Synbiotics preparation of lactobacillus helveticus as well as preparation method and application of synbiotics preparation

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a synbiotics preparation of lactobacillus helveticus as well as a preparation method and application of the synbiotics preparation. The synbiotics preparation of the lactobacillus helveticus, provided by the invention, is a microcapsule preparation prepared by taking the lactobacillus helveticus as probiotics and sialylated breast milk oligosaccharide as prebiotics and sequentially embedding by adopting sodium alginate and chitosan; wherein the lactobacillus helveticus is lactobacillus helveticus HL88. The lactobacillus helveticus HL88 adopted by the invention has better cracking activity on the sialylated breast milk oligosaccharide, and can effectively improve the utilization efficiency of the sialylated breast milk oligosaccharide. Furthermore, the lactobacillus helveticus and the sialylated breast milk oligosaccharide are subjected to composite embedding, so that the synergistic metabolism effect can be achieved, and the brain development process is effectively promoted. Therefore, the invention can provide a treatment thought for improving the brain development retardation, and can also provide a technical basis for developing probiotic medicines for promoting brain development.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to synbiotic preparations of Lactobacillus helveticus, their preparation methods, and applications. Background Technology

[0002] Brain development is a highly complex biological process encompassing the proliferation, migration, synapse formation, and myelination of neural progenitor cells. Simultaneously, brain development is one of the most important physiological processes in the human body, directly determining an individual's cognitive, emotional, and behavioral abilities. The period from fetal development to adolescence is the golden age of brain development, during which the formation, pruning, and functional differentiation of neuronal connections are highly active, laying the foundation for lifelong learning and environmental adaptation. However, the process of brain development is often influenced by multiple factors, including genetics, nutrition, and environmental stimuli, and developmental abnormalities can lead to learning disabilities, emotional problems, or neurological disorders.

[0003] Currently, many traditional drugs used to promote brain development (such as intervening in neurodevelopmental delays or improving cognitive function) not only have low bioavailability due to the blood-brain barrier, but also may produce potential systemic side effects in infants and young children who are in critical developmental stages. In contrast, probiotics, as natural colonizers in the mammalian gut, have been shown to exhibit greater safety and gentleness in regulating nervous system health.

[0004] Recent studies have found that certain strains of lactobacillus can significantly influence the development of the central nervous system through the gut-brain axis. For example, existing literature (Microbial reconstitution reverses maternal diet-induced social and synaptic deficits in offspring, Cell, 2016 JUN; 165(7):1762-1775) reports that some specific lactobacilli can improve nervous system function by regulating the gut microbiota. Therefore, developing brain-promoting products containing lactobacillus has become a research hotspot in the clinical and healthcare fields.

[0005] However, single Lactobacillus strains have low survival rates in the acidic and bile-salt environment of the stomach, and their regulatory effects on brain development are often limited by the intestinal colonization environment and nutrient supply, resulting in limited effects on improving brain development. Therefore, how to develop a synbiotic formulation with high activity and high survival rate, while effectively enhancing its effects on brain development, is the technical problem to be solved by this invention. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a synbiotic preparation of *Lactobacillus helveticus*, its preparation method, and its application. The synbiotic preparation provided by this invention utilizes sialylated human milk oligosaccharides to construct a complex system of probiotics and prebiotics with *Lactobacillus helveticus*, which can exert a synergistic effect, thereby effectively enhancing strain activity and promoting brain development, thus solving the problem of limited brain development promotion effects of single preparations.

[0007] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution: A synbiotic formulation of *Lactobacillus helveticus*, wherein the synbiotic formulation is a microcapsule preparation prepared by encapsulating *Lactobacillus helveticus* as a probiotic and sialylated human milk oligosaccharides as prebiotics using sodium alginate and chitosan sequentially; wherein *Lactobacillus helveticus* is *Lactobacillus helveticus* (… Lactobacillus helveticus HL88, with accession number CGMCC NO. 28126, is deposited at the China General Microbiological Culture Collection Center on August 9, 2023.

[0008] As a preferred embodiment, the sialylated human milk oligosaccharide is one or both of 3′-sialyl lactose and 6′-sialyl lactose.

[0009] The second aspect of the present invention adopts the following technical solution: A method for preparing a synbiotic preparation of Lactobacillus helveticus as described above includes the following steps: S1. Lactobacillus helveticus is activated and cultured to obtain a bacterial suspension of Lactobacillus helveticus; sialylated human milk oligosaccharides are dissolved in water to obtain a sialylated human milk oligosaccharide solution; the bacterial suspension of Lactobacillus helveticus and the sialylated human milk oligosaccharide solution are mixed evenly to obtain a synbiotic complex solution; S2. The synbiotic composite solution is mixed with sodium alginate solution to obtain a mixture to be embedded; the mixture to be embedded is prepared into droplets and then injected into calcium ion solution for cross-linking and solidification to obtain monolayer microcapsules; the monolayer microcapsules are then mixed with chitosan solution for coating treatment and finally dried to obtain the synbiotic preparation.

[0010] As a preferred embodiment, in step S1, the viable count of the *Lactobacillus helveticus* suspension is 1 × 10⁻⁶. 9 ~1×10 10 CFU / mL.

[0011] As a preferred embodiment, in step S1, the concentration of the sialylated human milk oligosaccharide solution is 4%~6% w / v, more preferably 5% w / v. The volume ratio of the *Lactobacillus helveticus* suspension to the sialylated human milk oligosaccharide solution is 1:(1~3), more preferably 1:2.

[0012] As a preferred embodiment, in step S2, the concentration of the sodium alginate solution is 1.5%~3% w / v. The volume ratio of the synbiotic complex solution to the sodium alginate solution is 1:(1~3), more preferably 1:2. The concentration of the calcium ion solution is 2%~5% w / v; the concentration of the chitosan solution is 0.2%~0.6% w / v. The volume ratio of the monolayer microcapsules to the chitosan solution is 1:(8~12), more preferably 1:10.

[0013] As a preferred embodiment, the diameter of the droplet is 30~50μm.

[0014] As a preferred method, in step S2, the crosslinking curing time is 20-60 min; the coating treatment time is 10-30 min; and the drying is freeze drying.

[0015] As a preferred method, the freeze-drying temperature is -10 to -20°C and the time is 10 to 30 hours.

[0016] The third aspect of the present invention adopts the following technical solution: The use of a synbiotic preparation as described above or a synbiotic preparation prepared by the method described above in the preparation of a drug for promoting brain development.

[0017] As a preferred approach, the brain-development-promoting drug works through one or more of the following mechanisms: promoting neurogenesis, increasing dendritic spine density, promoting synapse formation, accelerating myelination, and improving cognitive abilities.

[0018] The technical solution of the present invention has the following comprehensive advantages and beneficial effects: The synbiotic formulation provided by this invention uses *Lactobacillus helveticus* and sialylated human milk oligosaccharides to construct a prebiotic system, and is prepared by secondary encapsulation with calcium ion-crosslinked sodium alginate and chitosan. *Lactobacillus helveticus* HL88 used in this invention has been verified to have excellent cleavage activity against sialylated human milk oligosaccharides, possessing the ability to efficiently degrade and utilize sialylated human milk oligosaccharides, thereby effectively improving the utilization efficiency of sialylated human milk oligosaccharides.

[0019] Furthermore, this invention involves the co-encapsulation of *Lactobacillus helveticus* with sialylated human milk oligosaccharides, which enables a synergistic metabolic effect. On one hand, it enhances the ability of *Lactobacillus helveticus* to resist the harsh environment of the gastrointestinal tract, improving its survival rate and colonization capacity. On the other hand, sialylated human milk oligosaccharides, as targeted prebiotics, are preferentially utilized by *Lactobacillus helveticus*, promoting its proliferation in the intestine. Simultaneously, the sialic acid and its metabolites released by *Lactobacillus helveticus* serve as important substrates for the synthesis of nerve cell membranes, synaptic structures, and neurotransmitters, thereby significantly improving brain development. Simultaneously, this invention employs calcium ion cross-linking of sodium alginate and chitosan encapsulation for secondary encapsulation of the prebiotics, further enhancing the structural stability and encapsulation efficiency of the synbiotics, significantly improving brain development.

[0020] Animal experiments have also confirmed that the synbiotic formulation of this invention has significant advantages in promoting brain development, specifically in promoting neurogenesis, increasing dendritic spine density, promoting synapse formation, accelerating myelination, and improving cognitive abilities. Therefore, this invention can provide a therapeutic approach for improving delayed brain development and also provide a technical basis for developing drugs that promote brain development. Attached Figure Description

[0021] Figure 1 This shows the growth kinetics of different strains to be screened in this invention under conditions where sialylated human milk oligosaccharides are the main carbon source;

[0022] Figure 2 The new object recognition index is the index measured after each group of mice in the new object recognition experiment in this invention;

[0023] Figure 3 These are the latency results measured after the water maze experiment in each group of mice in this invention;

[0024] Figure 4 The results of the determination of synaptic protein (SYN) content in each group of mice in this invention;

[0025] Figure 5 The results show the content of postsynaptic dense protein-95 (PSD-95) in each group of mice in this invention.

[0026] Figure 6 The results show the content of myelin basic protein (MBP) in each group of mice in this invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below with reference to specific embodiments. The materials and process parameters used in the following embodiments are merely illustrative and should not be considered as limiting the scope of protection of this invention. The specific terms used below are the same as those understood by one of ordinary skill in the art, unless otherwise expressly defined.

[0028] The biological preservation information of this invention is as follows:

[0029] Preservation Name: Lactobacillus helveticus HL88; Preservation Number: CGMCC NO.28126; Preservation Institution: China General Microbiological Culture Collection Center; Preservation Address: No.3, No.1 Beichen West Road, Chaoyang District, Beijing; Preservation Date: August 9, 2023.

[0030] The modified MRS culture medium used in this invention is prepared as follows: 12g of peptone, 6g of yeast extract, 6g of beef extract, 18g of glucose, 5g of sodium acetate, 2.15g of ammonium citrate, 1g of Tween 80, 0.58g of magnesium sulfate, 0.05g of manganese sulfate, and 2g of dipotassium hydrogen phosphate are mixed, and pure water is added to bring the volume to 1L. The pH is adjusted to 6.5, and the medium is then sterilized by autoclaving at 121℃ for 15 minutes.

[0031] The preparation method of the human milk oligosaccharide modified MRS medium (sHMO-MRS) used in this invention is basically the same as the preparation method of the modified MRS medium mentioned above, except that: an equal amount of 3'-sialyl lactose is used to replace glucose, and other components and preparation parameters are the same as the preparation method of the modified MRS medium.

[0032] The phosphate-buffered saline (PBS) used in this invention is a conventional buffer in the art and can be prepared according to a standard formulation.

[0033] Example 1

[0034] This embodiment provides a synbiotic formulation of *Lactobacillus helveticus*, which is a microcapsule formulation constructed using *Lactobacillus helveticus* as the probiotic and sialylated human milk oligosaccharides as the prebiotic. The *Lactobacillus helveticus* is HL88, with accession number CGMCC NO. 28126, deposited at the China General Microbiological Culture Collection Center on August 9, 2023. The sialylated human milk oligosaccharide is specifically 3'-sialyl lactose.

[0035] The isolation, purification, and identification process of Lactobacillus helveticus HL88 involved in this embodiment is as follows:

[0036] Lactobacillus helveticus HL88 was isolated from fecal samples of healthy full-term infants in Harbin. During isolation, an appropriate amount of fecal sample was taken, diluted with sterile physiological saline, and the diluted solution was spread on mMRS agar plates and cultured under anaerobic conditions at 37°C for 48 hours. Typical round single colonies with smooth surfaces, neat edges, moist and raised, and milky white or grayish white color were selected.

[0037] The isolated and purified bacterial strain was inoculated into MRS solid medium and anaerobically cultured at 37°C for 48 h. Single colonies were selected for Gram staining, and their cell morphology was observed under a microscope. The results showed that the strain was Gram-positive, with slender rod-shaped cells arranged singly, in pairs, or in short chains, with blunt, rounded ends, and no spores or flagella. Gene identification was then performed using universal 16S rRNA primers and sequencing. The sequencing was completed by Shanghai Sangon Biotech Co., Ltd. After gene sequencing identification and comparison with NCBI phylogenetic analysis, the isolated strain was identified as *Lactobacillus helveticus*, and named *Lactobacillus helveticus* HL88.

[0038] The nucleotide sequence of Lactobacillus helveticus HL88 identified by 16S rRNA is shown in SEQ ID NO. 1:

[0039]

[0040] Example 2

[0041] This embodiment provides a method for preparing the synbiotic preparation of Lactobacillus helveticus from Example 1, specifically including the following steps:

[0042] (1) Preparation of bacterial suspension and sialylated human milk oligosaccharide solution

[0043] Lactobacillus helveticus HL88, frozen at -80℃, was inoculated into modified MRS medium and cultured anaerobically at 37℃ for 24 h. After culturing, the resulting culture was centrifuged at 3000 rpm for 10 min at 4℃, the supernatant was discarded, and the resulting bacterial cells were washed twice with sterile PBS buffer. Finally, the bacterial cells were resuspended in sterile PBS buffer and the bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL was used to obtain a bacterial suspension of Lactobacillus helveticus; sialylated human milk oligosaccharides (specifically 3'-sialyl lactose) were dissolved in sterile deionized water to obtain a 5% (w / v) sialylated human milk oligosaccharide solution.

[0044] (2) Preparation of Synbiotic Complex Solution

[0045] Under aseptic conditions, the sialylated human milk oligosaccharide solution prepared above was mixed with the bacterial suspension of Lactobacillus helveticus at a volume ratio of 2:1 to obtain the synbiotic complex solution.

[0046] (3) Preparation of Synbiotic Preparations

[0047] Sodium alginate was dissolved in deionized water and magnetically stirred at room temperature (25±5℃) until completely dissolved, yielding a sodium alginate solution with a mass fraction of 2.0% (w / v). The synbiotic composite solution prepared in step (2) was added to the sodium alginate solution at a volume ratio of 1:2 and stirred evenly to obtain the encapsulation mixture. The encapsulation mixture was prepared into 40μm droplets using a microdroplet preparation instrument (FluidicLab DG-01), and then the droplets were injected into a calcium chloride solution with a mass fraction of 3% (w / v). The mixture was allowed to stand and crosslink at room temperature for 30 min to obtain monolayer microcapsules. The monolayer microcapsules were collected by filtration and transferred to a chitosan solution with a mass fraction of 0.4% (w / v) (the volume ratio of the wet solution of the monolayer microcapsules to the chitosan solution was 1:10). The mixture was then coated for 15 min under stirring at 200 rpm. After encapsulation, the microcapsules were washed twice with sterile saline to remove unbound chitosan, resulting in bilayer microcapsules. The obtained bilayer microcapsules were then pre-frozen at -40°C for 5 hours, followed by freeze-drying at -15°C for 18 hours to obtain the synbiotic formulation of this embodiment.

[0048] Example 3

[0049] This embodiment provides a synbiotic preparation of Lactobacillus helveticus, which is prepared in a manner that is basically the same as that in Example 2, except that the sialylated human milk oligosaccharide used in this embodiment is 6′-sialyl lactose.

[0050] Comparative Example 1

[0051] This comparative example provides a microcapsule formulation of *Lactobacillus helveticus* that does not contain sialylated human milk oligosaccharides. The preparation method of the microcapsule formulation of *Lactobacillus helveticus* in this comparative example is basically the same as that in Example 2, except that: the preparation process of the sialylated human milk oligosaccharide solution in step (1) is omitted; step (2) is omitted; step (3) is adjusted to add the bacterial suspension of *Lactobacillus helveticus* to sodium alginate solution at a volume ratio of 1:2 and stir evenly to obtain the mixture to be encapsulated. The remaining processes and specific parameters are the same as in Example 2.

[0052] Comparative Example 2

[0053] This comparative example provides a synbiotic preparation of *Lactobacillus helveticus*, which is a monolayer microcapsule cross-linked with sodium alginate and does not contain chitosan. The preparation method of the synbiotic preparation of *Lactobacillus helveticus* in this comparative example is basically the same as that in Example 2, except that chitosan coating is not performed. That is, step (3) is adjusted to filter and collect the obtained monolayer microcapsules, wash the microcapsules twice with sterile physiological saline, and then freeze-dry the obtained microcapsules after pre-freezing to obtain the synbiotic preparation of *Lactobacillus helveticus* in this comparative example. The remaining processes and specific parameters are the same as in Example 2.

[0054] Comparative Example 3

[0055] This comparative example provides a synbiotic formulation of *Lactobacillus helveticus*, which is a single-layer microcapsule coated with chitosan and does not contain sodium alginate. The preparation method of the synbiotic formulation of *Lactobacillus helveticus* in this comparative example is basically the same as that in Example 2, except that sodium alginate cross-linking is not performed, that is, step (3) is adjusted to transfer the synbiotic complex liquid to a chitosan solution with a mass fraction of 0.4% (w / v) (the volume ratio of the synbiotic complex liquid to the chitosan solution is 1:10), and coat it for 15 min under stirring at 200 rpm. The remaining processes and specific parameters are the same as in Example 2.

[0056] Comparative Example 4

[0057] This comparative example provides a synbiotic formulation of *Bifidobacterium animalis* subsp. *lactis*, which is a microcapsule formulation prepared using conventional strains reported in existing technologies. The preparation method of this comparative example's synbiotic formulation of *Bifidobacterium animalis* subsp. *lactis* is basically the same as in Example 2, except that commercially available *Bifidobacterium animalis* subsp. *lactis* BB-12 is used instead of *Lactobacillus helveticus* HL88. The remaining processes and specific parameters are the same as in Example 2. *Bifidobacterium animalis* subsp. *lactis* BB-12 is from the Chr. Hansen brand.

[0058] The technical effects of this invention are illustrated below with specific experimental examples. All experimental data are expressed as mean ± standard deviation, and were repeated at least three times. One-way ANOVA was performed on the data between different groups using SPSS 18.0 software, and graphs were plotted using GraphPad Prism 5.0 and Origin 9.0 software. A p-value < 0.05 was considered statistically significant.

[0059] Experiment 1: Screening of different strains for the utilization of sialylated human milk oligosaccharides

[0060] 1. Determination of the strains to be screened

[0061] The strains to be screened include *Lactobacillus helveticus* HL85, *Lactobacillus helveticus* HL86, and *Lactobacillus helveticus* HL88 (Example 1 of this invention). *Lactobacillus helveticus* HL85 and HL86 were isolated from different infant fecal samples in Harbin using the same isolation method as *Lactobacillus helveticus* HL88, and then identified using 16S rRNA. The sample sources and identification results for the different strains to be screened are shown in Table 1.

[0062] Table 1. Strains' origin and identification results

[0063]

[0064] 2. Tests on the utilization capacity of different strains of sialylated human milk oligosaccharides

[0065] Before testing, each of the selected strains (Lactobacillus helveticus HL85, Lactobacillus helveticus HL86, and Lactobacillus helveticus HL88) was activated and cultured. The culture process was as follows: the selected strains were removed from cryovials at -80℃ and rapidly thawed at 37℃. Under aseptic conditions, the selected strains were inoculated at 5% (v / v) into sterile modified MRS medium and cultured anaerobically at 37℃ for 24 h. After culture, the cells were centrifuged at 2000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile PBS buffer. Finally, the cells were resuspended in PBS buffer, and the bacterial concentration was adjusted to 1×10⁻⁶. 7CFU / mL was used to obtain suspensions of each strain to be screened.

[0066] The suspensions of the selected strains prepared above were inoculated into human milk oligosaccharide modified MRS medium (sHMO-MRS) at an inoculation rate of 5% (v / v) and cultured anaerobically at 37°C for 24 h. The OD values ​​at different time points during the culture process were measured. 600 Values ​​and viable cell counts were used to plot growth curves, allowing for comparison of growth rates and stationary phase activity among different strains. Strains with rapid growth rates and high stationary phase viable cell counts were selected as target strains. The growth kinetics of different selected strains under conditions using sialylated human milk oligosaccharides as the primary carbon source are shown below. Figure 1 As shown.

[0067] Depend on Figure 1 It was found that different strains exhibited significant differences in growth rate and final biomass level. Overall, *Lactobacillus helveticus* HL88 demonstrated faster growth initiation and higher growth levels during culture. Specifically, *Lactobacillus helveticus* HL88 entered the logarithmic growth phase in the early stages of culture (0–6 h), and its OD... 600 The OD value increased significantly faster than that of HL85 and HL86, suggesting that HL88 can adapt more quickly to a culture environment using sialylated human milk oligosaccharides as a carbon source. Throughout the logarithmic growth phase and the subsequent stationary phase (8–24 h), HL88 consistently maintained the highest OD value. 600 The final bacterial count of HL85 and HL86 was significantly higher than that of other strains, indicating a stronger and more sustained capacity for utilizing sialylated human milk oligosaccharides. Furthermore, the growth rate and final OD values ​​of HL85 and HL86 were significantly higher than those of other strains. 600 The values ​​were relatively close and significantly lower, indicating limited utilization efficiency of sialylated human milk oligosaccharides. These results suggest that different strains of *Lactobacillus helveticus* exhibit specific differences in their ability to utilize sialylated human milk oligosaccharides.

[0068] Based on comprehensive indicators such as growth rate, logarithmic growth phase initiation time, and final biomass level, Lactobacillus helveticus HL88 exhibited the strongest utilization capacity of sialylated human milk oligosaccharides, providing experimental evidence for its role as a core probiotic strain in sialylated human milk oligosaccharide synbiotics.

[0069] Experiment Example 2: Strain Survival Test

[0070] This experimental example mainly tests the survival rate of bacterial strains in the microcapsules or synbiotic preparations prepared in Example 2 and Comparative Examples 1-3 of this invention. The specific testing method is as follows: The preparation samples of Example 2 and Comparative Examples 1-3 were respectively added to simulated gastric juice (containing 3.2 g / L pepsin, pH 2.0-3.0) preheated to 37°C, at a ratio of 1:10 (v / v), and continuously digested for 120 min in a constant temperature shaker (150 rpm) at 37°C. Samples were taken periodically during this period, and the microcapsules were depolymerized using 55 mmol / L sodium citrate solution to completely release the internal bacteria. Finally, the microcapsules were serially diluted with physiological saline and plated for counting to calculate the survival rate of the embedded bacterial strains in the simulated gastric juice. The bacterial survival rate results of different preparations in simulated gastric juice are shown in Table 2.

[0071] Table 2. Survival rate of strains in simulated gastric fluid for different formulations

[0072]

[0073] As shown in Table 2, different microcapsule formulations exhibited significantly different survival rates under the same treatment conditions. Specifically, the survival rate of the synbiotic formulation group in Example 2 of this invention was 89±2.35%, while the survival rate of the HL88 single-strain microcapsule group in Comparative Example 1 without prebiotics was 68±1.91%. Compared with microcapsules that only encapsulated *Lactobacillus helveticus* HL88, the introduction of sialylated human milk oligosaccharides into the synbiotic microcapsules significantly improved the bacterial survival rate (P<0.05), indicating that the presence of sialylated human milk oligosaccharides significantly enhanced the protective effect of the microcapsule system on probiotics. Furthermore, compared with the single-layer microcapsules of Comparative Examples 2 and 3, the survival rate of the synbiotic formulation obtained through double-layer encapsulation in Example 2 of this invention was also significantly improved. This demonstrates that the present invention, through the double-layer encapsulation of sialylated human milk oligosaccharides and sodium alginate-chitosan, can synergistically improve the survival rate. On the one hand, sodium alginate-chitosan and sialylated human milk oligosaccharides can serve as hydrophilic functional polysaccharides, participating in the formation of the internal network structure of microcapsules, improving the density and stability of the wall material, thereby reducing the damage to the bacteria caused by adverse external environments (such as acidity, digestive enzymes, or bile salts). On the other hand, sodium alginate-chitosan and sialylated human milk oligosaccharides, as specific substrates available to Lactobacillus helveticus HL88, can provide immediate energy support and metabolic buffering for the bacteria during encapsulation and processing, enhancing their environmental tolerance.

[0074] Experiment Example 3: Animal Experiment

[0075] 1. Experimental Design

[0076] The experimental materials were healthy newborn SPF-grade C57BL / 6J mice, purchased from the Second Affiliated Hospital of Harbin Medical University. Experiments began at 7 days of age. All animals were housed in a standard animal facility with a temperature controlled at 22±2℃ and a relative humidity of 50%–60%, using a 12-hour light / dark alternating lighting system, and with free access to food and water. The animals were randomly divided into the following experimental groups (n=6 per group), and each mouse underwent the following interventions:

[0077] HL88 bacterial suspension group: Lactobacillus helveticus HL88 bacterial suspension was administered by gavage (the dosage of Lactobacillus helveticus HL88 was 10...). 7 CFU (dissolved in sterile saline).

[0078] Sialized human milk oligosaccharide group (SA-HMO): Sialized human milk oligosaccharide (20 mg of 3'-sialyl lactose dissolved in sterile saline) was administered by gavage.

[0079] Comparative Example 1 Microcapsule Formulation Group: The microcapsule formulation prepared in Comparative Example 1 (Lactobacillus helveticus HL88) was administered by gavage at a dosage of 10 g / L. 7 CFU (to fully dissolve the microcapsule formulation in sterile saline).

[0080] Comparative Example 2 Synbiotic Preparation Group: The synbiotic preparation prepared in Comparative Example 2 (Lactobacillus helveticus HL88) was administered by gavage at a dosage of 10... 7 CFU (to fully dissolve the synbiotic preparation in sterile saline).

[0081] Comparative Example 3 Synbiotic Preparation Group: The synbiotic preparation prepared in Comparative Example 3 (Lactobacillus helveticus HL88) was administered by gavage at a dosage of 10... 7 CFU (to fully dissolve the synbiotic preparation in sterile saline).

[0082] Comparative Example 4 Synbiotic Preparation Group: The synbiotic preparation prepared in Comparative Example 4 (containing Bifidobacterium animalis subsp. lactis) was administered by gavage at a dosage of 10... 7 CFU (to fully dissolve the synbiotic preparation in sterile saline).

[0083] Example 2 Synbiotic preparation group: The synbiotic preparation prepared in Example 2 (Lactobacillus helveticus HL88) was administered by gavage at a dosage of 10... 7 CFU (to fully dissolve the synbiotic preparation in sterile saline).

[0084] Positive control group: 7,8-dihydroxyflavone was administered by gavage. The dosage was converted from human dosage to mouse equivalent dosage using the body surface area conversion method. The drug was fully dissolved in sterile physiological saline and administered at a dose of 5 mg / kg / day.

[0085] Blank control group: The same volume of sterile saline was administered by gavage.

[0086] All treatment groups received the same gavage volume for 4 consecutive weeks. After a 24-hour fast, all mice were euthanized. Body weight changes were recorded for each group, and whole brain tissue was rapidly extracted to record brain weight changes. The hippocampus and cerebral cortex were isolated for further use. Simultaneously, a portion of brain tissue was collected and fixed in 10% buffered formaldehyde for histological analysis.

[0087] 2. Changes in body weight and brain weight

[0088] Changes in body weight and brain weight are important indicators for assessing an individual's growth and development, as well as brain development. Body weight reflects the overall nutritional status and growth level of the organism, while brain weight, as an important morphological indicator of central nervous system development, is closely related to brain tissue growth, the number of neurons, and the maturation of neural structures. During the growth and development stage, both body weight and brain weight show a gradual increasing trend with increased nutrient supply and continuous development of the nervous system. Increased brain weight is usually accompanied by an increase in nerve cell volume, an increase in synaptic connections, and the maturation of brain tissue structure, until brain development approaches maturity. Therefore, simultaneously detecting changes in body weight and brain weight can provide a relatively intuitive reflection of an organism's growth and development, as well as brain development, from both the overall growth level and the development of the central nervous system. The changes in body weight and brain weight of mice in each group are shown in Table 3. Different lowercase letters in the same column indicate significant differences (P<0.05), and the same applies below.

[0089] Table 3. Effects of different interventions on mouse body weight and brain weight

[0090]

[0091] Table 3 shows that *Lactobacillus helveticus* intervention increased mouse body weight and brain weight to some extent. Compared with the blank control group, the HL88 bacterial suspension group and the SA-HMO group significantly increased mouse body weight and brain weight. Compared with the HL88 bacterial suspension group and the SA-HMO group, the body weight and brain weight of the synbiotic preparation group in Example 2 were significantly improved, reaching or even slightly exceeding the levels of the positive control group. Furthermore, compared with Comparative Example 1, the synbiotic preparation group in Example 2 significantly improved various growth and development indicators of mice, further validating the core value of sialylated human milk oligosaccharides in promoting body development and brain growth. Compared with Comparative Examples 2 and 3, the values ​​of the synbiotic preparation group in Example 2 were significantly improved, fully demonstrating the technical advantages of the double-layer microcapsule encapsulation process in ensuring the bioactivity of synbiotics and enhancing their developmental efficacy. Compared with the synbiotic preparation group in Comparative Example 4, Example 2 also showed a more significant trend in improving key developmental indicators such as brain weight.

[0092] 3. Cognitive Ability Test

[0093] Cognitive ability is a crucial manifestation of higher functions of the central nervous system, primarily encompassing learning ability, memory formation, and information retrieval. To evaluate the cognitive function and learning / memory abilities of mice in different treatment groups, behavioral tests, including the novel object recognition test and the water maze test, were conducted to systematically assess changes in cognitive abilities. The novel object recognition test primarily reflects the mouse's ability to recognize and retain novel stimuli, and its performance depends on the functional state of the hippocampus and related cortical regions. The water maze test is widely used to assess spatial learning and spatial memory abilities, comprehensively reflecting the mouse's learning efficiency and memory consolidation level in response to environmental cues. Generally, improvements in the novel object recognition index and indicators such as escape latency and time spent in the target quadrant during the water maze indicate enhanced cognitive function and learning / memory abilities in mice; conversely, decreases in these indicators may suggest cognitive impairment. Therefore, the combined testing of the novel object recognition test and the water maze test provides a more comprehensive evaluation of the cognitive abilities and neurological development of mice.

[0094] The testing method for the new object recognition experiment was as follows: Mice were placed in a 40cm×40cm×40cm experimental box for environmental adaptation on the first day; on the second day, they entered the familiarization stage, where two identical objects A1 and A2 were placed in opposite positions in the box, and the mice were allowed to explore them for 10 minutes; after a 24-hour interval, the testing stage began, and A2 was replaced with a new object B with a different shape and material. The exploration time of the mice for the old and new objects was recorded within 5 minutes. The recognition index (RI), which is the percentage of the total exploration time spent by the mice exploring the new object, was calculated to evaluate the mice's recognition and memory abilities.

[0095] The Morris water maze test was conducted by setting up a hidden platform in a 120cm diameter constant-temperature water tank. The experiment consisted of five consecutive days of navigation training and a sixth day of spatial exploration testing. On the sixth day, the time required for the mice to find and climb onto the platform from entering the water was recorded, i.e., the escape latency, to assess their spatial learning ability.

[0096] The new object recognition index measured after the new object recognition experiment in each group of mice is as follows: Figure 2 As shown in the figure. The latency results of each group of mice after the water maze test are as follows. Figure 3 As shown in the figure. In the figure, ns indicates no significant difference compared with Example 2; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate significant differences compared with Example 2, and the same applies below.

[0097] Depend on Figure 2It was found that, compared with the blank control group, the HL88 bacterial suspension group and the SA-HMO group significantly increased the novel object recognition index. Compared with the HL88 bacterial suspension group and the SA-HMO group, the synbiotic preparation group of Example 2 significantly improved, reaching a level comparable to the positive control. Furthermore, compared with Comparative Example 1, the synbiotic preparation group of Example 2 significantly increased the novel object recognition index, demonstrating the necessity of sialylated human milk oligosaccharides. Compared with Comparative Examples 2 and 3, the novel object recognition index of the synbiotic preparation group of Example 2 was significantly improved, highlighting the advantages of the synbiotic bilayer microcapsule process. Compared with the synbiotic preparation group of Comparative Example 4, the group of Example 2 also showed significantly higher scores.

[0098] Depend on Figure 3 It was found that, compared with the blank control group, the HL88 bacterial suspension group and the SA-HMO group significantly reduced the latency period in mice. Compared with the HL88 bacterial suspension group and the SA-HMO group, the latency period of the synbiotic preparation group in Example 2 was significantly reduced, reaching a level comparable to the positive control. Furthermore, compared with Comparative Example 1, the synbiotic preparation group in Example 2 significantly reduced the latency period, further demonstrating the key role of sialylated human milk oligosaccharides in improving learning and memory impairment. Moreover, the latency period of the synbiotic preparation group in Example 2 was significantly lower than that in Comparative Examples 2 and 3, reflecting the contribution of the double-layer encapsulation process to enhancing the bioavailability of the synbiotic. Compared with the synbiotic preparation group in Comparative Example 4, Example 2 also showed a significant advantage in shortening the latency period.

[0099] 4. Brain dendritic spine density test

[0100] To evaluate the development of neural structures in mice across different treatment groups, Golgi staining was used to stain brain tissue, and changes in the density of dendritic spines in neurons were observed and analyzed. Dendritic spines are tiny projections on the surface of neuronal dendrites and are an important structural basis for synapse formation and information transmission. Dendritic spine density typically reflects the number and maturity of synaptic connections in neurons. Higher dendritic spine density usually indicates richer synaptic connections and stronger neural plasticity between neurons, while decreased dendritic spine density may be associated with restricted neural development or weakened synaptic function. Therefore, detecting dendritic spine density under Golgi staining can effectively assess the structural development of the central nervous system and the maturity of neural networks.

[0101] The experimental procedure was as follows: Brain tissue from mice in each group was cut into appropriate sizes, then immersed in staining solution and kept out of light for a period of time. The tissue was then sectioned using a vibratory microtome (VT 1000S) to a thickness of 100 μm. The sections were then stained and dehydrated using a series of ethanol solutions of different concentrations (70%, 90%, and 100%), and cleaned with xylene. The density of dendritic spines was then assessed by analyzing the third dendrites in the dentate scattering granule layer. The dendritic spine density results for each group of mice are shown in Table 4.

[0102] Table 4. Effects of different treatments on dendritic spine density in mice

[0103]

[0104] Table 4 shows that, compared with the blank control group, the HL88 bacterial suspension group and the SA-HMO group significantly increased the dendritic spine density of neurons in the mouse brain. Compared with the HL88 bacterial suspension group and the SA-HMO group, the dendritic spine density of the synbiotic preparation group in Example 2 was significantly increased, reaching a level comparable to the positive control group. Furthermore, compared with Comparative Example 1, the synbiotic preparation group in Example 2 significantly increased the dendritic spine density, further verifying the key enhancing effect of adding sialylated human milk oligosaccharides on synaptic plasticity. Compared with Comparative Examples 2 and 3, the values ​​of the synbiotic preparation group in Example 2 were significantly improved, demonstrating the technical advantages of the double-layer microcapsule process in protecting the activity of core components and enhancing their physiological efficacy. Compared with the synbiotic preparation group in Comparative Example 4, the group in Example 2 showed better statistical results. This indicates that the synbiotic formulation of *Lactobacillus helveticus* HL88 and sialylated human milk oligosaccharides exhibits superior efficacy in increasing dendritic spine density.

[0105] 5. Synaptic and myelin marker determination

[0106] The determination of synaptic and myelin markers is an important means of assessing the development and functional status of the central nervous system. Among them, mouse synaptic protein (SYN) and postsynaptic density protein-95 (PSD-95) are important marker proteins reflecting synaptic structure and function, while myelin basic protein (MBP) is a key indicator for evaluating the formation and maturation of myelin in nerve fibers. Changes in the levels of these markers can provide important information about brain development and neural network maturation. SYN is a specific protein on the presynaptic vesicle membrane, and its expression level usually reflects the number of presynaptic structures and synaptic transmission capacity. During brain development and the enhancement of neural plasticity, SYN expression levels increase, suggesting increased synapse formation and neural connections. PSD-95 is a scaffold protein located on the postsynaptic membrane, involved in the assembly of the postsynaptic signaling complex, and its expression level is closely related to synaptic maturity and stability. Elevated PSD-95 levels generally indicate well-developed postsynaptic structures and enhanced synaptic function. Myelin sheathing (MBB) is an important component of the myelin sheath in the central nervous system, and its expression level reflects the degree of myelination in nerve fibers. During brain development, with the differentiation of oligodendrocytes and myelin formation, MBP levels gradually increase, suggesting improved neural conduction efficiency and neural network integration capabilities.

[0107] Therefore, simultaneously detecting the expression levels of SYN, PSD-95, and MBP allows for a comprehensive evaluation of the developmental status and functional maturity of the central nervous system from both synaptic structure and nerve fiber myelination perspectives. The testing procedure involved grinding an appropriate amount of mouse hippocampus in liquid nitrogen and then using an enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme Immunosorbent Assay Biotechnology Co., Ltd.) according to the manufacturer's instructions to determine the levels of mouse synaptic protein (SYN), postsynaptic density protein-95 (PSD-95), and myelin basic protein (MBP).

[0108] The results of the determination of mouse synaptic protein (SYN), postsynaptic density protein-95 (PSD-95), and myelin basic protein (MBP) are as follows: Figure 4 , 5 As shown in Figure 6.

[0109] Depend on Figures 4-6It was found that, compared with the blank control group, the HL88 bacterial suspension group and the SA-HMO group significantly increased the contents of SYN, PSD-95, and MBP in the mouse brain. Compared with the HL88 bacterial suspension group and the SA-HMO group, the contents of all the above indicators in the synbiotic preparation group of Example 2 were significantly increased, reaching levels comparable to the positive control. Furthermore, compared with Comparative Example 1, the HL88-SA-HMO synbiotic preparation group of Example 2 significantly increased the contents of SYN, PSD-95, and MBP in the brain, further demonstrating the key role of sialylated human milk oligosaccharides in promoting synaptic development and myelin formation. Compared with Comparative Examples 2 and 3, the contents of SYN, PSD-95, and MBP in the synbiotic preparation group of Example 2 were significantly increased, reflecting the significant advantages of the double-layer microcapsule technology in enhancing the bioavailability of active ingredients and strengthening neuroprotective function. Compared with the synbiotic preparation group of Comparative Example 4, Example 2 also showed superior effects in increasing the expression of synaptic-related proteins and myelin proteins.

[0110] The above results collectively demonstrate that the synbiotic formulation provided by this invention, which utilizes Lactobacillus helveticus HL88 and sialylated human milk oligosaccharides in synergy to construct a sodium alginate-chitosan bilayer microcapsule, can promote brain development by accelerating myelin formation and promoting neurogenesis, exhibiting a good effect on promoting brain development.

[0111] The above results also demonstrate that the synbiotic formulation of the present invention promotes brain development by influencing processes related to the development of the nervous system. Its effects include, but are not limited to: promoting neurogenesis, increasing dendritic spine density, promoting synapse formation, accelerating myelination, and improving cognitive abilities, thereby having a positive effect on the overall growth and development of the body.

[0112] Finally, without departing from the principles of this invention, those skilled in the art can make reasonable adjustments or substitutions to the raw material dosages, types of sialylated human milk oligosaccharides, the ratio of Lactobacillus helveticus to sialylated human milk oligosaccharides, and the preparation parameters of the synbiotic formulation of this invention. Such adjustments and substitutions should also be considered to fall within the protection scope of this invention.

Claims

1. A synbiotic preparation of Lactobacillus helveticus, characterized in that, The synbiotic formulation is a microcapsule preparation made by encapsulating the probiotics and prebiotics with sodium alginate and chitosan sequentially using *Lactobacillus helveticus* as the probiotic and sialylated human milk oligosaccharides as the prebiotic; the *Lactobacillus helveticus* is *Lactobacillus helveticus* (…). Lactobacillus helveticus HL88, with accession number CGMCC NO. 28126, is deposited at the China General Microbiological Culture Collection Center on August 9, 2023.

2. The synbiotic preparation of *Lactobacillus helveticus* according to claim 1, characterized in that, The sialylated human milk oligosaccharide is one or both of 3′-sialyl lactose and 6′-sialyl lactose.

3. A method for preparing a synbiotic formulation of *Lactobacillus helveticus* as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Lactobacillus helveticus is activated and cultured to obtain a bacterial suspension of Lactobacillus helveticus; sialylated human milk oligosaccharides are dissolved in water to obtain a sialylated human milk oligosaccharide solution; the bacterial suspension of Lactobacillus helveticus and the sialylated human milk oligosaccharide solution are mixed evenly to obtain a synbiotic complex solution; S2. Mix the synbiotic composite solution with sodium alginate solution to obtain the encapsulation mixture; prepare the encapsulation mixture into droplets, and then inject them into calcium ion solution for cross-linking and solidification to obtain monolayer microcapsules; The monolayer microcapsules were then mixed with a chitosan solution for encapsulation, and finally dried to obtain the synbiotic formulation.

4. The method for preparing the synbiotic preparation of Lactobacillus helveticus according to claim 3, characterized in that, In step S1, the viable count of the *Lactobacillus helveticus* suspension is 1 × 10⁻⁶. 9 ~1×10 10 CFU / mL.

5. The method for preparing the synbiotic preparation of Lactobacillus helveticus according to claim 3, characterized in that, In step S1, the concentration of the sialylated human milk oligosaccharide solution is 4%~6% w / v; the volume ratio of the Lactobacillus helveticus suspension to the sialylated human milk oligosaccharide solution is 1:(1~3).

6. The method for preparing a synbiotic preparation of *Lactobacillus helveticus* according to any one of claims 3 to 5, characterized in that, In step S2, the concentration of the sodium alginate solution is 1.5%~3% w / v; the volume ratio of the synbiotic complex solution to the sodium alginate solution is 1:(1~3); the concentration of the calcium ion solution is 2%~5% w / v; the concentration of the chitosan solution is 0.2%~0.6% w / v; and the volume ratio of the monolayer microcapsules to the chitosan solution is 1:(8~12).

7. The method for preparing a synbiotic preparation of *Lactobacillus helveticus* according to any one of claims 3 to 5, characterized in that, In step S2, the diameter of the droplet is 30~50μm.

8. The method for preparing a synbiotic preparation of *Lactobacillus helveticus* according to any one of claims 3 to 5, characterized in that, In step S2, the crosslinking curing time is 20-60 min; the coating treatment time is 10-30 min; and the drying is freeze drying.

9. The use of a synbiotic preparation as described in claim 1 or 2, or a synbiotic preparation prepared by any one of claims 3 to 8, in the preparation of a medicament for promoting brain development.

10. The use of the synbiotic preparation according to claim 9 in the preparation of a medicament for promoting brain development, characterized in that, The brain-development-promoting drugs work through one or more of the following mechanisms: promoting neurogenesis, increasing dendritic spine density, promoting synapse formation, accelerating myelination, and improving cognitive abilities.

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