A method for screening prebiotics for relieving obesity based on a human-derived fecal bacteria in vitro fermentation model and application thereof

Abstract

The application discloses a method for screening prebiotics for relieving obesity based on a human-source fecal bacteria in-vitro fermentation model and application thereof, and belongs to the technical field of microorganisms. The application utilizes healthy human-source and obese human-source fecal flora to perform in-vitro anaerobic fermentation on candidate prebiotics (such as inulins, beta-glucans, galactooligosaccharides and other oligosaccharides); the yield of short-chain fatty acids in the fermentation system is determined, microbial community dynamics are analyzed, and a LPS-induced macrophage inflammation model and an FFA-induced hepatocyte lipid deposition model are combined to screen prebiotics with significant anti-inflammatory and lipid metabolism disorder relieving activities. Animal experiments verify that the healthy human-source fecal bacteria fermentation product of the prebiotics can effectively reduce the body weight of high-fat-diet-induced obese mice, improve insulin resistance, reduce liver fat accumulation and regulate blood lipid disorders. The method for screening prebiotics for relieving obesity based on a human-source fecal bacteria in-vitro fermentation model and the method for preparing prebiotic fecal bacteria fermentation products with obesity relieving functions provided by the application have good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method for screening and evaluating oligosaccharide prebiotics with the potential to alleviate obesity through in vitro fermentation using healthy human fecal bacteria, and further obtaining prebiotic fecal bacteria fermentation metabolites through this method, which have the function of alleviating obesity and related metabolic diseases. Background Technology

[0002] Obesity and its associated metabolic syndromes (such as type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disease) have become a global public health crisis. Studies have shown that gut microbiota dysbiosis is one of the key drivers of obesity development. Regulating gut microbiota and its metabolites through dietary fiber / prebiotic intervention is an effective strategy for improving obesity and its associated metabolic syndrome.

[0003] Currently, methods for evaluating the obesity-alleviating potential of prebiotics mainly rely on animal models and clinical trials, which suffer from problems such as long cycles, high costs, significant individual variability, and unclear mechanisms. Furthermore, existing technologies largely remain at the level of describing the relationship between "microbial community structure" and "macrophenotype," failing to identify "who" is the core functional executor and "how" they exert their effects at the strain and gene levels. Therefore, there is an urgent need to develop a highly reproducible in vitro evaluation method and model that can simulate the human gut microenvironment and deeply analyze the causal relationships along the "microbiome-metabolite-host" axis, for the precise screening and validation of prebiotics and their metabolites with clear obesity-alleviating functions. Summary of the Invention

[0004] To address the problems of long evaluation cycles, high costs, large individual differences, and unclear mechanisms in existing methods for evaluating the effects of prebiotics on obesity, this invention aims to provide a method for screening prebiotics that alleviate obesity based on a human fecal microbiota in vitro fermentation model and its application. This method can screen oligosaccharide prebiotics with the potential to alleviate obesity and systematically verify the efficacy of human fecal microbiota fermentation products of prebiotics.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for screening prebiotics for alleviating obesity based on a human fecal microbiota in vitro fermentation model, comprising the following steps: (1) Fresh fecal samples were collected from healthy and obese individuals respectively. A reducing buffer solution was added under anaerobic conditions, homogenized, and large food residues and insoluble fiber were removed to prepare a fecal microbiota suspension. (2) Under anaerobic conditions, the fecal microbiota suspension obtained in step (1) was inoculated into a culture medium containing the prebiotic to be tested for anaerobic fermentation, and the fermentation liquid was collected. (3) The fermentation broth obtained in step (2) was subjected to short-chain fatty acid production detection and microbial community composition analysis to compare the SCFAs content and relative abundance of key genera produced by prebiotic fermentation in the microbial community of healthy and obese individuals. (4) Verify the function of the prebiotic human fecal bacteria fermentation liquid obtained in step (2) in alleviating inflammation and lipid accumulation using cell models: treat the LPS-induced macrophage inflammation model with the prebiotic human fecal bacteria fermentation supernatant to evaluate the anti-inflammatory activity of the fermentation product, and treat the FFA-induced hepatocyte lipid deposition model with the prebiotic human fecal bacteria fermentation supernatant to evaluate the lipid-lowering activity of the fermentation product. (5) Using high-fat diet-induced obese mice to verify the function of the prebiotic human fecal bacteria fermentation liquid obtained in step (2) in alleviating lipid metabolism disorder in obese model mice: The freeze-dried powder of the fermentation liquid of healthy human fecal bacteria-fermented prebiotics was administered to high-fat diet-induced obese mice by gavage. The effect of alleviating lipid metabolism disorder in high-fat diet-induced obese model mice was evaluated by detecting mouse weight, glucose and lipid metabolism indicators and organ index indicators, and prebiotics with the potential to alleviate obesity were screened.

[0006] Based on the above technical solution, further, the BMI of the healthy population mentioned in step (1) is 18.5~24 kg / m². 2 The obese population has a BMI ≥ 30 kg / m². 2 The donor should maintain a regular diet for at least one month before sampling and should not use antibiotics or probiotic / prebiotic preparations within three months.

[0007] Based on the above technical solution, further, the gas composition of the anaerobic conditions in step (1) is 85% N2, 10% CO2, and 5% H2.

[0008] Based on the above technical solution, further, the reducing buffer in step (1) is a PBS buffer containing 0.01~0.10% L-cysteine ​​hydrochloride, pH 7.0~7.5.

[0009] Based on the above technical solution, further, in step (1), each 100 mL of suspension contains 15~30 g of fecal sample.

[0010] Based on the above technical solution, further, the prebiotics to be tested in step (2) include inulin, pectin, fructooligosaccharides, galactooligosaccharides, β-glucan, stachyose, isomaltooligosaccharides and resistant starch; the concentration of the prebiotics to be tested in the culture medium is 0.5~5g / 100mL.

[0011] Based on the above technical solution, further, each liter of culture medium in step (2) contains the following components: 4.0~6.0g trypsin, 2.0~4.0g soybean peptone, 4.0~6.0g prion peptone, 8.0~12.0g digested serum powder, 2.0~3.0g yeast extract, 2.0~2.4g beef extract, 1.0~1.4g liver extract, 0.1~0.3g L-tryptophan, 0.2~0.4g L-cysteine, 0.2~0.4g sodium thioglycolate, 0.5~1.5g L-arginine, 1~10mg vitamin K, 5~15mg heme chloride, 2.0~3.0g potassium dihydrogen phosphate, and 2.0~4.0g sodium chloride.

[0012] Based on the above technical solution, further, in step (2), the inoculation volume ratio of the fecal microbiota suspension to the culture medium is 1:5~1:15; the fermentation conditions are anaerobic static fermentation at 36~38℃, and the fermentation time is 18~30h.

[0013] Based on the above technical solution, further, the short-chain fatty acid production detection in step (3) includes detecting the content of acetic acid, propionic acid and butyric acid; quantitative analysis is performed by liquid chromatography-mass spectrometry (LC-MS / MS), and the microbial community composition analysis specifically involves 16S rRNA gene amplicon sequencing to analyze α diversity, β diversity and compositional differences at the phylum, family and genus levels.

[0014] Secondly, the present invention provides a method for preparing a prebiotic fermentation product that can alleviate obesity, comprising the following steps: inoculating a suspension of fecal microbiota from healthy individuals into a culture medium containing prebiotics screened by the above method, performing in vitro anaerobic fermentation, and then preparing a powder by filtration, concentration, and freeze-drying.

[0015] Based on the above technical solution, the prebiotics further include inulin, pectin, fructooligosaccharides, galactooligosaccharides, β-glucan, stachyose, isomaltooligosaccharides, and resistant starch; the concentration of prebiotics in the culture medium is 0.5~5g / 100mL.

[0016] Based on the above technical solution, each liter of culture medium further contains the following components: 4.0-6.0g trypsin, 2.0-4.0g soybean peptone, 4.0-6.0g prion peptone, 8.0-12.0g digested serum powder, 2.0-3.0g yeast extract, 2.0-2.4g beef extract, 1.0-1.4g liver extract, 0.1-0.3g L-tryptophan, 0.2-0.4g L-cysteine, 0.2-0.4g sodium thioglycolate, 0.5-1.5g L-arginine, 1-10mg vitamin K, 5-15mg heme chloride, 2.0-3.0g potassium dihydrogen phosphate, and 2.0-4.0g sodium chloride.

[0017] Based on the above technical solution, the inoculation volume ratio of the fecal microbiota suspension to the culture medium is 1:5 to 1:15; the fermentation conditions are anaerobic static fermentation at 36 to 38°C, and the fermentation time is 18 to 30 hours.

[0018] Thirdly, the present invention provides a prebiotic fermentation product that alleviates obesity, prepared by the above-described method.

[0019] Fourthly, the present invention provides a composition for alleviating obesity, comprising the above-mentioned prebiotic fermentation product and food or pharmaceutically acceptable excipients.

[0020] Fifthly, the present invention provides the use of the above-mentioned prebiotic fermentation products or compositions in the preparation of products for the prevention or relief of obesity and related metabolic diseases; said related metabolic diseases include hyperlipidemia, non-alcoholic fatty liver disease, type 2 diabetes, insulin resistance, and chronic low-grade inflammation.

[0021] The fermentation products exert their effects through one or more of the following mechanisms: selectively regulating the intestinal flora, enriching beneficial bacteria such as Bifidobacteria, and inhibiting opportunistic pathogens; directly supplementing short-chain fatty acids, especially butyric acid and propionic acid; improving intestinal barrier function; inhibiting hepatic lipid accumulation; and regulating systemic low-grade inflammation.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes fecal microbiota from healthy and obese individuals to conduct in vitro anaerobic fermentation of candidate prebiotics (such as inulin, β-glucan, and galactooligosaccharides). By measuring short-chain fatty acid production, analyzing microbial community dynamics, and combining LPS-induced macrophage inflammation and FFA-induced hepatocyte lipid deposition models, prebiotics with significant anti-inflammatory and lipid-lowering activities, along with their fermentation products from healthy human fecal microbiota, were screened. Animal experiments verified that the fermentation products from healthy human fecal microbiota of prebiotics can effectively reduce the body weight of obese mice induced by a high-fat diet, improve insulin resistance, reduce liver fat accumulation, and regulate dyslipidemia. This function is related to the regulation of intestinal microbiota (increasing the relative abundance of beneficial microorganisms and reducing the relative abundance of harmful microorganisms) and the increase in short-chain fatty acid production by the fermentation products from healthy human fecal microbiota of prebiotics, showing promising application prospects. Attached Figure Description

[0023] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0024] Figure 1 This is a comparison of the production of short-chain fatty acids in the supernatant of fecal microbiota fermentation of different oligosaccharides in obese individuals. In the figure, A represents the total production of short-chain fatty acids, B represents the production of acetic acid, C represents the production of propionic acid, and D represents the production of butyric acid. Figure 2 This is a comparison of the production of short-chain fatty acids in the supernatant of fecal microbiota fermentation of different oligosaccharides in healthy individuals. In the figure, A represents the total production of short-chain fatty acids, B represents the production of acetic acid, C represents the production of propionic acid, and D represents the production of butyric acid. Figure 3 The composition of the microbial community in the in vitro fermentation precipitate of fecal bacteria in healthy and obese individuals for different oligosaccharides is shown. Among them, A is the Chao1 index, B is the Simpson index, and C is the Shannon index. Figure 4 Venn diagram of common / unique OTUs in the microbial community of healthy individuals (A) and obese individuals (B) after fermentation; Figure 5 The effects of different oligosaccharide fermentations based on 16S rRNA sequencing on the composition of human fecal microbial communities are shown in the figure. A represents the phylum level, B the family level, and C the genus level. Figure 6 The figure shows the relative abundance changes of key SCFAs-producing genera in human fecal bacteria, where A represents Clostridium difficile, B represents Escherichia coli, C represents Clostridium paraclostridium, D represents Bifidobacterium, and E represents Collinsella spp. Figure 7 The effects of different oligosaccharides in human fecal microbiota in vitro fermentation supernatant on LPS-induced inflammatory factors and NO secretion in RAW264.7 cells were investigated. In this study, A represents IL-6 content, B represents IL-1β content, C represents IL-10 content, D represents TNF-α content, and E represents NO content. Figure 8 The effects of different oligosaccharides in human fecal microbiota fermentation supernatant on FFA-induced intracellular TC (A), TG (B), and relative lipid content (C) in HepG2 cells were investigated. Figure 9 A comparison graph showing the weight gain curves (A) and final weight (B) of mice in each group during animal experiments; Figure 10 The results of brown adipose tissue (BAT), epididymal fat index (eWAT), white adipose tissue (iWAT), and liver index for each group of mice are shown in the figure. Figure 11 The graph shows the results of the four lipid levels in the serum of mice in each group. In the graph, A is TG, B is TC, C is LDL-C, and D is HDL-C. Detailed Implementation

[0025] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0026] The oligosaccharide prebiotics used in the following examples include: Inulin: extracted from Jerusalem artichoke, with an average degree of polymerization (avDP) of approximately 10 and a molecular weight range of approximately 1500-3500 Da, the main glycosidic bond being β-(2,1)-fructoside. Pectin (POS): extracted from citrus peel, with a degree of esterification of approximately 70% and a molecular weight of approximately 80,000-120,000 Da, the main glycosidic bond being α-(1,4)-galacturonic acid. Fructooligosaccharides (FOS): obtained from sucrose via fructosyltransferase conversion, with an average degree of polymerization of approximately 4 and a molecular weight of approximately 666-828 Da, the main glycosidic bond being β-(2,1)-fructoside. Galacto-oligosaccharides (GOS): obtained from lactose via β-galactosidase-catalyzed transglycosylation reaction, with an average degree of polymerization of approximately 3-5 and a molecular weight of approximately 504-828 Da. Da, the main glycosidic bonds are β-(1,4)- and β-(1,6)-galactoside bonds. β-Glucan: extracted from oat bran by hot water, with a molecular weight of approximately 150,000-200,000 Da, and the main glycosidic bonds are β-(1,3)- and β-(1,4)-glucosidic bonds. Stachyose (SS): extracted from the roots and rhizomes of plants such as Rehmannia glutinosa, with a clearly defined chemical structure of galactose-galactose-glucose-fructose tetrasaccharide, a molecular weight of 666 Da, and the main glycosidic bond is α-(1,6)-galactoside bond. Isomaltooligosaccharide (IMO): obtained from starch through the co-conversion of α-amylase and α-glucosidase, the main components are isomaltose, panose, and isomalttriose, with an average degree of polymerization of approximately 3-5 and a molecular weight of approximately 504-828. Da, with the main glycosidic bonds being α-(1,6)-glucosidic bonds. Resistant starch (RS): prepared from high amylose corn starch through a pressure heating-cooling cycle, it is RS3 type retrograde starch with a molecular weight of approximately 50,000-150,000 Da, and the main glycosidic bonds being α-(1,4)- and α-(1,6)-glucosidic bonds. All products are commercially available food-grade or analytical-grade products; the formulation of the fermentation basal medium is based on a typical intestinal simulated medium with slight modifications; unless otherwise specified, all anaerobic operations are carried out in an anaerobic workstation (85% N2, 10% CO2, 5% H2).

[0027] Example 1: Construction of an in vitro fermentation system for human fecal bacteria and analysis of short-chain fatty acids 1. Fecal sample collection and processing We are recruiting healthy volunteers (BMI 18.5-24 kg / m²). 2 (n=30) and obese volunteers (BMI ≥ 30 kg / m²) 2(n=30) All volunteers maintained a regular diet for at least one month prior to sampling, did not use antibiotics, probiotics, or prebiotics within three months, and signed informed consent forms. Fresh morning stool samples were collected and immediately transferred to an anaerobic workstation (gas composition: 85% N2, 10% CO2, 5% H2). Under anaerobic conditions, 10.0 g of fresh fecal sample was weighed and 40 mL of pre-reduced sterile phosphate-buffered saline (PBS, pH 7.2, containing 0.05% L-cysteine ​​hydrochloride as a reducing agent) was added. The sample was homogenized at medium speed for 2-3 min to prepare a homogeneous slurry. The slurry was filtered through four layers of sterile gauze to remove large food particles and insoluble fiber, and the filtrate was collected. Pre-reduced PBS was added to the filtrate until the total volume reached 50 mL, yielding a 20% (w / v, i.e., 20 g of fecal sample per 100 mL suspension) fecal microbiota suspension. The suspension should be prepared and used immediately, and the entire operation should be completed within the anaerobic workstation to maximize the preservation of bacterial activity.

[0028] 2. In vitro fermentation Fermentation basal medium (g / L): Weigh out 5.0g tryptone, 3.0g soybean peptone, 5.0g prion peptone, 10.0g digested serum powder, 2.5g yeast extract, 2.2g beef extract, 1.2g liver extract, 0.2g L-tryptophan, 0.3g L-cysteine, 0.3g sodium thioglycolate, 1.0g L-arginine, 5mg vitamin K, 10mg heme chloride, 2.5g potassium dihydrogen phosphate, and 3.0g sodium chloride. Prepare 1L of intestinal microbial culture medium with purified water. Adjust the pH to 7.2 and sterilize at 115℃ for 15 minutes. Vitamin K and heme chloride are added after the culture medium has cooled and sterilized. Since high temperatures can destroy the structure of some dietary factors and affect the fermentation effect of fecal microbiota, the prepared culture medium was dissolved with dietary polysaccharides and then sterilized by filtration. 1% (w / v, i.e., 1g of dietary polysaccharide per 100 mL of culture medium) of inulin, pectin (POS), fructooligosaccharides (FOS), galactooligosaccharides (GOS), β-glucan (β-gulcan), stachyose (SS), isomaltooligosaccharides (IMO), and resistant starch (RS) were added to the basal culture medium to form the experimental groups.

[0029] Inoculate 20% of the fecal microbiota suspension into the above culture medium at a volume ratio of 1:9 (total fermentation volume 50 mL), place it in a 37 ℃ anaerobic workstation for static batch fermentation, and collect the fermentation broth at the 24 h endpoint. Centrifuge at 4 ℃ and 10,000 g for 15 min, and collect the supernatant and bacterial precipitate separately. Store at -80 ℃ for later use.

[0030] 3. Determination of short-chain fatty acids (SCFAs) The supernatant was filtered through a 0.22 μm filter membrane, and the concentrations of acetic acid, propionic acid, and butyric acid were quantitatively analyzed by gas chromatography (GC). Before the determination, an appropriate amount of filtrate was taken, an equal volume of 2-ethylbutyric acid internal standard solution was added, mixed well, and filtered again through a 0.22 μm filter membrane before being placed into a gas chromatograph vial. Chromatographic conditions: A gas chromatograph equipped with a flame ionization detector (FID) was used; the column was a DB-FFAP capillary column (30 m × 0.25 mm × 0.25 μm); the carrier gas was high-purity nitrogen at a flow rate of 1.0 mL / min; the injection port temperature was 250℃, and the detector temperature was 280℃; the column oven temperature program was as follows: initial temperature 80℃, held for 1 min, increased to 200℃ at 15℃ / min, and held for 2 min. The injection volume was 1 μL, and the split ratio was 10:1. Qualitative analysis was performed by comparing the retention time with that of the standard, and quantification was performed using the internal standard method.

[0031] POS represents pectin + healthy individuals, POS-F represents pectin + obese individuals, Inulin represents inulin + healthy individuals, Inulin-F represents inulin + obese individuals, GOS represents galactooligosaccharides + healthy individuals, GOS-F represents galactooligosaccharides + obese individuals, SS represents stachyose + healthy individuals, SS-F represents stachyose + obese individuals, β-gulcan represents β-glucan + healthy individuals, β-gulcan-F represents β-glucan + obese individuals, RS represents resistant starch + healthy individuals, RS-F represents resistant starch + obese individuals, FOS represents fructooligosaccharides + healthy individuals, FOS-F represents fructooligosaccharides + obese individuals, IMO represents isomaltooligosaccharides + healthy individuals, IMO-F represents isomaltooligosaccharides + obese individuals, STS represents glucose + healthy individuals, STS-F represents glucose + obese individuals (control group).

[0032] Fermentation results of gut microbiota in obese individuals, as follows Figure 1 As shown, in in vitro fermentation of gut microbiota in obese individuals, different dietary fibers exhibited significant differences in the production of short-chain fatty acids (SCFAs). The propionic acid production in the β-glucan group was significantly higher than that in the inulin and fructooligosaccharide groups. P <0.05), and the total SCFAs yield also showed a significant advantage. Inulin and fructooligosaccharide fermentation products mainly promoted butyric acid production, with relatively limited promotion of propionic acid. These results indicate that in vitro fermentation of β-glucan is highly effective in promoting propionic acid production and increasing total SCFAs yield. Fermentation results from healthy microbial communities are shown below. Figure 2 As shown, in in vitro fermentation by gut microbiota in healthy individuals, β-glucan and galactooligosaccharides significantly increased propionic acid production, while inulin significantly increased butyric acid production. Inulin and galactooligosaccharides significantly increased the production of total SCFAs (SCFAs). P<0.05), demonstrating prebiotic properties that comprehensively promote the generation of multiple beneficial SCFAs.

[0033] Example 2: Microbial community composition analysis of fermentation precipitate The bacterial pellet from Example 1, fermented for 24 h, was used to extract total DNA using the PowerSoil DNA Extraction Kit. The 16S rRNA gene V3-V4 region was amplified using primers 341F / 806R, and after library construction, paired-end sequencing was performed on the Illumina NovaSeq platform. The data were processed using QIIME2, clustered at 97% similarity as operational taxonomic units (OTUs), and annotated against the SILVA database. Alpha diversity (Chao1, Shannon, Simpson indices), β diversity (PCoA based on Weighted UniFrac), and species composition at each taxonomic level were analyzed; paired-samples t-tests and one-way ANOVA were used for intergroup comparisons.

[0034] The results of the microbial community composition of fecal microbiota in vitro fermentation precipitates of different oligosaccharides in healthy and obese individuals are as follows: Figure 3 As shown, Figure 3 In the diagram, POS represents pectin + healthy individuals, POS-F represents pectin + obese individuals, Inulin represents inulin + healthy individuals, Inulin-F represents inulin + obese individuals, GOS represents galactooligosaccharides + healthy individuals, GOS-F represents galactooligosaccharides + obese individuals, SS represents stachyose + healthy individuals, SS-F represents stachyose + obese individuals, β-gulcan represents β-glucan + healthy individuals, β-gulcan-F represents β-glucan + obese individuals, RS represents resistant starch + healthy individuals, RS-F represents resistant starch + obese individuals, FOS represents fructooligosaccharides + healthy individuals, FOS-F represents fructooligosaccharides + obese individuals, IMO represents isomaltooligosaccharides + healthy individuals, IMO-F represents isomaltooligosaccharides + obese individuals, STS represents glucose + healthy individuals, STS-F represents glucose + obese individuals. α-diversity analysis showed that, except for galactooligosaccharides, the Chao1 index of all oligosaccharides after fermentation was significantly lower than before fermentation. P <0.05, with the inulin and fructooligosaccharide groups showing the most significant decrease ( P <0.01). Conversely, the Shannon and Simpson indices of all treatment groups increased significantly after fermentation ( P <0.01 indicates that fermentation makes the species distribution within the community more even and improves overall diversity. After fermentation, there were significant differences in the Shannon index among different oligosaccharide groups ( P <0.01), among which the β-glucan group and resistant starch had the highest Shannon index, significantly higher than the oligofructose group (P <0.05).

[0035] The shared / unique OTUs in the gut microbiota of healthy individuals (A) and obese individuals (B) after fermentation are shown in the Venn diagram. Figure 4 As shown, Venn diagram analysis revealed the differential regulation of microbial species composition by fermentation of different oligosaccharides. In healthy individuals, the core OTUs shared by the fermentation products of fructooligosaccharides, galactooligosaccharides, and resistant starch accounted for only 15.2% of the total OTUs, while each group contained a large number of unique OTUs (fructooligosaccharide group 28%, galactooligosaccharide group 22%, resistant starch group 19%). Figure 4 A) indicates that healthy gut microbiota can produce highly specific species-level responses to different oligosaccharides. In obese individuals, the pattern of comparison of the same oligosaccharides changed significantly, with the proportion of shared core OTUs across the three groups increasing to 31.5%, while the proportion of group-specific OTUs decreased significantly (fructooligosaccharide group 12%, galactooligosaccharide group 10%, resistant starch group 8%). Figure 4 (B) This suggests that the response of the obese microbiota is generally dulled and its specificity is reduced. Notably, isomaltooligosaccharide exhibits uniqueness in the obese group. Its fermentation products have very few OTUs shared with all other oligosaccharide groups, but a high proportion of its own unique OTUs, far exceeding the level in the healthy group. This indicates that isomaltooligosaccharide enriches a very unique microbial set in obese individuals.

[0036] The compositional analysis results at the phylum, family, and genus levels are as follows: Figure 5 As shown, at the phylum level, Firmicutes and Bacteroides were the absolutely dominant phyla in all samples, with a combined relative abundance exceeding 70%. Figure 5 A) Different oligosaccharides exert differentiated regulation on the Firmicutes / Bacteroidetes ratio (F / B value): galactooligosaccharides, fructooligosaccharides, and isomaltooligosaccharides showed a decreasing F / B value after fermentation; while β-glucan and resistant starch showed an increasing F / B value after fermentation. The F / B ratio at the fermentation initiation point was generally higher in obese individuals than in healthy individuals. After intervention with fermentation using certain oligosaccharides (such as isomaltooligosaccharides), this ratio could be significantly reversed, converging towards the pattern observed in healthy individuals.

[0037] The results of the analysis at the family and genus levels are as follows: Figure 5 B and Figure 5 As shown in C, the targeted analysis of different functional bacterial genera ( Figure 6 This further indicates that Bifidobacterium ( ) Bifidobacterium It was uniformly enriched by inulin and isomaltooligosaccharide, while the promoting effect of galactooligosaccharide was more significant in the obese group. Escherichia coli ( Escherichia ) and Clostridium difficile ( ClostridiumThe abundance of ) in the initial microbiota of obese individuals was significantly higher than that in healthy individuals. P <0.001); fermentation of galactooligosaccharides, β-glucan, and resistant starch effectively inhibited the proliferation of these two bacteria in the obese group, maintaining their abundance at or below initial levels. *Collinsella* spp. Collinsella The abundance of these compounds was generally higher in the obese group, and it was further significantly increased after fermentation with inulin, galactooligosaccharides, and stachyose. P <0.05). Clostridium parasiticum ( Paraclostridium The abundance of ) remained relatively stable across all treatments. Isomaltooligosaccharide, inulin, and stachyose were all shown to significantly increase ) Bifidobacterium The relative abundance of.

[0038] In summary, the results of this embodiment demonstrate that the prebiotic effect of oligosaccharides is strain-specific and host-state dependent. Inulin and isomaltooligosaccharide are reliable enrichment agents for Bifidobacteria; galactooligosaccharides exhibit a dual property of promoting beneficial bacteria and inhibiting opportunistic pathogens in obese individuals. The aforementioned changes in microbial community structure are highly consistent with the production characteristics of SCFAs.

[0039] Example 3: In vitro cell activity verification of fermentation supernatant 1. Evaluation of anti-inflammatory activity The fermentation supernatant from Example 1, after 24 h, was sterilized by filtration through a 0.22 μm filter membrane and stored at -20°C for later use. RAW264.7 mouse mononuclear macrophages were routinely cultured in DMEM high-glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were then cultured at 2 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 12 h until cells adhered. The experimental groups were set up as follows: Blank control group: Added an equal volume of DMEM complete culture medium, without LPS or fermentation supernatant; LPS model group: Only LPS stimulation was added, with a final LPS concentration of 1 μg / mL; LPS + fermentation supernatant of each prebiotic group: 0.22 μm filtered and sterilized fermentation supernatant was added at a final concentration of 10% (V / V), pre-incubated for 4 h, and then LPS (final concentration 1 μg / mL) was added, for a total stimulation of 24 h. Each group was set up with 3 replicates, and the experiment was independently repeated 3 times. After culture, the cell culture supernatant of each group was collected, centrifuged at 300×g for 5 min at 4℃ to remove cell debris, and the concentrations of TNF-α, IL-6, IL-1β and IL-10 were determined according to the ELISA kit instructions. At the same time, the content of nitric oxide (NO) was detected by the Griess method.

[0040] The results are as follows Figure 7As shown, compared with the LPS model group, inulin fermentation supernatant significantly inhibited the excessive production of pro-inflammatory factors TNF-α, IL-1β and inflammatory mediator NO, while β-glucan fermentation supernatant significantly increased the level of anti-inflammatory factor IL-10. P <0.001); the fermentation supernatant of inulin, fructooligosaccharides, β-glucan and resistant starch also showed significant inhibitory effects on a variety of pro-inflammatory factors, indicating that the intestinal fermentation products of prebiotics such as β-glucan and inulin have clear anti-inflammatory potential.

[0041] 2. HepG2 cell lipid accumulation model and evaluation of lipid-lowering activity HepG2 human liver cancer cells were routinely cultured in MEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were harvested and cultured at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 cell / well in 24-well plates and cultured for 12 h until adherence. The following groups were set up: Blank control group: an equal volume of MEM complete medium was added, without FFA or fermentation supernatant; FFA model group: FFA (oleic acid:palmitic acid = 2:1, final concentration 0.5 mM) was added to induce lipid accumulation; FFA + prebiotic fermentation supernatant group: Fermentation supernatant filtered through a 0.22 μm membrane for sterilization was added to the culture system at a final concentration of 10% (V / V), along with FFA (final concentration 0.5 mM), and incubated for 36 h. Each group had 3 replicates, and the experiment was independently repeated 3 times. After incubation, the culture supernatant was discarded, and cells were washed twice with PBS. After washing with PBS, cell lysis buffer was added, and cells were lysed on ice for 30 min. The supernatant was collected by centrifugation at 12,000 × g for 10 min at 4 °C. The levels of triglycerides (TG) and total cholesterol (TC) in the lysis buffer were measured according to the instructions of the commercially available kit, and corrected for by the total cellular protein concentration. Results are as follows: Figure 8 As shown, compared with the FFA model group, the intracellular TG and TC contents in the fermentation supernatant treatment groups with β-glucan and inulin were significantly reduced. P <0.001). This result confirms at the cellular level that the screened prebiotic fermentation products can directly reduce lipid accumulation in hepatocytes.

[0042] Example 4: Verification of the in vivo anti-obesity efficacy of fermentation products 1. Establishment of the obesity model and experimental grouping Seventy 6-week-old SPF-grade male C57BL / 6J mice (weighing 18-20 g) were acclimatized for one week and then divided into a normal control group (normal maintenance diet, 10% fat energy supply) and a high-fat diet group (HFD, 60% fat energy supply). After 8 weeks of feeding, the obesity model was successfully established when the average body weight of the mice in the high-fat diet group exceeded that of the mice in the normal control group by 20%. During the experimental treatment, the mice were divided into the following groups: Normal control group (K group): fed a standard maintenance diet and administered 200 μL of sterile saline per mouse via gavage daily; Successfully modeled obese mice were randomly divided into the following 6 groups (n=10 per group): Model control group (M group): fed a high-fat diet and administered 200 μL of sterile saline per mouse via gavage daily; Positive drug group (DG group): fed a high-fat diet and administered orlistat suspension (75 mg / kg / day) via gavage daily, 200 μL per mouse; Inulin fermentation product group (Inulin group): fed a high-fat diet and administered lyophilized concentrate of the supernatant of fermented inulin from healthy human feces via gavage daily, 200 μL per mouse, equivalent to 10 mg / kg / day. mL of original fermentation broth / kg body weight; Galacto-oligosaccharide fermentation product group (GOS group): fed a high-fat diet, and administered daily by gavage a lyophilized concentrate of the supernatant of galacto-oligosaccharide fermentation from healthy individuals' feces, with the same gavage volume and dosage as above; β-glucan fermentation product group (β-glucan group): fed a high-fat diet, and administered daily by gavage a lyophilized concentrate of the supernatant of β-glucan fermentation from healthy individuals' feces, with the same gavage volume and dosage as above; Fructo-oligosaccharide fermentation product group (FOS group): fed a high-fat diet, and administered daily by gavage a lyophilized concentrate of the supernatant of fructo-oligosaccharide fermentation from healthy individuals' feces, with the same gavage volume and dosage as above. The intervention lasted for 6 weeks, during which patients had free access to food and water.

[0043] 2. Preparation of fermentation products Following the method in Example 1, inulin, β-glucan, fructooligosaccharides, and galactooligosaccharides were subjected to 24-hour in vitro anaerobic fermentation using fecal microbiota from healthy individuals. The supernatant was collected by centrifugation, filtered through a 0.22 μm membrane, concentrated by rotary evaporation, and freeze-dried to obtain fermentation product powder. The powder was then reconstituted with physiological saline to the required concentration for gavage.

[0044] 3. Indicator Testing Weight changes were recorded weekly. After the experiment, the animals were fasted for 12 hours. Blood was collected from the orbital rim to separate serum. After euthanasia, the liver, white fat (iWAT), brown fat (BAT), and epididymal fat were weighed and the organ indices were calculated. The levels of TG, TC, LDL-C, and HDL-C in the serum were detected using a fully automated biochemical analyzer. The levels of IL-6, IL-1β, IL-10, and TNF-α in the serum were measured using an ELISA kit.

[0045] 4. Results (1) Weight and body shape: such as Figure 9-10As shown in the figure, K represents the blank group, M represents the model blank group, DG represents the drug treatment group, Inulin represents the inulin fermentation product group, GOS represents the galactooligosaccharide fermentation product group, β-gulcan represents the β-glucan fermentation product group, and FOS represents the fructooligosaccharide fermentation product group. After 6 weeks of intervention, compared with the HFD model group, the mice in the inulin fermentation product group showed a significantly slower weight gain and a highly significant decrease in final weight. P <0.001, liver index, white fat (iWAT), brown fat (BAT), and epididymal fat index (eWAT) also showed a highly significant decreasing trend. P <0.001). Additionally, the mice in the β-glucan fermentation product group also showed a certain trend of decreased body weight, and the white fat, brown fat, and epididymal fat indices also showed a significant decreasing trend. P <0.01).

[0046] (2) Intervention with inulin fermentation products can significantly improve dyslipidemia induced by a high-fat diet. Figure 11 Compared with the model group, the serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels in the inulin fermentation product group were significantly lower. P <0.01, showing a clear beneficial regulatory trend. Simultaneously, the high-density lipoprotein cholesterol (HDL-C) level in this group was significantly higher than that in the control group ( P <0.01), suggesting that intervention with inulin fermentation products can promote a shift in the lipid profile toward cardiovascular protection, and significantly alleviate obesity-related lipid metabolism disorders as a whole.

[0047] Example 5: Preparation of anti-obesity fermentation products Taking inulin, which demonstrated the best efficacy in Example 4, as an example, fecal microbiota from healthy individuals was inoculated into a fermentation medium containing 1% inulin. Anaerobic fermentation was carried out at 37 °C for 24 h. The supernatant was collected by centrifugation (10,000 g, 15 min) and filtered sequentially through 0.45 μm and 0.22 μm microporous membranes. The supernatant was then freeze-dried to obtain a powdered fermentation product rich in active SCFAs such as butyric acid. This product can be used as a functional ingredient in weight management products in the form of tablets, powders, and capsules. The recommended dosage can be adjusted according to actual efficacy requirements.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; modifications or equivalent substitutions may be made to the technical solutions described in the foregoing embodiments, and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening prebiotics to alleviate obesity based on a human fecal microbiota in vitro fermentation model, characterized in that, Includes the following steps: (1) Fresh fecal samples were collected from healthy and obese individuals respectively. A reducing buffer solution was added under anaerobic conditions, homogenized, and large food residues and insoluble fiber were removed to prepare a fecal microbiota suspension. (2) Under anaerobic conditions, the fecal microbiota suspension obtained in step (1) was inoculated into a culture medium containing the prebiotic to be tested for anaerobic fermentation, and the fermentation liquid was collected. (3) The fermentation broth obtained in step (2) was subjected to short-chain fatty acid production detection and microbial community composition analysis to compare the SCFAs content and relative abundance of key genera produced by prebiotic fermentation in the microbial community of healthy and obese individuals. (4) Verify the function of the prebiotic human fecal bacteria fermentation liquid obtained in step (2) in alleviating inflammation and lipid accumulation using cell models: treat the LPS-induced macrophage inflammation model with the prebiotic human fecal bacteria fermentation supernatant to evaluate the anti-inflammatory activity of the fermentation product, and treat the FFA-induced hepatocyte lipid deposition model with the prebiotic human fecal bacteria fermentation supernatant to evaluate the lipid-lowering activity of the fermentation product. (5) Using high-fat diet-induced obese mice to verify the function of the prebiotic human fecal bacteria fermentation liquid obtained in step (2) in alleviating lipid metabolism disorder in obese model mice: The freeze-dried powder of the fermentation liquid of healthy human fecal bacteria-fermented prebiotics was administered to high-fat diet-induced obese mice by gavage. The effect of alleviating lipid metabolism disorder in high-fat diet-induced obese model mice was evaluated by detecting mouse weight, glucose and lipid metabolism indicators and organ index indicators, and prebiotics with the potential to alleviate obesity were screened.

2. The method according to claim 1, characterized in that, The BMI of the healthy individuals mentioned in step (1) is 18.5~24 kg / m². 2 The obese population has a BMI ≥ 30 kg / m². 2 ; Donors should maintain a regular diet for at least one month prior to sampling and should not use antibiotics or probiotic / prebiotic preparations within three months. The reducing buffer is a PBS buffer containing 0.01-0.10% L-cysteine ​​hydrochloride, pH 7.0-7.

5. Each 100 mL suspension contains 15-30 g of fecal sample.

3. The method according to claim 1, characterized in that, The prebiotics to be tested in step (2) include inulin, pectin, fructooligosaccharides, galactooligosaccharides, β-glucan, stachyose, isomaltooligosaccharides, and resistant starch; the concentration of the prebiotics to be tested in the culture medium is 0.5~5g / 100mL; the inoculation volume ratio of the fecal microbiota suspension to the culture medium is 1:5~1:15; the fermentation conditions are anaerobic static fermentation at 36~38℃ and the fermentation time is 18~30h.

4. The method according to claim 1, characterized in that, The short-chain fatty acid production detection in step (3) includes detecting the content of acetic acid, propionic acid, and butyric acid; quantitative analysis is performed using liquid chromatography-mass spectrometry (LC-MS / MS); the microbial community composition analysis specifically involves 16S rRNA gene amplicon sequencing to analyze α diversity, β diversity, and compositional differences at the phylum, family, and genus levels.

5. A method for preparing a prebiotic fermented product that alleviates obesity, characterized in that, The method includes the following steps: inoculating a suspension of fecal microbiota from healthy individuals into a culture medium containing prebiotics screened by the method described in any one of claims 1-4, performing in vitro anaerobic fermentation, and then preparing a powder by filtration, concentration, and freeze-drying.

6. The preparation method according to claim 5, characterized in that, The inoculation volume ratio of the fecal microbiota suspension to the culture medium is 1:5 to 1:15; the fermentation conditions are anaerobic static fermentation at 36 to 38°C, and the fermentation time is 18 to 30 hours.

7. The prebiotic fermentation product for relieving obesity prepared by the preparation method according to claim 5 or 6.

8. A composition for relieving obesity, characterized in that, It comprises the prebiotic fermentation product as described in claim 7 and food or pharmaceutically acceptable excipients.

9. The use of the prebiotic fermentation product of claim 7 or the composition of claim 8 in the preparation of products for the prevention or relief of obesity and related metabolic diseases.

Images