Lactobacillus delbrueckii subsp. Ld-sj 001 and its applications

Abstract

This invention belongs to the field of pet oral care technology, and particularly relates to *Lactobacillus delbrueckii* subsp. *lactotrichum* Ld-sj 001 and its applications. Specifically, *Lactobacillus delbrueckii* subsp. *lactotrichum* (…) Lactobacillus delbrueckii subsp. lactis Ld-sj 001, with accession number CGMCC No. 36909. This invention relates to a pet probiotic complex used in the preparation of products for preventing and / or improving oral health problems in pets. The fermentation broth compound proposed in this invention, comprising a mixture of fermentation broths of *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactam* Ld-sj 001, exhibits a unique antibacterial effect, particularly against stubborn anaerobic bacteria specific to pet oral tracts, such as *Porphyromonas canis gingivalis*, *C. feline*, and *C. canis*.

Description

Technical Field

[0001] This invention belongs to the field of pet oral care technology, and in particular relates to a subspecies of Lactobacillus delbrueckii Ld-sj 001 and its application. Background Technology

[0002] With the increasing status of pets in households, pet oral health is receiving more and more attention. Bad breath, plaque, gingivitis, and other oral problems are common ailments in pets, not only affecting their quality of life but also potentially leading to systemic infections. Currently, commercially available pet oral care products mainly consist of mouthwash, dental chews, and sprays. However, products containing chemical antibacterial agents such as chlorhexidine are limited in use due to potential side effects and the risk of disrupting the oral microecological balance.

[0003] Probiotic therapy, as a microecological regulation strategy, inhibits pathogenic bacteria through mechanisms such as competitive rejection, immune regulation, and the production of antimicrobial substances, providing a new direction for pet oral care.

[0004] Existing technologies have disclosed the application of strains such as *Lactobacillus plantarum* and *Lactobacillus salivarius* in oral care, but these technologies still have many limitations: First, the strains are not specific enough, mostly targeting common pathogenic bacteria in the human oral cavity, lacking inhibitory effects on canine and feline specific core periodontal pathogens (such as *Porphyromonas gingivalis* and *Fibrophyton floccosum*) and feline oral inflammation-related bacteria (such as *Fibrophyton floccosum*). Second, the protection system is imperfect, with insufficient attention paid to the survival rate and shelf-life stability of probiotics in liquid products. Third, the efficacy needs to be verified; compared with traditional chemical mouthwashes and single-strain products, the advantages in rapid onset and long-term maintenance of oral microecological balance have not been fully proven. Summary of the Invention

[0005] This invention proposes a subspecies of Lactobacillus delbrueckii, whose fermentation broth has special oral cleaning effects and exhibits special antibacterial activity against stubborn bacteria unique to pet oral cavity, such as Porphyromonas canis gingivalis, Fibrophytophaga felis, and Fibrophytophaga canis.

[0006] This invention proposes a strain of *Lactobacillus delbrueckii* subsp. *lactamella*, specifically *Lactobacillus delbrueckii* subsp. *lactamella* (… Lactobacillus delbrueckii subsp. lactis Ld-sj 001 ， The accession number is CGMCC No. 36909.

[0007] The present invention also proposes a bacterial agent comprising any of the above-mentioned Lactobacillus delbrueckii subsp. Ld-sj 001.

[0008] This invention also proposes the application of the above-mentioned Lactobacillus delbrueckii subsp. Ld-sj 001 in the preparation of pet probiotic complexes.

[0009] The present invention also proposes a pet probiotic complex, comprising a fermentation broth compound obtained by mixing fermentation broth of Lactobacillus plantarum LP-sj and fermentation broth of any of the above-mentioned Lactobacillus delbrueckii subsp. Ld-sj 001.

[0010] Furthermore, at least one of the following conditions must be met:

[0011] (1) The total viable bacteria concentration of the fermentation broth compound is 1×10⁻⁶. 8 ~1×10 9 CFU / mL; (2) In the fermentation broth compound, the ratio of viable bacteria of Lactobacillus plantarum LP-sj to Lactobacillus delbrueckii subsp. Ld-sj 001 is (1-2):1.

[0012] Further, it is prepared from raw materials comprising the following parts by weight: The fermentation broth mixture of *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactospirae* Ld-sj 001 is prepared in quantities of 5-20 parts. Plant extracts: 1-5 parts; prebiotics: 0.5-2 parts; moisturizers: 3-10 parts; hyaluronic acid: 0.01-0.05 parts; poloxamer 188: 0.1-0.5 parts; flavoring: 0.05-0.2 parts; deionized water; the total weight of all ingredients is 100 parts.

[0013] Furthermore, at least one of the following conditions must be met: (1) The plant extract includes at least one of licorice extract, tea extract or honeysuckle extract; (2) The prebiotic is at least one of xylooligosaccharide, fructooligosaccharide or galactooligosaccharide; (3) The moisturizer is at least one of glycerin, propylene glycol or polyethylene glycol 400.

[0014] This invention also proposes a method for preparing any of the above-described pet probiotic complexes, comprising the following steps: After heating the deionized water, add the moisturizer, prebiotic, hyaluronic acid, and poloxamer 188 in sequence, mix and cool to obtain the aqueous phase; After adding the fermentation broth compound, plant extracts, and flavoring agents to the above aqueous phase, the mixture is obtained as a pet probiotic complex.

[0015] The present invention also proposes the use of any of the above-described pet probiotic complexes in the preparation of products for the prevention and / or improvement of oral problems in pets; and the use of any of the above-described pet probiotic complexes in the preparation of products for regulating intestinal function in pets at specific physiological stages, wherein the specific physiological stages include the weaning period for puppies and the period of intestinal function decline in senior pets.

[0016] Furthermore, at least one of the following conditions must be met: (1) The oral problems mentioned include at least one of halitosis, dental plaque, gingivitis, dental caries, or oral microecological imbalance; (2) The oral problems are caused by pet-specific pathogens; the pet-specific pathogens include at least one of Porphyromonas gingivalis, Porphyromonas canis, or Porphyromonas felis.

[0017] This invention has the following advantages: (1) The Lactobacillus delbrueckii subsp. Ld-sj 001 provided by this invention was obtained through extensive screening and strain domestication. Through fermentation, it was found that the fermentation broth has a special antibacterial effect, especially against stubborn bacteria in the pet's oral cavity, such as Porphyromonas canis gingivalis, Fibrophytophaga felis, and Fibrophytophaga canis. These anaerobic bacteria are highly resistant to oxygen and chemical antibacterial agents, and existing oral care products have a very weak sterilization effect and are difficult to kill effectively.

[0018] (2) The pet probiotic complex provided by this invention enhances the survival and colonization ability of the strains in the pet's oral environment by fermenting and combining *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactotrichum* Ld-sj 001 separately. The two promote each other's growth and produce richer metabolites with synergistic antibacterial effects. Furthermore, the resulting complex has broad-spectrum antibacterial activity, effectively inhibiting not only a variety of common oral pathogens, including *Porphyromonas gingivalis* (such as *Fusobacterium nucleatum*, *Streptococcus mutans*, etc.), but also specifically and effectively inhibiting key oral pathogens in pets, including but not limited to: *Porphyromonas canis*, *Cyclophagocytosus felis*, *Cyclophagocytosus canis*, *Actinomyces filamentosa*, *Prevotella intermedia*, and oral ciliates. In particular, few existing pet probiotic complexes have a cleaning effect on these key pathogens in pets, such as *Porphyromonas canis*, *Cyclophagocytosus felis*, *Cyclophagocytosus canis*, and *Actinomyces filamentosa*.

[0019] (3) The pet probiotic complex provided by this invention uses a fermentation broth compound of Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. lactis Ld-sj 001 as its main components. It can colonize the pet's oral cavity and intestines, inhibit the proliferation of pathogenic bacteria, and regulate the balance of the flora. Plant extracts assist in antibacterial and anti-inflammatory effects, prebiotics provide nutrition for probiotics and enhance the regulatory effect, moisturizers maintain the moisture of the system, protect the activity of probiotics, and moisturize the oral mucosa, hyaluronic acid prolongs the residence time of the components and repairs the mucosal barrier, poloxamer 188 optimizes the solubility and stability of the formula, and flavoring agents improve the palatability for pets. The components work synergistically to target and solve pet oral problems, regulate the intestinal microecology, and enhance immunity.

[0020] (4) The pet probiotic complex provided by the present invention can quickly, effectively, and safely regulate the oral microecology of pets.

[0021] Rapidly improves bad breath: By significantly reducing the concentration of hydrogen sulfide (H2S), the main odor-causing gas in the oral cavity, it achieves a rapid improvement in bad breath. It can significantly improve bad breath problems in the initial stage of use and effectively inhibit the formation of oral pathogenic bacteria biofilm in a short period of time. Long-lasting and long-lasting: Overcomes the shortcomings of chemical mouthwashes, such as the reliance on frequent use and the rebound effect after discontinuation. The core efficacy is maintained by more than half even 48 hours after discontinuation, achieving a radical and long-lasting effect of microecological regulation. Furthermore, because the probiotics in this product can be swallowed, they can also provide systemic health benefits to pets, such as improving the intestinal barrier, enhancing immunity, and optimizing coat condition, which are not available in chemical mouthwashes.

[0022] (5) The pet probiotic complex provided by the present invention contains fermentation liquid compound, which can be used to prepare pet bite repair products. It can be directly applied to the wound. Since the wound is bitten by a pet, there are pet-specific pathogens. It can effectively inhibit bacteria.

[0023] (6) The pet probiotic complex provided by the present invention can also improve immunity and adaptability of the body by targeting and regulating the balance of oral and intestinal microecology according to the immune characteristics of pets at different stages (young pets have immature immunity and older pets have declining immunity), reducing the risk of infection and stress-related diseases, and helping pets to smoothly pass through special physiological stages. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. In the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0025] In a first aspect, one embodiment of the present invention provides a strain of *Lactobacillus delbrueckii* subsp. *lactobacter*, specifically *Lactobacillus delbrueckii* subsp. *lactobacter* (… Lactobacillus delbrueckii subsp. of milk Ld-sj 001 ， The accession number is CGMCCNo.36909.

[0026] In this embodiment of the invention, the *Lactobacillus delbrueckii* subsp. *lactotrichum* ( Lactobacillus delbrueckii subsp. milk Ld-sj 001, classified and named as: Lactobacillus delbrueckii subsp. lactis Lactobacillus delbrueckii subsp. MilkThe specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The accession number is CGMCCNo. 36909, and the deposit date is December 5, 2025.

[0027] In this embodiment of the invention, *Lactobacillus plantarum* ( Lactiplantibacillus plantarum LP-sj has been disclosed in patent CN104357348A. This strain is classified and named *Lactobacillus plantarum*. Lactobacillus plantarum The strain's accession number is CGMCC No. 9513; the depository is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is August 15, 2014.

[0028] Specifically, the 16S rRNA sequence of Lactobacillus delbrueckii subsp. Ld-sj 001 is shown in the sequence listing SEQ ID No. 1.

[0029] Secondly, an embodiment of the present invention also proposes a bacterial agent comprising Lactobacillus delbrueckii subsp. Ld-sj 001.

[0030] Thirdly, an embodiment of the present invention also proposes the application of Lactobacillus delbrueckii subsp. Ld-sj 001 in the preparation of pet probiotic complexes.

[0031] Fourthly, an embodiment of the present invention also proposes a pet probiotic complex, comprising a fermentation broth compound obtained by mixing fermentation broth of Lactobacillus plantarum LP-sj and fermentation broth of Lactobacillus delbrueckii subsp. lactis Ld-sj 001.

[0032] In one embodiment of the present invention, the total viable bacteria concentration of the fermentation broth compound is 1×10⁻⁶. 8 ~1×10 9 CFU / mL.

[0033] In one embodiment of the present invention, the ratio of viable bacteria of Lactobacillus plantarum LP-sj to Lactobacillus delbrueckii subsp. lactis Ld-sj 001 in the fermentation broth compound is (1-2):1.

[0034] In one embodiment of the present invention, the pet probiotic complex is prepared from raw materials comprising the following parts by weight: The fermentation broth mixture of *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactospirae* Ld-sj 001 is prepared in quantities of 5-20 parts. Plant extracts: 1-5 parts; prebiotics: 0.5-2 parts; moisturizers: 3-10 parts; hyaluronic acid: 0.01-0.05 parts; poloxamer 188: 0.1-0.5 parts; flavoring: 0.05-0.2 parts; deionized water; the total weight of all ingredients is 100 parts.

[0035] In this embodiment of the invention, the fermentation broth compound of *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactobacter* Ld-sj 001 can colonize the pet's oral cavity and intestines, inhibiting the proliferation of pathogenic bacteria and regulating the balance of the gut microbiota. Plant extracts assist in antibacterial and anti-inflammatory effects, prebiotics provide nutrition for probiotics and enhance the regulatory effect, moisturizers maintain the system's moisture, protect the activity of probiotics, and moisturize the oral mucosa, hyaluronic acid prolongs the retention time of the components and repairs the mucosal barrier, poloxamer 188 optimizes the solubility and stability of the formula, and flavoring agents improve palatability for pets. The synergistic effect of each component not only targets and solves pet oral problems but also regulates the gut microbiota and enhances immunity.

[0036] In one embodiment of the present invention, the plant extract includes at least one of licorice extract, tea extract, or honeysuckle extract.

[0037] In one embodiment of the present invention, the prebiotic is at least one of xylooligosaccharide, fructooligosaccharide, or galactooligosaccharide.

[0038] In one embodiment of the present invention, the moisturizer is at least one of glycerin, propylene glycol or polyethylene glycol 400.

[0039] In one embodiment of the present invention, the flavoring agent is a conventional chicken flavoring and / or fish flavoring.

[0040] Fifthly, an embodiment of the present invention also provides a method for preparing a pet probiotic complex, comprising the following steps: After heating the deionized water, add the moisturizer, prebiotic, hyaluronic acid, and poloxamer 188 in sequence, mix and cool to obtain the aqueous phase; After adding the fermentation broth compound, plant extracts, and flavoring agents to the above aqueous phase, the mixture is obtained as a pet probiotic complex.

[0041] In one embodiment of the present invention, the temperature is heated to 40-50°C, preferably 45°C.

[0042] In one embodiment of the present invention, the temperature is cooled to 20~30°C.

[0043] In one embodiment of the present invention, the fermentation broth compound is prepared by the following steps: Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. lactis Ld-sj were fermented separately under their respective fermentation conditions to obtain high-concentration fermentation broths. The two fermentation broths were then mixed to obtain a fermentation broth compound.

[0044] In one embodiment of the present invention, the fermentation is anaerobic fermentation.

[0045] In a preferred embodiment of the present invention, the fermentation temperature of *Lactobacillus plantarum* is 37°C, and the fermentation time is 18-24 hours. The viable count at the end of fermentation reaches 5.0 × 10⁻⁶. 10 ~1.0×10 11 CFU / mL.

[0046] In a preferred embodiment of the present invention, the fermentation temperature of *Lactobacillus delbrueckii* subsp. *lactamella* is 42°C, and the fermentation time is 16-20 hours. The viable count at the end of fermentation reaches 5.0 × 10⁻⁶. 10 ~1.0×10 11 CFU / mL.

[0047] In one embodiment of the present invention, the ratio of viable bacteria of Lactobacillus plantarum LP-sj to Lactobacillus delbrueckii subsp. lactis Ld-sj 001 in the fermentation broth compound is (1-2):1.

[0048] Sixthly, an embodiment of the present invention also proposes the use of any of the above-mentioned pet probiotic complexes in the preparation of products for the prevention and / or improvement of oral problems in pets.

[0049] In one embodiment of the present invention, the products include mouthwash, oral spray, dental gel, toothpaste, dental chews, teething sticks, etc. for pets.

[0050] In one embodiment of the present invention, the pet includes dogs or cats.

[0051] In one embodiment of the present invention, the oral problems include at least one of halitosis, dental plaque, gingivitis, dental caries, or oral microecological imbalance.

[0052] In a preferred embodiment of the present invention, the oral problem is caused by pet-specific pathogens.

[0053] Specifically, the pet-specific pathogens include at least one of *Porphyromonas canis gingivalis*, *Cyclophila canis*, or *Cyclophila felis*. *Porphyromonas canis gingivalis* and *Cyclophila canis* colonize dogs; *Cyclophila felis* colonizes cats.

[0054] The pet probiotic complex proposed in this invention can be used to improve, prevent and treat oral diseases such as gingivitis and dental caries caused by pathogenic bacteria, and can also be used to regulate the oral microecological balance of pets, prevent plaque formation, and improve halitosis symptoms.

[0055] In a seventh aspect, an embodiment of the present invention also proposes the application of any of the above-mentioned pet probiotic complexes in the preparation of products for regulating intestinal function in pets at specific physiological stages, wherein the specific physiological stages include the weaning period of young pets and the period of intestinal function decline in older pets.

[0056] The pet probiotic complex proposed in this invention also demonstrates significant advantages in regulating pet intestinal function at specific physiological stages. During weaning: Young pets are prone to oral flora imbalance and intestinal stress due to dietary changes and an immature immune system. The complex of this invention, applied through oral spray or added to drinking water, can quickly establish a healthy oral and intestinal flora, reducing the incidence of diarrhea and enhancing immunity. During the intestinal decline stage in senior pets: Senior pets experience slower intestinal motility and decreased flora diversity, often accompanied by worsened halitosis and constipation. The complex of this invention improves digestion and absorption by regulating both the oral and intestinal microecologies, thus slowing down the intestinal aging process.

[0057] As can be seen, the pet probiotic complex of the present invention enhances immunity and the body's adaptability by targeting and regulating the balance of oral and intestinal microecology. According to the immune characteristics of pets at different stages (the immune system of young pets is not mature and the immune system of older pets is declining), it enhances local and systemic immune function through microbial regulation, reduces the risk of infection and stress-related diseases, and helps pets smoothly pass through special physiological stages.

[0058] The present invention will now be described in detail with reference to the embodiments.

[0059] Explanation of the source of experimental materials: The basic MRS medium, model 02-293X (MRS agar medium, solid), was purchased from Beijing Aoboxing Biotechnology Co., Ltd. MRS liquid culture medium, model 02-291 (MRS broth medium, liquid), was purchased from Beijing Aoboxing Biotechnology Co., Ltd. Chicken flavoring, model number XG9441, was purchased from Yangjiang Xingguang Food Ingredients Co., Ltd. The commercially available Lactobacillus plantarum used in this invention is Lactobacillus plantarum LP05, which was purchased from Microcare Probiotics (Suzhou) Co., Ltd. The commercially available Lactobacillus delbrueckii in this invention is Lactobacillus delbrueckii subsp. bulgaricus LB2, which was purchased from Shanghai Nord Biotechnology Co., Ltd. BHI liquid culture medium (brain and heart infusion broth culture medium for microorganisms) was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd. The commercially available pet probiotic mouthwash, brand name Qiaobeili, was purchased from Guangzhou Aien Pet Products Co., Ltd.

[0060] Example 1 strain origin and domestication (1) Strain screening The Lactobacillus delbrueckii subsp. Ld-sj 001 used in this invention was isolated from a cheese sample.

[0061] The specific method includes: aseptically collecting 2g of cheese sample, homogenizing it with physiological saline, and serially diluting it to obtain a bacterial suspension; Then, a preliminary screening is carried out. Specifically, a bacterial suspension of appropriate dilution is spread on a modified MRS medium (0.05% bromocresol purple and 5% calcium carbonate are added to the basic MRS medium, all by mass percentage), and incubated at 37℃ for 48-72 hours. Suspected colonies with calcium dissolution zones and those that turn the medium yellow are screened out. Then, a second screening was performed. Specifically, the suspected single colonies obtained from the initial screening were purified by streaking 2-3 times on basic MRS medium to obtain pure colonies, which were then stored on slant at 4°C. BGI Genomics was commissioned to perform 16S rRNA sequencing and identification, which confirmed that the strain was *Lactobacillus delbrueckii* subsp. *lactotrichum*, which is genetically stable and has good acid production capacity.

[0062] (2) Strain domestication Obtain a saliva sample from a pet dog, centrifuge to collect the supernatant, filter to sterilize, and obtain a saliva extract. Culture Porphyromonas gingivalis and Fusobacterium nucleatum and collect cell-free fermentation supernatant (CFS); The original strain of Lactobacillus delbrueckii subsp. lactis obtained in step (1) was inoculated into MRS liquid medium containing 5% saliva extract (mass percentage) and cultured at 37°C for 24 hours. The resulting culture was transferred to MRS medium containing 5% saliva extract and 10% pathogenic CFS and cultured further. This step was repeated, with the concentration of pathogenic CFS gradually increased to 30%, and the culture was passaged for 10-15 generations. The domesticated strain with the highest antibacterial activity and the most vigorous growth was screened using the inhibition zone method. The obtained strain was deposited on December 5, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with accession number CGMCCNo.36909.

[0063] The 16S rRNA sequence of *Lactobacillus delbrueckii* subsp. *lactobacillus* Ld-sj 001 is shown in the sequence listing SEQ ID No. 1.

[0064] Example 2 Fermentation process (1) Fermentation medium The specific composition is as follows (g / L): Peptone: 10.0; Beef extract: 10.0; Yeast extract: 5.0; Glucose: 20.0; Tween 80: 1.0; Dipotassium hydrogen phosphate: 2.0; Sodium acetate: 5.0; Ammonium citrate: 2.0; Magnesium sulfate (MgSO4·7H2O): 0.1; Manganese sulfate (MnSO4·H2O): 0.05; The pH of the culture medium was adjusted to 6.5±0.2, and then sterilized at 121℃ for 15 minutes before use.

[0065] (2) Seed liquid preparation Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. lactis Ld-sj 001 were inoculated into MRS liquid medium, respectively. Lactobacillus plantarum was cultured anaerobically at 37°C for 18-24 hours, and Lactobacillus delbrueckii subsp. lactis was cultured anaerobically at 42°C for 16-20 hours until the logarithmic growth phase (viable count ≥1×10⁻⁶). 9 CFU / mL was used as a seed solution.

[0066] (3) Fermentation by Lactobacillus plantarum Inoculation amount: Inoculate the seed culture into the fermentation medium at a volume percentage of 3%-5%.

[0067] Fermentation conditions: Anaerobic fermentation is carried out at 37℃ for 18-24 hours. Gentle stirring is maintained throughout the process (100-150 rpm); pH is not controlled during fermentation, but pH changes are monitored (usually decreasing from 6.5 to 5.0-5.5).

[0068] Fermentation endpoint: The pH of the fermentation broth was monitored using an online pH sensor. Fermentation was terminated when the pH dropped to 5.0 ± 0.1 and remained stable, and the total fermentation time reached 18-24 hours. The viable cell count stabilized at 5.0 × 10⁻⁶. 10 ~ 1.0×10 11 CFU / mL.

[0069] Fermentation broth treatment: After fermentation, the whole fermentation broth of Lactobacillus plantarum can be used directly without centrifugation or purification in order to retain the metabolites.

[0070] (4) Fermentation of Lactobacillus delbrueckii subsp. lactis Inoculation amount: Inoculate the seed culture into the fermentation medium at a volume percentage of 3%-5%.

[0071] Fermentation conditions: Anaerobic fermentation is carried out at 42℃ for 16-20 hours. Gentle stirring is maintained throughout the process (100-150 rpm); pH is not controlled during fermentation, but pH changes are monitored (usually decreasing from 6.5 to 4.8-5.2).

[0072] Fermentation endpoint: The pH of the fermentation broth was monitored using an online pH sensor. Fermentation was terminated when the pH dropped to 4.8 ± 0.1 and remained stable, and the total fermentation time reached 16-20 hours. The viable cell count stabilized at 5.0 × 10⁻⁶. 10 ~ 1.0×10 11 CFU / mL.

[0073] Fermentation broth treatment: After fermentation, the whole fermentation broth of Lactobacillus delbrueckii subsp. lactis is used directly without centrifugation or purification to retain the metabolites.

[0074] Example 3 Preparation method of pet probiotic complex The raw materials are compounded according to the following weight percentages: Fermentation broth mixture of Lactobacillus plantarum and Lactobacillus delbrueckii subsp. lactis (live count ratio 1:1): 15%; A compound plant extract consisting of licorice extract (1%), tea extract (1%), and honeysuckle extract (0.5%): 2.5%; Xylooligosaccharides (prebiotics): 1%; Glycerin (humectant): 5%; Hyaluronic acid: 0.03%; Poloxamer 188: 0.3%; Chicken flavoring: 0.1%; Deionized water: 76.07%; Preparation methods include: (1) Lactobacillus plantarum and Lactobacillus delbrueckii subsp. lactis were inoculated into the fermentation medium, and Lactobacillus plantarum was anaerobic fermented at 37°C for 20 hours to obtain a viable count of approximately 5 × 10⁻⁶. 10 Fermentation broth containing CFU / mL; *Lactobacillus delbrueckii* subsp. *lactamella* was anaerobic fermented at 42°C for 18 hours, yielding a viable count of approximately 8 × 10⁻⁶. 10 Fermentation broth at CFU / mL. Mix at a 1:1 volume ratio to obtain a fermentation broth compound, wherein the total viable cell concentration of the obtained fermentation broth compound is 1×10⁻⁶. 9 CFU / mL; (2) Add deionized water (70% of the total amount) to the mixing tank, heat to 45°C, add glycerin, xylooligosaccharide, hyaluronic acid and poloxamer 188 in sequence while stirring, stir until completely dissolved, and cool to below 30°C; under slow stirring, continue to add the fermentation broth compound obtained in step (1) to the aqueous phase; continue to add the compounded plant extract and chicken flavor essence, stir slowly for 30 minutes to make it uniform, and use the remaining deionized water to make up to 100%, stir evenly to obtain the product; Sampling and testing for viable bacteria count (ensuring a count of 1×10⁻⁶) 8 After meeting the requirements of CFU / mL (above) and pH value (6.0-7.0), the product is filled and sealed.

[0075] test case 1 Study on the broad-spectrum antibacterial effect of single strain fermentation liquid This study verifies the broad-spectrum antibacterial effect of the single-strain fermentation broth of the present invention against a variety of common oral pathogens in pets, including but not limited to: *Actinomyces myxobolus* (…). Actinomyces viscosus ), canine bite carbon dioxide fibrinolytic bacteria ( Capnocytophaga dog-bitten ), Porphyromonas canis gingivalis ( Porphyromonas gulae ), feline carbon dioxide cellulose-eating bacteria ( Capnocytophaga felis Streptococcus canis ( Streptococcus canis ), Intermediate Prevotella ( Prevotella intermediate ), oral fibrillary bacteria ( Leptotrichia buccalis ), and Fusobacterium nucleatum ( Fusobacterium nucleated ), Porphyromonas gingivalis ( Porphyromonas gingivalis Helicobacter pylori ( Helicobacter pylori ), Streptococcus mutans ( Streptococcus mutans ) and Staphylococcus aureus ( Staphylococcus golden ).

[0076] 1. Antibacterial effect of fermentation broth of Lactobacillus delbrueckii subsp. Ld-sj 001 1.1 Test Samples Test sample: Lactobacillus delbrueckii subsp. lactis Ld-sj 001 fermentation broth (preparation method is the same as Lactobacillus delbrueckii subsp. lactis fermentation in Example 2, yielding a viable count of approximately 1 × 10⁻⁶). 9 Fermentation broth (CFU / mL); Negative control: sterile deionized water.

[0077] 1.2 Test strains The pathogens used in the experiments were all sourced from the China Center for Type Culture Collection (CCTCC) or the American Center for Type Culture Collection (ATCC), including: Actinomyces spp. (CCTCC AB 99001), Canine biting carbon dioxide fibrinolytic bacteria (ATCC 35979), Porphyromonas gingivalis (ATCC 51700), Feline carbon dioxide fibrinolytic bacteria (DSM 105099), Streptococcus canis (ATCC 43496), Prevotella intermedia (ATCC 15033), Oral fibrillary bacteria (ATCC 14201), Fusobacterium nucleatum (ATCC 25586), Porphyromonas gingivalis (ATCC 33277), Helicobacter pylori (ATCC 43504), Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 6538).

[0078] 1.3 Test Methods The antibacterial effect was evaluated using the agar diffusion method (inhibition zone method): Each pathogenic bacterium was inoculated separately into a suitable liquid culture medium and anaerobically cultured at 37°C for 24-48 hours until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL.

[0079] Take 100 μL of bacterial suspension and spread it evenly on the corresponding solid agar plate.

[0080] Place a sterile Oxford cup in the center of the plate and add 100 μL of the test sample to each cup.

[0081] Place the plates in an anaerobic incubator at 37°C and incubate for 24-48 hours.

[0082] Measure the diameter of the inhibition zone (including the outer diameter of the Oxford cup, unit: mm), repeat 3 times for each sample, and take the average value.

[0083] The results of the inhibition zone diameter are shown in Table 1 (unit: mm, Oxford cup outer diameter is 8 mm).

[0084] Table 1

[0085] As shown in Table 1, the fermentation broth of *Lactobacillus delbrueckii* subsp. *lactamella* Ld-sj 001 exhibited clear antibacterial activity against all tested pathogenic bacteria, with inhibition zone diameters ranging from 12.9 to 18.3 mm. The inhibitory effects against *Porphyromonas gingivalis*, *Prevotella intermedia*, and *Fusobacterium nucleatum* were relatively prominent (inhibition zone diameter ≥ 16 mm), while the inhibitory effects against *Porphyromonas canis*, *Carbohydratea canis*, or *Carbohydratea felis* were still relatively good.

[0086] 2. Antibacterial effect of Lactobacillus plantarum LP-sj fermentation broth 2.1 Test Samples Test sample: Lactobacillus plantarum LP-sj fermentation broth (prepared using the same method as Lactobacillus plantarum fermentation in Example 2, yielding a viable count of approximately 1×10⁻⁶). 9 Fermentation broth (CFU / mL); Negative control: sterile deionized water.

[0087] 2.2 Test strains The pathogens used in the experiments were all sourced from the China Center for Type Culture Collection (CCTCC) or the American Center for Type Culture Collection (ATCC), including: Actinomyces spp. (CCTCC AB 99001), Canine biting carbon dioxide fibrinolytic bacteria (ATCC 35979), Porphyromonas gingivalis (ATCC 51700), Feline carbon dioxide fibrinolytic bacteria (DSM 105099), Streptococcus canis (ATCC 43496), Prevotella intermedia (ATCC 15033), Oral fibrillary bacteria (ATCC 14201), Fusobacterium nucleatum (ATCC 25586), Porphyromonas gingivalis (ATCC 33277), Helicobacter pylori (ATCC 43504), Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 6538).

[0088] 2.3 Test Methods The antibacterial effect was evaluated using the agar diffusion method (inhibition zone method): Each pathogenic bacterium was inoculated separately into a suitable liquid culture medium and anaerobically cultured at 37°C for 24-48 hours until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL.

[0089] Take 100 μL of bacterial suspension and spread it evenly on the corresponding solid agar plate.

[0090] Place a sterile Oxford cup in the center of the plate and add 100 μL of the test sample to each cup.

[0091] Place the plates in an anaerobic incubator at 37°C and incubate for 24-48 hours.

[0092] Measure the diameter of the inhibition zone (including the outer diameter of the Oxford cup, unit: mm), repeat 3 times for each sample, and take the average value.

[0093] The results of the inhibition zone diameter are shown in Table 2 (unit: mm, Oxford cup outer diameter is 8 mm).

[0094] Table 2

[0095] As shown in Table 2, the fermentation broth of *Lactobacillus plantarum* LP-sj exhibited antibacterial activity against all tested pathogenic bacteria, with inhibition zone diameters ranging from 12.1 to 17.6 mm. The inhibitory effects on *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Prevotella intermedia* were relatively significant (inhibition zone diameter ≥ 15 mm). It also showed some effect against *Porphyromonas canis*, *Cyclophila canis*, or *Cyclophila felis*.

[0096] test case 2 Study on the broad-spectrum antibacterial effect of the complex formed by the fermentation broth of the compound strains in vitro This study verified the broad-spectrum antibacterial effect of the pet probiotic complex of the present invention against a variety of common oral pathogens in pets, including but not limited to: Actinobacterium myxobolus (…). Actinomyces viscosus ), canine bite carbon dioxide fibrinolytic bacteria ( Capnocytophaga dog-bitten ), Porphyromonas canis gingivalis ( Porphyromonas gulae ), feline carbon dioxide cellulose-eating bacteria ( Capnocytophaga felis Streptococcus canis ( Streptococcus canis ), Intermediate Prevotella ( Prevotella intermediate ), oral fibrillary bacteria ( Leptotrichia buccalis ), and Fusobacterium nucleatum ( Fusobacterium nucleated ), Porphyromonas gingivalis ( Porphyromonas gingivalis Helicobacter pylori ( Helicobacter pylori ), Streptococcus mutans ( Streptococcus mutans ) and Staphylococcus aureus ( Staphylococcus golden ).

[0097] 1.1 Test Samples The formulations and preparation processes of all test samples were consistent with those in Example 3, with the only difference being the strain composition: ① Example 3: Contains Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. Ld-sj 001; ② Control 1: Replace Lactobacillus plantarum LP-sj with commercially available Lactobacillus plantarum, while Lactobacillus delbrueckii subsp. lactis remains unchanged; ③ Control 2: Replace Lactobacillus delbrueckii subsp. lactis Ld-sj 001 with commercially available Lactobacillus delbrueckii, while Lactobacillus plantarum remains unchanged; ④ Control 3: Both commercially available Lactobacillus plantarum and commercially available Lactobacillus delbrueckii subsp. lactis were used simultaneously.

[0098] Positive control: 0.2% chlorhexidine mouthwash.

[0099] Negative control: sterile deionized water.

[0100] 1.2 Test strains The pathogens used in the experiments were all sourced from the China Center for Type Culture Collection (CCTCC) or the American Center for Type Culture Collection (ATCC), including: Actinomyces spp. (CCTCC AB 99001), Canine biting carbon dioxide fibrinolytic bacteria (ATCC 35979), Porphyromonas gingivalis (ATCC 51700), Feline carbon dioxide fibrinolytic bacteria (DSM 105099), Streptococcus canis (ATCC 43496), Prevotella intermedia (ATCC 15033), Oral fibrillary bacteria (ATCC 14201), Fusobacterium nucleatum (ATCC 25586), Porphyromonas gingivalis (ATCC 33277), Helicobacter pylori (ATCC 43504), Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 6538).

[0101] 1.3 Test Methods The antibacterial effect was evaluated using the agar diffusion method (inhibition zone method): Each pathogenic bacterium was inoculated separately into a suitable liquid culture medium and anaerobically cultured at 37°C for 24-48 hours until the logarithmic growth phase. The bacterial concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL.

[0102] Take 100 μL of bacterial suspension and spread it evenly on the corresponding solid agar plate.

[0103] Place a sterile Oxford cup in the center of the agar plate and add 100 μL of the test sample (samples ①-④, positive control, and negative control).

[0104] Place the plates in an anaerobic incubator at 37°C and incubate for 24-48 hours.

[0105] Measure the diameter of the inhibition zone (including the outer diameter of the Oxford cup, unit: mm), repeat 3 times for each sample, and take the average value.

[0106] The results of the inhibition zone diameter are shown in Table 3 (unit: mm, Oxford cup outer diameter is 8 mm).

[0107] Table 3 Comparison of the diameter of the in vitro inhibition zone

[0108] Table 3 shows the broad-spectrum antibacterial effect: Sample ① (Example 3) exhibited obvious inhibition zones against all tested pathogens, with inhibition zone diameters all greater than 16 mm, indicating broad-spectrum and strong antibacterial activity. The inhibitory effects against *Porphyromonas gingivalis*, *Porphyromonas gingivalis*, *Cyclophagocytosus feli*, *Cyclophagocytosus canis*, *Fusobacterium nucleatum*, and *Prevotella intermedia* were particularly prominent (inhibition zone diameters all exceeded 18 mm). No existing probiotic product has ever demonstrated such good inhibitory effects against pet-specific *Cyclophagocytosus feli*, *Cyclophagocytosus canis*, and *Porphyromonas gingivalis*. The effect achieved by this invention is unattainable by other probiotic complexes in the prior art.

[0109] Synergistic effect of strains: Compared with the control sample, sample ① showed significantly better antibacterial effect than other groups. The inhibition zone diameters of samples ② and ③ were generally smaller, indicating that *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactobacter* Ld-sj 001 have a synergistic effect in antibacterial activity. Sample ④ (common dual strains) showed the weakest antibacterial effect, further proving that common strains cannot achieve the antibacterial performance of the strains of this invention.

[0110] Compared with chemical antibacterial agents: Although the antibacterial effect of sample ① of the present invention is slightly lower than that of the positive control chlorhexidine, the difference is small. Moreover, the product of the present invention is a probiotic preparation, which has the advantages of high safety and no disruption of the microecological balance.

[0111] As can be seen, the pet probiotic complex of this invention exhibits significant broad-spectrum antibacterial activity against a variety of common pet oral pathogens (including Gram-positive bacteria, Gram-negative bacteria, and anaerobic bacteria) and a variety of pet-specific pathogens (Cyclophagocytosus felis, Cyclophagocytosus canis, and Porphyromonas gingivalis). This effect is mainly attributed to the combination of specific strains (Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. lactis Ld-sj 001) and the strong antibacterial activity of the strains themselves. This provides a solid basis for its application in preventing and improving oral problems such as halitosis, dental plaque, and gingivitis in pets.

[0112] Test example 3 Strains' stability, biofilm inhibition effect, and oral odor removal effect The formulations and processes of all test samples are completely consistent with those of Example 3 of this invention, except that one or two strains are replaced.

[0113] Test sample: ①(Example 3): Contains Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. Ld-sj 001.

[0114] ②(Control 1): Replace Lactobacillus plantarum LP-sj with commercially available Lactobacillus plantarum, while Lactobacillus delbrueckii subsp. lactis remains unchanged.

[0115] ③ (Control 2): ​​Lactobacillus delbrueckii subsp. lactis Ld-sj 001 was replaced with commercially available Lactobacillus delbrueckii, while Lactobacillus plantarum remained unchanged.

[0116] ④ (Control 3): Both commercially available common Lactobacillus plantarum and commercially available common Lactobacillus delbrueckii were used simultaneously.

[0117] 1. Comparison of strain stability (survival rate of live bacteria after 30 days of storage at 37℃) Methods: The four samples were stored at 37℃ under accelerated storage conditions for 30 days. The number of viable bacteria before and after storage was measured, and the survival rate was calculated. The results are shown in Table 4.

[0118] Table 4 Comparison of strain stability data

[0119] As shown in Table 4, the combination of Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. Ld-sj 001 in this invention has stronger tolerance to liquid environments, which is the fundamental guarantee for the stable shelf life of the product.

[0120] 2. Comparison of biomembrane inhibition effects (inhibition rate after 24 hours of treatment) The standard in vitro static biofilm model was used, and the specific steps are as follows: 2.1 Culture and establishment of biofilms Strains and culture medium: Streptococcus mutans (Streptococcus mutans) Streptococcus mutans ) and Porphyromonas gingivalis ( Porphyromonas gingivalis The bacteria were revived in BHI liquid medium. Streptococcus mutans was cultured at 37°C and 5% CO2 for 24 hours; Porphyromonas gingivalis was cultured at 37°C under anaerobic conditions (85% N2, 10% H2, 5% CO2) for 48 hours.

[0121] Preparation of bacterial suspensions: The cultured Streptococcus mutans and Porphyromonas gingivalis bacterial suspensions were centrifuged (4000 rpm, 10 min) and resuspended in preheated fresh BHI medium (containing 1% sucrose to enhance biofilm formation). The bacterial suspension concentration was adjusted to 1×10⁻⁶. 6 CFU / mL yielded a suspension of Streptococcus mutans and a suspension of Porphyromonas gingivalis.

[0122] Mixed microbial inoculation: The two bacterial suspensions mentioned above are mixed in a 1:1 volume ratio to prepare a mixed microbial inoculation.

[0123] Biofilm formation: Add 200 μL of mixed bacterial inoculum to each well of a sterile 96-well cell culture plate. Incubate the plate under anaerobic conditions at 37°C for 24 hours to allow the formation of a mature mixed bacterial biofilm.

[0124] 2.2 Test Samples The formulations and preparation processes of all test samples are consistent with those of Example 3 of this invention, except for the strain composition: ① Example 3 (Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. Ld-sj 001) ② Control 1 (commercially available Lactobacillus plantarum + Lactobacillus delbrueckii subsp. Ld-sj 001) ③ Control 2 (Lactobacillus plantarum LP-sj + commercially available Lactobacillus delbrueckii) ④ Control 3 (commercially available Lactobacillus plantarum + commercially available Lactobacillus delbrueckii) A blank control group was set up (only an equal volume of sterile phosphate-buffered saline PBS was added, without any probiotic samples).

[0125] 2.3 Quantitative detection of biofilm quantity (crystal violet staining method) Pretreatment: After 24 hours of culture and establishment of the biofilm as described in section 2.1, carefully aspirate the supernatant from each well to remove airborne bacteria. Then, gently add 200 μL of PBS buffer to each well to dilute to the working concentration (approximately 1 × 10⁻⁶ viable bacteria). 8 The corresponding test samples (CFU / mL) were added. An equal volume of PBS was added to the blank control group. The culture plates were incubated under anaerobic conditions at 37°C for 24 hours.

[0126] Fixation and staining: After 24 hours of incubation, carefully aspirate the liquid from each well. Gently rinse each well twice with 300 μL of sterile PBS to remove any unattached bacteria. Then, add 200 μL of methanol to each well and fix the biofilm for 15 minutes. Discard the methanol and allow the plate to air dry in a clean bench. Afterward, add 200 μL of 0.1% (w / v), i.e., 0.1 g / 100 ml, crystal violet solution to each well and stain at room temperature for 20 minutes.

[0127] Washing and destaining: After staining, slowly pour off the crystal violet staining solution and gently rinse the 96-well plate with running ultrapure water until the outflowing water is colorless to wash away unbound dye. After air drying, add 200 μL of 95% ethanol to each well and shake for 15 minutes to destain, ensuring that the crystal violet bound to the biofilm is fully dissolved in the ethanol.

[0128] Absorbance measurement: The absorbance (OD) of the solution in each well was measured at a wavelength of 570 nm using a microplate reader. 570 (nm). This OD value is positively correlated with the biomass of the biofilm.

[0129] 2.4 Data Processing and Inhibition Rate Calculation Six replicates (n=6) were set up for each sample group and blank control group, and the results were taken as mean ± standard deviation.

[0130] The biofilm inhibition rate is calculated using the following formula: Biomembrane inhibition rate (%) = [1 - (OD) 570 Sample group / OD 570 [Blank control group] × 100% The results are shown in Table 5.

[0131] Table 5. Comparison of biofilm inhibition rates (24 hours)

[0132] As shown in Table 5, sample ① exhibited the highest biofilm inhibition rate, significantly superior to the control group where any part or all of the strains were replaced with common strains. The *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactotrichum* Ld-sj 001 used in this invention demonstrate a significant synergistic effect in inhibiting the formation of mixed biofilms of oral pathogens. This synergistic effect is not present in commercially available common strains, further confirming the uniqueness and technical advantages of the strain combination of this invention.

[0133] 3. Verification of H2S inhibition effect and persistence in simulated pet oral environment The oral environment of a pet was simulated to evaluate the ability and persistence of the pet probiotic complex of the present invention to inhibit hydrogen sulfide (H2S), the main component of halitosis, during simulated "use period" and "disuse period".

[0134] 3.1 Experimental Model Construction Simulated saliva culture medium: Based on published studies on the composition of canine saliva, simulated saliva (containing mucin, urea, electrolytes, etc.) is prepared.

[0135] Oral microbiota inoculum: Dental plaque from healthy dogs was collected and mixed with specific pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia) to construct an inoculum containing a complex microbiota.

[0136] Reaction system: Sterile anaerobic culture flasks were used as "simulated oral reactors". Each reactor was filled with 50 mL of simulated saliva, 5 mL of oral microbiota inoculum, and a small amount of food residue simulation.

[0137] 3.2 Experimental Grouping and Treatment The formulations and preparation methods of all test samples were the same as those in Example 3, with the only difference being the strain composition.

[0138] ① Example 3: Contains Lactobacillus plantarum LP-sj and Lactobacillus delbrueckii subsp. Ld-sj 001; ② Control 1: Replace Lactobacillus plantarum LP-sj with commercially available Lactobacillus plantarum, while Lactobacillus delbrueckii subsp. lactis remains unchanged; ③ Control 2: Replace Lactobacillus delbrueckii subsp. lactis Ld-sj 001 with commercially available Lactobacillus delbrueckii, while Lactobacillus plantarum remains unchanged; ④ Control 3: Both commercially available Lactobacillus plantarum and commercially available Lactobacillus delbrueckii subsp. lactis were used simultaneously.

[0139] All reactors were placed in a constant-temperature anaerobic environment at 37°C for incubation.

[0140] 3.3 Test Plan Simulated usage period (days 1-14): Every 24 hours, 1 mL of old culture medium was taken from each reactor and 1 mL of the corresponding sample and an equal amount of fresh simulated saliva and food residue were added to simulate daily mouthwash.

[0141] Simulated withdrawal period (days 15-16): Stop adding any samples, only replenish with an equal amount of fresh simulated saliva and food residue, and observe the persistence of the effect.

[0142] Detection indicators and methods: On day 0 (start), day 7 (middle of use), day 14 (end of use), day 15 (24h of discontinuation), and day 16 (48h of discontinuation), the concentration of hydrogen sulfide (H2S) in the headspace gas of each reactor was accurately measured using a portable hydrogen sulfide (H2S) detector as a representative indicator of halitosis gas.

[0143] Detailed operation steps: Instrument calibration: Before each measurement, calibrate the detector using H2S gas of standard concentration according to the instrument manual.

[0144] Headspace sampling: Using a sterile, airtight glass syringe (e.g., 50 mL), slowly draw 40 mL of headspace gas through the sampling port of the reactor.

[0145] Concentration determination: Quickly connect the syringe needle to the air inlet of the H2S detector, and push all the gas in the syringe into the detector at a uniform and slow speed.

[0146] Reading Recording: After the detector reading stabilizes, record the H2S concentration value (unit: ppb) displayed on the screen. Each reactor was measured three times, and the average value was taken as the final concentration at that time point. Results are shown in Table 6.

[0147] Table 6. H2S concentration changes in simulated oral environment (relative to initial concentration)

[0148] As shown in Table 6, regarding the effects during the usage period: throughout the entire usage period, Example 3 of the present invention consistently exhibited the fastest rate of H2S concentration decrease and the lowest endpoint concentration (30% of the initial concentration on day 14). Any control group that replaced the common strain showed significantly inferior results compared to the group of the present invention.

[0149] In terms of durability: during the simulated shutdown period, after 48 hours of shutdown, the H2S concentration in the group of this invention only recovered to 55% of the initial concentration, indicating that the healthy microecology it established has a strong self-sustaining ability.

[0150] Test example 4 Synergistic Regulation of Oral and Gut Microbiota During Weaning in Young Pets This study verifies the dual regulatory effect of the probiotic complex of the present invention on oral and intestinal flora in a simulated weaning environment for young pets, and its effect on reducing the incidence of diarrhea.

[0151] 1.1 Experimental Design Thirty healthy weaned puppies (8 weeks old) were selected and randomly divided into 3 groups of 10 each: Experimental group: The probiotic complex of the present invention (obtained in Example 3) was sprayed into the oral cavity daily for 14 consecutive days.

[0152] Positive control group: Used commercially available pet probiotic mouthwash.

[0153] Blank control group: Used an equal volume of physiological saline.

[0154] All puppies are fed standard puppy food, and their daily fecal characteristics and oral odor scores are recorded (0-5 points, 0 for no odor).

[0155] 1.2 Detection Indicators Oral swabs and fecal samples were collected on days 0, 7, and 14 for 16S rRNA sequencing to analyze microbial diversity (Shannon index).

[0156] Record the number of diarrhea episodes (stool score ≥4 is considered diarrhea). Results are shown in Tables 7 and 8.

[0157] Table 7. Changes in oral and gut microbiota diversity in young pets (Shannon index)

[0158] Table 8. Incidence of diarrhea and oral odor score

[0159] As shown in Tables 7 and 8, the probiotic complex of the present invention can significantly improve the diversity of oral and intestinal flora in young pets, reduce the incidence of diarrhea, improve oral odor, and is suitable for the microecological regulation of weaned young pets.

[0160] Test example 5 Regulatory effects on the decline of intestinal function in older pets The effects of the product of this invention on improving intestinal function, fecal quality, and overall health in older pets (≥10 years old) were evaluated.

[0161] 1.1 Experimental Design Twenty senior dogs were selected and randomly divided into two groups: Experimental group: The probiotic complex of the present invention (obtained in Example 3) (0.5 mL / kg body weight) was added to the drinking water daily for 28 consecutive days.

[0162] Control group: Equal volume of deionized water.

[0163] Record weekly weight, stool characteristics, appetite score, and coat luster.

[0164] 1.2 Detection Indicators Fecal short-chain fatty acid (SCFA) content (gas chromatography).

[0165] Serum levels of inflammatory factors IL-6 and TNF-α (ELISA method).

[0166] The results are shown in Table 9.

[0167] Table 9 Changes in Health Indicators of Senior Pets (Day 28)

[0168] As shown in Table 9, the complex of the present invention can significantly increase the level of SCFA in the intestine of elderly pets, reduce systemic inflammation, improve coat condition and constipation problems, and is suitable for health management of elderly pets in the intestinal decline period.

[0169] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus delbrueckii subsp. lactis ( Lactobacillus delbrueckii subsp. lactis Ld-sj 001, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36909.

2. An inoculum agent comprising Lactobacillus delbrueckii subsp. Ld-sj 001 as described in claim 1.

3. The application of Lactobacillus delbrueckii subsp. Ld-sj 001 as described in claim 1 in the preparation of a pet probiotic complex.

4. A pet probiotic complex, characterized in that, The fermentation broth compound includes the fermentation broth of Lactobacillus plantarum LP-sj and the fermentation broth of Lactobacillus delbrueckii subsp. Ld-sj 001 as described in claim 1, wherein the Lactobacillus plantarum LP-sj has the accession number CGMCC No. 9513.

5. The pet probiotic complex according to claim 4, characterized in that, At least one of the following conditions must be met: (1) The total viable bacteria concentration of the fermentation broth compound is 1×10⁻⁶. 8 ~1×10 9 CFU / mL; (2) In the fermentation broth compound, the ratio of viable bacteria of Lactobacillus plantarum LP-sj to Lactobacillus delbrueckii subsp. Ld-sj 001 is (1-2):

1.

6. The pet probiotic complex according to claim 4 or 5, characterized in that, It is prepared from raw materials comprising the following parts by weight: The fermentation broth mixture of *Lactobacillus plantarum* LP-sj and *Lactobacillus delbrueckii* subsp. *lactospirae* Ld-sj 001 is prepared in quantities of 5-20 parts. Plant extracts: 1-5 parts; prebiotics: 0.5-2 parts; moisturizers: 3-10 parts; hyaluronic acid: 0.01-0.05 parts; poloxamer 188: 0.1-0.5 parts; flavoring: 0.05-0.2 parts; deionized water; the total weight of all ingredients is 100 parts.

7. The pet probiotic complex according to claim 6, characterized in that, At least one of the following conditions must be met: (1) The plant extract includes at least one of licorice extract, tea extract or honeysuckle extract; (2) The prebiotic is at least one of xylooligosaccharide, fructooligosaccharide or galactooligosaccharide; (3) The moisturizer is at least one of glycerin, propylene glycol or polyethylene glycol 400.

8. A method for preparing the pet probiotic complex according to any one of claims 4 to 7, comprising the following steps: After heating the deionized water, add the moisturizer, prebiotic, hyaluronic acid, and poloxamer 188 in sequence, mix and cool to obtain the aqueous phase; After adding the fermentation broth compound, plant extracts, and flavoring agents to the above aqueous phase, the mixture is obtained as a pet probiotic complex.

9. The use of the pet probiotic complex according to any one of claims 4 to 7 in the preparation of a product for preventing and / or improving oral problems in pets; and / or the use of the pet probiotic complex according to any one of claims 4 to 7 in the preparation of a product for regulating intestinal function in pets at specific physiological stages; in, The oral problems are caused by pet-specific pathogens; the pet-specific pathogens are selected from at least one of Porphyromonas caninum, Carbohydrate-loving bacteria of the canine bite, or Carbohydrate-loving bacteria of the feline. The specific physiological stages are selected from the weaning period of young pets and the period of intestinal function decline in senior pets. The products mentioned are selected from pet mouthwash, oral spray, toothpaste, dental chews, and teething sticks; The pet is selected from dogs or cats.

10. The application according to claim 9, characterized in that, The oral problem described is halitosis (bad breath).

Images