Clostridium baumannii preparation and application thereof in pet disease treatment
By developing Clostridium boulardii preparations, combined with freeze-drying technology and prebiotics, the gut microbiota of pets is regulated, solving the problem that existing technologies for treating osteoarthritis in pets only address the symptoms and not the root cause. This achieves safe and effective intervention at the root of the pathology, significantly reducing inflammatory responses and improving gut health.
Patent Information
- Application Number
- CN202610063283.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-19
- Publication Date
- 2026-05-22
AI Technical Summary
Existing technologies for treating osteoarthritis in pets suffer from problems such as treating the symptoms but not the root cause, significant side effects, major trauma, and high medical costs. Furthermore, traditional understanding is limited to local mechanical damage to joints and lacks intervention methods that target the root cause of the pathology.
Develop a Clostridium boulardii preparation, including a lyophilized bacterial agent and prebiotics, to regulate the intestinal flora of pets. The preparation method includes the preparation of bacterial suspension and lyophilization, the addition of protective agents such as proteins, sugars, polyols, and antioxidants, and the combination of prebiotics and other auxiliary additives to make various dosage forms for treatment.
It effectively inhibits the inflammatory response caused by arthritis, reduces inflammatory damage, improves the gut health of pets, significantly reduces the expression of inflammatory factors, improves treatment efficacy, has high safety, and has no obvious toxic side effects.
Smart Images
Figure CN122070923A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the technical field of biomedicine, specifically relating to a Clostridium boulardii preparation and its application in the treatment of pet diseases. Background Technology
[0002] With the global rise in companion animal ownership, pet health management has become a significant area of public concern. Osteoarthritis, a common degenerative joint disease in pets, especially older animals, is facing increasing demands for prevention and treatment. Osteoarthritis is the most common type of joint disease in veterinary clinics, with a consistently high incidence in companion animals such as dogs and cats. The incidence is significantly higher in large dogs, older dogs, and overweight pets than in the general population. The disease is characterized by progressive degenerative degradation of articular cartilage, subchondral bone hyperplasia and sclerosis, chronic synovial inflammation, and osteophyte formation at the joint margins. Typical clinical symptoms include joint pain during movement, stiffness upon initiation, and reduced range of motion. In later stages, joint deformities, lameness, and even paralysis can occur, severely reducing the pet's quality of life. Furthermore, the chronic pain can lead to irritability, loss of appetite, and other behavioral abnormalities, further increasing the burden of care for pet owners.
[0003] Current clinical treatments for osteoarthritis in pets primarily focus on symptomatic intervention, encompassing conventional systems such as drug therapy, surgery, and physical therapy, as well as emerging biological therapies like stem cell therapy and platelet-rich plasma (PRP) therapy. However, these techniques suffer from drawbacks such as treating the symptoms but not the root cause, significant side effects, considerable trauma, and high medical costs. Therefore, developing a safe, efficient, affordable, and easily implemented treatment that addresses the underlying pathology of osteoarthritis is of great importance to pet health.
[0004] In recent years, the discovery of the "gut-joint axis" regulatory mechanism has opened up a completely new research perspective for the treatment of osteoarthritis, breaking the traditional understanding that osteoarthritis is caused solely by local mechanical damage to the joints. Methods for treating and intervening in osteoarthritis through gut microbiota are constantly emerging.
[0005] Through a series of screenings, this invention discovered the positive effects of Clostridium botulinum (C. bolteae), a relatively little-studied bacterium in the pet gut, on the treatment of diseases such as osteoarthritis in pets such as dogs and cats, and developed corresponding dosage forms. Summary of the Invention
[0006] To address the aforementioned problems, the present invention aims to provide a Clostridium boulardii preparation and its application in the treatment of pet diseases.
[0007] The technical content of this invention is as follows: This invention provides a Clostridium botulinum preparation comprising Clostridium botulinum (C. bolteae), wherein the ATCC number of Clostridium botulinum is BAA-613; Furthermore, the Clostridium boulardii preparation is a lyophilized bacterial agent, which is prepared from Clostridium boulardii including a lyophilization protectant and Clostridium boulardii. The lyophilization protectant includes protein protectants, sugar protectants, polyols, antioxidants, and solvents, etc. By mass fraction, protein protectants account for 10-15%, carbohydrate protectants account for 5-10%, polyols account for 4-8%, antioxidants account for 0.1-2%, and the remainder is solvent; The protein-based protective agents include one or more of the following protein-containing substances: skim milk powder, whey powder, gelatin, casein peptone, gum arabic, etc. The sugar preservatives include one or more of sucrose, lactose, trehalose, sorbitol, and xylitol; The polyol includes glycerol; The antioxidant includes L-cysteine hydrochloride; The solvent includes sterile saline solution.
[0008] The preparation method of the above-mentioned freeze-dried bacterial agent includes the following steps: 1) Preparation of bacterial suspension: A protective agent solution is prepared by mixing protein-based protective agents, sugar-based protective agents, polyols, antioxidants, and solvents, and then sterilized by autoclaving for later use. The activated and purified Clostridium boulardii cell precipitate was added to the protective agent solution and stirred thoroughly to obtain a cell suspension. The viable cell count was controlled at 1×10⁻⁶. 11 ~1×10 12 CFU / mL; 2) Preparation of freeze-drying: The bacterial suspension was dispensed into sterile freeze-drying bottles and placed in a freeze dryer for freeze-drying. The freeze-drying procedure was as follows: first, pre-freeze at -40℃ for 2-4 hours, then turn on the vacuum pump, control the vacuum degree to 10-30 Pa, gradually raise the temperature to -10℃, sublimate and dry for 8-12 hours, then raise the temperature to 25℃ and dry for 4-6 hours. After freeze-drying, the freeze-drying bottles were quickly sealed to obtain Clostridium botulinum freeze-dried bacterial agent.
[0009] Furthermore, the Clostridium boulardii preparation includes the above-mentioned lyophilized bacterial agent and prebiotics; By mass fraction, freeze-dried bacterial agents account for 70-90%, and prebiotics account for 10-30%; The prebiotics include one or more of the following: fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrophyllum, trachomatis polysaccharides, stachyose, and polydextrose.
[0010] Furthermore, the Clostridium boulardii preparation also includes other auxiliary additives, including neutralizing agents, gelling agents, polysaccharides, etc. By mass fraction, the freeze-dried bacterial agent accounts for 60-70%, prebiotics for 10-15%, neutralizing agent for 1-3%, gelling agent for 0.3-0.5%, and polysaccharides for 8-12%. The neutralizing agent includes calcium carbonate; The gelling agent includes xanthan gum; The polysaccharides include one or more of maltodextrin, amylose, and amylopectin.
[0011] The Clostridium boulardii preparation may also include other bacteria, including one or more of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium infantis, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Clostridium butyricum, Enterococcus faecalis, and Saccharomyces boulardii.
[0012] The Clostridium boulardii preparation may also include other drugs, such as ursodeoxycholic acid (UDAC), which is used to treat osteoarthritis in pets.
[0013] The Clostridium boulardii preparations are presented in the form of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, tablets, capsules, aerosols, suppositories, and chewing gum.
[0014] This invention also provides the application of the above-mentioned Clostridium boulardii preparation in the preparation of products for treating pet diseases; The pet diseases mentioned include osteoarthritis; The products include pharmaceuticals or laboratory reagents.
[0015] The beneficial effects of this invention are as follows: This invention relates to a Clostridium botulinum preparation, which is prepared by freeze-drying Clostridium botulinum (C. bolteae) and combined with prebiotics. The prebiotics provide nutritional support and enhance the bioactivity of Clostridium botulinum; they also regulate the body's microecological balance. Furthermore, the protein and sugar preservatives added to the freeze-dried preparation effectively maintain the activity of Clostridium botulinum, extend the shelf life of the preparation, and ensure the stability and reliability of its clinical efficacy. Through screening different proportions of ingredients, this invention has obtained a dosage form with superior efficacy, which shows better results in treating osteoarthritis in dogs and cats. It can effectively inhibit the inflammatory response caused by arthritis and reduce inflammatory damage. Furthermore, combining the Clostridium botulinum preparation with other probiotics can also improve the intestinal health of pets. The raw materials used in this invention's Clostridium botulinum preparation are natural, highly safe, and have no obvious toxic side effects, making it suitable for various individual pets. Attached Figure Description
[0016] Figure 1 A graph showing the comparison of inflammatory factor expression levels between the experimental group and the control group in the mouse osteoarthritis model. Figure 2 A graph showing the expression levels of inflammatory factors in the serum of mice fed with the Clostridium botulinum preparation of the present invention; Figure 3 This is a graph showing the effect of the Clostridium boulardii preparation of the present invention on inflammatory factors in the serum of diseased cats; Figure 4 This is a graph showing the effect of the Clostridium boulardii preparation of the present invention on inflammatory factors in the serum of diseased dogs. Detailed Implementation
[0017] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.
[0018] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.
[0019] Example 1 Cultivation of Clostridium boulardii and preparation of lyophilized bacterial agents 1. Cultivation of Clostridium boulardii Culture: Take out a frozen C. bolteae (ATCC BAA-613) and anaerobic culture it in the appropriate culture medium (10 g tryptone, 10 g beef extract, 5 g glucose, 5 g sodium chloride, 3 g yeast extract, 3 g sodium acetate, 1 g soluble starch, 0.5 g L-cysteine hydrochloride, mix the above components and add water to make up to 1 L, and adjust the pH to 6.8). If a solid culture medium is prepared, an additional 15 g of agar should be added and the culture should be scaled up under anaerobic conditions.
[0020] Centrifugation to collect bacterial cells: Place the fermentation broth in a high-speed refrigerated centrifuge and centrifuge at 4℃, 5000-8000r / min for 10-15min. Discard the supernatant and collect the bacterial cell precipitate. Wash the bacterial cell precipitate 2-3 times with sterile physiological saline. Repeat the centrifugation operation after each wash to obtain purified Clostridium botulinum cells.
[0021] 2. Preparation of Clostridium boulardii freeze-dried inoculum Optimization of the ratio of freeze-drying protectant: In order to screen a suitable ratio of freeze-drying protectant, the present invention optimizes the ratio of sucrose, skim milk powder and glycerin according to Table 1.
[0022] Bacterial cell suspension: Preparation of the protective agent solution: Add skim milk powder, sucrose, and glycerol according to Table 1, and 0.5% L-cysteine hydrochloride. The solvent is sterile physiological saline. Adjust the pH to 6.2-6.5. Autoclave at 115℃ for 15-20 minutes and then cool for later use. Add the purified bacterial cell precipitate to the protective agent solution and stir thoroughly to prepare a bacterial cell suspension. The viable cell count in the bacterial cell suspension should be controlled at 1×10⁻⁶. 11 ~1×10 12 CFU / mL.
[0023] Freeze-drying preparation: The bacterial suspension was dispensed into sterile freeze-drying bottles and placed in a freeze dryer for freeze-drying. The freeze-drying procedure was as follows: first, pre-freeze at -40℃ for 2-4 hours, then turn on the vacuum pump, control the vacuum degree to 10-30 Pa, gradually raise the temperature to -10℃, sublimate and dry for 8-12 hours, then raise the temperature to 25℃ and dry for 4-6 hours. After freeze-drying, the freeze-drying bottle was quickly sealed to obtain the freeze-dried Clostridium botulinum bacterial agent.
[0024] Test of the effect of freeze-dried bacterial agent: Take 0.1g of each component freeze-dried bacterial agent and dissolve it quickly. Take the same volume of dissolved bacterial solution and culture it in 125mL shake flasks under the above culture conditions. After more than half of the shake flasks become obviously turbid, take the bacterial solution and detect the OD600 value with a spectrophotometer. The test results are shown in Table 1-1 and Table 1-2.
[0025] As shown in the table below, the freeze-dried bacterial agent prepared with 10-15% skim milk powder, 5-10% sucrose, and 4-8% glycerol exhibits high activity. Similarly, using the optimized ratio of skim milk powder, sucrose, and glycerol as described above, and setting L-cysteine hydrochloride at 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, and 2%, the freeze-drying protectant containing 0.25-0.75% L-cysteine hydrochloride showed better performance after screening.
[0026] Table 1-1 Effect of different component ratios of freeze-dried bacterial agents on bacterial activity (OD600 value)
[0027] Table 1-2 Effect of different component ratios of freeze-dried bacterial agents on bacterial activity (OD600 value)
[0028] In the following scheme, the preferred freeze-drying protectant is composed of physiological saline containing 10% skim milk powder, 7.5% sucrose, 6% glycerol, and 0.5% L-cysteine hydrochloride, with a pH of 6.2-6.5.
[0029] Experimental Example 1 Treatment of OA mice with Clostridium boulardii preparations To determine whether the Clostridium boulardii preparation had a positive therapeutic effect on OA mice, and to aid in the colonization of Clostridium boulardii, this invention added prebiotic components to the freeze-dried bacterial agent in Example 1. The prebiotic components were selected as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), with a prebiotic component to freeze-dried bacterial agent ratio of 1:4 and a prebiotic component FOS to GOS ratio of 1:1. The resulting preparation was named Clostridium boulardii preparation 1. Simultaneously, cellulose was used to replace the prebiotic components in Clostridium boulardii preparation 1, and the resulting preparation was named Clostridium boulardii preparation 2.
[0030] Osteoarthritis (OA) Modeling in Mice: Male C57BL / 6 mice (purchased from the Experimental Animal Management Center of Southern Medical University) were used for OA modeling experiments. The mice were 10 weeks old. Mice were first placed in an acclimatization environment with a temperature of 20-26℃, humidity of 40%-60%, and a 12-hour light / 12-hour dark cycle. Afterward, they were grouped for modeling. Mice were anesthetized, and the surgical sites were disinfected with iodine. The experimental group underwent anterior cruciate ligament (ACLT) transection, while the control group underwent the same procedure as the surgical group, except that the ACL was not transcribed. The incisions were rinsed with saline to remove tissue debris and then closed. The modeling effect was assessed 8 weeks post-surgery. The difference in expression of relevant inflammatory factors (IL-6, TNF-α, and IL-1β) between the experimental and control groups was used to evaluate the modeling effect.
[0031] The results are as follows Figure 1 As can be seen, compared with the control group, the expression of IL-6, TNF-α, and IL-1β in the serum of mice in the experimental group was significantly upregulated. Simultaneously, bone and joint swelling was observed in the corresponding mice, indicating successful model establishment. In this invention, Prism 8.0 software was used to process data from different groups. A P < 0.05 indicated a significant difference between the experimental group and the control group, while a P > 0.05 indicated no significant difference. Subsequent embodiments were the same.
[0032] Using the above method, a batch of male mice (n=30) were used to establish a model. Twenty-four mice with significant joint swelling were selected and randomly divided into three groups of eight mice each. Group 1 was fed Clostridium botulinum preparation 1, Group 2 was fed Clostridium botulinum preparation 2, and Group 3 served as the control group, differing from the first two groups only in that it did not receive Clostridium botulinum preparations. Serum samples were collected on days 7, 14, and 21 to detect the expression levels of IL-6, TNF-α, and IL-1β.
[0033] The results are as follows Figure 2As shown, on day 7, the expression levels of IL-6, TNF-α, and IL-1β in the serum of mice in the Clostridium boulardii preparation group 1 were the lowest. On days 14 and 21, the expression levels in groups 1 and 2 were similar, both significantly lower than the control group. This indicates that intervention with Clostridium boulardii preparations reduces the expression levels of IL-6, TNF-α, and IL-1β in serum, suggesting that Clostridium boulardii preparations can effectively treat osteoarthritis. Furthermore, Clostridium boulardii preparations containing prebiotic components can achieve therapeutic effects more quickly.
[0034] Experimental Example 2 Comparison of the activity of different formulations of Clostridium botulinum in simulated environments Evaluation of strain tolerance to gastric acid environment: To ensure a higher survival rate of Clostridium boulardii in the acidic environment of the stomach, the following simulated gastric juice was prepared: a 0.5% NaCl solution was prepared, 0.3% pepsin was added, the pH was adjusted to 2.0 with 1 mol / L HCl, and after thorough dissolution, the solution was filtered through a 0.22 μm filter for sterilization and stored at 4 degrees Celsius. The protective effect of different formulations on the bacteria was verified by simulating gastric juice. After multiple rounds of testing, one excellent formulation was selected to counteract the gastric juice. This example only provides a few examples of the formulations for comparison to demonstrate the superior effect of this formulation.
[0035] Table 2 Composition of different Clostridium boulardii preparations
[0036] After treating the different formulations in Table 2 in simulated gastric fluid for 2 hours, the samples were washed with physiological saline, centrifuged, and the precipitate was collected. This process was repeated three times. Equal volumes of each sample were added to the culture medium from Example 1 for incubation. After culturing for 24-48 hours until the bacterial solution became turbid, the OD600 value of the bacterial solution was measured using a turbidimetric method to determine the bacterial activity of different formulations after treatment with simulated gastric fluid. This example was tested three times.
[0037] Table 3 OD600 values of Clostridium botulinum bacterial suspension
[0038] After treatment with simulated gastric juice, some Clostridium boulardii preparations were inactivated under acidic conditions, while the remaining viable bacteria continued to grow in the culture medium. The comparison of the number of remaining viable bacteria in different Clostridium boulardii preparations can be reflected by the OD600 values of the culture media cultured for the same period. As shown in Table 3, the Clostridium boulardii preparation group 1e exhibited the best bacterial activity after treatment with simulated gastric acid.
[0039] Experimental Example 3 Treatment of sick cats
[0040] Experimental plan: The selected cats were 12 male Persian cats aged 8-10 years, who were all in the middle stage of OA (and had no other diseases that might affect the experiment; this stage was strictly screened). The cats were in the middle stage of OA and beginning to progress to the late stage. The specific manifestations were: significantly decreased activity level, only active when eating and defecating, slow to get up (>3 seconds), occasional mild lameness (only after exercise), and when the affected joints were touched, the cats would flinch and growl. The joints may be slightly swollen, and the cats would occasionally curl up abnormally during sleep.
[0041] Feeding conditions: Temperature 20-26℃, humidity 40%-60%, lighting 12h light / 12h darkness, good ventilation, and low noise.
[0042] SPF-grade experimental complete pelleted feed (compliant with GB 14924.3-2010 standard, protein content 28%-32%, calcium-to-phosphorus ratio 1.2:1-1.5:1, fat content ≤15%) was used to feed the sick cats uniformly. After two weeks of feeding, and after observation and quarantine, the sick cats were found to have no other diseases that could affect the experiment, and the experiment began.
[0043] The cats were divided into two groups based on their average weight and age: Experimental group: 6 cats were fed 1g of Clostridium botulinum preparation daily (the preparation was dissolved in a small amount of cooled boiled water and mixed with the cat food) for 30 consecutive days.
[0044] Control group: 6 animals, except that the conditions were the same as those in the experimental group, except that there was no Clostridium botulinum preparation.
[0045] After the experiment began, the cats' activity was observed daily. Serum was collected on days 15 and 30 for ELISA detection of the inflammatory factors IL-6, TNF-α, and IL-1β. Results are as follows: Figure 3 As shown, the data were analyzed using Prism 8.0 software. At D15 and D30, the experimental group showed significant differences from the control group in terms of IL-6, TNF-α, and IL-1β (P < 0.001), indicating that the experimental group and the control group had significantly reduced inflammation related to osteoarthritis.
[0046] Meanwhile, the Von Frey pain meter was used to detect the cat's paw retraction threshold. During the detection process, a quiet environment was ensured to avoid interference.
[0047] The results are as follows Figure 3 and Table 4 below: (1) Inflammatory factors: The data were analyzed for significance using Prism 8.0 software. At D15 and D30, the experimental group showed significant differences from the control group in three factors: IL-6, TNF-α and IL-1β (P<0.001), indicating that the experimental group was significantly different from the control group and that the inflammation caused by osteoarthritis was significantly reduced.
[0048] (2) Paw retraction threshold: The data were analyzed for significance using Prism 8.0 software. At D15, the experimental group showed a significant difference from the control group (P<0.05). At D30, the experimental group showed a significant difference from the control group (P<0.01). Compared with the control group, the experimental group showed a significantly higher paw retraction threshold, indicating a significant reduction in pain at the cat's joints.
[0049] Table 4. Cat paw retraction threshold (mean ± SD, g)
[0050] * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and so on.
[0051] Test Example 4 Treatment of sick dogs Experimental plan: Selection of affected dogs: 12 Labrador Retrievers aged 8-10 years who were diagnosed with OA (without other diseases that might affect the experiment, and this step was strictly screened) and were in the middle to late stage. The main symptoms of the disease were pain and retraction of the joints of the limbs when pressed, joint swelling, difficulty in standing and walking, limping or lameness, and slow movement.
[0052] Feeding conditions: Temperature 20-26℃, humidity 40%-60%, lighting 12 hours of light / 12 hours of darkness, good ventilation, and low noise.
[0053] SPF-grade experimental complete pelleted feed (as above) was used to feed the sick dogs uniformly. After two weeks of feeding, the dogs were observed and quarantined, and it was confirmed that the sick dogs had no other diseases that could affect the experiment, so the experiment began.
[0054] Based on reference weight and age group, the animals were randomly divided into two groups of 6 each. Group 1 was the experimental group: Group 1 was fed normal basic dog food and 1e 2g of Clostridium boulardii preparation was given daily (the preparation was dissolved in a small amount of cooled boiled water and mixed with the dog food) for 30 consecutive days.
[0055] Group 2 was the control group: except for the absence of Clostridium botulinum preparation, all other conditions were the same as those in the experimental group.
[0056] After the experiment began, the dogs' activities were observed daily, and the knee joint circumference was counted and recorded every 15 days. If the difference between the knee joint and D0 was positive, it was recorded as a positive value; if the difference was negative, it was recorded as a negative value. At the same time, venous blood was collected and serum was separated for ELISA detection of inflammatory factors IL-6, TNF-α and IL-1β.
[0057] The results are as follows Figure 4 and Table 5 below: (1) Inflammatory factors: The data were analyzed for significance using Prism 8.0 software. At D15 and D30, the experimental group showed significant differences in IL-6, TNF-α and IL-1β compared with the control group, with P<0.001, indicating that there were significant differences between the experimental group and the control group, and the inflammation related to osteoarthritis was significantly reduced.
[0058] (2) Changes in knee joint circumference in dogs: Compared with the control group, the experimental group showed a decrease in joint circumference on day 15 of feeding, with dogs becoming more natural at standing and walking, and their activity level increasing; by day 30, the joint circumference had decreased further compared to day 15. Data were analyzed for significance using Prism 8.0 software. At D15 and D30, the experimental group showed a significant difference compared to the control group (P < 0.001), indicating a statistically significant difference between the two groups.
[0059] Table 5. Increase or decrease in knee joint circumference (mean ± SD, mm)
[0060] Experimental Example 5 The therapeutic effect of compound preparations on osteoarthritis in dogs and cats 1. The therapeutic effect of compound probiotic preparations on osteoarthritis in dogs and cats. To explore the effect of microbial cells in commonly used microbial feed additives on the Clostridium boulardii preparation in this invention, this experiment used a compound microbial agent to intervene in the treatment of osteoarthritis in dogs and cats. Commonly used microbial feed additives for dogs and cats can be one or more of the following: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium infantis, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Clostridium butyricum, Enterococcus faecalis, and Saccharomyces boulardii, etc.
[0061] One revival strain of Bacillus subtilis (CICC 24713) was cultured in LB liquid medium (formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2) at 37℃ on an aerobic shaker at 180 r / min for 16–24 h, with a cell concentration ≥1×10⁻⁶ at the end of the culture. 9CFU / mL; Resuscitate one strain of *Saccharomyces boulardii* (CNCM I-745): Cultured in YPD liquid medium (formulation: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, pH 6.0) at 30℃ on an aerobic shaker at 150 rpm for 24 h, with a final cell concentration ≥1×10⁻⁶. 8 CFU / mL. The two bacterial cells were washed and collected according to the method in Example 1. The two bacterial cells were mixed with Clostridium boulardii at a ratio of 3:7. After mixing, the bacterial cells were prepared according to the preparation method of Clostridium boulardii preparation 1e to obtain compound probiotic preparation 1 and compound probiotic preparation 2 respectively.
[0062] 2. The therapeutic effect of Clostridium boulardii / drug combination preparation on osteoarthritis in dogs and cats. Furthermore, to explore the efficacy of combined drug and Clostridium boulardii treatment for osteoarthritis in dogs and cats, this invention developed a compound preparation of Clostridium boulardii and a drug. Ursodeoxycholic acid (UDCA) was selected as one of the drugs and compounded with the aforementioned Clostridium boulardii preparation 1e at a mass ratio of 1:9 to obtain the Clostridium boulardii / drug compound preparation. Referring to the aforementioned experimental examples 3 and 4, intervention treatments were administered to cats and dogs suffering from osteoarthritis. Groups were set up as follows: compound probiotic preparation 1, compound probiotic preparation 2, Clostridium boulardii preparation 1e, and a blank control group. Each group consisted of 6 cats or dogs, and all were fed the same amount of probiotic preparation as in the aforementioned examples. A separate Clostridium boulardii / drug compound preparation was also prepared, with the feeding amount being half that of the aforementioned examples.
[0063] Table 6. Cat paw retraction threshold (mean ± SD, g)
[0064] As shown in Table 6, compared with the control group, the claw retraction threshold of cats began to increase at D15 in different probiotic preparation groups; and continued to increase at D30. Data significance analysis using Prism 8.0 software showed significant differences between each group and the control group, indicating that other microbial additives in the compound probiotic preparation did not antagonize the therapeutic effect of Clostridium botulinum. It is achievable that different probiotics combined with Clostridium botulinum can influence the feline gut microbiota and thus improve the cat's health. Furthermore, the combined use of ursodeoxycholic acid and Clostridium botulinum also significantly improved the health of sick cats.
[0065] Table 7. Increase or decrease in knee joint circumference (mean ± SD, mm)
[0066] As shown in Table 7, compared with the control group, the knee joint circumference of dogs began to shorten at D15 and further decreased at D30. Data significance analysis using Prism 8.0 software showed significant differences between each group and the control group, indicating that other microbial additives in the compound probiotic preparations did not antagonize the therapeutic effect of Clostridium botulinum. It is achievable that different probiotics combined with Clostridium botulinum can influence the canine gut microbiota and thus improve the health of dogs. Furthermore, the combined use of ursodeoxycholic acid and Clostridium botulinum preparations also significantly improved the health of sick dogs.
Claims
1. A Clostridium boulardii preparation for treating osteoarthritis in pets, characterized in that, It includes Clostridium botulinum.
2. The Clostridium boulardii preparation according to claim 1, characterized in that, The Clostridium botulinum is a freeze-dried bacterial agent, which is prepared by including a freeze-drying protectant and Clostridium botulinum. The freeze-drying protectant includes protein protectants, sugar protectants, polyols, antioxidants, and solvents; By mass fraction, protein protectants account for 10-15%, sugar protectants account for 5-10%, polyols account for 4-8%, antioxidants account for 0.1-2%, and the remainder is solvent.
3. The Clostridium boulardii preparation according to claim 2, characterized in that, The protein-based protective agents include one or more of the following protein-containing substances: skim milk powder, whey powder, gelatin, casein peptone, gum arabic, etc. The sugar preservatives include one or more of sucrose, lactose, trehalose, sorbitol, and xylitol; The polyol includes glycerol; The antioxidants include L-cysteine hydrochloride.
4. The Clostridium boulardii preparation according to claim 1, characterized in that, The Clostridium boulardii preparation includes a lyophilized bacterial agent and a prebiotic, wherein the lyophilized bacterial agent includes Clostridium boulardii. By mass fraction, freeze-dried bacterial agents account for 70-90%, and prebiotics account for 10-30%.
5. The Clostridium boulardii preparation according to claim 4, characterized in that, The prebiotics include one or more of the following: fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrophyllum, trachomatis polysaccharides, stachyose, and polydextrose.
6. The Clostridium boulardii preparation according to any one of claims 1 to 4, characterized in that, The Clostridium boulardii preparation also includes other auxiliary additives, including neutralizing agents, gelling agents, and polysaccharides; The polysaccharides include one or more of maltodextrin, amylose, and amylopectin.
7. The Clostridium boulardii preparation according to claim 2, characterized in that, In the preparation of the freeze-dried bacterial agent, *Clostridium botulinum* is pre-activated and cultured to obtain a bacterial suspension, and the viable count of *Clostridium botulinum* in the obtained bacterial suspension is 1 × 10⁻⁶. 11 ~1×10 12 CFU / mL.
8. The Clostridium boulardii preparation according to claim 2, characterized in that, The Clostridium boulardii preparation also includes other fungi and / or other drug components; The other bacteria include one or more of the following: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium infantis, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Clostridium butyricum, Enterococcus faecalis, and Saccharomyces boulardii. The other drug ingredients include medications for treating osteoarthritis in pets, including ursodeoxycholic acid.
9. The Clostridium boulardii preparation according to claim 2, characterized in that, The Clostridium boulardii preparations are presented in the form of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, tablets, capsules, aerosols, suppositories, and chewing gum.
10. The use of the Clostridium boulardii preparation according to any one of claims 1-9 in the preparation of products for treating pet diseases, characterized in that, The pet diseases mentioned include osteoarthritis; The products include pharmaceuticals or laboratory reagents.