Whole intestinal flora transplantation strategy and nano packaging method and application thereof

Abstract

The invention relates to a whole intestine flora transplantation strategy and a nanometer packaging method and application thereof. According to the method disclosed by the invention, the transplantation of the whole intestinal flora is realized by mainly utilizing the combined transplantation of the intestinal fluid flora and the fecal flora, so that the micro-ecology of the whole intestinal tract is more comprehensively, accurately and efficiently regulated and controlled, and a better flora transplantation treatment effect is achieved. The microflora (microcapsules) is encapsulated by a safe biological material, the encapsulating process is simple in process and short in time consumption, intestinal bacteria in different shapes can be simultaneously encapsulated, and the method is not only suitable for encapsulating oxygen-resistant bacteria and facultative anaerobic bacteria, but also suitable for encapsulating intestinal flora including strict anaerobic bacteria, and has the advantages of being simple in process and low in cost. According to the preparation method, death of intestinal bacteria in the preparation process can be reduced, erosion of gastric acid and the like to the intestinal bacteria can be resisted, the survival rate of the bacteria and the colonization efficiency of the bacteria in host intestinal tracts can be remarkably improved, and the microbial transplantation treatment effect is further enhanced.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology and relates to a whole intestinal flora transplantation strategy and a nano-encapsulation method and application thereof. Background Art

[0002] The intestinal microbiota, including the small intestine, cecum and colon microbiota and their interactions with the host environment, are extremely important for maintaining intestinal homeostasis and human health and disease. Microbiota biomarkers can be used as potential non-invasive diagnostic tools for diseases, and microbiota-based therapies such as fecal microbiota transplantation (FMT) and probiotic supplementation have emerged as promising adjuvant treatments for various dysbiosis diseases to restore their intestinal microbial and metabolic balance, especially for recurrent Clostridium difficile infection and inflammatory bowel disease (IBD).

[0003] Studies have shown that the colonization density of the entire gastrointestinal tract has a gradually increasing trend, with the lowest density in the stomach being <10 3 CFU / mL, jejunum: 10 4 -10 5 CFU / mL, ileum is 10 7 -10 8 CFU / mL, reaching a maximum density of 10 12 CFU / mL. There is a lot of digestive juice in the small intestine cavity. Although it is nutrient-rich, the transmission speed is fast. Therefore, compared with the colon, the diversity and colonization density of the small intestinal microbiota (SIM) are lower. Existing evidence shows that common bacterial genera in the small intestine include Lactobacillus, Clostridium, Staphylococcus, Streptococcus, Bacteroides, Veillonella, Prevotella, Fusobacterium and Haemophilus. The function of the colon is mainly the formation, storage and excretion of feces, so FMT also mainly refers to the transplantation of fecal flora from the colon. However, for the small intestine rich in a large number of facultative anaerobes and aerobic bacteria, FMT has limited therapeutic effects on various small intestinal diseases (including small intestinal bacterial overgrowth, irritable bowel syndrome, short bowel syndrome and pouchitis, etc.).

[0004] The small intestine and its microbiota play an important role in nutrient digestion and absorption, host metabolism, and immune regulation. Unlike colonic or fecal microbiota, SIM has been less studied due to sampling difficulties and ethical issues. Clinically, small intestinal samples can be obtained through negative pressure suction of an indwelling nasogastric tube or enteroscopy. With the advancement of technology, samples can also be collected through less invasive ingestible sampling capsules, which will facilitate the study of the dynamic characteristics of SIM and provide potential clinical feasibility for SIM transplantation. We believe that whole gut microbiota transplantation (WMT) that simultaneously transplants small intestinal fluid flora and fecal flora to regulate the whole intestinal microecology may be a more reasonable and more clinically promising method.

[0005] In addition, the current clinical administration methods of FMT mainly include bacterial capsules and bacterial suspensions. The bacteria themselves are intolerant to gastric acid. In order to avoid erosion by gastric juice, bacterial liquid is often delivered through a nasogastric tube or retrograde colon administration through a colonoscope. This invasive administration method increases patient discomfort and has low acceptance, which is not conducive to the promotion and application of FMT. Encapsulating the bacterial flora in capsules has the problem of cumbersome capsule preparation process, which requires processes such as centrifugation, freezing, and vacuum drying, which is time-consuming. The long preparation process will lead to the death of a large number of strict anaerobic bacteria, as well as the loss of certain key beneficial bacteria, which greatly reduces the therapeutic effect of FMT.

[0006] In recent years, nanocapsule technology, as an emerging bacterial surface modification technology, uses safe biomaterials to modify or encapsulate bacteria to form nanocapsules, which can greatly improve the survival rate of bacteria in harsh environments (such as gastric acid). However, the existing live bacterial microcapsule construction technology has little research on SIM and FMT containing a large number of strict anaerobes. In order to avoid the death of strict anaerobes during the encapsulation process, the development of a transplanted bacterial nanocapsule construction strategy that is short in time, simple to operate, and has a high survival rate of anaerobic bacteria will significantly improve patient acceptance and clinical treatment effects of WMT. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems existing in the prior art and to provide a whole intestinal flora transplantation strategy and a nano-encapsulation method and application thereof.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A whole intestinal flora transplantation strategy and a nano-encapsulation method thereof, comprising the following steps:

[0010] 1) Preparation of fecal flora from fresh mouse feces;

[0011] 2) Preparation of small intestinal flora from rat small intestinal fluid;

[0012] 3) preparing chitosan and sodium alginate solutions and sterilizing them by high pressure steam;

[0013] 4) After centrifuging the bacterial solutions in steps 1) and 2), the bacterial colonies were resuspended in the sterilized chitosan solution in step 3) and shaken;

[0014] 5) Centrifuging the bacterial solution after shaking in step 4), resuspending and washing with PBS buffer to remove excess chitosan;

[0015] 6) centrifuging the resuspended and washed bacterial solution in step 5) and resuspending them in the sterilized sodium alginate solution in step 3) and shaking them;

[0016] 7) Centrifuging the bacterial solution after shaking in step 6), resuspending and washing with PBS buffer to remove excess sodium alginate;

[0017] 8) Centrifuge the resuspended and washed bacterial solution in step 7) and resuspend it in PBS to complete the packaging of intestinal flora.

[0018] Preferably, the chitosan solution prepared in step 3) has a concentration of 1 mg / ml, dissolved in 0.2% acetic acid solution, and adjusted to pH=6.0 using NaOH solution. Too low or too high a pH value will cause bacterial death; wherein, the concentration of the sodium alginate solution is 2 mg / ml, dissolved in deionized water. If the concentration is too high, the solution is more viscous and easily forms flocculation, and if the concentration is too low, it is not easy to assemble onto the bacterial surface.

[0019] Preferably, the preparation of the chitosan and the sodium alginate solution comprises the following steps:

[0020] 1) Weigh the chitosan powder, dissolve it in 0.2% acetic acid solution, shake well to make it fully dissolved, adjust the pH with NaOH solution, and the pH is 6.0 as measured by test paper;

[0021] 2) Weigh the sodium alginate powder, dissolve it in deionized water, and shake well to fully dissolve it;

[0022] 3) The chitosan and sodium alginate solutions prepared in steps 1) and 2) are placed in a blue-mouthed glass bottle, the bottle mouth is loosened, and the bottle is sterilized in a high-pressure steam cooker and then cooled at room temperature for later use.

[0023] Preferably, the intestinal bacteria representative used is the Gram-negative bacillus Escherichia coli Nissle 1917 strain or the Gram-positive coccus Pediococcus acidilactici or the unicellular fungus yeast.

[0024] Preferably, the bacterial liquid of the fecal flora is prepared by feces of an organism, wherein the organism includes a mouse.

[0025] Preferably, the bacterial liquid of the fecal flora is prepared as follows:

[0026] Take fresh mouse feces, add PBS solution and mix well, then use a filter to filter out impurities to obtain the fecal flora solution.

[0027] Preferably, the bacterial liquid of the small intestinal flora is prepared from the small intestinal fluid of an organism, and the organism includes a rat.

[0028] Preferably, the bacterial liquid of the small intestinal flora is prepared as follows:

[0029] Fresh small intestine of rats was taken, and the small intestinal fluid was collected by squeezing. Impurities were filtered out by a filter, and the mixture was centrifuged and resuspended in PBS solution to obtain the bacterial fluid of small intestinal flora.

[0030] Preferably, whole intestinal flora transplantation can be achieved by sequentially administering fecal bacterial fluid and small intestinal bacterial fluid to experimental mice by gavage.

[0031] The present invention also provides an oral intestinal probiotic nanocapsule, and the prepared intestinal probiotic nanocapsule is used in intestinal flora transplantation.

[0032] The encapsulation principle of the intestinal bacteria or flora of the present invention is as follows: the bacteria are negatively charged, and chitosan is positively charged. Under gentle vibration, the two can adhere to the surface of the bacteria through the electrostatic effect of mutual attraction between the positive and negative charges, forming a chitosan positively charged nanoshell. Sodium alginate is negatively charged. After being added to the chitosan-coated bacterial solution, it adheres to the surface of the chitosan shell through electrostatic adsorption, achieving further reinforcement of the nanoshell, and finally completing the encapsulation of the intestinal bacteria or flora, that is, the preparation of nanocapsules, which can improve the tolerance of transplanted bacteria to the harsh environment of the gastrointestinal tract.

[0033] Due to the adoption of the above technical solution, the beneficial effects obtained by the present invention include:

[0034] The present invention mainly utilizes the combined transplantation of small intestinal fluid flora and fecal flora to achieve the transplantation of whole intestinal flora, thereby more comprehensively, accurately and efficiently regulating the microecology of the whole intestine, and achieving a better flora transplantation treatment effect. And by encapsulating the flora (microcapsules) with safe biological materials, the encapsulation process is simple and time-saving, and intestinal bacteria of various forms can be encapsulated at the same time. It is not only suitable for encapsulating aerobic bacteria and facultative anaerobic bacteria, but also suitable for encapsulating intestinal flora including strict anaerobic bacteria. It can reduce the death of intestinal bacteria during the preparation process, and can withstand the erosion of intestinal bacteria by gastric acid, etc., which can significantly improve the survival rate of bacteria and their colonization efficiency in the host intestine, and further enhance the therapeutic effect of microbial transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The morphology of Escherichia coli before and after nanoencapsulation in the embodiment is compared.

[0036] Figure 2The morphology of Pediococcus acidilactici before and after nanoencapsulation in the example is compared.

[0037] Figure 3 The figure is a comparison of the yeast morphology before and after nanoencapsulation in the example.

[0038] Figure 4 The effect of the nanoencapsulation on the survival of Escherichia coli in simulated gastric fluid is shown in the example.

[0039] Figure 5 1 is the effect of the nanoencapsulation on the survival of Pediococcus acidilactici in simulated gastric fluid.

[0040] Figure 6 The effect of nanoencapsulation on the survival of yeast in simulated gastric fluid is shown in the example.

[0041] Figure 7 The effect of the nanocapsules in the examples on the survival of intestinal flora in simulated gastric fluid. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The present invention will be further described below in conjunction with the accompanying drawings.

[0044] Example 1: Nano-encapsulation of Escherichia coli

[0045] 1) Escherichia coli Nissle 1917 was cultured in LB medium at 37°C and 200 rpm for 6 h at a 2% inoculum, and then washed three times with PBS buffer (pH 6.0);

[0046] 2) Weigh the chitosan powder, dissolve it in 0.2% acetic acid solution, shake well to fully dissolve, and the final concentration is 1 mg / ml. Use NaOH solution to adjust the pH, and the test paper determines it to be 6.0;

[0047] 3) Weigh the sodium alginate powder, dissolve it in deionized water, shake well to make it fully dissolved, and the final concentration is 2 mg / ml;

[0048] 4) sterilizing the prepared chitosan and sodium alginate solutions in a high pressure steam cooker;

[0049] 5) The washed bacterial solution was centrifuged at 10,000 rpm for 5 min, resuspended in chitosan solution, and shaken at 100 rpm for 10 min;

[0050] 6) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess chitosan;

[0051] 7) After centrifugation, the bacterial solution was resuspended in sodium alginate solution and shaken at 100 rpm for 10 min;

[0052] 8) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess sodium alginate;

[0053] 9) The shaken bacterial solution is centrifuged, resuspended and washed three times with PBS to complete the encapsulation of intestinal bacteria.

[0054] 10) Then, both the naked bacteria and the encapsulated bacteria were resuspended in simulated gastric fluid (pH 2.0, containing 10 g / L pepsin) and incubated at 37°C and 200 rpm for 2 h and 4 h. The morphology of E. coli before and after encapsulation is shown in the attached Figure 1 The transmission electron microscope (TEM) image shows obvious morphological differences, and the atomic force microscope (AFM) image characterizes the difference in roughness of the bacterial surface morphology before and after packaging. Figure 4 ,The results showed that the number of surviving E. coli after packaging was significantly higher than that of naked bacteria.

[0055] Example 2: Encapsulation of Pediococcus acidilactici

[0056] 1) Pediococcus acidilactici DQ2 was cultured in MRS medium at 37°C and 200 rpm for 12 h with a 5% inoculum, and then washed three times with PBS buffer (pH 6.0);

[0057] 2) Weigh the chitosan powder, dissolve it in 0.2% acetic acid solution, shake well to fully dissolve, and the final concentration is 1 mg / ml. Use NaOH solution to adjust the pH, and the test paper determines it to be 6.0;

[0058] 3) Weigh the sodium alginate powder, dissolve it in deionized water, shake well to make it fully dissolved, and the final concentration is 2 mg / ml;

[0059] 4) sterilizing the prepared chitosan and sodium alginate solutions in a high pressure steam cooker;

[0060] 5) The washed bacterial solution was centrifuged at 10,000 rpm for 5 min, resuspended in chitosan solution, and shaken at 100 rpm for 10 min;

[0061] 6) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess chitosan;

[0062] 7) After centrifugation, the bacterial solution was resuspended in sodium alginate solution and shaken at 100 rpm for 10 min;

[0063] 8) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess sodium alginate;

[0064] 9) The shaken bacterial solution is centrifuged, resuspended and washed three times with PBS to complete the encapsulation of intestinal bacteria.

[0065] 10) Then, both naked bacteria and encapsulated bacteria were resuspended in simulated gastric fluid (pH 2.0, containing 10 g / L pepsin) and incubated at 37°C and 200 rpm for 2 h and 4 h. The morphology of Pediococcus acidilactici before and after encapsulation is shown in the attached figure. Figure 2 The survival in simulated gastric fluid is shown in the attached Figure 5 The results showed that the number of surviving lactic acid bacteria after packaging was significantly higher than that of naked bacteria.

[0066] Example 3: Encapsulation of Yeast

[0067] 1) Saccharomyces cerevisiae CICC 1308 was cultured in YPD medium at 37°C and 200 rpm for 24 h at a 10% inoculum, and then washed three times with PBS buffer (pH 6.0);

[0068] 2) Weigh the chitosan powder, dissolve it in 0.2% acetic acid solution, shake well to fully dissolve, and the final concentration is 1 mg / ml. Use NaOH solution to adjust the pH, and the test paper determines it to be 6.0;

[0069] 3) Weigh the sodium alginate powder, dissolve it in deionized water, shake well to make it fully dissolved, and the final concentration is 2 mg / ml;

[0070] 4) sterilizing the prepared chitosan and sodium alginate solutions in a high pressure steam cooker;

[0071] 5) The washed bacterial solution was centrifuged at 10,000 rpm for 5 min, resuspended in chitosan solution, and shaken at 100 rpm for 10 min;

[0072] 6) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess chitosan;

[0073] 7) After centrifugation, the bacterial solution was resuspended in sodium alginate solution and shaken at 100 rpm for 10 min;

[0074] 8) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess sodium alginate;

[0075] 9) The shaken bacterial solution is centrifuged, resuspended and washed three times with PBS to complete the encapsulation of intestinal bacteria.

[0076] 10) Afterwards, both the naked bacteria and the encapsulated bacteria were resuspended in simulated gastric fluid (pH 2.0, containing 10 g / L pepsin) and incubated at 37°C and 200 rpm for 2 h and 4 h. The morphology of yeast before and after encapsulation is shown in the attached Figure 3 The survival in simulated gastric fluid is shown in the attached Figure 6 The surviving bacteria of encapsulated yeast were significantly higher than those of naked yeast.

[0077] Example 4: Encapsulation of fecal flora

[0078] 1) Take 1 g of fresh mouse feces, add PBS (pH 7.0) at a mass ratio of 1:4 and mix well. Filter impurities with a 70 μm filter to obtain a bacterial solution, and wash with PBS three times;

[0079] 2) Weigh the chitosan powder, dissolve it in 0.2% acetic acid solution, shake well to fully dissolve, and the final concentration is 1 mg / ml. Use NaOH solution to adjust the pH, and the test paper determines it to be 6.0;

[0080] 3) Weigh the sodium alginate powder, dissolve it in deionized water, shake well to make it fully dissolved, and the final concentration is 2 mg / ml;

[0081] 4) sterilizing the prepared chitosan and sodium alginate solutions in a high pressure steam cooker;

[0082] 5) The washed bacterial solution was centrifuged at 10,000 rpm for 5 min, resuspended in chitosan solution, and shaken at 100 rpm for 10 min;

[0083] 6) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess chitosan;

[0084] 7) After centrifugation, the bacterial solution was resuspended in sodium alginate solution and shaken at 100 rpm for 10 min;

[0085] 8) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess sodium alginate;

[0086] 9) The shaken bacterial solution is centrifuged, resuspended and washed three times with PBS to complete the packaging of fecal flora.

[0087] 10) Then, the naked bacterial colonies and the encapsulated bacterial colonies were resuspended in simulated gastric fluid (pH 2.0, containing 10 g / L pepsin) and incubated at 37°C and 200 rpm for 2 h and 4 h. The survival of the bacterial colonies in simulated gastric fluid is shown in the attached Figure 7 The results showed that the number of surviving bacteria in the encapsulated bacterial colony was significantly higher than that in the naked bacterial colony.

[0088] Example 5: Encapsulation of small intestinal flora

[0089] 1) Take 1 ml of fresh small intestinal fluid from rats, filter out impurities with a 70 μm filter to obtain a bacterial solution, and wash with PBS three times;

[0090] 2) Weigh the chitosan powder, dissolve it in 0.2% acetic acid solution, shake well to fully dissolve, and the final concentration is 1 mg / ml. Use NaOH solution to adjust the pH, and the test paper determines it to be 6.0;

[0091] 3) Weigh the sodium alginate powder, dissolve it in deionized water, shake well to make it fully dissolved, and the final concentration is 2 mg / ml;

[0092] 4) sterilizing the prepared chitosan and sodium alginate solutions in a high pressure steam cooker;

[0093] 5) The washed bacterial solution was centrifuged at 10,000 rpm for 5 min, resuspended in chitosan solution, and shaken at 100 rpm for 10 min;

[0094] 6) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess chitosan;

[0095] 7) After centrifugation, the bacterial solution was resuspended in sodium alginate solution and shaken at 100 rpm for 10 min;

[0096] 8) Centrifuge the shaken bacterial solution, resuspend and wash with PBS buffer to remove excess sodium alginate;

[0097] 9) The shaken bacterial solution is centrifuged, resuspended and washed three times with PBS to complete the packaging of fecal flora.

[0098] 10) Then, the naked bacterial colonies and the encapsulated bacterial colonies were resuspended in simulated gastric fluid (pH 2.0, containing 10 g / L pepsin) and incubated at 37°C and 200 rpm for 2 h and 4 h. The survival of the bacterial colonies in simulated gastric fluid is shown in the attached Figure 7 The results showed that the number of surviving bacteria in the encapsulated bacterial colony was significantly higher than that in the naked bacterial colony.

[0099] In addition, the present invention also provides an application of oral intestinal probiotic nanocapsules, specifically, an application of intestinal probiotic nanocapsules in intestinal flora transplantation.

[0100] Compared with the existing technology, the present invention has the following advantages:

[0101] (1) Propose a whole-gut microbiota transplantation strategy that combines fecal microbiota and small intestinal microbiota transplantation

[0102] FMT mainly refers to the transplantation of fecal flora from the colon, but for the small intestine rich in a large number of facultative anaerobic and aerobic bacteria, FMT has limited therapeutic effects on various small intestinal diseases (including small intestinal bacterial overgrowth, irritable bowel syndrome, short bowel syndrome and pouchitis, etc.). The whole intestinal flora transplantation (WMT) strategy of simultaneously transplanting small intestinal fluid flora and fecal flora to more comprehensively and accurately regulate the microecology of the entire intestine may be a more reasonable and more clinically promising method.

[0103] (2) Achieve uniform encapsulation of a single intestinal bacteria or flora

[0104] Traditional oral enterobacteria capsules are mostly freeze-dried enterobacteria powder wrapped in a gelatin shell, and most anaerobic bacteria will die during the complex freeze-drying process. In addition, the bacteria in the intestinal flora have different forms, and no research reports have been conducted on their simultaneous encapsulation. The method of the present invention can uniformly encapsulate bacteria of different forms in the intestinal flora at the same time through the electrostatic interaction between microorganisms and biomaterials, thereby improving the tolerance of the orally administered transplanted enterobacteria to the harsh gastrointestinal environment and its bioavailability.

[0105] (3) Improve the proportion of live bacteria and intestinal colonization rate of oral intestinal flora capsules

[0106] The clinical use of enteric capsules still has shortcomings such as poor therapeutic effect and repeated illness of patients. The reason may be that too many anaerobic bacteria (mostly probiotics, such as Bifidobacterium) die inside the capsules, which cannot meet the requirements of the number and proportion of live bacteria colonization (1×10 10 CFU, survival rate ≥ 90%). The preparation process of the present invention is simple, with fewer procedures and short time consumption, which can greatly reduce the death of intestinal bacteria during the preparation process. The tolerance of intestinal bacteria in microcapsules to the adverse gastrointestinal environment is improved by coating with biological materials, thereby further improving the proportion of live bacteria and intestinal colonization rate of oral intestinal flora microcapsules, laying a foundation for the quantity and quality of bacteria for exerting the therapeutic effect of microorganisms.

[0107] The above-mentioned related explanations and descriptions of the embodiments are for the convenience of those skilled in the art to understand and apply the present invention. It is obvious that those familiar with the art can easily make various modifications to these contents and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above-mentioned related explanations and descriptions of the embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A whole intestinal flora transplantation strategy and a nano-encapsulation method thereof, characterized in that: The following steps are involved: 1) Preparation of fecal flora from fresh mouse feces; 2) Preparation of small intestinal flora from rat small intestinal fluid; 3) preparing chitosan and sodium alginate solutions and sterilizing them by high pressure steam; 4) After centrifuging the bacterial solutions in steps 1) and 2), the bacterial colonies were resuspended in the sterilized chitosan solution in step 3) and shaken; 5) Centrifuging the bacterial solution after shaking in step 4), resuspending and washing with PBS buffer to remove excess chitosan; 6) centrifuging the resuspended and washed bacterial solution in step 5) and resuspending them in the sterilized sodium alginate solution in step 3) and shaking them; 7) Centrifuging the bacterial solution after shaking in step 6), resuspending and washing with PBS buffer to remove excess sodium alginate; 8) Centrifuge the resuspended and washed bacterial solution in step 7) and resuspend it in PBS to complete the packaging of intestinal flora.

2. The whole intestinal flora transplantation strategy and nano-encapsulation method thereof according to claim 1, characterized in that: The chitosan solution prepared in step 3) has a concentration of 1 mg / ml and is dissolved in 0.2% acetic acid solution, and the pH is adjusted to 6.0 using NaOH solution; wherein the sodium alginate solution has a concentration of 2 mg / ml and is dissolved in deionized water.

3. The whole intestinal flora transplantation strategy and nano-encapsulation method according to claims 1 and 2, characterized in that: The preparation of the chitosan and sodium alginate solution comprises the following steps: 1) Weigh the chitosan powder, dissolve it in 0.2% acetic acid solution, shake well to make it fully dissolved, adjust the pH with NaOH solution, and the pH is 6.0 as measured by test paper; 2) Weigh the sodium alginate powder, dissolve it in deionized water, and shake well to fully dissolve it; 3) The chitosan and sodium alginate solutions prepared in steps 1) and 2) are placed in a blue-mouthed glass bottle, the bottle mouth is loosened, and the bottle is sterilized in a high-pressure steam cooker and then cooled at room temperature for later use.

4. The whole intestinal flora transplantation strategy and nano-encapsulation method thereof according to claim 1, characterized in that: The intestinal bacteria used are represented by the Gram-negative bacillus Escherichia coli Nissle 1917 strain or the Gram-positive coccus Pediococcus acidilactici or the unicellular fungus yeast.

5. The whole intestinal flora transplantation strategy and nano-encapsulation method thereof according to claim 1, characterized in that: The bacterial liquid of the fecal flora is prepared from feces of an organism, wherein the organism includes a mouse.

6. The whole intestinal flora transplantation strategy and nano-encapsulation method thereof according to claim 5, characterized in that: The bacterial liquid of the fecal flora is prepared as follows: Take fresh mouse feces, add PBS solution and mix well, then use a filter to filter out impurities to obtain the fecal flora solution.

7. The whole intestinal flora transplantation strategy and nano-encapsulation method thereof according to claim 1, characterized in that: The bacterial liquid of the small intestinal flora is prepared from the small intestinal fluid of an organism, and the organism includes a rat.

8. The whole intestinal flora transplantation strategy and nano-encapsulation method thereof according to claim 7, characterized in that: The bacterial liquid of the small intestinal flora is prepared as follows: Fresh small intestine of rats was taken, and the small intestinal fluid was collected by squeezing. Impurities were filtered out by a filter, and the mixture was centrifuged and resuspended in PBS solution to obtain the bacterial fluid of small intestinal flora.

9. The whole intestinal flora transplantation strategy and nano-encapsulation method thereof according to claim 1, characterized in that: By continuously gavaging the experimental mice with fecal bacterial fluid and small intestinal bacterial fluid, whole intestinal flora transplantation can be achieved.

10. An oral intestinal probiotic nanocapsule, characterized in that: Prepared by adopting a whole intestinal flora transplantation strategy and nano-encapsulation method according to any one of steps 1 to 8; Among them, the prepared intestinal probiotic nanocapsules are used in intestinal flora transplantation.

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