CN114209818A - Composition containing hydrolase and polyglutamic acid for preventing and removing periodontal biofilm - Google Patents
Composition containing hydrolase and polyglutamic acid for preventing and removing periodontal biofilm Download PDFInfo
- Publication number
- CN114209818A CN114209818A CN202111566823.7A CN202111566823A CN114209818A CN 114209818 A CN114209818 A CN 114209818A CN 202111566823 A CN202111566823 A CN 202111566823A CN 114209818 A CN114209818 A CN 114209818A
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- CN
- China
- Prior art keywords
- alpha
- beta
- polyglutamic acid
- glucosidase
- acid
- Prior art date
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- Pending
Links
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- 108010020346 Polyglutamic Acid Proteins 0.000 title claims abstract description 97
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 9
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- CHUGKEQJSLOLHL-UHFFFAOYSA-N 2,2-Bis(bromomethyl)propane-1,3-diol Chemical compound OCC(CO)(CBr)CBr CHUGKEQJSLOLHL-UHFFFAOYSA-N 0.000 claims description 5
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- PUTDIROJWHRSJW-UHFFFAOYSA-N Quercitrin Natural products CC1OC(Oc2cc(cc(O)c2O)C3=CC(=O)c4c(O)cc(O)cc4O3)C(O)C(O)C1O PUTDIROJWHRSJW-UHFFFAOYSA-N 0.000 claims description 3
- OXGUCUVFOIWWQJ-XIMSSLRFSA-N acanthophorin B Natural products O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-XIMSSLRFSA-N 0.000 claims description 3
- OEKUVLQNKPXSOY-UHFFFAOYSA-N quercetin 3-O-beta-D-glucopyranosyl(1->3)-alpha-L-rhamnopyranosyl(1->6)-beta-d-galactopyranoside Natural products OC1C(O)C(C(O)C)OC1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OEKUVLQNKPXSOY-UHFFFAOYSA-N 0.000 claims description 3
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/74—Synthetic polymeric materials
- A61K31/785—Polymers containing nitrogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The present invention relates to a composition for preventing and removing a polyglutamic acid and a hydrolase enzyme formed in a periodontal biofilm of an oral cavity. By adding polyglutamic acid into single hydrolase or hydrolase composition, the dosage of single hydrolase or hydrolase composition is effectively reduced while the effect of the hydrolase or hydrolase composition per unit mass is improved.
Description
Technical Field
The present invention relates to a composition for preventing and removing a polyglutamic acid and a hydrolase enzyme formed in a periodontal biofilm of an oral cavity.
Background
Biofilm (Biofilm) is a means by which most bacteria adhere to a membrane structure that is focused on a solid surface. The biological membrane has stronger resistance to a host defense system, has low sensitivity to antibiotics, and has greatly higher pathogenic capability to human bodies than planktonic bacteria.
The oral cavity is one of four major bacterial banks of the human body, the bacteria in the oral cavity are various and high in quantity, and most of the bacteria are adhered to the surfaces or the inner parts of teeth, gums and oral mucosa in the form of biomembranes, which are often called dental plaques.
How to remove the biofilm has been one of the concerns of oral medicine. The oral periodontal biofilm is controlled by a mechanical physical method in clinic, and antibiotics are also used for treatment in severe cases.
Medical workers have looked at enzymes due to reduced sensitivity of biofilms to various antibiotics. The use of multiple enzymes, particularly hydrolases, alone or in combination, is effective in preventing and removing periodontal biofilm in the oral cavity. The use of one or a combination of several hydrolases to prevent and remove the formation of periodontal biofilm in the oral cavity has become a common technique.
The patents (application numbers: 200380102321.2, 200880121578.5, 200780026850.7) relate to methods for preventing and removing biofilms using solutions of single enzymes or enzyme mixtures. However, in practice we have found that the use of some commercial enzymes is limited due to their high cost.
Disclosure of Invention
In order to solve the cost problem of certain enzymes and compositions thereof, the inventor adds polyglutamic acid as a synergist into a single hydrolase or a mixture of hydrolases after a large number of experiments, improves the effect of the hydrolase or the mixture of hydrolases per unit mass, and effectively reduces the dosage of the single hydrolase or the mixture of the hydrolases, thereby better solving the cost problem.
The single hydrolase is selected from one of glycosidase, peptidase (protease) and esterase.
The hydrolase composition of the present invention is a composition comprising at least two or more members selected from the group consisting of glycosidases, peptidases (proteases), and esterases.
Compositions containing polyglutamic acid and a hydrolase are used to prevent or remove biofilms.
The polyglutamic ACID English name is gamma-POLY-GLUTAMIC ACID, is called gamma-PGA for short, is water-soluble, biodegradable and nontoxic, and is a biological macromolecule prepared by using a microbial fermentation method. Polyglutamic acid is a special anionic natural polymer, and is a polyamide with the same type formed by amide bonding between alpha-amino (alpha-amino) and gamma-carboxyl (gamma-carboxyl), and the molecular weight is between 5 ten thousand and 2 million daltons. Are collectively referred to as γ - (D, L) -PGA, γ - (D) -PGA, γ - (L) -PGA, and the like.
The polyglutamic acid is prepared by fermenting microorganisms, particularly Bacillus subtilis, and preferably Bacillus subtilis var natto.
The polyglutamic acid of the present invention includes monovalent or divalent salts such as sodium salt or calcium salt of γ -PGA, such as sodium γ -polyglutamate, potassium γ -polyglutamate, calcium γ -polyglutamate, zinc γ -polyglutamate, etc.
The dosage of the polyglutamic acid is 0.1-10%, and preferably 0.2-2%.
The glycosidase of the invention is selected from the group consisting of alpha-amylase (alpha-amylase, EC 3.2.1.1), beta-amylase (beta-amylase, EC 3.2.1.2), glucan1, 4-alpha-glucosidase (glucan 1,4-a-glucosidase, EC 3.2.1.3), cellulase (cellulose, EC 3.2.1.4), endo-1,3 (4) -beta-glucanase (endo-1, 3 (4) -beta-glucanase, EC3.2.1.6), inulase (inulase, EC3.2.1.7), endo-1, 4-beta-xylanase (endo-1, 4-beta-glucanase, EC 3.2.1.8), oligo-1, 6-glycosidase (oligo-1, 6-glucanase, EC3.2.1.10), glucanase (dextranase, EC3.2.1.11), chitinase (chitinase), exose (EC29, alpha-glucanase, EC3.2.1.15, alpha-glucanase, amylase, alpha-glucanase, EC3, 4-glucanase, lacosase, 3625, polysaccharidase (sialidase, alpha-glucanase, alpha-1.25, g-glucanase, amylase, alpha-glucanase, lacosase, alpha-glucanase, alpha-1, EC3.2.1.7, EC3.2.1.18), alpha-glucosidase (alpha-glucosidase, EC3.2.1.20), beta-glucosidase (beta-glucosidase, EC3.2.1.21), alpha-galactosidase (alpha-galactosidase, EC3.2.1.22), beta-galactosidase (beta-galactosidase, EC3.2.1.23), alpha-mannosidase (alpha-mannosidase, EC3.2.1.24), beta-mannosidase (beta-mannosidase, EC3.2.1.25), beta-fructofuranosidase (beta-fructofuranosidase, EC3.2.1.26), alpha-trehalase (alpha, alpha-trehalase, EC3.2.1.28), beta-glucuronidase (beta-glucuronidase, EC3.2.1.31), hyaluronidase (hyaluronan glucosidase, EC3.2.1.35), hyaluronidase (fucosidase, 678663), pullulanase (fucosidase, 67 EC3.2.1.44), pullulanase (fucosidase, 6741, 6778), pullulanase (fucosidase, 6778), EC3.2.1.57), mycodextranase (EC3.2.1.61), fructosidase (levanase, EC3.2.1.65), quercitrin (quercitrinase, EC3.2.1.66), isoamylase (isoamylase, ec 3.2.1.68), beta-agarase (beta-agarase, EC3.2.1.81), lactase (lactase, EC3.2.1.108), alpha-glucuronidase (alpha-glucuronidase, EC3.2.1.139), alpha-agarase (alpha-agarase, EC3.2.1.158).
The glycosidase of the invention is preferably selected from the group consisting of alpha-amylase (α -amylase, EC 3.2.1.1), beta-amylase (β -amylase, EC 3.2.1.2), cellulase (cellulose, EC 3.2.1.4), glucanase (dextranase, EC3.2.1.11), chitinase (EC3.2.1.14), lysozyme (lysozyme, EC3.2.1.17), exo- α -sialidase (exo- α -sialidase, EC 3.2.1.18), α -glucosidase (α -glucosidase, EC 3.2.1.20), β -glucosidase (β -glucosidase, EC 3.2.1.21), α -galactosidase (α -galactosidase, EC3.2.1.22), β -galactosidase (β -glucosidase, EC 3.2.1.23), α -mannosidase (α -mannosidase, EC 32), pullulanase (EC 3.32 ), pullulanase (pullulanase, EC 3.2.1.41), pullulanase (β -glucosidase, EC 3.32), pullulanase (amylopectin, EC 3.32, C3.32), pullulanase (fructose (fructosidase, 389), pullulanase, EC 3.41), fructosidase, EC 3.32, C, D, E, D, E, D, E, D, E, isoamylase (isoamylase, ec 3.2.1.68), lactase (lactase, EC3.2.1.108), alpha-glucuronidase (alpha-glucuronidase, EC3.2.1.139), alpha-agarase (alpha-agarase, EC3.2.1.158).
The peptidase (protease) of the invention is selected from Subtilisin (Subtilisin, EC 3.4.21.62), serralysin (EC 3.4.24.40), and trypsin (trypsin, EC 3.4.21.4).
The esterase of the invention is selected from the group consisting of carboxylesterase (EC 3.1.1.1), aroyl esterase (aryl esterase, EC 3.1.1.2), triacylglycerol lipase (EC 3.1.1.3), phospholipase A2 (phosphoesterase A2, EC 3.1.1.4), lysophospholipase (lysophospholipase, EC 3.1.1.5), acetyl esterase (acetylesterase, EC 3.1.1.6), gluconolactonase (glucolactonase, EC 3.1.1.17), phospholipase A1 (phosphoesterase A1, EC 3.1.1.32), lipoprotein lipase (lipocalin lipase, PAS EC3.1.1.34), alpha-amino acid esterase (alpha-amino-esterase, acetylesterase (acetylesterase, phosphoesterase, EC 3.1.1.32), phosphoesterase (phosphoesterase, phosphoesterase (EC 3.3.1.3.1.3), cutinase, phosphoesterase (phosphoesterase, EC 3.3.3.1.1.6), phosphoesterase (phosphoesterase, EC3. EC3.1.1.34), phosphoesterase (phosphoesterase, C3.3.3.1.1.1.1.1.17, 3674), phosphoesterase (phosphoesterase.
The esterase of the invention is preferably selected from the group consisting of carboxylesterase (EC 3.1.1.1), phospholipase A2 (phospholipase A2, EC 3.1.1.4), lysophospholipase (lysophospholipase, EC 3.1.1.5), phospholipase A1 (phospholipase A1, EC 3.1.1.32), lipoprotein lipase (lipoprotein lipase, EC3.1.1.34), alpha-amino acid esterase (alpha-amino-acid esterase, EC3.1.1.43), cutinase (cutinase, EC 3.1.1.74), alkaline phosphatase (alkaline phosphatase, EC3.1.3.1), acid phosphatase (acid phosphatase, EC 3.1.3.2), nucleotidase (nucleotidase, EC3.1.3.31), phospholipase C (phospholipase C, EC3.1.4.3), phospholipase D (phospholipase D, EC3.1.4.4).
In the first embodiment of the present invention, the present invention uses the common strains of periodontitis, such as fusobacterium nucleatum subspecies thetaiotaomicron, actinomyces viscosus, actinomycetemcomitans, porphyromonas gingivalis, which are cultured to establish a biofilm, and the strains can be obtained by the China general microbiological culture Collection center or the China Industrial microbiological culture Collection center.
In a second embodiment of the present invention, after the biofilm is established, the hydrolase composition with different dosage is added or not added with polyglutamic acid for treatment, then the fluorescent dye solution is added for culture, then the biofilm is scanned under a laser confocal scanning microscope, the three-dimensional reconstruction is carried out on the biofilm through NIS-Elements AR Analysis software, Extracellular Polymers (EPS) are quantified, and the average value is taken. Among them, extracellular polymer is a basic substance which is generated by microorganisms in the biofilm formation process and causes the pathogenicity of bacteria in the biofilm to be enhanced, plays an important role in changing the behaviors, the toxicity, the drug resistance and the like of the microorganisms in the biofilm, and the determination of EPS can evaluate the pathogenicity of the biofilm so as to reflect the progress of periodontal diseases.
In the third embodiment of the present invention, SPSS statistical software is used to perform statistical analysis on the total EPS amount of each group, and the effect of adding polyglutamic acid is determined. The results show that 0.01% hydrolase composition +0.5% polyglutamic acid is superior to 0.02% hydrolase composition, indicating that the dosage of the hydrolase composition can be significantly reduced after adding polyglutamic acid, and the effect of removing the biofilm is better.
In a fourth example of the present invention, we have exemplified by a number of experiments a combination of a polyglutamic acid and a single hydrolase or hydrolase effective according to the present invention according to the methods of example 1, example 2, and example 3.
The polyglutamic acid and the hydrolase or the hydrolase composition of the present invention are added to the biofilm in an amount to prevent or effectively remove the biofilm. The precise dosage is not critical to the present invention and can be adjusted depending on the nature of the oral periodontal biofilm to be treated. The amount of enzyme used may be up to 0.01% of the total amount, and in some cases, up to 10% of the total amount.
In applying the present invention, the composition is applied to the biofilm on the periodontal surface of the oral cavity for a period of time sufficient to substantially reduce Extracellular Polymeric Substance (EPS) levels.
The Nomenclature of the enzymes according to the invention is cited from the Committee for the Nomenclature of International Biochemistry and Molecular Biology (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology) (3-month version in 2019).
Detailed Description
Example 1
Establishment of a biomembrane of a plurality of strains related to periodontitis. Collecting frozen stock solutions of Fusobacterium nucleatum subspecies pleomorph, Actinomyces viscosus, Actinomyces actinomycetemcomitans and Porphyromonas gingivalis, respectively recovering to logarithmic phase, and adjusting the concentration of each solution to 1 × 108CFU/mL, mixing 4 bacterial solutions in equal proportion, and shaking and mixing uniformly to form a mixed bacterial solution.
Under the condition of keeping out of the sun, the pretreated sterile cover glass is placed into a sterile 6-hole plate, 200 mu L of mixed bacteria liquid and 1.8mL of fresh sterile BHI liquid culture medium (containing 1 mass percent of sucrose and 1 volume percent of chlorhematin-vitamin K)1Solution of AlexaFluor with volume fraction of 1 ‰TM647 fluorescent dye solution), anaerobic culture at 37 deg.C (80% N)2,10%CO2,10%H2) The culture medium was changed every other day and cultured for 48 hours.
After 48 hours, the 6-well plate was taken out in the dark, washed 3 times with sterile distilled water, 200. mu.L of viable bacteria fluorescent dye was added, and incubated at 37 ℃ under anaerobic conditions (80% N)2,10%CO2,10%H2) Standing for 15 min. The 6-well plate was removed and washed 3 times with sterile distilled water. After naturally drying, 200. mu.L of the anti-fluorescence quencher is dropped on the glass slide, the cover glass is placed on the glass slide upside down, and the slide is sealed for inspection.
Example 2
The effect of the enzyme solution on the extracellular polymeric substances of the biomembrane of the periodontitis-related multiple strains is observed by a laser confocal scanning microscope (CLSM).
The periodontitis-associated multi-bacterial biofilm prepared in example 1 was used for the test in the following manner.
(1) The enzyme solution is prepared immediately, and is preheated for 30min in a water bath kettle at 37 ℃ before the experiment.
(2) The grouping is as follows: group a is blank: sterile distilled water; group B was a 0.02% aqueous solution of an enzyme composition (α -amylase, subtilisin, lipase, endoglucanase, mannanase), and group C was an aqueous solution containing 0.5% polyglutamic acid and 0.01% of an enzyme composition (α -amylase, subtilisin, lipase, endoglucanase, mannanase).
The sources of the above enzyme compositions are as follows:
extracting alpha-amylase from bacillus licheniformis; extracting subtilisin from Bacillus subtilis; extracting endoglucanase from Bacillus; the lipase is extracted from Thermomyces lanuginosus; the mannanase is beta-mannanase.
Polyglutamic acid is prepared by fermenting Bacillus subtilis and has a molecular weight of about 70 ten thousand daltons.
(3) The biofilm prepared according to example 1 was removed under protection from light and washed 3 times with sterile distilled water. After the above experiments were performed in groups, the cells were washed 3 times with sterile distilled water, 200. mu.L of viable bacteria SYTO9 fluorescent stain (SYTO 9 to distilled water ratio 1.5. mu.L: 1 ml) was added, and the cells were incubated at 37 ℃ under anaerobic conditions (80% N)2,10%CO2,10%H2) Standing for 15 min. The 6-well plate was removed and washed 3 times with sterile distilled water. After naturally drying, 200. mu.L of the anti-fluorescence quencher is dropped on the glass slide, the cover glass is placed on the glass slide upside down, and the slide is mounted for inspection.
(4) 3 regions were randomly selected for each sample and the experiment was repeated 3 times. Alexa FluorTM647 the excitation/emission wavelength of the fluorescent dye solution is 650/668nm, and the excitation/emission wavelength of the SYTO9 green fluorescent nucleic acid dye is 480/500 nm. Scanning under a laser confocal scanning microscope, performing three-dimensional reconstruction on the biological membrane through NIS-Elements AR Analysis software, quantifying Extracellular Polymeric Substances (EPS), and taking an average value.
Example 3
Statistical analysis and results:
and (4) carrying out normal hypothesis test on the total EPS amount of each group by adopting SPSS statistical software. The results show that the data are in accordance with normal distribution and analyzed according to statistical requirements and measurement data. The total EPS amount of each group is expressed by mean +/-standard deviation, the homogeneity of variance test is carried out, the results show that the variances are uniform, pairwise comparison is carried out between the groups through two independent samples-t test, the total EPS amount in the experimental group is compared by adopting one-factor variance analysis, P is less than 0.05, and the statistical significance is shown.
TABLE 1 comparison of the total EPS amounts of the blank control groups with the respective treatment groups (X. + -. S, n = 3)
Indicates that the difference was statistically significant (P < 0.05) compared to the blank group;
indicates that the difference was statistically significant (P < 0.05) compared to the 0.02% enzyme composition group.
The above results indicate that, when polyglutamic acid is added to the enzyme composition, the dosage of the enzyme composition can be effectively reduced, and the effect of preventing or effectively removing the biofilm is better.
Example 4
The method of examples 1, 2 and 3 is adopted, and the composition of polyglutamic acid and hydrolase confirmed to be effective by the test of the invention at least comprises the following combinations.
(1) Polyglutamic acid, alpha-amylase (alpha-amylase, EC 3.2.1.1)
(2) Polyglutamic acid, beta-amylase (beta-amylase, EC 3.2.1.2)
(3) Polyglutamic acid, cellulase (cellulose, EC 3.2.1.4)
(4) Polyglutamic acid, dextranase (EC3.2.1.11)
(5) Polyglutamic acid, lysozyme (lysozyme, EC3.2.1.17)
(6) Polyglutamic acid, dextran 1, 4-alpha-glucosidase (glucan 1,4-a-glucosidase, EC 3.2.1.3)
(7) Polyglutamic acid, endo-1,3 (4) -beta-glucanase (endo-1, 3 (4) -beta-glucanase, EC3.2.1.6)
(8) Polyglutamic acid, inulinase (inulinase, EC3.2.1.7)
(9) Polyglutamic acid, endo-1, 4-beta-xylanase (endo-1, 4-beta-xylanase, EC 3.2.1.8)
(10) Polyglutamic acid, oligo-1, 6-glycosidase (oligo-1, 6-glucopyranosase, EC3.2.1.10)
(11) Polyglutamic acid, chitinase (chitinase, EC3.2.1.14)
(12) Polyglutamic acid, Polygalacturonase (polygalactatron, EC 3.2.1.15)
(13) Polyglutamic acid, exo-alpha-sialidase (exo-alpha-sialidase, EC 3.2.1.18)
(14) Polyglutamic acid, alpha-galactosidase (alpha-galactositase, EC3.2.1.22)
(15) Polyglutamic acid, beta-galactosidase (beta-galactositase, EC 3.2.1.23)
(16) Polyglutamic acid, beta-fructofuranosidase (EC 3.2.1.26)
(17) Polyglutamic acid, alpha-trehalase (alpha, alpha-trehalase, EC3.2.1.28)
(18) Polyglutamic acid, beta-glucuronidase (beta-glucuronidase, EC3.2.1.31)
(19) Polyglutamic acid, hyaluronic acid glucosaminidase (EC3.2.1.35)
(20) Polyglutamic acid, hyaluronic acid glucuronidase (EC3.2.1.36)
(21) Polyglutamic acid, fucosidase (EC3.2.1.44)
(22) Polyglutamic acid, cyclomaltodextrin enzyme (EC3.2.1.54)
(23) Polyglutamic acid, mycodextranase (mycodextranase, EC3.2.1.61)
(24) Polyglutamic acid, fructosan enzyme (levana, EC3.2.1.65)
(25) Polyglutamic acid, quercitrin (quercitrinase, EC3.2.1.66)
(26) Polyglutamic acid, isoamylase (isoamyylase, EC 3.2.1.68)
(27) Polyglutamic acid, beta-agarase (beta-agarase, EC3.2.1.81)
(28) Polyglutamic acid, lactase (lactase, EC3.2.1.108)
(29) Polyglutamic acid, alpha-glucuronidase (alpha-glucuronidase, EC3.2.1.139)
(30) Polyglutamic acid, alpha-agarase (alpha-agarase, EC3.2.1.158)
(31) Polyglutamic acid, alpha-glucosidase (EC 3.2.1.20)
(32) Polyglutamic acid, beta-glucosidase (EC 3.2.1.21)
(33) Polyglutamic acid, alpha-mannosidase (alpha-mannosidase, EC3.2.1.24)
(34) Polyglutamic acid, beta-mannosidase (beta-mannosidase, EC3.2.1.25)
(35) Polyglutamic acid, pullulanase (pullulanase, EC 3.2.1.41)
(36) Polyglutamic acid, isopullulanase (isopululanase, EC3.2.1.57)
(37) Polyglutamic acid, Subtilisin (Subtilisin, EC 3.4.21.62)
(38) Polyglutamic acid, serralysin (EC 3.4.24.40)
(39) Polyglutamic acid, carboxylesterase (EC 3.1.1.1)
(40) Polyglutamic acid, phospholipase A2 (phosphoipase A2, EC 3.1.1.4)
(41) Polyglutamic acid, lysophospholipase (lysophosphoipase, EC 3.1.1.5)
(42) Polyglutamic acid, phospholipase A1 (phosphoipase A1, EC 3.1.1.32)
(43) Polyglutamic acid, cutinase (cutinase, EC 3.1.1.74)
(44) Polyglutamic acid, carboxylesterase (EC 3.1.1.1)
(45) Polyglutamic acid, aryl esterase (EC 3.1.1.2)
(46) Polyglutamic acid, triacylglycerol lipase (triacylglycerol lipase, EC 3.1.1.3)
(47) Polyglutamic acid, acetyl esterase (EC 3.1.1.6)
(48) Polyglutamic acid, gluconolactonase (EC 3.1.1.17)
(49) Polyglutamic acid, lipoprotein lipase (EC3.1.1.34)
(50) Polyglutamic acid, alpha-amino acid esterase (alpha-amino-acid esterase, EC3.1.1.43)
(51) Polyglutamic acid, acetyl xylan esterase (acetylxylan esterase, EC3.1.1.72)
(52) Polyglutamic acid, alkaline phosphatase (alkaline phosphatase, EC3.1.3.1)
(53) Polyglutamic acid, acid phosphatase (EC 3.1.3.2)
(54) Polyglutamic acid, nucleotidase (EC3.1.3.31)
(55) Polyglutamic acid, phospholipase C (phospholipase C, EC3.1.4.3)
(56) Polyglutamic acid, phospholipase D (phospholipase D, EC3.1.4.4)
(57) Polyglutamic acid, alpha-glucosidase (EC 3.2.1.20), beta-glucosidase (EC 3.2.1.21)
(58) Polyglutamic acid, pullulanase (EC 3.2.1.41), isoamylase (isoamyylase, EC 3.2.1.68)
(59) Polyglutamic acid, alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2) (60) polyglutamic acid, cellulase (cellulose, EC 3.2.1.4), dextranase (dextranase, EC3.2.1.11)
(61) Polyglutamic acid, dextranase (EC3.2.1.11), phospholipase A1 (phosphoipase A1, EC 3.1.1.32), phospholipase A2 (phosphoipase A2, EC 3.1.1.4)
(62) Polyglutamic acid, Subtilisin (EC 3.4.21.62), alpha-amylase (α -amylase, EC 3.2.1.1), cellulase (cellulose, EC 3.2.1.4), glucanase (dextranase, EC3.2.1.11), cutinase (cutinase, EC 3.1.1.74).
Through a large number of experiments, the inventor selects several hydrolases to be combined with the polyglutamic acid alone, and proves that the polyglutamic acid has the effect of reducing the dosage of a single hydrolase but remarkably enhancing the effect. The polyglutamic acid is combined by two or more than two hydrolytic enzymes, so that the effect of reducing the dosage of the hydrolytic enzymes is obviously improved. The molecular weight of the polyglutamic acid is between 5 and 200 kilodaltons, and the polyglutamic acid comprises a monovalent salt or a divalent salt thereof.
The above experiments were combined to show that the use of hydrolytic enzymes alone or in combination with polyglutamic acid can reduce the amount of hydrolytic enzymes and significantly enhance the effect of preventing or effectively removing biofilms.
Claims (14)
1. A composition for preventing or removing biofilm from periodontal surfaces of the oral cavity, comprising a hydrolase enzyme and polyglutamic acid.
2. The composition of claim 1, wherein the polyglutamic acid is at least one of gamma- (D, L) -polyglutamic acid, gamma- (D) -polyglutamic acid, or gamma- (L) -polyglutamic acid prepared by fermentation of a microorganism, and has a molecular weight of between 5 and 200 kilodaltons.
3. The composition of claim 2, wherein the polyglutamic acid comprises a monovalent or divalent salt thereof.
4. The composition of claim 2, wherein the polyglutamic acid is prepared by fermentation of Bacillus subtilis.
5. The composition of claim 4 wherein the polyglutamic acid is preferentially produced by fermentation of Bacillus subtilis var natto.
6. The composition of claim 1, wherein the polyglutamic acid is present in an amount between 0.1% and 10%.
7. The composition according to claim 1, wherein the polyglutamic acid is preferably used in an amount of 0.2% to 2%.
8. The composition of claim 1, wherein the hydrolase enzyme is selected from at least one of a glycosidase, a peptidase, an esterase.
9. The composition of claim 8, wherein the glycosidase is selected from the group consisting of alpha-amylase (alpha-amylase, ec 3.2.1.1), beta-amylase (beta-amylase, ec 3.2.1.2), glucan1, 4-alpha-glucosidase (glucan 1,4-a-glucosidase, ec 3.2.1.3), cellulase (cellulose, ec 3.2.1.4), endo-1,3 (4) -beta-glucanase (endo-1, 3 (4) -beta-glucanase, EC3.2.1.6), inulase (inulase, EC3.2.1.7), endo-1, 4-beta-xylanase (endo-1, 4-beta-glucanase, ec 3.2.1.8), oligo-1, 6-glycosidase (oligo-1, 6-glucanase, EC3.2.1.10), glucanase (dextranase, EC3.2.1.11), chitinase (ecronase), Polygalacturonase (EC3.2.1.14.32.3.32), Polygalacturonase (Polygalacturonase) Lysozyme (lysozyme, EC3.2.1.17), exo- α -sialidase (exo- α -sialidase, EC 3.2.1.18), α -glucosidase (α -glucosidase, EC 3.2.1.20), β -glucosidase (β -glucosidase, EC 3.2.1.21), α -galactosidase (α -galactosidase, EC3.2.1.22), β -galactosidase (β -galactosidase, EC 3.2.1.23), α -mannosidase (α -mannosidase, EC3.2.1.24), β -mannosidase (β -mannosidase, EC3.2.1.25), β -fructofuranosidase (β -fructofuranosidase, EC 3.2.1.26), α -trehalase (α, α -glucosidase, EC3.2.1.28), β -glucuronidase (β -glucuronidase, EC 2.1.26), hyaluronic acid (fucosidase, hyaluronic acid) (fucosidase, hyaluronic acid (EC3.2.1.36, fucosidase, hyaluronic acid, fucosidase, EC3.2.1.35, hyaluronic acid (hyaluronic acid, fucosidase, hyaluronic acid, EC3.2.1.35, fucosidase, EC3.2.1.44), cyclomaltodextrinase (EC3.2.1.54), isopullulanase (EC3.2.1.57), mycodextranase (EC3.2.1.61), fructosylase (levanase, EC3.2.1.65), quercitrin (quercitrinase, EC3.2.1.66), isoamylase (isoamylase, EC 3.2.1.68), beta-agarase (beta-agarase, EC3.2.1.81), lactase (lactase, EC3.2.1.108), alpha-glucuronidase (alpha-glucuronidase, EC3.2.1.139), alpha-agarase (alpha-agarase, EC3.2.1.158).
10. The composition according to claim 9, wherein the glycosidase is preferably selected from the group consisting of alpha-amylase (alpha-amylase, EC 3.2.1.1), beta-amylase (beta-amylase, EC 3.2.1.2), cellulase (cellulose, EC 3.2.1.4), glucanase (dextranase, EC3.2.1.11), chitinase (chitinase, EC3.2.1.14), lysozyme (lysozyme, EC3.2.1.17), exo-alpha-sialidase (exo-alpha-sialidase, EC 3.2.1.18), alpha-glucosidase (alpha-glucosidase, EC 3.2.1.20), beta-glucosidase (beta-glucosidase, EC 3.2.1.21), alpha-galactosidase (alpha-galactosidase, EC3.2.1.22), beta-galactosidase (beta-galactosidase, EC 3.2.1.23), alpha-mannosidase (alpha-galactosidase, EC3.2.1.24), pullulanase (EC3.82, EC3.2.1.25), EC3.2.1.57), fructosidase (EC3.2.1.65), isoamylase (isoamyase, EC 3.2.1.68), lactase (lactase, EC3.2.1.108), alpha-glucuronidase (alpha-glucuronidase, EC3.2.1.139), alpha-agarase (alpha-agarase, EC3.2.1.158).
11. The composition according to claim 8, wherein the peptidase is selected from Subtilisin (Subtilisin, EC 3.4.21.62), serrapeptase (serralysin, EC 3.4.24.40).
12. The composition according to claim 8, wherein the esterase is selected from the group consisting of carboxylesterase (EC3.1.1.1), aroyl (aromatase) esterase (EC3.1.1.2), triacylglycerol lipase (triacylglycerol lipase, EC3.1.1.3), phospholipase A2 (phospholipase A2, EC3.1.1.4), lysophospholipase (lysophospholipase, EC3.1.1.5), acetylesterase (acetylesterase, EC3.1.1.6), gluconolactonase (gluconolactonase, EC3.1.1.17), phospholipase A1 (phospholipase A7, EC3.1.1.32), lipoprotein lipase (lipocalin, EC3.1.1.34), alpha-amino acid esterase (alpha-aminoesterase, EC3.1.1.43), xylanase (phosphoesterase, EC3.3568), phosphoesterase (phosphoesterase, phosphoesterases, 3623.26), phosphoesterases (phosphoesterases, EC3.3.3.1.26, 3623), phosphoesterases (phosphoesterases, EC3.3.1.26), phosphoesterases (phosphoesterases, EC2, phosphoesterases (C).
13. A composition according to claim 12, wherein the esterase is preferably selected from the group consisting of carboxylesterase (ec 3.1.1.1), phospholipase a2 (phosphoipase a2, ec 3.1.1.4), lysophospholipase (lysophosphoipase, ec 3.1.1.5), phospholipase a1 (phosphoipase a1, ec 3.1.1.32), lipoprotein lipase (lipoprotein lipase, EC3.1.1.34), alpha-amino acid esterase (alpha-amino-acid esterase, EC3.1.1.43), cutinase (cutinase, ec 3.1.1.74), alkaline phosphatase (alkaline phosphatase, EC3.1.3.1), acid esterase (acid phosphatase, ec 3.1.3.2), nucleotidase (nucleotidase, EC3.1.3.31), phospholipase C (phosphoipase C, EC3.1.4.3), phospholipase D (24D, EC3.1.4.4D).
14. The composition of claim 8, wherein the hydrolytic enzyme comprises an alpha-amylase, a subtilisin, a lipase, an endoglucanase, a mannanase, wherein the alpha-amylase is extracted from Bacillus licheniformis; extracting subtilisin from Bacillus subtilis; extracting endoglucanase from Bacillus; the lipase is extracted from Thermomyces lanuginosus; the mannanase is beta-mannanase.
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