A compound probiotic preparation with the efficacy of regulating intestinal flora and a preparation method thereof

Abstract

The present application relates to the technical field of probiotic preparation, in particular to a compound probiotic preparation with the efficacy of regulating intestinal flora and a preparation method thereof.The compound probiotic preparation with the efficacy of regulating intestinal flora is characterized in that it comprises Cordyceps sinensis extract from Ophiocordyceps unilateralis, probiotics and prebiotics, wherein the Cordyceps sinensis extract from Ophiocordyceps unilateralis is 2-5 parts by weight; the probiotics are 0.8-1.2 parts by weight; and the prebiotics are 1.6-2.4 parts by weight.The compound probiotic preparation prepared by the present application can effectively regulate the structure of intestinal flora and improve the effects of constipation and diarrhea in mice.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotic preparations, in particular to a compound probiotic preparation with the efficacy of regulating intestinal flora and a preparation method thereof. BACKGROUND

[0002] Probiotics are active microorganisms that are beneficial to the body by improving the balance of the host flora, activating the host endogenous microbiota or immune system. Most of the current research probiotics come from Lactobacillus and Bifidobacterium. Lactobacillus and Bifidobacterium are important members of the normal microflora in the gastrointestinal tract and are associated with the host throughout life. In the proximal and distal intestine, from the stomach, duodenum, jejunum, ileum, cecum, colon to rectum, there are lactobacillus. Probiotics can restore gastrointestinal function, regulate the immune system, and inhibit the growth of pathogenic bacteria and the production of endotoxins, reduce the level of inflammatory factors, and improve the body's immunity and intestinal mucosal barrier by correcting the imbalance of the flora. Some metabolites produced by probiotics, such as secreted proteins and organic acids, can promote mucus secretion, increase the production of antibacterial peptides, or enhance the expression of tight junctions to protect the epithelial barrier of the intestine.

[0003] Prebiotics can provide nutrition for probiotics (such as Bifidobacterium and Lactobacillus), promote their proliferation and metabolic activity, and inhibit the production of harmful bacteria. By increasing the number of beneficial bacteria, prebiotics can competitively inhibit the colonization of pathogenic bacteria (such as Escherichia coli and Salmonella). After being fermented by probiotics, prebiotics produce lactic acid and short-chain fatty acids (such as butyric acid), which can lower the pH of the intestinal tract, inhibit the growth of pathogenic bacteria, provide energy for intestinal epithelial cells, repair the intestinal mucosal barrier, regulate the immune system, and reduce inflammation. At the same time, prebiotics can promote the absorption of minerals such as calcium, magnesium, and iron, which are beneficial to bone health.

[0004] Cordyceps militaris mycelium extract is a dual-purpose medicinal and edible fungus containing cordycepin, cordyceps polysaccharides and other active substances, and has wide medicinal value. Cordycepin has a significant impact on intestinal flora and liver metabolite profile, and can improve the body's antioxidant level.

[0005] The existing market products are single probiotic preparations and single Cordyceps militaris mycelium extract preparations, and there is no combination of probiotics and Cordyceps militaris mycelium extract for better immune-enhancing combination preparations. SUMMARY

[0006] In view of the above-mentioned deficiencies in the prior art, the present application provides a compound probiotic preparation with the efficacy of regulating intestinal flora and a preparation method thereof.

[0007] To solve the above technical problems, the technical scheme adopted by the present application is:

[0008] A complex probiotic preparation with the efficacy of regulating intestinal flora, comprising Cordyceps sinensis extract from Cordyceps militaris, probiotics, prebiotics, the Cordyceps sinensis extract from Cordyceps militaris 2-5 parts by weight; probiotics 0.8-1.2 parts by weight; prebiotics 1.6-2.4 parts by weight.

[0009] Cordyceps sinensis extract is rich in cordycepin, cordyceps polysaccharide and various amino acids, and has multiple biological activities such as anti-inflammatory, antioxidant and immune regulation. When applied to probiotic preparations, it can not only directly inhibit intestinal pathogenic bacteria and relieve intestinal mucosal inflammation to create a healthy environment for probiotic colonization, but also promote the colonization and proliferation of probiotics as prebiotics. Probiotics further inhibit harmful bacteria through mechanisms such as acid production and competitive exclusion, and repair the intestinal mucosal barrier. The combination of the two, on the one hand, reduces intestinal damage by inhibiting harmful bacteria, and on the other hand, enhances the balance of the flora and the function of the barrier by promoting beneficial bacteria. In both constipation and diarrhea, the dual action of inhibiting harmful bacteria and promoting beneficial bacteria produces a significant synergistic effect with probiotics, enhancing the intestinal barrier function and regulating the balance of the flora, thereby comprehensively improving the comprehensive conditioning ability of the preparation for intestinal disorders.

[0010] Preferably, the probiotics include at least one of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus casei; and the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus casei is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2).

[0011] Preferably, the prebiotics are at least one of fructooligosaccharides, galactooligosaccharides, stachyose, and inulin, and the mass ratio of fructooligosaccharides, galactooligosaccharides, stachyose, and inulin is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2).

[0012] Preferably, the preparation method of the Cordyceps sinensis extract is as follows: Cordyceps militaris mycelium and 10-25 times the amount of water are loaded into an extraction tank, decocted for 1-4 hours, and the first filtrate is reserved; the filtrate and 10-20 times the amount of water are subjected to second decoction for 1-4 hours, and the second filtrate is reserved; the two filtrates are combined and filtered through a filter, and the filtrate is concentrated under reduced pressure to a flow extract with a density of 1.05-1.20 g / cm 3 , and then spray dried to obtain the Cordyceps sinensis extract.

[0013] Preferably, it further includes an active additive, the amount of the active additive being 20-50% of the probiotics; and the active additive is nano-sprayed by cross-linking modification of amino acids and hydroxyl carboxylic acids and loading of mineral elements.

[0014] In order to achieve the purpose of promoting the health of the host, it is necessary to maintain the viability of probiotics, and the minimum concentration of probiotics in the intestine must reach 10 6 The modified nano corn starch has a small particle size and a high specific surface area, can efficiently coat probiotics, and form a physical barrier. The surface thereof is crosslinked by amino acids and hydroxyl carboxylic acids, and the stability and biological adhesion are enhanced, which can effectively resist the erosion of gastric acid and improve the survival rate of probiotics. Meanwhile, the mineral elements loaded therein can synergistically enhance intestinal health, and are an ideal functional protective carrier. Oxygen, gastric acid and bile are effectively isolated, the stability and gastrointestinal tolerance of probiotics are significantly improved, and sufficient live bacteria are ensured to target the intestine and colonize, so that the biological efficacy of regulating the flora is maximized.

[0015] By adding the active additive, firstly, the protection efficiency of probiotics is significantly improved through the encapsulation process. The crosslinking modification of amino acids and hydroxyl carboxylic acids forms a dense microcapsule wall material, which can effectively block the erosion of gastric acid and bile salts, and release live bacteria in the intestine, thereby greatly improving the survival rate and stability of probiotics in processing, storage and digestive tract environment, and ensuring sufficient live bacteria to reach the intestine to play a role. Secondly, the additive brings functional synergistic gain. The mineral elements loaded therein are provided in the form of high bioavailability, which not only directly participates in the immune regulation and epithelial repair of the intestine, but also produces a synergistic effect with the metabolic and active ingredients of cordyceps extract, thereby collectively enhancing the comprehensive conditioning ability for intestinal flora balance, mucosal barrier protection and inflammation relief, so that the preparation shows more optimal and stable efficacy in improving intestinal problems such as constipation and diarrhea.

[0016] In summary, the addition of the active additive can effectively overcome the core shortcomings of probiotics in the prior art, such as easy inactivation due to gastric acid, processing and storage factors, and single functional components and insufficient synergistic effect.

[0017] Preferably, the active additive is prepared by a method comprising the following steps:

[0018] S1, mixing starch, water and an antioxidant, and after ultrasonic treatment, separation and drying, obtaining nano starch;

[0019] S2, dispersing the nano starch in water, adjusting the pH to 7.5-9.5 with a base, adding amino acids and hydroxyl carboxylic acids, and reacting at 100-120°C for 2-6h, and after separation and drying, obtaining modified nano starch;

[0020] S3, preparing the modified nano starch into an aqueous solution, adding a heat-resistant protective agent, and after mixing, freeze-drying and crushing, obtaining the active additive.

[0021] Further, the preparation method of the active additive is as follows:

[0022] S1, 3-8 parts by weight of corn starch, 50-120 parts by weight of water and 0.5-2 parts by weight of antioxidant are mixed, treated under ultrasonic power of 100-300 W, ultrasonic frequency of 40-100 kHz for 30-70 min, centrifuged, washed, and freeze-dried to obtain nano corn starch;

[0023] S2, the above nano corn starch is dispersed in water to prepare a nano corn starch water suspension with a mass fraction of 20-30 wt%, and a 0.5-2 mol / L sodium hydroxide aqueous solution is used to adjust the pH to 7.5-9.5; 0.2-0.8 parts by weight of a 20-30 wt% amino acid aqueous solution and 0.1-0.3 parts by weight of a 20-30 wt% hydroxyl carboxylic acid aqueous solution are added, stirred at 100-120℃ and 300-500 rpm for 2-6 h, centrifuged, washed, and freeze-dried to obtain modified nano corn starch;

[0024] S3, the above modified nano corn starch is prepared into a 2-6 wt% modified nano starch aqueous solution by adding water, 0.5-2 parts by weight of a heat-resistant protective agent is added, and stirring is continued for 10-30 min, and then cooled to room temperature, and then frozen at -60℃ for 18 h and freeze-dried, and then pulverized to a particle size of 0.02-0.2 mm to obtain the active additive.

[0025] Preferably, the antioxidant is ascorbic acid.

[0026] The amino acid is selected from one or more of L-cysteine, glutamic acid, and threonine; the hydroxyl carboxylic acid is selected from one or more of hydroxyl citric acid, tartaric acid, and malic acid; and the heat-resistant protective agent is one or more of ferric glycinate, zinc glycinate, and magnesium glycinate.

[0027] In the complex probiotic preparation, the total number of viable bacteria is 10 10 -10 11 CFU / mL.

[0028] A preparation method of the complex probiotic preparation for regulating the efficacy of intestinal flora, comprising the following steps: stirring the above active additive, soy lecithin and sterile water into an emulsion, adding probiotics to wet granulation, drying at 37℃ to obtain probiotic granules; then uniformly mixing with prebiotics, and then adding cordyceps sinensis extract to mix uniformly to obtain the complex probiotic preparation for regulating the efficacy of intestinal flora.

[0029] The beneficial effects of the present application are: 1. The present application provides a compound probiotic preparation with the effect of regulating intestinal flora and a preparation method thereof. The synergistic effect between the ingredients is achieved by compounding batryobasidium hynorum extract, probiotics and prebiotics, and the problem of single function in the prior art is overcome. The batryobasidium hynorum extract is introduced into the probiotic system for the first time, and the active ingredients such as cordycepin and cordyceps polysaccharide contained in the batryobasidium hynorum extract can not only directly resist inflammation and oxidation, but also create a good intestinal colonization environment for probiotics, and can also promote the proliferation of probiotics as prebiotics. This forms a two-way regulation mechanism with the effect of inhibiting harmful bacteria and repairing intestinal mucosal barrier, so that both constipation and diarrhea, two opposite intestinal disorder symptoms, can be significantly improved.

[0030] 2. The present application introduces an active additive prepared by a specific process, which greatly improves the stability and gastrointestinal survival rate of probiotics, and solves the technical bottleneck that probiotic preparations are easily inactivated during processing, storage and digestive tract transmission. The active additive described in the present application can form a dense microcapsule wall material to provide a physical barrier for probiotics, effectively resist the external adverse environment, and effectively improve the constipation and diarrhea effect of mice when applied to probiotic preparations. DETAILED DESCRIPTION

[0031] The above summary of the application will be further described in detail in combination with specific embodiments, but it should not be understood that the scope of the above subject matter of the application is limited to the following examples.

[0032] In the present application, some raw materials are introduced, and other raw material substances not introduced are all commercially available:

[0033] Lactobacillus bifidus was purchased from China General Microbiological Culture Collection Center, and the strain number was CGMCC NO.18093.

[0034] Lactobacillus plantarum was purchased from China Agricultural Microbial Culture Collection Center, and the strain number was ACCC11016.

[0035] Lactobacillus acidophilus was purchased from China Agricultural Microbial Culture Collection Center, and the strain number was ACCC00159.

[0036] Lactobacillus casei was purchased from China Agricultural Microbial Culture Collection Center, and the strain number was ACCC03948.

[0037] Corn starch was purchased from Shaanxi Ranguan Biological Technology Co., Ltd., and the article number was 03311654.

[0038] Fructooligosaccharide was purchased from Shaanxi Ranguan Biological Technology Co., Ltd., and the article number was 10-763-99.

[0039] Galacto-oligosaccharide was purchased from Shaanxi Ranfeng Biotechnology Co., Ltd., item number: 03281438.

[0040] Stachyose was purchased from Shaanxi Ranfeng Biotechnology Co., Ltd., item number: 10-66-88.

[0041] Inulin was purchased from Shaanxi Ranfeng Biotechnology Co., Ltd., batch number: RK20210521.

[0042] Soybean lecithin was purchased from Shaanxi Ranfeng Biotechnology Co., Ltd., item number: 1011.

[0043] Example 1

[0044] A complex probiotic preparation with the effect of regulating intestinal flora, which is composed of Cordyceps sinensis extract, probiotics and prebiotics, and the mass ratio of the Cordyceps sinensis extract, probiotics and prebiotics is 3:1:2; the probiotics are composed of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei, and the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei is 1:1:1:1; the prebiotics are composed of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin, and the mass ratio of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin is 1:1:1:1.

[0045] The preparation method of the Cordyceps sinensis extract is as follows: the cordyceps militaris mycelium and 15 times the amount of water are loaded into an extraction tank, decocted at 100℃ for 2h, and the first filtrate is reserved; the filter residue and 15 times the amount of water are subjected to second decoction, decocted at 100℃ for 2h, and the second filtrate is reserved; the two filtrates are combined and filtered through a filter, and the filtrate is concentrated under reduced pressure to a density of 1.15g / cm 3 of flow extract, spray dried to obtain the Cordyceps sinensis extract.

[0046] The preparation method of the complex probiotic preparation with the effect of regulating intestinal flora is as follows: the probiotics and the prebiotics are first mixed uniformly according to the mass ratio; then the Cordyceps sinensis extract is added and mixed uniformly, filled, to obtain the complex probiotic preparation with the effect of regulating intestinal flora.

[0047] Example 2

[0048] The compound probiotic preparation with the efficacy of regulating intestinal flora is composed of Cordyceps sinensis extract, probiotics, prebiotics and active additives, and the mass ratio of the Cordyceps sinensis extract, the probiotics, the prebiotics and the active additives is 3:1:2:0.4; the probiotics are composed of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei, and the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei is 1:1:1:1; the prebiotics are composed of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin, and the mass ratio of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin is 1:1:1:1.

[0049] The preparation method of the active additives is as follows: 5 parts by weight of corn starch, 80 parts by weight of water and 1 part by weight of ascorbic acid are mixed, treated under the ultrasonic power of 200 W and the ultrasonic frequency of 70 kHz for 50 min, centrifuged, washed, freeze-dried and pulverized to the particle size of 0.05 mm to obtain the active additives.

[0050] The preparation method of the Cordyceps sinensis extract is as follows: the cordyceps sinensis mycelium and 15 times the amount of water are loaded into an extraction tank, decocted at 100 DEG C for 2 h, and the first filtrate is reserved; the residue and 15 times the amount of water are subjected to second decoction, decocted at 100 DEG C for 2 h, and the second filtrate is reserved; the two filtrates are combined, filtered through a filter, and the filtrate is concentrated under reduced pressure to the density of 1.15 g / cm 3 of the flow extract, spray-dried to obtain the Cordyceps sinensis extract.

[0051] The preparation method of the compound probiotic preparation with the efficacy of regulating intestinal flora is as follows: 0.4 parts by weight of the active additives, 0.2 parts by weight of soybean lecithin and 30 parts by weight of sterile water are stirred into an emulsion, 1 part by weight of probiotics is added for wet granulation, dried at 37 DEG C to obtain probiotic granules, mixed with prebiotics according to the mass ratio, added with the Cordyceps sinensis extract, filled and obtained as the compound probiotic preparation with the efficacy of regulating intestinal flora.

[0052] Example 3

[0053] The compound probiotic preparation with the efficacy of regulating intestinal flora is composed of Cordyceps sinensis extract, probiotics, prebiotics and active additives, and the mass ratio of the Cordyceps sinensis extract, the probiotics, the prebiotics and the active additives is 3:1:2:0.4; the probiotics are composed of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei, and the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei is 1:1:1:1; the prebiotics are composed of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin, and the mass ratio of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin is 1:1:1:1.

[0054] The preparation method of the active additive is as follows:

[0055] S1, 5 parts by weight of corn starch, 80 parts by weight of water and 1 part by weight of ascorbic acid are mixed, treated under ultrasonic power of 200 W and ultrasonic frequency of 70 kHz for 50 min, centrifuged, washed, freeze-dried, and nano corn starch is obtained;

[0056] S2, the above nano corn starch is dispersed in water to prepare a 25wt% nano corn starch aqueous suspension, and the pH is adjusted to 8.5 with 1mol / L sodium hydroxide aqueous solution; 0.7 parts by weight of 25wt% L-cysteine aqueous solution is added, stirred at 110℃ and 400rpm for 4h, centrifuged, washed, freeze-dried, and crushed to a particle size of 0.05mm to obtain the active additive.

[0057] The preparation method of the cordyceps extract is as follows: batrachomyomycetes mycelium and 15 times the amount of water are loaded into an extraction tank, decocted at 100℃ for 2h, and the first filtrate is reserved; the filtrate and 15 times the amount of water are subjected to second decoction, decocted at 100℃ for 2h, and the second filtrate is reserved; the two filtrates are combined and filtered through a filter, and the filtrate is concentrated under reduced pressure to a density of 1.15g / cm 3 of flow extract, spray dried to obtain the cordyceps extract.

[0058] The preparation method of the complex probiotic preparation with the function of regulating intestinal flora is as follows: 0.4 parts by weight of the above active additive, 0.2 parts by weight of soybean lecithin and 30 parts by weight of sterile water are stirred into an emulsion, 1 part by weight of probiotics is added for wet granulation, dried at 37℃ to obtain probiotic granules, mixed uniformly with prebiotics according to a mass ratio; then the cordyceps extract is added and mixed uniformly, filled, and the complex probiotic preparation with the function of regulating intestinal flora is obtained.

[0059] Example 4

[0060] A complex probiotic preparation with the function of regulating intestinal flora, which is composed of cordyceps extract, probiotics, prebiotics and active additive, and the mass ratio of the cordyceps extract, probiotics, prebiotics and active additive is 3:1:2:0.4; the probiotics are composed of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei, and the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei is 1:1:1:1; the prebiotics are composed of fructooligosaccharide, galactooligosaccharide, stachyose and inulin, and the mass ratio of fructooligosaccharide, galactooligosaccharide, stachyose and inulin is 1:1:1:1.

[0061] The preparation method of the active additive is as follows:

[0062] S1, 5 parts by weight of corn starch, 80 parts by weight of water and 1 part by weight of ascorbic acid are mixed, treated under ultrasonic power of 200W and ultrasonic frequency of 70kHz for 50min, centrifuged, washed, and freeze-dried to obtain nano corn starch;

[0063] S2, the above nano corn starch is dispersed in water to prepare a 25wt% nano corn starch aqueous suspension, the pH is adjusted to 8.5 with 1mol / L sodium hydroxide aqueous solution; 0.7 parts by weight of 25wt% L-cysteine aqueous solution is added, stirred at 110℃ and 400rpm for 4h, centrifuged, washed, and freeze-dried to obtain modified nano corn starch;

[0064] S3, the above modified nano corn starch is prepared into a 4wt% modified nano starch aqueous solution by adding water, 1 part by weight of ferric glycinate is added, and stirring is continued for 20min, and then the mixture is cooled to room temperature, frozen at-60℃ for 18h, and then freeze-dried and pulverized to a particle size of 0.05mm to obtain an active additive.

[0065] The preparation method of the cordyceps extract is as follows: the cordyceps sinensis mycelium and 15 times the amount of water are loaded into an extraction tank, decocted at 100℃ for 2h, and the first filtrate is reserved; the filter residue and 15 times the amount of water are subjected to second decoction, decocted at 100℃ for 2h, and the second filtrate is reserved; the two filtrates are combined and filtered through a filter, and the filtrate is concentrated under reduced pressure to a density of 1.15g / cm 3 of a flow extract, and spray dried to obtain the cordyceps extract.

[0066] The preparation method of the complex probiotic preparation for regulating intestinal flora is as follows: 0.4 parts by weight of the above active additive, 0.2 parts by weight of soybean lecithin and 30 parts by weight of sterile water are stirred into an emulsion, 1 part by weight of probiotics is added for wet granulation, and the granules are dried at 37℃ to obtain probiotic granules, which are uniformly mixed with prebiotics in a mass ratio; the cordyceps extract is added and uniformly mixed, and then filled to obtain the complex probiotic preparation for regulating intestinal flora.

[0067] Example 5

[0068] The preparation method of the active additive is as follows:

[0069] S1, 5 parts by weight of corn starch, 80 parts by weight of water and 1 part by weight of ascorbic acid are mixed, treated under ultrasonic power of 200W and ultrasonic frequency of 70kHz for 50min, centrifuged, washed, and freeze-dried to obtain nano corn starch;

[0070] S2, the above nano corn starch is dispersed in water to prepare a 25wt% nano corn starch water suspension, and the pH is adjusted to 8.5 by using 1 mol / L sodium hydroxide aqueous solution; 0.7 parts by weight of 25wt% hydroxyl citric acid aqueous solution is added, stirring at 110℃, 400rpm for 4h, centrifugation, washing, and freeze-drying to obtain modified nano corn starch;

[0071] S3, the above modified nano corn starch is prepared into a 4wt% modified nano starch aqueous solution by adding water, 1 part by weight of iron glycinate is added, and stirring is continued for 20min, and then the mixture is cooled to room temperature, frozen at-60℃ for 18h, and then freeze-dried and crushed to a particle size of 0.05mm to obtain an active additive.

[0072] Example 6

[0073] The difference between the example 6 and the example 4 is that the preparation method of the active additive is as follows:

[0074] S1, 5 parts by weight of corn starch, 80 parts by weight of water and 1 part by weight of ascorbic acid are mixed, and treated under ultrasonic power of 200W and ultrasonic frequency of 70kHz for 50min, centrifuged, washed, and freeze-dried to obtain nano corn starch;

[0075] S2, the above nano corn starch is dispersed in water to prepare a 25wt% nano corn starch water suspension, and the pH is adjusted to 8.5 by using 1 mol / L sodium hydroxide aqueous solution; 0.7 parts by weight of 25wt% hydroxyl citric acid aqueous solution is added, stirring at 110℃, 400rpm for 4h, centrifugation, washing, and freeze-drying to obtain modified nano corn starch;

[0076] S3, the above modified nano corn starch is prepared into a 4wt% modified nano starch aqueous solution by adding water, 1 part by weight of iron glycinate is added, and stirring is continued for 20min, and then the mixture is cooled to room temperature, frozen at-60℃ for 18h, and then freeze-dried and crushed to a particle size of 0.05mm to obtain an active additive.

[0077] Example 7

[0078] The difference between the example 6 and the example 4 is that the preparation method of the active additive is as follows:

[0079] Example 8

[0080] The difference between the example 6 and the example 4 is that the preparation method of the active additive is as follows:

[0081] Example 9

[0082] The same as example 6, except that the mass ratio of Cordyceps sinensis extract, probiotics, prebiotics and active additives is 3:1:2:0.5.

[0083] Comparative example 1

[0084] A complex probiotic preparation with the efficacy of regulating intestinal flora, which is composed of probiotics and prebiotics, and the mass ratio of the probiotics and prebiotics is 1:2; the probiotics are composed of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei, and the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei is 1:1:1:1; the prebiotics are composed of fructooligosaccharide, galactooligosaccharide, stachyose and inulin, and the mass ratio of fructooligosaccharide, galactooligosaccharide, stachyose and inulin is 1:1:1:1.

[0085] The preparation method of the complex probiotic preparation with the efficacy of regulating intestinal flora is as follows: first, mix the probiotics and prebiotics uniformly according to the mass ratio, then fill them to obtain the complex probiotic preparation with the efficacy of regulating intestinal flora.

[0086] Comparative example 2

[0087] The same as example 2, except that the active additive is corn starch.

[0088] Comparative example 3

[0089] The same as example 4, except that a complex probiotic preparation with the efficacy of regulating intestinal flora is composed of probiotics, prebiotics and active additives, and the mass ratio of the probiotics, prebiotics and active additives is 1:2:0.4.

[0090] Comparative example 4

[0091] The same as example 6, except that the mass ratio of Cordyceps sinensis extract, probiotics, prebiotics and active additives is 3:1:2:0.1.

[0092] Comparative example 5

[0093] The same as example 6, except that the mass ratio of Cordyceps sinensis extract, probiotics, prebiotics and active additives is 3:1:2:0.6.

[0094] Test example

[0095] Experimental study on regulating intestinal flora

[0096] 1Materials and methods

[0097] 1.1 Test drug: Test sample 1: Cordyceps sinensis extract of bat hair fungus mycelium. The administration dose is 0.3 g / kg;

[0098] Test sample 2: Probiotics, prebiotics ratio is 1:2. The administration dose is 0.3 g / kg;

[0099] Test sample 3: Probiotics, prebiotics, Cordyceps sinensis extract of bat hair fungus mycelium ratio is 1:2:3, the administration dose is 0.3 g / kg.

[0100] 1.2 Experimental animals

[0101] SPF level male SD mice several, body weight 18-22 g. Room temperature 20~26℃, relative humidity 40%~70%, feeding SPF mouse growth and reproduction feed, free drinking water and food.

[0102] 2 Experimental process

[0103] Constipation model

[0104] Test samples 1, 2, and 3 are divided into 3 groups, 1 blank control group and 1 model control group, 15 animals in each group, test samples 1, 2, and 3 are configured into 30 mg / ml concentration of gavage liquid with purified water, the gavage amount is 0.2 ml / one, and the gavage amount is adjusted every 2d, the specific test arrangement and grouping are shown in Table 1.

[0105] Blank control group: physiological saline

[0106] Model control group: loperamide hydrochloride (10 mg / kg BW)

[0107] Table 1 Test arrangement and grouping of constipation model mice

[0108]

[0109] Experimental content:

[0110] 1: Ink push rate: by gavage drug loperamide, establish mouse constipation model, calculate the ink push rate of small intestine in a certain time, to judge the gastrointestinal peristalsis function of model mice. 0.5h after administration of loperamide, test samples 1.2.3 are respectively given ink containing test samples (containing 5% activated carbon powder, 10% gum arabic), the blank control group and the model control group mice are given ink gavage. Immediately after 25 min, the animals are sacrificed by cervical dislocation, the mesentery is separated, the intestinal canal from the upper end of the pylorus to the lower end of the ileocecal junction is cut, placed on a tray, and gently pulled into a straight line. The length of the intestinal canal is the total length of the small intestine, and the length of the ink push is from the pylorus to the ink front. And calculate the ink push rate according to formula (1).

[0111] Formula (1): M = L1 / L*100%

[0112] In the formula: M----Ink propulsion rate, %

[0113] L1----Ink propulsion length, cm;

[0114] L----Total length of small intestine, in cm;

[0115] 2. Defecation time, number of fecal particles, and fecal weight determination: The defecation status of the model mice was analyzed by measuring the time of the first black stool, the number of fecal particles, and the weight of feces within 6 hours. The intestinal regulatory effect and constipation-relieving effect of the probiotic compound formula were determined by gavage administration of formulations 1, 2, and 3.

[0116] Experimental Results and Analysis:

[0117] Table 2. Effects of compound probiotic preparations on ink propulsion rate in mouse small intestine.

[0118]

[0119] Note: **p<0.01 compared with the blank control group; compared with the model control group. # p<0.05 ## p<0.01.

[0120] Table 3. Effects of compound probiotic preparations on the time, number, and weight of the first black stool in mice.

[0121]

[0122] Note: **p<0.01 compared with the blank control group and the model control group. # p<0.05 ## p<0.01

[0123] Table 2 shows the effect on small intestinal ink propulsion rate. Compared with the blank control group, the ink propulsion rate of the model control group was significantly reduced (P < 0.01), indicating that the constipation model was established. Compared with the model control group, sample 1 had a certain effect on improving the ink propulsion rate of mice (P < 0.05), but the effect was not very significant compared with the blank control group. Sample 2 showed a significant difference compared with the model control group (P < 0.01), indicating that the combination of probiotics and prebiotics also had a good effect on improving the ink propulsion rate of mice. Sample 3 significantly improved the propulsion rate compared with the model control group, and the data was close to that of the blank control group, indicating that sample 3 could significantly improve the ink propulsion rate (P < 0.01). From the above data, it can be concluded that under the intervention of the interaction between the extract of *Hepialus hygroscopicus* mycelium and probiotics and prebiotics, the small intestinal ink propulsion rate can approach the normal level, indicating that our compound probiotic combination can significantly improve the intestinal propulsion rate of mice under constipation conditions.

[0124] From Table 3, compared with the blank control group, the model control group has significant differences in the first black stool time, black stool particle number and black stool weight (P<0.01), which indicates that the constipation model is successfully established. Compared with the model control group, sample 1 has certain influence on the intestinal function of mice, which can shorten the first black stool formation time (P<0.05), increase the black stool particle number within 6 hours (P<0.05) and the black stool weight (P<0.01); sample 2 can shorten the first black stool formation time (P<0.05), and has no significant difference in the black stool particle number, which can increase the black stool weight (P<0.05); sample 3 can significantly shorten the first black stool formation time (P<0.01) compared with the model control group, and significantly increase the black stool particle number and weight within 6 hours (P<0.01), which is even close to the level of the blank control group, which indicates that the simple bat moth hair fungus mycelium extract or the probiotic prebiotic combination can improve the constipation of mice, but the effect is not ideal, and the compound probiotic combination of sample 3 can significantly promote the intestinal peristalsis of mice, and then promote the defecation effect of mice, which basically can restore the mice to the normal level.

[0125] Diarrhea model

[0126] The diarrhea model caused by the bacterial flora disorder caused by mixed antibiotics is used, the test sample is given, and the change of the diarrhea stool frequency and intestinal mucosa of mice is observed, so as to determine the influence of the test sample on the diarrhea of mice.

[0127] Sample 1, 2 and 3 are used to set three groups, one blank control group and one model control group, 15 animals in each group, and the specific test arrangement and grouping are shown in Table 4.

[0128] Blank control group: physiological saline

[0129] Model control group: gentamicin sulfate injection and cefradine capsule are mixed with sterile physiological saline to form an antibiotic mixture with a mass concentration of 62.5 g / L.

[0130] Table 4 Test arrangement and grouping of diarrhea mice

[0131]

[0132] Table 5 Influence of the compound probiotic preparation on the colon mucosa injury of mice during diarrhea

[0133]

[0134] Note: compared with the blank control group, **p<0.01 compared with the model control group # p<0.05 ## p<0.01

[0135] From table 5, compared with the blank control group, the intestinal mucosa injury of other groups had significant difference (P<0.05), which indicated that the diarrhea model was successfully made. Compared with the model control group, the intestinal mucosa injury grade of sample 1 and 2 showed a decreasing trend, which indicated that sample 1 and 2 had the effect of protecting intestinal mucosa (P<0.05). Compared with the intestinal mucosa injury grade of the model control group, the intestinal mucosa injury grade of sample 3 was significantly decreased (p<0.01), and the data was close to the blank control group. It can be seen that the intestinal mucosa recovery ability of sample 1 and 2 was weaker than that of sample 3, which indicated that the combination of the complex probiotic preparation, the cordyceps cicadae mycelium extract, the probiotic and the prebiotic could more effectively repair the intestinal injury, and the effect was significant.

[0136] The mechanism of antibiotic-induced diarrhea is as follows: ① antibiotics directly cause intestinal mucosa damage, causing intestinal epithelial cilia atrophy, reducing intracellular enzyme activity, causing bile synthesis disorder, reducing fat absorption, and ultimately causing diarrhea; ② the use of antibiotics leads to the killing of sensitive bacteria, resulting in intestinal microecological imbalance, increased pathogenic bacteria, damaged intestinal barrier, and further leading to diarrhea. The complex probiotic preparation has the function of inhibiting antibiotic diarrhea, which may inhibit the growth of pathogenic microorganisms to a certain extent and maintain the growth of normal intestinal flora in the small intestine. At the same time, the probiotic can colonize in the intestinal tract of mice under certain conditions and produce a series of active mechanisms to maintain the ecological environment of the intestinal tract. The test shows that the complex formula of the cordyceps cicadae mycelium extract and the probiotic can significantly reduce the colon mucosa injury score and relieve antibiotic-induced diarrhea.

[0137] In summary, the test shows that under the symptoms of constipation in mice, the complex formula of the cordyceps cicadae mycelium extract and the probiotic can promote intestinal peristalsis by increasing the number and weight of defecation, improving the ink propulsion rate and other indicators, and effectively relieving constipation; under the symptoms of diarrhea, the complex formula of the cordyceps cicadae mycelium extract and the probiotic can improve diarrhea and inflammation by reducing the intestinal mucosa injury score, recovering the crypt structure and protecting the intestinal mucosa. At the same time, it can be known from the above experiments that the model control group and the sample 3 group have a significant effect on regulating the intestinal tract, and the present application has the effect of regulating the intestinal tract and improving constipation and diarrhea. The sample 3 group uses a traditional Chinese medicine-probiotic preparation without any toxic side effects, and the effect is more significant. For patients with weak intestinal function, the traditional Chinese medicine preparation is more reliable and safe. It is beneficial to the long-term treatment of such patients and has good clinical application prospect.

[0138] The mice were adapted for one week, and the animals were allowed to freely eat and drink. The experimental animals were randomly divided into 16 groups, 15 in each group, which were blank control group, model control group, examples 1-9 and comparative examples 1-5. Each group was pre-administered with 6 days of liquid, wherein the examples and comparative examples were administered with 0.2 mL of 25 mL / kg / d of the complex probiotic preparation aqueous solution for adjusting intestinal flora efficacy per day, the blank control group and the model control group were administered with the same amount of normal saline once a day. The animals were fasted for 12 hours before the last administration, and then administered with the antibiotic mixture at a dose of 62.5 g / L after 1 hour of administration, to establish the diarrhea type irritable bowel syndrome model, and the blank control group was administered with the same volume of deionized water. The diarrhea of the mice was counted from 3 hours after administration, and the number of diarrhea in 3 hours and 6 hours was calculated, and the statistical differences of the groups were compared. Soft stool, water-like stool, mucus stool and the like were called diarrhea stool, and the test results are shown in Table 6.

[0139] Model control group: gentamicin sulfate injection and cefradine capsule were mixed with sterile normal saline to form an antibiotic mixture with a mass concentration of 62.5 g / L.

[0140] Table 6 Effect on intestinal movement of diarrhea mice

[0141]

[0142] From the data in Table 6 above, in the antibiotic-induced diarrhea model, the intervention effects of each example and comparative example showed significant gradient differences, which indicated that the presence of active additives, the preparation process and the ratio of components had a great influence on the anti-diarrhea efficacy of the preparation. The number of 3h diarrhea stool of examples 4, 6 and 9 was 3.5±0.8, 3.2±0.7 and 3.8±0.8, respectively, which showed the best anti-diarrhea effect, and their common characteristics were that they all contained the active additive prepared by the complete process of cross-linking modification of L-cysteine or hydroxyl carboxylic acid and loading of glycine iron, and the best mass ratio of the additive to the probiotic was 0.4:1. The results showed that the hydroxyl citric acid in example 6 was esterified with the hydroxyl group of starch under the condition of heating and dehydration, to first establish a stable covalent cross-linking skeleton; L-cysteine was cross-linked with the hydroxyl group of starch through -NH2 / -COO -Electrostatic association and -SH-involved hydrogen bonds further tighten the network and provide a certain degree of reducing protection. In the presence of glycine iron, carboxyl sites in the network coordinate with the metal center, accompanied by electrostatic interactions, restricting chain segment migration and swelling. The resulting dense shell provides stronger barriers to the diffusion of gastric acid and bile salts, resulting in a higher survival rate of probiotics in the stomach and passive selective release under the pH and bile salt conditions of the small intestine. The gut microbiota recovers faster and exhibits more significant competitive inhibition of pathogens, ultimately leading to a significant decrease in the frequency of diarrhea. Compared to the single modification in Examples 4 or 5, this method more effectively isolates probiotics from the erosion of gastric acid and bile salts, thereby ensuring the survival of a higher number of active probiotics and their targeted delivery to the intestines. This rapid restoration of gut microbiota balance and competitive inhibition of pathogens is the key mechanism behind the lowest frequency of diarrhea.

[0143] On the other hand, the coordinated ratio of components is another key to achieving the best synergistic effect. In Comparative Example 4, the ratio of active additive to probiotics was 0.1:1. Due to insufficient additive dosage, the probiotic coating rate and degree of protection were significantly reduced. Its effect of 6.8±1.6 times / 3h was even worse than that of Comparative Example 1 (6.5±1.5 times / 3h without any additives), proving that an excessively low additive ratio cannot provide effective protection. Conversely, although the effect of Comparative Example 5 (5.5±1.0 times / 3h) was better than the model control group, it was inferior to the optimal example, indicating that an excessively high additive ratio may limit the colonization efficiency of probiotics due to excessive volume occupation of the carrier material or influence on the release kinetics of probiotics in the intestine. Therefore, there is an optimal mass ratio range between active additive and probiotics, with 0.4:1 being the best. Within this range, sufficient protection can be achieved without hindering the biological function of probiotics. In summary, this compound preparation achieves efficient delivery and maximizes the function of probiotics through the precise ratio of active additive and each component, thereby significantly alleviating diarrhea symptoms.

Claims

1. A complex probiotic formulation having modulating efficacy on gut microbiota, characterized in that, The extract of Cordyceps sinensis, probiotics, prebiotics and active additives, the mass ratio of the extract of Cordyceps sinensis, probiotics, prebiotics and active additives is 3:1:2:0.4; The probiotics are composed of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei, and the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus casei is 1:1:1:1; the prebiotics are composed of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin, and the mass ratio of fructo-oligosaccharide, galacto-oligosaccharide, stachyose and inulin is 1:1:1:1; The preparation method of the active additive is as follows: S1, 5 parts by weight of corn starch, 80 parts by weight of water and 1 part by weight of ascorbic acid are mixed, treated under ultrasonic power of 200 W and ultrasonic frequency of 70 kHz for 50 min, centrifuged, washed and freeze-dried to obtain nano corn starch; S2, the above nano corn starch is dispersed in water to prepare a 25wt% nano corn starch water suspension, and the pH is adjusted to 8.5 with 1mol / L sodium hydroxide solution; 0.5 parts by weight of 25wt% L-cysteine aqueous solution and 0.2 parts by weight of 25wt% hydroxyl citric acid aqueous solution are added, stirred at 110℃ and 400rpm for 4h, centrifuged, washed and freeze-dried to obtain modified nano corn starch; S3, the above modified nano corn starch is prepared into a 4wt% modified nano starch aqueous solution by adding water, 1 part by weight of ferric glycinate is added and stirred for 20 min, cooled to room temperature, frozen at-60℃ for 18h and then freeze-dried, and ground to a particle size of 0.05mm to obtain the active additive.

2. The probiotic formulation having modulating efficacy on gut microbiota as claimed in claim 1, wherein, The preparation method of the Cordyceps sinensis extract is as follows: Cordyceps sinensis mycelium and 10-25 times amount of water are loaded into an extraction tank, decocted for 1-4 hours, and the first filtrate is reserved; the residue and 10-20 times amount of water are subjected to second decoction, decocted for 1-4 hours, and the second filtrate is reserved; the two filtrates are combined, filtered through a filter, and the filtrate is concentrated under reduced pressure to a density of 1.05-1.20 g / cm 3 of a flow extract, spray dried to obtain the Cordyceps sinensis extract.

3. The probiotic formulation having efficacy in modulating gut microbiota as claimed in claim 1, wherein, The total number of viable bacteria in the complex probiotic preparation is 10 10 -10 11 CFU / mL.

4. A method of preparing a complex probiotic formulation having modulating the efficacy of gut flora as claimed in any one of claims 1-3, characterized in that, The following steps are included: the probiotics, prebiotics and active additives are first mixed uniformly according to the mass ratio, and then the extract of Cordyceps sinensis is added and mixed uniformly to obtain a composite probiotic preparation with the function of regulating intestinal flora.

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