CN117357468B - Probiotic gel preparation capable of being stored for long time, and use method and application thereof - Google Patents

Abstract

The invention provides a probiotic gel preparation capable of being stored for a long time, and a use method and application thereof; the probiotic hydrogel preparation comprises a solid probiotic preparation and a liquid preparation respectively; the solid preparation and the liquid preparation are mixed and then change along with temperature to form hydrogel with a three-dimensional network structure; before use, the solid probiotic preparation and the liquid preparation are uniformly mixed to obtain the hydrogel containing probiotics. According to the technical scheme provided by the invention, the probiotics are in the form of a solid preparation and are stored separately from a liquid preparation, and the solid preparation containing the probiotics is mixed with the liquid preparation before use to form a hydrogel product with adhesive property; the probiotic powder preparation and the hydrogel preparation are stored separately, and the existing preparation technology effectively avoids the unstable and easy-to-inactivate condition of the probiotics in the water environment, prolongs the preservation time of the probiotic preparation to the maximum extent, and prolongs the shelf life of the product.

Description

A probiotic gel preparation capable of long-term storage and its use method and application

Technical Field

The present invention relates to the technical field of biomedical materials, and in particular to a probiotic gel preparation capable of long-term storage and a method for using and application thereof, and in particular to a probiotic hydrogel preparation applied to skin and mucous membranes.

Background technique

Bacterial vaginosis is a common gynecological disease. There are several bacterial pathogens that can cause vaginal diseases, including vaginal Bacillus, Escherichia coli, Gardnerella, etc. (Machado A et al. J Infect Dis, 2015). Bacterial vaginosis causes increased vaginal discharge in patients, sometimes accompanied by a foul odor, or accompanied by vulvar itching and burning sensation, causing vaginal discomfort and affecting the patient's normal life; for pregnant women, bacterial vaginosis can cause premature rupture of membranes, leading to premature birth, and severe cases can cause fetal infection or even intrauterine fetal death (Emmanuel Amabebe et al. Frontiers in Medicine, 2018). Traditional treatments for bacterial vaginosis usually use antibiotics such as metronidazole, clindamycin, tinidazole, and antimicrobial agents such as povidone iodine and benzalkonium chloride to eliminate pathogenic bacteria.

Recently, there has been a surge in interest in probiotic products containing specific strains of bacteria that occur naturally in the vaginal flora and can help maintain vaginal health by preventing overgrowth of pathogenic bacteria. Vaginal probiotic products have the potential to restore the natural vaginal microbiota, and their mechanism of action involves multiple factors: first, probiotics can colonize in the vaginal epithelium and attach to the vaginal wall to form a protective biofilm. This colonization process helps them surpass competition and inhibit the growth of pathogenic bacteria by occupying binding sites and utilizing available resources (Coudeyras S et al. Infect Dis Obstet Gynecol, 2009); second, the lactic acid produced by probiotic metabolism acidifies the vaginal microenvironment (pH 3.8-4.5), inhibiting the overgrowth of harmful bacteria while promoting the proliferation of beneficial bacteria (Charlier C et al. Int J Food Microbiol, 2009); finally, probiotics can produce antimicrobial substances such as hydrogen peroxide (Wasiela M et al. Int J Gynecol Obstetr, 2009), bacteriocins (Aroutcheva AA et al. Infect Dis Obstet Gynecol, 2001) and organic acids, which can directly inhibit the growth and activity of pathogenic bacteria and help create a healthier vaginal environment. By restoring the natural balance of the vaginal microbiome, vaginal probiotic products can relieve symptoms of vaginal disorders and prevent recurrence of bacterial vaginal infections.

There are currently a variety of available administration methods that can deliver beneficial strains to the vagina. Vaginal capsules and tablets are common methods: the probiotic strains are encapsulated in gelatin or other suitable materials in a solid form, deeply inserted into the vagina, dissolved, released and colonized in the vagina (Komatsu A et al. Vaccine, 2018). However, these solid probiotic preparations may not be suitable for the soft tissue in the vagina, involving factors such as size, shape, dispersibility, comfort and convenience. To address these problems, more comfortable probiotic vaginal suppositories have been developed. These suppositories are made of water-soluble, anhydrous or oil-based materials, which can melt or dissolve after insertion to release the probiotic strains. However, formulas containing oil may cause allergic reactions, and some patients may also feel uncomfortable with the oil. In addition, compared with capsules and tablets, aqueous probiotic vaginal suppositories usually have a shorter shelf life. The reason is that the water in the suppository provides a high humidity environment, which will activate the enzymes in the bacteria and reduce their survival rate and stability. The probiotics in the suppository are suitable for weak acid environments. Under neutral pH conditions, it is not suitable for the survival of probiotics, and the activity of probiotics cannot be maintained for a long time. Or, probiotics lack protective factors and cannot protect the strains from the influence of the external environment. Therefore, the solid probiotic preparations in the prior art have the problems of short shelf life and insufficient activity.

In the prior art, a method of administering a drug on the surface of a mucosal tissue using a hydrogel as a carrier material is used. For example, Chinese invention patent CN110585246A discloses a vaginal antibacterial preparation and a preparation method thereof. The patent provides a vaginal antibacterial preparation having a multilayer structure, which includes a probiotic layer, an isolation layer, and an antibacterial layer from the inside to the outside, wherein the isolation layer includes gelatin; the antibacterial layer includes a hydrogel and an antibacterial composition. After vaginal administration, under the stimulation of the vaginal environment and vaginal secretions, the hydrogel disintegrates, releases the antibacterial composition, and inhibits harmful bacteria in the vagina. After the gelatin is completely degraded, the probiotic layer is exposed, and the matrix material carrying the probiotics disintegrates to release the probiotics. However, the aqueous environment in the hydrogel preparation is not suitable for long-term storage, even if gelatin, polyethylene glycol or polyvinyl alcohol is used as an isolation layer to prevent the probiotics from contacting the hydrogel, resulting in an extremely short shelf life. On the other hand, in order to extend the shelf life, hydrogel antibacterial preparations in the prior art usually use bacteria (non-live bacteria) or metabolites as antibacterial ingredients, and their antibacterial effects are often not ideal; and hydrogel products using live bacteria preparations usually have a very short shelf life. In particular, the activity of probiotics in the products is generally low, and even only contains some metabolites. During use, they cannot form a dominant flora, so the antibacterial effect is greatly limited. In particular, the hydrogel products directly used in the prior art are very easy to adhere to the coating tools due to their viscosity, and the operation convenience is poor. After covering, they are also very easy to fall off from the skin and mucous membrane surface.

Furthermore, Chinese invention patent CN104888224A discloses an amphiphilic polysaccharide derivative/poloxamer thermosensitive in situ hydrogel and its preparation method. The thermosensitive in situ hydrogel is prepared by the interaction of amphiphilic polysaccharide derivatives and poloxamer series polymers, as well as hydrophobic drugs to form a stable in situ hydrogel, and its gelation temperature is 34 to 37°C, and the gelation time of the in situ hydrogel formed at the gelation temperature is 1 to 3 minutes; the poloxamer series polymer is a mixture of poloxamer 407 and any other type of poloxamer. Based on this, the present invention applies poloxamer 407 to probiotic hydrogel preparations on the basis of its technical solution, and at the same time improves the technical problems existing in the prior art.

The present invention provides a probiotic hydrogel preparation that can be stored for a long time, and a method for using and application thereof. The probiotic preparation is based on a solid probiotic preparation and a liquid preparation, and contains a gel component that can form a hydrogel. The storage time of the probiotics in the liquid preparation is significantly increased by adopting a strategy of storing the two separately and preparing them for use immediately, and the probiotics still have 70% activity after 8 weeks.

Summary of the invention

In view of this, in order to solve the above problems, the present invention provides a probiotic gel preparation that can be stored for a long time, and a preparation method and application thereof, aiming to solve the problem that probiotic products such as tablets, capsule solid preparations and suppositories and other soft preparations in the prior art cannot have both stability and comfort, while avoiding the problem that probiotics cannot be stored for a long time in aqueous solution.

In order to achieve the above-mentioned object, the present invention provides a probiotic gel preparation, comprising a solid preparation and a liquid preparation respectively containing gel components, wherein the solid preparation contains a probiotic component; before use, the solid preparation and the liquid preparation are mixed to obtain a hydrogel containing probiotics.

After the solid preparation and the liquid preparation are mixed, they undergo physical cross-linking as the temperature changes to form a hydrogel with a three-dimensional network structure. Probiotics are loaded in the three-dimensional network structure. When the hydrogel is applied to the surface of the skin and mucous membranes, the adhesion of the hydrogel causes it to adhere to the skin and mucous membranes, and the probiotics form a dominant bacterial flora to kill harmful bacteria.

Preferably, the solid preparation comprises at least a first gel and probiotic powder; by weight, the solid preparation comprises 5 to 20 parts of the first gel and 1 to 5 parts of the probiotic powder; the first gel is in a solid powder form.

The solid preparation can be stored for more than 8 weeks at 4° C. in an inert gas atmosphere, and the activity of the probiotics is maintained at least 70%.

Preferably, the first gel comprises one or more of polyoxyethylene-polyoxypropylene block copolymers, cellulose and its derivatives, synthetic high molecular polymers, natural and modified polysaccharides, starch and its derivatives, and the like.

Preferably, the polyoxyethylene-polyoxypropylene block copolymer includes but is not limited to one or more of poloxamer 407 (P407, F127), poloxamer 338, poloxamer 188 and the like.

Preferably, the cellulose and its derivatives include one or more of methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose and the like.

Preferably, the synthetic high molecular polymer includes one or more of povidone, copovidone, polyethylene glycol, polyester-polyethylene glycol copolymer, polyvinyl alcohol, poly(N-isopropylacrylamide), and the like.

Preferably, the natural and modified polysaccharides include one or more of gelatin, hyaluronic acid, chitosan, agarose, xanthan gum, carrageenan, sodium alginate, pectin, dextran and the like.

Preferably, the starch and its derivatives include one or more of starch, carboxymethyl starch, pregelatinized starch, hydroxyethyl starch, cross-linked starch, starch phosphate and the like.

Preferably, the probiotic powder includes one or more of lactobacillus, bifidobacterium, bacillus, clostridium butyricum and the like.

Preferably, the lactobacillus includes one or more of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus bulgaricus, Lactobacillus johnsonii and the like.

Preferably, the bifidobacterium includes one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium infantis.

Preferably, the Bacillus includes one or more of Bacillus subtilis, Bacillus cereus, Bacillus licheniformis and Bacillus coagulans.

As one of the more preferred embodiments, the solid preparation further comprises an auxiliary agent.

Preferably, the solid probiotic preparation comprises 5 to 20 parts of the first gel, 1 to 5 parts of the probiotic powder, and 30 to 200 parts of the auxiliary agent, calculated by mass.

Preferably, the auxiliary agent includes one or more of prebiotics, flow aids, protein auxiliary agents, sugar auxiliary agents, alcohol auxiliary agents, amino acid auxiliary agents, and pH regulators.

Preferably, the prebiotics include one or more of inulin, isomaltooligosaccharide, fructooligosaccharide, galacto-oligosaccharide, xylooligosaccharide, lactofructooligosaccharide, soybean oligosaccharide and the like.

Preferably, the glidant comprises one or more of fumed silicon dioxide Aerosil, colloidal silicon dioxide, talc, and kaolin.

Preferably, the protein additive includes one or more of skim milk powder, egg white and soy protein.

Preferably, the carbohydrate auxiliary agent includes one or more of trehalose, sucrose, lactose, glucose and xylan.

Preferably, the alcohol auxiliary agent includes one or more of glycerol, mannitol and sorbitol.

Preferably, the amino acid auxiliary agent includes one or more of sodium glutamate and L-cysteine.

Preferably, the pH adjuster includes one or more of disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium citrate, citric acid, potassium citrate, trisodium phosphate, sodium carbonate, and sodium tripolyphosphate.

Preferably, the liquid formulation comprises a solvent and a second gel.

Preferably, the liquid preparation comprises 20 to 60 parts of the solvent and 5 to 10 parts of the second gel, by mass.

Preferably, the solvent in the liquid preparation includes one or more of sterile water, physiological saline, phosphate buffered saline (PBS), and 4-morpholineethanesulfonic acid buffer (MES).

As a preferred embodiment, the solvent is sterile water; the concentration of the second gel aqueous solution is preferably 18-24 wt %, and most preferably, the concentration of the second gel aqueous solution is 22 wt %.

The second gel includes one or more of poloxamer 407, poloxamer 338, poloxamer 188, and poly (N-isopropylacrylamide).

The probiotic hydrogel preparation provided based on the above technical solution can significantly increase the storage time of probiotics in liquid preparations, and still has 70% activity after 8 weeks. It can be prepared and used immediately when in use, so that the probiotics can maintain long-term activity.

As one of the preferred embodiments, the solid probiotic preparation is stored at 4° C. in a nitrogen atmosphere.

The present invention also provides a method for using the same, comprising the following steps:

S1. The solid preparation and the liquid preparation are stored separately, and the solid preparation is stored in an inert gas atmosphere at -20 to 25°C;

S2. Before use, the solid probiotic preparation and the liquid preparation are mixed in proportion to obtain a liquid gel mixture;

S3. placing the liquid gel mixture on the surface of the skin and mucosa; the skin and mucosa include the reproductive tract, rectum or nasal cavity;

S4. The liquid gel mixture undergoes in-situ gelation by physical cross-linking as the external temperature changes, thereby obtaining a hydrogel with a three-dimensional network structure, wherein probiotics are loaded in the three-dimensional network structure of the hydrogel; and the hydrogel is attached to the surface of the skin and mucous membrane.

Among them, in S2, the mixing method includes adding the liquid preparation to the solid preparation, dissolving the solid preparation in the liquid preparation by external physical extrusion, syringe mixing, etc., and the dissolution time is about 10 to 60 seconds.

As another object, the present invention also provides a probiotic hydrogel, comprising mixing the solid probiotic preparation and the liquid preparation in the probiotic hydrogel preparation provided in the above technical scheme, and then physically crosslinking the mixture under a temperature condition of 25 to 40°C; wherein the mass ratio of the solid preparation and the liquid preparation is 10 to 50 mg: 3 to 8 g.

Based on the above technical scheme, a hydrogel is obtained by mixing a solid probiotic preparation and a liquid preparation, which has adhesive properties and can be used to treat skin and mucosal diseases. The probiotic hydrogel or solid probiotic preparation and liquid preparation are applied to drugs for treating skin and mucosal diseases, and can have a stable antibacterial function. Compared with ordinary polysaccharide/poloxamer in situ hydrogels, the in situ hydrogel liquid preparation of the present invention can quickly dissolve the solid preparation, realize ready-to-use preparation, and can be applied to mucosal administration, transdermal administration and injection administration systems.

Beneficial technical effects obtained by the present invention:

1. The technical solution provided by the present invention adopts the probiotics in the probiotic hydrogel in the prior art in the form of a solid preparation, and is stored separately from the liquid preparation. Before use, the solid preparation containing probiotics is mixed with the liquid preparation to form a hydrogel product with adhesion properties, which is placed on the surface of the skin mucosa, and its adhesion properties are utilized to enable the probiotics to act on the mucosal tissue; the probiotic powder preparation and the hydrogel preparation are stored separately and prepared on a ready-to-use basis, which effectively avoids the instability and easy inactivation of probiotics in a water environment, and maximizes the shelf life of the probiotic preparation, so that its activity is maintained at 70% in the 8th week.

2. By adopting the technical solution of the present invention, the probiotic hydrogel preparation is divided into two parts, namely, a solid preparation and a liquid preparation. The solid preparation can achieve a longer storage time, extend the shelf life, and the activity of the probiotics therein is almost unaffected; on the other hand, by adopting the liquid preparation, it can be ensured that the solid preparation and the liquid preparation can be directly mixed, the dissolution time of the solid preparation can be reduced, and the preparation can be used immediately, thereby ensuring the activity of the probiotics to the greatest extent, so that the probiotics can form a dominant bacterial flora and give full play to the effect of inhibiting bacteria and killing harmful bacteria.

3. The present invention greatly improves and prolongs the activity of probiotics by adding prebiotics, adjuvants and other ingredients to the solid preparation. Prebiotics, as the growth raw materials of probiotics, can activate and promote the proliferation of probiotics; adjuvants can reduce the interference of environmental changes on the activity of probiotics; and flow aids can improve the uniform dispersibility of solid preparations.

4. The poloxamer series polymers selected for the solid preparation and the liquid preparation in the formula of the present invention, especially the thermosensitive gel P407, are polymers that can be used in the human body, are non-toxic, non-irritating to the skin and mucous membranes, and are non-allergenic. The gel state of its hydrogel product has strong adhesion and can maintain a certain adhesion time at the mucous membrane site of administration, thereby achieving a continuous therapeutic effect.

5. The liquid preparation used in the present invention has a temperature-sensitive property. It is liquid at low temperatures and can be quickly mixed with solid preparations, reducing the reduction in bacterial activity during the preparation of probiotic gel; when it is mixed with the solid preparation under low temperature conditions, it is a liquid gel mixture, which can be evenly applied to the administration site, and is easy to operate; it can quickly form a hydrogel with a three-dimensional network structure by in situ gelation at human body temperature, and adhere to the administration site with strong adhesion, and can continuously and efficiently achieve probiotic delivery therapy at the administration site.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG. 1 is a temperature-viscosity curve of different concentrations of P407 aqueous solution at 20 to 45° C. in Examples 1 to 4 of the present invention.

FIG. 2 is an adhesion strength curve of P407 aqueous solution of different concentrations at 37° C. in Examples 1-4 of the present invention.

FIG3 is a comparative curve of the activity of the probiotic powder preparation stored in dry state and stored in hydrogel according to Example 6 of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

The invention provides a hydrogel preparation containing probiotics, comprising a solid preparation and a liquid preparation respectively containing probiotic components; the solid preparation and the liquid preparation are mixed and then subjected to a physical cross-linking reaction to form a hydrogel with a three-dimensional network structure; before use, the solid preparation and the liquid preparation are mixed to obtain a hydrogel containing probiotics.

Preferably, the solid preparation comprises at least a first gel and probiotic powder.

Preferably, the solid preparation comprises 5 to 20 parts of the first gel and 1 to 5 parts of the probiotic powder, calculated by mass.

Preferably, the liquid formulation comprises a solvent and a second gel.

Preferably, the liquid preparation comprises 20 to 60 parts of the solvent and 5 to 10 parts of the second gel, by mass.

Preferably, the first gel comprises one or more of polyoxyethylene-polyoxypropylene block copolymers, cellulose and its derivatives, synthetic high molecular polymers, natural and modified polysaccharides, starch and its derivatives, and the like.

Preferably, the second gel comprises one or more of poloxamer 407, poloxamer 338, poloxamer 188, and poly (N-isopropylacrylamide).

When the solid probiotic preparation and the liquid preparation are mixed and applied to the skin and mucous membrane surface, the first gel and/or the second gel undergoes physical cross-linking as the ambient temperature changes to form a hydrogel with a three-dimensional network structure. The probiotics are loaded in the three-dimensional network structure of the hydrogel and released and colonized on the skin and mucous membrane surface to form a dominant bacterial flora, thereby exerting an antibacterial/bactericidal effect.

Preferably, when used, the solid preparation and the liquid preparation are mixed, and the mass ratio of the two is 10-50 mg: 3-8 g.

The technical solution of the present invention is further described in detail below through specific embodiments.

Example 1

This embodiment provides a preparation of a liquid preparation, and the raw material ratio is shown in Table 1. The preparation of the liquid preparation includes mixing poloxamer P407 (hereinafter referred to as P407) powder with pure water, and the specific method includes: mixing the raw materials according to the ratio shown in Table 1, placing them in a 4°C environment overnight, and preparing a 22% P407 aqueous solution.

Table 1 Raw material ratio of liquid preparation

Example 2

The difference between this embodiment and embodiment 1 is that the content of P407 in the liquid preparation is different. The content of P407 in the final liquid preparation is 15 wt %.

Example 3

The difference between this embodiment and embodiment 1 is that the content of P407 in the liquid preparation is different. The content of P407 in the P407 aqueous solution finally obtained is 18 wt %.

Example 4

The difference between this embodiment and embodiment 1 is that the content of P407 in the liquid preparation is different. The content of P407 in the P407 aqueous solution finally obtained is 24 wt %.

Testing of viscosity and adhesion strength of liquid preparations:

Take appropriate amounts of the liquid preparations provided in Examples 1-4 respectively, and drop them onto the sample stage of the rotational rheometer, set the temperature change range to 20-45°C, and measure the change in viscosity of the hydrogel with temperature; drop samples of different concentrations between two glass plates, and use a universal tensile testing machine to measure the adhesion strength of the samples at 37°C.

The test results are shown in Figures 1 and 2.

Referring to FIG. 1 and FIG. 2 , the temperature-viscosity change curves of the hydrogel products with different contents of P407 prepared in the above Examples 1-4 in the temperature range of 20 to 45° C. The adhesion properties of the aqueous solution can be analyzed by the curves. As can be seen from the figure, as the concentration of P407 increases, the viscosity and adhesion strength of the gel increase accordingly, while the gel temperature continues to decrease.

Furthermore, the gelling temperature of the gel should not be too low, and operating time should be reserved for the extrusion and mixing of the liquid preparation and the probiotic powder preparation. At the same time, it needs to have a certain viscosity after gelling to ensure adhesion to the tissue mucosa.

Based on the above comprehensive considerations, a 22% concentration of P407 solution was selected as the formula of the liquid preparation. On the one hand, sufficient operation time was reserved to ensure that the liquid preparation and the probiotic powder preparation were evenly mixed; at the same time, at 37°C, there was sufficient adhesion strength to allow the probiotic preparation to adhere to the administration site, thereby achieving a sustained therapeutic effect.

Example 5

This embodiment provides a preparation of a solid preparation containing probiotics. The probiotics selected in this embodiment are Lactobacillus paracasei.

The ingredients of the solid preparation containing probiotics in this embodiment only contain probiotics and P407 powder, and the proportion is shown in Table 2. The preparation method comprises placing poloxamer 407 (P407) powder and probiotic powder in a nitrogen (or air) atmosphere and mixing them according to the proportion shown in Table 2.

Table 2 Raw materials of solid preparations containing probiotics in Examples and Comparative Examples

Example 6

The ingredients of the solid preparation containing probiotics in this embodiment are shown in Table 2. The preparation method includes: mixing P407 powder, trehalose, sodium alginate, Aerosil powder, probiotic powder and inulin in a nitrogen (or air) atmosphere according to a certain proportion.

Example 7

The ingredients of the solid preparation containing probiotics in this embodiment are shown in Table 2. The preparation method includes: mixing P407 powder, skim milk powder, inulin, trehalose and probiotic powder in a nitrogen (or air) atmosphere according to proportion.

Comparative Example 1

The ingredients of the probiotic-containing solid preparation of this comparative example only contain probiotic powder, and the proportion is shown in Table 2.

The solid preparations containing probiotics obtained in the above examples and comparative examples were subjected to stability tests, and the test conditions included storage at 4° C., 22° C. or 40° C., and the storage environment included nitrogen or air, respectively.

Method of stability experiment: a solid preparation containing 1 part by weight of probiotic powder is placed in the above-mentioned different storage conditions, and samples are taken at 0 day, 1 week, 2 weeks, 4 weeks and 8 weeks, respectively, and a uniform gel is prepared according to the ratio of 1 part by weight of probiotics: 100 parts by weight of liquid preparation, and diluted with a diluent (0.9% NaCl saline) to a final dilution of 3000 CFU/mL, 100 μL of bacterial solution is taken, dropped onto MRS agar medium, and evenly spread with a coating rod, placed in an anaerobic workstation (80% N2+10% _CO2 +10% _H2 ), cultured at 37°C for 48 hours, and the colony growth of each flat plate is observed, the number of colonies on the plate is counted, and the probiotic activity and the number of viable bacteria are tested. The calculation formula of probiotic activity is:

The formula for calculating the number of viable bacteria is:

Viable bacteria count (CFU/g) = sum of colony counts on 3 plates/3 × 10 × final dilution factor

Table 3 Probiotic activity (%) test results of solid preparations of examples and comparative examples

Table 4 Test results of viable probiotic bacteria count (average colony count per dish) of solid preparations in the examples

Advantages of solid preparations on probiotic stability: See Tables 3 and 4, which are the test results of probiotic activity and viable count in the solid preparations of Examples 5-7 and the solid preparation of Comparative Example 1, respectively.

Referring to Table 3, the probiotic activities of Examples 5-7 and Comparative Example 1 in the second week of storage in nitrogen and air atmospheres, respectively, show that under the same storage conditions, when only probiotic powder is added (Comparative Example 1), the activity and viable count after 2 weeks of storage are significantly lower than those of the solid preparations to which auxiliary agents and/or gel and probiotic powder are added at the same time (Examples 5-7), indicating that the solid preparation formulas of Examples 5-7 can significantly improve the activity of probiotics during long-term storage and extend the effective shelf life of probiotic products.

Further analysis shows that Example 7 can still maintain 70% activity after 8 weeks of storage at 4°C and N2; followed by Example 5, which can maintain 56% activity after 8 weeks.

Refer to Table 4, the effect of prebiotics and adjuvants on the stability (viable count) of probiotics in the solid preparation formula. Compared with the solid adjuvant without gel (Comparative Example 1), the probiotics have almost no viable bacteria within 1 week; further analysis of the solid preparation without prebiotics and other adjuvants (Example 5) and the solid preparation with prebiotics and other adjuvants (Examples 6 and 7) shows that under the same storage conditions, the addition of prebiotics and adjuvants has a stabilizing and even activating effect on the activity of probiotics. Under the same storage conditions, the viable count results in Examples 6 and 7 are not only far better than those in Example 5, but also the viable count of probiotics in Examples 6 and 7 in the first week shows an upward trend, indicating that the probiotics proliferate under the solid preparation formula conditions, which is conducive to the storage of probiotic powder.

Storage temperature has a significant effect on the activity of probiotics. Probiotics stored at 40°C almost lost their activity in the first week, while probiotics stored at 4°C in a nitrogen atmosphere maintained 70% activity in the eighth week. Combined with the test results of sample activity and viable count, the 4°C, nitrogen storage condition in Example 7 is the best condition in the present invention, which has a maintaining effect on the activity of probiotics. Compared with other conditions, under this condition, the activity and viable count of probiotics are always maintained at the highest level.

The present invention also provides the effects of different use methods on the stability of probiotic powder preparations, taking Example 6 as an example, specifically including:

1. 1.1 g of P407 powder and 3.9 g of water were mixed to prepare a liquid preparation with a concentration of 22%, and the solid preparation of Example 6 was mixed at 4° C. to detect the activity of the probiotics in the mixture; the mass ratio of the solid preparation to the liquid preparation was 30 mg: 5 g; the mixing time was about 30 s to form a uniform, non-gelled liquid;

2. 1.1 g of P407 powder was mixed with 3.9 g of water and the solid preparation of Example 6, and the mixture was refrigerated and dissolved overnight at 4°C, and the activity of the mixture after overnight refrigeration and dissolution was tested. The mixing time was 8 to 12 hours, and the solid components were completely dissolved to form a uniform, non-gelled liquid.

See Table 5 for the results.

Table 5 Effect of different use methods on the stability of probiotic powder preparations in Example 6

Advantages of liquid preparations: P407 powder usually takes a long time to dissolve in a solvent (generally more than 10 hours). The thermosensitive gel component, water and probiotic powder are mixed and dissolved. The activity and number of viable bacteria in the dissolution process are significantly reduced (see the overnight refrigerated dissolution results in Table 5); and the liquid gel preparation (liquid preparation provided in Example 6) is prepared in advance. Before use, only a small amount of thermosensitive gel in the solid preparation for probiotic preservation needs to be dissolved. The dissolution time is 10 to 60 seconds, which greatly shortens the dissolution time and can reduce the impact on the activity and number of viable bacteria in the dissolution process; due to the thermosensitive viscosity of P407, it is liquid at 4°C, which can quickly dissolve the solid preparation of bacterial powder, reduce the time required for configuration, and ensure the activity of bacterial powder. At the same time, this temperature is also the optimal storage temperature for solid preparations, which can effectively maintain the activity of probiotics in the preparation. The method of using the probiotic liquid preparation and solid preparation for immediate use has obvious advantages in ensuring the activity of probiotics.

Furthermore, the present invention also compares the storage of the solid preparation containing probiotics in Example 6 with the storage after forming a hydrogel, specifically including: the hydrogel product obtained by mixing the solid preparation containing probiotics prepared in Example 6 with the liquid preparation is stored in the same environment as the solid preparation containing probiotics, including 4°C, in air or N2 atmosphere for 0 days, 1 day, 3 days, 7 days, 21 days, and 28 days to detect the active ingredients of probiotics in the samples.

The storage of the mixed hydrogel and solid preparations was compared, and the activity of the probiotics within 28 days was tested respectively. The test results are shown in Figure 3. The probiotics are very easy to be inactivated in the hydrogel. After only one day of storage in the hydrogel, the activity of the sample dropped to less than 60%, and the activity of some samples even dropped to less than 20%; after three days of storage, all the probiotics in the hydrogel product were inactivated; while the probiotic preparations and the hydrogel preparations were stored separately, and the probiotic activity of the samples was significantly improved, with the activity remaining at least 50% after 28 days, and up to 70% after 56 days. Obviously, the separate storage of probiotic powder and hydrogel preparation significantly extended the effective activity period of the probiotics.

It should be noted that the advantages of the probiotic gel preparation stored by solid-liquid separation provided by the present invention and its method of use also include that the probiotic gel provided by the present invention is in a liquid state at low temperature (usually below 10°C), and the solid preparation and the liquid preparation are mixed at low temperature to obtain a liquid gel mixture, which is applied to the surface of the skin and mucosa. As the ambient temperature rises, the liquid gel mixture quickly gels to form an in-situ hydrogel on the surface of the skin and mucosa, which has a stronger binding force with the skin and mucosa, and can prolong the action time of the hydrogel on the skin. At the same time, it can be directly mixed in a device that is easy to mix, such as a syringe, a sealed bag, etc., and then the liquid gel mixture can be applied to the surface of the skin and mucosa by a method of physical extrusion by external force, a syringe mixing method, etc., which is easy to operate and can avoid pollution from the external environment.

The probiotic hydrogel preparations in the prior art are different from those in the prior art in that they are all hydrogel products. When the preparations are applied to the surface of the skin and mucous membrane, they are easy to fall off. On the other hand, due to the viscosity of the hydrogel, they are more likely to adhere to the operating tools during operation, which is not convenient for operation. In particular, they are easily polluted by the environment. Moreover, the number of live bacteria in the hydrogel products in the prior art is generally very low, and they even contain only a small amount of probiotics or metabolites. After being contaminated by the outside world, they cannot form a dominant flora in the later metabolism, which reduces the therapeutic effect.

In summary, the present invention can significantly increase the storage time of probiotics and extend the shelf life of the product by storing the solid preparation containing probiotics separately from the liquid; at the same time, after adding prebiotics and other auxiliary agents, the number of viable probiotics in the solid preparation can be significantly increased; and adding gel powder to the solid preparation can also increase the activity of the probiotics.

In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims (7)

1. A probiotic gel formulation capable of long-term storage, comprising a solid formulation and a liquid formulation;

the solid preparation at least comprises probiotic powder and first gel;

The liquid formulation contains at least a solvent and a second gel;

The solid preparation and the liquid preparation are mixed to obtain a liquid gel mixture; the liquid gel mixture is physically crosslinked along with the temperature change to form hydrogel containing a three-dimensional network structure of probiotics;

The solid preparation comprises, by mass, 1-5 parts of the probiotic powder and 5-20 parts of first gel; the solid probiotic preparation can be stored for at least 8 weeks at 4 ℃ under the inert gas atmosphere;

The liquid preparation comprises 20-60 parts by mass of the solvent and 5-10 parts by mass of the second gel;

the mass ratio of the solid probiotic preparation to the liquid preparation is 10-50 mg: 3-8 g;

in the solid preparation, the first gel is one or more of poloxamer 407, poloxamer 338 and poloxamer 188; the probiotic powder is one or more of lactobacillus, bifidobacterium and bacillus;

in the liquid preparation, the concentration of the second gel is 18-24 wt%; the second gel is one or more of poloxamer 407, poloxamer 338 and poloxamer 188.

2. The probiotic gel formulation according to claim 1, wherein in the liquid formulation the solvent is one or more of sterile water, physiological saline, phosphate Buffered Saline (PBS), 4-morpholinoethanesulfonic acid buffer (MES).

3. The probiotic gel formulation according to claim 1, wherein the lactobacillus is one or more of lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus casei, lactobacillus paracasei, lactobacillus plantarum, lactobacillus grignard, lactobacillus reuteri, lactobacillus bulgaricus, lactobacillus johnsonii;

The bifidobacterium is one or more of bifidobacterium longum, bifidobacterium breve, bifidobacterium bifidum and bifidobacterium infantis;

The bacillus is one or more of bacillus subtilis, bacillus cereus, bacillus licheniformis and bacillus coagulans.

4. A probiotic gel formulation according to any one of claims 1 to 3, further comprising an auxiliary agent; the solid preparation comprises, by mass, 5-20 parts of the first gel, 1-5 parts of the probiotic powder and 30-200 parts of the auxiliary agent.

5. The probiotic gel formulation according to claim 4, characterized in that the auxiliary agent comprises one or more of prebiotics, glidants, proteinaceous auxiliary agents, sugar auxiliary agents, alcohol auxiliary agents, amino acid auxiliary agents, pH adjusting agents.

6. The probiotic gel formulation according to claim 5, characterized in that the prebiotic comprises one or more of inulin, isomaltooligosaccharide, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, lactulose oligosaccharides, soy oligosaccharides;

the glidant comprises one or more of Aerosil, colloidal silicon dioxide, talcum powder and kaolin;

The protein auxiliary agent comprises one or more of skim milk powder, egg white and soybean protein;

the saccharide auxiliary agent comprises one or more of trehalose, sucrose, lactose, glucose and xylan;

The alcohol auxiliary agent comprises one or more of glycerol, mannitol and sorbitol;

The amino acid auxiliary agent comprises one or more of sodium glutamate and L-cysteine;

The pH regulator comprises one or more of disodium hydrogen phosphate, monopotassium phosphate, sodium citrate, citric acid, potassium citrate, trisodium phosphate, sodium carbonate and sodium tripolyphosphate.

7. A probiotic hydrogel, which is characterized by being obtained by uniformly mixing a solid preparation and a liquid preparation in the probiotic gel preparation according to any one of claims 1-6 and then physically crosslinking at a temperature of 25-40 ℃.