PH response type lactobacillus plantarum gel ball as well as preparation method and application thereof

Abstract

The invention discloses a pH response type lactobacillus plantarum gel ball as well as a preparation method and application thereof, and belongs to the technical field of probiotic embedding and delivery. The pH response type lactobacillus plantarum gel ball provided by the invention has a compact gel network structure, so that the survival rate of probiotics in gastrointestinal fluid can be remarkably improved; the effect of targeted release in intestinal tracts is achieved; the high-quality prebiotics are contained and have a synergistic effect with the probiotics, so that the effect of relieving constipation is obviously superior to that of free probiotics and no-load gel; the preparation method is simple and easy to operate, the used reagents and materials are high in safety, toxic and harmful reagents are not used in the preparation process, toxic and harmful substances are not generated, and the preparation method is environment-friendly and has no potential safety hazard.

Description

Technical Field

[0001] This invention relates to the field of probiotic encapsulation and delivery technology, and in particular to a pH-responsive Lactobacillus plantarum gel ball, its preparation method, and its application. Background Technology

[0002] With increasing health awareness, probiotics are receiving more and more attention due to their beneficial effects such as regulating gut microbiota, enhancing immunity, and improving gut health. Lactobacillus plantarum, a common probiotic, has multiple health benefits, including regulating gut microbiota, enhancing immunity, and relieving constipation. Studies suggest that only when the ingested live bacteria count exceeds 10... 6 A CFU / g level is required to produce beneficial effects on the host. However, orally administered free probiotics must undergo erosion by the strong acid (pH 1.5-2.0) and digestive enzymes in the stomach before reaching the intestines, which may lead to significant inactivation and prevent them from fully exerting their probiotic effects.

[0003] Currently, encapsulation techniques are commonly used to protect probiotics. Common probiotic encapsulation techniques include spray drying, freeze drying, emulsification, extrusion, and electrostatic spraying. Among these, hydrogels prepared by extrusion are widely used for tissue repair and drug delivery due to their ease of operation and good nutrient retention. However, single protein / polysaccharide gels suffer from low encapsulation rates and are prone to lysis, leading to premature release of probiotics, thus limiting their protective effect. Composite gel systems can effectively improve these shortcomings. Sodium alginate-calcium ion crosslinking systems are one of the most common methods (e.g., patent CN114672480A), but their protective ability is limited in acidic environments, and the release behavior is not well-controllable. To further improve the protective effect, existing technologies often employ multi-layer encapsulation (e.g., the sodium alginate-chitosan-hydroxypropyl methylcellulose three-layer structure used in patent CN119366639A). However, such methods are complex, involve numerous steps, and require organic solvents (e.g., acetic acid to dissolve chitosan), which is not conducive to large-scale production and food applications.

[0004] Therefore, developing a probiotic delivery system that is simple in process, uses safe materials, can withstand gastrointestinal digestion, and can be precisely released into the intestine has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing pH-responsive Lactobacillus plantarum gel spheres, in order to solve the problems of low encapsulation rate, poor stability, complex process, cumbersome steps, and potential food safety hazards in existing encapsulation technologies.

[0006] To achieve the above objectives, the present invention provides a pH-responsive Lactobacillus plantarum gel ball, its preparation method and application, wherein the gel ball uses Lactobacillus plantarum as the core material and pectin, kelp polysaccharide and whey protein as composite wall materials, and is formed by double ionic cross-linking and curing through internal calcium carbonate and external calcium chloride solutions.

[0007] The pH-responsive Lactobacillus plantarum gel spheres described above, their preparation method, and applications are as follows: (1) After activating Lactobacillus plantarum, the bacterial concentration was adjusted with sterile distilled water to obtain free Lactobacillus plantarum stock solution; (2) To prepare the wall material solution, dissolve pectin and kelp polysaccharide in sterile distilled water, then mix them evenly according to the volume ratio, add whey protein powder, mix well, add calcium carbonate powder, stir and mix well, and then eliminate the bubbles to obtain the wall material solution. (3) Encapsulation of Lactobacillus plantarum: The free Lactobacillus plantarum mother liquor prepared in (1) is mixed evenly with the wall material solution prepared in (2), and then dripped into the curing solution by a microfluidic injection pump for cross-linking and curing to obtain pH-responsive Lactobacillus plantarum gel balls.

[0008] Preferably, in step (1), Lactobacillus plantarum is activated using MRS broth medium and cultured at 37°C; the bacterial concentration is adjusted with sterile distilled water and centrifuged at 8000×g for 10-15 min at 4°C, and then washed 2-3 times with sterile distilled water.

[0009] Preferably, the MRS broth culture medium consists of the following components per liter: 10.0 g casein digest, 10.0 g beef extract, 4.0 g yeast extract, 2.0 g triammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate (MgSO4·7H2O), 0.05 g manganese sulfate (MnSO4·4H2O), 2.0 g dipotassium hydrogen phosphate, 20.0 g glucose, and 1.08 g Tween-80 (final pH 5.7 ± 0.2).

[0010] Preferably, the adjusted bacterial concentration is 1-3 × 10⁻⁶. 10 CFU / mL.

[0011] Preferably, the pectin in (2) contains ≥74.0% galacturonic acid (dry basis), and the kelp polysaccharide has a molecular weight of 10-260kDa and a total sugar content of 62-78%.

[0012] Preferably, in the (2) process, the mass-volume ratio of pectin: kelp polysaccharide: whey protein powder: calcium carbonate powder: sterile distilled water is 1g:1g:1g:0.1g:100mL-3g:3g:3g:0.2g:100mL, and the bubbles are eliminated by standing at room temperature.

[0013] Preferably, the volume ratio of the free Lactobacillus plantarum mother liquor to the wall material solution in (3) is 1:5.

[0014] Preferably, the method of cross-linking and curing by dripping the microfluidic injection pump into the curing liquid in (3) is as follows: the advance speed of the microfluidic injection pump is 0.5-5 mL / min, the needle diameter is 0.2-0.8 mm, and the distance between the needle and the surface of the curing liquid is 10-20 cm; cross-linking and curing are carried out at room temperature.

[0015] The application of pH-responsive Lactobacillus plantarum gel balls as described above in the treatment and / or relief of constipation.

[0016] Therefore, the present invention provides a pH-responsive Lactobacillus plantarum gel ball, its preparation method, and its application, the specific technical effects of which are as follows: (1) The pH-responsive Lactobacillus plantarum gel ball provided by the present invention uses Lactobacillus plantarum as the core material and pectin, kelp polysaccharide, whey protein and calcium carbonate as composite wall material, and is formed by double cross-linking and curing through a two-step method of internal gelation and external ionic cross-linking. (2) The pH-responsive Lactobacillus plantarum gel spheres prepared by the method provided in this invention have a dense gel network structure, which can significantly resist gastric acid digestion and thus significantly improve the survival rate of Lactobacillus plantarum in gastrointestinal fluid. (3) A pH-responsive Lactobacillus plantarum gel ball prepared by the method provided in this invention has the effect of targeted release in the intestine; it can not only deliver Lactobacillus plantarum, but its wall material itself is a high-quality prebiotic, which has a synergistic effect with probiotics, and its effect in relieving constipation is significantly better than that of free probiotics and empty gel. (4) The preparation method provided by the present invention is simple and easy to operate. The reagents and materials used are highly safe. The preparation process does not use toxic or harmful reagents and does not generate any toxic or harmful substances. It is environmentally friendly and has no safety hazards.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 To verify the effectiveness, the survival rate of some free Lactobacillus plantarum Lp and the prepared bacterial-loaded gel spheres was statistically analyzed after digestion with simulated gastric juice. Figure 2 To verify the effectiveness, the statistical results of the release and survival rate of Lactobacillus plantarum Lp and the prepared bacterial-loaded gel spheres after digestion with simulated intestinal fluid were obtained. Figure 3 To verify the effectiveness, the results of the in vitro pH-responsive release experiment of partially loaded bacterial gel spheres were analyzed. Figure 4 To verify the effectiveness, the survival rate of some free Lactobacillus plantarum Lp and the prepared bacterial-loaded gel spheres was statistically analyzed after simulated pasteurization. Figure 5 To verify the effectiveness, the survival rate of some free Lactobacillus plantarum Lp and the prepared bacterial-loaded gel spheres was statistically analyzed after freeze-drying. Figure 6 To verify the effectiveness, statistical results were obtained on the time to the first black stool in constipated mice under different treatments. Figure 7 To verify the effectiveness, the number of fecal pellets excreted within 6 hours was statistically analyzed in constipated mice under different treatments; Figure 8 To verify the effectiveness, statistical results of intestinal propulsion rate were obtained from constipated mice under different treatments. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] All instruments, equipment, and reagents used in the examples were obtained commercially; methods not described in detail in the examples are conventional techniques in the art; the composition of the culture media used in the examples is as follows: MRS broth medium: 10.0g casein digest, 10.0g beef extract, 4.0g yeast extract, 2.0g triammonium citrate, 5.0g sodium acetate, 0.2g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·4H2O), 2.0g dipotassium hydrogen phosphate, 20.0g glucose, 1.08g Tween-80 (final pH 5.7±0.2); MRS solid medium: 10.0g peptone, 5.0g beef meal, 4.0g yeast extract, 20.0g glucose, 1.0mL Tween 80, 2.0g K2HPO4·7H2O, 5.0g sodium acetate·3H2O, 2.0g triammonium citrate, 0.2g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·4H2O), 15.0g agar (final pH 6.2±0.2).

[0023] Example 1 The specific steps for preparing a pH-responsive Lactobacillus plantarum composite gel sphere (ternary gel sphere: pectin + whey protein + calcium carbonate) are as follows: (1) Culture of Lactobacillus plantarum.

[0024] In a clean bench, a single colony of *Lactobacillus plantarum* was picked and inoculated into MRS broth medium and incubated at 37°C for 24 hours; this was the first activation. The culture was then transferred to fresh MRS broth medium at a 1% (v / v) inoculation rate and incubated for another 24 hours under the same conditions to complete the second activation. The activated bacterial solution was then centrifuged at 8000×g for 10 minutes at 4°C. The bacterial pellet was washed twice with sterile distilled water and resuspended to obtain a free *Lactobacillus plantarum* stock solution with a bacterial concentration of approximately 3×10⁻⁶. 10 CFU / mL.

[0025] (2) Preparation of wall material solution.

[0026] Dissolve 1g of pectin in 50mL of sterile distilled water and stir at 50℃ and 500rpm until fully dissolved to prepare a 2% (w / v) pectin solution. Let stand at room temperature for 30min to eliminate air bubbles. Add 1.5g of whey protein powder to bring the final whey protein concentration to 3% (w / v) and stir at 500rpm for 1h to ensure thorough mixing. Add 0.05g of calcium carbonate powder (final concentration 0.1% (w / v)) and stir at 600rpm for 2h to ensure thorough mixing. Let stand at room temperature overnight to eliminate air bubbles to obtain the wall material solution.

[0027] (3) Encapsulation of Lactobacillus plantarum.

[0028] The *Lactobacillus plantarum* stock solution prepared in step (1) and the wall material solution prepared in step (2) were mixed at a volume ratio of 1:5 and stirred at 500 rpm for 2 hours until homogeneous. The mixture of bacterial culture and wall material solution was then dripped into a curing solution (2% CaCl2 (w / v)) using a microfluidic injection pump for cross-linking and curing for 30 minutes (room temperature). The microfluidic injection pump was used at a flow rate of 4 mL / min, with a needle diameter of 0.45 mm and a distance of 15 cm between the needle and the surface of the curing solution. The mixture was washed three times with sterile distilled water to obtain ternary (pectin + whey protein + calcium carbonate) gel spheres loaded with *Lactobacillus plantarum*, denoted as A1.

[0029] Example 2 A pH-responsive Lactobacillus plantarum composite gel sphere (quaternary gel sphere: pectin + kelp polysaccharide + whey protein + calcium carbonate) was prepared using the following specific steps: (1) Culture of Lactobacillus plantarum.

[0030] In a clean bench, a single colony of *Lactobacillus plantarum* was picked and inoculated into MRS broth medium and incubated at 37°C for 24 hours; this was the first activation. The culture was then transferred to fresh MRS broth medium at a 1% (v / v) inoculation rate and incubated for another 24 hours under the same conditions to complete the second activation. The activated bacterial solution was then centrifuged at 8000×g for 10 minutes at 4°C. The bacterial pellet was washed twice with sterile distilled water and resuspended to obtain a free *Lactobacillus plantarum* stock solution with a bacterial concentration of approximately 1×10⁻⁶. 10 CFU / mL.

[0031] (2) Preparation of wall material solution.

[0032] Dissolve 0.4 g of pectin and 0.1 g of kelp polysaccharide in 40 mL and 10 mL of sterile distilled water, respectively, and stir at 500 rpm at 50°C to prepare 1% (w / v) pectin and kelp polysaccharide solutions. Mix the pectin and kelp polysaccharide solutions thoroughly at a volume ratio of 4:1, stir well, and let stand at room temperature for 30 min to eliminate air bubbles. Add 0.5 g of whey protein powder to bring the final whey protein concentration to 1% (w / v), and stir at 500 rpm for 1 h to mix thoroughly. Add 0.05 g of calcium carbonate powder (final concentration 0.1% (w / v)), stir at 600 rpm for 2 h to mix thoroughly, and let stand at room temperature overnight to eliminate air bubbles to obtain the wall material solution.

[0033] (3) Encapsulation of Lactobacillus plantarum.

[0034] The *Lactobacillus plantarum* stock solution prepared in step (1) and the wall material solution prepared in step (2) were mixed at a volume ratio of 1:5 and stirred at 500 rpm for 2 hours until homogeneous. Then, the mixture of *Lactobacillus plantarum* stock solution and wall material solution was dripped into a curing solution (2% CaCl2 (w / v)) using a microfluidic injection pump for cross-linking and curing for 30 minutes (room temperature). The microfluidic injection pump was used at a speed of 0.5 mL / min, the needle diameter was 0.2 mm, and the distance between the needle and the surface of the curing solution was 10 cm. The mixture was washed three times with sterile distilled water to obtain quaternary gel spheres loaded with *Lactobacillus plantarum*, denoted as A2.

[0035] Example 3 A pH-responsive Lactobacillus plantarum composite gel sphere (quaternary gel sphere: pectin + kelp polysaccharide + whey protein + calcium carbonate) was prepared using the following specific steps: (1) Culture of Lactobacillus plantarum.

[0036] In a clean bench, a single colony of *Lactobacillus plantarum* was picked and inoculated into MRS broth medium and incubated at 37°C for 24 hours; this was the first activation. The culture was then transferred to fresh MRS broth medium at a 1% (v / v) inoculation rate and incubated for another 24 hours under the same conditions to complete the second activation. The activated bacterial solution was then centrifuged at 8000×g for 10 minutes at 4°C. The bacterial pellet was washed twice with sterile distilled water and resuspended to obtain a free *Lactobacillus plantarum* stock solution with a bacterial concentration of approximately 2×10⁻⁶. 10 CFU / mL.

[0037] (2) Preparation of wall material solution.

[0038] 0.67 g of pectin and 0.33 g of kelp polysaccharide were dissolved in 33.33 mL and 16.67 mL of sterile distilled water, respectively, and stirred at 500 rpm at 50°C to prepare 2% (w / v) pectin and kelp polysaccharide solutions. The pectin and kelp polysaccharide solutions were then thoroughly mixed at a volume ratio of 2:1 and allowed to stand at room temperature for 30 min to eliminate air bubbles. 1 g of whey protein powder was then added to bring the final whey protein concentration to 2% (w / v), and the mixture was stirred for 1 h to ensure thorough mixing. Next, 0.075 g of calcium carbonate powder was added to bring the final concentration to 0.15% (w / v), and the mixture was stirred at 600 rpm for 2 h to ensure thorough mixing. The mixture was then allowed to stand at room temperature overnight to eliminate air bubbles, yielding the wall material solution.

[0039] (3) Encapsulation of Lactobacillus plantarum.

[0040] The *Lactobacillus plantarum* stock solution prepared in step (1) and the wall material solution prepared in step (2) were mixed at a volume ratio of 1:5 and stirred at 500 rpm for 2 hours until homogeneous. The mixture of *Lactobacillus plantarum* stock solution and wall material solution was then dripped into a curing solution (2% CaCl2 (w / v)) using a microfluidic injection pump for cross-linking and curing for 30 minutes (room temperature). The microfluidic injection pump was used at a flow rate of 4 mL / min, with a needle diameter of 0.45 mm and a distance of 15 cm between the needle and the surface of the curing solution. The mixture was washed three times with sterile distilled water to obtain quaternary gel spheres loaded with *Lactobacillus plantarum*, denoted as A3.

[0041] Example 4 A pH-responsive Lactobacillus plantarum composite gel sphere (quaternary gel sphere: pectin + kelp polysaccharide + whey protein + calcium carbonate) was prepared using the following specific steps: (1) Culture of Lactobacillus plantarum.

[0042] In a clean bench, a single colony of *Lactobacillus plantarum* was picked and inoculated into MRS broth medium and incubated at 37°C for 24 hours; this was the first activation. The culture was then transferred to fresh MRS broth medium at a 1% (v / v) inoculation rate and incubated for another 24 hours under the same conditions to complete the second activation. The activated bacterial solution was then centrifuged at 8000×g for 10 minutes at 4°C. The bacterial pellet was washed twice with sterile distilled water and resuspended to obtain a free *Lactobacillus plantarum* stock solution with a bacterial concentration of approximately 3×10⁻⁶. 10 CFU / mL.

[0043] (2) Preparation of wall material solution.

[0044] Dissolve 0.75g of pectin and 0.75g of kelp polysaccharide separately in 25mL of sterile distilled water, and stir at 500rpm at 50℃ to prepare 3% (w / v) pectin and kelp polysaccharide solutions respectively. Mix the pectin and kelp polysaccharide solutions thoroughly at a volume ratio of 1:1, stir well, and let stand at room temperature for 30min to eliminate air bubbles. Add 1.5g of whey protein powder to bring the final whey protein concentration to 3% (w / v), and stir at 600rpm for 1h to mix thoroughly. Add 0.1g of calcium carbonate powder (final concentration 0.2% (w / v)), stir at 600rpm for 2h to mix thoroughly, and let stand at room temperature overnight to eliminate air bubbles to obtain the wall material solution.

[0045] (3) Encapsulation of Lactobacillus plantarum.

[0046] The *Lactobacillus plantarum* stock solution prepared in step (1) and the wall material solution prepared in step (2) were mixed at a volume ratio of 1:5 and stirred at 500 rpm for 2 hours until homogeneous. The mixture of *Lactobacillus plantarum* stock solution and wall material solution was then dripped into a curing solution (2% CaCl2 (w / v)) using a microfluidic injection pump for cross-linking and curing for 30 minutes (room temperature). The injection speed of the microfluidic injection pump was 5 mL / min, the needle diameter was 0.8 mm, and the distance between the needle and the surface of the curing solution was 20 cm. The mixture was washed three times with sterile distilled water to obtain quaternary gel spheres loaded with *Lactobacillus plantarum*, denoted as A4.

[0047] Comparative Example 1 The specific steps for preparing a bacterial-carrying gel sphere that does not contain kelp polysaccharide, whey protein, or calcium carbonate are as follows: The preparation of the wall material solution in step (2) of Example 1 was modified as follows: 1g of pectin was dissolved in 50mL of sterile distilled water, stirred evenly at 50℃ and 500rpm to prepare a 2% (w / v) solution, and allowed to stand at room temperature overnight to eliminate bubbles, thus obtaining the wall material solution. The remaining steps were exactly the same as in Example 1, and the obtained bacterial-loaded gel spheres were named B1.

[0048] Comparative Example 2 The specific steps for preparing a bacterial-carrying gel sphere that does not contain kelp polysaccharides and calcium carbonate are as follows: The preparation of the wall material solution in step (2) of Example 1 was modified as follows: 1g of pectin was dissolved in 50mL of sterile distilled water and stirred at 500rpm at 50℃ to prepare a 2% (w / v) solution. The solution was then allowed to stand at room temperature for 30min to eliminate bubbles. 1.5g of whey protein powder was then added to bring the final whey protein concentration to 3% (w / v), and the solution was stirred at 500rpm for 1h to mix thoroughly. The solution was allowed to stand at room temperature overnight to eliminate bubbles, thus obtaining the wall material solution. The remaining steps were exactly the same as in Example 1, and the resulting bacterial-loaded gel spheres were named B2.

[0049] Comparative Example 3 The specific steps for preparing a bacterial-carrying gel sphere that does not contain calcium carbonate are as follows: The preparation of the wall material solution in step (2) of Example 3 was modified as follows: 0.67g of pectin and 0.33g of kelp polysaccharide were dissolved in 33.33mL and 16.67mL of sterile distilled water, respectively, and stirred evenly at 50℃ and 500rpm to prepare 2% (w / v) pectin and kelp polysaccharide solutions, respectively. The pectin solution and kelp polysaccharide solution were mixed at a volume ratio of 2:1, stirred evenly, and allowed to stand at room temperature for 30min to eliminate bubbles. Then, 1g of whey protein powder was added to make the final whey protein concentration 2% (w / v), and stirred at 500rpm for 1h to mix thoroughly. The mixture was allowed to stand at room temperature overnight to eliminate bubbles, thus obtaining the wall material solution. The remaining steps were exactly the same as in Example 3, and the obtained bacterial-loaded gel spheres were named B3.

[0050] Comparative Example 4 The specific steps for preparing an empty-loaded gel sphere are as follows: (1) Preparation of pectin-kelp polysaccharide-whey protein-calcium carbonate dispersion.

[0051] 0.67 g of pectin and 0.33 g of kelp polysaccharide were dissolved in 33.33 mL and 16.67 mL of sterile distilled water, respectively, and stirred at 500 rpm at 50°C to prepare 2% (w / v) pectin and kelp polysaccharide solutions. The pectin and kelp polysaccharide solutions were then mixed at a volume ratio of 2:1 and stirred thoroughly. The mixture was allowed to stand at room temperature for 30 min to eliminate air bubbles. Then, 1.5 g of whey protein powder was added to bring the final whey protein concentration to 3% (w / v), and the mixture was stirred at 500 rpm for 1 h to ensure thorough mixing. Next, 0.075 g of calcium carbonate powder was added (final concentration 0.15% (w / v)), and the mixture was stirred at 600 rpm for 2 h to ensure thorough mixing. The mixture was then allowed to stand at room temperature overnight to eliminate air bubbles, yielding a composite solution.

[0052] (2) Preparation of empty gel spheres.

[0053] The composite solution obtained in step (1) was dripped into the curing solution (2% CaCl2 (w / v)) using a microfluidic injection pump for cross-linking and curing for 30 min (room temperature). The injection rate of the microfluidic injection pump was 4 mL / min, the needle diameter was 0.45 mm, and the distance between the needle and the surface of the curing solution was 15 cm. The solution was washed three times with sterile distilled water to obtain empty gel spheres, denoted as B4.

[0054] Effect verification The encapsulation efficiency of *Lactobacillus plantarum*-loaded gel spheres prepared in Examples 1-4 and those prepared in Comparative Examples 1-3 were examined, along with the survival rate of probiotics during simulated gastric digestion, the release and survival rate of probiotics during simulated intestinal digestion, the survival rate of probiotics under simulated pasteurization conditions, the survival rate of probiotics during freeze-drying, and the in vitro pH-responsive release characteristics. Furthermore, to further illustrate the constipation-relieving effect of the *Lactobacillus plantarum*-loaded composite gel spheres on mice, the constipation-relieving effects of free *Lactobacillus plantarum* Lp, the empty gel sphere B4 prepared in Comparative Example 4, and the gel sphere A3 prepared in Example 3 on mice were compared. The specific steps are as follows: (a) Culture and counting of Lactobacillus plantarum.

[0055] Activation and culture of Lactobacillus plantarum: Lactobacillus plantarum frozen at -80℃ ( Lactiplantibacillus plantarum subsp. Plantarum Lactobacillus plantarum (hereinafter referred to as *Lactobacillus plantarum*) was cultured on MRS solid medium at 37°C for more than 48 hours using the streak plating method. Single colonies of *Lactobacillus plantarum* were then inoculated into MRS broth and incubated statically at 37°C for 24 hours; this was the first activation. The culture was then transferred to fresh MRS broth at a 1% (v / v) inoculation rate and cultured for another 24 hours under the same conditions to complete the second activation. The bacterial cells were then collected by centrifugation at 8000×g for 10 minutes at 4°C. The cells were washed twice with sterile distilled water, and the precipitate was resuspended in sterile distilled water to obtain a *Lactobacillus plantarum* suspension with a bacterial concentration of approximately 1-3 × 10⁻⁶. 10 CFU / mL.

[0056] Counting of *Lactobacillus plantarum*: Following the lactic acid bacteria counting method specified in GB 4789.35-2023 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination", the viable bacteria count in the *Lactobacillus plantarum* solution was determined using the plate count method. The specific procedure is as follows: Using a sterile pipette or micropipette, 1 mL of *Lactobacillus plantarum* solution was drawn and injected into a test tube containing 9 mL of sterile physiological saline or sterile distilled water. The mixture was thoroughly shaken to prepare a 1:10 homogenate. Then, based on the estimated concentration of the stock solution, a 10-fold serial dilution was prepared, using a different sterile pipette tip for each dilution. Based on the estimated bacterial concentration of the sample, 2-3 consecutive suitable dilutions were selected. 100 μL of each dilution was spread onto the surface of an MRS agar plate, with three replicates for each dilution. The plates were allowed to stand until the bacterial solution was absorbed, then inverted and incubated at 37°C for 72 h ± 2 h. After incubation, plates with colony counts between 15 and 150 CFU were selected for counting. The final results are expressed as the logarithm of colony-forming units (CFU) per milliliter of sample (Log CFU / mL).

[0057] (ii) Determination of the encapsulation rate of Lactobacillus plantarum.

[0058] Dissolve 2.0 g of gel beads in 18.0 mL of 10% sterile sodium citrate solution and stir at 300 rpm until the gel beads completely disintegrate and Lactobacillus plantarum is completely released. Measure the number of Lactobacillus plantarum in the gel beads using the method described in section (I). Spread 100 µL of the diluted solution onto an MRS agar plate and incubate at 37 °C for 72 h ± 2 h. Calculate the encapsulation efficiency (EE) using formula (1).

[0059] EE%=N'' / N0'' 100(1), Where EE is the encapsulation efficiency (%), N'' is the number of live cells released from the gel beads (Log CFU / mL), and N0'' is the initial number of live cells used for encapsulation (Log CFU / mL).

[0060] The results are shown in Table 1. Compared with the single polysaccharide system, the addition of whey protein, kelp polysaccharide, and calcium carbonate all improved the encapsulation efficiency of the encapsulation system to varying degrees. Overall, the quaternary system (pectin + kelp polysaccharide + whey protein + calcium carbonate) showed a higher encapsulation efficiency for *Lactobacillus plantarum* than the ternary system (pectin + kelp polysaccharide + whey protein), the ternary system showed a higher encapsulation efficiency for *Lactobacillus plantarum* than the binary system (pectin + whey protein), and the binary system showed a higher encapsulation efficiency for *Lactobacillus plantarum* than the single polysaccharide system (pectin). In the quaternary encapsulation system, the gel beads achieved the highest encapsulation efficiency for *Lactobacillus plantarum* (98.89 ± 0.02%) when the volume ratio of pectin solution to kelp polysaccharide solution was 2:1 (corresponding to A3).

[0061] Table 1. Statistical results of encapsulation efficiency of different gel beads for Lactobacillus plantarum

[0062] (III) Determination of the survival rate of Lactobacillus plantarum in simulated gastric juice in bacterial-loaded gel spheres.

[0063] Dissolve 3.0 g of pepsin in 1000 mL of sterile distilled water, and adjust the pH to 2.0 with 1 M HCl to obtain simulated gastric juice (SGF). Weigh 2 g of gel beads and add them to 18 mL of SGF, then simulate gastrointestinal motility at 37ºC and 100 rpm. Use an equal amount of free Lactobacillus plantarum as a control.

[0064] After digestion in SGF for 2 hours, the gel beads were removed and 18 mL of 10% sterile sodium citrate solution was added. The gel beads lysed to release the *Lactobacillus plantarum*, which was then serially diluted with sterile physiological saline. The number of *Lactobacillus plantarum* was measured using the method in (I). 100 µL of the diluted solution was spread onto MRS agar plates and incubated at 37 °C for 72 h ± 2 h. The survival rate (SR) in the stomach was calculated using formula (2).

[0065] SR%=N / N0 100 (2), Wherein, SR is the survival rate (%), N is the number of live Lactobacillus plantarum after simulated gastric digestion (Log CFU / mL), and N0 is the number of Lactobacillus plantarum in the gel spheres before simulated digestion (Log CFU / mL).

[0066] The results are as follows Figure 1As shown, the survival rate of free *Lactobacillus plantarum* (Lp) after 2 hours of simulated gastric digestion was only 35.91%. Encapsulation significantly improved the survival rate of Lp. Comparing B1, B2, and B3, it was found that the addition of whey protein and kelp polysaccharides significantly enhanced the protective effect of gel beads on Lp during simulated gastric digestion. Compared with B1, B2, and B3, gel beads A1, A2, A3, and A4 showed more significant protective effects on Lp, indicating that the introduction of calcium carbonate can greatly enhance the gel beads' resistance to gastric acid digestion. Furthermore, the protective effect increased with increasing kelp polysaccharide content.

[0067] (iv) Determination of the release and survival rate of Lactobacillus plantarum in simulated intestinal fluid from gel beads.

[0068] The simulated intestinal fluid (SIF) contained 1.0% (w / v) trypsin, 0.05% (w / v) sodium cholate, 0.05% (w / v) sodium deoxycholate, and 0.68% (w / v) KH2PO4. The pH of the SIF was adjusted to 6.8 using sterile NaOH solution. The sample digested in the simulated gastric juice was transferred to the SIF, and then intestinal peristalsis was simulated at 37ºC and 100rpm. After digestion for 2 hours, the release rate (RR) of Lactobacillus plantarum after digestion in the simulated intestinal juice was calculated using formula (3).

[0069] RR%=N1 / N0 100 (3), Wherein, RR is the release survival rate (%), N1 is the number of live Lactobacillus plantarum released into the simulated intestinal fluid after digestion (Log CFU / mL), and N0 is the number of Lactobacillus plantarum in the gel spheres before digestion (Log CFU / mL).

[0070] The results are as follows Figure 2 As shown, gel spheres A1, A2, A3, and A4 not only exhibit good resistance to gastric acid digestion but also effectively target and release *Lactobacillus plantarum* (Lp) into the intestine, with an Lp release survival rate exceeding 80%. Furthermore, the Lp release survival rate increases with the increase in the amount of kelp polysaccharide added. In contrast, gel spheres B1, B2, and B3, which did not contain added calcium carbonate, showed a significantly reduced Lp release survival rate after 2 hours of simulated intestinal digestion.

[0071] (v) In vitro pH-responsive release experiment of bacterial-loaded gel balls.

[0072] 2g of gel beads were placed in centrifuge tubes containing 20mL of sterile physiological saline buffer at specific pH values ​​(2.0, 4.0, 5.5, 6.8, 7.4), and the samples were incubated at 37°C and 100rpm with shaking. Samples were taken for testing after incubation at pH 2.0 for 120 min; subsequently, the buffer was changed sequentially to pH 4.0, 5.5, 6.8, and 7.4, and samples were taken for testing at 60, 120, and 180 min after each change. The number of viable bacteria released at each sampling point was determined using the plate count method described in (I), and the cumulative release rate relative to the initial total number of bacteria in the gel beads was calculated. Results are expressed as mean ± standard deviation of three parallel samples.

[0073] The results are as follows Figure 3 As shown, gel beads A1, A2, A3, and A4 all exhibit significant pH-targeted release and disintegration characteristics, demonstrating a clear response pattern of "low pH protection, high pH release": In a simulated gastric acid environment (pH 2.5-4.5), the release amount of all gel beads remains at an extremely low level, effectively protecting the embedded bacteria from strong acid damage, achieving "targeted protection." In an intestinal environment (pH 6.0-7.5), the release amount of all gel beads increases sharply, indicating that they can respond rapidly and disintegrate under the target pH environment, achieving "targeted release of Lactobacillus plantarum." Based on these shared targeting characteristics, gel bead A3 performed the best.

[0074] (vi) Stability of bacterial-loaded gel spheres.

[0075] (1) Thermal stability.

[0076] Take 2.0g of gel beads and 2.0mL of Lactobacillus plantarum suspension (bacterial concentration approximately 1×10⁻⁶). 10 The sample (CFU / mL) was placed in 5 mL of 0.85% sterile physiological saline (preheated to 85ºC) and treated at 85ºC for 1 min. Then, the sample was cooled in an ice-water bath for 10 min, the physiological saline was discarded, and the sample was dissolved in 18.0 mL of sterile 10% sterile sodium citrate solution. The sample was uncapsulated at 240 rpm and 37ºC for 30 min. After the complete release of *Lactobacillus plantarum*, the number of *Lactobacillus plantarum* in the gel beads was measured using the standard plate count method. The stability of unencapsulated and encapsulated *Lactobacillus plantarum* under simulated pasteurization conditions was evaluated. The survival rate of free and encapsulated *Lactobacillus plantarum* after simulated pasteurization was calculated using formula (4).

[0077] SR%=N1 / N2 100 (4), Wherein, SR is the survival rate (%), N1 is the number of viable bacteria after simulated pasteurization heat treatment (Log CFU), and N2 is the number of viable bacteria before simulated pasteurization heat treatment (Log CFU).

[0078] The results are as follows Figure 4 As shown, the survival rate of free *Lactobacillus plantarum* (Lp) after simulated pasteurization heat treatment was only 45.46%. After encapsulation, the survival rate of Lp in the gel spheres was significantly improved. Comparison of the thermal stability data of gel spheres B1, B2, and B3 revealed that the addition of whey protein and kelp polysaccharide significantly improved the thermal stability of the gel spheres. Overall, the thermal stability of gel spheres A1, A2, A3, and A4 was better than that of gel spheres B1, B2, and B3, indicating that the introduction of calcium carbonate can significantly improve the protective effect of the gel system on *Lactobacillus plantarum*, with gel sphere A3 exhibiting the best thermal stability.

[0079] (2) Freeze-drying stability.

[0080] 2.0g of gel beads were dried in a freeze dryer for 48h. The freeze-dried sample was collected and the viable number of Lactobacillus plantarum in the freeze-dried gel beads was determined by the method in (I). The survival rate of Lactobacillus plantarum after freeze-drying was calculated by formula (5).

[0081] SR%=N' / N0' 100 (5), Wherein, SR is the survival rate (%), N′ is the number of viable bacteria after freeze-drying (Log CFU), and N0′ is the number of viable bacteria before freeze-drying (Log CFU).

[0082] The results are as follows Figure 5 As shown, freeze-drying of bacterial-loaded gel spheres for 48 hours increased the survival rate of *Lactobacillus plantarum* in B1, B2, and B3 by 54.31%, 56%, and 59.25%, respectively, compared to free *Lactobacillus plantarum*. Encapsulation significantly improved the survival rate of *Lactobacillus plantarum* after freeze-drying. Gel spheres with added calcium carbonate showed even higher *Lactobacillus plantarum* survival rates after freeze-drying, with gel sphere A3 showing the best results.

[0083] (vii) The effect of bacterial-loaded gel balls on relieving constipation induced by loperamide hydrochloride in mice.

[0084] (1) Construction of a mouse model of constipation and intervention of drugs in each group.

[0085] Sixty male Babl / c mice (6-8 weeks old, 20±2g) were randomly divided into four groups (n=10 per group) after 7 days of acclimatization: a blank control group, a constipation model group, a positive control group (mosapride group), a free bacteria group, an empty gel ball group (B4, prepared from Comparative Example 4), and a bacterial gel ball group (A3, prepared from Example 3). From day 7 to 14, the model group, positive control group, and experimental groups (free bacteria group, empty gel ball group, and bacterial gel ball group) were subjected to constipation modeling. Each mouse was administered 10 mg / kg / day of loperamide hydrochloride by gavage, while the blank control group was administered an equal volume of physiological saline by gavage. This treatment continued for 7 days. From day 14 to 28, the model group, positive control group, and experimental group were administered 10 mg / kg / day of loperamide hydrochloride by gavage, while the blank control group was administered an equal volume of physiological saline by gavage. Half an hour later, the mice in the free bacteria group, empty gel ball group, and bacterial gel ball group were administered free Lactobacillus plantarum bacterial solution (1×10⁻⁶) by gavage. 8 CFU / animal / day, empty gel (300 mg / kg / day), and bacterial-loaded gel (1 × 10⁻⁶). 8 The mice were administered mosapride solution (10 mg / kg / day) via gavage to the positive control group, while the model group and blank control group were administered physiological saline via gavage to the same volume. Mice had free access to food and sterile distilled water during this period.

[0086] (2) Mouse defecation experiment.

[0087] On day 28, a defecation experiment was conducted in mice. The experimental method was as follows: Mice were fasted for 16 hours the night before, but allowed free access to water. Except for the blank control group, all other groups were administered 10 mg / kg loperamide hydrochloride by gavage. The blank control group was administered an equal volume of physiological saline by gavage. 0.5 hours later, the blank control group and the model group were administered activated charcoal juice by gavage, while the positive control group, free bacteria group, empty gel ball group, and bacterial gel ball group were administered activated charcoal juice containing the corresponding sample by gavage. Timing was started immediately after gavage. The mice were placed in metabolic cages and allowed to resume a normal diet. The time of the first black stool excreted by each mouse was observed and recorded. Feces were collected within 6 hours, and the number of excreted particles was recorded. The statistical results of the time of the first black stool excreted by constipated mice are as follows: Figure 6 As shown in the figure, the results of the fecal particle count are as follows: Figure 7 As shown.

[0088] The time it takes for the first black stool to pass is one of the most direct ways to assess the speed of intestinal peristalsis. Figure 6 It can be seen that, compared with the control group, the time to the appearance of the first black stool was significantly prolonged in the model group. p <0.05). However, after intervention with positive control drugs, free bacteria, B4 gel balls, and A3 gel balls, the time to the first black stool was significantly shortened compared to the model group. Among them, the positive control group and the A3 gel group showed the most significant effects, with no statistically significant difference.

[0089] The number of fecal particles within 6 hours is a characterizing indicator of fecal volume in mice. After modeling with loperamide hydrochloride, the number of fecal particles significantly decreased. p <0.05, Figure 7 ), while after intervention with positive drugs and A3 gel balls, the number of fecal particles increased significantly ( p <0.05), while free bacteria and B4 gel balls had no significant effect on the number of fecal particles.

[0090] (3) Small intestine motility experiment.

[0091] Thirty minutes after mice were modeled by gavage with loperamide, the blank control group and the model group were gavage with activated charcoal juice, while the positive control group, the free bacteria group, the empty gel ball group, and the bacterial gel ball group were gavage with activated charcoal juice containing the corresponding sample. Each mouse was gavage with 0.2 mL. Twenty-five minutes after administration of activated charcoal juice, the mice were euthanized by cervical dislocation, and the intestinal tract from the pylorus to the cecum was quickly removed and straightened without changing the length of the intestine. The intestinal propulsion rate was calculated according to formula (6).

[0092] Intestinal propulsion rate = L1 / L0 100 (6), Where L1 is the length of activated charcoal juice propulsion in cm; L0 is the length from the pylorus to the cecum in cm.

[0093] The results are as follows Figure 8 As shown. Small intestinal propulsion rate is also one of the intuitive methods for assessing fecal propulsion speed. Under the modeling effect of loperamide hydrochloride, the small intestinal propulsion rate of mice in the model group was significantly reduced ( p <0.05), while after intervention with positive drugs, free bacteria, B4 gel balls, and A3 gels, the small intestinal propulsion rate significantly increased ( p <0.05). Among them, the positive drug and A3 gel balls showed the most significant effects, with no significant difference.

[0094] Therefore, the pH-responsive Lactobacillus plantarum gel spheres provided by this invention have a dense gel network structure that can resist gastrointestinal digestion and significantly improve the survival rate of probiotics in gastrointestinal fluid; they have a targeted release effect in the intestine; they contain high-quality prebiotics, which have a synergistic effect with probiotics, and are significantly more effective than free probiotics and empty gels in relieving constipation; the preparation method is simple and easy to operate, the reagents and materials used are highly safe, the preparation process does not use toxic or harmful reagents, and no toxic or harmful substances are generated, making it environmentally friendly and without any safety hazards.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A pH-responsive Lactobacillus plantarum gel sphere, its preparation method and application, characterized in that: The gel spheres are made with Lactobacillus plantarum as the core material and pectin, kelp polysaccharide and whey protein as composite wall materials, and are formed by double ionic cross-linking and curing through internal calcium carbonate and external calcium chloride solutions.

2. The preparation method of pH-responsive Lactobacillus plantarum gel spheres as described in claim 1, comprising the following steps: (1) After activating Lactobacillus plantarum, the bacterial concentration was adjusted with sterile distilled water to obtain free Lactobacillus plantarum stock solution; (2) To prepare the wall material solution, dissolve pectin and kelp polysaccharide in sterile distilled water, then mix them evenly in a certain volume ratio, add whey protein powder, mix well, add calcium carbonate powder, stir and mix well, and then eliminate bubbles to obtain the wall material solution. (3) Encapsulation of Lactobacillus plantarum: The free Lactobacillus plantarum mother liquor prepared in (1) is mixed evenly with the wall material solution prepared in (2), and then dripped into the curing liquid by a microfluidic injection pump for cross-linking and curing, thus obtaining a pH-responsive Lactobacillus plantarum gel ball.

3. The method for preparing pH-responsive Lactobacillus plantarum gel spheres according to claim 2, characterized in that: In step (1), Lactobacillus plantarum was activated using MRS broth medium and cultured at 37°C. Before adjusting the bacterial concentration with sterile distilled water, the bacteria were centrifuged at 8000×g for 10-15 minutes at 4°C and washed 2-3 times with sterile distilled water.

4. The method for preparing pH-responsive Lactobacillus plantarum gel spheres according to claim 3, characterized in that, MRS broth medium consists of the following components per liter: 10.0 g casein digest, 10.0 g beef extract, 4.0 g yeast extract, 2.0 g triammonium citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate (MgSO4·7H2O), 0.05 g manganese sulfate (MnSO4·4H2O), 2.0 g dipotassium hydrogen phosphate, 20.0 g glucose, and 1.08 g Tween-80 (final pH 5.7 ± 0.2).

5. The method for preparing pH-responsive Lactobacillus plantarum gel spheres according to claim 2, characterized in that: The adjusted bacterial concentration is 1-3 × 10⁻⁶ free *Lactobacillus plantarum*. 10 CFU / mL.

6. The method for preparing pH-responsive Lactobacillus plantarum gel spheres according to claim 2, characterized in that: In (2), the pectin contains ≥74.0% galacturonic acid (dry basis), and the kelp polysaccharide has a molecular weight of 10-260 kDa and a total sugar content of 62-78%.

7. The method for preparing pH-responsive Lactobacillus plantarum gel spheres according to claim 2, characterized in that: In the (2) ingredient, the mass-volume ratio of pectin, kelp polysaccharide, whey protein powder, calcium carbonate powder, and sterile distilled water is 1g:1g:1g:0.1g:100mL-3g:3g:3g:0.2g:100mL. The bubbles are eliminated by letting the mixture stand at room temperature.

8. The method for preparing pH-responsive Lactobacillus plantarum gel spheres according to claim 2, characterized in that: The volume ratio of the free Lactobacillus plantarum mother liquor to the wall material solution in (3) is 1:

5.

9. The method for preparing pH-responsive Lactobacillus plantarum gel spheres according to claim 2, characterized in that, The method of cross-linking and curing in (3) by dripping the microfluidic injection pump into the curing liquid is as follows: the advance speed of the microfluidic injection pump is 0.5-5 mL / min, the needle diameter is 0.2-0.8 mm, and the distance between the needle and the surface of the curing liquid is 10-20 cm; cross-linking and curing are carried out at room temperature.

10. The use of the pH-responsive Lactobacillus plantarum gel balls as described in claim 1 in the treatment and / or relief of constipation.

Images