Rhamnosus lactis gg fermentum having a function of promoting healing of an oral ulcer wound and a method for preparing the same

Abstract

The application discloses a rhamnosus lactobacillus GG fermentation product with a function of promoting oral ulcer wound healing and a preparation method thereof. The rhamnosus lactobacillus GG fermentation product is obtained by fermenting a composition by rhamnosus lactobacillus GG, and the composition comprises the following components in parts by weight: camellia 33-47 parts, kiwi 33-35 parts, yeast beta-glucan 1-3 parts, fructooligosaccharide 15-33 parts and purified water 200-300 parts. The rhamnosus lactobacillus GG fermentation product with the function of promoting oral ulcer wound healing is obtained by fermenting the composition by rhamnosus lactobacillus GG, and the obtained fermentation product is a formula compound, can promote oral ulcer healing, accelerate tissue growth, has a remarkable effect and is healthy and safe.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and in particular to a Lactobacillus rhamnosus GG ferment that promotes the healing of oral ulcers and its preparation method. Background Technology

[0002] Oral ulcers, also known as canker sores, are ulcerative lesions that occur on the oral mucosa. They are characterized by redness, swelling, heat, and pain. They have a high incidence rate and can occur in any part of the oral mucosa, such as the lips, cheeks, tongue, palate, etc. When they occur, they are usually accompanied by a significant burning pain, which is persistent, prone to recurrence, and difficult to heal. This affects the patient's normal eating, speech, sleep, and mental state, thereby reducing the quality of life.

[0003] Intact skin and mucous membranes are the body's first line of defense against external pathogens, so timely epidermal healing is particularly important for tissue repair after they are damaged.

[0004] Most products on the market that relieve oral ulcers are anti-inflammatory and analgesic. While they can relieve oral ulcers by reducing inflammation, their ability to promote wound healing is somewhat inferior. The faster the wound heals, the better it is for the oral mucosal barrier to recover, thereby strengthening the oral mucosa's ability to resist pathogens and preventing the oral ulcer from worsening. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a Lactobacillus rhamnosus GG ferment that promotes the healing of oral ulcers.

[0006] The second objective of this invention is to provide a method for preparing Lactobacillus rhamnosus GG ferment that promotes the healing of oral ulcers.

[0007] One of the objectives of this invention is achieved through the following technical solution:

[0008] A fermented product of Lactobacillus rhamnosus GG that promotes the healing of oral ulcers is obtained by fermenting a composition with Lactobacillus rhamnosus GG. The composition comprises the following components in parts by weight: 33-47 parts of camellia, 33-35 parts of kiwi, 1-3 parts of yeast β-glucan, 15-33 parts of fructooligosaccharides, and 200-300 parts of purified water.

[0009] Camellia flowers contain a variety of active ingredients, such as polyphenols, triterpenoids, alkaloids, and flavonoids, which have various effects such as anti-oxidation, antibacterial, anti-allergy, anti-inflammatory, hemostasis, and promoting wound healing. They can reduce the inflammatory response of oral ulcers, eliminate superoxide free radicals, reduce the generation of free radicals in oral ulcers, and prevent the ulcers from worsening.

[0010] Kiwifruit is rich in various vitamins, organic acids, actinidin, polysaccharides, and a variety of essential amino acids and trace elements. It has high nutritional value and a long history of being used for food and medicine. It was recorded as early as 2,000 years ago in the "Erya" and was later included in "Ben Cao Yan Yi" and "Ben Cao Gang Mu". It can promote the growth of tissues in oral ulcers, repair the oral mucosal barrier function, and accelerate the recovery of oral ulcers.

[0011] Yeast β-glucan contains more β-1,6-glycosidic bonds than β-glucan from other sources, exhibiting stronger biological activity. It can regulate the levels of inflammatory factors, immune proteins, and other immune factors in the body, and improve the expression of inflammatory pathways to exert immunomodulatory effects, which is beneficial for improving oral mucosal immunity and preventing infection of oral ulcers.

[0012] Fructooligosaccharides can be fermented by beneficial oral bacteria (such as lactobacilli and bifidobacteria) and partially converted into short-chain fatty acids (mainly acetic acid, propionic acid and butyric acid) and a small amount of gas, which is beneficial to the host's oral health. In addition, as a prebiotic, fructooligosaccharides can also promote the growth and reproduction of beneficial oral bacteria, inhibit the proliferation of pathogenic bacteria at ulcer sites, regulate the balance of oral flora, and improve the oral microecology.

[0013] The composition of this invention is highly innovative in both the selection and proportion of its ingredients. With camellia, kiwi, yeast β-glucan, and fructooligosaccharides as core components, it encompasses sources from herbal, fruit, and microbial levels, promoting the healing of oral ulcers from multiple angles. Furthermore, the formulation complex, prepared through in vitro fermentation with *Lactobacillus rhamnosus* GG, is rich in vitamin C, short-chain fatty acids, amino acids, glycoproteins, and various probiotics. Upon contact with the mucosal tissue of the oral ulcer, it can be rapidly absorbed and utilized by cells, not only promoting wound healing but also strengthening the healed mucosa and preventing recurrence.

[0014] As a preferred embodiment of the present invention, the composition comprises the following components in parts by weight: 47 parts of camellia flower, 35 parts of kiwi fruit, 3 parts of yeast β-glucan, 15 parts of fructooligosaccharide, and 261 parts of purified water.

[0015] In a preferred embodiment of the present invention, during the preparation of the ferment, *Lactobacillus rhamnosus* GG activated bacterial solution is inoculated into the composition, and the concentration of the *Lactobacillus rhamnosus* GG activated bacterial solution is 2.0 × 10⁻⁶. 11 CFU / mL, inoculation amount 3-9%.

[0016] As a preferred embodiment of the present invention, the inoculation amount of Lactobacillus rhamnosus GG activated bacterial solution is 3-5%.

[0017] As a preferred embodiment of the present invention, after fermenting the composition with Lactobacillus rhamnosus GG to obtain the fermented product, the fermented product is pulverized using an ultrafine pulverizer.

[0018] The second objective of this invention is achieved by the following technical solution:

[0019] A method for preparing Lactobacillus rhamnosus GG ferment with the function of promoting the healing of oral ulcers includes the following steps:

[0020] Raw material selection: Camellia flowers, kiwi fruit, yeast β-glucan, and fructooligosaccharides were selected;

[0021] Pretreatment: Wash the camellia flowers with clean water, drain, and take the petals according to the formula; wash the kiwi fruit with clean water, peel it, take the pulp, cut it into pieces, and weigh it according to the formula; weigh the yeast β-glucan and oligofructose according to the formula.

[0022] Pulping: Add the processed camellia flowers to the first part of purified water and pulp to obtain camellia flower pulp A; add the processed kiwi fruit to the second part of purified water and break the cell wall for 1 minute to obtain kiwi fruit pulp B; add the processed yeast β-glucan to the third part of purified water, mix evenly, and homogenize to obtain base material C; add the processed oligofructose to the fourth part of purified water and stir to dissolve to obtain base material D;

[0023] Extraction: Place camellia flower pulp A in an ice water bath, stir, centrifuge, collect the supernatant, filter, and obtain camellia flower filtrate E;

[0024] Mixing: Mix camellia filtrate E, kiwi pulp B, base material C, and base material D evenly to obtain base material F;

[0025] Strain activation: Lactobacillus rhamnosus GG was activated to obtain bacterial culture G;

[0026] Fermentation: Inoculate bacterial culture G into substrate F, and incubate at a constant temperature to obtain fermentation product H.

[0027] As a preferred embodiment of the present invention, the method for preparing the Lactobacillus rhamnosus GG ferment with the function of promoting the healing of oral ulcers further includes the following steps:

[0028] Vacuum freeze drying: Fermentation product H is pre-frozen, and immediately placed in a freeze dryer for continuous freeze drying to obtain freeze-dried product I;

[0029] Pulverization: Place the freeze-dried material I in an ultra-micro pulverizer, pulverize, collect the powder, and obtain the formulated compound.

[0030] As a preferred embodiment of the present invention, the method for preparing the Lactobacillus rhamnosus GG ferment with the function of promoting the healing of oral ulcers includes the following steps:

[0031] Raw material selection: Select fresh, unrotten camellia flowers; select whole, 80% ripe kiwifruit; select yeast β-glucan and fructooligosaccharides that have not spoiled;

[0032] Pretreatment: Wash the camellia flowers with clean water, drain, and take the petals according to the formula; wash the kiwi fruit with clean water, peel it, take the pulp, cut it into pieces, and weigh it according to the formula; weigh the yeast β-glucan and oligofructose according to the formula.

[0033] Pulping: Add the treated camellia flowers to the first part of purified water and pulverize at 5500-6500 rpm for 2-4 minutes to obtain camellia flower pulp A; add the treated kiwi fruit to the second part of purified water and pulverize at 15000-20000 rpm for 1-3 minutes to obtain kiwi fruit pulp B; add the treated yeast β-glucan to the third part of purified water at 75-85℃ and mix evenly, homogenize at 5500-6500 rpm for 3-5 minutes to obtain base material C; add the treated oligofructose to the fourth part of purified water and stir to dissolve to obtain base material D; the sum of the amounts of purified water used in the first, second, third, and fourth parts is the formula amount;

[0034] Extraction: Place camellia flower pulp A in an ice-water bath at 2-4℃, stir at 400-600 r / min for 50-70 min, centrifuge at 3500-4500 r / min for 10-15 min, collect the supernatant, filter it with a filter screen to obtain camellia flower filtrate E.

[0035] Mixing: Mix camellia filtrate E, kiwi pulp B, base material C, and base material D evenly to obtain base material F;

[0036] Strain activation: Remove the *Lactobacillus rhamnosus* GG glycerol cryovials from the -80℃ freezer, and rapidly shake them in a water bath for 1–3 minutes. Use an inoculation loop to apply two loops of the solution to culture medium B, and incubate at 32–37℃ for 45–50 hours. Use an inoculation loop to pick up colonies and place them in culture medium A, incubating at 32–37℃ for 45–48 hours. Repeat this process three times, adjusting the lactic acid bacteria concentration to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture G;

[0037] Fermentation: Inoculate bacterial culture G into substrate F at an inoculation rate of 3-9%, and incubate at a constant temperature of 32-37℃ for 12-18 hours to obtain fermentation product H;

[0038] Vacuum freeze drying: Fermentation product H was pre-frozen at -80℃ for 3-5 hours, and immediately placed in a freeze dryer for 20-26 hours. The drying conditions were cold trap temperature -60℃ and vacuum degree of 1-2 Pa to obtain freeze-dried product I.

[0039] Pulverization: Place the freeze-dried material I in an ultra-micro pulverizer, adjust the speed of the cutter disc to 1000-1250 r / min and the speed of the classifier wheel to 800-950 r / min, pulverize for 10-20 min at 3-5℃, and collect the powder, which is the formulation complex.

[0040] As a preferred embodiment of the present invention, in the fermentation step, the bacterial solution G is inoculated into the substrate F at an inoculation amount of 3-5%.

[0041] As a preferred embodiment of the present invention, in the pulverization step, the freeze-dried material I is placed in an ultrafine pulverizer, the speed of the cutter disc is adjusted to 1232 r / min and the speed of the classifier wheel is 900 r / min, and pulverized for 15 min at 4°C. The powder is then collected to obtain the formulation complex.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] (1) The Lactobacillus rhamnosus GG fermentation product provided by the present invention has the function of promoting the healing of oral ulcers. The composition is fermented by Lactobacillus rhamnosus GG, and the resulting fermentation product is the formula complex. It can promote the healing of oral ulcers, accelerate tissue growth, and has significant effects, and is healthy and safe.

[0044] (2) The Lactobacillus rhamnosus GG fermentation product provided by the present invention, which has the function of promoting the healing of oral ulcer wounds, can not only promote the healing of oral ulcer wounds, but also strengthen the mucosa at the healing site and prevent the recurrence of oral ulcers. Detailed Implementation

[0045] The present invention will now be further described in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the raw materials, equipment, etc., used in the following embodiments can all be obtained through commercial channels.

[0046] A fermented product of Lactobacillus rhamnosus GG that promotes the healing of oral ulcers is obtained by fermenting a composition with Lactobacillus rhamnosus GG. The composition comprises the following components in parts by weight: 33-47 parts of camellia, 33-35 parts of kiwi, 1-3 parts of yeast β-glucan, 15-33 parts of fructooligosaccharides, and 200-300 parts of purified water.

[0047] A formulation complex with the function of promoting the healing of oral ulcer wounds is made from a probiotic fermentation composition, wherein the probiotic is Lactobacillus rhamnosus GG, and the composition comprises the following components by weight: 33-47 parts of camellia, 33-35 parts of kiwi, 1-3 parts of yeast β-glucan, 15-33 parts of fructooligosaccharide, and 200-300 parts of purified water.

[0048] Examples 1-6

[0049] A formulation compound with the function of promoting the healing of oral ulcer wounds is prepared according to the following method:

[0050] 1. Raw material selection: Select fresh, unrotten camellia flowers (roses); select whole, 80% ripe kiwifruit (oranges); select yeast β-glucan and fructooligosaccharides that have not spoiled;

[0051] 2. Pretreatment: Wash the camellia (rose) flowers with clean water, drain them, take the petals, and weigh them according to the weight parts in Table 1; wash the kiwi (orange) with clean water, peel it, take the pulp, cut it into pieces, and weigh it according to the weight parts in Table 1; weigh the yeast β-glucan and oligofructose according to the weight parts in Table 1.

[0052] 3. Pulping: Add the treated camellia (rose) flowers to 141 parts purified water, pulverize at 6000 rpm for 3 minutes to obtain camellia flower pulp A (rose pulp A); add the treated kiwi (orange) fruit to 70 parts purified water, break the cell walls at 18000 rpm for 1 minute to obtain kiwi fruit pulp B (orange pulp B); add the treated yeast β-glucan to 80℃ and 30 parts purified water, mix evenly, homogenize at 6000 rpm for 5 minutes to obtain base material C; add the treated oligofructose to 20 parts purified water and stir to dissolve to obtain base material D.

[0053] 4. Extraction: Place the camellia pulp A (rose pulp A) in an ice-water bath at 4°C, stir at 500 r / min for 60 min, centrifuge at 4000 r / min for 15 min, take the supernatant, filter it through a 300 mesh filter to obtain camellia filtrate E (rose filtrate E).

[0054] 5. Mixing: Mix camellia filtrate E (rose filtrate E), kiwi pulp B (orange pulp B), base material C, and base material D evenly to obtain base material F;

[0055] 6. Culture medium preparation:

[0056] (1) Culture medium A: 10.0g peptone, 10.0g beef powder, 5.0g yeast powder, 2.0g diammonium citrate, 20.0g glucose, 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1.0mL Tween 80, 1000mL distilled water;

[0057] (2) Culture medium B: Add 20g of agar to culture medium A and autoclave at 121℃ for 20min;

[0058] 7. Strain activation: Remove the *Lactobacillus rhamnosus* GG glycerol cryovials from the -80℃ freezer, and rapidly shake them in a 35℃ water bath for 3 minutes. Use an inoculation loop to apply two loops of the solution to culture medium B, and incubate at 35℃ for 48 hours. Use an inoculation loop to pick up colonies and place them in culture medium A, incubating at 35℃ for 48 hours. Repeat this process twice, adjusting the lactic acid bacteria concentration to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture G;

[0059] 8. Fermentation: Inoculate bacterial solution G into substrate F at an inoculation rate of 3%, and incubate at a constant temperature of 35℃ for 16 hours to obtain fermentation product H;

[0060] 9. Vacuum freeze drying: Fermentation product H was pre-frozen at -80℃ for 4 hours, and immediately placed in a freeze dryer for 24 hours. The drying conditions were cold trap temperature -60℃ and vacuum degree of 1 Pa to obtain freeze-dried product I.

[0061] 10. Pulverization: Place the freeze-dried material I in an ultra-micro pulverizer, adjust the speed of the cutter disc to 1232 r / min and the speed of the classifier wheel to 900 r / min, pulverize for 15 min at 4℃, and collect the powder, which is the formulation complex.

[0062] Table 1. Composition details of different embodiments

[0063]

[0064] Effect Verification 1: Wound Healing Test

[0065] 1. Experimental animals: 10-month-old zebrafish were randomly divided into 8 groups of 10 each, and were designated as group A, group B, group C, group D, group E, group F, group G and group H.

[0066] 2. Modeling: Sterilize a cylindrical metal rod with a diameter of 2 mm with 75% ethanol, then heat it over an open flame for 5 seconds, and then press it onto the lateral ventral surface of zebrafish from groups B to H for 1 second to obtain a zebrafish model with ulcer wound.

[0067] 3. Zebrafish were cultured in culture medium for 21 days. The corresponding culture media for each group are shown in Table 2. The water was changed every 2 days. The wound size was measured on days 2, 7, 14, and 21, and the wound healing rate W was calculated on days 7, 14, and 21. The average value was calculated, and the results are shown in Table 3. The wound area was first measured on day 2, and the wound healing rate W was calculated based on the wound area S on day 2.

[0068]

[0069] Table 2. Culture medium conditions for each group

[0070] Group Types of culture medium Group A Clear water Group B Clear water Group C Water containing 800 μg / L of the formulation complex from Example 1 Group D Water containing 800 μg / L of the formulation complex from Example 2 Group E Water containing 800 μg / L of the formulation complex from Example 3 Group F Water containing 800 μg / L of the formulation complex from Example 4 Group G Water containing 800 μg / L of the formulation complex from Example 5 Group H Water containing 800 μg / L of the formulation complex from Example 6

[0071] 4. Results and Analysis:

[0072] Table 3. Wound healing rate (%) of zebrafish in each group at different time points.

[0073]

[0074] Zebrafish are model organisms used to study wound healing, regeneration, and angiogenesis. In this study, we used zebrafish as the research subject to create a wound model and evaluate the effects of different formulations on wound healing. As shown in Table 3, except for group A, the wounds of zebrafish in the other groups gradually healed over time. Compared with group A, the zebrafish in group B showed successful modeling and a gradually increasing wound healing rate, while the healing rate in group A was 0, indicating that the zebrafish ulcer wound model was successfully established. Compared with group B, the wound healing rates of groups C to H all increased, indicating that the formulations of different embodiments all promoted wound healing in zebrafish. Among them, group E had the highest wound healing rate, indicating that the formulation of group E (i.e., the formulation of embodiment 3) had the best effect on promoting wound healing in zebrafish. Therefore, embodiment 3 was selected as the optimal formulation for promoting wound healing.

[0075] Examples 7-11

[0076] A formulation compound with the function of promoting the healing of oral ulcer wounds is prepared according to the following method:

[0077] 1. Raw material selection: Select fresh, unrotten camellia flowers; select kiwifruit with intact fruit that is 80% ripe; select yeast β-glucan and fructooligosaccharides that have not spoiled;

[0078] 2. Pretreatment: Wash the camellia flowers with clean water, drain them, take the petals, and weigh out 47 portions; wash the kiwi fruit with clean water, peel it, take the pulp, cut it into pieces, and weigh out 35 portions; weigh out 3 portions of yeast β-glucan and 15 portions of oligofructose.

[0079] 3. Pulping: Add 141 parts purified water to the treated camellia flowers and pulp at 6000 rpm for 3 minutes to obtain camellia flower pulp A; add 70 parts purified water to the treated kiwifruit and break the cell walls at 18000 rpm for 1 minute to obtain kiwifruit pulp B; add 30 parts purified water to the treated yeast β-glucan at 80℃ and mix evenly, then homogenize at 6000 rpm for 5 minutes to obtain base material C; add 20 parts purified water to the treated oligofructose and stir to dissolve to obtain base material D.

[0080] 4. Extraction: Place the camellia flower pulp A in an ice-water bath at 4°C, stir at 500 r / min for 60 min, centrifuge at 4000 r / min for 15 min, take the supernatant, filter it through a 300 mesh filter to obtain camellia flower filtrate E.

[0081] 5. Mixing: Mix the camellia filtrate E, kiwi pulp B, base material C, and base material D evenly to obtain base material F;

[0082] 6. Culture medium preparation:

[0083] (1) Culture medium A: 10.0g peptone, 10.0g beef powder, 5.0g yeast powder, 2.0g diammonium citrate, 20.0g glucose, 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1.0mL Tween 80, 1000mL distilled water;

[0084] (2) Culture medium B: Add 20g of agar to culture medium A and autoclave at 121℃ for 20min; 7. Activation of the strain: Take out the cryovials of Lactobacillus rhamnosus GG glycerol stored in a -80℃ freezer, shake rapidly in a 35℃ water bath for 3min, use an inoculation loop to take 2 loops and spread them on culture medium B, and incubate at 35℃ for 48h; pick up colonies with an inoculation loop and place them in culture medium A, incubate at 35℃ for 48h, repeat twice, and adjust the lactic acid bacteria concentration of the bacterial solution to 2.0×10 11 CFU / mL, yielding bacterial culture G;

[0085] 8. Fermentation: Inoculate bacterial solution G into substrate F according to the inoculation amount in Table 4, and incubate at a constant temperature of 35℃ for 16 hours to obtain fermentation product H;

[0086] 9. Vacuum freeze drying: Fermentation product H was pre-frozen at -80℃ for 4 hours, and immediately placed in a freeze dryer for 24 hours. The drying conditions were cold trap temperature -60℃ and vacuum degree of 1 Pa to obtain freeze-dried product I.

[0087] 10. Pulverization: Place the freeze-dried material I in an ultra-micro pulverizer, adjust the speed of the cutter disc to 1232 r / min and the speed of the classifier wheel to 900 r / min, pulverize for 15 min at 4℃, and collect the powder, which is the formulation complex.

[0088] Table 4. Inoculation amount of different formulation complexes

[0089] Example Example 7 Example 8 Example 9 Example 10 Example 11 Inoculation volume 1％ 3％ 5％ 7％ 9％

[0090] Effect Verification 2: Mucosal Strength and Toughness Test

[0091] 1. Experimental animals: 10-month-old zebrafish were randomly divided into 7 groups of 12 each, and were designated as group I, group J, group K, group L, group M, group N and group O.

[0092] 2. Modeling: Sterilize a cylindrical metal rod with a diameter of 2 mm with 75% ethanol, then heat it over an open flame for 5 seconds, and then press it onto the lateral ventral surface of zebrafish from groups J to O for 1 second to obtain a zebrafish model with ulcer wound.

[0093] 3. The zebrafish were cultured in the culture medium for 8 days. The corresponding culture medium for each group is shown in Table 5. The water was changed every 2 days.

[0094] Table 5. Culture medium conditions for each group

[0095] Group Types of culture medium Group I Clear water Group J Clear water Group K Water containing 800 μg / L of the formulation complex from Example 7 Group L Water containing 800 μg / L of the formulation complex from Example 8 Group M Water containing 800 μg / L of the formulation complex from Example 9 Group N Water containing 800 μg / L of the formulation complex from Example 10 Group O Water containing 800 μg / L of the formulation complex from Example 11

[0096] 4. Preparation of frozen sections:

[0097] (1) Preparation of glass slides

[0098] Immerse the untreated slides in acid for 12 hours, then rinse them thoroughly under running water to remove the acid. Wipe the slides dry with a silk cloth, then soak them in anhydrous ethanol for 6 hours. Remove them, air dry, and wipe them clean with a silk cloth again. Prepare a smear using APES adhesive and acetone at a ratio of 1:50, then rinse briefly with triple-distilled water and air dry for later use.

[0099] (2) Frozen slices

[0100] 1) Sample collection: Three zebrafish were taken and euthanized on days 2, 4, 6 and 8. Normal tissue from the wound and surrounding area of ​​groups J to O was collected and fixed in 4% paraformaldehyde solution for 16 hours. Normal tissue from the same site of group I was collected and fixed in 4% paraformaldehyde solution for 16 hours.

[0101] 2) Soak and wash the tissue three times with 1×PBS buffer, 5 min each time;

[0102] 3) Dehydrate the tissue with a 30% sucrose solution for 3 hours until it is in a good dehydration state.

[0103] 5. Tricolor dyeing:

[0104] (1) Remove the frozen sections and air dry the surface of the tissue sections at room temperature;

[0105] (2) Stain the tissue sections in Ponceau S and Fuchsia staining solution for 5 min, then wash them 3 times with 0.3% weak acid solution for 20 s each time.

[0106] (3) Wash the tissue sections in phosphomolybdic acid solution for 1 min, then wash them with a weak acid solution 3 times for 20 s each time.

[0107] (4) Immerse the tissue sections in aniline blue staining solution for 45 seconds, then wash them three times with a weak acid solution for 20 seconds each time.

[0108] (5) Soak the tissue sections in triple-distilled water for 1 minute;

[0109] (6) Quickly dehydrate the tissue sections in 95% ethanol;

[0110] (7) Place the tissue sections into three stages of 100% ethanol for dehydration, 5 seconds for each stage;

[0111] (8) The tissue sections were cleared in xylene three times, each time for 2 minutes. After removing the sections, they were mounted with neutral resin and allowed to dry. The collagen deposition area was measured under a microscope. The results are shown in Table 6.

[0112] 6. Results and Analysis:

[0113] Table 6. Collagen deposition area (μm) at ulcer wounds in zebrafish from different groups. 2 )

[0114]

[0115]

[0116] Wound healing requires collagen synthesis and deposition, and the synthesis and decomposition of collagen must be in dynamic equilibrium; otherwise, poor healing or over-healing will occur. Table 6 shows that, over time, except for group I, collagen deposition at the wound sites of zebrafish in the other groups increased or decreased to varying degrees, indicating that collagen deposition at the wound site is dynamically changing under different culture media. Compared to group I, the significant decrease in group J indicates that the skin tissue at the wound site of zebrafish in group J was destroyed, signifying successful modeling of zebrafish ulcer wounds. Compared to group J, the collagen deposition area at the ulcer wound sites of zebrafish in groups K to O all increased, and the trend within each group showed an initial increase followed by a decrease, indicating that the intervention of the formulated compound improved collagen deposition at the zebrafish ulcer wound sites. However, different formulated compounds have different quality due to variations in the amount of bacterial inoculation during their preparation, resulting in different effects on collagen deposition. Among them, the collagen deposition area at the ulcer wound of zebrafish in group L was the largest on days 2 and 4, and decreased to near that of group I on days 6 and 8. The fluctuation from day 2 to day 8 was the best, indicating that the formulation complex of group L (i.e., Example 8) promoted collagen deposition on days 2 and 4 and reduced collagen deposition on days 6 and 8, thereby strengthening the mucosa at the healing site and allowing the wounded skin to gradually recover its original structure and function, which is beneficial for preventing recurrence of oral ulcers. Therefore, Example 8 was selected as the optimal fermentation ratio of the formulation complex that can strengthen the mucosa at the healing site.

[0117] Examples 12-15

[0118] A formulation compound with the function of promoting the healing of oral ulcer wounds is prepared according to the following method:

[0119] I. Sample Preparation

[0120] 1. Raw material selection: Select fresh, unrotten camellia flowers; select kiwifruit with intact fruit that is 80% ripe; select yeast β-glucan and fructooligosaccharides that have not spoiled;

[0121] 2. Pretreatment: Wash the camellia flowers with clean water, drain them, take the petals, and weigh out 47 portions; wash the kiwi fruit with clean water, peel it, take the pulp, cut it into pieces, and weigh out 35 portions; weigh out 3 portions of yeast β-glucan and 15 portions of oligofructose.

[0122] 3. Pulping: Add 141 parts purified water to the treated camellia flowers and pulp at 6000 rpm for 3 minutes to obtain camellia flower pulp A; add 70 parts purified water to the treated kiwifruit and break the cell walls at 18000 rpm for 1 minute to obtain kiwifruit pulp B; add 30 parts purified water to the treated yeast β-glucan at 80℃ and mix evenly, then homogenize at 6000 rpm for 5 minutes to obtain base material C; add 20 parts purified water to the treated oligofructose and stir to dissolve to obtain base material D.

[0123] 4. Extraction: Place the camellia flower pulp A in an ice-water bath at 4°C, stir at 500 r / min for 60 min, centrifuge at 4000 r / min for 15 min, take the supernatant, filter it through a 300 mesh filter to obtain camellia flower filtrate E.

[0124] 5. Mixing: Mix the camellia filtrate E, kiwi pulp B, base material C, and base material D evenly to obtain base material F;

[0125] 6. Culture medium preparation:

[0126] (1) Culture medium A: 10.0g peptone, 10.0g beef powder, 5.0g yeast powder, 2.0g diammonium citrate, 20.0g glucose, 5.0g sodium acetate, 2.0g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1.0mL Tween 80, 1000mL distilled water;

[0127] (2) Culture medium B: Add 20g of agar to culture medium A and autoclave at 121℃ for 20min; 7. Activation of the strain: Take out the cryovials of Lactobacillus rhamnosus GG glycerol stored in a -80℃ freezer, shake rapidly in a 35℃ water bath for 3min, use an inoculation loop to take 2 loops and spread them on culture medium B, and incubate at 35℃ for 48h; pick up colonies with an inoculation loop and place them in culture medium A, incubate at 35℃ for 48h, repeat twice, and adjust the lactic acid bacteria concentration of the bacterial solution to 2.0×10 11 CFU / mL, yielding bacterial culture G;

[0128] 8. Fermentation: Inoculate bacterial solution G into substrate F at an inoculation rate of 3%, and incubate at a constant temperature of 35℃ for 16 hours to obtain fermentation product H;

[0129] 9. Vacuum freeze drying: Fermentation product H was pre-frozen at -80℃ for 4 hours, and immediately placed in a freeze dryer for 24 hours. The drying conditions were cold trap temperature -60℃ and vacuum degree of 1 Pa to obtain freeze-dried product I.

[0130] 10. Pulverization: Place the freeze-dried material I in an ultra-fine pulverizer, adjust the speed of the cutter disc to 1232 r / min and the speed of the classifier wheel to 900 r / min, and pulverize at 4℃. The pulverization time is shown in Table 7. Collect the powder, which is the formulation complex.

[0131] Table 7. Grinding time (min) for different formulations of the compound

[0132] Example Example 12 Example 13 Example 14 Example 15 Grinding time 5 10 15 20

[0133] Effect Verification III. Population Testing

[0134] 1. Subject population: Patients with oral ulcers lasting for 3 days, aged 16-50 years, were randomly divided into 4 groups of 10 people each, designated as group P, group Q, group R and group S.

[0135] (1) Inclusion criteria: All patients had oral ulcers; complete clinical data; no allergic symptoms to the drugs used; patients and their families were informed about the study and signed informed consent forms; and the study was approved by the hospital's ethics committee.

[0136] (2) Exclusion criteria: Patients with other major organic diseases and functional diseases; patients with mental illness, social impairment, and inability to communicate and express themselves normally; patients who withdraw from the study midway due to various uncontrollable reasons; patients who have allergic reactions to the selected products.

[0137] 2. Grouping: Patients in each group were treated with the formulated compound for 7 consecutive days. The grouping is shown in Table 8.

[0138] Table 8. Types of formulation compounds used in each patient group.

[0139] Group type Group P Formulation compound of Example 12 Q Group Formulation compound of Example 13 Group R Formulation compound of Example 14 Group S Formulation complex of Example 15

[0140] 3. Treatment method: Apply the formula compound to the oral ulcer site with a sterile cotton swab once a day, that is, rinse your mouth to keep the oral cavity clean before applying it before going to bed.

[0141] 4. Detection method:

[0142] (1) Particle size determination: The D10, D50, D90, D(4,3), and D(3,2) values ​​of the formulation complex (Examples 12 to 15) were determined using a Mastersizer 3000 laser particle size analyzer. The results are shown in Table 10.

[0143] (2) At 8:00 a.m. on the 15th day, the patients with oral ulcers were statistically analyzed according to the evaluation criteria for oral ulcer efficacy, and the total effective rate of oral ulcer treatment was calculated. The results are shown in Table 10. The recurrence rate was calculated on the 30th day after the end of the experiment. The results are shown in Table 11.

[0144] Table 9 Evaluation Criteria for Effective Healing Rate of Oral Ulcers

[0145]

[0146] 5. Results and Analysis:

[0147] Table 10. Particle size distribution of the formulation complexes in different embodiments.

[0148]

[0149]

[0150] D 10 D 50 and D 90 The values ​​refer to the particle size (D) corresponding to the cumulative particle size distribution percentages reaching 10%, 50%, and 90%, respectively. D(4,3) refers to the surface area average particle size, and D(3,2) refers to the volume average particle size. The closer the values ​​of D(4,3) and D(3,2) are, the more regular the shape of the sample particles and the more concentrated the particle size distribution. Insufficient grinding will reduce the bioavailability of the formulation complex, resulting in the inability of the active ingredients to be absorbed quickly; excessive grinding will destroy the structure of the active ingredients, resulting in a significant reduction in the effectiveness of the formulation complex. As shown in Table 10, with the increase of grinding time, the D values ​​of different embodiments... 10 D 50 D 90 The D(4,3) and D(3,2) values ​​gradually decrease. The D(4,3) value of Example 14 is approximately equal to 10 μm, which helps protect, dissolve and absorb the active ingredients of the formulation complex. The D(4,3) value of Example 14 is closest to the D(3,2) value, indicating that the formulation complex of Example 14 has the best uniformity and the best pulverization effect.

[0151] Table 11 Overall effective rate and recurrence rate of oral ulcers

[0152] Group Effective (number) Valid (number) Invalid (number) Overall effectiveness (%) Recurrence rate (%) Group P 5 20 15 62.50 5.00 Q Group 15 22 3 92.50 2.00 Group R 35 5 0 100.00 0.00 Group S 24 15 1 97.50 1.00

[0153] Table 11 shows that the overall effective rate of oral ulcer healing varied among different groups, indicating that the efficacy of the formulated compound differed among the groups. Specifically, the grinding time affected the effectiveness of the compound and also had a certain impact on the recurrence rate of oral ulcers. Group R showed the highest overall effective rate of oral ulcer healing and the lowest recurrence rate, indicating that the intervention with the formulated compound of Example 14 resulted in the best healing of the oral ulcer wounds, with strong and resilient mucosa, thus preventing recurrence. Therefore, Example 14 was selected as the optimal grinding time for the formulated compound.

[0154] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A fermented product of *Lactobacillus rhamnosus* GG that promotes the healing of oral ulcers, characterized in that, The composition is prepared by fermentation of Lactobacillus rhamnosus GG and consists of the following components in parts by weight: 47 parts of camellia, 35 parts of kiwi, 3 parts of yeast β-glucan, 15 parts of fructooligosaccharide, and 261 parts of purified water. The Lactobacillus rhamnosus GG ferment was prepared according to the following method: Raw material selection: Select fresh, unrotten camellia flowers; select whole, 80% ripe kiwifruit; select yeast β-glucan and fructooligosaccharides that have not spoiled; Pretreatment: Wash the camellia flowers with clean water, drain, and take the petals according to the formula; wash the kiwi fruit with clean water, peel it, take the pulp, cut it into pieces, and weigh it according to the formula; weigh the yeast β-glucan and oligofructose according to the formula. Pulping: Add 130-150 parts of purified water to the treated camellia flowers and pulp at 5500-6500 rpm for 2-4 minutes to obtain camellia flower pulp A; add 60-80 parts of purified water to the treated kiwifruit and break the cell walls at 15000-20000 rpm for 1-3 minutes to obtain kiwifruit pulp B; add 30-40 parts of purified water to the treated yeast β-glucan at 75-85℃ and mix evenly, then homogenize at 5500-6500 rpm for 3-5 minutes to obtain base material C; add 20-30 parts of purified water to the treated fructooligosaccharides and stir to dissolve to obtain base material D; Extraction: Place camellia flower pulp A in an ice-water bath at 2-4℃, stir at 400-600 r / min for 50-70 min, centrifuge at 3500-4500 r / min for 10-15 min, collect the supernatant, filter it with a filter screen to obtain camellia flower filtrate E. Mixing: Mix camellia filtrate E, kiwi pulp B, base material C, and base material D evenly to obtain base material F; Culture medium preparation: (1) Culture medium A: 10.0 g peptone, 10.0 g beef powder, 5.0 g yeast powder, 2.0 g diammonium citrate, 20.0 g glucose, 5.0 g sodium acetate, 2.0 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 1.0 mL Tween 80, 1000 mL distilled water; (2) Culture medium B: 20 ​​g agar was added to culture medium A and autoclaved at 121℃ for 20 min. Strain activation: Remove the *Lactobacillus rhamnosus* GG glycerol cryovials from the -80℃ freezer, and rapidly shake them in a water bath for 1-3 minutes. Use an inoculation loop to apply two loops of the solution to culture medium B, and incubate at 32-37℃ for 45-50 hours. Use an inoculation loop to pick up colonies and place them in culture medium A, incubating at 32-37℃ for 45-48 hours. Repeat this process three times, adjusting the lactic acid bacteria concentration to 2.0 × 10⁻⁶. 11 CFU / mL, yielding bacterial culture G; Fermentation: Inoculate bacterial culture G into substrate F at an inoculation rate of 3%, and incubate at a constant temperature of 32-37℃ for 12-18 hours to obtain fermentation product H; Vacuum freeze drying: Fermentation product H is pre-frozen at -80℃ for 3-5 hours, and then immediately placed in a freeze dryer for 20-26 hours. The drying conditions are cold trap temperature -60℃ and vacuum degree of 1-2 Pa to obtain freeze-dried product I. Pulverization: Place the freeze-dried material I in an ultra-micro pulverizer, adjust the speed of the cutter disc to 1232 r / min and the speed of the classifier wheel to 900 r / min, pulverize for 15 min at 4℃, and collect the powder, which is the fermentation product of Lactobacillus rhamnosus GG.

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