Complex probiotic formulation containing lactobacillus acidophilus wp285 and its use in the preparation of a product for inhibiting the formation of guano in pet birds

Abstract

The present application relates to a complex probiotic preparation containing Lactobacillus acidophilus WP285 and its use in the preparation of a product for inhibiting the formation of bird stone in pets, the strain in the complex probiotic preparation is composed of Bacillus coagulans Bacillus coagulans BC66 strain and Lactobacillus acidophilus Lactobacillus acidophilus WP285 strain. The present application finds that both BC66 strain and WP285 strain have excellent effect in inhibiting the formation of bird stone in pets, and they can synergistically enhance each other, the complex probiotic preparation can effectively reduce the pH value of the simulated stone formation environment and inhibit the formation and deposition of bird stone crystals in vitro; through pet clinical test, it is confirmed that the complex probiotic preparation can effectively and continuously adjust the urine pH value of susceptible cats to the weak acidic range which is not conducive to the formation of bird stone, and significantly reduces the number of bird stone crystals in urine.

Description

Technical Field

[0001] This invention belongs to the field of probiotic preparation technology, and relates to a compound probiotic preparation containing Lactobacillus acidophilus WP285 and its use in the preparation of products that inhibit the formation of guano in pet birds. Background Technology

[0002] Struvite stones are a common urinary tract disease in pets. Their main component is magnesium ammonium phosphate hexahydrate, and their formation is closely related to alkaline urine, urease-producing bacteria infection, and excessively high mineral content in the diet. Urease-producing bacteria, such as Staphylococcus and Proteus, decompose urea to produce ammonia, increasing the urine pH and promoting the crystallization and aggregation of magnesium ammonium phosphate to form stones.

[0003] Traditional treatments primarily involve surgery, but the recurrence rate is high, and surgical trauma can lead to complications.

[0004] Probiotics are live microorganisms with multiple benefits, including regulating gut microbiota balance, inhibiting pathogenic bacteria growth, and enhancing immunity. Probiotics can also lower urine pH by producing organic acids, inhibiting urease-producing bacteria activity, and reducing ammonia production, thereby reducing the risk of struvite formation. Furthermore, their metabolite, bacteriocin, has a strong inhibitory effect on common pathogens such as Staphylococcus aureus, effectively preventing urinary tract infections and further reducing factors that contribute to stone formation.

[0005] Therefore, it is of great significance to develop a probiotic product that can effectively prevent and improve bird droppings in pets. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a compound probiotic preparation containing Lactobacillus acidophilus WP285 and its use in the preparation of products that inhibit the formation of guano in pet birds.

[0007] To achieve this objective, the present invention employs the following technical solution:

[0008] In a first aspect, the present invention provides a compound probiotic preparation containing Lactobacillus acidophilus WP285, wherein the strain in the compound probiotic preparation consists of Bacillus coagulans BC66 strain with accession number CGMCC 21879 and Lactobacillus acidophilus WP285 strain with accession number CGMCC No. 35763.

[0009] This invention develops a novel probiotic compounding method and a novel strategy for inhibiting the formation of struvite stones in pets. It involves combining *Bacillus coagulans* BC66 strain and *Lactobacillus acidophilus* WP285 strain. The results show that these two strains work synergistically to inhibit struvite formation in pets. Specifically, in vitro, they effectively lower the pH of a simulated stone-forming environment and inhibit the formation and deposition of struvite crystals. Clinical trials in pets have demonstrated that this compound probiotic preparation effectively and continuously adjusts the urine pH of susceptible cats to a slightly acidic range unfavorable for struvite formation and significantly reduces the number of struvite crystals in the urine. Furthermore, the effect of this compound preparation is superior to that of a single strain.

[0010] Preferably, the ratio of viable bacteria count of the BC66 strain to the WP285 strain is 1:10-10:1, such as 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0011] Preferably, the live bacteria content in the compound probiotic preparation is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0012] Preferably, the dosage form of the compound probiotic preparation includes solution, powder, capsule, tablet, or granule. The compound probiotic preparation of the present invention can be formulated into various dosage forms to meet the needs of different usage scenarios and pet owners. For example, solutions are easy to add directly to a pet's drinking water; powders are easy to store and transport, and can be reconstituted with an appropriate amount of water before use; capsules, tablets, and granules are convenient for quantitative feeding.

[0013] In a second aspect, the present invention provides the use of the compound probiotic preparation according to the first aspect in the preparation of pet products.

[0014] Preferably, the pets include cats, dogs, and foxes.

[0015] Preferably, the product further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0016] Thirdly, the present invention provides the use of the compound probiotic preparation according to the first aspect in the preparation of a product that inhibits the formation of pet bird droppings.

[0017] Preferably, the pets include cats, dogs, and foxes.

[0018] Preferably, the product further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention develops a novel probiotic compounding method and a novel strategy for inhibiting the formation of struvite stones in pets. It involves combining *Bacillus coagulans* BC66 strain and *Lactobacillus acidophilus* WP285 strain. The results show that these two strains work synergistically to inhibit struvite formation in pets. Specifically, in vitro, they effectively lower the pH of a simulated stone-forming environment and inhibit the formation and deposition of struvite crystals. Clinical trials in pets have demonstrated that this compound probiotic preparation effectively and continuously adjusts the urine pH of susceptible cats to a slightly acidic range unfavorable for struvite formation and significantly reduces the number of struvite crystals in the urine. Furthermore, the effect of this compound preparation is superior to that of a single strain.

[0021] The BC66 strain involved in this invention is classified as Bacillus coagulans, deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 8, 2021, with accession number CGMCC 21879, and located at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0022] The WP285 strain involved in this invention is classified and named Lactobacillus acidophilus. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 28, 2025, with accession number CGMCC No. 35763. The address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0023] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0024] The BC66 strain involved in the following examples is classified as Bacillus coagulans, deposited on March 8, 2021, with accession number CGMCC 21879.

[0025] The WP285 strain involved in the following examples is classified as Lactobacillus acidophilus, deposited on August 28, 2025, with accession number CGMCC No. 35763.

[0026] The preparation method of the probiotic bacterial culture involved in the following examples is as follows: Activated Bacillus coagulans BC66 seed culture was inoculated into TGB liquid medium and cultured anaerobically at 45°C for 24 h; activated Lactobacillus acidophilus WP285 seed culture was inoculated into MRS liquid medium and cultured anaerobically at 37°C for 18 h. After culture, the bacterial sludge was collected by centrifugation. The sludge was washed twice with sterile physiological saline and resuspended to obtain a bacterial concentration of 1×10⁻⁶ cells. 9 CFU / mL bacterial suspension.

[0027] The preparation method of the probiotic powder involved in the following examples is as follows: Activated Bacillus coagulans BC66 seed culture was inoculated into TGB liquid medium and cultured anaerobically at 45°C; activated Lactobacillus acidophilus WP285 seed culture was inoculated into MRS liquid medium and cultured anaerobically at 37°C. After culture, the bacterial sludge was collected by centrifugation. The bacterial sludge was resuspended in a lyophilization protectant (100 g / L trehalose aqueous solution) at a mass ratio of 2:1 to obtain a resuspension; the resuspension was freeze-dried using a vacuum freeze-drying method to obtain freeze-dried bacterial powders of the two bacteria (both with a bacterial concentration of 1×10⁻⁶). 10 CFU / g).

[0028] Example 1

[0029] In vitro inhibition experiment of struvite crystallization:

[0030] (1) Preparation of simulated petrification solution: In an Erlenmeyer flask, add 0.1 mol / L magnesium chloride (MgCl2), diammonium hydrogen phosphate ((NH4)2HPO4), and urea solution respectively. Adjust the initial pH to 7.0 using HCl or NaOH so that the final reaction system contains Mg 2+ PO4 3- The concentrations of urea were 20 mmol / L, 20 mmol / L, and 50 mmol / L, respectively.

[0031] (2) Experimental Groups:

[0032] (S1) The prepared BC66 bacterial suspension and WP285 bacterial suspension were mixed at a live bacteria ratio of 1:1 to obtain a composite bacterial solution (bacterial concentration of 1×10⁻⁶). 9 (CFU / mL)

[0033] (S2) The prepared BC66 bacterial suspension and WP285 bacterial suspension were mixed at a live bacteria ratio of 10:1 to obtain a composite bacterial solution (bacterial concentration of 1×10⁻⁶). 9 (CFU / mL)

[0034] (S3) The prepared BC66 bacterial suspension and WP285 bacterial suspension were mixed at a live bacteria ratio of 1:10 to obtain a composite bacterial solution (bacterial concentration of 1×10⁻⁶). 9 (CFU / mL)

[0035] (S4) Single BC66 bacterial suspension (bacterial concentration 1×10⁻⁶) 9 (CFU / mL)

[0036] (S5) Single WP285 bacterial suspension (bacterial concentration 1×10⁻⁶) 9 (CFU / mL)

[0037] (S6) Blank control group.

[0038] (3) Add 5% (v / v) of the three compound bacterial solutions, a single Bacillus coagulans BC66 suspension, a single Lactobacillus acidophilus WP285 suspension, or an equal volume of sterile physiological saline (blank control group) to each component solution. Each group has 3 replicates. Place the above Erlenmeyer flasks in a constant temperature shaker at 37°C and 150 rpm for 48 h to simulate the dynamic environment of the urinary system.

[0039] (4) pH value measurement:

[0040] Immediately after the incubation period, the final pH value of each reaction solution was measured using a precision pH meter. The results are shown in Table 1.

[0041] (5) Quantitative analysis of struvite crystals:

[0042] Take 10 mL of culture medium from each group and centrifuge at 4°C and 8000 r / min for 10 min, discarding the supernatant. Wash the precipitate three times with pre-cooled deionized water to remove soluble ions. Dry the precipitate in a 60°C oven to constant weight. Dissolve the dried precipitate completely in 0.1 mol / L hydrochloric acid, and determine the phosphate (PO4) content in the solution using the molybdenum blue colorimetric method. 3- The concentration of struvite was converted into the amount of struvite formed (calculated as MgNH4HPO4·6H2O) using a standard curve. The results are shown in Table 1.

[0043] Table 1

[0044]

[0045] As shown in Table 1, the compound probiotic preparation composed of Bacillus coagulans BC66 and Lactobacillus acidophilus WP285 can effectively reduce the pH value of the simulated stone-forming environment in vitro and significantly inhibit the formation and deposition of struvite crystals. It shows clear potential in preventing and assisting in the improvement of struvite stones. Moreover, the effect of the compound probiotic preparation is better than that of a single Bacillus coagulans BC66 preparation or a single Lactobacillus acidophilus WP285 preparation with the same bacterial concentration. That is, the BC66 strain and the WP285 strain have a synergistic effect on the above-mentioned efficacy.

[0046] Example 2

[0047] Clinical trials on pet cats:

[0048] (1) Experimental groups:

[0049] (1.1) BC66 group: single BC66 mycelium powder;

[0050] (1.2) WP285 group: single WP285 bacterial powder;

[0051] (1.3) BC66+WP285 group: Mix BC66 powder and WP285 powder in equal mass to obtain mixed powder;

[0052] (1.4) ATCC7050+ WP285 group: Mix commercially available Bacillus coagulans ATCC7050 bacterial powder and WP285 bacterial powder in equal mass to obtain mixed bacterial powder;

[0053] (1.5) BC66+ ATCC4356 group: BC66 bacterial powder and commercially available Lactobacillus acidophilus bacterial powder of equal mass were mixed to obtain mixed bacterial powder;

[0054] (1.6) Blank control group.

[0055] (2) Laboratory animals:

[0056] Adult Chinese domestic cats weighing 3-5 kg ​​with similar weight, age, and mental state, but exhibiting slightly alkaline urine pH, were selected. Antibiotics and other medications were contraindicated within one month prior to the experiment. Cats were randomly assigned to groups with five replicates per group. There were no statistically significant differences in sex, age, or weight among the groups.

[0057] (3) Experimental procedure:

[0058] Except for the blank control group, all other groups of cats were given 1g of bacterial powder orally per cat daily for 28 days. All experimental cats were kept alone in independent cages at an ambient temperature of 20-25℃ and an ambient humidity of 40-60%, and were allowed to roam freely for 1 hour daily. During the experiment, they were kept in accordance with routine feeding and management procedures as well as disinfection and disease prevention procedures, and had free access to food and water.

[0059] During the experiment, the experimental cats were fed food at 8:00 AM every day, and their daily food intake was recorded. 300 mL of clean drinking water was provided to the cats at a fixed time each day, and any remaining drinking water was collected and measured at the same time the following day. On day 28 of the experiment, urine was collected using Danish KRUUSE non-absorbent cat litter, with a sample volume of approximately 5 mL. The pH value of the urine was measured using a Mindray UA-600T semi-automatic urine analyzer. Additionally, 10 mL of urine was centrifuged at 3000 rpm for 10 minutes, and 0.75 mL of urine and sediment were collected. Urine crystals were observed and counted using a standard urine sediment counting chamber. The results are shown in Table 2.

[0060] Table 2

[0061]

[0062] The data in Table 2 show that a 28-day clinical trial in pets confirmed that the compound probiotic preparation composed of Bacillus coagulans BC66 and Lactobacillus acidophilus WP285 can effectively and continuously adjust the urine pH of susceptible cats to a slightly acidic range unfavorable for struvite formation, and significantly reduce the number of struvite crystals in the urine. Furthermore, the compound probiotic preparation is more effective than either a single Bacillus coagulans BC66 preparation or a single Lactobacillus acidophilus WP285 preparation with the same bacterial concentration, indicating that the BC66 and WP285 strains have a synergistic effect on the aforementioned efficacy.

[0063] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0064] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0065] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A compound probiotic preparation containing Lactobacillus acidophilus WP285, characterized in that, The strains in the compound probiotic preparation consist of Bacillus coagulans BC66 strain, deposited at the China General Microbiological Culture Collection Center (CGMCC No. 21879), and Lactobacillus acidophilus WP285 strain, deposited at the China General Microbiological Culture Collection Center (CGMCC No. 35763). The ratio of viable cells of the BC66 strain to the WP285 strain is 1:10-10:

1.

2. The compound probiotic preparation according to claim 1, characterized in that, The live bacteria content in the compound probiotic preparation is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

3. The compound probiotic preparation according to claim 1, characterized in that, The dosage forms of the compound probiotic preparation include solutions, powders, capsules, tablets, or granules.

4. Use of the compound probiotic preparation according to any one of claims 1-3 in the preparation of pet products.

5. The use according to claim 4, characterized in that, The pets include cats, dogs, and foxes.

6. The use according to claim 4, characterized in that, The product also contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

7. Use of the compound probiotic preparation according to any one of claims 1-3 in the preparation of a product that inhibits the formation of pet bird droppings.

8. The use according to claim 7, characterized in that, The pets include cats, dogs, and foxes.

9. The use according to claim 7, characterized in that, The product also contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

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