CN111558033A

CN111558033A - Oral cavity cleaning composition and application thereof

Info

Publication number: CN111558033A
Application number: CN202010408941.4A
Authority: CN
Inventors: 王淋立; 陈月花; 李乾坤; 陈帝武; 王萍
Original assignee: Guangzhou Zhongyu Medical Biotechnology Co ltd
Current assignee: Wang Linli; Yamalaka Life Sciences Co ltd
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2020-08-21
Also published as: WO2021227200A1

Abstract

The invention provides an oral cleaning composition and application thereof, wherein the oral cleaning composition comprises proteolytic enzyme protein, non-proteolytic enzyme protein and probiotics. The oral cavity cleaning composition provided by the invention can destroy the living environment of harmful bacteria in the oral cavity by decomposing residues in the oral cavity, inhibit the growth of harmful bacteria in the oral cavity, provide a good growth environment for oral cavity probiotics, and indirectly or directly promote the growth of the oral cavity probiotics, so that the good oral cavity environment is maintained, and the occurrence of oral cavity diseases is reduced.

Description

Oral cavity cleaning composition and application thereof

Technical Field

The invention belongs to the field of oral preparations, and particularly relates to an oral cleaning composition and application thereof.

Background

Caries is commonly known as dental caries and decayed tooth and is a bacterial disease. The infectious diseases of hard tissues of teeth with high incidence rate and wide spread range of dental caries can directly or indirectly affect the whole body health of people, can cause secondary pulpitis and periapical periodontitis, and even can cause inflammation of alveolar bones and jaw bones. If not treated in time, the lesion continues to develop, forming a cavity, eventually the crown is completely destroyed and disappears, the end result of which is the loss of teeth. The widely accepted theory at present is that the triple factor theory proposed by Keyes says that caries can occur only when three factors of diet, bacteria and host exist simultaneously, and caries is not easy to occur when one factor is broken.

The utilization of food residues by polysaccharides, proteins, fats of the oral food residues and bacteria in the oral cavity creates a complex oral problem. The existing oral care products have various types and single functions, and cannot effectively deal with the complex oral environment and oral problems.

Disclosure of Invention

The invention aims to provide an oral cavity cleaning composition and application thereof, which destroy one of three factors of caries occurrence, thereby reducing the caries occurrence.

The invention concept of the invention is as follows:

the proteolytic enzyme protein can hydrolyze protein in food residue, destroy growth environment of harmful flora in oral cavity, and inhibit bacteria growth.

The non-proteolytic enzyme protein can decompose the membrane structure of bacteria and directly kill harmful bacteria.

The probiotics compete to inhibit the growth of harmful flora in the oral cavity.

The combination of the three can inhibit the growth of harmful flora in oral cavity, reduce oral diseases such as caries and inflammation, and maintain good environment of oral cavity.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

an oral cleaning composition comprising a proteolytic enzyme protein, a non-proteolytic enzyme protein and a probiotic.

Preferably, the probiotic bacteria include at least one of streptococcus salivarius M18, streptococcus salivarius K12, lactobacillus plantarum L-137, bacillus coagulans, lactobacillus reuteri, lactobacillus paracasei, and lactobacillus acidophilus.

Preferably, the probiotic bacteria comprise at least one of streptococcus salivarius M18, streptococcus salivarius K12 and lactobacillus reuteri.

Preferably, the proteolytic enzyme protein comprises at least one of papain, subtilisin, and bromelain.

Preferably, the non-proteolytic enzyme protein comprises at least one of lactoferrin, lactoperoxidase, lysozyme, glucose oxidase, amylase, lipase and superoxide dismutase.

In a second aspect of the present invention, there is provided:

an oral cavity cleaning agent comprises the oral cavity cleaning composition.

In a third aspect of the present invention, there is provided:

an application of the oral cleaning composition in preparing medicines for improving stomatitis is provided.

Preferably, the medicament is at least one of toothpaste, tooth powder, mouthwash, mouth rinse, dental tape and chewing gum.

In a fourth aspect of the present invention, there is provided:

The invention has the beneficial effects that:

(1) the oral cavity cleaning composition provided by the invention can destroy the living environment of harmful bacteria in the oral cavity by decomposing residues in the oral cavity, inhibit the growth of harmful bacteria in the oral cavity, provide a good growth environment for oral cavity probiotics, and indirectly or directly promote the growth of the oral cavity probiotics, so that the good oral cavity environment is maintained, and the occurrence of oral cavity diseases is reduced.

(2) The invention can decompose various starch and fat food residues in the oral cavity into glucose, short-chain fatty acid and other small molecular substances to increase the solubility of the substances by combining non-proteolytic enzyme protein, proteolytic enzyme protein and probiotics; the components in the proteolytic enzyme protein can hydrolyze protein in food residues into amino acid, and the small molecules can leave the oral cavity along with swallowing to reduce food residues in the oral cavity, so that the growth environment of harmful flora in the oral cavity is damaged, the adhesion of bacteria in the food residues is reduced, the utilization of bacteria on the food residues is reduced, the growth environment of the bacteria is damaged, and the growth of the bacteria is inhibited. The non-proteolytic enzyme protein combination can also act on harmful flora in the oral cavity, decompose the membrane structure of bacteria and directly kill the harmful bacteria; the probiotics can compete to inhibit the growth of harmful flora in the oral cavity, so that the growth of the harmful flora in the oral cavity is inhibited in many ways, the occurrence of oral diseases such as caries, inflammation and the like is reduced, and the good environment of the oral cavity is maintained.

(3) Plaque, tartar, traumatic occlusion, food impaction in the oral cavity and the like gradually cause inflammation and swelling of the gingiva, meanwhile, the plaque is gradually accumulated and is extended from the upper part of the gingiva to the lower part of the gingiva, and the invasion of the gingiva and periodontal tissues causes the occurrence of chronic inflammation, such as periodontitis. The invention reduces bacteria adhesion in food residues and utilization of the food residues by inhibiting and reducing the growth of harmful flora in the oral cavity, destroys the growth environment of the bacteria, reduces the occurrence of caries and simultaneously improves the oral inflammation.

Specifically, the invention has the following advantages:

(1) streptococcus salivarius M18, a probiotic developed by the university of ottaca, new zealand, is ubiquitous in every human, but streptococcus salivarius M18 is only naturally present in the mouth and throat of some healthy adults and children, producing exoglucanases and urease. Wherein the exoenzyme glucanase (an enzyme that hydrolyzes the 1, 6-glycosidic bond of glucan) inhibits the synthesis of water-insoluble glucan (a 1, 6-linked polymer with a high proportion of 1, 3-linked glucosyl residues) by Streptococcus mutans in the oral cavity, such that bacterial plaque does not accumulate on the tooth surface; the ammonia produced by urea metabolism by urease can alleviate glycolysis acidification of dental plaque, neutralize dental plaque acidity and create an environment unfavorable for acid substances to inhibit dental caries. Exoenzyme glucanase, urease and bacteriocin produced by streptococcus salivarius M18 can inhibit the growth of harmful flora in many aspects and improve the oral environment.

(2) Streptococcus salivarius K12 is also only found naturally in the mouth and throat of some healthy adults and children and produces two natural antimicrobial peptides, salivaricin a2 and salivaricin B, which are lantibiotic-type bacteriocins that inhibit the growth of harmful bacteria. The streptococcus salivarius K12 can inhibit growth of harmful bacteria such as gram-positive bacteria and Micromonospora associated with halitosis, reduce halitosis and inhibit growth of harmful bacteria in oral cavity.

The inventors found that the streptococcus salivarius M18 and K12 in (1) and (2) above were not ubiquitous, neither was present in the oral cavity of persons with oral problems, and that streptococcus salivarius M18 and K12 did not follow oral problems after they were treated. Therefore, the streptococcus salivarius M18 and K12 are important factors for improving the oral environment, and the active addition of the streptococcus salivarius M18 and K12 to the oral cleaning composition can bring the two probiotics to the oral cavity, thereby being beneficial to improving the oral cavity problems.

(3) Lactobacillus plantarum L-137 belongs to the genus Lactobacillus in the family Lactobacillus. Clinical experimental reports have shown that: the patient received 10 mg daily of heat-inactivated lactobacillus plantarum L-137 capsules for 12 weeks. The number of teeth or parts with exploratory Bleeding (BOP) and exploratory depth (PD) of more than or equal to 4mm is significantly reduced, and the intake of Lactobacillus plantarum L-137 which receives heat inactivation every day can reduce the depth of the periodontal pocket of patients receiving supportive periodontal treatment.

(4) The Bacillus coagulans is homolactic fermentation bacteria, and has inhibitory effect on Escherichia coli (NCTC-10418), Pseudomonas aeruginosa (NCIB-9016), Klebsiella pneumoniae (NCIB-9111), Bacillus subtilis (NCTC-6346), Staphylococcus aureus (Staphylococcus aureus) (NCTC7447) and Candida albicans (CBS-562).

(5) Lactobacillus reuteri is a lactic acid bacterium that produces a broad spectrum of antibacterial substances known as reuterin. Reuterin is a low molecular weight, neutral, water-soluble compound that inhibits the growth of escherichia, salmonella, shigella, proteus, pseudomonas, clostridium and staphylococcus.

(6) Lactobacillus paracasei is a gram-positive heterotypic lactobacillus fermentum that produces bacteriocins that change the cell membrane of porphyromonas gingivalis from a globular or coccoid shape to a rod shape and swell, causing the bacteria to die.

(7) Lactobacillus acidophilus belongs to the genus Lactobacillus. The bacteriocins produced by the bacteria inhibit the growth of gram-positive bacteria (Gardner and Streptococcus agalactiae) and gram-negative bacteria (Pseudomonas aeruginosa).

(8) Papain and bromelain belong to thiol proteases, and are pure natural biological enzyme products extracted from milk in immature papaya fruits, wherein bromelain is a generic name of a class of proteolytic enzymes extracted from pineapple plants, and subtilisin is a group of alkaline proteases derived from different strains of bacillus subtilis. The three proteases have wide protein hydrolysis capability, can decompose protein food residues in the oral cavity into small molecules such as short peptides, amino acids and the like, and the small molecules are dissolved in saliva and are discharged along with swallowing effect, so that the utilization and adhesion of harmful flora to the protein food residues in the oral cavity are reduced, the living environment of the harmful flora is damaged, and a certain inhibition effect on harmful bacteria in the oral cavity is achieved.

(9) Lactoferrin is a non-heme iron-binding glycoprotein with immunological functions, isolated from human milk, and its bacteriostatic action is attributed to the complexation of iron required for metabolism. Lactoferrin has a stronger binding effect on free iron, and inhibits the utilization of iron by bacteria, thereby inhibiting the growth of bacteria. In addition, lactoferrin can bind to lipid a on the cell wall of gram-negative bacteria, disrupt the cell membrane of gram-negative bacteria, alter the permeability of the membrane, and ultimately cause the bacteria to die. The lactoferrin can inhibit the growth of bacteria in the oral cavity by combining with iron, and can also act on the cell wall of gram-negative bacteria in the oral cavity to kill the gram-negative bacteria so as to achieve the bacteriostatic effect.

(10) Lactoperoxidase is an oxidoreductase. Lactoperoxidase together with thiocyanate ions or iodides or bromides and hydrogen peroxide forms a lactoperoxidase system. The mechanism of this system is that hydrogen peroxide oxidizes thiocyanate ions (also iodide and bromide) to hypothiocyanate ions (hypoiodide and hypobromide), which oxidize the sulfhydryl groups of amino acid residues of microbial proteins, resulting in impaired cell membrane function and thus microbial death. In the oral environment, this mechanism can effectively inhibit the growth and reproduction of bacteria.

(11) The natural substrate of lysozyme is the peptidoglycan cell wall of gram-positive and gram-negative bacteria, which consists of cross-linked oligosaccharides composed of alternating 2-acetamido-2-deoxy-glucopyranose (NAG) and 2-acetamido-2-deoxy-3-lactide-glucopyranose (NAM) residues. The peptidoglycan comprises 50-80% by dry weight of the cell wall of gram-positive bacteria and 5-20% by dry weight of the cell wall of gram-negative bacteria. Therefore, the lysozyme has broad-spectrum bactericidal capability and has the effects of inhibiting and killing gram-positive bacteria (such as streptococcus mutans) and gram-negative bacteria (porphyromonas gingivalis) in the oral cavity.

(12) Glucose oxidase is a flavoprotein, and takes oxygen molecules as an electron acceptor to catalyze beta-D-glucose to be oxidized into D-glucose-lactone and hydrogen peroxide. The reaction can be divided into reduction and oxidation steps, in the reduction reaction, the glucose oxidase catalyzes beta-D-glucose to be oxidized into D-gluconic acid-lactone. The Flavin Adenine Dinucleotide (FAD) loop of the glucose oxidase is then reduced to FADH 2. In the oxidation reaction, the reduced glucose oxidase is reoxidized by oxygen to produce H2O2, and Catalase (CAT) decomposes hydrogen peroxide to produce water and oxygen. In the oral environment, the glucose oxidase can work in cooperation with amylase, so that starch residues in food can be completely converted into small molecules, and the small molecules are dissolved in saliva and are discharged along with swallowing.

(13) Alpha-amylase and beta-amylase are common amylases. Starch is composed of two components, including glycosidic chains in which the glucose residues are bound by 1,4 α -D-glycosidic bonds, i.e. amylose, the majority of these chains being 1,4 α -D-glycosidic bonds. Some of the glucose units are also linked by 1,6 α -D-glucopyranoside linkages, i.e. amylopectin. When the alpha-amylase takes starch as a substrate, the starch is rapidly hydrolyzed to generate oligosaccharide, and the viscosity of the starch and the capability of color reaction with iodine are rapidly reduced at the stage. The oligosaccharides are then slowly hydrolyzed to yield the final products glucose and maltose. The amylase can decompose starch food residues in the oral cavity into small molecule compounds, and the small molecules are dissolved in saliva and are discharged along with swallowing effect, so that adhesion and utilization of harmful flora in the oral cavity in the starch food residues are reduced, and living environment of the harmful flora is damaged.

(14) Lipases are a generic term for a class of enzymes that catalyze the hydrolysis of triacylglycerols, a special class of ester bond hydrolases whose natural substrate is a long chain fatty acid ester. The lipase can decompose the grease in the oral cavity into short-chain fatty acid micromolecules which are dissolved in saliva and are discharged along with swallowing effect, so that the utilization of harmful flora in the oral cavity to the fat is reduced, and the living environment of the harmful flora is damaged.

(15) Superoxide dismutase is an enzyme that catalyzes the conversion of superoxide to oxygen and hydrogen peroxide by a disproportionation reaction. It is widely used in various animals, plants and microorganisms, and is an important antioxidant. The superoxide dismutase can be added into toothpaste, collutory, buccal tablet, etc., and has certain therapeutic effect on preventing oral diseases.

Drawings

Fig. 1 is a connection diagram of the artificial oral cavity components of the experimental design of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

In order to visually compare the caries-improving effects of the oral composition of the present invention, the following examples and comparative examples were prepared, and the respective component marks and mass ranges were as follows:

factor 1: proteolytic enzyme protein component

Factor 1.1: 2g of papain;

factor 1.2: 2g of bacillus subtilis enzyme powder;

factor 1.3: bromelain, 2g in mass.

Factor 2: non-proteolytic enzyme protein fraction

Factor 2.1: lactoferrin with the mass of 2 g;

factor 2.2: lactoperoxidase with a mass of 2 g;

factor 2.3: lysozyme, the mass is 2 g;

factor 2.4: glucose oxidase with the mass of 2 g;

factor 2.5: amylase, the mass is 2 g;

factor 2.6: 2g of lipase;

factor 2.7: the mass of the superoxide dismutase is 2 g.

Factor 3: probiotic compositions

Factor 3.1: streptococcus salivarius M18, mass 2 g;

factor 3.2: streptococcus salivarius K12 with mass of 2 g;

factor 3.3: lactobacillus plantarum L-137 with a mass of 2 g;

factor 3.4: bacillus coagulans with the mass of 2 g;

factor 3.5: lactobacillus reuteri with a mass of 2 g;

factor 3.6: lactobacillus paracasei with the mass of 2 g;

factor 3.7: lactobacillus acidophilus, 2g in mass.

Firstly, the toothpaste is prepared as follows:

(1) respectively and uniformly mixing the factors 1.1-1.3 with 45g of calcium carbonate, 30g of glycerol, 0.1g of saccharin sodium, 0.1g of sodium benzoate, 1g of silica powder, 1g of disodium lauroamphodiacetate and a proper amount of refined water, and homogenizing to obtain 100g of toothpaste containing the factors.

(2) Respectively and uniformly mixing the factors of 2.1-2.7 with 45g of calcium carbonate, 30g of glycerol, 0.1g of saccharin sodium, 0.1g of sodium benzoate, 1g of silica powder, 1g of disodium lauroamphodiacetate and a proper amount of refined water, and homogenizing to obtain 100g of toothpaste containing the factors.

(3) Respectively and uniformly mixing 3.1-3.7 of the factors with 45g of calcium carbonate, 30g of glycerol, 0.1g of saccharin sodium, 0.1g of sodium benzoate, 1g of silica powder, 1g of disodium lauroamphodiacetate and a proper amount of refined water, and homogenizing to obtain 100g of toothpaste containing the factors.

(4) Uniformly mixing the factor 1.1-1.3, 45g of calcium carbonate, 30g of glycerol, 0.1g of saccharin sodium, 0.1g of sodium benzoate, 1g of silica powder, 1g of disodium lauroamphodiacetate and a proper amount of refined water, and homogenizing to obtain 100g of toothpaste containing the factor 1.1-1.5, namely the combination 1.

(5) Uniformly mixing the factor 2.1-2.7, 45g of calcium carbonate, 30g of glycerol, 0.1g of saccharin sodium, 0.1g of sodium benzoate, 1g of silica powder, 1g of disodium lauroamphodiacetate and a proper amount of refined water, and homogenizing to obtain 100g of toothpaste containing the factor 2.1-2.7, namely the combination 2.

(6) Uniformly mixing the factor 3.1-3.7, 45g of calcium carbonate, 30g of glycerol, 0.1g of saccharin sodium, 0.1g of sodium benzoate, 1g of silica powder, 1g of disodium lauroamphodiacetate and a proper amount of refined water, and homogenizing to obtain 100g of toothpaste containing the factor 3.1-3.7, namely the combination 3.

II, an experimental method:

2.1 experimental strains:

porphyromonas gingivalis (P.gingivalis)

Streptococcus sanguis (S. sanguis)

Streptococcus mutans (S. mutans)

2.2 Experimental reagents:

hydroxyapatite tablets, Brain Heart Infusion (BHI) agar, Brain Heart Infusion (BHI) broth, P7000-25G porcine gastric mucin, micro sample genome DNA extraction kit, Carrier RNA, Platinum SYBR Green quantitative PCR supermix kit, RNase H, primer synthesis, lysozyme

2.3 preparation of improved Artificial saliva

The formula of the artificial saliva is an improved biomembrane culture medium BM-5: 2.5g/L of type III porcine gastric mucin; peptones 2 g/L; tryptone is decomposed by 1 g/L; 1g/L of yeast extract; 2.5g/L of potassium chloride (KCl); glucose 0.5 g/L; cystine hydrochloride 0.1 g/L; 1mg/L of hemin; dipotassium hydrogen phosphate (K)₂HPO₄·3H₂0)0.114 g/L; potassium dihydrogen phosphate (KH)₂PO₄)0.2 g/L. Adding 10g/L sucrose into the above components, adjusting pH to 7.5 with 1mol/L NaOH solution, autoclaving at 121 deg.C for 15min, and keeping.

2.4 recovery and passage of bacteria

And taking out the three experimental bacteria vacuum freeze-dried powder, sucking a proper amount of sterilized BHI liquid culture medium by using a sterile suction pipe to dissolve the bacteria powder, and transferring the bacteria powder into a sterile centrifuge tube filled with the BHI liquid culture medium for anaerobic recovery at 37 ℃ overnight. And streaking and inoculating the recovered 3 kinds of bacteria into a BHI solid culture medium, placing the BHI solid culture medium in an anaerobic culture box at 37 ℃, and carrying out anaerobic culture for 48 hours. The morphology and the staining were checked to be pure cultures, which were again inoculated into BHI solid medium and cultured for 48 hours, and the morphology was identified as pure cultures.

2.5 preparation of bacterial liquid

Respectively selecting three standard strains of bicyclic S.mutans, S.sanguis and P.gingivalis, dissolving the three standard strains in a centrifuge tube filled with 10mLBHI liquid culture medium, unscrewing a tube cover to ensure that bacteria are fully contacted with an anaerobic environment in the box, and carrying out anaerobic culture at 37 ℃ for 18 hours to obtain enriched liquid of the three standard strains of S.mutans, S.sanguis and P.gingivalis. Adjusting the concentration of each bacterium to A by using an ultraviolet spectrophotometer₅₅₀＝0.25±0.05(3.0x10⁸CFU/mL), 30mL of each bacterium was added to 500mL of modified artificial saliva for use.

2.6 preparation of hydroxyapatite tablets

Taking a proper amount of hydroxyapatite sheets, adhering and fixing one surface of the hydroxyapatite sheets on a perforated plastic tray orifice plate, and sterilizing the hydroxyapatite sheets for later use.

2.7 Artificial oral Cavity construction

As shown in figure 1, the artificial oral cavity designed in the experiment consists of an artificial saliva device, a bacterial suspension device, a constant-temperature culture chamber and a waste liquid device. The devices are connected by a silicone tube, wherein a digital display constant flow pump is arranged between the artificial saliva device and the bacterial suspension device, between the artificial saliva device and the constant temperature culture chamber, and between the bacterial suspension device and the constant temperature culture chamber. The bacterial suspension culture conditions, the constant-temperature culture chamber culture conditions and the preparation of the in-vitro dental plaque biomembrane in the constant-temperature culture chamber are as follows:

2.7.1 culture conditions for bacterial suspension

Temperature: maintaining the constant temperature of 37 ℃ and an anaerobic environment;

stirring: low speed, controlled by magnetic stirrer (about 90 rpm) to mix the bacterial suspension;

waste liquid clearance rate: 0.05 mL/min (1 drop/min), controlled by a flow regulator.

2.7.2 culture conditions in constant temperature culture Chamber

waste liquid clearance rate: 0.2 mL/min (4 drops/min), controlled by a flow regulator.

2.7.3 preparation of in vitro dental plaque biomembrane

Placing the prepared bacterial suspension in a bacterial suspension device, adding artificial saliva into the bacterial suspension device at a rate of 0.1 mL/min (2 drops/min), and carrying out anaerobic culture on the bacterial suspension for 24 hours at 37 ℃ to achieve a stable growth state; then, the bacterial suspension and the artificial saliva device are communicated with a constant temperature culture chamber (6 hydroxyapatite tablets are respectively suspended in the constant temperature culture chamber in advance and are all about 2cm lower than the liquid level), and the ratio of the bacterial suspension to the improved artificial saliva is determined according to the following formula: a9-ratio (i.e., 0.1 mL/min: 0.9 mL/min) was continuously pumped into the isothermal incubation chamber simultaneously, 24 hours later the pumping of the bacterial suspension was stopped, and the administration of modified artificial saliva (0.2 mL/min) was continued for 48 hours. The different reagents were administered at 8 and 20 points per day for 3 days throughout the constant current chamber, with 50mL per group (1 mL/min each for 50 minutes) while the waste was drained at the same rate to simulate oral swallowing. Waste gas is discharged from an anaerobic incubator in the experimental process, and whether the waste liquid is polluted by mixed bacteria or not is detected after the waste liquid is dyed every day.

2.8FQ-PCR assay

6 pieces of hydroxyapatite sheets were taken out from each experimental group at 6 hours, 24 hours, 48 hours, and 72 hours of continuous culture, all plaque on the surface of the hydroxyapatite sheets was scraped with a sterile scraper, collected in a 1.5mL centrifugal tube, 200. mu.L of lysozyme at a concentration of 20mg/mL was added, and reacted at 37 ℃ for 30 minutes or more, thereby sufficiently lysing cell walls and releasing nucleic acid substances in the cells.

2.9DNA extraction and primer design

And extracting DNA by using a trace sample genome DNA extraction kit. Amplifying the sample by adopting a two-step PCR method: firstly, a full-length 16SrDNA product is generated by adopting a bacterial universal primer, and then the product is used as a template to amplify the DNA of a corresponding target bacterium by using a specific primer of each bacterium. Specific PCR primers were designed using primer design software Primerpromier 5.0 based on the 16SrDNA gene sequence, and the sequences of the primers are shown in Table 1 below:

table 1s. mutans, s. sanguis, p. gingivalis, reference Universal primer sequences

2.10Real-timePCR amplification

(1) The reaction system was prepared as shown in Table 2

TABLE 2Real-timePCR amplification reaction System solution constitution

Components	Volume (μ L)
		2xSuper Real Pre Mix	10
Upstream primer (10. mu. mol/L)	0.6
		Downstream primer (10. mu. mol/L)	0.6
50x ROX Reference Dye	2
		cDNA template	1
dd H₂O	5.8
		General System	20

(2) Preparing a mixed solution of all components except the template according to a Real-timePCR reaction system;

(3) for each reaction, 19. mu.L of the mixture was added to each well of a 96-well PCR plate;

(4) adding 1 mu L of sample template into each reaction container, and sealing the reaction tube;

(5) lightly mixing to ensure that all components are at the bottom of the reaction container;

(6) the reaction system is placed in a well-set gene amplification instrument, data is collected, and results are analyzed.

2.11Real-timePCR reaction and detection

The PCR reaction conditions are shown in Table 3 (two-step, annealing extension in one step):

TABLE 3Real-timePCR reaction conditions

Number of cycles	Circulation conditions	Purpose(s) to
			1x	95℃，5min	Predeformation
45x	Step1：95℃，60s	Deformation of
				Step2：60℃，31s	Annealing/stretching

The whole sample adding process is operated on ice, a tube cover is covered, the mixture is tapped, and a palm centrifuge is instantly mixed to be convenient for gathering reagents to the bottom of the tube, the tube is placed on a PCR instrument, a circulation program is set according to the conditions, in order to ensure the validity of data, 3 duplicate holes are parallelly made on each sample, and then Real-timesPCR product detection is respectively carried out.

2.12 calculation mode

According to the result measured by Real-timePCR, the experiment adopts △△ Ct relative quantification method gene expression change, before using △△ Ct method to make quantification, it makes feasibility test to verify that the amplification efficiency of target gene and internal reference is basically equal, and uses Universal as internal reference to make a series of fluorescent quantitative PCR reactions, and its reaction system and reaction condition are identical to above-mentioned reactions, and the amplification slope difference value of target gene and internal reference is less than 0.1, and can use △△ Ct to make quantitative analysis, and has no need of making standard product absolute quantification or making relative standard curve quantification method^-△△Ct△ Ct is the average Ct value of the target gene-housekeeping gene, △△ Ct is △ Ct (test sample) - △ Ct (control sample), 2^-△△CtThe calculated relative expression level represents a multiple of the expression level of the target gene in the test sample relative to the expression level of the target gene in the control sample. Thus, 2 for this experiment^-(△△Ct)The method represents the relative content of certain bacteria in dental plaque biomembranes of the study object. Using a certain sample as a calibration sample and the rest samples as samples to be tested, 2^-(△△Ct)The value is the multiple of a certain bacteria calibration sample in the dental plaque biological film of the sample to be detected.

Third, clinical methods

3.1 study object

The sample is from volunteers, and the examination and diagnosis of patients with chronic periodontitis shows that plaque and tartar are accumulated, and the periodontal probing depth is more than 8mm, and the total number of the samples is 10.

3.2 periodontal pocket microorganism specimen Collection

A25 # sterile paper twist of about 10mm is inserted into the deepest periodontal pocket of the visit, each paper twist is left for 30 seconds, the paper twist is collected at different sites for each tooth, placed in an EP tube with 1mL of physiological saline at 4 ℃, and finally stored at-80 ℃.

3.3 detection of indicators

The periodontal probing depth before and after treatment, and the quantity of Porphyromonas gingivalis, Streptococcus sanguis, and Streptococcus mutans in periodontal pocket before and after treatment (the bacteria detection method is the same as the above detection method).

Experimental example 1: effect of proteolytic enzyme protein toothpaste formulation on growth of 3 bacteria

The factors 1.1-1.3 are combined to form experiment groups 1-6, the composition of each experiment group is shown in tables 4-6, and the + number in the table indicates that the experiment group contains a certain factor. The experimental samples were obtained by adding 50ml of water to 100g of the samples of experimental groups 1 to 6, respectively, and stirring them uniformly. The experiment was carried out according to the experimental method and the detection method described above.

TABLE 4 Effect of proteolytic enzyme protein toothpaste formulations on Porphyromonas gingivalis growth

TABLE 5 Effect of proteolytic enzyme protein toothpaste formulations on growth of Streptococcus sanguis

TABLE 6 Effect of proteolytic enzyme protein toothpaste formulations on growth of Streptococcus mutans

As can be seen from tables 4-6, the number of bacteria in the experimental group 6 at 6h, 24h, 48h and 72h is significantly less than that in the control group (P < 0.01). The results show that the combination of papain + bacillus subtilis enzyme + bromelain, which is the experimental group 6, can inhibit the growth of streptococcus mutans, streptococcus sanguis, and porphyromonas gingivalis.

Experimental example 2: effect of non-proteolytic enzyme protein toothpaste formulation on growth of 3 bacteria

The factors 2.1-2.7 are combined and then respectively corresponded to form experiment groups 7-34, the composition of each experiment group is shown in tables 7-9, and the + number in the table indicates that the experiment group contains a certain factor. The experimental samples were obtained by adding 50mL of water to 100g of samples of experimental groups 7 to 34, respectively, and stirring the mixture uniformly. The experiment was carried out according to the experimental method and the detection method described above.

TABLE 7 Effect of non-proteolytic enzyme protein toothpaste formulations on Porphyromonas gingivalis growth

TABLE 8 Effect of non-proteolytic enzyme protein toothpaste formulations on growth of Streptococcus sanguis

TABLE 9 Effect of non-proteolytic enzyme protein toothpaste formulations on growth of Streptococcus mutans

As can be seen from tables 7 to 9, the number of bacteria in the experimental group 34 at 6h, 24h, 48h and 72h is obviously less than that in the control group (P <0.01), and the results show that the experimental group 34, i.e., the combination of lactoferrin + lactoperoxidase + lysozyme + glucose oxidase + amylase + lipase + superoxide dismutase can inhibit the growth of Streptococcus mutans, Streptococcus sanguis and Porphyromonas gingivalis.

Experimental example 3: effect of probiotic toothpaste formulation on growth of 3 bacteria

The above-mentioned factors 3.1-3.7 are combined and respectively correspondent to form experimental group 35-62, and the composition of every experimental group is detailed in table 10-12, in the table + number indicates that said experimental group contains some factor. The experimental samples are obtained by adding 50mL of water to 100g of samples of experimental groups 35-62 respectively and stirring uniformly. The experiment was carried out according to the experimental method and the detection method described above.

TABLE 10 Effect of probiotic toothpaste formulations on Porphyromonas gingivalis growth

TABLE 11 Effect of probiotic toothpaste formulations on growth of Streptococcus sanguis

TABLE 12 Effect of probiotic toothpaste formulations on growth of Streptococcus mutans

As can be seen from tables 10-12, the number of bacteria in the experimental group 62 was significantly less than that in the control group (P <0.01) at 6h, 24h, 48h, and 72h, and the number of bacteria in most of the experimental groups was significantly less than that in the control group (P < 0.05). The results show that the experimental group 62, namely the combination of streptococcus salivarius M18+ streptococcus salivarius K12+ lactobacillus plantarum L-137+ bacillus coagulans + lactobacillus reuteri + lactobacillus paracasei + lactobacillus acidophilus, can inhibit the growth of streptococcus mutans, streptococcus sanguis and porphyromonas gingivalis, and most of the rest experimental groups also have a certain effect of inhibiting the growth of streptococcus mutans, streptococcus sanguis and porphyromonas gingivalis.

Example (b): effect of combined use of proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste and probiotic toothpaste on bacterial growth

1. Effect of the combination of the proteolytic enzyme protein fraction, the non-proteolytic enzyme protein fraction, and the probiotic fraction on the growth of Streptococcus mutans

The factors 3.1-3.7 are combined with the factor 1.1 and the factor 2.1 respectively to form examples 1-7, the composition of each experimental group is shown in table 13, and the + number in the table indicates that the experimental group contains a certain factor. Each of 100g samples of examples 1 to 7 was added to 50ml of water and stirred uniformly to obtain test samples. The experiment was carried out according to the experimental method and the detection method described above.

TABLE 13 proteolytic enzyme protein component, non-proteolytic enzyme protein component, probiotic component used in combination for growth of Streptococcus mutans bacteria

Influence of

As can be seen from Table 13, the bacteria counts of examples 1, 2 and 5 were significantly less than those of the control group (P <0.01) at 6h, 24h, 48h and 72h, and the bacteria counts of examples 1, 2 and 5 were significantly less than those of the control group (P < 0.05). The results show that the streptococcus salivarius M18+ papain + lactoferrin of example 1, the streptococcus salivarius K12+ papain + lactoferrin of example 2 and the lactobacillus reuteri + papain + lactoferrin combination of example 5 inhibit the growth of streptococcus mutans.

2. Effect of combined use of proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste and probiotic toothpaste on growth of 3 kinds of bacteria

Experimental group 63: use only combination 1;

experimental group 64: use only combination 2;

experimental group 65: use only combination 3;

experimental group 66: firstly using the combination 1, and then using the combination 2;

experimental group 67: firstly, using the combination 2, and then using the combination 1;

example 8: using combination 1, then combination 2, and finally combination 3;

example 9: combination 2 was used first, then combination 1 and finally combination 3.

And (3) adding 50ml of water into 100g of combined samples respectively and uniformly stirring to obtain experimental samples. Experiments were carried out according to the experimental methods and the detection methods described above, and the experimental data in tables 14 to 16 were obtained.

TABLE 14 proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste, probiotic toothpaste combinations for growth of Porphyromonas gingivalis

Influence of

TABLE 15 Effect of proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste, probiotic toothpaste in combination on growth of Streptococcus sanguis

Group of

6h

24h

48h

72h

P value (6h)

P value (24h)

P value (48h)

P value (72h)

Control group

1.000±0

-

Experimental group 63

0.614±0.254

0.610±0.184

0.590±0.169

0.613±0.202

2.12×10^-05

4.61×10^-07

9.88×10^-08

1.11×10^-06

Experimental group 64

0.344±0.056

0.341±0.065

0.330±0.034

0.339±0.059

4.48×10^-10

1.26×10^-12

3.63×10^-13

3.54×10^-12

Experimental group 65

0.435±0.266

0.428±0.200

0.402±0.207

0.421±0.208

1.61×10^-08

6.69×10^-11

9.64×10^-12

1.32×10^-10

Experimental group 66

0.315±0.063

0.338±0.052

0.335±0.068

0.337±0.058

1.45×10^-10

1.10×10^-12

4.47×10^-13

3.27×10^-12

Experimental group 67

0.353±0.046

0.333±0.043

0.346±0.049

0.341±0.037

6.23×10^-10

9.03×10^-13

7.53×10^-13

3.77×10^-12

Example 8

0.128±0.095

0.155±0.123

0.155±0.122

0.154±0.128

1.64×10^-13

5.83×10^-16

2.52×10^-16

1.97×10^-15

Example 9

0.105±0.032

0.103±0.059

0.091±0.037

0.088±0.022

7.44×10^-14

8.28×10^-17

2.25×10^-17

1.67×10^-16

TABLE 16 influence of proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste, probiotic toothpaste in combination on growth of Streptococcus mutans

Group of

6h

24h

48h

72h

P value (6h)

P value (24h)

P value (48h)

P value (72h)

Control group

1.000±0

-

Experimental group 63

0.624±0.228

0.627±0.204

0.663±0.202

0.636±0.186

3.74×10^-07

2.58×10^-07

3.98×10^-06

1.24×10^-07

Experimental group 64

0.336±0.029

0.356±0.035

0.340±0.037

0.343±0.037

2.47×10^-13

2.84×10^-13

5.37×10^-13

2.46×10^-14

Experimental group 65

0.412±0.180

0.424±0.192

0.422±0.218

0.437±0.181

8.49×10^-12

7.43×10^-12

2.29×10^-11

2.54×10^-12

Experimental group 66

0.361±0.053

0.337±0.040

0.366±0.040

0.353±0.054

7.76×10^-13

1.22×10^-13

1.71×10^-12

3.98×10^-14

Experimental group 67

0.352±0.041

0.366±0.049

0.352±0.042

0.368±0.039

5.14×10^-13

4.63×10^-13

9.14×10^-13

8.15×10^-14

Example 8

0.100±0.034

0.109±0.039

0.099±0.033

0.105±0.037

1.49×10^-17

8.71×10^-18

2.79×10^-17

9.38×10^-19

Example 9

0.097±0.034

0.104±0.048

0.094±0.039

0.096±0.053

1.33×10^-17

7.44×10^-18

2.33×10^-17

6.59×10^-19

Remarking: examples 8 and 9 compared to experimental groups 63-67 significance levels P <0.05

As can be seen from tables 14 to 16, the bacteria number of the samples of example 8 and example 9 at 6h, 24h, 48h and 72h is obviously less than that of the control group (P <0.001), the effect is better than that of the other experimental groups (P <0.05), and the results show that the samples of example 8 and example 9 inhibit the growth of Streptococcus mutans, Streptococcus sanguis and Porphyromonas gingivalis and are better than those of the other experimental groups.

Clinical examples: the combined use of protease protein toothpaste, non-protease protein toothpaste and probiotic toothpaste has effects on periodontal probing depth of patients with chronic periodontitis and growth of Porphyromonas gingivalis, Streptococcus sanguis and Streptococcus mutans in periodontal pocket

The patient brushes teeth 2 times a day in the morning and evening, and the oral cleaning products such as antibiotic medicines and other toothpaste or deep mouth wash cannot be used during the experiment. The experiment lasted 3 months, brushing teeth in a manner of using combination 1+ first, combination 2+ and combination 3 last. Before the start of the experiment and 3 months after the experiment, the weekly probing depth and the number of porphyromonas gingivalis, streptococcus sanguis and streptococcus mutans in the periodontal pocket are detected, as shown in tables 17-18.

TABLE 17 Effect of the combination of proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste, probiotic toothpaste on the depth of periodontal probing in patients with chronic periodontitis

	Mm before treatment	Mm after treatment	P value
				Depth of periodontal probing	10.5±1.56	4.43±1.02	5.45×10^-09

TABLE 18 influence of the combination of proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste, probiotic toothpaste on the growth of Porphyromonas gingivalis, Streptococcus sanguis, and Streptococcus mutans in periodontal pocket of patients with chronic periodontitis

As can be seen from tables 17 and 18, the brushing regimen of combination 1+ first followed by combination 2+ and finally combination 3 significantly reduced the depth of periodontal probing in patients with chronic periodontitis (P < 0.001); meanwhile, the number of porphyromonas gingivalis, streptococcus sanguis and streptococcus mutans is obviously reduced (P is less than 0.001).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. An oral cleaning composition characterized by: including proteolytic enzyme proteins, non-proteolytic enzyme proteins and probiotics.

2. The oral cleaning composition of claim 1, wherein: the probiotic bacteria comprise at least one of streptococcus salivarius M18, streptococcus salivarius K12, lactobacillus plantarum L-137, bacillus coagulans, lactobacillus reuteri, lactobacillus paracasei and lactobacillus acidophilus.

3. The oral cleaning composition of claim 2, wherein: the probiotic bacteria comprise at least one of streptococcus salivarius M18, streptococcus salivarius K12 and lactobacillus reuteri.

4. The oral cleaning composition of claim 1, wherein: the proteolytic enzyme protein comprises at least one of papain, bacillus subtilis and bromelain.

5. The oral cleaning composition of claim 1, wherein: the non-proteolytic enzyme protein comprises at least one of lactoferrin, lactoperoxidase, lysozyme, glucose oxidase, amylase, lipase and superoxide dismutase.

6. An oral cavity cleanser, comprising: comprising the oral cleaning composition of any one of claims 1 to 5.

7. Use of an oral cleaning composition according to any one of claims 1 to 5 in the manufacture of a medicament for ameliorating dental caries.

8. Use according to claim 7, characterized in that: the medicine is at least one of toothpaste, dentifrice, collutory, oral cavity cleaning agent, dental cleaning belt, and chewing gum.

9. Use of an oral cleaning composition according to any one of claims 1 to 5 in the manufacture of a medicament for ameliorating oral inflammation.

10. Use according to claim 9, characterized in that: the medicine is at least one of toothpaste, dentifrice, collutory, oral cavity cleaning agent, dental cleaning belt, and chewing gum.