A probiotic composition toothpaste for assisting in improving the state of the surface tissue of the oral cavity and a method for preparing the same

Abstract

The present application relates to the technical field of oral care products, and proposes a probiotic composition toothpaste for assisting in improving the state of oral surface tissue and a preparation method thereof.The toothpaste comprises a repair composition and a toothpaste carrier, the repair composition comprises inactivated lactobacillus powder, N-acetylneuraminic acid and bovine colostrum powder, the bovine colostrum powder is derived from colostrum secreted within 72 hours after a healthy cow gives birth to a calf, and the immunoglobulin IgG content is not less than 10%.The toothpaste is prepared by a mild foaming system, a suitable pH and low-temperature preparation conditions, so as to improve the compatibility stability of the three active ingredients in the toothpaste system, and is beneficial to maintaining the retention rate of IgG in the bovine colostrum powder.The toothpaste can assist in improving the state of oral surface tissue under the condition of short-time contact in daily tooth brushing, and is used for daily care of gum and oral mucosa discomfort.

Description

Technical Field

[0001] This invention relates to the field of oral care products technology, and in particular to a toothpaste containing an epigenetic composition that helps repair oral epidermal damage, including epigenetics, N-acetylneuraminic acid and bovine colostrum powder, and its preparation method. Background Technology

[0002] The oral mucosa, gums, and other oral surface tissues are constantly in a moist, bacteria-laden environment, subject to mechanical friction and food irritation, making them prone to minor damage, bleeding gums, gum discomfort, and oral mucosal discomfort. For these everyday oral problems, consumers typically rely on oral care products such as toothpaste and mouthwash for daily cleaning and supplementary care. As a frequently used oral care product, toothpaste would have significant convenience and practical value if it could provide gentle supplementary improvement to the condition of the oral surface tissues while cleaning teeth.

[0003] The term "oral surface tissue state" as used in this invention refers to the barrier state, mild discomfort, or repair-related state after minor injury of the oral surface tissues such as the gums and oral mucosa in oral care scenarios, and does not refer to a disease state requiring drug treatment. The term "auxiliary improvement" as used in this invention refers to providing auxiliary improvement to the aforementioned oral surface tissue state under routine oral care conditions.

[0004] Current toothpaste products primarily focus on cleaning, abrasive removal, fresh breath, plaque inhibition, or alleviating gum problems. Some toothpastes add plant extracts, antibacterial agents, film-forming agents, hyaluronic acid, chitosan, or other oral care active ingredients to improve gum discomfort, dry oral mucosa, or minor damage. However, many existing products still rely heavily on single antibacterial, single anti-inflammatory, or single film-forming moisturizing approaches, focusing more on regulating one aspect of the oral environment. They typically lack a well-defined combined design for factors such as the oral microecological environment, the state of the mucosal moisture barrier, and the supply of local protective ingredients involved in improving the condition of oral surface tissues.

[0005] Postbiotics are a type of biologically derived ingredient that has gained attention in the oral care field in recent years. They can include inactivated probiotics, probiotic fermentation products, or probiotic metabolites. Compared with live probiotics, postbiotics are easier to control in the formulation and storage of non-live bacteria preparations such as toothpaste, and can be used in oral care products to help regulate the oral microecological environment. N-acetylneuraminic acid, also known as sialic acid, is a component found in natural systems such as saliva and breast milk. It has good water solubility and compatibility with the oral environment and can be used as an auxiliary care ingredient in oral care products. However, when postbiotics are combined with N-acetylneuraminic acid alone, there is still room for improvement in their ability to improve the condition of oral surface tissues under short-term contact conditions with toothpaste.

[0006] Bovine colostrum powder is derived from the colostrum secreted by healthy cows within a certain period after calving. It typically contains immunoglobulins, lactoferrin, proteins, vitamins, and trace elements. Among these, immunoglobulin IgG is one of the important indicators for evaluating the quality of bovine colostrum powder. As a biologically derived raw material containing protein components, bovine colostrum powder has the potential to be used as an auxiliary ingredient in oral care. However, toothpaste systems usually contain abrasives, foaming agents, humectants, binders, preservatives, flavorings, and pH adjusting ingredients. Furthermore, the preparation process involves stirring, homogenization, and vacuum degassing. In such complex systems, the protein components such as IgG in bovine colostrum powder may be affected by temperature, pH, surfactants, free calcium and magnesium ions in the aqueous phase, abrasive adsorption, or shearing. Therefore, simply adding bovine colostrum powder to a conventional toothpaste system may not adequately maintain its detectable IgG content, nor can it stably exert its auxiliary care value in oral care.

[0007] Therefore, it is necessary to provide a new toothpaste composition and its preparation method, which introduces bovine colostrum powder with clear quality indicators on the basis of inactivated lactobacillus powder and N-acetylneuraminic acid, and through suitable toothpaste carrier and preparation conditions, makes it maintain good compatibility stability in the toothpaste system, thereby exhibiting better oral surface tissue state and related performance under simulated oral use conditions. Summary of the Invention

[0008] In view of this, the present invention proposes a postbiotic composition toothpaste for assisting in the improvement of oral surface tissue condition and its preparation method. The present invention uses "inactivated lactobacillus powder + N-acetylneuraminic acid" as the basic combination, and further introduces bovine colostrum powder with an immunoglobulin IgG content of not less than 10%, enabling the toothpaste to provide better auxiliary improvement to the oral surface tissue condition under the short-term contact conditions of daily brushing. Simultaneously, the present invention maintains good stability of the detectable IgG content in bovine colostrum powder during toothpaste preparation and storage by controlling the toothpaste carrier composition, pH conditions, foaming agent selection, preparation temperature, and feeding method.

[0009] The technical solution of the present invention is implemented as follows: The present invention provides a post-biotic composition toothpaste that helps improve the condition of oral surface tissues, including a repair composition and a toothpaste carrier; the repair composition includes inactivated lactobacillus powder, N-acetylneuraminic acid and bovine colostrum powder; the bovine colostrum powder is derived from the colostrum secreted by healthy cows within 72 hours after calving, and the content of immunoglobulin IgG in the bovine colostrum powder is not less than 10%.

[0010] In the above scheme, inactivated lactobacillus powder and N-acetylneuraminic acid constitute the basic repair combination, while bovine colostrum powder is introduced as a further enhanced biological source component. By limiting the source and IgG content of bovine colostrum powder, this component can be distinguished from ordinary milk powder, ordinary milk protein powder, or animal milk extracts of unknown origin, thereby giving the core active ingredient of this invention a clearer quality boundary and detectable indicators.

[0011] In some embodiments, the repair composition comprises, by weight: 0.01-2 parts of inactivated lactobacillus powder, 0.01-1 part of N-acetylneuraminic acid, and 0.01-2 parts of bovine colostrum powder. By using the above dosage range, the three active ingredients can maintain good compatibility in the toothpaste system, avoiding insufficient repair-related performance due to excessively low levels of active ingredients, and also avoiding the impact of excessively high levels of active ingredients on the stability, taste, or irritation of the toothpaste.

[0012] In some embodiments, the weight ratio of the inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder is 1:0.05-0.5:0.2-2; preferably, the weight ratio is 1:0.1-0.3:0.5-1.5. This ratio is designed based on the combination of "inactivated lactobacillus powder + N-acetylneuraminic acid" and keeps the bovine colostrum powder at an appropriate level as a synergistic ingredient, which is beneficial for demonstrating better repair-related performance under simulated short-term oral contact conditions, while also taking into account the stability of the toothpaste and its user experience.

[0013] In some embodiments, the repair composition comprises 0.1% to 5% of the total mass of the toothpaste. This range is applicable to conventional toothpaste formulations and allows the introduction of bioactive ingredients that help improve the condition of oral surface tissues without significantly altering the toothpaste's cleaning function and physical properties.

[0014] In some embodiments, the bovine colostrum powder is a powder obtained by processing colostrum secreted by healthy cows within 72 hours after calving through one or more of the following methods: defatting, concentration, membrane filtration, spray drying, and freeze drying. Preferably, the bovine colostrum powder contains at least 15% immunoglobulin IgG. Further, the bovine colostrum powder may also have one or more quality control indicators selected from total protein, lactoferrin, moisture, ash, microbial limits, heavy metal limits, or veterinary drug residue limits. By limiting the source, processing method, and IgG content of the bovine colostrum powder, batch stability of the raw materials can be improved, and it is also beneficial to ensure the repeatable addition and detectable evaluation of this ingredient in toothpaste.

[0015] In some embodiments, the inactivated lactobacillus powder is inactivated *Lactobacillus plantarum* powder. The inactivated *Lactobacillus plantarum* powder can be prepared by the following method: fermenting and culturing *Lactobacillus plantarum*, collecting the cells by centrifugation, heat-inactivating the cells at 80–90°C for 10–30 min, followed by washing and freeze-drying to obtain the inactivated *Lactobacillus plantarum* powder. Preferably, the heat inactivation conditions are 85°C for 15 min.

[0016] In some embodiments, the number of bacteria in the inactivated lactobacillus powder is calculated based on the viable count of the fermentation broth before inactivation, and the calculated value is not less than 1.0 × 10⁻⁶. 11 CFU / g; the converted value is the number of viable bacteria in the pre-inactivation fermentation broth determined by plate counting, and converted to the number of pre-inactivation viable bacteria per gram of inactivated powder based on the concentration and drying ratio. Furthermore, the inactivated Lactobacillus powder may also have one or more quality control indicators selected from cell count, free amino acids, total polysaccharides, short peptides, organic acids, or total solids.

[0017] In some embodiments, the content of N-acetylneuraminic acid is not less than 98% on a dry basis. N-acetylneuraminic acid is a sialic acid compound with good water solubility, suitable for co-dispersing with inactivated lactobacillus powder and bovine colostrum powder in the aqueous phase system of toothpaste. Using N-acetylneuraminic acid with clearly defined content requirements helps to ensure the actual amount of sialic acid compounds added to the formula, thereby improving the stability and repeatability of different batches of toothpaste products.

[0018] In some embodiments, the toothpaste carrier includes a humectant, an abrasive, a foaming agent, a binder, and water; based on the total mass of the toothpaste, the humectant comprises 20-70%, the abrasive 16-50%, the foaming agent 0.5-4%, the binder 0.8-3%, and the balance includes at least one of water, fragrance, sweetener, stabilizer, preservative, and colorant. This carrier system can meet the basic cleaning, dispersing, moisturizing, paste-forming, and usage requirements of toothpaste, and provides a suitable dispersion environment for inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder.

[0019] In some embodiments, the humectant includes at least one of sorbitol, glycerin, PEG-400, and propylene glycol; the abrasive includes at least one of hydrated silica, calcium carbonate, and calcium hydrogen phosphate; the foaming agent includes at least one of sodium lauroyl sarcosinate, cocamidopropyl betaine, sodium lauroyl glutamate, sodium methyl cocoyl taurate, and alkyl glycoside; and the adhesive includes at least one of cellulose gum, xanthan gum, carrageenan, hydroxyethyl cellulose, hydroxypropyl guar gum, and carbomer. Preferably, the foaming agent is free of sodium lauryl sulfate, or the content of sodium lauryl sulfate is not higher than 0.5% of the total mass of the toothpaste. Using a mild foaming system helps reduce the irritation of the toothpaste to the oral mucosa and also helps reduce the impact of strong surfactants on the stability of protein components in bovine colostrum powder.

[0020] In some embodiments, the toothpaste has a pH of 6.0–8.0. This pH range is suitable for oral use and also helps to maintain the stability of protein components such as N-acetylneuraminic acid, inactivated lactobacillus powder, and bovine colostrum powder, including IgG. Furthermore, the water-soluble salt in the toothpaste is preferably a sodium salt, and the free Ca2+ in the aqueous extract of the toothpaste is... 2+ and Mg 2+ The total content is not higher than 0.5%. The aqueous extract of the toothpaste can be obtained by mixing the toothpaste sample and phosphate buffer at a mass ratio of 1:5, shaking and extracting for 30 min, and centrifuging to collect the supernatant. This is achieved by reducing the free Ca in the aqueous phase. 2+ and Mg 2+ Increasing the content of divalent metal ions can reduce the likelihood of adverse interactions between them and protein-based or acidic active ingredients, thereby improving the compatibility and stability of the repair composition in the toothpaste system.

[0021] In some embodiments, the free Ca 2+ and Mg 2+ The content of [specific components] was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Specifically, 5.0 g of toothpaste sample was taken, 25 mL of deionized water was added, and the mixture was shaken and extracted for 30 min. After centrifugation at 10000 rpm for 15 min, the supernatant was filtered through a 0.22 μm filter membrane before injection for analysis. The detection wavelength for calcium was 317.933 nm, and for magnesium was 285.213 nm. Quantification was performed using the external standard method, with a standard curve concentration range of 0.1–10.0 μg / mL.

[0022] In some embodiments, when calcium carbonate or calcium dicalcium phosphate is selected as the abrasive, the amount of soluble calcium and magnesium salts, pH, and buffer system can be controlled to allow free Ca2+ in the aqueous extract of the toothpaste to be released. 2+ and Mg 2+ The total content meets the above range.

[0023] In some embodiments, after the toothpaste is prepared, the retention rate of IgG in the bovine colostrum powder is not less than 70%; and / or, after the toothpaste is stored at 40°C for 30 days, the retention rate of IgG in the bovine colostrum powder is not less than 60%. The IgG retention rate can be obtained by detecting the IgG content in the sample after toothpaste preparation or storage and comparing it with the theoretical IgG content corresponding to the addition of bovine colostrum powder. By setting an IgG retention rate index, the carrying and protection capacity of the toothpaste system of the present invention for key protein components in bovine colostrum powder can be reflected. It also helps to demonstrate that bovine colostrum powder is not merely added as a common nutritional powder, but retains detectable key quality indicators in the toothpaste system.

[0024] In some embodiments, the present invention also provides a method for preparing the above-mentioned toothpaste, comprising the following steps: S1. Inactivated lactobacillus powder, N-acetylneuraminic acid, bovine colostrum powder and water-soluble raw materials are added to water and dispersed or dissolved at a temperature not exceeding 45°C to obtain the first premix. S2. Premix the abrasive, foaming agent and adhesive to obtain a second premix; S3. After mixing the humectant with the first premix, the second premix is ​​added under vacuum conditions. The mixture is then homogenized and degassed to obtain the toothpaste. The above preparation method preferentially disperses the repair composition in a lower-temperature aqueous system, which helps to reduce the impact of temperature and shear on protein components such as IgG in bovine colostrum powder during the preparation process.

[0025] In some embodiments, in step S3, the vacuum degree is -0.08 to -0.1 MPa, the homogenization speed is 900 to 1100 rpm, and the toothpaste preparation temperature is 25 to 40°C. Preferably, the bovine colostrum powder is added during the aqueous phase premixing stage or at a low temperature before homogenization. By controlling the toothpaste preparation temperature, vacuum degree, and homogenization conditions, the adverse effects on the inactivated lactobacillus powder, N-acetylneuraminic acid, and key components in the bovine colostrum powder can be reduced while ensuring the uniformity of the toothpaste paste and the degassing effect.

[0026] The present invention has the following advantages over the prior art: This invention uses inactivated lactobacillus powder and N-acetylneuraminic acid as a basic combination, and further introduces bovine colostrum powder with an IgG content of not less than 10%. This allows the toothpaste to exhibit superior oral surface tissue improvement properties compared to the basic combination under simulated short-term oral contact conditions. The source and IgG content of the bovine colostrum powder are clearly defined, distinguishing it from ordinary milk powder or ordinary protein additives, thus ensuring better raw material controllability and detectability. This invention also improves the compatibility stability of inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder in the toothpaste system through a milder toothpaste carrier, a suitable pH range, a low-irritation foaming system, low-temperature preparation conditions, and reducing free calcium and magnesium ions in the aqueous phase. This is particularly beneficial for maintaining the retention rate of key protein components such as IgG in the bovine colostrum powder. Therefore, this invention can achieve a combination of cleaning and care with auxiliary improvement of oral surface tissue without changing the daily use properties of toothpaste, making it suitable for daily oral care scenarios such as gingival and oral mucosal discomfort. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Unless otherwise stated, "%" in the following embodiments and comparative examples refers to mass percentages, and "parts" refers to parts by weight.

[0028] All experimental results in this invention are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and Tukey multiple comparisons were used when necessary. p < 0.05 indicated statistical significance.

[0029] Raw material description The inactivated lactobacillus powder used in this embodiment of the invention is specifically inactivated Lactobacillus plantarum powder. The inactivated Lactobacillus plantarum powder is obtained by fermenting and culturing Lactobacillus plantarum, collecting the bacterial cells by centrifugation, heat-inactivating it by treating it at 85°C for 15 minutes, and then washing and freeze-drying it.

[0030] The main quality indicators of the inactivated lactobacillus powder used in this embodiment of the invention are as follows: the bacterial count is calculated based on the viable count of the pre-inactivation fermentation broth, with a calculated value ≥ 1.0 × 10¹¹ CFU / g; the calculated value is the viable count of the pre-inactivation fermentation broth measured by the plate count method, and converted to the pre-inactivation viable count per gram of inactivated powder according to the concentration and drying ratio. The total solids of the inactivated lactobacillus powder are ≥ 95%, the free amino acids (calculated as glycine) are ≥ 5.0%, the total polysaccharides (calculated as glucose) are ≥ 10.0%, and the moisture content is ≤ 5.0%.

[0031] The N-acetylneuraminic acid used in this embodiment of the invention is a white crystalline powder prepared by fermentation, with a purity of ≥98.0% on a dry basis. The N-acetylneuraminic acid content was detected by high performance liquid chromatography.

[0032] The bovine colostrum powder used in this embodiment of the invention is derived from the colostrum secreted by healthy cows within 72 hours after calving, and is obtained through defatting, membrane filtration, and freeze-drying. The bovine colostrum powder contains 15.2% immunoglobulin IgG, ≥40.0% total protein, ≥1.0% lactoferrin, ≤5.0% moisture, and ≤8.0% ash. Microbial limits, heavy metals, and veterinary drug residues meet the requirements for raw materials used in oral care products. The IgG content was determined using enzyme-linked immunosorbent assay (ELISA), and the kit used was a bovine IgG quantitative detection kit.

[0033] In addition to the active ingredients mentioned above, the sorbitol, glycerin, PEG-400, hydrated silica, sodium lauroyl sarcosinate, cocamidopropyl betaine, sodium methyl cocoyl taurate, cellulose gum, xanthan gum, fragrance, sweetener, and preservative used in the examples are all toothpaste ingredients.

[0034] Methods for determining IgG retention rate Weigh 5.0g of toothpaste sample, add 20mL of phosphate buffer (PBS, pH 7.2), and extract by shaking at 25℃ for 30min. Centrifuge at 10000rpm for 15min at 4℃, collect the supernatant, and determine the IgG content in the supernatant by ELISA.

[0035] To reduce the influence of the toothpaste matrix on IgG detection, the toothpaste extract was appropriately diluted before assay, and a spiked recovery experiment was used to verify the applicability of the detection method. Specifically, nine blank toothpaste extracts were taken and divided into three spiked concentration groups (low, medium, and high), with three replicates in each group. Bovine IgG standard was added to each group to achieve final spiked concentrations of 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. The samples were then analyzed using the same ELISA method as the original samples, and the spiked recovery rate was calculated. The experimental results showed that the average spiked recoveries for the low, medium, and high concentration levels were 94.2% ± 3.1%, 98.5% ± 2.8%, and 103.7% ± 4.2%, respectively, with an overall recovery rate range of 92.5%–106.8%, indicating that the detection method is suitable for determining the IgG content in toothpaste samples of this invention.

[0036] IgG retention rate is calculated using the following formula: IgG retention rate (%) = Measured IgG content in toothpaste sample / Theoretical IgG content added to toothpaste formula × 100%.

[0037] The measured and theoretical IgG contents in the toothpaste samples were both expressed as the mass of IgG per gram of toothpaste, in μg / g toothpaste. The theoretical IgG content was calculated based on the amount of bovine colostrum powder added to the formula and the measured IgG content in the bovine colostrum powder.

[0038] III. Preparation method of toothpaste extract The toothpaste sample to be tested was mixed with sterile phosphate buffer (PBS, pH 7.2) at a mass ratio of 1:5, and extracted by shaking for 30 min. After centrifugation, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original toothpaste extract.

[0039] For in vitro cell experiments, the toothpaste extract stock solution is diluted with PBS or a suitable cell culture medium to a working solution with a toothpaste equivalent concentration of 2%, as required by the experiment. The toothpaste equivalent concentration refers to the percentage of the original toothpaste mass in the working solution relative to the total mass of the working solution. Since toothpaste is diluted by saliva during brushing, this invention uses a 2% toothpaste equivalent concentration extract as an in vitro simulation evaluation condition to compare the relative repair-related properties between different formulations. In the following scratch repair, anti-inflammatory factor, and cell compatibility experiments, unless otherwise specified, the toothpaste extract working solution used is at a toothpaste equivalent concentration of 2%.

[0040] Example 1: Toothpaste containing inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder This embodiment provides a toothpaste based on a silica abrasive system, the formulation of which is as follows: This embodiment provides a toothpaste based on a silica abrasive system, the formulation of which is as follows: Table 1 Toothpaste Formulation for Example 1

[0041] The preparation method is as follows: S1. Add inactivated lactobacillus powder, N-acetylneuraminic acid, bovine colostrum powder, sodium saccharin, and sodium benzoate to a portion of deionized water, control the water temperature to not exceed 40℃, stir and disperse evenly to obtain the first premix. S2. Hydrated silica, cellulose gum, xanthan gum and sodium lauroyl sarcosinate powder are premixed evenly to obtain a second premix. S3. Add glycerin, sorbitol, PEG-400 and the first premix to a vacuum ointment making pot and mix evenly. Add the second premix under vacuum conditions, control the vacuum degree to -0.09MPa, the homogenization speed to 1000rpm, the ointment making temperature to 35℃, and homogenize for 20min. S4. Add cocamidopropyl betaine, sodium methyl cocoyl taurate and flavoring, and continue vacuum stirring and degassing for 10 minutes to obtain the toothpaste product.

[0042] Example 2: Low-addition bovine colostrum powder toothpaste This embodiment is basically the same as Example 1, except that the amount of bovine colostrum powder added is 0.15%, the amount of inactivated lactobacillus powder added is 0.50%, the amount of N-acetylneuraminic acid added is 0.10%, and the weight ratio of inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder is 1:0.2:0.3. The remaining components and preparation methods are the same as in Example 1, with deionized water added to bring the total to 100%.

[0043] Example 3: Toothpaste with high addition of bovine colostrum powder This embodiment is basically the same as Example 1, except that the amount of bovine colostrum powder added is 1.00%, the amount of inactivated lactobacillus powder added is 0.50%, the amount of N-acetylneuraminic acid added is 0.15%, and the weight ratio of inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder is 1:0.3:2.0. The remaining components and preparation methods are the same as in Example 1, with deionized water added to bring the total to 100%.

[0044] Comparative Example 1: Blank Base Toothpaste This comparative example is basically the same as Example 1, except that inactivated lactobacillus powder, N-acetylneuraminic acid and bovine colostrum powder are not added, and the reduced mass is made up by deionized water.

[0045] Comparative Example 2: Inactivated Lactobacillus Powder + N-acetylneuraminic Acid Toothpaste This comparative example is basically the same as Example 1, except that bovine colostrum powder is not added, the amount of inactivated lactobacillus powder added is 0.50%, and the amount of N-acetylneuraminic acid added is 0.10%. The reduced mass is made up by deionized water.

[0046] Comparative Example 3: Bovine Colostrum Powder Toothpaste This comparative example is basically the same as Example 1, except that inactivated lactobacillus powder and N-acetylneuraminic acid are not added, and only 0.50% bovine colostrum powder is added. The reduced mass is made up by deionized water.

[0047] Comparative Example 4: High-Temperature Formulation Three-Element Toothpaste This comparative example is basically the same as Example 1, except that the water temperature is controlled at 60°C in step S1 and the ointment-making temperature is controlled at 55°C in step S3. The remaining components and preparation methods are the same as in Example 1.

[0048] Comparative Example 5: Toothpaste containing sodium lauryl sulfate system This comparative example is essentially the same as Example 1, except that sodium lauroyl sarcosinate, cocamidopropyl betaine, and sodium methyl cocoyl taurate in Example 1 are all replaced with sodium lauryl sulfate, so that the content of sodium lauryl sulfate in the toothpaste is 2.5%. The remaining components and preparation methods are the same as in Example 1. This comparative example is not the preferred system of this invention, but is used to evaluate the effect of a high-content sodium lauryl sulfate foaming system on IgG stability and cell compatibility.

[0049] Comparative Example 6: Toothpaste with High Free Calcium and Magnesium System This comparative example is basically the same as Example 1, except that calcium chloride and magnesium sulfate are introduced into the toothpaste, so that free Ca²⁺ is present in the aqueous extract of the toothpaste. + and Mg² + The total content was 1.2%, of which free Ca²⁺ was 1.2%. + The free Mg²⁺ content is 0.8%. + The content is 0.4%. The remaining components and preparation methods are the same as in Example 1.

[0050] toothpaste basic performance test Basic performance tests were conducted on the toothpastes prepared in Examples 1-3 and Comparative Examples 1-6. The test items included appearance, pH, viscosity, water separation, odor, paste uniformity, and microbial limits.

[0051] Table 2. Results of basic toothpaste performance tests

[0052] The results showed that all toothpastes in the examples and comparative examples had satisfactory basic properties, indicating that the repair composition and carrier system of the present invention do not affect the basic usability properties of the toothpaste.

[0053] IgG retention rate test Following the aforementioned method for determining IgG retention rate, the IgG content in samples containing bovine colostrum powder immediately after paste preparation and samples stored at 40°C for 30 days was measured, and the IgG retention rate was calculated.

[0054] Table 3 Results of IgG Retention Rate Test

[0055] The results showed that, under the same or similar bovine colostrum powder addition conditions, the IgG retention rates of Examples 1-3 were significantly higher than those of Comparative Example 4 (high-temperature paste preparation), Comparative Example 5 (sodium lauryl sulfate system), and Comparative Example 6 (high free calcium and magnesium system). This indicates that the low-temperature paste preparation conditions and mild carrier system of the present invention are beneficial for maintaining the stability of IgG in bovine colostrum powder. Specifically, the IgG retention rates after paste preparation in Examples 1-3 remained above 80%, and after storage at 40°C for 30 days, they remained above 70%, meeting the retention rate targets set by the present invention.

[0056] Effects of different combinations of active ingredients on oral mucosal cell scratch repair Human oral keratinocytes (HOKs) were used as an in vitro evaluation model. Cells were seeded in 12-well plates and cultured until 90% confluence. Scratches were created using a 200 μL sterile pipette tip, followed by washing with PBS and treatment with 2% toothpaste extract (equivalent to toothpaste concentration). Treatment groups included Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and a blank culture control group. Treatment times were 12 h and 24 h. Inverted microscopes were used for photography, and measurements were taken from at least three fields of view for each group. The scratch area was measured, and the scratch closure rate was calculated.

[0057] Scratch closure rate is calculated using the following formula: Scratch closure rate (%) = (0h scratch area - treated scratch area) / 0h scratch area × 100%.

[0058] Table 4. Effects of different combinations of active ingredients on the closure rate of oral mucosal cell scratches ( ±SD, n=3)

[0059] Note: Compared with the blank control group, *p<0.05, **p<0.01; compared with comparative example 2, #p<0.05.

[0060] The results showed that, compared with the inactivated Lactobacillus powder + N-acetylneuraminic acid group and the bovine colostrum powder alone group, the toothpaste extract of Example 1 could further improve the scratch closure rate of oral mucosal cells. At 12 h, the closure rate of the ternary combination was 38.6%, higher than the 26.3% of the inactivated Lactobacillus powder + N-acetylneuraminic acid group; at 24 h, the closure rate of the ternary combination was 71.2%, higher than the 54.6% of the inactivated Lactobacillus powder + N-acetylneuraminic acid group. These results indicate that, under simulated oral contact conditions, the ternary combination of inactivated Lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder has further improved in vitro repair-related properties compared to the basic combination.

[0061] Effects of different combinations of active ingredients on the expression of inflammatory factors An in vitro inflammatory stimulation model was established using HOK cells. After stimulating the cells with 1 μg / mL lipopolysaccharide for 4 h, the cells were treated with 2% toothpaste extract (equivalent to toothpaste) for 24 h. The culture supernatant was collected, and the levels of IL-6, TNF-α, and IL-1β were detected by ELISA.

[0062] Table 5 Results of inflammatory factor level test ( ±SD, n=3, pg·mL - ¹)

[0063] Note: Compared with the model group, *p<0.05, **p<0.01; compared with Comparative Example 2, #p<0.05.

[0064] The results showed that the levels of IL-6, TNF-α, and IL-1β in the ternary combination treatment group of Example 1 were lower than those in the inactivated lactobacillus powder + N-acetylneuraminic acid group and the bovine colostrum powder alone group, indicating that the ternary combination has better performance in downregulating inflammatory factors in the in vitro inflammatory stimulation model.

[0065] Cell compatibility assessment The effects of different toothpaste extracts on the viability of oral mucosal cells were evaluated using the CCK-8 assay. Toothpaste extracts were diluted to a toothpaste equivalent concentration of 2%, treated with HOK cells for 24 h, and then incubated with CCK-8 reagent for 2 h. Absorbance at 450 nm was measured, and cell viability was calculated. Cell viability in the blank culture medium group was considered 100%.

[0066] Table 6. Cell viability test results ( ±SD, n=3)

[0067] Note: *p<0.05 compared with the blank control group.

[0068] The results showed that, at the tested concentration, the toothpaste extract of Example 1 had no significant inhibitory effect on oral mucosal cells, and the cell viability was 94.8%. Compared with the SLS-containing system, Comparative Example 5 had a cell viability of 78.3%, and Example 1 had better in vitro cell compatibility.

[0069] Effects of different preparation conditions and carrier systems on IgG retention rate To further verify the protective effect of the carrier system and preparation process of this invention on IgG stability, a comparative experiment was set up under different conditions to detect the IgG retention rate immediately after preparation and after storage at 40°C for 30 days.

[0070] Table 7. Effects of different preparation conditions and carrier systems on IgG retention rate ( ±SD, n=3)

[0071] Note: *p<0.01 compared to Example 1.

[0072] The results showed that the type of foaming agent, preparation temperature, and free calcium and magnesium ion content in the aqueous phase all affected the IgG retention rate. This invention, by selecting a mild foaming system, low-temperature preparation conditions, and a low-free calcium and magnesium environment, is beneficial to maintaining the stability of IgG in bovine colostrum powder in the toothpaste system.

[0073] The effect of different active ingredient ratios on repair efficacy and IgG retention rate Different weight ratios of inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder were set, and the total addition amount of the repair composition was fixed at 1.0%. The rest of the formulation was the same as in Example 1. Toothpaste samples were prepared, and the scratch closure rate and IgG retention rate were tested after 24 hours.

[0074] Table 8. Effects of different active ingredient ratios on 24h scratch closure rate and IgG retention rate ( ±SD, n=3)

[0075] The results showed that when the weight ratio of inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder was 1:0.1–0.3:0.5–1.5, the 24-hour scratch closure rate and IgG retention rate were both at optimal levels, with a ratio of 1:0.2:1.0 showing the best overall effect. When the proportion of bovine colostrum powder was further increased to 1:1:3, the IgG retention rate decreased, and the scratch closure rate also decreased, indicating that a reasonable ratio range is important for balancing stability and repair-related properties.

[0076] Effects of different amounts of repairing composition on the stability and efficacy of the ointment The total addition amounts of the repair composition were set to 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, and 8%, respectively, with a fixed ratio of 1:0.2:1.0. The rest of the formulation was the same as in Example 1. The stability of the ointment and the 24-hour scratch closure rate were evaluated.

[0077] Table 9. Effect of the amount of repair composition added on the stability of the ointment and scratch closure rate.

[0078] The results showed that when the amount of the repair composition added was in the range of 0.1% to 5%, the ointment exhibited good stability, and the repair-related properties generally improved with increasing addition amount. However, when the addition amount exceeded 5%, the ointment became rough or its dispersibility decreased, and the IgG retention rate also decreased. Therefore, this invention selected 0.1% to 5% as the suitable addition range.

[0079] Results Analysis The results above demonstrate that this invention introduces bovine colostrum powder with an IgG content of no less than 10% into the basic combination of inactivated lactobacillus powder and N-acetylneuraminic acid, and employs low-temperature paste preparation and a mild carrier system to maintain a high retention rate of IgG in the bovine colostrum powder during toothpaste preparation and storage. Simultaneously, under simulated oral use conditions, the toothpaste extract containing inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder showed superior effects in oral mucosal cell scratch repair and inflammatory factor evaluation compared to the combination of inactivated lactobacillus powder and N-acetylneuraminic acid and bovine colostrum powder alone, indicating that the introduction of bovine colostrum powder can further improve the oral surface tissue condition and related performance of the basic combination. Comparative experiments on the carrier system and preparation process further show that controlling factors such as the type of foaming agent, preparation temperature, and free calcium and magnesium ion content is beneficial for maintaining the stability of IgG in the toothpaste system. The ratio optimization and addition experiments provide experimental basis for the selection of the numerical range in the claims. In summary, the technical solution of this invention has good industrial applicability and application value for further improving the performance of oral care products.

Claims

1. A toothpaste containing a post-biotic composition that helps improve the condition of oral surface tissues, characterized in that, Including repair compositions and toothpaste carriers; The repair composition includes inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder; The bovine colostrum powder is derived from the colostrum secreted by healthy cows within 72 hours after calving, and the content of immunoglobulin IgG in the bovine colostrum powder is not less than 10%.

2. The toothpaste according to claim 1, characterized in that, The repair composition comprises, by weight: 0.01–2 parts of inactivated lactobacillus powder; 0.01 to 1 part of N-acetylneuraminic acid; Bovine colostrum powder 0.01 to 2 parts.

3. The toothpaste according to claim 1 or 2, characterized in that, The weight ratio of the inactivated lactobacillus powder, N-acetylneuraminic acid, and bovine colostrum powder is 1:0.05-0.5:0.2-2. Preferably, the weight ratio of the inactivated lactobacillus powder, N-acetylneuraminic acid and bovine colostrum powder is 1:0.1-0.3:0.5-1.

5.

4. The toothpaste according to any one of claims 1 to 3, characterized in that, The repair composition comprises 0.1% to 5% of the total mass of the toothpaste.

5. The toothpaste according to claim 1, characterized in that, The bovine colostrum powder is a powder obtained by processing colostrum secreted by healthy cows within 72 hours after calving through one or more of the following methods: defatting, concentration, membrane filtration, spray drying, and freeze drying. Preferably, the bovine colostrum powder contains no less than 15% immunoglobulin IgG.

6. The toothpaste according to claim 1, characterized in that, The inactivated lactobacillus powder is inactivated Lactobacillus plantarum powder; Preferably, the inactivated Lactobacillus plantarum powder is a powder obtained by heat inactivation of Lactobacillus plantarum; More preferably, the content of N-acetylneuraminic acid is not less than 98% on a dry basis.

7. The toothpaste according to claim 1, characterized in that, The toothpaste carrier includes a humectant, abrasive, foaming agent, adhesive, and water; Based on the total mass of the toothpaste, the humectant comprises 20-70%, the abrasive comprises 16-50%, the foaming agent comprises 0.5-4%, the binder comprises 0.8-3%, and the balance comprises at least one of water, fragrance, sweetener, stabilizer, preservative and colorant.

8. The toothpaste according to claim 7, characterized in that, The moisturizer includes at least one of sorbitol, glycerin, PEG-400 and propylene glycol; The abrasive includes at least one of hydrated silica, calcium carbonate, and calcium hydrogen phosphate. The foaming agent includes at least one of sodium lauroyl sarcosinate, cocamidopropyl betaine, sodium lauroyl glutamate, sodium methyl cocoyl taurate, and alkyl glycoside. The adhesive includes at least one of cellulose gum, xanthan gum, carrageenan, hydroxyethyl cellulose, hydroxypropyl guar gum, and carbomer. Preferably, the foaming agent does not contain sodium lauryl sulfate, or the content of sodium lauryl sulfate is not higher than 0.5% of the total mass of the toothpaste.

9. The toothpaste according to any one of claims 1 to 8, characterized in that, The toothpaste has a pH of 6.0–8.0; Preferably, the water-soluble salt in the toothpaste is a sodium salt, and the free Ca in the aqueous extract of the toothpaste is... 2+ and Mg 2+ The total content is not higher than 0.5%; More preferably, after the toothpaste is prepared, the retention rate of IgG in the bovine colostrum powder is not less than 70%; and / or, after the toothpaste is stored at 40°C for 30 days, the retention rate of IgG in the bovine colostrum powder is not less than 60%.

10. A method for preparing toothpaste according to any one of claims 1 to 9, characterized in that, Includes the following steps: S1. Inactivated lactobacillus powder, N-acetylneuraminic acid, bovine colostrum powder and water-soluble raw materials are added to water and dispersed or dissolved at a temperature not exceeding 45°C to obtain the first premix. S2. Premix the abrasive, foaming agent and adhesive to obtain a second premix; S3. After mixing the humectant with the first premix, add the second premix under vacuum conditions, homogenize and degas to obtain the toothpaste; In step S3, the vacuum degree is -0.08 to -0.1 MPa, the homogenization speed is 900 to 1100 rpm, and the paste preparation temperature is 25 to 40℃.

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