Buckwheat and rhizoma polygonati composition for improving type 2 diabetes mellitus and application of buckwheat and rhizoma polygonati composition in preparation of dairy products

Abstract

The invention discloses a buckwheat and polygonatum sibiricum composition for improving type 2 diabetes and application of the buckwheat and polygonatum sibiricum composition in preparation of dairy products, and relates to the technical field of food, the composition comprises the following raw materials in parts by weight: 30-40 parts of buckwheat, 20-30 parts of polygonatum sibiricum, 15-25 parts of barley and 10-20 parts of wheat bran, and the dairy products are prepared by mixing a composition extracting solution and cow milk and then performing low-temperature fermentation. Bidirectional regulation of an intestinal flora-insulin signal channel is realized through compounding of the composition, and different from a single target effect in the prior art, different components in the composition respectively target the intestinal flora and an insulin-mTOR-autophagy signal channel; and the two components can promote each other (the intestinal flora metabolite SCFA can enhance the activity of an insulin signal channel) to form a two-way regulation closed loop, which is a core innovation point for realizing the comprehensive blood glucose reducing effect, and the ratio is an optimal scheme verified by early animal experiments, and the blood glucose reducing effect can be reduced when the ratio is deviated.

Description

Technical Field

[0001] This invention relates to the field of food technology, specifically to a buckwheat and polygonatum composition for improving type 2 diabetes and its application in the preparation of dairy products. Background Technology

[0002] Currently, the core technology for functional dairy products targeting type 2 diabetes (T2DM) is generally a single hypoglycemic component-added preparation process, which means adding a single hypoglycemic active substance to conventional dairy products (such as milk and yogurt) to achieve the function of assisting in regulating blood sugar.

[0003] For example, the conventional method for preparing dairy products in the current technology involves incorporating a single hypoglycemic component into the dairy product through a simple dissolution-mixing-homogenization process. The specific process is as follows: In the raw material stage, raw milk or reconstituted milk is selected as the dairy product base, and a single hypoglycemic component is added, commonly a chemical hypoglycemic drug (such as metformin) or a single plant extract (such as extracted Polygonatum polysaccharide). Only one of the two is added. In the pretreatment stage, the dairy product raw materials are first pasteurized to avoid microbial contamination during subsequent mixing. At the same time, the single hypoglycemic component is dissolved in sterile water to prepare a low-concentration solution to improve its dispersibility in the dairy product. In the mixing and homogenization stage, the hypoglycemic component solution is mixed into the sterilized dairy product at a fixed ratio (usually 0.5-2%), and then high-pressure homogenization is performed to break up the fat globules in the raw materials, ensuring that the hypoglycemic component is evenly distributed and avoiding precipitation. In the finished product stage, the subsequent process is selected according to the type of dairy product. If yogurt is to be prepared, conventional fermentation strains are added for fermentation. If pure milk is to be prepared, it is directly packaged, and finally a dairy product with basic hypoglycemic function is obtained.

[0004] However, the above-mentioned existing technical solutions have the following three core drawbacks: (1) Single hypoglycemic mechanism and limited effect: The existing technology relies on a single hypoglycemic component, which can only act on a single pathological link of T2DM (such as metformin only inhibiting hepatic gluconeogenesis, and single Polygonatum polysaccharide only mildly regulating blood sugar), and cannot cover the multi-mechanism pathological features of T2DM such as "glucose and lipid metabolism disorder - intestinal flora imbalance - abnormal insulin signaling pathway"; for example, the single component added to existing dairy products cannot simultaneously regulate the intestinal flora structure (such as increasing the abundance of Bifidobacterium and decreasing the abundance of Klebsiella pneumoniae) and the insulin-mTOR-autophagy signaling pathway, resulting in the intervention effect on T2DM only remaining at the level of assisting in hypoglycemia, and unable to improve lipid metabolism (such as reducing TC and TG) and intestinal barrier function; (2) Lack of intestinal flora regulation ability: Existing technologies do not focus on the impact of gut microbiota on T2DM. The single ingredients added (such as metformin and single plant extracts) do not have the effect of regulating the diversity and structure of gut microbiota. Existing studies have confirmed that the gut microbiota of T2DM patients has the characteristics of increased Firmicutes / Bacteroidetes ratio, decreased Bifidobacteria, and increased pro-inflammatory bacteria. Existing dairy products cannot improve this imbalance, which means they cannot intervene in T2DM from the gut-metabolic axis level. (3) Poor taste and tolerance: If chemical hypoglycemic drugs (such as metformin) are added, the dairy products will have a bitter taste, and long-term consumption may cause gastrointestinal discomfort (such as diarrhea and bloating). If a single plant extract is added, its strong plant fishy smell will destroy the original milky flavor of the dairy products, resulting in low consumer acceptance and difficulty in long-term consumption to maintain the hypoglycemic effect.

[0005] To address the aforementioned problems, this invention provides a buckwheat and polygonatum composition for improving type 2 diabetes and its application in the preparation of dairy products. Summary of the Invention

[0006] This invention provides a buckwheat and polygonatum composition for improving type 2 diabetes and its application in the preparation of dairy products. This invention overcomes the limitations of existing technologies that rely on single-component additions by incorporating buckwheat and polygonatum extract, which has multi-target hypoglycemic activity, into dairy products, and optimizes the preparation process to preserve the compound's activity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A buckwheat and polygonatum composition for improving type 2 diabetes includes the following raw materials in parts by weight: 30-40 parts buckwheat, 20-30 parts polygonatum, 15-25 parts barley, and 10-20 parts wheat bran (wheat bran, also known as wheat husk, is sweet and cool in nature, and is the seed coat sieved out after milling wheat from the Triticum aestivum plant of the Poaceae family; wheat bran polysaccharides can promote insulin secretion by protecting pancreatic cells, thereby reducing blood sugar levels).

[0008] This invention achieves bidirectional regulation of the gut microbiota-insulin signaling pathway through the compounding of components. Unlike the single-target action of existing technologies, this invention targets the gut microbiota and the insulin-mTOR-autophagy signaling pathway respectively through different components in the compound, and the two can promote each other (the gut microbiota metabolite SCFA can enhance the activity of the insulin signaling pathway), forming a bidirectional regulatory closed loop. This is the core innovation of achieving a comprehensive hypoglycemic effect. Moreover, the ratio of this invention is the optimal solution verified by previous animal experiments. Deviating from this ratio will lead to a decrease in hypoglycemic effect.

[0009] Preferably, the composition is an extract.

[0010] Preferably, the preparation method of the extract is as follows: weigh the raw materials according to the specified weight parts, crush and mix them, add deionized water for extraction and concentration.

[0011] Preferably, the extraction is performed twice, and the steps are as follows: add 10-15 times the volume of deionized water to the pulverized raw material, extract at a constant temperature of 80-90℃ for 2-3 hours, filter and collect the first extract, add 8-10 times the volume of deionized water to the residue, extract at a constant temperature of 80-90℃ for 2-3 hours, filter and collect the second extract; combine the first extract and the second extract.

[0012] The extract of this invention contains a variety of active ingredients such as Polygonatum polysaccharide, buckwheat flavonoids, and D-fagopyrine.

[0013] Preferably, the pulverization is of 80-100 mesh, and the concentration is carried out under reduced pressure at 60-70℃ to a relative density of 1.1-1.2 (25℃), which can ensure that the concentration of active ingredients meets the standard and ensure that the extraction rate of active ingredients is ≥80%.

[0014] The application of the buckwheat-Polygonatum composition in the preparation of dairy products as described above.

[0015] Preferably, the method for preparing the dairy product includes the following steps: (1) The extract of buckwheat and polygonatum composition is diluted and added to sterilized milk and mixed evenly to obtain a mixture. Then, excipients are added to the mixture and homogenized to obtain a homogenized liquid. (2) Add fermentation bacteria to the homogenized liquid for fermentation, and cool it quickly after fermentation to obtain dairy products.

[0016] Preferably, the milk mentioned in step (1) is raw milk with a fat content of 3.0-3.5%, which can ensure a rich milky aroma and balanced nutrition; The dilution step is as follows: the extract is diluted with sterile water to a concentration of 20-30%, and the pH is adjusted to 6.5-7.0 with citric acid or sodium bicarbonate to make it consistent with the pH of milk, so as to avoid denaturation and precipitation of milk proteins due to pH difference after mixing. The amount of the diluted extract added is 5-8% of the mass of the milk. The mixing conditions are as follows: stirring at 100-150 r / min for 10-15 min at 30-40℃. Low-temperature stirring can avoid the degradation of active ingredients such as Polygonatum polysaccharide and D-fagopyrine in the extract caused by high temperature, while ensuring that the extract and milk are initially blended. The sterilization method is as follows: ultra-high temperature instantaneous sterilization at 75-80℃ for 15-20 seconds. Compared with the existing technology of pasteurization at 60-65℃ (15-30 minutes), this method can not only completely kill harmful bacteria in milk (sterilization rate ≥99.9%), but also shorten the heating time and reduce the damage to heat-sensitive active ingredients (such as buckwheat flavonoids) in the subsequently added compound extract. The excipient is sucrose, and the amount of sucrose added is 5-7% of the mass of the mixture, which adjusts the taste and avoids the slightly astringent taste brought by the compound. The homogenization pressure is 25 MPa, which can break down the fat globules in the milk, making the mixture system more stable and preventing stratification or precipitation during subsequent fermentation, while also ensuring that sucrose and compound ingredients are evenly dispersed.

[0017] Preferably, the fermentation strains in step (2) are Lactobacillus bulgaricus and Streptococcus thermophilus, and the inoculum amount is 2-3% of the mass of the homogenized liquid; The fermentation conditions are as follows: fermentation at 35-38℃ for 5-7 hours. Compared with the fermentation temperature of 37-42℃ in the prior art, this low temperature environment can ensure the normal reproduction of the strain (the pH at the end of fermentation is stably reduced to 4.2-4.5) while increasing the retention rate of heat-sensitive active ingredients in the compound by more than 30% (such as increasing the retention rate of D-fagopyrine from 65% in the prior art to more than 95%). The rapid cooling is as follows: after the fermentation reaches the end point, the temperature of the yogurt is reduced from the fermentation temperature (35 - 38°C) to below 10°C within 15 minutes, and the cooling rate is ≥1.8°C / minute, terminating the fermentation process to avoid further degradation of active ingredients caused by long-term high temperature. If the cooling time exceeds 15 minutes (cooling rate < 1.8°C / minute), the retention rate of polygonatum polysaccharide in the compound will decrease from 95% to below 88%, and the retention rate of D-buckwheat alkaloid will decrease from 96% to below 90%, directly affecting the hypoglycemic effect of the product. The jacket refrigeration system of a conventional food-grade fermenter can easily achieve this cooling rate without additional new equipment, meeting the actual requirements of industrial production. Those skilled in the art can precisely control the cooling rate by adjusting the cold water circulation flow rate; specifically, it is achieved through the rapid refrigeration system supporting the fermenter (such as jacketed cold water circulation, with the cold water temperature controlled at 0 - 4°C), quickly terminating the fermentation process to avoid further degradation of active ingredients caused by long-term high temperature.

[0018] Preferably, the mass ratio of the Lactobacillus bulgaricus to the Streptococcus thermophilus is 1:1, which can balance the yogurt flavor and fermentation efficiency.

[0019] Preferably, it further includes filling the cooled dairy product into a sterile yogurt cup in a sterile environment, sealing it, and storing it at 4°C in the refrigerator to complete the preparation of the whole functional yogurt; the refrigeration condition can further maintain the yogurt flavor and extend the preservation time of active ingredients (the loss of active ingredients within the shelf life ≤ 5%).

[0020] Through the optimal compounding of the buckwheat and polygonatum composition with the dairy product and the preparation process in the present invention, the retention rate of the compound active ingredients is ≥85%. If it exceeds the parameter range, such as the addition amount > 10% will cause astringent taste, and the fermentation temperature > 40°C will degrade D-buckwheat alkaloid, which will affect the product function or taste.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) Multiple-target hypoglycemia, more comprehensive effect: The buckwheat and polygonatum combination in the present invention contains various active ingredients, which can act on multiple pathological links of T2DM simultaneously. Polygonatum polysaccharide and barley extract synergistically regulate the insulin - mTOR - autophagy signaling pathway (such as promoting the expression of IRS and PI3K, inhibiting the high expression of mTOR), buckwheat flavonoids and wheat bran polysaccharides synergistically regulate the intestinal flora (such as increasing the abundance of Bacteroidetes and Bifidobacterium, reducing the abundance of Proteobacteria and Klebsiella), and the compound components can repair the intestinal barrier (up-regulating the expression of tight junction proteins Occludin and ZO1); compared with the existing single-component dairy products that can only reduce fasting blood glucose (FBG), the product of the present invention can reduce the FBG of T2DM model animals, while reducing TC and TG, and increasing the intestinal flora diversity index (Shannon index), achieving the comprehensive effects of hypoglycemia + lipid regulation + intestinal protection.

[0022] (2) Significantly enhanced gut microbiota regulation capacity: The single component of the existing technology cannot change the gut microbiota structure, while the buckwheat flavonoids in the compound of the present invention can promote the proliferation of beneficial bacteria such as Bifidobacterium and Mulliebacterium, and the wheat bran polysaccharide can inhibit the growth of pro-inflammatory bacteria such as Trichophyton spp. In addition, the short-chain fatty acid (SCFA) precursor substances in the compound extract can be fermented by gut microbiota to generate butyric acid, further improving the gut microenvironment; animal experiments have verified that after consuming the product of the present invention for 4 weeks, the abundance of Bifidobacterium in the intestine of T2DM model animals increased, the abundance of Klebsiella pneumoniae decreased, and the Firmicutes / Bacteroidetes ratio was close to the healthy level, effectively repairing the gut microbiota imbalance.

[0023] (3) Significantly improved taste and tolerance: This invention uses buckwheat and Solomon's seal compound to replace the chemical drugs or single extracts of the prior art. The buckwheat and barley in the compound have their own grain aroma, which can mask the slight astringency of Solomon's seal. After dilution, they blend with the milky aroma of milk to form a grain milky flavor. At the same time, the plant polysaccharides in the compound can protect the gastrointestinal mucosa and avoid discomfort caused by chemical drugs. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. The drawings in this description are merely embodiments of the present invention.

[0025] Figure 1 These are histopathological changes in the pancreatic tissue of mice in each group according to the present invention; Figure 2 These are histopathological changes in the liver tissue of mice in each group according to the present invention; Figure 3 These are histopathological changes in the colon tissue of mice in each group according to the present invention; Figure 4 This is a bar chart showing the changes in glucose and lipid metabolism levels in mice of each group in this invention; Figure 5 This is a graph showing the changes in the gut microbiota of mice in each group according to the present invention; Figure 6 This is a graph showing the expression of mTOR-autophagy pathway proteins in each group of mice in this invention. Detailed Implementation

[0026] Embodiments of the present invention are described below, examples of which are shown in the accompanying drawings. The embodiments described with reference to the drawings are exemplary and intended to explain the present invention, but are not to be construed as limiting the present invention.

[0027] Example 1 This invention provides a buckwheat and polygonatum composition for improving type 2 diabetes, specifically comprising 30 parts buckwheat, 20 parts polygonatum, 15 parts barley, and 10 parts wheat bran; The specific preparation method for using the relevant composition in the preparation of dairy products is as follows: (1) Raw material preparation: Extraction process of buckwheat and polygonatum composition: The above raw materials are crushed to 80 mesh, 10 times the volume of deionized water is added, and the mixture is extracted at 80℃ for 2 hours. The first extract is collected by filtration, and the residue is added to 8 times the volume of deionized water. The mixture is extracted at 80℃ for 2 hours, and the second extract is collected by filtration. The two extracts are combined and concentrated under reduced pressure at 60-70℃ to a relative density of 1.1 (measured at 25℃) to obtain the extract for later use. Raw milk with a fat content of 3.0% is selected as the base for dairy products to ensure a rich milky aroma and balanced nutrition; The fermentation strain was a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus in a 1:1 mass ratio. (2) Use 75℃ ultra-high temperature instantaneous sterilization for 15s, dilute the extract to 20% mass concentration with sterile water, and adjust the pH to 6.5 with citric acid. Add the diluted extract at 6wt% to the sterilized milk cooled to 30-40℃ while stirring at 100r / min for 10min to obtain the mixture. Add 5 wt% sucrose to the above mixture, and then homogenize it under a pressure of 25 MPa. Homogenization is only required once to obtain a homogenized solution. (4) Low-temperature fermentation and activation stage: Inoculate 2wt% of mixed fermentation bacteria into the homogenized liquid, stir evenly, transfer the inoculated homogenized liquid into the fermentation tank, control the temperature at 35-38℃, ferment for 5 hours, and periodically check the pH value. When the pH drops to 4.2-4.5, immediately lower the temperature to below 10℃ to terminate the fermentation. (5) Finished product filling stage: Under aseptic conditions, the cooled dairy products are filled into aseptic yogurt cups, sealed and stored at 4°C to complete the preparation of the entire functional yogurt.

[0028] Example 2 This invention provides a buckwheat and polygonatum composition for improving type 2 diabetes, specifically comprising 40 parts buckwheat, 30 parts polygonatum, 25 parts barley, and 20 parts wheat bran; The specific preparation method for using the relevant composition in the preparation of dairy products is as follows: (1) Raw material preparation: Extraction process of buckwheat and polygonatum composition: The above raw materials are crushed to 100 mesh, 15 times the volume of deionized water is added, and the mixture is extracted at 80℃ for 3 hours. The first extract is collected by filtration, and the residue is added to 10 times the volume of deionized water and extracted at 80℃ for 3 hours. The second extract is collected by filtration, and the two extracts are combined and concentrated under reduced pressure at 60-70℃ to a relative density of 1.15 (measured at 25℃) to obtain the extract for later use. Raw milk with a fat content of 3.5% is selected as the base for dairy products to ensure a rich milky aroma and balanced nutrition. The fermentation strain was a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus in a 1:1 mass ratio. (2) Use 80℃ ultra-high temperature instantaneous sterilization for 15s, dilute the extract to 30% mass concentration with sterile water, and adjust the pH to 6.5 with citric acid. Add the diluted extract at 8wt% to the sterilized milk cooled to 30-40℃ while stirring at a speed of 150r / min for 10min to obtain the mixture. Add 6 wt% sucrose to the above mixture, and then homogenize it under a pressure of 25 MPa. Homogenization is only required once to obtain a homogenized solution. (4) Low-temperature fermentation and activation stage: Inoculate 2wt% of mixed fermentation bacteria into the homogenized liquid, stir evenly, transfer the inoculated homogenized liquid into the fermentation tank, control the temperature at 35-38℃, ferment for 7 hours, and periodically check the pH value. When the pH drops to 4.2-4.5, immediately lower the temperature to below 10℃ to terminate the fermentation. (5) Finished product filling stage: Under aseptic conditions, the cooled dairy products are filled into aseptic yogurt cups, sealed and stored at 4°C to complete the preparation of the entire functional yogurt.

[0029] Example 3 This invention provides a buckwheat and polygonatum composition for improving type 2 diabetes, specifically comprising 35 parts buckwheat, 25 parts polygonatum, 25 parts barley, and 20 parts wheat bran; The specific preparation method for using the relevant composition in the preparation of dairy products is as follows: (1) Raw material preparation: Extraction process of buckwheat and polygonatum composition: The above raw materials are crushed to 100 mesh, 15 times the volume of deionized water is added, and the mixture is extracted at 90℃ for 3 hours. The first extract is collected by filtration, and the residue is added to 10 times the volume of deionized water. The mixture is extracted at 90℃ for 3 hours, and the second extract is collected by filtration. The two extracts are combined and concentrated under reduced pressure at 60-70℃ to a relative density of 1.2 (measured at 25℃) to obtain the extract for later use. Raw milk with a fat content of 3.5% is selected as the base for dairy products to ensure a rich milky aroma and balanced nutrition. The fermentation strain was a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus in a 1:1 mass ratio. (2) Use 80℃ ultra-high temperature instantaneous sterilization for 15s, dilute the extract to 30% mass concentration with sterile water, and adjust the pH to 6.5 with citric acid. Add the diluted extract at 5wt% to the sterilized milk cooled to 30-40℃ while stirring at a speed of 150r / min for 10min to obtain a mixture. Add 6 wt% sucrose to the above mixture, and then homogenize it under a pressure of 25 MPa. Homogenization is only required once to obtain a homogenized solution. (4) Low-temperature fermentation and activation stage: Inoculate 2wt% of mixed fermentation bacteria into the homogenized liquid, stir evenly, transfer the inoculated homogenized liquid into the fermentation tank, control the temperature at 35-38℃, ferment for 7 hours, and periodically check the pH value. When the pH drops to 4.2-4.5, immediately lower the temperature to below 10℃ to terminate the fermentation. (5) Finished product filling stage: Under aseptic conditions, the cooled dairy products are filled into aseptic yogurt cups, sealed and stored at 4°C to complete the preparation of the entire functional yogurt.

[0030] Effect verification Experimental subjects: Twenty-four 6-week-old male C57BL / 6J mice were selected. The normal group (n=6) was fed a standard diet; the model group (n=18) was fed a high-fat, high-sugar diet. After 4 weeks, all mice were fasted for 12 hours. Mice in the model group were injected intraperitoneally with 120 mg / kg streptozotocin (STZ), while mice in the normal group were simultaneously injected with an equal volume of citrate buffer. A fasting blood glucose level ≥11.1 mmol / L was used as the criterion for successful T2DM modeling. After successful modeling, the model group was divided into three groups: the control group (n=6) continued to be fed a high-fat, high-sugar diet; the metformin positive drug group (n=6) received 100 mg / kg metformin by gavage plus a high-fat diet; and the invention group (n=6) was fed a high-fat diet plus the dairy product from Example 1 (15 mL / kg / day). The intervention period was 12 weeks, and the mice's blood glucose and body weight were measured weekly. Experimental reagents and instruments Metformin hydrochloride tablets (0.5g×20 tablets, manufactured by Sino-American Shanghai Bristol-Myers Squibb Pharmaceutical Co., Ltd., National Drug Approval Number H20023370); Electronic balance (Sartorius GmbH, Germany); Blood glucose meter and matching blood glucose test strips (Bayer Healthcare GmbH, Germany); Low-temperature high-speed centrifuge (Thermo Fisher Scientific, USA); Optical microscope (OLYMPUS, Japan); Reagent kit (Tianjin Jiuding Medical Biotechnology Co., Ltd.); Specimen collection: After the last intervention, the mice were fasted for 8 hours and anesthetized with chloral hydrate. Blood was collected from the abdominal aorta and centrifuged to collect serum for later use. Pancreas, liver, colon and other tissues were collected from the mice. Half of the tissues were fixed in 4% formaldehyde and the other half were stored in a -80℃ refrigerator. HE staining of pancreatic, liver, and colon tissues: Ovaries fixed in 4% formaldehyde were dehydrated, embedded in paraffin blocks, and then cut into 5μm thick sections. Routine HE staining was performed to observe the pathological changes of pancreatic and liver tissues.

[0031] Serum glucose and lipid metabolism indicators were measured using radioimmunoassay: FBG (fasting blood glucose), FINS (fasting serum insulin), GSP (glycated serum protein), TC (total cholesterol), TG (triglycerides), HDL-C (high-density lipoprotein), and LDL-C (low-density lipoprotein). Impact analysis on gut microbiota characteristics: After collecting mouse fecal samples, they were transported at low temperature to Beijing Novogene Technology Co., Ltd. for preservation and subsequent analysis. The specific steps are as follows: Genomic DNA was extracted and diluted from fecal samples. Using the diluted genomic DNA as a template, the V3-V4 region was selected, and PCR amplification was performed using barcode-specific primers. The PCR products were detected by 2% agarose gel electrophoresis. Libraries were constructed using the TruSeq® DNA PCR Free Sample Preparation Kit. After the libraries passed quantification by Qubit and Q-PCR, they were sequenced using a NovaSeq 6000 sequencer. The raw sequencing data were spliced ​​and filtered to obtain valid data. Based on the valid data, α-diversity analysis and β-diversity analysis were performed. LEfSe and cluster heatmap analysis were used to obtain information on common and unique operational taxonomic units (OTUs) among different groups. Functional prediction analysis of the gut microbiota was performed.

[0032] Western blotting detection: 30 mg of partially frozen colon tissue was taken, and 300 μl of RIPA lysis buffer was added. After thorough homogenization, the mixture was centrifuged (3000 rpm, 10 minutes) and the supernatant was extracted to complete the extraction of liver and colon protein solutions. Protein concentration was determined using a BCA protein quantification kit, and the protein was denatured by heating in a 100℃ metal bath for 5 minutes. The protein loading volume per lane was uniformly 40 μg. Electrophoresis was started at 60 V, and after the protein entered the separating gel, the voltage was adjusted to 100 V until the protein reached the bottom of the separating gel. The protein was transferred to a PVDF membrane, washed three times with TBST solution for 10 minutes each time, blocked with 5% skim milk powder for 1 hour, and then incubated overnight at 4℃ with the appropriate primary antibody. The next day, the membrane was washed three times with TBST for 5 minutes each time, and then incubated at room temperature for 1 hour with the appropriate secondary antibody. ECL ultrasensitive luminescent solution was used for color development, and the membrane was exposed and developed in a gel imaging system. The antibodies used in the experiment (Claudin 1, catalog number GB152543, Servicebio; Occludin, catalog number GB111401, Servicebio; ZO1, catalog number GB115686, Servicebio; IRS2, catalog number F2167, Selleck; PI3 kinase, catalog number bs-10657R, Bioss; AKT1+2+3, catalog number bs-6951R, Bioss; GLUT4, catalog number A7637, Abclonal; ULK1, catalog number A8529, Abclonal; P62, catalog number GB11239-1019, Servicebio; mTOR, catalog number bs-1992R, Bioss; SIRT1, catalog number 3161-1-AP, Proteintech) were all used in accordance with the manufacturer's instructions. Tissue samples were observed and photographed under an optical microscope, and the experimental results were analyzed and statistically analyzed using ImageJ software. Statistical methods: Experimental data were processed using SPSS (version 20.0) software. Statistical results are presented in the form of mean ± SD. One-way ANOVA was used to compare groups. For groups with homogeneous variances, the t-test was used. For groups with heterogeneous variances, the Cochran & Cox t-test was used. A p-value < 0.05 was considered statistically significant.

[0033] Experimental results Improvement of pancreatic histopathological changes in type 2 diabetic mice H&E staining was used to study the pathological changes in mouse pancreatic tissue, such as... Figure 1As shown, from left to right, the mice are in the normal group, control group, yang drug group, and the present invention group. The islets of the normal group mice have a regular oval shape with clear boundaries and full cytoplasm. Compared with the normal group, the islets of the control group have blurred boundaries, and the cells are atrophied, deformed, and more severely damaged. Compared with the normal group, the islet cells in the yang drug group and the present invention group have increased in volume to varying degrees, the damage is alleviated, and the boundaries are significantly clearer. Modified liver histopathological changes in type 2 diabetic mice Liver H&E staining Figure 2 The data shows, from left to right, the normal group, the control group, the positive drug group, and the invention group. In the normal group, the mouse hepatocytes were normal in morphology, with clear nuclei, neat cell arrangement, and intact liver lobule structure, with no obvious pathological damage. Compared with the normal group, the control group showed swollen and deformed hepatocytes, disordered liver lobule structure, and visible fat vacuoles in the cells, indicating significant tissue damage. Compared with the control group, the positive drug group and the invention group showed reduced hepatocyte swelling, fewer fat vacuoles, liver lobule structure tending to be normal, gradual restoration of regularity in the morphology of intrinsic hepatocytes, and effective repair of tissue damage. Improvement of colonic histopathological changes in type 2 diabetic mice H&E staining results of colon tissue as follows Figure 3 As shown, from left to right, the mice are in the normal group, control group, yang drug group, and the present invention group. The colon cells of the mice in the normal group have normal morphology and are arranged regularly and orderly, and no obvious abnormal damage was observed. Compared with the normal group, the colon tissue of the control group was damaged and the cell morphology was deformed. Compared with the control group, the colon tissue of the yang drug group and the present invention group showed that the morphology of the intestinal gland cells in the lamina propria was relatively regular and the tissue damage was significantly repaired. Regulation of changes in glucose and lipid metabolism in type 2 diabetic mice Figure 4As shown, after 12 weeks of intervention, the body weight and serum biochemical indicators of mice were measured. Note: CON12: normal group; DM12: control group; MET12: metformin group; FF12: the present invention group; where AH represents the levels of fasting blood glucose (FBG), body weight, fasting insulin (FINS), glycated serum protein (GSP), total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in mice, respectively. The results showed that compared with the normal group, the levels of fasting blood glucose (FBG), body weight, fasting insulin (FINS), glycated serum protein (GSP), total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the control group were significantly increased (P<0.05). Compared with the control group, the levels of FBG, body weight, GSP, TG, and LDL-C in the positive drug group and the present invention group were significantly decreased (P<0.05), and the HDL-C level was higher than that in the control group. The above results indicate that the present invention can regulate the disorder of glucose and lipid metabolism in T2DM mice. Regulation of gut microbiota changes in type 2 diabetic mice High-throughput sequencing of 16S rDNA from mouse fecal tissue was performed strictly according to the operating procedure, and the results are as described above. Figure 5 As shown, note: control: normal group; T2DM: control group; MET: metformin group; SJ: invention group; in the figure, red blocks represent the abundance of the genus in the sample that is higher than in other samples, and blue blocks represent the abundance of the genus in the sample that is lower than in other samples; the genus-level heatmap changes of 16S rDNA high-throughput sequencing of mouse fecal tissue in each group, from Figure 5 As can be seen from this, the present invention alters the gut microbiota changes in type 2 diabetes, with a significant increase in some beneficial species, such as Lactobacillus, Butyricicoccus, and Lachnospira. Regulation of the mTOR-autophagy pathway in type 2 diabetic mice Figure 6 As shown, to further clarify the effect of the present invention on the mTOR signaling pathway in the colon tissue of type 2 diabetic mice, the expression of mTOR, ULK1, P62, and SIRT1 in the mTOR signaling pathway was detected by Western blotting. The results showed that compared with the normal group, the expression of mTOR and P62 in the colon of the control group mice was significantly increased, while the expression of SIRT1 and ULK1 was significantly decreased. Compared with the control group, the expression of mTOR and P62 in the colon of the present invention group and the positive drug group mice was significantly decreased, while the expression of SIRT1 and ULK1 was significantly increased, and the differences were statistically significant (P<0.01).

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A buckwheat and rhizoma polygonati composition for improving type 2 diabetes, characterized by, The composition comprises the following raw materials by weight: 30-40 parts of buckwheat, 20-30 parts of rhizoma polygonati, 15-25 parts of barley, and 10-20 parts of wheat bran.

2. The buckwheat and rhizoma polygonati composition for improving type 2 diabetes according to claim 1, wherein The composition is an extract.

3. The buckwheat and rhizoma polygonati composition for improving type 2 diabetes according to claim 2, characterized by, The preparation method of the extract comprises the following steps: after the raw materials are weighed according to the weight, the raw materials are crushed and uniformly mixed, deionized water is added for extraction and concentration.

4. The buckwheat and rhizoma polygonati composition for improving type 2 diabetes according to claim 2, wherein The extraction is performed twice, and the steps are as follows: 10-15 times of deionized water is added to the crushed raw materials, and the mixture is extracted at a constant temperature of 80-90 DEG C for 2-3 hours; the first extract is collected by filtration, and 8-10 times of deionized water is added to the residue; the mixture is extracted at a constant temperature of 80-90 DEG C for 2-3 hours; the second extract is collected by filtration; and the first extract and the second extract are combined.

5. The buckwheat and rhizoma polygonati composition for improving type 2 diabetes according to claim 3, wherein The crushed raw materials have a mesh number of 80-100, and the concentration is performed at 60-70 DEG C under reduced pressure until the relative density is 1.1-1.

2.

6. Use of the buckwheat rhizoma polygonati composition according to any one of claims 1-5 in the preparation of a dairy product.

7. Use according to claim 6, characterized in that, The preparation method of the dairy product comprises the following steps: (1) the extract of the buckwheat rhizoma polygonati composition is diluted and then added to sterilized cow milk to obtain a mixture, and then auxiliary materials are added to the mixture to obtain a homogenate; (2) a fermentation strain is added to the homogenate to perform fermentation, and the dairy product is obtained after rapid cooling after the fermentation is completed.

8. Use according to claim 7, characterized in that, In step (1), the cow milk is raw cow milk with a fat content of 3.0-3.5%; In the dilution step, the extract is diluted with sterile water to a concentration of 20-30%, and the pH is adjusted to 6.5-7.0 by adding citric acid or sodium bicarbonate; After dilution, the extract is added in an amount of 5-8% of the mass of the cow milk; The mixing is performed at 30-40 DEG C under stirring at 100-150 r / min for 10-15 min; The sterilization is performed by using ultra-high temperature instant sterilization at 75-80 DEG C for 15-20 s; The auxiliary material is sucrose, and the sucrose is added in an amount of 5-7% of the mass of the mixture; The homogenization is performed at a pressure of 25 MPa.

9. Use according to claim 7, characterized in that, In step (2), the fermentation strain is Lactobacillus bulgaricus and Streptococcus thermophilus, and the inoculation amount is 2-3% of the mass of the homogenate; The fermentation is performed at 35-38 DEG C for 5-7 h; The rapid cooling is performed by reducing the temperature of the yogurt from the fermentation temperature to below 10 DEG C within 15 min after the fermentation reaches the end point, and the cooling rate is ≥1.8 DEG C / min.

10. Use according to claim 9, characterized in that, The mass ratio of the Lactobacillus bulgaricus to the Streptococcus thermophilus is 1:1.

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