Lactobacillus rhamnosus microcapsule as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical material preparation, and particularly relates to a lactobacillus rhamnosus microcapsule as well as a preparation method and application thereof. 5-hydroxytryptamine is adopted as a raw material and is mixed with lactobacillus rhamnosus in an ammonia water environment; 5-hydroxytryptamine is subjected to an oxidative polymerization reaction in an alkaline environment, so that a nano-scale poly (5-hydroxytryptamine) shell is formed on the surface of lactobacillus rhamnosus, and the lactobacillus rhamnosus is wrapped to form the lactobacillus rhamnosus microcapsule. The lactobacillus rhamnosus microcapsule prepared by the invention can effectively resist gastric acid killing, ensure the activity of lactobacillus rhamnosus in a delivery process, and effectively remove a poly (5-hydroxytryptamine) nano shell wrapping the surface of lactobacillus rhamnosus by utilizing division and propagation of lactobacillus rhamnosus when the lactobacillus rhamnosus reaches a delivery site; the direct contact between the lactobacillus rhamnosus and intestinal mucosa is realized, so that the probiotic effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of medical material preparation, and particularly relates to a Lactobacillus rhamnosus microcapsule and a preparation method and application thereof. Background Art

[0002] Lactobacillus rhamnosus ( L. rhamnosus ) belongs to the genus Lactobacillus, is one of the normal flora of the human body, and has strong intestinal adhesion and colonization properties. Therefore, the human body's appropriate intake of Lactobacillus rhamnosus can effectively regulate the composition of intestinal flora, play an important physiological health care function such as lowering cholesterol, preventing and treating diarrhea, eliminating toxins, improving the body's immunity and anti-tumor.

[0003] However, during oral delivery, Lactobacillus rhamnosus will inevitably be killed by gastric acid, which greatly reduces the activity of Lactobacillus rhamnosus before it reaches the intestine for colonization, greatly reducing the bioavailability of Lactobacillus rhamnosus. Various attempts have been made to improve the survival rate of Lactobacillus rhamnosus in the stomach, such as Lactobacillus rhamnosus microcapsules prepared by extrusion, emulsification cross-linking, spray drying and coagulation, which can effectively improve the resistance of Lactobacillus rhamnosus to the adverse effects of the body.

[0004] In the prior art, such as the literature Kawai Lai, Yuhsuan How, Liewphing Pui. Microencapsulation of Lactobacillus rhamnosus GG with flaxseed mucilage using co-extrusion technique. J Microencapsul, 38: 134-148. The disclosed method uses gelatin and alginate beads as coating materials and calcium chloride as a cross-linking agent to encapsulate Lactobacillus rhamnosus to prepare microcapsules to achieve the delivery of Lactobacillus rhamnosus.

[0005] However, the above-mentioned prior art has the following problems: 1. The preparation process is relatively complicated, and the required microcapsules often use organic polymers as their raw materials, which affects the biocompatibility of Lactobacillus rhamnosus and further reduces the activity of Lactobacillus rhamnosus. 2. After the microcapsules reach the intestinal tissue, they cannot be degraded and ruptured or require a long time to degrade, resulting in Lactobacillus rhamnosus being eliminated from the intestine before direct contact with the intestinal mucosa. Therefore, there is an urgent need to develop a simple and effective method to effectively deliver Lactobacillus rhamnosus to the intestine, so that the biological properties of Lactobacillus rhamnosus are stable in the gastric acid environment and effectively contact the intestinal mucosa to improve its bioavailability. Summary of the invention

[0006] In view of the deficiencies of the prior art mentioned in the above background technology, the present invention provides a Lactobacillus rhamnosus microcapsule and a preparation method and application thereof. Through the oxidative polymerization of 5-hydroxytryptamine under alkaline conditions, a nano-scale protective shell can be formed on the surface of Lactobacillus rhamnosus, and the Lactobacillus rhamnosus is encapsulated to form a Lactobacillus rhamnosus microcapsule, which can effectively resist gastric acid damage and utilize the division and reproduction of Lactobacillus rhamnosus itself, effectively remove the poly 5-hydroxytryptamine nanoshell wrapped on the surface of Lactobacillus rhamnosus, achieve direct contact between Lactobacillus rhamnosus and intestinal mucosa, thereby exerting a prebiotic effect.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows: A method for preparing Lactobacillus rhamnosus microcapsules, characterized in that it comprises the following steps: Step S1: centrifuging a Lactobacillus rhamnosus bacterial solution, discarding the supernatant, adding a bacterial culture solution to resuspend the solution, and mixing the solution thoroughly to obtain a Lactobacillus rhamnosus resuspended solution; Step S2: stirring the Lactobacillus rhamnosus resuspended solution and adding ammonia water, and mixing thoroughly to obtain a mixed bacterial solution; Step S3: adding 5-hydroxytryptamine to the mixed bacterial solution, stirring for reaction, and collecting by centrifugation to obtain microcapsules encapsulating Lactobacillus rhamnosus.

[0008] Furthermore, the number of Lactobacillus rhamnosus in the Lactobacillus rhamnosus bacterial solution in step S1 is 3×10 10 ~4×10 10 CUF; the centrifugation time is 5 to 10 minutes, and the centrifugal force is 1000 to 1500g.

[0009] Furthermore, the volume ratio of ammonia water to bacterial culture solution in the mixed bacterial solution in step S2 is 1 to 2:100.

[0010] Furthermore, in step S3, the final concentration of 5-HT is 2-4 mg / ml; the stirring temperature is 20-25° C., and the stirring time is 60-120 min.

[0011] The present invention also provides a Lactobacillus rhamnosus microcapsule prepared by the preparation method.

[0012] Furthermore, the particle size of the Lactobacillus rhamnosus microcapsule is 1 to 2 μm, and the shell thickness is 50 to 100 nm.

[0013] Furthermore, the active content of Lactobacillus rhamnosus in the Lactobacillus rhamnosus microcapsules is 90-95%.

[0014] The present invention also provides an application of Lactobacillus rhamnosus microcapsules in preparing Lactobacillus rhamnosus microcapsule oral dosage form medicine.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The inventor of the present invention creatively uses 5-hydroxytryptamine as a raw material to solve the problems of existing Lactobacillus rhamnosus delivery materials. The inventor utilizes the property that 5-hydroxytryptamine is easily oxidized and polymerized to form poly-5-hydroxytryptamine in an alkaline environment, and mixes it with Lactobacillus rhamnosus in an alkaline environment to form a nanoshell layer on the surface of Lactobacillus rhamnosus, and encapsulates Lactobacillus rhamnosus to form Lactobacillus rhamnosus microcapsules, thereby effectively resisting gastric acid damage. At the same time, the division and reproduction of Lactobacillus rhamnosus itself are utilized, and the poly-5-hydroxytryptamine nanoshell wrapped on the surface of Lactobacillus rhamnosus can be effectively removed after delivery to the site, so that Lactobacillus rhamnosus can be directly contacted with the intestinal mucosa, thereby exerting a probiotic effect.

[0016] 2. Compared with other packaging materials, 5-hydroxytryptamine, as the main neurotransmitter secreted by the body, can effectively improve the biocompatibility of the in vivo transportation of Lactobacillus rhamnosus microcapsules, and reduce the damage to the normal tissues of the body. Different from the strong alkaline materials such as hydrogen peroxide, the ammonia water used in the present invention provides a gentle preparation process for the preparation of Lactobacillus rhamnosus microcapsules. The prepared Lactobacillus rhamnosus microcapsules have excellent biosafety and good dispersibility, and can be delivered by oral administration of Lactobacillus rhamnosus. After it reaches the gastric tissue, the direct contact of adverse factors such as gastric acid and enzyme with Lactobacillus rhamnosus is effectively prevented by the poly-5-hydroxytryptamine shell in the gastric tissue, and then the activity of Lactobacillus rhamnosus is maintained. In addition, the poly-5-hydroxytryptamine shell can also effectively slow down the direct contact of Lactobacillus rhamnosus with nutrients, and then delay the growth and reproduction of Lactobacillus rhamnosus. When the Lactobacillus rhamnosus microcapsules enter the intestinal tissue, the encapsulated Lactobacillus rhamnosus grows and differentiates, and then breaks through the encapsulated poly(5-hydroxytryptamine) shell to release the encapsulated Lactobacillus rhamnosus, thereby achieving the release of Lactobacillus rhamnosus in the intestine, and then promoting direct contact between Lactobacillus rhamnosus and intestinal mucosa, effectively improving the bioavailability of Lactobacillus rhamnosus.

[0017] 3. The present invention successfully synthesizes Lactobacillus rhamnosus microcapsules by a one-step method. The synthesis steps are relatively simple, the reaction conditions are mild, the preparation process has low equipment requirements, is easy to operate, is green and environmentally friendly, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a transmission electron microscopic image of Lactobacillus rhamnosus microcapsules prepared under different conditions in Experimental Example 1 of the present invention; Figure 2 It is a bar graph of the activity values ​​of Lactobacillus rhamnosus before and after the preparation of Lactobacillus rhamnosus microcapsules in Experiment 1 of Experimental Example 2 of the present invention; Figure 3 is a potential potential diagram of Lactobacillus rhamnosus and Lactobacillus rhamnosus microcapsules in Experiment 2 of Experimental Example 2 of the present invention; Figure 4 It is a line graph of OD values ​​corresponding to the growth curves of Lactobacillus rhamnosus and Lactobacillus rhamnosus microcapsules in Experiment 3 of Experimental Example 2 of the present invention; Figure 5 It is a bar graph of the survival rates of Lactobacillus rhamnosus and Lactobacillus rhamnosus microcapsules in simulated gastric acid in vitro in Experiment 4 of Experimental Example 2 of the present invention; Figure 6 These are transmission electron micrographs of the Lactobacillus rhamnosus microcapsules at different stages of growth in simulated intestinal fluid in vitro in Experiment 5 of Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0019] The present invention is further explained below in conjunction with examples. These examples are only used to illustrate the present invention and are not used to limit the scope of the present invention; the experimental methods without specific conditions in the examples are all based on conventional conditions, and the amounts of reagents in the kits used in the examples are only exemplary, and those skilled in the art can make corresponding adjustments according to actual conditions; the reagents and biological materials, unless otherwise specified, can all be obtained from commercial sources.

[0020] Example 1 This embodiment provides a method for preparing Lactobacillus rhamnosus microcapsules, comprising the following steps: Step S1: centrifuging a Lactobacillus rhamnosus bacterial solution, discarding the supernatant, adding a bacterial culture solution to resuspend the solution, and mixing the solution thoroughly to obtain a Lactobacillus rhamnosus resuspended solution; Specifically, the cultured Lactobacillus rhamnosus culture was centrifuged in a centrifuge with a centrifugal force of 1500 g for 5 min, wherein the number of Lactobacillus rhamnosus in the Lactobacillus rhamnosus culture was 3×10 10 After centrifugation, the supernatant was taken out, and 10 ml of bacterial culture solution was added to the Lactobacillus rhamnosus for re-suspending, and the Lactobacillus rhamnosus re-suspended solution was obtained after sufficient mixing.

[0021] Step S2: stirring the Lactobacillus rhamnosus resuspended solution and adding ammonia water, and mixing thoroughly to obtain a mixed bacterial solution; Further, 100 µl of aqueous ammonia was dripped into the Lactobacillus rhamnosus resuspended solution obtained in step S1, and stirred while dripping to obtain a mixed bacterial solution.

[0022] Step S3: adding 5-hydroxytryptamine to the mixed bacterial solution, stirring for reaction, and collecting by centrifugation to obtain microcapsules encapsulating Lactobacillus rhamnosus.

[0023] Specifically, 5-hydroxytryptamine was added to the mixed bacterial solution obtained in step S2 to a final concentration of 2 mg / mL, and then stirred at room temperature for 60 minutes to form Lactobacillus rhamnosus microcapsules. After the reaction was completed, the Lactobacillus rhamnosus microcapsules were collected by centrifugation.

[0024] This embodiment also provides a Lactobacillus rhamnosus microcapsule. The activity of Lactobacillus rhamnosus in the Lactobacillus rhamnosus microcapsule is 91.8%, the particle size is 1.58 μm, and the shell thickness is 88 nm.

[0025] This embodiment also provides an application of Lactobacillus rhamnosus microcapsules in the preparation of Lactobacillus rhamnosus microcapsule oral dosage forms, including its application in the treatment of enteritis and the delivery of Lactobacillus rhamnosus.

[0026] Example 2 This embodiment provides a method for preparing Lactobacillus rhamnosus microcapsules, comprising the following steps: Step S1: centrifuging a Lactobacillus rhamnosus bacterial solution, discarding the supernatant, adding a bacterial culture solution to resuspend the solution, and mixing the solution thoroughly to obtain a Lactobacillus rhamnosus resuspended solution; Specifically, the cultured Lactobacillus rhamnosus culture was centrifuged in a centrifuge with a centrifugal force of 1500 g for 5 min, wherein the number of Lactobacillus rhamnosus in the Lactobacillus rhamnosus culture was 3×10 10 After centrifugation, the supernatant was taken out, and 10 ml of bacterial culture solution was added to the Lactobacillus rhamnosus for re-suspending, and the Lactobacillus rhamnosus re-suspended solution was obtained after sufficient mixing.

[0027] Step S2: stirring the Lactobacillus rhamnosus resuspended solution and adding ammonia water, and mixing thoroughly to obtain a mixed bacterial solution; Further, 200 µl of aqueous ammonia was dripped into the Lactobacillus rhamnosus resuspended solution obtained in step S1, and the mixture was stirred while dripping to obtain a mixed bacterial solution.

[0028] Step S3: adding 5-hydroxytryptamine to the mixed bacterial solution, stirring for reaction, and collecting by centrifugation to obtain microcapsules encapsulating Lactobacillus rhamnosus.

[0029] Specifically, 5-hydroxytryptamine was added to the mixed bacterial solution obtained in step S2 to a final concentration of 2 mg / mL, and then stirred at room temperature for 60 minutes to form Lactobacillus rhamnosus microcapsules. After the reaction was completed, the Lactobacillus rhamnosus microcapsules were collected by centrifugation.

[0030] This embodiment also provides a Lactobacillus rhamnosus microcapsule, in which the activity of Lactobacillus rhamnosus in the Lactobacillus rhamnosus microcapsule is 90.02%, the particle size is 1.58 μm, and the shell thickness is 90 nm.

[0031] This embodiment also provides an application of Lactobacillus rhamnosus microcapsules in the preparation of Lactobacillus rhamnosus microcapsule oral dosage forms, including its application in the treatment of enteritis and the delivery of Lactobacillus rhamnosus.

[0032] Example 3 This embodiment provides a method for preparing Lactobacillus rhamnosus microcapsules, comprising the following steps: Step S1: centrifuging a Lactobacillus rhamnosus bacterial solution, discarding the supernatant, adding a bacterial culture solution to resuspend the solution, and mixing the solution thoroughly to obtain a Lactobacillus rhamnosus resuspended solution; Specifically, the cultured Lactobacillus rhamnosus culture was centrifuged in a centrifuge with a centrifugal force of 1500 g for 5 min, wherein the number of Lactobacillus rhamnosus in the Lactobacillus rhamnosus culture was 3×10 10 After centrifugation, the supernatant was taken out, and 10 ml of bacterial culture solution was added to the Lactobacillus rhamnosus for re-suspending, and the Lactobacillus rhamnosus re-suspended solution was obtained after sufficient mixing.

[0033] Step S2: stirring the Lactobacillus rhamnosus resuspended solution and adding ammonia water, and mixing thoroughly to obtain a mixed bacterial solution; Further, 100 µl of aqueous ammonia was dripped into the Lactobacillus rhamnosus resuspended solution obtained in step S1, and stirred while dripping to obtain a mixed bacterial solution.

[0034] Step S3: adding 5-hydroxytryptamine to the mixed bacterial solution, stirring for reaction, and collecting by centrifugation to obtain microcapsules encapsulating Lactobacillus rhamnosus.

[0035] Specifically, 5-hydroxytryptamine was added to the mixed bacterial solution obtained in step S2 to a final concentration of 4 mg / mL, and then stirred at room temperature for 60 minutes to form Lactobacillus rhamnosus microcapsules. After the reaction was completed, the Lactobacillus rhamnosus microcapsules were collected by centrifugation.

[0036] This embodiment also provides a Lactobacillus rhamnosus microcapsule. The activity of Lactobacillus rhamnosus in the Lactobacillus rhamnosus microcapsule is 91.2%, the particle size is 1.58 μm, and the shell thickness is 90 nm.

[0037] This embodiment also provides an application of Lactobacillus rhamnosus microcapsules in the preparation of Lactobacillus rhamnosus microcapsule oral dosage forms, including its application in the treatment of enteritis and the delivery of Lactobacillus rhamnosus.

[0038] Example 4 This embodiment provides a method for preparing Lactobacillus rhamnosus microcapsules, comprising the following steps: Step S1: centrifuging a Lactobacillus rhamnosus bacterial solution, discarding the supernatant, adding a bacterial culture solution to resuspend the solution, and mixing the solution thoroughly to obtain a Lactobacillus rhamnosus resuspended solution; Specifically, the cultured Lactobacillus rhamnosus culture was centrifuged in a centrifuge with a centrifugal force of 1500 g for 5 min, wherein the number of Lactobacillus rhamnosus in the Lactobacillus rhamnosus culture was 3×10 10 After centrifugation, the supernatant was taken out, and 10 ml of bacterial culture solution was added to the Lactobacillus rhamnosus for re-suspending, and the Lactobacillus rhamnosus re-suspended solution was obtained after sufficient mixing.

[0039] Step S2: stirring the Lactobacillus rhamnosus resuspended solution and adding ammonia water, and mixing thoroughly to obtain a mixed bacterial solution; Further, 100 µl of aqueous ammonia was dripped into the Lactobacillus rhamnosus resuspended solution obtained in step S1, and stirred while dripping to obtain a mixed bacterial solution.

[0040] Step S3: adding 5-hydroxytryptamine to the mixed bacterial solution, stirring for reaction, and collecting by centrifugation to obtain microcapsules encapsulating Lactobacillus rhamnosus.

[0041] Specifically, 5-hydroxytryptamine was added to the mixed bacterial solution obtained in step S2 to a final concentration of 2 mg / mL, and then stirred at room temperature for 120 minutes to form Lactobacillus rhamnosus microcapsules. After the reaction was completed, the Lactobacillus rhamnosus microcapsules were collected by centrifugation.

[0042] This embodiment also provides a Lactobacillus rhamnosus microcapsule. The activity of Lactobacillus rhamnosus in the Lactobacillus rhamnosus microcapsule is 90.8%, the particle size is 1.58 μm, and the shell thickness is 92 nm.

[0043] This embodiment also provides an application of Lactobacillus rhamnosus microcapsules in the preparation of Lactobacillus rhamnosus microcapsule oral dosage forms, including its application in the treatment of enteritis and the delivery of Lactobacillus rhamnosus.

[0044] Experimental Example 1: Effect of preparation conditions on the morphology and size of Lactobacillus rhamnosus microcapsules Experimental method: Referring to the preparation method of the embodiment, experiments were conducted on the three factors of 5-hydroxytryptamine concentration, ammonia addition amount and stirring time in the preparation parameters according to the controlled variable method to observe their effects on the morphology and size of Lactobacillus rhamnosus capsules.

[0045] Experimental results: as attached Figure 1 The TEM images in the first row are in the presence of 2 mg / mL 5-HT and 300 µl ammonia water, which further verifies whether the thickness of the poly-5-HT nanoshell increases with the increase of ammonia water (volume ratio with bacterial culture solution is 3:100) and 3× 10 10 The second row was obtained under the conditions of 2 mg / mL 5-HT, stirring time of 1 h and 3 × 10 10 The third behavior was obtained by adding different amounts of ammonia water under the conditions of CUF Lactobacillus rhamnosus, stirring for 1h and 3× 10 10 The results were obtained by adding different amounts of 5-HT under CUF Lactobacillus rhamnosus conditions.

[0046] Experimental results analysis: From the attached Figure 1It can be seen from the transmission electron microscope in the first row that when the stirring time is 30 minutes, micrometer-sized Lactobacillus rhamnosus microcapsules have basically been formed, but the shell thickness is obviously insufficient. When the stirring time reaches 60 minutes, it can be seen that the formed Lactobacillus rhamnosus microcapsules have a complete core-shell structure. When the stirring time is 240 minutes or even longer, the size and shell thickness of the formed Lactobacillus rhamnosus microcapsules have not changed significantly compared with the stirring time of 60 minutes.

[0047] From the attached Figure 1 It can be seen from the TEM in the second row that when the amount of ammonia added is 50µl (the volume ratio to the bacterial culture medium is 0.5:100), the complete core-shell structure of the Lactobacillus rhamnosus microcapsule cannot be formed. When the amount of ammonia added is 100µl or even higher, it can be seen that the Lactobacillus rhamnosus microcapsule has a complete core-shell structure. However, as the amount of ammonia added increases to 300µl, the size of the Lactobacillus rhamnosus microcapsule and the thickness of the shell layer do not change further.

[0048] From the attached Figure 1 It can be seen from the TEM in the third row that when the final concentration of 5-HT is 0.5 mg / ml and 1 mg / ml, although Lactobacillus rhamnosus microcapsules are formed, they do not have a complete core-shell structure or the shell thickness is very thin, which makes it impossible to protect the encapsulated Lactobacillus rhamnosus from gastric acid invasion in the gastric acid environment. When the final concentration of 5-HT is 2 mg / ml and 4 mg / ml, the Lactobacillus rhamnosus microcapsules have a complete core-shell structure and good size uniformity. When the final concentration of 5-HT reaches 8 mg / ml, the Lactobacillus rhamnosus microcapsules will undergo obvious agglomeration, which is not conducive to its dispersion during application.

[0049] Experimental Example 2: In vitro validation test of Lactobacillus rhamnosus microcapsules 1. Determination of the activity of Lactobacillus rhamnosus in Lactobacillus rhamnosus microcapsules Experimental method: Take 3×10 10 The Lactobacillus rhamnosus bacterial liquid of CUF and the Lactobacillus rhamnosus microcapsules prepared in Example 1 were diluted in multiple ratios to obtain a suitable concentration. The Lactobacillus rhamnosus bacterial liquid and the Lactobacillus rhamnosus microcapsules prepared in Example 1 were then evenly spread on an MRS plate and placed in a bacterial incubator at 37° C. After 12 h, the activity of Lactobacillus rhamnosus in the microcapsules was detected by the colony counting method, and then the corresponding bar graph was drawn.

[0050] Experimental results: as attached Figure 2As shown, the Lactobacillus rhamnosus microcapsules prepared according to the method of Example 1 were tested for the activity of Lactobacillus rhamnosus before and after preparation, wherein the survival rate of Lactobacillus rhamnosus after preparation can still reach 91.7%, indicating that the preparation method of the present invention and the reagent materials selected therein have good biocompatibility, will not inhibit the growth of Lactobacillus rhamnosus, and are suitable for preparing microcapsules for delivering Lactobacillus rhamnosus.

[0051] 2. Lactobacillus rhamnosus Encapsulation Assay Experimental method: Take 3×10 10 The zeta potential of the Lactobacillus rhamnosus bacterial solution of CUF and the Lactobacillus rhamnosus microcapsules prepared in Example 1 were tested respectively to observe whether the Lactobacillus rhamnosus was successfully embedded.

[0052] Experimental results: as attached Figure 3 As shown, the potential point of the Lactobacillus rhamnosus bacterial solution was -10.5 mV, and its potential point changed to -15.3 mV after the preparation of microcapsules, indicating that Lactobacillus rhamnosus and 5-hydroxytryptamine were successfully combined.

[0053] 3. Growth test of Lactobacillus rhamnosus capsules Experimental method: The same amount of active Lactobacillus rhamnosus bacterial liquid and the Lactobacillus rhamnosus microcapsules prepared in Example 1 were cultured in MRS medium respectively and their OD values ​​were detected by optical density meter at different time points.

[0054] Experimental results: as attached Figure 4 As shown, Lactobacillus rhamnosus can grow normally in a normal environment. When Lactobacillus rhamnosus microcapsules are exposed to a normal environment, the growth of Lactobacillus rhamnosus has a certain hysteresis, but it does not affect the growth of Lactobacillus rhamnosus. This shows that the presence of the shell layer in Lactobacillus rhamnosus microcapsules can effectively slow down the direct contact between external nutrients and rhamnose, thereby slowing down the growth and reproduction of rhamnose.

[0055] 4. In vitro gastric acid microenvironment simulation Experimental method: The same amount of active Lactobacillus rhamnosus bacterial liquid and Lactobacillus rhamnosus microcapsules prepared in Example 1 were placed in an in vitro simulated gastric acid microenvironment of pH 2.5, and the survival rates of Lactobacillus rhamnosus of the two were tested at different time periods.

[0056] Experimental results: as attached Figure 5 As shown in the figure, when Lactobacillus rhamnosus was exposed to the gastric acid environment for 3 hours, it was completely killed. When the Lactobacillus rhamnosus microcapsules were exposed to the simulated gastric acid microenvironment for 4 hours, the survival rate of Lactobacillus rhamnosus was still maintained at about 10%. This shows that the Lactobacillus rhamnosus microcapsules of the present invention can effectively resist the killing of Lactobacillus rhamnosus by gastric acid and effectively increase the survival rate of Lactobacillus rhamnosus in the stomach.

[0057] 5. Bioavailability of Lactobacillus rhamnosus microcapsules Experimental method: The Lactobacillus rhamnosus microcapsules prepared in Example 1 were placed in an in vitro simulated intestinal fluid microenvironment of pH 7.0, and the morphological characteristics of the Lactobacillus rhamnosus microcapsules were tested by transmission electron microscopy at different time periods.

[0058] Experimental results: as attached Figure 6 As shown in the figure, as the Lactobacillus rhamnosus grows and reproduces, the 5-hydroxytryptamine nanoshell wrapped on its surface gradually fades away, fully exposing the wrapped Lactobacillus rhamnosus. After 4 hours, the 5-hydroxytryptamine nanoshell basically fades away. The above results show that the Lactobacillus rhamnosus microcapsules can effectively achieve intestinal targeted delivery of Lactobacillus rhamnosus, and use their own growth and reproduction properties to achieve direct contact between Lactobacillus rhamnosus and intestinal mucosa, thereby playing a prebiotic role.

[0059] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be regarded as exemplary and non-restrictive from any point of view. In addition, it should be understood that although this specification is described in accordance with the embodiments, it does not contain only one technical solution. This narrative of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing Lactobacillus rhamnosus microcapsules, characterized in that: The method comprises the following steps: Step S1: centrifuging a Lactobacillus rhamnosus bacterial solution, discarding the supernatant, adding a bacterial culture solution to resuspend the solution, and mixing the solution thoroughly to obtain a Lactobacillus rhamnosus resuspended solution; Step S2: stirring the Lactobacillus rhamnosus resuspended solution and adding ammonia water, and mixing thoroughly to obtain a mixed bacterial solution; Step S3: adding 5-hydroxytryptamine to the mixed bacterial solution, stirring for reaction, and collecting by centrifugation to obtain microcapsules encapsulating Lactobacillus rhamnosus.

2. The method for preparing a Lactobacillus rhamnosus microcapsule according to claim 1, characterized in that: The number of Lactobacillus rhamnosus in the Lactobacillus rhamnosus bacterial solution in step S1 is 3×10 10 ~4×10 10 CUF; the centrifugation time is 5 to 10 minutes, and the centrifugal force is 1000 to 1500g.

3. The method for preparing a Lactobacillus rhamnosus microcapsule according to claim 1, characterized in that: In the step S2, the volume ratio of ammonia water to bacterial culture solution in the mixed bacterial solution is 1 to 2:

100.

4. The method for preparing a Lactobacillus rhamnosus microcapsule according to claim 1, characterized in that: In step S3, the final concentration of 5-HT is 2-4 mg / ml; the stirring temperature is 20-25° C., and the stirring time is 60-120 min.

5. A Lactobacillus rhamnosus microcapsule prepared by the preparation method according to any one of claims 1 to 4.

6. The Lactobacillus rhamnosus microcapsule according to claim 5, characterized in that: The particle size of the Lactobacillus rhamnosus microcapsule is 1-2 μm, and the shell thickness is 50-100 nm.

7. The Lactobacillus rhamnosus microcapsule according to claim 5, characterized in that: The active content of Lactobacillus rhamnosus in the Lactobacillus rhamnosus microcapsules is 90-95%.

8. Use of a Lactobacillus rhamnosus microcapsule prepared by the preparation method as claimed in any one of claims 1 to 4 or a Lactobacillus rhamnosus microcapsule as claimed in any one of claims 5 to 7 in the preparation of a Lactobacillus rhamnosus microcapsule oral dosage form drug.

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