A hoof bath bacterial liquid for preventing and treating hoof disease of cattle

Abstract

The application discloses hoof bath bacterial liquid for preventing and treating bovine hoof diseases, and belongs to the technical field of biotechnology. Sphingobacterium multivorum The hoof bath bacterial liquid comprises Sphingobacterium multivorum bacterial liquid and Lactobacillus reuteri bacterial liquid in a volume ratio of 6:1-2. Lactobacillus reuteri The effective viable cell number of the Sphingobacterium multivorum bacterial liquid and the Lactobacillus reuteri bacterial liquid is not less than 1×10 8 CFU / ml. The hoof bath bacterial liquid has good prevention and treatment effects on bovine hoof rot, warty dermatitis and hoof sole 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 *Sphingomyelin-Polyphagenin* 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 reuteri* suspensions are not less than 1 × 10⁻⁶. 8 CFU / ml.

[0005] Furthermore, the method for preparing the *Sphingosine monocytogenes* 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 reuteri includes the following steps: A1. *Lactobacillus reuteri* 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 in this invention ( 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 *Sphingosporobacter pylori* 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 24 hours to obtain the fermentation broth. After filtration and dilution, the fermentation broth yields the *Sphingospora polysaccharide* bacterial broth, with an effective viable count of approximately 1×10⁻⁶. 8 CFU / ml; the liquid fermentation medium contains: MgSO4·7H2O 0.5wt%, NaCl 0.5wt%, soybean meal 3wt%, and the remainder is water.

[0012] In this invention, the method for preparing Lactobacillus reuteri includes the following steps: A1. *Lactobacillus reuteri* 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 incubate at 30℃ and 180 rpm for 30 hours with stirring to obtain the fermentation broth. After filtration and dilution, the *Lactobacillus reuteri* broth is obtained. The effective viable count of the *Lactobacillus reuteri* broth is approximately 1 × 10⁻⁶. 8 CFU / ml; the liquid fermentation medium contained: 10.0 g / L dried tangerine peel powder, 10.0 g / L dried tangerine peel powder, 10.0 g / L dried Buddha's hand 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. Example 1

[0013] Prepare the hoof bath bacterial solution according to the proportions shown in Table 1 below. Table 1. Composition of Cattle Hoof Bath Bacterial Solution

[0014] Note: / indicates no addition, √ indicates that the ingredient is only present. Example 2

[0015] Antibacterial test

[0016] The main pathogens of bovine hoof disease, *Fusobacterium necrophorum*, *Corynebacterium pyogenes*, and *Bacteroides argentis*, were selected as experimental strains. The standard Oxford cup method was used in the antibacterial experiment. First, the selected experimental strains were prepared to a concentration of 1 x 10⁻⁶. 6 A bacterial suspension of CFU / ml was prepared, and then the suspension was added to the culture medium for spread culture. The diameter of the inhibition zone was measured by adding bovine hoof bath bacterial solutions numbered B1, B2, B3, C1, and C2 to Oxford cups, respectively. Each treatment was repeated three times during the experiment. The results are shown in Table 2 below. Table 2 Statistical table of antibacterial test results

[0017] As can be seen from Table 2 above, the bovine hoof bath bacterial solution of the present invention has a significant inhibitory effect on necrotic Fusobacterium, pyogenic Corynebacterium and Arthrozoon bacterium.

[0018] bovine hoof disease experiment

[0019] 1. The selection criteria for sick dairy cows are as follows: Foot rot: lameness of one or more limbs, preference for lying down, red or dark purple skin between the toes and the coronary band, congestion, swelling, and ulceration between the hooves.

[0020] Verrucous dermatitis: lameness in one or more limbs, preference for lying down, cauliflower-like true fibrous papillomas on the dorsal or palmar-plantar skin between the toes, redness and erosion of the hoof; lethargy, poor appetite, and decreased milk production; Sole ulcers: lameness of one or more limbs, preference for lying down, loss of horn on the sole of the hoof, leading to granulation tissue hyperplasia or even suppuration and infection of hoof tissue; decreased feed intake and milk production.

[0021] 2. Experimental Methods: A total of 360 dairy cows with hoof diseases from a farm in Yunnan Province were used as the research subjects. Among them, 120 cows each had hoof rot, wart dermatitis, and hoof ulcers. Each type of hoof disease cow was given a hoof bath with a hoof bath solution numbered B1, B2, B3, C1, and C2 (20 cows in each numbered group). A control group (20 cows) was set up for each type of hoof disease cow and used a sterile buffalo hoof bath.

[0022] 3. Judgment Criteria Table 3. Criteria for determining the effects of treatment on dairy cows with different hoof diseases.

[0023] 4. Based on the judgment criteria in Table 3, the treatment status of dairy cows with hoof diseases was recorded. The number of cows with effective, significantly effective, and ineffective treatments in each treatment group under each type of hoof disease was recorded, and the effectiveness rate was calculated as follows: Effective rate = (Effective + Significantly Effective) / Number of Cows * 100%. The specific results are shown in Table 4. Table 4 Comparison of treatment effects among different treatment groups

[0024] As can be seen from Table 4 above, the bovine hoof bath solution of this application has a good preventive and therapeutic effect on bovine foot rot, warty dermatitis and hoof ulcers.

[0025] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

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 reuteri* suspensions are not less than 1 × 10⁻⁶. 8 CFU / ml; wherein, the strain number of *Sphingosine monocytogenes* is BNCC139820, and the strain number of *Lactobacillus reuteri* is BMZ339697.

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 preparation method of the Lactobacillus reuteri includes the following steps: A1. *Lactobacillus reuteri* 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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