Composite IgY antibody freeze-dried powder for pet oral hygiene as well as preparation method and application of composite IgY antibody freeze-dried powder

Abstract

The invention belongs to the technical field of biological agents, and particularly relates to composite IgY antibody freeze-dried powder for pet oral hygiene and a preparation method and application thereof. Oral bacteria such as prevotella intermedia, porphyromonas gingivalis, streptococcus mutans, actinomyces viscosus and fusobacterium nucleatum are selected in a targeted manner, the bacteria are used for preparing a composite IgY antibody, the composite IgY antibody is combined with the freeze-drying agent optimized by the invention, and the antibody freeze-dried powder is prepared by immunizing laying hens, so that the range of the bacteria is wider, and the immunogenicity of the laying hens is improved. The compound IgY antibody obtained by the prepared compound IgY antibody freeze-dried powder is high in activity, and the compound IgY antibody freeze-dried powder has good re-dissolvability, stability, affordability, simplicity and convenience, can maintain titer at-20 DEG C for more than two years, and is convenient to store, transport and popularize. Compared with the prior art, the composite IgY antibody freeze-dried powder aims at wider flora, can effectively resist canine oral malignant bacteria, and can be used as an additive to be added into oral health products such as toothpaste and mouth wash.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological agents, and particularly relates to a composite IgY antibody freeze-dried powder for pet oral hygiene and a preparation method and application thereof. BACKGROUND

[0002] Yolk antibody (IgY) has the advantages of convenient acquisition, high specificity, high safety, good biocompatibility, and strong stability. As an oral antibacterial ingredient, IgY has advantages that chemical antibacterial agents such as chlorhexidine and fluoride do not have. In view of this, adding IgY in oral cleaning products to achieve the purpose of cleaning the oral cavity of pet dogs is a very promising product development direction.

[0003] There are many malignant bacteria in the oral cavity of dogs. Prevotella intermedia is a gram-negative anaerobic bacterium in the oral cavity of dogs, which is widely planted in the gingival sulcus and dental plaque biofilm, and its detection amount is significantly positively correlated with the progression of canine gingivitis to periodontitis. The bacterium secretes lipopolysaccharide, protease and other virulence factors, destroys the barrier function of gingival tissue, induces local inflammatory response, causes gingival swelling and bleeding, and is directly related to the production of volatile sulfide in the oral cavity of dogs, and is one of the core causes of dog halitosis. At present, the prevention and control of the bacterium mainly depends on mechanical cleaning (such as ultrasonic cleaning), but it cannot long-term inhibit its re-planting, and for dogs with moderate inflammation, mechanical cleaning can easily aggravate tissue damage, and there is an urgent need for biological control means that can specifically target the bacterium.

[0004] Porphyromonas gingivalis, as a key pathogenic bacterium of canine periodontitis, is a gram-negative anaerobic bacterium and a core member of the "red complex" of oral pathogenic bacteria, with a very low detection rate in the oral cavity of healthy dogs, but a detection rate of more than 90% in the periodontal pocket of dogs with periodontitis. The bacterium expresses gingivin, fibronectin binding protein and other virulence factors, not only can directly degrade gingival collagen tissue, but also can inhibit the phagocytic function of host immune cells, promote the spread of inflammation to alveolar bone, cause tooth loosening and alveolar bone absorption, and in severe cases, can cause secondary inflammation of the heart, kidney and other systemic organs of the dog through blood circulation. The existing chemical oral care products (such as antibacterial mouthwash) lack specificity in inhibiting the bacterium, and long-term use can easily destroy the balance of oral flora, and there is a certain risk of side effects.

[0005] Streptococcus mutans is a Gram-positive facultative anaerobic bacterium. Although the incidence of dental caries in dogs is lower than in humans, it remains a major pathogen causing enamel demineralization and cavity formation in dogs. Its pathogenic mechanism lies in its efficient breakdown of sucrose in food, producing large amounts of lactic acid, which lowers the local oral pH to below 5.5, eroding tooth enamel and dentin. Simultaneously, it synthesizes extracellular polysaccharides such as dextran, promoting the colonization of itself and other cariogenic bacteria on the tooth surface, forming cariogenic plaque. Current canine oral care products mostly focus on periodontitis prevention, with few specific products targeting Streptococcus mutans. Furthermore, traditional mechanical cleaning methods (such as brushing) cannot reach hidden areas like tooth fissures and pits, failing to effectively prevent tooth decay.

[0006] *Actinomyces mesophila* is a Gram-positive facultative anaerobic bacterium and a core colonizing bacterium in early dental plaque in dogs. It binds to the acquired membrane on the tooth surface through surface adhesion proteins, providing an attachment basis for subsequent colonization by bacteria such as Streptococcus and Fusobacterium. It is a key "starting bacterium" for the maturation of dental plaque biofilm from sparse to dense. Under normal circumstances, its proportion in the oral flora is stable, but when canine oral hygiene is poor (e.g., long-term lack of cleaning), its numbers can increase excessively. It combines with calcium and phosphate ions in saliva to form tartar precursors, accelerating tartar deposition. Simultaneously, its metabolites can irritate the gums, inducing mild gingivitis and creating conditions for the invasion of other pathogenic bacteria. Current prevention and control measures are insufficient to intervene in early dental plaque formation, making it difficult to inhibit the colonization and proliferation of this bacterium at its source.

[0007] *Fusobacterium nucleatum* is a Gram-negative anaerobic bacterium with a fusiform morphology. It has been detected in the oral cavity of both healthy and diseased dogs, but its abundance is negatively correlated with the severity of canine periodontitis. Although its own pathogenicity is relatively weak, it acts as a "bridging bacterium" in the oral flora—connecting early-colonizing bacteria (such as *Actinomyces myxobolus* and *Streptococcus*) with late-pathogenic bacteria (such as *Porphyromonas gingivalis* and *Prevotella intermedius*) through surface adhesion molecules, promoting the colonization and maturation of pathogenic bacteria in dental plaque biofilm and accelerating the inflammatory process. Furthermore, this bacterium can enhance the virulence of other pathogenic bacteria by secreting endotoxins, exacerbating inflammatory damage to gingival tissue. Currently, research on this bacterium is limited, and there is a lack of control technologies that can specifically block its "bridging role," resulting in limited overall inhibitory effects on dental plaque biofilm. Summary of the Invention

[0008] To address the above-mentioned problems, the present invention aims to provide a lyophilized compound IgY antibody powder for pet oral hygiene, its preparation method, and its application.

[0009] The technical content of this invention is as follows: This invention provides a compound IgY antibody lyophilized powder for pet oral hygiene, comprising a compound IgY antibody and a lyophilization protectant; The composite IgY antibody is obtained by separating the yolk of highly immunized eggs from laying hens after immunizing them with an inactivated vaccine containing Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces myxobolus and Fusobacterium nucleatum, and then extracting the antibody. The immunized laying hens were given long-chain inulin (degree of polymerization ≥23) added to the immunization adjuvant at a concentration of 0.5~3 mg / mL; The components of the freeze-drying protectant include trehalose, inulin, mannitol, and BSA; By mass fraction, the freeze-drying protectant contains 3-5% trehalose, 2-5% inulin, 2-4% mannitol, 1-2% BSA, and the remainder is water.

[0010] The volume ratio of the composite IgY antibody to the lyophilization protectant is (3~5):1.

[0011] This invention also provides a method for preparing the above-mentioned compound IgY antibody lyophilized powder for pet oral hygiene, comprising the following steps: 1) Preparation of composite IgY antibody ① Preparation of immunogens Inactivation of strains of Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces myxoides, and Fusobacterium nucleatum were carried out to prepare immunogens; ② Preparation of immune adjuvants Long-chain inulin was added to the oral bacterial antigen composition to a concentration of 0.5-3 mg / mL, and then emulsified with complete Freund's adjuvant to prepare the primary immunization emulsion. Long-chain inulin was added to the oral bacterial antigen composition to a concentration of 0.5-3 mg / mL, and then emulsified with incomplete Freund's adjuvant to prepare an enhanced immune emulsion. The oral bacterial antigen composition is a mixture of antigen solutions of Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces viscous, and Fusobacterium nucleatum. ②Immunization of laying hens For laying hens, the above-mentioned initial immunization emulsion and booster immunization emulsion were used for initial immunization and booster immunization, respectively. Collect immunized eggs, separate the yolks, and extract pure IgY solution from the yolks, which is the compound IgY antibody; 2) Preparation of freeze-drying protectant Trehalose, inulin, mannitol, and BSA are mixed in a specific ratio and dissolved in purified water to obtain a freeze-drying protectant. 3) Preparation of compound IgY antibody lyophilized powder The composite IgY antibody and the lyophilization protectant were mixed at a volume ratio of (3~5):1 to obtain a pre-lyophilized solution, and then lyophilized powder was obtained through a pre-freezing and drying process. The pH of the solution before lyophilization is 7.2~7.4; The pre-freezing temperature is -30~-40℃, and the temperature is maintained for 1~2 hours to ensure that the material is completely frozen; The drying process includes a main drying stage and a desorption drying stage. The temperature of the main drying process is -15 to -20°C, and the temperature is maintained for 6 to 10 hours. The desorption drying temperature is 30~35℃, and the temperature is maintained for 5~6 hours.

[0012] This invention also provides the application of the above-mentioned composite IgY antibody lyophilized powder in the preparation of pet oral health and hygiene products; The pet oral health and hygiene products include toothpaste, mouthwash, etc.

[0013] The beneficial effects of this invention are as follows: The compound IgY antibody lyophilized powder of this invention targets specific oral bacteria, including *Prevotella intermedius*, *Porphyromonas gingivalis*, *Streptococcus mutans*, *Actinomyces myxobolus*, and *Fusobacterium nucleatum*. These bacteria are used to prepare a compound IgY antibody, which is then combined with the lyophilizing agent of this invention. Immunization of laying hens is achieved through optimized reaction conditions, resulting in a broader range of targeted bacteria. The optimized antigen preparation process advances the peak antibody titer in laying hens, and the optimized lyophilizing agent composition and lyophilization process result in high activity of the compound IgY antibody. The compound antibody lyophilized powder exhibits good reconstitution, stability, affordability, and ease of use, meeting application requirements. The lyophilized powder maintains its titer for more than two years at -20°C, facilitating storage, transportation, and promotion. Compared to existing technologies, the compound IgY antibody lyophilized powder of this invention targets a broader range of bacteria, including those that are aggressive against canine oral malignancies, and can be added as an additive to toothpaste, mouthwash, and other oral health products. Furthermore, thanks to its unique technology, the composite antibody of this invention has high titer, good stability, and superior efficacy. Attached Figure Description

[0014] Figure 1 SDS-Page for IgY purified antibody; Figure 2 The effect of lyophilized compound IgY antibody powder on bacterial biofilm. Detailed Implementation

[0015] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0016] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0017] Example 1 Preparation of immunogens from Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces myxoides, and Fusobacterium nucleatum. 1.1 Bacterial culture and collection Cryopreserved standard strains of *Prevotella intermedius* (ATCC 25611), *Porphyromonas gingivalis* (ATCC 33277), *Streptococcus mutans* (ATCC 25175), *Actinomyces villosa* (ATCC 19246), and *Fusobacterium nucleatum* (ATCC 10953) were retrieved. They were streaked onto the corresponding agar plates and then cultured in the appropriate liquid medium under suitable conditions. The bacterial cells of each strain were collected by centrifugation (4°C, 8000×g, 15 min). The cells were resuspended in sterile PBS and washed by centrifugation, repeated three times to thoroughly remove any culture medium components.

[0018] After washing, the bacterial cell pellets of each strain were resuspended in an appropriate amount of PBS and inactivated using the following method: For Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces viscous, and Fusobacterium nucleatum, the formaldehyde inactivation method was used. Formaldehyde was added to the bacterial suspension to a final concentration of 0.5%, and the suspension was fixed at 4°C for 24 hours. After inactivation, the suspension was centrifuged again and washed three times with PBS to completely remove the formaldehyde.

[0019] Each inactivated bacterial suspension was adjusted to a concentration of 1×10 using physiological saline. 9 Five bacterial suspensions were mixed in equal volumes at CFU / mL to obtain an oral bacterial antigen composition. The inactivated bacterial solution was centrifuged at 8000g for 15 min at 4°C. A portion of the prepared immunogen was stored at 4°C and used for the first immunization of healthy hens within 24 hours. Another portion of the immunogen was stored at -80°C (for no more than one year) for booster immunization. The dosage of immunogen for booster immunization can be reduced to half of the dosage for the first immunization.

[0020] 1.2 Immunogen Preparation The effectiveness and efficiency of immunization in producing egg yolk antibodies in chickens are highly dependent on the strength and persistence of the chicken's own immune response, posing a significant challenge to traditional immunization protocols. To ensure that hens continuously produce and accumulate high titers of specific antibodies in their egg yolks, this invention conducted extensive efficacy tests on immune adjuvant compatibility. This embodiment presents only a few representative immune adjuvant protocols.

[0021] Adjuvant group 1: First immunization emulsion (complete Freund's adjuvant emulsion): Mix an equal volume of oral bacterial antigen composition with complete Freund's adjuvant, and repeatedly inject the mixture using a double syringe connected to a three-way valve until a stable "water-in-oil" emulsion is formed. Place a drop of the emulsion on a surface of cold water; if the droplet remains intact and does not spread, it indicates complete emulsification.

[0022] Enhanced immune emulsion (incomplete Freund's adjuvant emulsion): The preparation method is the same as above, except that the complete Freund's adjuvant is replaced with an incomplete Freund's adjuvant, and the concentration of the oral bacterial antigen composition is diluted by half.

[0023] The prepared immunoemulsion should be used immediately after preparation. If short-term storage is required, it should be stored at 4°C and used for animal immunization within 24 hours.

[0024] Adjuvant Group 2: The difference from Adjuvant Group 1 is that long-chain inulin is added to the oral bacterial antigen composition to make the final concentration of long-chain inulin 1 mg / mL. The other steps are the same as Adjuvant Group 1 to prepare the corresponding primary immunization emulsion and booster immunization emulsion.

[0025] Adjuvant Group 3: The difference from Adjuvant Group 2 is that the final concentration of long-chain inulin is 0.5 mg / mL, and the other procedures are the same.

[0026] Adjuvant Group 4: The difference from Adjuvant Group 2 is that the final concentration of long-chain inulin is 2 mg / mL, and the other procedures are the same.

[0027] Adjuvant Group 5: The difference from Adjuvant Group 2 is that long-chain inulin is replaced with chitosan, with a final chitosan concentration of 1 mg / mL; all other procedures are the same. Adjuvant Group 6: The difference from Adjuvant Group 2 is that the final concentration of long-chain inulin is 0.25 mg / mL; all other procedures are the same.

[0028] Adjuvant Group 7: The difference from Adjuvant Group 2 is that the final concentration of long-chain inulin is 4 mg / mL, and the other procedures are the same.

[0029] Example 2 Egg-laying hen immunization The immunization procedure for adjuvant group 1 is as follows: First immunization (D0 day): Disinfect the subcutaneous injection site in the neck of the laying hen with a 75% alcohol swab, and inject 0.5 mL of the first immunization emulsion. When injecting, push the injection slowly to avoid leakage of the drug solution. Booster immunization (D10): Using the same injection site disinfection method as the first immunization, administer subcutaneous booster immunization to the neck of the laying hens; inject 0.5 mL of booster immunization emulsion into each laying hen, and the injection procedure is the same as the first immunization; thereafter, administer booster immunization once every 10 days, for a total of 5 booster immunizations.

[0030] The immunization procedures for adjuvant groups 2 to 6 were the same as those for adjuvant group 1, with a total of 2 booster immunizations.

[0031] The immunization procedure for adjuvant group 7 was the same as that for adjuvant group 2. However, due to the significantly higher incidence of redness, swelling, and ulceration at the injection site in this group compared to other groups during the immunization process, the experiment in this group was abandoned.

[0032] Feeding management and egg collection Environmental control: Maintain chicken house temperature at 22-25℃, relative humidity at 50-60%, and 16 hours of light / 8 hours of darkness. Clean and disinfect regularly to ensure a stable growing environment for laying hens. Feed and water supply: Provide laying hens with complete compound feed for free access. Change drinking water twice a day. Egg collection: Starting 10 days after the first booster immunization, collect eggs twice a day at 9:00 AM and 3:00 PM. Record egg production, egg weight, and eggshell integrity. Number the eggs and store them at 4℃ for subsequent experimental analysis.

[0033] Example 3 Preparation of lyophilized compound IgY antibody powder 3.1 Raw material pretreatment Eggs immunized with adjuvant group 2 were collected in Example 2, and broken eggs were removed; the eggshells were rinsed with pure water, the eggshells were disinfected with 75% alcohol, rinsed once more with pure water, and drained.

[0034] In a clean environment, a sterile sieve is used to break the shell, and the egg liquid is poured into a sterile separating funnel and left to stand for 10 minutes to separate into layers. The lower layer of egg yolk liquid is collected and filtered through 100-mesh sterile gauze to obtain pure egg yolk liquid.

[0035] Add sterile deionized water to the egg yolk mixture at a ratio of 1:10 (v / v), slowly adjust the pH to 5.2 with 1 mol / L hydrochloric acid, and let it stand at 4℃ for 12 h; centrifuge at 4℃ and 8000 r / min for 20 min, and collect the supernatant.

[0036] The supernatant was treated with ammonium sulfate at a final concentration of 40% (w / v) and allowed to stand at 4°C for 8 hours; then centrifuged at 10,000 r / min for 30 minutes at 4°C, and the precipitate was gently resuspended in 0.9% sterile physiological saline to obtain crude IgY extract.

[0037] The crude extract was concentrated by ultrafiltration at 0.12 MPa and 30 °C using a 30 kDa polyethersulfone hollow fiber ultrafiltration membrane (low adsorption material). Simultaneously, it was desalted by dialysis three times with 5 times the volume of sterile physiological saline (4 hours each time to avoid sudden changes in ion concentration) until the conductivity of the permeate was <50 μS / cm, yielding pure IgY solution.

[0038] like Figure 1 The image shown is a gel electrophoresis diagram of the pure IgY solution. It can be seen that the light and heavy chain bands of the IgY antibody are of normal size, with few impurities. The grayscale analysis shows that the antibody purity is greater than 90%, indicating that the purification process of this invention is highly efficient and the obtained antibody has high purity.

[0039] 3.2 Freeze-drying In order to obtain higher quality compound IgY antibody lyophilized powder, the present invention optimized the grouping of lyophilization protectants and prepared the purified compound IgY antibody lyophilized powder based on the lyophilization experience accumulated by the applicant.

[0040] 3.2.1 Preparation of freeze-drying protectant Freeze-drying protectant 1: 6% trehalose + 3% mannitol + 1% glycine; Freeze-drying protectant 2: 6% trehalose + 3% mannitol + 1% BSA; Freeze-drying protectant 3: 3% trehalose + 3% inulin + 3% mannitol + 1% BSA.

[0041] Solvent and treatment: Dissolve the above components in purified water. After dissolution, filter sterilely using a 0.45 μm filter membrane, and temporarily store the filtrate in a clean environment at 4°C for later use.

[0042] 3.2.2 Preparation of Lyophilized Compound IgY Antibody Powder Mix the pure IgY solution with lyophilization protectants 1, 2, and 3 at a ratio of 4:1 (v / v). Mixing should be performed in a clean environment at a temperature ≤25℃. Gently invert the mixture to avoid foaming, mixing for approximately 15 minutes until a homogeneous pre-lyophilized solution is obtained. The pH of the pre-lyophilized solution should be 7.2-7.4.

[0043] Pre-freezing stage: Start the freeze dryer and rapidly reduce the temperature of the partition to -40°C at a rate of ≥5°C / min. Maintain this temperature for 2 hours to ensure the material is completely frozen.

[0044] Primary drying stage (main drying): Turn on the vacuum pump and stabilize the vacuum level in the freeze-drying chamber at <15 Pa.

[0045] The temperature of the partition is raised to -20°C at a rate of 10°C / h and held at this temperature for 6-10 hours.

[0046] Secondary drying (desorption drying) stage: Maintain a vacuum level of <15 Pa.

[0047] The temperature of the partition was increased to 35°C at a rate of 10°C / h and held at this temperature for 5 hours.

[0048] The freeze-drying process described above yields freeze-dried powder.

[0049] Tests showed that 0.1g of lyophilized powder from each of the above groups dissolved completely in 10mL of sterile physiological saline within 30 seconds, and the solution became clear.

[0050] Experimental Example 1 Valence testing After the aforementioned inactivated bacterial solution was broken up, the bacterial eluent was taken, diluted with 1× coating buffer, and coated onto an ELISA plate. Each well was coated with 10 µg of total bacterial protein. The composite IgY antibody was detected using a conventional indirect ELISA method. In this invention, we used the antibody dilution factor with an OD value greater than 1.0 as the efficacy value; this was applied to all groups in the following tests.

[0051] 1. Detection of purified complex IgY antibody titers in eggs collected at different immunization stages. To test the antibody titers of laying hens immunized with immunogens prepared with different adjuvants, this invention detects the titers of extracted composite IgY antibodies. To fully compare the immune differences among laying hens, antibodies from different adjuvant groups were purified using the same amount of egg yolk liquid. After purification, the samples were diluted to the same volume of antibody solution, and the results are as follows.

[0052] Table 1. IgY titer detection at different egg collection times (Prevotella intermedius)

[0053] Table 2. IgY titer detection at different egg collection times (Porphyromonas gingivalis)

[0054] Table 3. IgY titer detection at different egg collection times (Streptococcus mutans)

[0055] Table 4. IgY titer detection at different egg collection times (Actinomyces viscous)

[0056] Table 5. IgY titer detection at different egg collection times (Fusobacterium nucleatum)

[0057] As shown in Tables 1-5, the combined IgY antibody titers obtained from adjuvant groups 2, 3, and 4 were superior to those of the other groups, with adjuvant group 2 showing the best results. Its combined IgY antibody titer reached its peak more quickly, and its titer remained at a high level for a prolonged period after immunization was stopped. Therefore, the combined IgY antibody obtained using adjuvant group 2 (immunogens consisting of complete Freund's adjuvant and incomplete Freund's adjuvant + long-chain inulin) in this invention exhibits better immunizing effects.

[0058] 2. Detection of the titer of the compound IgY antibody before and after freeze-drying. The aforementioned adjuvant group 1 resulted in more immunizations and caused some tissue ulceration in the laying hens. Adjuvant group 3 was slightly worse than adjuvant group 2, and adjuvant group 4 was more irritating to the laying hens' tissues than adjuvant group 2. Therefore, this invention uses the aforementioned adjuvant group 2 immunization protocol to obtain eggs. Egg yolk samples were purified using IgY antibodies according to the previous examples. After purification, the antibodies were serially diluted, and the serially diluted samples were subjected to ELISA testing. The antibody solution concentration was further adjusted based on the test values ​​to ensure that the antibody solution titer at an OD value of 1.0 was 1:2000. Lyophilized samples were converted and diluted according to this dilution ratio and then subjected to ELISA titer testing.

[0059] 1) Comparison of potency of lyophilized samples with different freeze-drying preservatives The potency of lyophilized samples with three different lyophilization protectants was compared. As shown in the table below, the lyophilization protectant 3 maintained the best potency after lyophilization.

[0060] Table 6. Comparison of potency of lyophilized samples before and after lyophilization using different lyophilization protectants.

[0061] 2) Stability test of freeze-dried samples The stability of the lyophilized sample of lyophilization protectant 3 was tested, and the test results are as follows.

[0062] Table 7. Titer assay of purified IgY antibody before and after lyophilization

[0063] As shown in Table 7, the lyophilization protectant 3 of the present invention has stable titers before and after lyophilization, and the antibody titers are also relatively stable after storage for 6 months, 1 year and 2 years without serious decline. Furthermore, 0.1g of lyophilized powder dissolved in 10mL of sterile physiological saline completely dissolves within 30s, and the solution is clear.

[0064] Experimental Example 2 Antibacterial effect test of lyophilized compound IgY antibody powder after dissolution To evaluate the effect of lyophilized IgY antibody-containing compound powders against Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces viscous, and Fusobacterium nucleatum on bacterial biofilm formation, crystal violet staining was used for detection.

[0065] First, Prevotella intermedia, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces viscous, and Fusobacterium nucleatum were cultured under appropriate conditions. The OD600 value of the bacterial suspension was adjusted to 0.8 with the appropriate liquid culture medium and then diluted 50 times for later use. Add 50 µL of the corresponding bacterial suspension to each well of a 96-well microplate, mix well, and incubate under the corresponding conditions. After incubation, remove the culture medium, wash twice with distilled water, add 50 µL of 0.1% crystal violet solution to each well, and stain at room temperature for 15 minutes. Then, aspirate and discard the stain, wash three times with distilled water, air dry, add 200 µL of 95% ethanol solution to each well, mix well, and measure the OD590 absorbance using a microplate reader.

[0066] The experimental and control groups are set up as shown in Table 8: Table 8 Grouping of oral bacteria treatment for each dog

[0067] The results are as follows Figure 2 As shown, compared with the blank control group, the antibacterial control group can effectively inhibit the formation of biofilms of five bacteria, indicating that the experimental system is valid; the results of experimental group 1 and experimental group 2 show that 0.25 mg / mL and 1 mg / mL of compound egg yolk antibody can effectively inhibit the formation of bacterial biofilms.

Claims

1. A compound IgY antibody lyophilized powder for pet oral hygiene, characterized in that, It includes a complex IgY antibody and a lyophilization protectant; The composite IgY antibody is obtained by separating the yolk of highly immunized eggs from laying hens after immunizing them with an inactivated vaccine containing Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces myxobolus and Fusobacterium nucleatum, and then extracting the antibody. The components of the freeze-drying protectant include inulin.

2. The lyophilized compound IgY antibody powder according to claim 1, characterized in that, The immunized laying hens were given an immunization adjuvant containing long-chain inulin at a concentration of 0.5–3 mg / mL.

3. The lyophilized compound IgY antibody powder according to claim 1, characterized in that, The components of the freeze-drying protectant also include trehalose, mannitol, and BSA.

4. The lyophilized compound IgY antibody powder according to claim 1, characterized in that, The volume ratio of the composite IgY antibody to the lyophilization protectant is (3~5):

1.

5. A method for preparing the lyophilized compound IgY antibody powder for pet oral hygiene as described in any one of claims 1-4, characterized in that, Includes the following steps: 1) Preparation of composite IgY antibody ① Preparation of immunogens Inactivation of strains of Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces myxoides, and Fusobacterium nucleatum were carried out to prepare immunogens; ② Preparation of immune adjuvants Long-chain inulin was added to the oral bacterial antigen composition to a concentration of 0.5-3 mg / mL, and then emulsified with complete Freund's adjuvant to prepare the primary immunization emulsion. Long-chain inulin was added to the oral bacterial antigen composition to a concentration of 0.5-3 mg / mL, and then emulsified with incomplete Freund's adjuvant to prepare an enhanced immune emulsion. ②Immunization of laying hens For laying hens, the above-mentioned initial immunization emulsion and booster immunization emulsion were used for initial immunization and booster immunization, respectively. Collect immunized eggs, separate the yolks, and extract pure IgY solution from the yolks, which is the compound IgY antibody; 2) Preparation of freeze-drying protectant Trehalose, inulin, mannitol, and BSA are mixed in a certain proportion and dissolved in purified water to obtain a freeze-drying protectant. 3) Preparation of compound IgY antibody lyophilized powder The composite IgY antibody and the lyophilization protectant were mixed at a volume ratio of (3~5):1 to obtain a pre-lyophilized solution, which was then processed into lyophilized powder through a pre-freezing and drying process.

6. The method for preparing the lyophilized compound IgY antibody powder according to claim 5, characterized in that, The oral bacterial antigen composition is a mixture of antigen solutions of Prevotella intermedius, Porphyromonas gingivalis, Streptococcus mutans, Actinomyces viscerata, and Fusobacterium nucleatum.

7. The method for preparing the lyophilized compound IgY antibody powder according to claim 5, characterized in that, The freeze-drying protectant comprises, by mass fraction, 3-5% trehalose, 2-5% inulin, 2-4% mannitol, 1-2% BSA, and the remainder is water.

8. The method for preparing the lyophilized compound IgY antibody powder according to claim 5, characterized in that, The pH of the pre-lyophilized solution is 7.2-7.

4.

9. The use of the composite IgY antibody lyophilized powder according to any one of claims 1-4 in the preparation of pet oral health and hygiene products.

10. The application according to claim 9, characterized in that, The pet oral health and hygiene products include toothpaste and mouthwash.

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