Materials and Methods
S. ningpoensis was purchased from Yunnan Province, China. Acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, trans-2-butenoic acid, ribitol, and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) were bought from Sigma-Aldrich Inc. (St. Louis, MO, USA). Yeast extract, Casitone, and fatty acid (YCFA) medium were kindly provided by Hangzhou Hailu Biotechnology Co., Ltd (Hangzhou, China). Other chemical reagents were all ordered from Solarbio (Beijing, China).

Crude SNP was extracted using a method from previous research, albeit with certain modifications ( Chen et al., 2021 ). S. ningpoensis powder was mixed with deionized water (1:8, w/v) and heated to 90 °C for 7 h. After centrifuging the extract at 6,000 rpm for 10 min, the supernatant underwent deproteinization by using the Sevag method and precipitated using ethanol (95%, v/v). Thereafter, the precipitate was lyophilized to obtain crude SNP.

The method of phenol-sulfuric acid ( Dubois et al., 1951 ) and bovine serum albumin (BSA) as standard ( Chen et al., 2018 ) were separately used to estimate total carbohydrate and protein contents of SNP.

Simulated gastric and small intestinal digestions of SNP were analyzed with reference to previous approaches ( Minekus et al., 2014 ). In short, preparation of simulated gastric buffer followed Table S1 . The simulated gastric fluid was prepared by mixing 330 μL pepsin (3,000 U/mL), 4 mL simulated gastric buffer, and 25 μL CaCl 2 (0.3 M). For simulated gastric digestion, after mixing a polysaccharide solution of the same volume with the simulated gastric buffer, the mixture was incubated in a water bath at 37 °C, while maintaining the reaction solution at pH 3. Two hours after that, samples were taken, followed by inactivation at 100 °C for 5 min. Preparation of simulated small intestinal buffer followed the procedure outlined in Table S1 . This simulated intestinal fluid comprised 4 mL small intestinal buffer, 100 μL CaCl 2 (0.3 M), 250 μL pancreatin (4,000 U/mL), 400 μL bile salt (100 mg/mL), and 5 mL simulated gastric digestion. The final step was 2 h of digestion of the mixed solution in a water bath at 37 °C, and then enzyme inactivation was made at 100 °C for 5 min.

A diabetes diagnosis can be determined by the presence of diabetes-related symptoms such as polydipsia, polyuria and unexplained weight loss, along with one of the following diagnostic criteria: fasting plasma glucose levels of ≥7.0 mmol/L (whole blood of ≥6.1 mmol/L), glycated hemoglobin (HbA1c) of ≥6.5%, random venous plasma glucose of ≥11.1 mmol/L, or a 2-h plasma glucose concentration of ≥11.1 mmol/L, following a 75-g anhydrous glucose oral glucose tolerance test (OGTT) ( Society, 2024 ; World Health Organization and International Diabetes Federation, 2006 ).

The following inclusion criteria apply: 18–80 years old, the body mass index (BMI) of 18–35 and diagnosis of T2DM at least 6 months before the study.

The following exclusion criteria apply: antibiotic treatments in the 3 months before the research, pregnancy, or lactation.

The enrolment of 16 human volunteers was supervised, and the research was approved, by the Biomedical Ethics Committee of West China Hospital of Sichuan University, under protocol number 2018 (286). Written informed consent to participate in the study was obtained from volunteers. Eight females and eight males were included. The in-vitro fermentation was the same as that by Liu et al. (2020) . The 10% (w/v) stool suspension was formed by collected fresh stool samples and homogenized then using 0.1 M phosphate buffer (pH 7.0). After adding 1 mL fecal suspension, the 9 mL YCFA media containing or not containing SNP (50.0 mg) were transferred to a BSP-100 anaerobic box (Shanghai Boxun Industrial Co., Ltd, Shanghai, China) to be incubated at 37 °C for 24 h. Four treatments were used in this study: (1) original healthy control (HC); (2) original T2DM control (T2DM); (3) SNP group of fecal slurry from HC (HC+SNP); and (4) SNP group of fecal slurry from T2DM (T2DM+SNP).

The fermentation sample was centrifuged, followed by extraction of microbial genomic DNA through use of a Stool Genomic DNA Extraction Kit (Solarbio, Beijing, China) following the manufacturer protocols. Primer pairs 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were adopted to amplify hypervariable regions V3–V4 of the 16S rRNA gene of bacteria using an ABI GeneAmp ® 9700 PCR thermocycler (ABI, CA, USA). Majorbio Biotechnology Co., Ltd (Shanghai, China) was entrusted to perform 16S rRNA sequencing. Quality control was followed by grouping of selected sequences into operational taxonomic units (OTUs) through UPARSE (version 10.1 http://drive5.com/uparse/ ). OTUs were taxonomically annotated using Ribosomal Database Project (RDP). The microbiota compositions and diversity, as well as correlations of gas, short chain fatty acids (SCFAs) and significantly different metabolites were analyzed.

The media were collected after in - vitro fermentation for 24 h. A gas detector (Shanghai Yijie Industrial Safety Equipment Co., Shanghai, China) was used to measure the total gas volume in fermentation flasks, and the contents of methane (CH 4 ), carbon dioxide (CO 2 ), hydrogen (H 2 ) and hydrogen sulfide (H 2 S).

The gas chromatograph (GC, Shimadzu, GC-2010 Plus, Kyoto, Japan) equipped with a DB-FFAP column (Agilent Technologies, Santa Clara, CA, USA) and an H 2 flame ionization detector was adopted to measure the concentrations of SCFAs, such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in culture filtrates. The internal standard was trans-2-butenoic acid ( Bai et al., 2017 ).

The fermentation samples were centrifuged 12,000 g for 10 min at 4 °C. Subsequently, 200 μL of the supernatant was harvested, and ribitol was added as the internal standard at a concentration of 0.6 μg/mL. After using a reported approach ( Qi et al., 2022 ) for oximation and silylation derivatization, the metabolites of the samples were analyzed by an Agilent 7890B series gas chromatography (GC) system with an HP-5 column (30 × 0.25 µm × 0.25 mm) coupled to an Agilent 5977B quadrupole mass. The oven was programmed to have an initial temperature of 50 °C, a holding time of 2 min, a rapid temperature rise to 270 °C at 5 °C/min, another rise to 290 °C at 2.5 °C/min, a final rise to 310 °C at 10°C/min and a holding time of 4 min. The detector and injector temperatures were set to 250 °C, and the carrier gas was helium, which flowed at a rate of 1 mL/min. For mass spectrometry (MS) acquisition, the electron-impact mode was used to scan from 50–700 m / z according to programming. Each compound was identified by comparison with the NIST05a library.

Data analysis was performed using SPSS 22 (IBM, New York, NY, USA), with results shown as means ± standard deviations (SDs) ( n = 8). GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) was used for drawing the gas and SCFAs. The Majorbio Cloud Plat-form ( http://www.majorbio.com ) was adopted for analysis of 16 s rRNA sequencing data. Metabolic data were analyzed using Metaboanalyst 5.0 ( https://metaboanalyst.ca ) and SIMCA (Sartorius, Germany). The KEGG database was used for studying the metabolic pathways. Spearman correlation analysis was performed to ascertain the correlations of fecal microbiota with various metabolites.