Wheat bran araboxylan and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to wheat bran araboxylan and application thereof. The invention provides wheat bran AX with specific molecular weight and monosaccharide composition. The wheat bran AX comprises the following monosaccharides: 0.20%-0.22% of glucuronic acid, 0.58%-0.62% of galacturonic acid, 0.21%-0.23% of rhamnose, 0.6%-0.7% of glucose, 1.4%-1.5% of galactose, 18%-19.2% of xylose and 21%-23% of arabinose. Compared with commercially available wheat bran AX, inulin, fructo-oligosaccharide and other common dietary fibers, the self-made wheat bran AX has higher diversity of microbial communities in the fermentation process, has a better intestinal flora regulating effect, further has a good relieving effect on the abnormal glucose and lipid metabolism of the body, and can be used for preventing and treating the abnormal glucose and lipid metabolism of the body. Therefore, the traditional Chinese medicine composition has a good regulation effect on various diseases such as non-alcoholic fatty liver and the like caused by abnormal glucose and lipid metabolism of the organism.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a wheat bran arabinoxylan and its applications, particularly the application of this wheat bran arabinoxylan in the preparation of products for alleviating and / or treating diseases caused by abnormal glucose and lipid metabolism. Background Technology

[0002] Arabicaxylan (AX) is an important functional hemicellulose found in the cell walls of cereal bran and endosperm, and is widely distributed in grain crops such as wheat, oats, rice, and sorghum. Numerous studies have shown that AX, as a dietary fiber, exhibits excellent effects in regulating blood sugar, lowering blood lipids, anti-oxidation, promoting intestinal motility, anti-tumor activity, and enhancing immune activity.

[0003] However, AX (oxo-assortant) varies significantly in structure and the types of monosaccharides it contains due to different sources and preparation methods, resulting in substantial differences in their physiological effects. For example, AX obtained by treating wheat bran with a weak alkali followed by alkali extraction and acid precipitation in patent CN114874356A has a molar percentage of arabinose and xylose of 46.09% and 53.91%, respectively. AX prepared by a two-stage enzymatic hydrolysis method in patent CN111574640A contains three monosaccharide components: arabinoxylan, xylose, and galactose. Patent CN117264090A uses corn husks as raw material and employs an enzymatic hydrolysis method to prepare an AX containing monosaccharide components such as xylose, arabinose, glucose, galactose, rhamnose, mannose, glucuronic acid, galacturonic acid, and fucose.

[0004] In recent years, with the improvement of people's living standards and changes in lifestyle, the number of people with diabetes and non-alcoholic fatty liver disease has been increasing year by year. Although there have been reports on the role of AX in alleviating diabetes and non-alcoholic fatty liver disease, the effects of different types of AX vary significantly. Therefore, it is of great significance for people with diabetes and non-alcoholic fatty liver disease to find a better alternative to AX. This will not only help improve their metabolic status and alleviate the progression of the disease, but may also open up new avenues for the treatment and management of related diseases. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a wheat bran arabinoxylan and its applications, particularly relating to the use of this wheat bran arabinoxylan in the preparation of products for alleviating and / or treating diseases caused by abnormal glucose and lipid metabolism.

[0006] The first aspect of the present invention is to provide a wheat bran arabinoxylan with a weight-average molar mass of 3.86 × 10⁻⁶. 6 ±7.21×10 4The wheat bran arabinoxylan contains the following monosaccharides, in g / mol and by mass percentage: Glucuronic acid 0.20%-0.22%, galacturonic acid 0.58%-0.62%, rhamnose 0.21%-0.23%, glucose 0.6%-0.7%, galactose 1.4%-1.5%, xylose 18%-19.2%, arabinose 21%-23%.

[0007] Preferably, the wheat bran arabinoxylan contains the following monosaccharides: Glucuronic acid 0.21%, galacturonic acid 0.60%, rhamnose 0.22%, glucose 0.66%, galactose 1.46%, xylose 18.86%, arabinose 22.25%.

[0008] A second aspect of the present invention is the use of the wheat bran arabinoxylan in the preparation of products for alleviating and / or treating diseases caused by abnormal glucose and lipid metabolism, specifically, the use of the wheat bran arabinoxylan being added to products for alleviating and / or treating diseases caused by abnormal glucose and lipid metabolism.

[0009] Preferably, the wheat bran is black wheat bran.

[0010] Preferably, the diseases include, but are not limited to, the following: diabetes, cardiovascular disease, non-alcoholic fatty liver disease, obesity, and kidney disease.

[0011] Preferably, the wheat bran arabinoxylan works in the product by regulating the imbalance of the intestinal flora.

[0012] Preferably, the product is selected from food and pharmaceuticals, and the product includes, but is not limited to, any form of solid tablets, solid granules, liquids, or sprays. That is, when the product is a pharmaceutical, the form of the pharmaceutical can be any of solid tablets, solid granules, liquids, or sprays.

[0013] When the product is a food product, it includes compressed candies, compressed biscuits, effervescent tablets, instant granules, beverages, sauces, functional drinks, and preservative sprays containing the aforementioned wheat bran arabinoxylan.

[0014] The beneficial effects of this invention are as follows: This invention provides a wheat bran AX with a specific molecular weight and monosaccharide composition. Compared with commercially available AX and common dietary fibers such as inulin and fructooligosaccharides, it produces a higher diversity of microbial communities during fermentation, has a better intestinal flora regulation effect, and thus has a good alleviating effect on abnormal glucose and lipid metabolism in the body.

[0015] Furthermore, a non-alcoholic fatty liver disease (NAFLD) model was established by inducing a high-fat diet. The wheat bran AX provided in this invention significantly improved the liver structure and tissue lesions caused by a high-fat diet in mice, and regulated the serum and liver biochemical indicators of mice. Differential analysis of intestinal metabolites in mice was performed using fecal metabolomics technology. The study showed that, compared with the model group, wheat bran AX may regulate lipid metabolism by participating in signaling pathways such as arginine and proline metabolism, thereby showing a good alleviating effect on NAFLD. Attached Figure Description

[0016] Figure 1 The results of in vitro fermentation experiments on different types of dietary fiber in healthy and diabetic patients are shown in the figure. Figure 2 The results of analysis of OTU and Hcluster in samples from different carbon sources after 24 h of in vitro fermentation in healthy individuals and diabetic patients. Figure 3 The differences in the levels of gut microbiota phylum and genus in fermentation broth of healthy individuals and diabetic patients; Figure 4 PCA and PLS-DA score plots for fermentation broth samples from healthy individuals and diabetic patients; Figure 5 The effect of various dietary fibers on the metabolism of organic acids in fermentation broth of healthy individuals is shown in the figure. Figure 6 The effect of various dietary fibers on the metabolism of organic acids in the fermentation broth of diabetic patients is shown in the figure. Figure 7 Correlation analysis of fecal microbiota and metabolites in fermented samples from healthy individuals and diabetic patients; Figure 8 Changes in mouse body weight and food intake; Figure 9 The effect of wheat bran AX on mouse liver morphology; Figure 10 The effect of wheat bran AX on the morphology of mouse liver tissue; Figure 11 The effect of wheat bran AX on the morphology of mouse colon tissue; Figure 12 The effect of wheat bran AX on the morphology of mouse jejunal tissue; Figure 13 The regulation of serum and liver biochemical indicators by wheat bran AX in mice. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0018] Example 1 A type of wheat bran AX, with a number-average molar mass Mn =1.77×10 6 ±1.56×10 4 g / mol, weight-average molar mass M w =3.86×10 6 ±7.21×10 4 g / mol, dispersion coefficient M w / M n =2.19±0.02, and by mass percentage, the wheat bran AX contains the following monosaccharides: Glucuronic acid 0.21%, galacturonic acid 0.60%, rhamnose 0.22%, glucose 0.66%, galactose 1.46%, xylose 18.86%, arabinose 22.25%.

[0019] Since the general structure of wheat bran AX is composed of a linear β-(1→4) linked xylan backbone, and α-l-arabinuronic acid units are linked to the xylan backbone as side residues through α-(1→3) and α-(1→2) bonds, the degree of branching of wheat bran AX in this invention is represented by the Ara / Xyl (A / X) value, A / X=1.18.

[0020] The wheat bran AX provided in this embodiment is prepared using the following method: (1) Add 100 g of wheat bran to a 2 L beaker and autoclave at 121℃ for 15 min to destroy the activity of endogenous cell wall degrading enzymes (such as endogenous arabinoxylanase). Then add high-temperature α-amylase (7.5 g) and distilled water (1000 mL) in proportion. Incubate in a water bath at 95℃ for 2 h, stirring constantly during the process to reduce the starch content in the wheat bran. After the temperature drops to room temperature, adjust the pH to 7.5 with NaOH solution (1 mol / L). Add neutral protease (1.05 g) in proportion. Incubate in a water bath at 60℃ for 2 h, stirring constantly during the process to remove the protein in the bran. Filter the suspension with gauze and wash the residue with distilled water until the filtered liquid gradually becomes clear. Dry it overnight in a 60℃ oven until the mass remains constant to obtain starch- and protein-free wheat bran. (2) Steam explosion treatment was carried out on the destarched and deproteinized wheat bran (the steam explosion pressure was 0.8 MPa and the pressure holding time was 160 s). (3) Take 1 g of steam-exploded wheat bran powder, add 20 mL of 0.25 mol / L NaOH solution, and shake in a water bath at 85℃ and 200 r / min for 2 h. After cooling to room temperature, centrifuge at 4000 r / min for 15 min, retain the supernatant, repeat twice, and combine the supernatants. Then, adjust the pH value to 4.3 with 1 mol / L hydrochloric acid, centrifuge at 4000 r / min for 15 min to further precipitate the hemicellulose component that may be contained therein. Add anhydrous ethanol equivalent to three times its volume to the supernatant to precipitate wheat bran AX, so that the final volume fraction of the ethanol solution reaches 75%, and let it stand overnight at 4℃. After the ethanol precipitation, discard the supernatant, redissolve the precipitate in water, freeze dry, and obtain the wheat bran AX.

[0021] Example 2 Study on the hypoglycemic effect of wheat bran AX prepared in Example 1 of this invention.

[0022] 2.1 Feces from diabetic patients and healthy individuals were collected to construct in vitro fermentation models. The hypoglycemic effect of the prepared wheat bran AX was verified by using the wheat bran AX prepared in Example 1, commercially available wheat bran AX, inulin, and fructooligosaccharides as fermentation carbon sources.

[0023] The experimental results are attached. Figure 1 As shown.

[0024] The results showed that after 24 hours of fermentation using fecal microbial broth from different populations, several dietary fibers were beneficial in reducing the pH value of the colonic environment and improving intestinal health compared to the blank control group. However, the butyric acid content was highest in the fermentation broth of the healthy group and the diabetic group, which used the self-made AX as a carbon source in Example 1, at 2.09 mmol / L and 0.46 mmol / L, respectively. Moreover, the self-made AX had the lowest reducing sugar utilization rate during the fermentation process compared to other dietary fibers.

[0025] 2.2 Effects of different dietary fibers on gut microbiota during in vitro fermentation Appendix Figure 2 The results of OTU analysis in samples from different carbon sources after 24 h of fermentation in healthy individuals (A) and diabetic individuals (B), and the results of Hcluster analysis in samples from different carbon sources after 24 h of fermentation in healthy individuals (C) and diabetic individuals (D); where group H represents healthy individuals, H1: blank; H2: wheat bran AX (homemade); H3: wheat bran AX (commercially available); H4: inulin; H5: fructooligosaccharides; group D represents diabetic individuals, D1: blank; D2: wheat bran AX (homemade); D3: wheat bran AX (commercially available); D4: inulin; D5: fructooligosaccharides (the same below).

[0026] Figure 2The results showed that in the fermentation broth fermented with fecal microbial fluid from healthy individuals and diabetic patients, the Shannon index of each dietary fiber group was significantly lower than that of the control group. Among them, the microbial community diversity of H2 and D2 was significantly higher than that of the other dietary fibers.

[0027] Furthermore, the microbial communities of H4 and H5 in group H show certain similarities, the microbial communities of D1 and D2 in group D show certain similarities, and the microbial communities of D4 and D5 show certain similarities. This is consistent with the metabolic dataset, indicating that there is a correlation between microorganisms and metabolites.

[0028] Appendix Figure 3 The differences in the level of gut microbiota phylum in the fermentation broth of healthy individuals (A) and diabetic patients (B), and the differences in the level of gut microbiota genus in the fermentation broth of healthy individuals (C) and diabetic patients (D).

[0029] Figure 3 As can be seen, at the phylum level, Proteobacteria ( Proteobacteria Bacteroidetes ( Bacteroidota Firmicutes ( Firmicutes ), Actinobacteria ( Actinobacteriota These bacteria (including Fusobacteria) dominated the gut microbiota composition, accounting for over 95% of the gut microbiota in all groups. In group H, compared to the control group, several dietary fiber treatments altered the distribution of the microbial community at the phylum level to varying degrees. Compared to H1, dietary fibers H2, H3, H4, and H5 significantly reduced the number of Fusobacteria (including Fusobacteria). Fusobacteriota The relative abundance of beneficial bacteria (including Firmicutes) was significantly increased in group D compared to the control group. In group D, the addition of D2 and D3 significantly increased the relative abundance of Bacteroidetes, while D4 and D5 significantly increased the relative abundance of Proteobacteria. Compared to other dietary fibers, group D2 showed a higher relative abundance of beneficial bacteria (including Firmicutes). Firmicutes The proportion of butyric acid was the largest, which was also positively correlated with the production of butyric acid in the tested fermentation broth.

[0030] In addition, at the genus level, in group H, compared with the control group, several dietary fibers significantly downregulated harmful bacteria. Fusobacteriota and Norank_f_Lachnospiraceae The relative abundance of probiotics Lactobacillus (D2) was significantly upregulated in group D compared to other dietary fiber groups. Lactobacillus ) and Parabacterium genus ( Parabacteroides The relative abundance of ).

[0031] The differential analysis of gut microbiota at the phylum and genus levels shows that the self-made wheat bran AX of this invention has a greater regulatory effect on gut microbiota, promotes the growth of beneficial bacteria and the production of short-chain fatty acids, and regulates the gut environment.

[0032] Appendix Figure 4PCA score charts for fermentation broth samples from healthy individuals (A) and diabetic patients (B), and PLS-DA score charts for fermentation broth samples from healthy individuals (C) and diabetic patients (D).

[0033] The total variances of the PCA scores of the fermentation broth samples from healthy individuals (A) and diabetic patients (B) were 63.6% and 71.3%, respectively, indicating that there were significant differences in the organic acids among the various dietary fibers. Specifically, H1 was clearly separated from several dietary fiber groups, while H3, H4, and H5 were not clearly separated. D1 was clearly separated from several dietary fiber groups, while D3, D4, and D5 were not clearly separated.

[0034] The total variances of the PCA-DA scores of the fermentation broth samples from healthy individuals (A) and diabetic patients (B) were 82.3% and 71.3%, respectively, indicating that there were significant differences in organic acids among the various dietary fibers. H1 was clearly separated from several dietary fiber groups, while H4 and H5 were not clearly separated.

[0035] Appendix Figure 5 Appendix Figure 6 The graph shows the effects of various dietary fibers on the metabolism of organic acids in fermentation broth in healthy and diabetic patients. Red balls indicate upregulation, blue balls indicate downregulation, and white balls indicate no detection.

[0036] Figure 5 , Figure 6 As can be seen, soluble dietary fiber is the only carbon source that is broken down and utilized by gut microbes to obtain the energy required for growth. Figure 5 Except for PGA (3-phosphoglyceric acid), the content of glycolysis-related substances increased with the increase of organic acid and SCFA synthesis. After providing fiber for microorganisms, the PGA content decreased significantly in all fiber groups, and similarly... Figure 6 In addition to PGA, Malate, and Succinic acid, the levels of glycolysis-related substances increased with the increase of organic acid and SCFA synthesis. After providing fiber to microorganisms, the contents of PGA, Malate, and Succinic acid decreased significantly in several fiber groups.

[0037] Figure 7 Correlation analysis of fecal microbiota and metabolites in fermented samples from healthy individuals (A) and diabetic patients (B).

[0038] from Figure 7 A strong correlation can be observed between microorganisms and metabolites (including SCFAs and organic acids). Figure A shows that... g _ Bacteroides The relative abundance of malic acid was significantly negatively correlated with its content. g_Escherichia_Shigella The relative abundance of propionic acid was significantly negatively correlated with its abundance. g_Bifidobacterium The relative abundance of was significantly positively correlated with acetic acid, fructose-6-phosphate, pyruvate, glucose-6-phosphate, malic acid, and butyric acid. g_Veillonella The relative abundance was significantly positively correlated with glucose-6-phosphate, malic acid, butyric acid, and pyruvate. g_Lachnodostridium The relative abundance of is significantly negatively correlated with pyruvate.

[0039] As can be seen from Figure B, g_Bifidobacterium The relative abundance of these compounds was significantly positively correlated with malic acid and citric acid, and significantly negatively correlated with acetic acid and propionic acid. g_Escherichia_Shigella The relative abundance of was significantly positively correlated with acetic acid and propionic acid. g_Bacteroides The relative abundance of was significantly positively correlated with glucose-6-phosphate, pyruvate, and malic acid. g_ Lactobacillus The relative abundance was significantly negatively correlated with acetic acid and propionic acid, and significantly positively correlated with malic acid and succinic acid.

[0040] The addition of dietary fiber can regulate the structure of the gut microbiota and significantly change fecal metabolites. Furthermore, homemade wheat bran AX can alleviate changes in the structure and composition of the gut microbiota in diabetic patients and significantly alter the fecal metabolic profile.

[0041] Example 3 Study on the alleviating effect of wheat bran AX prepared in Example 1 of this invention on non-alcoholic fatty liver disease.

[0042] In this embodiment, the biochemical indicators of mouse serum and liver were detected, and the liver damage was assessed by combining histopathological examination, and the effects of wheat bran AX prepared in Example 1 on liver fat accumulation and liver damage were investigated.

[0043] The specific experimental procedures in this embodiment are as follows: C57BL / 6J mice were placed in clean cages, and the bedding was changed and disinfected regularly. The experimental animal room was maintained at a temperature of 25±2℃ and a humidity of 60%±10%, with a 12-hour light / dark cycle.

[0044] After 7 days of acclimatization and feeding, 50 male C57BL / 6J mice were randomly divided into 5 groups: control group (ND, n=10), high-fat diet-induced non-alcoholic fatty liver disease group (HFD, n=10), low-dose wheat bran AX group (LDG, n=10), medium-dose wheat bran AX group (MDG, n=10), and high-dose wheat bran AX group (HDG, n=10).

[0045] The ND group was fed a low-fat diet, while the HFD group and the wheat bran AX intervention groups (LDG, MDG, and HDG groups) were fed a high-fat diet. The wheat bran AX intervention groups were administered 100, 200, and 400 mg / kg body weight of wheat bran AX by gavage during the modeling process. The ND and HFD groups were administered 10 mL / kg body weight of physiological saline by gavage. After 8 weeks, liver pathological sections of 3 mice were randomly selected for observation. The NAFLD mouse model was considered to have been successfully established if more than 1 / 3 of the hepatocytes per unit area showed fatty degeneration and significant hepatocyte enlargement.

[0046] During this period, the mice's weekly weight changes and daily food intake were recorded.

[0047] After the experiment, mouse feces were collected using cryotubes and quickly frozen in liquid nitrogen, then stored in a -80°C freezer for subsequent fecal microbiota sequencing and fecal metabolite analysis.

[0048] The day before the end of the experiment, the mice were fasted for 12 hours, then anesthetized with chloral hydrate solution, and blood was drawn through the orbital cavity. The mice were then euthanized by dislocation of the neck, and liver, small intestine and colon specimens were collected.

[0049] Appendix Figure 8 The changes in mouse body weight and food intake during the experiment are shown.

[0050] Figure 8 As can be seen, compared with the ND group, the HFD group mice had a significantly increased body weight. The intake of different doses of wheat bran AX significantly reduced the weight gain induced by the high-fat diet, and there was no significant difference in the average daily food intake among the five groups of mice. Therefore, wheat bran AX can effectively reduce the weight gain caused by a high-fat diet without interfering with the mice's appetite.

[0051] Appendix Figure 9 The figure shows the effect of wheat bran AX on the liver morphology of mice. As can be seen from the figure, compared with the model group, the livers of LDG, MDG and HDG mice in the intervention group showed a certain degree of improvement in these characteristics.

[0052] Figure 10 The figure shows the effect of wheat bran AX on the morphology of mouse liver tissue. As can be seen from the figure, compared with the HFD group, the degree of hepatocyte steatosis in the three dose groups of LDG, MDG and HDG was significantly reduced. Only a few small lipid droplets could be observed, and some samples did not even have lipid droplets visible to the naked eye, which shows that wheat bran AX has an ameliorative effect on hepatic steatosis.

[0053] Appendix Figure 11 The effect of wheat bran AX on the morphology of mouse colon tissue is shown in the figure. As can be seen from the figure, compared with the HFD group, the submucosal edema of the colon tissue of mice in the LDG, MDG and HDG groups was reduced and improved.

[0054] Figure 12 To assess the effects of wheat bran AX on the morphology of mouse jejunal tissue, sections of jejunal tissue from mice fed the ND group showed normal structure and boundaries in the mucosa, submucosa, muscularis propria, and serosa. In contrast, the jejunal tissue from mice fed the HFD group showed mild edema in the lamina propria (red arrow) and local necrosis in the muscularis propria (black arrow). Compared with the HFD group, the lamina propria and local muscularis propria in the jejunal tissue of mice fed the LDG, MDG, and HDG groups were all improved.

[0055] Appendix Figure 13 The study showed the regulatory effects of wheat bran AX on serum and liver biochemical parameters in mice. The figure indicates that a high-fat diet significantly increased TG levels in both liver and serum, while wheat bran AX treatment effectively reversed this change, partially restoring TG levels to normal. Furthermore, wheat bran AX treatment also restored serum TC, ALT, AST, T-SOD levels, and liver T-SOD levels. Compared to the HFD group, mice fed with ND showed significantly reduced serum DAO activity and α-LA concentration, while mice fed with HFD showed significantly increased DAO and α-LA levels (19.61 U / L and 293.30 μmol / mL, respectively) compared to the intervention group fed with wheat bran AX. Therefore, wheat bran AX can reduce lipid accumulation in the liver and alleviate non-alcoholic fatty liver disease.

[0056] In this invention, the KEGG database was further used to analyze the metabolic pathways of the differential metabolites mentioned above. The differential metabolites of the NC and HFD groups are related to pathways such as choline metabolism, nucleotide metabolism, primary bile acid biosynthesis and glycophospholipid metabolism. Compared with the HFD group, the intervention in the HDG group may regulate lipid metabolism by participating in signaling pathways such as arginine and proline metabolism.

Claims

1. A wheat bran arabinoxylan, characterized in that, The weight-average molar mass of the wheat bran arabinoxylan was 3.86 × 10⁻⁶. 6 ±7.21×10 4 The wheat bran arabinoxylan contains the following components, in g / mol and by mass percentage: Glucuronic acid 0.20%-0.22%, galacturonic acid 0.58%-0.62%, rhamnose 0.21%-0.23%, glucose 0.6%-0.7%, galactose 1.4%-1.5%, xylose 18%-19.2%, arabinose 21%-23%.

2. The wheat bran arabinoxylan as described in claim 1, characterized in that, The wheat bran mentioned is black wheat bran.

3. The use of the wheat bran arabinoxylan of claim 1 in the preparation of products for alleviating and / or treating diseases caused by abnormal glucose and lipid metabolism.

4. The application as described in claim 3, characterized in that, The diseases mentioned include, but are not limited to, the following: diabetes, cardiovascular disease, non-alcoholic fatty liver disease, obesity, and kidney disease.

5. The application as described in claim 3, characterized in that, The wheat bran arabinoxylan mentioned above works in the product by regulating the imbalance of the intestinal flora.

6. The application as described in claim 3, characterized in that, The product is selected from any of the food and pharmaceutical products, and includes, but is not limited to, any form of solid tablets, solid granules, liquids, or sprays.

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