WO2021227200A1 - 一种口腔清洁组合物及其应用 - Google Patents

一种口腔清洁组合物及其应用 Download PDF

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WO2021227200A1
WO2021227200A1 PCT/CN2020/097247 CN2020097247W WO2021227200A1 WO 2021227200 A1 WO2021227200 A1 WO 2021227200A1 CN 2020097247 W CN2020097247 W CN 2020097247W WO 2021227200 A1 WO2021227200 A1 WO 2021227200A1
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oral
bacteria
oral cavity
proteolytic enzyme
growth
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PCT/CN2020/097247
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French (fr)
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王淋立
陈月花
李乾坤
陈帝武
王萍
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广州中昱医学生物科技有限公司
王淋立
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
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    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A61K9/0058—Chewing gums
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    • A—HUMAN NECESSITIES
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)
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Definitions

  • the invention belongs to the field of oral preparations, and particularly relates to an oral cleaning composition and applications thereof.
  • Caries commonly known as cavities and tooth decay, are bacterial diseases. Dental hard tissue infectious diseases with a high incidence of caries and a wide range of infections can directly or indirectly affect people's overall health. It can also cause pulpitis and periapical periodontitis, and even cause alveolar bone and jaw bone. Inflammation. If it is not treated in time, the disease will continue to develop and form cavities, and eventually the crown will be completely destroyed and disappear. The final result of its development is tooth loss.
  • the currently widely accepted theory is the triple factor theory proposed by Keyes. Caries will only occur when the three factors of diet, bacteria and host exist at the same time. If one of the factors is broken, caries will be less likely to occur.
  • the purpose of the present invention is to provide an oral cleaning composition and its application, which can destroy one of the three factors of caries, thereby reducing the caries.
  • Proteolytic enzyme protein can hydrolyze the protein in food residues, destroy the growth environment of harmful bacteria in the oral cavity, and inhibit the growth of bacteria.
  • Non-proteolytic enzyme protein can break down the membrane structure of bacteria and directly kill harmful bacteria.
  • Probiotics can compete to inhibit the growth of harmful bacteria in the oral cavity.
  • the combination of the three can inhibit the growth of harmful bacteria in the oral cavity from many aspects, reduce the occurrence of oral diseases such as caries and inflammation, and maintain a good oral environment.
  • the first aspect of the present invention provides:
  • An oral cleaning composition includes proteolytic enzyme protein, non-proteolytic enzyme protein and probiotics.
  • the above-mentioned probiotics include at least one of Streptococcus salivarius M18, Streptococcus salivarius K12, Lactobacillus plantarum L-137, Bacillus coagulans, Lactobacillus reuteri, Lactobacillus paracasei, and Lactobacillus acidophilus.
  • the above-mentioned probiotics include at least one of Streptococcus salivarius M18, Streptococcus salivarius K12, and Lactobacillus reuteri.
  • the proteolytic enzyme protein includes at least one of papain, subtilisin, and bromelain.
  • the aforementioned non-proteolytic enzyme protein includes at least one of lactoferrin, lactoperoxidase, lysozyme, glucose oxidase, amylase, lipase, and superoxide dismutase.
  • the second aspect of the present invention provides:
  • An oral cleaning agent comprising the above oral cleaning composition.
  • the third aspect of the present invention provides:
  • the above-mentioned medicine is at least one of toothpaste, tooth powder, mouthwash, mouthwash, tooth cleaning tape, and chewing gum.
  • the fourth aspect of the present invention provides:
  • the above-mentioned medicine is at least one of toothpaste, tooth powder, mouthwash, mouthwash, tooth cleaning tape, and chewing gum.
  • the oral cleansing composition of the present invention destroys the living environment of harmful oral bacteria by decomposing oral residues, inhibits the growth of harmful oral bacteria, and provides a good growth environment for oral probiotics, indirectly or directly promoting the growth of oral probiotics. Grow to maintain a good oral environment and reduce the occurrence of oral diseases.
  • the present invention can decompose various starches and fatty food residues in the oral cavity into small molecules such as glucose and short-chain fatty acids to increase their solubility by combining non-proteolytic enzyme proteins, proteolytic enzyme proteins, and probiotics;
  • the various components of proteolytic enzyme protein can hydrolyze the protein in food residues into amino acids.
  • These small molecules can leave the mouth with swallowing, reduce the food residues in the oral cavity, thereby destroying the growth environment of harmful bacteria in the oral cavity. , Reduce the adhesion of bacteria to food residues and reduce the use of bacteria to food residues, destroy the growth environment of bacteria, and inhibit the growth of bacteria.
  • the non-proteolytic enzyme protein combination can also act on the harmful bacteria in the oral cavity, decompose the bacterial membrane structure, and directly kill harmful bacteria; the addition of probiotics can compete to inhibit the growth of harmful bacteria in the oral cavity, thereby inhibiting the oral cavity in many ways
  • the growth of the harmful bacteria in the plant can reduce the occurrence of oral diseases such as caries and inflammation, and maintain a good oral environment.
  • the invention reduces the adhesion of bacteria in food residues and the utilization of food residues by bacteria, destroys the growth environment of bacteria, reduces the occurrence of caries, and can improve oral inflammation at the same time.
  • the present invention has the following advantages:
  • Streptococcus salivarius M18 is a probiotic developed by the University of Otago in New Zealand. Streptococcus salivarius is ubiquitous in everyone, but Streptococcus salivarius M18 only naturally exists in the mouth and throat of some healthy adults and children Among them, it produces exonuclease glucanase and urease. Among them, the exonuclease glucanase (an enzyme that hydrolyzes the 1,6-glycosidic bond of glucan) can inhibit the synthesis of water-insoluble glucan (with a high proportion of 1,3-linked glucan residues) from Streptococcus mutans.
  • Streptococcus salivarius K12 also exists naturally in the mouth and throat of some healthy adults and children, and can produce two natural antimicrobial peptides, namely sialin A2 and sialin B, which are lantibiotic bacteriocins , Can inhibit the growth of harmful bacteria. Streptococcus salivarius K12 can inhibit the growth of gram-positive bacteria, micromonas and other harmful bacteria related to bad breath, reduce oral odor and inhibit the growth of harmful oral flora.
  • Streptococcus salivarius M18 and K12 in the above (1) and (2) are not universal. Usually, people with oral problems do not have these two types of Streptococcus in the mouth, and after the oral problems are treated, the saliva The cocci M18 and K12 will not be produced. Therefore, Streptococcus salivarius M18 and K12 are important factors for improving the oral environment. Active addition to the oral cleaning composition can bring the above two probiotics to the oral cavity, which is beneficial to improve oral problems.
  • Lactobacillus plantarum L-137 belongs to the genus Lactobacillus in the family Lactobacillus. Clinical trials have reported that patients receive 10 mg of heat-inactivated Lactobacillus plantarum L-137 capsules daily for 12 weeks. The number of teeth or parts with blood detection (BOP) and detection depth (PD) ⁇ 4 mm are significantly reduced. Daily intake of heat-inactivated Lactobacillus plantarum L-137 can reduce the teeth of patients receiving supportive periodontal treatment. Perimeter pocket depth.
  • Bacillus coagulans is a homozygous lactic acid fermentation bacterium, which is resistant to Escherichia coli (NCTC-10418), Pseudomonas aeruginosa (NCIB-9016), Klebsiella pneumoniae (NCIB-9111), and Bacillus subtilis 6346), Staphylococcus aureus (Staphylococcus aureus) (NCTC7447) and Candida albicans (CBS-562) have inhibitory effects.
  • Lactobacillus reuteri is a kind of lactic acid bacteria, and the broad-spectrum antibacterial substance produced by this bacteria is called reuterin.
  • Reuterin is a low molecular weight, neutral water-soluble compound that can inhibit the growth of Escherichia, Salmonella, Shigella, Proteus, Pseudomonas, Clostridium and Staphylococcus.
  • Lactobacillus paracasei is a gram-positive hetero-fermentative lactic acid bacteria that produces bacteriocins that can change the cell membrane of Porphyromonas gingivalis from a spherical or cocci-like shape to a rod-like shape and swell, leading to the death of the bacteria.
  • Lactobacillus acidophilus belongs to the genus Lactobacillus.
  • the bacteriocin produced by this bacteria can inhibit the growth of Gram-positive bacteria (Gardnerella and Streptococcus agalactiae) and Gram-negative bacteria (Pseudomonas aeruginosa).
  • Papain and bromelain belong to the category of sulfhydryl proteases.
  • Papain is a pure natural biological enzyme product extracted from the milk of immature papaya fruit.
  • Bromelain is a type of proteolytic enzyme extracted from pineapple plants.
  • Bacillus subtilis proteases are a group of alkaline proteases derived from different strains of Bacillus subtilis. These three proteases have a wide range of ability to hydrolyze proteins, and can decompose protein food residues in the oral cavity into small molecules such as short peptides and amino acids. These small molecules are dissolved in saliva and excreted by swallowing, reducing harmful bacteria in the oral cavity. The utilization and adhesion of protein food residues destroys the living environment of harmful bacteria, and has a certain inhibitory effect on harmful bacteria in the oral cavity.
  • Lactoferrin is a non-heme iron-binding glycoprotein with immune function, which is isolated from human milk.
  • the antibacterial effect of lactoferrin is attributed to the complexation of iron required for metabolism.
  • Lactoferrin has a stronger binding effect on free iron and inhibits the use of iron by bacteria, thereby inhibiting the growth of bacteria.
  • lactoferrin can bind to lipid A on the cell wall of gram-negative bacteria, destroy the cell membrane of gram-negative bacteria, change the permeability of the membrane, and ultimately kill the bacteria.
  • Lactoferrin can inhibit the growth of bacteria in the oral cavity by binding to iron, and it can also act on the cell wall of gram-negative bacteria in the oral cavity to kill the gram-negative bacteria and achieve a bacteriostatic effect.
  • Lactoperoxidase is an oxidoreductase. Lactoperoxidase forms the lactoperoxidase system with thiocyanate ion or iodide or bromide and hydrogen peroxide.
  • the mechanism of the system is that hydrogen peroxide oxidizes thiocyanate ions (including iodide and bromide) to hypothiocyanate ions (hypoiodide and hypobromide), and hypothiocyanate ions oxidize microbial protein amino acids
  • hypothiocyanate ions include iodide and bromide
  • hypothiocyanate ions hypothiocyanate ions
  • hypothiocyanate ions hypothiocyanate ions
  • hypothiocyanate ions oxidize microbial protein amino acids
  • the natural substrate of lysozyme is the peptidoglycan cell wall of Gram-positive bacteria and Gram-negative bacteria.
  • the peptidoglycan cell wall consists of alternate 2-acetylamino-2-deoxy-glucopyranose (NAG) and 2-Acetylamino-2-deoxy-3-lactide-glucopyranose (NAM) residues consist of cross-linked oligosaccharides.
  • NAG 2-acetylamino-2-deoxy-glucopyranose
  • the peptidoglycan contained in the cell wall of gram-positive bacteria accounts for 50-80% of the dry weight
  • the peptidoglycan contained in the cell wall of the gram-negative bacteria accounts for 5-20% of the dry weight.
  • lysozyme has a broad-spectrum bactericidal ability and can inhibit and kill both Gram-positive bacteria (such as Streptococcus mutans) and Gram-negative bacteria (Porphyromonas gingivalis) in the oral cavity.
  • Gram-positive bacteria such as Streptococcus mutans
  • Gram-negative bacteria Porphyromonas gingivalis
  • Glucose oxidase is a flavoprotein that uses oxygen molecules as electron acceptors to catalyze the oxidation of ⁇ -D-glucose to D-glucose- ⁇ -lactone and hydrogen peroxide.
  • the reaction can be divided into reduction and oxidation steps.
  • glucose oxidase catalyzes the oxidation of ⁇ -D-glucose to D-gluconate- ⁇ -lactone.
  • the flavin adenine dinucleotide (FAD) ring of glucose oxidase is reduced to FADH2.
  • glucose oxidase 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.
  • catalase CAT
  • glucose oxidase can work in conjunction with amylase, so that starch residues in food can be completely converted into small molecules, which are dissolved in saliva and excreted with swallowing.
  • the common amylases are ⁇ -amylase and ⁇ -amylase.
  • Starch is composed of two components, including glycoside chains in which glucose residues are bonded by 1,4 ⁇ -D-glucosidic bonds, that is, amylose. Most of these chains are 1,4 ⁇ -D-glucosidic bonds. Some of the glucose units are also connected by 1,6 ⁇ -D-glucopyranoside bonds, that is, pullulan.
  • ⁇ -amylase uses starch as a substrate, the starch is rapidly hydrolyzed to produce oligosaccharides. At this stage, the viscosity of the starch and the ability to react with iodine for color reaction decline rapidly.
  • Amylase can decompose starchy food residues in the oral cavity into small molecular compounds, which are dissolved in saliva and excreted with swallowing, reducing the adhesion and utilization of harmful bacteria in the oral cavity in starchy food residues, and destroying harmful bacteria. Living environment.
  • Lipase refers to the general name of a class of enzymes that catalyze the hydrolysis of triacylglycerols. It is a special class of ester bond hydrolases whose natural substrate is long-chain fatty acid esters. Lipase can decompose the fat in the oral cavity into short-chain fatty acid small molecules. These small molecules are dissolved in saliva and excreted with swallowing, reducing the use of fat by harmful bacteria in the oral cavity and destroying the living environment of harmful bacteria.
  • Superoxide dismutase is an enzyme that can catalyze the conversion of superoxide into oxygen and hydrogen peroxide through a disproportionation reaction. It is widely present in various animals, plants, and microorganisms, and is an important antioxidant. Superoxide dismutase can be added to toothpaste, mouthwash, lozenges, etc., and has a certain effect on preventing oral diseases.
  • Figure 1 is a connection diagram of the artificial oral cavity designed by the experiment of the present invention.
  • Factor 1 Proteolytic enzyme protein component
  • Factor 1.2 Bacillus subtilis (Bacillus subtilis) enzyme powder, the quality is 2g;
  • Lactoperoxidase the mass is 2g
  • Amylase the mass is 2g;
  • Factor 3.4 Bacillus coagulans, the mass is 2g;
  • Toothpaste is made as follows:
  • Streptococcus mutan (S. mutans)
  • the artificial saliva formula is a modified biofilm medium BM-5: type III porcine gastric mucin 2.5g/L; peptide peptone 2g/L; trypsin decomposes casein peptone 1g/L; yeast extract 1g/L; potassium chloride ( KCl) 2.5g/L; glucose 0.5g/L; cystine hydrochloride 0.1g/L; hemin 1mg/L; dipotassium hydrogen phosphate (K 2 HPO 4 ⁇ 3H 2 0) 0.114g/L ; Potassium dihydrogen phosphate (KH 2 PO 4 ) 0.2g/L.
  • Add 10g/L sucrose to the above ingredients, adjust the pH to 7.5 with 1mol/L NaOH solution, 121°C, autoclave for 15min, and use it for later use.
  • the artificial oral cavity designed in this experiment is composed of an artificial saliva device, a bacterial suspension device, a constant temperature culture room, and a waste liquid device.
  • Each device is connected with a silicone tube, and a digital display constant flow pump is placed between the artificial saliva device and the bacteria suspension device, the artificial saliva device and the constant temperature culture room, and the bacteria suspension device and the constant temperature culture room.
  • the culture conditions of the bacterial suspension, the culture conditions of the constant temperature culture room and the preparation of in vitro dental plaque biofilm in the constant temperature culture room are as follows:
  • Temperature maintain a constant temperature of 37°C, an anaerobic environment
  • Waste liquid removal rate 0.05mL/min (1 drop/min), controlled by the flow rate regulator.
  • Temperature maintain a constant temperature of 37°C, an anaerobic environment
  • Waste liquid removal rate 0.2mL/min (4 drops/min), controlled by the flow rate regulator.
  • the pumping of bacterial suspension is stopped, and the modified artificial saliva (0.2mL/min) is continued for 48 hours .
  • different experimental reagents were given at 8 and 20 every day, and each group was given 50 mL (1 mL/min each time, 50 minutes in total), and the waste liquid was discharged at the same rate to simulate Oral swallowing effect.
  • the exhaust gas is removed from the anaerobic incubator, and the daily waste liquid is stained to check whether there is contamination by bacteria.
  • the DNA was extracted using a micro-sample genomic DNA extraction kit.
  • a two-step PCR method is used to amplify the sample: firstly, the general bacterial primer is used to generate a full-length 16S rDNA product, and then the product is used as a template to amplify the DNA of the corresponding target bacteria with the specific primers of each bacteria.
  • the primer design software Primerpremier5.0 is used to design specific PCR primers. The sequence of each primer is shown in Table 1 below:
  • PCR reaction conditions are shown in Table 3 (two-step method, annealing and extension in one step):
  • the entire sample loading process is operated on ice, cover the tube, tap and mix, and mix in a palm centrifuge instantaneously, so that the reagents can be gathered to the bottom of the tube, and the tube is placed on the PCR machine.
  • this experiment uses the ⁇ Ct relative quantification method of gene expression changes.
  • do a feasibility test to verify that the amplification efficiency of the target gene and the internal reference are basically equal.
  • Use Universal as the internal reference to perform a series of fluorescent quantitative PCR reactions.
  • the reaction system and reaction conditions are the same as the above reactions.
  • the difference between the amplification slope of the gene and the internal control is less than 0.1, and ⁇ Ct can be used for quantitative analysis, without the need for absolute quantification of standard products or relative standard curve quantification.
  • the samples were from volunteers, and patients who were diagnosed with chronic periodontitis after examination, had accumulation of plaque and calculus, and the depth of periodontal probing> 8mm, a total of 10 cases.
  • the depth of periodontal exploration before and after treatment the number of Porphyromonas gingivalis, Streptococcus sanguis, and Streptococcus mutans in the periodontal pocket before and after treatment (the bacterial detection method is the same as the aforementioned detection method).
  • the aforementioned factors 1.1-1.3 are combined to form experimental groups 1-6 respectively.
  • the composition of each experimental group is shown in Table 4-6.
  • the + sign in the table indicates that the experimental group contains certain factors.
  • 100g samples of the experimental group 1-6 were added to 50ml of water and stirred uniformly to obtain experimental samples. Perform the experiment in accordance with the aforementioned experimental methods and detection methods.
  • experimental group 7-34 The composition of each experimental group is shown in Table 7-9. The + sign in the table indicates that the experimental group contains certain factors. 100g samples of experimental group 7-34 were added to 50mL water and stirred uniformly to obtain experimental samples. Perform the experiment in accordance with the aforementioned experimental methods and detection methods.
  • the aforementioned factors 3.1-3.7 are combined to form experimental groups 35-62 respectively.
  • the composition of each experimental group is shown in Table 10-12.
  • the + sign in the table indicates that the experimental group contains certain factors.
  • 100g samples of the experimental group 35-62 were added to 50mL of water and stirred uniformly to obtain experimental samples. Perform the experiment in accordance with the aforementioned experimental methods and detection methods.
  • Examples 1-7 The composition of each experimental group is shown in Table 13. The + sign in the table indicates that the experimental group contains a certain factor. 100g of samples of Examples 1-7 were added to 50ml of water and stirred uniformly to obtain experimental samples. Perform the experiment in accordance with the aforementioned experimental methods and detection methods.
  • Example 8 Use combination 1 first, then use combination 2, and finally use combination 3;
  • Example 9 Use combination 2 first, then use combination 1, and finally use combination 3.
  • Example 8 and Example 9 compared with the experimental group 63-67 is P ⁇ 0.05
  • Example 8 and Example 9 can significantly reduce the number of bacteria at 6h, 24h, 48h, 72h than the control group (P ⁇ 0.001), and the effect is better than other experimental groups (P ⁇ 0.05), the results show that Example 8 and Example 9 inhibit the growth of Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalis, and are better than other experimental groups.
  • Proteolytic enzyme protein toothpaste, non-proteolytic enzyme protein toothpaste, probiotic toothpaste combined use Effects of Streptococcus mutans growth
  • the patients brushed their teeth twice a day in the morning and evening. During the experiment, antibiotics and other toothpastes or saliva and other similar oral cleaning products could not be used. The experiment lasted for 3 months, and brushed the teeth according to the method of using combination 1+ first, then using combination 2+ and finally using combination 3. Before the start of the experiment and 3 months after the experiment, respectively, the detection depth of the week and the number of Porphyromonas gingivalis, Streptococcus sanguis, and Streptococcus mutans in the periodontal pocket were measured, as shown in Tables 17-18.
  • Periodontal probing depth 10.5 ⁇ 1.56 4.43 ⁇ 1.02 5.45 ⁇ 10 -09

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Abstract

一种口腔清洁组合物及其应用，包括蛋白水解酶蛋白、非蛋白水解酶蛋白和益生菌。口腔清洁组合物通过分解口腔残留物，破坏口腔有害细菌的生存环境，抑制口腔有害菌群生长的同时为口腔益生菌提供了良好的生长环境，间接或直接促进口腔益生菌的生长，从而维持良好的口腔环境，降低口腔疾病的发生。

Description

一种口腔清洁组合物及其应用 技术领域
本发明属于口腔制剂领域，尤其涉及一种口腔清洁组合物及其应用。
背景技术
龋病俗称虫牙、蛀牙，是细菌性疾病。龋病发病率高、波及范围广的牙体硬组织感染性疾病，能直接或间接的影响人们的全身健康，可继发牙髓炎和根尖周炎，甚至能引起牙槽骨和颌骨炎症。如不及时治疗，病变继续发展，形成龋洞，终至牙冠完全破坏消失，其发展的最终结果是牙齿丧失。目前广泛被接受的理论是Keyes提出的三联因素学说，饮食、细菌和宿主三种因素同时存在时龋病才会发生，打破其中一个因素则龋病不易发生。
口腔食物残渣的多糖、蛋白质、脂肪及口腔中的细菌对食物残渣的利用形成了复杂的口腔问题。现有的口腔护理产品种类繁多，功能单一，无法有效地应对复杂的口腔环境及口腔问题。
发明内容
本发明的目的在于提供一种口腔清洁组合物及其应用，破坏龋病发生的三种因素之一，从而减少龋病的发生。
本发明的发明构思如下：
蛋白水解酶蛋白可以将食物残渣中的蛋白质水解，破坏口腔中的有害菌群的生长环境，抑制细菌的生长。
非蛋白水解酶蛋白可以分解细菌的膜结构，直接杀灭有害细菌。
益生菌能竞争抑制口腔中有害菌群的生长。
三者组合能从多方面抑制口腔中的有害菌群的生长，减少龋病、炎症等口腔疾病的发生，保持口腔良好的环境。
本发明所采用的技术方案为：
本发明的第一个方面，提供：
一种口腔清洁组合物，包括蛋白水解酶蛋白、非蛋白水解酶蛋白和益生菌。
优选的，上述益生菌包括唾液链球菌M18、唾液链球菌K12、植物乳杆菌L-137、凝结芽孢杆菌、罗伊氏乳杆菌、副干酪乳杆菌、嗜酸乳杆菌的至少一种。
优选的，上述益生菌包括唾液链球菌M18、唾液链球菌K12、罗伊氏乳杆菌的至少一种。
优选的，上述蛋白水解酶蛋白包括木瓜蛋白酶、枯草芽孢杆酶、菠萝蛋白酶的至少一种。
优选的，上述非蛋白水解酶蛋白包括乳铁蛋白、乳过氧化物酶、溶菌酶、葡萄糖氧化酶、淀粉酶、脂肪酶、超氧化物歧化酶的至少一种。
本发明的第二个方面，提供：
一种口腔清洁剂，包括上述口腔清洁组合物。
本发明的第三个方面，提供：
一种上述口腔清洁组合物在制备改善口腔炎症药物中的应用。
优选的，上述药物为牙膏、牙粉、漱口剂、口腔清洗剂、洁牙带、口香糖的至少一种。
本发明的第四个方面，提供：
一种上述口腔清洁组合物在制备改善口腔炎症药物中的应用。
优选的，上述药物为牙膏、牙粉、漱口剂、口腔清洗剂、洁牙带、口香糖的至少一种。
本发明的有益效果是：
(1)本发明口腔清洁组合物通过分解口腔残留物，破坏口腔有害细菌的生存环境，抑制口腔有害菌群生长的同时为口腔益生菌提供了良好的生长环境，间接或直接促进口腔益生菌的生长，从而维持良好的口腔环境，降低口腔疾病的发生。
(2)本发明通过将非蛋白水解酶蛋白、蛋白水解酶蛋白、益生菌组合使用可以将口腔中各种淀粉类、脂肪类食物残渣分解成葡萄糖、短链脂肪酸等小分子物质增加其溶解度；蛋白水解酶蛋白中的各组分可以将食物残渣中的蛋白质水解成氨基酸，这些小分子可以随着吞咽作用离开口腔，减少口腔中食物的残渣，从而破坏了口腔中的有害菌群的生长环境，减少细菌黏附在食物残渣中以及减少细菌对食物残渣的利用，破坏了细菌的生长环境，抑制细菌的生长。非蛋白水解酶蛋白组合也能作用于口腔中的有害菌群，分解细菌的膜结构，直接杀灭有害细菌；益生菌的加入能竞争抑制口腔中的有害菌群的生长，从而多方面抑制口腔中的有害菌群的生长，减少龋病、炎症等口腔疾病的发生，保持口腔良好的环境。
(3)口腔中的菌斑、牙石、创伤性咬合、食物嵌塞等原因逐渐使牙龈发炎肿胀，同时牙菌斑堆积逐渐加重，并由龈上向龈下扩延生长，侵犯牙龈和牙周组织导致慢性炎症的发生，如牙周炎的产生。本发明通过抑制和减少口腔有害菌群的生长，减少细菌黏附在食物残渣中以及减少细菌对食物残渣的利用，破坏了细菌的生长环境，减少了龋病的发生，同时能改善口腔炎症。
具体来说，本发明具有如下优点：
(1)唾液链球菌M18是由新西兰奥塔哥大学研发的益生菌，唾液链球菌普遍存在于每个人中，但唾液链球菌M18却只天然存在于一些健康的成年人和儿童的口腔和咽喉中，其会产生外切酶葡聚糖酶和脲酶。其中，外切酶葡聚糖酶(水解葡聚糖的1，6-糖苷键的酶)可以抑制口腔变异链球菌合成水不溶性葡聚糖(具有高比例的1，3-连接的葡萄糖基残基的1，6-连接的聚合物)，使细菌斑块不在牙齿表面的积累；而脲酶代谢尿素可以产生的氨可以缓和牙菌斑的糖酵解酸化作用，中和牙菌斑酸性并营造一个不利于酸类物质出现的环境来抑制龋齿的发生和发展。唾液链球菌M18产生的外切酶葡聚糖酶、脲酶、细菌素能多方面抑制有害菌群的生长，改善口腔环境。
(2)唾液链球菌K12同样只天然存在于一些健康的成年人和儿童的口腔和咽喉中，可产生两种天然抗菌肽，即唾液素A2和唾液素B，它们是羊毛硫抗生素型细菌素，可以抑制有害细菌生长。唾液链球菌K12可以抑制与口臭有关的革兰阳性菌、微单胞菌等有害菌的生长，减少口腔异味和抑制口腔有害菌群的生长。
发明人发现，以上(1)和(2)中的唾液链球菌M18和K12并非普遍存在，通常有口腔问题的人的口腔中都没有这两种链球菌，而且口腔问题得到治疗后，唾液链球菌M18和K12也不会随之产生。因此，唾液链球菌M18和K12是改善口腔环境的重要因素，主动添加到口腔清洁组合物中能为口腔带来上述两种益生菌，有利于改善口腔问题。
(3)植物乳杆菌L-137属于乳杆菌科中的乳杆菌属。已有临床实验报道表明：患者接受每日10毫克的热灭活的植物乳杆菌L-137胶囊，持续12周。探查出血(BOP)和探查深度(PD)≥4mm的牙齿或部位的数目均显着减少，每天接受热灭活的植物乳杆菌L-137摄入可以减少接受支持性牙周治疗的患者的牙周袋深度。
(4)凝结芽孢杆菌为同型乳酸发酵菌，该菌对大肠杆菌(NCTC-10418)、铜绿假单胞菌(NCIB-9016)、肺炎克雷伯菌(NCIB-9111)、枯草杆菌(NCTC-6346)、金黄色葡萄球菌(金黄色葡萄球菌)(NCTC7447)和白色念珠菌(CBS-562)具有抑制作用。
(5)罗伊氏乳杆菌是一种乳酸菌，该菌产生的广谱抗菌物质，称为罗伊氏菌素。罗伊氏菌素是一种低分子量，中性的水溶性化合物，能够抑制埃希氏菌，沙门氏菌，志贺氏菌，变形杆菌，假单胞菌，梭菌和葡萄球菌的生长。
(6)副干酪乳杆菌是一种革兰氏阳性异型发酵乳酸菌，产生的细菌素能将牙龈卟啉单胞菌的细胞膜从球状或球菌状改变为杆状并肿胀，导致细菌死亡。
(7)嗜酸乳杆菌属于乳杆菌属。该菌产生的细菌素可抑制革兰氏阳性菌(加德纳菌和无乳链球菌)和革兰氏阴性菌(铜绿假单胞菌)的生长。
(8)木瓜蛋白酶和菠萝蛋白酶属于巯基蛋白酶类，木瓜蛋白酶从未成熟的番木瓜果实中的乳汁中提炼出来的纯天然生物酶制品，菠萝蛋白酶是从菠萝植株中提取的一类蛋白水解酶的总称，枯草芽孢杆菌蛋白酶是一组源自枯草芽孢杆菌不同菌株的碱性蛋白酶。这三种蛋白酶均有广泛的水解蛋白质的能力，可以将口腔中蛋白类食物残渣分解为短肽和氨基酸等小分子，这些小分子溶于唾液中随吞咽作用排走，减少口腔中有害菌群对蛋白类食物残渣的利用和黏附，破坏有害菌群的生存环境，对口腔有害菌有一定的抑制作用。
(9)乳铁蛋白是一种具有免疫功能的非血红素铁结合糖蛋白，从人乳汁中从分离得到，乳铁蛋白的抑菌作用归因于代谢所需的铁的络合。乳铁蛋白对游离的铁有更强的结合作用，抑制了细菌对铁的利用，从而抑制了细菌的生长。此外，乳铁蛋白能与革兰氏阴性菌的细胞壁上的脂质A结合，破坏革兰氏阴性菌细胞膜，改变膜的通透性，最终使细菌死亡。乳铁蛋白能通过与铁的结合来抑制口腔中细菌的生长，也能作用于口腔中革兰氏阴性菌的细胞壁，使革兰氏阴性菌死亡，达到抑菌的效果。
(10)乳过氧化物酶是一种氧化还原酶。乳过氧化物酶与硫氰酸根离子或碘化物或溴化物以及过氧化氢一起形成乳过氧化物酶系统。该系统的机理是过氧化氢将硫氰酸盐离子(也包括碘化物和溴化物)氧化为次硫氰酸盐离子(次碘化物和次溴化物)，次硫氰酸根离子氧化微生物蛋白氨基酸残基的巯基，导致细胞膜功能受损，从而使微生物死亡。在口腔环境中，该机制可以有效地抑制细菌的生长繁殖。
(11)溶菌酶的天然底物是革兰氏阳性菌和革兰氏阴性菌的肽聚糖细胞壁，肽聚糖细胞壁由交替的2-乙酰氨基-2-脱氧-吡喃葡萄糖(NAG)和2-乙酰氨基-2-脱氧-3-丙交酯-吡喃葡萄糖(NAM)残基组成的交联寡糖组成。革兰氏阳性细菌胞壁所含的肽聚糖占干重的50-80％，革兰氏阴性细菌胞壁所含的肽聚糖占干重的5-20％。因此，溶菌酶具有广谱的杀菌能力，对口 腔中革兰氏阳性菌(如变异链球菌)和革兰氏阴性菌(牙龈卟啉单胞菌)均有抑制和杀灭的作用。
(12)葡萄糖氧化酶是一种黄素蛋白，利用氧分子作为电子受体催化β-D-葡萄糖氧化为D-葡萄糖-δ-内酯和过氧化氢。该反应可分为还原和氧化步骤，在还原反应中，葡萄糖氧化酶催化β-D-葡萄糖氧化为D-葡萄糖酸-δ-内酯。随后葡萄糖氧化酶的黄素腺嘌呤二核苷酸(FAD)环被还原为FADH2。在氧化反应中，减少的葡萄糖氧化酶被氧再氧化生成H2O2，过氧化氢酶(CAT)分解过氧化氢生成水和氧。在口腔环境中，葡萄糖氧化酶可以与淀粉酶配合工作，使食物中淀粉残渣能较彻底的转化为小分子，这些小分子溶于唾液中随吞咽作用排走。
(13)淀粉酶常见的有α-淀粉酶、β-淀粉酶。淀粉由两种成分组成，其中包括葡萄糖残基由1,4α-D-葡萄糖苷键结合的糖苷链，即直链淀粉，这些链中的大部分是1,4α-D-葡萄糖苷键。其中一些葡萄糖单元也通过1,6α-D-吡喃葡萄糖苷键连接，即支链淀粉。α-淀粉酶以淀粉为底物时，淀粉快速地水解，产生低聚糖，此阶段淀粉的黏度及与碘发生呈色反应的能力迅速下降。然后，低聚糖缓慢水解生成最终产物葡萄糖和麦芽糖。淀粉酶可以将口腔中的淀粉类食物残渣分解为小分子化合物，这些小分子溶于唾液中随吞咽作用排走，减少口腔中有害菌群在淀粉类食物残渣中黏附和利用，破坏有害菌群的生存环境。
(14)脂肪酶是指催化三酯酰甘油水解的一类酶的总称,是一类特殊的酯键水解酶，其天然底物是长链脂肪酸酯。脂肪酶可以将口腔中的油脂分解为短链的脂肪酸小分子，这些小分子溶于唾液中随吞咽作用排走，减少口腔中有害菌群对脂肪的利用，破坏有害菌群的生存环境。
(15)超氧化物歧化酶是一种能够催化超氧化物通过歧化反应转化为氧气和过氧化氢的酶。它广泛存在于各类动物、植物、微生物中，是一种重要的抗氧化剂。超氧化物歧化酶可以加入到牙膏、漱口水、含片等里面，对预防口腔疾病有一定的疗效。
附图说明
图1为本发明实验设计的人工口腔组成连接图。
具体实施方式
为了使本发明的发明目的、技术方案及其技术效果更加清晰，以下结合具体实施方式，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的具体实施方式仅仅是为了解释本发明，并非为了限定本发明。
为直观比对本发明口腔组合物的改善龋病效果，以下实施例和对比例中，各组分标记及质量范围如下：
因素1：蛋白水解酶蛋白组分
因素1.1：木瓜蛋白酶，质量为2g；
因素1.2：枯草(芽孢)杆菌酶粉，质量为2g；
因素1.3：菠萝蛋白酶，质量为2g。
因素2：非蛋白水解酶蛋白组分
因素2.1：乳铁蛋白，质量为2g；
因素2.2：乳过氧化物酶，质量为2g；
因素2.3：溶菌酶，质量为2g；
因素2.4：葡萄糖氧化酶，质量为2g；
因素2.5：淀粉酶，质量为2g；
因素2.6：脂肪酶，质量为2g；
因素2.7：超氧化物歧化酶，质量为2g。
因素3：益生菌组分
因素3.1：唾液链球菌M18，质量为2g；
因素3.2：唾液链球菌K12，质量为2g；
因素3.3：植物乳杆菌L-137，质量为2g；
因素3.4：凝结芽孢杆菌，质量为2g；
因素3.5：罗伊氏乳杆菌，质量为2g；
因素3.6：副干酪乳杆菌，质量为2g；
因素3.7：嗜酸乳杆菌，质量为2g。
一、牙膏制作如下：
(1)分别将因素1.1～1.3与碳酸钙45g，甘油30g，糖精钠0.1g，苯甲酸钠0.1g，硅石粉1g，月桂酰两性基二乙酸二钠1g及适量精制水混合均匀，均质后分别得到含有各因素的100g牙膏。
(2)分别将因素2.1～2.7与碳酸钙45g，甘油30g，糖精钠0.1g，苯甲酸钠0.1g，硅石粉1g，月桂酰两性基二乙酸二钠1g及适量精制水混合均匀，均质后分别得到含有各因素的100g牙膏。
(3)分别将因素3.1～3.7与碳酸钙45g，甘油30g，糖精钠0.1g，苯甲酸钠0.1g，硅石粉1g，月桂酰两性基二乙酸二钠1g及适量精制水混合均匀，均质后分别得到含有各因素的100g牙膏。
(4)将因素1.1-1.3与碳酸钙45g，甘油30g，糖精钠0.1g，苯甲酸钠0.1g，硅石粉1g，月桂酰两性基二乙酸二钠1g及适量精制水混合均匀，均质后得到含有因素1.1-1.5的100g牙膏，为组合1。
(5)将因素2.1-2.7与碳酸钙45g，甘油30g，糖精钠0.1g，苯甲酸钠0.1g，硅石粉1g，月桂酰两性基二乙酸二钠1g及适量精制水混合均匀，均质后得到含有因素2.1-2.7的100g牙膏，为组合2。
(6)将因素3.1-3.7与碳酸钙45g，甘油30g，糖精钠0.1g，苯甲酸钠0.1g，硅石粉1g，月桂酰两性基二乙酸二钠1g及适量精制水混合均匀，均质后得到含有因素3.1-3.7的100g牙膏，为组合3。
二、实验方法：
2.1实验菌种：
牙龈卟啉单胞菌Porphyromonas gingivalis(P.gingivalis)
血链球菌Streptococcus sanguis(S.sanguis)
变异链球菌Streptococcus mutan(S.mutans)
2.2实验试剂：
羟基磷灰石片，脑心浸液(BHI)琼脂，脑心浸液(BHI)肉汤，P7000-25G猪胃粘蛋白，微量样品基因组DNA提取试剂盒，Carrier RNA，Platinum SYBR Green定量PCR supermix试剂盒，RNase H、引物合成，溶菌酶
2.3改良人工唾液配制
人工唾液配方为改良生物膜培养基BM-5：Ⅲ型猪胃黏蛋白2.5g/L；肽胨2g/L；胰酶分解酪蛋白胨1g/L；酵母提取物1g/L；氯化钾(KCl)2.5g/L；葡萄糖0.5g/L；胱氨酸盐酸盐0.1g/L；氯化血红素1mg/L；磷酸氢二钾(K 2HPO 4·3H 20)0.114g/L；磷酸二氢钾(KH 2PO 4)0.2g/L。上述成分加入10g/L蔗糖，用1mol/L NaOH溶液调整其pH值为7.5，121℃，高压灭菌15min，备用。
2.4细菌复苏、传代
取出三种实验细菌真空冻干粉，用无菌吸管吸取适量经灭菌的BHI液体培养基溶解菌粉，转移至装有BHI液体培养基的无菌离心管中厌氧37℃过夜复苏。将复苏后的3种细菌划线接种于BHI固体培养基中，置于37℃厌氧培养箱中，厌氧培养48小时。形态学及染色检查为纯培养物，再次接种于BHI固体培养基，培养48小时，形态学鉴定为纯培养物。
2.5菌液制备
分别挑取二环S.mutans，S.sanguis，P.gingivalis三种标准菌株溶于装有10mL BHI液体培养基的离心管中，拧松管盖，使细菌与箱内厌氧环境充分接触，37℃厌氧培养18小时，获得S.mutans，S.sanguis，P.gingivalis三种标准菌株的增菌液。使用紫外分光光度计调整各菌浓度为A 550＝0.25±0.05(3.0x10 8CFU/ml)，每种细菌分别取30mL加入500mL的改良人工唾液中备用。
2.6羟基磷灰石片的制备
取适量羟基磷灰石片，一面粘固于有孔塑料托盘孔板上，灭菌后备用。
2.7人工口腔搭建
如图1所示，本实验设计的人工口腔由人工唾液装置、菌悬液装置、恒温培养室、废液装置构成。各装置间用硅胶管连接，其中人工唾液装置与菌悬液装置，人工唾液装置与恒温培养室、菌悬液装置与恒温培养室之间安放数显恒流泵。菌悬液培养条件、恒温培养室培养条件和在恒温培养室中制备体外牙菌斑生物膜如下：
2.7.1菌悬液培养条件
温度：维持恒温37℃，厌氧环境；
搅拌：低速，由磁力搅拌仪控制(约90转/分钟)，以使菌悬液混匀；
废液清除率：0.05mL/分钟(1滴/分钟)，由流速调节器控制。
2.7.2恒温培养室培养条件
温度：维持恒温37℃，厌氧环境；
废液清除率：0.2mL/分钟(4滴/分钟)，由流速调节器控制。
2.7.3制备体外牙菌斑生物膜
将预先制备好的菌悬液放置在菌悬液装置中，人工唾液以0.1mL/分钟(2滴/分钟)加入菌悬液装置中，菌悬液37℃厌氧培养24小时，达到稳定生长状态；然后，将菌悬液与人工唾液装置接通恒温培养室(分别取6片羟基磷灰石片预先悬吊于恒温培养室中均低于液面约2cm)，按菌悬液与改良人工唾液1：9比例(即0.1ml/分钟：0.9mL/分钟)持续同时泵入恒温培养室中，24小时后停止泵入菌悬液，继续给予改良人工唾液(0.2mL/分钟)48小时。在整个恒流培养室培养的3天，每天的8点、20点给予不同的实验试剂，每组给予50mL(每次1mL/分钟，共50分钟)，同时以相同的速率排出废液，模拟口腔吞咽作用。实验过程中废气由厌氧培养箱排除，每天的废液染色后检测是否有杂菌污染。
2.8 FQ-PCR检测
在连续培养的6小时、24小时、48小时、72小时分别从实验组各取出6片羟基磷灰石片，用无菌挖器刮取羟基磷灰石片表面的全部菌斑，收集到1.5mL离心管内，加入200μL浓度为20mg/mL的溶菌酶，37℃反应30分钟以上，充分裂解细胞壁，释放细胞内的核酸物质。
2.9 DNA提取和引物设计
使用微量样品基因组DNA提取试剂盒提取DNA。采用二步法PCR法对样本进行扩增：首先采用细菌通用引物产生全长的16SrDNA产物，然后再以该产物为模板，用各细菌的特异引物扩增相应目标细菌的DNA。根据16SrDNA基因序列，应用引物设计软件Primer premier5.0设计特异性PCR引物，各引物序列见下表1：
表1 S.mutans，S.sanguis，P.gingivalis，内参Universal引物序列
Figure PCTCN2020097247-appb-000001
2.10 Real-timePCR扩增
(1)按表2配备反应体系
表2 Real-timePCR扩增反应体系溶液构成
组分 体积(μL)
2xSuper Real Pre Mix 10
上游引物(10μmol/L) 0.6
下游引物(10μmol/L) 0.6
50x ROX Reference Dye 2
cDNA模板 1
dd H 2O 5.8
总体系 20
(2)按Real-timePCR反应体系准备除模板外所有组分的混合液；
(3)每次反应时，将19μL的混合液加入到96孔PCR板的每个孔中；
(4)加入1μL的样品模板至各反应容器中，密封反应管；
(5)轻轻混匀，确保所有组分都在反应容器底部；
(6)将反应体系置于己设置好的基因扩增仪中，收集数据并分析结果。
2.11 Real-timePCR反应与检测
PCR反应条件如表3所示(两步法，退火延伸合一步)：
表3 Real-timePCR反应条件
循环数 循环条件 目的
1x 95℃，5min 预变形
45x Step1：95℃，60s 变形
  Step2：60℃，31s 退火/延伸
整个加样过程在冰上操作，盖上管盖，轻敲混匀，手掌离心机瞬时混匀，以便于将试剂聚集到管底，将管子置于PCR仪上，按上述条件设置循环程序，为确保数据的有效性，每个样品均平行做3个复孔，再分别进行Real-timePCR产物检测。
2.12计算方式
根据Real-timePCR所测的结果本实验采用△△Ct相对定量法基因的表达变化。在应用△△Ct方法进行定量之前，做可行性试验来验证目的基因与内参扩增效率基本相等，以Universal为内参，进行一系列荧光定量PCR反应，反应体系及反应条件与以上反应相同，目的基因与内参的扩增斜率差值小于0.1，可以应用△△Ct进行定量分析，不必做标准品绝对定量或行相对标准曲线定量法。样本中目的基因相对表达量的计算公式：F＝2 -△△Ct，△Ct＝目的基因平均ct值-管家基因平均ct值，△△ct＝△ct(待测样本)-△ct(对照样本)，2 -△△Ct所计算的相对表达量表示待测样本中目的基因的表达量相对对照样本中目的基因表达量的倍数。因此，本实验用2 -(△△Ct)法表示研究对象牙菌斑生物膜某种细菌的相对含量。以某一样本为校准样本，其余样本为待测样本，2 -(△△Ct)值即为待测样本牙菌斑生物膜中某种细菌校准样本的倍数。
三、临床方法
3.1研究对象
样本来源于志愿者，经检查诊断为慢性牙周炎的患者，菌斑牙石堆积，牙周探诊深度>8mm，共10例。
3.2牙周袋微生物标本采集
在探诊最深的牙周袋中插入25#无菌纸捻约10mm，每根纸捻停留30秒，每颗牙在不同位点采集纸捻，置于4℃、1mL生理盐水的EP管中，最后在-80℃保存。
3.3检测指标
治疗前后牙周探诊深度，治疗前后牙周袋中牙龈卟啉单胞菌、血链球菌、变异链球菌数量(细菌检测方法同前述检测方法)。
实验例1：蛋白水解酶蛋白牙膏配方对3种细菌生长的影响
将前述因素1.1-1.3经组合后分别对应形成实验组1-6，各实验组组成详见表4-6，表中+号表示该实验组中含有某种因素。将实验组1-6的100g样品分别加入50ml水搅拌均匀，得到实验样品。按照前述实验方法及检测方法进行实验。
表4蛋白水解酶蛋白牙膏配方对牙龈卟啉单胞菌生长的影响
Figure PCTCN2020097247-appb-000002
表5蛋白水解酶蛋白牙膏配方对血链球菌生长的影响
Figure PCTCN2020097247-appb-000003
Figure PCTCN2020097247-appb-000004
表6蛋白水解酶蛋白牙膏配方对变异链球菌生长的影响
Figure PCTCN2020097247-appb-000005
由表4～6可看出，实验组6在6h、24h、48h、72h细菌数量明显少于对照组(P<0.01)。结果表明实验组6，即木瓜蛋白酶+枯草(芽孢)杆菌酶+菠萝蛋白酶的组合能抑制变异链球菌、血链球菌、牙龈卟啉单胞菌的生长。
实验例2：非蛋白水解酶蛋白牙膏配方对3种细菌生长的影响
将前述因素2.1-2.7经组合后分别对应形成实验组7-34，各实验组组成详见表7-9，表中+号表示该实验组中含有某种因素。将实验组7-34的100g样品分别加入50mL水搅拌均匀，得到实验样品。按照前述实验方法及检测方法进行实验。
表7非蛋白水解酶蛋白牙膏配方对牙龈卟啉单胞菌生长的影响
Figure PCTCN2020097247-appb-000006
Figure PCTCN2020097247-appb-000007
Figure PCTCN2020097247-appb-000008
表8非蛋白水解酶蛋白牙膏配方对血链球菌生长的影响
Figure PCTCN2020097247-appb-000009
Figure PCTCN2020097247-appb-000010
Figure PCTCN2020097247-appb-000011
表9非蛋白水解酶蛋白牙膏配方对变异链球菌生长的影响
Figure PCTCN2020097247-appb-000012
Figure PCTCN2020097247-appb-000013
Figure PCTCN2020097247-appb-000014
由表7～9可看出，实验组34在6h、24h、48h、72h细菌数量明显少于对照组(P<0.01)，结果表明实验组34，即乳铁蛋白+乳过氧化物酶+溶菌酶+葡萄糖氧化酶+淀粉酶+脂肪酶+超氧化物歧化酶的组合能抑制变异链球菌、血链球菌、牙龈卟啉单胞菌的生长。
实验例3：益生菌牙膏配方对3种细菌生长的影响
将前述因素3.1-3.7经组合后分别对应形成实验组35-62，各实验组组成详见表10-12，表中+号表示该实验组中含有某种因素。将实验组35-62的100g样品分别加入50mL水搅拌均匀，得到实验样品。按照前述实验方法及检测方法进行实验。
表10益生菌牙膏配方对牙龈卟啉单胞菌生长的影响
Figure PCTCN2020097247-appb-000015
Figure PCTCN2020097247-appb-000016
表11益生菌牙膏配方对血链球菌生长的影响
Figure PCTCN2020097247-appb-000017
Figure PCTCN2020097247-appb-000018
表12益生菌牙膏配方对变异链球菌生长的影响
Figure PCTCN2020097247-appb-000019
Figure PCTCN2020097247-appb-000020
Figure PCTCN2020097247-appb-000021
从表10～12可看出，实验组62在6h、24h、48h、72h细菌数量明显少于对照组(P<0.01)，且绝大部分实验组细菌数量明显少于对照组(P<0.05)。结果表明实验组62，即唾液链球菌M18+唾液链球菌K12+植物乳杆菌L-137+凝结芽孢杆菌+罗伊氏乳杆菌+副干酪乳杆菌+嗜酸乳杆菌的组合能抑制变异链球菌、血链球菌、牙龈卟啉单胞菌的生长，其余绝大部分实验组也有一定的抑制变异链球菌、血链球菌、牙龈卟啉单胞菌生长的作用。
实施例：蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对细菌生长的影响
1.蛋白水解酶蛋白组分、非蛋白水解酶蛋白组分、益生菌组分组合使用对变异链球菌细菌生长的影响
将前述因素3.1-3.7分别与因素1.1和因素2.1经组合后对应形成实施例1-7，各实验组组成详见表13，表中+号表示该实验组中含有某种因素。将实施例1-7的100g样品分别加入50ml水搅拌均匀，得到实验样品。按照前述实验方法及检测方法进行实验。
表13蛋白水解酶蛋白组分、非蛋白水解酶蛋白组分、益生菌组分组合使用对变异链球菌细菌生长的影响
Figure PCTCN2020097247-appb-000022
Figure PCTCN2020097247-appb-000023
从表13可看出，实施例1、2、5在6h、24h、48h、72h细菌数量明显少于对照组(P<0.01)，实施例1、2、5的细菌数量明显少于对照组(P<0.05)。结果表明实施例1唾液链球菌M18+木瓜蛋白酶+乳铁蛋白，实施例2唾液链球菌K12+木瓜蛋白酶+乳铁蛋白，实施例5罗伊氏乳杆菌+木瓜蛋白酶+乳铁蛋白组合能抑制变异链球菌的生长。
2.蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对3种细菌生长的影响
实验组63：仅使用组合1；
实验组64：仅使用组合2；
实验组65：仅使用组合3；
实验组66：先使用组合1，再使用组合2；
实验组67：先使用组合2，再使用组合1；
实施例8：先使用组合1，然后使用组合2，最后使用组合3；
实施例9：先使用组合2，然后使用组合1，最后使用组合3。
取组合的100g样品分别加入50ml水搅拌均匀，得到实验样品。按照前述实验方法及检测方法进行实验，得到表14～16的实验数据。
表14蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对牙龈卟啉单胞菌生长的影响
Figure PCTCN2020097247-appb-000024
表15蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对血链球菌生长的影响
Figure PCTCN2020097247-appb-000025
Figure PCTCN2020097247-appb-000026
表16蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对变异链球菌生长的影响
组别 6h 24h 48h 72h P值(6h) P值(24h) P值(48h) P值(72h)
对照组 1.000±0 1.000±0 1.000±0 1.000±0 - - - -
实验组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
实验组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
实验组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
实验组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
实验组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
实施例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
实施例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
备注：实施例8和实施例9与实验组63-67相比显著性水平为P<0.05
由表14～16可看出，实施例8和实施例9均能在6h、24h、48h、72h时细菌数量明显少于对照组(P<0.001)，而且效果好于其他实验组(P<0.05)，结果表明实施例8和实施例9抑制变异链球菌、血链球菌、牙龈卟啉单胞菌的生长，而且优于其他实验组。
临床实施例：蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对慢性牙周炎的患者牙周探诊深度和牙周袋中牙龈卟啉单胞菌、血链球菌、变异链球菌生长的影响
患者每天早晚刷牙2次，实验期间不能使用抗生素类药物和其它牙膏或涑口水等同类口腔清洁用品。实验持续3个月，按照先使用组合1+后使用组合2+最后使用组合3的方式刷牙。分别在实验开始前和实验3个月后，检测周探诊深度和牙周袋中牙龈卟啉单胞菌、血链球菌、变异链球菌数量，如表17～18所示。
表17蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对慢性牙周炎的患者牙周探诊深度的影响
  治疗前mm 治疗后mm P值
牙周探诊深度 10.5±1.56 4.43±1.02 5.45×10 -09
表18蛋白水解酶蛋白牙膏、非蛋白水解酶蛋白牙膏、益生菌牙膏组合使用对慢性牙周炎的患者牙周袋中牙龈卟啉单胞菌、血链球菌、变异链球菌生长的影响
  治疗前 治疗后 P值
牙龈卟啉单胞菌 0.913±0.212 0.349±0.137 1.37×10 -06
血链球菌 0.873±0.187 0.350±0.216 1.73×10 -05
变异链球菌 0.805±0.230 0.357±0.128 4.08×10 -05
从表17和表18可看出，先使用组合1+后使用组合2+最后使用组合3的刷牙方式显著减少了慢性牙周炎患者牙周探诊深度(P<0.001)；同时牙龈卟啉单胞菌、血链球菌、变异链球菌数量明显减少(P<0.001)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种口腔清洁组合物，其特征在于：包括蛋白水解酶蛋白、非蛋白水解酶蛋白和益生菌。
  2. 根据权利要求1所述的口腔清洁组合物，其特征在于：所述益生菌包括唾液链球菌M18、唾液链球菌K12、植物乳杆菌L-137、凝结芽孢杆菌、罗伊氏乳杆菌、副干酪乳杆菌、嗜酸乳杆菌的至少一种。
  3. 根据权利要求2所述的口腔清洁组合物，其特征在于：所述益生菌包括唾液链球菌M18、唾液链球菌K12、罗伊氏乳杆菌的至少一种。
  4. 根据权利要求1所述的口腔清洁组合物，其特征在于：所述蛋白水解酶蛋白包括木瓜蛋白酶、枯草芽孢杆酶、菠萝蛋白酶的至少一种。
  5. 根据权利要求1所述的口腔清洁组合物，其特征在于：所述非蛋白水解酶蛋白包括乳铁蛋白、乳过氧化物酶、溶菌酶、葡萄糖氧化酶、淀粉酶、脂肪酶、超氧化物歧化酶的至少一种。
  6. 一种口腔清洁剂，其特征在于：包括权利要求1～5任一项所述的口腔清洁组合物。
  7. 一种权利要求1～5任一项所述的口腔清洁组合物在制备改善口腔龋病药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于：所述药物为牙膏、牙粉、漱口剂、口腔清洗剂、洁牙带、口香糖的至少一种。
  9. 一种权利要求1～5任一项所述的口腔清洁组合物在制备改善口腔炎症药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于：所述药物为牙膏、牙粉、漱口剂、口腔清洗剂、洁牙带、口香糖的至少一种。
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CN114504646B (zh) * 2022-02-18 2022-11-04 深圳南粤药业有限公司 一种含粘蛋白的人工唾液组合物及其制备方法与应用
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