Probiotic composition for improving immunity and preparation method thereof

Abstract

The invention provides a probiotic composition for improving immunity and a preparation method thereof, and belongs to the technical field of probiotics. The preparation method comprises the following steps: fermenting selenium-enriched nostoc sphaeroides through selenium-enriched saccharomycetes to extract selenium-enriched nostoc sphaeroides polysaccharide and selenium-enriched nostoc sphaeroides protein peptide, mixing with traditional Chinese medicine polysaccharide obtained by extracting astragalus membranaceus, rhodiola rosea, codonopsis pilosula and pseudo-ginseng, and reacting with ferric salt to prepare a polysaccharide-Fe / selenium compound; solid residues are mixed and fermented, and active protein peptide is prepared from the solid residues and selenium-rich nostoc sphaeroides protein peptide; the probiotic composition capable of improving immunity is prepared by extracting a polysaccharide-Fe / selenium compound and a polysaccharide-Fe / selenium compound through an organic solvent, extracting a permeate through an organic solvent to obtain an active component, collecting a bacterial liquid, mixing and embedding the bacterial liquid in the polysaccharide-Fe / selenium compound and sodium alginate, and uniformly mixing the bacterial liquid with the active protein peptide, the active component, taurine and ginsenoside Rg3, so that the probiotic composition capable of improving immunity can play good anti-inflammatory, anti-oxidation, immunoregulation, anti-tumor, anti-virus and other roles, and has a good application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] This invention relates to the field of probiotic technology, specifically to a probiotic composition for enhancing immunity and its preparation method. Background Technology

[0002] Immunity is the body's ability to resist disease and maintain its health. The immune system performs this function within the body. It comprises immune organs, immune tissues, and immune molecules, which interact to form a vast network of bodily defenses. Although our understanding of the immune system is not yet complete, current knowledge has shown that 70% of the body's immune cells reside in the gut, and the development of the immune system is closely related to the gut microbiota.

[0003] The intestinal mucosa of the human body is home to thousands of species and hundreds of billions of gut microbiota. These gut microbiota maintain a dynamic balance with the human mucosal immune system. On the one hand, the human mucosal system adjusts and maintains the quantity and variety of gut microbiota; on the other hand, gut microbiota also "train" our immune system, helping it to mature.

[0004] Chinese invention patent application CN107752040A discloses a compound probiotic powder preparation for promoting the immunity of infants and young children. It screens five probiotic strains: Bifidobacterium infantis, Bifidobacterium animalis V9, Lactobacillus bulgaricus PZLB5, Lactobacillus plantarum PP12, and Streptococcus thermophilus QH. The culture medium and conditions for each strain are optimized, utilizing the unique synergistic effects between the strains to enhance immune cell activity and improve the immunity of infants and young children. However, the isolation and screening process for the five strains used in this patent is complex and the production cost is high, placing a significant economic burden on consumers.

[0005] Chinese invention patent application CN105641209A discloses a probiotic composition for enhancing immunity, comprising probiotic powder, astragalus extract powder, and wolfberry extract powder, supplemented with nutrients such as vitamin K, vitamin B12, pyridoxine, biotin, folic acid, niacin, and thiamine. This method utilizes the rational combination of traditional Chinese medicine and probiotics to synergistically enhance immunity and promote overall health. However, the complex composition of traditional Chinese medicine may contain components that can inactivate probiotics and reduce efficacy.

[0006] Chinese invention patent CN103478530B discloses an oligosaccharide probiotic composition and its application, as well as a method for preparing capsules using the composition. This document mainly uses oligosaccharide powder and hydroxypropyl methylcellulose. Chinese invention patent CN104983746B discloses a compound probiotic and its preparation method, which mainly uses prebiotics, dietary fiber, amino acids and vitamin B as fillers, but both have the problem of easy inactivation. Summary of the Invention

[0007] The purpose of this invention is to propose a probiotic composition for enhancing immunity and its preparation method, which can play a good role in anti-inflammatory, antioxidant, immunomodulatory, anti-tumor, and antiviral effects, improve the storage time of probiotics, greatly increase the number of live bacteria after entering the human intestine, and enhance the immunomodulatory effect of the probiotic composition through multiple pathways, and has broad application prospects.

[0008] The technical solution of this invention is implemented as follows:

[0009] This invention provides a method for preparing a probiotic composition to enhance immunity. Selenium-enriched *Gnaphalium affine* is fermented with selenium-enriched yeast to extract selenium-enriched *Gnaphalium affine* polysaccharides and protein peptides. These are mixed with traditional Chinese medicine polysaccharides extracted from *Astragalus membranaceus*, *Rhodiola rosea*, *Codonopsis pilosula*, and *Panax notoginseng*, and reacted with iron salts to prepare a polysaccharide-Fe / selenium complex. The solid residue is then mixed and fermented to obtain active protein peptides from the selenium-enriched *Gnaphalium affine* protein peptides. The permeate is extracted with an organic solvent to obtain active components. The bacterial solution is collected and encapsulated in a mixture of the polysaccharide-Fe / selenium complex and sodium alginate. This mixture is then thoroughly mixed with the active protein peptides, active components, taurine, and ginsenoside Rg3 to obtain the probiotic composition that enhances immunity.

[0010] As a further improvement to the present invention, the following steps are included:

[0011] S1. Fermentation of selenium-enriched kudzu root: Add selenium-enriched kudzu root powder to water, stir and mix evenly, sterilize, inoculate with selenium-enriched yeast seed solution, ferment and culture, filter, wash the solid, collect the bacterial solution to obtain selenium-enriched yeast solution, and keep the solid for use; add ethanol to the filtrate to precipitate, collect the polysaccharide solid, wash, dry to obtain selenium-enriched kudzu root polysaccharide, dialyze the liquid, freeze-dry the dialysate to obtain selenium-enriched kudzu root protein peptide, and keep the permeate for use;

[0012] S2. Preparation of Chinese herbal extracts: Astragalus membranaceus, Rhodiola rosea, Codonopsis pilosula, and Panax notoginseng were washed, dried, and pulverized to obtain Chinese herbal powder. Water was added for boiling extraction, and the powder was filtered. The solid was retained. Ethanol was added to the filtrate to precipitate the polysaccharide solid. The solid was collected, washed, and dried to obtain Chinese herbal polysaccharide. The filtrate was retained.

[0013] S3. Preparation of polysaccharide-Fe / selenium complex: The selenium-enriched kudzu polysaccharide obtained in step S1 and the traditional Chinese medicine polysaccharide obtained in step S2 were added to water, iron salt was added, the mixture was stirred and reacted, ethanol was added to precipitate, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex.

[0014] S4. Fermentation: Add the solids from step S1 and step S2 to water, sterilize, inoculate with Bacillus coagulans and Bifidobacterium bifidum seed liquid, ferment, filter, wash the solids, collect the bacterial liquid, mix it evenly with the selenium-enriched yeast liquid obtained in step S1, concentrate, and obtain concentrated bacterial liquid; dialyze the filtrate, freeze-dry the dialysate, mix it evenly with the selenium-enriched kudzu root protein peptide obtained in step S1, and obtain active protein peptide; retain the permeate;

[0015] S5. Extraction of active components: Mix the permeate from step S1 and the permeate from step S4 evenly, add organic solvent for extraction, collect the organic layer, remove the organic solvent under reduced pressure, wash, dry, and obtain the active components.

[0016] S6. Preparation of sodium alginate-probiotic / polysaccharide microcapsules: Add sodium alginate, polysaccharide-Fe / selenium complex and lecithin obtained in step S3 to the concentrated bacterial solution obtained in step S4, stir and mix evenly, add to fish oil, emulsify, add calcium chloride solution dropwise, solidify at room temperature, centrifuge, wash and dry to obtain sodium alginate-probiotic / polysaccharide microcapsules;

[0017] S7. Preparation of probiotic composition to enhance immunity: The active protein peptide obtained in step S4, the active component obtained in step S5, the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, taurine, and ginsenoside Rg3 are mixed evenly to obtain a probiotic composition to enhance immunity.

[0018] As a further improvement of the present invention, the selenium content of the selenium-enriched kudzu root powder in step S1 is 300-500 μg / 100g, the solid-liquid ratio of the selenium-enriched kudzu root powder to water is 1:5-7 g / mL, and the bacterial count of the inoculum seed solution is 10. 8 -10 9 The inoculum concentration of the selenium-enriched yeast seed solution is 1-3 v / v%, the fermentation conditions are 50-55℃, 100-200 r / min, and fermentation culture for 36-48 h, the dialysis bag pore size is 2-5 kDa, and the dialysis time is 3-5 h.

[0019] As a further improvement of the present invention, the mass ratio of Astragalus membranaceus, Rhodiola rosea, Codonopsis pilosula, and Panax notoginseng in step S2 is 5-7:10-12:3-5:2-4, the solid-liquid ratio of the herbal powder and water is 1:7-10 g / mL, and the boiling extraction time is 2-4 h.

[0020] As a further improvement of the present invention, the mass ratio of selenium-enriched kudzu root protein peptide, traditional Chinese medicine polysaccharide, and iron salt in step S3 is 10-12:7-10:1-3, and the iron salt is selected from at least one of ferric chloride, ferric sulfate, and ferric nitrate.

[0021] As a further improvement of the present invention, in step S4, the mass ratio of the solid in step S1 to the solid in step S2 is 10-15:8-12, and the bacterial content of the inoculum seed solution is 10. 8 -10 9 The inoculum concentration of *Bacillus coagulans* and *Bifidobacterium bifidum* was 1-3 v / v% and 2-4 v / v%, respectively. The fermentation conditions were 38-40℃, 100-200 r / min, and 48-56 h. The volume ratio of the collected bacterial solution to the selenium-enriched yeast solution obtained in step S1 was 12-17:8-12. The bacterial count of the concentrated bacterial solution was 10... 11 -10 12 The concentration of cfu / mL is specified. The dialysis bag used for the dialysis has a pore size of 2-5 kDa, the dialysis time is 3-5 h, and the mass ratio of the protein peptide to the selenium-enriched kudzu root protein peptide is 5-7:2-4.

[0022] As a further improvement of the present invention, the mass ratio of the permeate in step S1 to the permeate in step S4 in step S5 is 10-12:15-20, and the organic solvent is a mixed solution of cyclohexane and ethyl acetate with a volume ratio of 3-5:7-10.

[0023] As a further improvement of the present invention, the mass ratio of the concentrated bacterial solution, sodium alginate, polysaccharide-Fe / selenium complex, and lecithin in step S6 is 100-120:15-17:5-7:0.5-1, and the solidification time at room temperature is 20-40 min; the mass ratio of the active protein peptide, active component, sodium alginate-probiotic / polysaccharide microcapsules, taurine, and ginsenoside Rg3 in step S7 is 5-7:3-5:15-20:1-2:0.5-1.

[0024] As a further improvement to the present invention, the specific steps include:

[0025] S1. Fermentation of selenium-enriched kudzu root: Selenium-enriched kudzu root powder is added to water, with a solid-liquid ratio of 1:5-7 g / mL. The mixture is stirred and sterilized. Selenium-enriched yeast seed solution is inoculated at a rate of 1-3 v / v%, fermented at 50-55℃ and 100-200 r / min for 36-48 h. The mixture is filtered, the solid is washed, and the bacterial solution is collected to obtain selenium-enriched yeast solution. The solid is retained for use. Ethanol is added to the filtrate until the ethanol content of the system is 75-85 wt%. Precipitation is carried out for 3-5 h. The polysaccharide solid is collected, washed, and dried to obtain selenium-enriched kudzu root polysaccharide. The liquid is dialyzed using a dialysis bag with a pore size of 2-5 kDa for 3-5 h. The dialysate is freeze-dried to obtain selenium-enriched kudzu root protein peptides. The permeate is retained for use.

[0026] The bacterial count of the selenium-enriched yeast seed solution is 10. 8 -10 9 cfu / mL;

[0027] S2. Preparation of Traditional Chinese Medicine Extract: 5-7 parts by weight of Astragalus membranaceus, 10-12 parts by weight of Rhodiola rosea, 3-5 parts by weight of Codonopsis pilosula, and 2-4 parts by weight of Panax notoginseng were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was extracted by boiling in water for 2-4 hours, with a solid-liquid ratio of 1:7-10 g / mL. The mixture was filtered, and the solid was retained. Ethanol was added to the filtrate until the ethanol content reached 75-85 wt%, and precipitation was allowed for 3-5 hours. The polysaccharide solid was collected, washed, and dried to obtain the traditional Chinese medicine polysaccharide. The filtrate was retained.

[0028] S3. Preparation of polysaccharide-Fe / selenium complex: 10-12 parts by weight of selenium-enriched kudzu polysaccharide obtained in step S1 and 7-10 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were added to 100 parts by weight of water, 1-3 parts by weight of iron salt were added, the mixture was stirred and reacted for 20-30 min, ethanol was added until the ethanol content of the system was 75-85 wt%, precipitation was carried out for 2-4 h, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex.

[0029] S4. Fermentation: Add 10-15 parts by weight of the solid from step S1 and 8-12 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans and Bifidobacterium bifidum seed solutions. The inoculation amounts of Bacillus coagulans and Bifidobacterium bifidum seed solutions are 1-3 v / v% and 2-4 v / v%, respectively. Ferment at 38-40℃ and 100-200 r / min for 48-56 h. Filter, wash the solid, collect the bacterial solution, and mix 12-17 parts by volume of the bacterial solution with 8-12 parts by volume of the selenium-enriched yeast solution obtained in step S1. Concentrate to obtain a bacterial count of 10. 11 -10 12 Concentrated bacterial culture at cfu / mL; the filtrate was dialyzed for 3-5 h using a dialysis bag with a pore size of 2-5 kDa, the dialysate was freeze-dried, and 5-7 parts by weight of the obtained protein peptides were mixed evenly with 2-4 parts by weight of the selenium-enriched kudzu root protein peptides obtained in step S1 to obtain active protein peptides; the permeate was retained.

[0030] The bacterial count of the Bacillus coagulans and Bifidobacterium bifidum seed solution was 10. 8 -10 9 cfu / mL;

[0031] S5. Extraction of active components: Mix 10-12 parts by weight of the permeate from step S1 and 15-20 parts by weight of the permeate from step S4 evenly, add 50-70 parts by weight of organic solvent for extraction, collect the organic layer, remove the organic solvent under reduced pressure, wash, dry, and obtain the active components.

[0032] The organic solvent is a mixed solution of cyclohexane and ethyl acetate in a volume ratio of 3-5:7-10;

[0033] S6. Preparation of sodium alginate-probiotic / polysaccharide microcapsules: Add 15-17 parts by weight of sodium alginate, 5-7 parts by weight of polysaccharide-Fe / selenium complex prepared in step S3, and 0.5-1 parts by weight of lecithin to 100-120 parts by weight of the concentrated bacterial solution obtained in step S4. Stir and mix evenly, add to 200 parts by weight of fish oil, emulsify, add 5-10 parts by weight of 3-5wt% calcium chloride solution, solidify at room temperature for 20-40 min, centrifuge, wash, and dry to obtain sodium alginate-probiotic / polysaccharide microcapsules.

[0034] S7. Preparation of a probiotic composition for enhancing immunity: Mix 5-7 parts by weight of the active protein peptide obtained in step S4, 3-5 parts by weight of the active component obtained in step S5, 15-20 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, 1-2 parts by weight of taurine, and 0.5-1 parts by weight of ginsenoside Rg3 evenly to obtain a probiotic composition for enhancing immunity.

[0035] The present invention further protects an immune-enhancing probiotic composition prepared by the above-described preparation method.

[0036] The present invention has the following beneficial effects:

[0037] Nostoc commune is a freshwater cyanobacterium rich in polysaccharides, proteins, and trace elements. Selenium-enriched Nostoc commune is produced by adding an inorganic selenium source during its cultivation. This process converts inorganic selenium into organic selenium products such as polysaccharide-selenium complexes and polypeptide-selenium complexes, resulting in a high selenium content. This invention further enhances the organic selenium content through fermentation with selenium-enriched yeast. Simultaneously, it degrades high-molecular-weight polysaccharides and proteins into easily absorbed low-molecular-weight polysaccharides and protein peptides, significantly improving the body's absorption efficiency of selenium. Selenium polysaccharides exhibit high bioactivity, significantly lower toxicity than inorganic selenium, and are easily absorbed by the body. Polysaccharide selenium and polypeptide selenium can simultaneously exert the effects of selenium, polysaccharides, and polypeptides, with higher activity than selenium, polysaccharides, or polypeptides alone. The functional properties of selenium polysaccharides and selenium polypeptides include a variety of properties such as anti-oxidation, anti-tumor, anti-inflammation, liver protection, and enhanced immune function. They have stronger immunomodulatory activity and can enhance immunity by promoting the proliferation of T lymphocytes, improving the function of B lymphocytes, increasing the number of neutrophils, enhancing the phagocytic function of macrophages, and secreting cytokines.

[0038] Furthermore, the traditional Chinese medicine composition of this invention includes Astragalus membranaceus, Rhodiola rosea, Codonopsis pilosula, and Panax notoginseng, which are rich in polysaccharide components. These polysaccharides can effectively regulate the body's immune function, enhance the activity of immune cells such as the reticuloendothelial system, macrophages, natural killer cells, and cytotoxic T cells, promote the production of antibodies and complement, and induce the production of interferon. When mixed with selenium-enriched Ge Xian Mi polysaccharide, based on the presence of free alcohol hydroxyl, carboxyl, and amino groups in the polysaccharide molecular backbone that can provide lone pairs of electrons, these groups act as ligands, releasing lone pairs of electrons into the empty orbitals of metal ions, and reacting with Fe. 3+ Coordination reactions generate stable polysaccharide-metal complexes, which have good antioxidant, anti-anemic, antibacterial, antitumor, and immunomodulatory activities. They can remove excess metal ions and reactive oxygen species in the body, alleviate cellular oxidative damage, maintain genome stability, inhibit cell mutation and thus delay aging, regulate the proliferation of T lymphocytes and the secretion of immune factors, maintain the polarization balance of monocytes / macrophages M1 and M2, and promote the specific immunoglobulin cascade enzyme reaction in serum, thereby activating the complement system.

[0039] Then, the solid residue from the fermentation of selenium-enriched *Ge Xian Mi* (a type of medicinal herb) and the solid residue from the water extraction of the traditional Chinese medicine composition were mixed and inoculated with *Bacillus coagulans* and *Bifidobacterium bifidum*. This process promotes cell wall disruption by the fermenting bacteria, dissolving abundant proteins, amino acids, trace elements, flavonoids, triterpenes, sterols, polyphenols, and organic acids from the *Ge Xian Mi* and other medicinal plant cells, significantly improving raw material utilization efficiency. The waste residue after fermentation can be used as animal feed or plant fertilizer. Furthermore, it promotes the proliferation of probiotics and the release of beneficial substances. Subsequently, the fermenting bacteria were eluted and mixed with selenium-enriched yeast solution for concentration to obtain a high concentration of probiotics. *Bifidobacterium bifidum* significantly reduced the self-inducing substances secreted by pathogenic *Escherichia coli*, thereby reducing the adhesion ability of this pathogen to host cells. *Bacillus coagulans* can interact with dendritic cells, enhancing T cell proliferation and immune response, causing T cells to differentiate into Th1, Th2, and Treg cells, activating the immune system, producing cytokines, and improving anti-infection capabilities. Selenium-enriched yeast can regulate epithelial tissue function, such as mucus secretion from goblet cells, defensin release from Paneth cells, and tight junction protein synthesis in normal epithelial cells, thus regulating and preventing epithelial cell apoptosis. These three mechanisms have a synergistic effect. Furthermore, these probiotics produce antitoxins such as organic acids, bacteriocins, glycoproteins, enzymes, and H2O2 to inhibit the growth of harmful microorganisms. Organic acids can increase intestinal acidity, thereby inhibiting the growth of pathogenic bacteria. The produced alcohols, diacetone, lysozyme, H2O2, and other antimicrobial substances (such as lactobacillus and nisin) can also inhibit the growth of intestinal pathogens.

[0040] This invention utilizes the synergistic encapsulation of concentrated bacterial culture with a polysaccharide-Fe / selenium complex and sodium alginate. On one hand, the polysaccharide-Fe / selenium complex forms a relatively dense and stable shell with sodium alginate under the cross-linking effect of calcium chloride, encapsulating probiotics within microspheres. This effectively protects the probiotics from the effects of gastric acid, choline, and intestinal fluid, increasing the viable count of probiotics and significantly enhancing their regulatory effect. On the other hand, the polysaccharide-Fe / selenium complex and sodium alginate are also excellent prebiotics. After being degraded and broken down in the intestines, they can be directly utilized by probiotics, promoting their proliferation. While the polysaccharide-Fe / selenium complex is utilized, the selenium-enriched *Ge Xian Mi* polysaccharide and traditional Chinese medicine polysaccharides (including *Astragalus membranaceus* polysaccharide, *Rhodiola rosea* polysaccharide, *Codonopsis pilosula* polysaccharide, and *Panax notoginseng* polysaccharide) produced by its decomposition exert excellent antioxidant, anti-inflammatory, immunomodulatory, and anti-tumor effects. Simultaneously, it releases Fe ions that are directly absorbed by the intestines, further enhancing the antioxidant and immunomodulatory effects.

[0041] Protein peptides are structural fragments of proteins that function as active groups within proteins. They possess advantages such as low molecular weight, simple structure, easy water solubility, strong hygroscopicity, good emulsification, good thermal stability, low viscosity, low immunogenicity, strong permeability, high utilization efficiency, and ease of modification and transformation. The protein peptides obtained from the fermentation extraction of traditional Chinese medicine and the selenium-enriched kudzu root protein peptides obtained from the fermentation extraction of selenium-enriched kudzu root can effectively regulate the body's immunity and inhibit tumor growth. They directly kill pathogens through macrophages, presenting antigens to trigger an immune response and enhance the body's immune capacity against tumors.

[0042] In addition, the present invention collects the permeate after fermentation, which contains abundant active substances, including flavonoids, triterpenes, sterols, polyphenols, organic acids, etc., and enriches and purifies them under the extraction of organic solvents, which greatly increases the content of active substances in the active components and further enhances the immunomodulatory effect.

[0043] The composition of this invention also includes taurine and ginsenoside Rg3, which have good immunomodulatory effects. Taurine is a good immune stabilizer and membrane stabilizer, protecting nerve cells and enabling them to perform normal physiological functions. It has a good protective effect on lymphocytes, promoting lymphocyte proliferation and antibody production, promoting macrophages to produce interleukin-1, enhancing the phagocytic and bactericidal function of neutrophils, and improving the specific immune function of humans and animals. Ginsenoside Rg3 has the effects of improving the body's immunity, antibacterial activity, improving insufficient blood supply to the heart and brain, regulating the central nervous system, anti-fatigue, delaying aging, anti-tumor activity, and rapid recovery of physical condition. The addition of these two has a synergistic effect.

[0044] The probiotic composition for enhancing immunity prepared in this invention can play a good role in anti-inflammatory, antioxidant, immunomodulatory, anti-tumor, and antiviral effects. It can improve the storage time of probiotics and greatly increase the number of live bacteria after entering the human intestine. It enhances the immunomodulatory effect of the probiotic composition through multiple pathways and has broad application prospects. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Selenium-enriched Ge Xian rice powder is produced by adding sodium selenite to the culture medium during the cultivation of Ge Xian rice, followed by drying after harvesting. Its moisture content is less than 0.5%, and its selenium content is 300-500 μg / 100g. It was purchased from Hunan Yandi Bioengineering Co., Ltd.

[0047] Preparation method of selenium-enriched yeast (purchased from Angel Yeast Co., Ltd.) seed culture: Selenium-enriched yeast was inoculated into Gao's medium and activated at 50℃ and 120 r / min for 24 h to obtain a bacterial count of 10. 8 -10 9 CFU / mL bacterial seed solution.

[0048] Preparation method of Bacillus coagulans (purchased from Shandong Pingju Biotechnology Co., Ltd.) inoculum seed solution: Inoculate Bacillus coagulans into Gao's medium, incubate at 45℃ and 120 r / min for 24 h to activate the culture, yielding a culture with a bacterial count of 10... 8 -10 9 CFU / mL bacterial seed solution.

[0049] Preparation method of Bifidobacterium bifidum (purchased from Shandong Pingju Biotechnology Co., Ltd.) inoculum seed solution: Bifidobacterium bifidum was inoculated into Gao's medium and activated under anaerobic conditions at 37℃ and 150 r / min for 24 h to obtain a bacterial count of 10. 8 -10 9 CFU / mL bacterial seed solution.

[0050] Example 1

[0051] This embodiment provides a method for preparing a probiotic composition to enhance immunity, specifically including the following steps:

[0052] S1. Fermentation of selenium-enriched kudzu root: Selenium-enriched kudzu root powder was added to water, with a solid-liquid ratio of 1:5 g / mL. The mixture was stirred and sterilized. Selenium-enriched yeast seed solution was inoculated at a rate of 1 v / v%, fermented at 50℃ and 100 r / min for 36 h. The mixture was filtered, the solid was washed, and the bacterial solution was collected to obtain selenium-enriched yeast solution. The solid was retained. Ethanol was added to the filtrate until the ethanol content of the system was 75 wt%. The mixture was precipitated for 3 h, and the polysaccharide solid was collected, washed, and dried to obtain selenium-enriched kudzu root polysaccharide. The liquid was dialyzed using a dialysis bag with a pore size of 2 kDa for 3 h. The dialysate was freeze-dried to obtain selenium-enriched kudzu root protein peptides. The permeate was retained.

[0053] S2. Preparation of Traditional Chinese Medicine Extract: 5 parts by weight of Astragalus membranaceus, 10 parts by weight of Rhodiola rosea, 3 parts by weight of Codonopsis pilosula, and 2 parts by weight of Panax notoginseng were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was extracted by boiling in water for 2 hours. The solid-liquid ratio of the traditional Chinese medicine powder to water was 1:7 g / mL. The mixture was filtered, and the solid was retained. Ethanol was added to the filtrate until the ethanol content of the system was 75 wt%. The mixture was allowed to precipitate for 3 hours. The polysaccharide solid was collected, washed, and dried to obtain traditional Chinese medicine polysaccharide. The filtrate was retained.

[0054] S3. Preparation of polysaccharide-Fe / selenium complex: 10 parts by weight of selenium-enriched kudzu polysaccharide obtained in step S1 and 7 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were added to 100 parts by weight of water, 1 part by weight of ferric chloride was added, the mixture was stirred and reacted for 20 min, ethanol was added until the ethanol content of the system was 75 wt%, precipitation was carried out for 2 h, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex.

[0055] S4. Fermentation: Add 10 parts by weight of the solid from step S1 and 8 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans and Bifidobacterium bifidum seed solutions. The inoculation amounts of Bacillus coagulans and Bifidobacterium bifidum seed solutions are 1 v / v% and 2 v / v%, respectively. Ferment at 38°C and 100 r / min for 48 h, filter, wash the solid, collect the bacterial solution, mix 12 parts by volume of the bacterial solution with 8 parts by volume of the selenium-enriched yeast solution obtained in step S1, concentrate, and obtain a bacterial count of 10. 11 -10 12 The concentrated bacterial culture was prepared at cfu / mL; the filtrate was dialyzed for 3 hours using a dialysis bag with a pore size of 2 kDa, and the dialysate was freeze-dried. 5 parts by weight of the obtained protein peptide were mixed evenly with 2 parts by weight of the selenium-enriched kudzu root protein peptide prepared in step S1 to obtain the active protein peptide; the permeate was retained.

[0056] S5. Extraction of active components: Mix 10 parts by weight of the permeate from step S1 and 15 parts by weight of the permeate from step S4 evenly, add 50 parts by weight of organic solvent for extraction, collect the organic layer, remove the organic solvent under reduced pressure, wash, dry, and obtain the active components.

[0057] The organic solvent is a mixed solution of cyclohexane and ethyl acetate in a volume ratio of 3:7;

[0058] S6. Preparation of sodium alginate-probiotic / polysaccharide microcapsules: Add 15 parts by weight of sodium alginate, 5 parts by weight of polysaccharide-Fe / selenium complex prepared in step S3, and 0.5 parts by weight of lecithin to 100 parts by weight of the concentrated bacterial solution obtained in step S4. Stir and mix evenly, add to 200 parts by weight of fish oil, emulsify at 10000 r / min for 15 min, add 5 parts by weight of 3wt% calcium chloride solution, solidify at room temperature for 20 min, centrifuge, wash, and dry to obtain sodium alginate-probiotic / polysaccharide microcapsules;

[0059] S7. Preparation of probiotic composition for enhancing immunity: Mix 5 parts by weight of the active protein peptide obtained in step S4, 3 parts by weight of the active component obtained in step S5, 15 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, 1 part by weight of taurine, and 0.5 parts by weight of ginsenoside Rg3 evenly to obtain a probiotic composition for enhancing immunity.

[0060] Example 2

[0061] This embodiment provides a method for preparing a probiotic composition to enhance immunity, specifically including the following steps:

[0062] S1. Fermentation of selenium-enriched kudzu root: Selenium-enriched kudzu root powder was added to water, with a solid-liquid ratio of 1:7 g / mL. The mixture was stirred and sterilized. Selenium-enriched yeast seed solution was inoculated at a rate of 3 v / v%, fermented at 55℃ and 200 r / min for 48 h. The mixture was filtered, the solid was washed, and the bacterial solution was collected to obtain selenium-enriched yeast solution. The solid was retained. Ethanol was added to the filtrate until the ethanol content of the system was 85 wt%. The mixture was allowed to precipitate for 5 h, and the polysaccharide solid was collected, washed, and dried to obtain selenium-enriched kudzu root polysaccharide. The liquid was dialyzed using a 5 kDa dialysis bag for 5 h. The dialysate was freeze-dried to obtain selenium-enriched kudzu root protein peptides. The permeate was retained.

[0063] S2. Preparation of Traditional Chinese Medicine Extract: 7 parts by weight of Astragalus membranaceus, 12 parts by weight of Rhodiola rosea, 5 parts by weight of Codonopsis pilosula, and 4 parts by weight of Panax notoginseng were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was extracted by boiling in water for 4 hours. The solid-liquid ratio of the traditional Chinese medicine powder to water was 1:10 g / mL. The mixture was filtered, and the solid was retained. Ethanol was added to the filtrate until the ethanol content of the system was 85 wt%. The mixture was allowed to precipitate for 5 hours. The polysaccharide solid was collected, washed, and dried to obtain traditional Chinese medicine polysaccharide. The filtrate was retained.

[0064] S3. Preparation of polysaccharide-Fe / selenium complex: 12 parts by weight of selenium-enriched kudzu polysaccharide obtained in step S1 and 10 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were added to 100 parts by weight of water, 3 parts by weight of ferric nitrate were added, the mixture was stirred and reacted for 30 min, ethanol was added until the ethanol content of the system was 85 wt%, precipitation was carried out for 4 h, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex.

[0065] S4. Fermentation: Add 15 parts by weight of the solid from step S1 and 12 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans and Bifidobacterium bifidum seed solutions. The inoculation amounts of Bacillus coagulans and Bifidobacterium bifidum seed solutions are 3v / v% and 4v / v%, respectively. Fermentate at 40°C and 200 r / min for 56 h, filter, wash the solid, collect the bacterial solution, mix 17 parts by volume of the bacterial solution with 12 parts by volume of the selenium-enriched yeast solution obtained in step S1, concentrate, and obtain a bacterial count of 10. 11 -10 12 The concentrated bacterial culture was prepared at cfu / mL; the filtrate was dialyzed for 5 hours using a dialysis bag with a pore size of 5 kDa, and the dialysate was freeze-dried. 7 parts by weight of the obtained protein peptides were mixed evenly with 4 parts by weight of the selenium-enriched kudzu root protein peptides prepared in step S1 to obtain active protein peptides; the permeate was retained.

[0066] S5. Extraction of active components: Mix 12 parts by weight of the permeate from step S1 and 20 parts by weight of the permeate from step S4 evenly, add 70 parts by weight of organic solvent for extraction, collect the organic layer, remove the organic solvent under reduced pressure, wash, dry, and obtain the active components.

[0067] The organic solvent is a mixed solution of cyclohexane and ethyl acetate in a volume ratio of 5:10;

[0068] S6. Preparation of sodium alginate-probiotic / polysaccharide microcapsules: Add 17 parts by weight of sodium alginate, 7 parts by weight of polysaccharide-Fe / selenium complex prepared in step S3, and 1 part by weight of lecithin to 120 parts by weight of the concentrated bacterial solution obtained in step S4. Stir and mix evenly, add to 200 parts by weight of fish oil, emulsify at 10000 r / min for 15 min, add 10 parts by weight of 5 wt% calcium chloride solution, solidify at room temperature for 40 min, centrifuge, wash, and dry to obtain sodium alginate-probiotic / polysaccharide microcapsules;

[0069] S7. Preparation of the probiotic composition for enhancing immunity: 7 parts by weight of the active protein peptide obtained in step S4, 5 parts by weight of the active component obtained in step S5, 20 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, 2 parts by weight of taurine, and 1 part by weight of ginsenoside Rg3 are mixed evenly to obtain the probiotic composition for enhancing immunity.

[0070] Example 3

[0071] This embodiment provides a method for preparing a probiotic composition to enhance immunity, specifically including the following steps:

[0072] S1. Fermentation of selenium-enriched kudzu root: Selenium-enriched kudzu root powder was added to water, with a solid-liquid ratio of 1:6 g / mL. The mixture was stirred and sterilized. Selenium-enriched yeast seed solution was inoculated at a rate of 2 v / v%, fermented at 52℃ and 150 r / min for 42 h, filtered, and the solid was washed. The bacterial solution was collected to obtain selenium-enriched yeast solution, and the solid was retained. Ethanol was added to the filtrate until the ethanol content of the system was 80 wt%, and precipitation was carried out for 4 h. The polysaccharide solid was collected, washed, and dried to obtain selenium-enriched kudzu root polysaccharide. The liquid was dialyzed using a dialysis bag with a pore size of 3 kDa for 4 h. The dialysate was freeze-dried to obtain selenium-enriched kudzu root protein peptides, and the permeate was retained.

[0073] S2. Preparation of Traditional Chinese Medicine Extract: 6 parts by weight of Astragalus membranaceus, 11 parts by weight of Rhodiola rosea, 4 parts by weight of Codonopsis pilosula, and 3 parts by weight of Panax notoginseng were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was extracted by boiling in water for 3 hours. The solid-liquid ratio of the traditional Chinese medicine powder to water was 1:8 g / mL. The mixture was filtered, and the solid was retained. Ethanol was added to the filtrate until the ethanol content of the system was 80 wt%. The mixture was allowed to precipitate for 4 hours. The polysaccharide solid was collected, washed, and dried to obtain traditional Chinese medicine polysaccharide. The filtrate was retained.

[0074] S3. Preparation of polysaccharide-Fe / selenium complex: 11 parts by weight of selenium-enriched kudzu polysaccharide obtained in step S1 and 8.5 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were added to 100 parts by weight of water, 2 parts by weight of ferric sulfate were added, the mixture was stirred and reacted for 25 min, ethanol was added until the ethanol content of the system was 80 wt%, precipitation was carried out for 3 h, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex.

[0075] S4. Fermentation: Add 12 parts by weight of the solid from step S1 and 10 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans and Bifidobacterium bifidum seed solutions. The inoculation amounts of Bacillus coagulans and Bifidobacterium bifidum seed solutions are 2v / v% and 3v / v%, respectively. Ferment at 39°C and 150 r / min for 52 h, filter, wash the solid, collect the bacterial solution, mix 15 parts by volume of the bacterial solution with 10 parts by volume of the selenium-enriched yeast solution obtained in step S1, concentrate, and obtain a bacterial count of 10. 11 -10 12 The concentrated bacterial culture was prepared at cfu / mL; the filtrate was dialyzed for 4 hours using a dialysis bag with a pore size of 3 kDa, and the dialysate was freeze-dried. Six parts by weight of the obtained protein peptides were mixed evenly with three parts by weight of the selenium-enriched kudzu root protein peptides prepared in step S1 to obtain active protein peptides; the permeate was retained.

[0076] S5. Extraction of active components: Mix 11 parts by weight of the permeate from step S1 and 17 parts by weight of the permeate from step S4 evenly, add 60 parts by weight of organic solvent for extraction, collect the organic layer, remove the organic solvent under reduced pressure, wash, dry, and obtain the active components.

[0077] The organic solvent is a mixed solution of cyclohexane and ethyl acetate in a volume ratio of 4:8.5;

[0078] S6. Preparation of sodium alginate-probiotic / polysaccharide microcapsules: Add 16 parts by weight of sodium alginate, 6 parts by weight of polysaccharide-Fe / selenium complex prepared in step S3, and 0.7 parts by weight of lecithin to 110 parts by weight of the concentrated bacterial solution obtained in step S4. Stir and mix evenly, add to 200 parts by weight of fish oil, emulsify, add 7 parts by weight of 4wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain sodium alginate-probiotic / polysaccharide microcapsules;

[0079] S7. Preparation of the probiotic composition for enhancing immunity: 6 parts by weight of the active protein peptide obtained in step S4, 4 parts by weight of the active component obtained in step S5, 17 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, 1.5 parts by weight of taurine, and 0.7 parts by weight of ginsenoside Rg3 are mixed evenly to obtain the probiotic composition for enhancing immunity.

[0080] Comparative Example 1

[0081] The difference compared to Example 3 is that selenium-enriched kudzu polysaccharide was not added in step S3.

[0082] Specifically as follows:

[0083] S3. Preparation of polysaccharide-Fe / selenium complex: 19.5 parts by weight of the traditional Chinese medicine polysaccharide obtained in step S2 were added to 100 parts by weight of water, 2 parts by weight of ferric sulfate were added, the mixture was stirred for 25 min, ethanol was added until the ethanol content of the system was 80 wt%, precipitation was carried out for 3 h, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex.

[0084] Comparative Example 2

[0085] The difference from Example 3 is that no traditional Chinese medicine polysaccharides were added in step S3.

[0086] Specifically as follows:

[0087] S3. Preparation of polysaccharide-Fe / selenium complex: 19.5 parts by weight of selenium-enriched kudzu polysaccharide obtained in step S1 was added to 100 parts by weight of water, 2 parts by weight of ferric sulfate was added, the mixture was stirred for 25 min, ethanol was added until the ethanol content of the system was 80 wt%, precipitation was carried out for 3 h, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex.

[0088] Comparative Example 3

[0089] The difference from Example 3 is that ferric sulfate was not added in step S3.

[0090] Specifically as follows:

[0091] S3. Preparation of polysaccharide mixture: 11 parts by weight of selenium-enriched kudzu root polysaccharide obtained in step S1 and 8.5 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 are mixed evenly to obtain polysaccharide mixture.

[0092] Comparative Example 4

[0093] The difference from Example 3 is that Bacillus coagulans seed solution was not inoculated in step S4.

[0094] Specifically as follows:

[0095] S4. Fermentation: Add 12 parts by weight of the solid from step S1 and 10 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bifidobacterium bifidum seed solution at an inoculation rate of 5 v / v%, ferment at 39°C and 150 r / min for 52 h, filter, wash the solid, collect the bacterial solution, mix 15 parts by volume of the bacterial solution with 10 parts by volume of the selenium-enriched yeast solution obtained in step S1, concentrate, and obtain a bacterial count of 10. 11 -10 12 The concentrated bacterial culture was prepared at cfu / mL; the filtrate was dialyzed for 4 hours using a dialysis bag with a pore size of 3 kDa, and the dialysate was freeze-dried. Six parts by weight of the obtained protein peptide were mixed evenly with three parts by weight of the selenium-enriched kudzu root protein peptide prepared in step S1 to obtain the active protein peptide; the permeate was retained.

[0096] Comparative Example 5

[0097] The difference from Example 3 is that Bifidobacterium bifidum seed solution was not inoculated in step S4.

[0098] Specifically as follows:

[0099] S4. Fermentation: Add 12 parts by weight of the solid from step S1 and 10 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans seed solution. The inoculation amount of the Bacillus coagulans seed solution is 5v / v%, fermentation is carried out at 39℃ and 150r / min for 52h, filtered, solid washed, and bacterial solution collected. Mix 15 parts by volume of bacterial solution with 10 parts by volume of selenium-enriched yeast solution obtained in step S1 evenly, concentrate, and obtain a bacterial count of 10. 11 -10 12The concentrated bacterial culture was prepared at cfu / mL; the filtrate was dialyzed for 4 hours using a dialysis bag with a pore size of 3 kDa, and the dialysate was freeze-dried. Six parts by weight of the obtained protein peptide were mixed evenly with three parts by weight of the selenium-enriched kudzu root protein peptide prepared in step S1 to obtain the active protein peptide; the permeate was retained.

[0100] Comparative Example 6

[0101] The difference from Example 3 is that selenium-enriched kudzu root protein peptides were not added to the active protein peptides in step S4.

[0102] Specifically as follows:

[0103] S4. Fermentation: Add 12 parts by weight of the solid from step S1 and 10 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans and Bifidobacterium bifidum seed solutions. The inoculation amounts of Bacillus coagulans and Bifidobacterium bifidum seed solutions are 2v / v% and 3v / v%, respectively. Ferment at 39°C and 150 r / min for 52 h, filter, wash the solid, collect the bacterial solution, mix 15 parts by volume of the bacterial solution with 10 parts by volume of the selenium-enriched yeast solution obtained in step S1, concentrate, and obtain a bacterial count of 10. 11 -10 12 The concentrated bacterial culture was prepared at cfu / mL; the filtrate was dialyzed for 4 hours using a dialysis bag with a pore size of 3 kDa, and the dialysate was freeze-dried. Nine parts by weight of the obtained protein peptides were used as active protein peptides; the permeate was retained.

[0104] Comparative Example 7

[0105] The difference from Example 3 is that no protein peptides were added to the active protein peptides in step S4.

[0106] Specifically as follows:

[0107] S4. Fermentation: Add 12 parts by weight of the solid from step S1 and 10 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans and Bifidobacterium bifidum seed solutions. The inoculation amounts of Bacillus coagulans and Bifidobacterium bifidum seed solutions are 2v / v% and 3v / v%, respectively. Ferment at 39°C and 150 r / min for 52 h, filter, wash the solid, collect the bacterial solution, mix 15 parts by volume of the bacterial solution with 10 parts by volume of the selenium-enriched yeast solution obtained in step S1, concentrate, and obtain a bacterial count of 10. 11 -10 12 Concentrated bacterial culture (cfu / mL); 9 parts by weight of selenium-enriched kudzu root protein peptides obtained in step S1 were used as active protein peptides; the filtrate was retained.

[0108] Comparative Example 8

[0109] The difference from Example 3 is that organic solvent extraction was not performed in step S5.

[0110] Specifically as follows:

[0111] S5. Extraction of active components: Mix 11 parts by weight of the permeate from step S1 and 17 parts by weight of the permeate from step S4 evenly, freeze-dry, and obtain the active components.

[0112] Comparative Example 9

[0113] The difference from Example 3 is that the polysaccharide-Fe / selenium complex was not added in step S6.

[0114] Specifically as follows:

[0115] S6. Preparation of sodium alginate-probiotic microcapsules: Add 22 parts by weight of sodium alginate and 0.7 parts by weight of lecithin to 110 parts by weight of the concentrated bacterial solution obtained in step S4, stir and mix evenly, add 200 parts by weight of fish oil, emulsify, add 7 parts by weight of 4wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain sodium alginate-probiotic microcapsules.

[0116] Comparative Example 10

[0117] The difference from Example 3 is that no active protein peptides were added in step S7.

[0118] Specifically as follows:

[0119] S7. Preparation of probiotic composition for enhancing immunity: 4 parts by weight of the active component obtained in step S5, 17 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, 1.5 parts by weight of taurine, and 0.7 parts by weight of ginsenoside Rg3 are mixed evenly to obtain a probiotic composition for enhancing immunity.

[0120] Comparative Example 11

[0121] The difference from Example 3 is that no active ingredient was added in step S7.

[0122] Specifically as follows:

[0123] S7. Preparation of probiotic composition for enhancing immunity: 6 parts by weight of the active protein peptide obtained in step S4, 17 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, 1.5 parts by weight of taurine, and 0.7 parts by weight of ginsenoside Rg3 are mixed evenly to obtain a probiotic composition for enhancing immunity.

[0124] Comparative Example 12

[0125] The difference from Example 3 is that in step S7, the sodium alginate-probiotic / polysaccharide microcapsules are replaced by an equal amount of probiotic freeze-dried powder.

[0126] Specifically as follows:

[0127] S7. Preparation of a probiotic composition for enhancing immunity: 6 parts by weight of the active protein peptide obtained in step S4, 4 parts by weight of the active component obtained in step S5, 17 parts by weight of the probiotic freeze-dried powder (containing Bacillus coagulans, Bifidobacterium bifidum, and selenium-enriched yeast in a mass ratio of 2:3:2), 1.5 parts by weight of taurine, and 0.7 parts by weight of ginsenoside Rg3 are mixed evenly to obtain a probiotic composition for enhancing immunity.

[0128] Comparative Example 13

[0129] The difference from Example 3 is that taurine was not added in step S7.

[0130] Specifically as follows:

[0131] S7. Preparation of probiotic composition for enhancing immunity: 6 parts by weight of the active protein peptide obtained in step S4, 4 parts by weight of the active component obtained in step S5, 17 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, and 2.2 parts by weight of ginsenoside Rg3 are mixed evenly to obtain a probiotic composition for enhancing immunity.

[0132] Comparative Example 14

[0133] The difference from Example 3 is that ginsenoside Rg3 was not added in step S7.

[0134] Specifically as follows:

[0135] S7. Preparation of probiotic composition for enhancing immunity: 6 parts by weight of the active protein peptide obtained in step S4, 4 parts by weight of the active component obtained in step S5, 17 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, and 2.2 parts by weight of taurine are mixed evenly to obtain a probiotic composition for enhancing immunity.

[0136] Comparative Example 15

[0137] The difference from Example 3 is that taurine and ginsenoside Rg3 were not added in step S7.

[0138] Specifically as follows:

[0139] S7. Preparation of the probiotic composition for enhancing immunity: Mix 6 parts by weight of the active protein peptide obtained in step S4, 4 parts by weight of the active component obtained in step S5, and 17 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6 evenly to obtain the probiotic composition for enhancing immunity.

[0140] Test Example 1

[0141] The sodium alginate-probiotic / polysaccharide microcapsules prepared in Examples 1-3, the sodium alginate-probiotic microcapsules prepared in Comparative Example 9, and a commercially available probiotic agent (Bacillus coagulans, Bifidobacterium bifidum, and selenium-enriched yeast in a mass ratio of 2:3:2, without encapsulation) were subjected to tests for resistance to gastric acid, choline, and intestinal fluid. The results are shown in Table 1.

[0142] Choline tolerance test:

[0143] Add 0.3 wt% ox bile salt to LB liquid medium, sterilize, add 1 g microcapsule and commercially available probiotic agent to the above medium, and incubate at 37℃ and 180 r / min for 2 h. Take samples at 0 and 2 h for plate colony counting and calculate the survival rate.

[0144] Gastric acid resistance test:

[0145] Preparation of artificial gastric juice: Take 16.4 mL of 10% hydrochloric acid, dilute with water to make the pH value 2, sterilize, add 1 g of pepsin to every 100 mL, mix well, and the artificial gastric juice is prepared.

[0146] Add 1g of microcapsules and commercially available probiotics to artificial gastric fluid, and incubate at 37℃ and 180r / min for 2h. Take samples at 0 and 2h for plate colony counting and calculate the survival rate.

[0147] Intestinal fluid resistance test:

[0148] Preparation of artificial intestinal fluid: Dissolve 6.8g of Na2HPO4 in 500mL of water, adjust the pH to 6.8 with sodium hydroxide solution, dilute with water to 1000mL, sterilize, add 1g of trypsin to each 100mL of liquid, mix well, and the artificial intestinal fluid is obtained.

[0149] Add 1g of microcapsules to artificial intestinal fluid and incubate at 37℃ and 180r / min for 2h. Take samples at 0 and 2h for plate colony counting and calculate the survival rate.

[0150] Strain survival rate (%) = N t / N0×100%.

[0151] N t : Viable bacteria count after 2 hours; N0: Viable bacteria count after 0 hours.

[0152] Table 1

[0153]

[0154] As shown in the table above, the sodium alginate-probiotic / polysaccharide microcapsules prepared in Examples 1-3 of this invention have excellent resistance to gastric acid, choline, and intestinal fluid. Under the synergistic encapsulation effect of the polysaccharide-Fe / selenium complex and sodium alginate, the polysaccharide-Fe / selenium complex can form a relatively dense and stable shell with sodium alginate under the cross-linking effect of calcium chloride, thus encapsulating the probiotics in the microspheres. This effectively protects the probiotics from the effects of gastric acid, choline, and intestinal fluid, increases the number of viable probiotics, and greatly enhances the regulatory effect of probiotics.

[0155] Test Example 2

[0156] Female ICR mice, weighing 18-22g, were randomly divided into a blank group, Example 1-3 groups, and Comparative Example 1-15 groups, with 6 mice in each group. The mice were administered 1g / kg by gavage for 30 consecutive days and then sacrificed by cervical spine. Their organ indices, ConA-induced mouse lymphocyte transformation experiment, delayed-type pathological reaction test - ear swelling degree, and NK cell activity (lactate dehydrogenase activity) were measured.

[0157] 1. Organ Index

[0158] Before sacrifice, weigh the mice, dissect and remove the thymus and spleen, weigh them, and calculate the weight using the following formula:

[0159] Thymus index = thymus weight (mg) / mouse body weight (g)

[0160] Spleen index = Spleen weight (mg) / Mouse body weight (g)

[0161] The results are shown in Table 2.

[0162] Table 2

[0163]

[0164]

[0165] Note: * indicates P < 0.05 compared to the control group.

[0166] The thymus and spleen, as immune organs, can directly reflect the immune status. As shown in the table above, the probiotic compositions for improving immunity prepared in Examples 1-3 of this invention can significantly increase the thymus index and spleen index, thus having the effect of improving immunity.

[0167] 2. ConA-induced transformation of mouse lymphocytes experiment

[0168] Place a sterile mouse spleen into a small petri dish containing an appropriate amount of sterile Hanks' solution, shred it, filter, and obtain a single-cell suspension. Wash twice with Hanks' solution, centrifuge, and adjust the cell concentration to 4 × 10⁶ cells using trypan blue viable cell counting. 6Cells / mL. Cell suspension was added to two wells of a 24-well plate; 75 μL of concanavalin A (ConA) was added to one well, and the other well served as a control. After 72 h of culture, 4 h before the end of the culture period, 0.7 mL of supernatant was aspirated from each well, and 0.7 mL of RPMI 1640 (without fetal bovine serum) and 50 μL of MTT solution were added per well. Culture continued. After culture, 1 mL of acidic isopropanol was added to each well, and the mixture was stirred until the purple crystals dissolved. The OD value was measured at 570 nm. The results are shown in Table 3.

[0169] Table 3

[0170]

[0171]

[0172] Note: * indicates P < 0.05 compared to the control group.

[0173] Lymphocyte transformation can be detected in vitro by assessing lymphocyte responsiveness. As shown in the table above, the probiotic compositions for enhancing immunity prepared in Examples 1-3 of this invention can significantly enhance the transformation ability of ConA-induced mouse spleen lymphocytes.

[0174] 3. Delayed-type hypersensitivity test

[0175] On day 25 after gavage, hair was removed from a 3cm × 3cm area on the mouse abdomen, and sensitization was achieved by applying 50 μL of DNFB solution to the skin. Five days later, 10 μL of DNFB solution was applied to both sides of the mouse's right ear. Twenty-four hours later, the mice were sacrificed, and the left and right ear flaps were removed. A 6mm diameter disc was taken using a punch and weighed. The ear swelling rate (%) was calculated. The results are shown in Table 4.

[0176] Ear swelling rate (%) = (Right ear weight - Left ear weight) / Left ear weight × 100%

[0177] Table 4

[0178] Group Ear swelling rate (%) Blank group 9.92±3.12 Example 1 51.24±3.49 Example 2 51.09±3.95 Example 3 52.37±4.81 Comparative Example 1 47.52±3.57 Comparative Example 2 46.58±4.09 Comparative Example 3 48.57±3.82 Comparative Example 4 45.11±4.28 Comparative Example 5 44.47±3.76 Comparative Example 6 43.19±4.82 Comparative Example 7 42.98±3.85 Comparative Example 8 46.82±4.81 Comparative Example 9 42.29±4.19 Comparative Example 10 40.82±4.89 Comparative Example 11 41.27±5.77 Comparative Example 12 38.96±3.57 Comparative Example 13 46.22±4.28 Comparative Example 14 45.18±3.76 Comparative Example 15 42.39±3.42

[0179] Note: * indicates P < 0.05 compared to the control group.

[0180] As shown in the table above, the probiotic compositions for enhancing immunity prepared in Examples 1-3 of this invention can significantly increase the ear swelling rate and significantly improve the cellular immune function of mice.

[0181] 4. Mouse NK cell activity assay (lactate dehydrogenase (LDH) assay)

[0182] Blood was collected from mouse eyeballs and centrifuged immediately to obtain serum, which was then measured according to the lactate dehydrogenase (LDH) assay kit. The results are shown in Table 5.

[0183] Lactate dehydrogenase activity (U / L) = Measured OD value - Control OD value - Standard OD value - Blank OD value × Standard concentration (0.2 μmol / mL) × 1000.

[0184] Table 5

[0185] Group Lactate dehydrogenase activity (U / L) Blank group 262.66±70.15 Example 1 785.26±126.26* Example 2 789.15±155.19* Example 3 792.56±148.66* Comparative Example 1 712.26±132.18 Comparative Example 2 715.29±129.58 Comparative Example 3 725.18±148.81 Comparative Example 4 702.55±138.66 Comparative Example 5 697.83±152.68 Comparative Example 6 731.25±118.29 Comparative Example 7 734.58±125.81 Comparative Example 8 710.59±149.15 Comparative Example 9 684.26±142.95 Comparative Example 10 707.28±145.19 Comparative Example 11 711.92±138.57 Comparative Example 12 665.28±123.58 Comparative Example 13 686.21±143.32 Comparative Example 14 683.59±145.29 Comparative Example 15 672.15±139.16

[0186] Note: * indicates P < 0.05 compared to the control group.

[0187] As shown in the table above, the probiotic compositions for enhancing immunity prepared in Examples 1-3 of this invention can significantly increase the activity of serum lactate dehydrogenase in mice and significantly enhance the activity of NK cells in mice.

[0188] Compared with Example 3, Comparative Examples 1 and 2 did not include the addition of selenium-enriched *Gnaphalium affine* polysaccharide or other traditional Chinese medicine polysaccharides in step S3. Organ indices decreased, as did spleen lymphocyte transformation capacity, ear swelling rate, serum lactate dehydrogenase activity, and the ability to regulate immunity. Selenium-enriched *Gnaphalium affine* polysaccharide can enhance immunity by promoting T lymphocyte proliferation, improving B lymphocyte function, increasing the number of neutrophils, enhancing macrophage phagocytic function, and increasing cytokine secretion. The traditional Chinese medicine composition of this invention includes *Astragalus membranaceus*, *Rhodiola rosea*, *Codonopsis pilosula*, and *Panax notoginseng*, containing abundant polysaccharide components. These polysaccharides can effectively regulate human immune function, enhance the activity of immune cells such as the reticuloendothelial system, macrocells, natural killer cells, and cytotoxic T cells, promote antibody and complement production, and induce interferon production, exhibiting a synergistic effect.

[0189] Compared with Example 3, Comparative Example 3 did not include the addition of ferric sulfate in step S3. Splenic lymphocyte transformation capacity, serum lactate dehydrogenase activity, and immune regulation ability decreased. In this invention, the traditional Chinese medicine polysaccharide is mixed with selenium-enriched *Ge Xian Mi* polysaccharide. Based on the presence of free hydroxyl, carboxyl, and amino groups in the polysaccharide molecular backbone that can provide lone pairs of electrons, these groups act as ligands, releasing lone pairs of electrons into the empty orbitals of metal ions, and reacting with Fe... 3+ Coordination reactions generate stable polysaccharide-metal complexes, which have good antioxidant, anti-anemic, antibacterial, antitumor, and immunomodulatory activities. They can remove excess metal ions and reactive oxygen species in the body, alleviate cellular oxidative damage, maintain genome stability, inhibit cell mutation and thus delay aging, regulate the proliferation of T lymphocytes and the secretion of immune factors, maintain the polarization balance of monocytes / macrophages M1 and M2, and promote the specific immunoglobulin cascade enzyme reaction in serum, thereby activating the complement system.

[0190] Compared with Example 3, Comparative Examples 4 and 5 did not involve inoculation with Bacillus coagulans seed solution or Bifidobacterium bifidum seed solution in step S4. Organ indices decreased, as did spleen lymphocyte transformation capacity, ear swelling rate, serum lactate dehydrogenase activity, and immune regulation capacity. The solid residue from the fermentation of selenium-enriched *Ge Xian Mi* (a type of medicinal herb) and the solid residue from the water extraction of the traditional Chinese medicine composition were mixed and inoculated with *Bacillus coagulans* and *Bifidobacterium bifidum*. This process promotes cell wall disruption by the fermenting bacteria, dissolving abundant proteins, amino acids, trace elements, flavonoids, triterpenes, sterols, polyphenols, and organic acids from the *Ge Xian Mi* and other medicinal plant cells, significantly improving raw material utilization efficiency. The waste residue after fermentation can be used as animal feed or plant fertilizer. Furthermore, it promotes the proliferation of probiotics and the release of beneficial substances. Subsequently, the fermenting bacteria were eluted and mixed with selenium-enriched yeast solution for concentration to obtain a high concentration of probiotics. *Bifidobacterium bifidum* significantly reduced the self-inducing substances secreted by pathogenic *Escherichia coli*, thereby reducing the adhesion ability of this pathogen to host cells. *Bacillus coagulans* can interact with dendritic cells, enhancing T cell proliferation and immune response, causing T cells to differentiate into Th1, Th2, and Treg cells, activating the immune system, producing cytokines, and improving anti-infection capabilities. Selenium-enriched yeast can regulate epithelial tissue function, such as mucus secretion from goblet cells, defensin release from Paneth cells, and tight junction protein synthesis in normal epithelial cells, thus regulating and preventing epithelial cell apoptosis. These three mechanisms have a synergistic effect. Furthermore, these probiotics produce antitoxins such as organic acids, bacteriocins, glycoproteins, enzymes, and H2O2 to inhibit the growth of harmful microorganisms. Organic acids can increase intestinal acidity, thereby inhibiting the growth of pathogenic bacteria. The produced alcohols, diacetone, lysozyme, H2O2, and other antimicrobial substances (such as lactobacillus and nisin) can also inhibit the growth of intestinal pathogens.

[0191] Compared with Example 3, Comparative Examples 6 and 7 did not add selenium-enriched *Gnaphalium affine* protein peptides or other protein peptides to the active protein peptides in step S4. Compared with Example 3, Comparative Example 10 did not add active protein peptides in step S7. Organ indices decreased, splenic lymphocyte transformation capacity decreased, ear swelling rate decreased, serum lactate dehydrogenase activity decreased, and the ability to regulate immunity decreased. Protein peptides are structural fragments of proteins that can function as active groups of proteins. They have advantages such as low molecular weight, simple structure, easy water solubility, strong hygroscopicity, good emulsification, good thermal stability, low viscosity, low immunogenicity, strong permeability, high utilization efficiency, and ease of modification and transformation. The protein peptides obtained by fermentation extraction of traditional Chinese medicine in this invention, as well as the selenium-enriched *Gnaphalium affine* protein peptides obtained by fermentation extraction of selenium-enriched *Gnaphalium affine*, can effectively regulate the body's immunity and inhibit tumor growth. They directly kill pathogens through macrophages, presenting antigens to trigger an immune response and enhance the body's immune capacity against tumors.

[0192] Compared to Example 3, Comparative Example 8 did not involve organic solvent extraction in step S5. Compared to Example 3, Comparative Example 11 did not involve the addition of active components in step S7. Organ indices decreased, as did spleen lymphocyte transformation capacity, ear swelling rate, and immune-regulating ability. This invention also collects the permeate after fermentation, which contains abundant active substances, including flavonoids, triterpenes, sterols, polyphenols, and organic acids. These are enriched and purified through organic solvent extraction, significantly increasing the content of active substances in the active components and further enhancing the immunomodulatory effect.

[0193] Compared to Example 3, Comparative Example 9 did not add the polysaccharide-Fe / selenium complex in step S6. Compared to Example 3, in Comparative Example 12, the sodium alginate-probiotic / polysaccharide microcapsules were replaced with an equal amount of lyophilized probiotic powder in step S7. Organ indices decreased, as did spleen lymphocyte transformation capacity, ear swelling rate, serum lactate dehydrogenase activity, and immune regulation ability. This invention utilizes the synergistic encapsulation of concentrated bacterial culture with a polysaccharide-Fe / selenium complex and sodium alginate. On one hand, the polysaccharide-Fe / selenium complex forms a relatively dense and stable shell with sodium alginate under the cross-linking effect of calcium chloride, encapsulating probiotics within microspheres. This effectively protects the probiotics from the effects of gastric acid, choline, and intestinal fluid, increasing the viable count of probiotics and significantly enhancing their regulatory effect. On the other hand, the polysaccharide-Fe / selenium complex and sodium alginate are also excellent prebiotics. After being degraded and broken down in the intestines, they can be directly utilized by probiotics, promoting their proliferation. While the polysaccharide-Fe / selenium complex is utilized, the selenium-enriched *Ge Xian Mi* polysaccharide and traditional Chinese medicine polysaccharides (including *Astragalus membranaceus* polysaccharide, *Rhodiola rosea* polysaccharide, *Codonopsis pilosula* polysaccharide, and *Panax notoginseng* polysaccharide) produced by its decomposition exert excellent antioxidant, anti-inflammatory, immunomodulatory, and anti-tumor effects. Simultaneously, it releases Fe ions that are directly absorbed by the intestines, further enhancing the antioxidant and immunomodulatory effects.

[0194] Compared with Example 3, Comparative Examples 13 and 14 did not include taurine or ginsenoside Rg3 in step S7. Comparative Example 15, compared with Example 3, did not include taurine or ginsenoside Rg3 in step S7. Organ indices decreased, spleen lymphocyte transformation capacity decreased, ear swelling rate decreased, serum lactate dehydrogenase activity decreased, and the ability to regulate immunity decreased. The composition of this invention includes taurine and ginsenoside Rg3, which have good immunomodulatory effects. Taurine is a good immune stabilizer and membrane stabilizer, protecting nerve cells and enabling them to perform normal physiological functions. It has a good protective effect on lymphocytes, promoting lymphocyte proliferation and antibody production, promoting macrophage production of interleukin-1, enhancing the phagocytic and bactericidal function of neutrophils, and improving the specific immune function of humans and animals. Ginsenoside Rg3 has the effects of improving immunity, antibacterial activity, improving insufficient blood supply to the heart and brain, regulating the central nervous system, anti-fatigue, delaying aging, anti-tumor activity, and rapid recovery of physical condition. The addition of both has a synergistic effect.

[0195] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a probiotic composition to enhance immunity, characterized in that, Selenium-enriched *Ge Xian Ji* (a type of herb) was fermented with selenium-enriched yeast to extract selenium-enriched *Ge Xian Ji* polysaccharides and protein peptides. These were then mixed with polysaccharides extracted from *Huang Qi* (Astragalus membranaceus), *Rhodiola rosea*, *Codonopsis pilosula*, and *Panax notoginseng*, and reacted with iron salts to prepare a polysaccharide-Fe / selenium complex. The solid residue was then fermented together with the selenium-enriched *Ge Xian Ji* protein peptides to obtain active protein peptides. The permeate was extracted with an organic solvent to obtain active components. The bacterial solution was collected and encapsulated in a mixture of the polysaccharide-Fe / selenium complex and sodium alginate. This mixture was then thoroughly mixed with the active protein peptides, active components, taurine, and ginsenoside Rg3 to obtain a probiotic composition that enhances immunity.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Fermentation of selenium-enriched kudzu root: Add selenium-enriched kudzu root powder to water, stir and mix evenly, sterilize, inoculate with selenium-enriched yeast seed solution, ferment and culture, filter, wash the solid, collect the bacterial solution to obtain selenium-enriched yeast solution, and keep the solid for use; add ethanol to the filtrate to precipitate, collect the polysaccharide solid, wash, dry to obtain selenium-enriched kudzu root polysaccharide, dialyze the liquid, freeze-dry the dialysate to obtain selenium-enriched kudzu root protein peptide, and keep the permeate for use; S2. Preparation of Chinese herbal extracts: Astragalus membranaceus, Rhodiola rosea, Codonopsis pilosula, and Panax notoginseng were washed, dried, and pulverized to obtain Chinese herbal powder. Water was added for boiling extraction, and the powder was filtered. The solid was retained. Ethanol was added to the filtrate to precipitate the polysaccharide solid. The solid was collected, washed, and dried to obtain Chinese herbal polysaccharide. The filtrate was retained. S3. Preparation of polysaccharide-Fe / selenium complex: The selenium-enriched kudzu polysaccharide obtained in step S1 and the traditional Chinese medicine polysaccharide obtained in step S2 were added to water, iron salt was added, the mixture was stirred and reacted, ethanol was added to precipitate, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex. S4. Fermentation: Add the solids from step S1 and step S2 to water, sterilize, inoculate with Bacillus coagulans and Bifidobacterium bifidum seed liquid, ferment, filter, wash the solids, collect the bacterial liquid, mix it evenly with the selenium-enriched yeast liquid obtained in step S1, concentrate, and obtain concentrated bacterial liquid; dialyze the filtrate, freeze-dry the dialysate, mix it evenly with the selenium-enriched kudzu root protein peptide obtained in step S1, and obtain active protein peptide; retain the permeate; S5. Extraction of active components: Mix the permeate from step S1 and the permeate from step S4 evenly, add organic solvent for extraction, collect the organic layer, remove the organic solvent under reduced pressure, wash, dry, and obtain the active components. S6. Preparation of sodium alginate-probiotic / polysaccharide microcapsules: Add sodium alginate, polysaccharide-Fe / selenium complex and lecithin obtained in step S3 to the concentrated bacterial solution obtained in step S4, stir and mix evenly, add to fish oil, emulsify, add calcium chloride solution dropwise, solidify at room temperature, centrifuge, wash and dry to obtain sodium alginate-probiotic / polysaccharide microcapsules; S7. Preparation of probiotic composition to enhance immunity: The active protein peptide obtained in step S4, the active component obtained in step S5, the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, taurine, and ginsenoside Rg3 are mixed evenly to obtain a probiotic composition to enhance immunity.

3. The preparation method according to claim 2, characterized in that, The selenium-enriched *Ge Xian Mi* powder mentioned in step S1 has a selenium content of 300-500 μg / 100g, the solid-liquid ratio of the selenium-enriched *Ge Xian Mi* powder to water is 1:5-7 g / mL, and the bacterial count of the inoculum seed solution is 10. 8 -10 9 The inoculum concentration of the selenium-enriched yeast seed solution is 1-3 v / v%, the fermentation conditions are 50-55℃, 100-200 r / min, and fermentation culture for 36-48 h, the dialysis bag pore size is 2-5 kDa, and the dialysis time is 3-5 h.

4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of Astragalus membranaceus, Rhodiola rosea, Codonopsis pilosula, and Panax notoginseng is 5-7:10-12:3-5:2-4, the solid-liquid ratio of the herbal powder to water is 1:7-10 g / mL, and the boiling extraction time is 2-4 h.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of selenium-enriched kudzu root protein peptide, traditional Chinese medicine polysaccharide, and iron salt is 10-12:7-10:1-3, and the iron salt is selected from at least one of ferric chloride, ferric sulfate, and ferric nitrate.

6. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of the solid in step S1 to the solid in step S2 is 10-15:8-12, and the bacterial count of the inoculum solution is 10. 8 -10 9 The inoculum concentration of *Bacillus coagulans* and *Bifidobacterium bifidum* was 1-3 v / v% and 2-4 v / v%, respectively. The fermentation conditions were 38-40℃, 100-200 r / min, and 48-56 h. The volume ratio of the collected bacterial solution to the selenium-enriched yeast solution obtained in step S1 was 12-17:8-12. The bacterial count of the concentrated bacterial solution was 10... 11 -10 12 The concentration of cfu / mL is specified. The dialysis bag used for the dialysis has a pore size of 2-5 kDa, the dialysis time is 3-5 h, and the mass ratio of the protein peptide to the selenium-enriched kudzu root protein peptide is 5-7:2-4.

7. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of the permeate in step S1 to the permeate in step S4 is 10-12:15-20, and the organic solvent is a mixed solution of cyclohexane and ethyl acetate with a volume ratio of 3-5:7-10.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of concentrated bacterial solution, sodium alginate, polysaccharide-Fe / selenium complex, and lecithin is 100-120:15-17:5-7:0.5-1, and the solidification time at room temperature is 20-40 min; in step S7, the mass ratio of active protein peptide, active component, sodium alginate-probiotic / polysaccharide microcapsules, taurine, and ginsenoside Rg3 is 5-7:3-5:15-20:1-2:0.5-1.

9. The preparation method according to claim 2, characterized in that, Specifically, the following steps are included: S1. Fermentation of selenium-enriched kudzu root: Selenium-enriched kudzu root powder is added to water, with a solid-liquid ratio of 1:5-7 g / mL. The mixture is stirred and sterilized. Selenium-enriched yeast seed solution is inoculated at a rate of 1-3 v / v%, fermented at 50-55℃ and 100-200 r / min for 36-48 h. The mixture is filtered, the solid is washed, and the bacterial solution is collected to obtain selenium-enriched yeast solution. The solid is retained for use. Ethanol is added to the filtrate until the ethanol content of the system is 75-85 wt%. Precipitation is carried out for 3-5 h. The polysaccharide solid is collected, washed, and dried to obtain selenium-enriched kudzu root polysaccharide. The liquid is dialyzed using a dialysis bag with a pore size of 2-5 kDa for 3-5 h. The dialysate is freeze-dried to obtain selenium-enriched kudzu root protein peptides. The permeate is retained for use. The bacterial count of the selenium-enriched yeast seed solution is 10. 8 -10 9 cfu / mL; S2. Preparation of Traditional Chinese Medicine Extract: 5-7 parts by weight of Astragalus membranaceus, 10-12 parts by weight of Rhodiola rosea, 3-5 parts by weight of Codonopsis pilosula, and 2-4 parts by weight of Panax notoginseng were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was extracted by boiling in water for 2-4 hours, with a solid-liquid ratio of 1:7-10 g / mL. The mixture was filtered, and the solid was retained. Ethanol was added to the filtrate until the ethanol content reached 75-85 wt%, and precipitation was allowed for 3-5 hours. The polysaccharide solid was collected, washed, and dried to obtain the traditional Chinese medicine polysaccharide. The filtrate was retained. S3. Preparation of polysaccharide-Fe / selenium complex: 10-12 parts by weight of selenium-enriched kudzu polysaccharide obtained in step S1 and 7-10 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were added to 100 parts by weight of water, 1-3 parts by weight of iron salt were added, the mixture was stirred and reacted for 20-30 min, ethanol was added until the ethanol content of the system was 75-85 wt%, precipitation was carried out for 2-4 h, the solid was collected, washed, and dried to obtain polysaccharide-Fe / selenium complex. S4. Fermentation: Add 10-15 parts by weight of the solid from step S1 and 8-12 parts by weight of the solid from step S2 to 200 parts by weight of water, sterilize, and inoculate with Bacillus coagulans and Bifidobacterium bifidum seed solutions. The inoculation amounts of Bacillus coagulans and Bifidobacterium bifidum seed solutions are 1-3 v / v% and 2-4 v / v%, respectively. Ferment at 38-40℃ and 100-200 r / min for 48-56 h. Filter, wash the solid, collect the bacterial solution, and mix 12-17 parts by volume of the bacterial solution with 8-12 parts by volume of the selenium-enriched yeast solution obtained in step S1. Concentrate to obtain a bacterial count of 10. 11 -10 12 Concentrated bacterial culture at cfu / mL; the filtrate was dialyzed for 3-5 h using a dialysis bag with a pore size of 2-5 kDa, the dialysate was freeze-dried, and 5-7 parts by weight of the obtained protein peptides were mixed evenly with 2-4 parts by weight of the selenium-enriched kudzu root protein peptides obtained in step S1 to obtain active protein peptides; the permeate was retained. The bacterial count of the Bacillus coagulans and Bifidobacterium bifidum seed solution was 10. 8 -10 9 cfu / mL; S5. Extraction of active components: Mix 10-12 parts by weight of the permeate from step S1 and 15-20 parts by weight of the permeate from step S4 evenly, add 50-70 parts by weight of organic solvent for extraction, collect the organic layer, remove the organic solvent under reduced pressure, wash, dry, and obtain the active components. The organic solvent is a mixed solution of cyclohexane and ethyl acetate in a volume ratio of 3-5:7-10; S6. Preparation of sodium alginate-probiotic / polysaccharide microcapsules: Add 15-17 parts by weight of sodium alginate, 5-7 parts by weight of polysaccharide-Fe / selenium complex prepared in step S3, and 0.5-1 parts by weight of lecithin to 100-120 parts by weight of the concentrated bacterial solution obtained in step S4. Stir and mix evenly, add to 200 parts by weight of fish oil, emulsify, add 5-10 parts by weight of 3-5wt% calcium chloride solution, solidify at room temperature for 20-40 min, centrifuge, wash, and dry to obtain sodium alginate-probiotic / polysaccharide microcapsules. S7. Preparation of a probiotic composition for enhancing immunity: Mix 5-7 parts by weight of the active protein peptide obtained in step S4, 3-5 parts by weight of the active component obtained in step S5, 15-20 parts by weight of the sodium alginate-probiotic / polysaccharide microcapsules obtained in step S6, 1-2 parts by weight of taurine, and 0.5-1 parts by weight of ginsenoside Rg3 evenly to obtain a probiotic composition for enhancing immunity.

10. An immune-enhancing probiotic composition prepared by the method according to any one of claims 1-9.

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