Cattle hoof bath bacterial liquid for preventing and treating cattle hoof diseases

Abstract

The invention discloses a cattle hoof bath bacterial liquid for preventing and treating cattle hoof diseases, and belongs to the technical field of biology, the cattle hoof bath bacterial liquid comprises a sphingobacterium polyphaga bacterial liquid and a lactobacillus reuteri bacterial liquid with the volume ratio of 6: (1-2), and the volume ratio of the sphingobacterium polyphaga bacterial liquid to the lactobacillus reuteri bacterial liquid is 1: (1-2). The effective viable count of the sphingobacterium polyphaga liquid and the effective viable count of the lactobacillus Rosei liquid are both not less than 1 * 10 < 8 > CFU / ml. The cattle hoof bath bacteria liquid has good prevention and treatment effects on cattle foot rot, verruca dermatitis and foot ulcer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a bovine hoof bath bacterial solution for the prevention and treatment of bovine hoof diseases. Background Technology

[0002] As people's living standards improve, the demand for milk is increasing year by year, bringing considerable economic benefits to dairy farmers and farms. Bovine hoof diseases are a general term for diseases affecting the limbs and hooves of dairy cows. With the development of large-scale and intensive dairy farms, the workload of workers has greatly increased, making it easy to neglect the health care of the cows' hooves. This leads to infections and inflammation of the hooves caused by pathogens, increasing the incidence of bovine hoof diseases in large-scale dairy farms. Although the initial symptoms are mild, they are easily overlooked, and without timely treatment, can result in serious consequences such as slow growth, reduced milk production, and even death. Summary of the Invention

[0003] In order to overcome the problems existing in the prior art, the present invention proposes a bovine hoof bath bacterial solution for the prevention and treatment of bovine hoof diseases.

[0004] To achieve the above objectives, the first aspect of the present invention provides: a bovine hoof bath bacterial solution for preventing and treating bovine hoof diseases, comprising *Sphingomonas polyphaga sinensis* in a volume ratio of 6:1-2 (…). Sphingobacterium multivorum ) bacterial solution and Lactobacillus reuteri ( Lactobacillus reuteri The bacterial suspensions; the effective viable counts of both *Sphingosine monocytogenes* and *Lactobacillus repens* suspensions are not less than 1 × 10⁻⁶. 8 CFU / ml.

[0005] Furthermore, the method for preparing the *Sphingospora polyphaga* bacterial suspension includes the following steps: S1. *Sphingosine monocytogenes* was inoculated onto a solid culture medium and cultured at 30°C for 24 hours. The culture was then transferred to a liquid culture medium and cultured at 30°C and 220 rpm for 18 hours to obtain the liquid culture. The solid culture medium contained: yeast extract: 5.0 g / L, peptone: 10.0 g / L, sodium chloride: 10.0 g / L, agar: 15.0 g / L, pH: 7.0 ± 0.2; the liquid culture medium contained: yeast extract: 5.0 g / L, peptone: 10.0 g / L, sodium chloride: 10.0 g / L, pH: 7.0 ± 0.2. S2. Inoculate the liquid bacterial culture into the liquid fermentation medium at a volume ratio of 3%-5%, and incubate at 30℃ and 220rpm for 24h to obtain the fermentation liquid. After filtration and dilution, the fermentation liquid is obtained as the Sphingosine monophosphate bacterial culture. The liquid fermentation medium contains: 0.5wt% MgSO4·7H2O, 0.5wt% NaCl, 3wt% soybean meal, and the remainder is water.

[0006] Furthermore, the method for preparing the *Lactobacillus rhodopsinii* includes the following steps: A1. *Lactobacillus dorothelius* was inoculated onto a solid culture medium and cultured at 28°C for 30 h. The culture was then transferred to a liquid culture medium and cultured at 30°C and 180 rpm for 24 h to obtain the liquid culture. The solid culture medium contained: peptone: 10.0 g / L, beef extract powder: 8.0 g / L, yeast extract: 4.0 g / L, glucose: 20.0 g / L, magnesium sulfate: 0.2 g / L, sodium acetate: 5.0 g / L, diammonium citrate: 2.0 g / L, dipotassium hydrogen phosphate: 2.0 g / L, manganese sulfate: 0.04 g / L, Tween 80: 1.0 mL, agar: 14.0 g / L, pH: 5.7 ± 0.2. The liquid culture medium differed from the solid culture medium in that it did not contain agar. A2. Inoculate the liquid bacterial culture into the liquid fermentation medium at a volume ratio of 3%-5%, and culture at 30℃ and 180 rpm for 30 h with stirring to obtain the fermentation broth. After filtration and dilution, the fermentation broth yields the Lactobacillus reuteri bacterial broth. The liquid fermentation medium contains: 10.0 g / L dried tangerine peel powder, 10.0 g / L dried tangerine peel powder, 10.0 g / L dried citron powder, 20.0 g / L soybean peptide, 4.0 g / L yeast extract, 20.0 g / L glucose, 5.0 g / L sodium acetate, 2.0 g / L diammonium citrate, 2.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.0 mL Tween-80, pH: 6.2±0.2.

[0007] The second aspect of the present invention provides the application of the bovine hoof bath bacterial solution used in the first aspect for the prevention and treatment of bovine hoof diseases in the preparation of products for the prevention and treatment of bovine hoof diseases.

[0008] Through the above technical solution, the present invention can achieve at least the following beneficial effects: the bovine hoof bath bacterial solution of the present invention has a good preventive and therapeutic effect on bovine foot rot, wart dermatitis and hoof ulcers. Detailed Implementation

[0009] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0010] The polyphagocytic sphingomyelin (Sphingomonas polyphagocytogenobacter) in this Sphingobacterium multivorum Purchased from Beina Chuanglian Biotechnology Co., Ltd. in the online store, strain number: BNCC139820; Lactobacillus reuisei (in this invention) Lactobacillus reuteri Purchased from Ningbo Mingzhou Biotechnology Co., Ltd., strain number: BMZ339697.

[0011] In this invention, the method for preparing Sphingosine monocytogenes bacterial suspension includes the following steps: S1, inoculate the Sphingobium multivorum on solid culture medium, cultivate at 30 DEG C for 24h, then transfer to liquid culture medium, cultivate at 30 DEG C, 220rpm shaking bed for 18h, obtain liquid strain; wherein, solid culture medium contains: yeast extract: 5.0g / L, protein peptone: 10.0g / L, sodium chloride: 10.0g / L, agar: 15.0g / L, pH: 7.0±0.2; liquid culture medium contains: yeast extract: 5.0g / L, protein peptone: 10.0g / L, sodium chloride: 10.0g / L, pH: 7.0±0.2; S2, inoculate the liquid strain to liquid fermentation culture medium according to the volume ratio of 3%-5%, cultivate at 30 DEG C, 220rpm for 24h, obtain fermentation bacteria liquid, the fermentation bacteria liquid is diluted after filtration, obtain Sphingobium multivorum bacteria liquid, the effective viable count of Sphingobium multivorum bacteria liquid is about 1x10 8 CFU / ml; wherein, liquid fermentation culture medium contains: MgSO4.7H2O 0.5wt%, NaCl 0.5wt%, soybean cake powder 3wt%, the rest is water.

[0012] In the application, the preparation method of Lactobacillus rogosae comprises the following steps: A1, inoculate the Lactobacillus rogosae on solid culture medium, cultivate at 28 DEG C for 30h, then transfer to liquid culture medium, cultivate at 30 DEG C, 180rpm shaking bed for 24h, obtain liquid strain; wherein, solid culture medium contains: protein peptone: 10.0g / L, beef infusion powder: 8.0g / L, yeast powder: 4.0g / L, glucose: 20.0g / L, magnesium sulfate: 0.2g / L, sodium acetate: 5.0g / L, citric acid diammonium: 2.0g / L, potassium phosphate: 2.0g / L, manganese sulfate: 0.04g / L, Tween 80: 1.0mL, agar: 14.0g / L, pH: 5.7±0.2; the difference between liquid culture medium and solid culture medium is that liquid culture medium does not contain agar; A2, inoculate the liquid strain to liquid fermentation culture medium according to the volume ratio of 3%-5%, cultivate at 30 DEG C, 180rpm stirring for 30h, obtain fermentation bacteria liquid, the fermentation bacteria liquid is diluted after filtration, obtain Lactobacillus rogosae bacteria liquid, the effective viable count of Lactobacillus rogosae bacteria liquid is about 1x10 8 CFU / ml; wherein, liquid fermentation culture medium contains: dried pericarpium citri reticulatae powder 10.0 g / L, dried pericarpium citri reticulatae powder 10.0 g / L, dried pericarpium citri reticulatae powder 10.0 g / L, soybean peptide 20.0g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, diammonium citrate 2.0 g / L, potassium phosphate 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05g / L and Tween-80 1.0 mL, pH: 6.2±0.2.

[0013] Example one The hoof bath bacterium liquid was prepared according to the proportion in Table 1 below Table 1 hoof bath bacterium liquid ingredient table Note: / represents no addition, and represents only containing the ingredient.

[0014] Example two Bacteriostatic experiment The main pathogenic bacteria of hoof disease, Clostridium necrophorum, Corynebacterium pyogenes and Bacteroides nodosus were selected as experimental strains, and the conventional Oxford cup method was used in the bacteriostatic experiment. First, the selected experimental strains were configured into a bacterial suspension with a concentration of 1x10 6 CFU / ml, and then the above bacterial suspension was added to the culture medium for coating culture. The hoof bath bacterium liquids numbered B1, B2, B3, C1 and C2 were added to the Oxford cups to measure the diameter of the bacteriostatic ring; each treatment was repeated 3 times during the experimental determination, and the results are shown in Table 2 below: Table 2 bacteriostatic experiment result statistics table As can be seen from Table 2 above, the hoof bath bacterium liquid of the present application has a significant inhibitory effect on Clostridium necrophorum, Corynebacterium pyogenes and Bacteroides nodosus.

[0015] Hoof disease experiment 1. The selection criteria for sick cows are as follows: Corrosion hoof disease: one or more limbs are lame, like to lie down, the skin between the toes (fingers) and the hoof crown is red or dark purple, the skin between the hooves and the hoof crown is congested, red and swollen, and the skin between the hooves is ulcerated.

[0016] Wart dermatitis: one or more limbs are lame, like to lie down, the skin between the toes is a cauliflower-like true fibrous papillary tumor on the back or palm side, the hoof is red and swollen, and the skin is eroded; spirit is not good, appetite is poor, and milk yield is reduced; Hoof ulcer: one or more limbs are lame, like to lie down, the hoof horn is missing, leading to hyperplasia of granulation tissue and even suppuration of the hoof tissue; feed intake and milk yield are reduced.

[0017] 2. Experimental method: the sick cows in a breeding farm in Yunnan were taken as the research object, a total of 360 cows, of which 120 cows were sick with corrosion hoof disease, wart dermatitis and hoof ulcer, and each kind of hoof disease cow was treated with hoof bath bacterium liquid numbered B1, B2, B3, C1 and C2 (20 cows for each number group), and each kind of hoof disease cow was set up a control group (20 cows) for hoof bath with sterile water.

[0018] 3. Judgment criteria Table 3 judgment criteria for different hoof disease cows after treatment 4. According to the judgment criteria in Table 3, the treatment of the cows with hoof diseases was recorded, the number of effective, significant and ineffective cows in each treatment group under each hoof disease category was recorded respectively, and the effective rate was calculated, wherein the effective rate = (effective + significant) / the number of cows * 100%. The specific results are shown in Table 4: Table 4 Comparison of treatment effects of different treatment groups As can be seen from the above Table 4, the hoof bath bacterial liquid of the cow has good prevention and treatment effect on bovine corneal hoof disease, warty dermatitis and hoof ulcer.

[0019] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A bovine hoof bath solution for preventing and treating bovine hoof diseases, characterized in that: Including Sphingosine monocytogenes with a volume ratio of 6:1-2 ( Sphingobacterium multivorum ) bacterial solution and Lactobacillus reuteri ( Lactobacillus reuteri The bacterial suspensions; the effective viable counts of both *Sphingosine monocytogenes* and *Lactobacillus repens* suspensions are not less than 1 × 10⁻⁶. 8 CFU / ml.

2. The bovine hoof bath bacterial solution for preventing and treating bovine hoof diseases according to claim 1, characterized in that: The preparation method of the *Sphingomonas polyphaga sinensis* bacterial suspension includes the following steps: S1. *Sphingosine monocytogenes* was inoculated onto a solid culture medium and cultured at 30°C for 24 hours. The culture was then transferred to a liquid culture medium and cultured at 30°C and 220 rpm for 18 hours to obtain the liquid culture. The solid culture medium contained: yeast extract: 5.0 g / L, peptone: 10.0 g / L, sodium chloride: 10.0 g / L, agar: 15.0 g / L, pH: 7.0 ± 0.2; the liquid culture medium contained: yeast extract: 5.0 g / L, peptone: 10.0 g / L, sodium chloride: 10.0 g / L, pH: 7.0 ± 0.

2. S2. Inoculate the liquid bacterial culture into the liquid fermentation medium at a volume ratio of 3%-5%, and incubate at 30℃ and 220rpm for 24h to obtain the fermentation liquid. After filtration and dilution, the fermentation liquid is obtained as the Sphingosine monophosphate bacterial culture. The liquid fermentation medium contains: 0.5wt% MgSO4·7H2O, 0.5wt% NaCl, 3wt% soybean meal, and the remainder is water.

3. The bovine hoof bath bacterial solution for preventing and treating bovine hoof diseases according to claim 1, characterized in that: The method for preparing the Lactobacillus rhodopseudomonas includes the following steps: A1. *Lactobacillus dorothelius* was inoculated onto a solid culture medium and cultured at 28°C for 30 h. The culture was then transferred to a liquid culture medium and cultured at 30°C and 180 rpm for 24 h to obtain the liquid culture. The solid culture medium contained: peptone: 10.0 g / L, beef extract powder: 8.0 g / L, yeast extract: 4.0 g / L, glucose: 20.0 g / L, magnesium sulfate: 0.2 g / L, sodium acetate: 5.0 g / L, diammonium citrate: 2.0 g / L, dipotassium hydrogen phosphate: 2.0 g / L, manganese sulfate: 0.04 g / L, Tween 80: 1.0 mL, agar: 14.0 g / L, pH: 5.7 ± 0.

2. The liquid culture medium differed from the solid culture medium in that it did not contain agar. A2. Inoculate the liquid bacterial culture into the liquid fermentation medium at a volume ratio of 3%-5%, and culture at 30℃ and 180 rpm for 30 h with stirring to obtain the fermentation broth. After filtration and dilution, the fermentation broth is obtained as Lactobacillus reuteri broth. The liquid fermentation medium contains: 10.0 g / L dried tangerine peel powder, 10.0 g / L dried tangerine peel powder, 10.0 g / L dried citron powder, 20.0 g / L soybean peptide, 4.0 g / L yeast extract, 20.0 g / L glucose, 5.0 g / L sodium acetate, 2.0 g / L diammonium citrate, 2.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.0 mL Tween-80, pH: 6.2±0.

2.

4. The application of the bovine hoof bath bacterial solution for preventing and treating bovine hoof diseases as described in any one of claims 1-3 in the preparation of products for preventing and treating bovine hoof diseases.

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