A probiotic culture core ball prepared by polysaccharide confinement and its application

Abstract

This invention discloses a probiotic liquid core ball cultured under polysaccharide confinement, its preparation method, and its application in improving children's intestinal flora. Belonging to the field of probiotic preparation technology, it employs an extrusion method combined with calcium alginate cross-linking technology, using xanthan gum and other thickening polysaccharides to optimize the types and concentrations of polysaccharides to construct the confinement space. The effects of different oxygen conditions on the encapsulation of different proportions of *Lactobacillus rhamnosus* LRa05 and *Bifidobacterium animalis* BLa80 were investigated to obtain the optimal probiotic liquid core ball cultured under polysaccharide confinement. This process is simple, uses safe raw materials, and after confinement culture, the viable bacteria count is ≥10⁹ CFU / g. It can directionally increase the abundance of beneficial bacteria in children's intestines, inhibit opportunistic pathogens, enhance flora diversity and stability, and is suitable for children's dietary scenarios, providing a safe and efficient intestinal flora regulation solution.

Description

Technical Field

[0001] This invention belongs to the field of probiotic preparation technology, specifically relating to a probiotic liquid core ball cultured through polysaccharide confinement, its preparation method, and its application. Background Technology

[0002] Children aged 2-6 are in a critical period of gut microbiota development. Their microbiota structure is similar to that of adults, but their stability is extremely poor. Their intestinal mucosal barrier and immune-microbiota interaction system are not yet mature, making them susceptible to microbiota imbalance caused by factors such as diet and environment, leading to problems such as diarrhea, allergies, and recurrent infections. Exogenous probiotic supplementation has become a key way to regulate the balance of children's gut microbiota.

[0003] Traditional probiotic preparations often employ direct addition or simple encapsulation techniques. However, most probiotics are anaerobic or facultative anaerobic bacteria, which are sensitive to oxygen. In normoxic environments, the accumulation of reactive oxygen species can easily damage the bacteria, reducing their survival and functional activity. This makes them prone to inactivation during production and storage. Furthermore, the lack of a suitable microenvironment for growth results in insufficient live bacteria reaching the children's intestines and limited secretion of metabolites. In addition, the physiological characteristics of the children's intestines differ significantly from those of adults, making traditional preparations insufficiently targeted and unable to meet the needs for efficient regulation.

[0004] Polysaccharide confined culture technology is a form of cell confined culture, providing an innovative approach to address the technical challenges of probiotic preparations for children. This technology constructs closed or semi-closed confined microspaces using natural and safe polysaccharide-based materials, encapsulating probiotics within to form a stable micro-ecosystem. This provides a sanctuary for probiotics, free from external oxygen, gastric acid, and bile salt interference, while also enabling high-density aggregation and proliferation of probiotics through spatial confinement.

[0005] Therefore, how to prepare a probiotic preparation for children using polysaccharide confined culture technology, and solve the technical defects of existing probiotic preparations such as low viable bacteria count, low secretion of metabolites, and poor regulation efficiency of children's intestinal flora, which prevents industrial application, is an urgent problem to be solved. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing probiotic liquid core balls through polysaccharide confinement culture.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: including, Probiotic suspension diluted to 10 6 ~10 8 After CFU / mL, it is mixed with polysaccharide thickening core material solution at a volume ratio of 1:10~30, and then dripped into sodium alginate crosslinking solution by extrusion. Crosslinking is carried out at room temperature. Primary liquid core balls are collected and hardened in calcium chloride solution to obtain hardened liquid core balls. After hardening, the liquid core balls were transferred into MRS broth and cultured at a constant temperature under 0-10% O2 concentration conditions. After the culture was completed, they were taken out and washed to obtain probiotic liquid core balls cultured through polysaccharide confinement. The polysaccharide concentration in the polysaccharide-thickened core liquid is 0.3~0.9% w / v.

[0010] As a preferred embodiment of the method for preparing probiotic liquid core balls by polysaccharide confinement culture according to the present invention, wherein: the probiotics are selected from Lactobacillus rhamnosus LRa05 ( Lacticaseibacillus rhamnosus LRA05 Bifidobacterium animalis BLa80 ( Bifidobacterium animalis subsp. lactis BLa80 One or two of them.

[0011] As a preferred embodiment of the method for preparing probiotic liquid core balls by polysaccharide confinement culture according to the present invention, wherein the probiotics are Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80 in a mass ratio of 1~2:1~2.

[0012] As a preferred embodiment of the method for preparing probiotic liquid core balls by polysaccharide confinement culture according to the present invention, wherein the polysaccharide is selected from xanthan gum, hyaluronic acid, guar gum, and locust bean gum.

[0013] As a preferred embodiment of the method for preparing probiotic liquid cores by polysaccharide confinement culture according to the present invention, the method for preparing the probiotic suspension includes culturing probiotics to the logarithmic phase, collecting the bacterial cells by centrifugation, washing with PBS buffer and resuspending to obtain the probiotic suspension.

[0014] As a preferred embodiment of the method for preparing probiotic liquid core balls by polysaccharide confinement culture according to the present invention, wherein: the polysaccharide thickening core liquid further includes calcium chloride, and the concentration of calcium chloride is 1.0~2.5% w / v.

[0015] In a preferred embodiment of the method for preparing probiotic liquid core balls by polysaccharide confinement culture according to the present invention, the concentration of sodium alginate in the sodium alginate crosslinking solution is 0.5~0.8% w / v, and the concentration of calcium chloride in the calcium chloride solution is 1.0~2.5% w / v.

[0016] As a preferred embodiment of the method for preparing probiotic liquid core balls by polysaccharide confinement culture according to the present invention, the cross-linking time at room temperature is 2-4 min, the hardening time is 8-12 min, the culture temperature for isothermal culture is 36-38℃, and the culture time is 20-24 h.

[0017] Another object of the present invention is to provide a probiotic liquid core ball cultured by polysaccharide confinement.

[0018] Another object of the present invention is to provide an application of probiotic liquid cores cultured in a polysaccharide confinement environment in improving the gut microbiota of children, the application including the preparation of foods or dietary supplements for regulating the gut microbiota of children.

[0019] Beneficial effects of this invention: (1) This invention addresses the core characteristics of poor intestinal flora stability and immune sensitivity in children aged 2-6 years, achieving precise and efficient regulation of the intestinal flora. Through the polysaccharide-restricted culture-constructed micro-ecosystem, probiotics and metabolites work synergistically to significantly increase the abundance of beneficial bacteria such as Bifidobacterium and Lactobacillus in the children's intestines, effectively inhibiting the proliferation of opportunistic pathogens such as Klebsiella and Streptococcus. Simultaneously, this liquid-core pellet optimizes the structure of the children's intestinal flora, enhances α-flora diversity and stability, improves the balance of the intestinal microecology, and specifically addresses problems such as diarrhea, allergies, and recurrent infections caused by flora imbalance in children, meeting the needs of children's intestinal development.

[0020] (2) The polysaccharide confined culture technology of the present invention provides key support for the effect of microbial community regulation. The closed microenvironment constructed by it can not only protect probiotics from external interference and greatly improve the survival rate during production, storage and intestinal colonization, but also achieve high-density aggregation and growth of probiotics, so that the viable count of liquid core pellets is ≥10 9 The CFU / g concentration is significantly higher than that of traditional formulations. Simultaneously, the confined space and low-oxygen environment enhance interactions between probiotics, inducing the secretion of more extracellular polymers, short-chain fatty acids, and other beneficial metabolites. These metabolites further strengthen the intestinal mucosal barrier function and synergistically amplify the gut microbiota regulation effect with probiotics. Furthermore, the polysaccharide raw materials used in this technology are natural and safe, the process is stable and easily industrialized, and the probiotics used meet the relevant requirements of the "List of Strains that can be Used in Food" and the "List of Strains that can be Used in Infant Food." The prepared liquid-core pellets are suitable for children's dietary scenarios, providing a safe and efficient probiotic formulation solution for children's gut health. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. 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. Wherein: Figure 1 The apparent state of the primary liquid core spheres prepared in Example 2 of the present invention with xanthan gum concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5% (w / v) are shown.

[0022] Figure 2 The core liquid prepared by Comparative Example 2 of the present invention with 0.3%, 0.6%, and 0.9% (w / v) pectin (P) in the static state. Detailed Implementation

[0023] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0024] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0025] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0026] Unless otherwise specified, all raw materials used in this invention are commercially available in the field.

[0027] In a specific embodiment of the present invention, MRS broth, fermentation medium, 0.9% physiological saline and other experimental supplies were autoclaved at 121°C for 15 minutes before the experiment.

[0028] Example 1 This embodiment provides a method for culturing probiotic core pellets using polysaccharide confinement, specifically: 1) Preparation of probiotic suspension: Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80 were cultured to the logarithmic phase, and the bacterial cells were collected by centrifugation (4℃, 5000 g, 10 min). After washing twice with PBS buffer, the bacterial suspension was resuspended to obtain the probiotic suspension. 2) Core solution preparation: Xanthan gum (XG) and calcium chloride are dissolved in deionized water to obtain a polysaccharide thickening core solution with a xanthan gum concentration of 0.5% (w / v) and a calcium chloride concentration of 2.0% (w / v); 3) Preparation of cross-linking solution: Sodium alginate was dissolved in deionized water and allowed to stand to degas, resulting in a sodium alginate cross-linking solution with a concentration of 0.6% (w / v). 4) Dilute the two probiotic suspensions to 10 g / L respectively. 7 CFU / mL was mixed at a 1:1 ratio and then mixed with a polysaccharide thickening core solution at a 1:20 volume ratio of bacterial solution (probiotic suspension and polysaccharide thickening core solution). The mixture was then dripped into a 0.6% (w / v) sodium alginate crosslinking solution by extrusion and crosslinked at room temperature for 2 min. The primary liquid core balls were collected and hardened in a 2.0% (w / v) calcium chloride solution for 10 min to obtain hardened liquid core balls. 5) Restricted culture: The hardened liquid core balls were transferred into MRS broth and cultured at 37°C for 24 h in a 5% O2 (hypoxic incubator). After washing, the probiotic liquid core balls cultured by polysaccharide restriction in this embodiment were obtained.

[0029] Example 2 The difference between this embodiment and Embodiment 1 is that the concentration of xanthan gum in the polysaccharide thickening core liquid in step 2) is adjusted to 0.1, 0.2, 0.3, 0.4, and 0.5% (w / v), respectively. The remaining steps are the same as in Embodiment 1, resulting in probiotic liquid core balls with different xanthan gum concentrations.

[0030] Figure 1 The appearance of primary liquid core balls prepared with xanthan gum at different concentrations shows that liquid core balls formed with xanthan gum at too low concentrations have poor sphericity, which is not conducive to the subsequent growth of probiotics in the confined space and further applications. Liquid core balls prepared with 0.5% (w / v) xanthan gum as a thickening polysaccharide have the best morphology.

[0031] Example 3 The difference between this embodiment and Example 1 is that the 0.5% (w / v) xanthan gum (0.5% XG) in the polysaccharide thickening core solution in step 2) is replaced with 0.4% (w / v) xanthan gum (0.4% XG), 0.5% (w / v) hyaluronic acid (0.5% HA), 0.8% (w / v) hyaluronic acid (0.8% HA), 0.5% (w / v) guar gum (0.5% GG), 0.8% (w / v) guar gum (0.8% GG), 0.5% (w / v) locust bean gum (0.5% LBG), and 0.8% (w / v) locust bean gum (0.8% LBG). The remaining steps are the same as in Example 1. Core solutions and corresponding probiotic core balls with different polysaccharide types and concentrations were obtained in this embodiment. The viscosity of the core solution and the number of bacteria in the core balls after confined culture were measured. The results are shown in Table 1.

[0032] Table 1 As shown in Table 1, the probiotics cultured in confined space with polysaccharides prepared with 0.5% (w / v) xanthan gum had the highest bacterial count, which was most conducive to the growth of probiotics inside. Correspondingly, the viscosity of the core solution with 0.5% (w / v) xanthan gum was the highest at near-zero shear rate, i.e., at simulated rest, which was 1.42 Pa·s. This indicates that the viscosity of the core solution and different types of thickening polysaccharides also affect the survival of probiotics under confined space culture. A core solution with higher viscosity may be more conducive to the growth of probiotics inside.

[0033] Example 4 The difference between this embodiment and Example 1 is that the mass ratio of Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80 after dilution in step 4) is adjusted to 0:1 (pure BLa80), 1:0 (pure LRa05), 1:1, 1:2, and 2:1, respectively. The remaining steps are the same as in Example 1, and the confined culture medium core balls with different proportions of probiotics prepared under low-oxygen confined culture conditions in this embodiment are obtained.

[0034] Example 5 The difference between this embodiment and Embodiment 1 is that: Adjust the mass ratio of Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80 after dilution in step 4) to 0:1 (pure BLa80), 1:0 (pure LRa05), 1:1, 1:2, and 2:1, respectively. Adjust the oxygen conditions for confined culture in step 5) to 0% O2 (anaerobic workstation); The remaining steps are the same as in Example 1, and the confined culture medium core balls with different proportions of probiotic mixtures prepared under anaerobic confined culture conditions in this example are obtained.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that: Adjust the mass ratio of Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80 after dilution in step 4) to 0:1 (pure BLa80), 1:0 (pure LRa05), 1:1, 1:2, and 2:1, respectively. Adjust the oxygen conditions for confined culture in step 5) to 21% O2 (normative oxygen incubator); The remaining steps were the same as in Example 1, and the confined culture medium core balls of different proportions of probiotic mixtures prepared under normoxic confined culture conditions were obtained in this comparative example.

[0036] The number of surviving bacteria (logCFU / g) and the extracellular protein production (mg / g) after culturing the confined culture medium core balls prepared in Examples 4, 5 and Comparative Example 1 were determined, and the results are shown in Tables 2 and 3.

[0037] Table 2 Table 2 shows that under different oxygen conditions, the number of surviving bacteria in the hypoxia and anaerobic groups was significantly higher than that in the normoxic group (p<0.05). Under hypoxia, all groups with different ratios had the highest number of bacteria. This may be because the hypoxia environment avoids the stress damage of normoxic conditions and activates the metabolism of probiotics through moderate stimulation, making it more suitable for the growth of the two types of probiotics. The comparison of different mixing ratios shows that the 1:1 mixing group had a surviving number of 9.72±0.12 logCFU / g under hypoxia, which was significantly higher than that of the pure bacteria group and other mixing ratios (p<0.05). This indicates that there may be a synergistic growth effect when LRa05 and BLa80 are co-cultured at a 1:1 ratio, which can reduce the stress restriction of single bacterial species and promote the high-density proliferation of probiotics in confined spaces.

[0038] Table 3 Table 3 shows the extracellular protein production, which was determined using a non-interfering protein concentration assay kit. The extracellular protein production in the hypoxia group was significantly higher than that in the anaerobic and normoxic groups. The hypoxia group, with a 1:1 mixture, exhibited the highest extracellular protein secretion (12.8 ± 0.9 mg / g). This indicates that a hypoxic environment specifically stimulates probiotic metabolism. Combined with the interaction between the two probiotic communities, this can promote the synthesis of extracellular proteins such as adhesion proteins and EPS. These proteins can form a protective membrane, enhancing the colonization stability of probiotics and strengthening the intestinal mucosal barrier.

[0039] Example 6 This embodiment is used to verify the effect of the probiotic liquid core pellets obtained by polysaccharide confinement culture of the present invention on improving the intestinal flora of children. After the probiotic liquid core pellets were crushed, an in vitro fermentation experiment was carried out with the collected feces of 10 children. Specifically: 1) Preparation of fecal suspension: Fecal samples from ten children were mixed evenly by weight to reduce individual differences. The mixture was added to PBS solution at pH 7.4 at a ratio of 1:9 and vortexed to mix. The mixture was then filtered through 8 layers of sterile gauze to remove large particulate impurities from the feces. The filtrate was collected and inoculated within 2 hours.

[0040] 2) Preparation of fermentation medium: Prepare the fermentation medium according to the formula in Table 4. After mixing, adjust the pH to 7.0 with 0.5 mol / L HCl, autoclave at 121℃ for 15 min, and cool before use.

[0041] Table 4. Fermentation medium formulation (g / L) 3) Anaerobic fermentation: The fermentation medium was mixed with the fecal suspension at a ratio of 4:1. Then, 0.5 mL of a 1:1 mixture of *Lactobacillus rhamnosus* LRa05 and *Bifidobacterium animalis* BLa80 (naked bacteria) and 0.5 g of ruptured probiotics cultured in confinement with different polysaccharides were added (under both anaerobic and hypoxic conditions) (probiotic content was 10 g / L). 9 (Approximately CFU), and a blank group was set up. Each group had three replicates. After co-culturing and fermenting at 37℃ in an anaerobic workstation for 24 h, the samples were taken out and centrifuged at 10000 rpm for 10 min at 4℃. The precipitate was then used for 16S rRNA amplicon sequencing analysis. The results are shown in Table 5.

[0042] Table 5 As shown in Table 5, the probiotic liquid core pellets prepared with different polysaccharides and concentrations exhibited significantly different effects on intestinal flora regulation. The 0.5% XG group showed the best performance in enriching beneficial bacteria such as Bifidobacterium, inhibiting pathogenic bacteria such as Pseudomonas, and improving flora diversity. This may be because the 0.5% XG core material had the highest viscosity, resulting in liquid core pellets with good morphology and stable structure, and the highest number of viable probiotics among all groups. It also promoted high-density aggregation and synergistic interaction of LRa05 and BLa80, enhanced bacterial metabolic activity, secreted more beneficial metabolites, and strengthened colonization and flora regulation effects. Other polysaccharides and 0.4% XG were less effective due to insufficient viscosity and could not achieve the same intestinal flora regulation effect.

[0043] Table 6 The changes in the gut microbiota structure of children were simulated by in vitro fecal fermentation. As shown in Table 6, from the perspective of microbiota structure optimization, the 1:1 mixed microbiota group in polysaccharide-confined hypoxia culture can effectively inhibit the proliferation of potential pathogens. The abundance of Pseudomonas was only 5.1±0.7%, which was significantly lower than that of other groups. The abundance of conditionally pathogenic bacteria such as Klebsiella and Streptococcus also decreased to 2.9±0.5% and 2.4±0.4%, respectively, minimizing the risk of intestinal infection in children. At the same time, the abundance of Bifidobacterium reached 15.2±1.0%, Lactobacillus reached 5.8±0.5%, and Faecalibacterium reached 6.3±0.6%, which were the highest levels among all groups. In addition, the Chao1 index and Shannon index were significantly improved, reflecting better microbiota richness and evenness. This may be because the closed microenvironment constructed by polysaccharides reduces the damage of harmful substances in the gut to probiotics. The low-oxygen environment (5% O2) not only avoids the stress damage of reactive oxygen species under normoxic conditions, but also stimulates probiotics to secrete metabolites such as antimicrobial peptides and extracellular proteins. The confined space of polysaccharides promotes the high-density aggregation and interaction of LRa05 and BLa80. Low oxygen further activates metabolic pathways and enhances colonization ability. Among them, LRa05 specifically enriches Lactobacillus, BLa80 directionally enhances Bifidobacterium, and the increased abundance of Faecalibacterium can also strengthen the intestinal mucosal barrier function, which meets the needs of children's intestines.

[0044] Comparative Example 2 The difference between this comparative example and Example 1 is that the 0.5% (w / v) xanthan gum in the polysaccharide thickening core solution in step 2) is adjusted to 0.3%, 0.6%, and 0.9% (w / v) pectin (P). The remaining steps are the same as in Example 1, resulting in core solutions made with different concentrations of pectin in this comparative example.

[0045] Figure 2 The static states of core solutions prepared with different concentrations of pectin (P) show that although pectin is also a thickening polysaccharide, the presence of calcium ions in the core solution causes a cross-linking reaction with the carboxyl groups in the pectin, forming... Figure 2 The obvious flocculent precipitation in the polysaccharide prevents the formation of liquid core spheres with confined spaces. Therefore, not all polysaccharides can be used for confined culture in this application.

[0046] Comparative Example 3 The difference between this comparative example and Example 1 is that the oxygen conditions for confined culture in step 5) are adjusted to 0, 2, 5, 8, 10% O2 (hypoxia incubator). The remaining steps were the same as in Example 1, resulting in 1:1 probiotic mixture confined culture medium core balls prepared under different hypoxic confined culture conditions in this comparative example.

[0047] The number of surviving bacteria (logCFU / g) and the extracellular protein production (mg / g) of the confined culture medium core pellets prepared in Comparative Example 3 were determined, and the results are shown in Table 7.

[0048] Table 7 As shown in Table 7, further optimization of hypoxic conditions revealed that the number of viable probiotics and the corresponding extracellular protein production were inhibited with increasing oxygen concentration. It is generally believed that an anaerobic environment is more suitable for the growth of anaerobic bacteria. However, this invention found that under certain hypoxic stimulation and specific compound ratios, the extracellular protein production of anaerobic bacteria and the improvement effect on intestinal flora were significantly improved. In particular, under 5% O2 hypoxic conditions, the highest number of viable bacteria and extracellular protein production could be achieved. This indicates that moderate hypoxic stimulation can not only avoid oxidative damage under normoxic conditions, but also activate the metabolic pathways and antioxidant enzyme systems of probiotics, inducing the secretion of more extracellular polymers, antimicrobial peptides and other beneficial metabolites. This provides a dual synergistic effect of probiotics and metabolites for the regulation of children's intestinal flora, rather than simply increasing the number of viable bacteria.

[0049] In summary, this invention provides a polysaccharide-confined culture probiotic preparation for improving children's gut microbiota and its application. It employs an extrusion method combined with calcium alginate cross-linking technology to construct confined spaces for encapsulating specific probiotics using polysaccharides such as xanthan gum. Probiotic core pellets are prepared by optimizing the polysaccharide formulation, probiotic mixing ratio, and culture conditions with varying oxygen levels. This process is simple to operate, uses natural and safe raw materials, and the selected probiotics meet the requirements for food and infant food strains. The prepared core pellets have a viable count ≥10-1. 9 With a concentration of CFU / g, it can stably withstand the production and storage process, and is suitable for various dietary scenarios such as infant formula and complementary foods, providing a safe and efficient formulation option for children's intestinal health.

[0050] This invention constructs a stable micro-ecosystem through polysaccharide-confined culture, providing a sanctuary free from external interference for the growth of Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80 (optimal mixing ratio 1:1), promoting high-density aggregation and interaction of the strains. The hypoxic environment (5% O2) precisely simulates the actual gut microecology of children, avoiding stress damage to the bacteria caused by reactive oxygen species under normoxic conditions, while activating the strains' metabolic pathways and antioxidant enzyme systems. In conjunction with the polysaccharide confinement space, the microbial interaction is further enhanced, inducing the secretion of more extracellular proteins, short-chain fatty acids, antimicrobial peptides, and other beneficial metabolites. These metabolites work synergistically with probiotics to precisely regulate the structure of children's gut microbiota, effectively reduce the abundance of potentially pathogenic phyla such as Pseudomonas, optimize the balance between Firmicutes and Bacteroidetes, selectively enrich beneficial bacteria such as Bifidobacterium and Faecalibacterium, inhibit opportunistic pathogens such as Klebsiella and Streptococcus, and at the same time improve the diversity and stability of gut microbiota, thus addressing the core pain points of poor gut microbiota stability, imperfect mucosal barrier, and immune sensitivity in children.

[0051] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing probiotic liquid core pellets cultured in a polysaccharide confinement environment, characterized in that: include, Probiotic suspension diluted to 10 6 ~10 8 After CFU / mL, it is mixed with polysaccharide thickening core material solution at a volume ratio of 1:10~30, and then dripped into sodium alginate crosslinking solution by extrusion. Crosslinking is carried out at room temperature. Primary liquid core balls are collected and hardened in calcium chloride solution to obtain hardened liquid core balls. After hardening, the liquid core balls were transferred into MRS broth and cultured at a constant temperature under 0-10% O2 concentration conditions. After the culture was completed, they were taken out and washed to obtain probiotic liquid core balls cultured through polysaccharide confinement. The polysaccharide concentration in the polysaccharide-thickened core liquid is 0.3~0.9% w / v.

2. The method for preparing probiotic core pellets via polysaccharide confinement culture as described in claim 1, characterized in that: The probiotics are selected from Lactobacillus rhamnosus LRa05 ( Lacticaseibacillus rhamnosus LRA05 Bifidobacterium animalis BLa80 ( Bifidobacterium animalis subsp. lactis BLa80 One or two of them.

3. The method for preparing probiotic core pellets via polysaccharide confinement culture as described in claim 2, characterized in that: The probiotics are Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80 in a mass ratio of 1~2:1~2.

4. The method for preparing probiotic liquid core pellets by polysaccharide confinement culture as described in claim 1, characterized in that: The polysaccharide is selected from xanthan gum, hyaluronic acid, guar gum, and locust bean gum.

5. The method for preparing probiotic liquid core pellets via polysaccharide confinement culture as described in claim 2, characterized in that: The method for preparing the probiotic suspension includes culturing probiotics to the logarithmic phase, collecting the bacterial cells by centrifugation, washing with PBS buffer, and resuspending to obtain the probiotic suspension.

6. The method for preparing probiotic core pellets via polysaccharide confinement culture as described in claim 2, characterized in that: The polysaccharide thickening core liquid also includes calcium chloride, the concentration of which is 1.0~2.5% w / v.

7. The method for preparing probiotic liquid core pellets by polysaccharide confinement culture as described in claim 2, characterized in that: The sodium alginate concentration in the sodium alginate crosslinking solution is 0.5~0.8% w / v, and the calcium chloride concentration in the calcium chloride solution is 1.0~2.5% w / v.

8. The method for preparing probiotic core pellets via polysaccharide confinement culture as described in claim 2, characterized in that: The cross-linking time at room temperature is 2-4 min, the hardening time is 8-12 min, the constant temperature culture temperature is 36-38℃, and the culture time is 20-24 h.

9. The probiotic liquid core pellets prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the probiotic liquid core ball as described in claim 9 in improving the intestinal flora of children, characterized in that: The applications include the preparation of foods or dietary supplements for regulating the gut microbiota of children.

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