Application of dietary fiber and polyphenol composition for synergistically regulating intestinal flora, composition substance and preparation method

Abstract

The invention relates to the technical field of functional food, in particular to application of a dietary fiber and polyphenol composition for synergistically regulating intestinal flora, a composition substance and a preparation method. The composition comprises oat bran insoluble dietary fibers and gallic acid, and the obtained composition can directionally regulate the intestinal flora structure, reduce the abundance of phylum firmicalis and fusobacterium and promote the abundance of phylum bacteroides, so that the ratio (F / B) of the phylum firmicalis / phylum bacteroides of the intestinal tract is reduced, and metabolic syndromes such as diabetes and obesity can be improved; the abundance of beneficial bacteria such as bacteroides is synergistically promoted, meanwhile, the proliferation of harmful bacteria such as Escherichia and fusobacterium is inhibited, and the intestinal health is maintained. In addition, the composition has the characteristic of slow glycolysis gas production, can provide nutritional support for the whole intestine segment, and is beneficial to reducing gastrointestinal tract discomfort symptoms caused by rapid fermentation gas production.

Description

Technical Field

[0001] The present invention relates to the technical field of functional foods, and particularly to the use of dietary fiber and polyphenol compositions for synergistically regulating the intestinal flora, as well as the combined substances and preparation methods thereof. Background Art

[0002] The intestinal flora, as the largest microbial community in the human body, has a close relationship with human health. In the intestine, probiotics such as Bacteroides and Bifidobacterium can produce short-chain fatty acids by fermenting dietary substrates, and these fatty acids are crucial for maintaining intestinal barrier function and regulating immune responses. However, when pathogenic bacteria such as Escherichia coli and Clostridium difficile overgrow, they will disrupt the balance of the intestinal flora and release pro-inflammatory factors such as lipopolysaccharide, thereby increasing the risks of various diseases such as obesity, type 2 diabetes and other metabolic syndromes, as well as inflammatory bowel disease. The ratio of Firmicutes to Bacteroidetes (F / B value) in the intestine is a key indicator for evaluating the stability of the flora. It has been found that in patients with type 2 diabetes, the ratio of Firmicutes to Bacteroidetes (F / B) is significantly increased. The overgrowth of Firmicutes will enhance the host's energy uptake and induce insulin resistance, while Bacteroidetes generate short-chain fatty acids (SCFAs) by fermenting dietary fiber, and these fatty acids contribute to maintaining the balance of intestinal barrier and glycolipid metabolism. In addition, Fusobacteria and its genus Fusobacterium are positively correlated with the risk of colorectal cancer, and their metabolites can activate pro-inflammatory pathways and damage the integrity of the intestinal mucosa.

[0003] Polyphenolic substances, due to their antibacterial activity, can affect the composition and metabolic activities of the intestinal flora. However, due to the fermentation inertia of polyphenolic substances themselves, they cannot be efficiently utilized by the flora as substrates, so relatively few beneficial metabolites such as short-chain fatty acids are produced. In contrast, dietary fiber can be fermented and utilized as a carbon source for the metabolism of the intestinal flora, and at the same time produce short-chain fatty acids such as acetic acid, propionic acid and butyric acid, and these fatty acids can enhance the intestinal barrier function. In addition, dietary fiber can also improve the richness and diversity of the intestinal flora and regulate the composition of the intestinal flora. However, most dietary fibers may have the characteristic of rapid fermentation and are quickly consumed in the proximal colon, making it difficult to continuously transport to the distal colon, which may lead to the imbalance of the distal flora due to lack of nutrients. At the same time, dietary fiber will produce gases such as H2 and CO2 during the fermentation process, and these gases are likely to cause gastrointestinal discomfort symptoms such as abdominal distension and abdominal pain. The human dietary system is a complex multi-component system, and these two components, polyphenols and dietary fiber, usually exist simultaneously, and their synergistic effects on the intestinal flora are still unclear.

[0004] In summary, although a single dietary component has a positive impact on the gut microbiota, its regulatory effect has limitations and it is difficult to meet the dual requirements of slow fermentation and targeted regulation of the microbiota. Therefore, in order to more comprehensively promote gut health, it may be necessary to comprehensively consider the synergistic effects of multiple dietary components and their comprehensive impact on the gut microbiota. Therefore, developing a composition that can combine the advantages of dietary fiber and polyphenols to target the regulation of the gut microbiota structure, provide nutrition for the entire intestinal segment, and synergistically promote gut health is a key challenge that urgently needs to be solved in this field.

[0005] On April 14, 2025, using "oat bran and insoluble and dietary fiber and gallic acid" as the abstract keywords and checking the option to allow synonym expansion, a search was conducted in the Chinese Patent Publication Database, and no relevant literature was found.

[0006] On April 14, 2025, an abstract search was conducted on CNKI using "oat bran and insoluble and dietary fiber and gallic acid", and no relevant literature was found.

[0007] On April 14, 2025, a search was conducted on the website of the United States Patent and Trademark Office using "oat bran with insoluble with dietary fiber with gallic acid", and no relevant literature was found; the search URL is https: / / ppubs.uspto.gov / pubwebapp / .

[0008] On April 14, 2025, a search was conducted on https: / / patentscope2.wipo.int / of WIPO using "oat bran and insoluble and dietary fiber and gallic acid", and no relevant literature was found.

[0009] On April 14, 2025, a search was conducted on the website of the Japan Patent Office https: / / www.j-platpat.inpit.go.jp / using "oat bran and insoluble and dietary fiber and gallic acid", and no relevant literature was found. Summary of the Invention

[0010] Objective of the Invention: To provide the use, combination substances, and preparation methods of a dietary fiber and polyphenol composition that can synergistically regulate the gut microbiota with better effects. For specific objectives, see the multiple substantial technical effects in the specific implementation section.

[0011] To achieve the above object, the present invention adopts the following technical solutions: A combined substance of dietary fiber and polyphenol composition for synergistically regulating intestinal flora, characterized in that the insoluble dietary fiber of oat bran and gallic acid are in a mass ratio of 25:1.

[0012] A preparation method of a combined substance of dietary fiber and polyphenol composition for synergistically regulating intestinal flora, characterized in that the combined substance is composed of a mixture of insoluble dietary fiber of oat bran and gallic acid in a mass ratio of 25:1, and the mixing process is to incubate at 25 °C for 24 hours.

[0013] A further technical solution of the present invention lies in that The preparation method of the insoluble dietary fiber of oat bran is as follows: oat bran is hydrolyzed by an enzymatic method to obtain insoluble dietary fiber, and through ultrafine pulverization treatment, it is the insoluble dietary fiber of oat bran.

[0014] A further technical solution of the present invention lies in that (1) Hydrolyze oat bran with heat-resistant α-amylase and alkaline protease respectively; (2) The hydrolyzate obtained in step (1) is washed and then freeze-dried; (3) The freeze-dried product obtained in step (2) is ultrafinely pulverized to obtain insoluble dietary fiber of oat bran.

[0015] A further technical solution of the present invention lies in that In step (1), the dosage of heat-resistant α-amylase is 9% of the dry basis mass of oat bran, and the enzyme activity of heat-resistant α-amylase is 4000 U / mg; the hydrolysis conditions of heat-resistant α-amylase are: the solid-liquid ratio is 1:10, pH 6.0, 65 °C, 80 minutes; In step (1), the dosage of alkaline protease is 1% of the dry basis mass of oat bran, and the enzyme activity of alkaline protease is 100 - 400 U / mg; the hydrolysis conditions of alkaline protease are: the solid-liquid ratio is 1:10, pH 9.0, 45 °C, 80 minutes.

[0016] A further technical solution of the present invention lies in that In step (2), the washing described is washing with water and ethanol; the temperature of the water is 50 - 80 °C, and the number of washing times is 3 times; the ethanol is an ethanol aqueous solution with a volume fraction of 70 - 95%, the temperature of the ethanol is 50 - 60 °C, and the number of washing times is 2 times.

[0017] A further technical solution of the present invention lies in that In step (3): the ultrafine pulverization is carried out in a low-temperature environment of -5 °C to 0 °C, and the median particle size of the prepared insoluble dietary fiber of oat bran is 28 - 48 μm.

[0018] A use, characterized in that the use is any one of the following: 1. Use of the combination of oat bran insoluble dietary fiber and gallic acid in the preparation of a substance for synergistically and directionally regulating the intestinal flora; 2. Use of the combination of oat bran insoluble dietary fiber and gallic acid in the preparation of a substance that exerts a synergistic effect in promoting the colonization of intestinal probiotics and inhibiting the reproduction of harmful bacteria; 3. Use of the combination of oat bran insoluble dietary fiber and gallic acid in synergistically reducing the abundances of Firmicutes and Fusobacteria, promoting the abundance of Bacteroidetes, and reducing the Firmicutes / Bacteroidetes ratio F / B; 4. Use of the combination of oat bran insoluble dietary fiber and gallic acid in the preparation of a drug for preventing and improving obesity and diabetes; 5. Use of the combination of oat bran insoluble dietary fiber and gallic acid as a substance for offsetting the promoting effect of gallic acid on the abundance of Fusobacterium; 6. Use of the combination of oat bran insoluble dietary fiber and gallic acid as an inhibitor of Fusobacterium; 7. Use of the combination of oat bran insoluble dietary fiber and gallic acid as a substance that slowly ferments and metabolizes in the entire colon to produce short-chain fatty acids, providing more nutritional support for the distal colon; 8. Use of the combination of wheat bran insoluble dietary fiber and gallic acid in drugs, foods, and health products for preventing diseases related to the distal colon; 9. Use of the combination of wheat bran insoluble dietary fiber and gallic acid as a substance that effectively reduces gas production, thereby avoiding gastrointestinal discomfort symptoms such as abdominal distension and abdominal pain.

[0019] A preparation method for the synergistic regulation of the intestinal flora by a dietary fiber and polyphenol composition, characterized in that the oat bran insoluble dietary fiber and gallic acid are mixed at a mass ratio of 25:1 and incubated at 25 °C for 24 hours; The preparation method of the oat bran insoluble dietary fiber is as follows: (1) The oat bran is sequentially treated with 9% oat bran dry basis and heat-resistant α-amylase, the heat-resistant α-amylase is 4000 U / mg, pH 6.0, 65 °C, solid-liquid ratio 1:10, for 80 minutes; and 1% alkaline protease, the 1% alkaline protease is 100 - 400 U / mg, pH 9.0, 45 °C, solid-liquid ratio 1:10, for 80 minutes; (2) The hydrolysate obtained in step (1) is washed 3 times with hot water at 50 - 80 °C and 2 times with an ethanol aqueous solution with a volume fraction of 70% - 95% at 50 - 60 °C, and the filter residue is obtained by filtration. The filter residue is freeze-dried, the freeze-drying temperature is -80 °C, and the freeze-drying time is 72 h; (3)The freeze-dried product obtained in step (2) is ultramicro pulverized at -5 °C to 0 °C for 20 minutes, thereby obtaining oat bran insoluble dietary fiber with a median particle size Dx50 of 28 μm.

[0020] A further technical solution of the present invention lies in that The determination of the gas production characteristics in in vitro fermentation is carried out by the following method: First, prepare a carbonate-phosphate buffer solution and a 0.25 g / L cysteine hydrochloride solution. Use an autoclave to sterilize the supplies required for the experiment at 121 °C for 20 minutes. The required items are beakers, gauze, pipette tips, anaerobic culture bottles and the prepared solutions. After sterilization, dry the materials. The human fecal samples for fermentation experiments are obtained from three healthy volunteers aged 20 - 30 years. In the past month, all three volunteers have had good eating habits and no history of gastrointestinal diseases, and have not taken any antibiotics or probiotic products such as yogurt. Collect the feces in a sterile tube and transfer it to an anaerobic chamber and place it overnight. Mix the prepared buffer solution with the feces of the three volunteers at a ratio of 5:1 v:w, mL:g. Filter the mixture with gauze to obtain a fecal suspension. Pipette 1 mL of the fecal solution and 4 mL of the carbonate-phosphate buffer solution into an anaerobic culture bottle containing 50 mg of the example and the comparative example, and mix and seal it. After ensuring the anaerobic culture bottle is sealed, place it in a 37 °C water bath and incubate and ferment for 4, 8, 12, and 24 h respectively. Take out the anaerobic culture bottle at each time point, measure the gas production of the sample fermentation with a graduated syringe, and record the data according to the displacement scale of the syringe.

[0021] The present invention adopting the above technical solution has the following beneficial effects compared with the prior art: The dietary fiber-polyphenol composition provided by the present invention with the ability to directionally regulate the intestinal flora composition can directionally regulate the intestinal flora composition and play a synergistic effect in promoting the colonization of intestinal probiotics and inhibiting the reproduction of harmful bacteria. At the phylum level, the composition can synergistically reduce the abundances of Firmicutes and Fusobacteria, promote the abundance of Bacteroidetes, and reduce the Firmicutes / Bacteroidetes ratio (F / B), achieving a better effect than a single component. The reduction of this ratio is beneficial for preventing and improving metabolic syndromes such as obesity and diabetes. At the genus level, the composition promotes the abundance of Bacteroides, while inhibiting the proliferation of harmful bacteria such as Escherichia and Fusobacterium. The inventors accidentally found in the experiment that the combined use of oat bran insoluble dietary fiber and gallic acid can offset the promoting effect of gallic acid on the abundance of Fusobacterium, and the inhibition of Fusobacterium is better than using dietary fiber alone, indicating that the two have a synergistic effect on inhibiting the proliferation of Fusobacterium. The change in the species composition of the intestinal flora shows that the composition can maintain the balance of the intestinal flora.

[0022] Meanwhile, the composition can be used as a fermentation substrate to slowly ferment and metabolize in the entire colon to produce short-chain fatty acids, providing more nutritional support for the distal colon, thereby preventing diseases related to the distal colon. And the composition can effectively reduce the gas production, thus avoiding gastrointestinal discomfort symptoms such as abdominal distension and abdominal pain, bringing higher comfort and tolerance to the consumers. Description of the Drawings

[0023] To further illustrate the present invention, the following description is provided in conjunction with the accompanying drawings: Figure 1 shows the gas production during the in vitro fermentation process of the examples and comparative examples of the present invention; Figure 2 shows the pH value of the fermentation broth during the in vitro fermentation process of the examples and comparative examples of the present invention; Figure 3 shows the content of short-chain fatty acids in the fermentation broth during the in vitro fermentation process of the examples and comparative examples of the present invention; Figure 4 shows the initial in vitro microbial community abundance and the phylum-level microbial community abundance after 24 hours of fermentation of the examples and comparative examples of the present invention; Figure 5 shows the initial in vitro microbial community abundance and the genus-level microbial community abundance after 24 hours of fermentation of the examples and comparative examples of the present invention; Figure 6 is Table 1; Figure 7 is Table 2; Figure 8 is Table 3. Detailed Embodiments

[0024] The following embodiments are provided to better understand the present invention further. They are not limited to the best mode, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0025] For those experimental steps or conditions not specified in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. The raw materials or instruments used are all conventional products that can be obtained through commercial purchase, including but not limited to the raw materials or instruments used in the embodiments of the present application. Embodiment

[0026] This embodiment provides a composition with the function of regulating the intestinal flora, and its preparation raw materials are as follows: insoluble dietary fiber of oat bran and gallic acid are mixed at a mass ratio of 25:1 and incubated at 25 °C for 24 hours (labeled as GA+IDF20).

[0027] The preparation method of the insoluble dietary fiber of oat bran is as follows: (1) The oat bran was treated successively with 9% (dry basis of oat bran) heat-resistant α-amylase (4000 U / mg, pH 6.0, 65 °C, solid-liquid ratio 1:10, 80 minutes) and 1% alkaline protease (100 - 400 U / mg, pH 9.0, 45 °C, solid-liquid ratio 1:10, 80 minutes).

[0028] (2) The hydrolysate obtained from step (1) was washed three times with hot water at 50 - 80 °C and twice with an aqueous ethanol solution with a volume fraction of 70% - 95% at 50 - 60 °C, and the filter residue was obtained by filtration. The filter residue was freeze-dried (-80 °C, 72 h).

[0029] (3) The freeze-dried product obtained from step (2) was ultrafinely pulverized at -5 °C to 0 °C for 20 minutes, thereby obtaining oat bran insoluble dietary fiber with a median particle size Dx50 of 28 μm (labeled as IDF20). Example

[0030] This example provides a composition with the function of regulating intestinal flora (labeled as GA+IDF10). Its preparation method refers to Example 1, with the difference that the ultrafine pulverization time of the insoluble dietary fiber in step 3 is 10 minutes, and oat bran insoluble dietary fiber with a median particle size Dx50 of 35 μm is obtained (labeled as IDF10). Example

[0031] This example provides a composition with the function of regulating intestinal flora (labeled as GA+IDF5). Its preparation method refers to Example 1, with the difference that the ultrafine pulverization time of the insoluble dietary fiber in step 3 is 5 minutes, and oat bran insoluble dietary fiber with a median particle size Dx50 of 48 μm is obtained (labeled as IDF5). Example

[0032] This example provides a composition with the function of regulating intestinal flora (labeled as GA+IDF0). Its preparation method refers to Example 1, with the difference that it is not subjected to the ultrafine pulverization treatment in step 3, and oat bran insoluble dietary fiber with a median particle size Dx50 of 289 μm is obtained (labeled as IDF0).

[0033] This comparative example is an oat bran insoluble dietary fiber, and its preparation method refers to the preparation of oat bran insoluble dietary fiber in Example 1 (labeled as IDF20).

[0034] This comparative example is an oat bran insoluble dietary fiber, and its preparation method refers to the preparation of oat bran insoluble dietary fiber in Example 2 (labeled as IDF10).

[0035] This comparative example is an oat bran insoluble dietary fiber, and its preparation method refers to the preparation of oat bran insoluble dietary fiber in Example 3 (labeled as IDF5).

[0036] This comparative example is an oat bran insoluble dietary fiber, and its preparation method refers to the preparation of oat bran insoluble dietary fiber in Example 4 (labeled as IDF0).

[0037] This comparative example is gallic acid (labeled as GA).

[0038] This comparative example is a blank control, that is, no dietary fiber or polyphenol is added.

[0039] This comparative example provides a commercially available dietary fiber - fructooligosaccharide (labeled as FOS).

[0040] In the following examples, the gas production characteristics in in vitro fermentation were measured by the following method: First, prepare a carbonate-phosphate buffer solution and a 0.25 g / L cysteine hydrochloride solution. Use an autoclave to sterilize the experimental supplies (beakers, gauze, pipette tips, anaerobic culture bottles and the prepared solutions) at 121 °C for 20 minutes. Subsequently, dry the sterilized materials. The human fecal samples for fermentation experiments were collected from three healthy volunteers aged 20 - 30 years. In the past month, the three volunteers had good eating habits and no history of gastrointestinal diseases, and did not take any antibiotics or probiotic products such as yogurt. Collect the feces in a sterile tube, transfer it to an anaerobic chamber and place it overnight. Mix the prepared buffer solution with the feces of the three volunteers at a ratio of 5:1 (v:w, mL:g), and filter the mixture with gauze to obtain a fecal suspension. Pipette 1 mL of the fecal solution and 4 mL of the carbonate-phosphate buffer solution into an anaerobic culture bottle containing 50 mg of the example and the comparative example, and mix and seal it. After ensuring the anaerobic culture bottle is sealed, place it in a 37 °C water bath and incubate and ferment for 4, 8, 12, and 24 h respectively. Take out the anaerobic culture bottle at each time point, measure the gas production of the sample fermentation with a graduated syringe, and record the data according to the displacement scale of the syringe.

[0041] The gas production of the in vitro fermentation of the examples and the comparative examples is as Figure 1 shown in Table 1.

[0042] Table 1 Gas production of in vitro fermentation (mL) From Figure 1As shown in Table 1, in the blank group and GA group, since there was no carbohydrate as the substrate, almost no gas was produced during glycolysis. The gas production after 24 hours was only 1.87 mL and 2.07 mL respectively, indicating that gallic acid was difficult to be utilized by microorganisms for gas-producing metabolism. The gas production of the FOS group showed a trend of first increasing rapidly and then leveling off. The gas production reached 12.87 mL at 8 hours, proving that FOS had the characteristics of rapid fermentation. After 8 hours, the nutrient substrate was almost completely utilized, resulting in its rapid fermentation in the proximal colon and being unable to reach the distal colon, and it was prone to cause adverse symptoms related to intestinal gas production such as abdominal distension, abdominal pain, and belching. The gas production of different particle size IDF groups reached 2.87 - 4.87 mL after 24 hours, proving that dietary fiber could be metabolized by intestinal flora for gas production during glycolysis, indicating that the insoluble dietary fiber of oat bran had the characteristics of slow glycolysis, with less gas produced and not easily causing gastrointestinal discomfort such as abdominal distension and abdominal pain. And as the particle size of the insoluble dietary fiber decreased, the gas production during glycolysis increased, indicating that the particle size of dietary fiber was negatively correlated with its fermentation rate and gas production. Compared with the IDF group, the gas production of the GA + IDF group was lower, and the gas production after 24 hours was only 2.53 - 4.00 mL, indicating that the combination of oat bran IDF and gallic acid could further reduce the fermentation rate, was beneficial to better relieve gastrointestinal discomfort caused by dietary fiber fermentation, and could also transport nutrients to the distal colon to maintain the homeostasis of the intestinal microenvironment in the distal colon.

[0043] The pH value of the glycolysis solution in Experimental Example 1 was measured by the following method: The anaerobic bottles cultured and glycolyzed at 37 °C in a water bath for 4, 8, 12, and 24 h were taken out respectively. After measuring the gas production of the glycolysis of the samples, the pH value of the fermentation broth was measured using a pH meter and the data was recorded.

[0044] The pH value of the glycolysis solution during the in vitro glycolysis process of the examples and comparative examples was as Figure 2 shown.

[0045] The pH value of the fermentation broth is an important indicator indicating the change of the intestinal microenvironment caused by dietary fiber fermentation. The change of the pH value is closely related to the metabolism of intestinal microorganisms. From Figure 2It can be seen that the initial fermentation broth was alkaline (pH 9.0). After 24 h of fermentation, the pH values of the fermentation broth in the blank group and the GA group slightly decreased to 8.88 and 8.82, respectively. This indicates that it is difficult for the intestinal flora to ferment and utilize gallic acid during fermentation to produce short-chain fatty acids, resulting in only a slight decrease in the pH value. The pH value of the FOS group decreased the fastest, showing a trend of rapid decrease first and then slight increase. This is because FOS was rapidly fermented by intestinal microorganisms to produce short-chain fatty acids, causing a rapid decrease in the pH value during the early stage of fermentation. In the later stage, due to the continuous consumption of short-chain fatty acids by microorganisms, the pH value increased slightly. The pH value of the IDF group decreased smoothly, and the pH value decreased to 8.30 - 8.58 after 24 hours, indicating that oat bran IDF can ferment slowly and produce short-chain fatty acids at a uniform speed. Compared with the IDF group, the pH value of the GA + IDF group was higher, and the pH value of the fermentation broth was in the range of 8.46 - 8.63 after 24 hours of fermentation, indicating that the combination of gallic acid and oat bran IDF can effectively reduce the fermentation rate of the flora, thereby reducing the rate of short-chain fatty acid production.

[0046] The determination of the short-chain fatty acid content in the fermentation broth of Experimental Example 1 was carried out by the following method: First, the supernatant of the fermentation broth was centrifuged at 13000 rpm for 10 minutes, and 800 µL of the centrifuged supernatant was taken and mixed evenly with 200 µL of the internal standard mixture. Subsequently, a syringe was used to extract the sample and filtered through a 0.22 µm aqueous filter membrane and collected into a sample vial. A gas chromatograph equipped with a polar ZB-FFAP capillary column (30 m × 0.25 mm × 0.25 µm) and a flame ionization detector (FID) was used to analyze the types and contents of short-chain fatty acids in the fermentation broth. The initial temperature of the column oven was set at 80 °C, the temperatures of the front inlet and the front detector were 230 °C, and nitrogen was used as the carrier gas at a fixed flow rate of 1 mL / min. The types of short-chain fatty acids were determined according to the fatty acid standard. The correction factor was determined by the ratio of the peak areas of tetramethylvaleric acid (internal standard) to the target fatty acid in the fatty acid standard, and then the short-chain fatty acids in the fermentation broth were quantified.

[0047] The contents of short-chain fatty acids in the fermentation broth during the in vitro fermentation of the examples and comparative examples are as Figure 2 shown.

[0048] Short-chain fatty acids are the final products of the fermentation of dietary fiber by specific anaerobic bacteria in the intestine and have a positive impact on host physiology and energy homeostasis. Therefore, the concentration of short-chain fatty acids is often used as one of the indicators of the fermentability of substances. Short-chain fatty acids can promote the production of glucagon-like peptide-1 and peptide YY in the body, and then promote the synthesis and secretion of insulin. At the same time, they can regulate insulin sensitivity through multiple pathways, thereby regulating the blood glucose homeostasis of the human body.

[0049] From Figure 2It can be seen that the content of short-chain fatty acids after 24 hours of fermentation in the blank group was 15.94 mM. The GA group (22.10 mM) produced less short-chain fatty acids, indicating that it is difficult for intestinal microorganisms to utilize gallic acid for metabolism and produce beneficial metabolites such as short-chain fatty acids. The FOS group, as a positive control, had the highest production of short-chain fatty acids (64.24 mM), indicating that FOS can be rapidly fermented and utilized by the intestinal flora to produce short-chain fatty acids. The content of short-chain fatty acids in the IDF group was 31.36 - 44.04 mM after 24 hours of fermentation, and the smaller the IDF particle size, the more fatty acids were produced, indicating that there was a negative correlation between the IDF particle size and the rate of short-chain fatty acid production by microbial fermentation. Compared with IDF, the short-chain fatty acids in the GA+IDF0 / 5 / 10 / 20 groups were lower, being 25.51, 33.79, 38.72, and 39.98 mM respectively, indicating that compared with the single use of oat bran insoluble dietary fiber, the combined use of gallic acid and oat bran IDF can reduce the fermentation rate, and it is predicted that it can reach the posterior end of the colon at a gentle glycolysis rate, provide short-chain fatty acids for the distal colon, maintain the health of the distal colon, and at the same time the composition makes up for the disadvantage of the low production of short-chain fatty acids by gallic acid.

[0050] The change in the flora abundance in the fermentation broth of Experimental Example 1 was determined by the following method: Sequence data analysis was mainly carried out through the Quantitative Insights Into Microbial Ecology 2 (QIIME2) and R software package (v3.2.0). First, the original off-machine data of high-throughput sequencing was preliminarily screened according to the sequence quality. The original sequences passing the quality preliminary screening were divided into libraries and samples according to the index and Barcode information, and the barcode sequences were removed. Then the DADA2 method was used to de-prime, quality-filter, denoise, splice, and remove chimeras from the sequences. It no longer clusters by similarity, but only performs deduplication or equivalent to clustering with 100% similarity. Each deduplicated sequence generated after DADA2 quality control is called an Amplicon Sequence Variant (ASV). Metastats was used to compare the taxonomic unit abundance results at the phylum and genus levels for each sample and display them as bar charts.

[0051] The initial flora abundance in vitro and the flora abundance at the phylum level and genus level after 24 hours of glycolysis in the examples and comparative examples are as Figure 3 、 Figure 4 shown in Table 2, 3.

[0052] Table 2 Species abundance at the phylum level and F / B value after in vitro glycolysis As Figure 3As shown in Table 2, at the phylum level, the dominant phyla in the intestinal flora of the initial flora and each group after 24 hours of fermentation were Firmicutes ( Firmicutes ), Proteobacteria ( Proteobacteria ), Actinobacteria ( Actinobacteriota ), Bacteroidetes ( Bacteroidota ), and Fusobacteria ( Fusobacteriota ).

[0053] Fusobacteria is associated with intestinal inflammation and the risk of colorectal cancer. The enrichment of some bacteria in Fusobacteria may produce toxic metabolites such as lipopolysaccharides and endotoxins, which can damage the integrity of the intestinal mucosa and may promote the occurrence of colorectal cancer. Compared with the blank group, the abundance of Fusobacteria in the GA group increased from 14.54% to 21.31%, indicating that GA may increase the risk of diseases related to Fusobacteria. The abundance of Fusobacteria in the IDF group was relatively low, ranging from 4.09% to 12.04%. The smaller the IDF particle size, the lower the abundance of Fusobacteria, suggesting that IDF can reduce the likelihood of diseases related to Fusobacteria. However, the inventors unexpectedly found during the experiment that the abundance of Fusobacteria in the GA+IDF group was lower than that in the IDF group, ranging from 3.60% to 10.75%, and the inhibitory effect was still negatively correlated with the IDF particle size. This shows that when gallic acid and oat bran insoluble dietary fiber are used in combination, it offsets the proliferative effect of GA on Fusobacteria, avoids the negative impact on the human body caused by the excessive proliferation of gallic acid, and their inhibitory effect on Fusobacteria is better than that of dietary fiber alone.

[0054] Bacteroidetes is one of the key beneficial bacterial phyla in the intestine. It can produce short-chain fatty acids through the fermentation of carbon sources, which can not only provide energy for intestinal epithelial cells, enhance intestinal barrier function, but also inhibit inflammatory responses and improve insulin resistance by regulating host immunity and glycolipid metabolism pathways. Firmicutes can also play a key role in host and nutrient metabolism through the synthesis of short-chain fatty acids. However, the over-proliferation of this bacterial phylum is closely related to energy metabolism imbalance, and its increased abundance is likely to induce chronic low-grade inflammation and insulin resistance, which is a typical microbiota characteristic of metabolic syndromes such as obesity and type 2 diabetes. At the same time, the ratio of Firmicutes / Bacteroidetes (F / B) has a significant impact on the risk of metabolic diseases such as type 2 diabetes and obesity. A high F / B value will promote the gene expression related to inflammation and immune responses, thus increasing the risk of disease. Compared with the blank group, the abundance of Bacteroidetes in the GA group did not change significantly, the abundance of Firmicutes decreased from 25.76% to 19.83%, and the F / B value decreased from 1.79 to 1.37. The abundance of Bacteroidetes in the IDF group increased to 27.66% - 39.25%, with a maximum increase of 28.05%. The abundance of Firmicutes decreased to 18.95% - 20.17%, and the F / B value decreased significantly to 0.51 - 0.72. Moreover, IDF ultra-milled to 28 - 48 μm had a better effect than non-ultra-milled IDF. This indicates that oat bran IDF and gallic acid can reduce the F / B value by regulating the growth and proliferation of these two bacterial phyla respectively, thereby improving metabolic syndromes such as type 2 diabetes and obesity. Compared with the IDF5 / 10 / 20 group, the GA + IDF5 / 10 / 20 group had a higher abundance of Bacteroidetes, a lower abundance of Firmicutes, and a lower F / B value, indicating that the combination of oat bran IDF with a particle size of 28 - 48 μm after ultra-milling and GA had a synergistic promoting effect on the abundance of Bacteroidetes and a synergistic reducing effect on the abundance of Firmicutes and the F / B value. However, no synergistic effect was found when non-ultra-milled oat bran IDF was combined with GA.

[0055] Table 3 Species abundances at the genus level after in vitro fermentation Bacteroides () Escherichia () Fusobacterium () GA+IDF20 32.63% 9.98% 3.56% GA+IDF10 31.36% 10.28% 5.54% GA+IDF5 30.89% 11.55% 5.69% GA+IDF0 16.72% 12.22% 10.71% IDF20 26.07% 13.42% 4.06% IDF10 27.51% 11.78% 5.26% IDF5 27.53% 14.43% 8.35% IDF0 18.73% 13.16% 11.99% GA 6.74% 15.55% 21.28% Blank 5.70% 16.78% 14.52% FOS 3.83% 4.69% 0.81% As Figure 4 shown in and Table 3, at the genus level, the main bacterial species in the microbiota include Klebsiella , Escherichia , Enterobacter of Proteobacteria, Bacteroides , Phocaeicola , Parabacteroides of Bacteroidetes, Phascolarctobacterium , Faecalibacterium , Blautia of Firmicutes, and Fusobacterium of Fusobacteria.

[0056] Bacteroides genus ( Bacteroidesis one of the core beneficial bacterial genera in the intestine. It can metabolize carbohydrates to produce short-chain fatty acids, maintain the acidic environment of the intestine and inhibit the reproduction of pathogenic bacteria. At the same time, it can enhance the host's immune regulation function. Compared with the blank group, the abundance of Bacteroides in the GA group increased from 5.70% to 6.74%. The abundances of Bacteroides in the IDF0 / 5 / 10 / 20 groups were 18.73%, 27.53%, 27.51% and 26.07% respectively, indicating that after ultrafine grinding treatment, IDF with a particle size of 28-48 μm had a better promoting effect on the abundance of Bacteroides. Compared with the IDF5 / 10 / 20 groups, the abundances of Bacteroides in the GA+IDF5 / 10 / 20 groups were higher, being 30.89%, 31.36% and 32.63% respectively, suggesting that the combined use of ultrafine-ground oat bran IDF and GA could have a synergistic promoting effect on the abundance of the beneficial bacterial genus Bacteroides, while the combined use of IDF0 and GA did not have a synergistic promoting effect on the abundance of Bacteroides.

[0057] The overgrowth of Escherichia spp. ( Escherichia ), such as Escherichia coli, will release endotoxins such as lipopolysaccharide, induce intestinal inflammation and damage the intestinal barrier function. Its abundance level is closely related to diseases such as irritable bowel syndrome. Compared with the blank group, the GA group could reduce the abundance of Escherichia from 16.78% to 15.55%. The abundances of Escherichia in the IDF groups were 11.78%-14.43%, and there was no obvious connection with the IDF particle size. This indicates that oat bran IDF and gallic acid can respectively reduce the risk of intestinal inflammation by inhibiting the proliferation of Escherichia. Compared with the IDF0 / 5 / 10 / 20 groups, the abundances of Escherichia in the GA+IDF0 / 5 / 10 / 20 groups were lower, being 12.22%, 11.55%, 10.28% and 9.98% respectively. The smaller the particle size of IDF in the composition, the better the inhibitory effect on Escherichia, indicating that the combination of oat bran IDF and gallic acid can synergistically inhibit the abundance of the harmful bacterial genus Escherichia and exert a better effect than single components.

[0058] Fusobacterium ( Fusobacterium)( ) is a genus of bacteria in the phylum Fusobacteria, which is highly associated with diseases such as colorectal cancer and inflammatory bowel disease. It exacerbates mucosal damage by adhering to intestinal epithelial cells and secreting pro-inflammatory factors. Compared with the blank group (14.52%), the abundance of Fusobacterium in the GA group was significantly increased to 21.28%, indicating that gallic acid might increase the risk of intestinal diseases associated with this genus of bacteria. The abundance of Fusobacterium in the IDF group was 4.06% - 11.99%, all lower than that in the blank group, and there was a positive correlation between the IDF particle size and the abundance of Fusobacterium, indicating that oat bran IDF could inhibit the proliferation of Fusobacterium and exert health benefits, and the smaller the IDF particle size, the better the inhibitory effect. Surprisingly, it was found that the abundance of Fusobacterium in the GA + IDF group was 3.56% - 10.71%, all lower than that in each IDF group, and there was still a positive correlation between the abundance of Fusobacterium and the IDF particle size, indicating that the combination of gallic acid and oat bran IDF could offset the promoting effect of GA on this harmful genus of bacteria, Fusobacterium, and exert a better inhibitory effect than using IDF alone, which was consistent with the changes at the phylum level.

[0059] Therefore, the composition of oat bran insoluble dietary fiber and gallic acid prepared in the present invention can not only be slowly fermented to maintain the health of the distal colon and provide nutrition for the entire intestinal segment. At the phylum level, this composition can have a synergistic promoting effect on the abundance of Bacteroidetes, and a synergistic reducing effect on the abundance of Firmicutes and the intestinal F / B value. At the same time, it can offset the side effect of gallic acid in promoting Fusobacteria, and exert a better inhibitory effect on Fusobacteria than using dietary fiber alone. At the genus level, the composition of oat bran insoluble dietary fiber and gallic acid can synergistically promote the proliferation of the beneficial bacterium Bacteroides, synergistically inhibit the growth and reproduction of harmful bacteria such as Escherichia and Fusobacterium, and offset the promoting effect of gallic acid on Fusobacterium. The composition can targetedly regulate the intestinal flora structure and become a potential high-quality compound nutritional supplement.

[0060] Figure 1 shows the gas production changes of samples during the fermentation process, where Figure 1 A shows the addition of different particle size dietary fibers alone (IDF0, IDF5, IDF10, and IDF20 groups), gallic acid (GA group), and fructooligosaccharide (FOS group), Figure 1 B shows the addition of a composition of dietary fiber and gallic acid (GA + IDF0, GA + IDF5, GA + IDF10, and GA + IDF20 groups). Figure 1 In A, the IDF0 / 5 / 10 / 20 groups and Figure 1Comparison of the GA+IDF0 / 5 / 10 / 20 groups in B showed that the gas production during the fermentation process in the IDF group was 2.87 - 4.87 mL, while that of the oat bran IDF+GA composition was 2.53 - 4.00 mL, a decrease of 4.76% - 17.86% compared to the addition of IDF alone. This indicates that the IDF+GA composition can further reduce the fermentation rate, which is beneficial for better alleviating gastrointestinal discomfort caused by dietary fiber fermentation. It can also transport nutrients to the distal colon and maintain the homeostasis of the intestinal microenvironment in the distal colon.

[0061] Figure 2 Figure shows the change in the pH value of the fermentation broth during the fermentation process, where Figure 2 A shows the individual addition of dietary fibers with different particle sizes (IDF0, IDF5, IDF10, and IDF20 groups), gallic acid (GA group), and fructooligosaccharide (FOS group), Figure 2 B shows the addition of dietary fiber and gallic acid compositions (GA+IDF0, GA+IDF5, GA+IDF10, and GA+IDF20 groups). Figure 2 Comparison of the dietary fiber groups (IDF0 / 5 / 10 / 20) and the FOS group in A showed that the pH value in the IDF group decreased gently. After 24 hours, the pH value dropped to 8.30 - 8.58, which was 1.10 - 1.13 times the pH value of the fermentation broth in the FOS group, indicating that oat bran IDF can ferment slowly and produce short-chain fatty acids evenly. Therefore, the rate of pH value decrease is slower than that in the FOS group. Figure 2 In A, the individual IDF (IDF0 / 5 / 10 / 20) groups and Figure 2 Comparison of the GA+IDF compositions (GA+IDF0 / 5 / 10 / 20) groups in B showed that the pH values of the IDF and gallic acid compositions were higher. After fermentation, the pH values were in the range of 8.46 - 8.63, higher than 8.30 - 8.58 for the individual use of IDF, indicating that the composition can effectively reduce the fermentation rate of the flora, thereby reducing the rate of short-chain fatty acid production. Therefore, the pH value decreases more slowly than for the individual use of IDF. Moreover, all the figures can illustrate that this patent has achieved the effect that the systematic effect 1+1 is greater than 2.

[0062] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the claimed protection.

Claims

1. A combination substance of dietary fiber and polyphenol composition for synergistically regulating intestinal flora, characterized in that, The mass ratio of oat bran insoluble dietary fiber to gallic acid is 25:

1.

2. Preparation method of dietary fiber and polyphenol composition for synergistically regulating intestinal flora, characterized in that, The combined substance is composed of oat bran insoluble dietary fiber and gallic acid mixed at a mass ratio of 25:

1. The mixing process is incubation at 25 °C for 24 hours.

3. The preparation method for the synergistic regulation of the intestinal flora by the dietary fiber and polyphenol composition according to claim 2, wherein, The preparation method of the oat bran insoluble dietary fiber is as follows: oat bran is hydrolyzed by an enzymatic method to obtain insoluble dietary fiber, and then subjected to ultrafine grinding treatment, which is the oat bran insoluble dietary fiber.

4. The preparation method for the synergistic regulation of intestinal flora by the dietary fiber and polyphenol composition according to claim 3, wherein, Specifically, it includes the following steps: (1) Hydrolyze oat bran with heat-resistant α-amylase and alkaline protease respectively; (2) The hydrolyzate obtained in step (1) is washed and then freeze-dried; (3) The freeze-dried product obtained in step (2) is subjected to ultrafine grinding to obtain oat bran insoluble dietary fiber.

5. The preparation method of the dietary fiber and polyphenol composition for synergistically regulating the intestinal flora as described in claim 4, characterized in that In step (1), the dosage of heat-resistant α-amylase is 9% of the dry basis mass of oat bran, and the enzyme activity of heat-resistant α-amylase is 4000 U / mg; the hydrolysis conditions of heat-resistant α-amylase are: the solid-liquid ratio is 1:10, pH 6.0, 65 °C, 80 minutes; In step (1), the dosage of alkaline protease is 1% of the dry basis mass of oat bran, and the enzyme activity of alkaline protease is 100 - 400 U / mg; the hydrolysis conditions of alkaline protease are: the solid-liquid ratio is 1:10, pH 9.0, 45 °C, 80 minutes.

6. The preparation method of the dietary fiber and polyphenol composition for synergistically regulating the intestinal flora as described in claim 4, characterized in that In step (2), the washing is carried out using water and ethanol; the temperature of the water is 50 - 80 °C, and the number of washing times is 3 times; the ethanol is an ethanol aqueous solution with a volume fraction of 70 - 95%, the temperature of the ethanol is 50 - 60 °C, and the number of washing times is 2 times.

7. The preparation method of the dietary fiber and polyphenol composition for synergistically regulating the intestinal flora as described in claim 4, characterized in that In step (3): the ultrafine grinding is carried out in a low-temperature environment of -5 °C to 0 °C, and the median particle size of the prepared oat bran insoluble dietary fiber is 28 - 48 μm.

8. A use, characterized in that, The uses are any one of the following:

1. The use of the combination of oat bran insoluble dietary fiber and gallic acid in the preparation of a substance for synergistically and directionally regulating the intestinal flora; 2. The use of the combination of oat bran insoluble dietary fiber and gallic acid in the preparation of a substance that plays a synergistic effect in promoting the colonization of intestinal probiotics and inhibiting the reproduction of harmful bacteria; 3. The use of the combination of oat bran insoluble dietary fiber and gallic acid in synergistically reducing the abundances of Firmicutes and Fusobacteria, promoting the abundance of Bacteroidetes, and reducing the Firmicutes / Bacteroidetes ratio F / B; 4. The use of the combination of oat bran insoluble dietary fiber and gallic acid in the preparation of a drug for preventing and improving obesity and diabetes; 5. The use of the combination of oat bran insoluble dietary fiber and gallic acid as a substance to offset the promoting effect of gallic acid on the abundance of Fusobacterium; 6. The use of the combination of oat bran insoluble dietary fiber and gallic acid as an inhibitor of Fusobacterium; Use of a combination of oat bran insoluble dietary fiber and gallic acid as a substance that slowly ferments and metabolizes in the entire colon to produce short-chain fatty acids, providing more nutritional support for the distal colon; Use of a combination of wheat bran insoluble dietary fiber and gallic acid in drugs, foods, and health products for preventing diseases related to the distal colon; Use of a combination of wheat bran insoluble dietary fiber and gallic acid as a substance that effectively reduces gas production, thereby avoiding gastrointestinal discomfort symptoms such as abdominal distension and abdominal pain.

9. Preparation method of a dietary fiber and polyphenol composition for synergistically regulating the intestinal flora, characterized in that, The oat bran insoluble dietary fiber and gallic acid are mixed at a mass ratio of 25:1 and incubated at 25 °C for 24 hours; The preparation method of oat bran insoluble dietary fiber is as follows: (1) The oat bran is sequentially treated with 9% oat bran dry basis and heat-resistant α-amylase, the heat-resistant α-amylase is 4000 U / mg, pH 6.0, 65 °C, solid-liquid ratio 1:10, for 80 minutes; and 1% alkaline protease, the 1% alkaline protease is 100 - 400 U / mg, pH 9.0, 45 °C, solid-liquid ratio 1:10, for 80 minutes; (2) The hydrolysate obtained from step (1) is washed 3 times with hot water at 50 - 80 °C and 2 times with an aqueous ethanol solution with a volume fraction of 70% - 95% at 50 - 60 °C, and the filter residue is obtained by filtration. The filter residue is freeze-dried, the freeze-drying temperature is -80 °C, and the freeze-drying time is 72 h; (3) The freeze-dried product obtained from step (2) is ultrafinely pulverized at -5 °C to 0 °C for 20 minutes, thereby obtaining oat bran insoluble dietary fiber with a median particle size Dx50 of 28 μm.

10. The preparation method of the dietary fiber and polyphenol composition for synergistically regulating the intestinal flora according to claim 9, characterized in that, The measurement of gas production characteristics in in vitro fermentation is carried out by the following method: First, prepare a carbonate-phosphate buffer solution and a 0.25 g / L cysteine hydrochloride solution. Use an autoclave to sterilize the supplies required for the experiment at 121 °C for 20 minutes. The supplies include beakers, gauze, pipette tips, anaerobic culture bottles, and the prepared solutions. After sterilization, dry the materials. The human fecal samples for the glycolysis experiment were collected from three healthy volunteers aged 20 - 30 years. In the past month, all three volunteers had good eating habits and no history of gastrointestinal diseases, and had not taken any antibiotics or probiotic products such as yogurt. Collect the feces in sterile tubes and transfer them to an anaerobic chamber and let them stand overnight. Mix the prepared buffer with the feces of the three volunteers at a ratio of 5:1 v:w (mL:g). Filter the mixture with gauze to obtain a fecal suspension. Pipette 1 mL of the fecal solution and 4 mL of the carbonate-phosphate buffer into anaerobic culture bottles containing 50 mg of the examples and comparative examples, and mix well and seal. After ensuring the anaerobic culture bottles are sealed, place them in a 37 °C water bath and incubate for glycolysis for 4, 8, 12, and 24 h respectively. Take out the anaerobic culture bottles at each time point, use a graduated syringe to measure the gas production of the sample during glycolysis, and record the data according to the displacement scale of the syringe.

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