International Journal of Aquaculture, 2017, Vol.7, No.23, 143-158
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For isolation and enumeration of different extracellular enzyme-producing bacteria (e.g., amylase, cellulase, lipase,
phytase, protease and xylanase), the diluted gut homogenates were spread onto starch (ST),
carboxymethylcellulose (CMC), tributyrin (TB), modified phytase screening (MPS) media, peptone-gelatin (PG)
and xylan (XY) plates, respectively. ST, PG, TB and CMC media were prepared following (Bairagi et al., 2002),
where as XY and MPS media were prepared following (Ninawe et al., 2006) and (Howson et al., 1983),
respectively. After incubation (30°C, 48 h), appearence of bacterial colonies were counted following dilution plate
count method and presented as log viable counts g
-1
GI tract (LVC) (Mandal and Ghosh, 2013a). Number of
colonies reported in the present study was an average of three replicates. The prominent colonies were collected at
random, streaked separately on respective media plates and repeatedly sub-cultured to get pure cultures. Pure
cultures were preserved on slants in a refrigerator (4°C) for subsequent study.
2.3 Determination of yeast or bacterial strains
Colonies with different morphological appearances (e.g. configuration, colour, margin, opacity and surface) were
selected on TSA plates and respective media plates for assessment of diverse extracellular enzyme-producing
capacities (e.g. amylase, cellulase, lipase, phytase, protease and xylanase) and YPD plates were stained to
determine the isolates as bacteria or yeast. Gram staining was applied to detect isolates as bacteria and yeast
strains were determined by staining the isolates by Lacto phenol cotton blue.
2.4 Composition of the media used
Amylase media (g/L): Yeast extract 5; Peptone 5; NaCl 5; soluble starch 2; Agar 20; Cellulase media (g/L):
Carboxymethylcellulose 5; Yeast extract 5; Peptone 5; NaCl 5; Agar 20; Lipase media (g/L): Tributyrin agar base
23.00 grams in 990 mL distilled water; Tributyrin 10 mL ; Modified phytase screening media (MPSM) (g/L):
Glucose 10; (NH
4
)
2
SO
4
1.0; Urea 10; Citric acid 3.0; Sodium citrate 2.0; MgSO
4
·
7H
2
O 1.0; Sodium phytate 3.0;
FeSO
4
·
7H
2
O 0.01; Agar 20; Protease media (g/L): Peptone 5; Gelatin 4; Yeast extract 3; Agar 20; Xylanase media
(g/L): Peptone 5; Yeast extract 2; MgSO
4
·
7H
2
O 0.5; NaCl 0.5; CaCl
2
0.15; Birch wood xylan 20; Agar 20.
2.5 Extracellular enzyme production: qualitative assay
Selective media plates were used to determine extracellular enzyme-producing capacities of the bacteria and yeast
isolates. For yeasts, media plates were supplemented with the antibiotics as previously mentioned. Isolates were
grown on ST and PG plates (30°C, 24 h) for detection of amylase- and protease-producing ability, respectively.
Development of clear halo zone, while flooded with 1% Lugol’s iodine (ST plates) or 15% HgCl
2
solution (PG
plates), indicated amylolytic and proteolytic activities, respectively (Jacob and Gerstein, 1960). Cellulolytic
activity was determined on CMC plates flooded with Congo red dye prepared with 0.7% agarose, whereas,
xylanolytic strains were detected on XY plates overlaid with 0.1% aqueous Congo red and repeated washing with
1M NaCl (Teather and Wood, 1982). Lipase producing strains showed halo surrounding their colony in 1%
tributyrin plates (Sangiliyandi and Gunasekaran, 1996). Colonies grown on MPSM plates with clear zone of
utilization indicated phytase activity of the isolates (Howson and Davis, 1983). There were 3 replicates for each
experimental set. A brief description on determination of qualitative extracellular enzyme activities on the basis of
measurement halo (diameter in mm) around the colony has been presented in Banerjee and Ghosh (2014). Scores
were recorded as follows; 0, nil (no halo); 1, low (6-10 mm halo); 2, moderate (11-15 mm halo); 3, good (16-25
mm halo); 4, high (26-35 mm halo); 5, very high (
≥
35 mm halo).
2.6 Extracellular enzyme production: quantitative assay
Based on the qualitative assay, 15 exo-enzyme producing isolates (5 yeast isolates and 10 bacterial isolates) were
selected and further studied through quantitative assay. Exo-enzymes were produced in respective selective broth
media following Banerjee and Ghosh (2014). Seed cultures were grown (30°C, 48 h) either in YPG broth (yeast)
or in nutrient broth (bacteria), inoculated into the liquid production medium (25 mL) and the culture flasks were
incubated in a rotary shaker at 150 rpm (30±1°C, 72 h). Subsequently, the contents were centrifuged (10,000 g, 10
min, 4°C) and the cell-free supernatant was used as the source of enzyme.
