprises nutrient substances and a feed additive, wherein the feed additive is the Bacillus subtilis RX 05. The nutrient substances include soybean meal, wheat bran, corn flour, etc.
More specifically, in the application of producing spores, Bacillus subtilis RX05 is adopted for fermentation. The components of the culture medium for producing spores comprise, by mass, 1.5-2.5 parts of glucose, 0.5-1.5 parts of soybean meal, 0.05-0.15 part of peptone, 1.5-2.5 parts of sodium nitrate, 0.05-0.15 part of potassium dihydrogen phosphate, 0.03-0.07 part of disodium hydrogen phosphate, 0.01-0.03 part of magnesium sulfate and 0.01-0.03 part of manganese sulfate. The initial pH value of the fermentation is 6.9-7.1. The inoculation amount is 3-5%.
More specifically, the antibiotic-substituted compound can be used as a feed additive for replacing antibiotics and a medicament for replacing antibiotics in the application of antibiotic-free breeding.
More specifically, in the application of preparing the pathogenic bacteria inhibitor, the pathogenic bacteria comprise staphylococcus aureus, escherichia coli, salmonella typhimurium, salmonella choleraesuis, bacillus thuringiensis, septicemia pleurorum, klebsiella pneumoniae and the like.
More particularly, in the application of preparing the mycotoxin degrading agent, the mycotoxin comprises vomitoxin and/or aflatoxin.
More specifically, in the application of treating the foul smell of the excrement, a specific treatment method for treating the foul smell of the excrement is provided, bacterial liquid is sprayed into the excrement, and an effective strain in the bacterial liquid is the Bacillus subtilis RX 05. The liquid bacteria is sprayed in the feces moving process, so that the feces deodorization effect is improved.
More specifically, in the application of treating the aquaculture wastewater, a specific treatment method of the aquaculture wastewater is provided, and a bacterial liquid is sprayed into the aquaculture wastewater, wherein an effective strain in the bacterial liquid is the Bacillus subtilis RX 05.
More specifically, in the application of treating the cultivation waste, a specific method for treating the cultivation waste is provided, and the cultivation waste is subjected to fermentation treatment by using the Bacillus subtilis RX 05. The breeding waste of the invention is animal stump, poultry feather and the like.
In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the technical scheme of the present invention will be described in detail below by combining specific examples and comparative examples.
Example 1 isolation and characterization of Bacillus subtilis RX05
(1) Strain separation:
weighing 10g of sample from saline-alkali wetland sediment in national natural reserve of yellow river delta, grinding into fine powder, placing the powder into a sterilized triangular flask containing 90mL of 0.9% NaCl saline, shaking for 1h at 28 ℃, and fully mixing uniformly to prepare a soil suspension; putting 1mL of the culture medium into 50mL of nutrient broth liquid culture medium, and carrying out enrichment culture at 30 ℃ and 150r/min for 24 h. Incubating the obtained bacterial suspension in 90 deg.C water bath for 10min, and diluting 0.1mL with sterile water to 10 -6 、10 -7 And 10 -8 Three concentration gradients, spread on nutrient gravy agar plates, and incubate at 30 ℃ for 24 h. Colonies were picked for spore staining. And selecting bacterial colonies with spores, carrying out streaking culture by utilizing a nutrient gravy agar plate to obtain single bacterial colonies, and storing for later use. The nutrient gravy liquid culture medium comprises the following components: 3.0g of beef extract, 10.0g of peptone, 5.0g of NaCl, 1.0L of distilled water and 7.0 of pH, and if a nutrient meat juice agar culture medium is prepared, 20.0g of agar is added. The pH value is 7.0-7.2. Sterilizing at 121 deg.C for 15 min.
(2) And (3) strain identification:
genomic DNA was extracted using a bacterial DNA extraction kit (Omega, USA), and the 16S rRNA gene was amplified using bacterial universal primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). And (3) carrying out sequencing on the PCR product by being handed over to Shanghai biological products corporation, comparing NCBI and ISTH, finding that the bacterial strain RX05 is Bacillus subtilis (named as Bacillus subtilis) RX05, storing the bacterial strain in China Center for Type Culture Collection (CCTCC) with the preservation date of 2022 years, 6 months and 15 days, and the preservation number is as follows: CCTCC NO: M2022887. Meanwhile, whole genome sequencing work is carried out on Bacillus subtilis RX05, and a genome map schematic diagram is shown in FIG. 1.
(3) The strains were cultured and observed:
the strains were cultured upside down on nutrient broth agar medium (FIG. 2). RX05 is grown on nutrient broth culture medium rapidly, and has round colony with size of 1-2mm, neat edge, light yellow color and opacity; rough and dark surface, dry texture, rough surface, irregular edge, grey white or light color on the front surface, flat shape, easy picking and no pigment generation.
Example 2 Bacillus subtilis RX05 fermentation spore production experiment
Seed culture: selecting a plate culture medium with good growth state, selecting activated bacteria, inoculating into a nutrient gravy liquid culture medium, culturing at 30 ℃ at 150r/min for 24h, and collecting bacterial liquid as seed liquid.
The spore-forming culture medium comprises the following components: 2% of glucose, 1% of soybean meal, 0.1% of peptone, 0.2% of sodium nitrate, 0.1% of potassium dihydrogen phosphate, 0.05% of disodium hydrogen phosphate, 0.02% of magnesium sulfate and 0.02% of manganese sulfate.
The fermentation conditions are that the initial pH value is 7, the temperature is 30 ℃, the rotating speed is 250r/min, the liquid loading amount is 125mL/250mL, the inoculation amount is 4 percent,
the Bacillus subtilis RX05 has strong capability of producing anti-spore, and can grow rapidly in spore production culture medium, and the spore yield is 31.5 multiplied by 10 after 3 days of culture 6 cfu/mL, yield up to 77.1%.
Example 3 identification of stress resistance of Bacillus subtilis
(1) Salt tolerance test
Activating RX05 strain on nutrient gravy plate, culturing at 30 deg.C in dark for 24-48h, selecting plate culture medium with good growth state, and inoculating activated bacteria with inoculating needle onto nutrient gravy plate containing NaCl of different concentrations. The plates were placed at 30 ℃ for inverted culture and the growth of colonies was observed.
As a result, it was found that Bacillus subtilis RX05 has strong salt tolerance, and RX05 can maintain normal growth under 5% NaCl treatment (FIG. 3).
(2) Heat resistance test
Activating RX05 strain on a nutrient gravy plate, culturing for 24-48h at 30 ℃ in the dark, picking plate culture medium with good growth state, and picking activated bacteria by using an inoculating needle to inoculate the nutrient gravy plate. And (3) placing the plate with the bacterium blocks at 40-70 ℃ for culture, and observing the growth condition of the bacterial colonies. In addition, the plate with the bacterium block is placed at 70 ℃ for 3d culture, then the plate is transferred to 30 ℃ for culture, and the growth condition of the bacterial colony is observed.
As a result, it was found that: RX05 has strong heat resistance and can grow normally at 60 deg.C. Although not growing at 70 ℃, RX05 colonies remained viable after 3 days of treatment and grew normally when transferred to 30 ℃ (fig. 4).
(2) Acid and alkali resistance experiment
Activating RX05 strain on nutrient gravy plate, culturing at 30 deg.C in dark for 24-48h, selecting plate culture medium with good growth state, and inoculating the activated bacteria on nutrient gravy plate with different pH values by inoculating needle. The plate was placed in 30 ℃ for inverted culture and the growth of colonies was observed.
As a result, it was found that: RX05 cells had good acid and alkali resistance and could grow normally at pH 3 and pH 10 (FIG. 5).
Example 4 degradation of mycotoxins by Bacillus subtilis RX05
Activating an RX05 strain on a nutrient gravy flat plate, culturing for 24-48h at 30 ℃ in the dark, selecting a flat plate culture medium with good growth state, selecting activated bacteria, inoculating the activated bacteria into a nutrient gravy liquid culture medium, culturing for 24h at 30 ℃ at 150r/min, and collecting bacterial liquid for researching the degradation effect of vomitoxin in a wheat sample.
Respectively grinding and sterilizing feed samples with vomitoxin and aflatoxin B1 exceeding standards, drying at 40 ℃ to constant weight, and measuring the contents of vomitoxin and aflatoxin B1. Taking strain fermentation liquor, and mixing the strain fermentation liquor with a sample according to the volume-mass ratio of 5: 1; taking a feed sample added with the same volume of liquid culture medium as a blank control, incubating for 48h at 37 ℃, and measuring the residual quantity of vomitoxin and aflatoxin B1 in the sample.
TABLE 1 degradation of vomitoxin in feed samples by Bacillus subtilis RX05
TABLE 2 degradation of aflatoxin B1 in feed samples by Bacillus subtilis RX05
The results show that: after 48h of treatment, RX05 showed a degradation rate of 76.85% for vomitoxin (table 1) and 67.53% for aflatoxin B1 (table 2).
Example 5 identification of inhibitory potency of Bacillus subtilis RX05 on common livestock and poultry breeding pathogenic bacteria
Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Salmonella choleraesuis, Bacillus thuringiensis, Patrinia scabiosa, Klebsiella pneumoniae, Edwardsiella, Pseudomonas fluorescens, Aeromonas hydrophila and the like are selected as the indicator pathogenic bacteria.
TABLE 3 inhibitory Effect of Bacillus subtilis RX05 on pathogenic bacteria
Activating RX05 strain on a nutrient gravy plate, culturing for 24-48h at 30 ℃ in the dark, selecting a plate culture medium with good growth state, selecting activated bacteria by using an inoculating needle, inoculating RX05 strain on a plate coated with pathogenic bacteria, culturing at 30 ℃ in the dark, observing whether a bacteriostatic transparent area or a bacteriostatic coverage area appears around the seeding area after 48h, and measuring the size of a bacteriostatic zone. The bacteriostasis is expressed as the ratio of the diameter of the bacteriostasis zone to the diameter of a bacterial colony, the ratio of 1 represents no inhibition, the ratio of 1-2 represents general inhibition, and the ratio of more than 2 represents obvious inhibition.
The results show that: RX05 has significant inhibitory effect on Escherichia coli, Salmonella typhimurium, Salmonella choleraesuis, Patrinia scabiosa, Klebsiella pneumoniae, Edwardsiella, Pseudomonas fluorescens, and Aeromonas hydrophila, and also has inhibitory effect on Staphylococcus aureus and Bacillus thuringiensis (Table 3).
Example 6 application of Bacillus subtilis RX05 for producing digestive enzymes and improving feed utilization rate
Activating RX05 strain on nutrient gravy plate, culturing at 30 deg.C in dark for 24-48h, picking plate culture medium with good growth state, and inoculating activated bacteria with inoculating needle to screening culture medium plate containing different substrates. The plate was placed at 30 ℃ for 48h of inverted culture and the colony growth and hydrolysis conditions were observed. The protease screening culture medium comprises the following components: 5.0g of yeast powder, 10.0g of glucose, 4.0g of dipotassium phosphate, 20.0g of skimmed milk powder, 20.0g of agar, 1000mL of distilled water and 7.0-7.2 of pH; the cellulase screening culture medium comprises the following components: 5.0g of peptone, 5.0g of yeast powder, 20.0g of CMC-Na, 4.0g of dipotassium hydrogen phosphate, 20.0g of agar, 1000mL of distilled water and 7.0-7.2 of pH; the formula of the amylase screening culture medium is as follows: 10.0g of starch, 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl, 20.0g of agar powder, 1000mL of distilled water and pH 7.0-7.2.
The results show that: after 48h of culture, RX05 can grow normally on the protease screening culture medium, the amylase screening culture medium and the cellulase screening culture medium, and has obvious hydrolysis loop, which shows that RX05 has good characteristics of producing protease, amylase and cellulase (figure 6).
TABLE 4 Bacillus subtilis RX05 in vitro simulated digestion test
In vitro simulation digestion test, bean pulp, wheat bran and corn flour are dried at 80 deg.C for 24 hr, pulverized, sieved (80 mesh), and mixed into combined feed (A: corn flour + bean pulp; B: corn flour + wheat bran); . Weighing 10g of three combined feeds in a 250mL triangular flask, adding 50mL of sterile water after sterilization, adding 1mL of RX05 bacterial liquid, shaking up and standing, adjusting the pH to about 6, adding 10% of hyocholic acid salt solution, culturing at 37 ℃ and 50r/min for 72h, and measuring the dry matter digestibility, the crude fiber digestibility and the soluble protein increment.
The results show that: after 72h of treatment, RX05 effectively enhanced the dry matter digestibility, the group fiber digestibility of the feed, and significantly increased the soluble protein increase rate (Table 4).
Example 7 application of Bacillus subtilis RX05 to elimination of foul smell of breeding manure
Activating RX05 strain on a nutrient gravy plate, culturing for 24-48h at 30 ℃ in the dark, selecting a plate culture medium with good growth state, selecting activated bacteria, inoculating into 500mL of nutrient gravy liquid culture medium, culturing for 72h at 30 ℃ at 220r/min, and collecting bacterial liquid for later use.
Two manure pools of a certain pig farm are selected for experimental study. And (3) 20 tons of excrement are contained in the excrement pool A, 10 tons of excrement are transferred into the other cleaned excrement pool B, and bacterial liquid is sprayed in the excrement transfer process. Taking the fecal sewage tank B sprayed with the bacterial liquid as a test group, and taking the fecal sewage tank A with 10 tons of residual bacterial liquid as a control group. NH was detected once after 24h 3 、H 2 S and the concentration of the malodorous gas, and the deodorization effect is inspected.
TABLE 5 deodorizing effect of Bacillus subtilis RX05 on cultured manure
The results show that: NH in the culture manure after 24 hours of treatment by RX05 strain 3 、H 2 The concentration of S as well as malodorous gases decreased significantly (table 5).
Example 8 application of Bacillus subtilis RX05 to treatment of livestock and poultry farming wastewater
Activating RX05 strain on a nutrient gravy plate, culturing for 24-48h at 30 ℃ in the dark, selecting a plate culture medium with good growth state, selecting activated bacteria, inoculating into 500mL of nutrient gravy liquid culture medium, culturing for 72h at 30 ℃ at 220r/min, and collecting bacterial liquid for later use.
TABLE 6 purification Effect of Bacillus subtilis RX05 on cultivation wastewater
Collecting the breeding wastewater of pig farm, adding 10L into sterilized glass jar (volume 20L), covering the jar with gauze, and standing in shade for 2 days. Adding RX05 into the jar to make the bacterial liquid concentration in water body be 2 × 10 6 cfu/mL. Test deviceAnd taking a water sample from the tank 24 hours after the start, and measuring water quality indexes such as nitrite nitrogen, ammonia nitrogen, chemical oxygen demand and the like after filtering through a filter membrane of 0.22 mu m. And taking the static culture wastewater without adding the bacterial liquid as a control group.
The results show that: after 24 hours of treatment by RX05 strain, the ammonia nitrogen content, nitrite content and chemical oxygen demand in the aquaculture wastewater are all significantly reduced (Table 6).
Example 9 application of Bacillus subtilis RX05 in degradation of livestock and poultry breeding waste
The main wastes in the livestock and poultry industry include animal stumps, poultry feathers and the like, most of which are not effectively utilized and are discarded, thereby causing environmental pollution. The feather is mainly composed of more than 90% of keratin, is rich in microelements such as calcium and phosphorus and a plurality of amino acids, and is protein with potential application value.
Poultry feathers are from poultry farms. 100mL of fermentation medium is filled into a 250mL triangular flask, after sterilization and cooling, bacillus subtilis RX05 is inoculated into the fermentation medium according to the proportion of 4 percent, and the fermentation medium is cultured for 6 days at the temperature of 30 ℃ and at the speed of 200r/min, and then fermentation liquor is collected. Centrifuging at 4 deg.C at 8000 r/min to obtain supernatant, and measuring feather degradation rate, protein concentration, amino acid content, etc. The formula of the fermentation medium is as follows: k 2 HPO 4 0.15g、MgSO 4 ·7H 2 O 0.0012g、CaCl 2 0.0025g、FeSO 4 ·7H 2 O 0.0015g、ZnSO 4 ·7H 2 O0.0005 g, feather 5g, water 100mL, pH 8.0.
TABLE 7 degradation Effect of Bacillus subtilis RX05 on poultry feathers
The results show that: after the poultry feather is treated by the RX05 strain for 6d, the feather degradation rate can reach 81.31%, the content of soluble polypeptide in the collected fermentation supernatant is 20.95mg/mL, and the content of amino acid is 11.48mg/mL (Table 7).
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The Bacillus subtilis is characterized by being named as Bacillus subtilis RX05 and preserved in China center for type culture Collection with the preservation date of 2022 years, 6 months and 15 days, and the preservation numbers are as follows: CCTCC NO: M2022887.
2. A Bacillus subtilis preparation comprising the Bacillus subtilis of claim 1, a fermentation product of the Bacillus subtilis of claim 1, and/or a metabolite of the Bacillus subtilis of claim 1.
3. The use of any one of the following bacillus subtilis of claim 1 and/or the bacillus subtilis preparation of claim 2;
a. the application in feed addition;
b. the application in producing spores;
c. the application of replacing antibiotics in non-antibiotic breeding;
d. the application in the preparation of pathogenic bacteria inhibitor;
e. the application in the preparation of mycotoxin degradation agents;
f. the application in treating feces odor;
g. the application in treating breeding wastewater;
h. application in the treatment of breeding waste.
4. The use according to claim 3, wherein in the spore production, the spore production medium comprises, by mass, 1.5 to 2.5 parts of glucose, 0.5 to 1.5 parts of soybean meal, 0.05 to 0.15 part of peptone, 1.5 to 2.5 parts of sodium nitrate, 0.05 to 0.15 part of potassium dihydrogen phosphate, 0.03 to 0.07 part of disodium hydrogen phosphate, 0.01 to 0.03 part of magnesium sulfate, and 0.01 to 0.03 part of manganese sulfate. The initial pH of the fermentation is 6.9-7.1.
5. The use according to claim 3, wherein in the preparation of the inhibitor of pathogenic bacteria, the pathogenic bacteria is one or more of Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Salmonella choleraesuis, Bacillus thuringiensis, Patrinia scabiosaefolia, and Klebsiella pneumoniae.
6. Use according to claim 3, wherein in the preparation of a mycotoxin degradation agent, the mycotoxin comprises vomitoxin and/or aflatoxin.
7. A feed for livestock and poultry, which is characterized by comprising nutrient substances and a feed additive, wherein the feed additive is the bacillus subtilis of claim 1; preferably, the nutrient substance includes soybean meal, wheat bran, corn flour, etc.
8. A method for treating odor of feces, which is characterized in that a bacterial liquid is sprayed into the feces, wherein an effective strain in the bacterial liquid is the bacillus subtilis in claim 1; preferably, the bacterial liquid is sprayed during the movement of the manure.
9. A specific treatment method of aquaculture wastewater is characterized in that a bacterial solution is sprayed into the aquaculture wastewater, and an effective strain in the bacterial solution is the bacillus subtilis in claim 1.
10. A specific method for treating cultivation waste, which is characterized in that the bacillus subtilis of claim 1 is used for fermenting the cultivation waste.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210681316.6A CN114921385B (en) | 2022-06-16 | 2022-06-16 | Bacillus subtilis and application thereof in feed addition and antibiotic-free cultivation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210681316.6A CN114921385B (en) | 2022-06-16 | 2022-06-16 | Bacillus subtilis and application thereof in feed addition and antibiotic-free cultivation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114921385A true CN114921385A (en) | 2022-08-19 |
| CN114921385B CN114921385B (en) | 2023-06-20 |
Family
ID=82815363
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210681316.6A Active CN114921385B (en) | 2022-06-16 | 2022-06-16 | Bacillus subtilis and application thereof in feed addition and antibiotic-free cultivation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114921385B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851533A (en) * | 2022-12-05 | 2023-03-28 | 众乐(潍坊)生物科技有限公司 | Bacillus thuringiensis and application thereof in inhibition of livestock and poultry feed mould |
| CN117070428A (en) * | 2023-10-17 | 2023-11-17 | 北京大北农科技集团股份有限公司 | Application of bacillus subtilis BS-22 strain in improving cultivation environment |
| CN117106676A (en) * | 2023-10-24 | 2023-11-24 | 北京大北农科技集团股份有限公司 | Bacillus subtilis and application thereof in feed production |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684450A (en) * | 2008-09-22 | 2010-03-31 | 北京欣博阳科技有限公司 | Fodder premix and special bacillus subtilis thereof |
| CN104099276A (en) * | 2014-07-16 | 2014-10-15 | 湖南省微生物研究院 | Bacillus subtilis AP139 and fermented microbial inoculum thereof, and application method of bacillus subtilis AP139 and fermented microbial inoculum |
| CN108865928A (en) * | 2018-06-13 | 2018-11-23 | 铜仁职业技术学院 | A kind of bacillus subtilis and the method for being used to prepare fruit of negundo probiotics |
| CN113249255A (en) * | 2021-05-18 | 2021-08-13 | 山东康地恩生物科技有限公司 | Bacillus subtilis for preventing and controlling enteritis of poultry and application thereof |
-
2022
- 2022-06-16 CN CN202210681316.6A patent/CN114921385B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684450A (en) * | 2008-09-22 | 2010-03-31 | 北京欣博阳科技有限公司 | Fodder premix and special bacillus subtilis thereof |
| CN104099276A (en) * | 2014-07-16 | 2014-10-15 | 湖南省微生物研究院 | Bacillus subtilis AP139 and fermented microbial inoculum thereof, and application method of bacillus subtilis AP139 and fermented microbial inoculum |
| CN108865928A (en) * | 2018-06-13 | 2018-11-23 | 铜仁职业技术学院 | A kind of bacillus subtilis and the method for being used to prepare fruit of negundo probiotics |
| CN113249255A (en) * | 2021-05-18 | 2021-08-13 | 山东康地恩生物科技有限公司 | Bacillus subtilis for preventing and controlling enteritis of poultry and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 张昊雪: "枯草芽孢杆菌在反刍动物饲料中应用的研究进展", 《粮食与饲料工业》, no. 1 * |
| 樊梅娜等: "禁抗背景下益生菌制剂在畜禽养殖中的应用研究进展", 《广东饲料》, vol. 31, no. 04 * |
| 王国艳: "肉牛场环境中枯草芽孢杆菌的16S rDNA分离鉴定", 《现代畜牧兽医》, vol. 12 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851533A (en) * | 2022-12-05 | 2023-03-28 | 众乐(潍坊)生物科技有限公司 | Bacillus thuringiensis and application thereof in inhibition of livestock and poultry feed mould |
| CN115851533B (en) * | 2022-12-05 | 2023-06-27 | 众乐(潍坊)生物科技有限公司 | Bacillus thuringiensis and application thereof in inhibiting livestock and poultry feed mould |
| CN117070428A (en) * | 2023-10-17 | 2023-11-17 | 北京大北农科技集团股份有限公司 | Application of bacillus subtilis BS-22 strain in improving cultivation environment |
| CN117070428B (en) * | 2023-10-17 | 2024-02-27 | 北京大北农科技集团股份有限公司 | Application of bacillus subtilis BS-22 strain in improving cultivation environment |
| CN117106676A (en) * | 2023-10-24 | 2023-11-24 | 北京大北农科技集团股份有限公司 | Bacillus subtilis and application thereof in feed production |
| CN117106676B (en) * | 2023-10-24 | 2024-02-27 | 北京大北农科技集团股份有限公司 | Bacillus subtilis and application thereof in feed production |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114921385B (en) | 2023-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100588624C (en) | Microorganism renovation agent of water environment and preparation method thereof | |
| CN114921385B (en) | Bacillus subtilis and application thereof in feed addition and antibiotic-free cultivation | |
| CN109337841B (en) | Bacillus subtilis BYS2 with efficient antibacterial performance | |
| CN110699300B (en) | Preparation method and application of composite microorganism substrate modifier with aquatic pathogenic bacteria antagonistic property | |
| CN102286376B (en) | Microbial inoculum for high-efficiency fermenting bed and preparation method thereof | |
| CN108611299B (en) | Lactobacillus plantarum for producing antibacterial peptide and application thereof | |
| CN108949739A (en) | A kind of complex micro organism fungicide and preparation method thereof for advanced treating high concentration livestock breeding wastewater | |
| CN109762767B (en) | Sewage composite microbial treatment agent and preparation method and application thereof | |
| CN101921710B (en) | Repairing agent for microbes in water bodies of excessive culture zones | |
| CN106854627A (en) | A kind of composite bacteria agent for promoting the flat angle stratiomyiid larval growth of speck and application | |
| CN102517227B (en) | Enterococcus faecalis and applications and feed additive and leavening agent thereof | |
| CN111593010B (en) | Lactobacillus plantarum and method for producing fermented feed by using same | |
| CN103184174A (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN117070428B (en) | Application of bacillus subtilis BS-22 strain in improving cultivation environment | |
| CN114437975B (en) | Lactic acid-producing bacillus coagulans and application thereof | |
| CN103451117B (en) | Bacillus preparation for preventing and controlling pathogenic bacteria in soil environments as well as preparation method and application thereof | |
| CN113678962B (en) | Micro-ecological feed additive for aquaculture and use method thereof | |
| CN103966145B (en) | One strain lactobacillus lactis and the application in the antibacterial polypeptide of fermentation product thereof | |
| CN112481163A (en) | Bacterial strain for aquatic feed addition and application thereof | |
| CN117106676B (en) | Bacillus subtilis and application thereof in feed production | |
| CN113337447B (en) | Complex microbial inoculant, fermented feed, application and preparation method | |
| CN117343883B (en) | Lactobacillus plantarum and application thereof | |
| CN113729109B (en) | Fermented feed rich in antibacterial substances and preparation method thereof | |
| CN111944717B (en) | Bacillus amyloliquefaciens TL106 and application thereof | |
| CN114921384B (en) | Bacillus subtilis and application thereof in degradation of medium-chain and short-chain chlorinated paraffin |
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 |