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International Journal of Aquaculture, 2014, Vol.4, No.01
http://ija.sophiapublisher.com
2
digestive tract of flathead grey mullet (
Mugil
cephalus
)
(Lin et al., 2013), and therefore the present study
addressed to evaluate the diversity of LAB in the gut
of this fish and further addressed the antimicrobial
activity of LAB against 29 fish, shrimp and human
pathogens. The flathead grey mullet is a typical
estuarine fish found in coastal waters in the tropics
and subtropics (Saleh, 2006). The adult fish normally
feed on algae and attains a size of 30 to 75 cm.
Considering the nature of flat head grey mullet, it is
presumed that its gut microbiota also can tolerate wide
fluctuations in salinity. The ability of gut microbiota
to tolerate a wide range in salinity might be a useful
feature to be included in future evaluations of LAB
strains with probiotic potential to combat important
aquaculture pathogens under varied culture conditions;
from fresh to sea water.
1 Materials and methods
1.1 Isolation and screening for LAB from fish gut
Flat head grey mullet were procured from the landing
centre at Fort Kochi, Kerala and 85 individuals were
collected. The weight of the fish ranged from 34.2 –
140.0g, with average weight of 60g. The fishes were
packed in ice boxes and transported to the laboratory
within 2 hours for isolation of gut bacteria. The fish
surfaces were washed in running tap water, weighed
and aseptically eviscerated. Gut samples were surface
washed with sterile physiological saline to remove
extraneous matter. The weight of the gut ranged from
1.34 to 7.08g. Depending on the weight, the gut
samples (with digesta) were mixed with 80-110 ml of
sterile saline solution and were homogenized in a
Masticator (IUL Instruments, Barcelona, Spain) for
about 3-5 minutes, until the gut tissues appeared
visibly macerated.
Homogenized gut tissues were transferred into 1%
peptone broth containing 0.5% NaCl and were kept
for enrichment for 24 h (Lantz et al., 1998). The
enriched broth media were serially diluted to 10
-1
and
10
-2
dilutions and were plated onto de Man Rogosa
and Sharpe (MRS) agar (MV 641, Hi Media, Mumbai)
and incubated at 31-32
℃
for 2-3 days. White colored,
well separated colonies of 2-3 mm diameter with
round margin were obtained suspended within the
agar mass. Colonies were picked and transferred into
MRS broth (MV 369, Hi Media, Mumbai) incubated
at 31-32
℃
for 2-3 days and streaked on MRS agar
slants until purity for storage at room temperature of
31-32
℃
. Further analysis was carried out from the
stored cultures.
1.2 Physical and biochemical examination of the
gut isolates
Two hundred and forty three gut isolates were isolated
and these were tested for colony and cell
morphologies, cell grouping - cluster or chain
formation, Gram-staining, spore, catalase production
and pigmentation. Gut isolates suspected of belonging
to lactic acid bacteria (LAB) were inoculated into
MRS broth and incubated at different temperature
points over varied incubation period. Growth was
enumerated by measuring optical density at 600nm
using a spectrophotometer (Systronics, India) after
incubation at different temperatures (4 and 10
℃
for
12 days and at 15, 37 and 45
℃
for 5 days). Growth at
4, 6, 8 and 10% NaCl was observed after incubation at
30
℃
for 5 days. Growth was measured at 600 nm.
Any detectable turbidity more than 0.5 by the
spectrophotometric analysis was considered as
positive growth. The O.D values ranging between 0.5
and 1.0 was categorized as “++” whereas O.D values
more than 1.0 were categorized as “+++”. All isolates
were checked for their ability to produce acid in
purple carbohydrate fermentation broth base (Peptone
- 10 gL
-1
, NaCl - 5 gL
-1
and Bromo Cresol Purple-
0.02 gL
-1
) containing the various sugars (1% w/v)
such as L-arabinose, sucrose, mannose, melibiose,
lactose, aesculin, cellobiose, dextrose, rhamnose,
maltose, galactose, xylose, sorbitol, salicin, fructose,
mannitol, raffinose, trehalose, amygdalin, inositol,
glycerol and ribose as described by Abegaz (2007).
1.3 Antibiogram of the gut isolates
Twenty nine LAB isolates were screened for the
presence of antibacterial activity by using the disk
diffusion method of Kirby-Bauer (1966) and the agar
well diffusion method described by Perez et al.
(1990). Sterile culture broth of LAB grown in MRS
broth was used to test the antagonism against the
target bacteria. The test organisms were inoculated
into MRS broth and incubated at 37
℃
for 3 days.