*4.4. Animal Experiments*

Twenty-two male C57 mice (Specific-pathogen-free grade, six-week-old) were purchased from SPF (Beijing, China) Biotechnology Co., Ltd. (Beijing, China), and kept at the Animal Center of Zhejiang, University of Technology. All mice were randomly divided into four groups (the blank and control groups: *n* = 6; the SfW and SfA groups: *n* = 5) and fed a normal diet for one week to stabilize all the metabolic conditions. Each group was housed in one standard cage under a condition of 22 ± 1 ◦C, a humidity of 55 ± 5%, and a 12 h light/dark cycle. From the beginning of the experiment, mice in the control, SfW, and SfA groups were fed with an HFD (TP23300, 60 kcal% fat, Trophic Animal Feed High-Tech Co., Ltd, Nantong, China), and mice in the blank group were still fed a standard lab chow

diet. The mice in the SfW and SfA groups had free access to 1 mg/mL of SfW and SfA in drinking water, respectively, for a period of 16 weeks, and the other two groups were treated with sterile water.

During the experiments, the body weight was measured weekly, and fasting blood glucose was measured monthly via tail vein using a glucometer (Johnson and Johnson, New Brunswick, NJ, USA) according to the instruction after fasting for 16 h. A sugar tolerance test (OGTT) and insulin tolerance test (ITT) were measured at week 15 and 16, respectively, and the feces were collected before the mice were dissected. All mice were sacrificed from asphyxiation by carbon dioxide. The liver and epididymal fat were harvested and their weight was measured. The cecum contents were collected and stored at −80 ◦C. The protocol was approved by the Animal Ethics Committee of the Zhejiang University of Technology, China. All efforts were made to minimize the suffering of the mice.

#### *4.5. Gut Microbiota Analysis by 16S rRNA Gene Sequencing*

Fecal samples and cecal contents were used for gu<sup>t</sup> microbiota analysis by sequencing the 16S rRNA genes. The DNA extraction, PCR amplification, sequencing, and data analysis were conducted according to our previous study [11].
