2.2 | Determination of digestive enzymes activities
Three fish for each species were carefully dissected and intestine and hepatopancreas were sampled and transferred into an Eppendorf tube and immediately placed into liquid nitrogen. The tubes containing the samples were stored at −80 °C until needed for analysis of enzymes activities. On analysis day, the hepatopancreas and intestine samples were weighed and mixed with 9 times phosphate buffer saline (PBS) (w:v = 1:9), then homogenized by using an electric homogenizer (Ningbo Scientz Biotechnology) in ice bath for 15 s. The resulting homogenate was carefully pipetted and centrifuged at 12,000 rpm at 4 °C for 20 min. Finally, the liquid supernatant was removed for digestive enzymes analysis. The digestive enzymes activities including pepsin, trypsin, lipase and amylase, and total protein concentration of hepatopancreas and intestine were determined by using specific commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) based on instructions from the manufacturer.