Bacillus velezensis and application thereof in prevention of urinary calculi of Boer goats

Abstract

On one hand, the invention discloses bacillus velezensis fermentation liquor, and the content of lipopeptide in the fermentation liquor is 2.16 g / L. The bacillus velezensis disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the bacillus velezensis is 27459, wherein the preservation number of the bacillus velezensis is CGMCC No. Lipopeptides in the fermentation liquor disclosed by the invention comprise iturin, abinogen and surfactin. The bacillus velezensis fermentation liquor disclosed by the invention can well inhibit reproduction and growth of bacteria and fungi. Meanwhile, fermentation liquor spray-dried powder prepared on the basis of the bacillus velezensis fermentation liquor is added into drinking water of the sheep flock at the content of four hundred thousand, so that the morbidity of the urinary calculi of the goats can be effectively controlled, and the survival rate of the goats can also be increased. The fermentation liquor spray-dried powder has great significance for preventing the goat urinary tract lithiasis, and provides a good reference and direction for preventing and treating the goat urinary tract lithiasis.

Description

Technical Field

[0001] The invention belongs to the technical field of animal feed and relates to application of fermentation liquid spray-dried powder of Bacillus velezensis in preventing urinary tract stones in Boer goats. Background Art

[0002] Sheep urethral stones, also known as urolithiasis, refer to the hemorrhagic inflammation and urinary tract obstructive diseases caused by the crystallization of salts in the urinary tract into coagulations of varying sizes and quantities, which stimulate the urinary tract mucosa. Clinically, it is characterized by abdominal pain, urination disorders and hematuria. This disease can also occur in Boer goats. If we do not regularly disinfect and clean the sheep's pens, it is easy to breed bacteria. Sheep live in the pens for a long time, and bacteria will cause urinary tract infections in sheep. When sheep have urinary tract infections, the amount of shed epithelium and inflammatory reaction products in the urethra increases, and urinary stones will also be generated. It mainly occurs in rams, and the symptoms are mental depression, inability to urinate normally or dripping urine, no rumination, abnormal swelling of the genitals, and large areas of tissue lumps around the genitals.

[0003] With people's pursuit of goat fattening speed and the addition of various feeds, although short-term fattening and marketing have been achieved, it has also caused a large outbreak of goat urinary stones, with the number of goats suffering from stones as high as 30%, and the mortality rate caused by stones as high as 10%. It only takes five days from the discovery of symptoms to death, causing great economic losses to farmers.

[0004] Urolithiasis in goats has also become one of the main problems in the development of goat animal husbandry in my country. Therefore, providing a feed that can both ensure the health of goats and effectively prevent the incidence of urinary stones during the fattening period of goats has become an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a Bacillus Velezii and its application in preventing urinary tract stones in Boer goats.

[0006] Therefore, the present invention discloses a Bacillus Velez fermentation broth in one aspect, wherein the lipopeptide content in the fermentation broth is 2.16 g / L.

[0007] Preferably, the Bacillus Velezii described in the present invention is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and its CGMCC No. is 27459.

[0008] Preferably, the lipopeptides in the fermentation broth of the present invention include iturin, fengyrin and surfactin.

[0009] Preferably, the mass ratio of surfactin:iturin:fengycin in the lipopeptide in the fermentation broth of the present invention is 1:1:3.

[0010] In another aspect, the present invention also discloses a Bacillus Velez fermentation broth spray-dried powder, wherein the Bacillus Velez fermentation broth spray-dried powder is prepared by adding 10%-30% of starch auxiliary material to the Bacillus Velez fermentation broth and then spray-drying the mixture to obtain the Bacillus Velez fermentation broth spray-dried powder.

[0011] On the other hand, the present invention also discloses drinking water for preventing urinary stones in Boer goats, wherein the drinking water contains 0.4 parts per hundred thousand spray-dried powder of fermentation liquid of Bacillus Velez.

[0012] In another aspect, the present invention also discloses a method for preparing the Bacillus Velez fermentation broth, characterized in that the method comprises the following steps:

[0013] (1) Bacillus Velezii was inoculated into LB medium, pH value was 8.3, culture temperature was 28-30°C, cultured for 60-80h, and 9×10 8 CFU / ml of bacterial solution;

[0014] (2) adding the fermentation buffer into a fermentation tank; sterilizing with high-temperature steam at 115° C. for 30 min; and fermenting for 2 days at a pH of 8.3 after the sterilization is completed and the fermentation tank is cooled and kept warm to 30° C. to obtain a fermentation liquid of Bacillus velezii;

[0015] The fermentation buffer was deionized water, 3% sucrose, 0.05% NaCl, 0.025% MgSO4·7H2O, 0.05% KH2PO4, 1% NH4Cl and 0.1% NH4NO3;

[0016] (3) adjusting the pH of the fermentation liquid obtained in step (2) to 6.0, centrifuging at 1000 g for 10 min, and discarding the precipitate;

[0017] (4) Store the fermentation supernatant at below -20°C for future use.

[0018] In another aspect, the present invention also discloses an application of the spray-dried powder of the fermentation liquid of Bacillus Velez in preparing feed for preventing urinary stones in Boer goats.

[0019] The Velez bacillus fermented liquid of the present invention can well inhibit the reproduction and growth of bacteria and fungi. At the same time, the fermented liquid spray-dried powder prepared based on the Velez bacillus fermented liquid is added to the drinking water of the flock at a content of 4 parts per 100,000, which can not only effectively control the incidence of goat urinary stones but also improve the survival rate of goats. Therefore, the fermented liquid spray-dried powder of the present invention is of great significance for preventing goat urinary tract stones, and provides a good reference and direction for the prevention and treatment of goat urinary tract stones.

[0020] Collection Instructions

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Information related to PCR of Bacillus velez.

[0023] Figure 2 Electrophoresis results after PCR of Bacillus Velez.

[0024] Figure 3 Mass spectrometry of fermentation broth.

[0025] Figure 4 Reference table of relative molecular masses of known lipopeptides.

[0026] Figure 5 HPLC profile of purified lipopeptides.

[0027] Figure 6 HPLC analysis chart of the purified lipopeptide.

[0028] Figure 7 The results of inhibition of Staphylococcus aureus by Bacillus Velez fermentation broth.

[0029] Figure 8 The inhibition results of Bacillus Velez fermentation broth on BS168.

[0030] Fig. 9 The results of inhibition of Fusarium oxysporum by Bacillus Velez fermentation broth.

[0031] Fig.10 The results of the inhibition of Aspergillus niger by Bacillus Velez fermentation broth.

[0032] Fig.11 Picture of the experimental herd of Boer goats.

[0033] Fig.12 Statistics of goat stone-free rates in each quarter after prism analysis.

[0034] Fig.13 Statistics of goat survival rate every quarter after prism analysis. DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0036] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1: PCR detection of Bacillus velez

[0038] Bacterial antimicrobial peptides: also known as bacteriocins, including cationic peptides and neutral peptides, can be secreted by both Gram-positive and Gram-negative bacteria. There are four types of antimicrobial peptides found in bacteria: Bacitracin, Gramicidin S, Polymyxin E and Nisin. At present, there are 119 types of bacteriocins included in the APD database, among which Nisin is a short peptide containing 3 to 4 amino acid residues produced by lactococci. It is an acid-resistant substance with high stability even in a low pH environment such as the stomach, and can inhibit Gram-positive bacteria such as Clostridium, Listeria and fungi.

[0039] Lipopeptides are a class of antimicrobial peptides mainly produced by Gram-positive Bacillus through non-ribosomal synthesis pathways. They are generally cyclic peptides composed of a β-hydroxy fatty acid and a 7-10 amino acid peptide chain connected by an amide bond. They have antibacterial, antifungal, antiviral, and antitumor biological activities.

[0040] The strain used in the present invention is Bacillus Velezii (taken from the intestinal solubles and mucosal fluid of the spotted bamboo shark) and is stored in the General Microbiological Center of China National Microbiological Culture Collection Administration, and its CGMCC No. is 27459.

[0041] (1) Bacillus velezensis, find the corresponding lipopeptide DNA template from the database, and select appropriate primers (see Figure 1 ), 10×PCR buffer, dNTPmix containing dATP, dCTP, dGTP, dTTP, and Taq enzyme for PCR.

[0042] (2) After the PCR reaction is completed, the sample is subjected to electrophoresis detection, and the results obtained are as follows Figure 2 .

[0043] (3) The electrophoresis results fully prove that the strain used in the present invention contains the gene sequences of antimicrobial lipopeptides such as mycosubtilin, iturin, fengycin, lysobactin, bacillus, and surfactin.

[0044] Example 2: Preparation of Bacillus Velez fermentation broth

[0045] (1) The Velezella sp. of the present invention was inoculated into LB medium, the pH value was 8.3, the culture temperature was 28-30°C, and the culture was carried out for 60-80 hours to obtain 9×10 8 CFU / ml of bacterial solution;

[0046] (2) adding the fermentation buffer into a fermentation tank; sterilizing with high-temperature steam at 115° C. for 30 min; and fermenting for 2 days at a pH of 8.3 after the sterilization is completed and the fermentation tank is cooled and kept warm to 30° C. to obtain a fermentation liquid of Bacillus velezii;

[0047] The fermentation buffer was deionized water, 3% sucrose, 0.05% NaCl, 0.025% MgSO4·7H2O, 0.05% KH2PO4, 1% NH4Cl and 0.1% NH4NO3;

[0048] (3) adjusting the pH of the fermentation liquid obtained in step (2) to 6.0, centrifuging at 1000 g for 10 min, and discarding the precipitate;

[0049] (4) Store the fermentation supernatant at below -20°C for future use.

[0050] Example 3: Purification and content detection of lipopeptides in Bacillus Velez fermentation broth

[0051] (1) The fermentation broth of Bacillus velezensis prepared in Example 2 was centrifuged, and the precipitate was discarded as much as possible, and the supernatant was retained.

[0052] (2) The fermentation broth supernatant was filtered through a 0.22 μm filter membrane, the pH of the permeate was adjusted to 2.0, and the permeate was centrifuged to remove the supernatant.

[0053] (3) The precipitate was extracted with methanol, the solution was centrifuged and the supernatant was evaporated.

[0054] (4) The material after rotary evaporation was freeze-dried and weighed, and a total of 2.16 g of lipopeptide was obtained, that is, the lipopeptide content in the Bacillus Velez fermentation broth was 2.16 g / L.

[0055] Example 4: Identification of lipopeptides in fermentation broth of Bacillus velez

[0056] (1) The freeze-dried powder of Example 3 was sent to a third party for mass spectrometry analysis, and the results were as follows: Figure 3 .

[0057] (2) Based on literature references Figure 4 As can be seen from the chart, the lipopeptides prepared in Example 3 mainly include iturin, fengycin A, and surfactin.

[0058] Example 5: Detection of the content ratio of various antimicrobial peptides in lipopeptides in Bacillus Velez fermentation broth

[0059] (1) The purified lipopeptide in Example 3 was cleaved and then subjected to HPLC for total amino acid detection, and the following results were obtained: Figure 5 and Figure 6 .

[0060] (2) The three most common lipopeptides are iturin, fengycin, and surfactin. The amino acid sequence of iturin's heptapeptide is (L-)Asn-(D-)Tyr-(D-)Asn-(L-)Gln-(L-)Pro-(D)Asn-(L-)Ser. The homologs iturin A, C, D, and E differ in the different carbon atom lengths of their fatty acid chains. Surfactin: (L-)Glu-(L-)Leu-(D-)Leu-(L-)Val-(L-)Asp-(D-)Leu-(L-)Leu. Fengyuansu: Fengyuansu is also composed of the peptide chain (L-)Glu-(D-)Orn-(L-)Tyr-(D-)Thr-(L-)Glu-(D-)Ala-(L-)Pro-(L-)Gln-(D-)Tyr-(L-)Ile. The structure of some amino acids will change during derivatization, glutamine is converted to glutamic acid, and asparagine is converted to aspartic acid. Therefore, in iturin: Asp:Tyr:Glu:Pro:Ser is 3:1:1:1:1. In surfactin: Glu:Leu:Val:Asp is 1:4:1:1, and in Fengyuansu: Glu:Orn:Tyr:Thr:Ala:Pro:Ile is 3:1:2:1:1:1:1.

[0061] (3) Based on the total amino acid ratio in the purified lipopeptides and the amino acid ratio in the three major types of lipopeptides, it can be inferred that the mass ratio of surfactin: iturin: fengcin in the lipopeptides is 1:1:3, which indicates that the fengcin content in the fermentation broth of the strain of the present invention is the highest.

[0062] Example 6: Experiment on the inhibition of bacteria by fermentation broth of Bacillus velez

[0063] (1) Experimental principle: When the fermentation liquid comes into contact with the test indicator bacteria in a solid plate, the growth of the indicator strain around it is inhibited, forming a sterile reproduction area.

[0064] (2) The bacterial suspension of Staphylococcus aureus and BS168 was inoculated on a solid plate of PDA medium and cultured at 29°C for 48 hours to obtain the first-generation spore culture; the first-generation spore culture was inoculated on a solid plate of PDA medium and cultured at 29°C for 48 hours to obtain the second-generation spore culture; the second-generation spore culture was inoculated on a solid plate of PDA medium and cultured at 29°C for 48 hours to obtain the third-generation spore culture.

[0065] (3) The third generation spore culture was washed with 0.85% saline to remove spores, and the spores were counted using a hemocytometer. The spore concentration was adjusted to 2 × 10 6 CFU / mL.

[0066] (4) Heat the prepared PDA medium to dissolve, cool to 45°C to 50°C, add the prepared Staphylococcus aureus and BS168 bacterial suspensions to the PDA medium at a rate of 1%, mix thoroughly, take 20 mL and pour into a sterile plate, gently shake the plate to evenly spread it, and wait for it to solidify before use.

[0067] (5) Place two Oxford cups at equal distances on the test plates containing Staphylococcus aureus and BS168 and press gently. Slowly add 100 μl of fermentation medium and fermentation liquid into the Oxford cups. Then move the plates to a 4°C to 6°C refrigerator for pre-diffusion for 2 h. Take out the plates and place them in a 29°C constant temperature incubator and incubate them upright for 6 h.

[0068] (6) Antibacterial test results Figure 7 , Figure 8 The experimental results fully proved that the fermentation broth had a significant inhibitory effect on Staphylococcus aureus and BS168.

[0069] Example 7: Fungus inhibition experiment of Bacillus Velez fermentation broth

[0070] (1) Experimental principle: Use fermentation liquid to inhibit the growth of test indicator bacteria in a solid plate to form a sterile breeding area.

[0071] (2) Inoculate Fusarium oxysporum and Aspergillus niger on a solid plate of tiger red agar medium and culture at 28°C for 48 hours to prepare a solid fungal culture.

[0072] (3) Pour the tiger red agar medium into a culture dish, wait for it to solidify, punch a hole in the center of the tiger red agar medium, and inoculate the solid culture of Fusarium oxysporum and Aspergillus niger into the tiger red agar culture dish.

[0073] (4) Place 2-3 Oxford cups at equal distances on the test plates containing Fusarium oxysporum and Aspergillus niger and press them gently. Slowly add 100 μl of the fermentation liquid into the 2-3 Oxford cups. Take out the plates and put them in a 28°C constant temperature incubator. After culturing upright for 12 hours, take out the Oxford cups and continue to culture in a 28°C constant temperature incubator for 48 hours.

[0074] (5) Antibacterial test results Fig. 9 , Fig.10 The experimental results fully proved that the fermentation liquid had a significant inhibitory effect on Fusarium oxysporum and Aspergillus niger.

[0075] Example 8: Preventive effect of spray-dried powder of Bacillus Velez fermentation broth on goat urinary calculi

[0076] The goats used in the present invention are Boer goats, which are raised by farmers in Jiangou Town, Shou County, Anhui Province. 200 goats were selected as the experimental goat flock (such as Fig.11 ), the flock received routine immunization vaccines, and the experiment lasted from October 2023 to September 2024.

[0077] (1) Add 10%-30% of starch auxiliary material to the fermentation liquid of Example 2 and spray-dry to obtain spray-dried powder of Bacillus velez fermentation liquid.

[0078] (2) 200 goats were randomly divided into 5 pens, with 40 goats in each pen. The 5 pens were divided into pens 1, 2, 3, 4, and 5. All goats ate the same forage and drank drinking water containing fermented spray-dried powder in different proportions.

[0079] (3) Column 1 is the blank group, and columns 2, 3, 4, and 5 are the experimental groups. Column 1 is for drinking normal tap water without any additives, column 2 is for drinking tap water containing 1 part of fermentation spray-dried powder (50 kg of tap water plus 0.5 g of fermentation liquid spray-dried powder), column 3 is for drinking tap water containing 2 parts of fermentation spray-dried powder (50 kg of tap water plus 1 g of fermentation liquid spray-dried powder), column 4 is for drinking tap water containing 4 parts of fermentation spray-dried powder (50 kg of tap water plus 2 g of fermentation liquid spray-dried powder), and column 5 is for drinking tap water containing 8 parts of fermentation spray-dried powder (50 kg of tap water plus 4 g of fermentation liquid spray-dried powder). The drinking water of each group was placed in the columns of their respective groups and raised in separate pens.

[0080] (4) The number of goats with stones and the number of deaths from stones were counted in each quarter. The statistics were collected for four quarters: October 2023 to December 2023; January 2024 to March 2024; April 2024 to June 2024; and July 2024 to September 2024.

[0081] Prevalence rate = number of sheep suffering from calculi / total number of sheep in each pen*100%

[0082] The prevalence of stone-free patients = 1-prevalence of stone

[0083] Mortality rate due to stones = number of sheep killed due to stones / total number of sheep in each pen * 100%

[0084] Survival rate = 1-mortality rate caused by stones

[0085] The statistical results are shown in the following table:

[0086] Table 1: Number of goats with calculi and deaths per quarter

[0087]

[0088]

[0089] Table 2: Stone-free rate and survival rate of goats in each quarter

[0090]

[0091] The data was analyzed by prism, and the results are as follows Fig.12 and Fig.13 .

[0092] From the above results, it can be seen that the bacterial liquid of the present invention can well inhibit the reproduction and growth of bacteria and fungi, and the fermentation liquid spray-dried powder is added to the drinking water of the flock at a content of 0.4 parts per hundred thousand (i.e., 2 g of fermentation liquid spray-dried powder is added to 50 kg of tap water), which can not only effectively control the incidence of goat urinary stones but also improve the survival rate of goats. Therefore, the fermentation liquid spray-dried powder of the present invention is of great significance for preventing goat urinary tract stones.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Bacillus Velez fermentation broth, characterized in that: The lipopeptide content in the fermentation broth is 2.16 g / L.

2. The fermentation broth according to claim 1, characterized in that The Bacillus Velez is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, and its CGMCC No. is 27459.

3. The fermentation broth according to claim 1, characterized in that The lipopeptides in the fermentation broth include iturin, fengyrin and surfactin.

4. The fermentation broth according to claim 3, characterized in that The mass ratio of surfactin:iturin:fengycin in the lipopeptide in the fermentation broth is 1:1:

3.

5. A spray-dried powder of Bacillus Velez fermentation broth, characterized in that: The Bacillus Velez fermentation liquid spray-dried powder is prepared by adding 10%-30% of starch auxiliary material to the Bacillus Velez fermentation liquid of claim 1, and then spray-drying the mixture to obtain the Bacillus Velez fermentation liquid spray-dried powder.

6. A drinking water for preventing urinary stones in Boer goats, characterized in that: The drinking water contains 0.4 parts per hundred thousand of the spray-dried powder of the fermentation liquid of Bacillus Velezii according to claim 5.

7. A method for preparing the Bacillus Velez fermentation broth according to claim 1, characterized in that: The method comprises the following steps: (1) The Velez subtilis bacteria described in claim 2 were inoculated into LB medium, the pH value was 8.3, the culture temperature was 28-30°C, and the culture was carried out for 60-80 hours to obtain 9×10 8 CFU / ml of bacterial solution; (2) adding the fermentation buffer into a fermentation tank; sterilizing with high-temperature steam at 115° C. for 30 min; and fermenting for 2 days at a pH of 8.3 after the sterilization is completed and the fermentation tank is cooled and kept warm to 30° C. to obtain a fermentation liquid of Bacillus velezii; The fermentation buffer was deionized water, 3% sucrose, 0.05% NaCl, 0.025% MgSO4·7H2O, 0.05% KH2PO4, 1% NH4Cl and 0.1% NH4NO3; (3) adjusting the pH of the fermentation liquid obtained in step (2) to 6.0, centrifuging at 1000 g for 10 min, and discarding the precipitate; (4) Store the fermentation supernatant at below -20°C for future use.

8. Use of the spray-dried powder of Bacillus Velez fermentation broth according to claim 5 in preparing feed for preventing urinary stones in Boer goats.

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