International Journal of Aquaculture, 2017, Vol.7, No.23, 143-158
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The specific micro-ecological system in the digestive tract of every species consists of diverse species of both,
bacteria and yeasts. Amongst them, bacteria have been reported as the principal microbial colonizers within the GI
tract of fish (MacDonald et al., 1986; Pond et al., 2006). Although, presence of yeasts have also been indicated to
a lesser extent (Andlid et al., 1998; Gatesoupe, 2007; Mandal and Ghosh, 2013a; Das and Ghosh, 2014; Banerjee
and Ghosh, 2014). In fact, it is a general trend in several of the earlier studies to consider fish gut microflora with
special emphasis only on bacteria, characteristically the aerobic bacterial component, leaving out the eukaryotes
(Austin, 2002). Previously, GI tract of the
Oreochromis
spp. have been evaluated in several studies in course of
enumerating extracellular enzyme-producing (Sugita et al., 1997; Bairagi et al., 2002; Saha et al., 2006; Ray et al
2007; Sarkar and Ghosh, 2014; Sasmal and Ray, 2015), or pathogenic bacteria (Plumb, 1997; Son et al., 1997;
Marathe et al., 2016). Enzymatic potential of gut bacteria isolated from various fish species have been displayed
as the likely function that probiotics might accomplish. However, enzymatic properties together with
anti-pathogenic potential in a backdrop of developing probiotics have been less studied for tilapia.
Consequently, the presently reported study intended to isolate and screen novel probiotics from Nile tilapia,
Oreochromis niloticus
with ability to produce diverse extracellular enzymes, inhibitory activity against potential
fish pathogens, and bio-safety to the target fish.
2 Materials and Methods
2.1 Collection of fish and processing of gut sample
Specimens of tilapia,
Oreochromis niloticus
(Linnaeus) were procured from 3 composite culture ponds located at
or around Burdwan (23°24’N, 87°86’E), West Bengal, India. Specimens were carried to the wet-laboratory in
oxygen-packed bags. From each of the collection ponds, 3 specimens were collected (altogether 9 fish; average
weight 100±7.34 g) and kept in glass aquaria for 7 days for acclimatization. While in the culture pond, the fish
were fed a mixture of fish meal, rice bran and mustard oil cake (crude protein: 30%) as supplementary feed along
with natural feeding. The ranges of limnological parameters during the collection period were: temperature
27.1-28.3°C, pH 7.1-7.5 and dissolved oxygen 5.8-6.9 mg L
-1
.
Prior to sacrifice, the fish specimens were starved (48 h) so as to clear their GI tracts. Tricaine methanesulfonate
(MS-222) was applied as an anaesthetizing agent (0.03%); ventral surfaces of the fish were rubbed with 70%
ethanol and dissected aseptically to take out the intestine (Ghosh et al., 2010). The GI tract was separated into
proximal (PI) and distal (DI) segments and processed as described by Mandal and Ghosh (2013a) to isolate
autochthonous gut-microorganisms. Gut segments from 3 specimens were pooled together to form one replicate
(region-wise), thereby, there were three replicates in the study. Pooled samples were used to rule out conflicting
conclusions owing to individual difference in gut microbiota (Ringo et al., 1995).
2.2 Isolation of microbial strains
For isolation of gut bacteria, homogenized samples of the gut regions were serially (1:10) diluted (Beveridge et al.,
1991) up to 10
-5
and each diluted sample (0.1 mL) was poured aseptically onto sterilized Soybean Casein Digest
Agar (Tryptone Soya Agar, TSA; HiMedia, Mumbai, India) plates to determine the aerobic heterotrophic or
facultative anaerobic culturable autochthonous bacteria population.
Isolation of yeasts was done as described by Hirimuthugoda et al. (2007) and modified after Banerjee and Ghosh
(2014). Briefly, homogenized samples of the two gut regions were serially diluted (1:10, w/v), homogenized
samples (2 mL) were placed in liquid YPD (1% yeast extract, 2% peptone, 2% dextrose) culture medium (20 mL)
supplemented with antibiotics (i.e., chloramphenicol, 150 mg L
-1
; tetracycline, 150 mg L
-1
). As chloramphenicol
and tetracycline typically inhibit the growth of gram-positive and gram-negative bacteria, respectively, isolates
grew on the YPD media were likely to be yeasts (Andlid et al., 1995). The broth culture medium was incubated
for 5 days (pH 5-7, 30°C) and cells grown in YPD broth were transferred on YPD plates containing agar. Pure
yeast colonies thus appeared were transferred to YPD slants for subsequent study.
