Gastric acid resistant viable microcapsule, and preparation method and application thereof

Abstract

The application provides a gastric acid-resistant viable bacteria microcapsule and a preparation method and application thereof, and belongs to the technical field of microbial preparations and drug delivery. The gastric acid-resistant viable bacteria microcapsule comprises, from inside to outside, an inner capsule core, a calcium alginate gel protective layer, a chitosan layer and a pH-responsive enteric coating layer. The inner capsule core is a mixed system of probiotics, whey protein isolate and a freeze-drying protective agent. The calcium alginate gel protective layer is coated on the outside of the inner capsule core and is made of a crosslinking agent and sodium alginate. The chitosan layer is coated on the outside of the calcium alginate gel protective layer and is made of chitosan. The pH-responsive enteric coating layer is coated on the outside of the chitosan layer and is made of a pH-responsive enteric polymer, a plasticizer and an anti-adhesive agent. The gastric acid-resistant viable bacteria microcapsule provided by the application has a 94.7% embedding rate, excellent gastric acid resistance, precise intestinal target release advantages and excellent storage stability.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparations and drug delivery technology, specifically relating to a gastric acid-resistant live bacteria microcapsule, its preparation method, and its application. Background Technology

[0002] Probiotics are a class of live microorganisms that exert beneficial effects on the host by colonizing the host's intestines and regulating the balance of intestinal flora. Commonly used probiotic strains include Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, and Lactobacillus acidophilus. However, probiotics face multiple challenges during oral delivery: (1) Gastric acid barrier: The pH of human gastric juice is about 1.0 to 2.0 when fasting and about 3.0 to 5.0 after eating. Most probiotics have a very low survival rate in this acidic environment. In vitro simulated gastric juice experiments show that the survival rate of free lactic acid bacteria after incubation at pH 1.5 for 2 hours is usually less than 5%. (2) Bile salt stress: The concentration of bile salts in the small intestine can reach 0.3 to 2.0% (w / v), which has a significant destructive effect on bacterial cell membranes. (3) Processing and storage stability: Probiotics are easily inactivated during formulation processing (such as mixing, tableting, drying) and long-term storage. At room temperature, the colony count can decrease by 1 to 2 logs every 30 days. Therefore, there is an urgent need to develop a live bacteria microcapsule formulation and its preparation method that combines high tolerance to gastric acid, efficient intestinal release, and excellent storage stability. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a live bacteria microcapsule that combines high tolerance to gastric acid, efficient release into the intestine, and excellent storage stability. It consists of an inner core (probiotic bacteria encapsulated in a protein-polysaccharide complex gel) and an outer shell (pH-responsive enteric polymer coating membrane). Through a dual barrier structure, it achieves graded protection of probiotics and targeted release into the intestine.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a gastric acid-resistant live bacteria microcapsule, comprising, from the inside out, an inner core, a calcium alginate gel protective layer, a chitosan layer, and a pH-responsive enteric coating layer; the inner core is a mixture of probiotics, whey protein isolate, and a lyophilization protectant; the calcium alginate gel protective layer, coated on the outside of the inner core, is made of a cross-linking agent and sodium alginate; the chitosan layer, coated on the outside of the calcium alginate gel protective layer, is made of chitosan; and the pH-responsive enteric coating layer, coated on the outside of the chitosan layer, is made of a pH-responsive enteric polymer, a plasticizer, and an anti-adhesion agent.

[0005] Preferably, the freeze-drying protectant comprises fructooligosaccharides or a mixture of fructooligosaccharides and trehalose; the weight proportions of each raw material in the inner capsule are: 5-20 parts probiotics, 5-15 parts whey protein isolate, and 4-11 parts freeze-drying protectant; the concentration of probiotics in the mixed system is 10. 10 CFU / mL or higher; the crosslinking agent includes calcium carbonate, with a weight percentage of 2-5 parts; the sodium alginate has a weight percentage of 30-50 parts; the chitosan has a weight percentage of 3-8 parts; the pH-responsive enteric polymer includes at least one of Eudragit L100-55, Eudragit L100, Eudragit S100, HPMCP, and HPMCAS; the plasticizer includes triethyl citrate; the anti-adhesion agent includes talc; the pH-responsive enteric polymer has a weight percentage of 10-25 parts; the plasticizer has a weight percentage of 1-5 parts; the anti-adhesion agent has a weight percentage of 1-3 parts; the probiotics include one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium breve, and Streptococcus thermophilus.

[0006] Preferably, the pH-responsive enteric polymer is composed of Eudragit L100-55 and Eudragit S100, with a mass ratio of Eudragit L100-55 to Eudragit S100 of 7:3.

[0007] This invention also provides a method for preparing the above-mentioned gastric acid-resistant live bacteria microcapsules, comprising the following steps: The probiotics were resuspended in a buffer solution containing whey protein isolate and a lyophilization protectant to obtain a mixed system; The mixture was then mixed with a sodium alginate solution to obtain a sodium alginate-bacterial cell blend. Vegetable oil containing emulsifier is mixed with sodium alginate-bacterial cell blend to form water-in-oil emulsion. Then, the water-in-oil emulsion is mixed with vegetable oil suspension. After demulsification, microspheres are collected to obtain probiotic microspheres coated with calcium alginate gel protective layer. The vegetable oil suspension contains glacial acetic acid and cross-linking agent. Probiotic microspheres coated with calcium alginate gel protective layer were mixed with chitosan acidic solution, washed and dried to obtain chitosan-coated probiotic microspheres. A pH-responsive enteric polymer was dissolved in an ethanol solution to obtain a coating solution. The coating solution was then mixed with a plasticizer and an anti-adhesion agent to coat chitosan-coated probiotic microspheres, resulting in acid-resistant probiotic microcapsules.

[0008] Preferably, the concentration of whey protein isolate in the buffer is 2% w / v, the concentration of fructooligosaccharides is 1% w / v, the buffer includes PBS buffer, and the concentration of probiotics in the mixture is 10.10 CFU / mL or higher.

[0009] Preferably, the concentration of the sodium alginate solution is 3.0% w / v, and the solvent of the sodium alginate solution is water; the volume ratio of the mixed system to the sodium alginate solution is 1:4.

[0010] Preferably, in the vegetable oil containing the emulsifier, the concentration of the emulsifier is 1.5%~3.0% w / v; the volume ratio of the vegetable oil containing the emulsifier to the sodium alginate-bacterial cell blend is (2~4):1; the concentration of glacial acetic acid in the vegetable oil suspension is 0.4%~0.6% v / v; the vegetable oil includes soybean oil; the emulsifier includes Span 80; the demulsification is achieved by adding 0.1 M phosphate buffer; and the stirring and mixing conditions are stirring and emulsifying at 600~1200 rpm for 15~30 minutes.

[0011] Preferably, the concentration of the chitosan acidic solution is 0.5% w / v; the solvent of the chitosan acidic solution is 1% v / v acetic acid; the drying includes freeze drying, and the freeze drying conditions are -50°C, 0.1 mbar drying for 24 hours.

[0012] Preferably, the concentration of the pH-responsive enteric polymer in the coating solution is 6%~12% w / v, the concentration of the ethanol solution is 95% v / v, and the coating weight gain is 15~30% w / w.

[0013] This invention also provides the application of the above-mentioned acid-resistant live bacteria microcapsules in the preparation of functional foods, dietary supplements or probiotic pharmaceutical preparations.

[0014] The beneficial effects of this invention are: The gastric acid-resistant live bacteria microcapsules provided by this invention have a three-tiered protective structure: whey protein isolate in the inner core provides first-level protein matrix protection, calcium alginate gel network provides second-level gel embedding protection, and the outer pH-responsive enteric polymer coating provides third-level gastric acid barrier protection. This triple protection works synergistically to significantly improve the gastric acid tolerance of probiotics. The gastric acid-resistant live bacteria microcapsules provided by this invention achieve an encapsulation rate of 94.7% and exhibit excellent gastric acid tolerance: after incubation for 2 hours in simulated gastric juice (pH 1.2, containing pepsin, 37°C), the viable bacteria survival rate reaches 92.3%, which is significantly higher than that of uncoated microspheres (approximately 55%) and monolayer enteric-coated microspheres (approximately 75%) (p<0.01). The acid-resistant live bacteria microcapsules provided by this invention have the advantage of precise intestinal-targeted release, achieving staged release with open membrane pores in the initial segment of the duodenum (pH≥5.5) and complete dissolution in the terminal ileum (pH≥7.0). The release sites cover the entire small intestine, achieving a release rate of 96.8% within 2 hours in simulated intestinal fluid (pH 6.8). The acid-resistant live bacteria microcapsules provided by this invention also exhibit excellent storage stability; after 6 months of sealed storage at 4°C, the viable bacterial count remains at 3.2 × 10⁻⁶. 9 CFU / g (decreased by <0.5 log), significantly better than free bacteria (decreased by >3 log) and monolayer microcapsules (decreased by approximately 1.5 log). Attached Figure Description

[0015] Figure 1 This is a schematic diagram (section A-A') of the structure of the gastric acid-resistant live bacteria microcapsule (double-capsule microcapsule) of the present invention, wherein 1 is the probiotic cell; 2 is the whey protein-fructooligosaccharide protective layer (first level); 3 is the calcium alginate gel network (second level); 4 is the chitosan electrostatic composite transition layer; and 5 is the pH-responsive enteric polymer outer shell (third level).

[0016] Figure 2 This is a scanning electron microscope (SEM) image of gastric acid-resistant live bacteria microcapsules.

[0017] Figure 3 The image shows Fourier transform infrared (FTIR) spectra, where (a) represents calcium alginate microspheres, (b) represents chitosan-coated microspheres, and (c) represents Eudragit-coated microspheres.

[0018] Figure 4 Survival rate-time curves of live bacteria in simulated gastric fluid for microcapsules with different formulations.

[0019] Figure 5 The cumulative live bacteria release curve of the gastric acid-resistant live bacteria microcapsules prepared in Example 1 in a continuous simulated digestion model.

[0020] Figure 6The long-term storage stability curves of the gastric acid-resistant live bacteria microcapsules at 4℃ and 25℃ are shown.

[0021] Figure 7 The effect of different Eudragit L100-55 and Eudragit S100 ratios on the cumulative release rate of microcapsules in gradient pH (5.0-7.4) buffers over 2 h. Detailed Implementation

[0022] This invention provides a gastric acid-resistant live bacteria microcapsule, comprising, from the inside out, an inner core, a calcium alginate gel protective layer, a chitosan layer, and a pH-responsive enteric coating layer; the inner core is a mixture of probiotics, whey protein isolate, and a lyophilization protectant; the calcium alginate gel protective layer, coated on the outside of the inner core, is made of a cross-linking agent and sodium alginate; the chitosan layer, coated on the outside of the calcium alginate gel protective layer, is made of chitosan; and the pH-responsive enteric coating layer, coated on the outside of the chitosan layer, is made of a pH-responsive enteric polymer, a plasticizer, and an anti-adhesion agent.

[0023] The gastric acid-resistant live bacteria microcapsules provided by this invention adopt a double-layer structure of "core gel-outer coat" and are prepared through endogenous emulsification and gelation combined with fluidized bed coating process, which can achieve efficient protection of probiotics in the gastric acid environment and targeted release into the intestine. The encapsulation efficiency of the microcapsules of this invention is ≥88%, the viable bacteria survival rate is ≥85% in simulated gastric juice (pH 1.2, 37℃, 2 hours), and the viable bacteria release rate is ≥90% in simulated intestinal juice (pH 6.8, 37℃, 2 hours).

[0024] In this invention, the probiotics preferably include one or more of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Bifidobacterium longum*, *Lactobacillus acidophilus*, *Lactobacillus casei*, *Bifidobacterium breve*, and *Streptococcus thermophilus*; the freeze-drying protectant preferably includes fructooligosaccharides, or a mixture of fructooligosaccharides and trehalose. In the inner capsule of this invention, the weight percentage of probiotics is preferably 5-20 parts, more preferably 8-15 parts; the weight percentage of whey protein isolate is preferably 5-15 parts, more preferably 8-12 parts; the weight percentage of the freeze-drying protectant is preferably 4-11 parts, more preferably 5-8 parts; when the freeze-drying protectant is a mixture of fructooligosaccharides and trehalose, the weight percentage of fructooligosaccharides is preferably 3-10 parts, and the weight percentage of trehalose is preferably 1 part; the concentration of probiotics in the mixture is preferably 10. 10CFU / mL or higher. In this invention, the probiotics are used as the active ingredient, the whey protein isolate is used as the inner capsule protective protein and nutrient source, the fructooligosaccharide is used as a prebiotic and lyophilization protectant, the sodium alginate is used as the inner capsule gel matrix, and the chitosan is used as an intermediate transition layer (polycationic electrostatic composite). The degree of deacetylation of the chitosan is preferably ≥85%. In this invention, the process excipients preferably also include vegetable oil and emulsifier. The vegetable oil is preferably soybean oil, and the emulsifier is preferably Span 80 (sorbitan monooleate). Since soybean oil and Span 80 are process excipients, they leave almost no residue in the final product (removed by centrifugation, washing, and lyophilization), so they are not included in the calculation of the solids ratio.

[0025] In this invention, the crosslinking agent preferably includes calcium carbonate, and the weight percentage of the crosslinking agent is preferably 2-5 parts, more preferably 3-4 parts; the weight percentage of sodium alginate is preferably 30-50 parts, more preferably 35-45 parts; the weight percentage of chitosan is preferably 3-8 parts, more preferably 5-7 parts; the pH-responsive enteric polymer preferably includes at least one of Eudragit L100-55 (methyl methacrylate-ethyl acrylate copolymer), Eudragit L100, Eudragit S100, HPMCP (hydroxypropyl methylcellulose phthalate), and HPMCAS (hydroxypropyl methylcellulose acetate succinate), and the HPMCP preferably includes HPMCP. HP-55; the plasticizer preferably includes triethyl citrate; the anti-adhesion agent preferably includes talc; the pH-responsive enteric polymer is preferably 10-25 parts by weight, more preferably 13-22 parts by weight; the plasticizer is preferably 1-5 parts by weight, more preferably 2-4 parts by weight; the anti-adhesion agent is preferably 1-3 parts by weight, more preferably 2 parts by weight. In this invention, when the pH-responsive enteric polymer is composed of Eudragit L100-55 and Eudragit S100, the mass ratio of Eudragit L100-55 to Eudragit S100 is preferably 7:3 to achieve a graded release characteristic of swelling in the duodenum (pH 5.5-6.5) and complete dissolution in the jejunum to ileum (pH 6.5-7.5). This invention does not have a specific limitation on the specific source of the above raw materials; conventional commercially available products in the art can be used. A schematic diagram of the structure of the gastric acid-resistant live bacteria microcapsules provided by this invention is shown below. Figure 1 As shown.

[0026] This invention also provides a method for preparing the above-mentioned gastric acid-resistant live bacteria microcapsules, comprising the following steps: The probiotics were resuspended in a buffer solution containing whey protein isolate and a lyophilization protectant to obtain a mixed system; The mixture was then mixed with a sodium alginate solution to obtain a sodium alginate-bacterial cell blend. Vegetable oil containing emulsifier is mixed with sodium alginate-bacterial cell blend to form water-in-oil emulsion. Then, the water-in-oil emulsion is mixed with vegetable oil suspension. After demulsification, microspheres are collected to obtain probiotic microspheres coated with calcium alginate gel protective layer. The vegetable oil suspension contains glacial acetic acid and cross-linking agent. Probiotic microspheres coated with calcium alginate gel protective layer were mixed with chitosan acidic solution, washed and dried to obtain chitosan-coated probiotic microspheres. A pH-responsive enteric polymer was dissolved in an ethanol solution to obtain a coating solution. The coating solution was then mixed with a plasticizer and an anti-adhesion agent to coat chitosan-coated probiotic microspheres, resulting in acid-resistant probiotic microcapsules.

[0027] In the preparation method provided by this invention, when the probiotic is a freeze-dried probiotic powder, it preferably further includes a step of making a bacterial slurry from the freeze-dried probiotic powder, preferably: making a bacterial slurry from the freeze-dried probiotic powder (live bacteria count ≥ 10). 11 The probiotics (CFU / g) were resuspended in sterile physiological saline (0.85% NaCl) at a ratio of 1:10 (w / v), centrifuged at 5000×g for 10 minutes at 4°C, the supernatant was discarded, and the mixture was washed twice with sterile physiological saline to obtain bacterial sludge. The bacterial sludge was then resuspended in a buffer solution containing whey protein isolate and a lyophilization protectant. The whey protein isolate concentration in the buffer solution was preferably 2% w / v, and the lyophilization protectant concentration was preferably 1% w / v. The buffer solution preferably included PBS buffer, the pH of which was preferably 7.0. The concentration of probiotics in the mixture was preferably 10. 10 CFU / mL or higher.

[0028] In the preparation method provided by the present invention, the concentration of the sodium alginate solution is preferably 3.0% w / v, the solvent of the sodium alginate solution is preferably water, more preferably deionized water, and the volume ratio of the mixed system to the sodium alginate solution is preferably 1:4.

[0029] In the preparation method provided by the present invention, the concentration of the emulsifier in the vegetable oil containing the emulsifier is preferably 1.5%~3.0% w / v, more preferably 2%~2.5% w / v; the volume ratio of the vegetable oil containing the emulsifier to the sodium alginate-bacterial cell blend is preferably (2~4):1, more preferably 3:1; when mixing the vegetable oil containing the emulsifier with the sodium alginate-bacterial cell blend, it is preferably stirred and emulsified at 4°C and 600~1200 rpm for 15~30 minutes, and the emulsification temperature is preferably 4~10°C. In this invention, the concentration of glacial acetic acid in the vegetable oil suspension is preferably 0.4%~0.6% v / v; the vegetable oil preferably includes soybean oil; the emulsifier preferably includes Span 80; the demulsification is preferably performed by adding 0.1 M phosphate buffer, the pH of which is preferably 7.4. After demulsification, the microspheres are collected by centrifugation and washed with sterile deionized water to obtain probiotic microspheres coated with a calcium alginate gel protective layer. The centrifugation conditions are preferably 3000×g for 5 minutes.

[0030] In this invention, the concentration of the chitosan acidic solution is preferably 0.5% w / v; the solvent of the chitosan acidic solution is preferably 1% v / v acetic acid, and the pH is preferably 4.5; the probiotic microspheres coated with the calcium alginate gel protective layer are gently stirred with the chitosan acidic solution at 25°C for 30 minutes, so that chitosan is adsorbed onto the surface of the calcium alginate microspheres through electrostatic interaction to form a polyelectrolyte composite film, and then washed twice with sterile deionized water to remove free chitosan, and finally dried, the drying preferably including freeze drying, the freeze drying conditions preferably being -50°C, 0.1 mbar drying for 24 hours.

[0031] In this invention, the concentration of the pH-responsive enteric polymer in the coating solution is preferably 6%~12% w / v, more preferably 8%~10% w / v, and the concentration of the ethanol solution is preferably 95% v / v. After mixing the coating solution with the plasticizer and anti-adhesion agent, the mixture is preferably passed through a 200-mesh sieve, followed by coating. The coating is preferably carried out in a fluidized bed coating machine using a bottom-spray mode (Wurster column). The inlet air temperature is preferably 38~42℃, the outlet air temperature is preferably 30~34℃, the atomization pressure is preferably 1.2~1.5 bar, the spray rate is preferably 2.0~3.0 mL / min, the material temperature is preferably 28~32℃, and the coating weight gain is preferably 15~30% (w / w), more preferably 20~25% (w / w). After coating, the mixture is further dried in a fluidized bed for 15 minutes, and then passed through a 40-mesh sieve to remove agglomerated particles, thus obtaining acid-resistant live bacteria microcapsules.

[0032] The preparation method provided by this invention employs an endogenous emulsification-gelation method to prepare the inner capsule core, combined with a fluidized bed bottom-spray coating method to prepare the outer capsule shell. This process is mild and results in a high retention rate of bacterial activity. Furthermore, the particle size of the microcapsules provided by this invention is controllable. By adjusting the emulsification stirring speed and emulsifier concentration, the microcapsule particle size can be controlled within the range of 150~800 μm, meeting the needs of different dosage forms (capsule filling, powder, tablets).

[0033] This invention also provides the application of the above-mentioned acid-resistant live bacteria microcapsules in the preparation of functional foods, dietary supplements or probiotic pharmaceutical preparations.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Unless otherwise specified, the following embodiments are all conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] Statistical analysis of data in the following examples: All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.0. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA and Tukey's post-hoc test were used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0038] The sources of the main raw materials and equipment in the following embodiments are shown in Table 1 below: Table 1 Main Raw Materials and Equipment

[0039] Example 1 Acid-resistant Lactobacillus plantarum LP299v microcapsules From the inside out, it comprises an inner core, a calcium alginate gel protective layer, a chitosan layer, and a pH-responsive enteric coating layer. The inner core is a mixture of probiotics, whey protein isolate (WPI) 3 g, and fructooligosaccharides (FOS) 2 g. The calcium alginate gel protective layer, covering the outer side of the inner core, is made of cross-linking agent calcium carbonate (CaCO3) 1.5 g and sodium alginate 12 g. The chitosan layer, covering the outer side of the calcium alginate gel protective layer, is made of chitosan 2 g. The pH-responsive enteric coating layer, covering the outer side of the chitosan layer, is made of pH-responsive enteric polymer, plasticizer triethyl citrate (TEC) 1.2 g, and anti-adhesion agent talc 3.0 g. The probiotic is 5 g of lyophilized Lactobacillus plantarum LP299v powder (live count 5.2 × 10⁻⁶).11 The pH-responsive enteric polymer (CFU / g) consists of 4.2 g Eudragit L100-55 and 1.8 g Eudragit S100.

[0040] The preparation method is as follows: Step S1: Preparation of the mixed system (probiotic suspension) The lyophilized Lactobacillus plantarum LP299v powder was resuspended in sterile physiological saline (0.85% NaCl) at a ratio of 1:10 (w / v), centrifuged at 5000×g for 10 minutes at 4°C, the supernatant was discarded, and the sample was washed twice with sterile physiological saline to obtain bacterial sludge. The bacterial sludge was resuspended in sterile PBS buffer (pH 7.0) containing 2% (w / v) whey protein isolate and 1% (w / v) fructooligosaccharides, and the bacterial concentration was adjusted to 10. 10 CFU / mL yields a mixed system (probiotic-protectant suspension).

[0041] Step S2: Preparation of sodium alginate-bacterial cell blend Sodium alginate powder was slowly added to deionized water and stirred at 40°C for 2 hours to prepare a 3.0% (w / v) sodium alginate solution. The solution was then autoclaved at 121°C for 15 minutes and cooled to 25°C. Under aseptic conditions, the mixture obtained in step S1 was mixed with the sodium alginate solution at a volume ratio of 1:4 and gently stirred for 10 minutes to obtain a sodium alginate-bacterial cell blend (final sodium alginate concentration of 2.4% w / v, bacterial concentration of approximately 2 × 10⁻⁶). 9 (CFU / mL).

[0042] Step S3: Preparation of probiotic microspheres (inner capsule core microspheres, calcium alginate microspheres) coated with calcium alginate gel protective layer (endogenous emulsification-gelation method) Aseptic soybean oil containing 2.0% (w / v) Span 80 emulsifier was prepared as the continuous phase, with a volume ratio of 3:1 between the aseptic soybean oil containing 2.0% (w / v) Span 80 emulsifier and the sodium alginate-bacterial cell blend obtained in step S2. The sodium alginate-bacterial cell blend was slowly added to the aseptic soybean oil containing 2.0% (w / v) Span 80 emulsifier, and emulsified by stirring at 4°C and 800 rpm for 20 minutes to form a stable water-in-oil (W / O) emulsion. Subsequently, a CaCO3 soybean oil suspension containing glacial acetic acid (final concentration 0.5% v / v) was slowly added dropwise, and stirring was continued for 30 minutes to allow the CaCO3 to settle. 2+ Sodium alginate was slowly released and cross-linked. An equal volume of 0.1M phosphate buffer (pH 7.4) was added to break the emulsion, and the microspheres were collected by centrifugation at 3000×g for 5 minutes. The microspheres were washed three times with sterile deionized water to obtain probiotic microspheres (inner capsule core microspheres, calcium alginate microspheres) coated with a calcium alginate gel protective layer.

[0043] Step S4: Preparation of chitosan electrostatic composite layer The microspheres with inner capsule cores obtained in step S3 were suspended in a 0.5% (w / v) acidic chitosan solution (dissolved in 1% v / v acetic acid, pH 4.5) and gently stirred at 25°C for 30 minutes, allowing chitosan to be adsorbed onto the surface of the calcium alginate microspheres via electrostatic attraction to form a polyelectrolyte composite membrane. The microspheres were washed twice with sterile deionized water to remove free chitosan, and then freeze-dried (-50°C, 0.1 mbar, 24 hours) to obtain chitosan-coated probiotic microspheres.

[0044] Step S5: Preparation of enteric-coated outer shell (fluidized bed bottom spray coating method) A pH-responsive enteric polymer was prepared by compounding Eudragit L100-55 and Eudragit S100 at a mass ratio of 7:3. The pH-responsive enteric polymer was then dissolved in a 95% ethanol solution (the volume ratio of ethanol to water in the ethanol solution was 19:1) to prepare a coating solution with a pH-responsive enteric polymer concentration of 8% (w / v). Triethyl citrate (TEC) plasticizer and talc anti-adhesion agent were added, and the mixture was magnetically stirred until completely dissolved. The solution was then passed through a 200-mesh sieve. The chitosan-coated probiotic microspheres obtained in step S4 were placed in a fluidized bed coating machine using a bottom spray mode (Wurster column). The coating conditions were: inlet air temperature 40℃, spray rate 2.5mL / min, atomization pressure 1.35 bar, and coating weight gain of 20%. After coating, the microspheres were dried in the fluidized bed for 15 minutes and then passed through a 40-mesh sieve to remove agglomerated particles, thus obtaining the finished product of gastric acid-resistant live bacteria microcapsules (enteric-coated live bacteria double capsule microcapsules, Eudragit-coated microspheres).

[0045] Scanning electron microscope images of the gastric acid-resistant live bacteria microcapsules prepared according to the above preparation method are shown below. Figure 2 As shown, by Figure 2 As can be seen from (a) in the figure, the gastric acid-resistant live bacteria microcapsules prepared by the present invention have a regular spherical shape and a smooth surface, made of Figure 2 As can be seen from (b) in the present invention, the gastric acid-resistant live bacteria microcapsules prepared by the present invention have a clear inner and outer double-layer structure.

[0046] In the above preparation process, the calcium alginate microspheres obtained in step S3, the polysaccharide-coated microspheres obtained in step S4, and the Eudragit-coated microspheres obtained in step S5 were subjected to Fourier transform infrared spectroscopy detection, and the results are as follows. Figure 3 As shown, it can be seen that chitosan-coated microspheres at 1560 cm⁻¹ -1 NH3 appeared at the location + Characteristic peak for Eudragit-coated microspheres at 1730 cm⁻¹ -1 A stretching vibration peak of the ester carbonyl group C=O appears at 1450~1490 cm⁻¹.-1 The presence of a CH3 bending vibration peak indicates that the three-layer structure was successfully prepared in this invention, and that the layers are chemically bonded or strongly electrostatically combined, rather than simply physically mixed. Furthermore, the functional groups remain independent, without any destructive chemical reactions, meaning that the microenvironment in which the live bacteria reside is effectively protected.

[0047] The gastric acid-resistant live bacteria microcapsules prepared above were tested for each of the indicators listed in Table 2, wherein: Encapsulation efficiency determination: Take 100 mg of microcapsules and add 10 mL of decapsulation solution (0.2 M sodium phosphate buffer, pH 7.4, containing 0.5% w / v trypsin). Incubate at 37°C with shaking until the microcapsules completely disintegrate (approximately 60 minutes). Dilute serially with sterile physiological saline and spread on MRS agar plates. Incubate anaerobically at 37°C for 48 hours and count the colonies. Encapsulation efficiency (%) = (number of viable bacteria in microcapsules / initial number of viable bacteria) × 100%.

[0048] Preparation of simulated gastric juice (SGF): NaCl 2.0 g / L, pepsin 3.2 g / L, pH adjusted to 1.2±0.05 with 1 M HCl, and sterilized by filtration through a 0.22 μm filter membrane.

[0049] Simulated intestinal fluid (SIF) preparation: KH2PO4 6.8 g / L, trypsin 10 g / L, pH adjusted to 6.8±0.05 with 0.2 M NaOH, and sterilized by filtration through a 0.22 μm filter membrane.

[0050] The results are shown in Table 2.

[0051] Table 2 Product Characterization Results

[0052] Example 2 Lactobacillus rhamnosus LGG microcapsules The difference between the formulation and Example 1 is that the *Lactobacillus plantarum* LP299v lyophilized powder is replaced with 5 g of *Lactobacillus rhamnosus* LGG lyophilized powder (4.8 × 10⁻⁶ viable cells). 11 (CFU), the rest are the same as in Example 1.

[0053] The preparation method differs from Example 1 in that: the concentration of the sodium alginate solution in step S2 is increased to 3.5% (w / v), and the volume ratio of the mixture obtained in step S1 to the sodium alginate solution is adjusted to 1:3.5; the emulsification stirring speed in step S3 is adjusted to 1000 rpm, and the emulsification time is shortened to 15 minutes to obtain a smaller inner capsule core particle size. All other preparation methods are the same as in Example 1.

[0054] The gastric acid-resistant live bacteria microcapsules prepared in this embodiment were tested for each of the indicators listed in Table 3. The testing method was the same as in Example 1, and the results are shown in Table 3.

[0055] Table 3 Product Characterization Results

[0056] Example 3 Acid-resistant Bifidobacterium longum BB536 (anaerobic bacteria) microcapsules The difference between the formulation and Example 1 is that the *Lactobacillus plantarum* LP299v lyophilized powder is replaced with 5 g of *Bifidobacterium longum* BB536 lyophilized powder (5.2 × 10⁻⁶ viable bacteria). 11 CFU), and an additional 1g of trehalose was added to enhance the freeze-drying tolerance of anaerobic bacteria, otherwise the same as in Example 1.

[0057] The preparation method differs from that in Example 1 in that, since Bifidobacterium longum BB536 is a strict anaerobic bacterium, steps S1 to S4 are all performed in an anaerobic workstation (N2:H2:CO2=85:10:5). The remaining preparation methods are the same as in Example 1.

[0058] The gastric acid-resistant live bacteria microcapsules prepared in this embodiment were tested for each of the indicators listed in Table 4. The testing method was the same as in Example 1, and the results are shown in Table 4.

[0059] Table 4 Product Characterization Results

[0060] Example 4 Stomach acid resistant mixed live bacteria microcapsules The difference between the formulation and Example 1 is that the lyophilized powder of Lactobacillus plantarum LP299v is replaced with a mixture of 2 g of lyophilized powder of Lactobacillus plantarum LP299v, 2 g of lyophilized powder of Lactobacillus rhamnosus LGG and 2 g of lyophilized powder of Bifidobacterium longum BB536, while the other components are the same as in Example 1.

[0061] The preparation method is the same as in Example 1.

[0062] The gastric acid-resistant live bacteria microcapsules prepared in this embodiment were tested for each of the indicators listed in Table 5. The testing method was the same as in Example 1, and the results are shown in Table 5.

[0063] Table 5 Product Characterization Results

[0064] Experimental Example 1 Coating weight gain gradient experiment Based on Example 1, samples with coating weight gain of 0% (uncoated control), 10%, 15%, 20%, 25%, and 30% were prepared (meaning that the only difference from Example 1 is the coating weight gain in step S5; all other aspects are the same as in Example 1), and the SGF 2h survival rate and particle size were measured. The detection method was the same as in Example 1.

[0065] The results are shown in Table 6. As the coating weight gain increased from 0% to 20%, gastric acid tolerance showed a significant upward trend (p<0.01). When the coating weight gain exceeded 20%, the improvement in survival rate slowed down, but the particle size continued to increase. Considering both the protective effect and product suitability, the optimal coating weight gain was determined to be 20%.

[0066] Table 6. Weight gain results of different coatings

[0067] Experimental Example 2 Viable bacterial survival time test of different microcapsule formulations in simulated gastric fluid (pH 1.2, 37℃, containing 3.2 g / L pepsin). The following microbes were tested in simulated gastric juice (pH 1.2, 37°C, containing 3.2 g / L pepsin): Lactobacillus plantarum LP299v (free bacteria) from Example 1, calcium alginate microspheres obtained in step S3 of Example 1, chitosan-coated microspheres obtained in step S4 of Example 1, gastric acid-resistant live bacteria microcapsules (double capsules) obtained in step S5 of Example 1, and gastric acid-resistant live bacteria microcapsules obtained in Example 4. The survival rate of live bacteria at different times was determined.

[0068] The results are as follows Figure 4 As shown, the double-encapsulated microcapsules (Example 1) had a survival rate of up to 92.3% at 120 min, which was significantly better than free bacteria (0.1%) and calcium alginate microspheres (54.8%).

[0069] Experimental Example 3 The acid-resistant live bacteria microcapsules (double-capsule microcapsules) prepared in Example 1 were used in a continuous simulated digestion experiment: the acid-resistant live bacteria microcapsules prepared in Example 1 were first placed in simulated gastric juice (SGF) (the SGF was prepared as in Example 1) for 2 hours, and then placed in simulated intestinal juice (SIF) (the SIF was prepared as in Example 1). The live bacteria release rate was detected at different time points, and the results are as follows. Figure 5 As shown, only 1.2% was released during the SGF phase, but after switching to SIF, it was released to 56.8% within 1 hour and to 96.8% within 3 hours.

[0070] Test Example 4 Test curve of viable bacteria count change of the gastric acid-resistant microcapsules (double-capsule microcapsules) obtained in Example 1 during long-term storage (0-180 days) 1. Sample Preparation The experiment requires the preparation of two sets of key samples for comparison: Experimental group: Different batches of double-encapsulated microcapsules prepared according to the method of Example 1 were labeled as "Example 1" and "Example 5" respectively.

[0071] Control group (Free bacteria): Free probiotics without any encapsulation protection, used as a comparison baseline.

[0072] 2. Storage Condition Settings To comprehensively evaluate stability at different temperatures, two typical storage scenarios were set up in the experiment: Refrigeration conditions (4℃): Simulates an ideal cold chain storage environment.

[0073] Normal temperature conditions (25℃): Simulate room temperature or normal storage environment.

[0074] 3. Sampling and Testing Procedures Sampling and Viable Bacterial Counting: Within a time span of 0 to 180 days, aseptic sampling was performed on samples taken at predetermined time points (e.g., days 0, 25, and 50) at two different temperatures. After sampling, the samples were thoroughly dissolved and diluted, and the viable bacterial count in each sample was determined using a standard plate count method (e.g., spread plate method, incubated on a suitable culture medium). The results were expressed as logarithmic values. 10 Recorded in units of (CFU / g).

[0075] Key observation indicators: Viable bacteria count decay trend: Observe the rate of decrease in viable bacteria count over time in different samples.

[0076] The results are as follows Figure 6 As shown, the gastric acid-resistant live bacteria microcapsules prepared by this invention showed a decrease of only <0.5 log after storage at 4°C for 180 days, and a decrease of about 3 log after storage at 25°C, both of which are significantly better than free bacteria.

[0077] Experimental Example 5 Effect of different Eudragit L100-55 to Eudragit S100 ratios on the cumulative release rate of microcapsules in gradient pH (5.0-7.4) buffers over 2 h.

[0078] The difference from Example 1 is that the mass ratio of Eudragit L100-55 to Eudragit S100 in step S5 is adjusted to 10:0, 8:2, 7:3, 5:5 and 0:10 respectively, while the rest is the same as in Example 1.

[0079] The different microcapsules prepared above were placed in buffer solutions with pH values ​​ranging from 5.0 to 7.4 for 2 hours, and the cumulative release rate of different groups was then measured. The results are as follows: Figure 7 As shown, the 7:3 ratio exhibits ideal gradient release characteristics.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microcapsule containing gastric acid-resistant live bacteria, characterized in that, From the inside out, it comprises an inner core, a calcium alginate gel protective layer, a chitosan layer, and a pH-responsive enteric coating layer. The inner core is a mixture of probiotics, whey protein isolate, and a lyophilization protectant. The calcium alginate gel protective layer, which coats the outer side of the inner core, is made of a cross-linking agent and sodium alginate. The chitosan layer, which coats the outer side of the calcium alginate gel protective layer, is made of chitosan. The pH-responsive enteric coating layer, which coats the outer side of the chitosan layer, is made of a pH-responsive enteric polymer, a plasticizer, and an anti-adhesion agent.

2. The gastric acid-resistant live bacteria microcapsule according to claim 1, characterized in that, The freeze-drying protectant includes fructooligosaccharides, or a mixture of fructooligosaccharides and trehalose; the weight proportions of each raw material in the inner capsule are: 5-20 parts probiotics, 5-15 parts whey protein isolate, and 4-11 parts freeze-drying protectant; the concentration of probiotics in the mixed system is 10. 10 CFU / mL or higher; the crosslinking agent includes calcium carbonate, with a weight percentage of 2-5 parts; the sodium alginate has a weight percentage of 30-50 parts; the chitosan has a weight percentage of 3-8 parts; the pH-responsive enteric polymer includes at least one of Eudragit L100-55, Eudragit L100, Eudragit S100, HPMCP, and HPMCAS; the plasticizer includes triethyl citrate; the anti-adhesion agent includes talc; the pH-responsive enteric polymer has a weight percentage of 10-25 parts; the plasticizer has a weight percentage of 1-5 parts; the anti-adhesion agent has a weight percentage of 1-3 parts; the probiotics include one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium breve, and Streptococcus thermophilus.

3. The gastric acid-resistant live bacteria microcapsule according to claim 2, characterized in that, The pH-responsive enteric polymer is composed of Eudragit L100-55 and Eudragit S100, with a mass ratio of Eudragit L100-55 to Eudragit S100 of 7:

3.

4. The method for preparing the gastric acid-resistant live bacteria microcapsules according to any one of claims 1 to 3, characterized in that, Includes the following steps: The probiotics were resuspended in a buffer solution containing whey protein isolate and a lyophilization protectant to obtain a mixed system. The mixture was then mixed with a sodium alginate solution to obtain a sodium alginate-bacterial cell blend. Vegetable oil containing emulsifier is stirred and mixed with sodium alginate-bacterial cell blend to form water-in-oil emulsion. Then, the water-in-oil emulsion is mixed with vegetable oil suspension. After demulsification, microspheres are collected to obtain probiotic microspheres coated with calcium alginate gel protective layer. The vegetable oil suspension contains glacial acetic acid and cross-linking agent. Probiotic microspheres coated with calcium alginate gel protective layer were mixed with chitosan acidic solution, washed and dried to obtain chitosan-coated probiotic microspheres. A pH-responsive enteric polymer was dissolved in an ethanol solution to obtain a coating solution. The coating solution was then mixed with a plasticizer and an anti-adhesion agent to coat chitosan-coated probiotic microspheres, resulting in acid-resistant probiotic microcapsules.

5. The preparation method according to claim 4, characterized in that, The buffer solution contains 2% whey protein isolate (w / v) and 1% w / v fructooligosaccharides (FOS). The buffer solution includes PBS buffer. The concentration of probiotics in the mixture is 10%. 10 CFU / mL or higher.

6. The preparation method according to claim 4, characterized in that, The concentration of the sodium alginate solution is 3.0% w / v, and the solvent of the sodium alginate solution is water; the volume ratio of the mixed system to the sodium alginate solution is 1:

4.

7. The preparation method according to claim 4, characterized in that, The concentration of the emulsifier in the vegetable oil containing the emulsifier is 1.5%~3.0% w / v; the volume ratio of the vegetable oil containing the emulsifier to the sodium alginate-bacterial cell blend is (2~4):1; the concentration of glacial acetic acid in the vegetable oil suspension is 0.4%~0.6% v / v; the vegetable oil includes soybean oil; the emulsifier includes Span 80; the demulsification is achieved by adding 0.1 M phosphate buffer; the stirring and mixing conditions are stirring and emulsifying at 600~1200 rpm for 15~30 minutes.

8. The preparation method according to claim 4, characterized in that, The concentration of the chitosan acidic solution is 0.5% w / v; the solvent of the chitosan acidic solution is 1% v / v acetic acid; the drying includes freeze drying, and the freeze drying conditions are -50°C, 0.1 mbar drying for 24 hours.

9. The preparation method according to claim 4, characterized in that, The concentration of the pH-responsive enteric polymer in the coating solution is 6%~12% w / v, the concentration of the ethanol solution is 95% v / v, and the coating weight gain is 15~30% w / w.

10. The use of the gastric acid-resistant live bacteria microcapsules according to any one of claims 1 to 3 in the preparation of functional foods, dietary supplements or probiotic pharmaceutical preparations.

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