Targeted probiotic nano microcapsule as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a targeted probiotic nano microcapsule as well as a preparation method and application thereof, and the preparation method comprises the following steps: coating probiotics by using a photosensitive preparation ICG and polysaccharide in a covalent coupling chemical combination manner to prepare the probiotic nano microcapsule; the core material of the microcapsule is mainly probiotics, and the wall material of the microcapsule is carboxymethyl glucan and a photosensitive preparation ICG; the microcapsule is convenient to prepare, good in slow release and controlled release, non-toxic in material, and good in biocompatibility and degradability. According to the invention, the wall material of the probiotic microcapsule is improved, the preparation method and the whole flow process are correspondingly designed, and the capsule wall is formed by using polysaccharide and a photosensitive preparation, so that the obtained nano probiotics are acid-resistant, cholate-resistant, digestive enzyme-resistant and heat-resistant, the stability of the probiotics is improved, and the intestinal colonization effect is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a targeted probiotic nano-microcapsule and a preparation method and application thereof. Background Art

[0002] Probiotics are specialized bacteria that benefit the host, improving the host's intestinal microbial ecosystem by regulating intestinal dysbiosis, balancing the intestinal microbiota, promoting the proliferation of beneficial bacteria, and inhibiting the growth of harmful bacteria. However, delivering probiotics to the large intestine and maintaining their efficacy remain significant challenges in the development of effective functional foods. Probiotics are highly sensitive to industrial processing, transportation, and storage conditions, which limits their use in food products. Another factor affecting probiotic survival is their ability to withstand the harsh gastrointestinal environment. Low gastric pH and a hostile intestinal environment significantly reduce their viability and proliferation. Furthermore, the rapid transit of probiotics in the gastrointestinal tract results in a short residence time, leading to suboptimal therapeutic efficacy. To ensure the survival of probiotics and enhance their viability during processing and transport to distal intestinal regions, an alternative approach is to encapsulate them in biodegradable microparticles. Consequently, in the past few years, numerous efforts have been proposed to use food-grade polymers as probiotic microencapsulation materials, including alginates, gums, cellulose, soy protein, oligofructose, and whey protein. However, the probiotic nanoparticles prepared by the above method have the disadvantages of weak ability to carry the loaded bacteria to the colonization site and promote their beneficial effects in vivo and low targeting. Summary of the Invention

[0003] In view of this, improvements are made to the defects and existing problems of the prior art. The purpose of the present invention is to provide a method for preparing probiotic microcapsules using photosensitizer-polysaccharide as wall material in a covalent coupling manner to achieve targeted colonization of probiotics in the intestine. The probiotic microcapsules prepared by this method are acid-resistant, bile-resistant, digestive enzyme-resistant, and heat-resistant, and the stability of the probiotics is improved.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] The first aspect of the present invention provides a method for preparing targeted probiotic nanocapsules, comprising the following steps:

[0006] (1) Preparation of photosensitive nano-particle INPs: polylactic acid-glycolic acid copolymer (PLGA) is dissolved in an aqueous acetonitrile solution to obtain a PLGA solution; lecithin, distearoyl phosphatidyl ethanolamine PEG amine (DSPE-PEG-NH2) and indocyanine green (ICG) are added to an aqueous ethanol solution, after ultrasonic treatment, the PLGA solution is added dropwise, and INPs are obtained by centrifugation and washing, which are functionalized INPs with reactive amino groups (-NH2); the whole process is carried out in a light-proof environment;

[0007] (2) Dextran modification: dextran is mixed with isopropyl alcohol, first stirring is carried out, NaOH solution is added, second stirring is carried out, chloroacetic acid is added, warming is carried out, third stirring is carried out, after neutralization with hydrochloric acid, dialysis, rotary evaporation and freeze-drying, modified polysaccharide is obtained;

[0008] (3) Activation of modified polysaccharide: the modified polysaccharide in step (2) is dissolved in PBS, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide sodium salt (NHS) are added, and the pH is adjusted to 4-6 for activation to activate the carboxyl groups on the modified polysaccharide, and then excess EDC and NHS and small molecule intermediates are removed by dialysis bag to obtain activated modified polysaccharide;

[0009] (4) Activation of probiotics: the activated probiotics are prepared into a bacterial suspension, and the probiotics are probiotics capable of targeting the intestinal tract;

[0010] (5) Coupling of polysaccharide and probiotics to form polysaccharide-probiotic (CM-dextran-ECN): the activated modified polysaccharide obtained in step (3) is mixed with the bacterial suspension in step (4), and magnetic stirring is carried out for 2-3 h to allow the amino groups on the probiotics to react with the polysaccharide through a carbodiimide coupling reaction, and then excess polysaccharide solution and uncoated probiotics are removed using a high-speed centrifuge to obtain CM-dextran-ECN;

[0011] (6) Coupling of INPs and CM-dextran-ECN (CM-dextran-ECN-INP): the INPs obtained in step (1) and the CM-dextran-ECN obtained in step (5) are incubated to allow amide coupling, and after the reaction is completed, centrifugation and washing are carried out to obtain CM-dextran-ECN-INP.

[0012] In the present application, the motility of probiotics can enrich ICG in the intestinal tract, and single bacterial encapsulation can be achieved, and the polysaccharide layer on the surface can adhere to the intestinal mucosa to play a role.

[0013] Further, in step (1),

[0014] The acetonitrile aqueous solution is an 80% acetonitrile aqueous solution, and the concentration of the PLGA solution is 2-3 mg / ml;

[0015] The sum of the mass of the lecithin and the DSPE-PEG-NH2 is 10-20% of the mass of PLGA, and the mass ratio of the lecithin to the DSPE-PEG-NH2 is 2:3;

[0016] The ethanol aqueous solution is a 4% ethanol aqueous solution, and the amount of the ICG is 0.10-0.25% of the mass of the PLGA.

[0017] Furthermore, in step (1), the ultrasonic treatment time is 3 to 8 minutes, the ultrasonic frequency is 20 kHz, and the power is 130W.

[0018] Furthermore, in step (2),

[0019] The concentration of the NaOH solution is 15-25%;

[0020] The material-liquid ratio of the dextran to the isopropanol is 1 g: 25-30 ml; the ratio of the dextran to the sodium hydroxide is 1: 15-20; the mass ratio of the dextran to the chloroacetic acid is 1: 0.8-1.2;

[0021] The first stirring time is 20-40 minutes, and the temperature is room temperature;

[0022] The second stirring time is 1-2h, and the temperature is room temperature;

[0023] The third stirring time is 3-4h, and the temperature is 55-65℃;

[0024] Dialysis was performed with distilled water for 2-4 days.

[0025] Furthermore, in step (3), the activation time is 15 minutes to 4 hours, and the activation temperature is room temperature; the mass ratio of the modified polysaccharide to EDC and NHS is 5:2 to 4:2 to 4.

[0026] Furthermore, in step (4), the probiotic is Escherichia coli Nissle 1917.

[0027] Furthermore, in step (5), the volume ratio of the activated modified polysaccharide to the bacterial suspension is 1:10-20; and the pH of the system during the formation of CM-dextran-ECN is 7-8.

[0028] Furthermore, in step (6), the stirring incubation time is 1 to 3 hours, the incubation temperature is 37°C, the centrifugation speed is 3500 to 4000 rpm / min, and the centrifugation further includes freeze-drying to obtain solid CM-dextran-ECN-INP. Alternatively, after centrifugation and PBS washing, the sample can be resuspended in PBS at pH 7.4.

[0029] In a second aspect, the present invention provides targeted probiotic nanocapsules prepared by any of the above preparation methods.

[0030] The third aspect of the present invention further provides the use of the above-mentioned targeted probiotic nanocapsules in the preparation of products for improving the intestinal microbial ecological environment of a host.

[0031] The present invention also provides the use of the above-mentioned targeted probiotic nano-microcapsules as active ingredients in the preparation of food additives, medicines and health products.

[0032] The beneficial effects of the present invention include at least:

[0033] The present invention discloses a method for preparing probiotic nano-microcapsules by coating probiotics with a photosensitive agent ICG and a polysaccharide using a covalently coupled chemical combination method; the microcapsule core material is mainly probiotics, and the microcapsule wall material is carboxymethyl dextran and a photosensitive agent ICG; the targeted colonization of probiotics in the intestine is utilized to cause ICG to aggregate in the intestine and exert its effect to reduce intestinal inflammatory diseases, and the photosensitive agent nanoparticles and polysaccharides can protect the probiotics from gastric acid and bile salts. In addition, the microcapsules are easy to prepare, have good sustained and controlled release, are non-toxic in materials, and have good biocompatibility and degradability. The present invention improves the wall material of the probiotic microcapsules, and designs the preparation method and the overall process accordingly, and uses polysaccharides and photosensitive agents to form the capsule wall. The obtained nano-probiotics are acid-resistant, bile-resistant, digestive enzyme-resistant, and heat-resistant, the stability of the probiotics is improved, and the intestinal colonization effect is enhanced. The microcapsule particles have uniform particle size, small surface area differences, good oral effect, and can maintain good stability in gastric juice and intestinal juice, which is beneficial to improving the survival rate of probiotics. After oral administration, they can be released in the intestine in a targeted manner and reduce intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Survival of differently modified probiotics in simulated gastric fluid. Note: NC: naked bacteria; DeX-ECN: dextran-coated probiotics; CMDeX-ECN: carboxymethyl dextran-coated probiotics; INPs-ECN: photosensitive nanoparticles encapsulated probiotics; INPS-CMDeX-ECN: photosensitive nanoparticles and carboxymethyl polysaccharide-coated probiotics.

[0035] Figure 2 To investigate the survival rate of differently modified probiotics in bile salt solution.

[0036] Figure 3 The survival rates of probiotics with different modifications in ethanol solution were investigated; ae represents ± standard deviation with different superscript letters in the same row indicating significant differences (p<0.05). DETAILED DESCRIPTION

[0037] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0038] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0039] The solution proposed by the present invention is described in detail below through specific embodiments:

[0040] Example 1

[0041] 1. Preparation of probiotic microcapsule preparations

[0042] (1) Preparation of photosensitive nanogranulated INPs: PLGA, soybean lecithin, ICG and distearoylphosphatidylethanolamine PEG amine (DSPE-PEG-NH2) were used as raw materials. 0.36 g of PLGA was dissolved in 80% (final concentration 2.5 mg / mL) acetonitrile aqueous solution, soybean lecithin, DSPE-PEG-NH2 and 750 μg of ICG were added to 3 mL of 4% ethanol aqueous solution, wherein lecithin and DSPE-PEG-NH2 were mixed. 2, The mass ratio of lecithin to DSPE-PEG-NH2 was 2:3, and the combined weight of lecithin and DSPE-PEG-NH2 was 15% of the PLGA. Ultrasonication was performed for 5 minutes, and the PLGA solution was added dropwise to generate INPs. Finally, the INPs were washed three times using a centrifugal filter.

[0043] (2) Dextran modification: Dextran was added to isopropanol and stirred at room temperature for 30 min. A 20% by volume NaOH solution was added and stirred for another 1 h. Chloroacetic acid was then added and the temperature was raised to 60°C. After cooling to room temperature, the mixture was neutralized with dilute hydrochloric acid and dialyzed with distilled water for 3 days. The mixture was then lyophilized by rotary evaporation. The dextran modification was determined by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy, and the degree of substitution of the dextran was determined by complexometric titration.

[0044] (3) Activation of modified polysaccharide: 50 mg of modified polysaccharide after step (2) was dissolved in PBS, 30 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 30 mg of N-hydroxysuccinimide sodium salt (NHS) were added to the solution, and diluted hydrochloric acid was used to adjust the pH to 5 to activate the carboxyl group on the modified polysaccharide, the activation time was 2 h, and the temperature was room temperature; then the excess EDC and NHS and small molecule intermediates were dialyzed out with a dialysis bag.

[0045] (4) Activation of probiotic bacteria: the bacteria solution stored at -80°C was streaked and cultured for 1 generation, then a single colony was picked and cultured in a liquid medium to the logarithmic phase or the bacteria solution stored at 4°C was inoculated into a liquid medium at an inoculation amount of 1% and cultured to the logarithmic phase for activation, and the activated Escherichia coli Nissle 1917 bacteria slurry was prepared into a bacterial suspension;

[0046] (5) Coupling of polysaccharide and probiotic bacteria to form polysaccharide-probiotic bacteria (CM-dextran-ECN): the activated modified polysaccharide in step (3) was mixed with the bacterial solution in step (4) at a ratio of 1:15, and was stirred on a magnetic stirrer for 2-3 h to allow the amino groups on the probiotic bacteria to react with the polysaccharide through a carbodiimide coupling reaction, and then a high-speed centrifuge was used to remove excess polysaccharide solution and uncoated probiotic bacteria.

[0047] (6) Coupling of INPs and CM-dextran-ECN: functionalized INPs with reactive amino groups (-NH2) were immobilized and maintained on the carboxyl groups (-COOH) on the surface of the polysaccharide. The mixture was incubated in PBS for about 10 8 CFU of CM-dextran-ECN and INPs to achieve bioconjugation. The mixture was then incubated at 37°C with gentle stirring for 2 hours to allow amide coupling. CM-dextran-ECN-INP was separated from unbound INP using 3500 rpm centrifugation followed by 3 PBS washes. The sample was resuspended in PBS at pH 7.4.

[0048] 2. Quality research of probiotic microcapsule preparation

[0049] (1) Viability detection after de-capsulation

[0050] The probiotic microcapsules need to release the probiotic bacteria with a de-capsulation solution before measuring the viability of the probiotic bacteria due to the wrapping of polysaccharides and INPs. The de-capsulation solution is generally phosphate buffer.

[0051] (2) Survival of free and encapsulated probiotic bacteria in simulated gastric juice (SGJ)

[0052] Prepare artificial gastric fluid with distilled water. In a clean beaker, add 6.9 mL of 37.3 g / L potassium chloride, 0.9 mL of 68 g / L potassium dihydrogen phosphate, 12.5 mL of 84 g / L sodium bicarbonate, 11.8 mL of 117 g / L sodium chloride, 0.4 mL of 30.5 g / L magnesium chloride hexahydrate, 0.5 mL of 48 g / L ammonium carbonate, and 33 μL of 0.3 mol / L CaCl₂. Mix thoroughly and sterilize. Add 23,000 U of porcine pepsin, mix well, and adjust the pH to 2 with hydrochloric acid to prepare simulated gastric fluid.

[0053] Dissolve 0.5 mL of free cells and 0.5 g of encapsulated cells in 4.5 mL of sterile SGJ. Incubate the tubes in a shaker at 37°C. Check viability by sampling after 0, 30, 60, 90, 120, and 150 minutes.

[0054] The results are as follows Figure 1 As shown, compared with other groups, photosensitive nanogranulation and carboxymethyl polysaccharide can well encapsulate probiotics and ensure the survival of probiotics in gastric juice.

[0055] (3) Survival of free and encapsulated E. coli in bile salt solution

[0056] The stability of free and encapsulated probiotics was determined in porcine bile salt solution. Free cells (0.5 mL) and encapsulated probiotics (0.5 g) were added to test tubes containing 4.5 mL of bile salt solution (2% w / v), and the test tubes were incubated at 37°C with constant stirring (100 rpm). Viable cells were checked by sampling after 0, 30, 60, 90, 120, and 150 minutes.

[0057] The results are as follows Figure 2 As shown, photosensitive nano-granulation and carboxymethyl polysaccharide can better encapsulate probiotics compared with other groups, ensuring the survival of probiotics in bile salt solution.

[0058] (4) Survival of free and encapsulated probiotics in ethanol

[0059] Weigh 3 g of free and encapsulated probiotics, add 5 mL of ethanol and place in a constant temperature experimental box at 37°C. After 30 minutes, take samples to measure the number of viable bacteria, determine the survival rate of unencapsulated probiotics and freeze-dried microcapsules, and examine the ethanol resistance.

[0060] The results are as follows Figure 3 As shown in the figure, the survival rate of probiotics in freeze-dried microcapsules was significantly higher than that of unencapsulated probiotics, proving that microcapsule preparations can effectively improve the ethanol resistance of bacteria. In addition, it can be clearly seen that the ethanol resistance of microcapsules coated with photosensitizers and carboxymethyl polysaccharides was greatly improved.

[0061] Example 2

[0062] 1. Preparation of probiotic microcapsule preparations

[0063] (1) Preparation of photosensitive nanogranulated INPs: PLGA, soybean lecithin, ICG and distearoylphosphatidylethanolamine PEG amine (DSPE-PEG-NH2) were used as raw materials. 0.58 g of PLGA was dissolved in 80% (2.5 mg / mL) acetonitrile aqueous solution, and soybean lecithin, (DSPE-PEG-NH2), and 850 μg of ICG were added to 10 mL of 4% ethanol aqueous solution. 2, The mass ratio of lecithin to DSPE-PEG-NH2 was 2:3, and the combined weight of lecithin and DSPE-PEG-NH2 was 20% of the weight of PLGA. Ultrasonication was performed for 5 minutes, and the PLGA solution was added dropwise to generate INPs. Finally, the INPs were washed three times with a centrifugal filter.

[0064] 2) Dextran Modification: Dextran was added to isopropanol and stirred at room temperature for 30 minutes. 20% NaOH solution was added and stirred for an additional hour. Chloroacetic acid was then added and the temperature was raised to 60°C. After cooling to room temperature, the mixture was neutralized with dilute hydrochloric acid and dialyzed with distilled water for 3 days. The mixture was then rotary evaporated and freeze-dried. The dextran modification was then analyzed by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy, and the degree of substitution of the dextran was determined by complexometric titration.

[0065] 3) Activation of modified polysaccharide: 100 mg of the modified polysaccharide in step (2) was dissolved in PBS, 80 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 80 mg of N-hydroxysulfosuccinimide sodium salt (NHS) were added to the solution, and the pH was adjusted to 5 with dilute hydrochloric acid to activate the carboxyl groups on the modified polysaccharide. The activation time was 2 h at room temperature; then, excess EDC and NHS as well as small molecule intermediates were dialyzed away using a dialysis bag.

[0066] 4) Activation of probiotics: After streaking for one generation, single colonies were selected from a bacterial solution stored at -80°C and cultured in liquid culture medium until the logarithmic phase. Alternatively, 1% of the bacterial solution stored at 4°C was inoculated into liquid culture medium and cultured until the logarithmic phase for activation. The activated Escherichia coli Nissle 1917 slurry was prepared into a bacterial suspension.

[0067] 5) Coupling of polysaccharide and probiotics to form polysaccharide-probiotics (CM-dextran-ECN): The modified polysaccharide activated in step (3) was mixed with the bacterial solution in step (4) at a ratio of 1:10 and stirred overnight under magnetic stirring to allow the amino groups on the probiotics to undergo carbodiimide coupling reaction with the polysaccharide. Excess polysaccharide solution and uncoated probiotics were then removed using a high-speed centrifuge.

[0068] 6) INPs conjugation with CM-dextran-ECN: Functionalized INPs with reactive amino groups (-NH2) were immobilized and maintained onto the polysaccharide surface carboxyl groups (-COOH). The mixture was incubated in PBS for about 10 8 CM-dextran-ECN conjugation with INPs to achieve bioconjugation. The mixture was then incubated at 37°C with gentle agitation for 2 hours to allow amide coupling. CM-dextran-ECN-INPs were separated from unbound INPs using centrifugation at 3500 rpm followed by 3 washes with PBS. The microcapsules were lyophilized using a freeze dryer.

[0069] 2. Quality study of probiotic microcapsule formulation

[0070] 1) Survival of free and encapsulated probiotics in simulated gastric juice (SGJ)

[0071] Simulated gastric juice was prepared with distilled water, 6.9 mL of 37.3 g / L potassium chloride, 0.9 mL of 68 g / L potassium dihydrogen phosphate, 12.5 mL of 84 g / L sodium bicarbonate, 11.8 mL of 117 g / L sodium chloride, 0.4 mL of 30.5 g / L magnesium chloride hexahydrate and 0.5 mL of 48 g / L ammonium carbonate, 33 μL of 0.3 mol / L CaCl2 were sequentially added to a clean beaker and mixed well, sterilized. Then 23,000 U of porcine pepsin was added, mixed, and the pH was adjusted to 2 with hydrochloric acid to prepare the simulated gastric juice.

[0072] 0.5 mL of free cells and 0.5 g of encapsulated cells were dissolved in 4.5 mL of sterile SGJ, respectively. The test tubes were incubated in a shaker at 37°C. Survival rate was checked by sampling at 0, 30, 60, 90, 120 and 150 minutes.

[0073] Table 1 Survival rate of different modified probiotics in SGJ

[0074]

[0075] Note: a-e indicate that the standard deviation of different superscripts in the same row represents significant difference (p < 0.05), the same below.

[0076] The results are shown in Table 1. Compared with other groups, photosensitive nano-particle and carboxymethyl polysaccharide can well coat probiotics to ensure the colonization of probiotics in the gastrointestinal tract.

[0077] 2) Survival of free and encapsulated E. coli in bile salt solution

[0078] The stability of free and encapsulated probiotics was determined in porcine bile salt solution. Free cells (0.5 mL) and encapsulated probiotics (0.5 g) were added to test tubes containing 4.5 mL of bile salt solution (2% w / v), and the test tubes were incubated at 37°C with constant stirring (100 rpm). Viable cells were checked by sampling after 0, 30, 60, 90, 120, and 150 minutes.

[0079] Table 2 Study on the survival rate of different modified probiotics in bile salt solution

[0080]

[0081] The results are shown in Table 2. The survival rate of the probiotic freeze-dried microcapsules was significantly higher than that of the unencapsulated probiotics, demonstrating that the microcapsule formulation can effectively improve the bile salt tolerance of the bacteria. Furthermore, it is clear that the bile salt tolerance of the microcapsules encapsulated with the photosensitizer and carboxymethyl polysaccharide was significantly improved.

[0082] 3) Heat resistance of microcapsules

[0083] Weigh 3 g of microcapsules, seal them in aluminum bags, and store them in a constant temperature laboratory box at 70°C. After 60 minutes, take samples to measure the number of viable bacteria, determine the survival rate of unencapsulated probiotics and freeze-dried microcapsules, and examine the heat resistance.

[0084] Table 3 Study on heat resistance of probiotics with different modifications

[0085] Probiotic preparations <![CDATA[活菌数(×10 8 CFU)]]> Survival rate (%) Unencapsulated probiotics <![CDATA[0±0 e ]]> <![CDATA[0±0 e ]]> Glucan-coated probiotics <![CDATA[11.95±0.89 d ]]> <![CDATA[12.40±0.50 d ]]> Carboxymethyl dextran coated probiotics 70.60 ± 0.11 b ]] 75.40 ± 0.10 b ]] INPs coated probiotics <![CDATA[60.30±0.78 c ]]> 63.80 ± 0.72 c ]] INPs-carboxymethyl dextran coated probiotics <![CDATA[85.40±2.30 a ]]> <![CDATA[86.40±2.0 a ]]>

[0086] Note: ae represents ± standard deviation. Different superscript letters in the same column indicate significant differences (p<0.05).

[0087] The results are shown in Table 3. The survival rate of probiotic freeze-dried microcapsules was significantly higher than that of unencapsulated probiotics, demonstrating that microcapsule preparations can effectively improve the heat resistance of bacteria. In addition, it can be clearly seen that the heat resistance of microcapsules encapsulated with photosensitizers and carboxymethyl polysaccharides was significantly improved.

[0088] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0089] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0090] The above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced, without departing from the purpose and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. A method for preparing targeted probiotic nanocapsules, characterized in that: The following steps are involved: (1) Preparation of photosensitive nanogranulated INPs: PLGA was dissolved in an acetonitrile aqueous solution to obtain a PLGA solution; phosphatidylcholine, DSPE-PEG-NH2, and ICG were added to an ethanol aqueous solution under a dark environment, and after ultrasonic treatment, the PLGA solution was added dropwise, and INPs were obtained by centrifugation and washing; (2) Modification of dextran: dextran was mixed with isopropanol, stirred for the first time, NaOH solution was added, stirred for the second time, chloroacetic acid was added, the temperature was increased, stirred for the third time, cooled, neutralized with hydrochloric acid, dialyzed, rotary evaporated, and freeze-dried to obtain the modified polysaccharide; (3) Activating modified polysaccharides: dissolving the modified polysaccharide in step (2) in PBS, adding EDC and NHS, and adjusting the pH to 4-6 for activation to obtain activated modified polysaccharides; (4) Activating probiotics: preparing the activated probiotics into a bacterial suspension, wherein the probiotics are probiotics that can target the intestinal tract; (5) Preparation of CM-dextran-ECN: mixing the activated modified polysaccharide obtained in step (3) with the bacterial suspension obtained in step (4), and stirring to obtain CM-dextran-ECN; (6) Coupling of INPs with CM-dextran-ECN: The INPs obtained in step (1) and the CM-dextran-ECN obtained in step (5) were stirred and incubated. After the reaction was completed, the mixture was centrifuged to obtain CM-dextran-ECN-INP.

2. The preparation method according to claim 1, characterized in that In step (1), The acetonitrile aqueous solution is an 80% acetonitrile aqueous solution, and the concentration of the PLGA solution is 2-3 mg / ml; The sum of the mass of the lecithin and the DSPE-PEG-NH2 is 10-20% of the mass of PLGA, and the mass ratio of the lecithin to the DSPE-PEG-NH2 is 2:3; The ethanol aqueous solution is a 4% ethanol aqueous solution, and the amount of the ICG is 0.10-0.25% of the mass of the PLGA.

3. The preparation method according to claim 1, characterized in that In step (1), the ultrasonic treatment time is 3 to 8 minutes, the ultrasonic frequency is 20 kHz, and the power is 130W.

4. The preparation method according to claim 1, characterized in that In step (2), The concentration of the NaOH solution is 15-25%; The material-liquid ratio of the dextran to the isopropanol is 1 g: 25-30 ml; the ratio of the dextran to the sodium hydroxide is 1: 15-20; the mass ratio of the dextran to the chloroacetic acid is 1: 0.8-1.2; The first stirring time is 20-40 minutes, and the temperature is room temperature; The second stirring time is 1-2h, and the temperature is room temperature; The third stirring time is 3-4h, and the temperature is 55-65℃; Dialysis was performed with distilled water for 2-4 days.

5. The preparation method according to claim 1, characterized in that In step (3), the activation time is 15 minutes to 4 hours, and the activation temperature is room temperature; the mass ratio of the modified polysaccharide to EDC and NHS is 5:2 to 4:2 to 4.

6. The preparation method according to claim 1, characterized in that In step (5), the volume ratio of the activated modified polysaccharide to the bacterial suspension is 1:10-20; the pH of the system during the formation of CM-dextran-ECN is 7-8.

7. The preparation method according to claim 1, characterized in that In step (6), the stirring incubation time is 1 to 3 hours, the incubation temperature is 37° C., the centrifugal speed is 3500 to 4000 rpm / min, and after centrifugation, freeze-drying is performed to obtain solid CM-dextran-ECN-INP.

8. The targeted probiotic nanocapsules prepared according to the method of any one of claims 1 to 7.

9. Use of the targeted probiotic nanocapsules according to claim 8 in the preparation of products for improving the intestinal microbial ecological environment of a host.

10. Use of the targeted probiotic nanocapsules according to claim 8 as active ingredients in the preparation of food additives, medicines, and health products.

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