Enzyme composition as well as preparation method and application thereof in oral cavity cleaning

Abstract

The invention provides an enzyme composition as well as a preparation method and application thereof in oral cavity cleaning, and belongs to the technical field of oral cavity cleaning. Comprising the following steps: S1, preparing modified lysozyme; s2, preparing porous silica; s3, preparing the modified porous silica; s4, preparing a biological enzyme; and S5, preparing the enzyme composition. The enzyme composition prepared by the invention can be used for efficiently removing tooth stains, whitening and resisting bacteria, is safe and reliable, has good stability, has an efficient decayed tooth resisting effect, is low in price, can also play a better dry mouth resisting effect, has the effects of improving oral cavity lubrication and relieving oral inflammation, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of oral cleaning, and in particular to an enzyme composition and a preparation method thereof and application thereof in oral cleaning. Background Art

[0002] With the improvement of living standards, people pay more and more attention to oral hygiene and health. Among them, having neat and white teeth has become an important manifestation of health and beauty. The black and yellow teeth of most people are exogenous stains. There are many bacteria adhering to the salivary protein film on the surface of the teeth. They secrete a lot of sticky substances. Tea stains, cigarette stains, coffee stains in daily diet and certain minerals in drinking water are adsorbed on these sticky substances and cannot be effectively cleaned, gradually turning the teeth yellow or even black and precipitating into dental calculus through calcification.

[0003] Currently, there are two main ways to remove tooth stains and whiten teeth: one is the grinding method, which mainly removes tooth stains physically by adding abrasives with high friction values ​​(such as calcium carbonate, calcium hydrogen phosphate, silica, etc.), but long-term use of high-friction abrasives poses a risk of wearing away tooth enamel; the other is to use phosphate substances (such as tetrasodium pyrophosphate, tetrapotassium pyrophosphate, etc.) to complex the calcium ions in tooth stains to achieve the effect of removing tooth stains, but this method has the disadvantage of less than ideal whitening effect.

[0004] At present, enzyme toothpaste is an important development direction of toothpaste products. Since the effect of enzymes far exceeds that of abrasives, it can remove a large amount of gum on the tooth surface.

[0005] At the same time, the problem of insufficient saliva secretion and dry mouth is common in the middle-aged and elderly people who have entered menopause, patients with diabetes and mental anxiety, cancer patients undergoing head and neck radiotherapy, oral patients with salivary gland abnormalities, swollen gums, and oral mucosal inflammation. When the salivary glands secrete insufficiently and the mouth is dry, oral acidification and bacterial reproduction cannot be suppressed, and dental plaque and root caries will gradually appear below the gum line, and the gums will swell and turn red. If it is still not controlled, the enamel will gradually become thinner until perforated. At the same time, the further aggravation of bacteria and acidification corruption will eventually cause the teeth to lose all support and fall off, affecting chewing and the health of the whole body.

[0006] If a toothpaste can be developed that not only has the effect of removing tooth stains and whitening teeth, but also can assist in treating problems such as dry mouth, it will be beneficial to oral health and have broad application prospects. Summary of the invention

[0007] The purpose of the present invention is to provide an enzyme composition and a preparation method thereof and application thereof in oral cleaning, which can effectively remove tooth stains, whiten and resist bacteria, is safe and reliable, has good stability, has a high efficiency in resisting tooth caries, is low in price, can also play a good anti-dry mouth effect, has the effects of increasing oral lubrication and relieving oral inflammation, and has broad application prospects.

[0008] The technical solution of the present invention is achieved in this way:

[0009] The present invention provides a method for preparing an enzyme composition, comprising the following steps:

[0010] S1. Preparation of modified lysozyme: adding lysozyme to water, adding EDC and NHS, activating the reaction, adding N,N-dimethyldodecylamine, stirring the reaction, adding propane sultone, heating and stirring the reaction, dialyzing the product, washing, and freeze-drying to obtain the modified lysozyme;

[0011] S2. Preparation of porous silica: dissolving tetraethyl orthosilicate in ethanol, adding a porogen, water and ammonia, stirring the reaction, centrifuging, washing, and drying to obtain porous silica;

[0012] S3. Preparation of modified porous silica: adding porous silica to water, adding tannic acid and a catalyst, heating and stirring the reaction, centrifuging, washing, and drying to obtain modified porous silica;

[0013] S4. Preparation of biological enzyme: The modified lysozyme, glucanase, glucose oxidase and amylase were mixed and stirred to obtain a biological enzyme;

[0014] S5. Preparation of enzyme composition: adding modified porous silica into water, adding EDC and NHS, activating reaction, adding biological enzyme, stirring reaction, centrifuging, washing, drying, and preparing enzyme composition.

[0015] As a further improvement of the present invention, the mass ratio of lysozyme, EDC, NHS, N,N-dimethyldodecylamine and propane sultone in step S1 is 10-12:2-3:2-4:1-2:1.5-2.5, the activation reaction time is 20-30 min, the stirring reaction time is 10-12 h, the heating stirring reaction temperature is 40-50 ° C, the time is 20-24 h, and the dialysis bag pore size used for product dialysis is 5K-8KDa.

[0016] As a further improvement of the present invention, the mass ratio of tetraethyl orthosilicate, ethanol, porogen, water and ammonia water in step S2 is 15-20:90-120:1-2:7-12:15-20, the porogen is hexadecyltrimethylammonium chloride, and the stirring reaction time is 7-10 hours.

[0017] As a further improvement of the present invention, the mass ratio of the porous silica, tannic acid and catalyst in step S3 is 10-12:4-7:0.5-1, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 3-5h.

[0018] As a further improvement of the present invention, the mass ratio of the modified lysozyme, glucanase, glucose oxidase and amylase in step S4 is 10:3-5:2-4:3-4, and the amylase is a mixture of α-amylase and β-amylase with a mass ratio of 3-5:7.

[0019] As a further improvement of the present invention, in step S5, the mass ratio of the modified porous silica, EDC, NHS and biological enzyme is 10:1-2:0.8-1.2:3-5, the activation reaction time is 20-30 minutes, and the stirring reaction time is 12-15 hours.

[0020] The present invention further protects an enzyme composition obtained by the above preparation method.

[0021] The present invention further protects the use of the above enzyme composition in oral cleaning.

[0022] The present invention further protects a toothpaste containing the above enzyme composition.

[0023] As a further improvement of the present invention, the enzyme composition is prepared from the following raw materials in parts by weight: 20-60 parts of sorbitol, 0.2-0.4 parts of tetrasodium pyrophosphate, 0.1-0.3 parts of sodium saccharin, 4-5 parts of cellulose gum, 0.3-0.5 parts of xanthan gum, 1-1.2 parts of flavor, 1-2 parts of vitamins, 10-15 parts of enzyme composition, 20-40 parts of deionized water, and 5-10 parts of glycerol.

[0024] The present invention has the following beneficial effects:

[0025] The enzyme composition of the present invention contains abundant amylase, which is similar to the structure of saliva. After entering the oral cavity, it can jointly produce an efficient saliva-like biotransformation system through the action of enzyme and substrate, and play the role of helping digestion, inhibiting oral acidification, and killing bacteria just like healthy saliva. At the same time, it also has the effect of increasing oral lubrication and relieving oral inflammation, ensuring the dietary quality of normal people or patients with dry mouth symptoms.

[0026] The enzyme composition of the present invention also adds modified lysozyme, wherein lysozyme can inhibit the growth of microorganisms. Compared with antibiotics or other antibacterial substances, long-term use of lysozyme will not cause drug resistance, and it can selectively dissolve the cell wall of the target microorganism to inactivate it. Mammals have no cell wall, so lysozyme does not damage the oral tissue, but plays a protective role. Lysozyme achieves antibacterial effect by destroying the β-1,4-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in the bacterial peptidoglycan layer. Since the peptidoglycan content in Gram-positive bacteria reaches 80%, while there is only a small amount of peptidoglycan in Gram-negative bacteria, this affects its antibacterial effect on negative bacteria to a certain extent.

[0027] The present invention modifies lysozyme by N,N-dimethyldodecylamine, and transforms the side chain groups of natural lysozyme to obtain an amphiphilic enzyme molecule, which can improve the antibacterial effect of the enzyme molecule on Gram-negative bacteria by destroying the lipopolysaccharide layer in the cell wall structure of Gram-negative bacteria. At the same time, the antibacterial activity of the lysozyme is further improved by further reacting with propane sultone to form a quaternary ammonium salt.

[0028] In addition, dextranase is also added to the enzyme composition. Dextran is a viscous polysaccharide secreted by Streptococcus mutans. It is a linear polymer connected by β-1,3-glucosidic bonds and β-1,4-glucosidic bonds. Under the action of dextranase, these polysaccharides are gradually degraded into soluble cellobiose and cellotriose, as well as some oligosaccharides and other molecular sugars. Dextran is the basis of dental plaque formation and is gradually dissolved under the action of enzymes.

[0029] Glucose oxidase is also added to the enzyme composition, which acts on the glucose substrate to form delta-gluconolactone and then generates gluconic acid, so that some pathogens parasitic in the oral cavity cannot utilize glucose and thus cannot reproduce, thereby effectively inhibiting the proliferation and reaction of the pathogens.

[0030] The present invention prepares a porous silica in the presence of a porogen through a sol-gel reaction. The porous silica has a small particle size and is convenient for removing tooth stains through friction. At the same time, the surface of the porous silica is modified by tannic acid. On the one hand, it can fix multiple enzymes through the formation of hydrogen bonds or the formation of amides after activation, thereby increasing the loading amount of the enzymes and improving the stability of the enzymes. During the friction process, the enzymes can fall off and enter the oral cavity, thereby exerting multiple effects. On the other hand, after being activated, the tannic acid can react with lysozyme, thereby further modifying the lysozyme. The lysozyme after modification has a small loss of enzyme activity, further improving its inhibitory effect on Gram-negative bacteria, and having a synergistic effect with the previous modification.

[0031] The enzyme composition prepared by the invention can effectively remove tooth stains, whiten and resist bacteria, is safe and reliable, has good stability, has a high efficiency in resisting tooth caries, is low in price, can also play a good anti-dry mouth effect, has the effect of increasing oral lubrication and relieving oral inflammation, and has broad application prospects. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Lysozyme is microbial source lysozyme (activity 1000U / mg): Dalian Green Snow Egg Products Development Co., Ltd.; glucanase (activity 40,000U / g): Nanning Dongheng Huadao Biotechnology Co., Ltd.; glucose oxidase (activity 300U / g): Nanning Dongheng Huadao Biotechnology Co., Ltd.; α-amylase (activity 100,000U / g): Nanjing Chengna Chemical Co., Ltd.; β-amylase (activity 50U / mg): Shanghai Yuanye Biotechnology Co., Ltd.

[0034] Example 1

[0035] This embodiment provides a method for preparing an enzyme composition, comprising the following steps:

[0036] S1. Preparation of modified lysozyme: 10 g of lysozyme was added to 500 mL of water, 2 g of EDC and 2 g of NHS were added, activation reaction was carried out for 20 min, 1 g of N, N-dimethyldodecylamine was added, stirring reaction was carried out for 10 h, 1.5 g of propane sultone and 50 mL of acetone were added, heated to 40° C., stirring reaction was carried out for 20 h, the product was dialyzed for 12 h using a dialysis bag with a pore size of 5 KDa, washed, and freeze-dried to obtain modified lysozyme;

[0037] S2. Preparation of porous silica: 15 g of tetraethyl orthosilicate was dissolved in 90 g of ethanol, 1 g of hexadecyltrimethylammonium chloride, 7 g of water and 15 g of aqueous ammonia were added, the reaction was stirred for 7 h, centrifuged, washed and dried to obtain porous silica;

[0038] S3. Preparation of modified porous silica: 10 g of porous silica was added to 200 mL of water, 4 g of tannic acid and 0.5 g of catalyst were added, heated to 40 ° C, stirred for 3 h, centrifuged, washed, and dried to obtain modified porous silica;

[0039] The catalyst is a Tris-HCl solution with a pH of 8.5;

[0040] S4. Preparation of biological enzyme: 10 g of modified lysozyme, 3 g of glucanase, 2 g of glucose oxidase and 3 g of amylase were stirred and mixed for 20 min to obtain biological enzyme;

[0041] The amylase is a mixture of α-amylase and β-amylase, with a mass ratio of 3:7;

[0042] S5. Preparation of enzyme composition: 10 g of modified porous silica was added to 200 mL of water, 1 g of EDC and 0.8 g of NHS were added, the activation reaction was carried out for 20 min, 3 g of biological enzyme was added, the reaction was stirred for 12 h, centrifuged, washed, and dried to obtain an enzyme composition.

[0043] Example 2

[0044] This embodiment provides a method for preparing an enzyme composition, comprising the following steps:

[0045] S1. Preparation of modified lysozyme: 12 g of lysozyme was added to 500 mL of water, 3 g of EDC and 4 g of NHS were added, and the reaction was activated for 30 min. 2 g of N, N-dimethyldodecylamine was added and the reaction was stirred for 12 h. 2.5 g of propane sultone and 50 mL of acetone were added and the reaction was stirred for 24 h. The product was dialyzed for 12 h using a dialysis bag with a pore size of 8 KDa, washed, and freeze-dried to obtain modified lysozyme.

[0046] S2. Preparation of porous silica: 20 g of tetraethyl orthosilicate was dissolved in 120 g of ethanol, 2 g of hexadecyltrimethylammonium chloride, 12 g of water and 20 g of aqueous ammonia were added, the reaction was stirred for 10 h, centrifuged, washed and dried to obtain porous silica;

[0047] S3. Preparation of modified porous silica: 12 g of porous silica was added to 200 mL of water, 7 g of tannic acid and 1 g of catalyst were added, heated to 50 ° C, stirred for 5 h, centrifuged, washed, and dried to obtain modified porous silica;

[0048] The catalyst is a Tris-HCl solution with a pH of 9.5;

[0049] S4. Preparation of biological enzyme: 10 g of modified lysozyme, 5 g of glucanase, 4 g of glucose oxidase and 4 g of amylase were stirred and mixed for 20 min to obtain biological enzyme;

[0050] The amylase is a mixture of α-amylase and β-amylase, with a mass ratio of 5:7;

[0051] S5. Preparation of enzyme composition: 10 g of modified porous silica was added to 200 mL of water, 2 g of EDC and 1.2 g of NHS were added, the activation reaction was carried out for 30 min, 5 g of biological enzyme was added, the reaction was stirred for 15 h, centrifuged, washed, and dried to obtain an enzyme composition.

[0052] Example 3

[0053] This embodiment provides a method for preparing an enzyme composition, comprising the following steps:

[0054] S1. Preparation of modified lysozyme: 11 g of lysozyme was added to 500 mL of water, 2.5 g of EDC and 3 g of NHS were added, the reaction was activated for 25 min, 1.5 g of N, N-dimethyldodecylamine was added, the reaction was stirred for 11 h, 2 g of propane sultone and 50 mL of acetone were added, the reaction was stirred for 22 h, the product was dialyzed for 12 h using a dialysis bag with a pore size of 6 KDa, washed, and freeze-dried to obtain modified lysozyme;

[0055] S2. Preparation of porous silica: 17 g of tetraethyl orthosilicate was dissolved in 110 g of ethanol, 1.5 g of hexadecyltrimethylammonium chloride, 10 g of water and 17 g of aqueous ammonia were added, the reaction was stirred for 8 h, centrifuged, washed and dried to obtain porous silica;

[0056] S3. Preparation of modified porous silica: 11 g of porous silica was added to 200 mL of water, 5 g of tannic acid and 0.7 g of catalyst were added, heated to 45 ° C, stirred for 4 h, centrifuged, washed, and dried to obtain modified porous silica;

[0057] The catalyst is a Tris-HCl solution with a pH of 9;

[0058] S4. Preparation of biological enzyme: 10 g of modified lysozyme, 4 g of glucanase, 3 g of glucose oxidase and 3.5 g of amylase were stirred and mixed for 20 min to obtain biological enzyme;

[0059] The amylase is a mixture of α-amylase and β-amylase, with a mass ratio of 4:7;

[0060] S5. Preparation of enzyme composition: 10 g of modified porous silica was added to 200 mL of water, 1.5 g of EDC and 1 g of NHS were added, the activation reaction was carried out for 25 min, 4 g of biological enzyme was added, the reaction was stirred for 13 h, centrifuged, washed, and dried to obtain an enzyme composition.

[0061] Comparative Example 1

[0062] Compared with Example 3, the difference is that propane sultone is not added to react in step S1.

[0063] Comparative Example 2

[0064] Compared with Example 3, the difference is that step S1 is not performed.

[0065] Comparative Example 3

[0066] Compared with Example 3, the difference is that the amylase in step S4 is a single α-amylase.

[0067] Comparative Example 4

[0068] Compared with Example 3, the difference is that the amylase in step S4 is a single β-amylase.

[0069] Comparative Example 5

[0070] Compared with Example 3, the difference is that no amylase is added in step S4.

[0071] Comparative Example 6

[0072] Compared with Example 3, the difference is that step S3 is not performed.

[0073] Test Example 1 Minimum inhibitory concentration (MIC) determination:

[0074] The enzyme composition samples prepared in Examples 1-3 or Comparative Examples 1-2 and Comparative Example 6 were made into water suspensions of different concentrations for experiments. For each test bacterial solution, a test tube with only liquid culture medium was set as a negative control, and a test tube with the same volume of sample solution and bacterial suspension was set as a test group. After mixing, Escherichia coli (ATCC25922), Bacillus subtilis (ATCC6633), Staphylococcus aureus (ATCC6538), Salmonella (ATCC14028), Listeria monocytogenes (ATCC19115), Bacillus stearothermophilus (ATCC12980) were cultured at 37°C for 24h, Clostridium butyricum (ATCC19398) was cultured at 37°C under anaerobic conditions for 48h, and Aspergillus niger (ATCC10864) was cultured at 28°C for 36h, and their OD values ​​were measured respectively. 600 The results are shown in Table 1.

[0075] Table 1

[0076]

[0077] It can be seen from the above table that the enzyme compositions prepared in Examples 1-3 of the present invention have good antibacterial activity.

[0078] Test Example 2 In vitro anti-inflammatory activity determination:

[0079] Take RAW264.7 cells in the logarithmic growth phase, count them and adjust the cell concentration to 2×10 5cells / mL, connected to a 24-well plate, 1 mL of mixed cell suspension was added to each well, and the plate was placed in an incubator with 5% CO2 and 37°C saturated humidity for 24 hours. The culture medium in the well was removed, and a blank control group, a model group, a positive control group and a sample group were set up for testing. 1 mL of 80 mg / L of the enzyme composition sample water suspension prepared in Example 1-3 or Comparative Examples 1-2 and 6 was added to the sample group, 1 mL of culture medium was added to the blank control group and the model group, and 1 mL of 80 mg / L dexamethasone solution was added to the positive control group. Three duplicate wells were set up in each group and placed in an incubator with 5% CO2 and 37°C saturated humidity for 24 hours. A culture medium containing 1 mg / L lipopolysaccharide was added to the sample group and the model group, and an equal volume of culture medium was added to the blank control group, and the plate was continued to be placed in an incubator with 5% CO2 and 37°C saturated humidity for 24 hours. The cell culture supernatant was collected, and the Griess kit was used to determine the secretion of NO in the cells under different sample treatments. The secretion of inflammatory factor TNF-α in the cell supernatant was determined using a TNF-α kit. The results are shown in Table 2.

[0080] Table 2

[0081]

[0082]

[0083] It can be seen from the above table that the enzyme compositions prepared in Examples 1-3 of the present invention have good anti-inflammatory activity.

[0084] Example 4

[0085] The present embodiment provides a toothpaste, which is prepared from the following raw materials in parts by weight: 20 parts of sorbitol, 0.2 parts of tetrasodium pyrophosphate, 0.1 parts of sodium saccharin, 4 parts of cellulose gum, 0.3 parts of xanthan gum, 1 part of flavor, 1 part of vitamins, 10 parts of the enzyme composition prepared in Example 1, 20 parts of deionized water, and 5 parts of glycerin.

[0086] The vitamins include vitamin C and vitamin E with a mass ratio of 10:3.

[0087] The preparation method is as follows:

[0088] Dissolve saccharin sodium in deionized water and add it to the paste making pot. Add sorbitol and tetrasodium pyrophosphate to the paste making pot and turn on the paste making machine to stir.

[0089] Add glycerin to the pre-dispersion pot, then add cellulose gum and xanthan gum to make glycerin glue, pump it into the paste making pot, stir for 15 minutes to make glue.

[0090] Add the enzyme composition and vitamins into the paste pot, turn on the vacuum pump, stir, and scraper. Add the essence after 10 minutes, and turn off the colloid mill, stir, scraper, and vacuum pump in turn after 5 minutes. After passing the inspection, squeeze out and fill to make toothpaste.

[0091] Example 5

[0092] Compared with Example 4, the difference lies in that the raw material ratio and components are different.

[0093] The present embodiment provides a toothpaste, which is prepared from the following raw materials in parts by weight: 60 parts of sorbitol, 0.4 parts of tetrasodium pyrophosphate, 0.3 parts of sodium saccharin, 5 parts of cellulose gum, 0.5 parts of xanthan gum, 1.2 parts of flavor, 2 parts of vitamins, 15 parts of the enzyme composition prepared in Example 2, 40 parts of deionized water, and 10 parts of glycerin.

[0094] Example 6

[0095] Compared with Example 4, the difference is that the raw material ratio is different.

[0096] The present embodiment provides a toothpaste, which is prepared from the following raw materials in parts by weight: 40 parts of sorbitol, 0.3 parts of tetrasodium pyrophosphate, 0.2 parts of sodium saccharin, 4.5 parts of cellulose gum, 0.4 parts of xanthan gum, 1.1 parts of flavor, 1.5 parts of vitamins, 12 parts of the enzyme composition prepared in Example 3, 30 parts of deionized water, and 7 parts of glycerol.

[0097] Comparative Example 7

[0098] Compared with Example 6, the difference is that the enzyme composition is prepared from Comparative Example 1.

[0099] Comparative Example 8

[0100] Compared with Example 6, the difference is that the enzyme composition is prepared from Comparative Example 2.

[0101] Comparative Example 9

[0102] Compared with Example 6, the difference is that the enzyme composition is prepared from Comparative Example 3.

[0103] Comparative Example 10

[0104] Compared with Example 6, the difference is that the enzyme composition is prepared from Comparative Example 4.

[0105] Comparative Example 11

[0106] Compared with Example 6, the difference is that the enzyme composition is prepared from Comparative Example 5.

[0107] Comparative Example 12

[0108] Compared with Example 6, the difference is that the enzyme composition is prepared from Comparative Example 6 respectively.

[0109] Test Example 3 Anti-dry mouth test

[0110] The toothpastes prepared in Examples 4-6 and Comparative Examples 9-11 were tested.

[0111] The nasopharyngeal carcinoma patients (aged between 21 and 68 years old) who were given radiotherapy in the nursing center of the tumor hospital were selected, half of them were male and half were female, and they were randomly divided into 7 groups, namely the control group (using ordinary toothpaste), the embodiment 4-6 group, and the comparative example 9-11 group, with 10 people in each group. They were used three times a day in a normal way of brushing teeth, and the saliva secretion of the patients was detected. On the day before the start of radiotherapy and when the nasopharyngeal radiotherapy dose reached DT3000cGy, a dry yarn ball was placed in the cheek of the patient and chewed for 30 minutes before being taken out. The weight of the dry and wet yarn balls was measured with a balance, and the difference between the two was the saliva secretion. The results are shown in Table 3.

[0112] Table 3

[0113]

[0114] It can be seen from the above table that the toothpastes prepared in Examples 4-6 of the present invention have a good effect of promoting saliva secretion.

[0115] Test Example 4: Plaque Removal Experiment

[0116] Mutans Streptococcus (ATCC 700610) was cultured on soybean agar plate at 37°C under anaerobic conditions (5v / v% carbon dioxide, 10v / v% hydrogen, and the balance was nitrogen) for 3 days. The cultured strains were picked and inoculated into broth medium, cultured under the same conditions for 17 hours, 20μL of culture solution was taken and added to a test tube containing 2mL broth medium containing 1% sucrose, and cultured at 37°C under anaerobic conditions (same conditions as above) for 7 hours, and a biofilm was formed on the inner wall of the test tube. The culture medium was removed, and the biofilm formed on the inner wall was rinsed with PBS buffer as a dental plaque model.

[0117] The toothpastes prepared in Examples 4-6 and Comparative Examples 7-12 (experimental group), PBS buffer (control group), and commercially available similar toothpaste (positive control group) were prepared into a 0.1 g / mL suspension, heated at 37°C for 10 min, and 3.00 mL of the test solution was added to the dental plaque model for 3 min, the test solution was removed, PBS buffer was added for rinsing, and 3 mL of 0.5 mol / L KOH solution was added to disperse the residual dental plaque. The absorbance (550 nm) of the liquid in the test tube was detected to quantify the remaining dental plaque.

[0118] Plaque removal rate (%) = (BA) / B×100 (%)

[0119] Among them, the absorbance of the supernatant of the experimental group is A; the absorbance of the supernatant of the control group is B;

[0120] The results are shown in Table 4.

[0121] Table 4

[0122] Group Plaque removal rate (%) Example 4 86.8 Example 5 87.2 Example 6 87.5 Comparative Example 7 79.1 Example 8 72.5 Comparative Example 9 82.2 Comparative Example 10 81.4 Comparative Example 11 78.6 Comparative Example 12 77.9 Commercially available 65.2

[0123] It can be seen from the above table that the toothpastes prepared in Examples 4-6 of the present invention have better plaque removal effects.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an enzyme composition, characterized in that: The following steps are involved: S1. Preparation of modified lysozyme: adding lysozyme to water, adding EDC and NHS, activating the reaction, adding N,N-dimethyldodecylamine, stirring the reaction, adding propane sultone, heating and stirring the reaction, dialyzing the product, washing, and freeze-drying to obtain the modified lysozyme; S2. Preparation of porous silica: dissolving tetraethyl orthosilicate in ethanol, adding a porogen, water and ammonia, stirring the reaction, centrifuging, washing, and drying to obtain porous silica; S3. Preparation of modified porous silica: adding porous silica to water, adding tannic acid and a catalyst, heating and stirring the reaction, centrifuging, washing, and drying to obtain modified porous silica; S4. Preparation of biological enzyme: The modified lysozyme, glucanase, glucose oxidase and amylase were stirred and mixed to obtain biological enzyme; S5. Preparation of enzyme composition: adding modified porous silica into water, adding EDC and NHS, activating reaction, adding biological enzyme, stirring reaction, centrifuging, washing, drying, and preparing enzyme composition.

2. The preparation method according to claim 1, characterized in that: The mass ratio of lysozyme, EDC, NHS, N,N-dimethyldodecylamine and propane sultone in step S1 is 10-12:2-3:2-4:1-2:1.5-2.5, the activation reaction time is 20-30min, the stirring reaction time is 10-12h, the heating stirring reaction temperature is 40-50°C, the time is 20-24h, and the dialysis bag pore size used for product dialysis is 5K-8KDa.

3. The preparation method according to claim 1, characterized in that: In step S2, the mass ratio of tetraethyl orthosilicate, ethanol, porogen, water and ammonia water is 15-20:90-120:1-2:7-12:15-20, the porogen is hexadecyltrimethylammonium chloride, and the stirring reaction time is 7-10 hours.

4. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of the porous silica, tannic acid and catalyst is 10-12:4-7:0.5-1, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50° C., and the time is 3-5 hours.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the modified lysozyme, glucanase, glucose oxidase and amylase in step S4 is 10:3-5:2-4:3-4, and the amylase is a mixture of α-amylase and β-amylase with a mass ratio of 3-5:

7.

6. The preparation method according to claim 1, characterized in that: In step S5, the mass ratio of the modified porous silica, EDC, NHS and biological enzyme is 10:1-2:0.8-1.2:3-5, the activation reaction time is 20-30 minutes, and the stirring reaction time is 12-15 hours.

7. An enzyme composition obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the enzyme composition according to claim 7 in oral cleaning.

9. A toothpaste, characterized in that: A composition comprising the enzyme according to claim 7.

10. The toothpaste according to claim 9, characterized in that The invention is prepared from the following raw materials in parts by weight: 20-60 parts of sorbitol, 0.2-0.4 parts of tetrasodium pyrophosphate, 0.1-0.3 parts of sodium saccharin, 4-5 parts of cellulose gum, 0.3-0.5 parts of xanthan gum, 1-1.2 parts of flavor, 1-2 parts of vitamins, 10-15 parts of enzyme composition, 20-40 parts of deionized water and 5-10 parts of glycerol.

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