Bacillus subtilis LB-KC and its application in preparing anti-pathogenic microbial litter

Abstract

The present invention belongs to the field of cattle and sheep bedding, and specifically relates to Bacillus subtilis LB-KC and its application in preparing anti-pathogenic microorganism bedding. The present invention provides Bacillus subtilis LB-KC, which has a better effect of inhibiting Fusobacterium necroticum and Bacteroides nodosa compared to other Bacillus subtilis. Bacillus subtilis LB-KC is used for the fermentation of cattle and sheep bedding, which can significantly reduce the incidence of cattle and sheep foot rot, and the bacteria has the effect of degrading cow dung, sheep dung, etc., which can further reduce the probability of cattle and sheep foot rot caused by cow dung and sheep dung causing cattle and sheep horny softening.

Description

Technical Field

[0001] The invention belongs to the field of cattle and sheep bedding, and particularly relates to Bacillus subtilis LB-KC and application thereof in preparing anti-pathogenic microorganism bedding. Background Art

[0002] Hoof rot, also known as hoof erosion or hoof fork rot, is a disease in which the hoof fork keratin and its deep layers rot and necrotize, and gray-black foul-smelling liquid oozes out or is filled with gray-brown residue. It belongs to the category of leaky hoof in traditional Chinese veterinary medicine and is a common hoof disease in dairy cows. Modern veterinary research has found that Fusobacterium necroticum and Bacteroides nodosa are the main pathogens that induce hoof rot in cattle. Therefore, inhibiting Fusobacterium necroticum can significantly reduce the occurrence of hoof rot in cattle and sheep.

[0003] Bedding, also called "foot pads" or "mattresses", refers to various natural and artificial materials covering the surface of the shed to provide a comfortable habitat for cattle and sheep. Most of them are straw, leaves, sawdust, straw, rice husks, turf, soil, peat, etc., which are rich in organic matter and have strong absorption capacity. Their function is to absorb livestock feces and urine, store fertilizer, reduce volatilization and loss, and are beneficial to livestock health and environmental hygiene. Straw, leaves, sawdust, straw, rice husks, turf, soil, peat and other raw materials in the bedding are soaked in cattle and sheep feces and urine. After being trampled by cattle and sheep, the keratin of cattle and sheep will soften, and the sole of the hoof will be infected with necrotic Fusobacterium and nodular Bacteroides due to excessive wear, which will promote hoof rot.

[0004] The applicant "Inner Mongolia Autonomous Region Academy of Agricultural and Animal Husbandry Sciences" has applied for patents CN118178530A, a method for preparing and using a medicinal ointment for treating cow foot rot, CN117815181A, a hoof spray for cow and sheep foot rot and its preparation method and application, and CN116807894A, a method for preparing and using a hoof bath for cow foot rot. Although the above patents all involve cow and sheep foot rot, the raw materials used for treating cow and sheep foot rot are Chinese medicinal materials or active ingredients of Chinese medicinal materials, and no microorganisms are used.

[0005] After searching, the existing technology for treating cattle and sheep foot rot (by inhibiting Fusobacterium necroticum and Bacteroides nodosa) mainly uses Western medicine, Chinese herbal medicine or its active ingredients to inhibit Fusobacterium necroticum and Bacteroides nodosa. There are few studies on using the antagonism between bacteria, that is, using one or more strains of microorganisms to inhibit Fusobacterium necroticum and Bacteroides nodosa to achieve the effect of treating cattle and sheep foot rot. Existing technology such as CN108159395A, a preparation for preventing and treating dairy cow limb and hoof disease and its preparation method, discloses a preparation for treating dairy cow limb and hoof disease composed of minerals, probiotics, antimicrobial peptides and carriers, wherein the probiotics include: Bacillus licheniformis, Bacillus subtilis, Bacillus natto, lactic acid bacteria, Bifidobacterium, yeast One or more. CN 111053039 A cattle dung absorbing straw mat and its application, discloses spraying microbial liquid when preparing cattle dung absorbing straw mat, the microbial liquid contains at least one of Bacillus licheniformis, Enterococcus faecalis, Bacillus coagulans, deaminating and deodorizing bacteria, as well as Bacillus megaterium and Bacillus subtilis, and the incidence of foot rot and cattle gastroenteritis is greatly reduced. CN101690464A composite bedding for fermentation bed breeding and its preparation method, discloses the use of brewer's yeast, lactobacillus, bacillus subtilis and aspergillus oryzae fermentation to prepare bedding, reducing the disease rate of poultry and livestock; CN110248670A bacterial composition for the treatment of foot infection of ungulates, discloses the prevention and improvement of the symptoms of ungulates suffering from diseases involving foot parasites or bacterial infections, the composition includes: at least one Bacillus bacterial strain, especially at least one Bacillus subtilis, and at least one lactic acid bacteria strain, especially at least one Lactococcus lactis or at least one Pediococcus pentosaceus. Document 1 ("Optimization of microbial antagonism detection method and its application in straw fermentation strains", Inner Mongolia Autonomous Region Academy of Agricultural and Animal Husbandry Sciences, 2020), the applicant's prior article discloses the optimization of microbial antagonism detection method and its application in straw fermentation strains, and found that there is antagonism between some microorganisms. Document 2 (“Experimental study on the effect of Bacillus subtilis-type probiotic preparations in dairy cow diets”, Institute of Animal Nutrition, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, 2011) discloses an experimental study on the effect of Bacillus subtilis-type probiotic preparations in dairy cow diets.

[0006] It can be seen that although the prior art discloses the use of microbial fermentation bedding or antibacterial agents to reduce foot rot or the number of pathogenic bacteria in the hooves of cattle and sheep, the existing microorganisms are all composite microorganisms, and other inhibitory raw materials are optionally added. There is no specific study on the inhibitory or antagonistic effect of Bacillus subtilis on Fusobacterium necroticum and Bacteroides nodosa. The present invention screens out a specific strain of Bacillus subtilis LB-KC, specifically studies its inhibitory effect on Fusobacterium necroticum and Bacteroides nodosa, and uses it in the fermentation of cattle and sheep bedding, which can significantly reduce the incidence of foot rot in cattle and sheep. Summary of the invention

[0007] The present invention provides Bacillus subtilis LB-KC, which has a better effect of inhibiting Fusobacterium necroticum and Bacteroides nodosa compared with other Bacillus subtilis. Bacillus subtilis LB-KC is used for fermentation of cattle and sheep bedding, which can significantly reduce the incidence of cattle and sheep foot rot, and the bacteria has the effect of degrading cow dung and sheep dung, so the probability of cattle and sheep foot rot caused by cow dung and sheep dung causing keratin softening can be further reduced.

[0008] The first aspect of the present invention provides Bacillus subtilis LB-KC, whose preservation number is CGMCC NO.25562, the preservation address is: General Microbiology Center of China National Microbiological Culture Collection Administration, and the preservation date is: August 19, 2022.

[0009] The second aspect of the present invention provides the use of Bacillus subtilis LB-KC in inhibiting pathogenic microorganisms in bedding of cattle and sheep pens.

[0010] Preferably, the pathogenic microorganisms include Fusobacterium necroticum and Bacteroides nodosa.

[0011] The third aspect of the present invention provides a method for preparing Bacillus subtilis LB-KC for antagonizing pathogenic microorganisms in cattle and sheep shed bedding, comprising the following steps: mixing Bacillus subtilis LB-KC with brown sugar, corn flour or molasses, adding water to mix evenly, and fermenting for 1-3 hours to obtain the obtained product.

[0012] A fourth aspect of the present invention provides a method for preparing a bedding material for antagonizing pathogenic microorganisms in bedding materials for cattle and sheep pens, comprising the following steps:

[0013] (1) Select sawdust, rice husks, straw and turf as the raw materials for litter, mix them evenly and control the carbon-nitrogen ratio of the raw materials to 200-300:1;

[0014] (2) Mix Bacillus subtilis LB-KC with brown sugar, corn flour or molasses, add water and mix well, then ferment for 1-3 hours for later use;

[0015] (3) Spray the fermented Bacillus subtilis LB-KC obtained in step (2) on the mixed bedding material obtained in step (1) at a spraying rate of 50-150 ml / m 3 The mixed bedding raw materials are adjusted to have a moisture content of 45-60%, and the mixed bedding raw materials are piled and fermented for 24-35 hours to obtain bedding for antagonizing pathogenic microorganisms in cattle and sheep shed bedding, wherein the pathogenic microorganisms are mainly necrotic Fusobacterium and nodular Bacteroides that cause foot rot in cattle and sheep.

[0016] Preferably, the carbon-nitrogen ratio of the bedding material is controlled at 250:1.

[0017] Preferably, the carbon-nitrogen ratio of the bedding material is controlled at 200:1.

[0018] Preferably, the spraying amount is 150ml / m 3 The mixed bedding materials were adjusted to have a moisture content of 60% and then piled and fermented for 35 hours.

[0019] Preferably, the spraying amount is 100 ml / m 3 The mixed bedding materials were adjusted to have a moisture content of 50% and then piled and fermented for 30 hours.

[0020] The bedding for cattle and sheep pens is prepared by any of the methods described above.

[0021] The fifth aspect of the present invention provides the use of the aforementioned cattle and sheep shed bedding in inhibiting pathogenic microorganisms in cattle and sheep shed bedding, wherein the pathogenic microorganisms are mainly Fusobacterium necroticum and Bacteroides nodosa that cause foot rot in cattle and sheep.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention screened out a specific Bacillus subtilis LB-KC, which, compared with the existing Bacillus subtilis CX31 and Bacillus subtilis FX1 with antibacterial effects, showed stronger antibacterial activity against Fusobacterium necroticum and Bacteroides nodosa.

[0024] (2) The present invention screens out a specific Bacillus subtilis LB-KC, which has the effect of degrading cow dung and sheep dung. Therefore, it can further reduce the probability of cow dung and sheep dung causing keratin softening in cattle and sheep, thereby causing foot rot in cattle and sheep.

[0025] (3) The Bacillus subtilis of the present invention is used for the fermentation of cattle and sheep bedding to produce bedding, which can significantly reduce the probability of cattle and sheep contracting foot rot compared with bedding fermented by other microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is an electron micrograph of Bacillus subtilis LB-KC. DETAILED DESCRIPTION

[0027] The specific implementation of the present invention is described below with reference to the accompanying drawings. The experimental methods used in the implementation examples are all conventional methods unless otherwise specified; the materials and reagents used are all commercially available unless otherwise specified.

[0028] Example 1 Effects of different Bacillus subtilis strains on the antibacterial effects of Fusobacterium necroticum and Bacteroides nodosa

[0029] (1) Screening of strains

[0030] Bacillus subtilis LB-KC was collected and isolated from the soil in Hohhot, Inner Mongolia Autonomous Region. The separation and purification method is as follows:

[0031] a) Weigh 5 g of soil sample from a breeding farm in Tumotezuoqi, Hohhot, and place it in a 100 mL Erlenmeyer flask containing 50 mL of sterile water. Shake for 30 min and mix thoroughly to obtain a soil suspension with a concentration of 1 / 10.

[0032] b) Pipette 0.5 mL of the above soil suspension into a sterile centrifuge tube containing 4.5 mL of sterile water, mix well to make the solution concentration 1 / 100, and then take 100 μL of the diluted soil suspension and spread it on LB solid culture medium. Purify and culture at 25°C for 3-5 days, pick the bacterial colony with the largest number of colonies for separation and purification, obtain a single colony, and obtain Bacillus subtilis.

[0033] (2) Identification of Bacillus subtilis LB-KC strain

[0034] Its taxonomic characteristics were identified as: Bacillus, Gram-positive bacteria, slightly yellow colonies, rough surface forming wrinkles, shape: long oval column, with strong acid resistance, bile resistance, oxidation resistance, high temperature resistance, stress resistance and strong stability (see the attached figure of the specification) Figure 1 , electron microscope image of Bacillus subtilis LB-KC), which can be identified as Bacillus subtilis. The strain Bacillus subtilis LB-KC has a deposit number of CGMCC NO.25562, a deposit address of General Microbiology Center of China Microbiological Culture Collection Administration, and a deposit date of August 19, 2022.

[0035] (3) According to the method described in Example 1 of the applicant's prior patent CN118178530A, antibacterial tests on different Bacillus subtilis were carried out.

[0036] The present invention's Bacillus subtilis LB-KC, the existing Bacillus subtilis CX31 (CCTCC No: M 2012138), and the Bacillus subtilis FX1 (CCTCC No: M2014563) were selected and diluted with water to 10 6 cfμ / ml bacterial suspension, wherein Bacillus subtilis CX31 and Bacillus subtilis FX1 are strains known in the prior art and available for purchase.

[0037] The antibacterial activity was determined by the Oxford cup method: specifically, 10 6 cfμ / ml bacterial suspension, take 20 mL of sterilized 45℃ MH agar medium and pour it into a 90 mm sterile culture dish, repeat the operation to make 6 plates, after condensation, accurately transfer 0.10 mL of necrotic Fusobacterium suspension to the surface of the culture dish for 3 plates, spread it evenly, accurately transfer 0.1 mL of nodular Bacteroides suspension to the surface of the culture dish for 3 plates, spread it evenly, let it stand at room temperature for 30 minutes, and evenly place the sterilized Oxford cups on the surface of the culture dish, with 3 Oxford cups placed on each plate;

[0038] To the three plates inoculated with the suspension of Fusobacterium necroticum (a total of 9 Oxford cups), 150 μl of Bacillus subtilis LB-KC, the existing Bacillus subtilis CX31, and Bacillus subtilis FX1 were added to the Oxford cups in sequence;

[0039] To the three plates inoculated with the suspension of Bacillus nodosa (9 Oxford cups in total), add 150 μl of Bacillus subtilis LB-KC, existing Bacillus subtilis CX31, and Bacillus subtilis FX1 into the Oxford cups in the same way;

[0040] After the plate was placed in an incubator at 37°C for 24 h, the diameters of the inhibition zones of different Bacillus subtilis against Fusobacterium necroticum and Bacteroides nodosa were measured, and the results were repeated three times. The specific results are shown in Table 1.

[0041] Table 1 Antibacterial effects of different Bacillus subtilis

[0042]

[0043] As can be seen from Table 1, different strains of Bacillus subtilis have different antibacterial effects on Fusobacterium necroticum and Bacteroides nodosa. Among them, the best antibacterial effect is the Bacillus subtilis LB-KC screened by this application; followed by the Bacillus subtilis CX31 with antibacterial effect; Bacillus subtilis FX1 has relatively poor antibacterial effect on Fusobacterium necroticum and Bacteroides nodosa. Therefore, the Bacillus subtilis LB-KC screened by the applicant is selected as the antagonist of Fusobacterium necroticum and Bacteroides nodosa for the fermentation of cattle and sheep bedding.

[0044] Example 2

[0045] A method for preparing Bacillus subtilis LB-KC for antagonizing pathogenic microorganisms in bedding of cattle and sheep pens comprises the following steps: mixing Bacillus subtilis LB-KC with brown sugar, corn flour or molasses, adding water to mix evenly and then fermenting for 2 hours to obtain the obtained product.

[0046] Example 3

[0047] A method for preparing bedding for antagonizing pathogenic microorganisms in bedding for cattle and sheep pens comprises the following steps:

[0048] (1) Select sawdust, rice husks, straw and turf as the litter materials, mix them evenly and control the carbon-nitrogen ratio of the litter materials to 250:1;

[0049] (2) Mix Bacillus subtilis LB-KC with brown sugar, corn flour or molasses, add water and mix well, then ferment for 2 hours for later use;

[0050] (3) Spray the fermented Bacillus subtilis LB-KC obtained in step (2) on the mixed bedding material obtained in step (1) at a spraying rate of 100 ml / m 3 The mixed bedding raw materials are adjusted to have a moisture content of 50%, and the mixed bedding raw materials are piled and fermented for 30 hours to obtain bedding for antagonizing pathogenic microorganisms in bedding of cattle and sheep pens.

[0051] The main pathogenic microorganisms are Fusobacterium necroticum and Bacteroides nodosa, which can cause foot rot in cattle and sheep.

[0052] Experimental Example 1

[0053] Experimental method: 45 healthy dairy cows were selected and divided into two groups, A, B, and C, with 15 cows in each group. The feeding diet, eating habits, and environmental parameters of each group were the same. The difference was that the bedding in the dairy cow shed was different. Group A was paved with the bedding prepared in Example 3, and Group B was paved with the bedding prepared according to the method of Example 3 (wherein Bacillus subtilis CX31 was used instead of Bacillus subtilis LB-KC in Example 3, and the rest of the steps were the same as in Example 3); Group C was the bedding prepared according to Sections 1.2-1.3 of Document 1 (“A Brief Discussion on Ecological Breeding Technology of Beef Cattle Biological Bedding”, Lai Jingtao, et al., China Livestock and Poultry Seed Industry, 2021);

[0054] The cows were raked every 7 days, the bedding was changed every 2 months, and the corresponding fermentation bacteria were sprayed every 20 days to degrade cow dung (cow dung can soften cow keratin and cause cow hoof rot). After one year of application, the cows' hoof rot was observed. The specific results are shown in Table 2 below:

[0055] Table 2 Results of hoof rot in dairy cows

[0056]

[0057] Foot rot can be mild or severe. As long as the symptoms of foot rot appear, it is counted as having foot rot.

[0058] As can be seen from Table 2, the use of bedding fermented with Bacillus subtilis LB-KC can significantly reduce the incidence of foot rot in dairy cows. The possible reason is that Bacillus subtilis LB-KC has a strong inhibitory effect on the pathogenic bacteria Fusobacterium necroticum and Bacteroides nodosa, which cause foot rot.

[0059] Example 4

[0060] A method for preparing bedding for antagonizing pathogenic microorganisms in bedding for cattle and sheep pens comprises the following steps:

[0061] (1) Select sawdust, rice husks, straw and turf as the litter materials, mix them evenly and control the carbon-nitrogen ratio of the litter materials to 200:1;

[0062] (2) Mix Bacillus subtilis LB-KC with brown sugar, corn flour or molasses, add water and mix well, then ferment for 1 hour for later use;

[0063] (3) Spray the fermented Bacillus subtilis LB-KC obtained in step (2) on the mixed bedding material obtained in step (1) at a spraying rate of 50 ml / m 3 The mixed bedding raw materials are adjusted to have a moisture content of 45%, and the mixed bedding raw materials are piled and fermented for 24 hours to obtain bedding for antagonizing pathogenic microorganisms in bedding of cattle and sheep pens.

[0064] The main pathogenic microorganisms are Fusobacterium necroticum and Bacteroides nodosa, which can cause foot rot in cattle and sheep.

[0065] Example 5

[0066] A method for preparing bedding for antagonizing pathogenic microorganisms in bedding for cattle and sheep pens comprises the following steps:

[0067] (1) Select sawdust, rice husks, straw and turf as the litter materials, mix them evenly and control the carbon-nitrogen ratio of the litter materials to 300:1;

[0068] (2) Mix Bacillus subtilis LB-KC with brown sugar, corn flour or molasses, add water and mix well, then ferment for 3 hours for later use;

[0069] (3) Spray the fermented Bacillus subtilis LB-KC obtained in step (2) on the mixed bedding material obtained in step (1) at a spraying rate of 150 ml / m 3 The mixed bedding raw materials are adjusted to have a moisture content of 60%, and the mixed bedding raw materials are piled and fermented for 35 hours to obtain bedding for antagonizing pathogenic microorganisms in bedding of cattle and sheep pens.

[0070] The main pathogenic microorganisms are Fusobacterium necroticum and Bacteroides nodosa, which can cause foot rot in cattle and sheep.

[0071] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Bacillus subtilis LB-KC, characterized in that The classification name of the Bacillus subtilis LB-KC is Bacillus subtilis LB-KC, the preservation number is CGMCC NO.25562, the preservation address is: General Microbiology Center of China Microorganism Culture Collection Administration, and the preservation date is: August 19, 2022.

2. A method for preparing the Bacillus subtilis LB-KC according to claim 1 for antagonizing pathogenic microorganisms in bedding materials of cattle and sheep pens, characterized in that: The preparation method comprises the following steps: mixing Bacillus subtilis LB-KC with brown sugar, corn flour or molasses, adding water to mix evenly, and then fermenting for 1-3 hours to obtain the product.

3. A method for preparing bedding for antagonizing pathogenic microorganisms in bedding for cattle and sheep pens, characterized in that: The pad preparation method comprises the following steps: (1) Select sawdust, rice husks, straw and turf as the raw materials for litter, mix them evenly and control the carbon-nitrogen ratio of the raw materials to 200-300:1; (2) Mix the Bacillus subtilis LB-KC described in claim 1 with brown sugar, corn flour or molasses, add water to mix evenly, and ferment for 1-3 hours for later use; (3) Spray the fermented Bacillus subtilis LB-KC obtained in step (2) on the mixed bedding material obtained in step (1) at a spraying rate of 50-150 ml / m 3 The mixed bedding raw materials are adjusted to have a moisture content of 45-60%, and the mixed bedding raw materials are piled and fermented for 24-35 hours to obtain a bedding material for antagonizing pathogenic microorganisms in cattle and sheep shed bedding, wherein the pathogenic microorganisms are necrotic Fusobacterium and nodular Bacteroides that cause foot rot in cattle and sheep.

4. A method for preparing bedding for antagonizing pathogenic microorganisms in bedding for cattle and sheep pens according to claim 3, characterized in that: Control the carbon-nitrogen ratio of the litter raw materials at 250:

1.

5. A method for preparing bedding for antagonizing pathogenic microorganisms in bedding for cattle and sheep pens according to claim 3, characterized in that: The spraying volume is 150ml / m 3 The mixed bedding materials were adjusted to have a moisture content of 60% and then piled and fermented for 35 hours.

6. A method for preparing bedding for antagonizing pathogenic microorganisms in bedding for cattle and sheep pens according to claim 3, characterized in that: Spraying volume is 100ml / m 3 The mixed bedding materials were adjusted to have a moisture content of 50% and then piled and fermented for 30 hours.

7. The bedding for cattle and sheep sheds prepared according to the bedding preparation method according to any one of claims 3 to 6.

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