A bifidobacterium bifidum immune-enhancing composition for increasing iga levels

Abstract

The present invention discloses a Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels, belonging to the technical field of microecological preparations. The composition comprises an active probiotic component, an inactivated postbiotic component, and an excipient component. The active probiotic component includes Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus live freeze-dried powders, each present in 10–30 parts, with a total of 30–90 parts and a viable count of 1×10⁹–1×10¹¹ CFU / g. A synergistic system formed by combining multiple probiotics with a heat-inactivated postbiotic enhances IgA levels. A double-layer encapsulation structure and optimized excipients improve bioavailability. A standardized process ensures stability and activity, thereby promoting intestinal health and immunity, with broad application potential.

Description

[0001] DESCRIPTION

[0002] A Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels

[0003] TECHNICAL FIELD

[0004] The present invention relates to the technical field of microecological preparations, and in particular to a Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels.

[0005] BACKGROUND ART

[0006] Research on the relationship between the intestinal microecology and the immune function of the body has continued to deepen. As important substances for regulating the balance of the intestinal flora, probiotics have attracted extensive attention in the field of immune enhancement. Immunoglobulin A, as the core antibody of mucosal immunity, is an important line of defense by which the body resists invasion by external pathogens. Increasing its level has become a key direction for enhancing mucosal immunity and improving the overall immune capacity of the body. Probiotic strains such as bifidobacteria and lactobacilli have been demonstrated to regulate the structure of the intestinal flora, stimulate activation and proliferation of intestinal mucosal immune cells, and thereby affect the secretion level of immunoglobulin A. Postbiotics, as derivative products of probiotics, retain part of the biological activity of probiotics and can form synergistic effects with probiotics to further strengthen immunomodulatory efficacy. Accordingly, research and development of immune-enhancing compositions based on the compounding of probiotics and postbiotics has become an important research direction in the current field of microecological immunity.

[0007] Conventional immune-enhancing probiotic products generally employ a single strain or a simply compounded probiotic combination, lack scientific matching with postbiotics, and thus find it difficult to maximize the immunomodulatory effect. In addition, certain products do not effectively DESCRIPTION protect probiotics, and the probiotics are readily inactivated when passing through gastric acid, bile, and similar environments, thereby failing to reach the intestine for colonization and action and greatly reducing the actual application effect of the product. Meanwhile, conventional postbiotic preparation processes lack standardized control, readily resulting in loss of biologically active components, and the compounding ratio with probiotics lacks a scientific basis, making it difficult for the two to form a stable synergistic system. Furthermore, conventional excipient combinations generally consider only basic shaping and palatability and are not specifically designed in light of the characteristics of probiotics and postbiotics, thereby further affecting the stability and efficacy of the product and failing to satisfy market demand for efficient and stable immune-enhancing microecological products.

[0008] SUMMARY OF THE INVENTION

[0009] An object of the present invention is to overcome the deficiencies of the prior art by providing a Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels. The composition is formed by scientifically proportioning active probiotics, an inactivated postbiotic, and compounded excipients. The probiotics comprise a combination of Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus; the inactivated postbiotic is heat-inactivated Lactococcus lactis freeze-dried powder; and the excipients are proportionally compounded from multiple components and designed to form a double-layer encapsulation structure. Preparation is carried out through standardized anaerobic and aerobic fermentation processes combined with low-temperature centrifugation, precise heat inactivation, vacuum freeze-drying, and the like, thereby ensuring the activity and efficacy stability of the respective components. The composition realizes a synergistic effect between probiotics and postbiotics, effectively increases mucosal IgA levels in the body, is easy to industrialize, DESCRIPTION and can be widely used in foods, health foods, and related fields, thereby providing both practical utility and industrial value.

[0010] To solve the above technical problems, the present invention provides the following technical solutions. In one aspect, the present invention provides a Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels, wherein the composition comprises, in parts by weight, an active probiotic component, an inactivated postbiotic component, and an excipient component;

[0011] The active probiotic component comprises Bifidobacterium bifidum live freeze-dried powder, Bifidobacterium longum live freeze-dried powder, and Lactobacillus rhamnosus live freeze-dried powder, wherein the Bifidobacterium bifidum live freeze-dried powder is present in 10-30 parts, the Bifidobacterium longum live freeze-dried powder is present in 10-30 parts, and the Lactobacillus rhamnosus live freeze-dried powder is present in 10-30 parts; the total amount of the active probiotic component is 30-90 parts by weight, and the total viable count is 1x10A9 CFU / g-lxl0Al l CFU / g;

[0012] The inactivated postbiotic component is heat-inactivated Lactococcus lactis freeze-dried powder, present in an amount of 15-60 parts by weight, with a cell count of 5x10A8 cells / g-2xlOAl l cells / g;

[0013] The total amount of the excipient component is 20-80 parts by weight and comprises 5-30 parts of prebiotics, 3-25 parts of a freeze-drying protectant, 10-35 parts of a filler, 0.5-5 parts of a lubricant, 2-15 parts of a flavoring agent, and 4-25 parts of an encapsulation wall material.

[0014] Further, the weight ratio of the live freeze-dried powders of Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is from 1:1: 1 to 3:3:3, and the viable count in the live freeze-dried powder of each individual strain is lxl0A10 CFU / g-lxlOA12 CFU / g.

[0015] Furthermore, the ratio of the total cell count in the inactivated postbiotic component to the total viable count in the active probiotic component is DESCRIPTION

[0016] 0.5: 1-2:1; wherein the cell content of the heat-inactivated Lactococcus lactis freeze-dried powder is lxl0A10 CFU / g-2xlOA12 CFU / g.

[0017] Furthermore, the prebiotic is a proportional blend of fructooligosaccharide, galactooligosaccharide, and inulin at 1:1 :1; and the freeze-drying protectant is a proportional blend of skim milk powder, trehalose, and mannitol at 1:1 :1.

[0018] Furthermore, the filler is a proportional blend of microcrystalline cellulose and maltodextrin at 1:1; the lubricant is a proportional blend of magnesium stearate and talc at 1:1, with a total amount of 1-4 parts by weight; and the flavoring agent is a proportional blend of xylitol, erythritol, and natural fruit powder at 1:1: 1.

[0019] Furthermore, the encapsulation wall material is a proportional blend of hydroxypropyl methylcellulose, resistant starch, and gelatin at 1:1: 1, and is used to form a double-layer encapsulation structure in which the active probiotic component is encapsulated in the inner layer and the inactivated postbiotic component is encapsulated in the outer layer.

[0020] Furthermore, the inactivated postbiotic component is formed by compounding heat-inactivated Lactococcus lactis freeze-dried powder with fermentation metabolites of the strain at a weight ratio of 1 :(0.3-l), wherein the fermentation metabolites comprise exopolysaccharides and small-molecule peptides.

[0021] Furthermore, the specific steps of the preparation method of the composition are as follows:

[0022] 51. Preparation of wet active probiotic cells: activating and expanding each active probiotic strain, followed by centrifugation to collect and wash the cells;

[0023] 52. Preparation of active probiotic freeze-dried powder: adding a freeze-drying protectant to the wet active probiotic cells for resuspension, followed by vacuum freeze-drying to obtain freeze-dried powder of each DESCRIPTION strain;

[0024] 53. Preparation of inactivated postbiotic freeze-dried powder: activating and expanding the postbiotic strain, followed by centrifugation to collect and wash the cells; after heat-inactivation verification, adding a freeze-drying protectant and vacuum freeze-drying to obtain inactivated postbiotic freeze-dried powder;

[0025] 54. Preparation of the finished composition: compounding the freeze-dried powders of the active probiotic strains, mixing with the inactivated postbiotic freeze-dried powder and excipient components, and processing into a target dosage form.

[0026] Furthermore, after two-stage anaerobic activation at 37 C, each active probiotic strain is inoculated into a fermentation medium having an initial pH of 6.0-6.5 at an inoculation amount of 2%-5%, anaerobically fermented at 37 C under nitrogen with stirring at 150-250 r / min for 16-24 h, and the fermentation broth is centrifuged at 4 C and 6000-10000 r / min for 10-20 min, followed by resuspension and washing twice with sterile normal saline at a volume ratio of 1:2- 1:4.

[0027] Furthermore, after two-stage aerobic activation at 37 C, the postbiotic strain is inoculated into a fermentation medium at an inoculation amount of 3% and aerobically cultured at 37 C for 18 h; the fermentation broth is centrifuged at 4 C and 6000-8000 r / min for 10-15 min, washed twice with sterile normal saline, and resuspended to a bacterial suspension concentration of lxl0Al l-lxlOA12 CFU / mL, then treated at 95-105 C for 20-40 min; after plate verification confirming the absence of viable bacteria, a freeze-drying protectant is added and vacuum freeze-drying is carried out to obtain the inactivated postbiotic freeze-dried powder.

[0028] Compared with the prior art, the Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels has the following DESCRIPTION beneficial effects:

[0029] I. The present invention forms a synergistic system by compounding multiple active probiotics with a heat-inactivated Lactococcus lactis postbiotic, thereby specifically increasing IgA levels in the body and achieving an efficient immune-enhancing effect. The active probiotic component can regulate the balance of the intestinal microecology, while the inactivated postbiotic component can trigger an intestinal mucosal immune response by means of the bacterial cells themselves and their metabolites. The combination of the two greatly improves the specificity and effectiveness of immune activation. The design of the double-layer encapsulation structure realizes phase-separated protection of the probiotics and postbiotics, not only ensuring that the active probiotics successfully pass through the gastric acid environment to reach the intestine for colonization, but also allowing the postbiotics to be released at an appropriate site to exert their function, thereby significantly improving the bioavailability of the active components in the composition. Meanwhile, the scientifically compounded excipients further optimize the stability and palatability of the composition, making the immune-enhancing effect easier to sustain.

[0030] II. The present invention precisely controls the production process of probiotics and postbiotics through a standardized preparation process, thereby ensuring the activity and efficacy stability of the respective components. The active probiotics retain high activity through steps such as anaerobic fermentation and low-temperature centrifugation and washing, while the postbiotics undergo aerobic fermentation and precise heat inactivation, thereby completely retaining the immunological activity of the bacterial cells and metabolites while ensuring DESCRIPTION that no viable bacteria remain. The reasonable addition of the freeze-drying protectant during preparation effectively reduces activity loss of probiotics and postbiotics during freeze-drying and improves storage stability of the product. Meanwhile, the compounding of the respective components of the composition follows the mechanism of intestinal microecology and mucosal immunity, so that intestinal health and systemic immune capacity are improved from the dual dimensions of microecological regulation and immune activation. The composition is suitable for a wide range of application scenarios and its mechanism of action more closely accords with physiological needs.

[0031] Other advantages, objectives, and features of the present invention will be set forth in part in the following description, and in part will become apparent to those skilled in the art upon examination of the following disclosure, or may be learned from practice of the invention.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] To describe the technical solutions in the embodiments of the present invention or in the prior art more clearly, the drawings required for describing the embodiments or the prior art are briefly introduced below. Obviously, the drawings in the following description merely illustrate certain embodiments of the present invention, and those of ordinary skill in the art can derive other drawings therefrom without inventive effort.

[0034] Figure 1 is a flow chart of the preparation process of the Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels;

[0035] Figure 2 is a simplified flow chart of active probiotic preparation;

[0036] Figure 3 is a simplified flow chart of inactivated postbiotic preparation;

[0037] Figure 4 is a simplified flow chart of finished product processing.

[0038] DETAILED DESCRIPTION DESCRIPTION

[0039] In order to further illustrate the technical means and effects adopted by the present invention to achieve the intended objectives, the specific implementation, structure, features, and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0040] Example 1 :

[0041] Preparation of a Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels, the specific process being shown in Figure 1.

[0042] 51. Preparation of wet active probiotic cells:

[0043] Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus strains are each subjected to two-stage anaerobic activation at 37 C. After activation is completed, the strains are inoculated into a fermentation medium having an initial pH of 6.2 at an inoculation amount of 3% and anaerobically fermented at 37 C with stirring at 200 r / min under nitrogen for 20 h. After fermentation is completed, the fermentation broth is centrifuged at 4 C and 8000 r / min for 15 min, and after centrifugation is completed, the cells are resuspended and washed twice with sterile normal saline at a volume ratio of 1:3, thereby collecting wet cells of the respective active probiotics.

[0044] 52. Preparation of active probiotic freeze-dried powder:

[0045] A freeze-drying protectant is respectively added to the prepared wet cells of the active probiotics for resuspension, wherein the mass concentration of the freeze-drying protectant in the resuspension is 10%. The resuspended bacterial suspensions are placed in a vacuum freeze dryer and pre-frozen at -40 C for 4 h, followed by freeze-drying for 24 h under a vacuum degree of 10 Pa and a cold-trap temperature of -55 C, thereby obtaining Bifidobacterium bifidum live DESCRIPTION freeze-dried powder, Bifidobacterium longum live freeze-dried powder, and Lactobacillus rhamnosus live freeze-dried powder, as shown in Figure 2.

[0046] 53. Preparation of inactivated postbiotic freeze-dried powder:

[0047] A Lactococcus lactis strain is subjected to two-stage aerobic activation at 37 C. After activation is completed, the strain is inoculated into a fermentation medium at an inoculation amount of 3% and aerobically cultured at 37 C for 18 h. After cultivation is completed, the fermentation broth is centrifuged at 4 C and 7000 r / min for 12 min, and after centrifugation is completed, the cells are washed twice with sterile normal saline. The washed cells are resuspended with sterile normal saline to a bacterial suspension concentration of lxlOAll CFU / mL and then treated at 100 C for 30 min. After plate culture verification confirms the absence of viable bacteria, a freeze-drying protectant is added to the bacterial suspension in an amount of 8% by mass of the suspension. After thorough mixing, the suspension is pre-frozen at -40 C for 5 h and then vacuum freeze-dried for 22 h under a vacuum degree of 8 Pa and a cold-trap temperature of -60 C, thereby obtaining heat-inactivated Lactococcus lactis freeze-dried powder. The heat-inactivated Lactococcus lactis freeze-dried powder is mixed with fermentation metabolites of the strain at a weight ratio of 1 :0.6 and fully ground to uniformity, thereby obtaining inactivated postbiotic freeze-dried powder, as shown in Figure 3.

[0048] 54. Preparation of the finished composition:

[0049] In parts by weight, 20 parts of the prepared Bifidobacterium bifidum live freeze-dried powder, 20 parts of the prepared Bifidobacterium longum live freeze-dried powder, and 20 parts of the prepared Lactobacillus rhamnosus live DESCRIPTION freeze-dried powder are compounded to obtain active probiotic composite powder. The active probiotic composite powder is subjected to inner-layer encapsulation by using an encapsulation wall material, wherein the encapsulation process comprises dissolving the encapsulation wall material in deionized water to prepare a wall-material solution having a mass concentration of 15%, mixing the wall-material solution with the active probiotic composite powder at a mass ratio of 3:1, and performing encapsulation by spray granulation at an inlet air temperature of 120 C and an outlet air temperature of 60 C to obtain inner-layer-encapsulated active probiotic granules. The inactivated postbiotic freeze-dried powder is then subjected to outer-layer encapsulation by using the encapsulation wall material, with the encapsulation process being the same as that used for the inner layer. The inner-layer-encapsulated active probiotic granules are mixed with the outer-layer-encapsulated inactivated postbiotic granules, after which prebiotics, a filler, a lubricant, and a flavoring agent are added, and the mixture is blended in a mixer at 30 r / min for 30 min. After uniform mixing, granulation, sizing, and tableting are carried out to obtain tablets of the Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels, as shown in Figure 4.

[0050] Example 2:

[0051] Verification of the effect of active probiotic strain ratios on the IgA-enhancing effect of the composition.

[0052] This example is used to verify the effect of different weight ratios of the live freeze-dried powders of Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus on the IgA-enhancing effect of the composition. In all DESCRIPTION experimental groups, the total amount of the active probiotic component is kept at 60 parts by weight, and the types, amounts, and preparation process of the inactivated postbiotic component and the excipient component are the same as those in Example 1, thereby preparing immune-enhancing compositions having different ratios.

[0053] Five experimental groups are established, wherein the weight ratios of the live freeze-dried powders of Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus are set to 1: 1:1, 1:2:2, 2:2:2, 2:3:3, and 3:3:3, respectively. In all groups, the viable count in the live freeze-dried powder of each individual strain is kept the same, and each process parameter is kept uniform during preparation.

[0054] An in vitro intestinal epithelial cell co-culture model is used for the efficacy test. Each composition group is co-cultured with human intestinal epithelial cells for 48 h. An enzyme-linked immunosorbent assay is used to determine the secretory IgA content in the culture supernatant, and the proliferation quantity of probiotics in the co-culture system is simultaneously determined. Each experiment is repeated six times and the average value is used for statistical analysis.

[0055] Comparative table of the IgA-enhancing effects of compositions having different strain weight ratios: i i DESCRIPTION

[0056] The table data show that all compositions with different weight ratios can increase the content of secretory IgA in the co-culture system while simultaneously achieving effective proliferation of probiotics. Among these, Groups 1, 3, and 5, in which the three strains are compounded in equal proportions, show higher secretory IgA contents and higher probiotic proliferation multiples than Groups 2 and 4, in which the strains are not compounded in equal proportions.

[0057] Group 3, having a strain weight ratio of 2:2:2, achieves the highest secretory IgA content among all groups, representing an increase of 14.66% over Group 1 and 8.16% over Group 5, while the probiotic proliferation multiple is also at the optimum level. This indicates that the synergistic effect of the three strains is most significant at this ratio, so that intestinal epithelial cells can be stimulated to secrete IgA more efficiently while stable self-proliferation is also achieved.

[0058] In Groups 2 and 4, which use non-equal compounding ratios, both the secretory IgA content and the probiotic proliferation multiple decrease significantly. This is because, when the strain ratio is imbalanced, metabolic competition arises among the different strains, thereby suppressing the synergistic proliferation effect of the strains and consequently reducing stimulation of IgA secretion.

[0059] All experimental strain ratios can achieve an effective IgA-enhancing effect, wherein the equal-ratio compounding groups show effects significantly superior to those of the non-equal -ratio groups, and a weight ratio of 2:2:2 is the optimal ratio. DESCRIPTION

[0060] Example 3 :

[0061] Verification of the effect of the cell-count ratio between the inactivated postbiotic and the active probiotics on the performance of the composition.

[0062] This example is used to verify the effect of different ratios between the total cell count of the heat-inactivated Lactococcus lactis freeze-dried powder and the total viable count of the active probiotic component on the performance of the composition. All experimental groups use the 2:2:2 strain weight ratio determined in Example 2, keep the total viable count of the active probiotic component constant, and use the same types, amounts, and preparation process of the excipient component as in Example 1, thereby preparing immune-enhancing compositions having different ratios.

[0063] Four experimental groups are established, wherein the ratio of the total cell count of the heat-inactivated Lactococcus lactis freeze-dried powder to the total viable count of the active probiotic component is set to 0.5: 1, 1:1, 1.5: 1, and 2:1, respectively. In all groups, the cell content of the heat-inactivated Lactococcus lactis freeze-dried powder is kept the same, and each process parameter is kept uniform during preparation.

[0064] Two core performance tests are respectively carried out on each composition group. The first test is an in vitro IgA-induction capability test, in which the same in vitro intestinal epithelial cell co-culture model as in Example 2 is used, and the secretory IgA content in the culture supernatant is measured after 48 h of co-culture. The second test is a storage-stability test of bacterial cells, in which the compositions are stored at 37 C and 60% humidity for 30 days, and the retention rate of viable counts of the active probiotics before and after storage is measured. DESCRIPTION

[0065] Each experiment is repeated six times and the average value is used for statistical analysis.

[0066] Comparative table of composition performance at different cell-count ratios:

[0067] The table data show that all compositions having different cell-count ratios can effectively induce production of secretory IgA while maintaining storage stability of the active probiotics. As the proportion of inactivated postbiotic cells increases, the secretory IgA content first increases and then decreases, while the viable-count retention rate shows a continuously decreasing trend.

[0068] Group 2, having a cell-count ratio of 1 :1, achieves the highest secretory IgA content among all groups, representing an increase of 31.14% over Group 1 and 22.56% over Group 4, while the 30-day viable-count retention rate is also at the optimum level. This indicates that, at this ratio, the synergistic immune-enhancing effect of the inactivated postbiotic and the active probiotics is most significant and at the same time does not negatively affect the storage stability of the active probiotics.

[0069] Because the proportion of inactivated postbiotic cells is relatively low in Group 1, the synergistic immunostimulatory effect of the inactivated postbiotic cannot be fully exerted, and therefore the IgA-induction effect is weaker than that of the other groups, achieving only a basic IgA-enhancing effect.

[0070] In Groups 3 and 4, the proportion of inactivated postbiotic cells is DESCRIPTION excessively high. The excessive amount of inactivated cells changes the microenvironment of the composition and exerts a certain inhibitory effect on survival of the active probiotics, thereby causing a significant decrease in the viable-count retention rate. Meanwhile, the excessive amount of inactivated cells also affects contact between the active probiotics and intestinal epithelial cells, thereby reducing the IgA-induction effect.

[0071] Example 4:

[0072] Verification of the effect of excipient compounding schemes on the core performance of the composition.

[0073] This example is used to verify the effect of different compounding schemes of functional components in the excipient component on the core performance of the composition. All experimental groups use the optimal strain ratio and cell-count ratio determined in Examples 2 and 3, and the types, amounts, and preparation process of the active probiotic component and the inactivated postbiotic component are the same as those in Example 1 ; only the compounding ratios of the respective functional components in the excipient component are adjusted, thereby preparing immune-enhancing compositions having different compounding schemes.

[0074] Five experimental groups are established. Group 1 uses the excipient compounding scheme of Example 1, in which the prebiotics, freeze-drying protectant, filler, lubricant, flavoring agent, and encapsulation wall material all use the predetermined equal-ratio compounding scheme. Group 2 adjusts only the prebiotic compounding ratio, wherein fructooligosaccharide, galactooligosaccharide, and inulin are compounded at 2:1 :1. Group 3 adjusts only DESCRIPTION the freeze-drying protectant compounding ratio, wherein skim milk powder, trehalose, and mannitol are compounded at 2: 1:1. Group 4 adjusts only the filler compounding ratio, wherein microcrystalline cellulose and maltodextrin are compounded at 2:1. Group 5 adjusts only the encapsulation wall material compounding ratio, wherein hydroxypropyl methylcellulose, resistant starch, and gelatin are compounded at 2:1:1. In all groups, the total amount of the excipient component is kept the same, and each process parameter is kept uniform during preparation.

[0075] Three core performance tests are respectively carried out on each composition group. The first test is the survival rate of probiotics after freeze-drying, calculated as the ratio of the viable count of the active probiotics after completion of the freeze-drying process to the viable count before freeze-drying. The second test is tolerance to simulated gastric fluid, in which the compositions are treated in simulated gastric fluid for 2 h and the retention rate of viable counts of the active probiotics after treatment is determined. The third test is tablet disintegration performance, in which the disintegration time limit of the composition tablets is measured according to a standard method. Each experiment is repeated six times and the average value is used for statistical analysis.

[0076] Comparative table of composition performance under different excipient compounding schemes: DESCRIPTION

[0077] The table data show that Group 1, which uses the fully equal-ratio compounding scheme, exhibits performance indices superior to those of the other groups in which the compounding ratio of only one excipient is adjusted. This indicates that the equal-ratio compounding scheme used in Example 1 can simultaneously take into account protection of bacterial activity, tolerance to gastric fluid, and formulation performance of the composition.

[0078] In Group 3, the compounding ratio of the freeze-drying protectant is adjusted, and the survival rate of probiotics after freeze-drying decreases significantly, by 9.55% compared with Group 1. This is because an equal-ratio compounded freeze-drying protectant can form a stable glassy-state structure during freeze-drying while simultaneously exerting osmotic protection and membrane protection. When the proportion of a single component is excessively high, a complete protective structure cannot be formed, thereby causing damage to the bacterial cells during freeze-drying and reducing the survival rate.

[0079] In Group 5, the compounding ratio of the encapsulation wall material is adjusted, and the retention rate of viable counts after treatment with simulated gastric fluid decreases significantly, by 15.02% compared with Group 1. This is because an equal-ratio compounded encapsulation wall material can form a dense encapsulation structure having pH responsiveness, thereby remaining structurally stable in acidic gastric fluid and rapidly dissolving in neutral intestinal fluid. DESCRIPTION

[0080] When the proportion of a single component is excessively high, the compactness and pH responsiveness of the encapsulation structure decrease, so that the active probiotics cannot be effectively protected against damage caused by gastric acid.

[0081] In Group 4, the compounding ratio of the filler is adjusted, and the tablet disintegration time limit is significantly prolonged, by 33.6% compared with Group 1. This is because an equal -ratio compounded filler can balance the shaping hardness of the tablet and the disintegration performance thereof; when the proportion of a single component is excessively high, the pore structure of the tablet changes, thereby slowing the disintegration rate.

[0082] In Group 2, the compounding ratio of the prebiotics is adjusted, and each performance index decreases only slightly, indicating that the compounding ratio of the prebiotics mainly affects the intestinal proliferation effect of the probiotics and has relatively little effect on the fundamental formulation performance of the composition.

[0083] Example 5 :

[0084] Verification of the in vivo IgA-enhancing effect and immune activity of the composition.

[0085] This example is used to verify the in vivo IgA-enhancing effect and immune activity of the composition in animals, with SPF healthy mice being used as experimental animals. Before the experiment begins, all mice are adaptively fed for 7 days, during which food and water are provided ad libitum, and the feeding environment is maintained at a temperature of 25 C, a humidity of 50%, and a 12 h light-dark cycle.

[0086] Five experimental groups are established, namely a blank control group, a DESCRIPTION positive control group, a low-dose composition group, a medium-dose composition group, and a high-dose composition group. Each group contains 12 mice, with equal numbers of males and females. The blank control group is gavaged daily with normal saline, the positive control group is gavaged daily with a commercially available immune-enhancing probiotic preparation of the same type, and the low-dose, medium-dose, and high-dose composition groups are gavaged daily with the immune-enhancing composition prepared in Example 1 at doses of 100 mg / kg, 200 mg / kg, and 400 mg / kg, respectively. All groups are gavaged once daily for 28 consecutive days.

[0087] After the gavage period ends, all mice are fasted for 12 h, blood samples are collected by orbital blood sampling, and serum is separated for determination of total serum IgA content. After the mice are sacrificed, duodenal, jejunal, and ileal tissues are collected, intestinal mucosa is scraped, and homogenate supernatants are prepared for determination of intestinal mucosal secretory IgA content. Fresh fecal samples of the mice are collected to determine fecal secretory IgA content and colonization quantities of the active probiotics. The experimental data of each group are averaged for statistical analysis.

[0088] Comparative table of in vivo IgA contents in mice among different groups: DESCRIPTION

[0089] The table data show that, compared with the blank control group, the serum total IgA, intestinal mucosal secretory IgA, and fecal secretory IgA contents in mice of each composition dose group all increase significantly, and the magnitude of increase shows an obvious positive correlation with the gavage dose. This indicates that the composition can increase IgA levels in mice in a dose-dependent manner and exert a stable immune-enhancing effect.

[0090] The gavage dose of the medium-dose composition group is the same as that of the positive control group, while the serum total IgA content of the medium-dose composition group is increased by 39.68% compared with the positive control group, the intestinal mucosal secretory IgA content is increased by 56.52%, and the fecal secretory IgA content is increased by 53.01%. This indicates that the in vivo IgA-enhancing effect of the composition is significantly superior to that of commercially available products of the same type.

[0091] All IgA indices in the high-dose composition group reach the highest values among all groups, wherein the serum total IgA content is increased by 152.43% compared with the blank control group, the intestinal mucosal secretory IgA content is increased by 207.66%, and the fecal secretory IgA content is increased by 194.47%. This indicates that, at a high dose, the composition can fully exert the synergistic effect of the active probiotics and the inactivated postbiotic, maximally stimulate secretion of IgA by the mucosal immune system of the body, DESCRIPTION and build an intestinal immune barrier.

[0092] Intestinal mucosal secretory IgA is the core effector molecule of intestinal mucosal immunity, and the intestinal mucosal secretory IgA content of each composition dose group is significantly increased. This indicates that the composition can directly act on the intestinal mucosal immune system and promote synthesis and secretion of intestinal mucosal secretory IgA, which is also the core mechanism by which the composition exerts its immune-enhancing effect.

[0093] The increase in fecal secretory IgA content indicates that the composition can promote secretion of secretory IgA into the intestinal lumen along with intestinal mucus, thereby exerting the effects of neutralizing pathogens and preventing pathogenic bacteria from adhering to the intestine, and further improving the intestinal immune-protection function.

[0094] Example 6:

[0095] Verification of storage stability and consumption safety of the composition.

[0096] This example is used to verify the long-term storage stability and consumption safety of the composition, thereby providing data support for the practical production application and daily consumption of the composition. This example is divided into two parts: storage-stability verification and consumption- safety verification.

[0097] The first part is storage- stability verification. Tablets of the composition prepared in Example 1 are respectively stored under three different storage conditions, namely room-temperature conditions of 25 C and 60% humidity, low-temperature conditions of 4 C and 45% humidity, and accelerated conditions DESCRIPTION of 40 C and 75% humidity, with a storage period of 6 months. Samples are taken at 1 month, 3 months, and 6 months, respectively, and the viable-count retention rate of the active probiotics in the composition is determined. Six samples are taken for each group at each time point, and the average value is used for statistical analysis.

[0098] The second part is consumption-safety verification. SPF healthy mice are used for an acute oral toxicity test, and 40 healthy mice are selected and randomly divided into a blank control group and a high-dose composition group, with 20 mice in each group and equal numbers of males and females. The high-dose composition group is gavaged daily with the composition prepared in Example 1 at a dose of 800 mg / kg, which is four times the daily recommended consumption dose, while the blank control group is gavaged daily with the same amount of normal saline for 30 consecutive days. During the gavage period, the mental state, food and water intake, and body-weight changes of the mice are observed daily, and abnormal reactions and mortality are recorded. After the gavage period ends, the mice are sacrificed, and major organs such as the heart, liver, spleen, lung, and kidney are collected for histopathological examination, while serum biochemical indices related to liver function and kidney function are also determined.

[0099] Comparative table of viable-count retention rates of the composition under different storage conditions:

[0100] The table data show that, under different storage conditions, the viable-count DESCRIPTION retention rate of the active probiotics in the composition gradually decreases as the storage time is prolonged, and the viable-count retention rate under low-temperature storage conditions is always higher than that under room-temperature and accelerated storage conditions. This indicates that a low-temperature environment can better maintain the activity of the active probiotics and prolong the shelf life of the composition.

[0101] After storage for 6 months under room-temperature conditions of 25 C and 60% humidity, the viable-count retention rate of the composition still reaches 86.72% and always remains within the effective viable-count range. This indicates that the composition has good stability under conventional room-temperature storage conditions and can satisfy daily storage and circulation requirements.

[0102] After storage for 6 months under accelerated conditions of 40 C and 75% humidity, the viable-count retention rate of the composition can still be maintained at 75.18%. This indicates that the composition can tolerate extreme storage environments of high temperature and high humidity and can maintain relatively good bacterial activity even if environmental fluctuations occur during transportation and storage, thereby demonstrating strong resistance to risk.

[0103] The consumption-safety verification results show that, throughout the gavage period, no mortality occurs in the high-dose composition group, the mice remain in a good mental state, food and water intake are normal, and body-weight gain shows no significant difference from that of the blank control group, with no abnormal toxic reactions being observed. Histopathological examination of the major organs reveals no pathological damage, and serum biochemical indices of DESCRIPTION liver function and kidney function all remain within the normal range, with no significant difference from those of the blank control group. This indicates that the composition still has good consumption safety at a high dose, has no acute toxic effect, and is suitable for daily long-term consumption.

[0104] Based on the comprehensive verification results of storage stability and consumption safety, the composition not only has good long-term storage stability and can maintain stable product performance under conventional conditions, but also has excellent consumption safety, thus meeting the basic conditions for large-scale production and market promotion.

[0105] The foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention has been disclosed above with reference to preferred embodiments, the present invention is not limited thereto. Any person skilled in the art may make certain changes or modifications to the disclosed technical content without departing from the scope of the technical solutions of the present invention, and such changes or modifications may be made into equivalent embodiments having equivalent variations. Therefore, any brief modifications, equivalent variations, and modifications made to the above embodiments according to the technical spirit of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of the technical solutions of the present invention.

Claims

CLAIMS1. A Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels, characterized in that the composition comprises, in parts by weight, an active probiotic component, an inactivated postbiotic component, and an excipient component;The active probiotic component comprises Bifidobacterium bifidum live freeze-dried powder, Bifidobacterium longum live freeze-dried powder, and Lactobacillus rhamnosus live freeze-dried powder, wherein the Bifidobacterium bifidum live freeze-dried powder is present in 10-30 parts, the Bifidobacterium longum live freeze-dried powder is present in 10-30 parts, and the Lactobacillus rhamnosus live freeze-dried powder is present in 10-30 parts; the total amount of the active probiotic component is 30-90 parts by weight, and the total viable count is 1x10A9 CFU / g-lxl0Al l CFU / g;The inactivated postbiotic component is heat-inactivated Lactococcus lactis freeze-dried powder, present in an amount of 15-60 parts by weight, with a cell count of 5xlOA8 cells / g-2xlOAl 1 cells / g;The total amount of the excipient component is 20-80 parts by weight and comprises 5-30 parts of prebiotics, 3-25 parts of a freeze-drying protectant, 10-35 parts of a filler, 0.5-5 parts of a lubricant, 2-15 parts of a flavoring agent, and 4-25 parts of an encapsulation wall material.

2. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 1, characterized in that the weight ratio of the live freeze-dried powders of Bifidobacterium bifidum, Bifidobacterium longum, and Lactobacillus rhamnosus is from 1:1 :1 to 3:3:3, and the viable count in the live freeze-dried powder of each individual strain is lxl0A10 CFU / g-lxlOA12 CFU / g.

3. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 1, characterized in that the ratio of the total cell count in the inactivated postbiotic component to the total viable count in the active probiotic component is 0.5: 1-2:1; wherein the cell content of theCLAIMS heat-inactivated Lactococcus lactis freeze-dried powder is lxl0A10 CFU / g-2xlOA12 CFU / g.

4. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 1, characterized in that the prebiotic is a proportional blend of fructooligosaccharide, galactooligosaccharide, and inulin at 1:1 :1; and the freeze-drying protectant is a proportional blend of skim milk powder, trehalose, and mannitol at 1:1: 1.

5. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 1, characterized in that the filler is a proportional blend of microcrystalline cellulose and maltodextrin at 1:1; the lubricant is a proportional blend of magnesium stearate and talc at 1:1, with a total amount of 1-4 parts by weight; and the flavoring agent is a proportional blend of xylitol, erythritol, and natural fruit powder at 1:1: 1.

6. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 1, characterized in that the encapsulation wall material is a proportional blend of hydroxypropyl methylcellulose, resistant starch, and gelatin at 1:1 :1, and is used to form a double-layer encapsulation structure in which the active probiotic component is encapsulated in the inner layer and the inactivated postbiotic component is encapsulated in the outer layer.

7. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 1, characterized in that the inactivated postbiotic component is formed by compounding heat-inactivated Lactococcus lactis freeze-dried powder with fermentation metabolites of the strain at a weight ratio of l:(0.3-l), wherein the fermentation metabolites comprise exopolysaccharides and small-molecule peptides.

8. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 1, characterized in that the specific steps of the preparation method of the composition are as follows:CLAIMS51. Preparation of wet active probiotic cells: activating and expanding each active probiotic strain, followed by centrifugation to collect and wash the cells;52. Preparation of active probiotic freeze-dried powder: adding a freeze-drying protectant to the wet active probiotic cells for resuspension, followed by vacuum freeze-drying to obtain freeze-dried powder of each strain;53. Preparation of inactivated postbiotic freeze-dried powder: activating and expanding the postbiotic strain, followed by centrifugation to collect and wash the cells; after heat-inactivation verification, adding a freeze-drying protectant and vacuum freeze-drying to obtain inactivated postbiotic freeze-dried powder;54. Preparation of the finished composition: compounding the freeze-dried powders of the active probiotic strains, mixing with the inactivated postbiotic freeze-dried powder and excipient components, and processing into a target dosage form.

9. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 8, characterized in that, in step SI, after two-stage anaerobic activation at 37 C, each active probiotic strain is inoculated into a fermentation medium having an initial pH of 6.0-6.5 at an inoculation amount of 2%-5%, anaerobically fermented at 37 C under nitrogen with stirring at 150-250 r / min for 16-24 h, and the fermentation broth is centrifuged at 4 C and 6000-10000 r / min for 10-20 min, followed by resuspension and washing twice with sterile normal saline at a volume ratio of 1:2-1:4.

10. The Bifidobacterium bifidum immune-enhancing composition for increasing IgA levels according to claim 8, characterized in that, in step S3, after two-stage aerobic activation at 37 C, the postbiotic strain is inoculated into a fermentation medium at an inoculation amount of 3% and aerobically cultured at 37 C for 18 h; the fermentation broth is centrifuged at 4 C andCLAIMS6000-8000 r / min for 10-15 min, washed twice with sterile normal saline, and resuspended to a bacterial suspension concentration of 1x10A11-lxl 0A12 CFU / mL, then treated at 95-105 C for 20-40 min; after plate verification confirming the absence of viable bacteria, a freeze-drying protectant is added and vacuum freeze-drying is carried out to obtain the inactivated postbiotic freeze-dried powder.

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