A traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral flora and its preparation process

Abstract

The present invention discloses a traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and its preparation process, which relates to the related field of biotechnology. The traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation of the present invention combines the synergistic effects of probiotics and traditional Chinese medicine, and has multiple advantages such as regulating oral flora, antibacterial and anti-inflammatory, antioxidant, and relieving fatigue. Lactobacillus acidophilus NM can tolerate acid and bile salts, effectively inhibit pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella, reduce the occurrence of dental caries, gingivitis, and halitosis. It has no antibiotic resistance and is safer for long-term use. The extracts of Scutellaria baicalensis, honeysuckle, and green tea are rich in flavonoids and tea polyphenols, which can further antibacterial and anti-inflammatory and improve oral health. At the same time, this probiotic can reduce blood lactic acid, blood urea nitrogen, and blood ammonia, enhance the activities of SOD, CAT, and GSH-Px, and inhibit lipid peroxidation, thereby relieving body fatigue and enhancing immunity. The preparation forms are diverse.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and its preparation process. Background Art

[0002] In recent years, oral health problems have received increasing attention. The incidence of diseases such as dental caries, gingivitis, and bad breath has been rising year by year. The main reason is closely related to the imbalance of oral flora. Research shows that the excessive reproduction of pathogenic bacteria such as Streptococcus mutans, Porphyromonas gingivalis, Escherichia coli, Staphylococcus aureus, and Salmonella in the oral cavity will lead to the formation of dental plaque and the aggravation of gingival inflammation, which will further affect overall health. In addition, with the accelerating pace of modern social life, people generally have problems such as fatigue, decreased immunity, and increased oxidative stress. And oral health status is closely related to the overall health of the body. Therefore, the development of probiotic products with functions of oral regulation, antioxidant, and fatigue relief has important practical significance. Summary of the Invention

[0003] Therefore, in order to solve the above deficiencies, the present invention provides a traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora and its preparation process.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: A traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, the probiotic preparation is composed of Lactobacillus acidophilus NM freeze-dried powder, traditional Chinese medicine extract, prebiotic, and functional excipients. The Lactobacillus acidophilus NM freeze-dried powder is freeze-dried from Lactobacillus acidophilus NM, and the Lactobacillus acidophilus NM is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC NO.4472.

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

[0006] Preferably, the preparation method of the Lactobacillus acidophilus NM freeze-dried powder includes the following steps:

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

[0008] S2 Bacterial Strain Inoculation: After the Lactobacillus acidophilus NM is resuscitated, it is inoculated and anaerobically cultured at 37 °C for 18-24 h to make the cell concentration reach 1×10 9 CFU / mL;

[0009] Collection and lyophilization of S3 bacteria: Centrifuge at 4000 rpm for 10 min at 4°C to collect the bacteria; suspend them in 10% skim milk powder + 5% mannitol and lyophilize at -50°C, controlling the water content ≤ 5%.

[0010] Preferably, the traditional Chinese medicine extract is made of 5 - 10 parts of Scutellaria baicalensis extract, 3 - 8 parts of honeysuckle extract, 2 - 6 parts of licorice extract, 1 - 4 parts of mint extract, 3 - 6 parts of hawthorn extract, and 1 - 5 parts of green tea extract by weight; the preparation method of the traditional Chinese medicine extract includes the following steps:

[0011] S1 Clean and air-dry the medicinal materials;

[0012] S2 Extraction process, water extraction: Add 10 times the amount of water and decoct at 90°C for 1 hour, filter to obtain the supernatant; secondary decoction: Add 8 times the amount of water and decoct at 90°C for 45 minutes, filter to obtain the supernatant; combine and concentrate: Concentrate under reduced pressure at 80°C to a solid content of 15%;

[0013] S3 Spray drying: Feed at 45°C, atomization pressure 4 MPa, outlet temperature 80 - 90°C.

[0014] Preferably, the traditional Chinese medicine extract is made of 5 parts of Scutellaria baicalensis extract, 4 parts of honeysuckle extract, 3 parts of licorice extract, and 2 parts of green tea extract by weight.

[0015] Preferably, the traditional Chinese medicine extract is made of 5 parts of honeysuckle extract, 4 parts of mint extract, and 3 parts of green tea extract by weight.

[0016] Preferably, the traditional Chinese medicine extract is made of 6 parts of Scutellaria baicalensis extract, 5 parts of hawthorn extract, and 4 parts of green tea extract by weight.

[0017] Preferably, the prebiotic is fructooligosaccharide and inulin, and the ratio of the two is 1:1.5.

[0018] Preferably, the functional excipient is maltodextrin.

[0019] Preferably, the preparation method of the traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation is as follows:

[0020] S1 Weigh: Weigh the freeze-dried powder of Lactobacillus acidophilus, traditional Chinese medicine extract, prebiotic, and functional excipient;

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

[0022] S3 Preparation forming: Directly package into 2 g / bags and seal with aluminum foil.

[0023] Advantages of the present invention:

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

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

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

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

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

[0029] Lactobacillus acidophilus NM is not resistant to antibiotics such as acetylspiramycin, streptomycin, and penicillin. Long-term use will not cause the proliferation of drug-resistant strains, ensuring the safety and effectiveness of the product;

[0030] It is applicable to people with oral ulcers, gingivitis, bad breath, etc., and can also be used for those who are prone to fatigue and have low immunity. There are various dosage forms such as powder, tablets, and chewable tablets to meet the needs of different age groups and usage. Brief Description of the Drawings

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

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

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

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

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

[0036] Figure 6 It is a schematic diagram of blood lactic acid in mouse of the present invention;

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

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

[0039] Figure 9 It is a schematic diagram of SOD activity in mouse serum of the present invention;

[0040] Figure 10 It is a schematic diagram of CAT activity in mouse serum of the present invention;

[0041] Figure 11 It is a schematic diagram of GSH-Px in mouse of the present invention;

[0042] Figure 12 It is a schematic diagram of MDA content in mouse of the present invention;

[0043] Figure 13 It is a schematic diagram of the whole gene sequence map of the present invention. Detailed implementation mode

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

[0045] A traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora. The probiotic preparation is composed of freeze-dried powder of Lactobacillus acidophilus NM, traditional Chinese medicine extract, prebiotic and functional excipients. The freeze-dried powder of Lactobacillus acidophilus NM is freeze-dried from Lactobacillus acidophilus NM. Lactobacillus acidophilus NM is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC NO.4472, the preservation date is December 14, 2010, the preservation address: General Microbiology Center of the China Committee for Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation result: survived.

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

[0047] Refer to Figure 2 , colony morphology, Lactobacillus acidophilus NM MRS medium, the colonies are white, nearly round, with a moist surface, opaque, and neat edges.

[0048] The pH of gastric juice fluctuates greatly depending on the diet structure. Generally, the pH is around 3. When fasting or consuming acidic foods, the pH can reach 1.5, and when consuming alkaline foods, the pH can reach 4 - 5. The time for food to pass through the stomach is 1 - 2 hours, and the bile salt concentration in the duodenum is 0.3 - 3 g / kg. The time for food to pass through the duodenum is relatively extremely short. Therefore, the human gastrointestinal tract belongs to a high - acid and high - bile - salt environment. This experiment simulates the human gastrointestinal environment to study the tolerance of Lactobacillus acidophilus NM to gastric acid and bile salts. Referring to Figure 3 After treating with a culture medium of pH 3.0 for 2 hours, the survival rate of Lactobacillus acidophilus NM was 79.40%. After treating with a culture medium of pH 2.0 for 2 hours, the survival rate of Lactobacillus acidophilus NM was 9.20%. After treating with a culture medium of pH 4.0 for 2 hours, the survival rate of Lactobacillus acidophilus NM was 98.20%. The results indicate that Lactobacillus acidophilus NM has strong tolerance to acid;

[0049] Take 9 a suspension of Lactobacillus acidophilus NM at 10

[0050] Treatment time (h) Survival rate at pH 1.5 (%) Survival rate at pH 2.0 (%) Survival rate at pH 3.0 (%) Survival rate at pH 4.0 (%) 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] CFU / mL, and add it to artificial gastric juices with pH 1.5, 2.0, 3.0, and 4.0 respectively. Incubate with shaking at 37°C for 2 hours, sample every 30 minutes, perform plate counting, and calculate the survival rate. The experimental results are as follows: Figure 4 ;

[0052] Take 9 a suspension of Lactobacillus acidophilus NM at 10

[0053] Treatment time (h) Survival rate with 0.10% bile salts (%) Survival rate with 0.30% bile salts (%) Survival rate with 0.50% bile salts (%) 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 all three 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 metabolites that can exert probiotic properties, Lactobacillus acidophilus can also adhere to the host digestive tract, colonize and survive to form a biological barrier (occupation effect), inhibit the adhesion of pathogenic bacteria on the digestive tract mucosa, and inhibit the growth of pathogenic bacteria by competing for nutrients with pathogenic bacteria. In addition, it can prevent the absorption of harmful substances such as toxins and neutralize toxic substances;

[0055] The experimental methods are as follows:

[0056] For the antibacterial activity experiment by the agar diffusion method, take the supernatant of Lactobacillus acidophilus NM cultured in MRS medium at 37°C for 24 h, centrifuge at 8000 rpm for 10 min, and take the supernatant for the antibacterial experiment. Remove the bacteria with a sterile filter membrane (0.22 μm). Inoculate Escherichia coli, Staphylococcus aureus, and Salmonella into LB agar medium respectively. After spreading the plate, punch holes (diameter 6 mm) on the agar, add 100 μL of the supernatant of Lactobacillus acidophilus NM to each hole, and culture at 37°C for 24 h, then measure the diameter of the antibacterial zone;

[0057] For the detection of metabolites of Lactobacillus acidophilus NM, high performance liquid chromatography (HPLC) is used to detect the contents of lactic acid, acetic acid, formic acid, propionic acid, and butyric acid in the fermentation broth of Lactobacillus acidophilus NM, and a hydrogen peroxide detection kit is used to detect whether the NM strain produces hydrogen peroxide;

[0058] For the antibiotic sensitivity experiment by the K-B disk diffusion method, take the culture solution of Lactobacillus acidophilus NM, spread it on an MRS agar plate, place acetylspiramycin, streptomycin, and penicillin antibiotic disks in the center of the plate, and culture anaerobically at 37°C for 24 h, then measure the diameter of the antibacterial zone to determine the sensitivity;

[0059] Strain Inhibition zone diameter / mm Escherichia coli 16.67±0.45（++） Staphylococcus aureus 27.54±0.67（++++） Salmonella 15.21±0.32（+）

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

[0061] Antibiotic Inhibition zone diameter / mm Acetylspiramycin 24.31 ± 0.21 (high sensitivity) Penicillin 31.21 ± 0.31 (high sensitivity) Streptomycin 26.33 ± 0.45 (high sensitivity)

[0062] Select SPF-grade male mice (BALB / c), 6 weeks old, weighing 20±2 g, adaptively fed for 1 week, and randomly divided into 4 groups (n = 10). For the experimental grouping, the control group: do not supplement Lactobacillus acidophilus NM, only feed ordinary feed; the low-dose group: 1×10 8 CFU / mL Lactobacillus acidophilus NM, intragastrically administered 0.2 mL per day for 4 weeks; the medium-dose group: 1×109 Lactobacillus acidophilus NM at 0.2 mL of 5×10 9 CFU / mL was intragastrically administered to mice once a day for 4 weeks; high-dose group: 5×10

[0063] Creatine kinase is an important kinase directly related to energy transfer, muscle contraction, and ATP regeneration. After exercise, the creatine kinase in the body increases significantly and is positively correlated with the exercise intensity. For example, Figure 5 after 4 weeks of supplementation with Lactobacillus acidophilus NM, compared with the control group of mice, the serum creatine kinase activities of the low-dose, medium-dose, and high-dose groups of Lactobacillus acidophilus NM mice were significantly decreased. However, there was no significant difference between the medium-dose and high-dose groups. The serum creatine kinase activities of the mice decreased by 16.00%, 28.50%, and 33.90% respectively. Trend analysis showed that with the increase in the dose of Lactobacillus acidophilus NM, the creatine kinase activity decreased in a dose-dependent manner, indicating that Lactobacillus acidophilus NM can reduce the creatine kinase activity in mice and thus relieve physical fatigue.

[0064] Lactic acid in the blood is an inevitable product of sugar metabolism in the body. The increase in lactic acid will lead to a decrease in the pH of muscle tissue and blood, triggering biochemical and physiological processes harmful to human functions. Lactic acid is an important indicator for evaluating aerobic metabolism and fatigue of the body. For example, Figure 6 as shown, after 4 weeks of supplementation with Lactobacillus acidophilus NM, compared with the control group of mice, the blood lactic acid contents of the low-dose, medium-dose, and high-dose groups of Lactobacillus acidophilus NM mice were significantly decreased. However, there was no significant difference between the medium-dose and high-dose groups. Their lactic acid contents 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 lactic acid in the blood of mice and thus relieve physical fatigue.

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

[0066] Ammonia is an important metabolite produced during exercise energy metabolism, generated from different sources. The ammonia accumulated in the blood and brain during exercise can have a negative impact on the central nervous system and cause fatigue. Although exercise-induced ammonia intoxication is transient and reversible, depending on the disease state, it may affect the continuous coordinated activities of key areas of the central nervous system, and the central nervous system plays a crucial role in the development of physical fatigue, such as Figure 8 As shown, after 4 weeks of supplementation with Lactobacillus acidophilus NM, compared with the blood ammonia levels of the control group mice, the blood ammonia levels of the low-dose, medium-dose, and high-dose groups of Lactobacillus acidophilus NM mice were all significantly reduced, being 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 group and the high-dose group; this indicates that Lactobacillus acidophilus NM can reduce the blood ammonia level of mice, thereby alleviating physical fatigue.

[0067] More and more evidence shows that reactive oxygen species induce protein oxidation and strongly promote muscle fatigue. Probiotics can reduce oxidative stress, improve exercise ability, and reduce the degree of fatigue. Therefore, in this study, mouse oxidative stress indicators were used to evaluate the role of Lactobacillus acidophilus NM in improving exercise ability and alleviating fatigue. Superoxide dismutase (SOD) is a biologically active substance, it is an active protease containing metal elements, which can scavenge harmful substances generated during metabolism. The content of SOD can directly reflect the degree of aging and fatigue of the body, such as Figure 9 As shown, compared with the serum SOD activity of the control group mice, the serum SOD activities of the low-dose, medium-dose, and high-dose groups of Lactobacillus acidophilus NM mice were all significantly increased, by 6.82%, 23.17%, and 37.80% respectively; trend analysis showed that as the dose of Lactobacillus acidophilus NM increased, the serum SOD activity increased in a dose-dependent manner, which indicates that Lactobacillus acidophilus NM can improve the antioxidant capacity of the body by increasing the SOD activity, thereby alleviating fatigue.

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

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

[0070] Malondialdehyde (MDA) is an important metabolite of oxygen free radicals in the body, which reflects the potential antioxidant capacity of the body, the rate of lipid peroxidation, and the degree of tissue peroxidation damage. Exercise hypoxia can generate a large number of free radicals in myocardial tissue. The free radicals act on the unsaturated fatty acids on the cell membrane, causing peroxidation of membrane lipids, resulting in damage to myocardial cells and forming lipid peroxidation. Therefore, the concentration of malondialdehyde increases in the fatigue state, such as Figure 12 As shown, the MDA contents of mice in the medium-dose and high-dose groups of Lactobacillus acidophilus NM decreased by 14.71% and 21.69% respectively, and their changes showed an inverse growth. This indicates that Lactobacillus acidophilus NM can inhibit lipid peroxidation in the body, thereby playing an anti-fatigue role.

[0071] Example 1

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

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

[0074] S1 Medium preparation (per liter): 100 g of skim milk powder, 5 g of glucose, 3 g of yeast extract, 2 g of disodium hydrogen phosphate, 1 g of potassium dihydrogen phosphate, 1000 mL of distilled water, adjust the pH to 6.3, and sterilize at 121 °C under high pressure for 15 min; S2 Strain inoculation: After the resuscitation of Lactobacillus acidophilus NM, inoculate it into anaerobic culture at 37 °C for 21 h to make the cell concentration reach 1×109 CFU / mL; Collection and lyophilization of S3 bacteria: Centrifuge (4000 rpm, 10 min, 4°C) to collect the bacteria; Suspend the bacteria in the cryoprotectant (10% skim milk powder + 5% mannitol) and lyophilize at -50°C, controlling the water content ≤ 5%.

[0075] Preferably, the traditional Chinese medicine extract is made of 7 parts of Scutellaria baicalensis extract, 5 parts of Lonicera japonica extract, 4 parts of Glycyrrhiza glabra extract, 2 parts of Mentha haplocalyx extract, 4 parts of Crataegus pinnatifida extract, and 3 parts of green tea extract by weight; The preparation method of the traditional Chinese medicine extract includes the following steps: S1 Wash and air-dry the medicinal materials; S2 Extraction process, water extraction: Add 10 times the amount of water and decoct at 90°C for 1 hour, filter to obtain the supernatant; Second decoction: Add 8 times the amount of water and decoct at 90°C for 45 minutes, filter to obtain the supernatant; Combine and concentrate: Concentrate under reduced pressure at 80°C to a solid content of 15%.

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

[0077] The prebiotics are fructooligosaccharide and inulin, and the ratio of the two is 1:1.5. The functional excipient is maltodextrin.

[0078] The preparation method of the traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation is as follows:

[0079] S1 Weigh: Weigh the freeze-dried powder of Lactobacillus acidophilus, the traditional Chinese medicine extract, the prebiotics, and the functional excipient;

[0080] S2 Mix: Mix the materials described in S1 in a V-shaped mixer for 30 min, with the temperature ≤ 25°C and the humidity ≤ 50%;

[0081] S3 Preparation forming: Directly package into 2 g / bags and seal with aluminum foil.

[0082] Example 2

[0083] Refer to the preparation method in Example 1 and prepare it into a buccal powder according to the following formula, with an emphasis on antibacterial and anti-inflammatory functions.

[0084] Component Content (g / 100g) Freeze-dried powder of Lactobacillus acidophilus NM 10 Scutellaria baicalensis extract 5 Flos Lonicerae extract 4 Glycyrrhiza uralensis extract 3 Camellia sinensis extract 2 Fructooligosaccharide 5 Inulin 3 Maltodextrin 68

[0085] Example 3

[0086] Prepare it into a film tablet according to the following formula, mainly for fresh breath, and add the preparation steps of the film tablet, the steps are as follows:

[0087] Culture and lyophilize Lactobacillus acidophilus NM (same as Example 1);

[0088] Extract and dry the traditional Chinese medicine (same as Example 1);

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

[0090] Tabletting: Tablet under a pressure of 6 - 8 MPa, with each tablet weighing 0.5 g and the tablet shape being round and flat;

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

[0092] Packaging: Blister packaging, with 10 tablets per board, and stored sealed.

[0093] Component Content (g / 100g) Freeze-dried powder of Lactobacillus acidophilus NM 8 Flos Lonicerae extract 5 Mentha haplocalyx extract 4 Camellia sinensis extract 3 Fructooligosaccharide 6 Inulin 3 Microcrystalline cellulose 5 PVP 1 Maltodextrin 65

[0094] Example 4

[0095] Prepared as chewable tablets according to the following formula, suitable for children and the elderly, enhancing oral moisture retention, and adding the preparation steps of chewable tablets:

[0096] Culture and freeze-dry Lactobacillus acidophilus NM (same as Example 1);

[0097] Extract and dry the traditional Chinese medicine (same as Example 1);

[0098] Dry granulation: Weigh the freeze-dried probiotic powder, traditional Chinese medicine extract powder, fructooligosaccharide, inulin, and microcrystalline cellulose, mix them evenly, pass through a 14-mesh sieve, and maintain the powder uniformity;

[0099] Tabletting: Tablet under a pressure of 6 - 8 MPa, with each tablet weighing 0.5 g and the tablet shape being round and flat;

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

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

[0102] Component Content (g / 100g) Freeze-dried powder of Lactobacillus acidophilus NM 12 Scutellaria baicalensis extract 6 Fructus Crataegi extract 5 Camellia sinensis extract 4 Fructooligosaccharide 8 Inulin 4 Microcrystalline cellulose 8 PVP 1 Maltodextrin 52

[0103] Based on the preparations in the above four groups of examples, four-part 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 (aged 18 - 45), randomly grouped. Grouping and administration methods: Group A (powder for buccal use): 2 g per day, buccally administered once a day; Group B (film tablets): 2 tablets per day, orally disintegrated once a day; Group C (chewable tablets): 2 tablets per day, chewed once a day; Group D (control group): did not take probiotics, only used ordinary toothpaste. Experimental period: 4 weeks. Detection time points: Day 0, Day 14, Day 28. Detection methods: qPCR was used to quantitatively detect the number of pathogenic bacteria Streptococcus mutans and Porphyromonas gingivalis; 16S rRNA high-throughput sequencing was used to analyze the abundance changes of Lactobacillus and Bifidobacterium; pH electrode method was used to measure the salivary pH value.

[0106] Changes in oral flora are as follows in the table:

[0107] Group Time point Streptococcus mutans (log CFU / mL) Porphyromonas gingivalis (log CFU / mL) Lactobacillus (log CFU / mL) Bifidobacterium (log CFU / mL) Salivary 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 Day 14 5.21 ± 0.20 4.80 ± 0.21 6.11 ± 0.25 5.72 ± 0.21 6.7 ± 0.2 Group A Day 28 4.32 ± 0.15 4.10 ± 0.17 7.22 ± 0.28 6.81 ± 0.23 6.9 ± 0.2 Group B (film tablet) Day 0 6.50 ± 0.25 5.93 ± 0.22 4.98 ± 0.18 4.92 ± 0.22 6.4 ± 0.1 Group B Day 14 5.40 ± 0.18 4.90 ± 0.20 6.05 ± 0.24 5.68 ± 0.25 6.6 ± 0.2 Group B Day 28 4.41 ± 0.16 4.25 ± 0.19 7.15 ± 0.26 6.75 ± 0.20 6.8 ± 0.2 Group C (chewable tablet) Day 0 6.48 ± 0.24 5.95 ± 0.20 5.05 ± 0.22 4.85 ± 0.21 6.5 ± 0.1 Group C Day 14 5.28 ± 0.19 4.85 ± 0.18 6.02 ± 0.22 5.65 ± 0.24 6.6 ± 0.2 Group C Day 28 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 Day 14 6.35 ± 0.22 5.92 ± 0.19 5.05 ± 0.20 4.95 ± 0.21 6.5 ± 0.1 Group D Day 28 6.38 ± 0.23 5.98 ± 0.22 5.02 ± 0.22 4.90 ± 0.20 6.4 ± 0.1

[0108] Experimental conclusion: In groups A, B, and C, the levels of oral pathogenic bacteria Streptococcus mutans and Porphyromonas gingivalis were significantly decreased after 28 days (P < 0.05), the numbers of Lactobacillus and Bifidobacterium were significantly increased (P < 0.05), the balance of oral flora was improved, and the salivary pH value increased, indicating that probiotics are helpful in reducing the oral acidic environment and lowering the risk of dental caries.

[0109] The antioxidant capacity experiment is as follows:

[0110] Experimental animals: SD rats (n = 60), randomly grouped; Group A: powder for buccal use, Group B: film tablets, Group C: chewable tablets, Group D: control group; Experimental period: 6 weeks, and antioxidant indexes were detected weekly; Detection indexes: superoxide dismutase (SOD) activity to evaluate free radical scavenging ability, glutathione peroxidase (GSH-Px) activity to evaluate antioxidant ability, and malondialdehyde (MDA) content to reflect the level of lipid peroxidation.

[0111] The experimental data are as follows in the 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: In groups A, B, and C, the levels of SOD and GSH-Px were significantly increased (P < 0.05), and MDA was decreased (P < 0.05). The probiotic preparation can enhance antioxidant capacity and reduce free radical damage.

[0114] The experiment on relieving fatigue is as follows:

[0115] Experimental animals: SD rats (n = 60), randomly divided into 4 groups. Group A (powder for buccal use): intragastrically administered 2 mL of probiotic suspension (10 9 CFU / mL) per day, Group B (film tablets): intragastrically administered 2 tablets of crushed and dissolved film tablets (10 9CFU / mL), Group C (chewable tablets): intragastric administration of 2 chewable tablets dissolved after being crushed every day (10 9 CFU / mL), Group D (control group): intragastric administration of an equal amount of distilled water every day, without taking probiotics; the experimental period was 6 weeks; fatigue evaluation indicators, mouse swimming endurance test (the time of swimming to exhaustion in water at 25°C with a load of 10% of body weight); blood lactic acid (LA) content (detected 30 min 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, and NH3 were determined by enzymatic colorimetry;

[0116] The experimental data are as follows in the table:

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

[0118] Group Week 0 Week 2 Week 4 Week 6 Group A (oral powder) 5.1 ± 0.8 8.2 ± 1.0 12.4 ± 1.2 16.9 ± 1.3 Group B (film tablet) 5.0 ± 0.9 7.8 ± 0.9 11.8 ± 1.1 16.0 ± 1.2 Group C (chewable tablet) 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 lactic acid (LA, mmol / L)

[0120] Group Week 0 Week 2 Week 4 Week 6 Group A (oral powder) 12.3 ± 0.8 10.5 ± 0.7 8.1 ± 0.5 5.9 ± 0.4 Group B (film tablet) 12.5 ± 0.9 10.8 ± 0.8 8.3 ± 0.6 6.2 ± 0.5 Group C (chewable tablet) 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 Week 0 Week 2 Week 4 Week 6 Group A (oral powder) 7.2 ± 0.4 6.5 ± 0.3 5.9 ± 0.3 4.3 ± 0.2 Group B (film tablet) 7.1 ± 0.5 6.4 ± 0.4 6.0 ± 0.3 4.5 ± 0.2 Group C (chewable tablet) 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: The swimming endurance of mice in Groups A, B, and C was significantly improved (P<0.05). Among them, the oral powder had the best improvement effect, and the levels of lactic acid, urea nitrogen, and blood ammonia after exercise were significantly reduced (P<0.05), indicating that the probiotic preparation could relieve muscle fatigue and reduce metabolic toxins.

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

[0125] Strains: Escherichia coli, Staphylococcus aureus, Salmonella; experimental method, the diameter of the inhibition zone (mm) was measured by the agar diffusion method, comparing Group A (oral powder), Group B (film tablets), Group C (chewable tablets), and Group D (blank control); culture conditions, 37°C, cultured for 24 h, probiotic concentration: 10 9 CFU / mL;

[0126] The experimental data are as follows:

[0127] Strain Group Inhibition zone diameter (mm) Escherichia coli Group A (oral powder) 16.2 ± 0.5 Group B (film tablet) 15.8 ± 0.4 Group C (chewable tablet) 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). The antibacterial effect of the oral powder is the strongest, probably because the powder form is more easily dispersed and can contact bacteria more fully.

[0129] The above are only the preferred examples of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, characterized in that: The probiotic preparation is composed of freeze-dried powder of Lactobacillus acidophilus NM, Chinese herbal medicine extract, prebiotic and functional excipients. The freeze-dried powder of Lactobacillus acidophilus NM is obtained by freeze-drying Lactobacillus acidophilus NM. Lactobacillus acidophilus NM is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC NO.4472; The Chinese herbal medicine extract is made of 5-10 parts of Scutellaria baicalensis extract, 3-8 parts of Lonicera japonica extract, 2-6 parts of Glycyrrhiza glabra extract, 1-4 parts of Mentha haplocalyx extract, 3-6 parts of Crataegus pinnatifida extract, and 1-5 parts of green tea extract by weight. The preparation method of the Chinese herbal medicine extract includes the following steps: S1 Cleaning and air-drying the medicinal materials; S2 Extraction process: water extraction: adding 10 times of water, decocting at 90°C for 1 hour, filtering to obtain the supernatant; secondary decoction: adding 8 times of water, decocting at 90°C for 45 minutes, filtering to obtain the supernatant; combining and concentrating: concentrating under reduced pressure at 80°C to a solid content of 15%; S3 Spray drying, with the feeding temperature of 45°C, atomization pressure of 4MPa, and outlet temperature of 80-90°C; The probiotic preparation is made of 5-15 parts of freeze-dried powder of Lactobacillus acidophilus NM, 10-29 parts of Chinese herbal medicine extract, 5-14 parts of prebiotic and 42-80 parts of functional excipients by weight.

2. The traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 1, wherein: The preparation method of the freeze-dried powder of Lactobacillus acidophilus NM includes the following steps: S1 Preparation of the culture medium: 100g of skim milk powder, 5g of glucose, 3g of yeast extract, 2g of disodium hydrogen phosphate, 1g of potassium dihydrogen phosphate, 1000mL of distilled water, adjusting the pH to 6.2-6.5, and autoclaving at 121°C for 15 minutes; Inoculation of Strain S2: After the resuscitation of Lactobacillus acidophilus NM, inoculate it into anaerobic culture at 37 °C for 18 - 24 h to make the cell concentration reach 1×10 9 CFU / mL; S3 Collection and freeze-drying of the bacterial cells: centrifuging at 4000rpm for 10 minutes at 4°C to collect the bacterial cells; suspending in 10% skim milk powder + 5% mannitol, and freeze-drying at -50°C, controlling the water content ≤ 5%.

3. A traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, characterized in that: The probiotic preparation is composed of freeze-dried powder of Lactobacillus acidophilus NM, Chinese herbal medicine extract, prebiotic and functional excipients. The freeze-dried powder of Lactobacillus acidophilus NM is obtained by freeze-drying Lactobacillus acidophilus NM. Lactobacillus acidophilus NM is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC NO.4472; The Chinese herbal medicine extract is made of 5 parts of Scutellaria baicalensis extract, 4 parts of Lonicera japonica extract, 3 parts of Glycyrrhiza glabra extract and 2 parts of green tea extract by weight. The preparation method of the Chinese herbal medicine extract includes the following steps: S1 Cleaning and air-drying the medicinal materials; S2 Extraction process: water extraction: adding 10 times of water, decocting at 90°C for 1 hour, filtering to obtain the supernatant; secondary decoction: adding 8 times of water, decocting at 90°C for 45 minutes, filtering to obtain the supernatant; combining and concentrating: concentrating under reduced pressure at 80°C to a solid content of 15%; S3 Spray drying, with the feeding temperature of 45°C, atomization pressure of 4MPa, and outlet temperature of 80-90°C; The probiotic preparation is made of 5-15 parts of freeze-dried powder of Lactobacillus acidophilus NM, 10-29 parts of Chinese herbal medicine extract, 5-14 parts of prebiotic and 42-80 parts of functional excipients by weight.

4. A traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, characterized in that: The probiotic preparation is composed of freeze-dried powder of Lactobacillus acidophilus NM, traditional Chinese medicine extract, prebiotic and functional excipients. The freeze-dried powder of Lactobacillus acidophilus NM is obtained by freeze-drying Lactobacillus acidophilus NM. Lactobacillus acidophilus NM is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC NO.4472; The traditional Chinese medicine extract is made of 5 parts of honeysuckle extract, 4 parts of mint extract and 3 parts of green tea extract by weight. The preparation method of the traditional Chinese medicine extract includes the following steps: S1 Wash and air-dry the medicinal materials; S2 Extraction process, water extraction: add 10 times of water, decoct at 90 °C for 1 hour, filter to obtain the supernatant; secondary decoction: add 8 times of water, decoct at 90 °C for 45 minutes, filter to obtain the supernatant; combine and concentrate: concentrate under reduced pressure at 80 °C to a solid content of 15%; S3 Spray drying, feed temperature 45 °C, atomization pressure 4 MPa, outlet temperature 80 - 90 °C; The probiotic preparation is made of 5 - 15 parts of freeze-dried powder of Lactobacillus acidophilus NM, 10 - 29 parts of traditional Chinese medicine extract, 5 - 14 parts of prebiotic and 42 - 80 parts of functional excipients by weight.

5. A traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora, characterized in that: The probiotic preparation is composed of freeze-dried powder of Lactobacillus acidophilus NM, traditional Chinese medicine extract, prebiotic and functional excipients. The freeze-dried powder of Lactobacillus acidophilus NM is obtained by freeze-drying Lactobacillus acidophilus NM. Lactobacillus acidophilus NM is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC NO.4472; The traditional Chinese medicine extract is made of 6 parts of Scutellaria baicalensis extract, 5 parts of hawthorn extract and 4 parts of green tea extract by weight. The preparation method of the traditional Chinese medicine extract includes the following steps: S1 Wash and air-dry the medicinal materials; S2 Extraction process, water extraction: add 10 times of water, decoct at 90 °C for 1 hour, filter to obtain the supernatant; secondary decoction: add 8 times of water, decoct at 90 °C for 45 minutes, filter to obtain the supernatant; combine and concentrate: concentrate under reduced pressure at 80 °C to a solid content of 15%; S3 Spray drying, feed temperature 45 °C, atomization pressure 4 MPa, outlet temperature 80 - 90 °C; The probiotic preparation is made of 5 - 15 parts of freeze-dried powder of Lactobacillus acidophilus NM, 10 - 29 parts of traditional Chinese medicine extract, 5 - 14 parts of prebiotic and 42 - 80 parts of functional excipients by weight.

6. The traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral bacterial flora according to claim 1, characterized in that: The prebiotic is fructooligosaccharide and inulin, and the ratio of the two is 1:1.

5.

7. The traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral flora according to claim 1, wherein: The functional excipient is maltodextrin.

8. A method for preparing a traditional Chinese medicine compound Lactobacillus acidophilus NM probiotic preparation for improving oral microbiota according to any one of claims 1-7, characterized in that: The specific steps are as follows: S1 Weighing: Weigh the freeze-dried powder of Lactobacillus acidophilus, traditional Chinese medicine extract, prebiotic and functional excipient; S2 Mixing: Mix the materials described in S1 with a V-type mixer for 30 min, temperature ≤ 25 °C, humidity ≤ 50%; S3 Preparation forming: Directly package into 2 g / bags and seal with aluminum foil.

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