Pu'er raw tea extract as well as preparation method and application thereof

Abstract

The invention belongs to the field of oral care products, and discloses a raw Pu'er tea extract as well as a preparation method and application thereof, the preparation method of the raw Pu'er tea extract comprises the following steps: S1, crushing raw Pu'er tea into powder, sieving to obtain raw Pu'er tea powder, adding water for dissolving, and sterilizing to obtain raw Pu'er tea liquid; s2, inoculating compound bacteria into the Pu'er raw tea liquid, adding cellulase and pectinase at the same time, and fermenting for 36-60 hours; and S3, carrying out sterilization and enzyme deactivation treatment on the fermentation liquor, centrifuging, collecting supernate, and preparing freeze-dried powder, so as to obtain the Pu'er raw tea extract. The compound bacteria comprise bacillus velezensis MRS and streptococcus salivarius QS-20. The raw Pu'er tea extract is safe, healthy and free of side effects, has a good antibacterial effect on streptococcus mutans and can be used as an active ingredient for preparing anti-caries antibacterial medicines, oral washing products or oral care products, and the preparation method can obviously improve the yield of the antibacterial active ingredient and can be applied to industrial production.

Description

Technical Field

[0001] The invention relates to the field of oral care products, and in particular to a Pu'er raw tea extract and a preparation method and application thereof. Background Art

[0003] Dental caries, commonly known as tooth decay, is a progressive destructive disease of dental hard tissues caused mainly by infection with bacterial plaque microorganisms. It manifests as tooth pigmentation, sensitivity, pain, tooth defects, looseness, and even tooth loss. It can lead to complications such as pulpitis and apical periodontitis, and can even cause inflammation of the alveolar bone and jawbone. Dental caries has become one of the diseases that seriously affect human oral function and health.

[0004] Mutans Streptococcus is the main pathogen of dental caries. It can ferment a variety of carbohydrates to produce acid and is acid-resistant, which reduces the pH of the plaque. It can also use sucrose as a substrate to synthesize intracellular and extracellular polysaccharides. In addition, the acquired membrane on the tooth surface also has a high affinity, adheres to and aggregates with bacteria to form a mature biofilm, and then forms dental plaque. These characteristics determine its important role in the occurrence of dental caries, and it is considered to be the main pathogen of S. mutans It is an important virulence factor in cariogenesis. Therefore, it is necessary to study how to control S. mutans The number of cells, their acid production and acid resistance, adhesion and ability to synthesize extracellular polysaccharides are of great significance in reducing their cariogenicity.

[0005] At present, the common methods for daily prevention and control of Streptococcus mutans are mechanical methods such as brushing teeth and gargling, and the use of fluoride-containing drugs. However, these mechanical methods cannot effectively remove Streptococcus mutans in the gaps between teeth. Although current oral rinse products can more effectively remove oral bacteria, they contain chemical antibacterial agents such as quaternary ammonium salts, chlorides, and fluorides. Long-term use can easily lead to bacterial resistance, oral mucosal lesions, tooth staining and other side effects.

[0006] Therefore, the prior art needs to be further improved. Summary of the invention

[0007] In view of the above problems, the present invention provides a Pu'er raw tea extract and a preparation method and application thereof. The Pu'er raw tea extract is not only safe to use and has no side effects, but also has an excellent antibacterial effect on Streptococcus mutans, can effectively inhibit the production of extracellular polysaccharides by Streptococcus mutans, and effectively inhibits the formation of its biofilm, and can be used as an active ingredient in the preparation of anti-caries and antibacterial drugs, oral wash products or oral care products.

[0008] To solve the above problems, this application provides the following technical solutions: In a first aspect, the present application provides a method for preparing a Pu'er raw tea extract, comprising the following steps: S1, grinding Pu'er raw tea into powder, sieving to obtain Pu'er raw tea powder, adding water to dissolve and sterilizing to obtain Pu'er raw tea liquid; S2, inoculate composite bacteria into Pu'er raw tea liquid, add cellulase and pectinase at the same time, and ferment for 36-60 hours; S3. After sterilizing and inactivating the enzymes in the fermentation liquid, the supernatant is collected by centrifugation and made into freeze-dried powder to obtain the Pu'er raw tea extract.

[0009] Optionally, in step S2 of the method for preparing the Pu'er raw tea extract, the composite bacteria include: Bacillus velezensis MRS with a preservation number of CGMCC No. 18065 and Streptococcus salivarius QS-20 with a preservation number of CGMCC 7.456.

[0010] Preferably, the ratio of Bacillus Velez MRS to Streptococcus salivarius QS-20 is 1:1.

[0011] The relevant information of the strain Bacillus Velezii MRS is disclosed in patent application number 202010487194.8, and the relevant information of the Streptococcus salivarius QS-20 is disclosed in patent application number 202210562002.4.

[0012] Preferably, the ratio of cellulase to pectinase is 1:1.

[0013] Preferably, in the method for preparing the Pu'er raw tea extract, in the step S2, the mass ratio of Pu'er raw tea powder to water is 1 g: (50-70) mL, the total inoculation amount is 6%, the total enzyme inoculation amount is 6%, and the fermentation temperature is 33-37°C.

[0014] Further preferably, in the preparation method of the Pu'er raw tea extract, the ratio of Pu'er raw tea powder to water is 1:60, the total inoculation amount is 4-8%, the total enzyme inoculation amount is 4-8%, the fermentation time is 48h, and the fermentation temperature is 35°C.

[0015] Under the optimal fermentation extraction conditions, the extraction rate of Pu'er raw tea is 73.49%, which is 52.05% higher than the extraction rate of the existing non-fermentation extraction method.

[0016] The traditional non-fermented extraction method is: mix tea powder and water in a material-liquid mass ratio of 1:60 into a 100 mL system, sterilize at 121°C with high pressure for 15 min, ferment for 48 h, and ferment at 35°C.

[0017] In a second aspect, the present application also provides a Pu'er raw tea extract, which is prepared using the aforementioned preparation method.

[0018] In a third aspect, the present application also provides the use of the above-mentioned Pu'er raw tea extract in inhibiting Streptococcus mutans.

[0019] Experiments have shown that the Pu'er raw tea extract has a good inhibitory effect on Streptococcus mutans, and the size of the inhibition zone reaches about 16.7 mm. Its antibacterial effect on Streptococcus mutans is significantly better than that of the unimproved tea crude extract.

[0020] Experiments have shown that the Pu'er raw tea extract can effectively inhibit the production of extracellular polysaccharides by Streptococcus mutans, can effectively destroy the structure of the biofilm of Streptococcus mutans organisms, and can effectively inhibit the formation of biofilm of Streptococcus mutans.

[0021] In a fourth aspect, the present application also provides the use of the above-mentioned Pu'er raw tea extract in the preparation of anti-caries and antibacterial drugs. The drugs are various drugs for preventing and / or treating caries, periodontitis or pulpitis.

[0022] In a fifth aspect, the present application also provides the use of the above-mentioned Pu'er raw tea extract in the preparation of oral wash products or oral care products.

[0023] Optionally, the oral wash product or oral care product includes toothpaste, tooth powder, antibacterial gel, toothpaste additive, mouthwash, chewing gum or oral spray.

[0024] The present invention has the following beneficial effects: 1. The present invention provides a Pu'er raw tea extract, which is extracted from green and healthy Pu'er tea powder. The Pu'er raw tea extract is not only safe and healthy, without side effects, but also has an excellent antibacterial effect on Streptococcus mutans, can effectively inhibit the production of extracellular polysaccharides by Streptococcus mutans, and effectively inhibits the formation of its biofilm. The extract can be used as an active ingredient in the preparation of anti-caries and antibacterial drugs, oral washing products or oral care products, and has a good application prospect.

[0025] 2. The present invention also provides a method for preparing the above-mentioned Pu'er raw tea extract. The method is simple to operate and uses two unique bacteria (Bacillus Velez MRS and Streptococcus salivarius QS-20) in synergistic cooperation with two enzymes (cellulase and pectinase), which greatly improves the extraction efficiency, significantly increases the yield of the extract, has a high utilization rate of raw materials, and does not destroy its antibacterial activity against Streptococcus mutans, and can be applied to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The single factor experimental results for the optimization of the extraction process of Pu'er raw tea; Figure 2 The results of the inhibition zone of Streptococcus mutans in treatment 1; well 1 is 1 mg / mL chlorhexidine acetate, well 2 is unfermented Pu'er raw tea extract, and well 3 is Pu'er raw tea extract fermented with bacterial enzymes; Figure 3 The results of the inhibition zone of Streptococcus mutans in treatment 2; well 1 is 1 mg / mL chlorhexidine acetate, well 2 is unfermented Pu'er raw tea extract, and well 3 is Pu'er raw tea extract fermented with bacterial enzymes; Figure 4 The MBC test of Pu'er raw tea extract on Streptococcus mutans; A is the control group; B is 1.25 mg / mL of Pu'er raw tea extract prepared in Example 1; C is 0.625 mg / mL of Pu'er raw tea extract; Figure 5 The effect of Pu'er raw tea extract on the growth curve of Streptococcus mutans; Figure 6 To study the effect of Pu'er raw tea extract on acid production of Streptococcus mutans; Figure 7 Effects of Pu'er raw tea extract on exopolysaccharide production by Streptococcus mutans; Figure 8 Effects of Pu'er raw tea extract on biofilm of Streptococcus mutans; Fig. 9 The effect of Pu'er raw tea extract on the biofilm structure of Streptococcus mutans; A and B are the plaque biofilm morphology of Streptococcus mutans without drug, C and D are the biofilm morphology of Streptococcus mutans with drug; among them, A is the control group magnified 5000 times; B is the control group magnified 20000 times; C is the MIC group magnified 5000 times; D is the MIC group magnified 20000 times. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0028] Example 1 Preparation of Pu'er Raw Tea Extract 1. This embodiment provides a method for preparing a Pu'er raw tea extract, which comprises the following steps: S1. Powder the Pu'er raw tea with a powdering machine, sieve through 100 mesh to obtain Pu'er raw tea powder, add 100 ml of water to dissolve, the tea-water ratio is 1:60, and then sterilize at 121°C for 15 min to obtain Pu'er raw tea liquid; S2, inoculate composite bacteria into Pu'er raw tea liquid, add cellulase and pectinase in a ratio of 1:1, and ferment at 35℃ for 48 hours; The composite bacteria include: 1:1 Bacillus Velez MRS and Streptococcus salivarius QS-20.

[0029] The total inoculum amount is 6%, and the total enzyme inoculum amount is 6%. S3. Sterilize and inactivate the enzyme in the fermentation liquid in a boiling water bath for 15 min, centrifuge at 12000 r / min for 15 min, collect the supernatant by centrifugation, and prepare freeze-dried powder to obtain Pu'er raw tea extract.

[0030] 2. Determination of extraction rate (1) Determination method: An experimental group and a control group were set up, and the experimental group was measured using the Pu'er raw tea extract prepared in this example.

[0031] The extraction method of the control group was as follows: the solid-liquid ratio of tea water was 1:60, the volume was fixed to 100 mL, high pressure sterilization was performed at 121°C for 15 min, the inoculation amount was 0%, the inoculation amount was 0%, the fermentation time was 48 h, the fermentation temperature was 35°C, and other operations were the same as those in the example.

[0032] The lyophilized powders of Pu'er raw tea extract prepared in the experimental group and the control group were weighed respectively, and the extraction rate was calculated according to the following calculation formula.

[0033]

[0034] g1: Net weight of container before freeze-drying g2: Net weight of container after freeze-drying m: Dry weight of Pu'er raw tea (2) Measurement results The extraction rate of the Pu'er raw tea extract obtained by the above method is 73.49%. Compared with the extraction method of the control group, the extraction rate of the Pu'er raw tea extract prepared by the preparation method of this embodiment is increased by 52.05%, and the yield of the extract is greatly improved.

[0035] Subsequently, the Pu'er raw tea extract prepared in this example was subjected to various antibacterial activity tests of Example 2.

[0036] Example 2 Determination of antibacterial activity of Pu'er raw tea extract 2.1 Inhibition zone determination (1) Experimental methods: Will S. mutans Plate three activation cultures, use a sterile inoculation loop to take a loop of bacteria and inoculate into liquid culture medium for anaerobic culture for 12 h, and dilute with sterile saline to 10 6 -10 8 CFU / mL is reserved.

[0037] Add 0.1 mL of the test bacterial suspension to a sterile plate, add 15-25 mL of sterile BHI agar, mix thoroughly, and after solidification at room temperature, use a sterile hole puncher (5 mm in diameter) to punch holes in each plate, and draw 60 μL of the sample into the groove. Transfer the plate to a 37°C incubator for anaerobic culture for 8-12 h, and use a colony counter to measure the diameter of the inhibition zone.

[0038] The Pu'er raw tea extract extracted by the method of Example 1 was used as sample No. 1, and the extract extracted by the method without adding bacteria and enzymes was used as sample No. 2. At the same time, 1 mg / mL chlorhexidine acetate was used as a positive control sample to determine the inhibition zone.

[0039] Treatment 1: Samples 1 and 2 were diluted with the same volume of solvent and then subjected to the experiment.

[0040] Treatment 2: Take the same amount of sample 1 and sample 2 and dilute them with solvent to extracts of the same concentration, and then conduct the experiment.

[0041] (2) Experimental results and analysis The experimental results show that in treatment 1, under the condition of dissolving in the same volume of solvent, the size of the inhibition zone of the Pu'er raw tea extract extracted by the method of Example 1 against Streptococcus mutans is 16.7±0.2 b mm, while the inhibition zone size of sample 2 was 12.1±0.3 a mm, there was a significant difference ( p <0.05), such as Figure 2 shown.

[0042] In treatment 2, the inhibition zones of samples 1 and 2 against Streptococcus mutans were both 16.7±0.2amm, with no significant difference (p<0.05).

[0043] The above results indicate that the antibacterial active ingredients extracted by the method of the present application and the traditional non-bacterial enzyme fermentation extraction method are the same, so the antibacterial zones of extracts with the same concentration are comparable. However, the amount of antibacterial active ingredients extracted by the method of the present application is greater, the concentration of the extract obtained by dissolving in the same volume of solvent is higher, and the antibacterial effect is higher. Subsequent experiments used Pu'er raw tea extracts fermented by bacterial enzymes.

[0044] 2.2 Determination of MIC and MBC (1) Experimental methods: A. MIC The Pu'er raw tea extract extracted by the combined fermentation method of bacteria and enzymes in Example 1 was used as the experimental group, and the extract extracted by the method without adding bacteria and enzymes was used as the control group, and MIC determination was performed at the same time.

[0045] Using two-fold gradient dilution, 40 mg / mL Pu'er raw tea extract was diluted into a series of sample solutions with different concentration gradients. 100 μL of bacterial suspension was added to 100 μL of sample and incubated anaerobically at 37°C for 12 h. The absorbance at 600 nm was observed and measured. The lowest concentration at which turbidity was not visible to the naked eye in the well plate was the MIC.

[0046] B. MBC The Pu'er raw tea extract extracted by the combined fermentation method of bacteria and enzymes in Example 1 was used as the experimental group for the following tests.

[0047] Take 100 μL of the mixed solution of the MIC and the sample wells with a concentration higher than this in the 96-well plate after incubation, spread it on the BHI agar plate, and incubate it at 37°C for 12 h. The concentration of sterile growth on the plate is taken as the minimum bactericidal concentration.

[0048] (2) Experimental results and analysis A. The experimental results are shown in Table 3. The MIC of Pu'er raw tea extracts extracted by different methods against Streptococcus mutans was 1.25 mg / mL.

[0049] B. The experimental results show that the MBC of the Pu'er raw tea extract extracted from the experimental group against Streptococcus mutans is 1.25 mg / mL. Figure 4 .

[0050] The above experimental results show that the antibacterial active ingredients extracted by different methods are the same, so the MIC and MBC of extracts with the same concentration are the same, but the amount of active ingredient extracted in the experimental group is large. Subsequent experiments used Pu'er raw tea extracts fermented by bacterial enzymes.

[0051]

[0052] 2.3 Effect of Pu'er raw tea extract on the growth curve of Streptococcus mutans (1) Experimental methods: The Pu'er raw tea extract prepared in Example 1 was used as the experimental object, and was diluted to concentrations of MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC, respectively, and then the following experiments were performed.

[0053] Use a sterile inoculation loop to take a loop of bacteria and inoculate it into liquid culture medium for anaerobic culture for 12 h. Dilute it to 10 6 -10 8 After fully mixing, 500 μL was pipetted into a deep-well plate, and 500 μL of Pu'er raw tea extract (at various concentrations) was added. 1 mL of the above bacterial suspension was used as the control group, and the growth curve was determined using an automatic microbial growth curve instrument.

[0054] (2) Experimental results and analysis like Figure 5 As shown in the figure, from the overall trend, different concentrations of Pu'er raw tea extract inhibited the growth of Streptococcus mutans. The test bacteria in the MIC group hardly grew, and the lag period of the 1 / 2MIC test bacteria was significantly prolonged compared with the control group. The bacterial volume in the 1 / 4MIC and 1 / 8MIC groups was affected and the bacterial activity was significantly reduced. The experimental results show that Pu'er raw tea extract has a strong inhibitory effect on Streptococcus mutans. The higher the concentration of Pu'er raw tea extract, the better its inhibitory effect on Streptococcus mutans.

[0055] 2.4 Effect of Pu'er Tea Extract on Acid Production of Streptococcus mutans (1) Experimental methods: The Pu'er raw tea extract prepared in Example 1 was used as the experimental object, and was diluted to concentrations of MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC, respectively, and then the following experiments were performed.

[0056] Use a sterile inoculation loop to take a loop of bacteria and inoculate it into liquid culture medium for anaerobic culture for 12 h. Dilute it to 10 6 -10 8 After fully mixing, pipette 2.5 mL into a deep-well plate, add 2.5 mL of Pu'er raw tea extract (MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC), take 5 mL of the above bacterial suspension as the control group, culture anaerobically at 37°C for 24 h, and measure the pH value at 0 h, 6 h, 12 h, and 24 h, respectively.

[0057] (2) Experimental results and analysis like Figure 6 As shown, the experimental results showed that at the sub-inhibitory concentration (1 / 8MIC-1 / 2MIC), as the concentration of Pu'er raw tea extract increased, the ΔpH of the experimental group gradually decreased, which indicates that Pu'er raw tea extract at the sub-inhibitory concentration has an inhibitory effect on the acid production of Streptococcus mutans and shows concentration dependence.

[0058] 1.3.7 Effect of Pu'er Tea Extract on Exopolysaccharide Production by Streptococcus mutans Extracellular polysaccharide is a type of glucan synthesized by Streptococcus mutans, which helps bacteria adhere and aggregate, enhances the mechanical stability of the biofilm structure, and can form a local acidic environment leading to the occurrence of dental caries. Therefore, we further tested the effect of Pu'er raw tea extract on the content of extracellular polysaccharides synthesized by Streptococcus mutans.

[0059] (1) Experimental methods: The Pu'er raw tea extract prepared in Example 1 was used as the experimental object, and was diluted to concentrations of MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC, respectively, and then the following experiments were performed.

[0060] Use a sterile inoculation loop to take a loop of bacteria and inoculate it into liquid culture medium for anaerobic culture for 12 h. Dilute it to 10 6 -10 8 CFU / mL for later use. After fully mixing, pipette 5 mL into a deep-well plate, add 5 mL of Pu'er raw tea extract (MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC), and use 10 mL of the above bacterial suspension as the control group, and culture at 37°C for 12 h. Take 5 mL of fresh bacterial solution from each group, centrifuge at 10000g for 10 min (repeat 3 times), collect the supernatant, and place it in a clean centrifuge tube. Add 3 times the volume of anhydrous ethanol, place it at 20°C for 30 min to precipitate polysaccharides, and then centrifuge. Quickly add 2 mL of 5% phenol aqueous solution and 5 mL of concentrated sulfuric acid to the mixture, and let it stand in the dark at room temperature for 10 min. Then, vortex for 30 seconds, place it in a water bath at 25°C for 20 min, and read the absorbance at 490nm.

[0061] (2) Experimental results and analysis The experimental results are shown in Figure 7. At the MIC concentration, the Pu'er raw tea extract can effectively inhibit the production of extracellular polysaccharides by Streptococcus mutans. At the sub-inhibitory concentration, the inhibition rate of extracellular polysaccharides increases with the increase of concentration, indicating that the Pu'er raw tea extract can effectively inhibit the production of extracellular polysaccharides by Streptococcus mutans.

[0062] 1.3.8 Effect of Pu'er Tea Extract on Streptococcus mutans Biofilm Caries biofilm is the initiating factor of caries and the key factor in the occurrence of caries. Therefore, the effect of Pu'er raw tea extract on mutans Streptococcus biofilm was further tested.

[0063] (1) Experimental methods: The Pu'er raw tea extract prepared in Example 1 was used as the experimental object, and was diluted to concentrations of MIC, 1 / 2MIC, 1 / 4MIC, and 1 / 8MIC, respectively, and then the following experiments were performed.

[0064] Use a sterile inoculation loop to take a loop of bacteria and inoculate it into liquid culture medium for anaerobic culture for 12 h. Dilute it to 10 6 -10 8CFU / mL for later use. After fully mixing, 100 μL was aspirated into a deep-well plate, and 100 μL of Pu'er raw tea extract (MIC, 1 / 2MIC, 1 / 4MIC, 1 / 8MIC) was added. 200 μL of the above bacterial suspension was used as the control group, and cultured anaerobically at 37°C for 12 h. The bacterial suspension was slowly aspirated from the 96-well plate after 12 h of incubation, and slowly rinsed 2-3 times with PBS buffer. After elution, 100 μL of 0.1% crystal violet staining solution was added to each well for staining and allowed to stand for 30 min; the crystal violet staining solution was then aspirated, and slowly rinsed 2-3 times with sterile water to remove planktonic bacteria; each well was then dissolved with 30% acetic acid solution and transferred to a new plate; the OD value was measured at OD590nm by an ELISA instrument to determine the biofilm formation ability. The OD value of the blank control group was set as 100% biofilm formation, and the percentage of biofilm formation in the double dilution solution well was calculated based on this.

[0065] (2) Experimental results and analysis like Figure 8 As shown in the figure, after treatment with Pu'er raw tea extract at MIC concentration, the biofilm formation rate decreased significantly, which indicates that Pu'er raw tea extract can effectively inhibit the formation of biofilm of Streptococcus mutans.

[0066] 1.3.9 Scanning electron microscopy (1) Experimental methods: Use a sterile inoculation loop to take a loop of bacteria and inoculate it into liquid culture medium for anaerobic culture for 12 h. Dilute it to 10 6 -10 8 CFU / mL for later use. After thorough mixing, aspirate 500 μL into a 24-well plate, add 500 μL of Pu'er raw tea extract (MIC concentration), and use 1 mL of the above bacterial suspension as the control group. Slowly aspirate the bacterial suspension from the 24-well plate after 12 h of incubation, and slowly rinse 2-3 times with 0.1 M PBS buffer. After elution, fix the biofilm with 4% glutaraldehyde overnight. Dilute and dehydrate with 70%, 80%, 90% and 100% ethanol for 15 minutes each time, and then examine under a microscope.

[0067] (2) Experimental results and analysis Electron microscopy results Fig. 9 As shown in the figure, the results show that after 12 hours of anaerobic culture, the mutans streptococci without extract treatment adhered tightly, the three-dimensional structure of the biofilm had been formed, and the biofilm was thick. In the treatment group with Pu'er raw tea extract, the free mutans streptococci on the carrier were sparse, the bacteria were damaged, and the three-dimensional structure of the biofilm was not formed. The experimental results show that Pu'er raw tea extract can effectively destroy the structure of the biofilm.

[0068] Example 3 Optimization of the preparation process of Pu'er raw tea extract 3.1 Single factor experiment on Pu'er raw tea extraction Five factors, including solid-liquid ratio (1:20, 1:40, 1:60, 1:80, 1:100, 1:120), inoculum amount (0%, 2%, 4%, 6%, 8%, 10%), enzyme addition amount (0%, 2%, 4%, 6%, 8%, 10%), fermentation temperature (31℃, 33℃, 35℃, 37℃, 39℃, 41℃), and fermentation time (12 h, 24 h, 36 h, 48 h, 60 h, 72 h), were selected for the following single factor extraction experiment.

[0069] Strain activation: The Bacillus Velezii MRS plate was activated three times, and a loop of bacteria was taken with a sterile inoculation loop and inoculated into liquid culture medium for 12 h, and then diluted to 10 with sterile saline. 6 -10 8 CFU / mL is reserved.

[0070] The Streptococcus salivarius QS-20 plate was activated three times, and a loop of bacteria was taken with a sterile inoculation loop and inoculated into liquid culture medium for 48 h, and then diluted to 10 with sterile saline. 6 -10 8 CFU / mL is reserved.

[0071] The subsequent enzyme amount is added according to the percentage of dry matter.

[0072] 3.1.1 Experimental methods (1) Single factor of material-liquid ratio Weigh 5 g, 2.5 g, 1.67 g, 1.25 g, 1 g, and 0.83 g of Pu'er raw tea powder, add 100 mL of water, and then sterilize at 121℃ for 15 min. The inoculation amount is 6%, the enzyme amount is 6%, the fermentation temperature is 37℃, and the fermentation time is 48h. After the fermentation is completed, sterilize and inactivate the enzyme in a boiling water bath for 15 min, centrifuge at 12000 r / min for 15 min, take the supernatant, prepare it into freeze-dried powder, and calculate the extraction rate. Repeat 3 times.

[0073] (2) Single factor of inoculum amount: Weigh 6 portions of 1.67 g Pu'er raw tea powder, add 100 mL of water respectively, and then sterilize at 121℃ for 15 min. The inoculum amount is 0%, 2%, 4%, 6%, 8%, 10%, the enzyme amount is 6%, the fermentation temperature is 37℃, and the fermentation time is 48h. After fermentation, sterilize and inactivate the enzyme in a boiling water bath for 15 min, centrifuge at 12000 r / min for 15 min, take the supernatant, prepare it into freeze-dried powder, and calculate the extraction rate. Repeat 3 times.

[0074] (3) Single factor of enzyme inoculation amount: Weigh 6 portions of 1.67 g Pu'er raw tea powder, add 100 mL of water to each portion, and then sterilize at 121℃ for 15 min. Set aside. The inoculation amount is 6%, the enzyme inoculation amount is 0%, 2%, 4%, 6%, 8%, 10%, the fermentation temperature is 37℃, and the fermentation time is 48h. After fermentation, sterilize and inactivate the enzyme in a boiling water bath for 15 min, centrifuge at 12000 r / min for 15 min, take the supernatant, and prepare it into freeze-dried powder. Repeat 3 times.

[0075] (4) Fermentation time single factor: Weigh 6 portions of 1.67 g Pu'er raw tea powder, add 100 mL of water to each portion, and then sterilize at 121℃ for 15 min. The inoculation amount is 6%, the enzyme inoculation amount is 6%, the fermentation temperature is 37℃, and the fermentation time is 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. After fermentation, sterilize and inactivate the enzyme in a boiling water bath for 15 min, centrifuge at 12000 r / min for 15 min, take the supernatant, prepare it into freeze-dried powder, and calculate the extraction rate. Repeat 3 times.

[0076] (5) Fermentation temperature single factor: Weigh 6 portions of 1.67 g Pu'er raw tea powder, add 100 mL of water to each portion, and then sterilize at 121℃ for 15 min. The inoculation amount is 6%, the enzyme inoculation amount is 6%, the fermentation time is 48 h, and the fermentation temperatures are 31℃, 33℃, 35℃, 37℃, 39℃, and 41℃. After fermentation, sterilize and inactivate the enzyme in a boiling water bath for 15 min, centrifuge at 12000 r / min for 15 min, take the supernatant, prepare it into freeze-dried powder, and calculate the extraction rate. Repeat 3 times.

[0077] Calculation formula:

[0078] g1: Net weight of container before freeze-drying g2: Net weight of container after freeze-drying m: Dry weight of Pu'er raw tea 3.1.2 Experimental results and analysis The experimental results are as follows Figure 1 As shown in the figure, the results of the single factor experiment showed that when the inoculum amount was 6%, the extraction rate of Pu'er raw tea extract was the highest; when the inoculum amount was 6%, the extraction rate of Pu'er raw tea extract was the highest; when the fermentation time was 48 h, the extraction rate was the highest; when the fermentation temperature was 35℃, the extraction rate of Pu'er raw tea extract was the highest; when the solid-liquid ratio was 1:60, the extraction rate of Pu'er raw tea extract was the highest.

[0079] 3.2 Orthogonal experiment of Pu'er raw tea extraction 3.2.1 Experimental methods According to the results of the single factor experiment, a five-factor, four-level orthogonal extraction experiment was carried out with the following factors: solid-liquid ratio (1:40, 1:60, 1:80, 1:100), inoculum amount (4%, 6%, 8%, 10%), enzyme addition amount (4%, 6%, 8%, 10%), fermentation temperature (31℃, 33℃, 35℃, 37℃), and fermentation time (36 h, 48 h, 60 h, 72 h).

[0080]

[0081] 3.2.2 Experimental results and analysis Taking the extraction rate as an indicator, the results of the orthogonal experiment are shown in Table 2. The results show that the order of the range R values ​​is A>E>B>D>C, that is, the factor that has the greatest impact on the extraction rate of Pu'er raw tea is the amount of strain added, and the factor with the least impact is the fermentation time.

[0082] According to the orthogonal analysis, the optimal conditions for extracting Pu'er raw tea are A2B2C2D3E2, that is, the inoculum amount is 6%, the enzyme amount is 6%, the fermentation time is 48 h, the fermentation temperature is 35℃, and the solid-liquid ratio is 1:60.

[0083]

[0084] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A method for preparing a Pu'er raw tea extract, characterized in that: The following steps are involved: S1, beating Pu'er raw tea into powder, sieving to obtain Pu'er raw tea powder, adding water to dissolve and sterilizing to obtain Pu'er raw tea liquid; S2, inoculate composite bacteria into Pu'er raw tea liquid, add cellulase and pectinase at the same time, and ferment for 36-60 hours; S3. After sterilizing and inactivating the enzymes in the fermentation liquid, the supernatant is collected by centrifugation and made into freeze-dried powder to obtain the Pu'er raw tea extract.

2. The method for preparing the Pu'er raw tea extract according to claim 1, characterized in that: In step S2, the composite bacteria include: Bacillus Velez MRS with a preservation number of CGMCC No. 18065 and Streptococcus salivarius QS-20 with a preservation number of CGMCC 7.

456.

3. The method for preparing the Pu'er raw tea extract according to claim 2, characterized in that: In step S2, the ratio of Bacillus Velezii MRS to Streptococcus salivarius QS-20 was 1:

1.

4. The method for preparing the Pu'er raw tea extract according to claim 1, characterized in that: The mass ratio of Pu'er raw tea powder and water is 1: (50~70), the total inoculum amount is 4~8%, the total enzyme inoculum amount is 4~8%, and the fermentation temperature is 33~37℃.

5. The method for preparing the Pu'er raw tea extract according to claim 1, characterized in that: The ratio of Pu'er raw tea powder to water is 1:60, the total inoculum amount is 6%, the total enzyme inoculum amount is 6%, the fermentation time is 48 h, and the fermentation temperature is 35°C.

6. A Pu'er raw tea extract, characterized in that: The preparation method is as claimed in claim 1.

7. Use of the Pu'er raw tea extract as claimed in claim 3 in inhibiting Streptococcus mutans.

8. Use of the Pu'er raw tea extract according to claim 3 in the preparation of anticaries and antibacterial drugs.

9. Use of the Pu'er raw tea extract according to claim 3 in preparing oral wash products or oral care products.

10. The use according to claim 9, characterized in that: The oral washing product or oral care product includes toothpaste, tooth powder, antibacterial gel, toothpaste additive, mouthwash, chewing gum or oral spray.

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