Probiotic and polysaccharide composite composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine and functional food, and discloses a probiotic and polysaccharide compound composition as well as a preparation method and application thereof, the composition comprises the following raw materials in parts by weight: 0.5-2 parts of probiotic powder, 2-5 parts of oat beta-glucan, 1-3 parts of prebiotics and the balance of auxiliary materials; the auxiliary material is selected from at least one of maltodextrin, microcrystalline cellulose and magnesium stearate, and the auxiliary material accounts for 50-80% of the total mass of the probiotic powder, the oat beta-glucan and the prebiotics. The bifidobacterium animalis subsp. Lactis powder and the oat beta-glucan are conventional raw materials in the food industry, so that the cost is relatively low, and the production cost can be reduced. In the preparation process of the composition, conventional equipment such as an anaerobic fermentation tank and a freeze dryer is used, the production capacity of each batch is high, and the composition is suitable for large-scale production of small and medium-sized enterprises, so that industrialization is realized.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and functional food technology, and more specifically, it relates to a probiotic and polysaccharide complex composition, its preparation method and application. Background Technology

[0002] With improved living standards and changes in dietary structure, the prevalence of diabetes is rising year by year, especially type 2 diabetes, which has become a global public health problem. The main characteristic of diabetes is insufficient insulin secretion or insulin resistance, leading to ineffective blood glucose regulation. Studies have shown that regulating the gut microbiota and increasing GLP-1 (glucagon-like peptide-1) secretion have a positive effect on controlling blood glucose levels. Therefore, probiotics and prebiotics are widely used in the adjunctive treatment of diabetes.

[0003] Although a variety of probiotic and prebiotic products have been launched on the market and have improved blood sugar control in diabetic patients to some extent, the existing probiotic preparations require relatively complex equipment and have high production costs, which limits their application in small and medium-sized enterprises. Summary of the Invention

[0004] To address the problem that existing probiotic preparations require complex equipment and incur high production costs, limiting their application in small and medium-sized enterprises, this application provides a probiotic and polysaccharide composite composition, its preparation method, and its application.

[0005] In a first aspect, this application provides a probiotic and polysaccharide complex composition, employing the following technical solution:

[0006] A probiotic and polysaccharide complex composition comprising the following raw materials in parts by weight:

[0007] 0.5-2 parts probiotic powder, 2-5 parts oat beta-glucan, 1-3 parts prebiotics, and the remainder is excipients;

[0008] The excipients are selected from at least one of maltodextrin, microcrystalline cellulose, and magnesium stearate, and the excipients account for 50-80% of the total mass of probiotic powder, oat β-glucan, and prebiotics.

[0009] By adopting the above technical solution, probiotic powder, as a viable microbial component, can contact the intestinal environment and maintain its activity after entering the intestine. Oat β-glucan, as a soluble polysaccharide, can form a swollen system with a certain viscosity in the aqueous phase and coexist with the probiotic powder. Prebiotics, as oligosaccharides that can be utilized by probiotics, can provide a carbon source for the probiotic powder to support its survival in the intestine. After combination, a mixed system composed of microbial components, soluble polysaccharide components and fermentable oligosaccharide components can be formed, so that the probiotic powder is in a condition that simultaneously has a polysaccharide encapsulation environment and a usable carbon source.

[0010] The excipients consist of at least one of maltodextrin, microcrystalline cellulose, and magnesium stearate, which can provide dilution, excipient, flowability adjustment, and molding support in the system, enabling probiotic powder, oat β-glucan, and prebiotic substances to be uniformly dispersed and fixed in solid form. This ensures that the composite composition has a stable physical state and uniformity during the actual formulation process, achieving the conditions for the simultaneous existence and synergistic effect of probiotic powder, oat β-glucan, and prebiotics in oral formulations.

[0011] Preferably, the probiotic powder is Bifidobacterium animalis subsp. lactis, with a live bacteria count of not less than 1×10⁻⁶. 10 CFU / g;

[0012] The animal Bifidobacterium lactis subsp. is strain CCFM0611 or a food-grade isolate with equivalent GLP-1 secretion-promoting activity.

[0013] By adopting the above technical solution, the probiotic powder uses Bifidobacterium animalis subsp. lactis, which can colonize in the intestine and promote the secretion of GLP-1 by intestinal endocrine cells, especially L cells. Through interaction with the intestinal environment, it activates specific intestinal signaling pathways, especially Ca2+. 2+ The CaMKII signaling pathway enhances the synthesis and secretion of GLP-1, which helps regulate blood sugar levels and improve insulin sensitivity. This strain was chosen because its safety and functionality have been certified as food-grade, meet national food safety standards, have good feasibility for industrial production, and have a high survival rate, maintaining its biological activity during freeze-drying and other preparation processes.

[0014] The use of CCFM0611 strain or similar food-grade isolates with GLP-1 secretion-promoting activity can further ensure its efficacy in clinical applications and fully realize its functions of regulating metabolism, improving gut health, and controlling GLP-1 levels under existing production conditions. The activity of this strain has been verified in cell model experiments, showing that it can significantly increase GLP-1 secretion, thus having potential therapeutic effects on metabolic diseases such as diabetes.

[0015] Preferably, the oat β-glucan has a weight-average molecular weight of 2 × 10⁻⁶. 5 -3×10 5 The ratio of β-(1→3) to β-(1→4) glycosidic bonds in Da is 1:2.5-3.5, and the purity is not less than 90%.

[0016] The oat β-glucan has a water solubility of not less than 95%, and its inhibition rate against α-glucosidase is not less than 35% as verified by in vitro experiments.

[0017] The prebiotic is selected from at least one of fructooligosaccharides and galactooligosaccharides.

[0018] By adopting the above technical solution, oat β-glucan can significantly and slowly release its soluble fiber components in vivo, effectively delaying gastric emptying and carbohydrate absorption, thereby regulating blood sugar levels. In addition, in vitro experiments have verified that oat β-glucan has an inhibition rate of no less than 35% against α-glucosidase. It can effectively reduce carbohydrate breakdown and slow down the absorption rate of sugars, thereby reducing the rapid rise in postprandial blood sugar and enhancing the control effect on diabetes.

[0019] Prebiotics, by serving as a nutrient source for probiotics, can optimize the structure of the gut microbiota, promote the reproduction of beneficial bacteria, and thus help improve gut health and enhance immune function. The mechanism of action of prebiotics in the gut can complement that of probiotics, providing a suitable carbon source environment to support the survival and reproduction of probiotics in the gut, and further enhancing their effect in regulating gut metabolism and GLP-1 secretion. This allows the compound composition to achieve a comprehensive effect of lowering blood sugar, improving gut function, and enhancing GLP-1 secretion through synergistic action.

[0020] Meanwhile, the cost per ton of Bifidobacterium animalis subsp. lactis powder is about 40,000 yuan, and the cost per ton of oat β-glucan is about 60,000 yuan. Both are conventional raw materials in the food industry. The preparation uses conventional equipment such as anaerobic fermenters and freeze dryers, and the single batch production capacity can reach 100,000 tablets / cans, which is suitable for small and medium-sized enterprises to produce.

[0021] Secondly, this application provides a method for preparing a probiotic and polysaccharide complex composition, using the following technical solution:

[0022] A method for preparing a probiotic and polysaccharide complex composition includes the following steps:

[0023] S1. After activating and expanding the culture of Bifidobacterium animalis subsp. lactis to obtain fermentation broth, the probiotic powder is obtained by centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying.

[0024] S2. Oat β-glucan was obtained by defatting, hot water extraction, enzymatic hydrolysis, ethanol precipitation and column chromatography purification.

[0025] S3. Premix probiotic powder, oat β-glucan and prebiotics to form a premix, then add excipients to the premix and mix, and finally form the mixture into a formulation.

[0026] By adopting the above technical solution, in step S1, the fermentation broth of Bifidobacterium animalis subsp. lactis is obtained through activation culture and expansion culture. Then, the bacterial cells are collected by centrifugation and resuspended with a protectant to effectively maintain the activity of probiotics. In the subsequent pre-freezing and vacuum freeze-drying process, the stability and biological activity of the bacterial cells are ensured, and the probiotic powder obtained in the end has sufficient activity and is suitable for subsequent compounding.

[0027] In step S2, oat β-glucan is extracted and purified through a series of steps. First, impurities are removed by defatting and hot water extraction, effectively preserving the bioactive components of oat β-glucan. Subsequently, starch impurities are removed by α-amylase enzymatic hydrolysis, followed by purification by ethanol precipitation and column chromatography to obtain high-purity oat β-glucan. Oat β-glucan exhibits an α-glucosidase inhibition rate of no less than 35% in in vitro experiments. This characteristic helps to delay the digestion and absorption of carbohydrates, slow the rapid rise in blood glucose, and optimize glycemic control.

[0028] In step S3, probiotic powder, oat beta-glucan, and prebiotics are premixed to form a premix. The role of prebiotics is to provide the nutrients needed by probiotics, promote the colonization of probiotics in the intestine, and thus enhance the function of probiotics. Then, excipients are added and mixed to ensure that the formula is uniform. Finally, the mixture is formulated into powder, capsules, or tablets to ensure that the final product can effectively exert its effects in the intestine after oral ingestion, promote the secretion of GLP-1, and regulate blood sugar and weight.

[0029] Through the above steps, a probiotic and high-purity oat β-glucan complex with good biological activity can be stably produced, which has the potential for synergistic effects, effectively regulating GLP-1 secretion, improving glucose metabolism, and optimizing the gut microbiota.

[0030] Preferably, in step S1, the activation and culture step is as follows:

[0031] Bifidobacterium animalis subsp. lactis was inoculated into BBL solid medium and anaerobic cultured at 35-39℃ for 45-51h.

[0032] Single colonies were picked and inoculated into BBL liquid medium, and anaerobically cultured with shaking at 35-39℃ for 18-20 hours to obtain seed culture, in which the viable count of the seed culture was not less than 1×10⁻⁶. 9 CFU / mL;

[0033] The steps for scaling up the culture are as follows:

[0034] Inoculate the seed culture into the expansion medium at an inoculation rate of 3-7%, and anaerobic static culture at 35-39℃ for 22-26h. The viable count of the fermentation broth should not be less than 1×10¹¹ CFU / mL. The expansion medium consists of 99.5% BBL medium and 0.5% yeast extract.

[0035] The steps of centrifugation, resuspending in a protective agent, pre-freezing, and vacuum freeze-drying are as follows:

[0036] The fermentation broth was placed in a centrifuge and centrifuged at 3-5℃ and 7000-9000r / min for 10-20min to collect the cells. The cells were then resuspended in a protectant containing 15% skim milk and 5% maltodextrin and the cell concentration was adjusted to 1×10¹² CFU / mL.

[0037] After pre-freezing at -35-45℃ for 2-6 hours, freeze-dry under a vacuum of 10-20Pa for 20-28 hours, and then pulverize through a 60-100 mesh sieve.

[0038] By employing the above technical solution, through activation and expansion culture, the growth of *Bifidobacterium animalis* subsp. *lactamase* under optimal conditions can be ensured, and a high concentration of active bacteria can be obtained. First, *Bifidobacterium animalis* subsp. *lactamase* is inoculated onto BBL solid medium and subjected to anaerobic culture to ensure the strain fully recovers its metabolic activity. During this process, the strain colonizes in the solid medium and forms single colonies. Subsequently, single colonies are picked and transferred to BBL liquid medium for further culture, resulting in a seed culture with a viable count of no less than 1 × 10⁻⁶. 9 The CFU / mL concentration ensures high activity of probiotics in the inoculum, providing sufficient strains for large-scale culture.

[0039] The seed culture was inoculated into the expansion medium at an inoculation rate of 3-7%, using BBL medium containing yeast extract, and cultured under anaerobic static conditions until the viable cell count in the fermentation broth reached 1×10⁻⁶. 11 The concentration of CFU / mL or higher ensures that a sufficient high concentration of active probiotics is obtained, providing enough cells for the subsequent freeze-drying process;

[0040] The probiotic cells were then efficiently separated and recovered from the fermentation broth through centrifugation. Subsequently, the cells were resuspended using a preservative to ensure the probiotics maintained their structure and function during freeze-drying. The cell concentration was adjusted to 1×10¹² CFU / mL to obtain a sufficient number of live cells for subsequent use. Pre-freezing ensured effective protection of the cells during freeze-drying, preventing cell damage due to ice crystal formation. Finally, freeze-drying under vacuum removed moisture and preserved the probiotics' biological activity. After freeze-drying, the bacterial powder was pulverized to ensure good flowability and uniformity in the final product.

[0041] Through the above steps, the high activity, long-term stability and good application effect of probiotics are effectively guaranteed, and sufficient active probiotics are retained in the final product to ensure that they can exert their biological effects in the intestine.

[0042] Preferably, in step S2, the purification steps of defatting, hot water extraction, enzymatic hydrolysis, ethanol precipitation, and column chromatography are as follows:

[0043] Take oat bran, add 80% ethanol at a material-to-liquid ratio of 1:10, defatt the oat bran 2-3 times at room temperature, each time for 2-6 hours, and centrifuge to collect the supernatant.

[0044] Defatted oat bran was added to distilled water at a material-to-liquid ratio of 1:25, and extracted in a water bath at 85-95℃ for 2-3 times, each time for 2-3 hours. The extracts were then combined.

[0045] Add 0.05-0.15% α-amylase and hydrolyze at 45-65℃ for 0.5-1.5h to remove starch, then centrifuge and collect the supernatant.

[0046] Add 3.5-4.5 times the volume of anhydrous ethanol, let stand at 3-5℃ for 10-14 hours, centrifuge to collect the precipitate, and purify the precipitate by DEAE-52 cellulose column.

[0047] By adopting the above technical solution, the extraction and purification process of oat β-glucan ensures its high purity and good biological activity. First, through the defatting step, oat bran is added to 80% ethanol and defatted 2-3 times at room temperature, which effectively removes the fat-soluble components in the bran, reduces the interference of impurities, and retains the effective components of oat β-glucan. After centrifugation, the supernatant obtained can enter the next extraction process.

[0048] Add defatted oat bran to distilled water and extract 2-3 times. Combine the extracts. High-temperature water bath can be used to extract β-glucan from oat bran, which can effectively improve the dissolution rate of the target component and ensure efficient extraction.

[0049] In the enzymatic hydrolysis step, α-amylase is added to remove excess starch from the oats. α-amylase removes starch components by hydrolyzing the α-glycosidic bonds in starch molecules, further improving the purity of β-glucan. After this step, the supernatant obtained by centrifugation is further purified.

[0050] Next, the ethanol precipitation step involves adding anhydrous ethanol and allowing the mixture to stand to further remove impurities dissolved in water and promote the formation of β-glucan precipitate. After centrifugation and collection, the precipitate enters the final purification step, where it is purified by DEAE-52 cellulose column chromatography. This process further removes residual impurities and yields β-glucan with higher purity. The target polysaccharide is selectively separated and enriched through the principle of ion exchange.

[0051] Through the above steps, the final oat β-glucan has high purity and water solubility, and retains its effective biological activity, ensuring the efficacy of oat β-glucan in promoting blood sugar regulation and intestinal health.

[0052] Preferably, in step S3, the premixing step is as follows:

[0053] Add probiotic powder, oat β-glucan, and prebiotics to a sterile mixer and stir for 8-12 minutes at 20-30℃ and 20-40r / min to obtain a premix.

[0054] The steps of adding excipients and mixing are as follows:

[0055] After pulverizing the premixed material, pass it through a 60-100 mesh sieve and continue stirring for 15-25 minutes to mix it evenly.

[0056] By adopting the above technical solution, the premixing and excipient mixing process ensures the uniform distribution of probiotic powder, oat β-glucan, and prebiotics in the final formulation, thereby optimizing the synergistic effect of each active ingredient. In the premixing step, the probiotic powder, oat β-glucan, and prebiotics are first added to a sterile mixer and stirred to ensure that the components are fully mixed in the initial stage, forming a uniform premix, effectively avoiding uneven distribution among active ingredients and maintaining their biological activity.

[0057] Subsequently, during the addition of excipients and mixing, the premix is ​​pulverized and screened to ensure the fineness and uniformity of the material. The pulverized premix is ​​then sieved to further eliminate larger particles, ensuring a higher degree of uniformity for each component in the mixture. Mixing continues thereafter to ensure that all components, including probiotics, oat beta-glucan, prebiotics, and excipients, are fully integrated physically and chemically, ensuring the stability and consistency of each dosage form, improving the bioavailability of active ingredients, and enhancing the efficacy of the final product.

[0058] Preferably, the protectant in step S1 consists of 2% sucrose, 15% skim milk, and 5% maltodextrin, with the remainder being water, and is used to improve the survival rate of the freeze-dried cells.

[0059] In step S2, the oat β-glucan column chromatography purification also includes a step of adding 0.5% activated carbon for decolorization for 25-35 minutes to improve polysaccharide purity.

[0060] By adopting the above technical solution, in step S1, the protective agents used include 2% sucrose, 15% skim milk, and 5% maltodextrin, with the remainder being water. Their function is to improve the survival rate of probiotic cells during freeze-drying. Sucrose, as a common protective agent, can effectively improve the stability of cell membranes and reduce cell damage caused by water evaporation during freeze-drying. The combined action of skim milk and maltodextrin provides appropriate osmotic pressure and a stable environment, allowing the cells to better maintain their activity during freeze-drying. The use of this protective agent effectively improves the survival rate of freeze-dried probiotics and ensures the high activity of probiotics in the final product.

[0061] In the column chromatography purification process of oat β-glucan in step S2, the activated carbon decolorization step further improves the purity of the polysaccharide. Activated carbon can adsorb pigments, impurities and other small molecules in the aqueous solution, thereby removing impurities that affect the purity of the polysaccharide, resulting in purer oat β-glucan and improving its quality. When used in combination with DEAE-52 cellulose column chromatography, the polysaccharide purification process becomes more efficient, and the purity is further improved, ensuring the effectiveness of the final product.

[0062] Thirdly, this application provides an application of a probiotic and polysaccharide complex composition, employing the following technical solution:

[0063] Application of a probiotic and polysaccharide complex composition, wherein the formulated product includes pharmaceuticals, health foods or functional foods, the product being used to promote GLP-1 secretion, improve insulin resistance, reduce fasting and postprandial blood glucose, reduce body fat accumulation or regulate gut microbiota.

[0064] By employing the above technical solution, the composite composition synergistically regulates GLP-1 through a triple mechanism: First, *Bifidobacterium animalis* subsp. *lactum* stimulates the proliferation of intestinal L cells and activates Ca2+. 2+The CaMKII signaling pathway promotes GLP-1 synthesis and secretion; secondly, oat β-glucan inhibits dipeptidyl peptidase-4 activity, reducing GLP-1 degradation; finally, prebiotics promote probiotic colonization in the gut, increasing the production of short-chain fatty acids, such as acetic acid, propionic acid, and butyric acid, further enhancing the GLP-1 secretion effect. These three factors synergistically achieve efficient metabolic regulation. The application of probiotic and polysaccharide complexes in pharmaceuticals, health foods, or functional foods mainly works by promoting GLP-1 secretion, improving insulin resistance, reducing fasting and postprandial blood glucose, reducing body fat accumulation, or regulating intestinal flora. Through mechanisms such as gut microbiota, probiotics achieve their health benefits. By stimulating L cells to secrete GLP-1, probiotics help increase GLP-1 levels, thereby improving insulin secretion function, lowering blood sugar levels, and improving insulin resistance. The polysaccharide component, oat β-glucan, delays the inhibition of insulin secretion by inhibiting dipeptidyl peptidase-4 activity, further helping to lower postprandial blood sugar. At the same time, the addition of prebiotics enhances gut health by promoting the colonization of probiotics and the production of short-chain fatty acids, and indirectly regulates metabolic processes in the body by improving gut microbiota, helping to reduce body fat accumulation.

[0065] Preferably, the drug dosage form is capsules, tablets or granules, and the daily dosage is 0.6-2g of the active ingredient in the composition, taken orally in 1-2 divided doses, 30 minutes before meals; the health food dosage form is solid beverage, compressed candy or probiotic powder, and the daily consumption amount contains 0.4-1.2g of the active ingredient, dissolved in warm water or mixed into liquid food for consumption.

[0066] By adopting the above technical solutions, the administration or consumption methods of pharmaceuticals and health foods, and the dosage design of the active ingredients, meet different application needs, allowing users to choose the appropriate dosage form according to their requirements. Pharmaceutical dosage forms such as capsules, tablets, or granules typically have a daily dosage of 0.6-2g of the active ingredient, taken orally in 1-2 divided doses 30 minutes before meals. This ensures that the active ingredient is fully absorbed and exerts its effect in the body, especially in promoting GLP-1 secretion, improving insulin resistance, and regulating blood sugar, thus guaranteeing the best therapeutic efficacy. Health food dosage forms such as solid beverages, compressed candies, or probiotic powders contain 0.4-1.2g of the active ingredient daily. These can be dissolved in warm water or mixed into liquid foods, making them more convenient for daily intake. This allows consumers to ingest beneficial ingredients in their daily lives, achieving long-term regulation of intestinal flora and improving metabolic health, ensuring the maximum effect of the probiotic and polysaccharide complex composition.

[0067] After 6 months of storage at room temperature, the tablets showed low loss of live bacteria and high retention of oat β-glucan; in a simulated gastrointestinal environment, the survival rate of probiotics was high.

[0068] In summary, this application has the following beneficial effects:

[0069] 1. The *Bifidobacterium animalis* subsp. lactis powder and oat β-glucan used in this application are both conventional raw materials in the food industry, with low cost, which helps to reduce production costs. The preparation process of this composition uses conventional equipment such as anaerobic fermenters and freeze dryers, and has a high production capacity per batch, making it suitable for large-scale production by small and medium-sized enterprises, thereby achieving industrialization.

[0070] 2. This application can significantly promote the secretion of GLP-1. The results of cell experiments and animal experiments show that the regulatory effect of the composition is significantly improved compared with the single component group, showing its potential in improving the symptoms of diabetes.

[0071] 3. Under normal temperature conditions, the composition of this application still maintains good stability after 6 months of storage, with minimal loss of probiotic activity and high retention rate of oat β-glucan. In addition, in a simulated gastrointestinal environment, the survival rate of probiotics is significantly higher than that of the group without prebiotics, indicating that the composition has good tolerability in vivo.

[0072] 4. This application enhances the effect through a triple mechanism of synergistic action: probiotics promote the secretion of GLP-1 by L cells, polysaccharides inhibit dipeptidyl peptidase-4 activity, and prebiotics promote the colonization of probiotics and the production of short-chain fatty acids. Under the combined action, the composition can more effectively regulate sugar metabolism.

[0073] 5. The composition of this application can not only regulate the secretion of GLP-1 and improve the symptoms of diabetes, but also regulate the balance of intestinal microecology. The synergistic effect of probiotics and polysaccharides can enhance intestinal health and improve metabolic abnormalities caused by diabetes. Attached Figure Description

[0074] Figure 1 This is a flowchart of a method for preparing a probiotic and polysaccharide complex composition according to this application. Detailed Implementation

[0075] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0076] Example 1

[0077] This application provides a probiotic and polysaccharide complex composition, which, by weight, comprises the following components: 0.5 parts probiotic powder, 2 parts oat β-glucan, 1 part prebiotic, and the remainder being excipients;

[0078] The excipients are selected from maltodextrin, and the excipients account for 50% of the total mass of probiotic powder, oat β-glucan and prebiotics;

[0079] The probiotic powder contains Bifidobacterium animalis subsp. lactis, with a live bacteria count of 1×10¹. 0CFU / g;

[0080] Bifidobacterium animalis subsp. lactis is strain CCFM0611;

[0081] The weight-average molecular weight of oat β-glucan is 2 × 10⁻⁶. 5 The ratio of β-(1→3) to β-(1→4) glycosidic bonds in Da is 1:2.5, and the purity is 90%.

[0082] Oat β-glucan has a water solubility of 95% and its inhibition rate against α-glucosidase is 35% as verified by in vitro experiments.

[0083] The prebiotics are selected from fructooligosaccharides; the mass ratio of probiotic powder, oat β-glucan, and prebiotics is 1:3:2.

[0084] The preparation method of the above-mentioned probiotic and polysaccharide complex composition includes the following steps: S1, after activating and culturing Bifidobacterium animalis subsp. lactis and obtaining fermentation broth through expansion culture, the probiotic powder is obtained by centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying;

[0085] The activation culture step is as follows: Bifidobacterium animalis subsp. lactis is inoculated into BBL solid medium and anaerobic cultured at 35°C for 45 hours;

[0086] Single colonies were picked and inoculated into BBL liquid medium, and anaerobically cultured with shaking at 35°C for 18 hours to obtain a seed culture, in which the viable count of the seed culture was 1×10⁻⁶. 9 CFU / mL;

[0087] The steps for scaling up the culture are as follows: the seed culture is inoculated into the scaling up medium at an inoculation rate of 3%, and then anaerobic statically cultured at 35°C for 22 hours. The viable cell count in the fermentation broth is 1×10¹¹ CFU / mL. The scaling up medium consists of 99.5% BBL medium and 0.5% yeast extract.

[0088] The steps of centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying are as follows: Place the fermentation broth in a centrifuge and centrifuge at 3℃ and 7000r / min for 10min to collect the cells. Resuspend the cells with a protectant and adjust the cell concentration to 1×1012CFU / mL.

[0089] After pre-freezing at -45℃ for 2 hours, freeze-drying under 10Pa vacuum for 20 hours, and then pulverizing through a 60-mesh sieve;

[0090] The protectant consists of 2% sucrose, 15% skim milk, and 5% maltodextrin, with the remainder being water, and is used to improve the survival rate of the freeze-dried cells.

[0091] S2. Oat β-glucan was obtained by defatting, hot water extraction, enzymatic hydrolysis, ethanol precipitation and column chromatography purification.

[0092] In this process, oat bran was taken and 80% ethanol was added at a material-to-liquid ratio of 1:10. The oat bran was defatted twice at room temperature for 2 hours each time, and the supernatant was collected by centrifugation. Defatted oat bran was added to distilled water at a material-to-liquid ratio of 1:25 and extracted twice in an 85℃ water bath for 2 hours each time. The extracts were then combined.

[0093] Add 0.05% α-amylase and hydrolyze at 45℃ for 0.5h to remove starch, then centrifuge and collect the supernatant;

[0094] Add 3.5 times the volume of anhydrous ethanol, let stand at 3°C ​​for 10 hours, centrifuge to collect the precipitate, and purify the precipitate by DEAE-52 cellulose column.

[0095] During column chromatography purification, a step of adding 0.5% activated charcoal for 25 minutes for decolorization is also included to improve the purity of polysaccharides.

[0096] S3. Premix probiotic powder, oat β-glucan and prebiotics to form a premix, then add excipients to the premix and mix, and finally form the mixture into a formulation.

[0097] The premixing step is as follows: add probiotic powder, oat β-glucan, and prebiotics to a sterile mixer and stir for 8 minutes at 20℃ and 20r / min to obtain the premix.

[0098] The steps for adding and mixing the excipients are as follows: After crushing the premix, pass it through a 60-mesh sieve and continue stirring for 15 minutes to mix evenly.

[0099] Example 2

[0100] This application provides a probiotic and polysaccharide complex composition, comprising the following components by weight:

[0101] 1.25 parts probiotic powder, 3.5 parts oat beta-glucan, 2 parts prebiotics, and the remainder is excipients;

[0102] The excipients are selected from a mixture of maltodextrin and microcrystalline cellulose, and the excipients account for 65% of the total mass of probiotic powder, oat β-glucan and prebiotics;

[0103] The probiotic powder contains Bifidobacterium animalis subsp. lactis, with a live bacteria count of 5 × 10¹. 0 CFU / g;

[0104] Bifidobacterium animalis subsp. lactis is strain CCFM0611;

[0105] The weight-average molecular weight of oat β-glucan is 2.5 × 10⁻⁶.5 The ratio of β-(1→3) to β-(1→4) glycosidic bonds in Da is 1:3.0, and the purity is 95%.

[0106] The water solubility of oat β-glucan is 97%, and in vitro experiments have shown that it inhibits α-glucosidase by 45%.

[0107] Prebiotics are selected from a mixture of fructooligosaccharides and galactooligosaccharides;

[0108] The preparation method of the above-mentioned probiotic and polysaccharide complex composition includes the following steps:

[0109] S1. After activating and expanding the culture of Bifidobacterium animalis subsp. lactis to obtain fermentation broth, the probiotic powder is obtained by centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying.

[0110] The activation culture step is as follows: Bifidobacterium animalis subsp. lactis is inoculated into BBL solid medium and anaerobic cultured at 37°C for 48 hours;

[0111] Single colonies were picked and inoculated into BBL liquid medium, and anaerobically cultured with shaking at 37°C for 19 h to obtain a seed culture, in which the viable count of the seed culture was 5 × 10⁻⁶. 9 CFU / mL;

[0112] The steps for scaling up the culture are as follows: the seed culture is inoculated into the scaling up medium at an inoculation rate of 5%, and then anaerobic statically cultured at 37°C for 24 hours. The viable cell count of the fermentation broth is 5×10¹¹ CFU / mL. The scaling up medium consists of 99.5% BBL medium and 0.5% yeast extract.

[0113] The steps of centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying are as follows: Place the fermentation broth in a centrifuge and centrifuge at 4℃ and 8000r / min for 15min to collect the cells. Resuspend the cells with a protectant and adjust the cell concentration to 1×1012CFU / mL.

[0114] After pre-freezing at -40℃ for 4 hours, freeze-drying under a vacuum of 15Pa for 24 hours, and then pulverizing through an 80-mesh sieve;

[0115] The protectant consists of 2% sucrose, 15% skim milk, and 5% maltodextrin, with the remainder being water, and is used to improve the survival rate of the freeze-dried cells.

[0116] S2. Oat β-glucan was obtained by defatting, hot water extraction, enzymatic hydrolysis, ethanol precipitation and column chromatography purification.

[0117] Among them, oat bran was taken, 80% ethanol was added at a material-to-liquid ratio of 1:10, defatted twice at room temperature for 4 hours each time, and the supernatant was collected by centrifugation.

[0118] Defatted oat bran was added to distilled water at a material-to-liquid ratio of 1:25 and extracted twice in a 90°C water bath for 2.5 hours each time. The extracts were then combined.

[0119] Add 0.1% α-amylase and enzymatically hydrolyze at 55℃ for 1.0 h to remove starch, centrifuge and collect the supernatant; add 4.0 times the volume of anhydrous ethanol, let stand at 4℃ for 12 h, centrifuge and collect the precipitate, and purify the precipitate by DEAE-52 cellulose column.

[0120] During column chromatography purification, a step of adding 0.5% activated charcoal for 30 minutes for decolorization is also included to improve the purity of polysaccharides.

[0121] S3. Premix probiotic powder, oat β-glucan and prebiotics to form a premix, then add excipients to the premix and mix, and finally form the mixture into a formulation.

[0122] The premixing step is as follows: add probiotic powder, oat β-glucan, and prebiotics to a sterile mixer and stir for 10 minutes at 25°C and 30 r / min to obtain the premix.

[0123] The steps for adding and mixing the excipients are as follows: After crushing the premix, pass it through an 80-mesh sieve and continue stirring for 20 minutes to mix evenly.

[0124] Example 3

[0125] This application provides a probiotic and polysaccharide complex composition, comprising the following components by weight:

[0126] 2 parts probiotic powder, 5 parts oat beta-glucan, 3 parts prebiotics, and the remainder is excipients;

[0127] The excipients are selected from magnesium stearate, and the excipients account for 80% of the total mass of probiotic powder, oat beta-glucan and prebiotics;

[0128] Among them, the probiotic powder is Bifidobacterium lactis subsp. animalis, with a live bacteria count of 1×10¹¹ CFU / g;

[0129] Bifidobacterium animalis subsp. lactis is a food-grade isolate with equivalent GLP-1 secretion-promoting activity;

[0130] The weight-average molecular weight of oat β-glucan is 3 × 10⁻⁶. 5 The ratio of β-(1→3) to β-(1→4) glycosidic bonds in Da is 1:3.5, and the purity is 98%.

[0131] The water solubility of oat β-glucan is 99%, and in vitro experiments have verified that it inhibits α-glucosidase by 55%; the prebiotics are selected from galactooligosaccharides.

[0132] The preparation method of the above-mentioned probiotic and polysaccharide complex composition includes the following steps:

[0133] S1. After activating and expanding the culture of Bifidobacterium animalis subsp. lactis to obtain fermentation broth, the probiotic powder is obtained by centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying.

[0134] The activation culture step is as follows: Bifidobacterium animalis subsp. lactis is inoculated into BBL solid medium and anaerobic cultured at 39°C for 51 h.

[0135] Single colonies were picked and inoculated into BBL liquid medium, and anaerobically cultured with shaking at 39°C for 20 h to obtain a seed culture, in which the viable count of the seed culture was 1×10¹. 0 CFU / mL;

[0136] The steps for scaling up the culture are as follows: the seed culture was inoculated into the scaling up medium at an inoculation rate of 7%, and the culture was anaerobic statically cultured at 39°C for 26 hours. The viable cell count in the fermentation broth was 1×10¹² CFU / mL. The scaling up medium consisted of 99.5% BBL medium and 0.5% yeast extract.

[0137] The steps of centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying are as follows: Place the fermentation broth in a centrifuge and centrifuge at 5℃ and 9000r / min for 20min to collect the cells. Resuspend the cells with a protectant and adjust the cell concentration to 1×1012CFU / mL.

[0138] After pre-freezing at -35℃ for 6 hours, freeze-drying under a vacuum of 20Pa for 28 hours, and then pulverizing through a 100-mesh sieve;

[0139] The protectant consists of 2% sucrose, 15% skim milk, and 5% maltodextrin, with the remainder being water, and is used to improve the survival rate of the freeze-dried cells.

[0140] S2. Oat β-glucan was obtained by defatting, hot water extraction, enzymatic hydrolysis, ethanol precipitation and column chromatography purification.

[0141] Among them, oat bran was taken, 80% ethanol was added at a material-to-liquid ratio of 1:10, defatted at room temperature 3 times, 6 hours each time, and the supernatant was collected by centrifugation.

[0142] Defatted oat bran was added to distilled water at a material-to-liquid ratio of 1:25, and extracted three times in a 95°C water bath for 3 hours each time. The extracts were then combined.

[0143] Add 0.15% α-amylase and hydrolyze at 65℃ for 1.5h to remove starch, then centrifuge and collect the supernatant.

[0144] Add 4.5 times the volume of anhydrous ethanol, let stand at 5°C for 14 hours, centrifuge to collect the precipitate, and purify the precipitate by DEAE-52 cellulose column.

[0145] During column chromatography purification, a step of adding 0.5% activated charcoal for 35 minutes for decolorization is also included to improve the purity of polysaccharides.

[0146] S3. Premix probiotic powder, oat β-glucan and prebiotics to form a premix, then add excipients to the premix and mix, and finally form the mixture into a formulation.

[0147] The premixing step is as follows: add probiotic powder, oat β-glucan, and prebiotics to a sterile mixer and stir for 12 minutes at 30℃ and 40r / min to obtain the premix.

[0148] The steps for adding and mixing the excipients are as follows: After crushing the premix, pass it through a 100-mesh sieve and continue stirring for 25 minutes to mix evenly.

[0149] Comparative Example 1

[0150] The only difference from Example 2 is the absence of oat β-glucan.

[0151] Comparative Example 2

[0152] The only difference from Example 2 is that the prebiotics are missing.

[0153] Comparative Example 3

[0154] The only difference from Example 2 is that the probiotic powder is replaced with an equal amount of common Bifidobacterium lactis subsp. animalis.

[0155] Comparative Example 4

[0156] The only difference from Example 2 is that oat β-glucan is replaced with an equal amount of ordinary glucan.

[0157] Comparative Example 5

[0158] The only difference from Example 2 is that the prebiotics are replaced with an equal amount of xylooligosaccharides.

[0159] Comparative Example 6

[0160] The only difference from Example 2 is that the column chromatography purification step is missing in step S2.

[0161] Comparative Example 7

[0162] The only difference from Example 2 is that the activated carbon decolorization step is missing in step S2.

[0163] Comparative Example 8

[0164] The only difference from Example 2 is that the premixing step is missing in step S3.

[0165] I. Experiment to determine the regulatory effect of the composite composition GLP-1

[0166] 0.5 g of each of the composite composition samples from Examples 1-3 and Comparative Examples 1-8 were placed in 50 mL centrifuge tubes, and 20 mL of DMEM medium containing 10% fetal bovine serum was added. The samples were then magnetically stirred at 100 rpm for 5 minutes at 37 °C until completely dispersed to obtain sample solutions. At the same time, an in vitro blank control group was set up, containing only 20 mL of DMEM medium containing 10% fetal bovine serum. The in vivo model control group consisted of type 2 diabetes mice, which were fed a high-sugar, high-fat diet for 4 weeks and induced gestation by a single intraperitoneal injection of 100 mg / kg STZ, and were only given physiological saline by gavage.

[0167] Take 1 mL of sample solution and filter it through a 0.22 μm microporous membrane. Collect the filtrate as the test solution. The in vitro GLP-1 content is detected using enzyme-linked immunosorbent assay (ELISA). First, coat a 96-well microplate with a monoclonal antibody against GLP-1. Add 100 μL of coating buffer (4 μg / mL) to each well, incubate at 4°C for 10 hours, then discard the coating buffer. Wash three times with phosphate buffer containing 0.05% Tween-20, 2 minutes each time. Add 100 μL of the test solution to each well. Add an equal volume of culture medium to the in vitro blank control group. Incubate at 37°C for 1.5 hours, then discard the liquid and wash three more times. Finally, add 100 μL of lycopene to each well. After incubating with peroxidase-labeled secondary antibody at 37°C for 1 hour, the sample was washed 3 times. Finally, 100 μL of tetramethylbenzidine chromogenic solution was added to each well, and the sample was incubated at 37°C in the dark for 10 minutes. The reaction was terminated by adding 50 μL of 2 mol / L sulfuric acid. The absorbance of each well was measured at 450 nm using a microplate reader. A standard curve was plotted using standard GLP-1 solution. The GLP-1 content in each sample was calculated based on the standard curve. The formula for calculating the improvement rate compared to the blank group is: Improvement rate compared to the blank group (%) = (GLP-1 content in the sample group - GLP-1 content in the in vitro blank control group) / GLP-1 content in the in vitro blank control group × 100.

[0168] Sixty SPF-grade C57BL / 6 male mice were randomly divided into 12 groups of 5 mice each, corresponding to Examples 1-3, Comparative Examples 1-8, in vitro blank control group, and in vivo model control group, respectively. Examples 1-3 and Comparative Examples 1-8 were administered the sample solution via gavage at a dose of 0.8 g / kg body weight of active ingredient once daily for 8 consecutive weeks. After the intervention, mice were fasted for 12 hours, and fasting blood glucose was measured using a blood glucose meter at the tail tip. Body fat percentage was measured using a small animal body composition analyzer. After sacrifice, ileal tissue homogenate was collected, and ileal GLP-1 content was measured using the ELISA method described above. The change rate compared to the model group was calculated using the formula: Change rate compared to the model group (%) = (Sample group index value - In vivo model control group index value) / In vivo model control group index value × 100 (blood glucose and body fat percentages represent the decrease rate, while GLP-1 represents the increase rate).

[0169] II. Experiment on the stability and intestinal colonization ability of the composite composition

[0170] 10g of each of the composite composition samples from Examples 1-3 and Comparative Examples 1-8 were selected and made into tablets, with a tablet weight of 0.5g / tablet. 20 tablets from each group were placed in sealed aluminum-plastic blister packaging and stored for 6 months at room temperature and relative humidity ≤60%. An initial control group was also set up.

[0171] After the storage period expired, three samples from each group were ground into powder, and 0.1g of the powder was weighed out and added to 10mL of sterile physiological saline. The mixture was shaken at 37℃ and 150rpm for 10 minutes to prepare a bacterial suspension. After serial dilution, 100μL of the suspension was spread on BBL solid medium and anaerobically cultured at 37℃ for 48 hours. The viable count of probiotics was determined by plate counting. The viable count loss rate was calculated as follows: Viable count loss rate (%) = (Number of viable bacteria in the initial control group - Number of viable bacteria in the sample after storage) / Number of viable bacteria in the initial control group × 100. Another 0.1g of sample powder was added to 5mL of ultrapure water and dissolved in an 80℃ water bath for 30 minutes. After cooling, the mixture was filtered through a 0.22μm microporous membrane. The oat β-glucan content was determined by high performance liquid chromatography. The retention rate was calculated as follows: Retention rate (%) = Oat β-glucan content in the sample after storage / Oat β-glucan content in the initial control group × 100.

[0172] III. Industrialization Cost of Composite Composition and DPP-4 Inhibition Rate Determination Experiment

[0173] 0.2 g of each of the composite composition samples from Examples 1-3 and Comparative Examples 1-8 were placed in 20 mL centrifuge tubes, 10 mL of ultrapure water was added, and the mixture was magnetically stirred at 25 °C and 120 rpm for 8 minutes until completely dissolved. The mixture was then filtered through a 0.22 μm microporous membrane, and the filtrate was collected as the test solution. At the same time, an enzyme-substrate control group containing only DPP-4 enzyme, Gly-Pro-pNA substrate and Tris-HCl buffer and a blank control group containing only Tris-HCl buffer were set up.

[0174] Add 50 μL of the test solution and 50 μL of DPP-4 enzyme solution to each well. Incubate at 37°C for 10 minutes, then add 50 μL of Lly-Pro-pNA substrate. Continue incubation at 37°C for 30 minutes. Finally, add 50 μL of 1 mol / L sodium carbonate solution to terminate the reaction. Measure the absorbance of each well at 405 nm using a microplate reader. The formula for calculating the DPP-4 inhibition rate is: DPP-4 inhibition rate (%) = (Absorbance of enzyme-substrate control group - Absorbance of sample group) / (Absorbance of enzyme-substrate control group - Absorbance of blank control group) × 100.

[0175] Table 1: Experimental Results of the GLP-1 Regulation Effect of the Composite Composition

[0176]

[0177]

[0178] Table 2: Results of Experiments on the Stability and Intestinal Colonization Ability of the Composite Composition

[0179]

[0180]

[0181]

[0182] Table 3: Industrialization cost of the composite composition and experimental results of DPP-4 inhibition rate determination

[0183] Grouping DPP-4 inhibition rate (%, x±s) Enzyme-substrate control group 0.0±0.2 Example 1 36.5±2.1 Example 2 38.0±2.3 Example 3 37.2±2.2 Comparative Example 1 15.2±1.6 Comparative Example 2 20.5±1.7 Comparative Example 3 22.8±1.8 Comparative Example 4 17.4±1.7 Comparative Example 5 21.8±1.7 Comparative Example 6 23.2±1.9 Comparative Example 7 22.7±1.8 Comparative Example 8 20.8±1.7

[0184] By comparing the experimental data of the examples and the comparative examples, it can be seen that:

[0185] Combining Example 2 and Comparative Example 1 with Table 1, it can be seen that in Comparative Example 1, due to the absence of oat β-glucan, the in vitro GLP-1 content decreased from 22.1±1.7 pg / mL in Example 2 to 16.2±1.2 pg / mL, the in vivo fasting blood glucose increased from 9.2±1.1 mmol / L to 12.8±1.5 mmol / L, the body fat percentage increased from 26.8±1.7% to 30.1±2.0%, and the ileal GLP-1 content decreased from 92.5±7.5 pg / g to 72.5±6.8 pg / g. The absence of this component prevents the formation of a synergistic system of probiotic secretion promotion and polysaccharide degradation inhibition, directly leading to a significant weakening of GLP-1 regulation and blood sugar and fat reduction effects. This demonstrates that oat β-glucan is a necessary component for the composite composition to exert its core function.

[0186] Based on Example 2 and Comparative Example 2, and referring to Tables 1 and 2, it can be seen that, due to the absence of prebiotics, the in vitro GLP-1 content in Comparative Example 2 decreased from 22.1±1.7 pg / mL in Example 2 to 17.5±1.3 pg / mL, and the in vivo ileal GLP-1 content decreased from 92.5±7.5 pg / g to 70.2±6.5 pg / g; simultaneously, after 6 months of storage, the number of viable probiotics decreased from 8.7×10⁻⁶ in Example 2. 8 CFU / tablet reduced to 6.0×10 8 CFU / tablet, live bacteria loss rate increased from 13% to 40%. The absence of CFU reduced the colonization ability of probiotics and the insufficient production of SCFA, which weakened the sustained secretion effect of GLP-1 and reduced the storage stability of the product, proving that prebiotics are an important support for maintaining the synergistic mechanism of the composition.

[0187] Based on Examples 2 and 3, and referring to Tables 1 and 3, it can be seen that after replacing the common strain in Comparative Example 3, the in vitro GLP-1 content decreased from 22.1±1.7 pg / mL to 18.1±1.2 pg / mL, the in vivo fasting blood glucose increased from 9.2±1.1 mmol / L to 14.2±1.6 mmol / L, and the DPP-4 inhibition rate decreased from 38.0±2.3% to 22.8±1.8%. This demonstrates that the target strain is the exclusive core ingredient for the composition to exert its excellent GLP-1 regulatory function.

[0188] Combining Example 2 and Comparative Example 4 with Tables 2 and 3, it can be seen that after the substitution in Comparative Example 4, the oat β-glucan retention rate decreased from 95.8±1.2% in Example 2 to 88.5±1.8%, the DPP-4 inhibition rate decreased from 38.0±2.3% to 17.4±1.7%, and the body fat percentage increased from 26.8±1.7% to 30.8±2.0%. This demonstrates that the structural specificity of oat β-glucan is a prerequisite for its functional realization.

[0189] Based on Example 2 and Comparative Example 5, and referring to Tables 1 and 2, it can be seen that after the substitution in Comparative Example 5, the in vitro GLP-1 content decreased from 22.1±1.7 pg / mL to 18.0±1.2 pg / mL, the total cecal SCFA content decreased from 81.3±4.5 mmol / L to 72.5±4.2 mmol / L, and the relative abundance of Bifidobacterium decreased from 8.0±0.7% to 3.7±0.3%.

[0190] Combining Example 2 and Comparative Example 6 with Tables 2 and 3, it can be seen that in Comparative Example 6, omitting this step resulted in a decrease in the oat β-glucan retention rate from 95.8±1.2% in Example 2 to 88.5±1.8%, a decrease in DPP-4 inhibition rate from 38.0±2.3% to 23.2±1.9%, and an increase in fasting blood glucose from 9.2±1.1 mmol / L to 14.6±1.6 mmol / L. This is because column chromatography can effectively remove impurities such as starch and miscellaneous sugars from oat β-glucan, increasing the purity to over 95%. Impurities interfere with DPP-4 inhibitory activity and intestinal metabolic signals, leading to a weakening of GLP-1 regulation and hypoglycemic effects. This demonstrates that column chromatography purification is a necessary process to ensure the function of oat β-glucan.

[0191] Combining Example 2 and Comparative Example 7 with Tables 2 and 3, it can be seen that after Comparative Example 7 omitted this step, the oat β-glucan retention rate decreased from 95.8±1.2% in Example 2 to 89.2±1.7%, the DPP-4 inhibition rate decreased from 38.0±2.3% to 22.7±1.8%, and the in vitro GLP-1 content decreased from 22.1±1.7 pg / mL to 18.4±1.1 pg / mL. This proves that activated carbon decolorization is a key auxiliary process for optimizing the quality of oat β-glucan.

[0192] Based on Example 2 and Comparative Example 8, and referring to Tables 1 and 2, it can be seen that in Comparative Example 8, omitting this step resulted in a decrease in the in vitro GLP-1 content from 22.1 ± 1.7 pg / mL to 17.3 ± 1.2 pg / mL, and the number of viable probiotics after 6 months of storage increased from 8.7 × 10⁻⁶. 8 CFU / tablet decreased to 8.1×10 8 CFU / tablet resulted in an increase in body fat percentage from 26.8±1.7% to 30.8±1.9%. This demonstrates that the premixing step is essential for ensuring the uniformity and stability of the composition.

[0193] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A probiotic and polysaccharide complex composition, characterized in that, Composed of the following raw materials in parts by weight: 0.5-2 parts probiotic powder, 2-5 parts oat beta-glucan, 1-3 parts prebiotics, and the remainder is excipients; The excipients are selected from at least one of maltodextrin, microcrystalline cellulose, and magnesium stearate, and the excipients account for 50-80% of the total mass of probiotic powder, oat β-glucan, and prebiotics.

2. The probiotic and polysaccharide complex composition according to claim 1, characterized in that, The probiotic powder is Bifidobacterium lactis subsp. animalis, with a live bacteria count of not less than 1×10⁻⁶. 10 CFU / g; The animal Bifidobacterium lactis subsp. is strain CCFM0611 or a food-grade isolate with equivalent GLP-1 secretion-promoting activity.

3. The probiotic and polysaccharide complex composition according to claim 1, characterized in that, The oat β-glucan has a weight-average molecular weight of 2 × 10⁻⁶. 5 -3×10 5 The ratio of β-(1→3) to β-(1→4) glycosidic bonds in Da is 1:2.5-3.5, and the purity is not less than 90%. The oat β-glucan has a water solubility of not less than 95%, and its inhibition rate against α-glucosidase is not less than 35% as verified by in vitro experiments. The prebiotic is selected from at least one of fructooligosaccharides and galactooligosaccharides.

4. A method for preparing a probiotic and polysaccharide complex composition, characterized in that, The probiotic and polysaccharide complex composition according to any one of claims 1-3 comprises the following steps: S1. After activating and expanding the culture of Bifidobacterium animalis subsp. lactis to obtain fermentation broth, the probiotic powder is obtained by centrifugation, resuspension with a protectant, pre-freezing and vacuum freeze-drying. S2. Oat β-glucan was obtained by defatting, hot water extraction, enzymatic hydrolysis, ethanol precipitation and column chromatography purification. S3. Premix probiotic powder, oat β-glucan and prebiotics to form a premix, then add excipients to the premix and mix, and finally form the mixture into a formulation.

5. The method for preparing a probiotic and polysaccharide complex composition according to claim 4, characterized in that, In step S1, the activation and culture step is as follows: Bifidobacterium animalis subsp. lactis was inoculated into BBL solid medium and anaerobic cultured at 35-39℃ for 45-51h. Single colonies were picked and inoculated into BBL liquid medium, and anaerobically cultured with shaking at 35-39℃ for 18-20 hours to obtain seed culture, in which the viable count of the seed culture was not less than 1×10⁻⁶. 9 CFU / mL; The steps for scaling up the culture are as follows: Inoculate the seed culture into the expansion medium at an inoculation rate of 3-7%, and anaerobic static culture at 35-39℃ for 22-26h. The viable count of the fermentation broth should not be less than 1×10¹¹ CFU / mL. The expansion medium consists of 99.5% BBL medium and 0.5% yeast extract. The steps of centrifugation, resuspending in a protective agent, pre-freezing, and vacuum freeze-drying are as follows: The fermentation broth was placed in a centrifuge and centrifuged at 3-5℃ and 7000-9000r / min for 10-20min to collect the cells. The cells were then resuspended in a protectant containing 15% skim milk and 5% maltodextrin and the cell concentration was adjusted to 1×10¹² CFU / mL. After pre-freezing at -35-45℃ for 2-6 hours, freeze-dry under a vacuum of 10-20Pa for 20-28 hours, and then pulverize through a 60-100 mesh sieve.

6. The method for preparing a probiotic and polysaccharide complex composition according to claim 4, characterized in that, In step S2, the purification process, including defatting, hot water extraction, enzymatic hydrolysis, ethanol precipitation, and column chromatography, is as follows: Take oat bran, add 80% ethanol at a material-to-liquid ratio of 1:10, defatt the oat bran 2-3 times at room temperature, each time for 2-6 hours, and centrifuge to collect the supernatant. Defatted oat bran was added to distilled water at a material-to-liquid ratio of 1:25, and extracted in a water bath at 85-95℃ for 2-3 times, each time for 2-3 hours. The extracts were then combined. Add 0.05-0.15% α-amylase and hydrolyze at 45-65℃ for 0.5-1.5h to remove starch, then centrifuge and collect the supernatant. Add 3.5-4.5 times the volume of anhydrous ethanol, let stand at 3-5℃ for 10-14 hours, centrifuge to collect the precipitate, and purify the precipitate by DEAE-52 cellulose column.

7. The method for preparing a probiotic and polysaccharide complex composition according to claim 4, characterized in that, In step S3, the premixing step is as follows: Add probiotic powder, oat β-glucan, and prebiotics to a sterile mixer and stir for 8-12 minutes at 20-30℃ and 20-40r / min to obtain a premix. The steps of adding excipients and mixing are as follows: After pulverizing the premixed material, pass it through a 60-100 mesh sieve and continue stirring for 15-25 minutes to mix it evenly.

8. The method for preparing a probiotic and polysaccharide complex composition according to claim 4, characterized in that, In step S1, the protectant consists of 2% sucrose, 15% skim milk, and 5% maltodextrin, with the remainder being water, and is used to improve the survival rate of the freeze-dried cells. In step S2, the oat β-glucan column chromatography purification also includes a step of adding 0.5% activated carbon for decolorization for 25-35 minutes to improve polysaccharide purity.

9. The application of a probiotic and polysaccharide complex composition, characterized in that, The method for preparing a probiotic and polysaccharide complex composition according to any one of claims 4-8, wherein the product formed by the formulation includes pharmaceuticals, health foods or functional foods, and the product is used to promote GLP-1 secretion, improve insulin resistance, reduce fasting and postprandial blood glucose, reduce body fat accumulation or regulate intestinal flora.

10. The application of the probiotic and polysaccharide complex composition according to claim 9, characterized in that, The drug dosage form is capsules, tablets or granules, and the daily dosage is 0.6-2g of the active ingredient in the composition, taken orally in 1-2 divided doses, 30 minutes before meals; the health food dosage form is solid beverage, compressed candy or probiotic powder, and the daily consumption amount contains 0.4-1.2g of active ingredient, dissolved in warm water or mixed into liquid food for consumption.

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