Solid formulations for formulating probiotic hydrogel formulations, methods of making and use

Abstract

The application provides a solid preparation for preparing a probiotic hydrogel preparation, a preparation method and application. The solid preparation comprises probiotics, prebiotics, anionic polysaccharides and lyophilization protective agents; the molecular weight of the anionic polysaccharides is less than 400 KDa, and the Zeta potential is less than-20 mV; the anionic polysaccharides are anchored on the surface proteins of the probiotics through a π-π conjugate bond of hydrogen bonds, and the probiotics are uniformly dispersed in the prebiotics. The anionic polysaccharides in the solid preparation can penetrate the extracellular polysaccharide layer of the probiotics, form a physical protection through hydrogen bonds and anchor the surface proteins of the probiotics, and the anionic polysaccharides are negatively charged, so that the probiotic bacteria are prevented from gathering through electrostatic repulsion. The probiotic hydrogel preparation based on the solid preparation has excellent long-acting stability and long-acting antibacterial property, and solves the problems of poor uniformity and poor activity stability of the probiotic preparation in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a solid preparation for formulating probiotic hydrogel formulations, the probiotic hydrogel formulations, and the preparation method thereof. Background Technology

[0002] In the clinical treatment of vaginal infections, bacterial vaginosis and candidal vaginitis are mainly caused by the abnormal proliferation of pathogenic microorganisms such as Candida albicans and Gardnerella vaginalis. These can lead to vaginal mucosal damage, recurrent inflammation of the reproductive system, and even increase the risk of premature birth. Current treatment regimens primarily rely on antibiotics such as metronidazole and clindamycin. While these can rapidly inhibit pathogens, long-term use can disrupt the vaginal microecological balance, induce the formation of drug-resistant strains, and fail to rebuild the defense barrier dominated by lactobacilli, resulting in a persistently high recurrence rate. In recent years, probiotic-based microecological regulation methods have gained increasing attention. By colonizing functional lactobacilli, these methods can directly and competitively inhibit the adhesion of pathogenic bacteria, thereby restoring the vaginal acidic environment and regulating local immune responses. This provides a new direction for solving the problems of drug resistance and recurrence associated with traditional antibacterial treatments.

[0003] Because probiotic powders are still easily affected by external environmental factors in non-aqueous systems, leading to a decrease in their activity (such as temperature and humidity), the combined application of probiotics and prebiotics has gradually become a research hotspot. As indigestible dietary components, prebiotics can selectively stimulate the growth and metabolic activity of probiotics in the host's gut, forming a "bacteria-prebiotic synergistic effect".

[0004] However, in existing technologies, traditional probiotic solid dosage form processes (such as dry mixing and tableting) struggle to achieve uniform dispersion and long-lasting protection of the bacterial powder within the prebiotics. For example, prior art CN119174495A discloses a plant prebiotic, its composition, and its application, which involves mixing plant prebiotics, traditional prebiotics, and freeze-dried probiotic powder in powder form to obtain a probiotic solid dosage form. Prior art CN112999305A discloses a solid dosage form for regulating intestinal flora imbalance and its preparation method, which involves mixing traditional Chinese medicine powder, probiotics, and prebiotics in powder form in a three-dimensional mixer to obtain a solid dosage form. On the one hand, powder mixing makes it difficult to ensure uniform distribution of probiotics within the prebiotics, resulting in poor product uniformity. In practical applications, the ratio of probiotics to prebiotics may vary significantly between different batches or even between different parts of the same batch, greatly affecting the stability and effectiveness of the product quality. On the other hand, when powdered probiotics are directly mixed, some probiotics are not surrounded by prebiotics and other excipients, so the probiotics cannot absorb the prebiotics well to proliferate. Furthermore, the probiotics are easily affected by external environmental factors (such as temperature, humidity, oxygen, etc.), which can cause their activity to gradually decrease, thereby shortening the product's shelf life. Summary of the Invention

[0005] To solve all or part of the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] A first aspect of the present invention provides a solid dosage form for formulating probiotic hydrogel preparations, the solid dosage form comprising probiotics, prebiotics, anionic polysaccharides and lyophilization protectants;

[0007] The anionic polysaccharide has a molecular weight of less than 400 kDa and a zeta potential of less than -20 mV. The anionic polysaccharide forms a π-π conjugated anchoring structure with the protein on the surface of the probiotic through hydrogen bonds, and the probiotic is uniformly dispersed in the prebiotic.

[0008] The solid formulation provided by this invention contains anionic polysaccharides with a molecular weight below 400 kDa, exhibiting nanoscale penetration characteristics. Its molecular chains can penetrate the extracellular polysaccharide layer of probiotics, forming a π-π conjugated anchoring structure with the S-layer proteins on the bacterial surface through intermolecular hydrogen bonds, thus constructing a nanoscale protective barrier. Furthermore, the anionic polysaccharides have a Zeta potential below -20 mV, and the negatively charged flexible chains create a steric hindrance effect through electrostatic repulsion, allowing probiotics to be uniformly dispersed within the prebiotics. This enables the prebiotics to effectively encapsulate the probiotics, providing them with nutrients and physical protection. The solid formulation provided by this invention solves the problems of activity attenuation and dosage form inhomogeneity caused by bacterial aggregation in existing probiotic formulations.

[0009] In some embodiments, the solid dosage form is obtained by freeze-drying a homogeneous dispersion containing the probiotics, prebiotics, anionic polysaccharides, and a freeze-drying protectant. By uniformly dispersing the probiotics, prebiotics, anionic polysaccharides, and freeze-drying protectant in a solvent, the anionic polysaccharides can form π-π conjugated anchoring structures with proteins on the surface of the probiotics through hydrogen bonding, improving the uniformity of probiotic distribution within the prebiotics. The freeze-drying protectant provides protection during the freeze-drying process. However, if a dry mixing method is used, such as directly mixing the solid powders of probiotics, prebiotics, and anionic polysaccharides to form a solid dosage form, hyaluronic acid cannot form hydrogen bonds with the probiotics, leading to probiotic aggregation.

[0010] In some embodiments, the anionic polysaccharide includes one or more combinations of glycosaminoglycan derivatives, plant-derived anionic oligosaccharides, or chemically modified anionic polysaccharides.

[0011] In some preferred embodiments, the anionic polysaccharide includes glycosaminoglycan derivatives, and more preferably includes hyaluronic acid. When the anionic polysaccharide includes small molecule hyaluronic acid with a molecular weight of less than 400 kDa and a Zeta potential of less than -20 mV, the probiotics exhibit excellent uniform dispersion in the prebiotics.

[0012] Furthermore, the glycosaminoglycan derivative includes one or more combinations of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparin oligosaccharides.

[0013] Furthermore, the plant-derived anionic oligosaccharides include one or more combinations of sodium alginate oligosaccharides, low-esterified pectin oligosaccharides, and λ-carrageenan oligosaccharides.

[0014] Furthermore, the chemically modified anionic polysaccharide includes carboxymethyl chitosan and / or sulfonated cellulose nanocrystals.

[0015] In some preferred embodiments, the molecular weight of the anionic polysaccharide is below 10 kDa. Using anionic polysaccharides with a molecular weight below 10 kDa, such as hyaluronic acid with a molecular weight below 10 kDa, results in lower viscosity and easier homogenization with other components.

[0016] In some embodiments, the zeta potential of the anionic polysaccharide is -20mV to -40mV. This enables excellent steric occlusion, allowing probiotics to be uniformly dispersed within the prebiotics without aggregation.

[0017] In some embodiments, the solid dosage form comprises, by weight percentage, 1% to 5% probiotics, 10% to 30% prebiotics, 60% to 80% lyophilization protectant, and 1% to 5% hyaluronic acid.

[0018] In some embodiments, the solid dosage form comprises, by weight percentage, 1% to 5% probiotic powder, 10% to 30% inulin, 10% to 30% skim milk powder, 20% to 40% trehalose, 10% to 30% maltodextrin, and 1% to 5% hyaluronic acid.

[0019] In some embodiments, the probiotics include one or more combinations of lactobacillus, bifidobacterium, thermophilic streptococcus, and yeast.

[0020] In some embodiments, the prebiotic includes one or more combinations of oligosaccharides, natural polysaccharides and their derivatives, and human milk oligosaccharides.

[0021] In some embodiments, the freeze-drying protectant includes one or more combinations of sugars, alcohols, amino acids, salts, and proteins.

[0022] In some embodiments, the lactobacillus includes one or more combinations of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus curvatureii, Lactobacillus fermentum, Lactobacillus gasseri, and Lactobacillus janseri.

[0023] In some embodiments, the Bifidobacterium includes one or more combinations of Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum.

[0024] In some embodiments, the oligosaccharide includes one or more combinations of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, chitosan oligosaccharides, and mannan oligosaccharides.

[0025] In some embodiments, the natural polysaccharides and derivatives include one or more combinations of inulin, lactulose, and resistant dextrin.

[0026] In some embodiments, the human milk oligosaccharide includes one or more combinations of 2'-fucosylated lactose and 3'-sialylated lactose (3'-SL).

[0027] In some embodiments, the carbohydrates include one or more combinations of glucose, mannose, galactose, fructose, trehalose, maltose, sucrose, lactose, maltotriose, dextran, and dextrin.

[0028] In some embodiments, the alcohols include one or more combinations of sorbitol, mannitol, inositol, xylitol, and glycerol.

[0029] In some embodiments, the amino acid substance includes one or more combinations of glycine, arginine, and monosodium glutamate.

[0030] In some embodiments, the salts include one or more combinations of sodium sulfate, calcium lactate, monosodium glutamate, sodium chloride, sodium thiosulfate, ammonium acetate, and ammonium chloride.

[0031] In some embodiments, the protein-like substances include one or more combinations of skim milk powder, gelatin, serum, peptone, yeast, and broth.

[0032] A second aspect of the present invention provides a method for preparing a solid formulation of a probiotic hydrogel preparation as described in any of the technical solutions, the method comprising: uniformly dispersing anionic polysaccharides, probiotics, prebiotics and a freeze-drying protectant in a first solvent, and performing freeze-drying to obtain the solid formulation.

[0033] The preparation method provided by the present invention adds anionic polysaccharides in solution to anchor negatively charged long chains on the surface of probiotics. The electrostatic repulsion prevents the probiotic cells from aggregating, and the probiotics are uniformly distributed in the prebiotics in the resulting solid preparation.

[0034] In some embodiments, the preparation method specifically includes:

[0035] The anionic polysaccharide is dispersed in a first solvent to obtain a first dispersion;

[0036] The first dispersion is mixed with probiotics until a homogeneous phase is obtained to obtain the second dispersion.

[0037] The second dispersion is uniformly mixed with prebiotics and lyophilization protectant to obtain a homogeneous phase, and the third dispersion is then freeze-dried to obtain a solid formulation.

[0038] In some embodiments, the first solvent includes water.

[0039] In some typical embodiments, the preparation method specifically includes the following steps:

[0040] The anionic polysaccharide was dispersed in water and stirred at 200–600 rpm at room temperature for 10–30 min until a homogeneous phase was obtained, thus obtaining the first dispersion.

[0041] Add probiotics to the first dispersion and stir at room temperature for 10-30 minutes at 200-600 rpm until a homogeneous phase is obtained to obtain the second dispersion;

[0042] Add the prebiotics and freeze-drying protectant to the second dispersion, and stir at room temperature (200-600 rpm) for 10-30 minutes until a homogeneous phase is obtained to obtain the third dispersion;

[0043] The third dispersion was frozen solidified, then freeze-dried for 40-60 hours, and then crushed into powder to obtain the solid preparation.

[0044] A third aspect of the present invention provides a probiotic hydrogel formulation, which includes the solid formulation described in any of the technical solutions and also includes a liquid formulation, wherein the liquid formulation includes a gel matrix and a second solvent.

[0045] The probiotic hydrogel formulation provided by this invention exhibits uniform probiotic distribution. In some embodiments, the coefficient of variation (CV) of the statistical activity test results is ≤10%, and the preparation process is feasible for continuous production. The probiotic hydrogel formulation provided by this invention demonstrates long-lasting and stable activity, with excellent long-lasting antibacterial properties. In some embodiments, after 30 weeks of storage, the probiotic activity remains at least 70%, and the inhibition rate against Gardnerella vaginalis remains at least 99%, while the inhibition rate against Candida albicans remains at least 95%. This probiotic hydrogel formulation can be widely used in the clinical treatment and prevention of gynecological inflammations such as bacterial vaginosis and candidal vaginitis, showing significant industrial application prospects in the field of microecological preparations.

[0046] In some embodiments, the gel matrix comprises one or more combinations of polyoxyethylene-polyoxypropylene block copolymers, cellulose and / or its derivatives, synthetic polymers, polysaccharides, starch and / or its derivatives.

[0047] Furthermore, the polyoxyethylene-polyoxypropylene block copolymer includes, but is not limited to, one or more of poloxamer 407, poloxamer 338, and poloxamer 188.

[0048] Furthermore, the cellulose and / or its derivatives include, but are not limited to, one or more of methylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and ethylcellulose.

[0049] Furthermore, the synthetic polymer includes, but is not limited to, one or more of the following: povidone, copovidone, polyethylene glycol, polyester-polyethylene glycol copolymer, polyvinyl alcohol, and poly(N-isopropylacrylamide).

[0050] Furthermore, the polysaccharide includes, but is not limited to, one or more of gelatin, chitosan, agarose, xanthan gum, carrageenan, sodium alginate, pectin, and dextran.

[0051] Furthermore, the starch and its derivatives include, but are not limited to, one or more of starch, carboxymethyl starch, pregelatinized starch, hydroxyethyl starch, cross-linked starch, and starch phosphate.

[0052] In some embodiments, the second solvent includes water, but is not limited thereto.

[0053] In some embodiments, the content of the gel matrix in the liquid formulation is 18% to 24%.

[0054] In some embodiments, the solid and liquid formulations are prepared and used immediately upon application.

[0055] In some embodiments, the mass ratio of the solid dosage form to the liquid dosage form is 1:10 to 1:50.

[0056] In some embodiments, the probiotic hydrogel formulation exhibits an activity of not less than 70% after being stored at 4°C in a nitrogen environment for 30 weeks, and the CV value of the probiotic hydrogel formulation is ≤10%.

[0057] A fourth aspect of the present invention provides a method for preparing the probiotic hydrogel formulation, the method comprising:

[0058] The solid dosage form is prepared according to any of the methods described above;

[0059] The solid formulation and the liquid formulation are mixed uniformly at 0-4°C and gelled at 25°C-40°C to obtain a probiotic hydrogel formulation.

[0060] The fifth aspect of the present invention provides the use of the probiotic hydrogel formulation in the preparation of products having the function of inhibiting or killing Gardnerella vaginalis, Candida albicans, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus or Escherichia coli.

[0061] Compared with the prior art, the present invention has at least the following beneficial effects:

[0062] (1) In the solid preparation provided by the present invention, the molecular chain of small molecule anionic polysaccharide can penetrate the extracellular polysaccharide layer of probiotics and form a π-π conjugated bond anchoring structure with the surface protein of bacteria through intermolecular hydrogen bonds, thereby constructing a nanoscale protective barrier; and, since the anionic polysaccharide is negatively charged, it can form a steric hindrance effect through electrostatic repulsion, thereby improving the uniformity of the distribution of probiotics in prebiotics and reducing the aggregation of probiotics.

[0063] (2) As the spatial distribution uniformity of probiotics in prebiotics is improved, prebiotics can effectively encapsulate probiotics, thereby providing probiotics with nutrients and physical protection more effectively, improving their activity stability, and improving their uniformity. In some embodiments, the statistical activity test results show a coefficient of variation (CV) value ≤10%.

[0064] (3) The probiotic hydrogel preparation provided by this invention has good antibacterial effects against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, especially Gardnerella vaginalis which causes bacterial vaginosis, and Candida albicans which causes vulvovaginitis. In some embodiments, the inhibition rate against Gardnerella vaginalis can reach over 99%, and the inhibition rate is still not less than 99% after storage at 4°C in an inert gas environment for 30 hours; the inhibition rate against Candida albicans can reach over 95%, and the inhibition rate is still not less than 95% after storage at 4°C in an inert gas environment for 30 hours. Furthermore, after co-culturing the probiotic hydrogel preparation with Candida albicans and Gardnerella vaginalis, the probiotics still retain their activity, which has potential application prospects for recurrent vaginitis.

[0065] (4) In the technical solution provided by the present invention, the preparation method of solid dosage form only requires simple conventional operation steps such as stirring, freezing, and freeze drying, without complicated equipment and processes. In the whole preparation process, the raw materials are mixed evenly, the reaction conditions are mild and easy to control, which can meet the requirements of large-scale production, reduce production costs, and facilitate the industrial promotion and application of the product. Attached Figure Description

[0066] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0067] Figure 1a , Figure 1b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation of Example 1 are shown below. Figure 1a , Figure 1b (respectively ×20 and ×40).

[0068] Figure 2a , Figure 2b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation in Example 6 ( Figure 2a , Figure 2b (respectively ×20 and ×40).

[0069] Figure 3a , Figure 3b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation of Comparative Example 1 ( Figure 3a , Figure 3b (respectively ×20 and ×40).

[0070] Figure 4a , Figure 4b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation of Comparative Example 2 ( Figure 4a , Figure 4b (respectively ×20 and ×40).

[0071] Figure 5 The CV plot shows the statistical results of the reactive coating plates (MRS agar plates) of Example 1 and Comparative Example 1.

[0072] Figure 6 The results show the activity (MRS agar plate coating) and stability of the probiotic hydrogel formulation of Comparative Example 3 under different storage conditions.

[0073] Figure 7 The results show the long-term activity (MRS agar plate coating) stability of the probiotic hydrogel formulation of Comparative Example 1 under different storage conditions.

[0074] Figure 8 The results show the long-term activity (MRS agar plate coating) stability of the probiotic hydrogel formulation of Example 1 under different storage conditions.

[0075] Figure 9The results show the long-term activity (MRS agar plate coating) stability of the probiotic hydrogel formulation of Example 2 under different storage conditions.

[0076] Figure 10 The results show the long-term activity (MRS agar plate coating) stability of the probiotic hydrogel formulation of Example 3 under different storage conditions.

[0077] Figure 11 The hydrogel formulation of the probiotic composition in Example 1 demonstrates the antibacterial effect against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Escherichia coli.

[0078] Figure 12 This is the standard curve for the C. albicans RPB1 gene.

[0079] Figure 13 The results show the inhibition rate of the probiotic hydrogel formulations in Examples 1-4 against Candida albicans.

[0080] Figure 14 This is the standard curve for the G. vaginalis gyrB gene.

[0081] Figure 15 The results show the inhibition rate of the probiotic hydrogel formulations in Examples 1-4 against Gardnerella vaginalis.

[0082] Figure 16 The results of co-culturing the culture medium with Candida albicans and Gardnerella vaginalis in Example 1 on MRS agar plates are shown.

[0083] Figure 17 The results of in vivo antibacterial (qPCR) in mice of Examples 1, 4, and 5 and the model group are shown. Detailed Implementation

[0084] The technical solutions of the present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention. The specific functional details disclosed herein should not be construed as limiting, but are merely intended to form the basis of the claims and to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0085] In addition, unless otherwise specified, all raw materials used in the following embodiments can be purchased from the market or other sources, and all production and testing equipment used are known in the art, as are the testing methods used.

[0086] Example 1

[0087] This embodiment provides a probiotic gel formulation, which includes a solid formulation and a liquid formulation. The raw material composition of the solid formulation is shown in Table 1, and the raw material composition of the liquid formulation is shown in Table 2.

[0088] The preparation method of the probiotic gel formulation in this embodiment includes:

[0089] According to the raw material ratio shown in Table 1, small molecule hyaluronic acid was dispersed in water and stirred at 500 rpm at room temperature for 20 min until a homogeneous phase was formed to form the first dispersion.

[0090] Add Lactobacillus paracasei powder to the first dispersion, stir at 500 rpm at room temperature for 10 min until a homogeneous phase is formed to form the second dispersion;

[0091] Inulin, maltodextrin, trehalose, and skim milk powder were added to the second dispersion in the proportions shown in Table 1. The mixture was stirred at 500 rpm at room temperature for 30 min until a homogeneous phase was obtained.

[0092] The mixture was placed in a -80°C freezer for 2 hours and then freeze-dried for 48 hours. The solid preparation was then crushed with a glass rod.

[0093] When using, mix the solid formulation and the liquid formulation at a mass ratio of 1:33 to form a probiotic gel formulation.

[0094] Table 1. Raw material ratios for solid dosage forms in Example 1

[0095]

[0096] Table 2. Raw material ratios for liquid formulations in Example 1

[0097] raw material weight ratio Polosham 407 22％ Deionized water Replenish to 100%

[0098] Example 2

[0099] The only difference between Example 2 and Example 1 is that the solid dosage form raw material composition of Example 2 is shown in Table 3. The rest is the same as that of Example 1 and will not be repeated here.

[0100] Table 3

[0101]

[0102] Example 3

[0103] The only difference between Example 3 and Example 1 is that the solid dosage form raw material composition of Example 3 is shown in Table 4. The rest is the same as that of Example 1 and will not be repeated here.

[0104] Table 4

[0105]

[0106] Example 4

[0107] The only difference between Example 4 and Example 1 is that in Example 4, the solid formulation from Example 3 was stored at 4°C and in a N2 environment for 30 hours before being mixed with the liquid formulation to form a hydrogel formulation. The rest of the process was the same as in Example 1 and will not be repeated here.

[0108] Example 5

[0109] The only difference between Example 5 and Example 1 is that the solid dosage form raw material composition of Example 5 is shown in Table 5. The rest is the same as that of Example 1 and will not be repeated here.

[0110] Table 5

[0111]

[0112]

[0113] The hyaluronic acid used in this embodiment was purchased commercially and its molecular weight was marked as 40-100 kDa.

[0114] Example 6

[0115] The only difference between Example 6 and Example 1 is that the solid dosage form raw material composition of Example 6 is shown in Table 6. The rest is the same as that of Example 1 and will not be repeated here.

[0116] Table 6

[0117]

[0118] The hyaluronic acid used in this embodiment was purchased commercially, and the molecular weight of the product was marked as 200-400 kDa.

[0119] Example 7

[0120] The only difference between Example 7 and Example 1 is that the solid dosage form raw material composition of Example 7 is shown in Table 7. The rest is the same as that of Example 1 and will not be repeated here.

[0121] Table 7

[0122]

[0123]

[0124] Example 8

[0125] The only difference between Example 8 and Example 1 is that the solid dosage form raw material composition of Example 8 is shown in Table 8, and the rest is the same as that of Example 1, which will not be repeated here.

[0126] Table 8

[0127]

[0128] Example 9

[0129] The only difference between Example 9 and Example 1 is that the solid dosage form raw material composition of Example 9 is shown in Table 9, and the rest is the same as that of Example 1, which will not be repeated here.

[0130] Table 9

[0131]

[0132] Example 10

[0133] The only difference between Example 10 and Example 1 is that the solid dosage form raw material composition of Example 10 is shown in Table 10. The rest is the same as that of Example 1, and will not be repeated here.

[0134] Table 10

[0135]

[0136] Example 11

[0137] Example 11 is basically the same as Example 1, except that the raw material ratios for solid and liquid dosage forms are changed according to Table 11 and Table 12, respectively.

[0138] Table 11. Raw material ratios for solid dosage forms in Example 11

[0139] raw material weight ratio Lactobacillus paracasei powder 1％ Hyaluronic acid (10 kDa) 1％ Inulin 18％ maltodextrin 25％ Trehalose 30％ skim milk powder 25％

[0140] Table 12 Raw material ratios for liquid formulation in Example 11

[0141] raw material weight ratio Polosham 338 18％ Deionized water Replenish to 100%

[0142] In this embodiment, the solid dosage form and the liquid dosage form are mixed at a mass ratio of 1:10 to prepare a probiotic hydrogel preparation.

[0143] Example 12

[0144] Example 12 is basically the same as Example 1, except that the raw material ratios for solid and liquid dosage forms are changed according to Tables 13 and 14, respectively.

[0145] Table 13. Raw material ratios for solid dosage forms in Example 12

[0146] raw material weight ratio Lactobacillus paracasei powder 5％ Hyaluronic acid (10 kDa) 5％ Inulin 25％ maltodextrin 15％ Trehalose 35％ skim milk powder 15％

[0147] Table 14. Raw material ratios for liquid formulations in Example 12

[0148] raw material weight ratio Polosham 338 24％ Deionized water Replenish to 100%

[0149] In this embodiment, the solid dosage form and the liquid dosage form are mixed at a mass ratio of 1:50 to prepare a probiotic hydrogel preparation.

[0150] Comparative Example 1

[0151] The raw material ratios for the solid and liquid dosage forms in Comparative Example 1 are the same as in Example 1, as shown in Tables 1 and 2. The difference between Comparative Example 1 and Example 1 lies in the method of preparing the solid dosage form. The preparation method for Comparative Example 1 includes: according to the raw material ratios shown in Table 1, pouring all raw materials into a V-type mixer at once, and thoroughly mixing the powder to obtain the solid dosage form. The rest of the process is the same as in Example 1 and will not be repeated here.

[0152] Comparative Example 2

[0153] The raw material composition of the solid dosage form in Comparative Example 2 is shown in Table 15, and the liquid dosage form in Comparative Example 2 is the same as that in Table 2.

[0154] The preparation method of the solid dosage form of Comparative Example 2 includes: according to the raw material ratio shown in Table 15, probiotics, inulin, maltodextrin, trehalose, and skim milk powder are stirred and mixed in deionized water until homogeneous, without adding hyaluronic acid, and then placed in a -80℃ freezer for 2 hours. After freezing solid, it is freeze-dried for 48 hours, taken out and crushed with a glass rod. When used, it can be mixed with the liquid dosage form. The weight ratio of the solid dosage form to the liquid dosage form is 1:33.

[0155] Table 15. Raw material ratios for solid dosage forms in Comparative Example 2

[0156]

[0157]

[0158] Comparative Example 3

[0159] The solid formulation of Comparative Example 3 was 100% Lactobacillus paracasei powder, and the liquid formulation of Comparative Example 3 was the same as in Table 2.

[0160] Comparative Example 4

[0161] Commercially available product: Fuyanjie Lactic Acid Bacteria Female Antibacterial Gel

[0162] Comparative Example 5

[0163] Commercially available product: Bayer Canesten Lactic Acid Antibacterial Gel

[0164] Comparative Example 6

[0165] The difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, the 10kDa hyaluronic acid in Example 1 was replaced with 800kDa-1500kDa hyaluronic acid. After preparing the first dispersion, it was found that the hyaluronic acid with too large a molecular weight was too viscous, and the probiotic powder could not be dissolved and dispersed uniformly in the first dispersion, thus failing to obtain a solid preparation with uniformly dispersed probiotics.

[0166] 1. Verification of the uniformity of probiotic distribution in prebiotics

[0167] This invention uses fluorescent protein-labeled probiotics (sfGFP, green fluorescent protein) to observe the probiotic hydrogel preparations obtained in Examples 1, 6, Comparative Example 1 and Comparative Example 2 using an inverted fluorescence microscope.

[0168] Figure 1a , Figure 1b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation of Example 1 are shown below. Figure 1a , Figure 1b (×20 and ×40 respectively). In Example 1, it was found that the probiotic cells did not aggregate, and the distance between cells was not less than 10 μm, indicating that the probiotics were uniformly dispersed within the prebiotics. This is because small-molecule hyaluronic acid can penetrate the extracellular polysaccharide layer of probiotics, anchoring the surface proteins of the cells through hydrogen bonds. Furthermore, hyaluronic acid itself is negatively charged, enabling electrostatic repulsion to prevent the probiotic cells from aggregating. Figure 2a , Figure 2b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation in Example 6 ( Figure 2a , Figure 2b (×20 and ×40 respectively). Example 5 has a similar effect.

[0169] Figure 3a , Figure 3b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation of Comparative Example 1 ( Figure 3a , Figure 3b (×20 and ×40 respectively). As can be seen from Figure 3, a large number of probiotics aggregated in Comparative Example 1. This is because hyaluronic acid and probiotics cannot form hydrogen bonds in the non-solution state.

[0170] Figure 4a , Figure 4b The results of inverted fluorescence microscopy images of the probiotic hydrogel formulation of Comparative Example 2 ( Figure 4a , Figure 4b (The values ​​are ×20 and ×40, respectively). As shown in Figure 4, a large number of probiotics also appeared in the results of Comparative Example 2, which confirms the effect of low molecular weight hyaluronic acid.

[0171] Figure 5The coefficient of variation (CV) values ​​(CV = (standard deviation SD / mean AV) × 100%) for viable bacterial counts (MRS agar plate coating) of Example 1 and Comparative Example 1 are shown. Four groups of activity tests were performed for Example 1 and Comparative Example 1, with three replicates for each group. The CV values ​​were statistically calculated. The CV value for Example 1 was 2.3%, and the CV value for Comparative Example 1 was 62.9%, indicating that the uniformity of the probiotic composition hydrogel formulation based on small molecule hyaluronic acid-prebiotic enhanced protection of the present invention was significantly improved.

[0172] 2. Long-lasting activity and stability of probiotics

[0173] The probiotic hydrogel formulations of Examples 1-3, Comparative Examples 1 and 3 were stored under different conditions, including 22°C-air, 22°C-nitrogen, 4°C-air, and 4°C-nitrogen. After a predetermined storage time, their activity stability was tested.

[0174] Long-term activity stability test method: The probiotic hydrogel preparations of Examples 1, 3, and Comparative Examples 1 and 3 were diluted to 3000 CFU / ml with phosphate buffered saline (PBS (1×)) and the probiotic hydrogel preparation of Example 2 was diluted to 1500 CFU / ml.

[0175] 100 μL of each sample was added to MRS agar medium and plated using the glass bead method (3 replicates). The plates were placed in an anaerobic workstation (80% N2 + 10% CO2 + 10% H2) and incubated at 37°C for 48 h. Colony growth was observed on each plate, and the number of colonies on each plate was counted. The formula for calculating probiotic activity is:

[0176]

[0177] Figure 6 The results show the activity (MRS agar plate coating) and stability of the probiotic hydrogel formulation in Comparative Example 3 under different storage conditions. Figure 6 As shown, the activity of pure probiotic powder gel formulations almost drops to 0 after 1 week ("W" represents week) stored at room temperature and in air, and drops to about 20% after 3 weeks stored at 4°C and in air.

[0178] Figure 7 This is the long-term activity (MRS agar plate coating) stability result of the probiotic hydrogel formulation of Comparative Example 1 under different storage conditions. Figure 7 As shown, the activity of the powder-prepared sample decreased to almost zero when stored at 4°C under nitrogen for 16W.

[0179] Figure 8 , Figure 9 , Figure 10The results show the long-term probiotic activity (MRS agar plate coating) stability of the probiotic hydrogel formulations of Examples 1, 2, and 3 under different storage conditions. Figure 8-10 It can be seen that the activity of the samples in Examples 1 to 3 is still not less than 70% when stored at 4°C under nitrogen for 30W. This is because the probiotics in this invention do not aggregate, but are uniformly dispersed in the prebiotics and are more effectively encapsulated by the prebiotics. Thus, the prebiotics can more effectively provide nutrients and physical protection for the probiotics.

[0180] 3. Probiotics provide long-lasting antibacterial effects and maintain the gut microbiota microenvironment.

[0181] In this invention, the probiotic composition hydrogel formulation of Example 1 was co-cultured with Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Escherichia coli, respectively, and the ratio of probiotic concentration to pathogenic bacteria concentration in the co-culture solution was controlled to be 10:1. 8 CFU / ml: 10 5 CFU / ml. After overnight incubation on a shaker, the OD value of the control group was measured and diluted to 10. 4 CFU / ml, the experimental groups were simultaneously diluted by the same factor, and 100μL of the diluted solution was evenly spread on TSA plates (3 replicates per group). After incubation at 37℃ for 24 hours, the number of colonies was counted and the inhibition rate was calculated.

[0182]

[0183] The results are as follows Figure 11 As shown, the probiotic composition hydrogel formulation of Example 1 has antibacterial effects against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli, with antibacterial rates of 99.4%, 98.8%, and 69.3%, respectively.

[0184] Since probiotics can grow on specific sheep blood agar plates containing Gardnerella vaginalis, it is impossible to distinguish between probiotics and Gardnerella vaginalis. Therefore, a more accurate method is used to test the antibacterial ability against Gardnerella vaginalis and Candida albicans.

[0185] In this invention, the probiotic composition hydrogel formulations of Examples 1-4 were co-cultured with Candida albicans and tested with quantitative real-time polymerase chain reaction (qPCR); Examples 1-4 were co-cultured with Gardnerella vaginalis and tested with quantitative real-time polymerase chain reaction (qPCR), and the inhibition rate was calculated.

[0186] The specific operating method is as follows:

[0187] S1. Activated bacterial strains (Candida albicans and Gardnerella vaginalis);

[0188] S2. Test the OD value of the bacterial solution during use, and control the ratio of probiotic concentration to bacterial solution concentration to be 10:1. 8 CFU / ml: 10 5 CFU / ml, co-cultured in a shaker for 48h (control group without probiotics);

[0189] S3. qPCR Detection: This experiment used the non-specific SYBR Green I dye method. SYBR Green I is a green excitation wavelength dye that binds to the minor groove region of all dsDNA double helices. In its free state, it emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. The fluorescence signal intensity of SYBR Green I is related to the amount of double-stranded DNA; therefore, the amount of double-stranded DNA present in the PCR system can be detected based on the fluorescence signal.

[0190] The primer information for Candida albicans is as follows:

[0191]

[0192] The primer information for Gardnerella vaginalis is as follows:

[0193]

[0194] Figure 12 This is the standard curve for the C. albicans RPB1 gene. Figure 14 This is the standard curve for the G. vaginalis gyrB gene. Based on the corresponding standard curve, the copy numbers of Candida albicans in the co-culture media of Examples 1-4 and the positive control group were obtained; the copy numbers of Gardnerella vaginalis in the co-culture media of Examples 1-4 and the positive control group were calculated using the following formula to determine the inhibition rate.

[0195]

[0196] Figure 13 The results show the inhibition rate of the probiotic hydrogel formulations in Examples 1-4 against Candida albicans. Figure 15 These are the results of the antibacterial rate of the probiotic hydrogel formulations in Examples 1-4 against Gardnerella vaginalis. Figure 13 and Figure 15It can be seen that the inhibition rate of Candida albicans in Examples 1-3 can reach over 95%. The solid formulation of Example 3, after being stored at 4°C in a nitrogen environment for 30 hours, was mixed with the liquid formulation to obtain the gel formulation of Example 4. The inhibition rate of Candida albicans in Example 4 still reached over 95%. The inhibition rate of Gardnerella vaginalis in Examples 1-3 reached over 99%, and the inhibition rate of Gardnerella vaginalis in Example 4 still reached over 99%. Furthermore, the probiotic hydrogel formulation of Example 1, along with co-culture solutions of Candida albicans and Gardnerella vaginalis, were plated onto MRS agar plates. Figure 16 The image shows the results of co-culturing the bacteria (Candida albicans and Gardnerella vaginalis) in Example 1 on an MRS agar plate. Figure 16 As shown, the number of probiotics can reach 10. 9 Probiotics with a concentration of CFU / ml or higher, meaning they remain active after antibacterial treatment, can regulate the health of the vaginal microenvironment in the long term, which has potential application value for the treatment of recurrent vaginitis.

[0197] 4. In vivo antibacterial test

[0198] Five groups were set up: a blank control group, a model control group, a positive drug group 1, a positive drug group 2, and a test substance group, with six mice in each group. The blank control group was inoculated with PBS, and the drug was administered in PBS; the model control group was inoculated with Gardnerella vaginalis, and the drug was administered in a vehicle; the positive drug group 1 was inoculated with Gardnerella vaginalis, and the drug was administered in Comparative Example 4; the positive drug group 2 was inoculated with Gardnerella vaginalis, and the drug was administered in Comparative Example 5; the test substance group was inoculated with Gardnerella vaginalis, and the drug was administered in Example 1.

[0199] Starting 3 days before infection (D-3), mice were subcutaneously injected with 100 μL of estradiol valerate. From Day 0 to Day 4, mice in groups 2 to 5 were vaginally inoculated with 20 μL of LGV (10^ 8 (CFU / mL) The first group of mice were inoculated with PBS. Starting from day 5, each group of mice was vaginally instilled with 20 μL of the corresponding drug once a day until the end of the experiment. The mice were then sacrificed, and 150 μL of vaginal irrigation fluid was collected for qPCR testing. Figure 17 The results of in vivo antibacterial (qPCR) in mice of Examples 1, 4, and 5, and the model group are shown below. Figure 17 As shown, the antibacterial effect of Example 1 is significantly better than that of Comparative Examples 4 and 5 of commercially available products.

[0200] In addition, the inventors of this case also conducted experiments with other probiotics, prebiotics, freeze-drying protectants, raw materials, process operations, and process conditions mentioned in this specification, referring to the aforementioned embodiments, and were able to obtain probiotic hydrogel formulations with uniform dispersion of probiotics, long-lasting and stable activity, and long-lasting antibacterial effect.

[0201] In summary, the solid formulation for probiotic hydrogel preparations provided by this invention utilizes low-molecular-weight hyaluronic acid, which can penetrate the extracellular polysaccharide layer of probiotics and anchor the surface proteins of the bacteria through hydrogen bonds to form physical protection. Hyaluronic acid has a flexible chain structure and is negatively charged; its electrostatic repulsion prevents the probiotic cells from agglomerating. Together with prebiotics and lyophilization protectants, it forms a ternary synergistic system, ensuring uniform dispersion of probiotics within the prebiotics. The prebiotics effectively provide nutrients and physical protection for the probiotics. Based on this solid formulation, the probiotic hydrogel preparation achieves long-lasting and stable activity and long-lasting antibacterial effect. After 30 hours of storage, the probiotic activity remains at least 70%, with an inhibition rate of at least 99% against Gardnerella vaginalis and at least 95% against Candida albicans. Simultaneously, its uniformity is improved, with a coefficient of variation (CV) ≤ 10%. The solid formulation provided by this invention solves the problems of poor uniformity and poor activity stability in existing probiotic preparations. Furthermore, it can be easily prepared using a solvent dispersion-freeze-drying method, making it feasible for large-scale production and showing significant application prospects in the treatment of vaginitis.

[0202] All aspects, embodiments, features, and examples of this invention are to be regarded as illustrative in all respects and are not intended to limit the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0203] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A solid dosage form for formulating probiotic hydrogel preparations, characterized in that, The solid dosage form comprises, by weight percentage, 1% to 5% probiotics, 10% to 30% prebiotics, 1% to 5% hyaluronic acid, and 60% to 80% lyophilization protectant; the prebiotic is inulin, and the lyophilization protectant is selected from one or more combinations of skim milk powder, trehalose, maltodextrin, sorbitol, and monosodium glutamate. The hyaluronic acid has a molecular weight of less than 400 kDa and a zeta potential of less than -20 mV. The hyaluronic acid forms a π-π conjugated anchoring structure with the protein on the surface of the probiotics through hydrogen bonds, and the probiotics are uniformly dispersed in the prebiotics. The probiotics include one or more combinations of Lactobacillus and Bifidobacterium. The solid dosage form is obtained by uniformly dispersing the probiotics, prebiotics, hyaluronic acid and freeze-drying protectant in water and then freeze-drying them.

2. The solid dosage form for formulating probiotic hydrogel preparations according to claim 1, characterized in that, The solid dosage form comprises, by weight percentage, 1% to 5% probiotic powder, 10% to 30% inulin, 10% to 30% skim milk powder, 20% to 40% trehalose, 10% to 30% maltodextrin and 1% to 5% hyaluronic acid.

3. The solid dosage form for formulating probiotic hydrogel preparations according to claim 1, characterized in that: The lactobacilli include one or more combinations of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus curvaturei, Lactobacillus fermentum, Lactobacillus gasseri, and Lactobacillus janniae.

4. The solid dosage form for formulating probiotic hydrogel preparations according to claim 1, characterized in that: The Bifidobacteria include one or more combinations of Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum.

5. The method for preparing a solid dosage form for formulating a probiotic hydrogel preparation according to any one of claims 1-4, characterized in that, include: The hyaluronic acid, probiotics, prebiotics, and freeze-drying protectant are uniformly dispersed in a first solvent and then freeze-dried to obtain the solid preparation; the first solvent is water.

6. The preparation method according to claim 5, characterized in that, Specifically, it includes: The hyaluronic acid is dispersed in a first solvent to obtain a first dispersion; the first dispersion is then uniformly mixed with probiotics to form a homogeneous phase to obtain a second dispersion. The second dispersion is uniformly mixed with prebiotics and lyophilization protectant to obtain a homogeneous phase, and the third dispersion is then freeze-dried to obtain a solid formulation.

7. A probiotic hydrogel formulation, characterized in that, The invention includes solid formulations and liquid formulations according to any one of claims 1-4, wherein the liquid formulation comprises a gel matrix and a second solvent; the gel matrix is ​​a polyoxyethylene-polyoxypropylene block copolymer.

8. The probiotic hydrogel formulation according to claim 7, characterized in that: The polyoxyethylene-polyoxypropylene block copolymer includes one or more combinations of poloxamer 407, poloxamer 338, and poloxamer 188.

9. The probiotic hydrogel formulation according to claim 7, characterized in that: The second solvent includes water.

10. The probiotic hydrogel formulation according to claim 7, characterized in that: The content of the gel matrix in the liquid formulation is 18% to 24%.

11. The probiotic hydrogel formulation according to claim 7, characterized in that: The mass ratio of the solid dosage form to the liquid dosage form is 1:10 to 1:

50.

12. The probiotic hydrogel formulation according to claim 7, characterized in that: The probiotic hydrogel formulation has an activity of no less than 70% after being stored at 4°C in a nitrogen environment for 30 weeks, and the CV value of the probiotic hydrogel formulation is ≤10%.

13. The method for preparing the probiotic hydrogel formulation according to any one of claims 7-12, characterized in that, include: The solid dosage form is prepared according to the preparation method according to any one of claims 5-6; The solid formulation and the liquid formulation are mixed uniformly at 0-4℃ and gelled at 25℃~40℃ to obtain a probiotic hydrogel formulation.

14. The use of the probiotic hydrogel formulation according to any one of claims 7-12 in the preparation of products having the function of inhibiting or killing Gardnerella vaginalis, Candida albicans, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus or Escherichia coli.

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