A feline oral care probiotic composition and uses thereof

Abstract

The application discloses a cat oral cavity nursing probiotic composition and application thereof, and the probiotic composition comprises animal bifidobacterium and lactic acid bacteria, wherein the lactic acid bacteria is any one or a combination of multiple of salivarius, lactobacillus casei, enterococcus faecalis, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus acidophilus and lactobacillus reuteri; compared with single probiotic, the probiotic composition has better effect on oral cavity odor removal, can effectively improve cat oral cavity source halitosis, and can also reduce dental plaque formation, relieve gingivitis and dental caries, and can be applied to preparation of a cat oral cavity odor cleaner, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pet oral care technology, and more specifically, relates to a probiotic composition for cat oral care and its application. Background Technology

[0002] Currently, most pets experience oral malodors such as bad breath, yellow teeth, plaque, tartar, and even stomatitis. If left untreated, these issues can not only lead to illness in pets but also cause discomfort and even pose a risk of infection to people who come into close contact with them. Existing oral care products, such as Jian Kou Le tooth powder, primarily deodorize through plant extracts. Their ingredients include chelated microparticles and brown algae extract, which can be ingested and work in the digestive tract. The ingredients combine with saliva to work in the mouth, increasing friction against teeth for a cleaning effect. The brown algae extract also has anti-inflammatory and antibacterial properties. The addition of tea polyphenols and peppermint extract further deodorizes and freshens breath, but these methods cannot address the root cause of oral malodor.

[0003] In fact, halitosis includes both oral-origin and non-oral-origin halitosis, with 90% of pet halitosis being oral-origin. Oral-origin halitosis stems from a disruption of the oral microecological balance. Dysbiosis of the oral flora leads to an increase in the proportion of odor-producing anaerobic bacteria, resulting in increased release of sulfur-containing protein metabolites from odor-causing bacteria. For example, bacteria decompose sulfur-containing amino acids to produce volatile sulfur-containing odor-causing gases, thus causing bad breath. Adding probiotics to balance the bacterial structure and abundance in a pet's oral cavity helps to fundamentally solve the problem of oral-origin halitosis.

[0004] Current probiotic oral care products are primarily designed for human oral health, and the selection of probiotic strains is undoubtedly based on human probiotic standards. There is no direct experimental data to suggest whether they can be directly applied to pets. Furthermore, humans and cats are biologically different species, and different pet breeds require different probiotics. Currently, there are no probiotic products available for cat oral care on the market. Therefore, researching a probiotic product that can eliminate oral odor in cats would be beneficial for addressing oral health issues at their source. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a probiotic composition for cat oral care and its application. The purpose is to regulate the oral flora of cats by adding the probiotic composition, thereby resolving the problem of bad breath in cats at its source. The probiotic composition includes Bifidobacterium animalis and lactic acid bacteria. The lactic acid bacteria are one or more combinations of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri, thus solving the technical problem that existing products are unable to improve bad breath in cats at its source.

[0006] To achieve the above objectives, according to one aspect of the present invention, a probiotic composition for feline oral care is provided, comprising Bifidobacterium animalis and lactic acid bacteria, wherein the lactic acid bacteria are any one or more combinations of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri.

[0007] Preferably, the cat oral care probiotic composition comprises Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum.

[0008] Preferably, the cat oral care probiotic composition comprises Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri.

[0009] Preferably, the probiotic composition for cat oral care has a total live bacteria count of 1×10⁻⁶. 10 -2×10 11 CFU / g.

[0010] Preferably, the probiotic composition for cat oral care has a live bacteria count of 1.0 x 10⁻⁶. 9 -1.8*10 10 The CFU / g concentration of Bifidobacterium animalis and the viable count were 1.0*10⁻⁶. 8 -2.4*10 9 The CFU / g of Lactobacillus salivarius and the viable count are 1.0*10⁻⁶. 9 -1.4*10 10 The CFU / g Lactobacillus casei count was 9.2*10⁻⁶. 9 -5.0*10 10 The CFU / g of Enterococcus faecalis and the viable count were 1.0*10⁻⁶. 9 -2.3*10 10 The CFU / g of Lactobacillus rhamnosus has a viable count of 1.0*10⁻⁶. 9 -2.8*10 10 The CFU / g of *Lactobacillus plantarum* and the viable count were 8.3*102. 9 -1.0*10 10 Lactobacillus acidophilus CFU / g and viable count of 2*10 8- 3.1*10 9 It is obtained from one or more combinations of Lactobacillus reuteri CFU / g.

[0011] Preferably, the cat oral care probiotic composition comprises Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum, wherein the mass ratio of Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum is 1-2:1-2:1-2:1-2:1-2.

[0012] According to another aspect of the present invention, the application of the cat oral care probiotic composition as described herein in a cat oral care product is also provided.

[0013] According to another aspect of the present invention, a cat oral odor cleaner is also provided, comprising a dental matrix material and a probiotic composition as described in the present invention, wherein the dental matrix material includes, but is not limited to, dental powder matrix material.

[0014] Preferably, the cat oral odor remover contains a total of 5 × 10⁻⁶ live probiotics. 8 -2×10 9 CFU / g; The tooth-cleaning powder matrix material includes one or more combinations of yucca extract, green tea extract, sugarcane extract, zinc gluconate, sodium vitamin C phosphate, brown algae powder, and silica, wherein, by mass ratio, the yucca extract is 1%-2%, the green tea extract is 0.5%-1%, the sugarcane extract is 0.5%-1%, the zinc gluconate is 0.5%-1%, the sodium vitamin C phosphate is 0.5%-1%, the brown algae powder is 90%-95%, and the silica is 1%-3%.

[0015] Preferably, the cat oral odor remover, wherein the dental matrix material further includes flavoring agents and / or palatability enhancers.

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects:

[0017] Compared to single probiotics, the probiotic composition provided by this invention exhibits significantly better co-aggregation with harmful oral bacteria, adhering well to them and effectively inhibiting their growth. Since the main compounds causing oral odor are volatile sulfur compounds such as hydrogen sulfide and ammonia, the probiotic composition is significantly superior to single probiotics in inhibiting the production of hydrogen sulfide and ammonia by harmful oral bacteria. Furthermore, some probiotics, such as Bacillus subtilis and Bacillus coagulans, can even promote the production of hydrogen sulfide and ammonia by harmful bacteria. Therefore, the probiotic composition provided by this invention is significantly more effective than single probiotics in deodorizing the oral cavity, fundamentally solving the problem of halitosis originating from the mouth.

[0018] In addition, the cat oral odor remover prepared by adding the probiotic composition provided by the present invention has been verified by animal experiments to have a significant effect on removing cat oral odor. It can also reduce the formation of dental plaque and improve gingivitis and tooth decay. It is evident that it has good market application prospects. Attached Figure Description

[0019] Figure 1 This invention provides the aggregation rate of probiotics and Streptococcus mutans;

[0020] Figure 2 This invention provides the aggregation rate of probiotics and Fusobacterium nucleatum;

[0021] Figure 3 This invention provides the aggregation rate of probiotics and Porphyromonas gingivalis;

[0022] Figure 4 This is a comparison of the inhibition of hydrogen sulfide production by Streptococcus mutans by probiotics and the probiotic combinations in Examples 1-4;

[0023] Figure 5 This is a comparison of the inhibition of hydrogen sulfide production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0024] Figure 6 This is a comparison of the inhibition of hydrogen sulfide production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0025] Figure 7 This is a comparison of the inhibition of ammonia production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0026] Figure 8 This is a comparison of the inhibition of ammonia production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0027] Figure 9 It is a comparison of the amount of H2O2 produced by different probiotics and combinations of probiotics;

[0028] Figure 10 It is a comparison of how probiotics inhibit the formation of biofilms by Streptococcus mutans;

[0029] Figure 11 It is a comparison of how probiotics inhibit the formation of biofilms by Fusobacterium nucleatum;

[0030] Figure 12 It is a comparison of how probiotics inhibit the formation of biofilms by Porphyromonas gingivalis. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0032] This study collected oral microbiota data from 157 cats, including 45 healthy cats (gum health, oral health) and 112 cats in a non-healthy group (gingivitis, mild periodontitis, halitosis). Symbiotic networks were constructed for both groups. The symbiotic network of the healthy group was subtracted from that of the non-healthy group to obtain the difference network between the two groups. The networks were then sorted in descending order of node degree (which can be understood as sorting in descending order of importance). The results showed that most bacteria in the cat's oral cavity were closely related to halitosis, with Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis being the main harmful bacteria causing halitosis.

[0033] However, the most important nodes (species) in the final differential network are not all probiotics, or probiotics that have not been approved by the Ministry of Health and cannot be used as food additives. Some bacteria even have very few research reports, making it impossible to determine whether they are probiotics or harmful. This demonstrates that the oral microbiome of cats is extremely complex, and the limited research on related probiotics undoubtedly increases the challenge of finding effective probiotics.

[0034] This invention has found in experiments that a combination of animal Bifidobacterium and lactic acid bacteria can inhibit the production of volatile sulfur-containing odor-causing gases (hydrogen sulfide, ammonia), which helps improve the problem of bad breath in cats. The lactic acid bacteria include one or more of the following: Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri. In particular, the probiotic combination of Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, animal Bifidobacterium, and Lactobacillus plantarum can significantly inhibit the production of hydrogen sulfide and ammonia by Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans, while also reducing the formation of dental plaque and tartar, and alleviating the symptoms of gingivitis.

[0035] Based on this, the present invention provides a probiotic composition for cat oral care, comprising Bifidobacterium animalis and lactic acid bacteria, wherein the lactic acid bacteria are any one or more combinations of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri; wherein the probiotic composition comprises bacteria in a mass ratio of 1-2:1-2, and in some embodiments, the bacteria are in a 1:1 ratio.

[0036] Preferably, the probiotic composition includes Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum.

[0037] In some embodiments, the probiotic composition includes *Lactobacillus salivarius*, *Lactobacillus casei*, *Enterococcus faecalis*, *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri*, preferably *Lactobacillus salivarius*, *Lactobacillus casei*, *Enterococcus faecalis*, *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri*.

[0038] The probiotic composition preferably has a total live bacteria count of 1×10⁻⁶. 10 -2×10 11 CFU / g; more preferably, based on a viable count of 1.0*10⁻⁶. 9 -1.8*10 10 The CFU / g concentration of Bifidobacterium animalis and the viable count were 1.0*10⁻⁶. 8 -2.4*10 9 The CFU / g of Lactobacillus salivarius and the viable count are 1.0*10⁻⁶. 9 -1.4*10 10 The CFU / g Lactobacillus casei count was 9.2*10⁻⁶. 9 -5.0*10 10 The CFU / g of Enterococcus faecalis and the viable count were 1.0*10⁻⁶. 9 -2.3*10 10 The CFU / g of Lactobacillus rhamnosus has a viable count of 1.0*10⁻⁶. 9 -2.8*10 10 The CFU / g of *Lactobacillus plantarum* and the viable count were 8.3*102. 9 -1.0*10 10 Lactobacillus acidophilus CFU / g and viable count of 2*10 8- 3.1*10 9 The probiotic composition is obtained from one or more combinations of Lactobacillus reuteri CFU / g, wherein the mass ratio of each bacterium in the probiotic composition is 1-2:1-2.

[0039] In some embodiments, the probiotic composition includes Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum; wherein the mass ratio of Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum is 1-2:1-2:1-2:1-2:1-2.

[0040] In some embodiments, the probiotic composition includes *Lactobacillus salivarius*, *Lactobacillus casei*, *Enterococcus faecalis*, *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri*; wherein the mass ratio of *Lactobacillus salivarius*, *Lactobacillus casei*, *Enterococcus faecalis*, *Lactobacillus rhamnosus*, *Bifidobacterium animalis*, *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Lactobacillus reuteri* is 1–2:1–2:1–2:1–2:1–2:1–2:1–2:1–2:1–2.

[0041] In addition, the probiotic composition described in this invention can be directly applied to existing dental cleaning substrates, such as dental cleaning powder. This invention also provides an application of the probiotic composition described in this invention in cat oral care products.

[0042] This invention also provides a feline oral odor remover, comprising a dental matrix material and a probiotic composition as described in this invention. The dental matrix material includes, but is not limited to, a dental powder matrix material. The dental powder matrix material comprises one or more combinations of yucca extract, green tea extract, sugarcane extract, zinc gluconate, sodium vitamin C phosphate, brown algae powder, and silica; by mass ratio, the yucca extract is 1%-2%, the green tea extract is 0.5%-1%, the sugarcane extract is 0.5%-1%, the zinc gluconate is 0.5%-1%, the sodium vitamin C phosphate is 0.5%-1%, the brown algae powder is 90%-95%, and the silica is 1%-3%.

[0043] The dental cleaning matrix material preferably also includes flavoring agents and / or palatability enhancers.

[0044] Preferably, the total number of live probiotics in the cat oral odor remover is 5 × 10⁻⁶. 8 -2×10 9 CFU / g.

[0045] In some embodiments, the added probiotic composition is a combination of Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum. Preferably, the mass ratio of Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum in the cat oral odor cleaner is 1:1:1:1:1, and the live bacteria count ratio is 1-2:1-2:2-3:1-2:3-4.

[0046] In some embodiments, the added probiotic composition is a combination of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri. Preferably, the mass ratio of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri in the cat oral odor cleaner is 1:1:1:1:1:1:1:1, and the live bacteria count ratio is 2-3:1-2:1-2:2-3:1-2:3-4:1-2:1-2.

[0047] The following is an example:

[0048] Example 1: Probiotic Composition for Cat Oral Care

[0049] The cat oral care probiotic composition includes Lactobacillus salivarius (2.4*10⁻⁶). 9 CFU / g), Lactobacillus casei (1.4*10) 10 CFU / g), Enterococcus faecalis (9.2*10) 9 CFU / g), Lactobacillus rhamnosus (2.3*10) 10 CFU / g), Bifidobacterium animalis (1.8*10) 10 CFU / g), Lactobacillus plantarum (2.8*10) 10 CFU / g), Lactobacillus acidophilus (8.3*10) 9 CFU / g), Lactobacillus reuteri (3.1*10) 9 The concentration of CFU / g was 2:1:1:1:1:1:1:1, and the total viable count was 1.2 × 10⁻⁶. 10 CFU / g.

[0050] Example 2: Probiotic Composition for Cat Oral Care

[0051] The cat oral care probiotic composition includes Lactobacillus salivarius (2.4*10⁻⁶). 9 CFU / g), Lactobacillus casei (1.4*10) 10 CFU / g), Enterococcus faecalis (9.2*10) 9 CFU / g), Bifidobacterium animalis (1.8*10) 10 CFU / g), Lactobacillus plantarum (2.8*10) 10 CFU / g), Lactobacillus acidophilus (8.3*10) 9 CFU / g) and Lactobacillus reuteri (3.1*10 9 The concentration of CFU / g was 2:1:1:1:1:1:1, and the total viable count was 1.2 × 10⁻⁶. 10 CFU / g.

[0052] Example 3: Probiotic Composition for Cat Oral Care

[0053] The cat oral care probiotic composition includes Lactobacillus casei (1.4*10⁻⁶). 10 CFU / g), Enterococcus faecalis (9.2*10) 9 CFU / g), Lactobacillus rhamnosus (2.3*10) 10 CFU / g), Bifidobacterium animalis (1.8*10) 10 CFU / g), Lactobacillus plantarum (2.8*10) 10 The concentration of CFU / g was 1:1:1:1:1, and the total number of viable bacteria was 1.8 × 10⁻⁶. 10 CFU / g.

[0054] Example 4: Probiotic Composition for Cat Oral Care

[0055] The cat oral care probiotic composition includes Lactobacillus casei (1.4*10⁻⁶). 10 CFU / g), Enterococcus faecalis (9.2*10) 9 CFU / g), Bifidobacterium animalis (1.8*10) 10 CFU / g) and Lactobacillus plantarum (2.8*10) 10 The concentration of CFU / g was 1:1:1:1, and the total number of viable bacteria was 1.7 × 10⁻⁶. 10 CFU / g.

[0056] Example 5: Co-aggregation experiment of probiotics and harmful bacteria

[0057] The co-aggregation of probiotics and harmful bacteria is a crucial auxiliary function supporting the primary function of probiotics. Oral probiotics must adhere well to harmful bacteria in the mouth, allowing the harmful bacteria to be directly exposed to the antibacterial agents secreted by the probiotics, such as bacteriocins, lactic acid, and hydrogen peroxide, effectively inhibiting their growth and reducing oral diseases and halitosis.

[0058] This experiment compared the co-aggregation of single probiotics and the probiotic compositions in Examples 1-4 with Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis. The results are as follows: Figure 1-3 As shown, the details are as follows:

[0059] Experimental methods:

[0060] 1. Culture oral probiotics and pathogens separately, and harvest the bacterial cells by centrifugation at 4000×g for 15 minutes;

[0061] 2. Wash the bacterial cells once with Cisar's buffer and resuspend at approximately 10⁹ cells / mL (approximately OD₂O₅). 600 =1);

[0062] 3. Take 1ml of probiotics, 1ml of pathogens, and 1ml of the mixture (0.5ml probiotics + 0.5ml pathogens) and vortex for 10 seconds each to homogenize.

[0063] 4. Incubate at 37°C with gentle stirring at 110 rpm for 30 minutes, then remove and incubate at room temperature for 3 minutes;

[0064] 5. Take 0.5 ml of the supernatant and use an ELISA reader to measure the optical density (OD) value at 600 nm.

[0065] Coagulation (%) = [(Probiotic OD)] 600 + Pathogen OD 600 ) / 2-(Probiotics + Pathogens)OD 600 ×100] / [(Probiotic OD 600 + Pathogen OD 600 ) / 2].

[0066] Figure 1 It is the aggregation rate of probiotics and probiotic compositions with Streptococcus mutans;

[0067] Figure 2 It is the aggregation rate of probiotics and probiotic compositions with Fusobacterium nucleatum;

[0068] Figure 3 It is the aggregation rate of probiotics and probiotic compositions with Porphyromonas gingivalis;

[0069] Depend on Figure 1-3 It is known that probiotics have a certain co-aggregation ability with Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis. Among them, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus reuteri, and Bifidobacterium animalis have better co-aggregation effects with the three pathogens. The aggregation rate of the probiotic composition provided by this invention with Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis is significantly higher than that of single probiotics. This indicates that the co-aggregation of the probiotic composition provided by this invention with Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis is significantly better than that of single probiotics, and it can effectively exert the role of probiotics in inhibiting harmful bacteria, which is beneficial to oral care.

[0070] Example 6: The effect of probiotics on the production of oral odor gases (hydrogen sulfide, ammonia)

[0071] The compounds responsible for bad breath are mainly volatile sulfur compounds, produced by some periodontal bacteria. These compounds, such as hydrogen sulfide and ammonia, produce a noticeable odor. Among them, *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Streptococcus mutans* produce either hydrogen sulfide or ammonia. *Streptococcus mutans* produces only hydrogen sulfide, not ammonia, while both *Porphyromonas gingivalis* and *Fusobacterium nucleatum* produce both hydrogen sulfide and ammonia.

[0072] This experiment selected 10 probiotics, specifically Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Bacillus subtilis, and Bacillus coagulans, as well as the probiotic compositions obtained in Examples 1-4. The production of hydrogen sulfide and ammonia was measured after co-culturing the probiotics with pathogenic bacteria. The results are as follows: Figure 4-8 The culture medium for co-cultured strains is a mixture of co-cultured strain culture media in equal proportions.

[0073] Experimental methods:

[0074] 1. Culture probiotics to the logarithmic phase, then centrifuge at 7000g for 10 minutes to collect the bacterial cells;

[0075] 2. The collected bacterial cells were resuspended and washed once with PBS, and then centrifuged at 7000g for 10 minutes to collect the bacterial cells;

[0076] 3. The collected bacterial cells were resuspended in PBS and the absorbance was measured to be approximately 0.5 (about 10⁸).

[0077] 4. Add 1 mL of probiotic suspension to 9 mL of pathogen culture in the logarithmic phase and shake incubate for 30 minutes.

[0078] Use a 10mL syringe to draw 10mL of gas from the air in the culture container (anaerobic test tube). Measure the amounts of hydrogen sulfide and ammonia using a hydrogen sulfide detector and an ammonia detector, respectively. The hydrogen sulfide measurement results are as follows: Figure 4-6 As shown, the ammonia measurement results are as follows: Figure 7-8 As shown in the figure. In the experiment, it is also possible to place the detector in a small, transparent, sealed container, inject the gas to be measured, and then perform the measurement.

[0079] Figure 4 This is a comparison of the inhibition of hydrogen sulfide production by Streptococcus mutans by probiotics and the probiotic combinations in Examples 1-4;

[0080] Figure 5 This is a comparison of the inhibition of hydrogen sulfide production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0081] Figure 6 This is a comparison of the inhibition of hydrogen sulfide production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0082] Depend on Figure 4-6It is evident that, compared to individual probiotics, the probiotic combination provided by this invention significantly reduces the production of hydrogen sulfide and ammonia by three pathogenic bacteria: *Porphyromonas gingivalis*, *Fusobacterium nucleatum*, and *Streptococcus mutans*, thus improving oral odor. However, *Bacillus subtilis* and *Bacillus coagulans* among the probiotics promote hydrogen sulfide production, indicating significant differences in the effectiveness of different probiotics in improving oral odor. By adding the probiotic composition provided by this invention, the production of hydrogen sulfide by oral pathogens can be effectively reduced, thereby improving the problem of oral odor.

[0083] Figure 7 This is a comparison of the inhibition of ammonia production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0084] Figure 8 This is a comparison of the inhibition of ammonia production by probiotics and the probiotic combinations in Examples 1-4 by probiotics;

[0085] Depend on Figure 7-8 It is evident that, compared to individual probiotics, the probiotic combination provided by this invention significantly reduces the ammonia production of two pathogenic bacteria, *Porphyromonas gingivalis* and *Fusobacterium nucleatum* (while *Streptococcus mutans* does not produce ammonia), thus improving oral odor. However, *Bacillus subtilis* and *Bacillus coagulans* among the probiotics promote ammonia production, indicating significant differences in the effectiveness of different probiotics in improving oral odor. Furthermore, intervention by adding the probiotic composition provided by this invention can effectively reduce the production of hydrogen sulfide and ammonia by oral pathogens, enabling it to function as an oral odor cleanser for improving oral odor.

[0086] Example 7: Comparison of H2O2 production by probiotics

[0087] H2O2 can inhibit the growth of oral pathogens, thereby reducing the production of volatile sulfur compounds in the oral cavity and alleviating halitosis. The higher the H2O2 content produced by probiotics, the better their effect on improving halitosis.

[0088] This experiment selected 10 kinds of probiotics and the probiotic combinations obtained in Examples 1-4. The strains were cultured to the logarithmic phase and the amount of H2O2 produced was detected, as follows:

[0089] Reagent: (Solarbio BC3590 - Hydrogen peroxide (H2O2) content detection kit 190 / 50 times).

[0090] Experimental method: The strain was cultured to the logarithmic growth phase (OD2). 600Remove the sample at approximately 0.8 μL and centrifuge at 3500 rpm for 4 minutes at 4°C. Collect the supernatant, neutralize it to pH 7.0, and filter through a syringe (0.45 μm). Use a hydrogen peroxide detection kit (ab102500, Abcam, Cambridge, MA, USA) for colorimetric and fluorescence detection of horseradish peroxidase (HTS) to measure H2O2 in biological samples. The results are as follows: Figure 9 As shown.

[0091] Depend on Figure 9 It is known that different probiotics have significantly different abilities to produce H2O2. Among the 10 probiotics, Bifidobacterium animalis has a stronger ability to produce H2O2, while the probiotic composition provided by this invention has an even stronger ability to produce H2O2 than Bifidobacterium animalis.

[0092] Example 8: Inhibition of Biofilm Formation by Probiotic Composition

[0093] Dental caries is caused by the accumulation of dental plaque (microbial biofilm) on the tooth surface and gingival margin, the vast majority of which is composed of bacteria. Inhibiting the growth of biofilm-forming bacteria can reduce dental plaque in the oral cavity and improve the oral environment. This experiment uses probiotics and the probiotic compositions in Examples 1-4 as subjects to study the effects of probiotics on biofilm formation, as detailed below:

[0094] Experimental methods:

[0095] 1. Adjusting Streptococcus mutans cultured to the logarithmic phase to OD. 600 =0.5(5×10 8 (CFU / mL), and diluted 10 times with BHI containing 5% sucrose;

[0096] Adjusting nucleated Fusobacterium cultured to the logarithmic phase to OD 600 =0.5(5×10 8 (CFU / mL), and diluted 10 times with BHI containing 5% sucrose;

[0097] Porphyromonas gingivalis cultured to the logarithmic phase was adjusted to OD. 600 =0.5(5×10 8 (CFU / mL), and diluted 10 times with BHI containing 5% sucrose;

[0098] 2. The probiotics cultured to the logarithmic growth phase were adjusted to OD values. 600 =0.5 (5×10⁸ CFU / mL) and diluted 0.0625, 0.125 and 0.25 times with its culture medium (containing 5% sucrose);

[0099] 3. Inoculate 100 μL (5 × 10⁶ CFU / mL) of Streptococcus mutans culture (Fusobacterium nucleatum or Porphyromonas gingivalis) into a 96-well plate, and then add 100 μL (5 × 10⁶ CFU / mL) of serially diluted probiotic culture to (6.25 × 10⁵ CFU / mL) to the wells.

[0100] After incubating at 4.37℃ for 24 hours, discard the supernatant;

[0101] 5. Wash the wells of the plate once with sterile distilled water and air dry the plate for 10 minutes;

[0102] 6. Stain each well with 100 μL of 0.5% crystal violet aqueous solution for 15 minutes;

[0103] 7. After staining, wash the plate twice with sterile water;

[0104] The biofilm formed on the side of each well was dissolved in 99% ethanol, and the absorbance was measured at 595 nm using a microplate reader. The results are as follows: Figure 10-12 As shown.

[0105] Figure 10 It is a comparison of how probiotics inhibit the formation of biofilms by Streptococcus mutans;

[0106] Figure 11 It is a comparison of how probiotics inhibit the formation of biofilms by Fusobacterium nucleatum;

[0107] Figure 12 It is a comparison of the inhibition of biofilm formation by probiotics against Porphyromonas gingivalis;

[0108] Depend on Figure 10-12 It is known that different probiotics have significantly different inhibitory effects on the formation of biofilms by harmful bacteria. The probiotic composition provided by the present invention has the best inhibitory effect on biofilm formation, which is significantly better than the inhibitory effect of a single probiotic on biofilm formation. This indicates that the probiotic composition provided by the present invention can be used to prevent or improve dental caries, and its effect is better than that of a single probiotic.

[0109] Example 9: Animal Experiment Verification

[0110] In this embodiment, the dental cleaning powder matrix and the dental cleaning powder formula are the same, both consisting of 90% brown algae powder, 7% chicken liver powder, and 3% silica.

[0111] In experimental group 1, the probiotic composition was prepared with 8g of each type of bacteria and 680g of dental powder matrix. The probiotics and dental powder matrix were mixed evenly and then packaged into 0.5g bags.

[0112] In experimental group 2, the probiotic composition was the same as in Example 1. Specifically, each type of bacteria was added at a rate of 8g, and the tooth powder matrix was 680g. The probiotics and tooth powder matrix were mixed evenly and then packaged into 0.5g bags.

[0113] In experimental group 3, the probiotic composition was the same as in Example 3. Specifically, each type of bacteria was added in an amount of 8g, and the tooth powder matrix was 680g. The probiotics and tooth powder matrix were mixed evenly and then packaged into 0.5g bags.

[0114] Four groups of adult cats (3 cats in each group) were selected. Before the experiment, the cats' ages were recorded, and their oral health was examined and scored. Each group was fed 25g of cat food (the total daily feed for an adult cat). A control group was fed dental powder without probiotics. The cats were fed 0.5g of dental powder containing different probiotic compositions. The total number of live probiotics in the probiotic-containing dental powder was 5 × 10⁻⁶. 8 CFU / g, cats were fed probiotic-containing dental powder at regular intervals daily. Weight was recorded and oral health was assessed at days 0, 7, and 14. The specific composition of the dental powder fed to each group is shown in Table 1.

[0115] Table 1. Composition of the dental powder fed to each group

[0116]

[0117] The matrix of the dental cleaning powder in Table 1 is the same as the dental cleaning powder formula.

[0118] The cat's oral environment was scored using plaque index (PI), gingival index (GI), tartar index (CI), and sensory evaluation criteria, as follows:

[0119] Plaque Index (PI): Apply plaque to the tooth surface with a plaque display stick for 10 seconds, then rinse with physiological saline. Observe and record the staining of plaque on the tooth surface: 0 - no plaque; 1 - no visible punctate plaque at the tooth neck; 2 - punctate plaque visible at the tooth neck, width not exceeding 1 mm; 3 - plaque band at the tooth neck > 1 mm, but < 1 / 3 of the tooth surface; 4 - plaque covers more than 1 / 3 of the tooth surface, but not more than 2 / 3; 5 - plaque covers more than 2 / 3 of the tooth surface.

[0120] Gingival Index (GI): 0 - Normal gingiva; 1 - Mild inflammation, mild discoloration and edema; 2 - Moderate inflammation, redness, edema, bleeding on probing; 3 - Severe inflammation, significant redness and swelling or ulceration, with a tendency to bleed spontaneously.

[0121] Calculus Index (CI): 0 - No calculus above or below the gum line; 1 - Less than 1 / 3 of the tooth surface is covered by supragingival calculus; 2 - Between 1 / 3 and 2 / 3 of the tooth surface is covered by supragingival calculus, or there is scattered subgingival calculus at the cervical region of the tooth; 3 - More than 2 / 3 of the tooth surface is covered by supragingival calculus, or there is continuous and thick subgingival calculus at the cervical region of the tooth.

[0122] Sensory evaluation: The cat's oral odor was examined at least 30 minutes after eating. The examiner scored the odor from a distance of 10cm from the cat's mouth: 0 - no odor, i.e., no detectable oral odor; 5 - slight halitosis, halitosis reaching the threshold for detection; 10 - moderate halitosis, very distinct halitosis; 15 - strong halitosis, halitosis strong but tolerable; 20 - foul odor, extremely strong halitosis, the examiner covered their nose or turned their head away. See Table 2 below for details.

[0123] Table 2 Evaluation results for each experimental group

[0124]

[0125] As shown in Table 2, the oral conditions of different feeding groups were significantly different before feeding (0d) and at 7d and 14d. Compared with the dental powder without added probiotics and the dental cleaner with added probiotic composition, the oral odor of cats in groups 2 and 3, which were fed with the dental cleaner containing the probiotic composition described in this invention, was significantly improved after 7 days of feeding. In particular, the oral odor of cats in group 3 was resolved after 14 days of feeding, indicating that the probiotic composition provided by this invention can improve or solve the problem of oral odor in cats.

[0126] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A feline oral care probiotic composition, characterized in that, For improving halitosis and dental plaque in cats caused by Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis, the probiotic composition comprises Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum, wherein the mass ratio of Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, and Lactobacillus plantarum is 1~2:1~2:1~2:1~2:1~2; The viable count of the Lactobacillus casei was 1.0*10⁻⁶. 9 -1.4*10 10 The CFU / g count of the *Enterococcus faecalis* was 9.2*10⁻⁶. 9 -5.0*10 10 The viable count of the *Lactobacillus rhamnosus* was 1.0 * 10⁻⁶ CFU / g. 9 -2.3*10 10 The viable count of the animal Bifidobacterium was 1.0*10 CFU / g. 9 -1.8*10 10 The viable count of the *Lactobacillus plantarum* was 1.0 * 10⁻⁶ CFU / g. 9 -2.8*10 10 CFU / g.

2. A feline oral care probiotic composition characterized in that, The probiotic composition includes Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri, wherein the mass ratio of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri is 1~2:1~2:1~2:1~2:1~2:1~2:1~2; The viable count of the *Lactobacillus salivarius* was 1.0 * 103. 8 -2.4*10 9 CFU / g, the viable count of the *Lactobacillus casei* is 1.0*102. 9 -1.4*10 10 The CFU / g count of the *Enterococcus faecalis* was 9.2*10⁻⁶. 9 -5.0*10 10 The viable count of the animal Bifidobacterium was 1.0*10 CFU / g. 9 -1.8*10 10 The viable count of the *Lactobacillus plantarum* was 1.0 * 10⁻⁶ CFU / g. 9 -2.8*10 10 The viable count of the *Lactobacillus acidophilus* was 8.3*10 CFU / g. 9 -1.0*10 10 CFU / g, the viable count of the *Lactobacillus reuteri* is 2*10cFU / g. 8- 3.1*10 9 CFU / g.

3. A feline oral care probiotic composition characterized in that, The probiotic composition includes Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri, wherein the mass ratio of Lactobacillus salivarius, Lactobacillus casei, Enterococcus faecalis, Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus reuteri is 1~2:1~2:1~2:1~2:1~2:1~2:1~2:1~2:1~2; The viable count of the *Lactobacillus salivarius* was 1.0 * 103. 8 -2.4*10 9 CFU / g, the viable count of the *Lactobacillus casei* is 1.0*102. 9 -1.4*10 10 The CFU / g count of the *Enterococcus faecalis* was 9.2*10⁻⁶. 9 -5.0*10 10 The viable count of the *Lactobacillus rhamnosus* was 1.0 * 10⁻⁶ CFU / g. 9 -2.3*10 10 The viable count of the animal Bifidobacterium, expressed as CFU / g, is 1.0*10^6. 9 -1.8*10 10 The viable count of the *Lactobacillus plantarum* was 1.0 * 10⁻⁶ CFU / g. 9 -2.8*10 10 The viable count of the *Lactobacillus acidophilus* was 8.3*10 CFU / g. 9 -1.0*10 10 CFU / g, the viable count of the *Lactobacillus reuteri* is 2*10cFU / g. 8- 3.1*10 9 CFU / g.

4. The feline oral care probiotic composition according to any one of claims 1 to 3, characterized in that, The total number of viable probiotic bacteria of said probiotic composition is 1 x 10 10 -2 x 10 11 CFU / g.

5. The use of the feline oral care probiotic composition as described in any one of claims 1 to 4 in the preparation of a feline oral care product.

6. A cat oral cavity malodor cleaning agent, characterized by, It includes a dental cleaning matrix material and a probiotic composition as described in any one of claims 1 to 4, wherein the dental cleaning matrix material includes, but is not limited to, dental powder matrix materials.

7. The cat oral malodor cleaning agent according to claim 6, wherein The dental powder matrix material includes one or more of the following: yucca extract, green tea extract, sugarcane extract, zinc gluconate, sodium vitamin C phosphate, brown algae powder, and silica. By mass ratio, the yucca extract is 1%-2%, the green tea extract is 0.5%-1%, the sugarcane extract is 0.5%-1%, the zinc gluconate is 0.5%-1%, the sodium vitamin C phosphate is 0.5%-1%, the brown algae powder is 90%-95%, and the silica is 1%-3%.

8. The cat oral malodor cleaning agent according to claim 6 or 7, wherein The dental matrix material also includes flavoring agents and / or palatability enhancers.

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