Multifunctional lactobacillus paracasei strain for promoting growth and development and application of multifunctional lactobacillus paracasei strain

Abstract

The invention relates to the technical field of microorganisms, in particular to a multifunctional lactobacillus paracasei strain capable of promoting growth and development and application of the multifunctional lactobacillus paracasei strain. The Latin name of the multifunctional Lactobacillus paracasei strain is Lacticaseibacillus paracasei ZG1, the preservation number of the multifunctional Lactobacillus paracasei strain is CGMCC No.36863, the preservation date of the multifunctional Lactobacillus paracasei strain is December 5, 2025, and the preservation unit of the multifunctional Lactobacillus paracasei strain is China General Microbiological Culture Collection Center. Experiments prove that the multifunctional lactobacillus paracasei strain has the functions of promoting neurocognitive development, immune system maturation and bone growth, and can be added into an infant probiotic preparation to play a role in promoting growth and development. Compared with existing probiotics, the lactobacillus paracasei ZG1 has a multi-target and multi-system synergistic promotion function on growth and development, is expected to replace a traditional mixed probiotic scheme, and fundamentally solves the core problems of single function, mixed antagonism and the like of most of existing probiotics.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a multifunctional Lactobacillus paracasei strain that promotes growth and development and its applications. Background Technology

[0002] The early stages of life are a critical window for the growth and development of the nervous, immune, and skeletal systems, and the developmental status during this period directly determines an individual's long-term health. If problems such as delayed neurodevelopment, immature immune function, or poor skeletal development occur during this period, they often cause irreversible damage to the body and seriously threaten subsequent health.

[0003] Currently, nutritional intervention is the mainstream approach to promoting growth and development, but traditional nutritional supplementation programs have significant limitations: on the one hand, they are difficult to achieve synergistic improvement of multiple systems such as the nervous, immune, and skeletal systems, and can only play a role in a single system; on the other hand, their bioavailability is low and there are certain risks of side effects, which cannot meet the multidimensional developmental needs in early life.

[0004] Probiotics have become a research hotspot in the field of nutritional intervention due to their high safety and diverse regulatory potential on host health. However, the functions of probiotics are highly strain-specific, with significant differences in the target sites and effects among different strains. Currently, most probiotic products on the market focus on regulating the balance of gut microbiota, and strains that can simultaneously cover multiple needs such as promoting neurodevelopment, supporting immune function, and assisting bone growth are extremely rare.

[0005] More importantly, existing technologies often require the use of a mixture of multiple single-function probiotics to achieve multiple growth and development promotion functions. However, this mixed approach has significant drawbacks: different strains compete for nutrients in the host's gut, resulting in nutrient competitive inhibition and limiting the survival efficiency and function of each strain; some strains may also have functional antagonism, which not only fails to synergistically enhance the effect but may also weaken the overall intervention effect and even cause potential problems such as gut microbiota imbalance. Existing studies have confirmed the widespread antagonistic effect of probiotic mixtures. For example, among 366 strain combinations of *Lactobacillus bulgaricus* and *Streptococcus thermophilus* commonly used in yogurt fermentation, only 8 pairs showed synergistic effects, while 97.9% of the combinations exhibited non-synergistic growth. Hydrogen peroxide and bacteriocins produced by some *Lactobacillus bulgaricus* strains significantly inhibited the growth and reproduction of *Streptococcus thermophilus*. Interaction studies of nine commonly used probiotics showed that about one-third of the strains exhibited clear antagonism. Among them, *Bifidobacterium bifidum* showed an inhibition zone diameter of up to 19.3 mm against *Streptococcus thermophilus*, and *Lactobacillus plantarum* also showed significant inhibitory effects on *Lactobacillus rhamnosus* and *Lactobacillus casei*. Strong antagonistic effects were also found between *Lactobacillus* strains 26c and 24t, and 33tl and 24t, isolated from natural dairy products, severely affecting the functional stability of mixed formulations. These antagonistic phenomena further increase the application risk of mixed probiotic programs and limit their practical effectiveness in promoting growth and development in multiple systems.

[0006] Therefore, screening a single-function strain with multi-target and multi-system synergistic effects can replace the traditional mixed probiotic regimen, fundamentally solving core problems such as single function and mixed antagonism. Summary of the Invention

[0007] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the primary objective of the present invention is to provide a multifunctional Lactobacillus paracasei strain, which differs from existing single-function probiotics and can achieve multi-system synergistic promotion of growth and development.

[0008] Another objective of this invention is to systematically evaluate the growth-promoting effects of Lactobacillus paracasei and to explore its mechanism of action in depth, so as to provide a scientific basis for the application of this strain in related products and solve the problem of limited functionality and effectiveness of traditional nutritional programs.

[0009] (II) Technical Solution The technical solution of the present invention is as follows: In a first aspect, the present invention provides a multifunctional strain of *Lactobacillus paracasei*, whose Latin name is... Lacticaseibacillus paracaseiZG1, with accession number CGMCC No.36863, deposited on December 1, 2025, is deposited at the China General Microbiological Culture Collection Center of the China Microbiological Culture Collection Committee.

[0010] Preferably, the multifunctional *Lactobacillus paracasei* strain has the function of promoting neurocognitive development, immune system maturation, and bone growth.

[0011] In a second aspect, the present invention provides a probiotic preparation comprising the aforementioned *Lactobacillus paracasei* strain.

[0012] Preferably, the probiotic preparation is one or more of the following: food raw materials, probiotic powder, food additives, feed additives, liquid beverages, health products, or medicines.

[0013] Preferably, the probiotic preparation further includes excipients, which include one or more of fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, and inulin.

[0014] Preferably, the probiotic preparation is a solid or liquid preparation, and it contains 1 × 10⁻⁶ cells of Lactobacillus paracasei ZG1. 5 CFU~1×10 12 CFU / g or 1×10 5 CFU~1×10 12 CFU / mL.

[0015] Thirdly, the application of the aforementioned *Lactobacillus paracasei* ZG1 in the preparation of formulations for promoting growth and development.

[0016] Preferably, the formulation is a solid or liquid formulation, wherein the quantity of *Lactobacillus paracasei* ZG1 is 1 × 10⁻⁶. 5 CFU~1×10 12 CFU / g or 1×10 5 CFU-1×10 12 CFU / mL.

[0017] Preferably, the promotion of growth and development includes promoting the neurocognitive development, immune system maturation, and bone growth of the subject, especially promoting the neurocognitive development, immune system maturation, and bone growth of young animals or children.

[0018] (III) Beneficial Effects The present invention relates to *Lactobacillus paracasei* ZG1 (classified as *Lactobacillus paracasei*). Lacticaseibacillus paracasei(CGMCC No. 36863, deposited on December 5, 2025), compared to existing technologies, it has significant and irreplaceable beneficial effects. Its core innovation lies in breaking through the limitations of traditional probiotics' single function, achieving synergistic promotion in three key areas: neurocognitive development, immune system maturation, and bone growth. It also solves the inherent defects of mixed probiotic solutions, specifically in the following aspects: (1) Promote neurocognitive development and enhance core cognitive abilities Rigorous validation through open field and Y-maze experiments demonstrated that this strain significantly enhances the autonomous exploration behavior of larvae. ZG1-treated larvae spent significantly more time in the central region of the open field compared to the control group (p<0.05), and exhibited a marked reduction in anxiety-like behaviors. Simultaneously, it effectively improved spatial working memory, with a significantly higher spontaneous alternation rate in the Y-maze compared to the control group. This indicates that the strain can directly act on key aspects of early-life nervous system development, laying a solid foundation for long-term cognitive development, and its effects are superior to conventional probiotics that only target gut health.

[0019] (2) Promote the maturation of the immune system and build a stable immune barrier This strain can regulate the immune system function in multiple dimensions: on the one hand, it significantly increases the levels of immunoglobulins IgA and IgG in colon tissue, strengthens the defense capabilities of mucosal immunity and humoral immunity, and provides key support for larvae to resist pathogen invasion; on the other hand, it promotes the increase of interferon (IFN-γ) levels by precisely regulating the balance of cytokines, while downregulating the expression of interleukin 1β (IL-1β), promoting Th1 immune polarization, effectively maintaining the balance of Th1 / Th2 immune response, and avoiding immune dysregulation. Compared with probiotics that only regulate immunoglobulins or cytokines, it has a more comprehensive immune regulatory value.

[0020] (3) Promote bone growth and lay a solid foundation for bone development This strain synergistically promotes bone growth at three levels: morphology, metabolism, and genes. Morphologically, it significantly increases femur length and bone volume fraction (BV / TV) in young mice, with the ratios of femur length to body length and bone volume fraction to body weight being higher than those in the control group, excluding confounding factors such as body weight and confirming its direct promoting effect on bone mass increase. Metabolically, it significantly increases the levels of bone formation markers such as serum growth hormone (GH), osteocalcin (OCN), and type I procollagen N-terminal propeptide (PINP), while upregulating serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 levels, enhancing osteoblast differentiation and bone matrix mineralization, and accelerating bone formation metabolism. Genetically, it specifically upregulates the transcriptional expression of osteogenic-related genes such as bone morphogenetic protein (BMP2), osteoprotegerin (OPG), and RUNX2 in the femur, without significantly adverse effects on bone resorption-related genes. This molecular mechanism ensures the scientific validity and safety of bone growth, and compared to traditional nutritional supplements that only increase bone density, it has a more systematic effect on promoting bone development.

[0021] (4) Alternative probiotic regimens to avoid competitive antagonistic risks This invention achieves multiple development-promoting functions with a single strain, fundamentally solving the core pain point of existing technologies where "multi-functionality requires mixing multiple strains": First, it avoids the competitive inhibition of nutrients between different strains in the gut. This strain does not need to compete with other probiotics for nutrient resources, resulting in more stable survival efficiency and function in the host gut, and preventing partial functional failure due to competition between strains. Second, it completely eliminates the risk of functional antagonism between strains, eliminating concerns about mutual inhibition between probiotic combinations and ensuring that the three major promoting functions of nerve, immunity, and bone can all be effectively and synergistically exerted. Third, it simplifies the formulation development and application process. The fermentation and formulation process of a single strain is simpler, the dosage control is more precise, the production cost is lower, and the metabolites are clearly defined, eliminating the risk of unknown metabolite interactions that may be caused by mixed strains. Safety and stability are significantly improved, providing great convenience for industrial application.

[0022] In summary, the multifunctional Lactobacillus paracasei ZG1 of this invention not only achieves a breakthrough in promoting growth and development through multi-system synergy, but also solves the multiple drawbacks of traditional nutritional programs and mixed probiotic programs. It provides a safe, efficient, and comprehensive solution for early life growth and development, especially offering a targeted improvement pathway for children with growth retardation. At the same time, it has broad application prospects in the fields of food, health products, and feed additives, and possesses significant industrialization value, academic reference value, and public health significance. Attached Figure Description

[0023] Figure 1 The results of experiments on the effects of Lactobacillus paracasei ZG1 on open field behavior in young rats.

[0024] Figure 2 The results show the effect of Lactobacillus paracasei ZG1 on the spontaneous alternation rate in the Y-maze experiment of young mice.

[0025] Figure 3 The results show the effects of Lactobacillus paracasei ZG1 on colonic immunoglobulins in young mice.

[0026] Figure 4 The results show the effects of Lactobacillus paracasei ZG1 on colonic cytokine levels in young mice.

[0027] Figure 5 The results show the effects of Lactobacillus paracasei ZG1 on femoral length in young rats.

[0028] Figure 6 The results show the effects of Lactobacillus paracasei ZG1 on the bone volume fraction of young mice.

[0029] Figure 7 This is the experimental result of the effect of Lactobacillus paracasei ZG1 on serum bone turnover markers and bone metabolism-related hormone levels in young mice.

[0030] Figure 8 This is the experimental result regarding the effect of Lactobacillus paracasei ZG1 on the transcriptional level of femoral bone turnover marker genes in young mice. Detailed Implementation

[0031] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Unless otherwise specified, all methods used in the following examples are conventional methods, and all reagents used are commercially available unless otherwise specified. The ZG1 strain involved in the following examples is classified as *Lactobacillus paracasei*. Lacticaseibacillus paracasei The accession number is CGMCC No. 36863, and the accession date is December 5, 2025. In the embodiments of the present invention, the mouse is a conventional model organism in the prior art and is commercially available.

[0033] The *Bifidobacterium animalis* subsp. *lactospirum* BAL531, *Bifidobacterium bifidum* B11, and *Lactococcus lactis* subsp. *lactospirum* S28 used as controls in the following experiments are all existing technologies and can be obtained through purchase. The preservation number for *Bifidobacterium animalis* subsp. *lactospirum* BAL531 is CGMCC No. 17329, for *Lactococcus lactis* subsp. *lactospirum* S28 it is CGMCC No. 23686, and for *Bifidobacterium bifidum* B11 it is CGMCC No. 24381.

[0034] Example 1 This embodiment investigates the promoting effect of probiotics on the neurocognitive development of young mice. The experimental steps are as follows: (1) Experimental animals: Healthy SPF-grade 6-week-old Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were housed in a controlled environment at a temperature of 20-24℃ and a humidity of 50%, following a 12-hour light / dark cycle. Mating was carried out in a female-to-male ratio of 2:1, and mice with vaginal plugs were considered pregnant and used for future experiments. Female offspring after giving birth were used as experimental subjects.

[0035] (2) Animal grouping: Newborn female offspring mice were randomly divided into 5 groups, with 6 mice in each group: control group (referred to as Control group), Bifidobacterium lactis subsp. BAL531 group (referred to as BAL531 group), Bifidobacterium bifidum B11 group (referred to as B11 group), Lactococcus lactis subsp. S28 group (referred to as S28 group), and Lactobacillus paracasei ZG1 group (referred to as ZG1 group).

[0036] (3) Intervention methods: Newborn mice were allowed free access to breast milk from birth to 1 week of age; from the 2nd to the 3rd week, mice were allowed free access to breast milk, while simultaneously receiving physiological saline (control group) and probiotic supplements (intervention group) via gavage; at 4-5 weeks of age, mice were fed normal diet and drank sterile water, and were given physiological saline (control group) and probiotic supplements (intervention group) by gavage daily. The gavage dose for each probiotic intervention group was 1×10⁻⁶. 9 CFU / day / mouse was administered via gavage in the form of bacterial suspension; the control group was administered an equal volume of physiological saline daily. The following experimental tests were conducted on mice in each group when they reached 5 weeks of age.

[0037] (4) Analysis of indicators promoting neurocognitive development Open field experiment: The experimental setup is 50 cm. A 50cm square acrylic area was prepared, with the edges 50cm high. Each group of young mice was slowly placed 10cm from the edge, facing the center, and their movement was recorded (for 10 minutes). The central region was defined as a 20 cm area. The percentage of mice exploring the central region was calculated as the time spent in the central region divided by the total time spent exploring it. Compared to the control group, the ZG1 group showed a significantly increased time spent in the central region (p<0.05), indicating reduced autonomous exploration ability and anxiety-like behavior.

[0038] Y-maze experiment: The experimental setup consists of three wooden arms at 120° angles to each other, labeled A, B, and C. The test mice are placed at the intersection of the Y-shaped maze and allowed to explore freely for 8 minutes. The total number of times the mouse enters each arm (N) and the order in which they enter the arms are recorded. Three consecutive entries into different arms are counted as one correct alternation response. The number of correct alternation responses is recorded, and the alternation response rate is calculated. Alternation response rate (%) = Number of correct alternation responses / (N-2) 100%. The Y-maze experiment is based on the spontaneous exploration of novel spaces by mice, assessing working memory through three-arm spatial alternation behavior. The core indicator is the spontaneous alternation rate, i.e., the proportion of mice that enter three different arms consecutively. Compared with the blank control group, the spontaneous alternation rate of the ZG1 group of young mice was significantly improved, indicating that their spatial working memory ability was enhanced.

[0039] (5) Experimental results The experimental results of the open field experiment can be found in [link to relevant documentation]. Figure 1 . Figure 1 The horizontal axis represents different intervention groups, including the control group (administered via gavage with saline), the BAL531 group, the B11 group, the S28 group (all other probiotic intervention groups), and the ZG1 group. The vertical axis represents the "percentage of time spent in the central area," which refers to the percentage of time the young mice spent in the central area (20cm × 20cm) of the open field experiment, out of the total experimental time (10 minutes). The core significance of this indicator is that a higher percentage indicates stronger autonomous exploration ability and less anxiety-like behavior in the young mice. Figure 1 As shown, the central region dwell time percentage in the control group was approximately 10%, representing the baseline state of the young mice before effective probiotic intervention. Among the other probiotic groups (BAL531, B11, S28), only the percentage in the Bifidobacterium animalis subsp. lactis BAL531 group was slightly lower than that in the control group, indicating that BAL531 had no promoting effect on the "neurocognitive development" of young mice. Furthermore, compared to the control group, the B11 group had approximately 16.5%, while the S28 group had approximately 12%, indicating that Bifidobacterium bifidum B11 and Lactococcus lactis subsp. lactis S28 had some promoting effect on the "neurocognitive development" of young mice, but neither showed a significant improvement effect. Compared to the three existing probiotics BAL531, B11, and S28, the central region dwell time percentage in the ZG1 group was close to 20%, and the difference in this group compared to the control group was statistically significant. p<0.05).

[0040] The results of the Y-maze experiment can be found in [link to experiment]. Figure 2 The horizontal axis of the graph represents different intervention groups, including the control group (administered via gavage with saline), the BAL531 group, the B11 group, the S28 group (other probiotic intervention groups), and the ZG1 group. The vertical axis represents the "spontaneous alternation rate," a core indicator for assessing spatial working memory in the Y-maze experiment (calculated as "number of correct alternation responses / (total number of arm advances - 2) × 100%)." A higher rate indicates stronger exploratory memory of novel spaces and better spatial working memory in the young mice. Figure 2As shown, the spontaneous alternation rate in the control group was approximately 50%, representing the baseline spatial memory level of young mice without effective probiotic intervention. Other probiotic groups (BAL531, B11, S28) showed similar rates to the control group (approximately 50%), while the BAL531 group and S28 group had slightly higher rates (approximately 52%) compared to the control group. However, none of these groups showed significant differences compared to the control group, indicating no significant improvement in memory. The spontaneous alternation rate in the ZG1 group was close to 60%, and the difference was statistically significant compared to the control group. p<0.05).

[0041] The above experimental results show that the spontaneous alternation rate of young mice after intervention with Lactobacillus paracasei ZG1 was significantly increased, indicating that this strain can effectively enhance the spatial working memory ability of young mice and has the technical effect of "promoting the neurocognitive development of young mice".

[0042] Example 2 Newborn female offspring mice were grouped and subjected to probiotic gavage intervention according to the method in Example 1. The intervention dosage and method were the same as in Example 1. When the mice in each group reached 5 weeks of age, the effects of different probiotic intervention groups on the immune system development indicators of young mice were tested.

[0043] Colonic immunoglobulin levels were measured according to the kit instructions: immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin E (IgE); colonic cytokine levels were measured: tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). The experimental results are as follows: Figures 3-4 As shown.

[0044] See Figure 3 At 35 days of age, compared with the blank control group, the levels of immunoglobulin A (IgA) and immunoglobulin G (IgG) in the colon of young mice in the ZG1 group were significantly increased, reaching 0.7 ng / mg protein (compared to 0.4 ng / mg IgA and 150 ng / mg IgG in the control group, respectively). The experimental results indicate that *Lactobacillus paracasei* ZG1 promotes the maturation of the immune system in young mice. In particular, the increase in immunoglobulin A (IgA) in the colon of young mice treated with ZG1 was even higher than that of the commercially available *Bifidobacterium animalis* subsp. *lactospirae* BAL531.

[0045] like Figure 4As shown, at 35 days of age, compared with the control group, ZG1 significantly promoted an increase in interferon-γ (IFN-γ) levels and downregulated interleukin-1β (IL-1β) levels, indicating that it can promote Th1 immune polarization and help maintain Th1 / Th2 balance. Among these, from... Figure 4 As shown in (A), the serum level of IL-6 (pro-inflammatory factor) in the control group was approximately 80 pg / mg protein. Compared with the control group, the serum levels in the BAL531 and B11 groups were slightly decreased, while the serum levels in the S28 and ZG1 groups decreased to approximately 40 pg / mg protein and 50 pg / mg protein, respectively, and the differences from the control group were statistically significant. p<0.05). From Figure 4 As shown in (B), the serum IL-1β (pro-inflammatory factor) level in the control group was approximately 15 pg / mg protein. Compared with the control group, the serum levels in the BAL531, B11, and S28 groups were slightly decreased, but without significant change; while the serum level in the ZG1 group was significantly reduced to approximately 5 pg / mg protein, and the difference compared with the control group was statistically significant. p<0.05). From Figure 4 As shown in (C), the serum TNF-α (pro-inflammatory factor) level in the control group was approximately 8 pg / mg protein; the serum level in the BAL531 group showed almost no fluctuation, while the serum level in the B11 group showed a slight increase, though not significant; the serum levels in the S28 and ZG1 groups decreased to approximately 4 pg / mg protein and 5 pg / mg protein, respectively. These findings indicate that *Lactobacillus paracasei* ZG1 can reduce pro-inflammatory responses. Figure 4 As shown in (D), the serum IL-10 (anti-inflammatory factor) level in the control group was approximately 30 ng / mg protein; the serum level in the BAL531 group significantly increased to approximately 60 ng / mg protein (“ "(p<0.05) indicates that the serum protein levels in the B11 and ZG1 groups increased to approximately 42 ng / mg protein, and the anti-inflammatory capacity of the young mice was enhanced after intervention with probiotic ZG1. Figure 4 As shown in (E), the serum IFN-γ (Th1 cytokine) level in the control group was approximately 1.0 ng / mg protein, while the serum level in the BAL531 group significantly increased to approximately 1.3 ng / mg protein (*p<0.05). The serum levels in the S28 and B11 groups decreased, while the serum level in the ZG1 group increased to approximately 1.2 ng / mg protein, indicating that the Th1 immune polarization trend in young mice was enhanced after intervention with probiotic ZG1.

[0046] The above experimental conclusions, combined with cytokine function (IL-1β / IL-6 / TNF-α are pro-inflammatory factors, IFN-γ drives Th1 immunity, and IL-10 is an anti-inflammatory factor), verify the immunomodulatory effect of Lactobacillus paracasei ZG1: it can significantly inhibit the expression of pro-inflammatory factors, slightly increase the levels of anti-inflammatory factors and Th1 cytokines, help maintain immune balance, promote the maturation of the immune system, and has the effect of promoting the development of the immune system.

[0047] Example 3 Newborn female offspring mice were grouped and administered probiotics via gavage according to the method in Example 1. The intervention dosage and method were the same as in Example 1. From 3 weeks of age to the end of 5 weeks of age, the following bone growth and development indicators were analyzed: (1) Detection of femoral length and femoral volume fraction The weight of the young mice was measured weekly starting at 3 weeks of age. At the end of the experiment in week 5, the mice were euthanized, the right femur was dissected, and the soft tissue was separated. The length was measured using calipers. See [link to results]. Figure 5 .like Figure 5 As shown, the femur length of the control group mice was 11.75 mm, and there was no significant difference compared with the control group in the BAL531, B11, and S28 groups; while the femur length of the ZG1 group mice was approximately 12 mm, and the difference compared with the control group was statistically significant. (p<0.05). The results shown in the figure indicate that at 35 days of age, the femur length of the ZG1 group mice was significantly higher than that of the blank control group. Further significance analysis of the ratio of femur length to body length showed that the ratio of femur length to body length of the ZG1 group mice was higher than that of the blank control group.

[0048] To eliminate the influence of body weight on bone volume fraction, the ratio of bone volume fraction to body weight was further analyzed. For example... Figure 6 As shown, the bone volume fraction of the control group pups was approximately 10.65%, and there was no significant change compared to the control group in the BAL531, B11, and S28 groups; while the bone volume fraction of the ZG1 group pups was approximately 12%, and the difference compared to the control group was statistically significant. p<0.05).

[0049] The above results indicate that the femur length and bone volume fraction (BV / TV) of the Lactobacillus paracasei ZG1 group were significantly higher than those of the control group. These results demonstrate that Lactobacillus paracasei ZG1 intervention can significantly promote bone mass increase in young mice.

[0050] (2) Detection of bone turnover markers and bone metabolism-related hormone levels According to the kit instructions, changes in serum growth hormone (GH), osteocalcin (OC / OCN), type I procollagen N-terminal propeptide (PINP), type I collagen C-terminal peptide (CTX-I), and serum vitamin D and parathyroid hormone (PTH) levels were measured. For bone turnover markers and bone metabolism-related hormone level detection, please refer to [link to kit instructions]. Figure 7 .

[0051] At 35 days of age, the ZG1 group significantly promoted an increase in serum growth hormone (GH), osteocalcin (OCN), and serum bone formation marker type I procollagen N-terminal propeptide (PINP) levels in young mice. Specifically, Figure 7 Figure (A) shows the changes in GH (growth hormone, unit: ng / ml). Growth hormone is the core hormone that promotes bone growth, and the higher the serum level, the more conducive it is to bone formation. Among them, the control group was about 190 ng / ml; the BAL531 group was slightly lower, the B11 and S28 groups were slightly higher; and the ZG1 group was significantly higher to about 230 ng / ml. (p<0.01) indicates that ZG1 can significantly increase growth hormone levels, directly promoting bone growth. Figure 7 Figure (B) shows the changes in PTH (parathyroid hormone, unit: pg / ml). PTH promotes osteoblast differentiation and enhances bone matrix mineralization, and is a key regulatory hormone for bone development. The control group had approximately 65 pg / ml; the BAL531 and S28 groups showed no significant changes, the B11 group showed a slight increase, and the ZG1 group showed a significant increase to approximately 84 pg / ml. (p<0.05), effectively enhancing osteogenic physiological processes. For example... Figure 7 Figure (C) shows the changes in 1,25-dihydroxyvitamin D3 (active vitamin D, unit: pg / ml). 1,25-dihydroxyvitamin D3 participates in intestinal calcium absorption and bone mineralization, and is an important nutritional regulator of bone development; its control group had approximately 190 pg / ml; the BAL531 group showed a slight decrease, B11 remained unchanged, and the S28 group showed a slight increase to approximately 210 pg / ml; ZG1 significantly increased to approximately 220 pg / ml (*p<0.05), effectively promoting calcium deposition and mineralization in bone tissue. Figure 7 Figure (D) shows the changes in OCN (osteocalcin, unit: ng / ml). OCN is a specific marker of bone formation and directly reflects the activity of osteoblasts. In the figure, the control group is about 400 ng / ml; the BAL531 group is slightly decreased, the B11 and S28 groups are slightly increased; and the serum OCN level in the ZG1 group is significantly increased. (p<0.01) indicates that osteoblast activity was significantly activated. Figure 7Figure (E) shows the changes in PINP (N-terminal propeptide of type I procollagen, unit: μg / ml). PINP is a marker of bone collagen synthesis, representing the rate of bone matrix formation. In the figure, the control group had approximately 60 μg / ml; the BAL531 and B11 groups showed no significant changes, the S28 group showed a slight increase, and the ZG1 group had a significantly elevated serum PINP level. (p<0.05) indicates accelerated collagen synthesis and enhanced bone formation metabolism. For example... Figure 7 Figure (F) shows the changes in CTX-I (type I collagen cross-linked C-terminal peptide, unit: ng / ml). CTX-I is a marker of bone resorption, reflecting osteoclast activity. In the figure, the serum level in the control group was approximately 30 ng / ml, while it was significantly lower in the BAL531 group. (p<0.01) Compared with the control group, the B11 group showed almost no change, the S28 group showed a slight decrease, and the ZG1 group also showed a significant decrease, indicating that ZG1 did not over-activate bone resorption and maintained the balance between bone formation and bone resorption. Therefore, *Lactobacillus paracasei* ZG1 can significantly increase the levels of bone growth hormone (GH), bone formation markers (OCN, PINP), and bone mineralization regulators (PTH, 1,25-dihydroxyvitamin D3), while maintaining stable bone resorption, thus demonstrating a technical effect of promoting skeletal growth and development in young mice.

[0052] The above results indicate that *Lactobacillus paracasei* ZG1 can significantly promote bone formation metabolism in mice. 1,25-dihydroxyvitamin D3 can enhance osteoblast differentiation and promote bone matrix mineralization; short-term low-dose elevation of parathyroid hormone levels can promote osteoblast proliferation and differentiation, increasing bone formation. At 35 days of age, the serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 levels in the ZG1 group were significantly higher than those in the blank control group. These results suggest that *Lactobacillus paracasei* ZG1 may promote bone growth in young mice by enhancing osteoblast differentiation.

[0053] (3) Detection of transcription of femoral bone turnover marker genes The femur was ground and disrupted with liquid nitrogen. RNA was extracted, quantified using a template, reverse transcribed, and analyzed by RT-qPCR for changes in the transcriptional levels of genes such as bone morphogenetic protein (BMP), osteopontin (OPN), RANKL, RUNX2, TRAF6, and NFATc1. For the detection of femoral bone turnover marker gene transcription, please refer to [link to relevant documentation]. Figure 8 .

[0054] like Figure 8 As shown in (A), probiotic intervention in each group significantly upregulated the transcription level of the femoral bone morphogenetic protein (BMP2) gene in young mice, especially in the BAL531 and ZG1 groups, where the upregulation of BMP2 was very significant. The upregulation of BMP2 in groups B11 and S28 was also significant. ).like Figure 8 As shown in (B) and (C), the BAL531 group and the ZG1 group significantly promoted the upregulation of the relative expression levels of osteoprotegerin (OPG) and RUNX2 gene mRNA in the femur of young mice. ).like Figure 8 As shown in (D), BAL531 has a slight but not significant effect on the relative expression level of NFATc1 mRNA. Figure 8 As shown in (E), group B11 upregulated the relative expression level of TRAF6 mRNA. );like Figure 8 As shown in (F), the BAL531 group upregulated the relative expression level of RANKL mRNA. Other probiotic interventions (S28, B11, ZG1) had no significant effect on the expression of bone resorption-related genes such as RANKL, TRAF6, and NFATc1.

[0055] The results show that *Lactobacillus paracasei* ZG1 specifically upregulates the transcription of osteogenic-related genes such as BMP2, OPG, and RUNX2, while having no significant effect on bone resorption-related genes such as RANKL, TRAF6, and NFATc1. Thus, at the molecular level, it is demonstrated that *Lactobacillus paracasei* ZG1 primarily promotes bone growth by enhancing bone formation metabolism (rather than enhancing bone resorption), further validating its technical efficacy in promoting skeletal development in young mice and explaining the specificity and safety of its bone growth-promoting effect.

[0056] The results indicate that *Lactobacillus paracasei* ZG1 may promote bone growth primarily by stimulating bone formation metabolism in mice. These experimental data fully demonstrate that *Lactobacillus paracasei* ZG1 has a significant and comprehensive role in promoting neural development, immune maturation, and bone growth in young mice.

[0057] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multifunctional strain of *Lactobacillus paracasei*, its Latin name is Lacticaseibacillus paracasei ZG1, with accession number CGMCC No.36863, deposited on December 5, 2025, is deposited at the China General Microbiological Culture Collection Center of the China Microbiological Culture Collection Committee.

2. The *Lactobacillus paracasei* strain according to claim 1, characterized in that, The multifunctional Lactobacillus paracasei strain has the function of promoting neurocognitive development, immune system maturation and bone growth.

3. A probiotic preparation, characterized in that, It contains the Lactobacillus paracasei strain as described in claim 1.

4. The probiotic preparation according to claim 3, characterized in that, The probiotic preparation is one or more of the following: food raw materials, probiotic powder, food additives, feed additives, liquid beverages, health products, or medicines.

5. The probiotic preparation according to claim 3, characterized in that, The probiotic preparation also includes excipients, which include one or more of fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, and inulin.

6. The probiotic preparation according to claim 3, characterized in that, The probiotic preparation is a solid or liquid preparation, and it contains 1 × 10⁻⁶ Lactobacillus paracasei ZG1 cells. 5 CFU~1×10 12 CFU / g or 1×10 5 CFU-1×10 12 CFU / mL.

7. The use of Lactobacillus paracasei ZG1 as described in claim 1 in the preparation of a formulation for promoting growth and development.

8. The application according to claim 7, characterized in that, The formulation is a solid or liquid formulation, wherein the number of *Lactobacillus paracasei* ZG1 is 1 × 10⁻⁶. 5 CFU~1×10 12 CFU / g or 1×10 5 CFU-1×10 12 CFU / mL.

9. The application according to claim 7, characterized in that, The promotion of growth and development includes promoting neurocognitive development, immune system maturation, and bone growth.

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