2.4. Sample Measurement and Sequencing
All frozen samples were shipped on dry ice and entrusted to the Shanghai Institute of Quality Inspection and Technical Research (SQI) for unified testing.An enzyme-linked immunosorbent assay (ELISA) kit (Cusabio; CSB-E09235h, 96T, Houston, TX, USA) was used to determine sIgA levels in saliva and fecal samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine cortisol levels in saliva samples. The pH of fecal samples was detected with a solid and semi-solid pH meter. An FC test kit (immunofluorescence chromatography, Guangzhou Fengrun Biotechnology Co., Ltd., Guangzhou, China) in combination with a dry fluorescent immunoanalyzer (model FR-101) was used to detect FC. The fecal AAT content was determined with an α1-antiprotease assay kit (immune transmission turbidimetry, Weifang 3D Biology, Weifang, China).The 16S rDNA amplification method was used to amplify the v3–v4 variable region of 16S rDNA in samples using specific primers, construct a high-throughput sequencing library, and analyze the sequence of the variable region so as to identify the microbial composition and abundance. The sequencing process included sample DNA extraction and quality inspection, polymerase chain reaction (PCR) amplification, library preparation and detection, Miseq sequencing, and sequencing data quality control and optimization. The valid sequences obtained by sequencing on the Illumina MiSeq platform were clustered into operational taxonomic units (OTUs). OTUs were clustered at 97% sequence similarity, and the species distribution information was obtained based on Silva_138 database annotation. OTU tables were normalized to account for differences in sequence depth.