Ph.D in Biology Azizpour K.
Artemia and Aquatic Animals Research Institute,
Biochemical
characterization of lactic acid bacteria
isolated from fish
Summary
Lactic acid bacteria (LAB)
were isolated from various samples of fresh and frozen fish and prawn. Thirteen
species of Lactobacillus were identified among the 64% isolates. Among them, L.
plantarum was the dominant species. The remaining 36% isolates of Lactobacillus
could not be assigned to any species with the available taxonomic schemes.
Introduction
Lactic acid bacteria (LAB)
are characterized as Gram - positive, usually non-motile, non - sporulating
bacteria that produce lactic acid as a major or sole product of fermentative
metabolism. Ringoe and Gatesoupe have prepared a review of the LAB present in
fish intestine [4]. Taxonomic studies on LAB from poikilothermic animals are
rare [4].
The aim of the study was
the isolation of LAB from fresh and frozen fish their classification based on
the morphological and biochemical characteristics.
Materials and Methods
Isolation
of LAB. Fresh fish (22 Spp), frozen fish (15 Spp), fresh and frozen
prawn (5 Spp) were procured from retail markets and cold storage in Kochi,
packed in iceboxes and transferred to the laboratory within 2 h. MRS agar and
broth were used for enumeration and culture of LAB [2, 5]. The samples of fish
(skin with muscle) and prawns (peeled) were homogenized in a raffinose,
rhamnose, D-ribose, salicin, sorbitol, sucrose, trehalose and D-xylose in MRS
broth devoid of glucose and beef extract with chlorophenol red as indicator;
production of acid and gas from 1 % glucose (MRS broth without beef extract);
methyl red and Voges-Proskauer test in MRVP medium; H&L test in O/F medium;
production of ammonia from arginine; nitrate reduction in nitrate broth; indole
production in tryptone broth and growth on acetate agar stomacher blender using
saline, serially diluted and pour plated on MRS plates. The MRS plates overlaid
with MRS agar and incubated in 5 % CO2 at 37 DC for 48-72 h. Well - isolated
colonies with typical characteristics namely pure white, small (2-3 mm
diameter) with entire margins were picked from each plate and transferred to
MRS broth.
Identification of the
bacterial strains
The cultures were
identified according to their morphological, cultural, physiological and
biochemical characteristics [3, 6].
The used tests were: Gram
reaction; production of catalase, cytochrome oxidase and hydrogen peroxide;
growth at 15 DC and 45 DC in 1 week; acid production from carbohydrates (1 %
w/v) - L-arabinose, cellobiose, D-fructose, D-galactose, esculin, lactose,
maltose, melezitose, melebiose, mannitol, D-mannose, raffinose, rhamnose,
D-ribose, salicin, sorbitol, sucrose, trehalose and D-xylose in MRS broth
devoid of glucose and beef extract with chlorophenol red as indicator;
production of acid and gas from 1 % glucose (MRS broth without beef extract);
methyl red and Voges-Proskauer test in MRVP medium; H&L test in O/F medium;
production of ammonia from arginine; nitrate reduction in nitrate broth; indole
production in tryptone broth and growth on acetate agar.
Results and Discussion
The LAB isolates were
classified into the genera Streptococcus, Leuconostoc, Pediococcus and Lactobacillus
based on their morphology and biochemical characters [6]. We showed the
distribution of different genera of LAB in fresh and frozen fish of the
cultures, 60 % in fresh fish, and 80 % in frozen fish belonged to the genus Lactobacillus.
The species identified showed above 80 % or more similarity to the ATCC type
cultures. Only tests that gave reproducible results were included in the
classification scheme. The species identified were L. plantarum (138
isolates), L. brevis (66), L. diver gens (28), L. gasseri (24), L.
rhamnosus (21), L. fermentum (20), L. viridescens (10), L.
farciminis (7), L. buchneri (7), L. acidophilus (5), L.
alimentarius (4), L. animalis (4) and L. reuteri (2). A significant
fact is that 217 cultures (36.2 %) were found to belong to the genus Lactobacillus
but could not be assigned to any particular species by these characters.
However, the L. divergens cultures
we have isolated grew well on acetate agar. Finding that Lactobacillus formed
the major flora in fish substantiated the observations of several other workers
[1].
References
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Clausen, A. M. Sandberg, J. Raa, 1979. In: Advances in Fish Science and
Technology, J. J. Connel (Ed.),
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Fryer, D. G. Smith, 1979. Identification of Lactic Acid
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