Polydextrose butyrate and preparation and use thereof

Abstract

The application discloses a polyfructose butyrate as well as preparation and application thereof, and is prepared by combining n-butyric acid or vinyl butyrate with polyfructose to form an ester bond by using an enzymatic synthesis method. The polyfructose butyrate can be well degraded and utilized by intestinal flora, and butyric acid is simultaneously generated, so that the polyfructose butyrate not only can promote richness and diversity of intestinal flora, improve intestinal flora, but also can promote growth of probiotics and inhibit growth of harmful bacteria. The polyfructose butyrate can significantly improve intestinal inflammation of mice and relieve symptoms of intestinal inflammation. The polyfructose butyrate can make polyfructose and butyric acid exert bioactivity at specific positions in the intestinal tract, and does not cause other burdens on the body.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a polyfructose butyrate as well as preparation and application thereof. BACKGROUND

[0002] Inflammatory bowel diseases (IBDs) is a chronic idiopathic intestinal inflammatory disease, mainly manifested as intermittent inflammation of the gastrointestinal tract, and its pathological features are intestinal inflammation, reduced tight junction, atrophic gland and epithelial damage. Common clinical features include persistent diarrhea, vomiting, hematochezia, weight loss and pain, etc. IBDs can be subdivided into ulcerative colitis (UC) and Crohn's disease (CD), and there are different clinical and pathological features between CD and UC. UC is a chronic non-specific inflammatory bowel disease, which only occurs in the large intestine, and the inflammation is limited to the mucosa. Its characteristic is continuous mucosal ulceration in the rectum and colon, which generally starts from the rectum and extends to varying degrees, and can spread to the cecum. CD can occur anywhere in the digestive tract, from the mouth to the rectum and anus, and it affects the entire intestinal wall, forming abscesses and fistulas in the skin or internal organs. However, the same is that both UC and CD will have a great impact on people's quality of life and work ability.

[0003] At present, the treatment of IBDs is mainly anti-inflammatory and immunoregulation, supplemented by non-drug treatments such as biological agents, hyperbaric oxygen and stem cell transplantation. Although traditional western medicine and new biological agents can relieve the clinical symptoms of IBDs to some extent, they often have problems such as large side effects and high price. It has been reported that butyric acid can help regulate intestinal homeostasis and may regulate gene expression through epigenetic regulation, thereby reducing the production of pro-inflammatory factors in the body and relieving colonic inflammatory response. However, if relying on human diet to treat or prevent diseases, the production of butyric acid has potential limitations. Low consumption of fermentable fiber and the diversity of intestinal microbiota in individuals usually limit the production of butyric acid in the human intestine. Due to the rancid taste and unpleasant aroma of butyric acid, and its rapid metabolism in the upper digestive tract, oral administration of butyric acid is not desirable. In addition, butyric acid can also be rapidly absorbed before reaching the large intestine, making oral administration ineffective. Although rectal administration of butyrate can be used as a clinical treatment for colonic and rectal diseases, it is functionally cumbersome and has only a slight effect on mild inflammation (DOI: 10.3390 / ijms21020445).

[0004] Therefore, it is urgent to develop a new auxiliary relief strategy, especially for the early stage of IBD, to establish an effective nutritional intervention strategy to relieve disease symptoms by changing the microbial community structure and enhancing the immune activity of the body. The target supplement exerts its biological activity at a specific location in the intestine without causing other burdens to the body. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the deficiencies of existing inflammatory bowel disease treatment, and to provide a polyfructose butyrate and its preparation and application.

[0006] The present application aims to provide a polyfructose butyrate.

[0007] The present application also aims to provide a preparation method of the polyfructose butyrate.

[0008] The present application also aims to provide the application of the polyfructose butyrate in improving intestinal flora and / or promoting the growth of probiotics.

[0009] The present application also aims to provide the application of the polyfructose butyrate in preparing food or medicine for treating and / or relieving inflammatory bowel disease.

[0010] Fructo oligosaccharide (FOS) and inulin are common prebiotics, which are not easily digested in the upper digestive tract and can be utilized by intestinal flora to exert many health benefits after reaching the lower digestive tract, having immune regulation effect. Butyric acid is one of the metabolic products of dietary fiber degraded by intestinal flora, which has the effects of regulating intestinal flora structure and maintaining intestinal barrier, and has important influence on maintaining human intestinal health and even whole body health. In the present application, FOS or inulin is used as a carrier, and n-butyric acid or vinyl butyrate is combined with FOS or inulin to form an ester bond to prepare polyfructose butyrate.

[0011] The above-mentioned objects of the present application are achieved by the following technical means:

[0012] The present application provides a polyfructose butyrate, which is prepared by combining n-butyric acid or vinyl butyrate with polyfructose to form an ester bond by enzymatic synthesis method, and the degree of polymerization of the polyfructose is less than or equal to 60.

[0013] Preferably, the polyfructose butyrate is prepared by combining vinyl butyrate with polyfructose to form an ester bond by enzymatic synthesis method.

[0014] Preferably, the polyfructose is fructo oligosaccharide or inulin.

[0015] Further preferably, the polyfructose is fructo oligosaccharide.

[0016] A method for preparing polyfructose butyrate, which utilizes enzymatic synthesis to combine n-butyric acid or vinyl butyrate with polyfructose to form ester bond, and the polyfructose has a polymerization degree less than or equal to 60.

[0017] Preferably, the method for preparing polyfructose butyrate is: utilizing enzymatic synthesis to combine vinyl butyrate with polyfructose to form ester bond.

[0018] Preferably, the method for preparing polyfructose butyrate is: combining polyfructose solution with n-butyric acid or vinyl butyrate under catalysis of lipase, removing excess lipase, and then purifying.

[0019] Preferably, the purification is: after removing the solvent, separating the reaction solution by column chromatography.

[0020] Preferably, the removal of the solvent is specifically: using a vacuum rotary evaporator to remove the solvent to obtain a solid.

[0021] Preferably, the separation of the reaction solution by column chromatography is specifically: dispersing the solid obtained by removing the solvent with 5% methanol aqueous solution to obtain a suspension, subjecting the suspension to column chromatography, eluting the suspension with 3 column volumes of 5% methanol aqueous solution, then eluting with methanol, and collecting the yellow eluate.

[0022] Preferably, the column chromatography is C18 column chromatography.

[0023] Preferably, the solvent of the polyfructose solution is anhydrous pyridine, dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).

[0024] Further preferably, the solvent of the polyfructose solution is anhydrous pyridine.

[0025] Preferably, the polyfructose solution is combined with vinyl butyrate under catalysis of lipase.

[0026] Preferably, the lipase is lipase 435 or immobilized lipase TLIM.

[0027] Further preferably, the lipase is immobilized lipase TLIM.

[0028] Preferably, the sufficient reaction is a reaction at 50-55°C for 45-50 hours.

[0029] Further preferably, the sufficient reaction is a reaction at 55°C for 48 hours.

[0030] Preferably, the polyfructose is fructooligosaccharide or inulin.

[0031] Further preferably, the polyfructose is fructooligosaccharide.

[0032] The polyfructose butyrate prepared by the preparation method is also within the protection scope of the present application.

[0033] The application of the polyfructose butyrate in improving intestinal flora and / or promoting the growth of probiotics is also within the protection scope of the present application.

[0034] The application of the polyfructose butyrate in preparing food or medicine for treating and / or relieving inflammatory bowel disease is also within the protection scope of the present application.

[0035] Preferably, the polyfructose butyrate is a fructooligosaccharide butyrate or inulin butyrate.

[0036] Preferably, the polyfructose butyrate is a fructooligosaccharide butyrate.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] The present application uses an enzymatic synthesis method to combine n-butyric acid or vinyl butyrate with polyfructose to form an ester bond to prepare a polyfructose butyrate, which can be well degraded and utilized by intestinal flora, and at the same time, produce butyric acid, not only can promote the richness and diversity of intestinal flora, improve intestinal flora, but also can promote the growth of probiotics and inhibit the growth of harmful bacteria; the polyfructose butyrate can significantly improve intestinal inflammation of mice and relieve the symptoms of intestinal inflammation. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 TLC results of different ratios of developing agents on the reaction liquid of the fructooligosaccharide butyrate of the present application; A is the result of developing FOS and B-FOS using different ratios of dichloromethane and methanol developing agent, B is (1) n-butanol: n-propanol: water volume ratio 1:6:1, (2) n-butanol: isopropanol: water: acetic acid volume ratio 7:5:4:2, (3) n-butanol: acetic acid: water volume ratio 4:1:5, (4) n-butanol: acetic acid: water volume ratio 8:3:2, (5) n-propanol: ethyl acetate: water volume ratio 8:1:1 as developing agent to develop FOS, fructotriose standard (DP3), and B-FOS.

[0040] Figure 2 TLC analysis results of the developing agent n-butanol: n-propanol: water = 1:6:1 on the reaction liquid of the synthesized B-FOS of the present application; FOS represents the development effect of the FOS solution, and B-FOS represents the development effect of the B-FOS reaction liquid.

[0041] Figure 3 The infrared absorption spectrum of the fructooligosaccharide butyrate of the present application.

[0042] Figure 4Flight mass spectrum of fructo-oligosaccharide butyrate of the present application; wherein A is the flight mass spectrum of FOS, B is the flight mass spectrum of B-FOS, and C is the flight mass spectrum of A-FOS.

[0043] Figure 5 B-FOS of the present application 1 H nuclear magnetic resonance analysis spectrum.

[0044] Figure 6 Total sugar content determination result of fructo-oligosaccharide butyrate of the present application.

[0045] Figure 7 TLC result diagram of fructo-oligosaccharide butyrate (B-FOS) reaction liquid eluted by n-propanol system in isocratic elution, wherein A is the TLC result diagram, B is a schematic diagram of SPE column method; 1-10 are sequential numbers of eluent of 10 mL as a unit.

[0046] Figure 8 TLC result of fructo-oligosaccharide butyrate (B-FOS) reaction liquid eluted by n-propanol system in gradient elution, wherein 2, 3, 5-10 are sequential numbers of eluent of 10 mL as a unit.

[0047] Figure 9 TLC result diagram of fructo-oligosaccharide butyrate (B-FOS) reaction liquid eluted by n-propanol system in gradient elution, wherein 1-5, 8, 10, 12, 13-18, 20, 22, 24, 25, 27, 29, 31, 33, 35 are sequential numbers of eluent of 4 mL as a unit.

[0048] Figure 10 TLC detection result of fructo-oligosaccharide butyrate prepared in No. 2 and No. 4 of Example 4 of the present application.

[0049] Figure 11 In vitro fermentation gas production and pH change result of fructo-oligosaccharide butyrate; wherein A is gas production change, B is pH change; C is a blank control group, F is a FOS control group, FB is a FOS + n-butyric acid group, BF is a B-FOS group, and AF is an A-FOS group.

[0050] Figure 12 In vitro fermentation total sugar content change of fructo-oligosaccharide butyrate of the present application; wherein C is a blank control group, F is a FOS control group, FB is a FOS + n-butyric acid group, BF is a B-FOS group, and AF is an A-FOS group; the value in the diagram represents mean ± SEM, n = 3.

[0051] Figure 13The short-chain fatty acid content of the in-vitro fermentation liquid of the fructooligosaccharide butyrate of the present application; A is acetic acid, B is propionic acid, C is butyric acid, D is isobutyric acid, E is valeric acid, F is isovaleric acid, G is caproic acid, and H is the total short-chain fatty acid content; the values in the figure represent the mean ± SEM, the results are analyzed by One-way ANOVA and Duncan multiple comparison, n = 3, different letters represent significant differences, p < 0.05, and ns represents no significant difference.

[0052] Figure 14 The beta-diversity results of the microorganisms in the fermentation liquid of the present application; A is Weighted unifrac, B is Bray curtis, and C is Euclidean; one point in the figure represents one sample, different colors of different groups, and the farther the distance between samples, the greater the difference in microbial community composition.

[0053] Figure 15 The relative abundance and composition structure of the microorganisms at the door level in the in-vitro fermentation of the fructooligosaccharide butyrate of the present application; A-I are the differences in the relative abundance of microorganisms at the door level in the fermentation liquid, and J is the column chart of the relative abundance of microorganisms at the door level in the fermentation liquid; the values in the figure represent the mean ± SEM, the results are analyzed by One-way ANOVA and Duncan multiple comparison, n = 3, different letters represent significant differences, p < 0.05.

[0054] Figure 16 The relative abundance and composition structure of the microorganisms at the genus level in the in-vitro fermentation of the fructooligosaccharide butyrate of the present application; A-I are the differences in the relative abundance of microorganisms at the genus level in the fermentation liquid, and J is the column chart of the relative abundance of microorganisms at the genus level in the fermentation liquid; the values in the figure represent the mean ± SEM, the results are analyzed by One-way ANOVA and Duncan multiple comparison, n = 3, different letters represent significant differences, p < 0.05.

[0055] Figure 17 The effect of the fructooligosaccharide butyrate B-FOS of the present application on the growth curve of probiotics; A is the effect of B-FOS on the growth curve of Bifidobacterium lactis BB-12, and B is the effect of B-FOS on the growth curve of Lactobacillus casei strain Shirota.

[0056] Figure 18 The intervention flowchart of the IBD mouse modeling of the present application.

[0057] Figure 19 The body weight changes of the mice on the 0th, 8th, 12th, and 20th days of modeling.

[0058] Figure 20Disease activity index score of mice in the present application; the value in the figure represents the mean ± SEM, the results were analyzed using One-way ANOVA and Duncan multiple comparison, n = 6-12, different letters represent significant differences, p < 0.05, ns represents no significant difference.

[0059] Figure 21 Colon length change of mice in the present application; wherein A is the colon length statistics of mice in each group, B is the colon dissection of mice in each group; the value in the figure represents the mean ± SEM, n = 12, the results were analyzed using One-way ANOVA and Duncan multiple comparison, different letters represent significant differences, p < 0.05.

[0060] Figure 22 H&E staining and inflammation score of colon tissue of mice in the present application; wherein A is H&E staining of colon tissue section, B is inflammation score of colon tissue section, C is colon wall thickness.

[0061] Figure 23 Relative expression level of inflammatory factor mRNA of colon tissue of mice in the present application; wherein A is tumor necrosis factor alpha (TNF-α), B is interleukin 1 beta (IL-1β), C is interleukin 6 (IL-6), D is interleukin 13 (IL-13), E is interleukin 18 (IL-18), F is interleukin 10 (IL-10).

[0062] Figure 24 Alcian blue staining of colon tissue section and intestinal mucosa change in the present application; wherein A is alcian blue staining of colon tissue section, B is relative proportion of mucus in colon tissue section, C is colon crypt depth.

[0063] Figure 25 Relative expression level of tight junction protein mRNA of colon tissue of mice in the present application, wherein A is claudin-1, B is claudin-2, C is zonula occludens-2 (ZO-2).

[0064] Figure 26 SCFAs content of colon contents of mice in the present application; wherein A is acetic acid, B is propionic acid, C is butyric acid, D is isobutyric acid, E is valeric acid, F is isovaleric acid, G is total short-chain fatty acid content.

[0065] Figure 27 Alpha-diversity of microorganisms in colon contents of mice in the present application; wherein A is Chao1 index, B is Simpson diversity index, C is Shannon diversity index.

[0066] Figure 28Figure 1. Microbial composition of the colon content; wherein A is PCoA plot of the mouse gut microbiota, B is relative abundance of gut microbiota at the phylum level, C is relative abundance of gut microbiota at the genus level, n = 6.

[0067] Figure 29 Figure 1. Microbial composition of the colon content; wherein A is PCoA plot of the mouse gut microbiota, B is relative abundance of gut microbiota at the phylum level, C is relative abundance of gut microbiota at the genus level, n = 6.

[0068] Figure 30 Figure 1. Microbial composition of the colon content; wherein A is PCoA plot of the mouse gut microbiota, B is relative abundance of gut microbiota at the phylum level, C is relative abundance of gut microbiota at the genus level, n = 6. DETAILED DESCRIPTION

[0069] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0070] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0071] Fructooligosaccharides: Quantum Hi-Tech (China) Biological Co., Ltd., batch number: FOSP95S;

[0072] Inulin: Vifor Group Ltd., batch number: NanoST P90.

[0073] Example 1. Preparation of fructooligosaccharide butyrate

[0074] 1. Enzymatic synthesis of fructooligosaccharide butyrate

[0075] Accurately weigh 1 g of fructooligosaccharides into a round-bottom flask, and add a clean and dry magnetic stirring rotor. Add 10 mL of anhydrous pyridine to the flask, and stir on a magnetic stirrer until the fructooligosaccharides are dissolved. Add 1 g of immobilized lipase TLIM to the flask, and accurately measure 2.36 mL of vinyl butyrate into the reaction system. Finally, place the round-bottom flask in a constant-temperature water bath magnetic stirrer, and react at 55°C for 48 h. After the reaction is completed, the sample in the round-bottom flask is filtered under suction, and the immobilized lipase TLIM is separated, to obtain the enzyme method of fructooligosaccharide butyrate (Butyric-FOS, B-FOS) reaction solution.

[0076] 2. Enzymatic synthesis of inulin butyrate

[0077] Accurately weigh 1 g inulin into a round-bottom flask, and add a clean and dry magnetic stirring rotor. Add 10 mL of anhydrous pyridine to the flask, and place it on a magnetic stirrer to stir until the inulin is dissolved. Add 1 g of immobilized lipase TLIM to the flask, and accurately measure 2.36 mL of vinyl butyrate and add it to the reaction system. Finally, place the round-bottom flask in a constant-temperature water bath magnetic stirrer, and react at 55°C for 48 h. After the reaction is completed, perform suction filtration on the sample in the round-bottom flask, separate the immobilized lipase, and obtain the enzyme method inulin butyrate (Butyric-Inulin, B-Inulin) reaction solution.

[0078] Comparative Example 1. Preparation of polyfructose butyrate

[0079] 1. Acid anhydride method for preparing fructooligosaccharide butyrate

[0080] Accurately weigh 1 g of fructooligosaccharide into a round-bottom flask, and add a clean and dry magnetic stirring rotor. Add 10 mL of anhydrous pyridine to the flask, and place it on a magnetic stirrer to stir until the fructooligosaccharide is dissolved. After the fructooligosaccharide is completely dissolved, add 5 mL of butyric anhydride to the reaction system. Finally, place the round-bottom flask in a constant-temperature magnetic stirrer, and react at 25°C for 48 h to obtain the acid anhydride method fructooligosaccharide butyrate (n-Butyric anhydride-FOS, A-FOS) reaction solution.

[0081] 2. Acid anhydride method for preparing inulin butyrate

[0082] Accurately weigh 1 g of inulin into a round-bottom flask, and add a clean and dry magnetic stirring rotor. Add 10 mL of anhydrous pyridine to the flask, and place it on a magnetic stirrer to stir until the inulin is dissolved. After the inulin is completely dissolved, add 5 mL of butyric anhydride to the reaction system. Finally, place the round-bottom flask in a constant-temperature magnetic stirrer, and react at 25°C for 48 h to obtain the acid anhydride method inulin butyrate (n-Butyric anhydride-Inulin, A-Inulin) reaction solution.

[0083] Example 2. Identification of fructooligosaccharide butyrate

[0084] 1. Thin layer chromatography (TLC) identification of fructooligosaccharide butyrate

[0085] (1) Method

[0086] The silica gel thin layer chromatography plate was dried by hot air and reserved. Using 0.05mm capillary, FOS pyridine solution as control, and oligofructose butyrate reaction liquid together in the thin layer plate end about 5mm place for sample. Respectively, dichloromethane (DCM): methanol (MT) volume ratio 9:1, 1:1, 3:7, 1:9, 1:50 and n-butanol: n-propanol: water volume ratio 1:6:1, n-butanol: isopropanol: water: acetic acid volume ratio 7:5:4:2, n-butanol: acetic acid: water volume ratio 4:1:5, n-butanol: acetic acid: water volume ratio 8:3:2, n-propanol: ethyl acetate: water volume ratio 8:1:1 as developing agent, pour the developing agent into the chromatography cylinder, so that the liquid level in the chromatography cylinder is about 3mm. Carefully put the sample thin layer chromatography plate into the developing cylinder, when the solvent front reaches about 5mm at the top, take out the chromatography plate. After the developing agent is evaporated, evenly spray α-naphthol solution on the thin layer chromatography plate, and high temperature makes the sugar on the chromatography plate purple.

[0087] (2) Results

[0088] The TLC results of oligofructose butyrate reaction liquid with different proportions of developing agent are shown in Figure 1 ; A is the result of using different volume ratios of dichloromethane and methanol developing agent to develop FOS and B-FOS, B (1) n-butanol: n-propanol: water volume ratio 1:6:1, (2) n-butanol: isopropanol: water: acetic acid volume ratio 7:5:4:2, (3) n-butanol: acetic acid: water volume ratio 4:1:5, (4) n-butanol: acetic acid: water volume ratio 8:3:2, (5) n-propanol: ethyl acetate: water volume ratio 8:1:1 as developing agent to develop FOS, fructo-oligosaccharide (DP3) standard and B-FOS.

[0089] Oligofructose butyrate with butyric acid due to the existence of ester bond, its polarity is smaller than that of oligofructose, and the higher the degree of esterification, the smaller the polarity of oligofructose butyrate, and the higher the position on the silica gel chromatography plate.

[0090] Figure 1 Figure A shows that when DCM: MT = 9:1 is used as developing agent, the position of B-FOS synthesis liquid is still at the sample point, indicating that the polarity of the developing agent is much smaller than that of FOS and B-FOS, and the other four ratios of DCM and MT cannot separate the B-FOS reaction liquid, all with tailing phenomenon. Figure 1 Figure B shows that (2)-(4) cannot separate FOS and B-FOS, and cannot separate FOS and B-FOS with different polymerization degrees, while Figure 1Although B(5) appears to separate FOS from B-FOS, it results in abnormal coloring of one component of FOS (fructotriose). Only a developing solvent of n-butanol:n-propanol:water = 1:6:1 can separate the synthesized B-FOS from FOS, and also separate samples with different degrees of esterification. Similarly, the inulin butyrate reaction solution prepared in Example 1 can only separate the synthesized B-Inulin from Inulin using a developing solvent of n-butanol:n-propanol:water = 1:6:1.

[0091] However, due to the excessive esterification of A-FOS and A-Inulin prepared in Comparative Example 1, the samples shifted to the solvent front after using the above-mentioned developing solvent, making it difficult to determine the appropriate ratio of developing solvent.

[0092] The TLC analysis results of the synthesized B-FOS reaction solution using n-butanol:n-propanol:water = 1:6:1 as the developing solvent are as follows: Figure 2 As shown; FOS represents the development effect of FOS solution, and B-FOS represents the development effect of B-FOS reaction solution.

[0093] from Figure 2 It can be observed that, in addition to FOS, other products are generated in the B-FOS reaction solution development diagram, and the products are not unique. The products may be oligofructose monoesters with different degrees of polymerization, or they may be oligofructose diesters, etc. This indicates that the B-FOS prepared in this invention is a mixture.

[0094] 2. Isolation and purification of fructooligosaccharide butyrate

[0095] (1) Isolation and purification of B-FOS

[0096] The B-FOS reaction solution obtained by the enzymatic method in Example 1 was evaporated to a solid state using a vacuum rotary evaporator to remove most of the pyridine from the reaction solution. A 5% (v / v) methanol aqueous solution was prepared, and the solid was added to a round-bottom flask, continuously agitated and stirred to ensure uniform dispersion in the methanol aqueous solution.

[0097] The reaction solution was separated using C18 column chromatography. The C18 column was first washed with 5% acidified methanol (5% formic acid and 95% methanol) and then the residual acidified methanol was replaced with 5% methanol in water. The sample suspension dispersed in 5% methanol in water was carefully added to the column and eluted with 3 column volumes of 5% methanol in water to remove the residual pyridine, unreacted oligofructose and vinyl butyrate in the sample. Pyridine is colored under UV light, so when the eluent no longer colored under UV light, it means that the pyridine was removed. Finally, the sample was eluted with methanol and the yellow eluent collected. The collected eluent was identified by TLC using n-butanol:n-propanol:water = 1:6:1 as the developing agent and the eluent with FOS removed was combined. The collected sample eluent was dried using a vacuum rotary evaporator, and then pure water was added to the flask to dissolve the yellow sample. The sample was transferred to a centrifuge tube and the water removed using a vacuum freeze dryer. The yellow solid obtained was purified B-FOS. Oligofructose has different degrees of polymerization and the oligofructose butyrate obtained by the present application has different degrees of esterification. The reaction solution of inulin butyrate (B-Inulin) prepared in Example 1 was separated and purified using the separation and purification method of B-FOS to obtain purified B-Inulin.

[0098] (2) Separation and purification of A-FOS

[0099] The A-FOS reaction solution obtained by the anhydride method of Comparative Example 1 was placed in a 500 Da dialysis bag and dialyzed in pure water for about two days. The water was changed frequently on the first day to accelerate the exchange of the FOS pyridine solution in the dialysis bag with the pure water outside the dialysis bag. The water change frequency was reduced on the second day to ensure that the FOS pyridine solution was completely removed. The dialysis was stopped when the pyridine was completely removed. The A-FOS sample in the dialysis bag was collected and dissolved in a certain amount of anhydrous ethanol. The ethanol and water in the A-FOS were completely removed using a vacuum rotary evaporator to obtain a thick yellowish transparent liquid, which was purified A-FOS. The reaction solution of inulin butyrate (A-Inulin) prepared in Comparative Example 1 was purified using the separation and purification method of A-FOS to obtain purified A-Inulin.

[0100] 3. Infrared spectrum identification of oligofructose butyrate

[0101] The chemical bonds in the above purified B-FOS, purified A-FOS and raw material FOS were analyzed and compared by Fourier infrared spectrometer (FT-IR).

[0102] Take 1 mg of purified B-FOS sample, purified A-FOS sample after freeze-drying, respectively, and grind with 100 mg of dry potassium bromide solid in a grinder to a powder. Use a tablet press to press the sample powder, and use a Fourier infrared spectrometer (FT-IR) to scan the infrared absorption spectrum of the tablet in the 4000-500 cm -1 range.

[0103] The infrared absorption spectrum of the oligofructose butyrate is shown in Figure 3 .

[0104] Figure 3 It can be seen that the peak at 3350 cm -1 is the stretching vibration absorption peak of the hydroxyl group (-OH) present in large quantities in purified A-FOS, purified B-FOS and FOS, indicating that the basic properties of the synthetic product have not been changed, and the absorption intensity of A-FOS is lower due to the substitution of butyric acid; the peak at 2950 cm -1 is the characteristic absorption peak of the saturated C-H of the sugar ring, and the peak of A-FOS and B-FOS is larger than that of FOS due to the connection of butyric acid; compared with FOS, A-FOS and B-FOS both appear a characteristic absorption peak of ester bond (C=O) at 1730 cm -1 , and the absorption peak intensity of the ester bond of A-FOS is greater than that of B-FOS; 1600-1630 cm -1 is the stretching vibration characteristic absorption peak of the carbonyl group (COO-); 1300-1500 cm -1 is the bending vibration group (OCH, COH, CCH); 800-1200 cm -1 is the stretching vibration of the glycosidic bond.

[0105] An important indicator of whether the oligofructose butyrate is successfully synthesized is whether the ester bond formed by the combination of FOS and butyric acid is present in the product. From Figure 3 , it can be observed that in addition to the characteristic absorption peak of the ester bond at 1730 cm -1 , the two curves are highly similar. Therefore, the ester bond formed by the successful combination of FOS and butyric acid is present in the product, indicating that oligofructose butyrate B-FOS and A-FOS are synthesized, and the esterification degree of A-FOS is higher than that of B-FOS.

[0106] Using infrared spectroscopy, it is found that the ester bond formed by the successful combination of Inulin and butyric acid is present in the purified B-Inulin and purified A-Inulin products, indicating that the inulin butyrate is synthesized.

[0107] 4. Oligofructose butyrate mass spectrometry analysis

[0108] Before analysis, the sample was mixed with a small molecule matrix solution and added to the target plate, and after the solvent was volatilized, a co-crystal was formed. Referring to the method of Tian et al., the purified oligofructose butyrate sample was analyzed using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS) Ultraflextreme workstation. A nitrogen laser with a wavelength of 337 nm was used to obtain the mass spectrum in positive mode, and after a delay extraction of 200 ns, the ions were accelerated to a kinetic energy of 12 kV and detected using a reflectron mode. The sample was desalted using AG 50W-X4 resin, and 1 μL of desalted sample was added to 1 μL of 10 mg / mL 2,5-dihydroxybenzoic acid in 50% acetonitrile solution (v / v) on a MALDI plate.

[0109] The oligofructose butyrate flight mass spectrum is shown in Figure 4 ; wherein A is the flight mass spectrum of FOS, B is the flight mass spectrum of B-FOS, and C is the flight mass spectrum of A-FOS.

[0110] As shown in Figure 4 , according to the molecular ion peaks of m / z 527, 689, 851 and 1013 [M+Na] + , the true molecular weight of FOS should be 504, 666, 828 and 990, respectively, indicating that the FOS used in this experiment is mainly composed of GF2, GF3, GF4 and GF5; Figure 4 As shown in , the molecular ion peaks of the synthesized product B-FOS are 597, 667, 759, 829, 921, 991, 1083 [M+Na] + , which are 70 or 140 more than the molecular weight of FOS, i.e. the molecular weight of 1 or 2 butyric acid (B) after dehydration. Combined with the FT-IR spectrum, it is considered that B-FOS is formed by connecting 1 or 2 butyric acids through an ester bond with different degrees of polymerization of sugars in FOS.

[0111] The intensity of the molecular ion peak indicates that GF3 is the main component of FOS, followed by GF2, GF4 and GF5, and the proportion of each component in FOS is about 44.89%, 39.23%, 11.80% and 4.08%, respectively; the highest content of sugar in the purified B-FOS is also GF3, but the second highest sugar is changed from GF2 to GF4. Further analysis Figure 4In the middle B, it was found that the ratio of FOS with 1 butyric acid and FOS with 2 butyric acids was about 83.45:16.55, and the main components from high to low were: GF3-B (43.59%), GF4-B (25.41%), GF3-2B (8.57%), GF2-B (7.86%), GF5-B (6.59%), GF4-2B (5.29%), GF2-2B (1.50%), and GF5-2B (1.19%). In addition, no FOS was detected in Figure 4 In the middle B, FOS-related molecular ion peaks were detected, indicating that FOS was basically removed during the purification of B-FOS.

[0112] Comparison Figure 4 As can be seen from middle B and C, A-FOS is much more complex than B-FOS, and the components with the highest proportion in A-FOS are GF3-10B, GF3-11B, and GF3-9B. Different degrees of FOS tend to combine with different numbers of butyric acids. GF2 preferentially combines with 8 butyric acids, GF3 preferentially combines with 10 butyric acids, and GF4 preferentially combines with 12 butyric acids. The degree of esterification is much higher than that of B-FOS.

[0113] 5. Analysis of oligofructose butyrate by nuclear magnetic resonance hydrogen spectrum

[0114] 50 mg of dry and purified B-FOS sample was weighed and dissolved in 500 μL of deuterium water (D2O). After mixing, it was added to a nuclear magnetic tube. Nuclear magnetic detection 1 (H NMR) used Brüker Advance 400 1 (H: 400 MHz), and the chemical shift (δ) was in ppm, with the residual solvent peak of deuterated reagent as the reference (for example 1 (H NMR: D2O, 4.79). The 1 H nuclear magnetic resonance analysis spectrum is shown in Figure 5 .

[0115] Figure 5 It is shown that both FOS and B-FOS have characteristic C-H peaks on the fructose ring skeleton in the range of 3.5-5.5 ppm. Compared with FOS, absorption peaks appeared at 1.0 ppm, 1.5 ppm, and 2.2 ppm in the spectrum of B-FOS, which are the absorption peaks of -CH3, -CH2, and -CH2 on butyryl, indicating that butyryl is successfully connected to the fructose ring R group of FOS. Since the products of A-FOS in MALDI-TOF MS are multiple, the degree of esterification is different, and the solubility is poor, no 1 HNMR analysis was performed on A-FOS.

[0116] 6. Determination of total sugar content of oligofructose butyrate

[0117] The total sugar content (w / w) of FOS, purified B-FOS, and purified A-FOS was determined using the phenol-sulfuric acid method.

[0118] (1) Sample hydrolysis

[0119] Accurately weigh 10 mg of sample powder or liquid into a K-max tube and place it in an ice bath. While shaking, add 0.45 mL of 72% concentrated sulfuric acid and heat in a 30°C water bath for 1 hour, shaking the tube every 20 minutes to ensure the lyophilized sample powder reacts fully with the concentrated sulfuric acid. After the tube cools, add 4.95 mL of distilled water, shake well, and heat in a 100°C metal bath for 3 hours, shaking the tube every 20 minutes. After the reaction is complete, cool to room temperature.

[0120] (2) Construction of standard curve

[0121] Add 0, 10, 20, 30, 40, 50, and 60 μL of 150 μg / mL glucose standard solution to test tubes, respectively, and then replenish each tube with 2.5% phenol solution to a final volume of 200 μL. Shake to mix. Add 0.5 mL of concentrated sulfuric acid, mix well, and cool to room temperature. Measure the absorbance at 490 nm. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa.

[0122] (3) Determination of total sugar in samples

[0123] Dilute the hydrolyzed sample solution 5–10 times, add 40 μL of the diluted sample solution and 160 μL of 2.5% phenol solution to a test tube, and shake to mix. Measure the absorbance of the mixture at 490 nm according to step (2). Calculate the total sugar content of the sample using a standard curve.

[0124] The results of the determination of total sugar content of fructooligosaccharide butyrate are as follows: Figure 6 As shown, Figure 6 The data shows that FOS has a sugar content of 95.33±0.79%, B-FOS has a sugar content of 82.08±4.23%, and A-FOS has the lowest sugar content at 31.17±2.73%. The lower sugar content of B-FOS and A-FOS compared to FOS is due to the presence of butyric acid in B-FOS and A-FOS, which constitutes a larger proportion of the total sugar content than FOS. Compared to B-FOS, A-FOS has a higher degree of esterification, and butyric acid constitutes a larger proportion of the A-FOS mass. Therefore, in terms of total sugar content by mass percentage, A-FOS has a lower sugar content than B-FOS.

[0125] Example 3: Effect of different solvents on the preparation of fructooligosaccharide butyrate

[0126] Using dimethyl sulfoxide (DMSO), anhydrous pyridine, N,N-dimethylformamide (DMF), tert-butyl alcohol and ethanol as solvents for FOS, it was found that FOS could be dissolved in DMSO, anhydrous pyridine and DMF, but hardly soluble in tert-butyl alcohol and ethanol.

[0127] Using DMSO, anhydrous pyridine and DMF respectively, and using the method of Example 1, FOS butyrate solution was prepared. The reaction liquid was analyzed by TLC using n-butanol:n-propanol:water = 1:6:1 as the developing agent. It was found that anhydrous pyridine would not react with the color developing agent used in TLC, while DMSO would react with the color developing agent used in TLC and was more difficult to remove. At the same time, pyridine was easier to remove, so it was more advantageous than DMSO and DMF as a solvent.

[0128] Effect of separation method on separation and purification of FOS butyrate reaction liquid

[0129] 1. Separation and purification by SPE column combined with eluent n-propanol, n-butanol and water

[0130] (1) Using a SPE column packed with silica gel as a chromatographic column, and using n-propanol:n-butanol:water with a volume ratio of 1:6:1 as the mobile phase, the sample was isocratically eluted, and the FOS butyrate reaction liquid prepared in Example 1 was separated and purified. The eluent was collected in units of 10 mL and numbered in order as 1-10, and the sample was identified by TLC method. The TLC result of isocratic elution of FOS butyrate (B-FOS) reaction liquid in n-propanol system is shown in Figure 7 , wherein A is the TLC result, B is the schematic diagram of SPE column method, and 1-10 are the sequential numbers of the eluent in units of 10 mL. Figure 7 The results showed that FOS and the synthetic product FOS ester could not be separated.

[0131] (2) Gradient elution was performed on the SPE column using n-butanol, n-propanol and water as the mobile phase. The elution gradient is shown in Table 1. The eluent was collected in units of 10 mL and numbered in order as 1-10, and the sample was identified by TLC method. The TLC result of gradient elution of FOS butyrate (B-FOS) reaction liquid in n-propanol system is shown in Figure 8 , wherein 2, 3, 5-10 are the sequential numbers of the eluent in units of 10 mL. Figure 8 The results showed that gradient elution also failed to separate the sugar ester from the raw material sugar, but due to the limitation of the amount of sample, the concentration was low, and the sugar ester was not obvious in color.

[0132] Table 1

[0133]

[0134] (3) Adjust the proportion of n-butanol, n-propanol and water in the mobile phase, and perform gradient elution on the silica gel column SPE column. The elution gradient is shown in Table 2. Collect the eluate in units of 4 mL and number them in order as 1-35. Identify using the TLC method. The TLC result diagram of the oligofructose butyrate (B-FOS) reaction liquid in the n-propanol system gradient elution is shown in Table 2, wherein 1-5, 8, 10, 12, 13-18, 20, 22, 24, 25, 27, 29, 31, 33, 35 are the sequential numbers of the eluate in units of 4 mL. Figure 9 Figure 9 The results are similar to those in the comparative example 1 (2), indicating that the gradient elution of the mobile phase n-butanol, n-propanol and water in Table 2 cannot separate the sugar ester from the sugar.

[0135] Table 2

[0136]

[0137] 2. Separation and purification by activated carbon and diatomite

[0138] (1) Pretreatment of activated carbon: soak the activated carbon in 1% hydrochloric acid by volume overnight, filter, then wash the activated carbon with deionized water until it is neutral, dry and cool for use;

[0139] (2) Weigh 2:1 of pretreated activated carbon and diatomite by mass, and mix well;

[0140] (3) Column the mixture of (2), and add the oligofructose butyrate reaction liquid prepared in Example 1 to the chromatographic column. When the reaction liquid reaches the activated carbon and diatomite, close the piston;

[0141] In actual experiments, due to the slow flow rate of the eluate in the packing material and the long experimental period, it is difficult to screen multiple experimental conditions, so this method is abandoned.

[0142] 3. Anion column

[0143] DEAE-Sepharose FF (DEAE agarose gel) is selected. It exists in part or whole as an anion in a strong alkaline mobile phase, can be retained and separated on an anion exchange column. To improve the elution effect, water, sodium hydroxide and sodium acetate are used for ternary gradient elution. However, in the actual operation process, the addition of strong alkali will cause the degradation of ester materials. And pyridine cannot be completely removed, resulting in experimental failure.

[0144] Example 4 Influence of reaction raw materials on preparation of oligofructose butyrate

[0145] ​Using the method of Example 1, in accordance with the following Table 3, the reaction raw materials were replaced to prepare fructo-oligosaccharide butyrate respectively.

[0146] Table 3 Reaction raw materials

[0147]

[0148] The yield of fructo-oligosaccharide butyrate in the reaction solution was detected by thin layer chromatography (TLC) detection conditions in Example 2. The results showed that the yield of fructo-oligosaccharide butyrate prepared in No. 3 and No. 4 was significantly higher than that in No. 1 and No. 2, indicating that it was more advantageous to use vinyl butyrate as a raw material to prepare fructo-oligosaccharide butyrate. The yield of fructo-oligosaccharide butyrate prepared in No. 3 was lower than that in No. 4, and the immobilized lipase TLIM was more cost-effective than lipase 435, and the former was not so demanding on water in the reaction, so it was more advantageous to use immobilized lipase TLIM as a raw material. The TLC detection results of fructo-oligosaccharide butyrate prepared in No. 2 and No. 4 are shown in Figure 10 Figure 10 It is shown that the yield of fructo-oligosaccharide butyrate prepared from vinyl butyrate is higher.

[0149] Using the method of Example 1, in accordance with the following Table 3, the reaction raw materials were replaced to prepare inulin butyrate respectively. The results showed that the yield of inulin butyrate prepared from vinyl butyrate was higher.

[0150] Example 5 In vitro fermentation characteristics and probiotic activity of fructo-oligosaccharide butyrate

[0151] 1. Construction of in vitro simulated colon fermentation model

[0152] Reference Yang et al. (Yang Z X, Huang T, Li P, et al. Dietary fiber modulates the fermentation patterns of cyanidin-3-o-glucoside in a fiber-type dependent manner [J]. Foods, 2021, 10(6).) to construct an in vitro simulated colon fermentation model, and prepare the culture medium and inoculate the bacterial solution.

[0153] ​The samples were purified oligofructose butyrate B-FOS and A-FOS prepared according to Example 2. The SIEM medium was placed in an anaerobic operation platform 12 h in advance for oxygen replacement until the color of the medium changed from red to yellow, mixed with an appropriate amount of sample before inoculation, and dissolved by shaking. A total of 5 groups were set up, namely, a blank control group (group C), a FOS control group (group F), a FOS + n-butyric acid group (group FB), an enzymatically synthesized oligofructose butyrate group (B-FOS, group BF), and an acid anhydride synthesized oligofructose butyrate group (A-FOS, group AF), with 3 parallel samples for each time point in each group. On the basis of the sugar content of FOS, in order to ensure the same concentration of samples in the fermentation broth, the total sugar content was consistent among the groups as a standard, so that the final concentration of the fermentation broth was 6 mg / mL. The amount of butyric acid added in the FB group was equivalent to the amount of butyric acid contained in the B-FOS under the condition of equal sugar content. Fresh fecal bacteria solution was immediately transferred to the anaerobic operation platform for inoculation, and the medium with the sample and the fecal bacteria solution were added to the anaerobic fermentation tube at a ratio of 9:1 by volume, and then mixed evenly. Subsequently, it was placed in a constant temperature shaking incubator at 37°C for culture, and the fermentation broth was taken out at 0, 6, 12, 24, and 36 h, respectively, and then divided and stored in a -80°C refrigerator.

[0154] 2. Measurement of gas production and pH of fermentation broth

[0155] A sterile syringe was directly inserted into the rubber plug on the fermentation tube to detect and record the gas production in the fermentation tube at different times.

[0156] The pH of the fermentation broth at different times was measured and recorded using a micro pH meter.

[0157] The results of the gas production and pH changes of oligofructose butyrate in vitro fermentation are shown in Figure 11 A and B, respectively; C is the blank control group, F is the FOS control group, FB is the FOS + n-butyric acid group, BF is the B-FOS group, and AF is the A-FOS group.

[0158] Figure 11 It is shown that the cumulative gas volume in the fermentation tube generally increased as the in vitro fermentation proceeded, and the pH of the fermentation broth showed a downward trend. Figure 11 It is shown in Fig. A that during 0-12 h of in vitro fermentation, the cumulative gas production in the fermentation tube gradually increased, especially in groups F and BF, which reached 2.52 mL and 2.85 mL, respectively. However, the gas production in groups C and AF was not obvious, and the cumulative gas production in group FB was significantly lower than that in group F. When the fermentation proceeded to 12 h, groups F and FB stopped producing gas, and the gas production rate in group B-FOS also decreased significantly, but it was still being degraded and producing gas. When the fermentation ended, the cumulative gas production in group BF was the highest, reaching 4.58 mL, followed by group F, which was 2.66 mL.

[0159] Figure 11 As shown in FIG. 3B, the pH of the C group and the AF group had little change during fermentation, and the pH of the FB group, the F group and the BF group decreased continuously during 0-12 h of in vitro fermentation. Among them, the pH of the FB group and the F group decreased most rapidly, reaching 3.91 and 4.05, respectively, when the fermentation was carried out for 12 h. The pH of the fermentation broth of the BF group was slightly higher than that of the FB group and the F group, reaching 4.82. After 12 h, the pH of the fermentation broth of each group no longer changed significantly.

[0160] 3. Determination of total sugar content of fermentation broth

[0161] (1) Hydrolysis of sample

[0162] Accurately take 1 mL of fermentation broth into a K-max tube and freeze-dry. Place the freeze-dried dry K-max glass tube in an ice bath, add 0.45 mL of 72% concentrated sulfuric acid while shaking, heat in a 30°C water bath for 1 h, and shake the reaction tube every 20 min to ensure that the sample freeze-dried powder is fully reacted with concentrated sulfuric acid. After cooling the test tube, add 4.95 mL of distilled water, shake uniformly, heat in a 100°C metal bath for 3 h, shake the reaction tube every 20 min, and cool to room temperature after the reaction is completed.

[0163] (2) Preparation of standard curve

[0164] The same as 6(2) of Example 2.

[0165] (3) Determination of total sugar of sample

[0166] The same as 6(3) of Example 2.

[0167] (4) Results

[0168] The change of total sugar content of in vitro fermentation of fructo-oligosaccharide butyrate is shown in FIG. 4B; wherein C is the blank control group, F is the FOS control group, FB is the FOS+butyric acid group, BF is the B-FOS group, and AF is the A-FOS group. The values in the figure represent the mean ± SEM, n=3. Figure 12

[0169] Figure 12 ​As shown, the total sugar content in the fermentation broth showed a general downward trend as the in vitro fermentation proceeded. In the first 12 h of fermentation, the sugar in the fermentation broth was rapidly utilized by the microorganisms, and the total sugar content decreased significantly. The total sugar degradation rate in the F group and the FB group was similar, and both were more rapid than that in the BF group, which was almost completely degraded at 12 h. When the fermentation proceeded to 36 h, the total sugar content in the fermentation broth in the F group and the FB group was only 1.11 mg / mL and 1.77 mg / mL, respectively, and about 89.13% and 86.56% of the total sugar was degraded, respectively. The sugar in the BF group fermentation broth was still being degraded by the microorganisms at 12 h, but the degradation rate was greatly reduced compared to before, and about 74.47% of the sugar in the fermentation broth was ultimately degraded. This indicates that B-FOS promotes the growth of more gas-producing bacteria in the fermentation broth, and compared to FOS, B-FOS can be more slowly fermented by the intestinal flora, thereby helping B-FOS to have a sustained effect in the intestinal tract. The initial high concentration of free butyric acid did not have a significant effect on the FOS degradation rate, but it seemed to inhibit the growth of gas-producing bacteria in the fermentation broth. A-FOS was not well utilized by the intestinal flora, and its total sugar degradation rate was slow, with only 18.55% being degraded in 36 h of in vitro fermentation, and the gas production and pH results both showed that it was highly similar to the C group.

[0170] In summary, the above results show that B-FOS has good fermentability in the in vitro fermentation system, and compared to FOS, it can be more stably degraded by the intestinal flora, while A-FOS cannot be well degraded by the intestinal flora.

[0171] Similarly, it was found that the purified B-Inulin prepared in Example 2 and the purified A-Inulin had good fermentability in the in vitro fermentation system, and compared to Inulin, the B-Inulin could be more stably degraded by the intestinal flora, while the A-Inulin could not be well degraded by the intestinal flora.

[0172] 4. Measurement of short-chain fatty acids in the fermentation broth

[0173] (1) Sample preparation

[0174] 200 μL of the fermentation sample or 200 μL of the standard was mixed with 200 μL of 0.3 mg / mL diethyl acetic acid (dissolved in 0.2 M hydrochloric acid) and 50 μL of 0.15 M oxalic acid. The mixture was filtered through a 0.22 μm filter membrane into a liquid phase vial for GC analysis.

[0175] (2) Sample measurement

[0176] Gas chromatography was performed using a flame ionization detector (FID) with a CP-FFAP CB column (25m × 0.53mm × 1.00μm, Agilent). Nitrogen was used as the carrier gas. The flow rate was set to 10 mL / min, the make-up gas rate to 30 mL / min, the injection volume to 1 μL, and the injection port temperature to 250℃. The column temperature was set as follows: initial temperature 100℃, ramped up to 160℃ at a rate of 5℃ / min, and held for 4 min. The concentrations of short-chain fatty acids in samples and standards were determined using a splitless injection method.

[0177] (3) Results

[0178] Changes in short-chain fatty acid content in the in vitro fermentation broth of fructooligosaccharide butyrate are as follows: Figure 13 As shown in the figure; A is acetic acid, B is propionic acid, C is butyric acid, D isobutyric acid, E is valeric acid, F isovaleric acid, G is hexanoic acid, and H is the total short-chain fatty acid content; the values ​​in the figure are expressed as mean ± SEM. The results were analyzed using one-way ANOVA and Duncan multiple comparison analysis, n=3, different letters indicate significant differences, p<0.05, and ns indicate no significant differences.

[0179] Figure 13 The results showed that the content of various short-chain fatty acids in the fermentation broth gradually increased with the extension of fermentation time. At 36 hours of fermentation, the FB group had the highest acetic acid yield, followed by the F group. The BF group had the highest propionic acid content in the fermentation broth, significantly higher than the other four groups (p<0.05), exceeding 10 mM, followed by the F and FB groups. Figure 13 It was also easy to observe in group B that, with the extension of fermentation time, the butyric acid production in group BF gradually increased. At 12 hours of fermentation, its butyric acid concentration was significantly higher than the other four groups (p<0.05), while group FB, due to the presence of butyric acid from the beginning, maintained a consistently high butyric acid level. With the extension of fermentation time, the levels of isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid in all groups gradually increased. Group AF showed similar SCFAs production capacity to group C for most of the time. Considering the overall changes in total sugar, it is believed that A-FOS cannot be well degraded and utilized by the gut microbiota in the in vitro fermentation system.

[0180] In summary, FOS, a mixture of FOS and butyric acid, and B-FOS can all be fermented by gut microbiota to produce SCFAs, but A-FOS exhibits poor fermentability. Among these, B-FOS demonstrates a significant butyric acid production capacity under the influence of gut microbiota. Similarly, experiments showed that the purified B-Inulin prepared in Example 2 also exhibited a significant butyric acid production capacity under the influence of gut microbiota.

[0181] 5. 16S rRNA gene sequencing of fermentation broth

[0182] The fermentation broth at 36 h of in vitro fermentation was subjected to 16S rRNA gene sequencing to study the diversity and composition changes of intestinal flora in the fermentation broth, according to the method of Yang et al. (Yang Z X, Huang T, Li P, et al. Dietary fiber modulates the fermentation patterns of cyanidin-3-o-glucoside in a fiber-type dependent manner [J]. Foods, 2021, 10(6).

[0183] (1) In vitro fermentation microbial beta-diversity

[0184] The beta-diversity results of the fermentation broth microorganisms are shown in Figure 14 ; wherein A is Weighted unifrac, B is Bray curtis, and C is Euclidean; one point in the figure represents one sample, the colors of different groups are different, and the farther the distance between samples, the greater the difference in microbial community composition.

[0185] The PCoA two-dimensional graph calculated using A, B and C three different methods shows that the distance between the samples in the experimental group is close, indicating that the experiment has good repeatability. However, the samples in group C and group AF are close in the figure, indicating that the microbial composition proportions of the two groups are similar and have no obvious difference. Except for group C and group AF, the samples between groups are obviously separated and far apart, indicating that FOS, FOS+ n-butyric acid and B-FOS have affected the microbial composition in in vitro fermentation, and the influencing factors are different.

[0186] (2) Changes in microbial composition at the phylum level in vitro fermentation

[0187] The relative abundance and composition structure of phylum-level microorganisms in in vitro fermentation of fructo-oligosaccharide butyrate are shown in Figure 15 ; A-I are the differences in the relative abundance of phylum-level microorganisms in the fermentation broth, and A-I are the columnar graphs of the relative abundance of phylum-level microorganisms in the fermentation broth; the values in the figure represent the mean ± SEM, and the results were analyzed using One-way ANOVA and Duncan multiple comparison, n = 3, and different letters indicate significant differences, p < 0.05.

[0188] Figure 15Figures A and C show that the presence of BF significantly increased the abundance of Bacteroidota (p<0.05) and Firmicutes (p<0.05) in the fermentation broth, followed by group F. Compared with groups C and AF, the relative abundance of Bacteroidota and Firmicutes in group FB was also significantly increased (p<0.05), but the increase was not as significant as that in groups BF and F. Furthermore, it can be observed from the figures that the relative abundance of each microorganism in the fermentation broth of group AF was similar to that of group C.

[0189] from Figure 15 As shown in Figure B, compared with group C, groups F, BF, FB, and AF all significantly inhibited the growth of Proteobacteria (p<0.05), reducing the relative abundance of Proteobacteria in the fermentation broth. This helps restore the gut microbiota composition to a relatively healthy state. Group FB, in particular, showed the most significant improvement, indicating that butyric acid directly inhibits the growth of Proteobacteria. While the effect of B-FOS was not as strong as that of group FB (with butyric acid added directly), it was more pronounced than that of FOS, demonstrating a certain additive effect.

[0190] from Figure 15 As can be seen from the results, the abundance of Actinobacteriota was significantly increased in the FB group, indicating that the presence of FOS and butyric acid may play a role in regulating the composition of the intestinal flora in mammals with enteritis, and can help restore the balance of the intestinal flora. Furthermore, FOS and B-FOS can also play a good role in maintaining the balance of the intestinal flora (p<0.05).

[0191] from Figure 15 As can be seen from E, groups F, FB, and BF significantly reduced the relative abundance of Fusobacteriota in the fermentation broth, which may improve gut health by altering the relative abundance of harmful bacteria in the gut microbiota.

[0192] exist Figure 15 The results showed that groups F, BF, and FB could significantly improve the composition of the microbial community in the fermentation broth and reduce the proportion of Desulfobacterota. In particular, group FB showed that the presence of butyric acid could significantly improve the composition of Desulfobacterota. BF with butyric acid had a similar effect and was more effective than FOS.

[0193] B-FOS can significantly increase the proportion of dominant bacteria in the fermentation broth (p<0.05), reduce the proportion of Proteobacteria containing more pathogenic bacteria and Desulfobacterota containing more opportunistic pathogens in the fermentation broth (p<0.05), and improve the intestinal flora composition structure by increasing Actinobacteriota and reducing Fusobacteriota, so as to change the trend to be beneficial to human health. A number of data and previous studies have shown that the presence of butyric acid in the fermentation broth can make the composition of the microbial flora in the fermentation broth develop in the direction that people expect, but n-butyric acid cannot directly reach the intestine, and B-FOS connected with butyric acid can release more butyric acid in the intestine, so as to make the microbial flora develop in the direction beneficial to human health, and its effect of promoting the growth of beneficial bacteria and inhibiting the growth of harmful bacteria is more obvious than FOS.

[0194] (3) Changes in the composition of in vitro fermented microbial genera

[0195] The relative abundance and composition structure of oligofructose butyrate in vitro fermentation of microbial genera are shown in Figure 16 A-I are the differences in the relative abundance of fermentation broth microbial genera, and J is the column chart of the relative abundance of fermentation broth microbial genera. The values in the figure represent the mean ± SEM, and the results of One-way ANOVA and Duncan multiple comparison analysis are used. n=3, different letters indicate significant differences, p<0.05.

[0196] As can be seen from Figure 16 , the relative abundance of Escherichia-Shigella in the fermentation broth of the FB group is significantly lower than that of the other groups (p<0.05), indicating that the presence of butyric acid has a more obvious inhibitory effect on the growth of harmful bacteria Escherichia-Shigella. The increase of butyric acid is accompanied by the decrease of the relative abundance of Escherichia-Shigella, which is conducive to promoting a more healthy intestinal flora composition structure. FOS also has a certain inhibitory effect on the growth of Escherichia-Shigella in the fermentation broth, and B-FOS connected with butyric acid further enhances this health-promoting effect, exerting the functions possessed by butyric acid.

[0197] From Figure 16 , we can clearly see that FOS, B-FOS and FOS+n-butyric acid can significantly increase the relative abundance of beneficial bacteria Bifidobacterium in the fermentation broth (p<0.05), especially the combination of FOS and butyric acid has a significantly higher growth-promoting effect on Bifidobacterium than all other groups (p<0.05).

[0198] Figure 16Figs. F, H and I show that the relative abundance of Ruminococcus, Faecalibacterium and Collinsella in the fermentation broth of F group, BF group and FB group was significantly higher than that of C group and AF group (p<0.05), and the growth-promoting effect of BF group was significantly higher than that of the other four groups (p<0.05).

[0199] Figure 16 Fig. G shows that B-FOS has the ability to significantly down-regulate the relative abundance of harmful bacteria Sutterella (p<0.05).

[0200] In summary, in the statistics of the relative abundance of microorganism genera in the fermentation broth, it was found that B-FOS could significantly reduce the relative abundance of harmful bacteria Escherichia-Shigella and Sutterella in the system (p<0.05), and the improvement effect was significantly better than that of FOS. B-FOS could significantly increase the relative abundance of a series of butyric acid-producing bacteria such as Bifidobacterium, Ruminococcus, Faecalibacterium and Collinsella in the fermentation broth (p<0.05), and the growth-promoting effect was significantly better than that of all other groups.

[0201] 6. Single-bacterium probiotic activity determination

[0202] (1) Method

[0203] Bifidobacterium lactis BB-12 (B. lactis BB-12) is the most fully researched Bifidobacterium at present, which has the effects of maintaining healthy intestinal flora and improving immunity, and Lactobacillus casei strain Shirota can promote digestive system health, improve diarrhea, constipation, prevent digestive tract infection and improve immunity, both of which are relatively representative probiotics. Therefore, we co-cultured Bifidobacterium lactis BB-12 and Lactobacillus casei strain Shirota with the synthesized B-FOS as a carbon source, respectively, to explore the growth-promoting effect of B-FOS on common probiotics Bifidobacterium and Lactobacillus.

[0204] Bifidobacterium lactis BB-12 and Lactobacillus casei strain Shirota were isolated by the laboratory and sequenced and identified. The determination of the proliferation of B-FOS on the two probiotics was carried out in an anaerobic incubator, and the two strains were cultured in a 96-well V-shaped culture plate using sterile MRS medium. The medium without any carbon source was used as a blank control group, the medium added with FOS or FOS+butyric acid (FOS+butyrate) was used as two sample control groups, and the medium added with B-FOS was used as an experimental group. Under sterile conditions, 150 μL of medium was added to each culture plate, three replicate wells were set for each sample, 2% of the volume of the medium of the strain mother liquor was inoculated in each culture plate, and the culture was incubated in a 37°C anaerobic incubator for 24 h. The growth of the two strains was detected every 2 h using an enzyme marker at OD 600nm , and the growth curves of the two probiotics were drawn.

[0205] (2) Results

[0206] The effect of oligofructose butyrate B-FOS on the growth curve of probiotics is shown in Figure 17 . A is the effect of B-FOS on the growth curve of Bifidobacterium lactis BB-12 (B. lactis BB-12), and B is the effect of B-FOS on the growth curve of Lactobacillus casei strain Shirota.

[0207] As shown in Figure 17 A, the FOS group and the B-FOS group both have a significant promoting effect on the growth of B. lactis BB-12, and the FOS+butyrate group does not have an obvious promoting effect on the growth of Bifidobacterium. Especially when the anaerobic culture is carried out for 12 h, the promoting effect of the FOS group and the B-FOS group on the growth of B. lactis BB-12 is significantly different from that of the FOS+butyrate group. The FOS+butyrate group does not begin to have a promoting effect on the growth of B. lactis BB-12 until 38 h of culture. The promoting effect of the B-FOS group is similar to that of the FOS group, but at 38 h of culture, the B-FOS group shows a better promoting effect than the FOS group.

[0208] As shown in Figure 17As shown in FIG. 8, the FOS group, the B-FOS group and the FOS+Butyrate group all had significant promoting effects on the growth of Lactobacillus casei strain Shirota. The B-FOS group had the most significant promoting effect, followed by the FOS group and the FOS+Butyrate group.

[0209] In summary, the B-FOS has good fermentation characteristics and probiotic activity, increases the production of butyric acid in the fermentation broth and improves the composition of intestinal flora, and plays an important role in intestinal health.

[0210] Similarly, it was found that the purified B-Inulin prepared in Example 2 also has good fermentation characteristics and probiotic activity, increases the production of butyric acid in the fermentation broth and improves the composition of intestinal flora, and plays an important role in intestinal health.

[0211] Example 6: Improving effect of oligofructose butyrate on DSS-induced intestinal inflammation mice

[0212] 1. Establishment of mouse colitis (IBD) model

[0213] With the approval of the ethics committee of the animal management center of Jilin University, 72 C57BL / 6 male mice (18-22 g) aged 6 weeks were purchased from Zhejiang Weitong Lihua, and were raised in the animal management center of Jilin University under standard conditions. During the quarantine period, the mice were fed with feed provided by the animal house (Synergy Biological Mouse Feed), had free access to water, and the water, feed and bedding in the mouse cage were changed regularly.

[0214] After 10 days of quarantine, the mice were randomly divided into 6 groups, 12 in each group, namely the blank control group (Control group, CON), blank sample group (B-FOS group, BF), modeling control group (DSS group, DSS), oligofructose intervention group (DSS+FOS group, DF), oligofructose-n-butanoic acid mixture intervention group (DSS+FOS+Butyrate group, DFB), and oligofructose butyrate intervention group (DSS+B-FOS group, DBF). The total modeling time was 20 days. The CON group and the BF group drank sterile water provided by the animal center throughout the experiment. The DSS group, the DF group, the DFB group, and the DBF group drank 2.0(w:v)% DSS solution for the first 8 days of the modeling process, and then drank 2.3(w:v)% DSS solution for the last 8 days, with a 4-day recovery period in between. The volume of gavage was kept consistent among the groups during the experiment. The CON and DSS groups were given 0.01 mL / g (body weight) of normal saline (NS) by gavage every day during the modeling period. The BF and DBF groups were given 60 mg / mL of oligofructose butyrate dissolved in normal saline by gavage every day during the modeling period. The DF group was given 50 mg / mL of oligofructose dissolved in normal saline by gavage every day. The DFB group was given a mixture of oligofructose (50 mg / mL) and n-butanoic acid (6.9 μL / mL) dissolved in normal saline by gavage every day. The six groups of mice were allowed to freely eat the feed provided by the animal experiment center of Jinan University, and the feed and water were recorded and replaced every two days from the 0th day of modeling, and the bedding was replaced regularly.

[0215] The IBD mouse modeling intervention flowchart is shown in Figure 18 .

[0216] 2. Effect of B-FOS on the basic indicators of colitis mice

[0217] (1) Effect of B-FOS on the body weight of colitis mice

[0218] The body weight of the mice was measured every day from the 0th day of modeling. The changes in body weight of the mice on the 0th, 8th, 12th, and 20th days of modeling are shown in Figure 19 . Figure 19The Control group and the B-FOS group mice grew well throughout the experiment, and the body weight of the mice remained stable. When the modeling was carried out to the 8th day, the body weight change between the mice in each group was not obvious (p>0.05). But when the modeling was carried out to the 12th day, the body weight of the mice in the four groups that drank DSS solution decreased, especially in the DSS+FOS+Butyrate and DSS+B-FOS groups (p<0.05). After a short recovery period when the mice stopped drinking DSS solution, the body weight of the mice in each group that drank DSS solution recovered to some extent, so when the second round of DSS intervention period, the mice were modeled with DSS solution containing 2.3% DSS. When the experiment was carried out to the 20th day, it can be seen that the body weight of the mice in the modeling group decreased significantly (p<0.05), and the body weight of the mice that were gavaged with FOS and B-FOS was higher than that of the mice in the modeling group, but the body weight of the FOS+Butyrate intervention group did not recover significantly.

[0219] (2) Effect of B-FOS on the disease activity index of mice

[0220] From the 0th day of modeling, the body weight of the mice was weighed every day, and the state and feces of the mice were observed and recorded. Fresh mouse feces were collected every two days in ice boxes and stored in a -20°C refrigerator. Fresh feces were collected from each mouse every other day, and the hardness and blood in feces of the mice were scored according to Table 4. The collected fresh mouse feces were tested for occult blood using an occult blood kit, and the average of the three indicators of feces hardness, blood in feces, and body weight change index was used to obtain the disease activity index (Disease activity index, DAI) score of each mouse.

[0221] Table 4 DAI score table

[0222]

[0223] The DAI scores of the mice are shown in Figure 20 ; the mean value is represented as mean ± SEM, and the results were analyzed using One-way ANOVA and Duncan multiple comparison, n=6-12, different letters indicate significant differences, p<0.05, ns indicates no significant difference.

[0224] Figure 20As shown, no obvious health problems occurred in mice in the Control group and the B-FOS group as the experimental time was prolonged, and thus the DAI scores of the two groups were at a low level throughout the experiment. The DAI scores of the four groups of model mice and intervention groups that drank DSS solution were significantly increased (p<0.05), and the feces of the model mice showed obvious mucous stool and bloody stool, and the activity of the mice also decreased compared to before. On the 20th day of the experiment, the DAI between the model and intervention groups that drank DSS showed significant differences, and the disease conditions of mice in each group intervened by FOS, FOS+Butyrate, and B-FOS were significantly improved (p<0.05), and the improvement of intestinal inflammation in the B-FOS dietary intervention group was the best.

[0225] (3) Effect of B-FOS on colon length in mice with colitis

[0226] The next morning after the completion of the disease activity experiment, the mice were treated, weighed, and anesthetized with isoflurane. The eyeball was extracted to collect blood in an anticoagulant tube. The mice were immediately dissected after perfusion. The colon of the mice was isolated. The mouse colon was completely removed and placed on a thin plate with a grid line after disinfection. The length was measured using a scale and recorded. All mouse colons were photographed under the same light source conditions and angles. Carefully cut 0.5 cm long tissue in the middle of the colon and place it in a centrifuge tube containing 4% paraformaldehyde for subsequent paraffin sectioning and staining. Cut 0.5 cm long tissue in the middle of the colon and place it in a grinding tube, and quickly place it in liquid nitrogen for quick freezing, then transfer it to a -80°C refrigerator for storage, and then extract RNA from the tissue. Collect the contents of the remaining colon in a cryotube and rinse the remaining colon tissue with low-temperature sterile PBS, store it in two parts in a cryotube, and quickly freeze it in liquid nitrogen, then transfer it to a -80°C refrigerator for storage. After standing at room temperature for 30 min, centrifuge the blood at 4°C, 3000g for 15 min, and place it on ice. Carefully aspirate the supernatant serum into a new centrifuge tube, aliquot, and transfer to a -80°C refrigerator for storage.

[0227] The change in the length of the mouse colon is shown in Figure 21 ; A is the length of the colon of each group of mice, and B is the dissection of the colon of each group of mice. The value in the figure represents the mean ± SEM, n=12, and the results were analyzed using One-way ANOVA and Duncan multiple comparison. Different letters indicate significant differences, p<0.05.

[0228] Figure 21As shown in FIG. 2A, the colon length of the mice in the Control group and the B-FOS group was the longest, and the colon length of the mice that drank the DSS solution was significantly shorter than that of the blank control group (p<0.05). The colon length of the mice in the three disease intervention groups was improved to different degrees, and the colon length of the mice in the DSS+B-FOS group was significantly improved compared with that of the mice in the DSS group (p<0.05). Figure 21 As shown in FIG. 2B, the colon of the mice in the Control group and the B-FOS group was normal in color and had no edema, and the contents in the colon were in the form of rice. The colon of the mice in the DSS group was significantly thickened and shortened, had a blood color, and the contents in the colon could not be shaped, and had the characteristics of the colon tissue of the mice with obvious intestinal inflammation. It was also found during the dissection that the colon of the mice in the DSS group was more fragile and easy to break. The colon length and color of the mice in the intervention groups were improved to different degrees, and the contents were shaped. The results show that the B-FOS dietary intervention has no adverse changes on the colon length and content state of the mice, and can relieve the symptoms of colon inflammation such as shortening, edema and bleeding of the colon of the mice with colon inflammation.

[0229] 3. Improvement of B-FOS on the inflammatory response of mice with colon inflammation

[0230] (1) Preparation of paraffin sections

[0231] The colon tissue fixed with 4% paraformaldehyde for more than 24 h was dehydrated, embedded, trimmed, sectioned, and the paraffin sections with a thickness of 4 μm were prepared. Finally, the tissue was flattened in warm water at 40°C, and the tissue was lifted with a clean glass slide and placed in a 60°C oven to bake the section. After the water was baked dry, it was taken out for use.

[0232] (2) Hematoxylin-eosin staining (H&E staining)

[0233] The prepared paraffin sections were deparaffinized in xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, and 75% alcohol, respectively. The sections were stained with hematoxylin dye, differentiated with differentiation solution, and then returned to blue with blue return solution. The sections were dehydrated with 85% alcohol and 95% alcohol, and then stained again with eosin dye. The sections were placed in anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, xylene I, and xylene II, respectively, and finally mounted with neutral balsam. The stained sections were examined under a microscope, and the images were collected and analyzed.

[0234] (3) Histopathological scoring criteria for colon tissue

[0235] The pathological analysis score of the colon tissue sections was based on the international standard, as shown in Table 5. The results of the section examination were scored in a blinded manner.

[0236] Table 5 Histological scoring of colon inflammation

[0237]

[0238] (4) Histological analysis and inflammation score of mouse colon

[0239] H&E staining and inflammation score of mouse colon tissue as follows Figure 22 As shown; where A is H&E staining of colon tissue sections, B is inflammation score of colon tissue sections, and C is colon wall thickness.

[0240] like Figure 22 As shown in Figure A, the intestinal walls of the colon cross sections were intact in all groups. The Control and B-FOS groups showed intact mucosal layers, complete crypt morphology, orderly arrangement of goblet cells within the crypts, and no inflammatory infiltration. In contrast, the DSS group showed a large number of inflammatory cells filling the intestinal mucosa, and the crypt structure was significantly disrupted. Dietary interventions with FOS, FOS+Butyrate, and B-FOS improved colonic inflammatory cell infiltration in all three groups of mice, resulting in more prominent and regular crypt structures and more intact intestinal epithelial cell structures.

[0241] like Figure 22 As shown in Figure B, compared with the Control group, the inflammation score of colon tissue sections in the DSS group was significantly higher (p<0.05). The inflammation scores of DSS+FOS, DSS+FOS+Butyrate, and DSS+B-FOS in the dietary intervention group were all significantly lower than those in the DSS group (p<0.05), and there was no significant difference in the inflammation scores of FOS and B-FOS between the dietary intervention group and the Control group (p>0.05).

[0242] like Figure 22 As shown in Figure C, the colon wall thickness of mice in the B-FOS group was approximately 107.41 ± 4.07 μm, which was not significantly different from that in the Control group (p > 0.05). The colon wall thickness of mice in the DSS group was significantly thicker than that in the control group (p < 0.05). After dietary intervention with FOS, FOS+Butyrate, or B-FOS, the intestinal wall thickness decreased significantly (p < 0.05), although it was still higher than that in the control group, but the difference was no longer significant.

[0243] In summary, based on pathological sections, the intake of B-FOS did not adversely affect the internal structure of the mouse colon, crypt morphology, or colonic inflammation. DSS caused significant damage to the mouse colonic tissue and increased inflammatory infiltration. The intake of FOS, FOS+Butyrate, and B-FOS could improve the intestinal tissue damage and inflammatory infiltration caused by DSS in mice, with B-FOS showing the best effect.

[0244] 4. Improvement of β-FOS on the expression of inflammatory factor genes in colonic tissue

[0245] (1) Colonic tissue mRNA extraction and reverse transcription

[0246] The colon tissue was immersed in a grinding tube containing 1 mL Trizol, and two 3 mm enzyme-free grinding beads were added thereto, and the colon tissue was cryogenically ground at 60 Hz for 180 s in a grinder. After grinding, 200 μL of chloroform was added to the grinding tube, and after vortexing for 20 s, it was left to stand for 7 min. Cryogenic high-speed centrifugation was performed, the protein layer was avoided, and an appropriate volume of the upper aqueous phase was carefully aspirated into a new centrifuge tube, and one volume of isopropanol was added thereto, which was shaken and left to stand at 4°C for 10 min. Cryogenic high-speed centrifugation was again performed, and the supernatant was discarded to obtain the lower precipitate. The precipitate was washed with 75% ethanol prepared with DEPC water, and after cryogenic high-speed centrifugation, the supernatant was discarded, and the centrifuge tube was left to dry on filter paper. According to the amount of precipitate in the centrifuge tube, appropriate DEPC water was added for dissolution, and the RNA mother liquor purity and concentration were determined using a Nanodrop. According to the Evo M-MLV reverse transcription kit instructions, the RNA was subjected to DNA removal reaction and reverse transcription.

[0247] (2) Fluorescent quantitative PCR detection

[0248] The PCR reaction system is shown in Table 6, and the primer information is shown in Table 7.

[0249] Table 6 PCR reaction system

[0250]

[0251] Table 7 Primer information

[0252]

[0253] Before the experiment, the samples were diluted to 0.01 μg / μL in advance for standby, and a mixture of enough amount of all reagents except cDNA was prepared according to the above table PCR Mix, 9 μL of the mixture was dispensed into a 384 PCR well plate, and 1 μL of the diluted sample cDNA was added to the well containing the mixture, and 3 replicates were made for each sample. After the film was pasted and scraped flat, it was centrifuged at 4°C, 800g for 3 min. After centrifugation, the sample was detected on the machine. The detection conditions were: 95°C, 30 s pre-denaturation; 95°C, 5 s denaturation, 60°C, 30 s annealing and amplification, for 40 cycles; slowly increase from 60°C to 97°C, collect 5 times of fluorescence signal every 1°C increase.

[0254] After the reaction, the data was exported for subsequent expression calculation and melting curve analysis, and the expression calculation was performed using the 2 -ΔΔCt method. ΔCt = Ct (target gene) - Ct (internal reference gene), ΔΔCt = ΔCt (experimental sample) - ΔCt (control sample). Relative expression = 2 -ΔΔCtThe formula is as follows:

[0255]

[0256] (3) Expression levels of inflammatory factor genes in mouse colon tissue

[0257] The relative expression levels of inflammatory factor mRNA in mouse colon tissue are as follows: Figure 23 As shown; where A is tumor necrosis factor α (TNF-α), B is interleukin 1β (IL-1β), C is interleukin 6 (IL-6), D is interleukin 13 (IL-13), E is cytoleukin 18 (IL-18), and F is interleukin 10 (IL-10).

[0258] from Figure 23 The results from the study showed that the mRNA expression level of TNF-α in the colon tissue of mice in the DSS-treated model group was significantly increased and significantly higher than that in the control group (p<0.05). However, under the dietary intervention of FOS, FOS+Butyrate, and B-FOS, the gene expression level of TNF-α in the colon of mice was significantly reduced (p<0.05).

[0259] Figure 23 The results showed that IL-18 gene expression in the colon of mice in the DSS group was significantly higher than that in the group that did not drink DSS solution (p<0.05). In contrast, the colonic IL-18 levels in the three groups of mice treated with FOS, FOS+Butyrate, and B-FOS decreased. The relative expression levels of FOS+Butyrate and B-FOS were significantly lower in the DSS group than in the control group (p<0.05), and there was no significant difference compared to the control group (p>0.05).

[0260] Figure 23 Figures B, C, D, and F show that DSS modeling and interventions with FOS, FOS+Butyrate, and B-FOS had no significant effect on the expression of IL-1β, IL-6, IL-13, and IL-10 genes in mouse colon tissue (p>0.05). In conclusion, B-FOS may improve enteritis symptoms in mice by downregulating the expression levels of pro-inflammatory factors TNF-α and IL-18 genes.

[0261] 5. Effects of β-FOS on the intestinal barrier in colitis mice

[0262] (1) Alcian blue staining

[0263] The paraffin sections prepared in Example 5 were dewaxed by placing them in xylene I, xylene II, absolute ethanol I, absolute ethanol II, 75% ethanol, respectively. The sections were dyed with Alcian Blue dye A, and then immersed in Alcian Blue dye B. The sections were placed in absolute ethanol I, absolute ethanol II, absolute ethanol III, xylene I, xylene II, respectively, and then mounted with neutral balsam. The dyed sections were observed under a microscope, and the images were collected and analyzed.

[0264] (2) Alcian Blue staining of colon tissue sections and changes in intestinal mucosa

[0265] The Alcian Blue staining of colon tissue sections and changes in intestinal mucosa are shown in Figure 24 , where A is the Alcian Blue staining of colon tissue sections, B is the relative proportion of mucus in colon tissue sections, and C is the depth of colon crypts.

[0266] Figure 24 It can be seen that the colon tissue of mice in the Control group and the B-FOS group contains complete and abundant goblet cells and their secreted mucus, and the crypt structure is complete. However, the colon tissue sections of mice subjected to DSS-induced colitis show a significant decrease in the number of goblet cells and mucus, and the crypt structure is incomplete. Figure 24 Figure B shows that the number of goblet cells and the proportion of mucus in the Control group and the B-FOS group are large, but under the influence of DSS, the number of goblet cells in the model group decreases, and the proportion of mucus significantly decreases (p<0.05). However, after dietary intervention with FOS, FOS+Butyrate, and B-FOS, there is an improvement to varying degrees, and the intake of FOS and butyric acid mixture increases the number of goblet cells and mucus, with the best effect being B-FOS.

[0267] Figure 24 Figure C shows that the thickness of the colon crypts and the thickness of the mucus layer of mice in the DSS group are thin, and dietary intervention with FOS, FOS+Butyrate, and B-FOS can increase the thickness of the crypts and the mucosal layer, which corresponds to the improvement in the number of goblet cells and mucus, and the improvement effect is also most obvious in the B-FOS group.

[0268] (3) Relative expression of tight junction protein-related genes in mouse colon tissue

[0269] The relative expression level of tight junction protein mRNA in mouse colon tissue was determined according to Example 6-4, and the results are shown in Figure 25 , where A is Claudin-1, B is Claudin-2, and C is ZO-2.

[0270] Figure 25Figure 3 shows that the expression levels of Claudin-1 and Claudin-2 in the colon of each group of mice were decreased compared with the Control group, and the expression levels were increased after FOS and B-FOS intervention, but not significantly (p>0.05). Figure 25 Figure 3 shows that the expression levels of Claudin-1 and Claudin-2 in the colon of each group of mice were decreased compared with the Control group, and the expression levels were increased after FOS and B-FOS intervention, but not significantly (p>0.05).

[0271] In summary, the occurrence of DSS-induced intestinal inflammation in the present application is not highly correlated with the expression level of Claudins gene, and B-FOS dietary intervention has no significant effect on the expression of Claudins series protein gene (p<0.05), but B-FOS can significantly increase the mRNA expression level of ZO-2 in the colon of intestinal inflammation mice (p<0.05).

[0272] 6. Effect of B-FOS on the content of short-chain fatty acids (SCFAs) in the colon contents of mice

[0273] (1) Determination of SCFAs content in the colon contents of mice

[0274] 0.2M hydrochloric acid solution was used to prepare 0.15mg / mL diethyl acetic acid and 0.15M oxalic acid, which were mixed in a ratio of 4:1 to prepare the extraction solution before use. The standard samples of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, iso-valeric acid and caproic acid were prepared in a ratio of 1:10 (v / v) using the extraction solution, and a mixed standard curve was prepared.

[0275] The mouse intestinal feces were accurately weighed in a 2mL centrifuge tube, 2 glass beads were added respectively, and the above-mentioned extraction solution was added in a ratio of 1:10 (m / v), i.e. 100mg feces were added with 1mL extraction solution. After homogenization, low-temperature centrifugation was performed, and the supernatant was passed through a 0.22μm filter membrane and placed in a sample vial inner liner for determination. The SCFAs concentration in the sample was calculated by the standard curve, and the instrument settings and method were referred to the standard curve preparation in Example 2.

[0276] (2) Effect of B-FOS on the content of SCFAs in the intestinal contents of colitis mice

[0277] The SCFAs content in the colon contents of mice is shown in Figure 26 Figure 6, wherein A is acetic acid, B is propionic acid, C is butyric acid, D is isobutyric acid, E is valeric acid, F is iso-valeric acid, and G is the total short-chain fatty acid content.

[0278] From Figure 26As can be seen in FIG. 6, drinking DSS reduced the concentration of total SCFAs in the feces of mice, and dietary intervention of FOS, FOS+Butyrate and B-FOS improved the decrease in the concentration of SCFAs in the feces caused by drinking DSS.

[0279] Figure 26 As shown in FIG. 7B, the concentration of propionic acid in the feces of the mice in the model group that drank DSS solution was significantly lower than that in the other groups (p<0.05), and the decrease in the concentration of propionic acid was alleviated in the dietary intervention groups, and the concentration of propionic acid in the feces of the B-FOS intervention group was significantly higher than that in the other groups (p<0.05).

[0280] Figure 26 As shown in FIG. 7C, there was no significant difference in the content of butyric acid in the feces of the mice in each group, but the DSS+B-FOS group showed an effect of increasing the content of butyric acid in the feces of the mice, regulating the expression of inflammatory factors in the intestine, and reducing the inflammatory response.

[0281] Figure 26 As shown in FIGS. 7B-7F, for isobutyric acid, valeric acid and iso-valeric acid, FOS, FOS+Butyrate and B-FOS all improved the decrease in the concentration of SCFAs caused by DSS to varying degrees.

[0282] 7. Effect of B-FOS on intestinal flora of colitis mice

[0283] (1) Full-length sequencing of microorganisms in the colon contents of mice

[0284] 0.25-0.5 g of the colon contents of mice were weighed and lysed to extract DNA from the colon contents for standby. The concentration of nucleic acids was detected and amplified using a microplate reader. The PCR products were detected by electrophoresis using 1.8% agarose and quantified by Qubit and mixed, and the electrophoresis gel was manually adjusted. The Qubit determination value of the sample amplification product was combined with the electrophoresis gel to adjust the concentration of the PCR product.

[0285] The mixed product was subjected to damage repair, end repair and linker ligation on a PCR instrument using a library construction kit (SMRTbell Template Prep Kit) provided by PacBio, and the library was recovered to obtain the library for sequencing. After meeting the requirements for sequencing, the library for sequencing was combined with primers and polymerase using a Pac Bio Binding Kit, and the final reaction product was purified by AMpure PB Beads and placed on a sequencer for sequencing.

[0286] (2) Diversity and richness of intestinal flora of mice

[0287] The alpha-diversity of microorganisms in the colon contents of mice was as shown in FIG. 8A. Figure 27As shown, reflecting the diversity and richness of the mouse intestinal flora. Among them, the average Shannon index of the colon contents of the mice in the model group drinking DSS solution is lower than that of each group, and the species richness is significantly improved under the dietary intervention of B-FOS (p<0.05).

[0288] Studies have shown that the intestinal flora of the IBD group often shows lower richness and diversity, and the higher the richness and diversity of the intestinal flora, the easier it is to maintain the intestinal flora homeostasis. The alpha-diversity results of the intestinal content microorganisms in the present application show that there is no significant difference between the Chao1 and Simpson indexes of each group, but B-FOS can significantly increase the Shannon index of the intestinal content of the mice with intestinal inflammation, which is conducive to restoring the intestinal flora homeostasis affected by DSS.

[0289] (3) Differences in the composition of the mouse intestinal flora

[0290] The composition of the colon content microorganisms is shown in Figure 28 ; wherein A is the PCoA plot of the mouse intestinal flora, B is the relative abundance of the intestinal flora at the phylum level, and C is the relative abundance column chart of the intestinal flora at the genus level, n=6.

[0291] As shown in Figure 28 A, in the PCoA plot of the present experiment, there is no special difference in the characteristics of the intestinal flora between each group, but the samples of the CON group and the BF group are relatively concentrated, and the dietary intervention has a certain effect on changing the DSS-induced flora change.

[0292] Figure 28 B shows that DSS causes changes in the structure of the intestinal flora of mice.

[0293] Figure 28 C shows that the relative abundance of Muribaculaceae in the colon contents of the mice in the DSS group decreases, but it is improved after B-FOS dietary intervention. B-FOS maintains intestinal health by improving the relative abundance of Bacteroides, Parasutterella, Dubosiella, and Parabacteroides. B-FOS dietary intervention reduces the increase in the relative abundance of potentially harmful bacteria Parasutterella, uncultured Clostridiales bacterium, and Erysipelatoclostridium caused by DSS, and B-FOS can cause significant up-regulation of butyrate-producing bacteria Faecalibaculum (p<0.05), increase the concentration of butyric acid in the intestine, and relieve the symptoms of intestinal inflammation in mice by improving the intestinal barrier and reducing the level of inflammation.

[0294] (4) Relative abundance of colon content microbial phylum level

[0295] The relative abundance of colon content microbial phylum level is shown in Figure 29 ; wherein A is Firmicutes, B is Verrucomicrobiota, C is Bacteroidota, D is Proteobacteria, E is Desulfobacterota, and F is Deferribacterota.

[0296] Figure 29 It is shown that DSS causes changes in the intestinal microbial community structure of mice. The dietary intervention of B-FOS, FOS+Butyrate and FOS improves the increase in the relative abundance of Proteobacteria, which contains a large number of pathogenic bacteria, caused by DSS. The relative abundance of Proteobacteria in the three intervention groups is down-regulated to varying degrees, and the improvement effect of B-FOS is the most obvious. A large number of studies have shown that the relative abundance of Proteobacteria in the intestinal flora of patients with intestinal inflammation is higher than that of normal individuals, and the down-regulation of Proteobacteria by B-FOS dietary intervention can help improve intestinal inflammation symptoms and maintain intestinal health.

[0297] (5) Relative abundance of colon content genus level butyrate-producing bacteria

[0298] The relative abundance of colon content genus level butyrate-producing bacteria is shown in Figure 30 ; wherein A is Lachnospiraceae_NK4A136_group, B is unclassified_Lachnospiraceae, C is Faecalibaculum, and D is Blautia.

[0299] Figure 30 It is shown that the relative abundance of butyrate-producing bacteria Faecalibaculum in the intestinal contents of normal mice and intestinal inflammation mice ingesting B-FOS is higher than that of other groups, and the relative abundance of Faecalibaculum in the B-FOS group is significantly higher than that of the other four groups (p<0.05). B-FOS can promote the proliferation of butyrate-producing bacteria, thereby increasing the content of butyric acid in the in vivo and in vitro system, thereby playing a role in improving inflammation.

[0300] In summary, B-FOS has a good effect on improving intestinal inflammation in mice. Similarly, it is found that the purified B-Inulin prepared in Example 2 also has a good effect on improving intestinal inflammation in mice.

[0301] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A polyfructose butyrate, characterized in that, The polyfructose butyric ester is prepared by combining vinyl butyrate and polyfructose through ester bond by enzymatic synthesis method, wherein the polyfructose has a polymerization degree less than or equal to 60, and the polyfructose is fructooligosaccharide. The method for combining vinyl butyrate and polyfructose through ester bond by enzymatic synthesis method is as follows: polyfructose solution and vinyl butyrate are fully reacted under catalysis of lipase, and then the excess lipase is removed, and then purified. The lipase is immobilized lipase TLIM. The full reaction is carried out at 50-55℃ for 45-50h.

2. A method for preparing polyfructose butyrate, characterized by, The polyfructose butyric ester is prepared by combining vinyl butyrate and polyfructose through ester bond by enzymatic synthesis method, wherein the polyfructose has a polymerization degree less than or equal to 60. The polyfructose is fructooligosaccharide. The method for combining vinyl butyrate and polyfructose through ester bond by enzymatic synthesis method is as follows: polyfructose solution and vinyl butyrate are fully reacted under catalysis of lipase, and then the excess lipase is removed, and then purified. The lipase is immobilized lipase TLIM. The full reaction is carried out at 50-55℃ for 45-50h.

3. The preparation method according to claim 2, characterized in that, The solvent of the polyfructose solution is anhydrous pyridine, dimethyl sulfoxide or N,N-dimethylformamide.

4. The polyfructose butyric ester prepared by the preparation method of any one of claims 2-3.

5. Use of the polyfructose butyric ester of any one of claim 1 or claim 4 in the preparation of a drug for improving intestinal flora and / or promoting growth of probiotics.

6. Use of the polyfructose butyric ester of any one of claim 1 or claim 4 in the preparation of a drug for treating and / or relieving inflammatory bowel disease.

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