The SPH was prepared according to a previous method with slight modification. In briefly, soy protein isolate (9010-10-0) (Yuanye Biotechnology, Shanghai, China) was suspended in 10% (w/v) distilled water, and porcine pepsin (Yuanye Biotechnology, S10027) hydrolysis was conducted at an enzyme-to-substrate ratio of 1:100 (w/w), pH 2.0, and 37°C for 1 h. Thereafter, the pH of the solution was adjusted to 7.0 with 2N NaOH to terminate the reaction. The obtained hydrolysate was stored at -20°C and lyophilised in a FreeZone 4.5 L Freeze Dry System (Labconco Co., Kansas City, MO, USA) for further use.
Animals and experimental procedures
Sixteen castrated pigs (Duroc × Landrace × Large White, aged 50 days) with an initial weight of 14.5 ± 0.2 kg were obtained from a commercial farm in Jiangsu Province, China. The pigs were kept in separate metabolic cages under a constant temperature of 25 ± 2°C and given unlimited access to water and pig feed. After a week of acclimatisation, the pigs were fasted for 12 h before installing a simple T-cannula in the duodenum. The length, width, and inner diameter of the T-cannula were 8.2, 10.0, and 1.5 cm, respectively. After the surgery, all pigs were hypodermically injected with ceftriaxone sodium and the wound and adjacent skin were disinfected with iodine tincture for one week (twice a day) to avoid potential infection. After they fully recovered from the duodenal fistula surgery over a two-week recuperation period, a two-week short-term experiment of SPH on the secretion of an intestinal satiety hormone was performed. After a week of rest, all pigs were randomly allocated to the saline (control, n = 8) group and SPH group (n = 8) with no differences in body weight (35.2 ± 0.3 kg) and feed intake. The entire experiment was conducted over a period of 16 days, during which the pigs in the saline and SPH groups were infused with 10 ml sterile saline and 10 ml SPH solution (70 g/day), respectively, through a duodenal fistula at 8:00 am and 5:00 pm each day. The SPH solution was adjusted to pH 5.0, which is close to the native pH of porcine duodenum. The basal diet in the experiment was designed based on the National Research Council (NRC) (2012) (Table 1). The feed consumption of each pig was recorded every day to calculate average feed intake. In addition, the body weights of all pigs were recorded on days 1 and 16 to determine average weight gain.
After fasting for 12 h on day 16, all pigs were given general anaesthesia by an intravenous injection of sodium pentobarbital solution (40 mg/kg body weight), and were sacrificed by jugular exsanguination. The colons of all pigs were taken out within 5 minutes after slaughter and measured for length; fresh digesta from the colon was collected in a sterile tube and immediately stored at -20°C for the analysis of microbiota and metabolites. The colon was weighed after removing all the digesta. In addition, a small piece of the colon tissue was collected and washed with phosphate buffered solution (PBS, pH 7.0); then, the colonic mucosa was scraped and immediately frozen in liquid nitrogen, and used to determine the concentration of inflammatory cytokines and the protein expression of tight junction proteins.
Analysis of colonic microbial metabolites
The content of short-chain fatty acids (SCFAs) was determined using the method of Wang et al.. Briefly, 1.5 ml of double distilled water was added into 0.4 g of digesta. The mixture was mixed and centrifuged (13,400 × g, 4℃, 10 min) in order to obtain supernatants. 1 ml supernatant and 200 μL of 25% (w/v) metaphosphoric acid were mixed and kept overnight at -20°C. Then, the supernatant was centrifuged (13, 400 × g, 4℃, 10 min) and filtered with a 0.22 μL filter, and then measured with an Agilent 7890B gas chromatograph.
High-performance liquid chromatography (HPLC) was selected to detect the content of biogenic amines according to the method given by Yang et al.. For this purpose, 0.6 g of digesta was weighed and 1.5 ml of trichloroacetic acid solution was added to precipitate the proteins and peptides. The sample was extracted with n-hexane and derived with dansyl chloride. Gradient elution of two solvents was carried out in the following manner: solvent A and solvent B were HPLC grade water and acetonitrile respectively, and the flow rate was set to 1.0 mL/min.
In addition, 0.1 g of digesta was prepared and acidified with 0.9 ml of 0.2 mol/L HCl for the determination of ammonia content using a spectrophotometer (UV-2450; Shimadzu, Tokyo, Japan) following the previous method.
16S rRNA sequencing
DNA from different colonic digesta was extracted using a DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA concentration and purity were measured. The primers 515F and 907R were used to amplify the V4-V5 region of the 16S rRNA gene according to the previous methods. Thereafter, the amplicon libraries were established using NEB Next®Ultra™DNA Library Prep Kit for Illumina (NEB, USA) based on the manufacturer’s illustrations. Then, the library was sequenced on an Illumina MiSeq platform. QIIME (version 1.70) was used to demultiplex and quality-filter the raw FASTQ files. Sequences with ≥ 97% similarity were classified as one operational taxonomic unit (OTU). The representative sequence of each OTU was selected, and the Ribosomal Database Project (RDP) classifier was used to annotate taxonomic information. The OTU table was refined to calculate alpha diversity. Beta diversity was calculated based on principal coordinates analysis (PCoA) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Sequence alignment was performed with BLAST, and the feature sequences were annotated for each representative sequence using the SILVA database (Release 132) to determine the different taxonomies.
The levels of interleukin (IL)-6, IL-1β, IL-8, IL-10, and tumour necrosis factor-α (TNF-α) in the colon mucosa supernatants were quantified using ELISA kits (Nanjing Jiancheng Bioengineering Institution, Nanjing, China) under manufacturer’s recommendations.
Western blotting assay
The expression of tight junction proteins of the colon was determined by western blotting analysis based on the previous method. The primary antibodies used include anti-zonula occludens 1 (ZO-1) (1:2,000, Abcam, ab211737), anti-occludin (1:1,000, Abcam, ab167161), anti-claudin-1 (1:1,000, CST, #5406), and anti-β-actin (1:1,000, Abcam, ab8226). Horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L) (1:5,000, Thermo Pierce, 31160) or goat anti-rabbit IgG (H+L) (1:5,000, Jackson, Thermo Pierce, 31210) were used as the secondary antibody. Image J software (NIH, Bethesda, MD, USA) was used to perform densitometry analysis.
Data pertaining to the growth performance, intestinal index, microbial metabolites, protein expression of cytokines, and tight junction proteins were presented as mean ± standard error of mean (SEM). All data were compared in SPSS 20.0 (SPSS Inc., Chicago, USA) and graphs were drawn using GraphPad Prism 8.0.2 (La Jolla, CA, USA). Furthermore, the Student’s t-test was performed to analyze the difference significance, and significant differences were deemed when P < 0.05.
The alpha diversity was examined by the Student’s t-test. Different bacteria were compared by the Mann-Whitney U test with multiple comparisons adjusted by the Benjamini-Hochberg false discovery rate (FDR). The correlation among the abundance of different genera, the concentration of microbial metabolites, and mucosal inflammatory cytokines were analyzed according to Spearman’s correlation analysis (XLStat software; Addinsoft).