Traditional Chinese medicine composite lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and preparation process of traditional Chinese medicine composite lactobacillus acidophilus NM probiotic preparation

Abstract

The invention discloses a traditional Chinese medicine composite lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and a preparation process thereof, and relates to the related field of biotechnology. According to the traditional Chinese medicine composite lactobacillus acidophilus NM probiotic preparation, the synergistic effect of probiotics and traditional Chinese medicine is combined, the traditional Chinese medicine composite lactobacillus acidophilus NM probiotic preparation has the multiple advantages of regulating oral flora, resisting bacteria and diminishing inflammation, resisting oxidation, relieving fatigue and the like, lactobacillus acidophilus NM can tolerate acid and cholate, pathogenic bacteria such as escherichia coli, staphylococcus aureus and salmonella are effectively inhibited, and the traditional Chinese medicine composite lactobacillus acidophilus NM probiotic preparation is suitable for oral administration. The traditional Chinese medicine composition has the advantages that the traditional Chinese medicine composition can be used for reducing dental caries, gingivitis and ozostomia, is free of antibiotic drug resistance and is safer after being used for a long time, radix scutellariae, honeysuckle and green tea extracts are rich in flavonoids and tea polyphenols and can further resist bacteria and inflammation and improve oral health, and meanwhile, the probiotics can reduce blood lactic acid, blood urea nitrogen and blood ammonia, enhance SOD, CAT and GSH-Px vitality and inhibit lipid peroxidation, so that body fatigue is relieved, and the traditional Chinese medicine composition can be used for preventing and treating dental caries, gingivitis and ozostomia. The immunity is improved, and preparation forms are diversified.

Description

Technical Field

[0001] The present invention relates to biotechnology-related fields, and in particular to a traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and a preparation process thereof. Background Art

[0002] In recent years, oral health issues have received increasing attention. The incidence of diseases such as caries, gingivitis, and halitosis has increased year by year. The main reason is closely related to the imbalance of oral flora. Studies have shown that pathogenic bacteria such as Streptococcus mutans, Porphyromonas gingivalis, Escherichia coli, Staphylococcus aureus, and Salmonella multiply in large numbers in the oral cavity, which will lead to the formation of dental plaque and aggravated gingival inflammation, thereby affecting the health of the whole body. In addition, the pace of life in modern society is accelerated, and people generally have problems such as fatigue, decreased immunity, and increased oxidative stress. Oral health status is closely related to the overall health of the body. Therefore, the development of probiotic products with oral regulation, anti-oxidation, and fatigue relief functions is of great practical significance. Summary of the invention

[0003] Therefore, in order to solve the above-mentioned shortcomings, the present invention provides a traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and a preparation process thereof.

[0004] In order to achieve the above object, the present invention adopts the following technical scheme: a traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, wherein the probiotic preparation consists of Lactobacillus acidophilus NM freeze-dried powder, a traditional Chinese medicine extract, a prebiotic and a functional auxiliary material, wherein the Lactobacillus acidophilus NM freeze-dried powder is freeze-dried from Lactobacillus acidophilus NM, and the Lactobacillus acidophilus NM is deposited in the General Microbiology Center of China Committee for the Collection of Microorganisms, with a deposit number of CGMCC NO.4472.

[0005] Preferably, the probiotic preparation is prepared from 5-15 parts of Lactobacillus acidophilus NM freeze-dried powder, 10-29 parts of traditional Chinese medicine extracts, 5-14 parts of prebiotics and 42-80 parts of functional excipients in parts by weight.

[0006] Preferably, the method for preparing the Lactobacillus acidophilus NM freeze-dried powder comprises the following steps:

[0007] Preparation of S1 medium: 100 g skim milk powder, 5 g glucose, 3 g yeast extract, 2 g disodium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 1000 mL distilled water, adjust pH to 6.2-6.5, and sterilize at 121°C for 15 min;

[0008] S2 bacterial inoculation: After Lactobacillus acidophilus NM is revived, inoculate it to 37°C for anaerobic culture for 18-24h to make the bacterial concentration reach 1×10 9 CFU / mL;

[0009] S3 bacterial cell collection and freeze-drying: centrifuge at 4000rpm, 10min, 4°C to collect the cells; suspend in 10% skim milk powder + 5% mannitol, freeze-dry at -50°C, and control the water content to ≤5%.

[0010] Preferably, the Chinese herbal medicine extract is prepared by weight from 5-10 parts of scutellaria baicalensis extract, 3-8 parts of honeysuckle extract, 2-6 parts of licorice extract, 1-4 parts of peppermint extract, 3-6 parts of hawthorn extract, and 1-5 parts of green tea extract; the preparation method of the Chinese herbal medicine extract comprises the following steps:

[0011] S1 Wash and air dry the medicinal materials;

[0012] S2 extraction process, water extraction: add 10 times of water, boil at 90°C for 1 hour, filter and take the supernatant; secondary boiling: add 8 times of water, boil at 90°C for 45 minutes, filter and take the supernatant; combined concentration: concentrate at 80°C under reduced pressure to a solid content of 15%;

[0013] S3 spray drying feed 45°C, atomization pressure 4MPa, outlet temperature 80-90°C.

[0014] Preferably, the Chinese medicine extract is prepared by weight from 5 parts of Scutellaria baicalensis extract, 4 parts of Honeysuckle extract, 3 parts of Licorice extract and 2 parts of Green Tea extract.

[0015] Preferably, the Chinese herbal extract is prepared from 5 parts of honeysuckle extract, 4 parts of peppermint extract and 3 parts of green tea extract by weight.

[0016] Preferably, the Chinese herbal medicine extract is prepared by 6 parts of Scutellaria baicalensis extract, 5 parts of Crataegus pinnatifida extract and 4 parts of green tea extract in parts by weight.

[0017] Preferably, the prebiotics are oligofructose and inulin, and the ratio of the two is 1:1.5.

[0018] Preferably, the functional auxiliary material is maltodextrin.

[0019] Preferably, the preparation method of the traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation comprises the following specific steps:

[0020] S1 weighing: weighing Lactobacillus acidophilus freeze-dried powder, Chinese medicine extract, prebiotics and functional excipients;

[0021] S2 Mixing: V-type mixer mixes the materials described in S1 for 30 min, temperature ≤ 25°C, humidity ≤ 50%;

[0022] S3 preparation molding: Direct packaging 2g / bag, sealed with aluminum foil.

[0023] Beneficial effects of the present invention:

[0024] This preparation combines Lactobacillus acidophilus NM with active ingredients of traditional Chinese medicine, and effectively promotes oral and whole body health through multiple effects such as regulating oral flora, enhancing antioxidant capacity, and relieving fatigue. The main advantages are as follows:

[0025] Lactobacillus acidophilus NM has an inhibitory effect on a variety of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella, and can effectively reduce harmful oral bacteria and reduce the risk of gingivitis, dental caries, and bad breath. The Chinese medicinal ingredients Scutellaria baicalensis, honeysuckle, and green tea extract are rich in flavonoids and tea polyphenols, have broad-spectrum antibacterial and anti-inflammatory effects, and work with probiotics to maintain the balance of oral flora;

[0026] Lactobacillus acidophilus NM has strong tolerance to acidic environment and bile salts, and can maintain a high survival rate in the oral cavity and digestive tract, improve the colonization ability of probiotics, and enhance the stability and durability of the preparation;

[0027] Lactobacillus acidophilus NM can reduce blood lactic acid, blood urea nitrogen and blood ammonia levels, reduce the accumulation of metabolic waste, thereby relieving fatigue and improving physical vitality. It can enhance the body's antioxidant capacity by increasing the activity of SOD, CAT, and GSH-Px, inhibit lipid peroxidation in the body, improve immunity, and prevent oral and systemic chronic inflammation;

[0028] Hawthorn extract helps promote blood circulation, while licorice can regulate immunity and synergistically enhance the antioxidant and anti-fatigue effects;

[0029] Lactobacillus acidophilus NM has no resistance to antibiotics such as acetylspiramycin, streptomycin and penicillin. Long-term use will not cause the proliferation of resistant strains, ensuring the safety and effectiveness of the product.

[0030] It is suitable for people with oral ulcers, gingivitis, bad breath, etc. It can also be used for people who are susceptible to fatigue and those with low immunity. It comes in a variety of dosage forms such as powders, tablets, chewable tablets, etc. to meet the needs of different age groups and usage. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the morphology of Lactobacillus acidophilus NM bacteria of the present invention;

[0032] Figure 2 This is a schematic diagram of the colony morphology of Lactobacillus acidophilus NM culture medium of the present invention;

[0033] Figure 3 is a schematic diagram of the acid tolerance of Lactobacillus acidophilus NM of the present invention;

[0034] Figure 4 is a schematic diagram of the tolerance of Lactobacillus acidophilus NM of the present invention to bile salts;

[0035] Figure 5is a schematic diagram of mouse serum creatine kinase of the present invention;

[0036] Figure 6 is a schematic diagram of mouse blood lactate of the present invention;

[0037] Figure 7 is a schematic diagram of blood urea nitrogen of mice of the present invention;

[0038] Figure 8 is a schematic diagram of blood ammonia of mice of the present invention;

[0039] Fig. 9 This is a schematic diagram of the SOD activity of the mouse serum of the present invention;

[0040] Fig.10 This is a schematic diagram of the CAT activity of mouse serum of the present invention;

[0041] Fig.11 is a schematic diagram of mouse GSH-Px of the present invention;

[0042] Fig.12 This is a schematic diagram of the MDA content of mice of the present invention;

[0043] Fig.13 It is a schematic diagram of the complete gene sequence map of the present invention. DETAILED DESCRIPTION

[0044] In order to further explain the technical solution of the present invention, it is described in detail below through specific embodiments.

[0045] A traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, the probiotic preparation consisting of Lactobacillus acidophilus NM freeze-dried powder, traditional Chinese medicine extracts, prebiotics and functional excipients, the Lactobacillus acidophilus NM freeze-dried powder being freeze-dried Lactobacillus acidophilus NM, the Lactobacillus acidophilus NM being deposited in the General Microbiology Center of China Culture Collection of Microorganisms, with a deposit number of CGMCCNO.4472, a deposit date of December 14, 2010, a deposit address of: General Microbiology Center of China Culture Collection of Microorganisms, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit result of: survival.

[0046] Reference Figure 1 , Bacterial morphology, Lactobacillus acidophilus NM MRS medium, anaerobic culture at 37℃ for 48h, the bacteria are rod-shaped, 0.4-0.6μm×1.8-4.1μm, arranged singly or in pairs, Gram-positive bacteria;

[0047] Reference Figure 2 , Colony morphology, Lactobacillus acidophilus NM MRS culture medium, colonies are white, nearly round, moist surface, opaque, and have neat edges.

[0048] The pH of gastric juice fluctuates greatly due to different dietary structures. Generally, the pH is around 3. The pH can reach 1.5 when fasting or eating acidic food, and 4-5 when eating alkaline food. The time for food to pass through the stomach is 1-2 hours. The bile salt concentration in the duodenum is 0.3-3g / kg, and the time for food to pass through the duodenum is relatively short. Therefore, the human gastrointestinal tract belongs to a high-acid and high-bile salt environment. This experiment simulates the human gastrointestinal environment and studies the tolerance of Lactobacillus acidophilus NM to gastric acid and bile salts. Figure 3 , treated with pH3.0 culture medium for 2 hours, the survival rate of Lactobacillus acidophilus NM was 79.40%, treated with pH2.0 culture medium for 2 hours, the survival rate of Lactobacillus acidophilus NM was 9.20%, treated with pH4.0 culture medium for 2 hours, the survival rate of Lactobacillus acidophilus NM was 98.20%. The results show that Lactobacillus acidophilus NM has a strong tolerance to acid;

[0049] Take 10 9 CFU / mL of Lactobacillus acidophilus NM suspension was added with artificial gastric juice of pH 1.5, 2.0, 3.0, and 4.0, respectively, and cultured at 37°C for 2 hours with shaking. Samples were taken every 30 minutes, and plate counts were performed to calculate the survival rate. The experimental results are as follows:

[0050] Processing time (h) pH 1.5 survival rate (%) pH 2.0 survival rate (%) pH 3.0 survival rate (%) pH 4.0 survival rate (%) 0 100 100 100 100 0.5 25.5 ± 1.2 48.2 ± 1.4 72.5 ± 1.3 90.8 ± 1.2 1.0 10.2 ± 1.0 32.1 ± 1.3 65.4 ± 1.5 85.6 ± 1.3 2.0 3.1 ± 0.8 9.2 ± 1.0 79.4 ± 1.4 98.2 ± 1.1

[0051] The bile salt tolerance of Lactobacillus acidophilus NM decreased with the increase of bile salt mass fraction. When the bile salt mass fraction was 0.30%, the survival rate of strain NM was 10.20%, when the bile salt mass fraction was 0.50%, the survival rate of strain NM was 5.12%, and when the bile salt mass fraction was 0.10%, the survival rate of strain NM could reach 65%. The results showed that Lactobacillus acidophilus NM had a strong tolerance to bile salts. Figure 4 ;

[0052] Take 10 9 CFU / mL of Lactobacillus acidophilus NM bacterial suspension was added to MRS medium containing 0.10%, 0.30%, and 0.50% bile salts, respectively, and cultured at 37°C for 6 hours to simulate the duodenal environment. Samples were taken every 2 hours and the survival rate was calculated by plate counting method. The experimental results are as follows:

[0053] Processing time (h) 0.10% Bile salt survival rate (%) 0.30% Bile salt survival rate (%) 0.50% Bile salt survival rate (%) 0 100 100 100 2 85.4 ± 2.0 48.2 ± 1.6 26.5 ± 1.5 4 74.2 ± 1.8 25.6 ± 1.4 12.8 ± 1.3 6 65.0 ± 2.1 10.2 ± 1.5 5.12 ± 1.2

[0054] Lactobacillus acidophilus NM has an inhibitory effect on the three strains of pathogenic bacteria, and can better inhibit Escherichia coli and Staphylococcus aureus. From the experimental results, it can be seen that Lactobacillus acidophilus NM can produce some antibacterial metabolites, such as lactic acid, acetic acid, formic acid, propionic acid, butyric acid, etc., as well as bacteriocins, bacteriocin-like substances, hydrogen peroxide, etc. In addition to the probiotic properties of metabolites, Lactobacillus acidophilus can also adhere to the host's digestive tract to colonize and survive to form a biological barrier (space-occupying effect), inhibit the adhesion of pathogens to the digestive tract mucosa, and inhibit the growth of pathogens by competing with pathogens for nutrients. In addition, it can prevent the absorption of harmful substances such as toxins and neutralize toxic substances;

[0055] The experimental method is as follows:

[0056] Antibacterial activity experiment agar diffusion method, take the supernatant of Lactobacillus acidophilus NM cultured in MRS medium at 37°C for 24h, centrifuge at 8000rpm×10min, take the supernatant for antibacterial experiment, filter with sterile filter membrane (0.22μm) to remove bacteria, inoculate Escherichia coli, Staphylococcus aureus, and Salmonella into LB agar medium respectively, after plating, punch holes (6mm in diameter) on the agar, add 100μL of Lactobacillus acidophilus NM supernatant to each hole, culture at 37°C for 24h, and measure the diameter of the inhibition zone;

[0057] The metabolites of Lactobacillus acidophilus NM were detected by high performance liquid chromatography (HPLC) to detect the contents of lactic acid, acetic acid, formic acid, propionic acid and butyric acid in the fermentation broth of Lactobacillus acidophilus NM. A hydrogen peroxide detection kit was used to detect whether the NM strain produced hydrogen peroxide.

[0058] The antibiotic sensitivity test used the KB paper diffusion method, taking the culture fluid of Lactobacillus acidophilus NM, spreading it on the MRS agar plate, placing acetylspiramycin, streptomycin, and penicillin antibiotic paper in the center of the plate, and incubating it anaerobically at 37°C for 24 hours, measuring the diameter of the inhibition zone, and determining the sensitivity;

[0059] strain Diameter of inhibition zone / mm Escherichia coli 16.67±0.45（++） Staphylococcus aureus 27.54±0.67（++++） salmonella 15.21±0.32（+）

[0060] Lactobacillus acidophilus NM has no resistance to acetylspiramycin, streptomycin and penicillin, and the results tend to be highly sensitive, as shown in the following table;

[0061] antibiotic Diameter of inhibition zone / mm Acetylspiramycin 24.31±0.21 (high sensitivity) penicillin 31.21±0.31 (high sensitivity) Streptomycin 26.33±0.45 (high sensitivity)

[0062] SPF male mice (BALB / c), 6 weeks old, weighing 20±2g, were selected and adaptively fed for 1 week and randomly divided into 4 groups (n=10). The control group was not supplemented with Lactobacillus acidophilus NM and only fed with ordinary feed; the low-dose group was 1×10 8 CFU / mL Lactobacillus acidophilus NM, 0.2 mL per day, for 4 weeks; medium dose group: 1×109 CFU / mL Lactobacillus acidophilus NM, 0.2 mL per day, for 4 weeks; high-dose group: 5×10 9 CFU / mL Lactobacillus acidophilus NM, 0.2mL was gavaged daily for 4 weeks; Experimental methods: Exercise fatigue model, mice underwent forced swimming test, and the swimming time to exhaustion was set; Serum index determination, mice were anesthetized immediately after exercise, blood was collected from the eyeball, centrifuged at 4°C 3000rpm for 10min, and serum was taken for testing. Serum creatine kinase (CK), blood lactic acid (LA), blood urea nitrogen (BUN), and blood ammonia (NH3) were detected by enzyme-linked immunosorbent assay (ELISA), and antioxidant capacity: SOD, CAT, GSH-Px, and MDA levels were determined.

[0063] Creatine kinase is an important kinase directly related to energy transfer, muscle contraction and ATP regeneration. After exercise, creatine kinase in the body increases significantly and is positively correlated with exercise intensity, such as Figure 5 After 4 weeks of supplementation with Lactobacillus acidophilus NM, the serum creatine kinase activity of mice in the low-dose, medium-dose and high-dose groups of Lactobacillus acidophilus NM was significantly reduced compared with that in the control group, but there was no significant difference between the medium-dose group and the high-dose group. The serum creatine kinase activity of mice decreased by 16.00%, 28.50% and 33.90%, respectively; trend analysis showed that with the increase of Lactobacillus acidophilus NM dose, the creatine kinase activity decreased in a dose-dependent manner, which indicates that Lactobacillus acidophilus NM can reduce the creatine kinase activity in mice, thereby alleviating body fatigue.

[0064] Lactic acid in the blood is an inevitable product of sugar metabolism in the body. The increase of lactic acid will lead to a decrease in the pH of muscle tissue and blood, triggering biochemical and physiological processes that are harmful to human functions. Lactic acid is an important indicator for evaluating the body's aerobic metabolism and fatigue. Figure 6 As shown, after 4 weeks of supplementation with Lactobacillus acidophilus NM, the blood lactate levels of mice in the low-dose, medium-dose and high-dose groups of Lactobacillus acidophilus NM were significantly reduced compared with those in the control group, but there was no significant difference between the medium-dose group and the high-dose group, whose lactate levels were 10.13±1.00, 8.36±0.99 and 6.09±0.36 mol / L, respectively. This study shows that Lactobacillus acidophilus NM alone can reduce the lactic acid in the blood of mice, thereby relieving body fatigue.

[0065] Under normal physiological conditions, proteins and amino acids undergo metabolism to produce ammonia, which is further metabolized into urea by the liver, and finally enters the kidneys through the blood circulation and is excreted from the body with urine. Since the sugar content in the body cannot meet the energy demand of exercise during fatigue, the body's protein participates in the energy supply reaction. The occurrence of exercise fatigue is positively correlated with the blood urea nitrogen content, such as Figure 7As shown, after 4 weeks of supplementation with Lactobacillus acidophilus NM, the blood urea nitrogen content of mice in the low-dose, medium-dose and high-dose groups of Lactobacillus acidophilus NM was significantly reduced compared with that of mice in the control group, but there was no significant difference among the three dose groups, which were reduced by 13.40%, 14.90% and 31.50%, respectively; this indicates that Lactobacillus acidophilus NM can reduce the blood urea nitrogen of mice, thereby alleviating body fatigue.

[0066] Ammonia is an important metabolite produced during exercise energy metabolism and is produced from different sources. Ammonia accumulated in the blood and brain during exercise can negatively affect the central nervous system and cause fatigue. Although exercise-induced ammonia poisoning is transient and reversible, depending on the disease state, it may affect the sustained coordinated activity of key areas of the central nervous system. The central nervous system plays a vital role in the development of physical fatigue, such as Figure 8 As shown, after 4 weeks of supplementation with Lactobacillus acidophilus NM, the blood ammonia levels of mice in the low-dose, medium-dose and high-dose groups of Lactobacillus acidophilus NM were significantly reduced compared with those in the control group, which were 115.34±4.50, 99.67±4.50 and 94.33±4.72 μmol / L, respectively, but there was no significant difference between the medium-dose and high-dose groups; this indicates that Lactobacillus acidophilus NM can reduce the blood ammonia levels of mice, thereby alleviating body fatigue.

[0067] There is increasing evidence that reactive oxygen species induce protein oxidation and strongly promote muscle fatigue. Probiotics can reduce oxidative stress, improve exercise capacity, and reduce fatigue. Therefore, this study used mouse oxidative stress indicators to evaluate the effects of Lactobacillus acidophilus NM in improving exercise capacity and relieving fatigue. Superoxide dismutase (SOD) is an active substance derived from living organisms. It is an active protease containing metal elements that can remove harmful substances produced during metabolism. The content of SOD can directly reflect the degree of aging fatigue in the body, such as Fig. 9 As shown, compared with the serum SOD activity of mice in the control group, the serum SOD activity of mice in the low-dose group, medium-dose group and high-dose group of Lactobacillus acidophilus NM was significantly increased, by 6.82%, 23.17% and 37.80%, respectively; trend analysis showed that with the increase of Lactobacillus acidophilus NM dose, serum SOD activity increased in a dose-dependent manner, which indicates that Lactobacillus acidophilus NM can improve the body's antioxidant capacity by increasing SOD activity, thereby relieving fatigue.

[0068] Catalase (CAT) can catalyze the decomposition of H2O2 into H2O and O2, protecting the integrity of cell membrane structure and function. Therefore, CAT is often used to evaluate the body's antioxidant capacity, such as Fig.10As shown, the serum CAT activity of mice in the medium-dose and high-dose groups of Lactobacillus acidophilus NM was significantly increased compared with that of mice in the control group, increasing by 16.36% and 35.15%, respectively. This indicates that Lactobacillus acidophilus NM can improve the body's antioxidant capacity by increasing CAT activity, thereby relieving fatigue.

[0069] Glutathione peroxidase (GSH-Px) is one of the most important peroxidases in the human body and can be used to assess fatigue and health. It can reduce toxic hydrogen peroxide to non-toxic and harmless hydroxyl compounds in the body, decompose hydrogen peroxide, and protect the integrity of cell membrane structure and function. Exercise causes the body to produce strong oxidative reactions such as lipid oxidation and glucose metabolism, such as Fig.11 As shown, the GSH-Px activity of mice in the medium-dose and high-dose groups of Lactobacillus acidophilus NM increased significantly in direct proportion to the increase in the dose of Lactobacillus acidophilus NM, increasing by 24.13% and 38.96%, respectively. This indicates that Lactobacillus acidophilus NM can improve the body's antioxidant capacity by increasing the activity of GSH-Px, thereby relieving fatigue.

[0070] Malondialdehyde (MDA) is an important metabolite of oxygen free radicals in the body, reflecting the body's potential antioxidant capacity, the rate of lipid peroxidation, and the degree of tissue peroxidation damage. Hypoxia during exercise can produce a large number of free radicals in myocardial tissue. Free radicals act on unsaturated fatty acids on the cell membrane, causing membrane lipid peroxidation, leading to myocardial cell damage and lipid peroxidation. Therefore, under fatigue conditions, the concentration of malondialdehyde increases. Fig.12 As shown, the MDA content of mice in the medium-dose group and high-dose group of Lactobacillus acidophilus NM decreased by 14.71% and 21.69%, and the changes showed an inverse increase, which indicates that Lactobacillus acidophilus NM can inhibit lipid peroxidation in the body and thus play an anti-fatigue effect.

[0071] Embodiment 1

[0072] A traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora. The probiotic preparation is prepared by weight from 5-15 parts of Lactobacillus acidophilus NM freeze-dried powder, 10-29 parts of traditional Chinese medicine extracts, 5-14 parts of prebiotics and 42-80 parts of functional excipients.

[0073] The preparation method of Lactobacillus acidophilus NM freeze-dried powder comprises the following steps:

[0074] S1 medium preparation (per liter): 100g skim milk powder, 5g glucose, 3g yeast extract, 2g disodium hydrogen phosphate, 1g potassium dihydrogen phosphate, 1000mL distilled water, adjust pH to 6.3, and sterilize at 121°C for 15min; S2 bacterial inoculation: After Lactobacillus acidophilus NM is revived, inoculate it at 37°C for anaerobic culture for 21h to make the bacterial concentration reach 1×109 CFU / mL; S3 bacterial collection and freeze-drying: centrifuge (4000rpm, 10min, 4°C) to collect the bacteria; suspend in a protective agent (10% skim milk powder + 5% mannitol), freeze-dry at -50°C, and control the water content to ≤5%.

[0075] Preferably, the Chinese medicine extract is prepared by weight from 7 parts of scutellaria baicalensis extract, 5 parts of honeysuckle extract, 4 parts of liquorice extract, 2 parts of peppermint extract, 4 parts of hawthorn extract, and 3 parts of green tea extract; the preparation method of the Chinese medicine extract comprises the following steps: S1 washing and air-drying the medicinal materials; S2 extraction process, water extraction: adding 10 times of water, decocting at 90°C for 1 hour, filtering and taking the supernatant; secondary decoction: adding 8 times of water, decocting at 90°C for 45 minutes, filtering and taking the supernatant; combined concentration: concentrating under reduced pressure at 80°C to a solid content of 15%;

[0076] S3 spray drying (feed 45°C, atomization pressure 4MPa, outlet temperature 85°C).

[0077] Prebiotics are oligofructose and inulin, with a ratio of 1:1.5, and the functional excipient is maltodextrin.

[0078] The preparation method of the traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation comprises the following specific steps:

[0079] S1 weighing: weighing Lactobacillus acidophilus freeze-dried powder, Chinese medicine extract, prebiotics and functional excipients;

[0080] S2 Mixing: V-type mixer mixes the materials described in S1 for 30 min, temperature ≤ 25°C, humidity ≤ 50%;

[0081] S3 preparation molding: Direct packaging 2g / bag, sealed with aluminum foil.

[0082] Embodiment 2

[0083] Referring to the preparation method of Example 1, the oral powder is prepared according to the formula in the following table, focusing on the antibacterial and anti-inflammatory functions.

[0084] Element Content (g / 100g) Lactobacillus acidophilus NM freeze-dried powder 10 Scutellaria baicalensis extract 5 Honeysuckle Extract 4 Licorice Extract 3 Green Tea Extract 2 Fructo-oligosaccharides 5 Inulin 3 Maltodextrin 68

[0085] Embodiment 3

[0086] Prepare a film tablet according to the formula in the following table, which is mainly used for breath freshening, and add the film tablet preparation steps, the steps are as follows:

[0087] Cultivation and freeze-drying of Lactobacillus acidophilus NM (same as in Example 1);

[0088] Extraction and drying of Chinese medicine (same as in Example 1);

[0089] Wet granulation: weigh probiotic freeze-dried powder, Chinese medicine powder, microcrystalline cellulose, oligofructose, and inulin, mix evenly, add 5% PVP solution to moisten, make wet granules, pass through a 14-mesh sieve, and dry at 40°C for 3h. The moisture content should be controlled to ≤5%;

[0090] Tableting: Tableting pressure 6-8MPa, single tablet 0.5g, tablet shape is round and flat;

[0091] Coating: Eudragit L100 (5% ethanol solution) enteric coating to improve the survival rate of probiotics;

[0092] Packaging: Blister pack, 10 tablets per board, sealed for storage.

[0093] Element Content (g / 100g) Lactobacillus acidophilus NM freeze-dried powder 8 Honeysuckle Extract 5 Peppermint Extract 4 Green Tea Extract 3 Fructo-oligosaccharides 6 Inulin 3 Microcrystalline Cellulose 5 PVP 1 Maltodextrin 65

[0094] Embodiment 4

[0095] The following formula is used to prepare chewable tablets, which are suitable for children and the elderly, enhance oral moisturizing, and add chewable tablet preparation steps:

[0096] Cultivation and freeze-drying of Lactobacillus acidophilus NM (same as in Example 1);

[0097] Extraction and drying of Chinese medicine (same as in Example 1);

[0098] Dry granulation: weigh probiotic freeze-dried powder, Chinese medicine extract powder, oligofructose, inulin, and microcrystalline cellulose, mix evenly, and pass through a 14-mesh sieve to maintain powder uniformity;

[0099] Tableting: Tableting pressure 6-8MPa, single tablet 0.5g, tablet shape is round and flat;

[0100] Coating: Add a small amount of xylitol to enhance the taste, and spray a thin layer of edible film on the surface to prevent deliquescence;

[0101] Packaging: Aluminum foil sealed packaging, 60 tablets per bottle, stored at room temperature.

[0102] Element Content (g / 100g) Lactobacillus acidophilus NM freeze-dried powder 12 Scutellaria baicalensis extract 6 Hawthorn Extract 5 Green Tea Extract 4 Fructo-oligosaccharides 8 Inulin 4 Microcrystalline Cellulose 8 PVP 1 Maltodextrin 52

[0103] Based on the preparations in the above four groups of embodiments, four experiments were conducted on probiotic preparations of different dosage forms, namely: oral flora regulation experiment, antioxidant capacity experiment, fatigue relief experiment and in vitro antibacterial experiment.

[0104] The oral flora regulation experiment is as follows:

[0105] Subjects: 60 healthy volunteers (18-45 years old), randomly divided into groups, grouping and administration method: Group A (oral powder): 2g per day, buccal, once a day, Group B (film tablets): 2 tablets per day, orally dissolved, once a day, Group C (chewable tablets): 2 tablets per day, chewed, once a day, Group D (control group): no probiotics, only ordinary toothpaste, experimental period: 4 weeks, detection time points: day 0, day 14, day 28, detection method: qPCR quantitative detection of the number of pathogenic bacteria of Streptococcus mutans and Porphyromonas gingivalis; 16SrRNA high-throughput sequencing analysis of the abundance changes of Lactobacillus and Bifidobacterium; pH electrode method to measure saliva pH;

[0106] Changes in oral flora are shown in the following table:

[0107] Group Time Streptococcus mutans (log CFU / mL) Porphyromonas gingivalis (log CFU / mL) Lactobacillus (log CFU / mL) Bifidobacterium (log CFU / mL) Saliva pH Group A (oral powder) Day 0 6.45 ± 0.22 5.98 ± 0.18 5.02 ± 0.20 4.88 ± 0.19 6.5 ± 0.1 Group A 14 days 5.21 ± 0.20 4.80 ± 0.21 6.11 ± 0.25 5.72 ± 0.21 6.7 ± 0.2 Group A 28 days 4.32 ± 0.15 4.10 ± 0.17 7.22 ± 0.28 6.81 ± 0.23 6.9 ± 0.2 Group B (film tablets) Day 0 6.50 ± 0.25 5.93 ± 0.22 4.98 ± 0.18 4.92 ± 0.22 6.4 ± 0.1 Group B 14 days 5.40 ± 0.18 4.90 ± 0.20 6.05 ± 0.24 5.68 ± 0.25 6.6 ± 0.2 Group B 28 days 4.41 ± 0.16 4.25 ± 0.19 7.15 ± 0.26 6.75 ± 0.20 6.8 ± 0.2 Group C (chewable tablets) Day 0 6.48 ± 0.24 5.95 ± 0.20 5.05 ± 0.22 4.85 ± 0.21 6.5 ± 0.1 Group C 14 days 5.28 ± 0.19 4.85 ± 0.18 6.02 ± 0.22 5.65 ± 0.24 6.6 ± 0.2 Group C 28 days 4.38 ± 0.14 4.15 ± 0.16 7.10 ± 0.27 6.78 ± 0.22 6.85 ± 0.2 Group D (control group) Day 0 6.42 ± 0.21 5.96 ± 0.20 5.00 ± 0.21 4.89 ± 0.20 6.5 ± 0.1 Group D 14 days 6.35 ± 0.22 5.92 ± 0.19 5.05 ± 0.20 4.95 ± 0.21 6.5 ± 0.1 Group D 28 days 6.38 ± 0.23 5.98 ± 0.22 5.02 ± 0.22 4.90 ± 0.20 6.4 ± 0.1

[0108] Experimental conclusion: After 28 days, the levels of oral pathogenic bacteria Streptococcus mutans and Porphyromonas gingivalis in groups A, B, and C were significantly reduced (P<0.05), the number of Lactobacillus and Bifidobacterium was significantly increased (P<0.05), the balance of oral flora was improved, and the saliva pH value increased, indicating that probiotics can help reduce the acidic environment of the oral cavity and reduce the risk of dental caries.

[0109] The antioxidant capacity test is as follows:

[0110] Experimental animals: SD rats (n=60), randomly divided into groups; Group A: oral powder, Group B: film tablets, Group C: chewable tablets, Group D: control group; Experimental period: 6 weeks, antioxidant indexes were tested every week; Test indexes: superoxide dismutase (SOD) activity, to evaluate free radical scavenging ability, glutathione peroxidase (GSH-Px) activity, to evaluate antioxidant capacity, malondialdehyde (MDA) content, to reflect the level of lipid peroxidation;

[0111] The experimental data are shown in the following table:

[0112] Group SOD (U / mL) GSH-Px (U / mL) MDA (nmol / L) Group A 105.2 ± 5.8 76.1 ± 3.6 2.42 ± 0.18 Group B 101.8 ± 5.4 72.5 ± 3.8 2.55 ± 0.20 Group C 98.7 ± 5.0 70.9 ± 3.5 2.61 ± 0.22 Group D (control) 85.6 ± 4.9 60.2 ± 3.1 3.25 ± 0.26

[0113] Experimental conclusion: Groups A, B, and C significantly increased the levels of SOD and GSH-Px (P<0.05) and reduced MDA (P<0.05). Probiotic preparations can enhance antioxidant capacity and reduce free radical damage.

[0114] The fatigue relief experiment is as follows:

[0115] Experimental animals: SD rats (n=60) were randomly divided into 4 groups. Group A (oral powder): 2 mL of probiotic suspension was intragastrically administered daily (10 9 CFU / mL), Group B (film tablets): 2 pieces of crushed and dissolved film tablets were intragastrically administered daily (10 9CFU / mL), Group C (chewable tablets): 2 chewable tablets (10 9 CFU / mL), group D (control group): gavage of the same amount of distilled water every day, no probiotics; experimental period of 6 weeks; fatigue evaluation index, mouse swimming endurance test (load 10% of body weight, swimming to exhaustion in 25°C water); blood lactic acid (LA) content (detected 30 minutes after exercise, reflecting the degree of muscle fatigue); blood urea nitrogen (BUN) level (protein metabolism fatigue index); blood ammonia (NH3) concentration (nervous system fatigue index); detection method, swimming endurance test: record the time of swimming to exhaustion; LA, BUN, NH3 are determined by enzymatic colorimetry;

[0116] The experimental data are shown in the following table:

[0117] 1. Swimming endurance time (unit: min)

[0118] Group 0 Weeks 2 weeks 4 weeks 6 weeks Group A (oral powder) 5.1 ± 0.8 8.2 ± 1.0 12.4 ± 1.2 16.9 ± 1.3 Group B (film tablets) 5.0 ± 0.9 7.8 ± 0.9 11.8 ± 1.1 16.0 ± 1.2 Group C (chewable tablets) 5.2 ± 0.7 7.5 ± 1.1 11.5 ± 1.0 15.7 ± 1.1 Group D (control group) 5.1 ± 0.8 6.0 ± 0.7 8.3 ± 0.9 10.2 ± 1.0

[0119] 2. Post-exercise blood lactate (LA, mmol / L)

[0120] Group 0 Weeks 2 weeks 4 weeks 6 weeks Group A (oral powder) 12.3 ± 0.8 10.5 ± 0.7 8.1 ± 0.5 5.9 ± 0.4 Group B (film tablets) 12.5 ± 0.9 10.8 ± 0.8 8.3 ± 0.6 6.2 ± 0.5 Group C (chewable tablets) 12.4 ± 0.8 10.9 ± 0.7 8.5 ± 0.7 6.5 ± 0.5 Group D (control group) 12.6 ± 0.8 11.7 ± 0.9 9.8 ± 0.8 8.2 ± 0.6

[0121] 3. Post-exercise blood urea nitrogen (BUN, mmol / L)

[0122] Group 0 Weeks 2 weeks 4 weeks 6 weeks Group A (oral powder) 7.2 ± 0.4 6.5 ± 0.3 5.9 ± 0.3 4.3 ± 0.2 Group B (film tablets) 7.1 ± 0.5 6.4 ± 0.4 6.0 ± 0.3 4.5 ± 0.2 Group C (chewable tablets) 7.0 ± 0.4 6.6 ± 0.4 6.2 ± 0.4 4.6 ± 0.3 Group D (control group) 7.3 ± 0.4 7.1 ± 0.5 6.8 ± 0.5 5.8 ± 0.4

[0123] Experimental conclusion: Groups A, B, and C significantly improved the swimming endurance of mice (P<0.05), among which the oral powder had the best improvement effect. The levels of lactic acid, urea nitrogen, and blood ammonia after exercise were significantly reduced (P<0.05), indicating that probiotic preparations can relieve muscle fatigue and reduce metabolic toxins.

[0124] The in vitro antibacterial test is as follows:

[0125] Strains: Escherichia coli, Staphylococcus aureus, Salmonella; Experimental method: agar diffusion method was used to determine the diameter of the inhibition zone (mm), and group A (oral powder), group B (film tablet), group C (chewable tablet), and group D (blank control) were compared; culture conditions: 37°C, 24h culture, probiotic concentration: 10 9 CFU / mL;

[0126] The experimental data are as follows:

[0127] strain Group Diameter of inhibition zone (mm) Escherichia coli Group A (oral powder) 16.2 ± 0.5 Group B (film tablets) 15.8 ± 0.4 Group C (chewable tablets) 15.5 ± 0.5 Group D (control group) 0.0 Staphylococcus aureus Group A 27.4 ± 0.6 Group B 26.9 ± 0.5 Group C 26.5 ± 0.6 Group D 0.0 salmonella Group A 14.8 ± 0.4 Group B 14.5 ± 0.4 Group C 14.3 ± 0.5 Group D 0.0

[0128] Experimental conclusion: Probiotics can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus, and Salmonella (P<0.05). Oral powder has the strongest antibacterial effect, which may be because the powder form is easier to disperse and has more complete contact with bacteria.

[0129] The above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, characterized in that: The probiotic preparation is composed of Lactobacillus acidophilus NM freeze-dried powder, Chinese medicine extract, prebiotics and functional excipients, wherein the Lactobacillus acidophilus NM freeze-dried powder is freeze-dried from Lactobacillus acidophilus NM, and the Lactobacillus acidophilus NM is deposited in the General Microbiology Center of China Microbiological Culture Collection Committee with a deposit number of CGMCC NO.4472; The Chinese medicine extract is prepared by weight from 5-10 parts of scutellaria baicalensis extract, 3-8 parts of honeysuckle extract, 2-6 parts of licorice extract, 1-4 parts of peppermint extract, 3-6 parts of hawthorn extract, and 1-5 parts of green tea extract; the preparation method of the Chinese medicine extract comprises the following steps: S1 Wash and air dry the medicinal materials; S2 extraction process, water extraction: add 10 times of water, boil at 90°C for 1 hour, filter and take the supernatant; secondary boiling: add 8 times of water, boil at 90°C for 45 minutes, filter and take the supernatant; combined concentration: concentrate at 80°C under reduced pressure to a solid content of 15%; S3 spray drying, feed 45°C, atomization pressure 4MPa, outlet temperature 80-90°C.

2. A Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 1, characterized in that: The probiotic preparation is prepared from 5-15 parts of Lactobacillus acidophilus NM freeze-dried powder, 10-29 parts of traditional Chinese medicine extracts, 5-14 parts of prebiotics and 42-80 parts of functional excipients by weight.

3. A Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 2, characterized in that: The preparation method of the Lactobacillus acidophilus NM freeze-dried powder comprises the following steps: Preparation of S1 medium: 100 g skim milk powder, 5 g glucose, 3 g yeast extract, 2 g disodium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 1000 mL distilled water, adjust pH to 6.2-6.5, and sterilize at 121°C for 15 min; S2 bacterial inoculation: After Lactobacillus acidophilus NM is revived, inoculate it to 37°C for anaerobic culture for 18-24h to make the bacterial concentration reach 1×10 9 CFU / mL; S3 bacterial cell collection and freeze-drying: centrifuge at 4000 rpm for 10 min at 4°C to collect the bacterial cells; suspend in 10% skim milk powder + 5% mannitol, freeze-dry at -50°C, and control the water content to ≤5%.

4. The Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 1, characterized in that: The Chinese medicine extract is prepared from 5 parts of scutellaria baicalensis extract, 4 parts of honeysuckle extract, 3 parts of liquorice extract and 2 parts of green tea extract by weight.

5. The Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 1, characterized in that: The traditional Chinese medicine extract is prepared from 5 parts of honeysuckle extract, 4 parts of peppermint extract and 3 parts of green tea extract by weight.

6. The Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 1, characterized in that: The Chinese medicine extract is prepared from 6 parts of scutellaria baicalensis extract, 5 parts of hawthorn extract and 4 parts of green tea extract by weight.

7. The Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 2, characterized in that: The prebiotics are oligofructose and inulin, and the ratio of the two is 1:1.

5.

8. The Chinese medicinal composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 2, characterized in that: The functional auxiliary material is maltodextrin.

9. The method for preparing the traditional Chinese medicine composite Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to any one of claims 1 to 8, characterized in that: The specific steps are as follows: S1 weighing: weighing Lactobacillus acidophilus freeze-dried powder, Chinese medicine extract, prebiotics and functional excipients; S2 Mixing: V-type mixer mixes the materials described in S1 for 30 min, temperature ≤ 25°C, humidity ≤ 50%; S3 preparation molding: Direct packaging 2g / bag, sealed with aluminum foil.

Images