Lipase

The caseinate (protein content of 984.2 g/kg on dry basis) and D-lactose were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Both pepsin and trypsin (measured respective activities of 40 and 120 U/mg) were provided by Beijing Aoboxing Biotechnologies Inc. (Beijing, China). The galactose assay kit was purchased from BioAssay Systems (Hayward, CA, USA). The alkaline phosphatase and bicinchoninic acid protein assay kits were bought from Beyotime Institute of Biotechnology (Shanghai, China). The lipase and α-amylase assay kits were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). In addition, the water used in this study was ultrapure water generated by Milli-Q Plus (Millipore Corporation, New York, NY, USA). Other chemicals used in this study were of analytical grade.

All animals were sacrificed after anesthesia with 5% chloral hydrate. Samples of the pancreatic tissue were fixed in 4% paraformaldehyde for H&E and histochemical staining. The remaining pancreatic tissues were frozen at –80°C for later use. Blood samples were centrifuged, and the supernatants were preserved for hematological detection. Serum amylase (BioTechnology & Science Inc.), lipase (Nanjing Jiancheng Corp.), and IL-1β levels were measured.

Two grams of preprocessed jejunum contents were weighed and added to 18 ml of PBS (pH 7.4) to make a 10% homogenate. Then the samples were centrifuged at 3000 g for 20 min at low temperature, and the supernatant was collected. The activities of amylase, lipase and trypsin were measured according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China).

Plasma levels of lysozyme (LZM) were measured using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. LZM were measured by the colorimetric method. The activity of three enzymes in intestinal tissue was measured according to the instructions provided in the α amylase, lipase, and trypsin kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). α amylase was measured by starch-iodine colorimetry, lipase by colorimetry, and trypsin by ULTRAVIOLET colorimetry.

Candida utilis was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (No. DSM 2361). Total superoxide dismutase (SOD), total antioxidant capacity, aminotransferase aspartate, alanine aminotransferase, glutathione peroxidase, glucose, total cholesterol, total protein, trypsin, amylase, and lipase were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit-actin, occludin, ZO-1, β-defensin-2, and goat-rabbit all purchased from Cell Signaling Technology (Danvers, America). Seal fluid and developer were purchased from Millipore. AMV First Strand cDNA Synthesis Kit was purchased from Shanghai Bioengineering (Shanghai, China). SYBR^® Green PCR Kit and PowerFecal DNA Isolation Kit was purchased from Germany Qiagen reagent company (Dusseldorf, Germany).

Amylase, lipase, D-lactate, soluble tumor necrosis factor-α receptor (sTNF-αR), and secretory immunoglobulin A (sIgA) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). The chromogenic limulus amebocyte lysate kit was purchased from Bioendo Technology Co., Ltd (Xiamen, China). Rat interleukin-1beta (IL-1β) and transforming growth factor-β (TGF-β) ELISA kits were purchased from ExCell Biotech Co., Ltd (Shanghai, China). Rat TNF-α and IL-10 ELISA kits were purchased from Cusabio Biotech Co., Ltd (Wuhan, China). Rat IL-6 and IL-18 ELISA kits were purchased from Elabscience Biotech Co., Ltd (Wuhan, China). The rat IL-8 ELISA kit was purchased from Neobioscience Technology Co., Ltd (Shenzhen, China). The rat IL-4 ELISA kit was purchased from Invitrogen (Carlsbad, CA, USA). The specific antibodies against FITC anti-rat CD4, Alexa fluor^®647, anti-rat IFN-γ, PE anti-rat IL-4, and Alexa fluor^® 647 anti-rat Foxp3 were purchased from BioLegend (San Diego, CA, USA). Rabbit anti-rat CD68 and rabbit anti-rat CD103 were purchased from Abcam (Cambridge, MA, UK). PE anti-rat CD25 was purchased from BD Biosciences (San Jose, CA, USA). The sources of other reagents were indicated in the specified methods.