Preparation and use of polyfructropropionates

Abstract

The application discloses a preparation method and application of polyfructose propionate, and the preparation method is that polyfructose is esterified with a propionylating reagent under the catalysis of an enzyme to obtain polyfructose propionate.The polyfructose propionate is well degraded and utilized by intestinal flora, and propionic acid is generated at the same time, the ratio of bacteria Firmicutes and Bacteroidota can be reduced, and the polyfructose propionate has a weight loss effect; the polyfructose propionate can reduce obesity induced by a high-fat diet, and can also improve intestinal flora, promote the growth of probiotics and inhibit the growth of harmful bacteria.The polyfructose propionate disclosed by the application can be well degraded in the intestinal tract, and the generated propionic acid can further play a role in treating or relieving obesity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation and application of polyfructose propionate. Background Technology

[0002] Propionic acid is a major short-chain fatty acid, abundant in the human colon. It possesses biological activities such as lipid-lowering, cholesterol-lowering, anti-inflammatory, and anti-cancer properties. However, propionic acid cannot be directly utilized; propionic acid ingested in propionate form not only lacks lipid-lowering effects but can also lead to weight gain. Existing technologies utilize the combination of inulin and propionic anhydride to prepare inulin propionate esters via the anhydride method. Consuming inulin propionate esters can stimulate the release of more propionate esters in the intestines, enhancing satiety, reducing calorie intake, and having a stronger inhibitory effect on overeating, thus reducing the chance of obesity. However, inulin propionate esters prepared using the anhydride method are not well degraded by gut microbiota. Therefore, is it necessary to provide a new method for preparing inulin propionate esters that can be effectively degraded by gut microbiota?

[0003] Fructose oligosaccharides (FOS) are a class of indigestible oligosaccharides, consisting of fructotriose, fructotetraose, and fructopentose formed by β-(1→2) glycosidic bonds to 1 to 3 fructose molecules. Their molecular formula is G-Fn (n=1, 2, 3; G for glucose; F for fructose). They are linear heterooligosaccharides composed of fructose and glucose. They are mainly found in vegetables and fruits. The raw materials for synthesizing FOS are inulin and sucrose. FOS synthesized from inulin is a fructo-fructose type, where all units are fructose. FOS synthesized from sucrose is a sucrose-fructose type, where the non-reducing segment is glucose and the remaining sugar rings are fructose rings. FOS has many beneficial effects; it is low in sweetness, stable, and long-term consumption can lower cholesterol, improve lipid metabolism, and benefit gut microbiota. Fructooligosaccharides (FOS) can reach the colon intact without being hydrolyzed by endogenous enzymes, where they are utilized by the gut microbiota and selectively influence its composition and function. They also produce beneficial results for conditions such as hyperlipidemia, osteoporosis, impaired immune function, colon cancer, and ulcerative colitis.

[0004] Existing technologies have also revealed that inulin propionate can alleviate obesity to some extent and has anti-inflammatory effects. However, inulin propionate has a high degree of esterification, making it difficult for humans to fully utilize it. Therefore, it is necessary to prepare propionate with a low degree of esterification for the treatment of obesity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing obesity treatments and to provide a method for preparing and applying polyfructose propionate.

[0006] The purpose of this invention is to provide a method for preparing oligofructose propionate.

[0007] Another object of the present invention is to provide a polyfructose propionate prepared using the preparation method described above.

[0008] The present invention also aims to provide the application of the aforementioned fructosyl propionate in alleviating obesity, improving gut microbiota, and promoting the growth of probiotics.

[0009] Another object of the present invention is to provide the use of the polyfructose propionate in the preparation of medicaments for treating and / or alleviating obesity.

[0010] The above-mentioned objectives of the present invention are achieved through the following technical means:

[0011] This invention provides a method for preparing polyfructose propionate, wherein polyfructose and a propionylating agent are esterified by enzyme catalysis to obtain polyfructose propionate, wherein the degree of polymerization of the polyfructose is less than or equal to 60.

[0012] Preferably, the preparation method involves reacting a fructooligosaccharide solution with a propionylating reagent and an enzyme, filtering the reaction solution, and purifying the filtrate.

[0013] Preferably, the propionylating agent is propionic acid or vinyl propionate.

[0014] More preferably, the propionylating agent is vinyl propionate.

[0015] Preferably, the enzyme is an immobilized lipase TLIM.

[0016] Preferably, the fructooligosaccharide is an oligofructose or inulin.

[0017] More preferably, the fructooligosaccharide is an oligofructose.

[0018] Preferably, the solvent for the fructooligosaccharide solution is anhydrous pyridine or tetrahydrofuran.

[0019] More preferably, the solvent for the fructooligosaccharide solution is anhydrous pyridine.

[0020] Preferably, the purification is performed by concentrating the filtrate, dissolving it in ethanol, and then separating and purifying it using column chromatography.

[0021] More preferably, the purification is performed by concentrating the filtrate and dissolving it in 40%–50% ethanol, followed by separation and purification by column chromatography.

[0022] More preferably, the column chromatography is C18 column chromatography.

[0023] Preferably, the separation and purification using column chromatography specifically involves: loading a 40%–50% ethanol solution of the filtrate concentrate onto a chromatography column, eluting with 40%–50% ethanol and 70% ethanol respectively, collecting the eluent eluted with 70% ethanol, and drying it.

[0024] Preferably, the complete reaction is carried out at 40–45°C for 24–36 h.

[0025] More preferably, the complete reaction is carried out at 40°C for 36 hours.

[0026] The polyfructose propionate prepared using the above preparation method is also within the scope of protection of this invention.

[0027] The application of the polyfructose propionate in treating and / or alleviating obesity, improving gut microbiota, and promoting the growth of probiotics is also within the scope of protection of this invention.

[0028] The use of the polyfructose propionate in the preparation of medicaments for treating and / or alleviating obesity is also within the scope of protection of this invention.

[0029] Preferably, the polyfructose propionate is oligofructose propionate or inulin propionate.

[0030] Preferably, the polyfructose propionate is oligofructose propionate.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention utilizes immobilized lipase TLIM to promote the formation of ester bonds between vinyl propionate and fructooligosaccharides or inulin to prepare fructooligosaccharide propionate or inulin propionate (polyfructooligosaccharide propionate). The fructooligosaccharide propionate or inulin propionate (polyfructooligosaccharide propionate) is well degraded and utilized by intestinal flora, while simultaneously producing propionic acid, which can reduce the ratio of Firmicutes and Bacteroidota bacteria, thus having a weight loss effect; it can alleviate obesity induced by a high-fat diet, and can also improve intestinal flora, promote the growth of probiotics and inhibit the growth of harmful bacteria. Attached Figure Description

[0033] Figure 1 This is the infrared absorption spectrum of fructooligosaccharide propionate in Example 2 of the present invention.

[0034] Figure 2 The images show the flight mass spectra of fructooligosaccharide propionate in Example 2 of this invention, where A is the flight mass spectrum of FOS, B is the flight mass spectrum of P-FOS, and C is the flight mass spectrum of AP-FOS.

[0035] Figure 3 The oligofructose propionate in Example 2 of this invention 1H NMR spectrum.

[0036] Figure 4 The results show the total sugar content determination of fructooligosaccharide propionate in Example 2 of this invention.

[0037] Figure 5 The cumulative gas production and pH changes of samples at different time points during the in vitro fermentation of fructooligosaccharide propionate in Example 3 of the present invention are shown. A represents the change in gas production, B represents the change in pH; C represents the blank control group, F represents the FOS control group, PF represents the P-FOS group, APF represents the AP-FOS group, and FAP represents the FOS+propionic acid group.

[0038] Figure 6 The figure shows the changes in total sugar content during in vitro fermentation in Example 3 of this invention. C represents the blank control group, F represents the FOS control group, PF represents the P-FOS group, APF represents the AP-FOS group, and FAP represents the FOS+propionic acid group.

[0039] Figure 7 The graph shows the changes in short-chain fatty acid content in the in vitro fermentation broth of fructooligosaccharide propionate in Example 3 of this invention; A represents acetic acid, B represents propionic acid, C represents butyric acid, D represents isobutyric acid, E represents valeric acid, F represents isovaleric acid, G represents hexanoic acid, and H represents the total short-chain fatty acid content; the values ​​in the graph are expressed as average ± SEM, n = 3, and different letters indicate significant differences. p <0.05, ns indicates that the difference is not significant.

[0040] Figure 8 The NMDS analysis results of the beta diversity of microorganisms in the fermentation broth in Example 3 of the present invention are shown. In the figure, one point represents one sample, and different groups have different colors. The greater the distance between samples, the greater the difference in the composition of the microbial community.

[0041] Figure 9 The figures show the relative abundance and composition of microorganisms at the phylum level during the in vitro fermentation of fructooligosaccharide propionate in Example 3 of this invention; AF represents the difference in relative abundance of microorganisms at the phylum level in the fermentation broth, and G is a bar chart of the relative abundance of microorganisms at the phylum level in the fermentation broth; the values ​​in the figures are expressed as mean ± SEM, n = 3, and different letters indicate significant differences. p <0.05.

[0042] Figure 10 This figure shows the relative abundance and composition of microorganisms at the genus level during the in vitro fermentation of fructooligosaccharide propionate in Example 3 of this invention; AH represents the difference in relative abundance of microorganisms at the genus level in the fermentation broth, and I is a bar chart of the relative abundance of microorganisms at the genus level in the fermentation broth; the values ​​in the figure are expressed as mean ± SEM, n = 3, and different letters indicate significant differences. p <0.05.

[0043] Figure 11 The figure shows the effect of B-FOS on the growth curve of probiotics in Example 4 of this invention. A represents the effect of P-FOS on the growth curve of probiotics. Bifidobacterium lactis The effect of BB-12 on the growth curve, where B represents the effect of P-FOS on the growth curve. Lactobacillus casei The effect of strain Shirota on the growth curve.

[0044] Figure 12 Figure 5 shows the cumulative gas production and pH changes at different time points during the in vitro fermentation process of IBD patients in Example 5 of this invention. Figure A shows the change in gas production, and Figure B shows the change in pH. Among them, CON is the blank control group, F is the FOS control group, FB is the FOS + propionic acid group, PF is the P-FOS group, and APF is the AP-FOS group; R is the fecal sample of the patient in remission, and A is the fecal sample of the patient in the active phase.

[0045] Figure 13 The figure shows the changes in short-chain fatty acid content in the in vitro fermentation broth of IBD patients' feces in Example 5 of this invention. A represents acetic acid, B represents propionic acid, and C represents butyric acid. CON represents the blank control group, F represents the FOS control group, FB represents the FOS + propionic acid group, PF represents the P-FOS group, and APF represents the AP-FOS group. R represents fecal samples from patients in remission, and A represents fecal samples from patients in active phase. The values ​​in the figure are expressed as mean ± SEM, n=3, different letters indicate significant differences, p<0.05, and ns indicates no significant difference.

[0046] Figure 14 The figure shows the changes in total sugar content during the in vitro fermentation of IBD in Example 5 of this invention. CON is the blank control group, F is the FOS control group, FB is the FOS + propionic acid group, PF is the P-FOS group, and APF is the AP-FOS group; R is the fecal sample of the patient in remission and A is the fecal sample of the patient in the active phase.

[0047] Figure 15 The diagram shows a horizontal bar chart (A), a horizontal bar chart (B), and a horizontal heatmap (C) of microbial phyla for in vitro fermentation of feces from IBD patients in Example 5 of this invention.

[0048] Figure 16 The percentage change in mouse body weight in Example 6 of this invention.

[0049] Figure 17 This is a flowchart of the intervention process for creating an obese mouse model in Example 7 of the present invention.

[0050] Figure 18 This is the result of mouse weight gain in Example 7 of the present invention.

[0051] Figure 19 This is the result of Lee's index in Embodiment 7 of the present invention.

[0052] Figure 20 This is a morphological comparison of mice in the CON, HFD-C, and HFD-PF groups in Example 7 of the present invention. Detailed Implementation

[0053] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0054] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0055] Fructooligosaccharides: Quantum Hi-Tech (China) Biotechnology Co., Ltd., Batch No.: FOSP95S;

[0056] Inulin: Viloff Group Co., Ltd., Batch No.: NanoST P90.

[0057] Example 1 Preparation of fructosylpropionate

[0058] 1. Preparation of fructooligosaccharide propionate

[0059] 1 g of fructooligosaccharide was dissolved in 5 mL of pyridine. The dissolution was accelerated using a magnetic stirrer and a magnetic stirring rotor (d=1 cm). Then, 1 g of immobilized lipase TLIM and 1 mL of vinyl propionate were added. The resulting mixture was placed in an oil bath at 40 °C and reacted for 36 h. After the reaction was completed, the mixture was filtered, and the filtrate was collected. The remaining immobilized lipase TLIM was washed three times each with dichloromethane and ethanol, and the filtrates were collected. The filtrates were combined, and a total of 15 mL of liquid was obtained. The liquid was then rotary evaporated at 40 °C to obtain 2 mL of concentrated solution.

[0060] The concentrate was mixed with 5 mL of 50% ethanol, and the mixture was purified by C18 column chromatography. The eluent was eluted with 200 mL of 50% ethanol and 200 mL of 70% ethanol, and the eluent eluted with 200 mL of 70% ethanol was collected, rotary evaporated and freeze-dried to obtain propionyl-FOS (P-FOS).

[0061] 2. Preparation of inulin propionate

[0062] 6 g of inulin was dissolved in 40 mL of tetrahydrofuran. Dissolution was accelerated using a magnetic stirrer and a magnetic stirring rotor (d=2 cm). Then, 10 g of immobilized lipase TLIM and 5 mL of vinyl propionate were added. The resulting mixture was reacted in an oil bath at 40 °C for 24 h. After the reaction, the mixture was filtered, and the filtrate was collected. The remaining immobilized lipase TLIM was washed three times each with dichloromethane and ethanol, and the filtrates were collected. The filtrates were combined, yielding a total of 100 mL of liquid. This liquid was then rotary evaporated at 50 °C to obtain 5 mL of concentrated solution.

[0063] The concentrate was mixed with 10 mL of 40% ethanol, and the mixture was purified by C18 column chromatography. First, it was eluted with 500 mL of 40% ethanol, and then eluted with a mixture of 500 mL of 70% ethanol and 50 mL of n-propanol. 550 mL of the eluent eluted with the mixture of 70% ethanol and n-propanol was collected, rotary evaporated and freeze-dried to obtain inulin propionate (P-Inulin).

[0064] Comparative Example 1: Preparation of polyfructose propionate by acid anhydride method

[0065] 1. Fructooligosaccharide propionate

[0066] 1 g of fructooligosaccharide, 10 mL of pyridine, 0.8 mL of propionic anhydride, and a magnetically stirred rotor (d = 1 cm) were sequentially added to a 50 mL round-bottom flask, and the mixture was reacted in a water bath at 25 °C for 72 h. After the reaction was complete, the reaction solution was rotary evaporated at 40 °C to obtain 2 mL of concentrated solution. The concentrated solution was dissolved in 40% ethanol, and then purified by C18 column chromatography. The solution was eluted sequentially with 200 mL of 40% ethanol and 200 mL of 70% ethanol. The eluent of 200 mL of 70% ethanol was collected, and the eluent was rotary evaporated and lyophilized to obtain fructooligosaccharide propionate (AP-FOS).

[0067] 2. Inulin propionate

[0068] 1 g of inulin, 10 mL of pyridine, 0.8 mL of propionic anhydride, and a magnetically stirred rotor (d = 1 cm) were sequentially added to a 50 mL round-bottom flask, and the mixture was reacted in a water bath at 25 °C for 48 h. After the reaction was complete, the reaction solution was rotary evaporated at 40 °C to obtain 2 mL of concentrated solution. The concentrated solution was dissolved in 40% ethanol, and then purified by C18 column chromatography. First, 500 mL of 40% ethanol was used as the eluent, followed by 500 mL of a mixture of 70% ethanol and 50 mL of n-propanol. 550 mL of the eluent was collected, rotary evaporated, and freeze-dried to obtain inulin propionate (AP-Inulin).

[0069] Example 2 Identification of fructooligosaccharide propionate

[0070] 1. Infrared spectroscopy identification of fructooligosaccharide propionate

[0071] The chemical bonds in the P-FOS prepared in Example 1, the AP-FOS prepared in Comparative Example 1, and the raw material FOS were analyzed, identified, and compared using Fourier transform infrared spectroscopy (FT-IR).

[0072] Weigh 1 mg of purified P-FOS and AP-FOS samples respectively, and grind them with 100 mg of dry potassium bromide solid in a grinder until they become powdery. Compress the sample powder into tablets using a tablet press, and then use a Fourier transform infrared spectroscopy (FT-IR) instrument at 4000–500 cm⁻¹. -1 Scan the infrared absorption spectrum within the wavenumber range.

[0073] The infrared absorption spectrum of fructooligosaccharide propionate is shown below. Figure 1 As shown.

[0074] Figure 1 The results show that P-FOS, AP-FOS, and FOS all contain a large number of hydroxyl groups (-OH), which are represented by a peak value at 3350 cm⁻¹ in the infrared spectrum. -1 Absorption peaks due to stretching vibrations appearing on the left and right sides. 2700–3000 cm⁻¹ -1 The peak at 1600–1630 cm⁻¹ is the characteristic absorption peak of the stretching vibration of saturated CH in alkane chains. -1 It is the characteristic absorption peak of the stretching vibration of the carbonyl group (COO-), 1300–1500 cm⁻¹ -1 These are bending vibration groups (OCH, COH, CCH); 800–1200 cm -1 The region represents the stretching vibration of glycosidic bonds. Compared to FOS, P-FOS and AP-FOS exhibit vibrations at 1731 cm⁻¹. -1 The presence of a characteristic absorption peak of ester bond (C=O) indicates that propionic acid was successfully linked to fructooligosaccharides via ester bonds, and two fructooligosaccharide propionates, P-FOS and AP-FOS, were successfully synthesized.

[0075] from Figure 1 It can be observed that, except for P-FOS and AP-FOS, which are both at 1730 cm⁻¹, 1 Apart from the characteristic absorption peak of the ester bond appearing at the point of origin, the two curves are highly similar. Therefore, the product contains an ester bond formed by the successful combination of FOS and propionic acid, indicating the successful synthesis of oligofructose propionate esters P-FOS and AP-FOS, with AP-FOS having a higher degree of esterification than P-FOS.

[0076] Infrared spectroscopy revealed the presence of ester bonds formed by the successful combination of inulin and propionic acid in the P-Inulin and AP-Inulin products, indicating the successful synthesis of inulin propionate.

[0077] 2. Flight mass spectrometry analysis of fructooligosaccharide propionate

[0078] Prior to analysis, the oligofructose propionate sample was mixed with a small-molecule matrix solution and placed on the target plate, allowing co-crystallization to occur after solvent evaporation. Following the method of Tian et al., the purified oligofructose butyrate sample was analyzed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) Ultraflextreme workstation. Mass spectra were acquired using a 337 nm nitrogen laser in positive mode. After a 200 ns delay extraction, ions were accelerated to a kinetic energy of 12 kV and detected using reflector mode. The sample was desalted using AG 50W-X4 resin, and 1 μL of the desalted sample was added to 1 μL of a 10 mg / mL 50% acetonitrile solution (v / v) of 2,5-dihydroxybenzoic acid on a MALDI plate.

[0079] The mass spectrum of fructooligosaccharide propionate is as follows: Figure 2 As shown; where A is the flight mass spectrum of FOS, B is the flight mass spectrum of P-FOS, and C is the flight mass spectrum of AP-FOS.

[0080] according to Figure 2 The molecular ion peaks at m / z 527, 689, 851 and 1013 of A indicate that its true molecular weight should be 504, 666, 828 and 990, respectively, which is consistent with the molecular weight of FOS. In this experiment, the substrate FOS for the synthesis of P-FOS and AP-FOS is mainly composed of four sugars: GF2, GF3, GF4 and GF5. Figure 2 The results in B show that the molecular weights of the synthesized product P-FOS are 583, 639, 745, 801, 857, 907, and 963, which are 56 or 112 greater than the molecular weight of FOS, which is equivalent to 1 to 2 times the molecular weight of propionic acid after dehydration.

[0081] Figure 2 The results of the C-ray diffraction (C-C) analysis showed that the molecular weights of AP-FOS were 639, 695, 751, 801, 807, 857, 863, 913, 929, 969, 1019, 1025, 1075, 1081, 1131, 1137, 1187, 1237, 1243, 1293, 1299, and 1349, respectively, in conjunction with the infrared spectra. Figure 1 AP-FOS was determined to be mainly composed of 2 to 8 propionic acid molecules and FOS linked by ester bonds.

[0082] The results showed that propionic acid was successfully attached to fructooligosaccharides. P-FOS mainly attached two propionic acid molecules, while AP-FOS mainly attached six propionic acid molecules. The absence of FOS peaks in the P-FOS and AP-FOS plots indicated that FOS was completely removed.

[0083] 3. 1H NMR spectroscopy analysis of fructooligosaccharide propionate

[0084] Weigh 50 mg of dried FOS, P-FOS, and AP-FOS samples respectively, dissolve them in 500 μL of deuterated water (D2O), mix well, and then add them to an NMR tube. NMR detection (1H NMR) was performed using a Brüker Advance 400 (1H: 400 MHz), with chemical shifts (δ) expressed in ppm. The internal standard was determined by the residual solvent peak of the deuterated reagent (e.g., 1 H NMR: D2O, 4.79).

[0085] The composition of FOS, P-FOS, and AP-FOS was identified using MALDI-TOF MS, and the composition of fructooligosaccharide propionate was determined. 1 The H NMR spectrum is shown below. Figure 3 As shown.

[0086] Figure 3 The results showed that FOS exhibited characteristic CH peaks corresponding to the fructose ring backbone in the range of 3.0–5.30 ppm. However, the proton peaks on the fructose rings of P-FOS and AP-FOS shifted to the left because the carbonyl (C=O) electron group was attached after the reaction, reducing the electron cloud density around the hydrogen nucleus, resulting in decreased shielding effectiveness and increased chemical shift. Compared to FOS, P-FOS and AP-FOS spectra showed absorption peaks at 1.0 ppm and 2.5 ppm, respectively, which are the absorption peaks of -CH2-CH3 on the propionyl group. Therefore, we inferred that the propionyl group was successfully attached to the sugar ring of FOS.

[0087] Combination Figure 2 and Figure 3 It can be determined that P-FOS is mainly composed of one or two propionic acids and FOS linked by ester bonds.

[0088] 4. Determination of total sugar content of fructooligosaccharide propionate

[0089] (1) The total sugar content (w / w) of the raw material FOS, the purified P-FOS prepared in Example 1, and the purified AP-FOS prepared in Comparative Example 1 was determined using the phenol-sulfuric acid method. 10 mg of sample powder or liquid was accurately weighed into a K-max tube and placed in an ice bath. 0.45 mL of 72% concentrated sulfuric acid was added while shaking. The tube was heated in a 30°C water bath for 1 h, shaking the tube every 20 min to ensure complete reaction between the lyophilized sample powder and the concentrated sulfuric acid. After cooling, 6 mL of distilled water was added, and the tube was shaken thoroughly. The tube was then heated in a 100°C metal bath for 1 h, shaking the tube every 30 min. After the reaction was complete, the tube was cooled to room temperature.

[0090] (2) Construction of standard curve

[0091] Add 0, 10, 20, 30, 40, 50, and 60 µL of 150 µg / mL glucose standard solution to test tubes, respectively, and then replenish to 200 µL with 2.5% phenol solution, shaking to mix. Add 0.5 mL of concentrated sulfuric acid, mix well, cool to room temperature, and measure the absorbance at 490 nm. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa.

[0092] (3) Determination of total sugar in the sample

[0093] 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.

[0094] The results of the determination of total sugar content of fructooligosaccharide propionate are as follows: Figure 4 As shown, Figure 4 The results showed that FOS had a sugar content of 60.23%, P-FOS had a sugar content of 45.90%, and AP-FOS had a sugar content of 35.62%, with AP-FOS having the lowest sugar content.

[0095] Example 3: Determination of in vitro fermentation characteristics of fructooligosaccharide propionate

[0096] 1. Construction of an in vitro simulated colonic fermentation model

[0097] Following the method of Yang et al. (Yang ZX, Huang T, Li P, et al. Dietary fibermodulates the fermentation patterns of cyanidin-3-o-glucoside in a fiber-typedependent manner[J]. Foods, 2021, 10(6).), an in vitro simulated colonic fermentation model was constructed, and the culture medium and inoculum were prepared.

[0098] The fermentation characteristics of fructooligosaccharide propionate (FOS) were investigated using a constructed colonic fermentation model. Samples were collected at 0, 6, 12, 24, and 36 h of culture for subsequent processing. The basic fermentation characteristics of FOS were analyzed by monitoring changes in pH, gas production, and total sugar content. Furthermore, the effects of FOS on the gut microbiota were investigated by sequencing the microbial community in the fermentation broth, and the changes in its metabolites under the influence of the gut microbiota were analyzed.

[0099] The samples used were fructooligosaccharide propionate P-FOS and AP-FOS prepared and purified in Example 1. SIEM medium was placed in the anaerobic operating platform 12 h in advance for oxygen replacement until the medium color changed from red to yellow. Before inoculation, an appropriate amount of sample was mixed and shaken to dissolve. Five groups were set up: blank control group (Group C), FOS control group (Group F), FOS + propionic acid group (Group FAP), P-FOS group (Group PF), and AP-FOS group (Group APF). Each group was tested in triplicate at each time point. Based on the sugar content of FOS, to ensure the same concentration of the sample in the fermentation broth, the total sugar content was consistent across groups, resulting in a final concentration of 6 mg / mL for all groups. The amount of propionic acid added in the FAP group was the same as the amount of propionic acid contained in the AP-FOS with the same sugar content. Fresh fecal bacterial solution was immediately transferred to the anaerobic operating platform for inoculation after preparation. The medium containing the sample and the fecal bacterial solution were added to the anaerobic fermentation tube at a volume ratio of 9:1, sealed, and mixed thoroughly. It was then placed in a constant temperature shaking incubator at 37°C for incubation.

[0100] 2. Measurement of gas production and pH of fermentation broth

[0101] A sterile syringe was used to directly insert into the rubber stopper on the fermentation tube to detect and record the gas production in the fermentation tube at different times.

[0102] The pH of the fermentation broth at different times was measured and recorded using a miniature pH meter.

[0103] The cumulative gas production and pH changes of samples at different time points during the in vitro fermentation of fructooligosaccharide propionate are shown in the figure. Figure 5 As shown in the figure, A represents the change in gas production, B represents the change in pH; C represents the blank control group, F represents the FOS control group, PF represents the P-FOS group, APF represents the AP-FOS group, and FAP represents the FOS+propionic acid group.

[0104] Figure 5 As shown in Figure A, the cumulative gas production in the fermentation tubes gradually increased from 0 to 12 h during in vitro fermentation, especially in the PF and F groups, reaching 1.50 mL and 1.49 mL, respectively. However, gas production in the C and FAP groups was not significant, and the APF group produced only a small amount of gas, with its cumulative gas production significantly lower than that of the PF group. After 12 h of fermentation, the F group had stopped producing gas, and the gas production rate of the PF group also decreased significantly, although it was still being degraded and producing gas. At the end of fermentation, the PF group had the highest cumulative gas production, reaching 2.1 mL.

[0105] Figure 5 The results from Figure B show that the pH of group C remained almost unchanged during fermentation. From 0 to 12 h during in vitro fermentation, the pH of groups FAP, F, PF, and APF all decreased continuously. Groups FAP and F experienced the fastest pH decrease, reaching 4.14 and 4.36 respectively at 12 h. The pH of the PF fermentation broth was slightly higher than that of groups FAP and F, reaching 4.79. After 12 h, except for APF, the pH of the fermentation broths of the other groups no longer showed significant changes. The pH of the APF group remained relatively stable at approximately 5.17 at 24 h.

[0106] 3. Changes in total sugar content in the fermentation broth

[0107] (1) Sample hydrolysis

[0108] Same as 4(1) in Example 2.

[0109] (2) Construction of standard curve

[0110] Same as 4(2) in Example 2.

[0111] (3) Determination of total sugar in the sample

[0112] Same as 4(3) in Example 2.

[0113] (4) Results

[0114] The changes in total sugar content during in vitro fermentation are as follows: Figure 6 As shown, C represents the blank control group, F represents the FOS control group, PF represents the P-FOS group, APF represents the AP-FOS group, and FAP represents the FOS + propionic acid group.

[0115] Figure 6The results showed that the total sugar content in the fermentation broth generally decreased as in vitro fermentation progressed. In the first 12 hours of fermentation, the sugar in the broth was rapidly utilized by microorganisms, resulting in a significant decrease in total sugar content. The degradation rates of total sugar in groups F and FAP were similar, both faster than those in groups PF and APF, and were almost completely degraded by 12 hours. By 24 hours, the sugar in group PF was almost completely degraded, while the degradation rate in group APF slowed considerably. At 24 hours, the total sugar content in the fermentation broth of groups PF and APF was only 1.77 mg / mL and 4.03 mg / mL, respectively, representing degradation rates of approximately 75.06% and 59.57%. In summary, P-FOS promoted the growth of more gas-producing bacteria in the fermentation broth, while the high initial propionic acid concentration seemed to inhibit the growth of gas-producing bacteria. AP-FOS, possibly due to its high degree of esterification, had a lower total sugar degradation rate than P-FOS.

[0116] In summary, P-FOS exhibits better fermentability in in vitro fermentation systems and is more stably degraded by gut microbiota compared to FOS, while AP-FOS cannot be effectively degraded by gut microbiota.

[0117] Similarly, experiments showed that P-Inulin prepared in Example 1 and AP-Inulin prepared in Comparative Example 1 had better fermentability in the in vitro fermentation system and was more stably degraded by gut microbiota than Inulin, while AP-Inulin could not be well degraded by gut microbiota.

[0118] 4. Determination of short-chain fatty acids (SCFAs) in fermentation broth

[0119] Changes in short-chain fatty acid content in the in vitro fermentation broth of fructooligosaccharide propionate are as follows: Figure 7 As shown in the figure; A represents acetic acid, B represents propionic acid, C represents butyric acid, D represents isobutyric acid, E represents valeric acid, F represents isovaleric acid, G represents hexanoic acid, and H represents the total short-chain fatty acid content; the values ​​in the figure are expressed as mean ± SEM, n = 3, different letters indicate significant differences. p <0.05, ns indicates that the difference is not significant.

[0120] Figure 7 The results showed that the content of various short-chain fatty acids in the fermentation broth gradually increased with the extension of fermentation time. The FAP group had the highest acetic acid yield, followed by the F group. The PF 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 APF and FAP groups. With the extension of fermentation time, the butyric acid yield in the PF group gradually increased. By 12 h of fermentation, the butyric acid concentrations in the PF and APF groups were significantly higher than the other three groups (p<0.05).

[0121] In summary, FOS, a mixture of FOS and propionic acid, and P-FOS can all be fermented by gut microbiota to produce SCFAs, but AP-FOS exhibits poor fermentability. Among these, P-FOS demonstrates a significant propionic acid-producing capacity under the influence of gut microbiota. Similarly, experiments showed that P-Inulin prepared in Example 1 also exhibited a significant propionic acid-producing capacity under the influence of gut microbiota.

[0122] 5. 16S rRNA gene sequencing of fermentation broth

[0123] Following the method of Yang et al. (Yang ZX, Huang T, Li P, et al. Dietary fibermodulates the fermentation patterns of cyanidin-3-o-glucoside in a fiber-typedependent manner[J]. Foods, 2021, 10(6).), 16S rRNA gene sequencing was performed on the fermentation broth after 36 h of in vitro fermentation to study the diversity and compositional changes of the gut microbiota in the fermentation broth.

[0124] Differences between different samples can be identified through analytical methods such as non-metric multi-dimensional scaling (NMDS).

[0125] The NMDS analysis results of the beta diversity of microorganisms in the fermentation broth are as follows: Figure 8 As shown in the figure; one point in the figure represents one sample, different groups are different colors, and the greater the distance between samples, the greater the difference in the composition of the microbial community. Figure 8 The stress level in the NMDS analysis was 0.0388, indicating excellent representativeness. The close proximity of samples within each group suggests good reproducibility. The significant separation and large distance between samples between groups indicate that FOS, FOS+propionic acid, P-FOS, and AP-FOS all influence microbial composition in in vitro fermentation, with different influencing factors.

[0126] (2) Changes in the composition of microorganisms at the phylum level during in vitro fermentation

[0127] The relative abundance and composition of microorganisms at the phylum level in the in vitro fermentation of fructooligosaccharide propionate are as follows: Figure 9 As shown in the figure; AF represents the difference in relative abundance of microorganisms at the phylum level of the fermentation broth, and G is a bar chart of the relative abundance of microorganisms at the phylum level of the fermentation broth; the values ​​in the figure are expressed as mean ± SEM, n = 3, and different letters indicate significant differences. p <0.05.

[0128] Figure 9 The results showed that after 24 hours of in vitro fermentation, Bacteroidetes, Firmicutes, Proteobacteria, and Fusobacteria were the dominant bacterial phyla in the five sample groups, accounting for more than 82.29% of the total bacterial community in the fermentation broth. Bacteroidetes and Firmicutes are two major phyla constituting the human gut microbiota. Bacteroidetes are mainly anaerobic bacilli in the human gastrointestinal tract, and are primarily found in environments containing phagocytic chitinous bacteria, phagocytes, flavibacteria, and spirochetes, which can degrade polymeric organic matter. When the number of Bacteroidetes in the gut decreases or even disappears, it may lead to various infections and abscesses in the intestine, and even affect systemic tissues and organs. Some bacteria in Firmicutes can promote the hydrolysis and utilization of carbohydrates by encoding carbohydrate-activating enzymes.

[0129] Figure 9 The results in group A showed that the presence of PF significantly increased the abundance of Bacteroidota in the fermentation broth (p<0.05), followed by the APF group. Compared with group C, both groups F and FAP increased the relative abundance of Bacteroidota, but there was no significant difference compared with group C. The abundance of Firmicutes in the fermentation broth was highest in group F (p<0.05), followed by groups FAP and PF, all of which significantly increased the relative abundance of Firmicutes in the fermentation broth. Compared with group C, group APF also showed an increase, but there was no significant difference.

[0130] The ratio of firmicutes to bacteroidota (F / B ratio) is an important indicator of obesity biology. Obese individuals tend to have a higher F / B ratio in their gut, and a lower F / B ratio may reduce the risk of insulin resistance and weight gain. The F / B results for each group in the experimental and control groups are shown in Table 1 below.

[0131] Table 1

[0132]

[0133] Table 1 shows that, compared with the control, the F / B ratios of both the PF group and the APF group were reduced, with a significant decrease in the F / B ratio of the PF group. Conversely, the F / B ratios of the F and FAP groups were increased, indicating that both the PF and APF groups can reduce the risk of insulin resistance and weight gain, thus having a weight-loss effect. The PF group showed a more significant reduction and better weight-loss effect. Similarly, the experiment found that P-Inulin prepared in Example 1 also reduced the risk of insulin resistance and weight gain, exhibiting a weight-loss effect superior to AP-Inulin.

[0134] Proteobacteria are all Gram-negative bacteria, including many pathogens such as Escherichia coli, Escherichia coli, Salmonella, Shigella, Pseudomonas aeruginosa, and Helicobacter pylori. Overgrowth of this phylum in the intestine can lead to a range of diseases. Figure 9 The results showed that, compared with group C, groups F, PF, and FAP all significantly inhibited the growth of Proteobacteria (p<0.05). The APF group also reduced the relative abundance of Proteobacteria in the fermentation broth, which helps restore the gut microbiota composition to a relatively healthy state. The FAP group, in particular, showed the most significant improvement, indicating that the direct presence of propionic acid has a certain inhibitory effect on the growth of Proteobacteria. While the improvement effect of the PF group was not as significant as that of the FAP group, it was more pronounced than that of FOS. This suggests that the PF group combines the beneficial properties of both propionic acid and FOS.

[0135] Fusobacteria are common pathogens found in the digestive tract and oral cavity, which can cause various diseases such as appendicitis, oral inflammation and infectious diseases, and even cancer. Figure 9 The results showed that, compared with group C, groups PF, F, and FAP all significantly inhibited the relative abundance of Fusobacteria (p<0.05). Group APF also inhibited the growth of Fusobacteria. Among them, group PF had the best effect, and no Fusobacteria were detected in its fermentation broth.

[0136] Actinobacteria are Gram-positive bacteria. Most members of this phylum have complex hyphal lifestyles, forming aerial and substrate mycelia, characterized by pigmentation and high sporulation rates. They possess strong biosynthetic potential, producing secondary metabolites with broad structural diversity and commercial significance. Bifidobacterium is the most common in the human gut and is one of the main users of functional oligosaccharides such as FOS. Figure 9 The results showed that, compared with group C, groups FAP, PF, and F all significantly increased the abundance of Actinobacteria (p<0.05), promoting Actinobacteria growth. Furthermore, FOS and P-FOS also played a good role in maintaining gut microbiota balance. However, the abundance of Actinobacteria in the APF group was significantly lower than that in group C, indicating an inhibition of Actinobacteria abundance.

[0137] Desulfobacterota contains some pathogenic bacteria, and studies have shown that the relative abundance of Desulfobacterota in the gut microbiota of diabetic retinopathy patients is increased compared to healthy individuals. A high-fat diet promotes the growth of Desulfobacterota, thereby disrupting the gut microbiota structure in mice. Figure 9 The results showed that, compared with group C, groups F, PF, FAP, and APF all significantly inhibited the growth of Desulfobacterota, with groups F, PF, and FAP showing even better inhibitory effects than group APF. This indicates that the co-existence of FOS and propionic acid can inhibit the growth of Desulfobacterota.

[0138] (4) Changes in the composition of microorganisms at the genus level during in vitro fermentation

[0139] The relative abundance and composition of horizontal microorganisms in the in vitro fermentation of fructooligosaccharide propionate are as follows: Figure 10 As shown; AH represents the relative abundance difference of microorganisms at the genus level in the fermentation broth, and I is a bar chart of the relative abundance of microorganisms at the genus level in the fermentation broth; the values ​​in the figure are expressed as mean ± SEM, n = 3, and different letters indicate significant differences. p <0.05.

[0140] Figure 10 The results showed that after 24 hours of in vitro fermentation, Fusobacterium, Bacteroides, Escherichia_ Shigella, Sutterella, Phascolarctobacterium and Dialister It is the dominant genus in group C. Fusobacterium It is a Gram-negative anaerobic bacterium. Studies have shown that it is associated with the development of colorectal cancer, as well as ulcerative colitis (UC) and Crohn's disease (CD). Figure 10 The results from group A showed that, compared with group C, groups PF, FAP, and F all significantly reduced the fermentation broth temperature. Fusobacterium relative abundance ( p < 0.05), the relative abundance in the PF group was the lowest, and it was significantly different from that in the FOS group. The APF group... Fusobacterium Although the relative abundance of [a substance] also decreased, it was not significant.

[0141] Escherichia_Shigella These are common pathogenic bacteria with pro-inflammatory properties, including Escherichia coli and Salmonella. Compared with healthy individuals, they are more prevalent in patients with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver fibrosis. Escherichia_Shigella In NAFLD patients, there are a large number of Escherichia_ShigellaIt may lead to Escherichia_Shigella The surge in quantity penetrates the intestinal epithelial barrier, exacerbating intestinal leakage. Figure 10 The results in group B showed that, compared with group C, PF, FAP, and group F all effectively reduced the fermentation broth temperature. Escherichia_Shigella The relative abundance of the APF group was lowest (p < 0.05), followed by the PF group, both of which showed significant differences from the F group (p < 0.05). The APF group did not show significant differences from the C and F groups, but its inhibitory effect was significant. Escherichia_Shigella The growth capacity was between that of group C and group F. This indicates that propionic acid inhibits growth. Escherichia_ Shigella The growth effect is good, and FOS is also reduced to a certain extent. Escherichia_Shigella The effect of APF on growth inhibition Escherichia_Shigella Its growth ability is not as outstanding as that of the FOS and PF groups.

[0142] Klebsiella It is also a pathogenic bacterium that can produce enterotoxins and induce apoptosis in intestinal epithelial cells. Figure 10 The results showed that, compared with group C, groups FAP, PF, F, and APF all significantly reduced [the condition]. Klebsiella Relative abundance ( p <0.05). The PF group showed a better reduction effect than the APF group.

[0143] Dialister It belongs to the Firmicutes phylum and includes anaerobic, non-motile, and Gram-negative bacilli. Dialister Some species may play a role in the regulation of sugar metabolism. Dialister It is closely related to the increase in SCFAs levels. Figure 10 The results from the D-test showed that, compared with group C, groups FAP, PF, and F all significantly improved the concentration of phosphorus in the fermentation broth. Dialister The relative abundance (p<0.05) showed the greatest increase in group F, while that in group APF was significantly higher. Dialister The relative abundance was basically similar to that of group C.

[0144] Bifidobacterium It is a typical probiotic in the gut, which can ferment polysaccharides such as FOS to produce acetic acid and lactic acid, and has functional activities such as regulating intestinal flora, improving intestinal function, anti-tumor activity and improving nutrition. Figure 10 The results from the study showed that, compared with group C, groups FAP, PF, and F all significantly improved the concentration of phosphorus in the fermentation broth. Bifidobacterium Relative abundance ( p <0.05), FAP group pair Bifidobacterium The growth-promoting effect of the APF group was significantly higher than that of all other groups. The APF group, however, increased... Bifidobacterium The effect of relative abundance was significantly lower than that of the PF group.

[0145] Prevotela 9 is abundant and diverse in the human microbiome, distributed in different organs and ecological niches, such as the oral cavity and intestines. It has glycolytic function, metabolizing polysaccharides into succinic acid, and is closely related to the production of propionic acid. Zhao et al. used red yeast rice fermented ginseng (PM) to treat obesity induced by HFD in rats and found that after PM treatment, the F / B ratio of rats decreased and the relative abundance of Prevotela 9 increased significantly, effectively alleviating the intestinal microbiota disorder caused by metabolic diseases. Figure 10 The results showed that, compared with group C, groups FAP, PF, F, and APF all significantly increased the relative abundance of Prevotela_9. p <0.05). Among them, the PF group showed the most significant improvement.

[0146] Faecalibacterium and Collinsella These are all important bacteria that produce butyric acid. Figure 10 The results from the G-test showed that, compared with group C, the fermentation broths of groups F and PF contained... Faecalibacterium The relative abundance was significantly improved (p<0.05). Figure 10 The middle H shows that, compared with group C, all other groups Collinsella The relative abundance decreased. Collinsella The butyrate content decreased, while the butyrate content increased. There was a significant difference between the PF group and the F group (p<0.05), and the FOS group inhibited [the activity / effects]. Collinsella This also explains the high butyric acid content produced during fermentation in the PF and APF groups.

[0147] Example 4: Determination of the bioavailability of fructooligosaccharide propionate

[0148] The P-FOS prepared in Example 1 was used as a carbon source and reacted with Bifidobacterium ( ) Bifidobacterium lactis BB-12, B. lactis BB-12) and Lactobacillus casei ( Lactobacillus casei The study investigated the growth-promoting effect of P-FOS on common probiotics Bifidobacterium and Lactobacillus by co-culturing strain Shirota with a blank control group (CON group) without any added carbon source and positive controls with FOS (FOS group) and FOS + propionic acid (FAP group).

[0149] Bifidobacterium ( Bifidobacterium lactis BB-12), Lactobacillus casei ( Lactobacillus caseiThe strain *Shirota* was isolated in our laboratory and identified by sequencing. The effect of P-FOS on the proliferation of the two probiotics was determined in an anaerobic incubator using sterile MRS medium in 96-well V-type plates. The blank control group (CON group) was cultured in medium without any carbon source, and the positive controls were FOS (FOS group) and FOS + n-propionic acid (FAP group). The experimental group included P-FOS. Under sterile conditions, 150 μL of medium was added to each plate, with three replicates per sample. Each plate was inoculated with 2% of the culture medium volume of the stock solution. The plates were incubated at 37°C for 24 h in an anaerobic incubator, and the microplates were analyzed every 2 h using a microplate reader. 600nm The growth of the two strains was detected, and growth curves of the two probiotics were plotted.

[0150] (2) Results

[0151] The effect of B-FOS on the growth curve of probiotics, such as Figure 11 As shown, A is the P-FOS pair Bifidobacterium lactis The effect of BB-12 on the growth curve, where B represents the effect of P-FOS on the growth curve. Lactobacillus casei The effect of strain Shirota on the growth curve.

[0152] Figure 11 As shown in Figure A, the growth lag phase of *B. lactis* BB-12 was 0–6 h, the logarithmic growth phase was 6–16 h, and the growth rate of *B. lactis* BB-12 began to slow down after 16 h. Compared with the control group, the FOS, P-FOS, and FAP groups all significantly promoted the growth of *B. lactis* BB-12. P-FOS, like FOS, promoted good growth of *B. lactis* BB-12. At 6 h of anaerobic culture, the growth-promoting effect of the P-FOS group on *B. lactis* BB-12 was significantly different from that of the FOS and FAP groups. The P-FOS group showed a better growth-promoting effect than the FOS group throughout the entire culture process.

[0153] Figure 11 The B-mode indicates that 0–4 hours is… Lactobacillus casei strain Shirota The growth lag phase of Lactobacillus is 4–10 h, which is the logarithmic growth phase. After 10 h of culture, the growth rate of Lactobacillus begins to slow down until it stabilizes. It is clear that the FOS group, P-FOS group, and FAP group all have a significant promoting effect on the growth of Lactobacillus casei strain Shirota.

[0154] Example 5: The effect of fructooligosaccharide propionate on the stool of IBD patients.

[0155] 1. Determination of gas production and pH of fermentation broth

[0156] The gas production and pH of the fermentation broth were measured according to the method described in Example 3.

[0157] Cumulative gas production and pH changes at different time points during in vitro fermentation of IBD patients, as shown in the following figures. Figure 12 As shown in the figure, A represents the change in gas production, and B represents the change in pH; CON is the blank control group, F is the FOS control group, FB is the FOS + propionic acid group, PF is the P-FOS group, and APF is the AP-FOS group; R is the fecal sample of the patient in remission, and A is the fecal sample of the patient in the active phase.

[0158] Figure 12 The results showed that as in vitro fermentation progressed, the cumulative gas volume in the fermentation tube generally increased, while the pH of the fermentation broth, except for the CON group, showed a decreasing trend. Figure 12 The results showed that during in vitro fermentation from 0 to 24 hours, the gas production of each group gradually increased, especially CON-R, AP-FOS-R, and P-FOS-R, indicating an increase in the content of gas-producing bacteria. Comparing the gas production of feces from patients in the active and remission phases, it can be seen that the feces of patients in the active phase contained more gas-producing bacteria. Figure 12 The results in group B show that pH decreased with increasing fermentation time, with the FOS-R and FOS-A groups showing the largest decrease, followed by P-FOS-R and P-FOS-A. AP-FOS-A showed virtually no change. A decrease in pH indicates that fermentation is underway; without nutrients, the pH would decrease very slowly, as seen in group CON.

[0159] 2. Determination of short-chain fatty acids (SCFAs) in fermentation broth

[0160] The measurement method is the same as in Example 3.

[0161] Changes in short-chain fatty acid content in in vitro fermentation broth of feces from IBD patients are as follows: Figure 13 As shown, A represents acetic acid, B represents propionic acid, and C represents butyric acid; CON represents the blank control group, F represents the FOS control group, FB represents the FOS + propionic acid group, PF represents the P-FOS group, and APF represents the AP-FOS group; R represents fecal samples from patients in remission, and A represents fecal samples from patients in active phase; the values ​​in the figure are expressed as mean ± SEM, n=3, different letters indicate significant differences, p<0.05, and ns indicate no significant differences.

[0162] from Figure 13The results showed that short-chain fatty acids increased in all sample groups with prolonged fermentation time. P-FOS and AP-FOS produced significantly more propionic acid than other groups in both the active and remission phases, exceeding 20 mM.

[0163] 2. Changes in total sugar content in the fermentation broth

[0164] The method for determining the change in total sugar in the fermentation broth is the same as that in Example 2, section 4.

[0165] The changes in total sugar content during IBD in vitro fermentation are as follows: Figure 14 As shown, CON is the blank control group, F is the FOS control group, FB is the FOS + propionic acid group, PF is the P-FOS group, APF is the AP-FOS group; R is the fecal sample of the patient in remission, and A is the fecal sample of the patient in the active phase.

[0166] Figure 14 The results showed that the total sugar content in the fermentation broth generally decreased as in vitro fermentation progressed. After 24 hours of fermentation, FOS-R and FOS-A were consumed the most, followed by P-FOS-R and P-FOS-A. AP-FOS-R was also consumed to some extent, but AP-FOS-A was hardly consumed. This indicates that AP-FOS, due to its high degree of esterification, may be difficult for IBD patients to utilize in their feces.

[0167] A bar chart of microbial phyla in in vitro fermentation of feces from IBD patients is shown below. Figure 15 In the diagram of microbial genera A, the horizontal bar chart is shown below. Figure 15 In the B category, a horizontal heatmap of microbial genus is shown below. Figure 15 C.

[0168] Figure 15 The results showed that both P-FOS and FOS had beneficial effects on the gut microbiota of patients in the active phase. FOS promoted the growth of Bifidobacteria in the feces of patients in the active phase and promoted the growth of Lactobacillus in patients in the remission phase. P-FOS promoted the growth of both Bifidobacteria and Lactobacillus in the feces of patients in the active phase. Except for the CON group, the intervention treatments in the other groups inhibited the growth of Lachnoclostridium, a biomarker of colon cancer and colonoma.

[0169] In summary, the fructooligosaccharide propionate (PF) intervention group of this invention improved the fecal microbiota structure in the feces of patients during the active phase, promoted the growth of beneficial bacteria such as Bifidobacteria and Lactobacillus, and inhibited the growth of many harmful bacteria; demonstrating a certain ability to improve the fecal microbiota of IBD patients. Similarly, experiments found that P-Inulin prepared in Example 1 can also improve the fecal microbiota structure of IBD patients and promote the growth of beneficial bacteria such as Bifidobacteria and Lactobacillus.

[0170] Example 6 Effects of fructooligosaccharide propionate and fructooligosaccharide butyrate on body weight of normally fed mice

[0171] Preparation of fructooligosaccharide butyrate:

[0172] Accurately weigh 1 g of fructooligosaccharide into a round-bottom flask, add a clean and dry magnetic stir bar, and add 10 mL of anhydrous pyridine to the flask. Stir on a magnetic stirrer until the fructooligosaccharide dissolves. 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 with a magnetic stirrer and react at 55℃ for 48 h. After the reaction is complete, filter the sample in the round-bottom flask to separate the immobilized lipase TLIM, obtaining the enzymatic preparation solution of fructooligosaccharide butyrate (Butyric-FOS, B-FOS). Use a vacuum rotary evaporator to evaporate to a solid state to remove most of the pyridine from the reaction solution. Prepare a 5% (v / v) methanol aqueous solution, add the solid to the round-bottom flask, and continuously blow and stir to ensure it is evenly dispersed in the methanol aqueous solution.

[0173] The reaction solution was separated using a C18 column chromatography. First, the C18 column was washed with 5% (v / v) acidified methanol (a mixture of 5% formic acid and 95% methanol), and then the residual acidified methanol was replaced with a 5% (v / v) methanol-water solution. The sample suspension dispersed in the 5% (v / v) methanol-water solution was carefully added to the chromatography column, and the sample was eluted with 3 column volumes of 5% (v / v) methanol-water solution to remove residual pyridine, fructooligosaccharides not yet attached to butyric acid, and vinyl butyrate. Pyridine is colorimetric under UV light; therefore, when the collected eluent no longer shows color under UV light, pyridine has been completely removed. Finally, methanol was added to the chromatography column to elute the sample, and the resulting yellow eluent was collected. The collected solution was analyzed by TLC using n-butanol:n-propanol:water = 1:6:1 as the developing solvent, and the eluents with completely removed FOS were combined. The collected sample eluent was evaporated using a vacuum rotary evaporator. Then, pure water was added to the flask to dissolve the yellow sample. The sample was then transferred to a centrifuge tube and the water was removed using a vacuum freeze dryer. The resulting yellow solid was purified B-FOS.

[0174] With the approval of the Ethics Committee of the Animal Management Center of Jinan University, 72 six-week-old male C57BL / 6 mice (18–22 g) were purchased from Zhejiang Vital River and housed at the Animal Management Center of Jinan University under standard conditions. During the quarantine period, they were fed feed (co-produced biological rodent feed) provided by the animal facility, had free access to water, and had their water, feed, and bedding in their cages changed regularly.

[0175] After 10 days of quarantine, mice were randomly divided into three groups of 12 mice each: a blank control group (Control group, CON), a fructooligosaccharide butyrate control group (B-FOS group), and a fructooligosaccharide propionate intervention group (P-FOS group). The modeling process lasted 23 days, with all mice fed a standard D12450H diet for 23 days. All mice were randomly divided into three groups, all fed a standard D12450H diet (control group), and all groups received sterile water provided by the animal center throughout the process. The B-FOS group was administered a purified B-FOS solution dissolved in physiological saline via gavage daily during the modeling period. The P-FOS group was administered a P-FOS solution prepared in Example 1, dissolved in physiological saline, via gavage daily during the modeling period. All three groups of mice had free access to feed provided by the Animal Experiment Center of Jinan University daily. Starting from day 0 of the modeling process, feed and water were recorded and changed every two days, and bedding was changed regularly. Mouse weight was recorded over the 23 days of feeding, and the rate of weight change was calculated.

[0176] Mouse body weight change rate as Figure 16 As shown.

[0177] Figure 16 The results showed that the weight change rate of mice in the B-FOS group was significantly higher than that in the control group and the P-FOS group. At the same feeding time, the weight change rates of mice in the control group and the P-FOS group were basically similar. This indicates that fructooligosaccharide propionate is more effective than fructooligosaccharide butyrate in controlling mouse weight.

[0178] Example 7: The effect of fructooligosaccharide propionate on the improvement of obese mice fed a high-fat diet.

[0179] 1. Establishment of a mouse obesity model

[0180] With the approval of the Ethics Committee of the Animal Management Center of Jinan University, 72 six-week-old male C57BL / 6 mice (18–22 g) were purchased from Zhejiang Vital River and housed at the Animal Management Center of Jinan University under standard conditions. During the quarantine period, they were fed feed (co-produced biological rodent feed) provided by the animal facility, had free access to water, and had their water, feed, and bedding in their cages changed regularly.

[0181] After 10 days of quarantine, mice were randomly divided into 6 groups of 8 mice each: a blank control group (Control group, CON), a model control group (HFD group, HFD-C), an oligofructose intervention group (HFD+FOS group, HFD-FOS), an oligofructose-propionic acid mixture intervention group (HFD+FOS+P group, HFD-FAP), a P-FOS intervention group prepared in Example 1 (HFD+P-FOS group, HFD-PF), and an AP-FOS intervention group prepared in Example 1 (HFD+AP-FOS, HFD-APF). The modeling process lasted 11 weeks, with all mice fed a standard D12450H diet for 7 days. All mice were randomly divided into 6 groups: a normal control group fed a standard D12450H diet (control), and a high-fat diet group fed a high-fat D12451 diet (HED). All groups drank sterile water provided by the animal center throughout the process to establish a mouse obesity model. During the modeling period, the HFD-PF group was administered P-FOS solution (60 mg / mL in physiological saline) by gavage daily, while the HFD-APF group was administered AP-FOS solution (81 mg / mL in physiological saline) by gavage daily. The FOS group was administered fructooligosaccharide solution (50 mg / mL in physiological saline) by gavage daily, and the HFD-FAP group was administered a mixture of fructooligosaccharide (50 mg / mL) and propionic acid (7 μL / mL) in physiological saline by gavage daily. All six groups of mice were allowed free access to feed provided by the Animal Experiment Center of Jinan University. Starting from day 0 of modeling, feed and water were recorded and changed every two days, and bedding was changed regularly.

[0182] The flowchart of the intervention for establishing an obese mouse model is as follows: Figure 17 As shown.

[0183] 2. Effects of P-FOS on body weight in mice with colitis

[0184] The weight of the mice was recorded during the experiment. At the end of the experiment, the weight gain and Lee's index were calculated. The results of the mouse weight gain are as follows: Figure 18 As shown, the results of Lee's index are as follows: Figure 19 As shown.

[0185] and Figure 18 and Figure 19It was observed that with prolonged feeding time, the CON group mice experienced steady weight gain, while the HFD and high-fat diet intervention mice showed a greater trend of weight gain than the CON group, with a significant difference in weight between the HFD and CON groups at week 6 (p<0.05). After 11 weeks of feeding, the Lee's obesity index of the mice showed that the obesity index of the HFD mice was significantly higher than that of the CON group (p<0.05), indicating successful model establishment. The weight gain during the feeding process was statistically analyzed, showing that the weight gain of the HFD group was significantly higher than that of the CON group (p<0.05), while the HFD-PF group (p<0.05) and HFD-FOS group (p<0.05) significantly reduced the weight gain. However, direct gavage administration of propionic acid stimulated the mice, affecting their food and water intake, leading to abnormal weight loss.

[0186] Morphological comparisons of mice in the CON, HFD-C, and HFD-PF groups, for example Figure 20 As shown, Figure 20 The results showed that mice in the HFD-C group were significantly larger than mice in the CON group. Figure 18 It can be seen that the body weight of mice in the HFD-C group also increased significantly, indicating that the high-fat mouse model was successfully established. Mice in the HFD-PF group were significantly smaller than mice in the HFD-C group, combined with... Figure 18 It can be seen that the body weight of mice in the HFD-PF group was significantly lower than that in the HFD-C group, indicating that obesity in mice in the HFD-PF group was alleviated and that P-FOS played a role in alleviating obesity.

[0187] In conclusion, P-FOS has a significant effect on controlling weight gain in mice, indicating that daily intake of P-FOS can alleviate obesity induced by a high-fat diet to some extent.

[0188] Similarly, experiments showed that P-Inulin prepared in Example 1 also had a significant effect on controlling weight gain in mice, indicating that daily intake of P-FOS can alleviate obesity induced by a high-fat diet to a certain extent.

[0189] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a polyfructose propionate in the preparation of a medicament for treating and / or alleviating obesity, characterized in that, The preparation method of the polyfructose propionate is as follows: polyfructose and a propionylating agent are esterified by enzyme catalysis to obtain polyfructose propionate. The degree of polymerization of the polyfructose is less than or equal to 60. The propionylating agent is vinyl propionate. The polyfructose is oligofructose. The enzyme is immobilized lipase TLIM. The reaction conditions for catalytic esterification are 40-45℃ for 24-36 h.

2. The application according to claim 1, characterized in that, The fructooligosaccharide solution was reacted thoroughly with propionylating reagent and enzyme. The reaction solution was filtered, and the filtrate was purified.

3. The application according to claim 2, characterized in that, The solvent for the fructooligosaccharide solution is anhydrous pyridine or tetrahydrofuran.

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