A targeted probiotic nanocapsule, its preparation method and application

Abstract

This invention relates to the field of biomedical technology, disclosing a targeted probiotic nanocapsule, its preparation method, and its application. The method involves using a photosensitizing agent ICG and polysaccharides to covalently couple probiotics to prepare probiotic nanocapsules. The core material of the microcapsule is primarily probiotics, and the microcapsule wall material is carboxymethyl dextran and the photosensitizing agent ICG. These microcapsules are easy to prepare, exhibit good sustained-release and controlled-release properties, and the materials are non-toxic, biocompatible, and biodegradable. This invention improves the wall material of the probiotic microcapsules and designs the preparation method and overall process accordingly. By utilizing polysaccharides and photosensitizing agents to form the capsule wall, the resulting nano-probiotics are acid-resistant, bile salt-resistant, digestive enzyme-resistant, and heat-resistant, resulting in improved probiotic stability and enhanced intestinal colonization.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a targeted probiotic nanocapsule, its preparation method, and its application. Background Technology

[0002] Probiotics are special bacteria that benefit the host. They can improve the gut microbiota ecosystem by regulating gut microbiota imbalance, balancing gut 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 remains a significant challenge in developing effective functional foods. Probiotics are highly sensitive to industrial processing, transportation, and storage conditions, which limits their use in food. Another factor affecting probiotic survival is their ability to withstand the harsh gastrointestinal environment; low gastric pH and a harsh intestinal environment significantly reduce probiotic viability and proliferation. Furthermore, the rapid transport of probiotics in the gastrointestinal tract results in short residence times, leading to less than ideal therapeutic effects. To ensure probiotic survival and improve their viability during processing and transport to the distal intestinal region, an alternative approach is to encapsulate them in biodegradable microparticles. Therefore, in recent years, numerous developments have been made using food-grade polymers as probiotic microencapsulation materials, including alginate, gums, cellulose, soy protein, fructooligosaccharides, and whey protein. However, the probiotic nanoparticles prepared by the above methods have the disadvantages of weak ability to carry loaded bacteria to the colonization site and promote their beneficial in vivo effects, and low targeting. Summary of the Invention

[0003] In view of this, and in order to improve upon the deficiencies and existing problems of the prior art, the present invention aims to provide a method for preparing probiotic microcapsules by using photosensitizer-polysaccharide as the wall material in a covalent coupling manner, so as to achieve targeted colonization of probiotics in the intestine, and the probiotic microcapsules prepared by this method are resistant to acid, bile salts, digestive enzymes, and heat, thus improving the stability of probiotics.

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

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

[0006] (1) Preparation of photosensitive nanoparticles (INPs): Polylactic acid-glycolic acid copolymer (PLGA) was dissolved in acetonitrile aqueous solution to obtain PLGA solution; lecithin, distearate phosphatidylethanolamine PEG (DSPE-PEG-NH2) and indocyanine green (ICG) were added to ethanol aqueous solution, ultrasonicated, and then PLGA solution was added dropwise. After centrifugation and washing, INPs were obtained, which are functionalized INPs with reactive amino groups (-NH2); the whole process was carried out in a light-protected environment.

[0007] (2) Dextran modification: 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 raised, stirred for the third time, cooled, neutralized with hydrochloric acid, dialyzed, rotary evaporated, and freeze-dried to obtain the modified polysaccharide;

[0008] (3) Activation of modified polysaccharide: Dissolve the modified polysaccharide in PBS, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide sodium salt (NHS), adjust the pH to 4-6 for activation, so as to activate the carboxyl group on the modified polysaccharide. Then, dialyze the excess EDC and NHS and small molecule intermediates with a dialysis bag to obtain activated modified polysaccharide;

[0009] (4) Activating probiotics: The activated probiotics are prepared into a bacterial suspension, wherein the probiotics are probiotics that can target the intestines;

[0010] (5) Polysaccharide and probiotics are coupled to form polysaccharide-probiotics (CM-dextran-ECN): The activated modified polysaccharide obtained in step (3) is mixed with the bacterial suspension in step (4) and magnetically stirred for 2-3 hours so that the amino groups on the probiotics undergo a carbodiimide coupling reaction with the polysaccharide. After removing the excess polysaccharide solution and uncoated probiotics using a high-speed centrifuge, CM-dextran-ECN is obtained.

[0011] (6) Coupling of INPs with CM-dextran-ECN (CM-dextran-ECN-INP): Stir and incubate the INPs obtained in step (1) and the CM-dextran-ECN obtained in step (5) to allow amide coupling. After the reaction is complete, centrifuge and wash to obtain CM-dextran-ECN-INP.

[0012] In this invention, the motility of probiotics is utilized to enrich ICG in the intestines, and single-bacterial encapsulation is used, with the surface polysaccharide layer adhering to the intestinal mucosa to exert its effect.

[0013] Furthermore, 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-water solution is a 4% ethanol-water solution, and the amount of ICG used is 0.10 to 0.25% of the mass of 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 130 W.

[0018] Furthermore, in step (2),

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

[0020] The ratio of dextran to isopropanol is 1g:25-30ml; the ratio of dextran to sodium hydroxide is 1:15-20; and the mass ratio of dextran to 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 1-2 hours, and the temperature is room temperature;

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

[0024] Distilled water is used for dialysis for 2-4 days.

[0025] Further, in step (3), the activation time is 15 min to 4 h, 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 activated modified polysaccharide to bacterial suspension is 1:10-20; the pH of the system during the formation of CM-dextran-ECN is 7-8.

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

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

[0030] In a third aspect, the present invention also provides the application of the above-described targeted probiotic nanocapsules in the preparation of products that improve the intestinal microbial ecological environment of the host.

[0031] This invention also provides the application of the above-described targeted probiotic nanocapsules as active ingredients in the preparation of food additives, pharmaceuticals, and health products.

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

[0033] This invention discloses a method for preparing probiotic nanocapsules by covalently coupling probiotics with a photosensitizing agent ICG and polysaccharides. The core material of the microcapsules is mainly probiotics, and the wall material is carboxymethyl dextran and the photosensitizing agent ICG. By utilizing the targeted colonization of probiotics in the intestine, ICG accumulates and exerts its effects in the intestine to reduce intestinal inflammatory diseases. Furthermore, the photosensitizing agent nanoparticles and polysaccharides protect the probiotics from gastric acid and bile salts. In addition, the microcapsules are easy to prepare, have good sustained-release and controlled-release properties, and the materials are non-toxic, biocompatible, and biodegradable. This invention improves the wall material of the probiotic microcapsules and designs the preparation method and overall process accordingly. By using polysaccharides and photosensitizing agents to form the capsule wall, the resulting nano-probiotics are acid-resistant, bile salt-resistant, digestive enzyme-resistant, and heat-resistant, improving the stability of the probiotics and enhancing their intestinal colonization effect. Microcapsule particles have uniform particle size and small surface area differences, resulting in good oral efficacy. They maintain good stability in gastric and intestinal fluids, which helps improve the survival rate of probiotics. After oral administration, they can be released into the intestines and reduce intestinal diseases. Attached Figure Description

[0034] Figure 1 To investigate the survival rate of probiotics with different modifications in simulated gastric juice. Note: NC: naked bacteria group; DeX-ECN: probiotics coated with dextran; CMDex-ECN: probiotics coated with carboxymethyl dextran; INPs-ECN: probiotics encapsulated by photosensitive nanoparticles; INPS-CMDeX-ECN: probiotics encapsulated by photosensitive nanoparticles and carboxymethyl polysaccharides.

[0035] Figure 2 To investigate the survival rate of probiotics with different modifications in bile salt solution.

[0036] Figure 3 To investigate the survival rate of probiotics with different modifications in ethanol solution; ae indicates that the ± standard deviation of different superscript letters in the same row indicates a significant difference (p<0.05). Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0039] The following specific embodiments illustrate the solution proposed in this invention:

[0040] Example 1

[0041] 1. Preparation of probiotic microcapsule formulations

[0042] (1) Preparation of photosensitive nanoparticles (INPs): PLGA, soybean lecithin, ICG, and distearate-PEG-NH2 were used as raw materials. 0.36 g of PLGA was dissolved in an 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. The lecithin reacted with DSPE-PEG-NH2... 2, The mass ratio of lecithin to PEG-NH2 was 2:3, and the sum of the weights of lecithin and DSPE-PEG-NH2 was 15% of that of PLGA. The mixture was sonicated for 5 minutes using an ultrasonic processor, and PLGA solution was added dropwise to generate INPs. Finally, the INPs were washed three times using a centrifuge filter.

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

[0044] (3) Activation of modified polysaccharide: Take 50 mg of the modified polysaccharide from step (2) and dissolve it in PBS. Add 30 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 30 mg of N-hydroxythiosuccinimide sodium salt (NHS) to the solution and adjust the pH to 5 with dilute hydrochloric acid to activate the carboxyl groups on the modified polysaccharide. The activation time is 2 h and the temperature is room temperature. Then, dialyze the excess EDC and NHS and small molecule intermediates with a dialysis bag.

[0045] (4) Activating probiotics: After streaking culture of bacterial solution stored at -80℃ for 1 generation, single colonies are picked and cultured in liquid culture medium until the logarithmic phase. Alternatively, bacterial solution stored at 4℃ is inoculated into liquid culture medium at an inoculation rate of 1% and cultured until the logarithmic phase for activation. The activated Escherichia coli Nissle 1917 bacterial sludge is then prepared into a bacterial suspension.

[0046] (5) Polysaccharide and probiotics are coupled to form polysaccharide-probiotic (CM-dextran-ECN): The modified polysaccharide activated in step (3) is mixed with the bacterial solution in step (4) at a ratio of 1:15 and stirred magnetically for 2-3 hours to allow the amino groups on the probiotics to undergo a carbodiimide coupling reaction with the polysaccharide. Afterwards, a high-speed centrifuge is used to remove excess polysaccharide solution and uncoated probiotics.

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

[0048] 2. Quality Research on Probiotic Microcapsule Preparations

[0049] (1) Vitality test after clotting

[0050] Because probiotic microcapsules are encapsulated with polysaccharides and INPs, the probiotics need to be released using an unpacking solution before their activity can be measured. The unpacking solution is usually a phosphate buffer solution.

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

[0052] To prepare artificial gastric juice, distilled water was used. In a clean beaker, 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 were added sequentially. The mixture was thoroughly mixed and sterilized. Then, 23,000 U of porcine pepsin was added, and the mixture was adjusted to pH 2 with hydrochloric acid to create the simulated gastric juice.

[0053] Dissolve 0.5 mL of free cells and 0.5 g of encapsulated cells separately in 4.5 mL of sterile SGJ. Incubate the tubes in a shaker at 37 °C. Check survival 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 nanoparticles and carboxymethyl polysaccharides can effectively encapsulate probiotics and ensure their survival in gastric juice.

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

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

[0057] The results are as follows Figure 2 As shown, photosensitive nanoparticles and carboxymethyl polysaccharides can effectively encapsulate probiotics compared to other groups, ensuring the survival of probiotics in bile salt solutions.

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

[0059] Weigh 3g of free and encapsulated probiotics, add 5mL of ethanol, and place in a 37℃ constant temperature test chamber. After 30 minutes, take a sample to measure the number of viable bacteria, determine the survival rate of unencapsulated probiotics and lyophilized microcapsules, and investigate ethanol resistance.

[0060] The results are as follows Figure 3 As shown, the survival rate of lyophilized probiotic microcapsules was significantly higher than that of unencapsulated probiotics, proving that microcapsule formulations can effectively improve the ethanol resistance of bacteria. Furthermore, it is evident that microcapsules encapsulated with photosensitizers and carboxymethyl polysaccharides exhibited significantly enhanced ethanol resistance.

[0061] Example 2

[0062] 1. Preparation of probiotic microcapsule formulations

[0063] (1) Preparation of photosensitive nanoparticles (INPs): PLGA, soybean lecithin, ICG, and distearate phosphatidylethanolamine (DSPE-PEG-NH2) were used as raw materials. 0.58 g of PLGA was dissolved in an 80% (2.5 mg / mL) acetonitrile aqueous solution. Soybean lecithin, (DSPE-PEG-NH2), and 850 μg of ICG were added to 10 mL of 4% ethanol aqueous solution. The lecithin reacted with DSPE-PEG-NH2... 2, The mass ratio of lecithin to PEG-NH2 was 2:3, and the sum of the weights of lecithin and DSPE-PEG-NH2 was 20% of that of PLGA. The mixture was sonicated for 5 minutes using an ultrasonic processor, and PLGA solution was added dropwise to generate INPs. Finally, the INPs were washed three times using a centrifuge filter.

[0064] 2) Dextran modification: Dextran was added to isopropanol and stirred at room temperature for 30 min. 20% NaOH solution was added, and the mixture was stirred for another 1 h. Chloroacetic acid was then added, and the temperature was raised to 60℃. After cooling to room temperature, the mixture was neutralized with dilute hydrochloric acid, dialyzed with distilled water for 3 days, and then rotary evaporated and freeze-dried. The degree of dextran modification was then detected by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy, and the degree of substitution was determined by complexometric titration.

[0065] 3) Activation of modified polysaccharide: Take 100 mg of the modified polysaccharide from step (2) and dissolve it in PBS. Add 80 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 80 mg of N-hydroxythiosuccinimide sodium salt (NHS) to the solution and adjust the pH to 5 with dilute hydrochloric acid to activate the carboxyl groups on the modified polysaccharide. The activation time is 2 h and the temperature is room temperature. Then, dialyze the excess EDC and NHS and small molecule intermediates with a dialysis bag.

[0066] 4) Activating probiotics: After streaking culture of bacterial solution stored at -80℃ for one generation, single colonies are picked and cultured in liquid culture medium until the logarithmic phase. Alternatively, bacterial solution stored at 4℃ is inoculated into liquid culture medium at an inoculation rate of 1% and cultured until the logarithmic phase for activation. The activated Escherichia coli Nissle 1917 bacterial sludge is then prepared into a bacterial suspension.

[0067] 5) Polysaccharide and probiotics are coupled to form polysaccharide-probiotic (CM-dextran-ECN): The modified polysaccharide activated in step (3) is mixed with the bacterial solution in step (4) at a ratio of 1:10 and stirred overnight on a magnetic stirrer so that the amino groups on the probiotics undergo a carbodiimide coupling reaction with the polysaccharide. Then, a high-speed centrifuge is used to remove excess polysaccharide solution and uncoated probiotics.

[0068] 6) Coupling of INPs with CM-dextran-ECN: Functionalized INPs with reactive amino groups (-NH2) are immobilized and maintained onto carboxyl groups (-COOH) on the polysaccharide surface. Incubation in PBS for approximately 10... 8 CFU of CM-dextran-ECN was bioconjugated with INPs. The mixture was then incubated with gentle stirring at 37°C for 2 hours to allow amide coupling. CM-dextran-ECN-INP was separated from unbound INPs by centrifugation at 3500 rpm followed by washing three times with PBS. The microcapsules were then lyophilized.

[0069] 2. Quality Research on Probiotic Microcapsule Preparations

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

[0071] To prepare artificial gastric juice, distilled water was used. In a clean beaker, 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 were added sequentially. The mixture was thoroughly mixed and sterilized. Then, 23,000 U of porcine pepsin was added, and the mixture was adjusted to pH 2 with hydrochloric acid to create the simulated gastric juice.

[0072] Dissolve 0.5 mL of free cells and 0.5 g of encapsulated cells separately in 4.5 mL of sterile SGJ. Incubate the tubes in a shaker at 37 °C. Check survival by sampling after 0, 30, 60, 90, 120, and 150 minutes.

[0073] Table 1. Survival rate of probiotics with different modifications in SGJ.

[0074]

[0075] Note: ae indicates that the ± standard deviation of different superscript letters in the same row represents a significant difference (p<0.05), and the same applies below.

[0076] The results are shown in Table 1. Compared with other groups, photosensitive nanoparticles and carboxymethyl polysaccharides can effectively encapsulate probiotics and ensure their colonization in the gastrointestinal tract.

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

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

[0079] Table 2. Survival rate of probiotics with different modifications in bile salt solution.

[0080]

[0081] The results are shown in Table 2. The survival rate of the lyophilized probiotic microcapsules was significantly higher than that of the unencapsulated probiotics, demonstrating that microcapsule formulations can effectively improve the bile salt tolerance of bacteria. Furthermore, it is evident that microcapsules encapsulated with photosensitizers and carboxymethyl polysaccharides exhibited significantly enhanced bile salt tolerance.

[0082] 3) Heat resistance of microcapsules

[0083] Weigh 3g of microcapsules, seal them, put them in an aluminum bag, seal them, and store them in a 70℃ constant temperature test chamber. After 60 minutes, take the sample 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 the heat resistance of probiotics with different modifications.

[0085] probiotic preparations <![CDATA[Viable cell count (×10 8 CFU)]]> Survival rate (%) Uncoated 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 <![CDATA[70.60±0.11 b ]]> <![CDATA[75.40±0.10 b ]]> INPs-coated probiotics <![CDATA[60.30±0.78 c ]]> <![CDATA[63.80±0.72 c ]]> INPs-Carboxymethylglucan-coated probiotics <![CDATA[85.40±2.30 a ]]> <![CDATA[86.40±2.0 a ]]>

[0086] Note: ae indicates that the ± standard deviation of different superscript letters in the same column represents a significant difference (p<0.05).

[0087] The results are shown in Table 3. The survival rate of the lyophilized probiotic microcapsules was significantly higher than that of the unencapsulated probiotics, proving that microcapsule formulations can effectively improve the heat resistance of bacterial cells. In addition, it can be clearly seen that the heat resistance of microcapsules encapsulated with photosensitizers and carboxymethyl polysaccharides is significantly improved.

[0088] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0089] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for the preparation of targeted probiotic nanocapsules, characterized in that, Includes the following steps: (1) Preparation of photosensitive nanoparticles INPs: PLGA was dissolved in acetonitrile aqueous solution to obtain PLGA solution; lecithin, DSPE-PEG-NH2 and ICG were added to ethanol aqueous solution under light-protected environment, ultrasonic treatment was performed, PLGA solution was added dropwise, and INPs were obtained by centrifugation and washing. (2) Dextran modification: Dextran is mixed with isopropanol, stirred for the first time, NaOH solution is added, stirred for the second time, chloroacetic acid is added, heated, stirred for the third time, cooled, neutralized with hydrochloric acid, dialyzed, rotary evaporated, and freeze-dried to obtain the modified polysaccharide; (3) Activation of modified polysaccharide: Dissolve the modified polysaccharide in step (2) in PBS, add EDC and NHS, adjust the pH to 4-6 for activation, and obtain activated modified polysaccharide; (4) Activating probiotics: The activated probiotics are prepared into a bacterial suspension, wherein the probiotics are probiotics that can target the intestines; (5) Preparation of CM-dextran-ECN: The activated modified polysaccharide obtained in step (3) is mixed with the bacterial suspension in step (4) and stirred to obtain CM-dextran-ECN; (6) Coupling of INPs with CM-dextran-ECN: Stir and incubate the INPs obtained in step (1) and the CM-dextran-ECN obtained in step (5). After the reaction is complete, centrifuge to obtain CM-dextran-ECN-INP. 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-water solution is a 4% ethanol-water solution, and the amount of ICG used is 0.10~0.25% of the mass of PLGA; During ultrasonic treatment, the time is 3-8 minutes, the ultrasonic frequency is 20 kHz, and the power is 130 W. In step (2), The concentration of the NaOH solution is 15-25%; The ratio of dextran to isopropanol is 1g:25~30ml; the ratio of dextran to sodium hydroxide is 1:15~20; the mass ratio of dextran to 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 1-2 hours, and the temperature is room temperature; The third stirring time is 3-4 hours, and the temperature is 55-65℃; Dialysis is performed using distilled water for 2-4 days. In step (3), the activation time is 15 min to 4 h 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. In step (5), the volume ratio of activated modified polysaccharide to bacterial suspension is 1:10~20; the pH of the system during the formation of CM-dextran-ECN is 7~8; In step (6), the stirring and incubation time is 1~3h, the incubation temperature is 37℃, the centrifugation speed is 3500~4000 rpm / min, and after centrifugation, the solid CM-dextran-ECN-INP is obtained by freeze drying.

2. Targeted probiotic nanocapsules prepared by any of the methods described in claim 1.

3. The use of the targeted probiotic nanocapsules of claim 2 in the preparation of products that improve the intestinal microbial ecological environment of the host.

4. The application of the targeted probiotic nanocapsules as an active ingredient in the preparation of food additives, pharmaceuticals, and health products according to claim 2.

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