CN115838647B - Compound microecological preparation for preventing and treating bovine mastitis and application thereof - Google Patents
Compound microecological preparation for preventing and treating bovine mastitis and application thereof Download PDFInfo
- Publication number
- CN115838647B CN115838647B CN202210042069.5A CN202210042069A CN115838647B CN 115838647 B CN115838647 B CN 115838647B CN 202210042069 A CN202210042069 A CN 202210042069A CN 115838647 B CN115838647 B CN 115838647B
- Authority
- CN
- China
- Prior art keywords
- bacillus
- percent
- culture medium
- seed
- microecological preparation
- 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
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 38
- 150000001875 compounds Chemical class 0.000 title claims abstract description 29
- 208000031462 Bovine Mastitis Diseases 0.000 title claims description 6
- 241000283690 Bos taurus Species 0.000 claims abstract description 46
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 43
- 241000193749 Bacillus coagulans Species 0.000 claims abstract description 42
- 229940054340 bacillus coagulans Drugs 0.000 claims abstract description 42
- 208000004396 mastitis Diseases 0.000 claims abstract description 34
- 239000001963 growth medium Substances 0.000 claims description 40
- 239000000843 powder Substances 0.000 claims description 28
- 238000000855 fermentation Methods 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 238000012258 culturing Methods 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 14
- 229920001817 Agar Polymers 0.000 claims description 13
- 239000008272 agar Substances 0.000 claims description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 9
- 108010080698 Peptones Proteins 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 229940099596 manganese sulfate Drugs 0.000 claims description 9
- 239000011702 manganese sulphate Substances 0.000 claims description 9
- 235000007079 manganese sulphate Nutrition 0.000 claims description 9
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 9
- 235000019319 peptone Nutrition 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 238000001694 spray drying Methods 0.000 claims description 6
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 239000012137 tryptone Substances 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- -1 citric acid diamine Chemical class 0.000 claims description 3
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 abstract description 16
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 16
- 241000193985 Streptococcus agalactiae Species 0.000 abstract description 16
- 235000013365 dairy product Nutrition 0.000 abstract description 12
- 244000000010 microbial pathogen Species 0.000 abstract description 10
- 208000015181 infectious disease Diseases 0.000 abstract description 8
- 238000009395 breeding Methods 0.000 abstract description 7
- 230000001488 breeding effect Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 244000052616 bacterial pathogen Species 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 239000004480 active ingredient Substances 0.000 abstract 1
- 238000013329 compounding Methods 0.000 abstract 1
- 210000001082 somatic cell Anatomy 0.000 description 13
- 230000006651 lactation Effects 0.000 description 12
- 235000013336 milk Nutrition 0.000 description 12
- 239000008267 milk Substances 0.000 description 12
- 210000004080 milk Anatomy 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 230000001717 pathogenic effect Effects 0.000 description 9
- 235000020247 cow milk Nutrition 0.000 description 8
- 230000003385 bacteriostatic effect Effects 0.000 description 6
- 239000002131 composite material Substances 0.000 description 6
- 239000006041 probiotic Substances 0.000 description 6
- 235000018291 probiotics Nutrition 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 101150013736 gyrB gene Proteins 0.000 description 5
- 238000009629 microbiological culture Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000003042 antagnostic effect Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000000529 probiotic effect Effects 0.000 description 4
- 229910021536 Zeolite Inorganic materials 0.000 description 3
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 210000005075 mammary gland Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000010457 zeolite Substances 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102000014171 Milk Proteins Human genes 0.000 description 2
- 108010011756 Milk Proteins Proteins 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000021239 milk protein Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101100230413 Streptomyces niveus gyrBR gene Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007236 host immunity Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000021243 milk fat Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Abstract
The invention discloses a compound microecological preparation for preventing and treating cow mastitis and application thereof, which is prepared by compounding bacillus bailii and bacillus coagulans serving as active ingredients. The bacillus belicus and bacillus coagulans strain has a strong inhibition effect on pathogenic bacteria such as staphylococcus aureus, streptococcus agalactiae, escherichia coli and the like, and can inhibit pathogenic microorganisms in a breeding environment and improve the breeding environment on one hand, and can inhibit infection of pathogenic microorganisms on dairy cows on the other hand, so that the dairy cow mastitis is effectively prevented and treated.
Description
Technical Field
The invention relates to the technical field of feeds, in particular to a preparation method and an application method of a compound microbial ecological agent for preventing and treating bovine mastitis.
Background
Mastitis is one of the most common mammary gland diseases in the dairy cow feeding process, and can be divided into two types of clinical mastitis and recessive mastitis, wherein the two types of mastitis are usually cows in the early lactation period and high-yield cows in the lactation peak period are easy to develop. Research shows that 5% -10% of cows have clinical mastitis, not only can the lactation yield of the cows be reduced, but also fibrosis and small-sized swelling of the milk area can be caused, the level of plasmin in the cow milk is increased, the proteolysis of milk proteins such as casein is increased, the quality of milk and related dairy products is obviously reduced, and great economic loss is caused.
The occurrence of cow mastitis is the result of the combined action of a plurality of complex factors such as pathogenic microorganism infection, cow self and genetic factors, feeding management factors, nutrition factors, environmental factors and the like. Among them, pathogenic microorganism infection is the most major cause of bovine mastitis, and pathogenic microorganisms are mainly bacteria such as staphylococcus aureus, streptococcus agalactiae, escherichia coli, and the like, and in addition, some fungi and viruses. Staphylococcus aureus is a common pathogen of cow mastitis, can invade from a nipple canal and spread to the inside of mammary tissue, generates a plurality of pathogenic factors such as cell adhesion, toxic extracellular proteins, capsular polysaccharide and the like, causes acute mastitis or chronic mastitis, and has certain characteristics such as biofilm formation, intracellular survival, capsular expression and the like which are related to the occurrence of cow mastitis. Streptococcus agalactiae is an infectious pathogen, is highly infectious, mainly causes recessive infection of the mammary glands of cows, and can be rapidly spread in the cow groups under the condition of lack of management and prevention and control measures. Coli causes inflammation and immune defenses in the udder through various cytokines and chemokines, mainly causes escherichia coli cow mastitis in the perinatal period and early lactation period when the host immunity is inhibited, has different symptoms, is slightly and locally mastitis, and causes serious mastitis and even death.
Researchers in various countries in the world apply multidisciplinary theory and methods, and have conducted intensive basic research on diagnosis and prevention of cow mastitis from different angles, but as pathogenic factors are various, and meanwhile, the factors have interaction and synergistic effects, so far no effective method for preventing and treating cow mastitis exists. At present, various comprehensive measures are adopted in the prevention and treatment of cow milk Fang Yan, including disease-resistant breeding, nutrition regulation and control, enhanced feeding management, early detection, drug treatment and the like of the cows, and antibiotic treatment is still a main method for preventing and treating cow mastitis. In recent years, researchers have achieved many results in bacteriostasis of probiotics, regulation of intestinal health, improvement of the microecological environment of farms, improvement of animal immune functions and the like, and have also achieved good effects in disease control, improvement of production performance and the like. The probiotics can inhibit the proliferation of pathogenic microorganisms such as staphylococcus aureus, streptococcus agalactiae, escherichia coli and the like or directly kill the pathogenic microorganisms by competing for adhesion sites or producing substances with antibacterial activity such as bacteriocin and the like, and can regulate the environment of a farm and maintain animal health, thereby reducing the occurrence of cow mastitis.
Disclosure of Invention
The invention aims to provide a compound microecological preparation consisting of bacillus belicus and bacillus coagulans. The bacillus belicus and bacillus coagulans strain has a strong inhibition effect on pathogenic bacteria such as staphylococcus aureus, streptococcus agalactiae, escherichia coli and the like, and can inhibit pathogenic microorganisms in a breeding environment, improve the breeding environment, inhibit infection of pathogenic microorganisms on dairy cows and effectively prevent and treat dairy cow mastitis when being used in combination.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a compound microecological preparation for preventing and treating cow mastitis comprises Bacillus bailii (CGMCC 19349), bacillus coagulans (CGMCC No. 16244), and optionally carrier, preferably zeolite powder.
Preferably, the number of bacillus bailii viable bacteria in each gram of the composite microecological preparation is 5.0x10 9 -1.0×10 10 CFU/g。
Preferably, the viable count of bacillus coagulans in each gram of the composite microecological preparation is 5.0x10 9 -1.0×10 10 CFU/g。
The preparation method of the composite microecological preparation comprises the following steps:
the production method of bacillus belicus comprises the following steps:
(1) Inoculating Bacillus bailii strain on slant culture medium, and culturing at 30-40deg.C for 12-24 hr;
(2) Inoculating Bacillus bailii subjected to slant culture into a seed activation liquid culture medium, wherein the liquid loading amount of a 500mL triangular flask is 100-200mL, and culturing for 12-24 hours at 30-40 ℃ and 180-220 rpm;
(3) Inoculating Bacillus bailii seed solution in 0.5-2% by weight into a seed tank, culturing at 30-40deg.C and 180-220rpm for 12-24 hr with 500L seed tank liquid volume of 100-200L;
(4) Inoculating the seed tank culture solution into a fermentation tank at a weight ratio of 0.5-2%, wherein the tank pressure is 0.01-0.05MPa, the stirring rotation speed is 180-220rpm, the ventilation ratio is 0.5-1:0.1-0.5 in terms of volume ratio, and the fermentation time is 16-24 hours;
(5) Spray drying bacillus belicus fermentation liquor through a drying tower, wherein the air inlet temperature of an air blower is 140-170 ℃; the outlet temperature is 60-80 ℃, and the dried product passes through a 60-mesh vibrating screen.
The slant culture medium in the step (1) comprises the following components: 5.0-15.0g of tryptone, 1.0-5.0g of beef extract powder, 5.0-10.0g of sodium chloride, 3.0-8.0g of glucose, 15-20g of agar powder, 1000mL of distilled water and pH of 7.0-7.5;
the seed culture medium in the step (2) is the slant culture medium in the step (1) for removing the agar powder;
the seeding tank culture medium in the step (3) and the fermentation culture medium in the step (4) are composed of: 1.0 to 2.0 percent of soybean peptone, 1.0 to 2 percent of bean cake powder, 1.0 to 1.5 percent of glucose, 0.5 to 2 percent of corn flour, 0.5 to 2 percent of calcium carbonate, 0.001 to 0.01 percent of magnesium sulfate, 0.001 to 0.01 percent of manganese sulfate and pH of 7.0 to 7.5.
The production method of the bacillus coagulans comprises the following steps:
(1) Inoculating bacillus coagulans on the slant culture medium, and culturing for 24-48 hours under the facultative or anaerobic condition at the temperature of 35-45 ℃;
(2) Inoculating bacillus coagulans subjected to slant culture into a liquid seed activation culture medium, and culturing for 24-48 hours at 35-45 ℃ under facultative or anaerobic conditions;
(3) Inoculating the seed liquid obtained in the step (2) into a fermentation tank culture medium, and performing stationary culture for 24-48 hours under the facultative or anaerobic condition at the temperature of 35-45 ℃.
(4) Spray drying the bacillus coagulans fermentation liquor by a drying tower, wherein the inlet air temperature of a blower is 140-170 ℃; the outlet temperature is 60-80 ℃, and the dried product passes through a 60-mesh vibrating screen.
The slant culture medium in the step (1) comprises the following components: 10.0g of peptone, 5.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 1.0mL of tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of diammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 16g of agar powder, 1000mL of distilled water and sterilizing for 30min at 121 ℃;
removing agar powder from the seed culture medium in the step (2) which is a slant culture medium;
the fermentation medium in the step (3) comprises the following components: 1 to 2.0 percent of peptone, 0.5 to 1.0 percent of yeast powder, 0.5 to 1.0 percent of corn powder, 1.0 to 3.0 percent of glucose, 0.05 to 0.10 percent of citric acid diamine, 0.05 to 0.10 percent of dipotassium hydrogen phosphate, 0.05 to 0.10 percent of magnesium sulfate, 0.05 to 0.10 percent of manganese sulfate and 6.0 to 7.0 of pH value.
Mixing Bacillus bailii and Bacillus coagulans, and mixing with zeolite powder as carrierThe live bacterial number of the bacillus rice is 5.0 multiplied by 10 9 -1.0×10 10 CFU/g, viable count of Bacillus coagulans is 5.0X10 9 -1.0×10 10 CFU/g。
The invention also provides an application method of the compound microecological preparation for preventing and treating cow mastitis, wherein the compound microecological preparation is prepared according to the proportion of 5-10g/m 2 The usage amount is sprinkled in the dairy cow breeding environment or 50-100 g/head of the compound biological microecological preparation is added into daily ration, so that pathogenic bacteria such as staphylococcus aureus, streptococcus agalactiae, escherichia coli and the like can be inhibited or killed, the breeding environment is improved, the infection of pathogenic microorganisms on dairy cows is prevented, and the dairy cow mastitis is effectively prevented.
The bacillus belgium CA03001 (Bacillus velezensis) of the invention is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center, china academy of sciences of China, with a preservation number of CGMCC No. 19349) at 1 and 13 days of 2020. Bacillus coagulans CA04121 (Bacillus coagulans) has been deposited in China general microbiological culture Collection center (CGMCC) with a deposit number of CGMCC No.16244 at 8 and 10 of 2018.
Drawings
FIG. 1 is a comparison of the bacteriostatic action of different strains against Staphylococcus aureus as a pathogenic indicator.
FIG. 2 is a comparison of the bacteriostatic action of different strains against Streptococcus agalactiae as a pathogenic indicator.
FIG. 3 is a comparison of the bacteriostatic action of different strains against E.coli as a pathogenic indicator.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and examples:
EXAMPLE 1 screening and identification of antagonistic species of Staphylococcus aureus, streptococcus agalactiae and Escherichia coli Material 1
1.1 Test materials
1.1.1 Bacterial strain
Staphylococcus aureus, streptococcus agalactiae and escherichia coli are purchased from the China center for type culture collection of industrial microorganisms, and probiotic bacterial strains are isolated and preserved by Beijing as Bo Biotechnology institute.
1.1.2 Medium
(1) LB medium
10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 1000mL of distilled water, pH 7.0+/-0.1 and sterilizing for 30min.
Solid medium: 1.6% agar powder was added on the basis of the liquid medium.
1.2 Test method
1.2.1 Screening of staphylococcus aureus, streptococcus agalactiae and escherichia coli antagonistic strains
(1) Activation of pathogenic indicator bacteria species
Staphylococcus aureus, streptococcus agalactiae and escherichia coli strains are respectively inoculated into an LB liquid culture medium and are subjected to shaking culture for 16 hours at 37 ℃ and 180rpm for standby.
(2) Preparation of probiotic culture supernatant
The probiotic strains are respectively inoculated into LB liquid culture medium and are cultured for 24 hours under the conditions of 37 ℃ and 180rpm in a shaking way. And centrifuging the culture solution at 3500rpm for 10min, and placing into a refrigerator at 4 ℃ for standby.
(3) Preparation of pathogen indicator plate
And transferring about 5mL of LB agar medium, clockwise rotating to enable the medium to be flatly paved at the bottom of the flat plate, and placing 4 oxford cups symmetrically in sequence at the bottom of the flat plate by using sterile forceps until solidification. Mixing Staphylococcus aureus, streptococcus agalactiae and Escherichia coli with LB agar medium melted and cooled to 50deg.C at a ratio of 1:100, respectively transferring about 15mL of the culture medium into corresponding culture dishes with oxford cups, cooling and solidifying, and removing the oxford cups with sterile forceps.
(4) Loading sample
100. Mu.L of the probiotic culture supernatant was removed separately from the oxford cup well of the indicator plate, taking care to avoid spillage or splatter of the liquid.
(5) Culturing
The culture dish after the sample application is kept stand for 1 to 2 hours at room temperature, and then staphylococcus aureus, streptococcus agalactiae and escherichia coli indicator bacteria are cultured for 16 to 20 hours at 37 ℃.
(6) Observing and measuring
Photographing, measuring the diameter of each bacteriostasis zone by using a vernier caliper and recording.
1.2.2 Identification of staphylococcus aureus, streptococcus agalactiae and escherichia coli antagonistic strains
gyrB gene sequence analysis: the extraction of the total bacterial DNA adopts a bacterial genome DNA extraction kit for extraction. The gyrB gene amplification primers are as follows: gyrBF:5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGG NAARTTYGA-3'; gyrBR:5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGC RTCNGTCAT-3'. Reaction system (20 μl): 1 mu LDNA template, 0.5 mu L upstream primer, 0.5 mu L downstream primer, 0.2 mu L Easy Taq, 2 mu L dNTPs, 2 mu L10 Xeasy Taq Buffer, 14 mu L ddH 2 Adding the components on ice, uniformly mixing and centrifuging; the PCR reaction procedure is that the reaction is pre-denatured for 5min at 95 ℃; denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 2min,32 cycles, and extension at 72℃for 10min. The PCR product was detected by electrophoresis on a 1% agarose gel and sent to Beijing Productivity Co. The resulting gene sequences were subjected to BLAST homology alignment in the GenBank database.
2. Results
2.1 Screening of staphylococcus aureus, streptococcus agalactiae and escherichia coli antagonistic strains
The screened strain CA03001 has the strongest inhibitory effect on streptococcus agalactiae, the bacteriostatic diameter is 20.17mm, the inhibitory effect on staphylococcus aureus is strongest by CA04121, the bacteriostatic diameter is 19.24mm, and both strains have stronger inhibitory effect on escherichia coli, as shown in table 1, and the bacteriostatic effect is shown in figures 1-3.
Table 1 antibacterial diameter
2.2 identification of strains
CA03001 strain gyrB gene sequence (SEQ NO. 1):
acagtccgccagtttgcccggcagattggaaatctcaagcgcacttttgcggcgggtcaattcccgcgcttttttcgctgccatccg cgctcttgcggccattaaacctttttcaacgattttgcgggctgagtccggattttcaagaaggaatgtttccagcgcagaagaaaacagcgtatcagtgatcgttctcgcttcggagttgccgagcttcgttttcgtctgcccttcgaattgcggatcagggtgcttaattgaaata atggcagtcagcccttccctcacatcatccccgcttaaattcggatcattttctttgaaaatcccttttcttcttgcatagtcgtttataacacgggtcagaccggttttaaatccggcttcgtgcgtgccgccttcgtatgtgttgatattatttgtgaaagaataaatattgcttgtatagc tgtcgttgtattgcaatgcaacttcaaccgttatgccgtctttctcgccttcgatataaatcggctcttcatgaacgacttctttggaacggtttaagtactcaacatagcttttgattccgccttcgtagtggtactcgtttttccgttcttgtccttcacgtttgtcttcaatcgtgatgttta caccttttgtcaggaaggccaattcccggacacggtttgaaagcaggtcatagtcgtattcggttgtttctttgaaaatttccggatccggaacgaagtgcgtaatcgttccggtcttatcagtatcaccgatcacttcaagatcggccacaggtacaccgcgctcgtacgcctg atagtggatttttccgtcacgatgaaccgtaacgtcaagagtggtcgacaaggcgtttacga。
CA04121 strain gyrB gene sequence (SEQ NO. 2):
tggaaactcgggtggcggaataggtgtcggcggtctgcacgggttggtgcgtctgttgtgaatgccctgtctacagagctcgatgt ctatgtccaccgcgacgggcatatttactaccaaaaataccgccgcggcaaacctgcctttgacttaaaaatcattggcgaaacggatcgtaccggcacaacgactcacttcttgccggatccggaaatttttacggaaacgaccgagttcgactttgatattcttacaacccg gctccgtgaacttgcctttttaaataaagggatcaaaatcaccattgaggacaaacgcgaaaaaaatccgcgcatacaagaataccattacgaaggcgggatcaaatcgtacgtggagcatttgaaccgctcgaaagaagtgctccataaggaaccgatttatgtagaagg cgaaaaagacggcataaccgttgaagtatcccttcaatacaatgacgggtttgccagcaatatctattcttttgccaataatattcacacctacgaaggcggcacacctgagtccggttttaaaacggctttaacgcgcgtcatcaatgattatgcccggaaaaacaatatgatc aaagaaaatgaagccaatttaacgggcgaagatgtccgggaaggcctcactgcgattgtttccatcaaacacccggatccgcagtttgaaggacaaacgaaaacgaagcttggaaatacagaagcaagaacgattacagatgcggtattttccgagacgtttgagaagttt atgatggaaaatcctcaagtggcgcggaaaattgtcgaaaaaggggtgatggctgcccgggcaaggatggctgcgaaaaaagcgcgcgaactgacacggagaaaaagcgcgctcgaatcgacaagcctaccgggcaaacttgcagactgcacatcacgcgacccg gctgagagcgagctctacattgtcgaaggagactcggccggcggttctgccaagcagggaagggaccggatgtttcaggcgatcctgcctttaagagggaaaattttaaacgtagagaaagcgcgtttggataaaattttgtcaaatgcggaaatccggaccattattacc gcgcttggcacaggtgtcggagaagattcgacatctagaaggcgcgtatcatagaagtcgagatccatagc。
by gyrB gene sequencing and sequence analysis, the CA03001 strain was identified as Bacillus belicus (Bacillus velezensis) and the CA04121 strain was identified as Bacillus coagulans (Bacillus coagulans). The strains are respectively preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC No.1, 3 of North West Lu No.1 of the Korean area of Beijing, and by post code 100101 of the institute of microorganisms of the national academy of sciences of China) at 4 months of 2020 and 8 months of 2018, wherein the preservation numbers are CGMCC No.19349 and CGMCC No.16244 respectively.
Example 2 preparation of composite microecological preparation
1. The production method of bacillus belicus comprises the following steps:
(1) Inoculating Bacillus bailii strain on slant culture medium, and culturing at 30-40deg.C for 12-24 hr;
(2) Inoculating Bacillus bailii subjected to slant culture into a seed activation liquid culture medium, wherein the liquid loading amount of a 500mL triangular flask is 100-200mL, and culturing for 12-24 hours at 30-40 ℃ and 180-220 rpm;
(3) Inoculating Bacillus bailii seed solution in 0.5-2% by weight into a seed tank, culturing at 30-40deg.C and 180-220rpm for 12-24 hr with 500L seed tank liquid volume of 100-200L;
(4) Inoculating the seed tank culture solution into a fermentation tank at a weight ratio of 0.5-2%, wherein the tank pressure is 0.01-0.05MPa, the stirring rotation speed is 180-220rpm, the ventilation ratio is 0.5-1:0.1-0.5 in terms of volume ratio, and the fermentation time is 16-24 hours;
(5) Spray drying bacillus belicus fermentation liquor through a drying tower, wherein the air inlet temperature of an air blower is 140-170 ℃; the outlet temperature is 60-80 ℃, and the dried product passes through a 60-mesh vibrating screen.
The slant culture medium in the step (1) comprises the following components: 5.0-15.0g of tryptone, 1.0-5.0g of beef extract powder, 5.0-10.0g of sodium chloride, 3.0-8.0g of glucose, 15-20g of agar, 1000mL of distilled water and pH of 7.0-7.5;
the seed culture medium in the step (2) is the slant culture medium in the step (1) for removing the agar powder;
the seeding tank culture medium in the step (3) and the fermentation culture medium in the step (4) are composed of: 1.0 to 2.0 percent of soybean peptone, 1.0 to 2 percent of bean cake powder, 1.0 to 1.5 percent of glucose, 0.5 to 2 percent of corn flour, 0.5 to 2 percent of calcium carbonate, 0.001 to 0.01 percent of magnesium sulfate, 0.001 to 0.01 percent of manganese sulfate and pH of 7.0 to 7.5.
2. The production method of the bacillus coagulans comprises the following steps:
(1) Inoculating bacillus coagulans on the slant culture medium, and culturing for 24-48 hours under the facultative or anaerobic condition at the temperature of 35-45 ℃;
(2) Inoculating bacillus coagulans subjected to slant culture into a liquid seed activation culture medium, and culturing for 24-48 hours at 35-45 ℃ under facultative or anaerobic conditions;
(3) Inoculating the seed liquid obtained in the step (2) into a fermentation tank culture medium, and performing stationary culture for 24-48 hours under the facultative or anaerobic condition at the temperature of 35-45 ℃.
(4) Spray drying the bacillus coagulans fermentation liquor by a drying tower, wherein the inlet air temperature of a blower is 140-170 ℃; the outlet temperature is 60-80 ℃, and the dried product passes through a 60-mesh vibrating screen.
The slant culture medium in the step (1) comprises the following components: 10.0g of peptone, 5.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 1.0mL of tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of diammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 16g of agar powder, 1000mL of distilled water and sterilizing for 30min at 121 ℃;
removing agar powder from the seed culture medium in the step (2) which is a slant culture medium;
the fermentation medium in the step (3) comprises the following components: 1 to 2.0 percent of peptone, 0.5 to 1.0 percent of yeast powder, 0.5 to 1.0 percent of corn powder, 1.0 to 3.0 percent of glucose, 0.05 to 0.10 percent of citric acid diamine, 0.05 to 0.10 percent of dipotassium hydrogen phosphate, 0.05 to 0.10 percent of magnesium sulfate, 0.05 to 0.10 percent of manganese sulfate and 6.0 to 7.0 of pH value.
3. Mixing Bacillus bailii and Bacillus coagulans, and taking zeolite powder as carrier, wherein the number of viable bacteria of mixed Bacillus bailii is 5.0X10 9 -1.0×10 10 CFU/g, viable count of Bacillus coagulans is5.0×10 9 -1.0×10 10 CFU/g。
Example 3 Effect of composite microecologics on lactation performance and number of mammary gland cells in cows suffering from mastitis
1. Test materials
Bacillus belicus, bacillus coagulans and composite microecological preparation products are provided by Beijing for Bo Biotechnology Co.
2. Test method
2.1 Trial grouping design and feeding management
The method comprises the steps of selecting 80 Holstein cows with mastitis, which are similar in gestation (2-4 fetuses), lactation period (40 d+/-3 d of lactation days), lactation amount (25 kg+/-1 kg) and the like, randomly dividing the cows into 4 groups, wherein the first group is a control group, feeding basic ration, the second group is a bacillus berryis group, feeding the basic ration while adding 100 g/bacillus berryis preparation every day, the third group is a bacillus coagulans group, feeding the basic ration while adding 100 g/bacillus berryis preparation every day, and the fourth group is a compound microecological preparation group, and feeding the basic ration while adding 100 g/head compound microecologics preparation every day.
All cows are fed with full mixed ration, are fed with free food and drinking water, and are pre-fed for 7 days and tested for 28 days.
2.2 Measurement index and method
Test periods 0d, 7d, 14d, 21d and 28d record milk production and determine milk composition using a milk detector and milk somatic cell count using a milk somatic cell counter.
3. Test results
(1) Effects on lactation Performance and milk quality
As shown in table 2, the compound microecological preparation group showed significantly improved lactation (P < 0.05), and the bacillus coagulans group and bacillus bailii group showed a tendency to improve lactation (P > 0.05) compared with the control group; compared with the control group, the milk proteins and the milk fat rates of the compound microecological preparation group, the bacillus coagulans group and the bacillus bailii group all have an increasing trend (P & gt 0.05).
TABLE 2 influence on milk secretion performance and milk quality of cows
Note that: the same row of shoulder marks and different lower case letters indicate significant differences (P < 0.05), and the same letters indicate insignificant differences (P > 0.05), as follows.
(2) Effect on the number of mammary somatic cells
As shown in Table 3, the number of somatic cells in the cow's milk was 50 ten thousand/mL or more, which is a clinical condition of mastitis, and the number of somatic cells in the cow's milk in the control group was always higher than the threshold value and not decreased during the test period. After the bacillus belicus, the bacillus coagulans and the compound microecologics are fed for one week, the number of somatic cells in the cow milk of the test group is obviously reduced, the number of somatic cells in the cow milk of the compound microecologics is obviously reduced to 27.03 ten thousand per mL after the feeding for 3 weeks, the number of somatic cells in the cow milk of the test group is obviously lower than that of other groups (P is less than 0.05), and the number of somatic cells in the cow milk of each test group is smaller than 50 ten thousand per mL at the end of the test period. Therefore, bacillus belicus, bacillus coagulans and the compound microecological preparation are added into daily ration of the dairy cows, so that the somatic cell number in the dairy cows can be effectively reduced, and the mastitis of the dairy cows can be improved.
TABLE 3 influence on the number of bovine milk somatic cells (ten thousand/mL)
Conclusion 4
The test result shows that feeding the compound microecological preparation consisting of bacillus belicus and bacillus coagulans can improve the lactation yield and the milk quality of cows, reduce the number of somatic cells in cows and improve the mammitis of cows, wherein the compound microecological preparation has the best effect, and the reduction rate of the compound microecological preparation on the somatic cells of cows is larger than the sum of the reduction rates of bacillus belicus and bacillus coagulans when the bacillus belicus and the bacillus coagulans are singly used, so that the compound microecological preparation has the synergistic effect when the bacillus belicus and the bacillus coagulans are compounded.
The method can be realized by the upper and lower limit values of the interval and the interval value of the process parameters (such as temperature, time and the like), and the examples are not necessarily listed here.
The invention may be practiced without these specific details, using any knowledge known in the art.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and are not limiting. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the appended claims.
Sequence listing
<110> Beijing family Bo Biotech Co., ltd
<120> a compound microecological preparation for preventing and treating bovine mastitis and its application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 872
<212> DNA
<213> Bacillus bailii (Bacillus velezensis)
<400> 1
acagtccgcc agtttgcccg gcagattgga aatctcaagc gcacttttgc ggcgggtcaa 60
ttcccgcgct tttttcgctg ccatccgcgc tcttgcggcc attaaacctt tttcaacgat 120
tttgcgggct gagtccggat tttcaagaag gaatgtttcc agcgcagaag aaaacagcgt 180
atcagtgatc gttctcgctt cggagttgcc gagcttcgtt ttcgtctgcc cttcgaattg 240
cggatcaggg tgcttaattg aaataatggc agtcagccct tccctcacat catccccgct 300
taaattcgga tcattttctt tgaaaatccc ttttcttctt gcatagtcgt ttataacacg 360
ggtcagaccg gttttaaatc cggcttcgtg cgtgccgcct tcgtatgtgt tgatattatt 420
tgtgaaagaa taaatattgc ttgtatagct gtcgttgtat tgcaatgcaa cttcaaccgt 480
tatgccgtct ttctcgcctt cgatataaat cggctcttca tgaacgactt ctttggaacg 540
gtttaagtac tcaacatagc ttttgattcc gccttcgtag tggtactcgt ttttccgttc 600
ttgtccttca cgtttgtctt caatcgtgat gtttacacct tttgtcagga aggccaattc 660
ccggacacgg tttgaaagca ggtcatagtc gtattcggtt gtttctttga aaatttccgg 720
atccggaacg aagtgcgtaa tcgttccggt cttatcagta tcaccgatca cttcaagatc 780
ggccacaggt acaccgcgct cgtacgcctg atagtggatt tttccgtcac gatgaaccgt 840
aacgtcaaga gtggtcgaca aggcgtttac ga 872
<210> 2
<211> 1184
<212> DNA
<213> Bacillus coagulans (Bacillus coagulans)
<400> 2
tggaaactcg ggtggcggaa taggtgtcgg cggtctgcac gggttggtgc gtctgttgtg 60
aatgccctgt ctacagagct cgatgtctat gtccaccgcg acgggcatat ttactaccaa 120
aaataccgcc gcggcaaacc tgcctttgac ttaaaaatca ttggcgaaac ggatcgtacc 180
ggcacaacga ctcacttctt gccggatccg gaaattttta cggaaacgac cgagttcgac 240
tttgatattc ttacaacccg gctccgtgaa cttgcctttt taaataaagg gatcaaaatc 300
accattgagg acaaacgcga aaaaaatccg cgcatacaag aataccatta cgaaggcggg 360
atcaaatcgt acgtggagca tttgaaccgc tcgaaagaag tgctccataa ggaaccgatt 420
tatgtagaag gcgaaaaaga cggcataacc gttgaagtat cccttcaata caatgacggg 480
tttgccagca atatctattc ttttgccaat aatattcaca cctacgaagg cggcacacct 540
gagtccggtt ttaaaacggc tttaacgcgc gtcatcaatg attatgcccg gaaaaacaat 600
atgatcaaag aaaatgaagc caatttaacg ggcgaagatg tccgggaagg cctcactgcg 660
attgtttcca tcaaacaccc ggatccgcag tttgaaggac aaacgaaaac gaagcttgga 720
aatacagaag caagaacgat tacagatgcg gtattttccg agacgtttga gaagtttatg 780
atggaaaatc ctcaagtggc gcggaaaatt gtcgaaaaag gggtgatggc tgcccgggca 840
aggatggctg cgaaaaaagc gcgcgaactg acacggagaa aaagcgcgct cgaatcgaca 900
agcctaccgg gcaaacttgc agactgcaca tcacgcgacc cggctgagag cgagctctac 960
attgtcgaag gagactcggc cggcggttct gccaagcagg gaagggaccg gatgtttcag 1020
gcgatcctgc ctttaagagg gaaaatttta aacgtagaga aagcgcgttt ggataaaatt 1080
ttgtcaaatg cggaaatccg gaccattatt accgcgcttg gcacaggtgt cggagaagat 1140
tcgacatcta gaaggcgcgt atcatagaag tcgagatcca tagc 1184
Claims (7)
1. A compound microecological preparation for preventing and treating cow mastitis, which is characterized by comprising bacillus belicus (Bacillus velezensis), bacillus coagulans (Bacillus coagulans) and a carrier; the preservation number of bacillus belicus is CGMCC No.19349, and the preservation number of bacillus coagulans is CGMCC No.16244.
2. The compound microecological preparation for preventing and treating cow mastitis according to claim 1, wherein the number of bacillus bailii viable bacteria in the compound microecological preparation is 5.0×10 9 -1.0×10 10 CFU/g, viable count of Bacillus coagulans is 5.0X10 9 -1.0×10 10 CFU/g。
3. The compound microecological preparation for preventing and treating cow mastitis according to claim 1, wherein the production method of bacillus belicus comprises the following steps:
(1) Inoculating Bacillus bailii strain on slant culture medium, and culturing at 30-40deg.C for 12-24 hr;
(2) Inoculating Bacillus bailii subjected to slant culture into seed activation liquid culture medium, wherein the liquid volume of 500mL triangular flask is 100-200mL, and culturing at 30-40deg.C and 180-220rpm for 12-24 hr;
(3) Inoculating Bacillus bailii seed solution in an amount of 0.5-2% by weight into a seed tank, wherein the liquid amount in 500L seed tank is 100-200L, and culturing at 30-40deg.C and 180-220rpm for 12-24 hr;
(4) Inoculating the seed tank culture solution into a fermentation tank at a weight ratio of 0.5-2%, wherein the tank pressure is 0.01-0.05MPa, the stirring rotation speed is 180-220rpm, the ventilation ratio is 0.5-1:0.1-0.5 in terms of volume ratio, and the fermentation time is 16-24 hours;
(5) Spray drying bacillus belicus fermentation liquor through a drying tower, wherein the air inlet temperature of an air blower is 140-170 ℃; the outlet temperature is 60-80 ℃, and the dried product passes through a 60-mesh vibrating screen.
4. A compound microecological preparation for preventing and treating cow mastitis as set forth in claim 3, wherein,
the slant culture medium in the step (1) comprises the following components: 5.0-15.0g of tryptone, 1.0-5.0g of beef extract powder, 5.0-10.0g of sodium chloride, 3.0-8.0g of glucose, 15-20g of agar powder, 1000mL of distilled water and pH of 7.0-7.5;
the seed culture medium in the step (2) is the slant culture medium in the step (1) for removing the agar powder;
the seeding tank culture medium in the step (3) and the fermentation culture medium in the step (4) are composed of: 1.0 to 2.0 percent of soybean peptone, 1.0 to 2 percent of bean cake powder, 1.0 to 1.5 percent of glucose, 0.5 to 2 percent of corn flour, 0.5 to 2 percent of calcium carbonate, 0.001 to 0.01 percent of magnesium sulfate, 0.001 to 0.01 percent of manganese sulfate and pH of 7.0 to 7.5.
5. The compound microecological preparation for preventing and treating cow mastitis according to claim 1, wherein the production method of bacillus coagulans comprises the following steps:
(1) Inoculating bacillus coagulans on the slant culture medium, and culturing for 24-48 hours under the facultative or anaerobic condition at the temperature of 35-45 ℃;
(2) Inoculating bacillus coagulans subjected to slant culture into a liquid seed activation culture medium, and culturing for 24-48 hours under the facultative or anaerobic condition at the temperature of 35-45 ℃;
(3) Inoculating the seed liquid obtained in the step (2) to a fermentation tank culture medium, and performing stationary culture for 24-48 hours under the facultative or anaerobic condition at the temperature of 35-45 ℃;
(4) Spray drying the bacillus coagulans fermentation liquor by a drying tower, wherein the inlet air temperature of a blower is 140-170 ℃; the outlet temperature is 60-80 ℃, and the dried product passes through a 60-mesh vibrating screen.
6. The compound microecological preparation for preventing and treating cow mastitis as set forth in claim 5, wherein,
the slant culture medium in the step (1) comprises the following components: 10.0g of peptone, 5.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 1.0mL of tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of diammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 16g of agar powder, 1000mL of distilled water and sterilizing for 30min at 121 ℃;
removing agar powder from the seed culture medium in the step (2) which is a slant culture medium;
the fermentation medium in the step (3) comprises the following components: 1 to 2.0 percent of peptone, 0.5 to 1.0 percent of yeast powder, 0.5 to 1.0 percent of corn powder, 1.0 to 3.0 percent of glucose, 0.05 to 0.10 percent of citric acid diamine, 0.05 to 0.10 percent of dipotassium hydrogen phosphate, 0.05 to 0.10 percent of magnesium sulfate, 0.05 to 0.10 percent of manganese sulfate and 6.0 to 7.0 of pH value.
7. Use of a complex microecological formulation for the prevention and treatment of bovine mastitis according to any of claims 1 to 6, wherein the use is:
adding the compound raw microecological preparation into daily ration, and mixing uniformly, wherein the addition amount is 50-100 g/head.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210042069.5A CN115838647B (en) | 2022-01-14 | 2022-01-14 | Compound microecological preparation for preventing and treating bovine mastitis and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210042069.5A CN115838647B (en) | 2022-01-14 | 2022-01-14 | Compound microecological preparation for preventing and treating bovine mastitis and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115838647A CN115838647A (en) | 2023-03-24 |
| CN115838647B true CN115838647B (en) | 2024-02-13 |
Family
ID=85574634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210042069.5A Active CN115838647B (en) | 2022-01-14 | 2022-01-14 | Compound microecological preparation for preventing and treating bovine mastitis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115838647B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104510779A (en) * | 2013-09-29 | 2015-04-15 | 瑞普（天津）生物药业有限公司 | Traditional Chinese medicine micro-ecologic preparation for treating recessive mastitis of dairy cattle |
| CN108102991A (en) * | 2018-03-07 | 2018-06-01 | 山东戴盟得生物科技有限公司 | One plant of Bei Laisi bacillus and its application in watermelon blight is prevented |
| CN108220190A (en) * | 2017-12-29 | 2018-06-29 | 山东高龙生物科技有限公司 | A kind of preparation method for ruminating specific complex probiotics |
| CN109423465A (en) * | 2017-09-04 | 2019-03-05 | 北京科为博生物科技有限公司 | A kind of control aflatoxin B1Composite biological agent and its application |
| CN113170842A (en) * | 2021-04-26 | 2021-07-27 | 北京科为博生物科技有限公司 | Composite microecological preparation for preventing and treating necrotic enteritis of poultry and application thereof |
| CN114403307A (en) * | 2022-01-29 | 2022-04-29 | 北京科为博生物科技有限公司 | Compound feed additive for improving salpingitis of laying ducks |
| WO2023154183A1 (en) * | 2022-02-09 | 2023-08-17 | Jeffrey Robbins | Composition including effervescent agents, biostimulant, nutrient, and pesticide |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114007694A (en) * | 2019-03-25 | 2022-02-01 | 以色列国家农业部、农村发展农业研究组织·沃尔卡尼机构 | Method for treating bovine mastitis |
| US20230172194A1 (en) * | 2019-04-12 | 2023-06-08 | Jeffrey Hanson Robbins | Composition including effervescent agents, biostimulant, nutrient, and pesticide |
-
2022
- 2022-01-14 CN CN202210042069.5A patent/CN115838647B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104510779A (en) * | 2013-09-29 | 2015-04-15 | 瑞普（天津）生物药业有限公司 | Traditional Chinese medicine micro-ecologic preparation for treating recessive mastitis of dairy cattle |
| CN109423465A (en) * | 2017-09-04 | 2019-03-05 | 北京科为博生物科技有限公司 | A kind of control aflatoxin B1Composite biological agent and its application |
| CN108220190A (en) * | 2017-12-29 | 2018-06-29 | 山东高龙生物科技有限公司 | A kind of preparation method for ruminating specific complex probiotics |
| CN108102991A (en) * | 2018-03-07 | 2018-06-01 | 山东戴盟得生物科技有限公司 | One plant of Bei Laisi bacillus and its application in watermelon blight is prevented |
| CN113170842A (en) * | 2021-04-26 | 2021-07-27 | 北京科为博生物科技有限公司 | Composite microecological preparation for preventing and treating necrotic enteritis of poultry and application thereof |
| CN114403307A (en) * | 2022-01-29 | 2022-04-29 | 北京科为博生物科技有限公司 | Compound feed additive for improving salpingitis of laying ducks |
| WO2023154183A1 (en) * | 2022-02-09 | 2023-08-17 | Jeffrey Robbins | Composition including effervescent agents, biostimulant, nutrient, and pesticide |
Non-Patent Citations (5)
| Title |
|---|
| Bacillus velezensis AP183 Inhibits Staphylococcus aureus Biofilm Formation and Proliferation in Murine and Bovine Disease Models;Sayma Afroj等;Front Microbiol * |
| Exopolysaccharides produced by Bacillus spp. inhibit biofilm formation by Staphylococcus aureus strains associated with bovine mastitis;International Journal of Biological Macromolecules;International Journal of Biological Macromolecules;1-12 * |
| 一株具有抑菌活性的凝结芽孢杆菌的筛选、鉴定及其抗逆性研究;韩明渠;李富伟;潘兴亮;王玉田;郑瑞峰;;饲料博览(01);41-44 * |
| 益生菌在奶牛上的应用与研究进展;龚;张彬;;中国奶牛(01);6-12 * |
| 芽孢杆菌的作用机制及其在奶牛生产中应用进展;张建刚;侯玉洁;周美玲;夏晨;赵国琦;;中国奶牛(01);7-10 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115838647A (en) | 2023-03-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106282072B (en) | Compound lactobacillus microecological preparation and preparation method and application thereof | |
| CN113170842B (en) | Composite microecological preparation for preventing and treating necrotic enteritis of poultry and application thereof | |
| CN106906154B (en) | Lactobacillus plantarum strain, feed additive and feed thereof | |
| CN107312732B (en) | Probiotic feed additive | |
| CN113388550B (en) | Lactobacillus salivarius NHE-LsE33 and application thereof | |
| CN103060222A (en) | Bacillus subtilis B27 with probiotic effect and application thereof | |
| KR20090030661A (en) | A feed additive for raising fishes, a feed composition, and a breeding method thereof | |
| CN104206789A (en) | Feed additive for preventing cow mastitis and brucellosis and preparation method thereof | |
| WO2016046706A1 (en) | Probiotic fermented feed additives | |
| CN108546663B (en) | Porcine lactobacillus crispatus and application thereof | |
| CN113502243B (en) | Lactobacillus plantarum GBW-LP001 capable of highly producing lactic acid and antibacterial agent alternative thereof and application | |
| CN112574924B (en) | Bacillus subtilis strain, microecological preparation and application thereof | |
| CN112899186A (en) | Burkholderia and application thereof in biological control of fusarium graminearum | |
| CN115838647B (en) | Compound microecological preparation for preventing and treating bovine mastitis and application thereof | |
| KR20060130854A (en) | Novel strains, lactobacillus fermentum ib 282(kctc 18110p), and its application as antimicrobial probiotics | |
| Sibanda et al. | In vitro antibacterial activities of crude extracts of Garcinia kola seeds against wound sepsis associated Staphylococcus strains | |
| CN114891678B (en) | Bacillus polymyxa CPL258 and screening and application thereof | |
| RU2415604C2 (en) | Method to prepare silage | |
| CN113265352B (en) | Preparation method and application of enterococcus faecium powder | |
| CN115651860A (en) | Bacillus coagulans BC-HYC strain and application thereof | |
| CN112704153B (en) | Immunodeficient mouse feed and preparation method thereof | |
| KR101003740B1 (en) | Novel Streptococcus thermophilus and Probiotics Using feed additive fermented colostrum | |
| CN106811429B (en) | Bacillus subtilis strain, application of feed additive thereof and feed | |
| CN114717150A (en) | Lactobacillus plantarum CRS33 and application thereof | |
| CN105166397B (en) | Brevibacillus brevis microecological preparation |
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 |