CN114984065B - Probiotic composition for improving immunity and preparation method thereof - Google Patents
Probiotic composition for improving immunity and preparation method thereof Download PDFInfo
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Abstract
The invention provides a probiotic composition for improving immunity and a preparation method thereof, and belongs to the technical field of probiotics. The probiotic composition for improving immunity prepared by the invention has good effect of adjusting flora, promotes growth and reproduction of human probiotics, can stimulate in vivo immunoregulation, improves organism immunity, adjusts blood sugar, blood fat and blood pressure, prevents atherosclerosis and diabetes, reduces inflammation and infection, and promotes human health.
Description
Technical Field
The invention relates to the technical field of probiotics, in particular to a probiotic composition for improving immunity and a preparation method thereof.
Background
Immunity is the body's own defense mechanism, and is the body's recognition and elimination of any foreign bodies (viruses, bacteria, etc.) that invade externally. The ability to treat senescent, damaged, dead, denatured, autologous cells, and to recognize and treat mutant and virally infected cells in vivo. The normal body is under the action of the immune system, and the mutated cells are inhibited and decomposed. However, in the case of the population with low immunity, the immunocompetent cells cannot recognize the mutant cells, so that the mutant cells are randomly propagated, and finally, the disease is caused. Especially children, because of the imperfect physiological structure and function development, are more easily affected by environmental pathogens and the adverse effects of parents on the use of antibiotics by children in order to prevent infection, and the resistance to certain diseases needs to be improved by means of external immune preparations. Therefore, improving the immunity of the body, preventing the biological factors or allergic infection, finding safer microecological food with less side effects, and becoming a topic for urgent solving at present.
Probiotics are a class of bacteria that produce lactic acid when they use carbohydrates such as glucose. Probiotics have a common feature of being able to grow both aerobically and anaerobically, i.e. they are mostly facultative anaerobes. The probiotics are very closely related with human beings, are the most important beneficial flora in human intestinal tracts, and have important effects on nutrition, immunity, health care, aging resistance and longevity of human bodies. The higher the proportion of probiotics in the intestinal tract of a human body, the better the micro-ecological environment, and the younger and active intestinal tract is. The probiotics that have been found and used today are mainly of the following 3 classes: (1) lactobacillus, such as lactobacillus, lactobacillus rhamnosus, lactobacillus acidophilus, etc. (2) Bifidobacteria such as bifidobacterium longum, bifidobacterium breve, bifidobacterium bifidum and the like. (3) Other bacteria such as Streptococcus thermophilus, pediococcus acidilactici, and Saccharomyces. The above 3 major probiotics are derived from human or animal bodies, and the most used intestinal bacteria are mainly lactobacillus and bifidobacterium which can produce lactic acid, such as lactobacillus acidophilus, lactobacillus reuteri, lactobacillus rhamnosus, bifidobacterium longum, bifidobacterium breve and the like.
The Chinese patent application CN107752040A discloses a composite probiotic powder preparation for promoting the immunity of infants, 5 probiotics including bifidobacterium infantis, bifidobacterium animalis V9, lactobacillus bulgaricus PZLB5, lactobacillus plantarum PP12 and streptococcus thermophilus QH are screened out, the culture medium and the culture condition are respectively optimized, and the purposes of enhancing the immunocyte activity and improving the immunity of the infants are realized by utilizing the unique synergistic effect among strains. However, the above-mentioned 5 strains used in the patent have complicated separation and screening processes and high production cost, and bring great economic burden to consumers.
The Chinese patent application CN105641209A discloses a probiotic composition for improving immunity, which comprises probiotic dry powder, astragalus extract powder and medlar extract powder, and is supplemented with vitamin K, vitamin B12, pyridoxine, biotin, folic acid, nicotinic acid, thiamine and other nutritional ingredients, and the traditional Chinese medicine and the probiotic are reasonably combined for medication to perform synergistic action, obviously improve the immunity and promote the health of organisms. However, the traditional Chinese medicine has complex components, and the probiotic is not inactivated due to the existence of some components, so that the curative effect is reduced.
Chinese patent No. 103478530B discloses an oligosaccharide probiotic composition and application thereof, and a method for preparing capsules by using the composition, wherein the composition mainly comprises oligosaccharide powder and hydroxypropyl methylcellulose, and Chinese patent No. CN104983746B discloses a composite probiotic and a preparation method thereof, and the composite probiotic mainly comprises prebiotics, dietary fibers, amino acids and vitamin B as fillers, but has the defect of easy inactivation.
Disclosure of Invention
The invention aims to provide a probiotic composition for improving immunity and a preparation method thereof, and the probiotic composition contains rich probiotic components and other nutritional factors, including astragaloside IV, chinese yam polysaccharide, radix codonopsis, weeping forsythiae capsule water extract, soybean and fermentation products thereof.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a probiotic composition for improving immunity, which comprises the steps of inoculating bacillus subtilis to cooked soybeans for fermentation, adding the bacillus subtilis, a water extract of codonopsis pilosula and fructus forsythiae, astragaloside IV and Chinese yam polysaccharide into a culture medium, further inoculating bacillus coagulans TQ33 and selenium-enriched yeast, fermenting and culturing, sequentially adding vitamins and amino acids, and further fermenting and culturing to obtain the probiotic composition for improving immunity.
As a further improvement of the invention, the method comprises the following steps:
S1, activating strains: inoculating bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast into a Gao's medium respectively, and culturing into strain seed liquid under a micro-oxygen condition;
s2, soybean treatment: cleaning soybean, boiling, and pulverizing to obtain cooked soybean powder;
s3, primary fermentation treatment of soybeans: inoculating the bacillus subtilis strain seed liquid activated in the step S1 into the cooked soybean powder obtained in the step S2, and fermenting and culturing under a micro-oxygen condition to obtain primarily fermented soybean;
s4, extracting traditional Chinese medicines: cleaning radix Codonopsis and fructus forsythiae respectively, drying, pulverizing, extracting with water under microwave boiling, filtering, concentrating the filtrate, and drying to obtain Chinese medicinal water extract;
s5, preparing a liquid culture medium: dissolving astragaloside IV, rhizoma Dioscoreae polysaccharide, carbon source, nitrogen source and inorganic salt with sterile water, mixing, adjusting pH of culture medium with PBS solution, and sterilizing;
s6, fermenting: adding the primarily fermented soybeans obtained in the step S3 and the traditional Chinese medicine water extract obtained in the step S4 into the liquid culture medium obtained in the step S5, inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast seed liquid, and fermenting and culturing under a micro-aerobic condition to obtain a fermentation product;
S7, fermenting: and (3) adding vitamins into the fermentation product obtained in the step (S6), fermenting and culturing under the condition of micro-oxygen, adding amino acid, and fermenting and culturing under the condition of micro-oxygen for a second period of time to obtain the probiotic composition for improving the immunity.
As a further improvement of the invention, the micro-oxygen condition is that the oxygen content is 5-10%, the carbon dioxide content is 3-7%, and the balance is nitrogen, wherein the percentage is volume percent.
As a further improvement of the invention, the bacterial seed liquid in the step S1 has a bacterial content of 10 7 -10 9 cfu/mL; the inoculation amount of the bacillus subtilis in the step S3 is 5-10%; the fermentation culture temperature is 35-39 ℃ and the fermentation culture time is 12-24 hours; in the step S4, the mass ratio of the codonopsis pilosula to the weeping forsythia is (5-10): 2; the microwave boiling extraction times are 2-3 times, the microwave power is 1000-1500W, and the extraction time is 1-2h each time.
As a further improvement of the invention, the mass ratio of astragaloside IV, chinese yam polysaccharide, carbon source, nitrogen source, inorganic salt and sterile water in the step S5 is (1-2): (3-5): (15-25): (7-15): (1-3): 1000; the carbon source is at least one selected from molasses, glucose, maltose, lactose, sucrose, fructose and soluble starch; the nitrogen source is selected from at least one of ammonia water, urea, ammonium salt, nitrate and fish meal; the inorganic salt is at least one selected from sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferrous chloride, ferrous sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride; the PBS solution adjusts the pH value of the culture medium to 6.8-7.2.
As a further improvement of the invention, the mass ratio of the soybeans, the traditional Chinese medicine water extract and the liquid culture medium which are subjected to primary fermentation treatment in the step S6 is (5-12): (2-5): 20, a step of; the inoculation amount of the bacillus coagulans TQ33 is 2-5%; the inoculation amount of the selenium-enriched yeast is 4-7%; the fermentation culture condition is 35-38 ℃, and the fermentation culture is carried out for 24-36 hours; the fermentation culture condition in the step S7 is 35-38 ℃, the first time period is 8-12h, and the second time period is 24-48h.
As a further improvement of the present invention, the vitamin is at least one selected from the group consisting of vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin a, vitamin K, vitamin D, and vitamin E.
Preferably, the vitamin B6 and vitamin B12 compound is a compound mixture of vitamin B6 and vitamin B12, and the mass ratio is (3-5): 2.
as a further improvement of the present invention, the amino acid is at least one selected from glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine, phenylalanine, taurine.
Preferably, the compound mixture of taurine and methionine is prepared by the following components in percentage by mass: (2-3).
As a further improvement of the invention, the method specifically comprises the following steps:
s1, activating strains: inoculating Bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast into Gao's culture medium, and culturing under micro-oxygen condition to obtain strain seed solution with bacterial content of 10 7 -10 9 cfu/mL；
S2, soybean treatment: cleaning soybean, boiling, and pulverizing to obtain cooked soybean powder;
s3, primary fermentation treatment of soybeans: inoculating the bacillus subtilis strain seed liquid activated in the step S1 into the cooked soybean powder obtained in the step S2, wherein the inoculum size of bacillus subtilis is 5-10%, and fermenting and culturing for 12-24 hours at 35-39 ℃ under a micro-aerobic condition to obtain primarily fermented soybean;
s4, extracting traditional Chinese medicines: cleaning 5-10 parts by weight of radix codonopsis pilosulae and 2 parts by weight of fructus forsythiae respectively, drying, crushing, adding into 50 parts by weight of water, extracting for 2-3 times by microwave boiling at 1000-1500W for 1-2h each time, filtering, concentrating and drying filtrate to obtain a traditional Chinese medicine water extract;
s5, preparing a liquid culture medium: dissolving 1-2 parts by weight of astragaloside IV, 3-5 parts by weight of Chinese yam polysaccharide, 15-25 parts by weight of carbon source, 7-15 parts by weight of nitrogen source and 1-3 parts by weight of inorganic salt by using 1000 parts by weight of sterile water, uniformly mixing, regulating the pH value of a culture medium to 6.8-7.2 by using PBS solution, and sterilizing by ultraviolet rays for later use;
S6, fermenting: adding 5-12 parts by weight of the soybeans subjected to primary fermentation obtained in the step S3 and 2-5 parts by weight of the traditional Chinese medicine water extract obtained in the step S4 into 20 parts by weight of the liquid culture medium obtained in the step S5, and inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast strain seed liquid, wherein the inoculum size of the bacillus coagulans TQ33 is 2-5%; the inoculation amount of the selenium-enriched yeast is 4-7%, and fermentation culture is carried out for 24-36h at 35-38 ℃ under the condition of micro oxygen to obtain a fermentation product;
s7, fermenting: adding 0.5-1 weight part of vitamin into 100 weight parts of fermentation product in the step S6, fermenting and culturing for 8-12h at 35-38 ℃ under the condition of micro-oxygen, adding 1-2 weight parts of amino acid, fermenting and culturing for 24-48h at 35-38 ℃ under the condition of micro-oxygen, and obtaining the probiotic composition for improving immunity;
the micro-oxygen condition is that the oxygen content is 5-10%, the carbon dioxide content is 3-7%, and the balance is nitrogen, wherein the percentage is the volume percentage.
The present invention further provides a probiotic composition for enhancing immunity prepared by the preparation method of any one of claims 1 to 9.
The invention has the following beneficial effects: the bacillus coagulans TQ33 has the advantages of being good in stress resistance, high in temperature and pressure resistance, easy to store and the like of lactobacillus and bifidobacterium, can improve the structure of intestinal flora, regulate the functional disorder of the intestinal tract, maintain the balance of the intestinal flora, improve the immunity level of organisms, promote the metabolism and utilization of nutrient substances and the like, has good acid and alkali resistance, can survive for more than 75% after being treated in simulated gastric juice for 12 hours, and can survive for 85% after being treated in simulated intestinal juice for 12 hours, so that the probiotic has high survival rate in vivo, and meanwhile, fermentation supernatant and cell wall components of the bacillus coagulans TQ33 can improve the immunity of the intestinal tract by regulating cytokines, inhibiting active oxygen and enhancing cell phagocytosis, and in addition, the bacillus coagulans can promote the immune reaction of the mucous membrane of the digestive tract, thereby playing a main role in immunoregulation. Meanwhile, the bacillus coagulans TQ33 does not generate enterotoxin like other bacillus, does not contain enterotoxin genes, has no obvious cytotoxicity, does not generate mutagenesis effect, has excellent safety, and can be safely added into food for use.
The bacillus subtilis can secrete a large amount of various digestive enzymes (mainly amylase), so that the digestion and absorption of human bodies on nutrient substances in foods are promoted; the nutrient substances such as vitamins, amino acids and the like are secreted for human body to absorb and utilize, and promote growth and development; inhibit the growth and development of harmful bacteria in the body, thereby preventing various diseases and improving immunity; free oxygen in the environment in the digestive tract is rapidly consumed to form an anaerobic environment, and beneficial anaerobic bacteria growth is promoted.
Selenium is one of microelements necessary for human body, and has important effects in maintaining human health, and simultaneously, selenium has important effects in preventing cardiovascular diseases, resisting oxidation, aging, resisting virus, resisting cancer, regulating immunity, etc. The main metabolites of the selenium-enriched saccharomycete added by the invention comprise selenium-enriched biomass, selenocysteine, selenomethionine, selenocysteine, dimethyl selenium, dimethyl diselenide, elemental selenium and the like, and even comprise macromolecular compounds such as selenium-containing polysaccharide, selenium-containing phospholipid, selenium-containing nucleic acid and the like, so that a rich organic selenium source is provided for a human body, the balance of trace elements of the human body is promoted, and the effects of resisting cancer and improving immunity are achieved. Meanwhile, the selenium-enriched yeast is also a beneficial bacterium, and can play a role in promoting immunity improvement by adjusting the beneficial intestinal flora.
The bacillus coagulans TQ33, the bacillus subtilis and the selenium-enriched yeast have obvious antagonism on intestinal harmful bacteria after entering the body and normally growing and propagating in the body through synergistic interaction, can inhibit the growth and propagation of the harmful bacteria, prevent the infection of pathogenic bacteria and avoid a plurality of diseases, and simultaneously, bacteriocins, peptidoglycan, proteins and the like generated by metabolism can activate immune cells in the body, excite the body to generate immune antibodies and improve the immunity of the body. It can also enhance the immunity of the organism by regulating the nonspecific and specific immune response of the organism.
Meanwhile, the three probiotics interact, organic acids produced by self metabolism, such as bacillus coagulans TQ33, can decompose micromolecular saccharides to generate L-1 lactic acid, the pH value of an intestinal environment can be reduced, absorption of trace elements by the intestinal tract is promoted, the utilization rate of the trace elements ingested by a human body to the outside is improved, bacillus subtilis can synthesize various vitamins such as B vitamins, the vitamins and the trace elements can well promote the immunity of the human body, such as zinc can promote the immunity, appetite can be improved, anemia and the like, organic selenium produced by selenium-enriched saccharomycetes can enhance activities of macrophages, T lymphocytes and natural killer cells, so that the functions of an immune system are promoted, iron is an important component of a hemoglobin, cytochrome enzymes and oxidase system, the regulation effect of the immune function of the organism is participated, copper participates in the composition, activation and bioconversion of various enzymes, the phagocytosis of white blood cells is promoted, the effect of immune regulation is promoted, vitamin B1 can participate in metabolism in a coenzyme form, vitamin B12 can participate in the preparation of red blood cells, and the like, so that the immune function is improved.
According to the invention, bacillus subtilis is inoculated on cooked soybeans for fermentation, so that a large amount of anticancer substances soybean isoflavone are produced, the lipid substances nattokinase and the like are regulated, the proliferation of cancer cells is inhibited, the cancer cells are effectively destroyed or killed, the production of interferon can be induced by stimulating an immune system, the activity of macrophages of an organism can be improved, the phagocytic capacity of phagocytes is enhanced, and the immunity of the organism is enhanced.
The radix codonopsis and fructus forsythiae water extracts in the traditional Chinese medicine water extract promote the absorption and utilization of glycine in the intestinal tract of a mouse, increase the permeability of the intestinal barrier, increase glycine in the blood circulation, promote the synthesis of glutathione in lung tissues to play anti-inflammatory and antioxidant roles, and improve the anti-inflammatory and antiviral infection abilities of organisms such as lung/liver and the like;
the added astragaloside can regulate unbalance of immune cells Th1/Th2 in intestinal tracts, reduce eosinophil infiltration, increase secretion of sIgA (immunoglobulin) on intestinal mucosa, and reduce inflammatory reaction of intestinal tracts and improve immunity of organisms; the yam polysaccharide is a good prebiotic, promotes the growth of probiotics in intestinal tracts, inhibits the colonization of pathogenic bacteria, and can selectively promote the propagation of bacillus coagulans TQ33, bacillus subtilis, selenium-enriched yeast, bifidobacteria and lactobacillus added by the invention under the synergistic effect of the two, regulate secondary metabolites, recover intestinal barrier functions, intervene atherosclerosis and coronary heart disease, improve intestinal microecological environment, play a role in reducing blood pressure, enhance the oxidization capability of organisms and effectively improve blood fat.
The probiotic composition for improving immunity prepared by the invention contains abundant probiotic components and other nutritional factors, comprises astragaloside IV, chinese yam polysaccharide, radix codonopsis pilosulae, weeping forsythia water extract and soybean, and fermentation products thereof, has good effect of adjusting flora, promotes growth and propagation of human probiotics, can stimulate in vivo immunoregulation, improves organism immunity, adjusts blood sugar, blood fat and blood pressure, prevents atherosclerosis and diabetes, reduces inflammation and infection, and promotes human health.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Bacillus coagulans TQ33 was supplied by the Tianjin light industry institute using the microorganism teaching and research laboratory. Bacillus subtilis is supplied by Ai Baisen (beijing) biotechnology limited. Selenium enriched yeasts are supplied by Angel Yeast Co. Astragaloside IV has purity of above 98%, and is supplied by Guangzhou macro-range biotechnology Co. The purity of the yam polysaccharide is more than 90%, and the yam polysaccharide is provided by Vickers biotechnology Co Ltd.
In the following examples, the micro-oxygen condition was 7% oxygen, 5% carbon dioxide, and the balance nitrogen, wherein% is volume percent.
Example 1
The embodiment provides a preparation method of a probiotic composition for improving immunity, which specifically comprises the following steps:
s1, activating strains: inoculating Bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast into Gao's culture medium, culturing at 35deg.C under micro-oxygen condition for 16 hr to obtain strain seed solution with a bacterial content of 10 7 cfu/mL；
S2, soybean treatment: cleaning semen glycines, steaming at 100deg.C for 15min, and pulverizing to obtain cooked semen glycines powder;
s3, primary fermentation treatment of soybeans: inoculating the bacillus subtilis strain seed liquid activated in the step S1 into the cooked soybean powder obtained in the step S2, wherein the inoculum size of bacillus subtilis is 5%, and fermenting and culturing for 12 hours at 35 ℃ under a micro-aerobic condition to obtain primarily fermented soybean;
s4, extracting traditional Chinese medicines: respectively cleaning 5 parts by weight of radix codonopsis pilosulae and 2 parts by weight of fructus forsythiae, drying, crushing, adding into 50 parts by weight of water, extracting for 2 times by 1000W microwave boiling, wherein the extracting time is 1h each time, filtering, concentrating and drying filtrate to obtain a traditional Chinese medicine water extract;
S5, preparing a liquid culture medium: 1 part by weight of astragaloside IV, 3 parts by weight of Chinese yam polysaccharide, 10 parts by weight of glucose, 5 parts by weight of maltose, 7 parts by weight of urea, 0.5 part by weight of potassium chloride, 0.3 part by weight of calcium chloride and 0.2 part by weight of magnesium chloride are dissolved by 1000 parts by weight of sterile water, after uniform mixing, the pH value of a culture medium is regulated to 6.8 by using PBS solution, and ultraviolet sterilization is carried out for standby;
s6, fermenting: adding 5 parts by weight of the primarily fermented soybeans obtained in the step S3 and 2 parts by weight of the traditional Chinese medicine water extract prepared in the step S4 into 20 parts by weight of the liquid culture medium prepared in the step S5, and inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast strain seed liquid, wherein the inoculation amount of the bacillus coagulans TQ33 is 2%; the inoculation amount of the selenium-enriched yeast is 4 percent, and fermentation culture is carried out for 24 hours at 35 ℃ under the micro-oxygen condition to obtain a fermentation product;
s7, fermenting: and (2) adding 0.5 part by weight of vitamins into 100 parts by weight of fermentation products in the step (S6), fermenting and culturing for 8 hours at 35 ℃ under a micro-oxygen condition, adding 1 part by weight of amino acid, and fermenting and culturing for 24 hours at 35 ℃ under the micro-oxygen condition to obtain the probiotic composition for improving the immunity.
The vitamin is a compound mixture of vitamin B6 and vitamin B12, and the mass ratio is 3:2; the amino acid is a compound mixture of taurine and methionine, and the mass ratio is 2:2.
Example 2
The embodiment provides a preparation method of a probiotic composition for improving immunity, which specifically comprises the following steps:
s1, activating strains: inoculating Bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast into Gao's culture medium, culturing at 38deg.C under micro-oxygen condition for 24 hr to obtain strain seed solution with a bacterial content of 10 9 cfu/mL；
S2, soybean treatment: cleaning semen glycines, steaming at 100deg.C for 15min, and pulverizing to obtain cooked semen glycines powder;
s3, primary fermentation treatment of soybeans: inoculating the bacillus subtilis strain seed liquid activated in the step S1 into the cooked soybean powder obtained in the step S2, wherein the inoculum size of bacillus subtilis is 10%, and fermenting and culturing for 24 hours at 39 ℃ under a micro-aerobic condition to obtain primarily fermented soybean;
s4, extracting traditional Chinese medicines: respectively cleaning 10 parts by weight of radix codonopsis pilosulae and 2 parts by weight of fructus forsythiae, drying, crushing, adding into 50 parts by weight of water, extracting for 3 times by 1500W microwave boiling, wherein each extracting time is 2 hours, filtering, concentrating and drying filtrate to obtain a traditional Chinese medicine water extract;
s5, preparing a liquid culture medium: dissolving 2 parts by weight of astragaloside IV, 5 parts by weight of yam polysaccharide, 15 parts by weight of lactose, 5 parts by weight of sucrose, 5 parts by weight of fructose, 5 parts by weight of urea, 10 parts by weight of ammonium nitrate, 2 parts by weight of sodium chloride, 0.2 part by weight of copper sulfate, 0.3 part by weight of manganese sulfate, 0.2 part by weight of zinc chloride and 0.3 part by weight of ferric chloride in 1000 parts by weight of sterile water, uniformly mixing, regulating the pH value of a culture medium to 7.2 by using PBS solution, and sterilizing by ultraviolet rays for later use;
S6, fermenting: adding 12 parts by weight of the primarily fermented soybeans obtained in the step S3 and 5 parts by weight of the traditional Chinese medicine water extract prepared in the step S4 into 20 parts by weight of the liquid culture medium prepared in the step S5, and inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast strain seed liquid, wherein the inoculation amount of the bacillus coagulans TQ33 is 5%; the inoculation amount of the selenium-enriched yeast is 7%, and fermentation culture is carried out for 36 hours at 38 ℃ under the micro-oxygen condition, so as to obtain a fermentation product;
s7, fermenting: and (2) adding 1 part by weight of vitamin into 100 parts by weight of fermentation product obtained in the step (S6), fermenting and culturing for 12 hours at 38 ℃ under a micro-oxygen condition, and then adding 2 parts by weight of amino acid, fermenting and culturing for 48 hours at 38 ℃ under the micro-oxygen condition, so as to obtain the probiotic composition for improving the immunity.
The vitamin is a compound mixture of vitamin B6 and vitamin B12, and the mass ratio is 5:2; the amino acid is a compound mixture of taurine and methionine, and the mass ratio is 2:3.
example 3
The embodiment provides a preparation method of a probiotic composition for improving immunity, which specifically comprises the following steps:
s1, activating strains: inoculating Bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast into Gao's culture medium, culturing at 37deg.C under micro-oxygen condition for 20 hr to obtain strain seed solution with a bacterial content of 10 8 cfu/mL；
S2, soybean treatment: cleaning semen glycines, steaming at 100deg.C for 15min, and pulverizing to obtain cooked semen glycines powder;
s3, primary fermentation treatment of soybeans: inoculating the bacillus subtilis strain seed liquid activated in the step S1 into the cooked soybean powder obtained in the step S2, wherein the inoculum size of bacillus subtilis is 7%, and fermenting and culturing for 18 hours at 37 ℃ under a micro-aerobic condition to obtain primarily fermented soybean;
s4, extracting traditional Chinese medicines: respectively cleaning 7 parts by weight of radix codonopsis pilosulae and 2 parts by weight of fructus forsythiae, drying, crushing, adding into 50 parts by weight of water, extracting for 3 times by microwave boiling of 1200W, wherein the extraction time is 1.5 hours each time, filtering, concentrating and drying filtrate to obtain a traditional Chinese medicine water extract;
s5, preparing a liquid culture medium: 1.5 parts by weight of astragaloside IV, 4 parts by weight of yam polysaccharide, 15 parts by weight of glucose, 5 parts by weight of fructose, 2 parts by weight of urea, 8 parts by weight of ammonium nitrate, 2 parts by weight of fish meal, 1 part by weight of sodium chloride, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate, 0.2 part by weight of manganese sulfate and 0.2 part by weight of magnesium sulfate are dissolved by 1000 parts by weight of sterile water, after uniform mixing, the pH value of a culture medium is regulated to 7 by a PBS solution, and ultraviolet sterilization is carried out for standby;
S6, fermenting: adding 7 parts by weight of the primarily fermented soybeans obtained in the step S3 and 3.5 parts by weight of the traditional Chinese medicine water extract prepared in the step S4 into 20 parts by weight of the liquid culture medium prepared in the step S5, and inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast strain seed liquid, wherein the inoculation amount of the bacillus coagulans TQ33 is 3.5%; the inoculation amount of the selenium-enriched yeast is 5%, and fermentation culture is carried out for 30 hours at 37 ℃ under the micro-oxygen condition, so as to obtain a fermentation product;
s7, fermenting: adding 0.7 weight part of vitamin into 100 weight parts of fermentation product in the step S6, fermenting and culturing for 10 hours at 37 ℃ under a micro-oxygen condition, and then adding 1.5 weight parts of amino acid, fermenting and culturing for 36 at 37 ℃ under the micro-oxygen condition to obtain the probiotic composition for improving immunity.
The vitamin is a compound mixture of vitamin B6 and vitamin B12, and the mass ratio is 4:2; the amino acid is a compound mixture of taurine and methionine, and the mass ratio is 2:2.5.
example 4
In comparison with example 3, the vitamin was vitamin B6 alone, and the other conditions were not changed.
Example 5
In comparison with example 3, the vitamin was vitamin B12 alone, and the other conditions were not changed.
Example 6
In comparison with example 3, the amino acid was taurine alone, and the other conditions were not changed.
Example 7
In contrast to example 3, the amino acid was a single methionine, and the other conditions were not changed.
Comparative example 1
In contrast to example 3, step S7 was not performed, and the other conditions were not changed.
Comparative example 2
In contrast to example 3, bacillus coagulans TQ33 was not added in step S3, i.e., step S3 was not performed, and the other conditions were not changed.
Comparative example 3
In contrast to example 3, no Bacillus subtilis was added in step S6, and the other conditions were not changed.
Specifically, S6, fermenting: adding 7 parts by weight of the primarily fermented soybeans obtained in the step S3 and 3.5 parts by weight of the traditional Chinese medicine water extract obtained in the step S4 into 20 parts by weight of the liquid culture medium obtained in the step S5, inoculating the selenium-enriched yeast seed liquid activated in the step S1, and fermenting and culturing for 30 hours at 37 ℃ under the condition of micro oxygen, wherein the inoculation amount of the selenium-enriched yeast is 8.5%, so as to obtain a fermentation product.
Comparative example 4
In contrast to example 3, no selenium-enriched yeast was added in step S6, and the other conditions were not changed.
Specifically, S6, fermenting: adding 7 parts by weight of the primarily fermented soybeans obtained in the step S3 and 3.5 parts by weight of the traditional Chinese medicine water extract prepared in the step S4 into 20 parts by weight of the liquid culture medium prepared in the step S5, inoculating the seed liquid of the bacillus coagulans TQ33 strain activated in the step S1, wherein the inoculation amount of the bacillus coagulans TQ33 is 8.5%; fermenting and culturing for 30h at 37 ℃ under the condition of micro-oxygen to obtain a fermentation product.
Comparative example 5
In contrast to example 3, astragaloside IV was not added in step S5, and the other conditions were not changed.
S5, preparing a liquid culture medium: 5.5 parts by weight of yam polysaccharide, 15 parts by weight of glucose, 5 parts by weight of fructose, 2 parts by weight of urea, 8 parts by weight of ammonium nitrate, 2 parts by weight of fish meal, 1 part by weight of sodium chloride, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate, 0.2 part by weight of manganese sulfate and 0.2 part by weight of magnesium sulfate are dissolved in 1000 parts by weight of sterile water, after uniform mixing, PBS solution is used for regulating the pH value of a culture medium to 7, and ultraviolet sterilization is carried out for standby.
Comparative example 6
In comparison with example 3, the yam polysaccharide was not added in step S5, and the other conditions were not changed.
S5, preparing a liquid culture medium: 5.5 parts by weight of astragaloside IV, 15 parts by weight of glucose, 5 parts by weight of fructose, 2 parts by weight of urea, 8 parts by weight of ammonium nitrate, 2 parts by weight of fish meal, 1 part by weight of sodium chloride, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate, 0.2 part by weight of manganese sulfate and 0.2 part by weight of magnesium sulfate are dissolved in 1000 parts by weight of sterile water, and after uniform mixing, the pH value of a culture medium is regulated to 7 by a PBS solution, and ultraviolet sterilization is carried out for standby.
Comparative example 7
Compared with example 3, no water extract of the traditional Chinese medicine was added in step S6, and the other conditions were not changed.
Specifically, S6, fermenting: adding 7 parts by weight of the primarily fermented soybeans obtained in the step S3 into 20 parts by weight of the liquid culture medium prepared in the step S5, and inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast strain seed liquid, wherein the inoculum size of the bacillus coagulans TQ33 is 3.5%; the inoculation amount of the selenium-enriched yeast is 5%, and the fermentation product is obtained by fermenting and culturing for 30 hours at 37 ℃ under the condition of micro oxygen.
Test example 1
The probiotic compositions for improving immunity prepared in examples 1 to 7 and comparative examples 1 to 7 of the present invention were placed in a constant temperature and humidity cabinet having a relative humidity of 70.+ -. 5% at 38.+ -. 2 ℃ for 12 weeks, and the number of viable bacteria was sampled and detected weekly, and the results are shown in Table 1.
The method for detecting the number of viable bacteria is described in GB 4789.35-2016.
Table 1 number of viable bacteria (unit: cfu/g)
| Group of | 0 week | 4 weeks of | 8 weeks of | For 12 weeks |
| Example 1 | 6.72×10 10 | 6.45×10 10 | 6.12×10 10 | 5.77×10 10 |
| Example 2 | 7.24×10 10 | 7.02×10 10 | 6.75×10 10 | 6.32×10 10 |
| Example 3 | 8.25×10 10 | 7.97×10 10 | 7.46×10 10 | 7.15×10 10 |
| Example 4 | 5.65×10 9 | 8.25×10 8 | 2.41×10 8 | 7.65×10 7 |
| Example 5 | 6.23×10 9 | 9.42×10 8 | 3.17×10 8 | 8.17×10 7 |
| Example 6 | 1.45×10 10 | 7.84×10 9 | 2.14×10 9 | 8.73×10 8 |
| Example 7 | 2.11×10 10 | 8.02×10 9 | 2.56×10 9 | 8.91×10 8 |
| Comparative example 1 | 1.24×10 9 | 3.51×10 8 | 8.22×10 7 | 1.24×10 7 |
| Comparative example 2 | 4.72×10 9 | 9.17×10 8 | 4.25×10 8 | 1.21×10 8 |
| Comparative example 3 | 5.17×10 9 | 0.11×10 9 | 7.63×10 8 | 3.21×10 8 |
| Comparative example 4 | 4.82×10 9 | 9.54×10 8 | 5.67×10 8 | 2.17×10 8 |
| Comparative example 5 | 3.66×10 10 | 7.58×10 9 | 2.15×10 9 | 7.98×10 8 |
| Comparative example 6 | 2.67×10 10 | 6.82×10 9 | 1.57×10 9 | 6.79×10 8 |
| Comparative example 7 | 5.62×10 10 | 2.17×10 10 | 9.27×10 9 | 5.22×10 9 |
The number of live bacteria of the probiotics in the probiotic product directly influences the exertion of the efficacy of the product, and in general, the more the number of live bacteria of the probiotics is, the slower the attenuation is, and the more obvious the efficacy is. As can be seen from the above table, the probiotic compositions for improving immunity prepared in examples 1 to 3 of the present invention have the highest viable count content, and have good tolerance, and can maintain a higher viable count for a long period of time.
Test example 2
The probiotic compositions for enhancing immunity prepared in examples 1 to 7 and comparative examples 1 to 7 of the present invention were tested for crude polysaccharide, selenium and isoflavone content, and the results are shown in table 2.
The method for detecting the content of the crude polysaccharide comprises the step of drawing a standard curve according to a glucose standard curve in GB/T15672-2009 by a phenol-sulfuric acid method.
Selenium content detection method refers to DB 34/T2578-2015.
The isoflavone content detection method is described in GB/T23788-2009.
TABLE 2
| Example 1 | 722 | 32.22 | 756 |
| Example 2 | 747 | 33.45 | 764 |
| Example 3 | 755 | 34.17 | 769 |
| Example 4 | 434 | 22.23 | 522 |
| Example 5 | 475 | 23.17 | 537 |
| Example 6 | 525 | 26.52 | 574 |
| Example 7 | 547 | 25.78 | 582 |
| Comparative example 1 | 312 | 20.11 | 489 |
| Comparative example 2 | 582 | 26.98 | 568 |
| Comparative example 3 | 573 | 27.42 | 622 |
| Comparative example 4 | 602 | 0 | 645 |
| Comparative example 5 | 623 | 27.58 | 657 |
| Comparative example 6 | 637 | 26.92 | 672 |
| Comparative example 7 | 698 | 30.25 | 712 |
The crude polysaccharide has great influence on anti-inflammatory effect of human body, selenium element is one of trace elements necessary for human body, and participates in immune condition, and isoflavone is also a high-efficiency anticancer substance. As shown in the table above, the probiotic compositions for improving immunity prepared in examples 1-3 of the present invention are rich in polysaccharide and organic selenium, and are rich in isoflavone.
Test example 3 Effect on NK cell Activity in mice
Grouping and administration: kunming mice weighing 20.+ -.2 g were randomly divided into 15 groups, blank, examples 1-7 and comparative examples 1-7. The blank group was administrated with 10mL/kg physiological saline by stomach, and the groups of examples 1-7 and comparative examples 1-7 were administrated with 10g/kg of the probiotic composition for enhancing immunity prepared in the corresponding group of the present invention once daily for 15 consecutive days.
Preparation of chicken erythrocyte suspension: washing chicken blood with physiological saline for 3 times, centrifuging at 3000r/min for 10min, removing supernatant, and preparing into chicken erythrocyte suspension with volume fraction of 20% with physiological saline.
1. Determination of phagocytic function
The chicken erythrocyte suspensions are respectively injected into the abdominal cavity of each group of mice, after 1 mL/mouse, 30min, the mice are killed by cervical dislocation, the abdomen is cut off, 2mL of physiological saline is injected into the abdominal cavity, after 10 times of rotation, 1mL of abdominal cavity washing liquid is sucked out, the mice are dripped on a glass slide, the glass slide is dried, the mice are stained, and macrophages are counted under an oil lens, and each mouse counts 100 mice. Percent phagocytosis was calculated:
percent phagocytosis = macrophages phagocytosed chicken erythrocytes/counted macrophages x 100%
The results are shown in Table 3.
| Group of | Percent phagocytosis (%) |
| Blank group | 24.25±1.24 |
| Example 1 | 74.52±7.23 * |
| Example 2 | 75.71±8.21 * |
| Example 3 | 76.57±7.89 * |
| Example 4 | 56.73±5.67 |
| Example 5 | 55.25±6.13 |
| Example 6 | 58.38±6.24 |
| Example 7 | 52.26±5.36 |
| Comparative example 1 | 49.25±5.74 |
| Comparative example 2 | 59.23±6.43 |
| Comparative example 3 | 62.14±5.26 |
| Comparative example 4 | 61.25±6.14 |
| Comparative example 5 | 63.55±5.37 |
| Comparative example 6 | 64.15±5.82 |
| Comparative example 7 | 67.25±6.72 |
Annotation: * For comparison with the blank, there was a significant difference (P < 0.05).
2. Effect on NK cell Activity in mice
Taking mouse spleen aseptically, placing into sterile Hanks solution, shredding spleen to obtain single cell suspension, filtering, washing, centrifuging at 10000r/min for 10min, floating cells in culture solution, and adjusting cell concentration to 10 6 And obtaining effector cells at a concentration of each mL. Target cells are treated(YAC-l cells) subculturing, and adjusting cell concentration to 10 with culture solution 5 Target cells were obtained at a concentration of each mL.
Preparing an LDH mixed solution: a mixed solution containing 0.05mol/L lithium lactate, 0.05mol/L nitrotetrazolium chloride, 0.00066mol/L phenazine methosulfate, 0.00028mol/L oxidized coenzyme I and 0.0013mol/L oxidized coenzyme I was prepared by using a Tris-HCl solution with pH=8.2 to obtain an LDH mixed solution.
NK cell activity assay:
reaction group: adding target cells and effector cells into a 96-well culture plate according to a target effect ratio of 1:50, wherein each of the target cells and the effector cells is 100 mu L;
target cell natural release group: adding 100 mu L of each of the target cells and the culture solution into a 96-well culture plate;
target cell maximum release well group: target cells and 1% NP40 lysates were added at 100. Mu.L each to 96-well plates.
Each group is provided with 5 groups which are parallel and 7 percent of CO at 37 DEG C 2 Culturing in an incubator for 4 hours, centrifuging, absorbing 100 mu L of supernatant liquid in each hole, adding LDH mixed solution for reaction for 5 minutes, adding 10 mu L of 2mol/LHCl solution, measuring 490nm absorbance by using an enzyme-labeling instrument, and calculating NK cell activity:
NK cell activity (%) = (absorbance of reaction well-absorbance of natural release well)/(absorbance of maximum release well-absorbance of natural release well) ×100%
The results are shown in Table 4.
| Group of | NK cell Activity (%) |
| Blank group | 14.52±3.10 |
| Example 1 | 64.53±6.73 * |
| Example 2 | 65.75±7.23 * |
| Example 3 | 66.64±7.52 * |
| Example 4 | 42.52±5.26 |
| Example 5 | 43.15±4.98 |
| Example 6 | 48.67±6.11 |
| Example 7 | 40.27±5.27 |
| Comparative example 1 | 37.52±4.67 |
| Comparative example 2 | 54.25±4.91 |
| Comparative example 3 | 50.27±5.15 |
| Comparative example 4 | 52.45±5.32 |
| Comparative example 5 | 57.24±4.98 |
| Comparative example 6 | 56.98±5.37 |
| Comparative example 7 | 60.21±4.76 |
Annotation: * For comparison with the blank, there was a significant difference (P < 0.05).
From the table, the probiotic composition for improving immunity prepared in the embodiments 1-3 can obviously improve the phagocytic percentage, improve the NK cell activity and have good effect of improving immunity.
Examples 4 and 5 were compared with example 3, in which the vitamins were single vitamin B6 or vitamin B12, the number of viable bacteria was decreased, the attenuation of viable bacteria was accelerated, and the metabolite content was decreased. The addition of vitamins can promote the proliferation of probiotics, prolong the logarithmic phase and improve the stress resistance of active bacteria, thereby reducing the attenuation of the active bacteria. The addition of vitamins B6 and B12 also has a synergistic effect.
Examples 6 and 7 were compared with example 3, in which the amino acid was methionine or taurine alone, the number of viable bacteria was decreased, the content of metabolites was decreased, and the phagocytic percentage and NK cell activity were decreased in example 7. The addition of amino acid provides abundant nitrogen source for probiotics, increases the activity of the probiotics, generates more beneficial metabolites, and taurine serving as an active amino acid required by human body can also obviously improve the immunity of the organism, and the addition of the taurine and the taurine has a synergistic effect.
Comparative example 1 compared with example 3, step S7 was not performed, and the number of viable bacteria was decreased, the attenuation of viable bacteria was accelerated, and the metabolite content was decreased, and the phagocytic percentage and NK cell activity were decreased. It can be seen that the step S7 of promoting fermentation can significantly improve the overall effect of the composition of the present invention.
Comparative examples 2, 3 and 4 showed a decrease in the number of viable bacteria, a decrease in the content of metabolites, a significant decrease in the percentage of phagocytosis and NK cell activity, as compared with example 3, without adding Bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast. The three probiotics interact, organic acids produced by self metabolism, such as bacillus coagulans TQ33, can decompose micromolecular saccharides to generate L-1 lactic acid, can reduce the pH value of an intestinal environment, promote the absorption of trace elements by the intestinal tract, improve the utilization rate of the trace elements ingested by a human body to the outside, bacillus subtilis can synthesize various vitamins such as B vitamins, the vitamins and the trace elements can well promote the immunity of the human body, such as zinc can promote the immunity, appetite can be improved, anemia and the like, organic selenium produced by selenium-enriched saccharomycetes can enhance the activities of macrophages, T lymphocytes and natural killer cells, thereby promoting the functions of an immune system, iron is an important component of hemoglobin, cytochrome enzymes and oxidase systems, participates in the regulation effect of the immune function of the organism and the like, copper participates in the composition, activation and bioconversion of various enzymes, promotes the phagocytosis of white blood cells, and the effect of immune regulation is mentioned, vitamin B1 can participate in the metabolism of sugar in the form of coenzyme, vitamin B12 can participate in the bone marrow erythrocyte and the like, so that the immune function is improved.
In comparative examples 5 and 6, as compared with example 3, astragaloside IV or yam polysaccharide was not added in step S5, the viable count was decreased, the crude polysaccharide content was decreased, and the phagocytic percentage and NK cell activity were decreased. The added astragaloside can regulate unbalance of immune cells Th1/Th2 in intestinal tracts, reduce eosinophil infiltration, increase secretion of sIgA (immunoglobulin) on intestinal mucosa, and reduce inflammatory reaction of intestinal tracts and improve immunity of organisms; the yam polysaccharide is a good prebiotic, promotes the growth of probiotics in intestinal tracts, inhibits the colonization of pathogenic bacteria, and can selectively promote the propagation of bacillus coagulans TQ33, bacillus subtilis, selenium-enriched yeast, bifidobacteria and lactobacillus added by the invention under the synergistic effect of the two, regulate secondary metabolites, recover intestinal barrier functions, intervene atherosclerosis and coronary heart disease, improve intestinal microecological environment, play a role in reducing blood pressure, enhance the oxidization capability of organisms and effectively improve blood fat.
Comparative example 7 compared with example 3, the phagocytic percentage and NK cell activity were decreased without adding the aqueous extract of the traditional Chinese medicine in step S6. The radix codonopsis and fructus forsythiae water extracts in the traditional Chinese medicine water extract promote absorption and utilization of glycine in the intestinal tract of a mouse, increase the permeability of the intestinal barrier, increase glycine in blood circulation, promote synthesis of glutathione in lung tissues to play anti-inflammatory and antioxidant roles, improve the immunity of organisms, and have a synergistic effect by adding the radix codonopsis and fructus forsythiae water extracts.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (7)
1. A method of preparing a probiotic composition for enhancing immunity, comprising the steps of:
s1, activating strains: inoculating bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast into a Gao's medium respectively, and culturing into strain seed liquid under a micro-oxygen condition; the bacterial seed liquid has a bacterial content of 10 7 -10 9 cfu/mL；
S2, soybean treatment: cleaning soybean, boiling, and pulverizing to obtain cooked soybean powder;
s3, primary fermentation treatment of soybeans: inoculating the bacillus subtilis strain seed liquid activated in the step S1 into the cooked soybean powder obtained in the step S2, and fermenting and culturing under a micro-oxygen condition to obtain primarily fermented soybean; the inoculation amount of the bacillus subtilis is 5-10%; the fermentation culture temperature is 35-39 ℃ and the fermentation culture time is 12-24 hours; the method comprises the steps of carrying out a first treatment on the surface of the
S4, extracting traditional Chinese medicines: cleaning radix Codonopsis and fructus forsythiae respectively, drying, pulverizing, extracting with water under microwave boiling, filtering, concentrating the filtrate, and drying to obtain Chinese medicinal water extract; the mass ratio of the codonopsis pilosula to the weeping forsythia is (5-10): 2;
S5, preparing a liquid culture medium: dissolving astragaloside IV, rhizoma Dioscoreae polysaccharide, carbon source, nitrogen source and inorganic salt with sterile water, mixing, adjusting pH of culture medium with PBS solution, and sterilizing; the mass ratio of the astragaloside IV to the Chinese yam polysaccharide to the carbon source to the nitrogen source to the inorganic salt to the sterile water is (1-2): (3-5): (15-25): (7-15): (1-3): 1000;
s6, fermenting: adding the primarily fermented soybeans obtained in the step S3 and the traditional Chinese medicine water extract obtained in the step S4 into the liquid culture medium obtained in the step S5, inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast seed liquid, and fermenting and culturing under a micro-aerobic condition to obtain a fermentation product; the mass ratio of the soybean, the traditional Chinese medicine water extract and the liquid culture medium which are subjected to primary fermentation treatment is (5-12): (2-5): 20, a step of; the inoculation amount of the bacillus coagulans TQ33 is 2-5%; the inoculation amount of the selenium-enriched yeast is 4-7%; the fermentation culture condition is 35-38 ℃, and the fermentation culture is carried out for 24-36 hours;
s7, fermenting: adding vitamins into the fermentation product obtained in the step S6, fermenting and culturing for a first time period under a micro-oxygen condition, and then adding amino acid, fermenting and culturing for a second time period under the micro-oxygen condition to obtain a probiotic composition for improving immunity;
The fermentation culture condition is 35-38 ℃, the first time period is 8-12h, and the second time period is 24-48h;
the micro-oxygen condition is that the oxygen content is 5-10%, the carbon dioxide content is 3-7%, and the balance is nitrogen, wherein the percentage is volume percent.
2. The method according to claim 1, wherein the number of times of microwave boiling extraction in step S4 is 2-3, the microwave power is 1000-1500W, and the time for each extraction is 1-2h.
3. The method according to claim 1, wherein the carbon source in step S5 is at least one selected from the group consisting of molasses, glucose, maltose, lactose, sucrose, fructose, and soluble starch; the nitrogen source is selected from at least one of ammonia water, urea, ammonium salt, nitrate and fish meal; the inorganic salt is at least one selected from sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferrous chloride, ferrous sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride; the PBS solution adjusts the pH value of the culture medium to 6.8-7.2.
4. The method according to claim 1, wherein the vitamin is at least one selected from the group consisting of vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin a, vitamin K, vitamin D, and vitamin E.
5. The method according to claim 1, wherein the amino acid is at least one selected from the group consisting of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine, phenylalanine, and taurine.
6. The preparation method according to claim 1, characterized by comprising the following steps:
s1, activating strains: inoculating Bacillus coagulans TQ33, bacillus subtilis and selenium-enriched yeast into Gao's culture medium, and culturing under micro-oxygen condition to obtain strain seed solution with bacterial content of 10 7 -10 9 cfu/mL；
S2, soybean treatment: cleaning soybean, boiling, and pulverizing to obtain cooked soybean powder;
s3, primary fermentation treatment of soybeans: inoculating the bacillus subtilis strain seed liquid activated in the step S1 into the cooked soybean powder obtained in the step S2, wherein the inoculum size of bacillus subtilis is 5-10%, and fermenting and culturing for 12-24 hours at 35-39 ℃ under a micro-aerobic condition to obtain primarily fermented soybean;
s4, extracting traditional Chinese medicines: cleaning 5-10 parts by weight of radix codonopsis pilosulae and 2 parts by weight of fructus forsythiae respectively, drying, crushing, adding into 50 parts by weight of water, extracting for 2-3 times by microwave boiling at 1000-1500W for 1-2h each time, filtering, concentrating and drying filtrate to obtain a traditional Chinese medicine water extract;
S5, preparing a liquid culture medium: dissolving 1-2 parts by weight of astragaloside IV, 3-5 parts by weight of Chinese yam polysaccharide, 15-25 parts by weight of carbon source, 7-15 parts by weight of nitrogen source and 1-3 parts by weight of inorganic salt by using 1000 parts by weight of sterile water, uniformly mixing, regulating the pH value of a culture medium to 6.8-7.2 by using PBS solution, and sterilizing by ultraviolet rays for later use;
s6, fermenting: adding 5-12 parts by weight of the soybeans subjected to primary fermentation obtained in the step S3 and 2-5 parts by weight of the traditional Chinese medicine water extract obtained in the step S4 into 20 parts by weight of the liquid culture medium obtained in the step S5, and inoculating the bacillus coagulans TQ33 activated in the step S1 and selenium-enriched yeast strain seed liquid, wherein the inoculum size of the bacillus coagulans TQ33 is 2-5%; the inoculation amount of the selenium-enriched yeast is 4-7%, and fermentation culture is carried out for 24-36h at 35-38 ℃ under the condition of micro oxygen to obtain a fermentation product;
s7, fermenting: adding 0.5-1 weight part of vitamin into 100 weight parts of fermentation product in the step S6, fermenting and culturing for 8-12h at 35-38 ℃ under the condition of micro-oxygen, adding 1-2 weight parts of amino acid, fermenting and culturing for 24-48h at 35-38 ℃ under the condition of micro-oxygen, and obtaining the probiotic composition for improving immunity;
the micro-oxygen condition is that the oxygen content is 5-10%, the carbon dioxide content is 3-7%, and the balance is nitrogen, wherein the percentage is the volume percentage.
7. A probiotic composition for enhancing immunity prepared by the preparation method of any one of claims 1 to 6.
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