Preparation method and application of mint probiotic composition with effect of preventing and / or relieving halitosis

Abstract

The invention provides a preparation method and application of a mint probiotic composition with the effect of preventing and / or relieving ozostomia, mint, green tea and sea buckthorn seeds are used as plant raw materials, supercritical CO2 fluid is adopted for efficient wall breaking, then an ultrasonic technology is used for secondary extraction, then the mixture is compounded with compound probiotics, and the mint probiotic composition is prepared. According to the present invention, the effective components in the plant raw materials can be efficiently extracted, and the prepared tablet has good oxidation resistance, can inhibit the life activity of fusobacterium nucleatum, and has effects of halitosis prevention and / or alleviation.

Description

Technical Field

[0001] The invention relates to the technical field of composition preparation, and in particular to a preparation method and application of a mint probiotic composition capable of preventing and / or alleviating halitosis. Background Art

[0002] Bad breath, also known as oral odor, refers to the bad smell in the mouth, which is usually related to indigestion, oral diseases, fatigue, pathogenic heat, internal heat stagnation or certain digestive system diseases. Bad breath not only affects appetite and aggravates oral diseases, but also seriously affects people's social activities and mental health. Conventional methods to relieve bad breath mainly include improving oral hygiene habits, such as brushing teeth, using dental floss, rinsing mouth, etc. These methods can often only provide temporary relief and are difficult to fundamentally solve the problem of bad breath, especially for bad breath caused by oral flora imbalance. Traditional methods cannot effectively regulate the balance of oral microecology, and thus cannot achieve long-term and stable treatment effects.

[0003] The monoterpenoid compounds contained in mint can reach the lungs through blood circulation, making the breath feel fresh; green tea is rich in catechins, which can help remove oral bacteria and reduce bad breath; sea buckthorn seeds have antibacterial effects and can fight against bacteria in the mouth; probiotics can regulate intestinal flora and enhance metabolic capacity, and have potential value in the prevention and treatment of bad breath. By combining mint, green tea, sea buckthorn seeds and compound probiotics in a scientific ratio, their synergistic effects can be fully utilized to provide a more lasting and effective bad breath control effect. Summary of the invention

[0004] The present invention provides a preparation method and application of a mint probiotic composition having the function of preventing and / or alleviating bad breath, using mint, green tea and sea buckthorn seeds as plant raw materials, and adopting supercritical CO 2 The fluid breaks the wall efficiently, and then ultrasonic technology is used for secondary extraction, and then compounded with compound probiotics. This can not only efficiently extract the effective ingredients in the plant raw materials, but also the oral preparation has good antioxidant properties, can inhibit the life activities of Fusobacterium nucleatum, and has the effect of preventing and reducing bad breath.

[0005] The technical solution of the present invention is:

[0006] A method for preparing a mint probiotic composition having the effect of preventing and / or alleviating bad breath comprises the following steps:

[0007] S1: According to weight, 50 parts of mint, 20-30 parts of green tea and 20-30 parts of sea buckthorn seeds were washed and dried, and then heated to 400 °C using supercritical CO 2 Extraction, the extraction temperature is 30-50°C, the extraction pressure is 30-50MPa, and the plant mixed oil and plant residue are obtained;

[0008] S2: mixing the plant residues described in step S1 with purified water and then performing ultrasonic extraction to obtain a plant extract;

[0009] S3: Evenly mix the plant extract described in step S2, the plant mixed oil described in step S1, purified water, glucose, isomalt oligosaccharide, xylitol, sorbitol, fructo-oligosaccharide, microcrystalline cellulose, magnesium stearate, composite probiotic powder and sucrose ester to obtain a mint probiotic composition.

[0010] As a further improvement of the above scheme, in step S1, the green tea is 30 parts and the sea buckthorn seeds are 20 parts.

[0011] As a further improvement of the above scheme, in step S1, the extraction temperature is 40° C. and the extraction pressure is 30 MPa.

[0012] As a further improvement of the above scheme, the preparation method comprises the following steps:

[0013] S1: According to weight, 50 parts of mint, 20-30 parts of green tea and 20-30 parts of sea buckthorn seeds were washed and dried, and then heated to 400 °C using supercritical CO 2 Extraction, the extraction temperature is 30-50°C, the extraction pressure is 30-50MPa, and the plant mixed oil and plant residue are obtained;

[0014] S2: adding purified water to the plant residue in step S1 at a solid-liquid ratio of 1 g:15 mL, and performing ultrasonic extraction for 30 min at a temperature of 40° C. and a power of 400 W to obtain a plant extract;

[0015] S3: According to the weight ratio, 13.5 parts of pure water, 6 parts of glucose and 3 parts of oligofructose are stir-fried at 60°C to make syrup, 30 parts of the plant extract described in step S2 are added and stirred, and then 3 parts of xylitol, 3 parts of sorbitol and 6 parts of oligofructose are added and stirred until they are completely dissolved; the temperature is lowered to 40°C, 12 parts of microcrystalline cellulose, 3 parts of magnesium stearate, 10 parts of composite probiotic powder and 1 part of sucrose ester are added and stirred; wherein the composite probiotic powder is composed of maltodextrin, Lactobacillus plantarum HH-LP56, Lactococcus lactis lactis subsp. lactis HH-LLL39, Streptococcus thermophilus HH-ST08, Lactobacillus rhamnosus PB-LR76, Lactobacillus paracasei HH-LP58, Bifidobacterium longum HH-BL18 and Bifidobacterium lactis HH-BA68; finally, 15 parts of the plant mixed oil described in step S1 are added and stirred evenly to obtain a mint probiotic composition.

[0016] An application of the mint probiotic composition obtained by any one of the above preparation methods comprises using the mint probiotic composition to inhibit Fusobacterium nucleatum from producing volatile sulfides.

[0017] An application of the mint probiotic composition obtained by any one of the above preparation methods comprises using the mint probiotic composition as a raw material to prepare an oral preparation for preventing and / or alleviating halitosis.

[0018] As a further improvement of the above solution, the oral preparation is a tablet.

[0019] The beneficial effects of the present invention are:

[0020] (1) Combining supercritical extraction technology with ultrasonic extraction technology to extract components from plant raw materials such as mint, green tea and sea buckthorn seeds, and achieving efficient extraction of volatile oils and polyphenol compounds by overall control of plant raw material ratios and supercritical extraction pressure, extraction temperature and other conditions; compared with traditional steam distillation extraction technology, this extraction process can avoid the destruction of the structure of effective components in plant raw materials during the extraction process to a greater extent, thereby improving extraction efficiency;

[0021] (2) By combining various auxiliary materials, the plant raw material extract and the probiotic component are processed to form a composition and a raw material package, which is convenient for subsequent processing into various oral preparations for freshening breath; experiments have shown that the oral preparation prepared by the present invention has good antioxidant properties and can inhibit the formation of biofilms and the production of volatile sulfides by Fusobacterium nucleatum, thereby preventing and alleviating the symptoms of bad breath from the root. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] The weight of the relevant components mentioned in the embodiments of the present invention description can not only refer to the specific content of each component, but also represent the proportional relationship of the weights of the components. Therefore, as long as the content of the relevant components in the embodiments of the present invention description is proportionally enlarged or reduced, it is within the scope disclosed in the embodiments of the present invention description. Specifically, the mass described in the embodiments of the present invention description can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0024] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and carefully in combination with preferred embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0025] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0027] Example 1

[0028] S1: According to weight, 50 parts of mint, 20 parts of green tea and 30 parts of sea buckthorn seeds were washed with clean water, dried at 50°C for 15 hours, crushed, and sieved through 30 mesh to obtain plant raw material powder. The plant raw material powder was sieved using supercritical CO 2 extraction, extraction temperature is 40℃, extraction pressure is 30MPa, CO 2 The flow rate is 30 L / h, the pressure holding time is 1.5 h, and the vegetable mixed oil L1 and the vegetable residue L1 are obtained;

[0029] S2: Plant residue L1 was mixed with pure water at a solid-liquid ratio of 1 g:15 mL, and ultrasonically extracted for 30 min at a temperature of 40 °C and a power of 400 W. After the extraction, it was purified by centrifugal filtration at a speed of 8000 r / min and a processing time of 12 min. A 200 nm ceramic membrane and an 800 Da nanofiltration membrane were used for filtration to obtain plant extract L1;

[0030] S3: According to the weight ratio, put 13.5 parts of pure water into the pot in advance and heat it to boiling, cool it down and keep the temperature at about 60°C, add 6 parts of glucose and 3 parts of oligomaltose to stir into syrup, add 30 parts of plant extract L1 and stir for 40 minutes, then add 3 parts of xylitol, 3 parts of sorbitol, and 6 parts of oligofructose and stir until completely dissolved; cool it down to 40°C, add 12 parts of microcrystalline cellulose, 3 parts of magnesium stearate, 10 parts of compound probiotic powder and 1 part of sucrose ester , stirring for 30 minutes; wherein the composite probiotic powder consists of maltodextrin, Lactobacillus plantarum HH-LP56, Lactococcus lactis lactis subspecies HH-LLL39, Streptococcus thermophilus HH-ST08, Lactobacillus rhamnosus PB-LR76, Lactobacillus paracasei HH-LP58, Bifidobacterium longum HH-BL18 and Bifidobacterium lactis HH-BA68; finally, 15 parts of plant mixed oil L1 were added and stirred for 20 minutes to obtain a mint probiotic composition L1.

[0031] Example 2

[0032] The supercritical extraction temperature in step S1 of Example 1 was adjusted to 30° C., and the parameters of the other steps were the same as those of Example 1, to obtain plant mixed oil L2 and plant extract L2.

[0033] Example 3

[0034] The supercritical extraction temperature in step S1 of Example 1 was adjusted to 50° C., and the parameters of the other steps were the same as those of Example 1, to obtain plant mixed oil L3 and plant extract L3.

[0035] Example 4

[0036] The supercritical extraction pressure in step S1 of Example 1 was adjusted to 40 MPa, and the parameters of the other steps were the same as those of Example 1, to obtain plant mixed oil L4 and plant extract L4.

[0037] Example 5

[0038] The supercritical extraction pressure in step S1 of Example 1 was adjusted to 50 MPa, and the parameters of other steps were the same as those of Example 1, to obtain plant mixed oil L5 and plant extract L5.

[0039] Example 6

[0040] The weight proportions of the components in step S1 of Example 1 were adjusted to 50 parts of mint, 30 parts of green tea, and 20 parts of sea buckthorn seeds, and the remaining step parameters were the same as those of Example 1, to obtain plant mixed oil L6 and plant extract L6.

[0041] Example 7

[0042] The mint probiotic composition L1 obtained in Example 1 was subjected to vacuum freeze-drying treatment, the temperature was set to minus 60°C, the pressure was 50 Pa, the treatment time was 8 hours, and then ground and pulverized to obtain a freeze-dried powder; the freeze-dried powder was placed in a mold and tableted using a tablet press. The tablet press equipment parameters were: a filling depth of 75% of the mold volume, a pre-compression pressure of 20 MPa, a main compression pressure of 100 MPa, a holding time of 10 seconds, and a demolding pressure of 8 MPa. The tablets after tableting were taken out to obtain Cuikouqing Tablets S1.

[0043] Comparative Example 1

[0044] According to weight, 50 parts of mint, 20 parts of green tea and 30 parts of sea buckthorn seeds were washed, dried at 50°C for 15 hours, crushed and sieved through 30 mesh to obtain plant raw material powder, the plant raw material powder was mixed with pure water at a solid-liquid ratio of 1g:15mL, and then steam distilled for 6 hours; after the distillation, the solid-liquid mixture in the distillation flask and the oily mixed liquid in the condenser were centrifuged and filtered to obtain plant extract D1 and plant mixed oil D1; then the mint probiotic composition D1 was obtained according to the method described in step S3 of Example 1.

[0045] Comparative Example 2

[0046] The supercritical extraction temperature in step S1 of Example 1 was adjusted to 60° C., and the parameters of the other steps were the same as those of Example 1, to obtain plant mixed oil D2 and plant extract D2.

[0047] Comparative Example 3

[0048] The supercritical extraction pressure in step S1 of Example 1 was adjusted to 20 MPa, and the parameters of the other steps were the same as those of Example 1, to obtain plant mixed oil D3 and plant extract D3.

[0049] Comparative Example 4

[0050] The weight proportions of the components in step S1 of Example 1 were adjusted to 50 parts of mint, 10 parts of green tea, and 40 parts of sea buckthorn seeds, and the remaining step parameters were the same as those of Example 1, to obtain plant mixed oil D4 and plant extract D4.

[0051] Comparative Example 5

[0052] The mint probiotic composition L1 in Example 7 was replaced by the mint probiotic composition D1, and the remaining step parameters were the same as in Example 7 to obtain Cuikouqing Tablet D1.

[0053] Test Example 1: Effects of different extraction processes on the extraction effect of plant raw materials

[0054] The amount of plant mixed oil obtained and the content of polyphenol compounds in the plant extract were used as indicators to evaluate the extraction effects of different extraction processes, wherein the content of polyphenol compounds was determined by the Folin phenol method, using a UV-1900i series ultraviolet spectrophotometer at a wavelength of 765 nm. The calculation results of the total polyphenol yield and the plant mixed oil yield in the plant extracts obtained in Examples 1-6 and Comparative Examples 1-4 are shown in Table 1.

[0055] Table 1 Determination results of polyphenols in plant mixed oils and plant extracts

[0056] Group Yield of mixed vegetable oil (mg / g) Total polyphenol yield in plant extract (mg / g) Example 1 16.8 127.5 Example 2 13.4 125.3 Example 3 13.3 126.5 Example 4 15.8 124.9 Example 5 13.6 124.4 Example 6 16 130.6 Comparative Example 1 9.8 80.6 Comparative Example 2 10.5 119.3 Comparative Example 3 11.1 118.5 Comparative Example 4 17.4 118.2

[0057] It can be seen from the test results that compared with the traditional steam distillation extraction technology, the supercritical CO 2 Extraction combined with ultrasonic extraction can improve the extraction efficiency of volatile oils in plant raw materials and reduce the decomposition of polyphenol compounds. When the supercritical extraction temperature is increased from 30°C to 50°C, the extraction amount of plant mixed oil first gradually increases and then begins to decline, but can basically maintain a high extraction efficiency, and when the temperature rises to 60°C, the extraction effect of plant extracted oil significantly deteriorates. Moreover, changes in supercritical extraction conditions will also affect the effect of subsequent ultrasonic extraction. When the supercritical extraction pressure and temperature exceed the limit range, not only does it lead to a decrease in the extraction efficiency of plant mixed oil, but it also reduces the extraction efficiency of active ingredients in plants during the subsequent ultrasonic process. In addition, changes in plant raw material components also affect the composition of the extract, and the plant raw material ratio of Example 1 is preferred.

[0058] Test Example 2: Antioxidant Performance Test

[0059] 1. DPPH free radical scavenging test

[0060] The oral preparations Cuikouqing Tablets S1 and Cuikouqing Tablets D1 prepared in Example 7 and Comparative Example 5 were respectively subjected to DPPH free radical scavenging tests according to the following methods:

[0061] Set up sample tube (T), sample background (T 0 ), DPPH tube (C) and solvent background (C 0 ), three parallel tubes were set up for each sample tube (T) of Cuikouqing tablets, and water was used as the diluent to prepare Cuikouqing solutions of different concentrations (0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL).

[0062] For example, taking 5 mg / ml extract solution as an example, the following sample tube (T), sample background (T 0 ), DPPH tube (C) and solvent background (C 0 ):

[0063] Sample tube (T): 1 mL of extract solution (concentration of 5 mg / mL) + 2 mL of water + 1 mL of DPPH ethanol solution (concentration of 0.04 mg / mL);

[0064] Sample background tube (T 0 ): 1 mL of extract solution (concentration of 5 mg / mL) + 2 mL of water + 1 mL of 95 vol% ethanol solution;

[0065] DPPH tube (C): 3 mL of water + 1 mL of DPPH ethanol solution (concentration 0.04 mg / mL);

[0066] Solvent background tube (C 0 ): 3mL water + 1mL 95% vol ethanol solution;

[0067] The solutions in the above reaction tubes were transferred into cuvettes, the absorbance was measured at 517 nm, and the DPPH free radical scavenging rate was calculated by formula I. The specific results are shown in Table 2.

[0068]

[0069] The results show that compared with the Cuikouqing tablets obtained in Comparative Example 5, the Cuikouqing tablets obtained in Example 7 have a better scavenging effect on DPPH free radicals.

[0070] Table 2 DPPH free radical scavenging rate test results

[0071] concentration 0.3125mg / mL 0.625mg / mL 1.25mg / mL 2.5mg / mL 5mg / mL Cuikouqing Tablets S1 36.33％ 42.54％ 63.55％ 75.30％ 97.24％ Cuikouqing Tablets D1 28.57％ 37.21％ 49.04％ 58.53％ 73.37％

[0072] 2. ABTS +Free Radical Scavenging Test

[0073] The oral preparations Cuikouqing Tablets S1 and Cuikouqing Tablets D1 prepared in Example 7 and Comparative Example 5 were subjected to ABTS respectively according to the following method: + Free Radical Scavenging Test:

[0074] Set up the sample tube (A S ), sample background (A b ), sample blank tube (A 0 ), sample tubes for each tested concentration of each sample (A S ) Set up three parallel tubes, use water as diluent, prepare different concentrations of extract solution (0.125mg / mL, 0.25mg / mL, 0.5mg / mL, 1mg / mL, 2mg / mL), and at the same time, the sample blank tube (A 0 ) also set up 3 parallel tubes. S ) and sample background (A b ) were added with 0.2 mL of sample solution of the same concentration, and the sample blank tube (A 0 ) add 0.2mL PBS buffer. S ) and sample blank tube (A 0 ) were added with 0.8 mL ABTS + Working solution, sample background (A b ) then add 0.8 mL PBS buffer and react for 6 min in the dark. Transfer the solution in each reaction tube into a 1 cm cuvette, measure the absorbance at 734 nm, and calculate ABTS according to formula II + The free radical scavenging rate is shown in Table 3.

[0075]

[0076] The results showed that compared with the Cuikouqing tablets obtained in Comparative Example 5, the Cuikouqing tablets obtained in Example 7 had a significantly higher + Free radicals have better scavenging effect.

[0077] Table 3 ABTS + Free Radical Scavenging Rate Test Results

[0078] concentration 0.125mg / mL 0.25mg / mL 0.5mg / mL 1mg / mL 2mg / mL Cuikouqing Tablets S1 19.38％ 32.85％ 54.24％ 70.57％ 92.37％ Cuikouqing Tablets D1 12.26％ 25.43％ 37.57％ 52.37％ 80.07％

[0079] 3. Hydroxyl free radical scavenging test

[0080] The oral preparations Cuikouqing Tablets S1 and Cuikouqing Tablets D1 prepared in Example 7 and Comparative Example 5 were subjected to a hydroxyl radical scavenging test according to the following method:

[0081] Set up the sample tube (A 样品 )、Loss pipe (A损 )、Undamaged(A 未损 ), three parallel tubes were set up for each sample tube (T) of Cuikouqing tablets, and water was used as the diluent to prepare Cuikouqing solutions of different concentrations (0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL).

[0082] A 样品 : 1mL of o-phenanthroline ethanol solution (concentration of 5mmol / L) + 2mL of phosphate buffer (concentration of 0.2mol / L) + 1mL of extract solution + 1mL of ferrous sulfate solution (5mmol / L) + 1mL of H 2 O 2 Solution (concentration 0.1% v / v);

[0083] A 损 : 1mL of o-phenanthroline ethanol solution (concentration of 5mmol / L) + 2mL of phosphate buffer (concentration of 0.2mol / L) + 1mL of ultrapure water + 1mL of ferrous sulfate solution (5mmol / L) + 1mL of H 2 O 2 Solution (concentration 0.1% v / v);

[0084] A 未损 : 1 mL of o-phenanthroline ethanol solution (concentration of 5 mmol / L) + 2 mL of phosphate buffer (concentration of 0.2 mol / L) + 1 mL of ferrous sulfate solution (5 mmol / L) + 2 mL of ultrapure water.

[0085] The solutions in the above reaction tubes were transferred into cuvettes, the absorbance was measured at 536 nm, and the hydroxyl radical scavenging rate was calculated by formula III. The specific results are shown in Table 4.

[0086]

[0087] The results show that compared with the Cuikouqing tablets obtained in Comparative Example 5, the Cuikouqing tablets obtained in Example 7 have a better scavenging effect on hydroxyl free radicals.

[0088] Table 4 Hydroxyl radical scavenging rate test results

[0089] concentration 0.125mg / mL 0.25mg / mL 0.5mg / mL 1mg / mL 2mg / mL Cuikouqing Tablets S1 21.05％ 33.46％ 47.33％ 63.13％ 82.45％ Cuikouqing Tablets D1 13.84％ 24.37％ 34.21％ 51.07％ 76.28％

[0090] Test Example 3: Test on the production of volatile sulfides by Fusobacterium nucleatum

[0091] Volatile sulfides (VSCs) are the main culprit of halitosis, which not only reflects the degree of halitosis but also damages gingival tissue. The effect of the oral preparation Cuikouqing Tablets prepared in the present invention on the production of VSCs by Fusobacterium nucleatum was tested to evaluate its effect on preventing and alleviating halitosis.

[0092] Determination method: MRS medium ( / L) was selected for negative control: glucose 20g, tryptone 10g, beef extract 10g, yeast extract 5g, potassium phosphate trihydrate 2.6g, diammonium citrate 2g, sodium acetate 2g, magnesium sulfate heptahydrate 0.1g, manganese sulfate monohydrate 0.05g, Tween 80 1mL, pH = 6.2-6.4, autoclave at 115℃ for 20min. BHI medium was selected, and 0.05% hemin and 0.1% vitamin K1 were added. Solid medium was added with 5% sterile defibrinated sheep blood, pH = 7.2-7.4, autoclave at 121℃ for 20min, for the cultivation of Fusobacterium nucleatum. MSA medium was selected, and 0.28mL of 1% potassium tellurite solution was added to each 100mL, and autoclave at 121℃ for 20min, for the isolation and cultivation of Streptococcus salivarius. Fusobacterium nucleatum and Streptococcus salivarius K12 in frozen glycerol tubes were activated with BHI and MSA, respectively. Single colonies were picked and activated in BHI and MSA liquid culture media for 2 generations. Fusobacterium nucleatum was cultured anaerobically at 37°C for 24 hours, and Streptococcus salivarius K12 was cultured in a constant temperature incubator at 37°C for 24 hours. The cells were collected to prepare a bacterial suspension and the concentration of the bacterial suspension was adjusted to 10 7 CFU / mL. After culturing Streptococcus salivarius K12 at 37°C for 24 hours, centrifuge at 8,000 r / min and 4°C for 10 minutes, take the supernatant, filter it through a 0.22 μm sterile filter membrane, and store it at -20°C for later use.

[0093] The oral preparations of Cuikouqing Tablets S1 and Cuikouqing Tablets D1 prepared in Example 7 and Comparative Example 5 were diluted with water to make Cuikouqing S1 solution and Cuikouqing D1 solution with concentrations of 0.5 mg / mL, 1 mg / mL and 2 mg / mL, respectively. 7 A total of 2 mL of a suspension of Fusobacterium nucleatum with CFU / mL and different concentrations of Cuikouqing S1 solution, Cuikouqing D1 solution, MRS culture medium, and 15% (v / v) supernatant of Streptococcus salivarius K12 were added to a Hungate tube and cultured for 9 hours. Two 50 mL syringe needles were inserted into the tube, one end was connected to the Halimeter halitosis meter, and the other end was used to balance the air pressure. The oscillator was used to oscillate while measuring. The gas in the control group was diluted and measured. The specific operation is: after oscillation and mixing, 1 mL of gas was drawn with a 1 mL syringe, and immediately added to an empty Hungate tube, while oscillating and measuring. When the value stabilizes and begins to decrease, the highest value is read, and then substituted into the standard curve to obtain the value, repeated 3 times, and the test results are shown in Table 5.

[0094] The results showed that Cuikouqing tablets had a good inhibitory effect on the production of VSCs by Fusobacterium nucleatum, and the inhibitory effect was stronger than that of the positive control Streptococcus salivarius K12. Among them, the inhibitory effect of Cuikouqing tablets obtained in Example 7 on the production of VSCs by Fusobacterium nucleatum was significantly better than that of Cuikouqing tablets obtained in Comparative Example 5.

[0095] Table 5 Effect of Cuikouqing Tablets on VSCs production by Fusobacterium nucleatum

[0096] Group VSCs value / ppb Inhibition rate (%) MRS culture group 5891 / Streptococcus salivarius K12 culture group 1753 70.24 0.5mg / mL Cuikouqing S1 solution culture group 952 83.84 1mg / mL Cuikouqing S1 solution culture group 669 88.64 2mg / mL Cuikouqing S1 solution culture group 531 90.99 0.5mg / mL Cuikouqing D1 solution culture group 1304 77.86 1mg / mL Cuikouqing D1 solution culture group 962 83.67 2mg / mL Cuikouqing D1 solution culture group 735 87.52

[0097] Test Example 4: Fusobacterium nucleatum biofilm formation test

[0098] Periodontal pockets and tongue coating provide an ideal growth environment for bacteria that produce VSCs and other odor molecules, and microorganisms usually exist in the periodontal pockets and tongue coating in the form of biofilms. Fusobacterium nucleatum is an intermediate bridge bacteria in the formation of dental plaque, and its biofilm formation is the key to causing bad breath. The Cuikouqing tablets obtained in Example 7 and Comparative Example 5 were prepared into solutions, and the crystal violet staining method was used to determine their effects on the biofilm formation of Fusobacterium nucleatum.

[0099] Detection method: Crystal violet staining was used to adjust the concentration of activated pathogenic bacteria to 10 7 CFU / mL, and static culture for 9h as bacterial liquid, add 180μL of bacterial suspension to each well of 96-well plate, then add 20μL of different concentrations (0.5mg / mL, 1mg / mL, 2mg / mL, diluted with ultrapure water) of Cuikouqing S1 solution, Cuikouqing D1 solution and 15% (v / v) of S. salivarius K12 supernatant, so that the total volume of the two reaches 200μL, 37℃ anaerobically cultured for 48h, and 6 parallel wells are set for each supernatant. MRS group is used as negative control group, and the same volume of MRS culture medium is added. After culture, wash with PBS twice, then fix with 99% methanol for 15min, discard the supernatant, and dry at room temperature. After drying completely, add 100μL of 0.1% crystal violet solution to each well, stain for 5min, wash twice with sterile water after staining, and then place at room temperature until completely dry. Finally, 200 μL of 33% acetic acid solution was added for dissolution, and after mixing with a gun, 175 μL was transferred from each well to a new 96-well plate, and the absorbance was read at 570 nm on a microplate reader. The test results are shown in Table 6. The reduction amount after biofilm mediation was calculated as follows: inhibition rate (%) = (negative control biofilm-supernatant intervention biofilm amount) / negative control biofilm amount.

[0100] The results showed that the mint probiotic composition obtained by the present invention has a good inhibitory effect on the formation of Fusobacterium nucleatum biofilm, and the inhibitory effect is stronger than that of the positive control Streptococcus salivarius K12. Among them, the inhibitory effect of the Cuikouqing tablets obtained in Example 7 on the formation of Fusobacterium nucleatum biofilm is significantly better than that of the Cuikouqing tablets obtained in Comparative Example 5.

[0101] Table 6 Inhibitory effect of Cuikouqing tablets on biofilm of Fusobacterium nucleatum

[0102] Group OD570 Inhibition rate MRS culture group 3.34 / Streptococcus salivarius K12 culture group 2.62 21.56 0.5mg / mL Cuikouqing S1 solution culture group 2.22 33.53 1mg / mL Cuikouqing S1 solution culture group 1.91 42.81 2mg / mL Cuikouqing S1 solution culture group 1.73 48.20 0.5mg / mL Cuikouqing D1 solution culture group 2.39 28.44 1mg / mL Cuikouqing D1 solution culture group 2.04 38.92 2mg / mL Cuikouqing D1 solution culture group 1.88 43.71

[0103] Test Example 5: Fresh Breath Test

[0104] A Halimeter breath tester was selected, and 36 testers were selected. The breath tester was used to test and record the bad breath value of each person. Before the test, the tester rinsed his mouth with normal saline and kept not eating for 6 hours, then took a simple work meal (make sure to eat it all), and tested the bad breath value 1 hour after the meal and recorded it; then chewed a piece of Cuikouqing Tablet S1 or Cuikouqing Tablet D1 (0.6g), and tested the bad breath value 20 minutes later and recorded it, calculated the difference in bad breath value before and after the same person used the Cuikouqing Tablet, and statistically calculated the average value, and the results are shown in Table 7. The results show that both types of Cuikouqing Tablets can effectively freshen the breath after chewing, and the breath freshening effect of Cuikouqing Tablet S1 obtained in Example 7 is more significant.

[0105] Table 7 Breath test results

[0106] Group Average reduction in bad breath value (VSCs / ppb) Cuikouqing Tablets S1 Group 60.67 Cuikouqing Tablets D1 Group 48.35

[0107] The oral preparation finally prepared in the embodiment of the present invention is a chewable tablet, which is easy to carry and eat, has a sweet taste and a minty fragrance, and the composite probiotic powder added in the formula can accelerate the decomposition of food in the gastrointestinal tract, thereby promoting intestinal absorption, and fundamentally achieves the purpose of reducing bad breath.

[0108] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a mint probiotic composition having the effect of preventing and / or alleviating bad breath, characterized in that: The following steps are involved: S1: According to weight, 50 parts of mint, 20-30 parts of green tea and 20-30 parts of sea buckthorn seeds are washed and dried, and then extracted with supercritical CO2 at an extraction temperature of 30-50° C. and an extraction pressure of 30-50 MPa to obtain a plant mixed oil and plant residue; S2: mixing the plant residues described in step S1 with purified water and then performing ultrasonic extraction to obtain a plant extract; S3: Evenly mix the plant extract described in step S2, the plant mixed oil described in step S1, purified water, glucose, isomalt oligosaccharide, xylitol, sorbitol, fructo-oligosaccharide, microcrystalline cellulose, magnesium stearate, composite probiotic powder and sucrose ester to obtain a mint probiotic composition.

2. The preparation method according to claim 1, characterized in that In step S1, the green tea is 30 parts and the sea buckthorn seeds are 20 parts.

3. The preparation method according to claim 1, characterized in that: In step S1, the extraction temperature is 40° C. and the extraction pressure is 30 MPa.

4. The preparation method according to claim 1, characterized in that: The following steps are involved: S1: According to weight, 50 parts of mint, 20-30 parts of green tea and 20-30 parts of sea buckthorn seeds are washed and dried, and then extracted with supercritical CO2 at an extraction temperature of 30-50° C. and an extraction pressure of 30-50 MPa to obtain a plant mixed oil and plant residue; S2: adding purified water to the plant residue in step S1 at a solid-liquid ratio of 1 g:15 mL, and performing ultrasonic extraction for 30 min at a temperature of 40° C. and a power of 400 W to obtain a plant extract; S3: According to the weight ratio, 13.5 parts of pure water, 6 parts of glucose and 3 parts of oligofructose are stir-fried at 60°C to make syrup, 30 parts of the plant extract described in step S2 are added and stirred, and then 3 parts of xylitol, 3 parts of sorbitol and 6 parts of oligofructose are added and stirred until they are completely dissolved; the temperature is lowered to 40°C, 12 parts of microcrystalline cellulose, 3 parts of magnesium stearate, 10 parts of composite probiotic powder and 1 part of sucrose ester are added and stirred; wherein the composite probiotic powder is composed of maltodextrin, Lactobacillus plantarum HH-LP56, Lactococcus lactis lactis subsp. lactis HH-LLL39, Streptococcus thermophilus HH-ST08, Lactobacillus rhamnosus PB-LR76, Lactobacillus paracasei HH-LP58, Bifidobacterium longum HH-BL18 and Bifidobacterium lactis HH-BA68; finally, 15 parts of the plant mixed oil described in step S1 are added and stirred evenly to obtain a mint probiotic composition.

5. An application of a mint probiotic composition obtained by the preparation method according to any one of claims 1 to 4, characterized in that: The invention comprises using the mint probiotic composition to inhibit Fusobacterium nucleatum from producing volatile sulfide.

6. An application of a mint probiotic composition obtained by the preparation method according to any one of claims 1 to 4, characterized in that: The invention comprises using the mint probiotic composition as a raw material to prepare an oral preparation for preventing and / or alleviating halitosis.

7. The use according to claim 6, characterized in that The oral preparation is a tablet.

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