JOURNAL
OF CULTURE COLLECTIONS
Volume 4,=
2004-2005,
pp. 48-52
BIOCHEMIC=
AL
CHARACTERIZATION OF LACTIC ACID BACTERIA ISOLATED FROM FISH AND PRAWN
Parvathy Seema Nair* and Puthuvallil Kumaran Surend=
ran
Division of Microbiology, Fermentation =
and
Biotechnology, Central Institute of Fisheries Technology, Matsyapuri. =
P. O.
Kochi, Kerala- 682 029, India; E-mail: pads@sancharnet.in
Summary
Lactic acid bacteria (LAB) were isolated from va=
rious
samples of fresh and frozen fish and prawn. Thirteen species of Lactobacill=
us
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. Kandler and Weiss have classified Lactobacillus isolates from temperate regions according to their
morphology, physiology and molecular characters [7]. Schleifer classified L=
AB
based on the molecular characteristics [16]. LAB from food and their current
taxonomical status have been described by many [6, 13, 14, 1=
9].
Ringoe and Gatesoupe have prepared a review of the LAB present in fish inte=
stine
[13]. Taxonomic studies on LAB from poikilothermic animals are rare
[6, 13].
The aim of the study was the isolation of LAB fr=
om
fresh and frozen fish and prawn and their classification based on the
morphological and biochemical characteristics.
Materials and Meth=
ods
Isolation of LAB. Fresh fish (22 Spp), frozen fish (15 Spp), fres=
h 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 [3]. The
samples of fish (skin with muscle) and prawns (peeled) were homogenized in a
stomacher oblender using sali=
ne,
serially diluted and pour plated on MRS plates. The MRS plates overlaid with
MRS agar and incubated in 5 % CO2 at 37 °C =
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 accord=
ing to
their morphological, cultural, physiological and biochemical characteristics
[7, 18]. The used tests were: Gram reaction; production of catalase,
cytochrome oxidase and hydrogen peroxide; growth at 15 oC a=
nd
45 oC 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 <=
/b>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 with=
out
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 ag=
ar.
Results and Discus=
sion
The LAB isolates were classified in=
to the
genera Streptococcus, Leuconostoc, Pediococcus and Lactobacillus =
based
on their morphology and biochemical characters [18]. Table 1 shows the
distribution of different genera of LAB in fresh and frozen fish and prawn.=
Of
the cultures, 60 % in fresh fish, 65 % in fresh prawn and 80 =
;%
each in frozen fish and prawn belonged to the genus Lactobacillus. The predominant Lactobacillus
sp. was further classified to the species level [7]. The differentiating
characteristics of Lactobacillus species
are given in Table 2. Each strain showed variation in their sugar fermentat=
ion
pattern. The species identified showed above 80 % or more similarity to
the ATCC type cultures. Only tests that gave reproducible results were incl=
uded
in the classification scheme. The species identified were L. plantarum (138 isolates), L. brevis (66), L. dive=
rgens
(28), L. gasseri (24), L. rhamnosus (21), L. fermentum (20), L.
viridescens (10), L. farci=
minis (7), L. buchneri (7), L. acidophilus (5), L.
alimentarius (4), L. animalis <=
/i>(4) and
L. reuteri (3). A significant fact is that 217 cultures (36.2 =
;%)
were found to belong to the genus L=
actobacillus
but could not be assigned to any particular species by these characters=
.
Table 1. The percentage distributio=
n of
different genus of LAB in fresh and frozen fish/prawn samples.
|
Sample |
Lactic
acid bacteria (%) |
|||
Streptococcus |
Leuconostoc
|
Pediococcus
|
Lactobacillus |
|
|
Fresh
fish |
20 |
10 |
10 |
60 |
|
Fresh
prawn |
20 |
10 |
5 |
65 |
|
Frozen
fish |
5 |
5 |
10 |
80 |
|
Frozen
prawn |
10 |
5 |
5 |
80 |
Table 2. Differentiating characteri=
stics of
Lactobacillus species.
|
No |
Lactobacillus spp |
Morphology |
Growth at |
Acid and gas from glucos=
e |
NH3 from arginine |
Sugar fermentation
|
|||||||||||||||
|
15 ºC only |
45 ºC only |
15 and 45 ºC |
Arabinose |
Cellobiose |
Mannitol |
Mannose |
Melebiose |
Raffinose |
Ribose |
Salicin |
Lactose |
Melezitose |
Rhamnose |
Sorbitol |
Xylose |
Trehalose |
|||||
|
1. |
L. plantarum
|
SR |
+ |
- |
+ |
- |
- |
|
+ |
+ |
+ |
+ |
+ |
+ |
+ |
|
|
|
|
|
|
|
2. |
L. brevis |
SR |
+ |
- |
+ |
+ |
+ |
+ |
|
|
|
|
|
+ |
|
+ |
- |
- |
- |
+ |
|
|
|
L. divergens |
SR |
+ |
- |
|
+ |
+ |
|
+ |
|
+ |
- |
- |
|
+ |
- |
|
|
|
+ |
+ |
|
4. |
L. gasseri |
SR |
- |
+ |
|
- |
- |
- |
+ |
|
+ |
|
|
- |
+ |
|
|
- |
+ |
|
+ |
|
5. |
L. rhamnosus |
SR |
- |
- |
+ |
- |
- |
|
+ |
+ |
+ |
- |
|
|
+ |
+ |
+ |
|
+ |
+ |
+ |
|
6. |
L. fermentum |
SR |
|
|
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
+ |
- |
- |
+ |
+ |
+ |
|
7. |
L. viridescens |
SR |
|
|
+ |
|
|
- |
+ |
+ |
+ |
- |
- |
- |
- |
+ |
+ |
- |
- |
- |
+ |
|
8. |
L. farciminis |
SR |
|
|
+ |
|
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
+ |
- |
+ |
- |
+ |
+ |
+ |
|
9. |
L. buchneri |
SR |
+ |
|
|
+ |
+ |
- |
+ |
- |
- |
+ |
- |
+ |
- |
+ |
- |
- |
- |
+ |
- |
|
10. |
L. acidophilus |
SR |
- |
- |
|
|
|
+ |
+ |
+ |
+ |
- |
- |
- |
+ |
+ |
+ |
- |
- |
+ |
+ |
|
11. |
L. alimentarius |
C |
+ |
|
|
|
|
+ |
+ |
+ |
+ |
+ |
- |
+ |
+ |
- |
+ |
- |
+ |
+ |
+ |
|
12. |
L. animalis |
C |
- |
- |
|
|
|
+ |
+ |
+ |
+ |
+ |
+ |
- |
+ |
+ |
- |
+ |
+ |
- |
+ |
|
13. |
L. reuteri |
R |
|
|
+ |
+ |
+ |
+ |
- |
- |
- |
+ |
- |
+ |
- |
+ |
- |
- |
- |
+ |
- |
![3D"Текс&=](3D"JCC0546NB_files/image002.gif")
Legend: 80 % or more of strains are
positive (+); 80 % or more of strains are negative (-).
It is int=
eresting
to note that majority of the Lactob=
acillus
sp. that have been isolated from fresh and frozen fish/prawns were those
species which were commonly found on meat, animals and human [7]. There wer=
e a
few reports of isolation of LAB from fresh and seawater fish [2, 12]. L.
plantarum have been isolated from herring, Arctic krill and chilled cha=
nnel
catfish fillets [4, 17]. The L. divergens is now classified as =
Carnobacterium divergens [1].
According to them, the Carnobacteri=
um
cultures would not grow on acetate agar. However, the L. divergens cultures we have isolated grew well on acetate agar
and so did not conform to the classification adopted by Collins et al for C. divergens [1].
Finding t=
hat Lactobacillus formed the major flo=
ra in
fish substantiated the observations of several other workers
[1, 4, 5, 8-12, 15, 20, 21]. However, Maugin =
and
Novel found that Lactococcus wa=
s the
major flora isolated from fish [9].
The occur=
rence of
“typical” lactobacilli as described by Kandler and Weiss [7] we=
re
rare in fish and prawn. This signified the need for a proper classification
scheme for LAB from tropical fish and prawn. In our studies we attempted to
classify LAB on the basis of the available classification schemes. However
further studies are needed in order to include other atypical Lactobacillus cultures in the
classification scheme.
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