CN115948285A - Biological disinfectant for lactobacillus salivarius BMC-06 and livestock breeding and application - Google Patents
Biological disinfectant for lactobacillus salivarius BMC-06 and livestock breeding and application Download PDFInfo
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Abstract
The invention discloses a Lactobacillus salivarius BMC-06 and biological disinfectant for livestock breeding and application, and relates to the technical field of improving livestock breeding environment by environment-controlled probiotics, wherein the strain is stored in China Center for Type Culture Collection (CCTCC) at 8.23.2021, is classified and named as Lactobacillus salivarius BMC-06, and has the storage number of CCTCC NO: m20211070, the preservation place is Wuhan, china, the strain is prepared into a biological disinfectant for disinfecting poultry house air, poultry house appliances and the like.
Description
Technical Field
The invention belongs to the technical field of improving livestock and poultry breeding environment by environment-controlled probiotics, and particularly relates to lactobacillus salivarius BMC-06, a biological disinfectant for livestock and poultry breeding and application thereof.
Background
The livestock and poultry breeding industry develops rapidly in recent years. However, environmental pollution caused by livestock and poultry breeding becomes a serious social problem, and one of the persistent concerns is the influence of air particulate pollution in houses on animals, and the pollution can infect through instruments, feed, water tanks, air and other channels, thereby causing economic damage to livestock and poultry breeding. At present, the method for controlling the environmental particulate pollution of the livestock and poultry breeding house mainly uses antibiotics and chemical disinfectants in the livestock and poultry breeding process. However, with the use of antibiotics and chemical disinfectants in large quantities, the problems of potential bacterial resistance, chemical toxicity, instability, instrument corrosiveness and the like are increasingly highlighted. Therefore, the natural nontoxic functional biological disinfectant is researched and developed to improve the pollution of environmental particles in the livestock and poultry environment, and has important significance for the healthy growth of livestock and poultry and the sustainable development of the breeding industry.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides a lactobacillus salivarius BMC-06 and a biological disinfectant for livestock and poultry breeding.
The technical scheme is as follows: in order to achieve the aim, the invention provides a Lactobacillus salivarius BMC-06 which is preserved in China Center for Type Culture Collection (CCTCC) at 23/8 in 2021, is classified and named as Lactobacillus salivarius BMC-06, and has a preservation number of CCTCC NO: m20211070, the preservation place is Wuhan, china.
Preferably, the nucleotide sequence of 16s rDNA of the strain is shown in SEQ ID NO. 3.
The lactobacillus salivarius BMC-06 can be applied to disinfection.
The invention also provides a biological disinfectant for livestock breeding, which comprises lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and lactobacillus paracasei MIT-16.
Preferably, the viable bacteria ratio of the lactobacillus salivarius BMC-06, the lactobacillus plantarum RS-09 and the lactobacillus paracasei MIT-16 is (30-35): (30-35): (30 to 35).
Preferably, the dosage form of the biological disinfectant is a liquid preparation and/or a solid preparation.
The invention also provides a preparation method of the biological disinfectant, when the dosage form of the biological disinfectant is a liquid preparation, the preparation method is to concentrate fermentation liquor of lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and lactobacillus paracasei MIT-16, and the concentration of effective bacterial colony of the concentrated liquid preparation is more than or equal to 10 10 CFU/mL, wherein the viable bacteria ratio of the lactobacillus salivarius BMC-06, the lactobacillus plantarum RS-09 and the lactobacillus paracasei MIT-16 is (30-35): (30-35): (30-35);
when the dosage form of the biological disinfectant is a solid preparation, the preparation method comprises freeze-drying fermentation broth of Lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and Lactobacillus paracasei MIT-16, wherein the effective flora concentration of the solid preparation obtained by freeze-drying is more than or equal to 10 10 CFU/mL, wherein the viable bacteria ratio of the lactobacillus salivarius BMC-06, the lactobacillus plantarum RS-09 and the lactobacillus paracasei MIT-16 is (30-35): (30-35): (30-35).
The invention also provides a using method of the biological disinfectant, when the dosage form of the biological disinfectant is a liquid preparation, the diluting method of the biological disinfectant comprises the following steps: mixing and diluting a biological disinfectant with water, wherein the volume ratio of the biological disinfectant to the water is 1: (400 to 500); when the dosage form of the biological disinfectant is a solid preparation, the dilution method of the biological disinfectant comprises the following steps: mixing and diluting a biological disinfectant with water, wherein the mass ratio of the biological disinfectant to the water is 1g: (400-500) mL.
The applications of the biological disinfectant at least comprise the application of conditional pathogenic bacteria pollution in air particles in the livestock and poultry breeding process and the application of inhibiting the growth of environmental particle bacteria on the surfaces of instruments, objects, water tanks, troughs, wastes or drainage ditches in livestock and poultry houses.
Preferably, when the biological disinfectant is used for reducing the conditioned pathogenic bacteria pollution in air particles in the livestock and poultry breeding process, the biological disinfectant is sprayed on the surface of an object, the surface of a device or the air, and the spraying amount is 20-50 mL/m 3 (ii) a The spraying frequency is 1 time/day, and after continuously spraying for 3d, the spraying frequency is 2 times/week;
when the biological disinfectant is used for inhibiting the growth of environmental particulate bacteria on the surfaces of instruments, objects, water tanks, troughs, wastes or drainage ditches of the livestock and poultry house, diluent of the biological disinfectant is added into the water tanks, the troughs, the wastes and the drainage ditches of the livestock and poultry house or is smeared on the surfaces of the objects and floor drainage devices, and the smearing dosage is 3-5 mL/m 2 (ii) a The frequency of the smearing is 1 time/24 h.
The lactobacillus salivarius BMC-06, the biological disinfectant for livestock and poultry breeding and the application thereof have at least the following technical effects:
(1) The fermentation liquor of the lactobacillus salivarius BMC-06 contains various natural antibacterial active substances, is a natural, pollution-free and safe disinfectant, can effectively improve the livestock and poultry breeding environment, and reduces the spread of pathogenic bacteria in livestock and poultry houses; meanwhile, the problems of bacterial drug resistance, drug residue in the environment, food safety and the like caused by using antibiotics or chemical disinfectants can be solved;
(2) The lactobacillus salivarius BMC-06 has a superior disinfection effect, and the biological disinfectant prepared on the basis contains antibacterial peptide, bacteriocin, bacteroid antagonistic substances and other fermentation broth with an antibacterial effect, so that the occurrence of respiratory tract and digestive tract diseases in livestock and poultry houses can be effectively reduced, the environment in the houses can be controlled, and the livestock and poultry breeding benefit can be improved;
(3) The lactobacillus salivarius BMC-06 can effectively reduce the concentration of particulate matters PM10 and PM2.5 in the air, reduce the total concentration of bacteria on the surface of an apparatus, reduce the types of pathogenic bacteria in the air and reduce the spread of the pathogenic bacteria; meanwhile, the biological disinfectant containing the lactobacillus salivarius BMC-06 can also achieve the technical effects, and on the basis of the disinfection effect of the lactobacillus salivarius BMC-06, the disinfection effect of the lactobacillus salivarius BMC-06 can be enhanced through specific proportioning of various bacteria.
Drawings
FIG. 1 is a gram-stained optical microscope photograph of Lactobacillus salivarius BMC-06;
FIG. 2 is a 16s rDNA agarose electrophoresis of Lactobacillus salivarius BMC-06;
FIG. 3 is the change in concentration of PM2.5 and PM10 before and after sterilization with Lactobacillus salivarius BMC-06 alone;
FIG. 4 shows the change of the total concentration of bacteria on the surface of the apparatus before and after the disinfection of livestock houses by Lactobacillus salivarius BMC-06 alone;
FIG. 5 shows the change of the diversity of the bacterial flora in the air after the poultry and livestock house is disinfected by Lactobacillus salivarius BMC-06 alone;
FIG. 6 shows the change of the diversity of the bacterial flora on the surface of the apparatus after the lactobacillus salivarius BMC-06 is used for disinfecting livestock houses;
FIG. 7 shows the concentration changes of particulate matter PM10 and PM2.5 in the air before and after the biological disinfectant is sprayed to the livestock and poultry house;
FIG. 8 shows the change of the total concentration of bacteria in the air before and after the biological disinfectant is sprayed to the livestock and poultry house;
FIG. 9 shows the variation of the diversity of bacterial flora in the air before and after spraying the biological disinfectant on the livestock and poultry house;
FIG. 10 shows the change of the diversity of the bacterial flora on the surface of the apparatus before and after the biological disinfectant is sprayed on the livestock and poultry house.
Detailed Description
The principles and features of the present invention are described below in conjunction with fig. 1-10, which are provided by way of example only and are not intended to limit the scope of the present invention.
1. Separation, purification, identification and preservation of lactobacillus salivarius BMC-06
1. Separation and purification of Lactobacillus salivarius BMC-06 strain
Taking intestinal contents of healthy mice, adding 1mL of normal saline, and diluting with 10-fold gradient to 10 -6 150 μ L of each dilution was applied to a coating containing 1% CaCO 3 Culturing in anaerobic jar at 37 deg.C for 48 hr. Single colonies on the plate were picked and cultured in MRS solid medium. And performing gram stain identification on the purified colonies. The strain of the invention contains CaCO 3 The MRS culture medium is white colony, convex and has obvious calcium dissolving rings. The bacterial strain is gram-positive through observation, and the round end is straight rod-shaped. Gram staining characteristics of the cells are shown in FIG. 1.
2. Lactobacillus salivarius strain 16srDNA sequencing identification
The obtained strain was subjected to extraction of genomic DNA. Adding 50 microliter of lysozyme with the concentration of 20mg/mL into a water bath kettle at 37 ℃ for treatment for 2 hours, and then extracting DNA by using an artificial bacteria genome kit. 16s universal primers (27F and 1492R,27F primer sequences shown in SEQ ID NO. 1: 5'-AGAGTTTGATCCTGGCTCAG-3';
1492R primer sequence is shown as SEQ ID NO.2, 5'-GGTTACCTTGTTACGACTT-3') to carry out PCR amplification on the strain cDNA gene sequence, and electrophoresis is carried out on the PCR amplification product, wherein the electrophoresis result is shown as figure 2.
The nucleotide sequence of 16s rDNA of the strain is shown in SEQ ID NO.3 through sequencing. Through 16srDNA gene comparison, the homology with Lactobacillus salivarius strain in Genebank reaches 99 percent with Lactobacillus salivarius 2019NCHU414, so that the strain is a Lactobacillus salivarius strain named as Lactobacillus salivarius BMC-06.
SEQ ID NO.3：ctggctccttgcggttaccccaccggctttgggtgttacaaactctcatggtgtgacggg cggtgtgtacaaggcccgggaacgtattcaccgcgacatgctgattcgcgattactagcgattccgacttcatgtaggcgagttgcagcctacaatccgaactgagaacggctttaagagattagctaaacctcgcggtctcgcgactcgttgtaccgtccattgtagcacgtgtgtagcccaggtcataaggggcatgatgacttgacgtcgtccccaccttcctccggtttgtcaccggcagtctcgccagagtgcccaacttaatgctggcaactgacaacaagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacagccatgcaccacctgtcactttgtccccgaagggaaaacctaatctcttaggtggtcaaaggatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcaaccttgcggtcgtactccccaggcggaatgcttattgcgttagctgcggcactgaagggcggaaaccctccaacacctagcattcatcgtttacggcgtggactaccagggtatctaatcctgtttgctacccacgctttcgaacctcagcgtcagttacagaccagagagccgctttcgccactggtgttcttccatatatctacgcatttcaccgctacacatggagttccactctcctcttctgcactcaagtcttccagtttccaatgcactactccggttaagccgaaggctttcacatcagacttaaaagaccgcctgcgttccctttacgcccaataaatccggacaacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtgacttgctggttagataccgtcatcgaatgaacagttactctcactcgtgttcttctctaacaacagagttttacgatccgaagaccttcttcactcacgcggcgttgctccatcagacttgcgtccattgtggaagattccctactgctgcctcccgtaggagtttgggccgtgtctcagtcccaatgtggccgatcaacctctcagttcggctacgtatcatcaccttggtaggccgttaccccaccaactagttaatacgccgcgggtccatctaaaagcgatagcagaaccatctttcatctaaggatcatgcgatccttagagatatacggtattagcacctgtttccaagtgttatccccttcttttaggcaggttacccacgtgttactcacccgtccgccactcaacttcttacgatgaatgcaagcattcggtgtaagaaagtttcgttcgactg
3. Preservation of Lactobacillus salivarius Strain 16srDNA
Lactobacillus salivarius BMC-06 has been deposited in China Center for Type Culture Collection (CCTCC) at 23.8.2021, and is classified and named as Lactobacillus salivarius BMC-06 with the collection number of CCTCC NO: m20211070, the preservation place is Wuhan, china.
2. Application of lactobacillus salivarius BMC-06 in disinfection
Example one, the effect of Lactobacillus salivarius BMC-06 on the concentration of particulate matter PM10 and PM2.5 in air.
Taking 1mL of effective flora with a concentration of 10 10 CFU/mL lactobacillus salivarius BMC-06 fermentation broth is mixed with 400mL water to prepare the microbial inoculum. The microbial inoculumAnd (4) carrying out spray disinfection by using an ultrasonic atomization disinfector, and calculating the amount of the disinfectant required by spraying according to 20 ml per cubic meter of chicken house. Spraying once a day, continuously spraying for three days, changing to twice a week, and respectively spraying PM2.5 and PM10 (unit is μ g/m) in air of livestock and poultry house before and after sterilization by using fixed particle sampler after two weeks 3 ) And (5) collecting. As shown in FIG. 3, in the closed breeding mode, PM2.5 (186.88 μ g/m) is in the livestock and poultry house 3 ) And PM10 (200.52. Mu.g/m) 3 ) The concentration of the particulate matter is higher, and after the microbial disinfectant is used, the PM2.5 (60.56 mu g/m) in the house can be obviously reduced 3 ) And PM10 (75.42. Mu.g/m) 3 ) The concentration of particulate matter.
Example two, the effect of Lactobacillus salivarius BMC-06 on the total concentration of bacteria on the surface of animal house equipment.
The invention uses an International Standard Andersen-6 stage air microorganism collector for sampling, the sampler simulates the anatomical structure and aerodynamic characteristics of a human respiratory tract, is designed by utilizing the inertial impaction principle and is divided into 6 stages, 400 holes are arranged in each stage, the diameter of the holes from top to bottom is gradually reduced, the air flow is constant, and the flow speed is gradually increased, so that bacteria-carrying particles in the air are respectively collected on culture dishes of each stage of the sampler according to different particle sizes. Respectively collecting air samples in the livestock and poultry house before and after the microbial inoculum is disinfected, wherein the air flow rate is 28.3 L.min -1 The sampling time is 2min, the sampling medium is 5% sheep blood soybean agar culture medium, and the culture conditions are 37 ℃ and 24h respectively. Calculating the number of airborne colonies according to the following formula: c = (N × 1000)/(t × F); in the formula: c is the bacterial aerosol concentration, cfu m -3 (ii) a N number of colonies at each level; t is sampling time, min; f is gas flow at sampling time, L.min -1 。
Results analysis, the detection results are shown in fig. 4, and it can be seen from fig. 4 that the distribution ratio of airborne aerobic bacteria in the poultry house after the microbial inoculum treatment is in a descending trend on the Anderson-6 grade collector, and the microbial inoculum can significantly reduce the concentration of airborne aerobic bacteria (P) in the chicken house compared with the concentration before the disinfection<0.05 The concentration of the bacterial aerosol before and after disinfection is 185.85cfu/m respectively 3 And 70.42cfu/m 3 。
Example three, effect of lactobacillus salivarius BMC-06 on the diversity of bacterial flora in the air of poultry houses.
A Biosampler sampler is used for collecting total bacterial aerosol in the air, 50mL of sampling liquid is placed in a sampling bottle, the air flow rate is 12.8L/min, and the sampling time is 2h. The ultracentrifuge is centrifuged at 70000rpm for 2h. Total bacterial RNA was extracted using the EasyPure Viral DNA/RNA Kit (King of Beijing, china). cDNA was synthesized by reverse transcription using the reverse transcription kit EasyScript One-Step gRNA Removal and cDNA Synthesis SuperMix (Beijing, china, all-purpose gold) and stored at-20 ℃. And (3) carrying out high-throughput sequencing on the DNA sample, and analyzing the change of the diversity of the bacterial flora.
The analysis result is shown in fig. 5, and the microbial clustering chart belongs to the level, so that microbial flora in the air of the livestock and poultry house is rich before the microbial inoculum treatment, and the air of the livestock and poultry house contains various conditional pathogenic bacteria such as staphylococcus and pseudomonas, the types of the pathogenic bacteria in the air are obviously reduced after the microbial inoculum treatment, the abundance of various beneficial bacteria such as lactobacillus is increased, and the diversity of the bacterial flora in the air of the livestock and poultry house is improved.
Example four, effect of Lactobacillus salivarius BMC-06 on the diversity of bacterial flora on the surface of poultry house appliances.
And wiping the surface of the instrument cage in the livestock and poultry house by using a sterile cotton swab, and placing the cotton swab in a penicillin bottle containing 5mL of sampling solution for later use. The ultracentrifuge is centrifuged at 70000rpm for 2h. Total bacterial RNA was extracted using the EasyPure Viral DNA/RNA Kit (King of Beijing, china). Reverse transcription was performed using EasyScript One-Step gRNA Removal and cDNA Synthesis Supermix (Kyoto King, china) to synthesize cDNA, and the cDNA was stored at-20 ℃. And (3) carrying out high-throughput sequencing on the DNA sample, and analyzing the change of the diversity of the bacterial flora.
The analysis result is shown in fig. 6, from the level of the microbial clustering heatmap, it can be known that microbial flora in the surface of the livestock and poultry house apparatus is rich before the microbial inoculum treatment, and the apparatus contains various conditional pathogenic bacteria such as staphylococcus aureus, and after the microbial inoculum treatment, the types of the pathogenic bacteria on the surface of the apparatus are obviously reduced, the abundance of various beneficial bacteria such as lactobacillus is increased, and the propagation of the conditional pathogenic bacteria in the livestock and poultry house is reduced.
3. Biological disinfectant for livestock breeding
In the first embodiment, the biological disinfectant comprises lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and lactobacillus paracasei MIT-16, and the ratio of viable bacteria to colony is 35.
In the second embodiment, the biological disinfectant comprises lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and lactobacillus paracasei MIT-16, and the ratio of viable bacteria to colony is 30.
In the third embodiment, the biological disinfectant comprises lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and lactobacillus paracasei MIT-16, and the ratio of viable bacteria to colony is 32.
The biological disinfectants of the first embodiment to the third embodiment can be prepared into liquid preparations or solid preparations.
4. A preparation method of a biological disinfectant for livestock breeding.
Example one, a method for preparing a liquid formulation type biological disinfectant.
Concentrating fermentation liquid of Lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and Lactobacillus paracasei MIT-16, and obtaining liquid preparation with effective colony concentration not less than 10 10 CFU/mL, wherein the viable count ratio of the lactobacillus salivarius BMC-06, the lactobacillus plantarum RS-09 and the lactobacillus paracasei MIT-16 is 35:35:30.
example two, a method for preparing a solid formulation type biological disinfectant.
Freeze drying the fermentation liquid of Lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and Lactobacillus paracasei MIT-16, and freeze drying to obtain solid preparation with effective flora concentration of 10 or more 10 CFU/mL, wherein the viable count ratio of the lactobacillus salivarius BMC-06, the lactobacillus plantarum RS-09 and the lactobacillus paracasei MIT-16 is 35:35:30.
5. a method for using biological disinfectant for livestock breeding is provided.
Example one
Taking a liquid preparation type biological disinfectant, and mixing and diluting the biological disinfectant and water, wherein the volume ratio of the biological disinfectant to the water is 1.
Example two
Taking a biological disinfectant in a liquid preparation form, and mixing and diluting the biological disinfectant with water, wherein the volume ratio of the biological disinfectant to the water is 1.
EXAMPLE III
Taking a liquid preparation type biological disinfectant, and mixing and diluting the biological disinfectant with water, wherein the volume ratio of the biological disinfectant to the water is 1.
Example four
Taking a solid preparation type biological disinfectant, and mixing and diluting the biological disinfectant with water, wherein the mass ratio of the biological disinfectant to the water is 1g.
EXAMPLE five
Taking a solid preparation type biological disinfectant, and mixing and diluting the biological disinfectant with water, wherein the mass ratio of the biological disinfectant to the water is 1g.
Example six
Taking a solid preparation type biological disinfectant, mixing and diluting the biological disinfectant with water, wherein the mass ratio of the biological disinfectant to the water is 1g.
6. A method for using biological disinfectant is provided.
Example one Effect of biological disinfectants on particulate matter PM10 and PM2.5 concentrations in air
Adding 1mL of liquid preparation type biological disinfectant into 400mL of water for dilution, then carrying out spray disinfection on the diluted disinfectant by using an ultrasonic atomization disinfector, and calculating the amount of the disinfectant required by spraying according to 20 mL of the disinfectant per cubic meter of the chicken house. Spraying once a day, changing into twice a week after continuously spraying for three days, and respectively using fixed particle sampler to obtain data (unit is μ g/m) of PM2.5 and PM10 in air of livestock and poultry house before and after sterilization after two weeks 3 ) And (5) collecting. As shown in FIG. 7, the result of the detection is PM2.5 (171.88 μ g/m) in the poultry house in the closed breeding mode 3 ) And PM10 (202.52. Mu.g/m) 3 ) The concentration of the particulate matter is higher, and after the biological disinfectant is used, the PM2.5 (66.39 mu g/m) in the house can be obviously reduced 3 ) And PM10 (70.24. Mu.g/m) 3 ) The concentration of particulate matter.
Example two Effect of biological disinfectants on Total concentration of bacteria in poultry House air
1mL of liquid preparation type biological disinfectant is taken and added into 400mL of water for dilution, then the diluted disinfectant is sprayed and disinfected by an ultrasonic atomization disinfector, and the amount of the disinfectant required by spraying is calculated according to 20 mL of the disinfectant in each cubic meter chicken house. Spraying once a day, changing to twice a week after continuously spraying for three days, and then sampling by adopting an international standard Andersen-6 grade air microorganism collector. The sampler is designed by utilizing the inertial impact principle and is divided into 6 stages, wherein 400 holes in each stage are gradually reduced in diameter from top to bottom, the air flow is constant, and the flow speed is gradually increased, so that bacteria particles in the air are respectively collected on each stage of culture dish of the sampler according to different particle sizes. Respectively collecting air samples in the livestock and poultry house before and after the microbial inoculum is disinfected, wherein the air flow rate is 28.3 L.min -1 The sampling time is 2min, the sampling medium is 5% sheep blood soybean agar culture medium, and the culture conditions are respectively 37 ℃ and 24h. Calculating the number of airborne colonies according to the following formula: c = (N × 1000)/(t × F); in the formula: c is the bacterial aerosol concentration in cfu.m -3 (ii) a N number of colonies at each level; t is sampling time in min; f is the gas flow during sampling, and the unit is L.min -1 。
The detection results are shown in fig. 8. As can be seen from FIG. 8, the distribution ratio of airborne aerobic bacteria in the poultry house after the biological disinfectant treatment showed a tendency to decrease on the Anderson-6 collector, and the disinfectant significantly decreased the concentration of airborne aerobic bacteria (P) in the chicken house compared to that before the disinfection<0.05 The concentration of the aerosol before and after sterilization is 3.27X 10 respectively 3 cfu/m 3 And 9.33X 10 2 cfu/m 3 。
EXAMPLE III Effect of biological disinfectants on the biodiversity of bacterial communities on the surfaces of livestock and poultry housing appliances
Adding 1mL of liquid preparation type biological disinfectant into 400mL of water for dilution, then carrying out spray disinfection on the diluted disinfectant by using an ultrasonic atomization disinfector, and calculating the amount of the disinfectant required by spraying according to 20 mL of the disinfectant per cubic meter of the chicken house. Spraying is carried out once a day, and after three days of continuous spraying, the spraying is carried out twice a week.
And wiping the surface of the instrument cage in the livestock and poultry house by using a sterile cotton swab, and placing the cotton swab in a penicillin bottle containing 5mL of sampling solution for later use. The ultracentrifuge is centrifuged for 2h at 70000 rpm. Total bacterial RNA was extracted using the EasyPure Viral DNA/RNA Kit (King of Beijing, china). cDNA was synthesized by reverse transcription using the reverse transcription kit EasyScript One-Step gRNARemoval and cDNA Synthesis SuperMix (Beijing, china, all-purpose gold) and stored at-20 ℃. And (3) carrying out high-throughput sequencing on the DNA sample, and analyzing the change of the diversity of the bacterial flora.
The detection results are shown in fig. 10. According to the level of the microbial clustering heatmap, microbial flora in the surface of the livestock and poultry house instrument is rich before the biological disinfectant is treated, and the livestock and poultry house instrument contains various conditional pathogenic bacteria such as vibrio, bronchial bacillus and the like, after the lactobacillus disinfectant is treated, the types of the pathogenic bacteria in the air are obviously reduced, the abundance of various beneficial bacteria such as lactobacillus, lactococcus and the like is increased, and the propagation of the conditional pathogenic bacteria in the livestock and poultry house is reduced.
The liquid preparation type biological disinfectant in the above embodiments is replaced by a solid preparation type biological disinfectant, and the use effect thereof is not different from that of the liquid preparation type biological disinfectant.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The Lactobacillus salivarius BMC-06 is characterized in that the strain is preserved in China Center for Type Culture Collection (CCTCC) at 8/23/2021, is classified and named as Lactobacillus salivarius BMC-06, and has a preservation number of CCTCC NO: m20211070, the preservation place is Wuhan, china.
2. Lactobacillus salivarius BMC-06 according to claim 1 wherein: the nucleotide sequence of 16srDNA of the strain is shown in SEQ ID NO. 3.
3. Use of lactobacillus salivarius BMC-06 as claimed in claim 1 for disinfection.
4. The biological disinfectant for livestock breeding is characterized by comprising lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and lactobacillus paracasei MIT-16.
5. The biological disinfectant for livestock breeding according to claim 4, wherein the viable bacteria ratio of the Lactobacillus salivarius BMC-06, the Lactobacillus plantarum RS-09 and the Lactobacillus paracasei MIT-16 is (30-35): (30-35): (30 to 35).
6. The biological disinfectant for livestock breeding according to any one of claims 4 to 5, wherein the biological disinfectant is in the form of a liquid preparation and/or a solid preparation.
7. A preparation method of biological disinfectant is characterized in that when the dosage form of the biological disinfectant is liquid preparation, fermentation liquor of lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and lactobacillus paracasei MIT-16 is concentrated, and the effective bacterial colony concentration of the concentrated liquid preparation is more than or equal to 10 10 CFU/mL, wherein the viable bacteria ratio of the lactobacillus salivarius BMC-06, the lactobacillus plantarum RS-09 and the lactobacillus paracasei MIT-16 is (30-35): (30-35): (30-35);
when the biological disinfectant is in the form of solid preparation, freeze-drying the fermentation liquor of Lactobacillus salivarius BMC-06, lactobacillus plantarum RS-09 and Lactobacillus paracasei MIT-16, wherein the effective flora concentration of the solid preparation obtained by freeze-drying is more than or equal to 10 10 CFU/mL, wherein the viable bacteria ratio of the lactobacillus salivarius BMC-06, the lactobacillus plantarum RS-09 and the lactobacillus paracasei MIT-16 is (30-35): (30-35): (30 to 35).
8. A method of using a biological disinfectant as claimed in claim 7, wherein when the dosage form of the biological disinfectant is a liquid formulation, the biological disinfectant is diluted by: mixing and diluting a biological disinfectant with water, wherein the volume ratio of the biological disinfectant to the water is 1: (400-500); when the dosage form of the biological disinfectant is a solid preparation, the dilution method of the biological disinfectant comprises the following steps: mixing and diluting a biological disinfectant with water, wherein the mass to volume ratio of the biological disinfectant to the water is 1g: (400-500) mL.
9. Use of a biological disinfectant according to claim 4, characterized in that the use comprises at least the use for opportunistic pathogen contamination of air particles in livestock breeding processes and the use for inhibiting the growth of environmental particulate bacteria on surfaces of apparatus, objects, sinks, troughs, wastes or gutters in livestock houses.
10. Use of a biological disinfection machine according to claim 9, characterised in that when the biological disinfectant is used for reducing the contamination of opportunistic pathogens in air particles during the cultivation of livestock, the biological disinfectant is sprayed onto the surface of an object, onto the surface of a device or into the air, the amount of spraying being 20-50 mL/m 3 (ii) a The spraying frequency is 1 time/day, and after continuously spraying for 3 days, the spraying frequency is 2 times/week;
when the biological disinfectant is used for inhibiting the growth of environmental particulate bacteria on the surfaces of instruments, objects, sinks, troughs, wastes or drainage ditches of a livestock and poultry house, diluent of the biological disinfectant is added into the sinks, troughs, wastes and drainage ditches of the livestock and poultry house or is smeared on the surfaces of the objects and floor drainage devices, and the smearing dosage is 3-5 mL/m 2 (ii) a The frequency of the smearing was 1 time/24 h.
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