Fermented mucus lactobacillus BN11 for improving oral health and application thereof

Abstract

The present invention discloses a fermented mucus lactobacillus for improving oral health and its application, wherein the fermented mucus lactobacillus is fermented mucus lactobacillus (Limosilactobacillus fermentum) BN11, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on November 22, 2023, with a deposit number of CGMCC No.29108. The fermented mucus lactobacillus BN11 for improving oral health provided by the present invention, its lysate can promote the proliferation of probiotics, regulate the balance of oral microecology, and promote oral mucosal repair. The probiotic fermented product provided by the present invention can increase the content of pectin, vitamin B2, vitamin C, and flavonoids in the composition, and the obtained postbiotics are rich in a variety of active natural small molecules, can be quickly absorbed by oral cells, reduce oral inflammation, inhibit the growth and reproduction of pathogenic bacteria, and improve oral health.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to a fermented mucus lactobacillus BN11 for improving oral health and an application thereof. Background Art

[0002] The oral cavity is the main entrance for food to enter the human body, and its health is closely related to human health. When people eat spicy food, sour food, sweet food, food with hard / sharp surface, unhygienic food, carbonated drinks, maocai, hot drinks and iced drinks and other irritating foods, it is easy to cause oral sub-health, which manifests as dental caries, gingivitis, oral ulcers, bad breath and other uncomfortable symptoms.

[0003] Most oral anti-inflammatory products on the market use mint or antibiotics to sterilize the oral cavity, which results in the elimination of beneficial bacteria in the oral cavity while sterilizing, causing an imbalance in the oral flora, damaging the oral immune barrier, and preventing the mucous membrane of oral ulcers from being repaired in a timely manner, making them more susceptible to invasion by pathogenic bacteria. Oral anti-inflammatory products need to be used repeatedly, and the treatment time is long, and they cannot fundamentally improve oral health. Therefore, it is necessary to develop a healthy and efficient product that can promote oral mucosal repair, regulate the balance of oral flora, and fundamentally improve oral health while reducing inflammation.

[0004] Fermentation is the process of using microorganisms to convert the functional factors and nutrients of the substrate into small molecules that can be directly absorbed by the human body. Products that directly use small molecules can reduce the transportation and loss of small molecules in the human body, have a shorter action time, more significant effects, and stronger affinity for the human body. Therefore, postbiotic products made by fermentation are becoming more and more popular. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to provide a fermented mucus Lactobacillus BN11 for improving oral health, the lysate of which can promote the proliferation of probiotics, regulate the balance of oral microecology, and promote the repair of oral mucosa.

[0006] A second object of the present invention is to provide a product for improving oral health.

[0007] The third object of the present invention is to provide a probiotic fermentation product.

[0008] A fourth object of the present invention is to provide a method for preparing a probiotic fermentation product.

[0009] One of the purposes of the present invention is achieved by the following technical solution:

[0010] A fermented mucus lactobacillus for improving oral health, wherein the fermented mucus lactobacillus is fermented mucus lactobacillus (Limosilactobacillus fermentum) BN11, and the fermented mucus lactobacillus (Limosilactobacillusfermentum) BN11 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on November 22, 2023, with a deposit number of CGMCC No.29108.

[0011] As a preferred embodiment of the present invention, the fermented Lactobacillus mucilaginosus BN11 improves oral health through at least one of the following items (I) to (II):

[0012] (Ⅰ) Promote the growth of beneficial bacteria Escherichia coli Nissle1917;

[0013] (Ⅱ) Reduce the levels of inflammatory factors interleukin-6, tumor necrosis factor, and interleukin-1β.

[0014] The second object of the present invention is achieved by adopting the following technical solution:

[0015] A product for improving oral health, wherein the active ingredients of the product include live bacteria and / or inactivated bacteria of Limosilactobacillus fermentum BN11, and the preservation number of the Limosilactobacillus fermentum BN11 is CGMCC No.29108.

[0016] As a preferred embodiment of the present invention, the product comprises probiotic fermentation precipitate and postbiotics.

[0017] As a preferred embodiment of the present invention, the postbiotics are lysates of probiotic fermentation products.

[0018] The third object of the present invention is achieved by adopting the following technical solution:

[0019] A probiotic fermented product, which is obtained by fermenting a composition with Limosilactobacillus fermentum BN11. The composition comprises the following components in weight percentage: 25-50 parts of kiwi fruit, 25-50 parts of peach, and 20-40 parts of low-temperature slightly fermented white tea; the preservation number of the fermented Lactobacillus fermentum BN11 is CGMCC No.29108.

[0020] The raw materials of the probiotic fermented product provided by the present invention are all fresh plant raw materials, including fruits and herbs. Kiwi fruit is rich in carbohydrates, dietary fiber, protein, organic acid, 17 kinds of amino acids, unsaturated fatty acids, multiple minerals and vitamins and other nutrients and functional factors for maintaining human health. Among them, it is famous for its high content of vitamin C and is known as the "crown of vitamin C". It has the functions of enhancing immunity, anti-inflammatory and anti-swelling, anti-oxidation, and repairing cell damage.

[0021] When ripe, peaches are rich in nutrients such as water-soluble pectin, iron, protein, sugars, vitamins, etc., which are beneficial for repairing oral mucosa and promoting wound healing.

[0022] White tea is a slightly fermented tea. According to the "Shennong Bencao Jing", "Shennong tasted hundreds of herbs and encountered seventy-two poisons every day, which he solved with tea." The "tea" here refers to white tea. The tea soup is rich in protein, amino acids, alkaloids, catechins, flavonoids, vitamins and other functional ingredients. It has a strong ability to scavenge free radicals and can be anti-inflammatory, antibacterial, anti-oxidant, anti-radiation, anti-viral, anti-tumor and enhance immunity.

[0023] The composition of the present invention is made of carefully selected fresh plant raw materials and undergoes two rounds of fermentation, i.e., low-temperature micro-fermentation + secondary fermentation. It not only retains and improves the content of functional ingredients such as pectin, vitamin B2, vitamin C, flavonoids, etc., but also, although it is a food formula, every drop of it is an essence with mild and significant effects. After entering the oral cavity, it can be quickly absorbed by oral cells, inhibit the growth and reproduction of pathogenic bacteria, reduce oral inflammation, promote oral mucosal repair, and improve oral health.

[0024] As a preferred embodiment of the present invention, the low-temperature micro-fermented white tea is white tea fermented by Limosilactobacillus fermentum BN11, and the fermentation conditions are as follows: under the conditions of a constant temperature of 22-30°C and a constant humidity of 55-80%, the fermented Lactobacillus fermentum BN11 bacterial liquid is evenly sprayed on the tea leaves, stirred evenly, and micro-fermented for 25-35 hours, stirring every 5-7 hours.

[0025] As a preferred embodiment of the present invention, the low-temperature slightly fermented white tea is low-temperature slightly fermented Silver Needle Baihao.

[0026] As a preferred embodiment of the present invention, the composition comprises the following components in percentage by weight: 50 parts of kiwi fruit, 25 parts of peach, and 25 parts of low-temperature slightly fermented white tea.

[0027] The fourth object of the present invention is achieved by adopting the following technical solution:

[0028] A method for preparing a probiotic fermentation product comprises the following steps:

[0029] Raw material selection: Choose kiwi fruit and peach;

[0030] Pretreatment: weigh the formula amount of kiwi fruit, wash, peel, take the pulp, and cut into pieces; weigh the formula amount of peaches, soak them in salt water, wash, and cut into pieces;

[0031] Picking fresh leaves: Select the first round of strong seedlings of spring tea, and only pick the fat and strong single seedlings on the new shoots to get the green tea leaves;

[0032] Opening green tea: weigh the formula amount of green tea, and spread it evenly on the water sieve, and dry it in natural sunlight to obtain base material A;

[0033] Strain activation: activating Limosilactobacillus fermentum BN11 to obtain an activated bacterial solution; centrifuging the activated bacterial solution, discarding the supernatant, and taking the precipitate to obtain activated bacterial mud of Limosilactobacillus fermentum BN11;

[0034] Preparation of bacterial liquid: adding activated bacterial sludge of fermented Lactobacillus fermentum BN11 into physiological saline, and mixing evenly to obtain a suspension, namely, fermented Lactobacillus fermentum BN11 bacterial liquid;

[0035] Micro-fermentation: transfer base material A indoors, cool it to room temperature, and evenly spray the fermented Lactobacillus fermentum BN11 bacterial solution on the tea leaves at a constant temperature of 22-30°C and a constant humidity of 55-80%, stir evenly, and micro-ferment for 25-35 hours, stirring every 5-7 hours to obtain base material B;

[0036] Drying: Place base material B in a drying room and dry it until the dryness reaches 2-8%, thereby obtaining Silver Needle White Needle C;

[0037] Grinding: Grind Silver Needle Baihao C with a grinder to obtain Silver Needle Baihao D;

[0038] Extraction: Place Baihao Yinzhen D in boiling water, stir, cool to room temperature, filter, and obtain base material E;

[0039] Wall breaking: put the diced kiwifruit and peach into a wall breaking machine, add base material E, break the wall, filter, adjust the pH, and obtain base material F;

[0040] Sterilization: sterilize and cool base material F to obtain base material G;

[0041] Fermentation: weigh the fermented mucus lactobacillus (Limosilactobacillus fermentum) BN11 bacterial liquid, add it to the base material G, and ferment it anaerobically for 16 hours to obtain the base material H;

[0042] Lysis: Lyse the base material H to obtain base material I;

[0043] Vacuum freeze drying: freeze the base material I to obtain a frozen product J, and vacuum freeze dry the frozen product J to obtain a dried product K;

[0044] Rehydration: Add the dried substance K into distilled water, stir to dissolve, adjust the pH, and put it into a spray bottle to obtain a postbiotic freeze-dried liquid oral spray, which is a fermented product of Limosilactobacillus fermentum BN11 for improving oral health.

[0045] The micro-fermentation technology of the present invention uses natural sunlight and indoor fermentation in combination, that is, firstly, the tea leaves are evenly exposed to sunlight under suitable sunlight outdoors to reduce water vapor on the surface and inside of the fresh tea leaves, reduce the cell respiration of the tea leaves, and are more conducive to the subsequent lactic acid bacteria micro-fermentation to transform nutrients; under certain external temperature and humidity conditions indoors, with the gradual loss of water and the action of lactic acid bacteria, the changes in leaf cell concentration and cell membrane permeability, as well as the activation of various enzymes, organic acids, tea polyphenols, amino acids, sugars, caffeine, chlorophyll and other substances change, thereby forming the unique flavor quality of white tea, and also allowing the nutrients to be more fully released when consumed later.

[0046] The high-pressure homogenization and crushing method of the present invention belongs to a physical crushing method, which is to impact and shear the cells in a high-pressure chamber, so that the cells are subjected to physical actions and thermal effects such as convection impact, high-speed shear, high-frequency oscillation, cavitation, etc., thereby destroying the cell wall structure and achieving the purpose of crushing the cells. It has the advantages of short crushing time, continuous operation, no secondary pollution, and large processing capacity.

[0047] The concentration technology of the present invention adopts vacuum freeze drying + rehydration. The feed liquid is first made into a dry product by vacuum freeze drying, and then the dry product is dissolved with a small amount of water to achieve the purpose of concentration. Without high temperature, the vitamins, flavonoids, minerals, amino acids, dietary fiber, active proteins, sugars and other nutrients of the original liquid are very well and completely retained, so that the nutritional content of the postbiotic freeze-dried liquid oral spray is higher and the effect is better. When the human body uses this oral spray, the oral cells can quickly and directly absorb and utilize these functional ingredients that have undergone biotransformation, promote oral mucosal repair, regulate the balance of oral flora, inhibit the growth and reproduction of harmful bacteria, reduce oral inflammation, and strengthen the oral immune barrier.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The fermented mucus Lactobacillus BN11 for improving oral health provided by the present invention has a lysate that can promote the proliferation of probiotics, regulate the balance of oral microecology, and promote the repair of oral mucosa.

[0050] (2) The probiotic fermentation product provided by the present invention can increase the content of pectin, vitamin B2, vitamin C, and flavonoids in the composition. The obtained postbiotics are rich in a variety of active natural small molecules, can be quickly absorbed by oral cells, reduce oral inflammation, inhibit the growth and reproduction of pathogenic bacteria, and improve oral health.

[0051] Biomaterial preservation information: Fermented mucus Lactobacillus BN11, the preservation number is CGMCC No.29108, the classification name is: Fermented mucus Lactobacillus Limosilactobacillus fermentum, it was deposited on November 22, 2023 in the General Microbiology Center of China Culture Collection Administration (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101), and the abbreviation of the preservation unit is CGMCC. DETAILED DESCRIPTION

[0052] The present invention is further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments. The raw materials, equipment, etc. used in the following embodiments can be purchased through commercial channels unless otherwise specified.

[0053] System 1: Screening for strains of postbiotics that can promote the proliferation of beneficial bacteria

[0054] 1. Experimental strains

[0055] (1) Lactic acid bacteria: Bifidobacterium animalis subsp. lactis, Lactobacillus casei, Pediococcus pentosaceus, Lactobacillus mucosa fermentum, Lactobacillus plantarum, Streptococcus salivarius subsp. thermophilus.

[0056] (2) Beneficial bacteria: Escherichia coli Nissle1917.

[0057] 2. Culture medium preparation

[0058] (1) MRS liquid medium: peptone 10 g / L, beef extract 5 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 mL, pH 6.2, make up to 1000 mL with distilled water, and sterilize at 121°C for 15 min.

[0059] (2) MRS solid medium: Add 2% agar to MRS liquid medium and sterilize at 121°C for 15 min for viable bacteria counting.

[0060] (3) NB liquid culture medium: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, pH = 7.2, sterilized at 121°C for 15 min.

[0061] (4) NB solid medium: Add 15.0 g / L agar to NB nutrient broth medium and sterilize at 121°C for 15 min.

[0062] 3. Bacteria activation

[0063] (1) Lactic acid bacteria: After the strains frozen in -80°C glycerol storage were thawed at room temperature, the bacterial solution was dipped on MRS solid medium under sterile conditions for streaking, and cultured in a constant temperature incubator at 37°C for 24 hours. A single colony was picked and inoculated into MRS liquid medium, and cultured at 37°C for 24 hours. Then, it was inoculated into MRS liquid medium at an inoculum of 2%, and activated for two generations to obtain activated bacterial solution. The activated bacterial solution was centrifuged at 4°C at a speed of 4000 r / min for 5 minutes, and then the supernatant was discarded and the precipitate was taken to obtain activated bacterial mud.

[0064] (2) Beneficial bacteria: After the strains frozen in -80°C glycerol storage were thawed at room temperature, the bacterial solution was dipped into NB solid culture medium for streaking under sterile conditions, and cultured in a constant temperature incubator at 37°C for 24 hours. A single colony was picked and inoculated into NB liquid culture medium, and cultured at 37°C for 24 hours. Then, it was inoculated into NB liquid culture medium at an inoculum of 2%, and activated for two generations to obtain activated bacterial solution. The activated bacterial solution was centrifuged at 4°C at a speed of 4000 r / min for 5 minutes, and then the supernatant was discarded and the precipitate was taken to obtain activated bacterial mud.

[0065] 4. Experimental methods

[0066] (1) Preparation of lysate: Add 10% of activated bacterial slurry of lactic acid bacteria to physiological saline, and mix well with a vortex mixer to obtain a suspension; place the suspension in a high-pressure homogenizer to lyse the cells, and repeat 5 times at a homogenization pressure of 1200 bar and an interval of 5 minutes to obtain a lysate, which is a lactic acid bacteria postbiotic.

[0067] (2) Culture medium preparation

[0068] Liquid A: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, animal Bifidobacterium lactis subsp. postbiotics 4 mg / mL, pH = 7.2, sterilized at 121°C for 15 min.

[0069] Solution B: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, Lactobacillus casei postbiotics 4 mg / mL, pH = 7.2, sterilized at 121°C for 15 min.

[0070] Liquid C: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, Pediococcus pentosaceus postbiotics 4 mg / mL, pH = 7.2, sterilized at 121°C for 15 min.

[0071] Liquid D: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, fermented mucus lactobacillus postbiotics 4 mg / mL, pH = 7.2, sterilized at 121°C for 15 min.

[0072] Liquid E: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, Lactobacillus plantarum postbiotics 4 mg / mL, pH = 7.2, sterilized at 121°C for 15 min.

[0073] Liquid F: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, Streptococcus salivarius thermophilus subsp. postbiotics 4 mg / mL, pH = 7.2, sterilized at 121°C for 15 min.

[0074] (3) Vaccination

[0075] The activated bacterial sludge of Escherichia coli Nissle1917 was inoculated at an inoculation rate of 1% into the liquid culture medium added with postbiotics, i.e., A to F liquids, as experimental groups, recorded as Examples 1 to 6; the activated bacterial sludge of Escherichia coli Nissle1917 was inoculated at an inoculation rate of 1% into the NB liquid culture medium, as a blank group, recorded as Example 7.

[0076] (4) Cultivation

[0077] At the same time, it was placed in a 37°C constant temperature water bath shaker (120 rpm) for shaking culture. Samples were taken at different time points within 10 hours after inoculation and culture (0h, 2h, 4h, 6h, 8h, 10h), and the growth changes of probiotics were determined by turbidimetry. The effects of different postbiotics on the proliferation of E. coli Nissle1917 were compared. The results are shown in Table 1.

[0078] The turbidimetric test method is as follows: 200 μL of the sample to be tested is pipetted into a 96-well plate, and its OD value (OD600nm) at a wavelength of 600nm, ie, the growth change of E. coli Nissle1917, is measured using an enzyme marker.

[0079] 5. Results and Analysis

[0080]

[0081] Escherichia coli Nissle1917, referred to as EcN, is a beneficial bacterium that participates in the immune regulation of the host body, relieves the body's inflammatory response, and enhances human immunity. As shown in Table 1, compared with the blank group, the OD600nm values ​​of the experimental groups increased, indicating that the postbiotics in the experimental groups have different degrees of proliferation effects on the beneficial bacteria, among which Example 4 has the most significant effect and the strongest proliferation effect on the beneficial bacteria. Therefore, the strain of Example 4 is selected as the best strain for postbiotics to promote the proliferation of beneficial bacteria.

[0082] System 2: Screening for strains of postbiotics that promote the proliferation of beneficial bacteria

[0083] 1. Experimental strains: fermented mucus Lactobacillus BN11, fermented mucus Lactobacillus CICC 21800 (preservation number: CICC21800), fermented mucus Lactobacillus CICC 21828 (preservation number: CICC 21828)

[0084] 2. Experimental method: The experimental strains were used according to the method of system 1 to determine their effect on the proliferation of beneficial bacteria.

[0085] 3. Experimental results: The results are shown in Table 2.

[0086]

[0087] It can be seen from Table 2 that different strains of postbiotics have different effects on the proliferation of beneficial bacteria. Among them, the effect of fermented Lactobacillus mucilaginosus BN11 is the most significant and has the strongest effect on the proliferation of beneficial bacteria.

[0088] System 3: Screening of postbiotic strains that can promote oral mucosal repair

[0089] 1. Experimental strains: fermented Lactobacillus mucilaginosus BN11, fermented Lactobacillus mucilaginosus CICC 21800 (preservation number: CICC21800), fermented Lactobacillus mucilaginosus CICC 21828 (preservation number: CICC 21828).

[0090] 2. Experimental animals: 60 SPF SD rats, half male and half female, 8 weeks old, weighing 200 g.

[0091] 3. Experimental methods

[0092] (1) Sample preparation

[0093] 1) Culture medium preparation

[0094] MRS liquid culture medium: peptone 10g / L, beef extract 5g / L, yeast extract powder 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, sodium acetate 5g / L, triammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, Tween 80 1mL, pH 6.2-6.4, make up to 1000mL with distilled water, sterilize at 121℃ for 15min.

[0095] MRS solid medium: Add 2% agar to MRS liquid medium and sterilize at 121°C for 15 minutes for viable bacteria counting.

[0096] 2) Strain activation

[0097] After the fermented mucus lactobacillus frozen in -80°C glycerol storage is thawed at room temperature, the bacterial solution is dipped on the MRS solid culture medium under sterile conditions for streaking, and the culture is cultured in a constant temperature incubator at 37°C for 24 hours, a single colony is picked and inoculated into the MRS liquid culture medium, and the culture is statically cultured at 37°C for 24 hours, and then inoculated into the MRS liquid culture medium with an inoculum amount of 2%, and activated for two generations to obtain an activated bacterial solution. The activated bacterial solution is centrifuged at 4°C at a speed of 4000r / min for 5 minutes, and then the supernatant is discarded and the precipitate is taken to obtain the activated bacterial mud of the fermented mucus lactobacillus.

[0098] 3) Preparation of bacterial solution

[0099] The activated bacterial mud of fermented Lactobacillus mucilaginosus was added into physiological saline at an addition rate of 10%, and mixed evenly with a vortex mixer to obtain a suspension, which was the fermented Lactobacillus mucilaginosus bacterial liquid.

[0100] 4) Lysis

[0101] The fermented Lactobacillus mucilaginosus bacterial liquid is placed in a high-pressure homogenizer to lyse the cells. The homogenization pressure is 1200 bar, the interval is 5 minutes, and this process is repeated 5 times to obtain a lysed product, which is the postbiotic.

[0102] (2) Animal treatment

[0103] 1) Rats grouping

[0104] The rats were randomly divided into 5 groups according to body weight and gender, namely blank group, model group, group A, group B, and group C.

[0105] 2) Rat modeling

[0106] Except for the blank group, rats in all groups were anesthetized by intraperitoneal injection of 3 mL / kg 10% chloral hydrate solution, and 10% acetic acid solution was injected under the buccal mucosa on the left side of the mouth, with each rat being injected with 0.05 mL. Rats in the blank group were injected with an equal amount of normal saline at the same position of the oral mucosa.

[0107] 3) Administration

[0108] Each group of rats was subjected to the modeling 24 hours after the modeling. The blank group and the model group used a sterile cotton swab to dip 0.5 mL of 0.9% sodium chloride injection and evenly applied it on the oral ulcer of the rats. The rats in group A used a sterile cotton swab to dip 0.5 mL of fermented mucus Lactobacillus BN11 postbiotics and evenly applied it on the oral ulcer of the rats; the rats in group B used a sterile cotton swab to dip 0.5 mL of fermented mucus Lactobacillus CICC 21800 postbiotics and evenly applied it on the oral ulcer of the rats; the rats in group C used a sterile cotton swab to dip 0.5 mL of fermented mucus Lactobacillus CICC 21828 postbiotics and evenly applied it on the oral ulcer of the rats. The frequency of administration was 2 times / day, and the administration was continued for 14 days.

[0109] (3) Detection indicators

[0110] 1) Body weight changes: The changes in the oral mucosa of the animals, the general behavioral activities of the animals, and the food intake of the animals in each group were observed daily. The animals in each group were weighed on D4, D7, D10, and D14, and the body weight growth rate (%) was calculated. The results are shown in Table 3.

[0111]

[0112] 2) Detection of oral mucosal cytokines: After the last administration, rats in each group were anesthetized by intraperitoneal injection of 60 mg / mL of Zotal 50, and then euthanized by bleeding from the abdominal aorta. The left oral mucosal tissue was obtained and added with 0.9% sodium chloride injection at a ratio of 1:9 (g / mL). The mixture was homogenized at high speed to prepare oral mucosal homogenate. The mixture was centrifuged at 3000 r / min at 4°C for 15 min, and the supernatant was collected and used for ELISA kit.

[0113] The contents of IL-6, TNF-α, and IL-1β in the supernatant were detected. The results are shown in Table 3.

[0114] 3) Histopathological examination: The right oral mucosal tissue was fixed in 10% neutral formaldehyde solution for 24 h and stained with HE. The pathological changes of the ulcer mucosal tissue were quantitatively scored. The results are shown in Table 6.

[0115]

[0116]

[0117] 4. Results and Analysis

[0118] 1) Weight changes

[0119]

[0120] On D1 after modeling, except for the blank group, redness and swelling were visible in the mouths of rats in all groups. On D3 after modeling, the rats in the model group gradually reduced their food intake, and their daily activities decreased and accompanied by drooling. On D4-D10 after modeling, the oral mucosa of the rats in the model group turned white, forming a certain pseudomembrane, accompanied by drooling. Under the intervention of postbiotics, the symptoms of oral ulcers in rats were improved to varying degrees, among which the effect of group A was the most significant. As shown in Table 3, compared with the blank group, the weight of rats in the model group increased slowly, and the weight of rats in groups A, B and C increased to varying degrees. Among them, the weight of rats in group A increased the most, indicating that the postbiotics in group A had an effect on the recovery of oral ulcers in rats, making the daily diet of rats gradually return to normal.

[0121] 2) Oral mucosal cytokine detection

[0122]

[0123] As shown in Table 4, compared with the blank group, the levels of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and interleukin-1β (IL-1β) in the model group were significantly increased, indicating that the oral inflammation of the rats was more severe; the levels of inflammatory factors in groups A, B, and C decreased to varying degrees, indicating that under the intervention of postbiotics, the oral inflammation of rats could be improved, among which the postbiotics in group A had the best effect, which could inhibit the expression of cytokines, thereby alleviating and inhibiting the oral inflammatory response.

[0124] 3) Histopathological examination

[0125]

[0126] It can be seen from Table 6 that compared with the blank group, the oral ulcer mucosal tissue pathology score of the rats in the model group was higher, indicating that the oral ulcer symptoms of the rats were more severe and a large number of oral cells were damaged; the oral ulcer mucosal tissue pathology scores of groups A, B, and C were all reduced to varying degrees, indicating that under the intervention of postbiotics, the oral ulcer mucosa of the rats was repaired relatively well, among which the effect of group A was the most significant, indicating that the postbiotics in group A can promote the repair of oral mucosa, accelerate wound healing, and avoid persistent infection in ulcers.

[0127] In summary, the strains in group A were selected as the best strains to promote oral mucosal repair, namely, fermentative Lactobacillus mucosa BN11, whose lysate is rich in nutrients and efficacy factors, and can promote the proliferation of beneficial oral bacteria and oral mucosal repair.

[0128] System 4: Screening of a postbiotic formula to improve oral health

[0129] 1. Sample preparation

[0130]

[0131] (1) Raw material selection: Select 80% ripe and soft kiwi fruit, relatively mature, fragrant and non-moldy peaches, and ripe and non-rotten oranges;

[0132] (2) Pretreatment: Weigh kiwi fruit, honey peach, and orange according to the weight parts in Table 7, wash the kiwi fruit and orange, peel them, take out the pulp, and cut them into 3×3 cm small pieces; weigh the honey peach, soak it in salt water for 30 minutes, wash it, and cut it into 3×3 cm small pieces;

[0133] (3) Picking fresh leaves: Select the first round of strong seedlings of spring tea, and only pick the plump single seedlings on the new shoots to get the green tea. The single seedlings picked in this way are plump and thick, with high quality. After sufficient brewing, the buds are plump and the front seedlings are upright;

[0134] (4) Opening the green tea leaves: Weigh the green tea leaves according to the weight parts in Table 7, spread the green tea leaves evenly on the water sieve to a thickness of 3 mm, and expose them to natural sunlight for 6 hours at an outdoor temperature of 30°C to obtain base material A;

[0135] (5) Culture medium preparation:

[0136] 1) MRS liquid medium: peptone 10 g / L, beef extract 5 g / L, yeast extract powder 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 mL, pH 6.2, dilute to 1000 mL with distilled water, sterilize at 121 °C for 15 min;

[0137] 2) MRS solid medium: Add 2% agar to MRS liquid medium and sterilize at 121°C for 15 min for viable bacteria counting;

[0138] (6) Strain activation: After the fermented mucus lactobacillus BN11 frozen in -80°C glycerol storage is thawed at room temperature, the bacterial solution is dipped on the MRS solid culture medium for streaking under sterile conditions, and cultured in a constant temperature incubator at 37°C for 24 hours. A single colony is picked and inoculated into the MRS liquid culture medium, and then statically cultured at 37°C for 24 hours, and then inoculated into the MRS liquid culture medium with an inoculum of 2%, and activated for two generations to obtain an activated bacterial solution. The activated bacterial solution is centrifuged at 4°C and 4000r / min for 5 minutes, and then the supernatant is discarded and the precipitate is taken to obtain the activated bacterial mud of the fermented mucus lactobacillus BN11;

[0139] (7) Preparation of bacterial solution: adding 10% of activated bacterial sludge of fermented Lactobacillus mucilaginosus BN11 to physiological saline, and mixing with a vortex mixer to obtain a suspension, namely, the fermented Lactobacillus mucilaginosus BN11 bacterial solution;

[0140] (8) Micro-fermentation: transfer base material A to a room, cool it to room temperature, and evenly spray the fermented Lactobacillus mucilaginosus BN11 bacterial solution on the tea leaves at a constant temperature of 25° C. and a constant humidity of 68%, stir evenly, and micro-ferment for 30 hours, stirring every 6 hours, to obtain base material B;

[0141] (9) Drying: placing base material B in a drying room at 45° C. and drying for 10 h until the dryness reaches 6%, thereby obtaining Baihao Yinzhen C. At this temperature, the biological activity of the enzyme is the strongest, which can promote the rapid oxidation of some polyphenols in a short time, thus facilitating further oxidation of the contents;

[0142] (10) Grinding: Grind the Silver Needle White Needle C into 60-mesh particles using a grinder to obtain Silver Needle White Needle D;

[0143] (11) Extraction: Place 5 times the amount of Baihao Yinzhen D in 100°C boiling water, keep stirring for 15 minutes, cool to room temperature, and filter with a 100-mesh sieve to obtain base material E;

[0144] (12) Wall breaking: Place the diced kiwifruit and peach in a wall breaking machine, add base material E, and break the wall at a speed of 24,000 r / min for 3 min. Filter with a 100-mesh sieve and adjust the pH to 6.2 to obtain base material F;

[0145] (13) Sterilization: sterilize base material F at 80° C. for 30 min, and cool to 37° C. to obtain base material G;

[0146] (14) Fermentation: Weigh 5 parts of fermented Lactobacillus mucilaginosus BN11 bacterial solution, add it to base material G, and ferment it anaerobically at 37° C. for 16 hours to obtain base material H;

[0147] (15) Lysis: Place base material H in a high-pressure homogenizer to lyse the cells. Repeat 5 times at a homogenization pressure of 1200 bar and an interval of 5 min to obtain base material I.

[0148] (16) Vacuum freeze drying: base material I was placed on a freeze drying box shelf in a vacuum freeze dryer (the shelf temperature was previously reduced to -10°C), with a thickness of 6 mm. After cooling the water trap to -40°C, the vacuum pump was turned on to directly evacuate and freeze for 15 min, with a vacuum degree of 500 Pa, to obtain frozen product J. The frozen product J was further evacuated and vacuum freeze-dried for 730 min at -5°C and a vacuum chamber pressure of 10 Pa, to obtain dried product K.

[0149] (17) Rehydration: Add 2 times distilled water to the dried substance K, stir to dissolve, adjust the pH to 6.6, and put it into a spray bottle to obtain a postbiotic freeze-dried liquid oral spray, which is the fermented Lactobacillus mucilaginosus BN11 postbiotic for improving oral health, and is used as a test sample.

[0150] In addition, Example 12 is specially added as a blank group, and the other examples are experimental groups. Example 12 is equal to the composition formula of Example 9, except that, in Example 12, fermented mucus Lactobacillus BN11 is not added for micro-fermentation and secondary fermentation (steps 5, 6, 7, and 14 are not included; the spraying liquid in step 8 is changed to water, and step 9 remains unchanged, and the whole process is to remove the fermentation strain).

[0151] 2. Detection method

[0152] (1) The pectin content of the test samples was tested according to "NY / T 2016 Determination of Pectin Content in Fruits and Their Products by Spectrophotometry", and the results are shown in Table 8;

[0153] (2) The vitamin B2 content of the test samples was tested according to "GB 5009.85 National Food Safety Standard Determination of Vitamin B2 in Foods", and the results are shown in Table 8;

[0154] (3) The vitamin C content of the test samples was tested according to "GB 14754 National Food Safety Standard Food Additive Vitamin C (Ascorbic Acid)", and the results are shown in Table 8;

[0155] (4) The flavonoid content of the test samples was tested according to “SN / T 4592-2016 Determination of total flavonoids in exported food”. The results are shown in Table 8.

[0156] 3. Results and analysis

[0157]

[0158] Pectin in postbiotics is a soluble dietary fiber. Studies have shown that soluble dietary fiber has prebiotic-like effects, can promote the proliferation of oral probiotics, and is beneficial for regulating the balance of the bacterial flora.

[0159] Studies have shown that vitamin B2 and vitamin C can promote the development of human cells and tissue regeneration, promote the healing of oral ulcer wounds, and enhance the immunity of oral mucosa.

[0160] Excessive free radicals produced by the imbalance of metabolism and biochemical reactions in the human body can damage biomacromolecules and cell structures such as DNA, proteins, and mitochondria, leading to inflammation. Studies have shown that flavonoids have strong antioxidant capacity and can remove free radicals in the oral cavity, thereby reducing the body's inflammatory response and helping to reduce oral inflammation.

[0161] As shown in Table 8, compared with Example 12, the pectin, vitamin B2, vitamin C and flavonoid contents of the experimental groups (Examples 8-11, 16) are increased, indicating that after the composition of the appropriate ratio is fermented by the strain, various nutrients and functional ingredients can be well converted and released, but the fermentation effects of different prescriptions are significantly different. Due to the lack of prescription types in Examples 13-14, the content of nutrients is greatly affected. Therefore, in order to ensure the fermentation effect, none of the prescriptions can be deleted. The pectin, vitamin B2, vitamin C and flavonoid contents of Example 9 are the highest, and the fermentation effect is the best, so the prescription of Example 9 is selected as the best prescription for fermenting Lactobacillus mucilaginosus BN11 postbiotics.

[0162] The fermented mucus Lactobacillus BN11 postbiotics provided by the embodiment of the present invention are made from a carefully selected plant formula combined with the best oral lactic acid bacteria using new processes such as two rounds of deep fermentation, lysis, and low-temperature concentration. The resulting postbiotic essence is rich in fermentation metabolites of the strain, pectin, vitamin B2, vitamin C, flavonoids, and a variety of nutrients and efficacy factors after bacterial lysis. After entering the oral cavity, it can be quickly absorbed and utilized by cells, thereby significantly improving oral inflammation and promoting mucosal healing of oral ulcers. In addition, it can also promote the proliferation of beneficial bacteria, inhibit the growth and reproduction of harmful bacteria, and improve the oral microenvironment. In particular, it also removes organic acids, balances the intestinal flora, and reduces the impact of acids on teeth. The dosage form presented is a spray, which is easy to use, has a wide atomization area in the oral cavity, can fully cover the oral cavity, and improve oral health.

[0163] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A fermented mucus lactobacillus for improving oral health ( Limosilactobacillus fermentum ), characterized in that, The fermented mucus Lactobacillus is fermented mucus Lactobacillus BN11, and the fermented mucus Lactobacillus BN11 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 22, 2023, with a deposit number of CGMCC No.29108.

2. A product for improving oral health, characterized in that: The active ingredients of the product include the lysate of fermented Lactobacillus mucilaginosus BN11, and the preservation number of the fermented Lactobacillus mucilaginosus BN11 is CGMCC No.29108.

3. A probiotic fermentation product, characterized in that: The fermented product is a lysate obtained by fermenting a composition with fermented Lactobacillus mucilaginosus BN11. The composition comprises the following components in weight percentage: 25-50 parts of kiwi fruit, 25-50 parts of honey peach, and 20-40 parts of low-temperature slightly fermented white tea. The preservation number of the fermented Lactobacillus mucilaginosus BN11 is CGMCC No.29108.

4. The probiotic fermented product according to claim 3, characterized in that: The low-temperature micro-fermented white tea is white tea fermented with fermented Lactobacillus mucilaginosus BN11, and the fermentation conditions are as follows: under the conditions of constant temperature of 22-30°C and constant humidity of 55-80%, the fermented Lactobacillus mucilaginosus BN11 bacterial liquid is evenly sprayed on the tea leaves, stirred evenly, and micro-fermented for 25-35 hours, stirring every 5-7 hours.

5. The probiotic fermented product according to claim 3, characterized in that: The low-temperature slightly fermented white tea is low-temperature slightly fermented Silver Needle Baihao.

6. The probiotic fermented product according to claim 3, characterized in that: The composition comprises the following components in percentage by weight: 50 parts of kiwi fruit, 25 parts of honey peach, and 25 parts of low-temperature slightly fermented white tea.

7. A method for preparing a probiotic fermented product according to any one of claims 3 to 6, characterized in that: The following steps are involved: Raw material selection: Choose kiwi fruit and peach; Pretreatment: weigh the formula amount of kiwi fruit, wash, peel, take the pulp, and cut into pieces; weigh the formula amount of peaches, soak them in salt water, wash, and cut into pieces; Picking fresh leaves: Select the first round of strong seedlings of spring tea, and only pick the fat and strong single seedlings on the new shoots to get the green tea leaves; Opening green tea: weigh the formula amount of green tea, and spread it evenly on the water sieve, and dry it in natural sunlight to obtain base material A; Strain activation: activating the fermented Lactobacillus mucilaginosus BN11 to obtain an activated bacterial solution; centrifuging the activated bacterial solution, discarding the supernatant, and taking the precipitate to obtain an activated bacterial mud of the fermented Lactobacillus mucilaginosus BN11; Preparation of bacterial solution: adding the activated bacterial sludge of fermented Lactobacillus mucilaginosus BN11 into physiological saline, and mixing evenly to obtain a suspension, which is the fermented Lactobacillus mucilaginosus BN11 bacterial solution; Micro-fermentation: transfer base material A to the room, cool it to room temperature, and evenly spray the fermented Lactobacillus mucilaginosus BN11 liquid on the tea leaves at a constant temperature of 22-30°C and a constant humidity of 55-80%, stir evenly, and micro-ferment for 25-35 hours, stirring every 5-7 hours to obtain base material B; Drying: Place base material B in a drying room and dry it until the dryness reaches 2-8%, and obtain Baihao Yinzhen C; Grinding: Grind Silver Needle Baihao C with a grinder to obtain Silver Needle Baihao D; Extraction: Place Baihao Yinzhen D in boiling water, stir, cool to room temperature, filter, and obtain base material E; Wall breaking: put the diced kiwifruit and peach into a wall breaking machine, add base material E, break the wall, filter, adjust the pH, and obtain base material F; Sterilization: sterilize and cool base material F to obtain base material G; Fermentation: Weigh the fermented Lactobacillus mucilaginosus BN11 bacterial liquid, add it to the base material G, and ferment it anaerobically for 16 hours to obtain the base material H; Lysis: Lyse the base material H to obtain base material I; Vacuum freeze drying: freeze the base material I to obtain a frozen product J, and vacuum freeze dry the frozen product J to obtain a dried product K; Rehydration: Add the dried substance K into distilled water, stir to dissolve, adjust the pH, and put it into a spray bottle to obtain a postbiotic freeze-dried liquid oral spray, which is a fermented product of Lactobacillus mucilaginosus BN11 for improving oral health.

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