Enzymatic activity of intestinal bacteria in roach Rutilus rutilus L.

Abstract

The digestive tract of fish is colonized by a great number of heterotrophic bacteria. Reported numbers of viable aerobic and anaerobic bacteria range between 10 and 10 and 6.6 ¥ 10-1.6 ¥ 10 CFU g-1, respectively. Consequently, it is widely accepted that fish intestines provide favorable ecologic niches for various taxa of microflora. Resident intestinal bacteria, which could be important for fish digestion, may produce extracellular enzymes supporting food decomposition. If this is the case, intestinal bacteria should exhibit enzymatic activity in relation its food composition. This study examined in vitro proteolytic and amylolytic activities of microflora from the intestinal tract of adult roach Rutilus rutilus L. (widespread in Lithuanian lakes fish, which feed mainly on mollusks and macrophytes). A total of five fish specimens of roach from Dringis Lake, located in the Aukstaitija National Park, Lithuania, were collected by fishing and transported on ice to the laboratory. Intestinal tracts were aseptically removed, divided and separated into three regions (foregut, midgut, hindgut). The roach is a stomachless fish, its intestinal tract comprised from a relatively straight tube with several loops. The foregut comprised the segment from the esophagus until the first loop; midgut was intestine and the rectum was hindgut. The gut content was squeezed from each region and homogenized in a nine-fold volume of a diluent of phosphate buffer (pH 7.0). From five fish specimens were made three integrated intestinal contents samples from foregut, midgut and hindgut. Each sample was serially diluted and 0.05 mL portions of diluents were spread on meat-peptone agar (Oxoid). A total of 60 aerobic and facultative anaerobic bacteria (20 from each part of intestine) were isolated at random for further analysis. Isolates were identified to the genus/family using methods and schemes described by Sakata and Ringo. Each isolate was incubated in a rotary shaker (100 r.p.m.) at 22°C in a mineral salts medium, which contained (l-1): NH4Cl, 9.0 g; K2HPO4, 0.5 g; MgSO4 7H2O, 0.5 g; CaCO3, 3.0 g; glucose, 20.0 g; and tap water (pH 7.0) enriched 10 g of peptone for detection of proteolytic activity (PA), and 10 g of soluble starch for detection of amylolytic activity (AA). Extracellular PA was measured every 24 h for 3 days, AA for 4 days. PA and AA were determined using methods described in ‘Enzyme preparation’. PA was determined by a modified method of Anson. This method is based on the protein (casein) hydrolysis under the action of the protease in the investigated solution, inactivation of protease and precipitation of unhydrolyzed protein with trichloroacetic acid. Neutral protease was tested because intestinal contents pH in carp fish fluctuated between 6.8 and 7.2. A PA unit, used for activity assessment, is the enzyme amount that in 1 min at 30°C converts the amount of casein corresponding to 1 mmol of tyrosine to a form non-precipitable by thrichloroacetic acid. PA was calculated in unit mL-1 (U mL-1) using formula: