A kind of inulin fermentation product for reducing blood sugar and its preparation and application

Abstract

The present invention provides a blood sugar lowering inulin fermentation product and its preparation and application, belonging to the field of microbial fermentation technology. The blood sugar lowering inulin fermentation product is obtained by fermenting inulin with a specific fermentation bacterium, Schleiferilactobacillus harbinensis JNDM, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 11, 2023, with a deposit number of GDMCC No. 64143. The inulin fermentation product can significantly enhance the blood sugar lowering effect of inulin, reduce complications of hyperglycemia, improve anti-inflammatory function, and effectively relieve pancreatic and renal tissue lesions; it can regulate the homeostasis of intestinal flora, and significantly increase the diversity of intestinal microorganisms and the content of beneficial metabolites in the intestine. It can be used as a food, health product, medicine or functional beverage for preventing and treating type 2 diabetes and its complications or for lowering blood sugar.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to an inulin fermentation product capable of lowering blood sugar, and a preparation method and application thereof. Background Art

[0002] Diabetes is a common endocrine and metabolic disorder characterized by persistent hyperglycemia. Typical diabetic patients show symptoms of polydipsia, polyuria, polyphagia, and weight loss. Clinically, diabetes is divided into two categories: type 1 diabetes, an autoimmune disease with absolute insulin deficiency; and type 2 diabetes, caused by relative insulin deficiency or insulin resistance. According to the latest report of the International Diabetes Federation, there are about 463 million adult diabetics in the world, and it is expected to reach 578 million by 2030 and 702 million by 2045. Long-term high blood sugar can damage body organs and cause chronic complications such as diabetic nephropathy, retinopathy, and peripheral neuropathy. It seriously threatens human life and health and imposes a huge economic burden on society and patients.

[0003] At present, the treatment for diabetic patients is mainly to use oral hypoglycemic drugs alone or in combination to achieve control of hyperglycemia. However, most hypoglycemic drugs are expensive, and long-term use may cause adverse effects such as severe hypoglycemia, lactic acidosis, liver damage, permanent neurological defects, digestive discomfort, headache, dizziness and even death. Probiotic fermentation products are more of a dietary regulation effect, and there are fewer products with better therapeutic effects. The Chinese invention patent with publication number CN114196562A discloses a strain of Bacillus coagulans with blood sugar lowering effect and its application, and it is found that Bacillus coagulans can treat hyperglycemia. The Chinese invention patent with publication number CN109363023A discloses a probiotic beverage with blood sugar lowering function and a preparation method thereof, which is prepared by mixed fermentation of plant lactobacillus and multiple substances such as vitamins, lactose, inulin, etc., to obtain a composition with blood sugar lowering function. With the development and development of probiotics related to hypoglycemia, it is necessary to develop more probiotic fermentation products that can achieve blood sugar lowering effect and can prevent and treat its complications. Summary of the invention

[0004] The purpose of the present invention is to solve the above technical problems and provide an inulin fermentation product that can effectively lower blood sugar; another purpose of the present invention is to provide a method for preparing the blood sugar-lowering inulin fermentation product; a further purpose of the present invention is to provide the use of the blood sugar-lowering inulin fermentation product in the preparation of blood sugar-lowering drugs.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A blood sugar-lowering inulin fermentation product is obtained by fermenting inulin with a fermentation bacterium, wherein the fermentation bacterium is Harbin Schleifer Lactobacillus (Schleiferilactobacillus harbinensis), named JNDM, and the Harbin Schleifer Lactobacillus JNDM has been preserved in the Guangdong Provincial Microbiological Culture Collection Center GDMCC on December 11, 2023. The preservation address is: 5th Floor, Compound Experimental Building (Building 59), No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the preservation number is: GDMCC No. 64143.

[0007] The present invention discovers and preserves a Harbin Schleifer Lactobacillus JNDM. The fermented product obtained by fermenting Harbin Schleifer Lactobacillus with inulin as a raw material can produce a significant blood sugar lowering effect, and can significantly reduce complications of hyperglycemia and improve insulin inhibition; at the same time, the fermented product has an anti-inflammatory function, can reduce the level of pro-inflammatory factors such as TNF-a in tissues, and significantly increase the IL-10 content; and effectively alleviates pancreatic and kidney tissue lesions; at the same time, the inulin fermented product can significantly regulate the intestinal flora homeostasis of diabetic mice, improve the diversity of intestinal microorganisms, and increase the content of various short-chain fatty acids in the intestine, and can be used to prepare blood sugar lowering drugs.

[0008] Furthermore, the present invention provides a blood sugar-lowering inulin fermentation product, which is obtained by fermenting the following raw materials in parts by weight with the above-mentioned fermentation bacteria:

[0009] Inulin 30-60;

[0010] Sucrose 10-20;

[0011] Yeast extract 5-15;

[0012] Lactate 10-20.

[0013] Furthermore, the lactate is selected from calcium lactate, magnesium lactate, sodium lactate, and potassium lactate.

[0014] The present invention also provides a method for preparing the above-mentioned inulin fermentation product, comprising the following steps:

[0015] S1. Mixing the above raw materials and water to form a mixed solution;

[0016] S2. The above-mentioned fermentation bacteria are inoculated into the mixed solution, and anaerobic fermentation is carried out at 33-37° C. for 3-7 days to obtain inulin fermentation product.

[0017] Furthermore, the weight ratio of the raw material to water in the mixed solution in step S1 is 1:15-30.

[0018] Furthermore, the inoculation amount of the fermentation bacteria in step S2 is 0.05-2% of the weight of the fermentation raw material.

[0019] Furthermore, the mixed solution in step S2 needs to be sterilized before inoculating the fermentation bacteria.

[0020] Furthermore, in step S2, the anaerobic fermentation needs to stir the mixed aqueous solution every 12 hours.

[0021] Furthermore, after the anaerobic fermentation in step S2 is completed, the fermentation bacteria and sediment need to be removed.

[0022] Preferably, the removal of fermentation bacteria is performed by high temperature instantaneous sterilization or filtration sterilization.

[0023] The present invention claims to protect the use of the above-mentioned inulin fermentation product for reducing blood sugar in the preparation of medicines for preventing and treating type 2 diabetes and its complications.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) The present invention uses inulin rich in dietary fiber as a fermentation raw material, and uses the newly discovered strain Harbin Schleifer Lactobacillus to perform anaerobic fermentation on it to prepare an inulin fermentation product with hypoglycemic function. The inulin fermentation product of the present invention contains a variety of beneficial metabolites such as organic acids, oligosaccharides, and amino acids, which can significantly reduce the blood sugar level of type 2 diabetic mice and improve their insulin suppression.

[0026] (2) This inulin fermentation product has anti-inflammatory function, can reduce the level of pro-inflammatory factors such as TNF-a in tissues, and significantly increase the content of IL-10; it can effectively alleviate pancreatic and kidney tissue lesions.

[0027] (3) The inulin fermentation product regulated the intestinal flora homeostasis of diabetic mice, significantly improved the intestinal microbial diversity, and increased the content of various short-chain fatty acids in the intestine.

[0028] (4) The inulin fermentation product can be used as a drug with good blood sugar lowering effect in preventing or treating type 2 diabetes and its complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a photo of the colony streak isolation culture medium.

[0030] Figure 2 It is the graph of blood glucose changes in each group of mice.

[0031] Figure 3 are the pathological photos of mice in each group;

[0032] Figure 3 In the figure, a represents the kidney pathological photos of mice in each group, and b represents the pancreatic pathological photos of mice in each group.

[0033] Figure 4 It is the result diagram of urine index determination of each group of mice. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0035] Inulin and yeast extract were purchased from Shanghai Yuxin Biotechnology Co., Ltd., and lactate was purchased from Jiangsu Duoyang Bioengineering Technology Co., Ltd.

[0036] Example 1 Isolation, screening and identification of Harbin Lactobacillus Schleifer JNDM

[0037] 1. Experimental Methods

[0038] (1) Discovery separation and screening steps:

[0039] 1) Take 1 g of the collected fermented kimchi sample, inoculate it into 200 mL of MRS liquid culture medium under sterile conditions, and culture it statically in an anaerobic incubator at 37° C. for 48 h.

[0040] 2) Dilute the mixed bacterial solution after culture to an appropriate gradient, spread it on MRS solid culture medium, place it in an anaerobic incubator at 37°C and culture it for 24 hours. When there is obvious colony growth, pick a single bacterium and streak it on the MRS solid culture medium for isolation and culture. The culture conditions are consistent.

[0041] 3) When obvious colonies grow on the streaked plate, pick a single bacterium and inoculate it into MRS liquid culture medium. After incubation at 37°C in an anaerobic incubator for 48 hours, collect the bacteria by centrifugation and extract the bacterial DNA for strain identification.

[0042] The above culture medium formula is as follows:

[0043] MRS solid medium: 10g tryptone; 5g yeast powder; 10g beef extract; 20g glucose; 2g diamine hydrogen citrate; 5g sodium acetate; 2.6g K2HPO4·3H2O; 0.2g MgSO4·7H2O; 0.05g MnSO4·4H2O; 1mL Tween 80; 1.5-2% agar powder. Adjust pH to 6.8±0.2; make up to 1L, and sterilize at 121℃ for 15min.

[0044] MRS liquid culture medium: tryptone 10g; yeast powder 5g; beef extract 10g; glucose 20g; diamine hydrogen citrate 2g; sodium acetate 5g; K2HPO4·3H2O 2.6g; MgSO4·7H2O 0.2g; MnSO4·4H2O 0.05g; Tween 80 1mL. Adjust pH to 6.8±0.2; make up to 1L, and sterilize at 121℃ for 15min.

[0045] (2) Identification of bacterial species

[0046] Identification method: 16S rDNA sequencing, the specific operation is: collect the cultured bacteria, use the DNA extraction kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) to extract DNA, perform PCR amplification after extraction, and finally send it to Shanghai Biotechnology for sequencing analysis. The PCR primers are universal primers: P0 (27F), P6 (1492R), the amplification system is shown in Table 1, and the amplification steps and parameter conditions are shown in Table 2.

[0047] Table 1 16S sequencing PCR amplification system

[0048] Reagents 25μL reaction system Taq enzyme 12.5μL Upstream primer (p0) 1μL Downstream primer (p6) 1μL DNA 1μL <![CDATA[ddH2O]]> Make up to 25 μL with water

[0049] Table 2 16S sequencing PCR amplification steps and parameter conditions

[0050]

[0051] 2. Experimental results

[0052] Photo of the culture medium for colony streaking isolation Figure 1 As shown by Figure 1It can be seen that the Harbin Shleifer Lactobacillus JNDM is a milky white colony, which is opaque. The Harbin Shleifer Lactobacillus JNDM can be cultured anaerobically in MRS medium. Using the universal primers for PCR amplification of the 16SrRNA gene as primers, PCR amplification was carried out to obtain an amplified band with a length of 1476bp. The 16s sequencing sequence is shown in SEQ ID NO.1, and the measured 16S rDNA sequence was compared and analyzed with the GenBank database using BLAST software, and finally the species of the bacteria was determined at the molecular level; it was determined to be Harbin Schleiferilactobacillus harbinensis after comparison with the NCBI database. This Harbin Schleiferilactobacillus harbinensis (Schleiferilactobacillus harbinensis) was named JNDM, and the Harbin Schleiferilactobacillus JNDM was deposited in the Guangdong Provincial Microbial Culture Collection Center GDMCC on December 11, 2023, and the preservation address is: 5th Floor, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the preservation number is: GDMCC No.64143.

[0053] Example 2

[0054] A method for preparing an inulin fermentation product having a blood sugar lowering function is as follows:

[0055] The fermentation equipment uses a 1000L fermentation tank for preparation, and the specific operation steps are as follows:

[0056] (1) Weigh 25 kg of inulin, 10 kg of sucrose, 5 kg of yeast extract, 2 kg of sodium lactate, 2 kg of magnesium lactate, 2 kg of potassium lactate, and 2 kg of calcium lactate and put them into a fermentation tank, add 800 L of pure water, stir until completely dissolved, and sterilize at 115°C for 30 min;

[0057] (2) After the liquid in the fermentation tank was cooled to room temperature, 0.5 kg of Harbin Schleifer Lactobacillus JNDM powder was added under sterile conditions, and the fermentation tank was sealed. The fermentation tank was maintained at 37° C. for anaerobic fermentation for 3 days, and the fermentation was stirred for 20 minutes every 12 hours.

[0058] (3) After fermentation, the fermentation broth was centrifuged at 5000 r / min to remove the precipitate, and the supernatant was filtered and sterilized to obtain the inulin fermentation product.

[0059] Example 3

[0060] A method for preparing an inulin fermentation product having a blood sugar lowering function is as follows:

[0061] The fermentation equipment uses a 1000L fermentation tank for preparation, and the specific operation steps are as follows:

[0062] (1) Weigh 20 kg of inulin, 8 kg of sucrose, 5 kg of yeast extract, 3 kg of sodium lactate, 2 kg of potassium lactate, and 1 kg of calcium lactate and put them into a fermentation tank. Add 850 L of pure water and stir until completely dissolved. Sterilize at 115°C for 30 min.

[0063] (2) After the liquid in the fermentation tank is cooled to room temperature, 0.3 kg of Harbin Schleifer Lactobacillus JNDM powder is added under sterile conditions, and the fermentation tank is sealed. The fermentation tank is maintained at 35° C. for anaerobic fermentation for 7 days, and the fermentation is stirred for 20 minutes every 12 hours.

[0064] (3) After fermentation, the fermentation broth was centrifuged at 5000 r / min to remove the precipitate, and the supernatant was filtered and sterilized to obtain the inulin fermentation product.

[0065] Example 4

[0066] A method for preparing an oral liquid medicine of inulin fermentation product with blood sugar lowering function is as follows:

[0067] The fermentation equipment uses a 1000L fermentation tank for preparation, and the specific operation steps are as follows:

[0068] (1) Weigh 30 kg of inulin, 6 kg of sucrose, 10 kg of yeast extract, 5 kg of sodium lactate, and 3 kg of calcium lactate and put them into a fermentation tank. Add 900 L of pure water and stir until completely dissolved. Sterilize at 115°C for 30 min.

[0069] (2) After the liquid in the fermentation tank was cooled to room temperature, 0.8 kg of Harbin Schleifer Lactobacillus JNDM powder was added under sterile conditions, and the fermentation tank was sealed. The fermentation tank was maintained at 33° C. for anaerobic fermentation for 5 days, and the fermentation was stirred for 20 minutes every 12 hours.

[0070] (3) After the fermentation is completed, the fermentation liquid is centrifuged at 5000 r / min to remove the precipitate, and the supernatant is sterilized at high temperature to obtain the inulin fermentation product.

[0071] Example 5

[0072] A method for preparing an inulin fermentation product having a blood sugar lowering function is as follows:

[0073] The fermentation equipment uses a 1000L fermentation tank for preparation, and the specific operation steps are as follows:

[0074] (1) Weigh 60 kg of inulin, 20 kg of sucrose, 15 kg of yeast extract, 10 kg of sodium lactate, and 10 kg of calcium lactate and put them into a fermentation tank. Add 900 L of pure water and stir until completely dissolved. Sterilize at 115°C for 30 min.

[0075] (2) After the liquid in the fermentation tank was cooled to room temperature, 0.8 kg of Harbin Schleifer Lactobacillus JNDM powder was added under sterile conditions, and the fermentation tank was sealed. The fermentation tank was maintained at 33° C. for anaerobic fermentation for 7 days, and the fermentation was stirred for 20 minutes every 12 hours.

[0076] (3) After the fermentation is completed, the fermentation liquid is centrifuged at 5000 r / min to remove the precipitate, and the supernatant is sterilized at high temperature to obtain the inulin fermentation product.

[0077] Comparative Example 1

[0078] The preparation method refers to the steps of Example 1, the only difference being that inulin is not contained, and other components and preparation steps remain unchanged to prepare a fermentation product.

[0079] Example 6

[0080] The preparation method is the same as that in Example 1, except that only the amount of inulin is changed to 80 kg, and the other components and preparation steps remain unchanged to obtain the inulin fermentation product.

[0081] Comparative Example 2

[0082] The preparation method is similar to the steps in Example 1, except that the fermentation bacteria is commercially available (purchased from Ningbo Mingzhou Biotechnology Co., Ltd.) Harbin Schleifer lactobacillus (Schleiferilactobacillus harbinensis) to prepare the inulin fermentation product.

[0083] Experimental Example 1 Application in the treatment of type Ⅱ diabetes

[0084] 1. Experimental Grouping

[0085] Normal mice: db / m mice, 6 weeks old male, 6 mice.

[0086] Diabetic mice: db / db mice, 6 weeks old male, 60 mice.

[0087] db / db mice carry a mutation in the Leptin gene, which results in symptoms of type 2 diabetes. The db / db (C57BL / KsJ) mice have a point mutation in the Leptin receptor that causes a disorder in the leptin signaling pathway, which results in obesity, insulin resistance, hyperglycemia, fatty liver and other symptoms in the mice. Obvious obesity and increased fasting blood sugar, increased water intake and urine output can occur in mice 6 weeks after birth, which is most obvious at 8-12 weeks, and complications such as diabetic nephropathy can occur. db / db mice are usually used as the control group for experiments with db / m mice.

[0088] The mice were randomly divided into 6 groups, 6 mice in each group, including normal group, model group, control group, drug administration groups 1-3; drug administration group 4 (unfermented product raw material filtrate in Example 1); drug administration groups 5-8. The grouping and drug administration relationship are shown in Table 3.

[0089] Table 3 Grouping and administration of mice

[0090] Grouping drug Normal group Normal saline Model Group Normal saline Control group Metformin Treatment group 1 Product of Example 2 of the present invention Treatment group 2 Product of Example 3 of the present invention Treatment group 3 This product embodiment 4 product Treatment group 4 Unfermented product raw materials of the present invention Treatment group 5 Embodiment 5 of the present invention product Treatment group 6 Product of Example 6 of the present invention Treatment group 7 Comparative Example 1 product of the present invention Treatment group 8 Comparative Example 2 product of the present invention

[0091] The mice were adapted to the barrier environment for one week. In the 2nd to 9th week, the mice in the normal group and the model group were gavaged with 10 mL / kg sterile saline every day, the mice in the control group were gavaged with 100 mg / kg metformin every day, the treatment groups 1-3 and the treatment groups 5-6 were gavaged with 10 mL / kg inulin fermentation every day, the treatment group 4 was gavaged with an equal amount of unfermented product raw materials; the treatment groups 7-8 were gavaged with 10 mL / kg of the comparative example 1-2 product. During this period, the blood glucose changes of the mice were detected every week. After the end of the 9th week, the mice were anesthetized and sacrificed, and the serum, kidneys, pancreas, and feces of the mice were collected. The content of inflammatory factors in the serum of the mice, the pathological changes of the kidneys and pancreas, the content of short-chain fatty acids in the feces, and the changes in intestinal flora were detected.

[0092] 2. Experimental Methods

[0093] (1) Mouse blood glucose detection

[0094] After the treatment, the mice were fasted for 8 h, blood (50 μl) was collected from the tail using a blood collection needle, and the blood glucose level was measured using a blood glucose meter and recorded.

[0095] (2) Serum inflammatory factor detection

[0096] After the mice were anesthetized, blood was collected, the blood was kept at room temperature for 45 minutes, and centrifuged at 4°C, 3000 rpm / min for 15 minutes to obtain mouse serum. The ELISA enzyme-linked immunosorbent assay kit (purchased from Nanjing Jiancheng Bioengineering Institute) was used to detect the content of TNF-α, IL-1β, IL-6, and IL-10 inflammatory factors in mouse serum.

[0097] (3) HE staining analysis of mouse kidney and pancreatic tissue

[0098] Kidney and pancreatic tissues of mice were fixed in 4% paraformaldehyde for 24 hours, and then dehydrated by gradient ethanol (30%-100%), xylene clearing and wax immersion, paraffin embedded, tissue sectioned, and stained with hematoxylin-eosin (H&E). The staining results were observed under a microscope.

[0099] (4) Mouse urine assay

[0100] After 8 weeks of drug administration, mice in each group were placed in metabolic cages, and urine and feces of each mouse were collected. The 24-hour urine output of each mouse was recorded, and the urine protein, creatinine, and microalbumin content in the urine of the mice were measured using a detection kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0101] (5) Determination of changes in short-chain fatty acid content in mouse feces

[0102] Weigh 0.1 g of mouse feces collected from each group, add 0.9 mL of pure water to fully dissolve, centrifuge at 8000 rpm for 5 min, take the supernatant, remove the protein using potassium ferrocyanide and zinc sulfate solution, centrifuge at 12000 rpm for 2 min, take the supernatant, and filter through a 0.22 μm filter membrane to obtain the short-chain fatty acid test sample.

[0103] The HPLC method was used to determine the content of acetic acid, propionic acid, butyric acid, and valeric acid in the sample filtrate. The chromatographic column Waters XBridge TM C18 (4.6×250mm, 5μm) was heated to 35°C. The mobile phase was acetonitrile-water (30:70), and the pH of the mobile phase was adjusted to 3.0 using phosphoric acid. The sample injection volume was 10μL, the flow rate was 0.7mL·min-1, the detection UV wavelength was set to 210nm, and the single sample running time was set to 20min.

[0104] (6) Mouse intestinal microbial analysis

[0105] After the mice were killed and dissected, the cecum and its contents were collected into sterile EP tubes and frozen in liquid nitrogen. The quick-frozen samples were transferred to -80°C ultra-low temperature refrigerators for storage. The bacterial DNA in the cecum contents and mouse feces was extracted using a bacterial DNA rapid extraction kit, and the V3-V4 hypervariable region of the isolated bacterial DNA was amplified. The universal primer pair used for 16S rRNA analysis is as follows: primer 338F, whose sequence is shown in SEQ ID NO.2, specifically: 5'-ACTC CTACGGGAGGCAGCAG-3'; primer 806R, whose sequence is shown in SEQ ID NO.3, specifically: 5'-GGA CTACNNGGGTATCTAAT-3'. According to the standard procedures provided by the sequencing company, the PCR products of the V3-V4 gene sequence were electrophoresed, stained, purified and quantified. The purified polymerase chain reaction products were sequenced by BGI on the Illumina MiSe q platform. The DADA2 (Divisive Amplicon Denoising Algor ithm) method in the software QIIME2 was used for denoising to obtain Amplicon Sequence Variants (ASV), where ASV is a 100% similar sequence, and then a feature table was obtained. The comparison database was Silva V1382019-12-16, and the bioinformatics analysis was performed using R language (version number v3.4.1).

[0106] 3. Experimental results

[0107] (1) After treatment, the fasting blood glucose levels of mice in each group were tested. Figure 2 As shown, compared with the model group, the fasting blood glucose of diabetic mice was reduced in drug administration groups 1, 2, 3, 5, and 6, and the effect was better than that of the control group, while there was no obvious therapeutic effect in drug administration groups 4, 6, and 7. The above results show that the inulin fermentation products of Examples 2, 3, 4, 5, and 6 can effectively alleviate the increase of blood glucose in diabetic mice.

[0108] (2) The content of inflammatory factors in the serum of each group of mice was analyzed. The results are shown in Table 4. The content of TNF-α, IL-1β, and IL-6 in the serum of the model group mice increased, while the content of the anti-inflammatory factor IL-10 decreased. Compared with the model group, the content of pro-inflammatory factors in the administration groups 1, 2, 3, 5, and 6 was significantly downregulated, while the anti-inflammatory factors were significantly increased. This shows that the inulin fermentation products in Examples 2, 3, 4, 5, and 6 significantly improved the inflammatory response caused by diabetes.

[0109] Table 4 Contents of inflammatory factors in mouse serum

[0110]

[0111] (3) Pathological analysis of the kidneys and pancreas of mice in each group was performed. HE staining of kidney tissue was performed. Figure 3 As shown in (a), the model group, control group, and drug-treated groups 4, 7, and 8 all showed enlarged renal tubules, and the model group showed watery degeneration of renal tubular epithelial cells, while drug-treated groups 1, 2, 3, 5, and 6 showed significant improvement and no enlarged renal tubules were found. Figure 3 As shown in (b), no obvious pathological changes were observed in the pancreatic tissue of the normal group. The pancreas of the model group and the treatment groups 4, 7, and 8 showed swelling of the acinus volume and enlargement of the volume, and the number of islets decreased and the volume was small. The number of islets in the control group increased slightly and the volume was large, but the islet shape was not considered, and the cytoplasm of the islet cells was loose and lightly stained. The number of islets in the treatment groups 1, 2, 3, 5, and 6 increased and the volume was large, and no swelling of the acinus was observed. This indicates that the inulin fermentation products in Examples 2, 3, 4, 5, and 6 can alleviate the pathological damage of the kidney and pancreas caused by diabetes.

[0112] (4) Collect urine from each group of mice, record their 24-hour urine output, and analyze it. The results are as follows: Figure 4 As shown. The 24h urine volume of mice in each group was significantly different. All diabetic mice had significantly higher urine volume than normal mice. Compared with the model group, the urine volume of mice in drug administration groups 1, 2, 3, 5, and 6 decreased significantly, while there was no significant difference between drug administration groups 4, 7, and 8 and the model group. The 24h urine protein content and microalbumin showed that the urine protein content of diabetic mice increased, which may be caused by kidney damage in diabetic mice. The urine protein of drug administration groups 1, 2, 3, 5, and 6 decreased significantly, and the microalbumin in drug administration groups 1, 2, and 3 decreased significantly. The microalbumin in drug administration groups 5 and 6 also showed a downward trend. The determination of creatinine content in urine showed that it showed an upward trend in diabetic mice, while compared with the model group, drug administration groups 1, 2, 3, 5, and 6 showed a downward trend. The above shows that the inulin fermentation products in Examples 2, 3, 4, 5, and 6 can reduce the urine output of diabetic mice, and the results of urine protein, microalbumin, and creatinine determination show that the inulin fermentation products in Examples 2, 3, 4, 5, and 6 can reduce kidney damage in diabetic mice.

[0113] (5) After the treatment, the feces of the mice were collected to detect the changes in the content of short-chain fatty acids in the feces. The results are shown in Table 5. Compared with the model group, the treatment groups 1, 2, 3, 5, and 6 significantly increased the levels of acetic acid, propionic acid, butyric acid, and valeric acid, while there was no significant difference between the treatment group 4 and the control group, indicating that the inulin fermentation products in Examples 2, 3, 5, and 6 can increase the content of some short-chain fatty acids in diabetic mice.

[0114] Table 5 Changes of short-chain fatty acids in mouse feces

[0115]

[0116]

[0117] (6) In order to explore the effect of inulin fermentation on intestinal flora after administration, 16SrRNA sequencing was used to analyze the changes in microbial diversity in mouse feces. The ACE index, Chao1 index, and Shannon index were used to characterize the richness and diversity of mouse microbial intestinal tract, and the results are shown in Table 6. Compared with the model group, the richness and diversity of intestinal microorganisms in groups 1, 2, 3, 5, and 6 were significantly improved, and there was no significant difference between the administration groups 4, 7, and 8 and the model group. It shows that the inulin fermentation in Examples 2, 3, 5, and 6 can significantly increase the diversity of intestinal microorganisms in diabetic mice, thereby alleviating the symptoms of type 2 diabetes.

[0118] Table 6 Diversity of mouse intestinal flora

[0119]

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the solution. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents based on the understanding of the present solution without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A blood sugar lowering inulin fermentation product, characterized in that: Obtained by fermenting the following raw materials with fermentation bacteria; The raw materials are calculated by weight: Inulin 20-60; Sucrose 10-20; Yeast extract 5-15; Lactate 10-20; The inulin fermentation product is prepared by the following method: S1. mixing the raw material and water to form a mixed solution; S2. Inoculate the fermentation bacteria into the mixed solution, and perform anaerobic fermentation at 33-37°C for 3-7 days to obtain inulin fermentation product; The fermentation bacteria is Schleiferilactobacillus harbinensis, named JNDM. The Harbin Schleiferilactobacillus JNDM was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 11, 2023, and the preservation number is: GDMCC No.64143.

2. The inulin fermentation product according to claim 1, characterized in that The lactate is selected from calcium lactate, magnesium lactate, sodium lactate and potassium lactate.

3. The inulin fermentation product according to claim 1, characterized in that The weight ratio of the raw material to water in the mixed solution in step S1 is 1:15-30.

4. The inulin fermentation product according to claim 1, characterized in that The inoculation amount of the fermentation bacteria in step S2 is 0.05-2% of the weight of the fermentation raw material.

5. The inulin fermentation product according to claim 1, characterized in that The mixed solution in step S2 needs to be sterilized before inoculating the fermentation bacteria.

6. The inulin fermentation product according to claim 1, characterized in that The anaerobic fermentation in step S2 requires stirring the mixed aqueous solution every 12 hours.

7. The inulin fermentation product according to claim 1, characterized in that After the anaerobic fermentation in step S2 is completed, the fermentation bacteria and sediment need to be removed.

8. Use of the inulin fermentation product according to any one of claims 1 to 7 in the preparation of medicines for treating type 2 diabetes or having a blood sugar lowering effect.

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