A probiotic composition, a preparation method thereof, and application in mouthwash

Abstract

The present invention proposes a probiotic composition and a preparation method thereof and an application in mouthwash, which belongs to the field of oral hygiene technology. Lysozyme is modified by tannic acid and then heated with glucose and cinnamaldehyde to obtain modified lysozyme; Centella asiatica, Forsythia suspensa leaves and green tea are subjected to enzymatic fermentation by cellulase and fermentation bacteria, and probiotics are collected respectively, and the products are dried to obtain fermentation products; the modified lysozyme and probiotics are mixed and heated for enzymolysis to obtain a complex, and the complex is subjected to ultrasonic assisted heating treatment and dried to obtain a probiotic composition. The probiotic composition prepared by the present invention has a good inhibitory, destructive and competitive colonization effect on cariogenic bacteria, thereby greatly reducing the damage of cariogenic bacteria to teeth. At the same time, it also has anti-inflammatory, antioxidant, breath freshening and other effects. The mouthwash prepared by adding it has low cost, good effect, easy to realize industrial application, and has broad application prospects.

Description

Technical Field

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

[0002] The oral cavity is the beginning of the digestive tract. The human oral cavity is a complete and complex ecosystem. The appropriate temperature, humidity, pH value and nutrient supply provide good environmental conditions for the growth, settlement and reproduction of various oral microorganisms. There are many kinds of microorganisms in the oral cavity, up to more than 700 species, including various bacteria, fungi, viruses and mycoplasmas. They parasitize on the oral mucosa, teeth, tongue and saliva. Under normal circumstances, the oral flora maintains a relative balance between the colonies in an intricate symbiotic way, and a dynamic balance is also maintained between the microorganisms and the host body. This balance maintains the health of the human oral cavity. Once the balance is broken by external factors or internal factors of the body, the various flora in the oral cavity will be out of balance, which will affect oral health and even induce various oral diseases, such as caries, periodontitis, gingivitis, bad breath and oral ulcers.

[0003] In the past, the research on probiotics was mainly focused on the gastrointestinal field. In recent years, probiotics have begun to be used in the prevention and treatment of various oral diseases, becoming an emerging field of research on oral disease prevention and treatment technology. At present, there are nearly 20 known probiotics, which can be roughly divided into five categories: Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus and other bacteria. Among them, Lactobacillus and Bifidobacterium are the first oral probiotics discovered and studied. Streptococcus salivarius is also a good oral probiotic, which plays an important role in the prevention and treatment of oral diseases such as pharyngitis, halitosis, and oral candidiasis. Through people's unremitting efforts, the use of probiotics in the prevention and treatment of periodontitis, gingivitis, halitosis, etc. has achieved certain results. Summary of the invention

[0004] The purpose of the present invention is to provide a probiotic composition and a preparation method thereof and an application in a mouthwash, which has a good inhibitory, destructive and competitive colonization effect on cariogenic bacteria, thereby greatly reducing the damage of cariogenic bacteria to teeth. At the same time, it also has anti-inflammatory, antioxidant, breath freshening and other effects. The mouthwash prepared by adding the probiotic composition has low cost, good effect, is easy to realize industrial application, and has broad application prospects.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The invention provides a preparation method of a probiotic composition. The method comprises the following steps: modifying lysozyme with tannic acid and heating the modified lysozyme with glucose and cinnamaldehyde to obtain a modified lysozyme; subjecting Centella asiatica, forsythia leaf and green tea to enzymatic fermentation with cellulase and fermentation bacteria, collecting probiotics respectively, and drying the products to obtain a fermentation product; mixing the modified lysozyme and the probiotics and heating and enzymolyzing them to obtain a composite; subjecting the composite to ultrasonic-assisted heating treatment and drying to obtain a probiotic composition.

[0007] As a further improvement of the present invention, the following steps are included:

[0008] S1. Preparation of tannic acid-modified lysozyme: dissolving tannic acid in water, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activating the reaction, adding lysozyme, stirring at room temperature, dialyzing, and freeze-drying to obtain tannic acid-modified lysozyme;

[0009] S2. Preparation of modified lysozyme: Glucose and cinnamaldehyde were added to water, tannic acid was added to modify lysozyme, heated and stirred for reaction, dialyzed, and freeze-dried to obtain modified lysozyme;

[0010] S3. Fermentation: Mix and crush Centella asiatica, Forsythia suspensa leaves, and green tea, add to water, add cellulase, sterilize, inoculate thermophilic Streptococcus and Lactobacillus salivarius seed solution, culture by enzymatic fermentation, filter, wash and collect the solid solution, freeze-dry, and obtain probiotics; dry the filtrate to obtain a fermentation product;

[0011] S4. Enzymatic hydrolysis: mixing the modified lysozyme and the probiotics, adding them to water, heating and enzymatic hydrolysis to obtain a complex;

[0012] S5. Ultrasonic heat treatment: The complex is subjected to ultrasonic-assisted heating treatment and dried to obtain a probiotic composition.

[0013] As a further improvement of the present invention, the mass ratio of tannic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and lysozyme in step S1 is 10-12:15-18:12-14:25-30, the temperature of the activation reaction is 0-4°C, the time is 30-50min, and the time of the room temperature stirring reaction is 12-15h.

[0014] As a further improvement of the present invention, the mass ratio of glucose, cinnamaldehyde and tannic acid modified lysozyme in step S2 is 10-12:5-7:25-30, the temperature of the heating and stirring reaction is 65-75°C, and the time is 20-24h.

[0015] As a further improvement of the present invention, the mass ratio of Centella asiatica, Forsythia suspensa leaves, green tea and cellulase in step S3 is 3-5:4-7:2-4:0.1-0.2, the inoculation amounts of the thermophilic Streptococcus and Lactobacillus salivarius seed solutions are 1-3 v / v% and 0.5-1.5 v / v%, respectively, and the bacterial content of the seed solution is 10 8 -10 9 cfu / mL, and the conditions of the enzymatic fermentation culture are 36-38°C, 100-150r / min, and the enzymatic fermentation culture is carried out under anoxic conditions for 24-48h.

[0016] As a further improvement of the present invention, in step S4, the mass ratio of the modified lysozyme to the probiotics is 2-4:7-10, the temperature of the heating enzymolysis is 36-38° C., and the time is 20-40 min.

[0017] As a further improvement of the present invention, the ultrasonic power of the ultrasonic-assisted heating treatment in step S5 is 1200-1500W, the heating temperature is 50-60°C, and the treatment time is 0.5-1h.

[0018] The present invention further protects a probiotic composition prepared by the above preparation method.

[0019] The present invention further protects a use of the probiotic composition in preparing an oral cleaning product, wherein the oral cleaning product is at least one of a mouthwash, a toothpaste, and a tooth cleaning powder.

[0020] The present invention further protects a mouthwash containing 0.5-10 wt % of the probiotic composition.

[0021] The present invention has the following beneficial effects:

[0022] Generally, the oral cavity is a weakly alkaline environment (pH=7.4). Dental caries is the result of the combined effects of bacteria, host, food, and time. Generally, sweet foods are prone to cause dental caries, mainly because sugar will become an acidic substance after fermentation, and the acidic environment has a good growth-promoting effect on cariogenic bacteria. Probiotics, such as thermophilic streptococci and salivarius lactobacillus, have good adhesion to hydroxyapatite in the oral saliva environment and the ability to antagonize the proliferation of other pathogens. However, these probiotics are also prone to fermenting the food residues remaining in the oral cavity to produce acidic short-chain fatty acids, which has an adverse effect on the prevention of dental caries. The present invention uses ultrasonic assisted heat treatment to inactivate probiotics, lose metabolic acid production ability but still retain adhesion ability, which can reduce the cariogenicity of mutans streptococci in the dentin cavity, thereby playing a role in controlling dental caries.

[0023] The present invention also adds lysozyme, although it can destroy the probiotic bacterial wall, but still retains the main structure of the probiotic pili, retains the pili protein, mucus binding protein, polysaccharide and other structures that promote adhesion on teeth, thereby can not affect the adhesion of probiotics to teeth after the destruction, has a certain competitive effect on the colonization of cariogenic bacteria, reduces the colonization of cariogenic bacteria. Simultaneously, it can also promote the dissolution of active substances such as antimicrobial peptides and bacteriocins, can suppress the growth of pathogenic microorganisms to a great extent. Bacteriocins have a wide antimicrobial spectrum and can suppress and kill multiple pathogenic bacteria. In addition, thermophilic streptococci can also secrete a kind of thermophilic protein, can suppress the formation of mutans streptococcus biofilm, and indirectly suppress the colonization of mutans streptococci in the oral cavity.

[0024] At the same time, lysozyme can also destroy the lipopolysaccharide and phospholipid in the membrane structure of pathogenic bacteria, causing the cell membrane to rupture and kill, which has a great destructive effect on caries-causing bacteria, reduces bacterial glycolysis and acid production, and thus reduces the risk of caries. By regulating the steady-state balance of the oral environment and reducing inflammatory reactions, it helps maintain oral health. At the same time, lysozyme, as one of the nonspecific immune factors, can enhance the body's immunity and improve the defense against oral pathogens. Lysozyme can also effectively treat periodontitis, reduce periodontal swelling and gingival bleeding, and can significantly improve symptoms and shorten the course of the disease when used in recurrent oral ulcers.

[0025] However, there are many problems in directly adding lysozyme, such as lysozyme may cause allergic reactions, lack the characteristics of high temperature resistance, and only have a killing effect on Gram-positive bacteria, thereby limiting its antibacterial effect. The present invention modifies lysozyme with tannic acid, and further connects it with glucose and cinnamaldehyde through the Maillard reaction, thereby improving the high temperature resistance of the modified lysozyme, and the denaturation temperature is increased to more than 90 degrees. At the same time, the killing and destruction ability of the modified lysozyme on Gram-negative bacteria is also improved, greatly improving its antibacterial and antibacterial effect.

[0026] Ultrasonic heat treatment can improve the binding ability of modified lysozyme to microbial cell membranes, especially to the intracellular membrane, causing cell membrane damage and cell death, thereby greatly improving the antibacterial ability of lysozyme. At the same time, it can cause protein unfolding, destroy or eliminate allergic epitopes, thereby reducing the allergenicity of modified lysozyme.

[0027] The active ingredients of Centella asiatica are mainly triterpenes, including asiatica glycoside, madecassoside, asiatic acid and madecassoside, which can reduce the content of inflammatory factors and protect gums and soothe. Forsythia suspensa leaves are rich in forsythia glycosides, forsythia glycosides, rutin, forsythiaside, etc., which have antibacterial, liver protection, and antioxidant effects. Green tea is also rich in tea polyphenols, tea polysaccharides, theanine, protocatechuic acid and other active components, which have good antioxidant, antibacterial and anti-inflammatory effects, and also have the effect of refreshing breath. After cellulase-assisted fermentation, the cell walls of Centella asiatica, Forsythia suspensa leaves, and green tea plants are destroyed, promoting the dissolution of active components. At the same time, the polysaccharide structure of the cell wall can also promote the proliferation of fermented bacteria, thereby obtaining a large amount of probiotic mud and improving economic benefits.

[0028] The probiotic composition prepared by the present invention has good inhibitory, destructive and competitive colonization effects on cariogenic bacteria, thereby greatly reducing the damage of cariogenic bacteria to teeth. At the same time, it also has anti-inflammatory, antioxidant, breath freshening and other effects. The mouthwash prepared by adding the probiotic composition has low cost, good effect, easy industrial application, and broad application prospects. DETAILED DESCRIPTION

[0029] 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.

[0030] Streptococcus thermophilus, 10 billion cfu / g, was purchased from Xi'an Bai Nian Kang Biotechnology Co., Ltd. Lactobacillus salivarius, 20 billion cfu / g, was purchased from Guangzhou Huijian Biotechnology Co., Ltd.

[0031] Preparation method of bacterial seed solution: inoculate the bacterial strain into the slant culture medium, culture at 37℃, 100r / min, and culture for 24h to obtain a bacterial liquid with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution.

[0032] Cellulase, 20,000 U / g, was purchased from Shenzhen Fusheng Biotechnology Co., Ltd.; lysozyme, 20,000 U / g, was purchased from Xi'an Yatu Biotechnology Co., Ltd.

[0033] Example 1

[0034] This embodiment provides a method for preparing a probiotic composition, comprising the following steps:

[0035] S1. Preparation of tannic acid modified lysozyme: 10 g tannic acid was dissolved in 500 mL water, 15 g N-hydroxysuccinimide and 12 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the reaction was activated at 0°C for 30 min, 25 g lysozyme was added, the reaction was stirred at room temperature for 12 h, dialyzed, and freeze-dried to obtain tannic acid modified lysozyme;

[0036] S2. Preparation of modified lysozyme: 10 g of glucose and 5 g of cinnamaldehyde were added to 500 mL of water, 25 g of tannic acid-modified lysozyme was added, heated to 65 ° C, stirred for 20 h, dialyzed, and freeze-dried to obtain modified lysozyme;

[0037] S3. Fermentation: 3 g of Centella asiatica, 4 g of Forsythia suspensa leaves, and 2 g of green tea were mixed and crushed, added to 200 mL of water, 0.1 g of cellulase was added, sterilized, inoculated with thermophilic Streptococcus and Lactobacillus salivarius seed liquid, the inoculation amounts were 1 v / v% and 0.5 v / v%, respectively, at 36 ° C, 100 r / min, under anoxic conditions, enzymatic fermentation was cultured for 24 h, filtered, the solid solution was washed and collected, freeze-dried, and probiotics were obtained; the filtrate was dried to obtain a fermentation product;

[0038] S4. Enzymatic hydrolysis: 2 g of modified lysozyme and 7 g of probiotics were mixed, added to 200 mL of water, heated to 36 ° C, and enzymatically hydrolyzed for 20 min to obtain a complex;

[0039] S5. Ultrasonic heat treatment: The composite was subjected to ultrasonic assisted heating treatment, the ultrasonic power was 1200 W, the heating temperature was 50° C., the treatment time was 0.5 h, and then dried to obtain a probiotic composition.

[0040] Example 2

[0041] This embodiment provides a method for preparing a probiotic composition, comprising the following steps:

[0042] S1. Preparation of tannic acid modified lysozyme: 12 g of tannic acid was dissolved in 500 mL of water, 18 g of N-hydroxysuccinimide and 14 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the reaction was activated at 4°C for 50 min, 30 g of lysozyme was added, the reaction was stirred at room temperature for 15 h, dialyzed, and freeze-dried to obtain tannic acid modified lysozyme;

[0043] S2. Preparation of modified lysozyme: 12 g of glucose and 7 g of cinnamaldehyde were added to 500 mL of water, 30 g of tannic acid-modified lysozyme was added, heated to 75 ° C, stirred for 24 h, dialyzed, and freeze-dried to obtain modified lysozyme;

[0044] S3. Fermentation: 5 g of Centella asiatica, 7 g of Forsythia suspensa leaves, and 4 g of green tea were mixed and crushed, added to 200 mL of water, 0.2 g of cellulase was added, sterilized, inoculated with thermophilic Streptococcus and Lactobacillus salivarius seed liquid, the inoculation amounts were 3 v / v% and 1.5 v / v%, respectively, at 38 ° C, 150 r / min, under anoxic conditions, enzymatic fermentation was cultured for 48 h, filtered, the solid solution was washed and collected, freeze-dried, and probiotics were obtained; the filtrate was dried to obtain a fermentation product;

[0045] S4. Enzymatic hydrolysis: 4 g of modified lysozyme and 10 g of probiotics were mixed, added to 200 mL of water, heated to 38 ° C, and enzymatically hydrolyzed for 40 min to obtain a complex;

[0046] S5. Ultrasonic heat treatment: The composite was subjected to ultrasonic assisted heating treatment, the ultrasonic power was 1500W, the heating temperature was 60°C, the treatment time was 1h, and then dried to obtain a probiotic composition.

[0047] Example 3

[0048] This embodiment provides a method for preparing a probiotic composition, comprising the following steps:

[0049] S1. Preparation of tannic acid modified lysozyme: 11 g of tannic acid was dissolved in 500 mL of water, 17 N-hydroxysuccinimide and 13 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the reaction was activated at 2°C for 40 min, 27 g of lysozyme was added, the reaction was stirred at room temperature for 13 h, dialyzed, and freeze-dried to obtain tannic acid modified lysozyme;

[0050] S2. Preparation of modified lysozyme: 11 g glucose and 6 g cinnamaldehyde were added to 500 mL water, 27 g tannic acid modified lysozyme was added, heated to 70 ° C, stirred for 22 h, dialyzed, and freeze-dried to obtain modified lysozyme;

[0051] S3. Fermentation: 4 g of Centella asiatica, 5.5 g of Forsythia suspensa leaves, and 3 g of green tea were mixed and crushed, added to 200 mL of water, 0.15 g of cellulase was added, sterilized, inoculated with thermophilic Streptococcus and Lactobacillus salivarius seed liquid, the inoculation amount was 2 v / v% and 1 v / v%, respectively, at 37 ° C, 125 r / min, under anoxic conditions, enzymatic fermentation was cultured for 36 h, filtered, the solid was washed and the bacterial solution was collected, freeze-dried, and probiotics were obtained; the filtrate was dried to obtain a fermentation product;

[0052] S4. Enzymatic hydrolysis: 3 g of modified lysozyme and 8.5 g of probiotics were mixed, added to 200 mL of water, heated to 37 ° C, and enzymatic hydrolyzed for 30 min to obtain a complex;

[0053] S5. Ultrasonic heat treatment: The composite was subjected to ultrasonic assisted heating treatment, the ultrasonic power was 1350W, the heating temperature was 55°C, the treatment time was 1h, and then dried to obtain a probiotic composition.

[0054] Comparative Example 1

[0055] Compared with Example 3, the difference is that step S1 is not performed.

[0056] The details are as follows:

[0057] S1. Preparation of modified lysozyme: 11 g glucose and 6 g cinnamaldehyde were added to 500 mL water, 27 g lysozyme was added, heated to 70°C, stirred for 22 h, dialyzed, and freeze-dried to obtain modified lysozyme;

[0058] S2. Fermentation: 4 g of Centella asiatica, 5.5 g of Forsythia suspensa leaves, and 3 g of green tea were mixed and crushed, added to 200 mL of water, 0.15 g of cellulase was added, sterilized, inoculated with thermophilic Streptococcus and Lactobacillus salivarius seed liquid, the inoculation amounts were 2 v / v% and 1 v / v%, respectively, at 37 ° C, 125 r / min, under anaerobic conditions, enzymatic fermentation was cultured for 36 h, filtered, the solid solution was washed and collected, freeze-dried, and probiotics were obtained; the filtrate was dried to obtain a fermentation product;

[0059] S3. Enzymatic hydrolysis: 3 g of modified lysozyme and 8.5 g of probiotics were mixed, added to 200 mL of water, heated to 37 ° C, and enzymatic hydrolyzed for 30 min to obtain a complex;

[0060] S4. Ultrasonic heat treatment: The composite was subjected to ultrasonic assisted heating treatment, the ultrasonic power was 1350 W, the heating temperature was 55° C., the treatment time was 1 h, and then dried to obtain a probiotic composition.

[0061] Comparative Example 2

[0062] Compared with Example 3, the difference is that glucose is not added in step S2.

[0063] The details are as follows:

[0064] S2. Preparation of modified lysozyme: Add 17 g of cinnamaldehyde to 500 mL of water, add 27 g of tannic acid-modified lysozyme, heat to 70°C, stir to react for 22 hours, dialyze, and freeze-dry to obtain modified lysozyme.

[0065] Comparative Example 3

[0066] Compared with Example 3, the difference is that cinnamaldehyde is not added in step S2.

[0067] The details are as follows:

[0068] S2. Preparation of modified lysozyme: Add 17 g of glucose to 500 mL of water, add 27 g of tannic acid-modified lysozyme, heat to 70°C, stir to react for 22 h, dialyze, and freeze-dry to obtain modified lysozyme.

[0069] Comparative Example 4

[0070] Compared with Example 3, the difference is that the thermophilic Streptococcus seed liquid is not inoculated in step S3.

[0071] The details are as follows:

[0072] S3. Fermentation: Mix and grind 4g of Centella asiatica, 5.5g of Forsythia suspensa leaves, and 3g of green tea, add to 200mL of water, add 0.15g of cellulase, sterilize, inoculate with Lactobacillus salivarius seed liquid, the inoculation amount is 3v / v%, 37℃, 125r / min, under anaerobic conditions, culture for 36h by enzymatic fermentation, filter, wash the solid and collect the bacterial liquid, freeze-dry to obtain probiotics; dry the filtrate to obtain the fermentation product.

[0073] Comparative Example 5

[0074] Compared with Example 3, the difference is that the Lactobacillus salivarius seed liquid is not inoculated in step S3.

[0075] The details are as follows:

[0076] S3. Fermentation: Mix and grind 4g of Centella asiatica, 5.5g of Forsythia suspensa leaves, and 3g of green tea, add to 200mL of water, add 0.15g of cellulase, sterilize, inoculate with thermophilic Streptococcus seed solution, the inoculation amount is 3v / v%, 37℃, 125r / min, under anaerobic conditions, culture for 36h by enzymatic fermentation, filter, wash the solid and collect the bacterial solution, freeze-dry to obtain probiotics; dry the filtrate to obtain the fermentation product.

[0077] Comparative Example 6

[0078] Compared with Example 3, the difference is that step S4 is not performed.

[0079] The details are as follows:

[0080] S1. Preparation of tannic acid modified lysozyme: 11 g of tannic acid was dissolved in 500 mL of water, 17 N-hydroxysuccinimide and 13 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the reaction was activated at 2°C for 40 min, 27 g of lysozyme was added, the reaction was stirred at room temperature for 13 h, dialyzed, and freeze-dried to obtain tannic acid modified lysozyme;

[0081] S2. Preparation of modified lysozyme: 11 g glucose and 6 g cinnamaldehyde were added to 500 mL water, 27 g tannic acid modified lysozyme was added, heated to 70 ° C, stirred for 22 h, dialyzed, and freeze-dried to obtain modified lysozyme;

[0082] S3. Fermentation: 4 g of Centella asiatica, 5.5 g of Forsythia suspensa leaves, and 3 g of green tea were mixed and crushed, added to 200 mL of water, 0.15 g of cellulase was added, sterilized, inoculated with thermophilic Streptococcus and Lactobacillus salivarius seed liquid, the inoculation amount was 2 v / v% and 1 v / v%, respectively, at 37 ° C, 125 r / min, under anoxic conditions, enzymatic fermentation was cultured for 36 h, filtered, the solid was washed and the bacterial solution was collected, freeze-dried, and probiotics were obtained; the filtrate was dried to obtain a fermentation product;

[0083] S4. Enzymatic hydrolysis: 3 g of modified lysozyme and 8.5 g of probiotics were mixed, and subjected to ultrasonic-assisted heating treatment with an ultrasonic power of 1350 W, a heating temperature of 55° C., a treatment time of 1 h, and dried to obtain a probiotic composition.

[0084] Comparative Example 7

[0085] Compared with Embodiment 3, the difference is that step S5 is not performed.

[0086] The details are as follows:

[0087] S1. Preparation of tannic acid modified lysozyme: 11 g of tannic acid was dissolved in 500 mL of water, 17 N-hydroxysuccinimide and 13 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the reaction was activated at 2°C for 40 min, 27 g of lysozyme was added, the reaction was stirred at room temperature for 13 h, dialyzed, and freeze-dried to obtain tannic acid modified lysozyme;

[0088] S2. Preparation of modified lysozyme: 11 g glucose and 6 g cinnamaldehyde were added to 500 mL water, 27 g tannic acid modified lysozyme was added, heated to 70 ° C, stirred for 22 h, dialyzed, and freeze-dried to obtain modified lysozyme;

[0089] S3. Fermentation: 4 g of Centella asiatica, 5.5 g of Forsythia suspensa leaves, and 3 g of green tea were mixed and crushed, added to 200 mL of water, 0.15 g of cellulase was added, sterilized, inoculated with thermophilic Streptococcus and Lactobacillus salivarius seed liquid, the inoculation amount was 2 v / v% and 1 v / v%, respectively, at 37 ° C, 125 r / min, under anoxic conditions, enzymatic fermentation was cultured for 36 h, filtered, the solid was washed and the bacterial solution was collected, freeze-dried, and probiotics were obtained; the filtrate was dried to obtain a fermentation product;

[0090] S4. Enzymatic hydrolysis: 3 g of modified lysozyme and 8.5 g of probiotics were mixed, added into 200 mL of water, heated to 37° C., and subjected to enzymatic hydrolysis for 30 min to obtain a complex, namely, a probiotic composition.

[0091] Test Example 1

[0092] The probiotic composition prepared in Examples 1-3 and Comparative Examples 1-7 was added into the formula in an amount of 2 wt % to prepare a mouthwash. The formula of the mouthwash is shown in Table 1 below.

[0093] Table 1

[0094] Element Mass fraction (%) glycerin 10 Methylparaben 0.2 Probiotic composition 2 Dipotassium glycyrrhizinate 0.2 Sorbitol 10 Sucralose 0.01 essence 0.05 Poloxamer 407 1.0 Sodium Citrate 0.02 PEG-60 Hydrogenated Castor Oil 0.2 Deionized water margin

[0095] Stability test of mouthwash:

[0096] The prepared mouthwashes were stored at high temperature (45°C), room temperature, light, refrigeration (4°C), and freezing (-18°C). The inspection indicators were observed for 3 months in accordance with the requirements of the mouthwash standard "Oral Cleansing and Care Solution (QBT2945-2012)".

[0097] Results: The mouthwashes prepared by adding the probiotic compositions prepared in Examples 1-3 and Comparative Examples 1-7 were subjected to stability tests on sensory indicators, physicochemical indicators and microbiological indicators. The results showed that the mouthwashes were qualified in terms of appearance, aroma, pH value, microbiological indicators and other indicators, and the overall product antiseptic challenge test was passed, meeting the requirements of QBT2945 for mouthwash products.

[0098] Mouthwash antibacterial test:

[0099] The antibacterial test was outsourced to Guangzhou Institute of Microbiology Co., Ltd. The antibacterial test method referred to the "Evaluation Method for Antibacterial and Antimicrobial Effects of Daily Chemical Products (QB / T2738-2012)". The bacterial strain selected was Porphyromonas gingivalis, an important periodontal pathogen that has been widely studied and has sufficient evidence. According to the habit of mouthwash use, the contact time between mouthwash and bacteria was selected as 30 seconds.

[0100] The results are shown in Table 2.

[0101] Table 2

[0102] Group Antibacterial rate (%) Example 1 >99.9 Example 2 >99.9 Example 3 >99.9 Comparative Example 1 93.11 Comparative Example 2 91.06 Comparative Example 3 90.75 Comparative Example 4 86.94 Comparative Example 5 87.37 Comparative Example 6 82.56 Comparative Example 7 92.65

[0103] It can be seen from the above table that the mouthwash prepared by adding the probiotic composition prepared in Examples 1-3 of the present invention has a good antibacterial rate against Porphyromonas gingivalis.

[0104] Mouthwash gum protection effect test:

[0105] Based on the IL-1α stimulated 3D oral mucosal epithelial model provided by Shaanxi Boxi General Testing Technology Co., Ltd. An inflammatory response model was established, and the in vitro oral gingival protection effect of the mouthwash was evaluated by detecting the changes in the secretion of proinflammatory factors (IL-6 and IL-8). When there is a problem with the health of the gums, such as an outbreak of inflammation, the inflammatory factors IL-6 and IL-8 will be secreted in large quantities. Gingival inflammation can induce symptoms such as gingival redness, swelling, and pain. Therefore, the in vitro oral gingival protection and soothing effect of the test sample can be evaluated by detecting the expression of the proinflammatory factors IL-6 and IL-8. A blank group, a model group, a positive drug group (0.1wt% dexamethasone solution), Example 1-3 groups, and Comparative Example 1-7 groups were established. The sample size of each group was 6. Except for the blank group, the other groups all used IL-1α to stimulate the 3D oral mucosal epithelial model Establish an inflammatory response model.

[0106] The results are shown in Table 3.

[0107] Table 3

[0108] Group IL-6 content (pg / mL) IL-8 content (pg / mL) Blank group 2.19±0.12* 359.63±32.13* Model Group 6.22±0.29# 859.26±73.66# Positive drug group 3.24±0.15# 612.96±9.45# Example 1 3.57±0.11# 610.45±14.67# Example 2 3.60±0.13# 612.16±11.73# Example 3 3.55±0.09# 607.37±12.46# Comparative Example 1 4.09±0.16 718.93±16.66 Comparative Example 2 4.14±0.22 729.27±17.71 Comparative Example 3 4.10±0.27 723.72±19.16 Comparative Example 4 3.78±0.18 689.47±21.93 Comparative Example 5 3.82±0.21 680.26±22.15 Comparative Example 6 4.01±0.27 704.77±18.94 Comparative Example 7 4.19±0.24 712.15±20.67

[0109] Note: * compared with the blank group, P < 0.05; # compared with the model group, P < 0.05.

[0110] 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 a probiotic composition, characterized in that: The following steps are involved: S1. Preparation of tannic acid modified lysozyme: dissolving tannic acid in water, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activating the reaction, adding lysozyme, stirring at room temperature, dialyzing, and freeze-drying to obtain tannic acid modified lysozyme; the mass ratio of tannic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and lysozyme is 10-12:15-18:12-14:25-30; S2. Preparation of modified lysozyme: adding glucose and cinnamaldehyde to water, adding tannic acid-modified lysozyme, heating and stirring the reaction, dialyzing, and freeze-drying to obtain modified lysozyme; the mass ratio of glucose, cinnamaldehyde, and tannic acid-modified lysozyme is 10-12:5-7:25-30; S3. Fermentation: Mix and crush Centella asiatica, Forsythia suspensa leaves, and green tea, add to water, add cellulase, sterilize, inoculate thermophilic Streptococcus and Lactobacillus salivarius seed solution, culture by enzymatic fermentation, filter, wash and collect the bacterial solution, freeze-dry to obtain a probiotic; dry the filtrate to obtain a fermentation product; the mass ratio of the Centella asiatica, Forsythia suspensa leaves, green tea, and cellulase is 3-5:4-7:2-4:0.1-0.2, and the inoculation amounts of the thermophilic Streptococcus and Lactobacillus salivarius seed solution are 1-3v / v% and 0.5-1.5v / v% respectively; S4 enzymatic hydrolysis: The modified lysozyme and the probiotics were mixed, added to water, heated and hydrolyzed to obtain a complex; the mass ratio of the modified lysozyme and the probiotics was 2-4:7-10; S5. Ultrasonic heat treatment: The composite is subjected to ultrasonic-assisted heating treatment and dried to obtain a probiotic composition.

2. The preparation method according to claim 1, characterized in that: The activation reaction in step S1 is carried out at a temperature of 0-4°C for 30-50 min, and the reaction is stirred at room temperature for 12-15 h.

3. The preparation method according to claim 1, characterized in that: The temperature of the heating and stirring reaction in step S2 is 65-75° C. and the time is 20-24 h.

4. The preparation method according to claim 1, characterized in that: The bacterial content of the bacterial seed liquid in step S3 is 10 8 -10 9 cfu / mL, and the conditions of the enzymatic fermentation culture are 36-38°C, 100-150r / min, and the enzymatic fermentation culture is carried out under anoxic conditions for 24-48h.

5. The preparation method according to claim 1, characterized in that: The temperature of the heating enzymolysis in step S4 is 36-38° C. and the time is 20-40 min.

6. The preparation method according to claim 1, characterized in that: The ultrasonic power of the ultrasonic-assisted heating treatment in step S5 is 1200-1500W, the heating temperature is 50-60°C, and the treatment time is 0.5-1h.

7. A probiotic composition prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the probiotic composition according to claim 7 in preparing oral cleaning products, characterized in that: The oral cleaning product is at least one of mouthwash, toothpaste and tooth powder.

9. A mouthwash, characterized in that: Containing 0.5-10wt% of the probiotic composition as claimed in claim 7.