A composite probiotic composition with the function of biodegrading dander and its application

Abstract

The present application belongs to the field of pet food and biomedicine, and discloses a compound probiotic composition with the effect of biodegradation of hair balls and application thereof. The composition comprises Weizmannia coagulans, Bacillus subtilis and oil-tea camellia seed extract. The oil-tea camellia seed extract from a specific source is used as a biological surfactant to emulsify and break the "oil seal" sebum layer on the surface of pet hair, significantly reduce the surface tension of the hair, induce keratinase secreted by Bacillus subtilis to be efficiently wetted and adsorbed on the hair fiber, and realize deep biodegradation of keratin. At the same time, lactic acid produced by Weizmannia coagulans promotes intestinal peristalsis, and the decomposition and excretion of hair balls are realized in cooperation. The experimental results show that the cat hair dry weight loss rate of the composition is 65%-78%, the frequency of vomiting can be effectively reduced, and the composition has excellent acid stability, palatability and safety.

Description

Technical Field

[0001] This invention belongs to the field of pet food and biomedicine technology, specifically relating to a composite particle containing Weizmann's coagulans, Bacillus subtilis and camellia seed extract, and its application in promoting the degradation and excretion of pet hairballs. Background Technology

[0002] Pets such as cats and rabbits have a habit of licking their fur. After the fur is ingested, the keratin in it is extremely difficult for digestive enzymes to break it down, and it easily forms hairballs (trichobezoars) in the gastrointestinal tract. Hairballs can cause vomiting and loss of appetite, and in severe cases, can lead to intestinal obstruction (hairball syndrome) and even endanger life.

[0003] Currently, commercially available hairball removal products are mainly divided into two categories: one is physical hairball removal products, which mainly increase intestinal peristalsis by adding dietary fiber (such as psyllium husk and cat grass), but they lack the ability to break down already formed tight hairballs; the other is mineral oil / oil products, which lubricate the intestines with petroleum jelly or mineral oil, but long-term consumption will interfere with the pet's absorption of fat-soluble vitamins (such as VA, D, and E), and the non-natural ingredients do not meet the health needs of modern pets.

[0004] In attempts to address hairballs using biotechnology, the use of probiotics such as Bacillus subtilis to secrete keratinase to biodegrade hairballs has become a research hotspot. However, practical applications show that the degradation effect of Bacillus subtilis alone on pet hairballs is significantly limited. This is because the hydrophobic sebum layer on the surface of a pet's fur acts like an 'oil seal' (i.e., a highly hydrophobic sebum barrier on the fur surface), preventing water-soluble keratinase from contacting hair keratin. Within the extremely limited intestinal transit time of a pet (usually 12-24 hours), the enzyme solution produced by Bacillus subtilis often cannot break through the sebum barrier, resulting in a very low degradation rate for tightly packed hairballs. On the other hand, Weizmann's coagulans is often used to regulate intestinal motility in pets, and the lactic acid it produces can moderately stimulate intestinal smooth muscle peristalsis. However, when treating stubborn hairballs, the use of Weizmann's coagulans alone has obvious drawbacks: it can only physically move the hairball, but cannot break down the physical entanglement structure of the hairball. For large hairballs that have already formed, simply increasing intestinal motility often makes it difficult to expel them smoothly. In fact, the friction between the strong peristalsis and the hard hairball may cause damage to the intestinal mucosa or aggravate the risk of obstruction.

[0005] Furthermore, while a simple physical mixture of the two probiotics can achieve a balance between "degradation" and "removal" to some extent, the lack of biochemical means to break through the sebum barrier in the hair means that the enzyme production efficiency of Bacillus subtilis is still greatly inhibited, resulting in an overall hair removal effect that is less than the sum of its parts (1+1<2). Therefore, finding a comprehensive solution that can synergistically enhance the degradation efficiency of probiotics, overcome sebum barriers, and safely remove hair is a pressing technical challenge in the pet health field.

[0006] Further research revealed that the underlying reason hindering the efficiency of probiotic hair removal lies in the microscopic physicochemical properties of the hairball surface. The sebum secreted by the cat's sebaceous glands (such as cholesterol esters and free fatty acids) tightly wraps around the outer edge of the hair cuticle layer, forming a highly hydrophobic film. This 'oil film' not only prevents water-soluble keratinases from directly contacting the substrate, but also creates extremely high surface tension between the intestinal fluid and the hairball, causing the effective components produced by probiotic metabolism to be 'rejected' from the hairball structure.

[0007] Although chemically synthesized surfactants (such as sodium lauryl sulfate) are commonly used to remove grease in industrial cleaning or leather processing, these ingredients have strong cytotoxicity and mucosal irritation, and should never be ingested by pets. Furthermore, the natural emulsifiers commonly used in pet food (such as soy lecithin) do not have stable emulsifying abilities in the complex acid-base environment of the intestinal tract, and they are insufficient to generate enough wetting power to penetrate the tightly tangled structure of fur.

[0008] Therefore, finding a natural component that can effectively reduce surface tension, emulsify the sebum barrier, possess extremely high intestinal safety, and synergistically enhance the effects of *Weizmannii* and *Bacillus subtilis* has become the key to overcoming the bottleneck in probiotic hairball reduction efficiency. Inspired by this scientific principle, this invention introduces bioactive components from specific sources, aiming to fully unleash the degradation potential of probiotics by altering the wettability of the hairball surface. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a compound probiotic composition with biodegradable hairball effects, the composition comprising: *Weizmannii coagulans* (… Weizmannia coagulans Bacillus subtilis ( Bacillus subtilis The product contains camellia seed extract. Preferably, the *Weizmannii coagulans* has the accession number CGMCC No. 35843, and the *Bacillus subtilis* has the accession number CGMCC No. 35844.

[0010] Preferably, the ratio of viable counts of *Weizmannii coagulans* to *Bacillus subtilis* is 2:1 to 5:1; preferably, the ratio of viable counts is 3.6:1; preferably, the weight percentage of tea saponin in the camellia seed extract is 60% to 70%. Preferably, by weight, the composition comprises: 10-20 parts of *Weizmannii coagulans* powder, 3-10 parts of *Bacillus subtilis* powder, and 1-5 parts of camellia seed extract. Preferably, the composition further comprises 60-80 parts of a protective agent. Preferably, the protective agent is selected from one or more of goat milk powder, maltodextrin, trehalose, and skim milk powder. This invention constructs a highly efficient synergistic degradation system by precisely controlling the proportions of each component.

[0011] Another aspect of the present invention provides a composite particle comprising a core material and a wall material, wherein the core material comprises the composition and the wall material comprises sodium alginate and whey protein; preferably, the moisture content of the microcapsule-encapsulated particles is ≤4.5%; more preferably, the composite particle is a microcapsule-encapsulated particle.

[0012] Another aspect of the present invention provides a method for preparing the composition, comprising the following steps: (1) culturing *Weizmannii coagulans* and *Bacillus subtilis* respectively to obtain a number of viable bacteria of not less than 1 × 10⁻⁶. 11 (2) Prepare tea seed extract with tea saponin content ≥ 60% by using ethanol ultrasonic extraction process; (3) Mix the bacterial powder and the tea seed extract to obtain the composition.

[0013] Another aspect of the present invention provides a method for preparing composite particles, comprising the following steps: (1) culturing *Weizmannii coagulans* and *Bacillus subtilis* respectively, to obtain effective viable bacteria counts of not less than 1 × 10⁻⁶. 11 (2) Prepare tea seed extract with tea saponin content ≥60% by using ethanol ultrasonic extraction process; (3) Mix the bacterial powder and the tea seed extract as core material; (4) Coat the core material with wall material to obtain composite particles with moisture content ≤4.5%.

[0014] Another aspect of the present invention provides the application of the compound probiotic composition or the compound granules in the preparation of pet hair removal products; preferably, the product is pet food or pet health products; more preferably, the pet food includes dry pet food, baked pet food, wet food canned food, hair removal paste or hair removal probiotic powder.

[0015] Another aspect of the invention provides the use of the compound probiotic composition or the compound granules in the preparation of a medicine or nutritional product for pet hairballs, constipation, difficulty in defecation or intestinal obstruction; preferably, the pet includes cats, rabbits, dogs, chinchillas or guinea pigs.

[0016] Biological Preservation Information:

[0017] Bacterial species name: Weizmann's coagulation bacteria ( Weizmannia coagulans )

[0018] Latin name: Weizmannia coagulans

[0019] Classification and nomenclature: Weizmann's coagulation bacteria ( Weizmannia coagulans )

[0020] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0021] Collection institution abbreviation: CGMCC

[0022] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0023] Deposit date: September 5, 2025

[0024] CGMCC Registration Number: CGMCC No. 35843

[0025] Bacterial strain name: Bacillus subtilis ( Bacillus subtilis )

[0026] Latin name: Bacillus subtilis

[0027] Classification and nomenclature: Bacillus subtilis ( Bacillus subtilis )

[0028] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0029] Collection institution abbreviation: CGMCC

[0030] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0031] Deposit date: September 5, 2025

[0032] CGMCC Registration Number: CGMCC No. 35844

[0033] The technical principle of this invention is as follows: First, by utilizing the bio-emulsifying effect of tea saponins in camellia seed extract, the hydrophobic sebum layer on the hair surface is rapidly emulsified and peeled off, significantly reducing the surface tension of the substrate and providing a wetting path for the water-soluble enzyme solution. Second, Bacillus subtilis generates highly efficient adsorption on the hair surface after breaking the "oil seal," and its secreted specific keratinase precisely acts on the disulfide bonds of keratin, inducing the disintegration and degradation of the hair's physical structure. During this process, Weizmann's coagulates produce lactic acid through metabolism to regulate the intestinal environment to a slightly acidic state, which not only optimizes the catalytic activity range of keratinase but also synergistically stimulates intestinal smooth muscle peristalsis, accelerating the excretion of degraded hair residue with feces. To ensure the stability of the active components, this composition is microencapsulated using sodium alginate and whey protein as wall materials to prepare composite particles with a core-shell structure.

[0034] Compared with existing technologies, this invention has significant technical advantages and clinical practical value.

[0035] First, in terms of synergistic effect, the present invention adopts a whole-chain layout of "barrier breaking-hair dissolving-hair removal", which has a degradation efficiency of 65%~78% for hair keratin, which is significantly better than the effect of simply adding single components or strains, and achieves a qualitative leap.

[0036] Secondly, it has significant clinical efficacy. Experiments have shown that this composition can increase the amount of hair excreted in pet feces by more than 100% and reduce the frequency of vomiting hairballs by more than 80%.

[0037] Furthermore, this invention exhibits excellent safety; long-term use does not interfere with the normal absorption of fat-soluble vitamins, and it is non-irritating to the intestinal mucosa, with excellent palatability. Finally, thanks to the specific microencapsulation process, the product possesses extremely strong processing adaptability, maintaining over 85% of its activity even at 90°C, making it suitable for large-scale application as a pet food, treat, or health supplement additive. Attached Figure Description

[0038] Figure 1 The in vitro degradation ability of the composite particles in this invention on hair is shown. (A) shows the hair dry weight loss rate of each experimental group; (B) shows the amino acid content in the degradation solution of each experimental group. P < 0.001, ***; P < 0.01: **; P < 0.05: *. Detailed Implementation

[0039] Example 1: Preparation, extraction and processing technology of each component of the composite particles

[0040] This embodiment illustrates the preparation process of each active component of the composition of the present invention and the molding process of the final product.

[0041] (1) Preparation of freeze-dried strain powder:

[0042] *Weizmannii coagulans* and *Bacillus subtilis* were inoculated into MRS liquid medium for fed-batch fermentation, respectively. After 24-36 hours of fermentation, samples were taken for analysis: the viable count of the *Bacillus subtilis* B4D55 fermentation broth reached 3.4 × 10⁻⁶. 10 CFU / mL; the viable count of *Weizmannii* B2N21 fermentation broth reached 1.8 × 10⁻⁶. 10 CFU / mL. The bacterial sludge was then collected by centrifugation, and after adding trehalose as a preservative, it was freeze-dried under vacuum to obtain a highly active bacterial powder. The effective viable count of the prepared *Weizmannia coagulans* B2N21 bacterial powder was determined to be 1.5 × 10⁻⁶ CFU / mL. 11 The effective viable count of Bacillus subtilis B4D55 bacterial powder is 1.0 × 10⁻⁶ CFU / g. 11 CFU / g.

[0043] (2) Refining of Camellia oleifera seed extract (tea saponin):

[0044] Dried camellia seed meal was pulverized and extracted three times with 75% ethanol solution at 55℃ using ultrasonic assistance. The extracts were combined. After recovering the ethanol by vacuum distillation, the extract was subjected to chromatography using an overloaded D101 macroporous adsorption resin column. Impurities were first eluted with water, followed by elution with 70% ethanol solution to remove the target component. The eluent was collected, dried, and pulverized to obtain a purified camellia seed extract powder. High-performance liquid chromatography (HPLC) analysis showed that the tea saponin content in the extract was 65.5% (weight percentage).

[0045] (3) Proportioning and encapsulation of composite particles:

[0046] This process achieves a synergistic wool-reducing effect by precisely controlling the proportions of each component. By weight, 15 parts of Weizmannii coagulans B2N21 bacterial powder, 5 parts of Bacillus subtilis B4D55 bacterial powder (at this point, the ratio of viable bacteria of B2N21 to B4D55 is approximately 3.6:1), 2 parts of camellia seed extract powder, 20 parts of goat milk powder as a carrier and protectant, and 58 parts of maltodextrin are weighed.

[0047] The above components were thoroughly mixed to obtain the composition. This composition was then used as the core material and microencapsulated using fluidized bed coating technology. A 1.5% sodium alginate and 2.0% whey protein solution was used as the wall material. The wall material solution was uniformly coated onto the surface of the core material particles using a bottom spray device within a fluidized bed. The inlet air temperature was controlled at 45°C and the outlet air temperature at 32°C. After coating, the particles were dried until the moisture content was below 4.5%, resulting in composite particles with a core-shell structure.

[0048] To comprehensively verify the basic properties and high-temperature environmental adaptability of the prepared composite particles, systematic tests were conducted, including moisture content, particle angle of repose, high-temperature stability, viable bacteria count, and retention rate of active components. The high-temperature stability and active component retention effect of the composite particles were comprehensively evaluated.

[0049] Table 1. Process parameters of composite particles

[0050]

[0051] The experimental results are shown in Table 1. The prepared composite particles had a moisture content of 3.9% and an angle of repose of less than 35°, exhibiting good flowability. The high-temperature extrusion process (90℃, 1 min) in pet food production was simulated. The composite particles were plate-counted using bromocresol purple glucose agar medium. Based on colony color and morphology, colonies with a yellow discoloration zone and a smooth surface were identified as *Weizmannii coagulans* B2N21; colonies without a discoloration zone and with a rough, wrinkled surface were identified as *Bacillus subtilis* B4D55. The viable cell survival rates of B2N21 and B4D55 in the particles were 88% and 86%, respectively. The retention rate of the active component in the composite particles was 87%, providing a stable test material for subsequent experiments.

[0052] Example 2: In vitro degradation kinetics of cat hair keratin by composite particles

[0053] This embodiment utilizes the composite particles prepared in Example 1 to verify the synergistic effect of the combined components. Four groups were set up: a blank group (PBS buffer at pH 7.5), a single extract group (containing the same amount of Camellia oleifera seed extract as in Example 1), a two-strain control group (containing the same doses of *Weizmannii coagulans* B2N21 and *Bacillus subtilis* B4D55 as in Example 1), and the experimental group of this invention (the composite particles prepared in Example 1). Each group was incubated with 1.0 g of natural cat hair at a constant temperature of 38.5°C with shaking for 24 hours. After cultivation, residual hair was collected, washed, dried, and weighed to calculate the hair dry weight loss rate. Degradation solutions from each group were collected, and the amino acid content in the degradation solution was determined using the ninhydrin colorimetric method to quantify the hair degradation rate and demonstrate that the hair was converted into soluble nutrients. A portion of residual hair was taken, and the disulfide bond (SS) breakage rate in the hair was detected using a thiol-based quantitative analysis method, demonstrating from a biochemical perspective that the keratin structure was destroyed. Another sample of residual hair was taken, and the microscopic morphology of the hair surface was observed using scanning electron microscopy. The degree of hair damage was scored according to a pre-defined scoring standard, providing a direct reflection of the hair degradation status.

[0054] Table 2. In vitro degradation capacity of composite particles

[0055]

[0056] Note: Hair damage rating: 1: Hair surface is intact and smooth, without damage; 2: Surface is slightly rough, without obvious cracks; 3: Surface is rough, with a few small cracks; 4: Complete disintegration and breakage. P value: vs. control group, P < 0.001, ***; P < 0.01: **; P < 0.05: *.

[0057] The results are as follows Figure 1 As shown in Table 2, the hair dry weight loss rate in the blank group and the single extract group was less than 10%. Figure 1 A), the content of degraded amino acids is less than 0.5 mg / mL ( Figure 1 B), the disulfide bond breakage rate was less than 5.0%, the hair damage score was 1-2 points, and the hair surface was basically intact under scanning electron microscopy; compared with the blank group, the in vitro degradation ability of the double-bacterial control group was improved; due to the barrier of the hair sebum layer, the hair dry weight loss rate of the double-bacterial control group was 40% ( Figure 1 A), the level of degraded amino acids increased slightly to 0.6 mg / mL ( Figure 1 B), with a disulfide bond breakage rate of 15.8%, a hair damage score of 3.4, and slightly rough hair surface under scanning electron microscopy; while the composite particle group showed a hair dry weight loss rate of 78.0% ( Figure 1 A), the content of hair degradation amino acids was significantly increased to 1.5 mg / mL ( Figure 1 Group B showed a disulfide bond breakage rate as high as 65.0%, significantly better than other groups. The hair damage score was 4.6, and scanning electron microscopy revealed severely rough hair surfaces, dense cracks, and even fiber breakage. Experimental data demonstrate that the combined use of camellia seed extract and specific bacterial strains exhibited a significant synergistic multiplier effect on degradation efficiency, effectively disrupting keratin disulfide bond structures, accelerating hair degradation, and converting hair into soluble nutrients.

[0058] Example 3: Verification of the contact angle of camellia seed extract in breaking through the hydrophobic barrier of hair.

[0059] This embodiment aims to reveal the biochemical mechanism by which the product of Example 1 efficiently degrades hair. Using a video contact angle meter, the wetting properties of deionized water (blank group), the degradation solution from the dual-bacterial group in Example 2 (dual-bacterial group), and the degradation solution from the experimental group of this invention (composite particle group) were tested on the surface of a dry single cat hair.

[0060] Dry, single cat hairs of uniform size were selected and fixed onto a glass slide, ensuring the hair surface was flat and undamaged. The video contact angle measuring instrument was adjusted to room temperature and pressure. Equal amounts of samples from the blank group, the two-bacterial group, and the composite particle group were dropped onto the cat hair surface at the same rate. The contact angle change of the droplets after 30 seconds of contact with the hair was precisely recorded using a high-speed camera. Each group was tested in triplicate, and the average contact angle was calculated. Wetting performance was evaluated based on the contact angle and the time to complete immersion (a smaller contact angle and faster spreading indicate a better wetting effect and a better breaking effect on the hydrophobic barrier of the hair). Additionally, the keratinase in the degradation solutions of the two-bacterial group and the composite particle group were fluorescently labeled, and then immersed in the cat hair for the same time. The fluorescence intensity on the hair surface was observed using a fluorescence microscope to analyze the relative keratinase adsorption density of each group; higher fluorescence intensity indicates a greater adsorption density.

[0061] Table 3. Verification results of hair hydrophobic barrier contact angle

[0062]

[0063] Note: P-value: vs. blank group, P<0.001, ***; P<0.01: **; P<0.05: *.

[0064] Table 3 shows that the initial contact angles of both the blank group and the double-bacterial group were greater than 100°, and the droplets showed no spreading trend within 30 seconds, with no significant change in contact angle. Fluorescence detection showed that the fluorescence intensity on the hair surface of the double-bacterial group was weak, and the keratinase adsorption density was low. In contrast, the contact angle of the composite particle group containing camellia seed extract rapidly dropped to below 10° at the moment of contact with the hair (0 seconds), and it completely spread and infiltrated within 3 seconds. Fluorescence detection showed that the fluorescence intensity of keratinase produced by Bacillus subtilis B4D55 in the composite particle group on the hair fiber was significantly higher than that in the double-bacterial group, and the adsorption amount was twice that of the double-bacterial group. This experiment demonstrates that camellia seed extract provides a highly efficient infiltration and enzyme production site for Bacillus subtilis B4D55, thus laying the foundation for subsequent efficient hair degradation.

[0065] Example 4: Component stability experiment under simulated gastrointestinal sequential treatment

[0066] This embodiment simulates the activity retention of the composite particles prepared in Example 1 in the pet's digestive tract. First, the particles were placed in simulated gastric fluid at pH 2.0 for 2 hours; then, the pH of the solution was adjusted to 7.5, and bile salts and trypsin were added, followed by further treatment for 4 hours to simulate the intestinal environment. The detection indicators included: the viable cell survival rate (%) of *Weizmannii coagulans* B2N21 and *Bacillus subtilis* B4D55 at each stage, and the secretion level of keratinase after entering the intestinal environment; simultaneously, the keratinase activity level at 1h, 2h, 4h, and 6h of intestinal environment treatment was detected using the casein / keratin substrate method, and the peak enzyme activity during the reaction was recorded; in addition, the emulsification activity retention rate at the reaction endpoint was detected, and the emulsification activity was determined using a turbidimetric method.

[0067] Table 4. Tolerance to artificial gastrointestinal fluids of B2N21 and B4D55

[0068]

[0069] Table 5 Results of keratinase activity and emulsifying activity in simulated intestinal environment

[0070]

[0071] After strong acid treatment, the survival rates of *Weizmannii coagulans* B2N21 and *Bacillus subtilis* B4D55 in the particles of Example 1 remained above 83.0% after treatment in a simulated gastrointestinal environment (Table 4). Within one hour of entering the simulated intestinal environment, *Bacillus subtilis* B4D55 secreted keratinase, reaching a peak enzyme activity of 550 U / mL (Table 5), and maintained a high enzyme activity level thereafter. Simultaneously, the emulsification activity retention rate at the experimental endpoint was excellent, remaining >90% after treatment with simulated gastrointestinal fluid. This demonstrates that the combined strains possess excellent acid resistance and intestinal restart ability, and the camellia seed extract can stably retain viability, jointly ensuring the product's effectiveness in the pet's digestive tract.

[0072] Example 5: Clinical feeding and fecal / hair dry weight statistical experiment of domestic cats

[0073] This embodiment verifies the hair removal effect of the composite granules prepared in Example 1 in real organisms. Twenty adult cats were randomly divided into a control group and an experimental group, with 10 cats in each group, ensuring that there were no significant differences in age, weight, and grooming frequency between the two groups. The control group was fed only ordinary cat food, while the experimental group was fed ordinary cat food with the composite granules prepared in Example 1 added at an appropriate dose. The feeding amount was the same for both groups, and they had free access to water. They were fed continuously for 14 days. All feces of the cats in both groups were collected daily at regular intervals. Hair residue in the feces was separated by washing and filtering. The feces were repeatedly rinsed with deionized water until no feces residue remained. The feces were dried in a 60°C constant temperature oven until constant weight. The dry weight of the hair was accurately weighed and recorded, and the hair length was measured. The vomiting of the cats in both groups was observed and recorded daily, and the frequency of vomiting hairballs was counted weekly.

[0074] Table 6 Clinical hair removal effectiveness indicators

[0075]

[0076] Note: P-value: vs. control group, P<0.001, ***; P<0.01: **; P<0.05: *.

[0077] The results showed that the average dry weight of hair excreted in the feces of cats in the experimental group was 35-45 mg / g, significantly higher than that in the control group (15-25 mg / g, as shown in Table 6), indicating a significant increase in hair excretion in the experimental group. Microscopic observation revealed that the hair excreted in the experimental group was severely damaged due to degradation by Bacillus subtilis B4D55, with a rough surface, obvious cracks, and some broken hair fibers, while the hair excreted in the control group had an intact structure and a smooth surface. In addition, the frequency of hairball vomiting in the experimental group decreased from an average of 1.0 times / week at the beginning of the experiment to less than 0.2 times / week, which was significantly different from the vomiting frequency in the control group. This demonstrates the synergistic effect of B2N21 promoting intestinal peristalsis and B4D55 biodegrading hair, which can effectively reduce hairball vomiting in cats and promote the excretion of hair from the body.

[0078] Example 6: Verification of product palatability and long-term safety

[0079] This embodiment evaluated the safety and palatability of products containing *Weizmannii coagulans* B2N21 and *Bacillus subtilis* B4D55 in practical applications. The palatability test employed a "double-basin method," selecting 10 healthy adult cats. Two equal-volume food bowls were placed in each test group, one containing regular cat food (control group) and the other containing regular cat food plus the compound pellets from Example 1 (experimental group). The cats were placed in the same environment and allowed free access to feed. The amount of food consumed by both groups was recorded over 2 hours, and the feed selection rate of the experimental group was calculated. Simultaneously, fecal samples were collected daily from both groups, and the fecal characteristics were scored using the Waltham 5-point scale. The daily scores were recorded, and the average was taken to assess the product's impact on the cats' intestinal condition. The palatability and fecal score tests were repeated three times, and the average value was used. For the safety test, 15 healthy adult cats were fed an overdose of feed containing the compound pellets from Example 1 for 28 consecutive days (the dose was twice the normal dose). After 28 days of feeding, blood samples were collected from the cats, and the serum levels of fat-soluble vitamins A and E and inflammatory factors (IL-6, TNF-α) were detected by ELISA. The changes in each indicator were compared and analyzed to assess the long-term effects of intake on the intestinal mucosa and the safety of the product.

[0080] In the palatability test (Table 7), the palatability indices of the two groups were similar, proving that the encapsulation process of Example 1 successfully masked the odor of the components, the product had good palatability, and did not affect the cats' willingness to eat; the fecal characteristics score results showed that, compared with the control group, the fecal characteristics of the experimental group with added compound particles were better, and the average fecal score of the experimental group was maintained between 2.3 and 3.3 points, which was significantly better than that of the control group.

[0081] Table 7 Palatability Indicators

[0082]

[0083] Note: A palatability selection index close to 0.5 indicates no odor; fecal characteristics score: 2.5 ± 0.5 points for ideal formed stool.

[0084] P-value: vs. control group, P < 0.001, ***; P < 0.01, **; P < 0.05, *.

[0085] Table 8 Safety indicators after 28 days of feeding

[0086]

[0087] Safety data (Table 8) showed that, for 28 days, both cats that ingested products containing strains B2N21 and B4D55 and control cats maintained serum fat-soluble vitamin levels and intestinal mucosal inflammatory factors (IL-6, TNF-α) levels within the ideal physiological range, with no significant differences between the two groups. This demonstrates that the product containing this strain combination achieves highly effective hair removal while exhibiting extremely high clinical safety, making it safe for long-term use without inducing intestinal mucosal inflammation.

Claims

1. A compound probiotic composition with biodegradable hairball effects, said composition comprising: Weizmann's coagulation bacteria ( Weizmannia coagulans Bacillus subtilis ( Bacillus subtilis The composition comprises: 10-20 parts of *Weizmannii coagulans* powder, 3-10 parts of *Bacillus subtilis* powder, and 1-5 parts of *Camellia oleifera* seed extract; wherein the preservation number of *Weizmannii coagulans* is CGMCC No. 35843, and the preservation number of *Bacillus subtilis* is CGMCC No. 35844; wherein the ratio of the number of viable bacteria of *Weizmannii coagulans* to *Bacillus subtilis* is 2:1 to 5:1; the weight percentage of tea saponin in the *Camellia oleifera* seed extract is 60% to 70%; and, by weight, the composition comprises: 10-20 parts of *Weizmannii coagulans* powder, 3-10 parts of *Bacillus subtilis* powder, and 1-5 parts of *Camellia oleifera* seed extract.

2. The compound probiotic composition according to claim 1, wherein the composition further comprises a protectant selected from one, two or more of goat milk powder, maltodextrin, trehalose, and skim milk powder.

3. A composite particle, said composite particle comprising a core material and a wall material, wherein, The core material comprises the composition of claim 1 or 2, the wall material comprises sodium alginate and whey protein; the composite particles are microencapsulated particles; the moisture content of the microencapsulated particles is ≤4.5%.

4. A method for preparing the composition according to claim 1 or 2, characterized in that, Includes the following steps: (1) Culture *Weizmannii coagulans* and *Bacillus subtilis* separately to obtain effective viable bacteria counts of not less than 1 × 10⁻⁶. 11 CFU / g bacterial powder; (2) Camellia seed extract with tea saponin content ≥60% was prepared by ethanol ultrasonic extraction process; (3) The bacterial powder is mixed with the camellia seed extract to obtain the composition.

5. A method for preparing the composite particles of claim 3, characterized in that, Includes the following steps: (1) Culture *Weizmannii coagulans* and *Bacillus subtilis* separately to obtain effective viable bacteria counts of not less than 1 × 10⁻⁶. 11 CFU / g bacterial powder; (2) Camellia seed extract with tea saponin content ≥60% was prepared by ethanol ultrasonic extraction process; (3) The bacterial powder and the camellia seed extract are mixed as core material; (4) The core material is coated with the wall material to obtain composite particles with a moisture content of ≤4.5%; the wall material includes sodium alginate and whey protein.

6. The application of the compound probiotic composition of claim 1 or 2, or the compound particles of claim 3, in the preparation of pet hair removal products; the product is pet food or pet health products.

7. The application according to claim 6, wherein, The pet food includes dry pet food, baked pet food, wet food canned food, hairball remedy paste, or hairball remedy probiotic powder.

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