Measurement of gastro-intestinal pH and total colony count of LAB
Two hours postprandial, five fish/replicate were opened, and their gastrointestinal tracts were removed. The full stomach was opened, and the respective pH was determined directly using a digital pH meter (HANNA HI 2210 benchtop pH meter supplied with HI 1131B glass body pH electrode, HI7662 temperature electrode). The intestinal tract was divided into three equal parts (upper gut, middle and lower gut). A 0.5 g sample of the content of each part (fluid and solids) was mixed with 4.5 ml of distilled water for pH measurement [10].
For the total colony count of LAB, one gram of intestinal content was homogenised with 9 ml of sterile normal saline and mixed for 1 min. Subsequently, a dilution series was prepared in sterile saline from 10−1 to 10−5. One millilitre of each dilution was transferred and mixed with 20 ml of deMan-Rogosa-Sharpe (MRS) (Conda, Spain). The plates were incubated anaerobically at 37 °C for 48–72 h [26]. The averages of triplicate plates were used to express the counts as log CFU (colony forming units) per gram of sample [27]. The isolates were examined for cellular morphology and gram staining and for catalase and oxidase activity.