Medium-chain inulin and a preparation method and application thereof

Abstract

The application discloses a kind of medium chain inulin and its preparation method and application, belong to plant extraction technical field.The unique inulin structure and chain length disclosed in the application remodel intestinal microbiome and metabolome, especially selectively regulate key microorganisms such as coprococcus, bifidobacterium, parabacteroides, Clostridium_sensu_stricto_1 and Akkermansia, and metabolites such as nicotinamide, taurocholic acid, aldosterone, corticosterone, 2-(1H-indole-3-yl)acetic acid, 5-hydroxy-L-tryptophan, 5alpha-DHT and maltose, promote the improvement of host obesity and overall health status.The core microorganisms and metabolites identified by the technical scheme represent potential therapeutic targets for preventing or treating obesity and related metabolic symptoms.These findings support the development of inulin-based nutritional strategies to effectively manage weight by regulating specific microbiota and metabolome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, in particular to a medium-chain inulin and a preparation method and application thereof. BACKGROUND

[0002] Obesity is a global health problem affecting millions of people worldwide, and the prevalence of obesity continues to rise globally. According to the World Health Organization (WHO), in 2022, more than 2.5 billion adults were overweight, of which more than 890 million were classified as obese. In addition, obesity is always accompanied by a variety of metabolic diseases, including diabetes, hypertension, cardiovascular disease (CVD) and musculoskeletal diseases. The main cause of obesity is the imbalance between energy intake and consumption, leading to excessive fat storage in white adipose tissue. High-fat diet (HFD) is common in both developed and developing countries and is a major factor in the global obesity epidemic. HFD is usually deficient in sufficient dietary fiber, which can disrupt the gut microbiota, affect physiological processes and lead to obesity, hyperlipidemia and other metabolic problems. Therefore, in contemporary society, there has been increasing concern about preventing obesity and its complications caused by high-fat diets and insufficient dietary fiber.

[0003] Inulin is a natural dietary fiber composed of linear beta-2,1-linked fructose and terminal alpha-1,2-linked glucose residues with a degree of polymerization (DP) ranging from 2 to 60. Due to the special glycosidic bond, inulin is not easily digested by amylase in the proximal gastrointestinal tract. However, almost 90% of inulin can reach the colon and be fermented by beneficial bacteria, thereby producing a variety of metabolites, including short-chain fatty acids (SCFAs), amino acids and vitamins, which are believed to be the main way in which inulin exerts its biological activity. However, the specific regulation of microbial flora by inulin of different chain lengths (different DP values) varies, resulting in different metabolites and exhibiting different biological effects. The biological activity of inulin is closely related to its DP value, and current research in the field believes that medium-chain inulin with a chain length between 10 and 20 has a wide range of biological effects. However, the targeted extraction of medium-chain inulin and the exploration of its biological effects are still in the blank field. Exploring the directional extraction technology of medium-chain inulin and its biological effects, especially for the treatment effect of obesity and metabolic syndrome, has important significance for the development of natural functional foods and biological medicines. SUMMARY

[0004] The purpose of the present application is to provide a medium-chain inulin and a preparation method and application thereof to solve the problems existing in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0006] One of the technical solutions of the present application is a preparation method of medium-chain inulin, comprising the following steps:

[0007] (1) grinding the Jerusalem artichoke to obtain a slurry, adding water to extract, and then filtering, concentrating, removing protein, and alcohol precipitation to obtain a crude polysaccharide;

[0008] (2) dialyzing the crude polysaccharide through a semi-permeable membrane, concentrating, and freeze-drying to obtain the medium-chain inulin.

[0009] In the second technical solution of the present application, the medium-chain inulin prepared by the above preparation method is composed of fructose connected by 2,1 glycosidic bonds, and has an average degree of polymerization of 12.

[0010] In the third technical solution of the present application, the medium-chain inulin is applied to the preparation of a product for preventing and treating obesity.

[0011] In the fourth technical solution of the present application, a product for preventing and treating obesity comprises the medium-chain inulin.

[0012] In the fifth technical solution of the present application, the medium-chain inulin is applied to the preparation of a product for regulating intestinal flora.

[0013] In the sixth technical solution of the present application, the medium-chain inulin is applied to the preparation of a product for up-regulating Faecalibaculum, Bifidobacterium, Parasutterella, Muribaculum, Clostridium_sensu_stricto_1 and Akkermansia.

[0014] Based on the above technical solutions, the present application has the following technical effects:

[0015] The unique inulin structure and specific chain length disclosed in the present application reshape the intestinal microbiome and metabolome, particularly selectively regulate key microorganisms such as Faecalibaculum, Bifidobacterium, Parasutterella, Clostridium_sensu_stricto_1 and Akkermansia, and metabolites such as nicotinamide, taurocholic acid, aldosterone, corticosterone, 2-(1H-indol-3-yl)acetic acid, 5-hydroxy-L-tryptophan, 5alpha-DHT and maltose, thereby promoting the improvement of obesity and overall health. In addition, the identified key microorganisms and metabolites represent promising therapeutic targets for preventing or treating obesity. These findings support the development of inulin-based nutritional strategies aimed at regulating specific microbiota and metabolome, thereby effectively managing body weight. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only show some of the embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort based on these drawings.

[0017] Figure 1 Structural characteristics of medium-chain inulin. Wherein, A is ion chromatography (IC) of medium-chain inulin. B is the change trend of multi-angle laser scattering (LS), refractive index (RI) and fitting molar mass signal of medium-chain inulin. The red line represents the change trend of LS with the retention time, and the green line represents the RI trend of medium-chain inulin. The trend of the red line and the green line indicates the size and relative proportion of the polysaccharide molecules contained in the test sample. The blue line is the change trend of the molar mass of the LS and RI signal fitting of inulin with the retention time. C is the Fourier transform infrared (FT-IR) spectrum of medium-chain inulin. D is the proposed chemical and structural formula of medium-chain inulin. E is the scanning electron microscope (SEM) image of inulin. E.1 is the overall appearance of the scanning electron microscope of inulin; E.2 is the inulin sphere composition observed by scanning electron microscope at 200 times magnification; E.3 is the inulin observed by scanning electron microscope at 1000 times magnification. F is the three-dimensional spatial structure of the inulin image fitted with the determined structural parameters. F.1 is spherical inulin; F.2 is the three-dimensional structure of inulin molecules with DP=12; F.3 is long-chain inulin with DP=60; G is the transmission electron microscope (TEM) scanning image of inulin, G.1 is the spherical conformation coiled by inulin with different chain lengths at 70,000 times magnification; G.2 is the network woven by long-chain inulin at 30,000 times magnification; G.3 is a single long-chain inulin at 30,000 times magnification.

[0018] Figure 2 Supplementing medium-chain inulin for 12 weeks in C57BL / 6 mice can prevent hfd-induced obesity and improve lipid deposition. Wherein, A and B are the experimental design and the obesity phenotype of white rats fed with HFD or HFD+; C is the body weight change and body weight of rats fed with inulin at the end of the experiment, D is the change of tissue weight during the experiment, white adipose tissue (inguinal fat pad), and brown adipose tissue (bat); E is the serum lipid level, F is the fasting blood glucose (FBG) and serum urea level; G is the oil red staining section showing the degree of liver fat accumulation and distribution. H indicates the percentage (%) of liver oil red staining positive area in the corresponding section (G), and I is the liver lipid profile. The asterisk indicates significant difference, *P<0.05, **P<0.01, ***P<0.001.

[0019] Figure 3SCFA levels were restored in C57BL / 6 mice by medium-chain inulin, and intestinal damage caused by HFD feeding was reduced. Among them, A. SCFA content in feces. B. BCFA content in feces. C. Overview of total fatty acids (TFA), short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) in feces. D-F. Ileum morphology and quantitative analysis. After hematoxylin and eosin (H&E) staining, the ileal villus height, crypt depth and villus height / crypt depth ratio (V / C) were observed under a light microscope at x100 magnification. G-I: Quantitative determination of colon morphology and related parameters, crypt depth, intestinal wall thickness, intestinal contents, pH value and representative images of colon sections of 5 test group mice. *P<0.05, **P<0.01.

[0020] Figure 4 The intestinal microbiome profile and differential microorganisms after adding inulin were screened. Among them, A is the Shannon index representing the diversity index of the intestinal microbiome, B is the Beta diversity of the intestinal microbiome, C is the abundance ratio of the main intestinal bacteria Firmucute and Bacteroidota, D is the differential microorganism at the door level, E is the differential microorganism at the genus level, F is the differential microorganism column chart obtained by LEfse analysis method, and G is the evolutionary branch diagram of the intestinal microbiome obtained by LEfse analysis method.

[0021] Figure 5 Metabolome profiles and differential metabolites between experimental groups. Among them, A. Heat map showing the effects of HFD feeding and adding inulin on intestinal metabolites in mice. B. Orthogonal projection of potential structural discriminant analysis (OPLS-DA) shows the distribution of intestinal metabolites in different groups. C. S plot of OPLS-DA shows the distribution of differential metabolites. Red dots represent metabolites with VIP≥1, and blue dots represent metabolites with VIP<1. D-H. Volcano plot showing the differences in metabolites between HFD, ND and inulin supplemented groups. I. Classification of differential metabolites up-regulated and down-regulated in inulin supplemented group and HFD fed group.

[0022] Figure 6The correlation between inulin addition altering health parameters, gut microbiota, and metabolites was investigated. In Figures A, B, and C, red text indicates significant upregulation of gut microbiota or metabolites after inulin addition. A. Correlation between health parameters and different gut microbiota. B. Correlation between health parameters and diverse metabolites. C. Correlation between different gut microbiota and metabolites. D. Analysis of metabolic pathway regulation by HFD feeding and / or inulin addition, comparing the ND group with the HFD group, and between the HFD+1%, HFD+3%, or HFD+5% inulin groups and the HFD group. The metabolic pathways in D are shown below. In HFD vs. ND, 1 represents biotin metabolism, 2 represents the interconversion of pentose and gluconate, 3 represents tryptophan metabolism, 4 represents arachidonic acid metabolism, 5 represents steroid hormone biosynthesis, 6 represents tyrosine metabolism, 7 represents starch and sucrose metabolism, 8 represents lysine degradation, 9 represents galactose, 10 represents glutathione, and 11 represents cysteine ​​and methionine metabolism. In the HFD vs. HFD+1% inulin group, 1 represents starch / sucrose metabolism, 2 represents arachidonic acid metabolism, and 3 represents steroid hormone biosynthesis. In the HFD vs. HFD+3% inulin group, 1 represents arachidonic acid metabolism, 2 represents nicotinic acid / nicotinamide metabolism, 3 represents steroid hormone metabolism, and 4 represents primary bile acid biosynthesis. In the HFD vs. HFD+5% inulin group, 1 represents arachidonic acid metabolism, 2 represents tryptophan metabolism, and 3 represents primary bile acid biosynthesis.

[0023] Figure 7 Based on differential metabolites and KEGG pathways, inulin supplementation significantly altered the correlations between metabolic pathways. Metabolites connected by solid lines represent direct conversions, while those connected by dashed lines represent indirect conversions via biochemical processes. Red text indicates increased concentrations, while blue text indicates decreased concentrations due to dietary inulin supplementation. Rounded rectangles of different colors represent pathways enriched by KEGG analysis. Pink-marked metabolic pathways are upregulated, while light blue indicates downregulated pathways. Light yellow rectangles represent physiological and / or biochemical processes affected by inulin-regulated metabolic pathways.

[0024] Figure 8 This is a co-occurrence network of gut microbiota and metabolites with body weight (BW) and short-chain fatty acids (SCFAs). Light blue dashed lines indicate negative correlations (mutually exclusive, mu-) between two nodes, while light pink solid lines indicate positive correlations (co-) between two nodes. A. Correlation between microbiota, body weight, and short-chain fatty acids (SCFAs). B. Correlation between metabolites, body weight, and SCFAs. Detailed Implementation

[0025] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather as a description of certain aspects, features and embodiments of the application.

[0026] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included in the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to the extent allowed by law. In the case of conflict between the description herein and the incorporated material, the description herein shall control.

[0028] In the description of the application specific embodiments, numerous specific details are set forth in order to provide a thorough understanding of the application. However, those of ordinary skill in the art will realize that the application can be practiced without many of these details. In other instances, well-known methods, procedures and components have not been described in detail so as not to unnecessarily obscure aspects of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from a review of the description of the application and practice of the application.

[0029] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0030] The technical solutions described in the present application are conventional solutions in the art, and the reagents or raw materials used are commercially available or have been disclosed, unless otherwise specified.

[0031] The present application provides a preparation method of medium-chain inulin, comprising the following steps:

[0032] (1) grinding Jerusalem artichoke to obtain a slurry, adding water to extract, and then filtering, concentrating, removing protein, and alcohol precipitation to obtain crude polysaccharide;

[0033] (2) dialyzing the crude polysaccharide through a semi-permeable membrane, concentrating, and freeze-drying to obtain the medium-chain inulin.

[0034] In some specific embodiments, in step (1), the water extraction conditions are as follows: the volume ratio of the slurry to water is 1:30, the extraction temperature is 80°C, and the time is 3 h.

[0035] The concentration is specifically concentrated to 30% of the original volume;

[0036] The protein removal method is: adding an equal volume of 5% trichloroacetic acid solution, standing for 60 minutes, centrifuging at 8000 rpm, 20 DEG C for 10 minutes, and removing the precipitate;

[0037] The alcohol precipitation method is: adding 4 volumes of anhydrous ethanol, standing for 8-12 hours, and then centrifuging at 6000 rpm, 20 DEG C for 10 minutes, and discarding the supernatant.

[0038] In some specific embodiments, in step (2), the dialysis condition is that the membrane pore diameter D = 3500 daltons.

[0039] The present application also provides the medium-chain inulin prepared by the preparation method.

[0040] The present application also provides the application of the medium-chain inulin in the preparation of an obesity-preventing product.

[0041] The present application also provides an obesity-preventing product comprising the medium-chain inulin.

[0042] The present application also provides the application of the medium-chain inulin in the preparation of a product for regulating intestinal flora.

[0043] The present application also provides the application of the medium-chain inulin in the preparation of a product for up-regulating Faecalibaculum, Bifidobacterium, Parasutterella, Muribaculum, Clostridium_sensu_stricto_1 and Akkermansia.

[0044] The present application extracts inulin from Jerusalem artichoke and removes small molecules, including monosaccharides or partial oligosaccharides with a DP value less than 10, through dialysis by a semi-permeable membrane (molecular weight > 3,000D). Structural feature analysis shows that the extraction process produces medium-chain inulin (DP = 12). The present application proposes the following hypotheses: (1) inulin with a DP in the range of 10 to 20 can selectively promote beneficial intestinal microorganisms and affect the metabolome / metabolic pathways, (2) inulin can alleviate obesity and its related complications in HFD-fed mice. The purpose of the present application is to explore the effects of medium-chain inulin supplementation on obesity and its mechanisms, especially by regulating the intestinal microbiome and metabolome of diet-induced obese (DIO) mice.

[0045] Inulin is a natural dietary fiber with multiple biological activities. However, few studies have focused on the effects of medium-chain inulin (degree of polymerization = 12) on high-fat diet (HFD)-induced obesity in mice. The present invention verifies whether medium-chain inulin intake (1%, 3%, and 5% in the diet) can counteract obesity in HFD-fed mice by regulating the gut microbiota and metabolome. The structural characteristics and spatial conformation of inulin are investigated by determining the structural parameters of inulin. Forty 3-week-old C57BL / 6 male mice are randomly divided into five groups (n = 8 / group) and fed with standard chow or HFD supplemented with 1%, 3%, and 5% inulin for 12 consecutive weeks. Obesity and health status are detected by examining body weight, serum biochemical and physiological indicators, and the gut microbiota and metabolome of the intestinal contents to interpret the underlying mechanisms. Medium-chain inulin is mainly composed of fructose linked by 2,1 glycosidic bonds, with an average degree of polymerization of 12. Inulin supplementation (3% or 5%) can prevent diet-induced obesity (DIO) and metabolic disorders in mice. In addition, the addition of inulin to the diet reshapes the gut microbiota, increasing the abundance of Bacteroidetes, Fecalibacteres, Bifidobacterium, Parabacteroides, Lactobacillaceae, Bacteroidetes, Clostridia, and Akkermansia. At the same time, the addition of inulin to the diet selectively increases the concentration of 11 key metabolites in the intestinal contents of HFD-fed mice, which are directly related to energy utilization pathways. Overall, medium-chain inulin alleviates DIO by specifically reshaping key gut microbes and metabolites, regulating metabolic pathways related to energy utilization. Therefore, medium-chain inulin can be used as a potential functional food or therapeutic agent to prevent or treat host DIO.

[0046] Example 1

[0047] 1 Inulin extraction and determination of structural characteristics

[0048] The medium-chain inulin used in this experiment was extracted from Jerusalem artichoke and dialyzed through a semi-permeable membrane (molecular weight > 3,000 D) to remove free monosaccharides or partial oligosaccharides with a DP value less than 10. The structural characteristics of monosaccharide composition, molar mass, glycosidic bond, and C and H spectra were determined.

[0049] The specific steps are as follows: (1) water extraction at a suitable temperature: dry or fresh Jerusalem artichoke slices are ground into a pulp, distilled water is added to a stainless steel pot (material to water volume ratio 1:30), and extracted at 80°C for 3 hours. After cooling, filter twice with a polyester mesh (mesh <1 cm) to remove residues, and mix the filtrates. (2) Concentration: use a vacuum low-temperature concentration tank to concentrate the filtrate to 30% of the original extraction volume at 60-80°C. (3) Protein removal: after the concentrated solution cools, add an equal volume of 5% trichloroacetic acid solution and let it stand for 60 minutes. Centrifuge at 8000 rpm and 20°C for 10 minutes to remove the precipitate. The supernatant is a polysaccharide solution without protein. (4) Alcohol precipitation of polysaccharides: based on the property of polysaccharides being insoluble in ethanol solution, transfer the polysaccharide solution without protein to a 5000 ml beaker, and add 4 times the volume of anhydrous ethanol to the polysaccharide solution. Let the alcohol soak for 8 hours or overnight, then centrifuge at 6000 rpm and 20°C for 10 minutes. Discard the supernatant and the precipitate is the crude polysaccharide. (5) Dialysis: dissolve the extracted crude polysaccharide solution in deionized water at a dose of 1:1.5. Place the crude polysaccharide solution in a treated dialysis bag (membrane pore diameter D = 3500 daltons, intercept molecular weight > 3500D), rinse the surface of the dialysis bag, clamp one end of the dialysis bag with a dialysis bag special clamp, add 70% of the volume of the crude polysaccharide solution to the dialysis bag, then clamp the other end with a large clamp, and place the sealed dialysis bag in deionized water for dialysis. Change the deionized water at 3, 9, and 18 hours, and collect the solution in the dialysis bag after 40 hours. (6) Freeze-drying: after dialysis, collect the liquid in the dialysis bag, concentrate to one-tenth of the volume, and freeze-dry to obtain medium-chain inulin with a degree of polymerization greater than 10.

[0050] 2. Design of biological function verification test of medium-chain inulin

[0051] A total of 40 male C57BL / 6 mice (3 weeks old, 11-13 g; SPF level) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). All experiments were approved by the Animal Use Ethics Committee of Shandong Agricultural University (protocol number 20200526). The mice were raised in the environmental control shed of the animal center of Shandong Agricultural University (temperature 22 ± 2°C, relative humidity 55-60%, 12 / 12 hour light / dark cycle), and were allowed free access to water and food during the experiment. Body weight was measured once a week, and food intake was measured daily. All experiments were approved by the Animal Use Ethics Committee of Shandong Agricultural University (protocol number 20200526).

[0052] Forty male C57BL / 6 mice (3 weeks old, 11-13 g; SPF grade) were purchased from the Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Jinan, China). All experiments were approved by the Animal Use Ethics Committee of Shandong Agricultural University (Agreement No: 20200526). Mice were housed in environmentally controlled rooms (temperature, 22 ± 2 °C; relative humidity 55-60%; 12 / 12 h light / dark cycle was regularly performed at the animal center of Shandong Agricultural University), with free access to water and chow diet (normal diet) during the experiment.

[0053] After the purchased mice were adapted to the mouse house environment and feed for 1 week, they were randomly divided into 5 groups (n = 8, 4 / cage), and fed with normal diet (normal group), high-fat diet (HFD), HFD added with 1%, 3%, and 5% inulin, respectively, for 12 weeks. Mice could eat and drink freely, and were weighed once a week, and food intake was monitored daily.

[0054] 3 Sample collection and determination

[0055] During the last three days of the experiment, fresh feces were collected, and the short-chain fatty acid (SCFA) level was determined by high-performance gas chromatography (HPGC97, Zhejiang Fuli). At the end of the experiment, fasting blood glucose (FBG) was measured from the tail vein using a rapid blood glucose meter before sacrifice. Blood samples were collected from the eyeball of each mouse to determine serum biochemical parameters, and then the mice were sacrificed by cervical dislocation. After opening the abdominal cavity, the hindgut contents (from the cecum to the rectum) were collected into two sterile tubes for each mouse and immediately frozen in liquid nitrogen to determine the microbiome and metabolome. The liver, abdominal white adipose tissue (WAT), and scapular brown adipose tissue (BAT) were collected, weighed, immediately frozen in liquid nitrogen, and then stored at -80 °C for further analysis. A 3-cm intestinal segment was cut from the middle of the colon and ileum using surgical scissors, and histological sections were prepared. The collected intestinal samples were rinsed with normal saline (9 g / L, w / v) and fixed in formaldehyde-phosphate buffer (100 g / L, w / v) for not less than 24 hours, followed by paraffin sectioning. For each intestinal sample, 5-10 observation samples (sections with a thickness of 5 pm) were successively cut using a microtome and stained with hematoxylin and eosin (H&E). The intestinal morphology was evaluated by measuring the villus height (V), crypt depth (C), and villus-to-crypt ratio (V:C) using an optical microscope (BX-51, Olympus, Tokyo, Japan) equipped with Image-Pro Plus software (version 6.0, Motic Images Software, Motic China Group Co., Ltd., Xiamen, China). Each sample was measured 10 times in duplicate.

[0056] 4 Determination of cecal microbiota using 16S rDNA amplicon sequencing

[0057] Microbiota determination was performed using Ion S5™ XL Sequencing Platform (Novogene, Beijing, China). DNA was extracted from cecal chyme samples using QIAamp DNA Stool Mini Kit (Qiagen Inc., Hilden, Germany); then, the V3-V4 hypervariable region of the 16S rRNA gene was amplified. Amplicon libraries were sequenced on Ion S5™ XL Sequencing Platform (Novogene, Beijing, China) with 400 and 600 bp single-end reads (SE400 and SE600). Detailed procedures are listed in the Supplementary Materials.

[0058] 5. Determination of cecal metabolome

[0059] Cecal metabolome was quantitatively measured using liquid chromatography tandem mass spectrometry (LC-MS / MS) (Beijing Novogene Bioinformatics Technology Co., Ltd.). The measurement process contains three main steps: (1) metabolite extraction; (2) metabolites in chyme were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS); (3) data processing and metabolite identification.

[0060] 6. Statistical analysis

[0061] Statistical analysis was performed using one-way analysis of variance (ANOVA) according to Dunn's multiple comparisons using IBM SPSS Statistics 23. Histograms were generated using GraphPad Prism 8.0 to represent the results of the analysis. Linear discriminant analysis (LDA) effect size analysis was performed on the intestinal microbiota changes using the BIC online program (http: / / www.ehbio.com / Cloud_Platform / front / # / ). Differences were considered statistically significant when P < 0.05. Metabolic pathways enrichment and differential metabolic pathways analysis were performed using MetaboAnalyst4.0 online software and KEGG pathway database (https: / / www.metaboanalyst.ca / ). Spearman analysis was used to analyze the correlation between differentiated microorganisms, metabolites, and health parameters, and the heatmap illustrator program (version 1.0.3.7) was used to present the results in the form of a heat map.

[0062] 7. Experimental results

[0063] 7.1 Structural characteristics of medium-chain inulin

[0064] The molecular structural characteristics of inulin are shown in Figure 1 Ion chromatography (IC) analysis showed that inulin molecules were composed of two monosaccharides, fructose (Fru) and glucose (Glc), with a molar ratio of 82.61%: 17.39% (Fru: Glc). Figure 1(See Table 1). The average molecular weight (Mw), number-average molecular weight (Mn), and Z-average molecular weight (Mz) of inulin were 1.30 × 10⁻⁶. 3 1.80×10 3 and 2.40×10 3 g / mol (Table 1). The polydispersity indices of inulin were 1.35 (Mw / Mn) and 2.10 (Mz / Mn), indicating a relatively narrow molar mass distribution range. Furthermore, the weight-average radius (Rw), number-average radius (Rn), and Z-average radius (Rz) of inulin were calculated to be 14.1, 14.0, and 13.6 nm, respectively. The molar mass variation trend curve ( Figure 1 In section B), the molecular weight characteristics of inulin were examined. The results showed that the laser scattering (LS) curve (red line) decreased rapidly, while the refractive index (RI) curve (green line) gradually increased. With prolonged retention time, the molar mass line (blue line) decreased at 10... 3 ~10 4 Variation within the g / mol range. Fourier transform infrared spectroscopy (FT-IR) indicates the presence of hydroxyl (-OH) groups in the inulin structure (3,600–3,200 cm⁻¹). -1 3,490cm -1 The peak at the peak clearly indicates the OH stretching vibration, further confirming the presence of polysaccharides. According to GC-MS analysis, the inulin molecule consists of 12 monosaccharide residues linked by three different types of glycosidic bonds, involving non-reducing terminal t-Fruf and t-Glcp (molar ratios of 16.44% and 16.33%, respectively), and intrachain residues of 2,1-Fruf and 1,6-Fruf (molar ratios of 66.68% and 0.55%, respectively) (Table 2). This indicates that the ratio of Fru to Glc residues is 83.68%:16.33%, consistent with the compositional analysis. NMR spectroscopy analysis revealed the inulin molecule to be a medium-length PS (Pyramid Fiber) chain with 12 monosaccharide residues. Figure 1 (D). Combining all structural parameters, the potential spatial conformation of the inulin molecule was further fitted. The three-dimensional molecular structure showed that the inulin molecule is spherical, with few branches, and contains a small number of rod-shaped macromolecules (D). Figure 1 (F.1–F.3). Furthermore, images obtained from scanning electron microscopy (SEM) show that inulin molecules are spherical with smooth surfaces. Figure 1 (E.1–E.3). Furthermore, transmission electron microscopy (TEM) images revealed that the inulin samples contained both long-chain and short-chain molecules. Inulin molecules of different chain lengths formed spherical or interconnected network spatial conformations through folding and coiling. Figure 1 (G.1~G.3).

[0065] Table 1 Monosaccharide composition and molecular weight parameters

[0066]

[0067]

[0068] Table 2. Glycosidic bond residue structure and molar ratio

[0069]

[0070] 7.2 Effects of medium-chain inulin supplementation on diet-induced obesity (DIO) in mice

[0071] like Figure 2 As shown in Figure A, C57BL / 6 mice were fed a normal diet (ND), HFD, or HFD supplemented with different concentrations (w / w: 1%, 3%, and 5%) of medium-chain inulin, respectively. After a 12-week experimental period, compared with ND-fed mice, HFD-fed mice had a significantly increased body weight (BW) (P<0.05), while inulin supplementation significantly inhibited HFD-induced BW increase in a dose-dependent manner. Figure 2 (B, C) For example Figure 2 As shown in Figure D, compared with ND-fed mice, HFD-fed mice had a significantly increased liver weight (liver weight to body weight ratio, w / w), while inulin supplementation inhibited the increase in liver weight in HFD-fed mice (P<0.05). Similarly, HFD feeding led to an increase in white adipose tissue (WAT) and epididymal fat pad weight, but a decrease in brown adipose tissue (BAT) weight, while inulin supplementation reduced the proportion of WAT and increased the proportion of BAT in the body.

[0072] In addition, changes in blood lipid profile, glucose, and urea levels, such as Figure 2 As shown in Figures E and F, the levels of triglycerides (TG), total cholesterol (TCHO), and urea in the inulin-supplemented group were significantly lower than those in the HFD group. Furthermore, inulin supplementation increased serum high-density lipoprotein (HDL) levels and decreased serum low-density lipoprotein (LDL) levels, thereby improving the HDL / LDL ratio. Figure 2 As shown in Figure G, liver sections were stained with modified Oil Red O to observe the degree of lipid accumulation in the liver. Figure 2 (G in the middle, red staining area represents lipid droplets). Hepatic lipid accumulation in the HFD group was significantly higher than in the ND group (P<0.01), while inulin supplementation significantly reduced hepatic lipid accumulation in a dose-dependent manner (P<0.05). Dietary supplementation with 3% inulin reduced HFD-induced lipid deposition to a level similar to that in the ND group, while 5% inulin supplementation significantly reduced the lipid accumulation area compared to the ND group (P<0.05). Figure 2H) Finally, the present application evaluated the hepatic lipid profile and found that inulin supplementation decreased the concentrations of triglycerides (TG), total cholesterol (TCHO) and low-density lipoprotein (LDL) (P<0.05), while increasing the levels of high-density lipoprotein (HDL) and the HDL / LDL ratio in a dose-dependent manner Figure 2 I).

[0073] 7.3 Effect of medium-chain inulin on intestinal development and short-chain fatty acids (SCFA) production

[0074] As shown in Figures 6A-6C, feeding mice with HFD decreased (P<0.05) the intestinal SCFAs content compared to the normal group (ND), while inulin supplementation restored the content of acetic, propionic and butyric acids in a dose-dependent manner. In contrast, HFD feeding significantly increased (P<0.05) the levels of branched-chain fatty acids (BCFAs), such as isobutyric and isovaleric acids (P<0.05). However, dietary inulin supplementation decreased the production of BCFAs in a dose-dependent manner (P<0.05). Figure 3 In addition, intestinal morphological studies showed that HFD feeding increased the crypt depth, while decreasing the villus height and V / C ratio (P<0.05) compared to ND-fed mice (Figures 7D-7F). Dietary inulin supplementation prevented this damage, instead promoting the growth and development of the ileum, as shown by the increase in ileal villus height and V / C ratio (P<0.05) and the decrease in crypt depth (P<0.05). Similarly, HFD feeding increased the crypt depth and pH, while decreasing the colon wall thickness. In contrast, inulin supplementation reversed these effects, increasing the intestinal wall thickness and decreasing the crypt depth and pH in a dose-dependent manner (P<0.05) (Figures 7G-7I).

[0075] Figure 3 7.4 Intestinal microbiota characteristics and different microorganisms between groups Figure 3 Intestinal microbiota analysis showed that the intestinal microorganisms of each group were mainly dominated by Firmicutes and Bacteroidetes at the phylum level. However, HFD feeding reshaped the different intestinal microbiota, decreasing the alpha diversity (P<0.05) and increasing the Firmicutes richness and F / B ratio (P<0.05) (Figures 8A, 8B and 8C); in contrast, dietary inulin supplementation enhanced (P<0.05) the microbial diversity, especially the Bacteroidetes richness and F / B ratio (Figures 8C and 8D). In addition, principal coordinate analysis (PCoA) and group clustering showed that the ND group was completely separated from the other groups, while the 3% and 5% inulin supplementation groups presented partial overlap, and the 1% inulin group and the HFD group were clustered together (Figures 8B).

[0076]

[0077] Intestinal microbiota analysis showed that the intestinal microorganisms of each group were mainly dominated by Firmicutes and Bacteroidetes at the phylum level. However, HFD feeding reshaped the different intestinal microbiota, decreasing the alpha diversity (P<0.05) and increasing the Firmicutes richness and F / B ratio (P<0.05) (Figures 8A, 8B and 8C); in contrast, dietary inulin supplementation enhanced (P<0.05) the microbial diversity, especially the Bacteroidetes richness and F / B ratio (Figures 8C and 8D). In addition, principal coordinate analysis (PCoA) and group clustering showed that the ND group was completely separated from the other groups, while the 3% and 5% inulin supplementation groups presented partial overlap, and the 1% inulin group and the HFD group were clustered together (Figures 8B). Figure 4 Figure 4 Intestinal microbiota analysis showed that the intestinal microorganisms of each group were mainly dominated by Firmicutes and Bacteroidetes at the phylum level. However, HFD feeding reshaped the different intestinal microbiota, decreasing the alpha diversity (P<0.05) and increasing the Firmicutes richness and F / B ratio (P<0.05) (Figures 8A, 8B and 8C); in contrast, dietary inulin supplementation enhanced (P<0.05) the microbial diversity, especially the Bacteroidetes richness and F / B ratio (Figures 8C and 8D). In addition, principal coordinate analysis (PCoA) and group clustering showed that the ND group was completely separated from the other groups, while the 3% and 5% inulin supplementation groups presented partial overlap, and the 1% inulin group and the HFD group were clustered together (Figures 8B). Figure 4 Intestinal microbiota analysis showed that the intestinal microorganisms of each group were mainly dominated by Firmicutes and Bacteroidetes at the phylum level. However, HFD feeding reshaped the different intestinal microbiota, decreasing the alpha diversity (P<0.05) and increasing the Firmicutes richness and F / B ratio (P<0.05) (Figures 8A, 8B and 8C); in contrast, dietary inulin supplementation enhanced (P<0.05) the microbial diversity, especially the Bacteroidetes richness and F / B ratio (Figures 8C and 8D). In addition, principal coordinate analysis (PCoA) and group clustering showed that the ND group was completely separated from the other groups, while the 3% and 5% inulin supplementation groups presented partial overlap, and the 1% inulin group and the HFD group were clustered together (Figures 8B).​​​

[0078] In addition, inulin supplementation inhibited the HFD-induced increase in the abundance of Lachnospiraceae_NK4A136_group, Alistipes, Dubosiella, and Colidextribacter (P < 0.05) and significantly increased the abundance of Faecalibaculum, Lactobacillus, Bifidobacterium, Akkermansia, Bacteroides, and Clostridium_sensu_stricto_1 (P < 0.05). Figure 4 Central E). Linear discriminant analysis (LDA) effect size (LefSe) analysis Figure 4 Central F and G) showed that feeding HFD increased the relative abundance of Fibrobacter, Fibrobacteres, Desulfobacterota, and unclassified bacteria (LDA score > 4.0), while inulin supplementation increased the abundance of Verrucomicrobiota, Muribaculaceae, Lactobacillaceae, and Bifidobacteriaceae (LDA score > 4.0), while the HFD-fed group did not. Finally, a cladogram showed the phylogenetic distribution of the differentially abundant bacteria in the different experimental groups Figure 4 Central G).

[0079] 7.5 Intestinal metabolome analysis and differential metabolites

[0080] The intestinal content metabolome was analyzed by LC-MS and 1,503 metabolites were found: 946 positive ions and 557 negative ions. Overall correlation analysis of all metabolites showed that the HFD + 1% inulin and HFD groups clustered together, while the HFD + 3% inulin and HFD + 5% inulin groups formed a separate cluster Figure 5 Central A). Further analysis using orthogonal projection to latent structure discriminant analysis (OPLS-DA) showed that there was complete separation between all five treatment groups Figure 5 Central B). These findings indicate that the intestinal metabolome was significantly altered by HFD feeding and / or dietary inulin supplementation. The contribution of each metabolite to the discrimination between groups was assessed using the variable importance in the projection (VIP) value. The S-plot of the OPLS-DA analysis identified 505 metabolites that showed a significant impact in this trial (P < 0.05, VIP > 1) Figure 5 Central C). The differential metabolites between the ND, HFD, and inulin-supplemented groups were presented in a volcano plot Figure 5 Central D-H). There were 265 differential metabolites between the HFD and ND groups, of which 245 were upregulated and 19 were downregulated.

[0081] Likewise, 1%, 3% and 5% inulin groups showed 40, 44 and 71 differential metabolites compared to HFD group (P < 0.05) (Table 2, Figure 2). Figure 5 Among them, inulin supplementation led to the up-regulation of 18, 22 and 30 metabolites in 1%, 3% and 5% groups, respectively (P < 0.05). Regardless of inulin dose, the regulated metabolites in inulin supplemented groups mainly included indole / derivatives (5-hydroxytryptophan, 2-(1H-indol-3-yl)acetic acid), steroids / derivatives (taurocholic acid, corticosterone, aldosterone, 5a-dihydrotestosterone (5a-DHT)), organic oxygen compounds (maltopentaose and maltose), fatty acids (arachidonic acid, prostaglandin D2, docosapentaenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, ethyl laurate, propionyl-L-carnitine and docosahexaenoic acid) and pyridine / derivatives (nicotinamide) (Table 2, Figure 2). Figure 5 In addition, similar metabolites and regulatory effects were observed in the three inulin supplemented groups.

[0082] 7.6 Correlation between inulin supplementation regulated physiological parameters, differential microorganisms, metabolites and metabolic pathways

[0083] Spearman rank correlation and metabolic pathway analysis were performed to assess the potential correlation between mice physiological parameters, gut microbiota, metabolites and altered metabolic pathways. The inulin selectively promoted microorganisms included Bacteroidetes, Faecalibacterium, Lactobacillus, Bifidobacterium, Akkermansia, Bacteroides, Clostridium_sensu_stricto_1 and Parasutterella, which were positively correlated with mice fatty acid (acetic acid, propionic acid, butyric acid, SCFAs and TFAs) concentration, ileal villus height, HDL content and HDL / LDL ratio in liver and serum (P < 0.05), but negatively correlated with mice body weight, TG, TCHO and LDL content in liver and serum and the degree of fat accumulation in liver (P < 0.05) (Table 3, Figure 3). Figure 6 Likewise, the inulin supplemented improved metabolites mainly included nicotinamide, taurocholic acid, 5a-DHT, aldosterone, corticosterone, 5-hydroxy-L-tryptophan, 2-(1H-indol-3-yl)acetic acid, propionyl-L-carnitine, MN-18N-(5-hydroxypentyl) metabolite, maltopentaose and maltose, which were positively correlated with mice fatty acid in liver and serum, ileal villus height, HDL content and HDL / LDL ratio (P < 0.01), but negatively correlated with mice body weight, TG, TCHO and LDL content in liver and serum and the degree of fat accumulation in liver (P < 0.05) (Table 3, Figure 3). Figure 6 In addition, those increased microorganisms due to inulin supplementation were positively correlated with specific metabolites increased due to inulin supplementation (Figure 6 arachidonic acid, lauric acid ethyl ester, docosahexaenoic acid, and retinoic acid (P < 0.05, Figure 6 Medium C).

[0084] In addition, by matching the differential metabolites in the KEGG pathway database of Mus musculus (HFD vs. ND; 1%, 3%, and 5% inulin vs. HFD), further identified the metabolic pathways significantly regulated by HFD feeding and / or dietary inulin supplementation ( Figure 6 Medium D). The results showed that HFD feeding upregulated physiological processes related to inflammation and energy metabolism, including steroid hormone biosynthesis, lysine degradation, tryptophan, arachidonic acid, tyrosine, starch / sucrose metabolism, and downregulated biotin metabolism (impact > 0.02, Figure 6 Medium D).

[0085] However, all inulin-supplemented groups downregulated the arachidonic acid metabolic pathway ( Figure 6 Medium D). The intrinsic correlation of metabolic pathways regulated by inulin supplementation ( Figure 7 ) further indicated that inulin supplementation significantly upregulated the metabolism of tryptophan, nicotinic acid, nicotinamide, starch, sucrose, taurine / sulfocholine, primary bile acids, and steroid hormone biosynthesis, while downregulated the biosynthesis of unsaturated fatty acids and arachidonic acid metabolism. Nicotinamide, taurocholic acid, aldosterone, corticosterone, 5-hydroxy-L-tryptophan, 2-(1H-indol-3-yl)acetic acid, 5a-DHT, and maltose were observed to be involved in the upregulation of metabolic pathways. Therefore, dietary inulin supplementation can downregulate inflammation-related metabolic pathways while improving glucose and lipid metabolism, which can be the potential mechanism of inulin affecting physiological health and alleviating obesity symptoms.

[0086] 7.7 Identification of inulin-mediated core microbes and metabolites directly related to body weight (BW) of HFD-fed mice

[0087] With BW and SCFAs as environmental factors, a co-occurrence network was constructed to further identify the core microbes and metabolites related to the BW of mice. As Figure 8As shown in Figure A, SCFAs have an inhibitory effect on HFD-induced weight gain. Faecalibaculum, Bifidobacterium, Prevotellaceae_UCG-001, Parasutterella, Ruminococcus, Muribauculum, Lachnospiraceae_UCG_006, Clostridium_sensu_stricto_1, Akkermansia, and Bacteroides were also negatively correlated with weight. Among these microorganisms, Faecalibaculum, Bifidobacterium, Parasutterella, Muribauculum, Clostridium_sensu_stricto_1, and Akkermansia had relatively high abundance (>0.5%), and were significantly upregulated by dietary inulin supplementation. Therefore, these microorganisms may be potential core bacteria in inulin intake-mediated inhibition of HFD-induced obesity.

[0088] Similarly, the metabolites regulated by inulin supplementation are also directly or indirectly related to body weight. For example... Figure 8 As shown in Figure B, arachidonic acid, lysergic acid diacetamide D3 (LSD-d3), ethyl laurate, 16-hydroxyhexadecanoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and docosapentaenoic acid were directly and positively correlated with body weight, and all of these metabolites were reduced in the inulin-supplemented group. Therefore, these metabolites represent a subset of metabolites that promote body weight gain in HFD-fed mice. Figure 8 (Medium B, light purple node). Conversely, the inulin-supplemented group showed increased levels of nicotinamide, taurine, aldosterone, corticosterone, 2-(1H-indole-3-yl)acetic acid, MN-18N-(5-hydroxypentyl) metabolite, 5α-DHT, maltopentose, 5-hydroxytryptophan, propionyl-L-carnitine, and maltose, exhibiting a repulsive relationship with these metabolites compared to the subgroup promoting weight gain. Furthermore, short-chain fatty acids were directly correlated with weight gain and showed a repulsive relationship. However, tocopherol quinoline (TQH), 2-oxoindole, and 2-(1H-indole-3-yl)acetic acid were positively correlated with the production of short-chain fatty acids. Therefore, these metabolites may interfere with weight gain by affecting the production of short-chain fatty acids. Considering differences and pathway enrichment, eight key metabolites, including nicotinamide, taurocholic acid, aldosterone, corticosterone, 2-(1H-indole-3-yl)acetic acid, 5-hydroxy-L-tryptophan, 5α-DHT, and maltose, formed a subset of BW inhibitory metabolites. Figure 8 (Middle B, light pink node).

[0089] In summary, the present invention investigated the effects of inulin (DP=12) from Jerusalem artichoke on HFD-induced obesity and related metabolic disorders in mice. The unique cross-linked and / or globular space conformation of inulin might contribute to its observed biological activities. The inulin supplement effectively reversed the negative effects of HFD, leading to a dose-dependent decrease in BW and WAT mass. At the same time, it increased the BAT index. Second, HFD feeding resulted in higher levels of TG, TCHO, LDL, FBG, and urea in serum, while the inulin supplement restored these physiological parameters to normal levels. Inulin supplementation led to an increase in the production of SCFAs, mainly acetate, propionate, and butyrate. These SCFAs are an important energy source for intestinal cells and play a key role in activating the host immune system. In contrast, HFD feeding led to an increase in the level of branched-chain fatty acids (BCFAs), which can cause insulin resistance. In addition, SCFAs bind to receptors and improve the production of glucagon-like peptide-1 (GLP-1) and peptide tyrosine, thereby affecting the host's energy balance and lipid metabolism. Therefore, in the present invention, the increase in SCFA production by inulin supplementation was the basis for improving obesity-related metabolic complications. In the present invention, the observed improvements in metabolic parameters were associated with changes in the gut microbiota and the metabolome. HFD feeding significantly reduced the diversity of the gut microbiota and increased the F / B ratio, indicating the presence of dysbiosis. In contrast, inulin supplementation significantly increased the diversity of the microbiota, reduced the F / B ratio, and enriched beneficial bacteria, including Bacteroidetes, Akkermansiaceae, Bacteroidaceae, Lactobacillaceae, Faecalibacterium, Clostridium_sensu_stricto_1, Bifidobacterium, and Parasutterella. In addition, the present invention also found that the inulin-promoted microorganisms were positively correlated with key metabolites that drive the regulation of metabolic pathways involved in fatty acid, amino acid, and energy metabolism. In addition, the gut microbial structure (diversity and composition) has a significant impact on the host's energy absorption, storage, and consumption.

[0090] Metabolomics analysis revealed that the medium-chain inulin supplement specifically decreased the concentration of arachidonic acid, LSD-d3, ethyl laurate, 16-hydroxyhexadecanoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and docosapentaenoic acid in the gut content compared to the HFD-fed group. These metabolites were negatively correlated with improved health parameters (HDL, BAT, and SCFAs) and positively correlated with weight gain. Therefore, these metabolites constitute a functional subgroup of core metabolites that promote weight gain. In contrast, supplementing inulin to HFD-fed mice increased the content of nicotinamide, taurocholic acid, aldosterone, corticosterone, 2-(1H-indol-3-yl)acetic acid, MN-18 N-(5-hydroxypentyl) metabolite, 5a-DHT, maltopentose, 5-hydroxytryptophan, propionyl-L-carnitine, maltose, and 4-hydroxyretinoic acid, which inhibited weight gain in HFD-fed mice. Therefore, these metabolites constitute a subgroup of metabolites that inhibit weight gain and play an important role in preventing weight gain in HFD-fed mice. Furthermore, these inulin-supplementation-altered metabolites modulated key metabolic pathways, including downregulation of arachidonic acid metabolism and upregulation of niacin / nicotinamide metabolism, primary bile acid biosynthesis, and tryptophan metabolism. These metabolic pathways are closely related to inflammation, lipid, and energy metabolism, providing insights into the mechanisms by which inulin exerts its beneficial effects. In the present invention, co-occurrence network analysis showed that specific microbial groups (Faecalibaculum, Bifidobacterium, Parasutterella, Clostridium_sensu_stricto_1, and Akkermansia) directly inhibited weight gain, highlighting their potential role in weight management. Therefore, these microorganisms form a functional subgroup that can ferment indigestible inulin into key metabolites, including SCFAs, and exert an inhibitory effect on weight gain through the "diet-microbiota-metabolite-phenotype" axis. Regarding different metabolites, SCFAs, as the main microbial-derived metabolites, can play a key role in resisting weight gain. In addition, an essential intermediate in the tryptophan metabolism process, 2-(1H-indol-3-yl)acetic acid, is positively correlated with SCFAs but negatively correlated with weight. Furthermore, 2-(1H-indol-3-yl)acetic acid is also positively correlated with Bacteroidetes, Faecalibaculum, Bifidobacterium, Parasutterella, Clostridium_sensu_stricto_1, and Akkermansia, and negatively correlated with TG, TCHO, and LDL content in the mouse liver and serum. This finding implies that 2-(1H-indol-3-yl)acetic acid can be a core metabolite in the physiological process by which inulin affects systemic health through the "diet (inulin)-gut microbiota / metabolites-phenotype" axis.

[0091] Inulin alleviates HFD-induced obesity and metabolic disorders by modulating the “diet (inulin)-gut microbiota / metabolites-phenotype” axis. Specifically, inulin promotes the enrichment of key microbes (Faecalibaculum, Bifidobacterium, Parasutterella, Clostridium_sensu_stricto_1, and Akkermansia) and the production of specific metabolites, notably 2-(1H-indol-3-yl)acetic acid, which collectively contribute to the improvement of metabolic health in DIO mice.

[0092] In summary, the present application provides compelling evidence for the beneficial effects of medium-chain inulin (DP=12) in alleviating HFD-induced obesity and metabolic disorders. The unique inulin structure and specific chain length reshaped the gut microbiome and metabolome, particularly selectively modulating key microbes such as Faecalibaculum, Bifidobacterium, Parasutterella, Clostridium_sensu_stricto_1, and Akkermansia, and metabolites such as nicotinamide, taurocholic acid, aldosterone, corticosterone, 2-(1H-indol-3-yl)acetic acid, 5-hydroxy-L-tryptophan, 5a-DHT, and maltose, thereby promoting the improvement of obesity and overall health status. Moreover, the identified key microbes and metabolites represent promising therapeutic targets for the prevention or treatment of obesity. These findings support the development of inulin-based nutritional strategies aimed at modulating specific microbiota and metabolome for effective management of body weight.

[0093] Obviously, the above-mentioned embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, it is not necessary and impossible to enumerate all the embodiments. Any modification, equivalent replacement, and improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.

Claims

1. A method for preparing medium-chain inulin, characterized by, The method comprises the following steps: (1) grinding jerusalem artichoke to obtain pulp, adding water to extract, then filtering, concentrating, removing protein, and alcohol precipitation to obtain crude polysaccharide; (2) dialyzing the crude polysaccharide through a semi-permeable membrane, concentrating, and freeze-drying to obtain the medium-chain inulin; In step (1), the water extraction conditions are as follows: the volume ratio of the pulp to water is 1:30, the extraction temperature is 80°C, and the time is 3h; The concentration is specifically to concentrate to 30% of the original volume; The method for removing protein is as follows: adding an equal volume of 5% trichloroacetic acid solution, standing for 60 minutes, centrifuging at 8000 rpm and 20°C for 10 minutes, and removing the precipitate; The method for alcohol precipitation is as follows: adding 4 times the volume of anhydrous ethanol, standing for 8-12 hours, and then centrifuging at 6000 rpm and 20°C for 10 minutes, and discarding the supernatant; In step (2), the dialysis conditions are as follows: the membrane pore diameter D is 3500 daltons; The medium-chain inulin is composed of fructose connected by 2,1 glycosidic bonds, and the average degree of polymerization is 12.

2. The medium-chain inulin prepared by the preparation method of claim 1.

3. The use of the medium-chain inulin of claim 2 in the preparation of a product for regulating intestinal flora and relieving obesity and metabolic disorders caused by HFD.

4. A product of a product for modulating gut microbiota for alleviating HFD-induced obesity and metabolic disorder, characterized in that, The product comprises the medium-chain inulin of claim 2.

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