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.