Bacteria isolated from raw (untreated and unprocessed) prawn (Penaeus indicus) stored at 28±2°C, 4°C and-18°C were tested for spoilage potential, namely,  production of protease, lipase, amylase, reduction of trimethylamineoxide (TMAO) to trimethylamine (TMA), production of off odours from flesh broth and halo zone around the colony grown on flesh agar. About 63 % of the total isolates tested were potential spoilers. Members of Vibrio, Pseudomonas and Acinetobacter were found to be dominant potential spoilers at all temperatures

Chandramohan, D.

Chandrasekaran, M.

Lakshmanaperumalsamy, P.

Aquatic Commons

S oilage Bacteria Penaeus indicus 
CHANDRASEKARAN, M*., P. LAKSHMANAPERUMALSAMY and 
D. CHANDRAMOHAN** 
School of Marine Sciences, University of Cochin, Cochin - 682 016 
Bacteria isolated from raw (untreated and unprocessed) prawn (Penaeus indicus) 
stored at 28±2°C, 4°C and-18°C were tested for spoilage potential, namely, production 
of protease, lipase, amylase, reduction of trimethylamineoxide (TMAO) to trimethyla-
mine (TMA), production of off odours from flesh broth and halo zone around the colony 
grown on flesh agar. About 63 % of the total isolates tested were potential spoilers. 
Members of Vibrio, Pseudomonas and Acinetobacter were found to be dominant poten-
tial spoilers at all temperatures. 
Fish spoilage is attributed to bacterial 
action on flesh substrates and several authors 
have reported various means to assess spoi-
lage potential of bacteria isolated from fish 
or prawn. Bacteria isolated from spoiled fish 
that could hydrolyse fat, reduce trimethyla-
mine oxide (TMAO) to trimethylamine 
(TMA), decompose proteins like gelatin and 
casein (Casten & Mapplebeck, 1952), grow 
at low temperatures and exhibit proteolytic 
property (Shewan et al., 1960), produce off 
odours, volatile reducing substances (VRS), 
TMA, total volatile vases (TVB) (Lerke et 
al., 1965; Shaw & Shewan, 1968), volatile 
sulphide compounds from flesh (Herbert 
et al.,1971) and produce clearing area around 
the colony grown on flesh agar (Chandra-
sekaran et al., 1985) are considered to be 
potential spoilage bacteria. We report here 
the existence of potential :flesh spoiling 
bacteria as a commensal :flora in Penaeus 
indicus stored as raw, untreated and unpro-
cessed samples at three different temperatures. 
Materials and Methods 
Penaeus indicus collected live from Cochin 
backwaters were killed by shock treatment, 
thoroughly washed with sterile saline and 
stored in raw unprocessed conditions as 
whole, headless, peeled and undeveined 
*Department of Applied Chemistry, University of 
Cochin, Cochin - 682 022 
**National Institute of Oceanography, Donapaula, 
Goa - 403 004 
(PUD) and peeled and deveined (PD), 
at three different temperatures (28° + 2°C, 
4°C and -l8°C). Samples were periodically 
drawn and analysed for spoilage charac-
teristics. 
Bacteriological analyses 
Total heterotrophic bacteria (THB) was 
estimated using ZoBell's 2216e agar emplo-
ying pour plate technique and incubated for 
5-7 days at room temperature l28 + 2°C). 
Bacterial cultures were isolated randomly, 
checked for their purity and maintained on 
ZoBell's agar slants. All the isolates were 
identified to various genera based on their 
morphological and biochemical characteris-
tics (Bunchanan & Gibbons, 1974; Cowan, 
1974). 
Assessment of spoilage potential of bacteria 
Bacteria selected for assessment of spoilage 
potential were checked for their proteolytic, 
lipolytic and amylolytic properties(Harrigan& 
McCance, 1972), reduction of trimethylamine 
oxide (TMAO) to trimethylamine (TMA) 
(Wood & Baird, 1943; Laycock & Reiger, 
1971) and for the production of off odours 
in flesh broth and clearing zone around the 
colony grown on flesh agar prepared accor-
ding to Chandrasekaran et al. (1985). lOOg 
of Penaeus indicv.s flesh was homogenized 
with an equal volume of 1 % NaCl solution in 
a homogenizer. The homogenate was made 
upto 1000 ml and centrifuged at 1000 g 
for 30 min at room temperature (28° + 2°C). 
FISHERY TECHNOLOGY 
< ~ 
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Cl 
- LT.I ~ Table 1. Percentage composition of potential spoilers isolated from prawns with respect to storage at three different temperatures t::d 
~ > 
Storage temperature 28 ±2° C 4°C -18°C Total q 
LT.I 
General Total No. of % of Total No. of % of Total No. of % of No. of No. of % of ~ 
isolates spoilers spoilers isolates spoilers spoilers isolates spoilers spoilers isolates spoilers spoilers > 
Vibrio 21 16 72.0 9 8 89.0 10 10 100 40 34 85.00 ~ 
Aeromonas 1 1 100.0 0 0 0 0 0 0 1 1 100.00 tg 
Pseudomonas 6 5 83.3 39 27 69.2 32 14 43.8 77 46 59.74 =<: 
Alcaligenes 0 0 0 2 2 100.0 0 0 0 2 2 100.00 ~ 
Acinetobacter 9 2 22.2 21 14 66.7 20 13 65 50 29 58.00 23; 
Enterobacteriaceae 2 1 50.0 9 3 60.0 2 1 50 9 5 55.56 ~ 
Micrococcus 0 0 0 8 5 62.5 12 4 33.3 20 9 45.00 ~ 
Bacillus 0 0 0 7 4 57.1 8 5 62.5 15 9 60.00 (::) 
Corynebacterium 0 0 0 5 3 60.0 0 0 0 5 3 60.00 ~ 
Total 39 25 64.1 96 66 68.8 84 47 56 219 138 63.00 
-N w 
124 M. CHANDRASEKARAN, P. LAKSHMANAPERUMALSAMY 
AND D. CHANDRAMOHAN 
The supernatant was boiled to remove coa-
gulable protein and used as broth after auto-
claving at 121°C for 15 min. For solidifed 
media 1.5 ~~ agar (Difeo) was added to the 
broth. When cultures were tested, a loopful 
of 18 h old broth culture (103 /ml), previously 
cultured in Zobell's broth, was inoculated 
to 10 ml of flesh broth in test tubes and spot 
inocufated in flesh agar medium. 
Results and Discussion 
Among 219 isolates tested, 138 represen-
ting species of Vibrio, Aeromonas, Pseudo-
monas, Alcaligenes, Acinetobacter, Entero-
bacteriaceae, Micrococcus, Bacillus and 
Corynebacterium were positive for produ-
ction of protease, lipase and amylase enzymes 
and reduction of TMAO to TMA. These 
219 strains were selected and tested for off 
odour production and halozone formation 
in flesh broth and flesh agar respectively. 
They were originally recorded as dominant 
groups among the bacterial flora existed on 
prawns during storage at the three different 
temperatures (Chandrasekaran et al. unpubli-
shed data). Results indicated that 63 % 
of the 219 isolates examined were capable 
of producing off odours and halozone for-
mation. AU the isolates of Aeromonas and 
Alcaligenes tested were found as spo1lers. 
Maximum number of Vibrio (85 %) followed 
by Bacillus ( 60% ), Corynebacterium ( 60 %), 
Pseudomonas (59.7 %), Acinetobacter (58 %), 
Fnterobacteriaceae (55.6 %) and Micrococcus 
(45 %) were spoilers. Members of Vibrio, 
Aeromonas, Pseudomonas, Acinetobacter, 
Alcalzgenes, Enterobacteriaceae and Micro-
coccus were reported earlier to produce off 
odours from flesh juice (Lerke et al. 1965; 
Cox & Lovell, 1973; van Spreekens, 1977). 
In the present study an the above said groups 
and species of Bacillus and Corynebacterium 
were recorded to produce off odours in 
flesh broth and produce clearing area around 
their colony grown on flesh agar indicating 
utilisation of flesh substrates, mainly pro-
tein and lipid (Chandrasekaran et al., 1985) 
of Penaeus indicus. Their ability to produce 
hydrolytic enzymes and halo formation on 
flesh agar confirms the existence of spoilage 
bacteria in P. indicus during storage and its 
·1 sp01.age. 
From the results summarised in Table 1 
it is dear that maximum percentage of 
spoilers were present in prawns stored at 
4°C (68.8 %) than 28±2° C (64.l %) and 
at -l8°C (56 %). At all the three storage 
temperatures, strains of Vibrio, J!seudomonas, 
Acinetobacter and Enterobactenaceae show-
ing spoilage activity were recorded. During 
storage at 28 + 2°c, Vibrio sp. formed the 
major dominant group in all the four types 
of samples. At 4°C Pseudomonas formed 
the major dominant group. However at 
-18°C both Acinetobacter and Pseudomonas 
formed the major group (Chandrasekaran, 
1985). At 4°C and -l8°C, when 
one of these groups was dominant, the 
rest of the groups formed second dominant 
groups. However, when they were assessed 
for their spoilage potential Vibri~ sp. ~nd 
Acinetobacter sp. represented mcreas1~g 
number of spoilers with decrease m 
storage temperature. However, Pseudo-
mona'S recorded a decrease in the percentage 
of spoilers along with decrease in storage 
temperature which was rather unus~al. 
According to Adams et al. (1964), durmg 
storage, a wide variety of organisms may be 
present, of which only a small percentage 
cause spoilage. The rest of the groups 
probably exist as free riders. or pe~haps are 
involved in some synergism with weak 
spoiler. One should not say an organism 
isolated from spoiled fish or prawn a~ ': non-
spoiler just because in pure cultur~ it is u~­
able to spoil fish. The same orgamsm may m 
mixed culture, play an important role (Adams 
et al., 1964). Hence in the present study 
although only 63 % of the isolates tested 
showed them to be active spoilers of prawn 
.flesh, the rest of the groups also might play 
an unknown role in the spoilage of prawns 
during storage. 
The authors express their gratitude to the authorities 
of the University of Cochin for pr?viding n~cessary 
facilities and to the .Indian Council of Agricultural 
Research for research grant support No. 4 (ii)/78 
ASR-I. 
References 
Adams, R., Farber, L. & Lerke, P. (1964) 
Appl. Microbial. 12, 277 
Bunchanan, R. E. & Gibbons, N. E. (1974) 
Beray's Manual of Determinative Bacte-riol~gy, 8th edn. Williams & Wilkins 
Co., Baltimore 
FISHERY TECHNOLOGY 
SPOILAGE BACTERIA OF PENAEUS INDICUS 125 
Castell, C. H. & Mapplebeck, E. G. (1952) 
J. Fish. Res. Bd Can. 9, 148 
Chandrasekaran, M. (1985) Ph. D. Thesis, 
University of Cochin 
Chandrasekaran, M., Lakshmanaperumal-
samy, P. & Chandramohan, D. (1985) 
Antonie van Leeuwenhoek, 51, 219 
Cowan, S. T. (1974) Cowan and Steel's 
Manual for the Identification of Medical 
Bacteria. 2nd edn. Cambridge Univer-
sity Press. 
Cox, N. A. & Lovell, R. T. (1973) J. Fd 
Sci. 38, 679 
Harrigan, W. F. & McCance, M. E. (1972) 
Laboratory Methods of Microbiology 
Academic Press, London 
Vol. 24, 1987 
Herbert, R. A., Hendrie, M. S., Gibson, 
D. M. & Shewan, J. M. (1971) J. 
Appl. Bacterial. 34, 41 
Laycock, R. A. & Reiger. L. W. (1971) J. 
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Lerke, P., Adams, R. & Farber. L. (1965) 
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Shaw, B. G. & Shewan, J. M. (1968) J. 
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Van Spreekens. K. J. A. (1977) Antonie 
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Spoilage bacteria of Penaeus indicus

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Spoilage bacteria of Penaeus indicus

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