Probiotic oral soluble film with oral adhesiveness as well as preparation method and application of probiotic oral soluble film

Abstract

The invention discloses a probiotic oral soluble film with oral adhesiveness and a preparation method and application thereof, and belongs to the technical field of oral care and functional delivery systems. According to the preparation method provided by the invention, the technical limitation of the traditional probiotic carrier on the aspects of activity maintenance and local retention is broken through by combining'probiotic structured packaging 'and'quick release membrane carrier', and a novel delivery strategy with in-situ release and protection functions is provided; the polyelectrolyte composite liquid drop encapsulating the probiotics still keeps certain structural stability in an oral environment, has good surface adhesion ability, forms a biofilm-like structure locally, prolongs residence time, enhances colonization ability and has an ecological intervention function on pathogenic bacteria; the probiotics maintain high activity in the preparation, storage and application processes; and the capability maintaining material is safe, the process is mild, and industrialization feasibility is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oral care and functional delivery system, and particularly relates to a probiotic film with oral adhesion and a preparation method and application thereof. BACKGROUND

[0002] As an important barrier between human body and external environment, the oral cavity is covered with a diverse and complex microbial community, which constitutes a unique oral microecological system. This system plays an irreplaceable role in maintaining oral health and defending against the invasion of exogenous pathogens. In recent years, with the continuous development of microecology and microbial intervention methods, the application of probiotics in oral microecological regulation and disease prevention has attracted more and more attention.

[0003] Previous studies have shown that specific oral symbiotic strains can effectively inhibit pathogenic bacteria such as Streptococcus mutans and Candida albicans through various mechanisms such as secreting antibacterial substances, competing for adhesion sites, and regulating local pH environment, thereby achieving the effects of regulating microecology, preventing dental caries, and preventing oral Candida infection. For example, Streptococcus salivarius K12 has been confirmed to have good inhibitory effect on Candida albicans through mechanisms such as secreting antibacterial peptides (such as Salivaricin A and B) and inhibiting fungal hyphae formation. However, due to the continuous existence of barrier factors such as saliva flushing, microbial competition, and mucosal immune rejection in the oral environment, the colonization efficiency of probiotics on the surface of the oral cavity is often low, and the functional expression time is limited. It is difficult to achieve sustained effect by relying on traditional lozenges or spray forms of "passive delivery".

[0004] Existing probiotic preparations mainly exist in the form of capsules and tablets, and their original design is mainly aimed at intestinal delivery, which is difficult to meet the needs of efficient colonization in the oral cavity. Even probiotic lozenges, sprays, mouthwashes and chewing gums designed for the oral cavity are often difficult to achieve targeted delivery and long-term adhesion due to slow release speed, weak adhesion, short residence time under saliva flushing, and other reasons. Under this background, oral dissolving films as a delivery carrier that can quickly disintegrate in the oral cavity and strongly adhere to the mucosa have attracted attention. However, the oral dissolving films in the current market or existing technology lack adhesion-enhancing structures, and after dissolving, they cannot provide a local protective barrier for probiotics, which leads to the loss of bacteria with saliva, affecting the maintenance of biological activity and colonization effect.

[0005] In recent years, in the research of drug delivery to the oral cavity, new adhesive dosage forms such as so-called "buccal adhesive oral thin films" or "orodispersible films" have appeared, which mainly have the following problems:

[0006] 1) The whole film body is adhered, and there is no structure reservation after release: in the prior art, the adhesion comes from the whole film body, and has nothing to do with the rapid release of probiotics in the film body into the saliva after the film body is disintegrated, so it cannot provide the protection of "in-situ retention" after disintegration;

[0007] 2) Probiotics are easily washed away by saliva: the film body dissolves quickly, the bacteria lack an independent retention mechanism, resulting in a large number of probiotics being quickly washed away after release, and a low colonization rate;

[0008] 3) Lack of microstructure or interface separation function: the current dissolving film is mainly homogeneous dispersion, lacks a structural carrier simulating the natural extracellular environment of microorganisms, and is difficult to effectively protect the activity of bacteria or simulate the microecology of biofilm.

[0009] Therefore, there is still a lack of a delivery strategy for probiotics that can achieve rapid release and generate a microstructure with oral adhesion and biological protection function in-situ. Especially when a high-retention, high-activity, and high-positioning probiotic barrier environment needs to be established, the traditional film body adhesion strategy is difficult to meet the actual needs, and a fundamental innovation is needed in terms of material structure and delivery method. SUMMARY

[0010] The purpose of the present application is to provide a probiotic oral dissolving film with oral adhesion and its preparation method and application, so as to solve the problems that the existing oral dissolving film is difficult to be retained in the local area of the oral mucosa for a long time, cannot provide a local protection barrier for probiotics after dissolving, is easy to be washed away by saliva, has low activity, and has poor function.

[0011] To achieve the above-mentioned purpose, the present application provides a preparation method of a probiotic oral dissolving film with oral adhesion, and the steps are as follows:

[0012] S1, activating and culturing probiotics to the logarithmic growth phase, collecting the bacteria to prepare a bacterial suspension again;

[0013] S2, mixing the bacterial suspension prepared in S1 with a negatively charged polyelectrolyte solution and a positively charged polyelectrolyte solution in sequence, adjusting the pH, and continuing to react to prepare a polyelectrolyte complex droplet encapsulating probiotics;

[0014] S3, dissolving the film-forming matrix, then adding a plasticizer, a disintegrant, and the polyelectrolyte complex droplet encapsulating probiotics prepared in S2 in sequence, mixing uniformly, then pouring into a mold, drying, demolding, and cutting to obtain a probiotic oral dissolving film with oral adhesion.

[0015] Preferably, the probiotics in S1 are single strains or mixed strains; the bacterial cells are resuspended in sterile distilled water, PBS or liquid medium; the concentration of the bacterial suspension is 10 8 -10 10 CFU / mL.

[0016] Preferably, the negatively charged polyelectrolyte in S2 is one or more of sodium hyaluronate, sodium carboxymethyl cellulose and polyglutamic acid; the positively charged polyelectrolyte is one or more of chitosan, type A gelatin and polylysine.

[0017] Preferably, the volume ratio of the bacterial suspension, the negatively charged polyelectrolyte solution and the positively charged polyelectrolyte solution in S2 is 1:4:5; the mass concentration of the negatively charged polyelectrolyte solution and the positively charged polyelectrolyte solution is 0.2-0.3%; the pH is adjusted to 5.5-6.0.

[0018] Preferably, the plasticizer and the disintegrant in S3 are both low-toxic or non-toxic materials; the plasticizer is one or more of glycerol, sorbitol, propylene glycol, polyethylene glycol, maltitol, glyceryl triacetate, triethyl citrate, tributyl citrate; the disintegrant is one or more of croscarmellose sodium, microcrystalline cellulose, crospovidone, sodium starch glycolate, low-substituted hydroxypropyl cellulose, mannitol.

[0019] Preferably, the film-forming matrix in S3 is one or more of hydroxypropyl methylcellulose, pullulan, gelatin, soluble starch derivatives and polyvinyl alcohol.

[0020] Preferably, the mass concentration of the film-forming matrix solution in S3 is 5-8%.

[0021] Preferably, the drying temperature in S3 is 25-40℃, and the drying time is 8-24 hours; the mold is made of inert materials.

[0022] The probiotic buccal film with oral adhesion prepared by the preparation method described above has or does not have auxiliary materials; the auxiliary materials are substances for improving the taste and film performance, including sweeteners, colorants.

[0023] The probiotic buccal film with oral adhesion described above is used in the application of oral microecological regulation and oral disease prevention and control.

[0024] The method provided by the application has the following characteristics:

[0025] 1) replaceability of probiotic species

[0026] The probiotics are not limited to specific strains, and can be selected from various probiotics with oral colonization potential and functional expression capacity according to actual application requirements, such as gram-positive cocci (such as Streptococcus salivarius, Streptococcus oralis, Streptococcus thermophilus); Lactobacillus (such as Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus casei); other functional strains (such as Weissella cibaria, Bacillus subtilis, Bifidobacterium longum, etc.).

[0027] The above strains can be used alone, or can be used in combination according to the principle of functional complementation, so as to enhance the microecological regulation effect.

[0028] 2) Regulation and replacement of droplet structure.

[0029] The selection and proportion of polyelectrolytes can be flexibly adjusted according to the physicochemical properties and adhesion characteristics of the target probiotics. For example: when the oral adhesion of the droplets needs to be enhanced, high molecular weight sodium hyaluronate or chitosan can be preferably used; when the dry stability needs to be improved, polyglutamic acid or gelatin can be added as an auxiliary composite material; the droplet particle size can be controlled in the range of 10-100 μm by adjusting the shear rate, polyelectrolyte concentration and pH value.

[0030] Without departing from the core idea of the present application, the droplet structure can also be used to encapsulate other bioactive ingredients (such as antibacterial peptides, adhesion proteins, mucosal repair factors, etc.) other than probiotics, to expand its functional attributes.

[0031] 3) Flexible adjustment of membrane material and auxiliary system.

[0032] The film liquid matrix and auxiliary components described in the present application have good universality, and can be personalized designed according to the target population, physiological conditions and taste requirements, including: the film forming agent can be replaced by polyvinylpyrrolidone (PVP), soluble starch derivatives, etc.; the type and concentration of plasticizer can be adjusted to improve the mechanical properties and oral disintegration rate; flavoring agents, pigments, mucosal soothing factors, etc. can be added to improve user acceptance.

[0033] 4) Adapt to different clinical and daily application scenarios.

[0034] The oral dissolving film is suitable for the following typical application scenarios: clinical use: used for the auxiliary intervention of oral infections or micro-ecological imbalance diseases such as oral candidiasis, denture stomatitis, radiotherapy-related oral inflammation and the like; daily care: used for the micro-ecological regulation of sub-healthy people such as gingival sensitivity, oral odor, xerostomia syndrome and the like; special groups: such as children, the elderly, postoperative patients and the like who are inconvenient to take capsules or swallow capsules.

[0035] In addition, the film sheet can also be adjusted into a strip-shaped film, a tablet, a foldable or sublingual patch and the like according to requirements.

[0036] Therefore, the present application provides a probiotic oral dissolving film with oral adhesion and a preparation method and application thereof, and the specific technical effects are as follows:

[0037] (1) The present application constructs a new strategy for structured probiotic delivery, by pre-encapsulating probiotics in polyelectrolyte droplets with oral adhesion, and further embedding them in a fast-disintegrating oral dissolving film carrier, realizing the transition from "traditional film release" to "structured droplet point residence", and the released droplets can form a micro-scale in-situ residence structure on the oral mucosa, significantly improving the local colonization ability and effective residence time of probiotics;

[0038] (2) Enhancing the stability and survival rate of probiotics during the preparation process, the polyelectrolyte complex droplets encapsulating probiotics formed by the complex of positively charged polyelectrolytes and negatively charged polyelectrolytes in a mild aqueous phase can encapsulate a variety of probiotics with significant differences in morphology, structure and ecological niche, forming a quasi-three-dimensional microenvironment, effectively resisting the influence of environmental stress (such as dehydration, temperature fluctuation) on the activity of the bacterial body, thereby improving the activity maintenance ability of probiotics during the preparation, storage and application process, and increasing its functional stability;

[0039] (3) Enhancing adhesion, the prepared probiotic oral dissolving film, after release, the polyelectrolyte complex droplets encapsulating probiotics still maintain a certain structural stability in the oral environment, have good surface adhesion ability, can adhere to the tongue coating, oral epithelium or gingiva and the like through charge adsorption, hydrogen bond interaction or polysaccharide-protein interaction mechanism, form a biofilm-like structure locally, prolong the residence time, enhance the colonization ability and the ecological intervention function to pathogenic bacteria; by combining "structured encapsulation of probiotics" with "fast-release film carrier", the technical limitations of traditional probiotic carriers in activity maintenance and local residence are broken through, and a new delivery strategy with in-situ release and protection function is provided;

[0040] (4) The rapid disintegration of the film body and the coupling of the site-specific release function of the droplets are achieved, and the prepared probiotic oral dissolving film can rapidly disintegrate in the oral cavity within 10-60 s to release intact droplet microcapsules, so that the probiotics can be released and accurately gathered at the target site in a short time, and the bioavailability is improved;

[0041] (5) The oral microecological regulation and anti-infection potential are improved, the encapsulated probiotics still have physiological activity after being released, and can inhibit the hyphal transformation and biofilm formation of oral pathogenic bacteria (such as Candida albicans) in an in vitro co-culture model, and show obvious microecological intervention and anti-infection ability, thereby providing an auxiliary intervention means for diseases such as thrush and denture stomatitis;

[0042] (6) The material is safe, the process is mild, and the invention has industrial feasibility. The polyelectrolyte and film-forming material used in the present application are food-grade or medical-grade degradable polymers. The preparation process does not involve organic solvents or high-temperature inactivation steps, and the overall process conditions are mild, which is suitable for large-scale production and the use needs of sensitive groups such as children and the elderly.

[0043] The technical solutions of the present application will be further described below by means of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the description of the embodiments of the present application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0045] Figure 1 is a micrograph of the polyelectrolyte complex droplet encapsulating probiotics prepared in Example 1 of the present application;

[0046] Figure 2 is a photo of the probiotic oral dissolving film based on Streptococcus salivarius K12 prepared in Example 1 of the present application;

[0047] Figure 3 is a fluorescence image of the experimental group in Test Example 3 of the present application;

[0048] Figure 4 is a fluorescence image of the control group in Test Example 3 of the present application. DETAILED DESCRIPTION

[0049] The technical solutions of the present application will be further described below by means of the accompanying drawings and examples.

[0050] In order to make the purpose, technical scheme and advantages of the present application more clear, thorough and complete, the technical scheme of the present application is described clearly and completely below through the drawings and examples. The following detailed description is the description of examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0051] The instrument equipment and reagent materials used in the examples are obtained through commercial channels; the method steps not described in detail in the examples are conventional technical means in the art.

[0052] The preparation method of artificial saliva simulation solution used in the examples is as follows: 0.35 g of NaCl, 0.20 g of KCl, 0.147 g of CaCl2·2H2O, 0.249 g of Na2HPO4·2H2O, 0.496 g of NaH2PO4·H2O, 1.0 g of urea, 2.0 g of mucin are added into about 800 mL of pure water for dissolution, and 0.5 M HCl or NaOH is used for fine adjustment to pH 6.8, and pure water is added to make up the volume to 1 L.

[0053] Example 1

[0054] A probiotic film based on Streptococcus salivarius K12 is prepared, and the specific steps are as follows:

[0055] (1) Preparation of bacterial solution.

[0056] Streptococcus salivarius K12 strain is inoculated in BHI liquid medium and cultured at 37°C and 160 rpm for logarithmic growth phase (OD 600 about 0.6), centrifuged to collect bacterial cells, resuspended with sterile PBS, and adjusted to a concentration of 10 9 CFU / mL for standby.

[0057] (2) Preparation of droplets.

[0058] 0.25 g of sodium hyaluronate (HA) is added to 100 mL of pure water and stirred at room temperature at 200 rpm to dissolve overnight to obtain a HA solution with a mass concentration of 0.25%.

[0059] 0.25 g of chitosan (Chitosan) is added to 80 mL of 0.1% acetic acid aqueous solution, stirred and dissolved, and NaOH powder is added while stirring to adjust the pH to 5.8. After adding pure water to make up to 100 mL, a chitosan (Chitosan) solution with a mass concentration of 0.25% is prepared.

[0060] Take 10 mL (1) prepared bacteria suspension and 40 mL prepared HA solution (volume ratio 1:4) and mix evenly, then mix with chitosan solution at a volume ratio of 1:1, continue to stir at room temperature for 10 minutes to form polyelectrolyte complex droplets encapsulating probiotics. Micrograph of polyelectrolyte complex droplets encapsulating probiotics is shown in Figure 1 .

[0061] (3) Preparation of mouth dissolving film.

[0062] Mix 0.3 g of hydroxypropyl methylcellulose (HPMC, type E5) and 0.3 g of pullulan (mass ratio 1:1) evenly, then dissolve in 10 mL of warm water at 30°C, the total mass concentration of hydroxypropyl methylcellulose and pullulan is 6% (w / v), then add 0.15 g of glycerol (1.5% of the total mass of the solution), 0.15 g of cross-linked sodium carboxymethylcellulose (1.5% of the total mass of the solution), then add 1.5 mL (2) of polyelectrolyte complex droplets encapsulating probiotics (15% of the total solution), mix thoroughly and pour into a polytetrafluoroethylene flat mold, dry at 30°C for 12 hours, peel off the film, cut into 2 cm x 2 cm film pieces (see photo Figure 2 ), and store in a sealed, light-proof container. The film thickness is 70 microns.

[0063] Example 2

[0064] A Lactobacillus reuteri-based probiotic mouth dissolving film was prepared according to the following steps:

[0065] (1) Bacteria solution preparation.

[0066] Inoculate Lactobacillus reuteri DSM 17938 strain into MRS liquid medium, cultivate at 37°C under anaerobic conditions until logarithmic growth phase (OD 600 about 0.6), centrifuge to collect the bacteria, resuspend with sterile PBS buffer, adjust the bacteria concentration to about 10 9 CFU / mL, and reserve for use.

[0067] (2) Droplet preparation.

[0068] Dissolve 0.6 g of polyglutamic acid (γ-PGA) in 100 mL of pure water to obtain a γ-PGA solution with a mass concentration of 0.6%.

[0069] Dissolve 0.5 g of polylysine in 100 mL of pure water to obtain a polylysine solution with a mass concentration of 0.5%.

[0070] The 10 mL (1) prepared bacterial suspension was mixed evenly with 40 mL of the γ-PGA solution (volume ratio of 1:4), and then an equal volume of polylysine solution was added under stirring at 160 rpm. After mixing, the pH was adjusted to 6.0 with 0.5 M HC1, and then the reaction was continued under stirring at room temperature for 10 minutes to form the polyelectrolyte complex droplets encapsulating probiotics.

[0071] (3) Preparation of mouth dissolving film.

[0072] 0.35 g of HPMC and 0.35 g of polyvinyl alcohol (PVA) (mass ratio of 1:1) were dissolved in 10 mL of warm water at 30°C, and the total mass concentration of HPMC and PVA was 7% (w / v). Then 0.2 g of glycerol (2% of the total mass of the solution) and 0.1 g of cross-linked sodium carboxymethyl cellulose (1% of the total mass of the solution) were added. Subsequently, the polyelectrolyte complex droplets encapsulating probiotics prepared in (2) were added (about 15% of the total solution), and the mixture was stirred evenly at 300 rpm. Then the mixture was poured into a silicone mold and dried at 28°C for 16 hours. After peeling off the film, it was cut into 2 cm x 2 cm pieces and stored in the dark after drying. The thickness of the obtained film was about 120 microns.

[0073] Example 3

[0074] A Bacillus subtilis-based probiotic mouth dissolving film was prepared according to the following specific steps:

[0075] (1) Preparation of bacterial solution.

[0076] Bacillus subtilis strain was inoculated into LB liquid medium and cultured at 37°C and 220 rpm until the logarithmic growth phase (OD 600 was about 0.6). The bacterial cells were collected by centrifugation, resuspended with sterile water, and the concentration was adjusted to 10 9 CFU / mL for standby use.

[0077] (2) Preparation of droplets.

[0078] 0.75 g of sodium carboxymethyl cellulose (CMC-Na) was dissolved in 100 mL of pure water to obtain a CMC-Na solution with a mass concentration of 0.75%.

[0079] 1 g of type A gelatin was dissolved in 100 mL of 45°C deionized water to obtain a type A gelatin solution with a mass concentration of 1%.

[0080] 10 mL of the bacteria suspension prepared in (1) was mixed with 40 mL of the CMC-Na solution (volume ratio of 1:4) to form a mixture, and then an equal volume of the gelatin solution of type A was slowly added. After mixing, the temperature of the mixture was maintained at no higher than 40°C, and the pH was adjusted to 5.5 using a 0.5M HC1 solution to form the polyelectrolyte complex droplets encapsulating the probiotic bacteria.

[0081] (3) Preparation of the oral dissolving film.

[0082] 0.3 g of HPMC and 0.3 g of pullulan (mass ratio of 1:1) were dissolved in 10 mL of warm water at 30°C to form a film-forming solution with a total mass concentration of 6% of HPMC and pullulan. Then, 0.15 g of glycerol (1.5% of the total mass of the solution) and 0.1 g of microcrystalline cellulose (1% of the total mass of the solution) were added. Subsequently, 1.5 mL of the polyelectrolyte complex droplets encapsulating the probiotic bacteria obtained in (2) (about 15% of the total mass of the solution) were added, and the mixture was mixed to obtain a film solution. The film solution was poured into a flat mold to spread into a layer, and after drying at 30°C for 12 hours, the film was peeled off and cut into standard pieces of 2 cm x 2 cm for use. The thickness of the obtained film was about 170 microns.

[0083] Test Example 1

[0084] The encapsulation efficiency of the polyelectrolyte complex droplets encapsulating the probiotic bacteria prepared in Examples 1-3 was determined to evaluate the wrapping efficiency of the prepared polyelectrolyte complex droplets for the probiotic bacteria, as follows:

[0085] (1) Preparation of the droplet sample.

[0086] The polyelectrolyte complex droplets encapsulating Streptococcus salivarius, Lactobacillus reuteri, and Bacillus subtilis were prepared according to the methods of (1)-(2) of Examples 1, 2, and 3, respectively.

[0087] (2) Determination of the free bacteria content.

[0088] 5 mL of the polyelectrolyte complex droplets encapsulating the probiotic bacteria prepared in (1) were centrifuged at 1000 x g for 5 minutes, and the supernatant (containing unencapsulated free bacteria) was collected. The supernatant was diluted by ten-fold gradient dilution, and 100 μL of each dilution was inoculated on the corresponding solid culture medium (BHI solid culture medium for Streptococcus salivarius, MRS solid culture medium for Lactobacillus reuteri, and LB solid culture medium for Bacillus subtilis). After incubation at 37°C for 24 hours, the number of colonies was counted to obtain the number of free bacteria.

[0089] (3) Determination of the total bacteria content.

[0090] Another 5 mL of the polyelectrolyte complex droplets encapsulating probiotic bacteria prepared in (1) was added with 0.05% Triton X-100 solution and shaken for 10 minutes to fully destroy the droplet structure and release all the encapsulated bacteria, and then gradient dilution and plate counting were performed by the method in (2) to obtain the total number of bacteria.

[0091] (4) Calculation of encapsulation efficiency.

[0092] The encapsulation efficiency was calculated by the following formula:

[0093] Encapsulation efficiency (%) = (total number of bacteria - free bacteria) / total number of bacteria x 100%.

[0094] Each group of samples was determined in triplicate, and the average value was taken as the result, as shown in Table 1.

[0095] Table 1 shows the results of the determination of the encapsulation efficiency of probiotic bacteria

[0096]

[0097] In Examples 1-3, three types of typical probiotic bacterial strains were selected: gram-positive coccus Streptococcus salivarius, Lactobacillus reuteri, and Bacillus subtilis with spore formation ability, to systematically verify the adaptability of the method provided by the present application to different types of bacteria. Using the method provided by the present application, the three types of strains were successfully encapsulated, and all showed good encapsulation efficiency. The results show that the method provided by the present application is not only suitable for probiotic species with significant differences in morphology, structure and ecological niche, but also suitable for constructing diversified functional combinations, providing experimental evidence for realizing personalized microecological intervention or multi-target synergistic treatment.

[0098] Test Example 2

[0099] The number of viable probiotic bacteria in the probiotic oral dissolving films prepared in Examples 1-3 was determined as follows:

[0100] (1) Sample treatment.

[0101] One piece of the probiotic oral dissolving film prepared in each of Examples 1-3 (specification: 2 cm x 2 cm) was cut into pieces and added into 10 mL of sterile phosphate buffer (PBS) in a rotary shaker at a speed of 100 rpm for 15 minutes to fully disintegrate the film and release the encapsulated probiotic bacteria droplets. The obtained suspension was vortexed for 30 seconds and ready for use.

[0102] (2) Viable bacteria counting.

[0103] The bacterial suspension corresponding to each membrane was subjected to ten-fold serial gradient dilution, and 100 μL of each was inoculated on the corresponding solid medium plate: Streptococcus salivarius used BHI medium, Lactobacillus reuteri used MRS medium, and Bacillus subtilis used LB medium. After inoculation, the plates were incubated at 37°C for 24 hours, and the colony forming units (CFU) were counted, and the number of viable probiotics in each membrane was calculated using the following formula:

[0104] Number of bacteria (CFU / piece) = number of plated bacteria × dilution factor × 100.

[0105] Each group of samples was detected in triplicate, and the results were averaged. The results are shown in Table 2.

[0106] Table 2: Statistical results of the number of viable probiotics

[0107]

[0108]

[0109] It can be seen that the probiotic oral dissolving film prepared by the method provided by the present application can effectively maintain the physiological activity of the probiotics. The survival rate of probiotics in the oral dissolving films prepared in Examples 1 to 3 is more than 80%, which shows that the method provided by the present application not only realizes the encapsulation and release of probiotics, but also provides a certain microenvironment protection effect under dry conditions, reducing the damage of temperature and water loss stress to bacteria. It shows that the preparation method provided by the present application has good stability of probiotics under actual preparation and storage conditions, which lays a foundation for its application in industrial production.

[0110] Test Example 3

[0111] The adhesion behavior of the probiotic droplets formed after the release of the probiotic oral dissolving films prepared in Examples 1-3 on the simulated oral surface was evaluated, and compared with the control oral dissolving film without droplet encapsulation, and only with probiotics uniformly dispersed in the film body, to verify the functional contribution of the polyelectrolyte complex droplet structure provided by the present application in improving the adhesion ability, as follows:

[0112] (1) Construction of an oral cavity substrate.

[0113] A three-dimensional microstructure substrate simulating the oral epithelial surface was constructed using PDMS (polydimethylsiloxane) material. The periodic convex structure on the substrate surface was constructed by a soft etching mold (microsphere array imprinting), and the individual convexity was square-shaped with a side length of 100 μm and a pitch of 20 μm, simulating the geometric characteristics of the tongue back or oral mucosa surface microprotrusions, thereby providing a more realistic surface roughness and local fluid perturbation environment.

[0114] The PDMS substrate was treated with oxygen plasma for 30 seconds to enhance hydrophilicity, and then immediately immersed in a 0.5% (w / v) porcine gastric mucin (PGM) aqueous solution and incubated at 4°C for 16 hours to construct a glycoprotein-rich simulated oral mucosal layer. After modification, the PDMS substrate was gently washed with deionized water three times and naturally air-dried for use.

[0115] (2) Membrane release and adhesion experiment.

[0116] The probiotic oral dissolving film prepared in Example 1 was cut into 1 cm x 1 cm pieces and placed in the center of the PDMS microstructure substrate prepared in (1). 20 μL of pre-warmed artificial saliva simulation solution was added. After standing at 37°C for 5 minutes, the film was allowed to disintegrate and release the probiotic droplets, which were recorded as the experimental group.

[0117] Control group: In (2) of Example 1, 10 mL of the bacterial suspension prepared in (1) was mixed with 90 mL of PBS to obtain a non-encapsulated probiotic suspension. The oral dissolving film was prepared in the same way as in Example 1, except that 1.5 mL of the polyelectrolyte complex droplet prepared in (2) was replaced with the non-encapsulated probiotic suspension prepared by the above method.

[0118] (3) Confocal microscope observation and fluorescence analysis.

[0119] To achieve visualization, all probiotics were first stained with Syto9 green fluorescent dye (Invitrogen LIVE / DEAD BacLight Kit) before the preparation of the oral dissolving film. After labeling, the probiotics were washed and used to prepare the polyelectrolyte complex droplet or the oral dissolving film. The porcine gastric mucin on the simulated oral substrate was labeled with Cascade Blue NHS ester (blue fluorescence) before confocal imaging to visualize the mucin layer and facilitate the determination of the position of the droplet and bacteria relative to the simulated oral interface.

[0120] After the release in (1), the retention behavior of the droplets on the simulated substrate surface was observed using a confocal laser scanning microscope (CLSM). Three-dimensional adhesion images were obtained by Z-stack scanning, focusing on the following observations:

[0121] 1) Whether the droplets remain in place and adhere to the protrusions between them;

[0122] 2) Whether the bacteria form clusters in the droplet structure;

[0123] 3) Whether a more obvious adhesion is formed compared to the scattered and released bacteria in the control film.

[0124] (4) Quantitative analysis and comparison.

[0125] The number of adherent bacteria was counted by image analysis of the total fluorescence intensity (fluorescence integral per unit area) of the adherent surface and the number of significantly aggregated regions. Image J was used to analyze the fluorescence pixel distribution of the adherent area, and the adherent area and average fluorescence signal intensity were counted to compare the adherent density of the droplet group and the non-droplet group.

[0126] As shown in Figure 3 and Figure 4 , all the orally dissolving films in the experimental group formed uniform adherent plaques on the PDMS microstructure substrate after release, and the adherent bacteria aggregated into groups, showing a biofilm-like distribution. The adherent area was mainly distributed near the microstructure recess gap Figure 3 . The bacteria released by the control group showed scattered distribution, most of which were washed away by artificial saliva, and the total fluorescence signal on the surface was significantly lower than that of the experimental group Figure 4 , with an average reduction of about 95-97%.

[0127] It is shown that the structure of the orally dissolving film prepared by the present application not only helps to protect the integrity of probiotics during release, but also promotes the "in situ adhesion" behavior on the microstructure interface, which is the key mechanism to achieve site-specific retention.

[0128] Therefore, the preparation method provided by the present application combines "structured encapsulation of probiotics" with "fast-release film carrier", breaks through the technical limitations of traditional probiotic carriers in terms of activity maintenance and local retention, and provides a new delivery strategy with in-situ release and protection functions. The polyelectrolyte complex droplets encapsulating probiotics still maintain a certain structural stability in the oral environment, have good surface adhesion capacity, form a biofilm-like structure locally, prolong the retention time, enhance the colonization ability and ecological intervention function on pathogenic bacteria. The probiotics maintain high activity during preparation, storage and application. The material safety and mild process have industrialization feasibility.

[0129] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method of preparing a probiotic orally dissolving film having oral adhesion, characterized in that, The steps are as follows: S1, activating the probiotics to logarithmic growth phase, collecting the bacteria to prepare the bacteria suspension again; S2, mixing the bacteria suspension prepared in S1 with the negatively charged polyelectrolyte solution and the positively charged polyelectrolyte solution in turn, adjusting the pH and continuing to react to prepare the polyelectrolyte composite droplets encapsulating the probiotics; S3, dissolving the film-forming matrix, then adding the plasticizer, disintegrant and the polyelectrolyte composite droplets encapsulating the probiotics prepared in S2 in turn, mixing uniformly, then pouring into the mold, drying, demolding and cutting to obtain the probiotic oral dissolving film with oral adhesion.

2. A process for the preparation of a probiotic film with oral adhesive properties according to claim 1, characterized in that: The probiotics in S1 are single strains or mixed strains; the bacterial cells are resuspended in sterile distilled water, PBS or liquid medium; the bacterial concentration in the bacterial suspension is 10 8 ~ 10 10 CFU / mL.

3. The process for the preparation of a probiotic film with oral adhesive properties according to claim 1, characterized in that: The negatively charged polyelectrolyte in S2 is one or more of sodium hyaluronate, sodium carboxymethyl cellulose and polyglutamic acid; the positively charged polyelectrolyte is one or more of chitosan, type A gelatin and polylysine.

4. The process for the preparation of a probiotic film with oral adhesive properties according to claim 1, characterized in that: The volume ratio of the bacteria suspension, the negatively charged polyelectrolyte solution and the positively charged polyelectrolyte solution in S2 is 1:4:5; the mass concentration of the negatively charged polyelectrolyte solution and the positively charged polyelectrolyte solution is 0.2-0.3%; the pH is adjusted to 5.5-6.

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5. The process for the preparation of a probiotic film with oral adhesive properties according to claim 1, characterized in that: The plasticizer and the disintegrant in S3 are both low-toxic or non-toxic materials; the plasticizer is one or more of glycerol, sorbitol, propylene glycol, polyethylene glycol, maltitol, glyceryl triacetate, triethyl citrate, tributyl citrate; the disintegrant is one or more of cross-linked sodium carboxymethyl cellulose, microcrystalline cellulose, cross-linked povidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and mannitol.

6. The process for the preparation of a probiotic film with oral adhesive properties according to claim 1, characterized in that: The film-forming matrix in S3 is one or more of hydroxypropyl methylcellulose, pullulan, gelatin, soluble starch derivatives and polyvinyl alcohol.

7. A process for the preparation of a probiotic film with oral adhesive properties according to claim 1, characterized in that: The mass concentration of the film-forming matrix solution in S3 is 5-8%.

8. A process for the preparation of a probiotic film with oral adhesive properties according to claim 1, characterized in that: The drying temperature in S3 is 25-40℃, and the drying time is 8-24 hours; the mold is made of inert base material.

9. The probiotic film having oral adhesion prepared by the preparation method according to any one of claims 1 to 8, wherein: The probiotic oral dissolving film with oral adhesion can be added with or without auxiliary materials; the auxiliary materials are substances for improving the taste and film performance, including sweeteners and colorants.

10. The probiotic oral dissolving film with oral adhesion according to claim 9 in the application of oral microecological regulation and oral disease prevention and control.

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