142

Journal of Food Protection, Vol. 49, No. 2, Pages 142-145 (February 1986)
Copyright® International Association of Milk, Food, and Environmental Sanitarians

Effects of Radurization and Refrigerated Storage

on Quality and Shelf-Life of Freshwater
Prawns, Macrobrachium rosenbergii1
S. ANGEL2, B. J. JUVEN2, Z. G. WEINBERG2,
P. LINDNER2 and E. EISENBERG3

Department of Food Science, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel, and Soreq Nuclear
Research Center, Yavne, Israel

(Received for publication June 6, 1985)

ABSTRACT Raw fish and shellfish may also be contaminated with

The effects of radurization on bacteriological, chemical,
physical and sensory changes were studied in iced-stored fresh-
water prawns of the species Macrobrachium rosenbergii. At
both 145 and 230 krad, counts of potential spoilage bacteria
were reduced. Irradiation at 230 krad resulted in increased
TVBN values. No significant differences in texture (mushiness)
were observed between irradiated and nonirradiated samples
throughout the storage period. Atypical streptobacteria (D = 59
krad) were the predominant organisms isolated from irradiated
prawns after 4 weeks of refrigerated storage. No connection
was found between development of mushiness and total or pro-
teolytic bacterial counts.
Fresh raw prawns contain a broad spectrum of microor-
ganisms coming from the live animal and the processing
operations involving equipment and human contact. If not
inhibited or destroyed, these microorganisms may cause
rapid undesirable quality changes. Control of microor-
ganisms in prawns consists mainly of good manufacturing
practices, sanitation, packaging plus processing and mar-
keting at low temperatures.
Use of low doses of gamma radiation (i.e., raduriza-
tion or radiation pasteurization), in conjunction with re-
frigeration, is likely to extend the shelf-life of fresh
prawns through reduction of the population of spoilage
bacteria which are quite radiosensitive. In previous work
(/), Pseudomonas spp. were found to constitute the pre-
dominant spoilage organisms in the freshwater prawn
Macrobrachium rosenbergii stored at refrigerator temper-
atures. Low radiation D values in the range of less than
10 krad (17) and up to 23 krad (9) have been reported
for Pseudomonas species.
'Contribution from the Agricultural Research Organization, The Volcani
Center, Bet Dagan, Israel, No. 1432-E.1985 series.
2
The Volcani Center.
3
Soreq Nuclear Research Center.
certain nonsporeforming bacterial pathogens including
Salmonella, Shigella, enteropathogenic Escherichia coli,
Staphylococcus aureus and Vibrio parahaemolyticus (15).
This can occur when the seafood is taken from polluted
waters or as a result of postharvest contamination. De-
pending on the radiation dose and degree of contamina-
tion of the raw material, radiation pasteurization may be
also effective in eradication of those potential nonsporing
pathogenic contaminants (7). Although it is recommended
that radiopasteurized seafoods be held at temperatures
below 6°C, which are inhibitory to those pathogens, their
presence in the irradiated product should be reduced to
an insignificant level to decrease the potential hazard of
food poisoning.
Ronsivalli et al. (13) and Rhodes (11) found that ir-
radiation doses of 200 and 300 krad extended the shelf-
life of refrigerated shrimp and prawns. Those studies did
not report on mushiness or any other textural problems
in the irradiated shrimp and prawns. Nip and Moy (8),
Rowland et al. (14), and Angel et al. (7) found that
prawns stored at 0CC developed mushiness within 7 to
9d.
The aim of the present research was to increase the
shelf-life of M. rosenbergii by means of radurization plus
refrigerated storage.
MATERIALS AND METHODS
Source of material
Two batches of live prawns, raised at ca 2000 prawns per
1000 m2 (0.25 acre) in poly cultured ponds, were harvested on
two dates. After catching, the prawns were placed in an aerated
basin for ca 1/2 h, rinsed, and transported on ice to the labora-
tory. All soft unshelled prawns were discarded. Following re-
moval of the appendages, the whole unshelled (with the shells
on) prawns were packed in a single layer in sealed, double,
low-density-polyethylene bags, ca 30 prawns per bag, and the
bags were stored on ice in a cold room (1 ± 1 °C). In the second

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

 RADURIZED AND REFRIGERATED PRAWNS
batch, a number of bags were also frozen to serve as references
in taste panel sessions.
Irradiation procedure
Immediately following packaging, the bags for irradiation
were taken on ice to the Sorek Nuclear Research Center, and
placed in metal containers sandwiched in ice. The containers
were irradiated with a ^Co source at an average dose rate of
60 krad per hour as measured by ferrous-ferric sulfate
dosimetry. The radiation doses tested were 145 krad (1.45 kGy)
and 230 krad (2.3 kGy). Nonirradiated prawns in bags on ice
served as the control. The nonirradiated and irradiated sealed
bags of prawns were stored on ice in a 1 ± 1°C cold room.
The day following irradiation, the first tests for shelf-life were
performed (hence, this was designated as day one of shelf life).
Microbiological, sensory and chemical tests were carried out
on days 1, 7 or 8, 13 or 14, 19, 20 or 21 and 27-29 for each
of the two batches.
Microbiological sampling
Prawns were stored with the shells on (unshelled). After
selected periods, headless prawns were analyzed either unshel-
led or with the shells off (shelled). Homogenates were prepared
for plating by blending ca 20 g of prawns with nine times the
quantity (in ml) of 0.5% peptone solution in 0.05 M phosphate
buffer (PBD, pH 7.0) for 2 min in a blender. Three replicates
were analyzed per sample, and the averages are reported.
Bacterial counts and isolates
Decimal dilutions of homogenates were prepared in peptone
diluent and surface-plated on both plate count agar (PCA) and
milk-nutrient agar (MNA) plates, which were then incubated for
3-5 d at 20°C. Total aerobic counts on PCA were considered
as counts of potential spoilage organisms. Colonies producing
a clear zone of casein hydrolysis on MNA (confirmed by a
negative reaction with trichloroacetic acid) were recorded as
proteolytic. A full correlation was found between the ability of
the bacteria isolated from prawns to hydrolyze prawn extract
proteins and their ability to hydrolyze casein in MNA (1).
Bacterial counts are given as logarithms of colony-forming units
(CFU) per gram.
Potential spoilage bacteria were isolated from irradiated
prawns after 28 d of storage. To determine microbial types, all
the colonies were picked from PCA plates which had 20 to
100 colonies and identified to the genus. The isolates^ were
tested for gram reaction, colony and cellular morphology, spore
formation, production of catalase at 22 and 30°C, oxidase reac-
tion, nitrate reduction, and for their ability to grow in the MRS
medium (3), in the citric acid (CA) medium (J) and in the
selective acetate medium of Rogosa et al. (12). Altogether, ca
100 isolates were tested.
Radiation resistance of Lactobacillus spp.
Suspensions were prepared from 2-d-old cultures in modified
(acetate omitted) MRS broth at 20°C; the cells were harvested
by centrifuge, washed with peptone buffered diluent (PBD), the
resuspended in PBD to a final concentration of ca 108 viable
cells per ml. The bacterial suspensions were exposed to irradia-
tion doses of 0, 100, 200 and 400 krad at ca 0°C. The number
of survivors was determined by plating appropriate dilutions on
modified MRS agar and incubating the plates for up to 5 d at
20°C. The logarithms of counts were plotted against irradiation
doses and decimal reduction values calculated from the dose-
survival curves.
JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986
143
Sensory evaluation
As soon as the bags were opened and the samples for
microbiological analysis were taken, opened bags were sniffed
and the odor of the uncooked prawns was noted. Then the rest
of the prawns in the bags were cooked for 5 min in boiling
water, chilled, drained, headed, and the tails were peeled. The
peeled cooked tails were placed on trays for panel evaluation
in a random manner. Four panelists were chosen among the
students and staff of the Agricultural Research Organization.
The panelists evaluated the textural changes by placing the tails
between their fingers and feeling the degree of firmness, mushi-
ness or disintegration, segment by segment. Each panelist
examined two or three prawn tails from each treatment and
gave a verbal description of the finger feel of the segments.
The wording used was converted into a scale ranging from 1
(indicating firm segment) to 3 (very mushy). When only part
of the segment was mushy (top bottom or halfway along the
segment only), the segment was assigned a mushiness score of
2.
Total volatile basic nitrogen
The tests were carried out on four prawns per treatment on
each day of the shelf life tests, according to the official
methods (2).
Statistical analysis
The panel scores for mushiness and flavor were subjected to
an analysis of variance and Duncan's Multiple Range Test,
using a GLM procedure with a SAS program on a 4143 IBM
computer.
RESULTS
Microbiological changes
Figure 1 shows the effect of irradiation (145 and 230
krad) on total bacterial counts and proteolytic bacterial
counts in (Batch I) raw prawns stored at 1°C. In this
batch, prawns were analyzed with the shells on (unshel-
led). About 1-log reduction in total counts and 1.3-log
reduction in proteolytic counts occurred in prawns treated
with a dose of 145 krad; in samples treated with 230
krad, reductions of about 2.5- and 2.2-log were observed
in total counts and proteolytic counts, respectively.
Trends of changes in counts of proteolytic bacteria fol-
lowed those of total counts. During 28 d of storage, total
counts increased by 2.0 to 2.5 log, whereas proteolytic
counts increased by 1 to 3 log in irradiated and nonir-
radiated samples, respectively.
The changes in total and proteolytic counts were also
determined in prawns of Batch II. In this batch, prawns
were stored unshelled and after selected periods, samples
were analyzed either unshelled or without the shells (shel-
led). During 20 d of storage, only minor changes in total
counts were observed in irradiated samples. In nonir-
radiated prawns, total counts increased after 20 d of stor-
age by 0.4- to 1.4-log in shelled and unshelled samples,
respectively. In this second batch, irradiation caused a re-
duction in proteolytic counts of ca 1 to 3 log. In prawns
of Batch II, almost no changes in proteolytic counts were
found in irradiated prawns during 20 d of storage. In
nonirradiated samples, an increase of about 0.2 (unshel-
144 ANGEL ET AL.

TOTAL COUNTS
# NONIRRADIATEO
o
en O 145 KRAOS
o A 230 KRAOS
I Batch I; A and B refer to segments 1 and 2, respectively.
3 At the lower (145 krad) and higher dose (230 krad) the
O
O scores for mushiness were 1.7 and 2.0, respectively, at
m 2.3 at 29 d. None of the irradiated samples were signific-
< antly different from the controls on any storage date. The
>
STORAGE TIME (days)
Fi gure 1. Total aerobic and proteolytic bacterial counts in un-
shelled raw prawns following radiation treatment and storage
at 1°C. Batch I.
led) to 2.4 log (shelled) was observed in proteolytic
counts after an identical storage period. Total counts as
well as proteolytic counts were 10-48% (average 29%)
lower in prawns tested shelled than unshelled.
In Batch I, the reduction in bacterial counts which fol-
lowed irradiation at 230 krad was somewhat larger than
that observed with 145 krad, whereas in Batch II minor
differences in total and proteolytic counts were found be-
tween samples irradiated at 145 krad as compared with
at 230 krad.
After the 28-d storage period gram-positive, non-
motile, non sporeforming, catalase-negative, oxidase-
negative short rods, further classified as Lactobacillus
spp., constituted most of the microbial colonies isolated
from irradiated prawns on PC A plates. They did not re-
duce nitrate, grew well at 2-4°C and 25°C but failed to
grow at 45°C. They did not grow neither in the selective
Sensory changes
Sniffing of the open bags gave mixed results with re-
gard to the odor of the uncooked prawns. The panelists
noted differences in odor between the non-irradiated and
the irradiated samples. At the beginning of the experi-
ment, prawns which had received the higher irradiation
dose (230 krad) had burnt or cooked odors. From day
13 onwards such odors were not apparent, while in the
control bags fishy odors appeared. Weaker fishy odors
appeared also in the irradiated bags from day 13 onward.
Figure 2 shows the mushiness scores for prawns of
8 d; 2.4 and 2.3 at 14, 2.7 and 2.5 at 20 and 2.0 and
mushiness scores for irradiated and nonirradiated prawns
in Batch II followed a similar trend to those in Batch
1, except that all the scores were 10% lower. In segment
2, at the lower and higher doses, the scores for mushi-
ness were 1.2 and 1.0, respectively at 8 d; 1.7 for both
doses at 14; 2.0 and 1.8 at 20, and 1.4 and 2.0 at 29 d.
None of the irradiated samples were significantly differ-
ent from the controls up to 14 d. On day 20, the lower
and higher doses resulted in scores of 1.7 and 1.9,
whereas the controls received a significantly lower socre
of 1.3 for mushiness. On the 29th day of storage, scores
for the lower dose of irradiation rose to 2.0. The score
for prawns irradiated at 230 krad fell to 1.3, whereas the
controls were 1.7. The scores for irradiated samples
were, however, not significantly different from the con-
trols.
Chemical changes
In Batch I, TVBN values (Fig. 3) were ca 35-40 mg
of N/100 g of sample up to day 14 for all three treat-
ments, and then increased to ca 45-50 mg of N at day
• NONIRRADIATEO
O 145 KRAOS
A 230 KRAOS
D NONIRRADIATED, FROZEN

acetate medium of Rogosa et al. (72), in citric acid (CA)

agar (pH 5.5), or in the STAA medium recommended
by Gardner (4) for the isolation of Brochothrix thermos-
phacta. Following growth in MRS broth at 27°C, the pH
of the medium was reduced from 6.4 to 5.4. On the basis
of their homofermentative pattern and those characteris- DAYS IN ICE

tics reported above, the isolated lactobacilli were further Figure 2. Panel scores for mushiness for segment 1 (A) and
classified as atypical streptobacteria (70). They had a D segment 2 (B) of raw prawns following radiation treatment and
value of 59 krad in buffered peptone diluent. storage at I°C. Batch I.

JOURNAL OF FOOD PROTECTION, VOL. 49, FEBRUARY 1986

 RADURIZED AND REFRIGERATED PRAWNS
DAYS IN ICE
Figure 3. Total volatile base nitrogen (TVBN) values of raw
prawns following radiation treatment and storage at PC. Batch cle food are likely to be connected with changes in pro-
I.
19. At the end of the storage period (day 29), the values
reached ca 50 mg of N for the irradiated prawns, and
above 60 mg of N/100 g of sample for the control. The
values for the frozen reference prawns remained constant
at under 30 mg of N/100 g of sample throughout the ex-
periment. Up to day 14 the TVBN values for the ir-
radiated samples (especially with 230 krad) remained
slightly higher than the control, while from day 14 on-
ward the values for irradiation with 230 krad remained
lower, compared with the other treatments.
DISCUSSION
Irradiation at doses of 145 and 230 krad caused a sig-
nificant reduction in the load of potential spoilage bac-
teria and a delay in the increase of counts of those bac-
teria which survived the irradiation treatment. The prawns
used in this study had relatively high (ca 106/g) initial
microbial counts. Variations in the type of feed used, in
the feeding rate and in harvesting practices, are likely to
cause differences in the level of microbial contamination
found on pond-reared prawns. Initial bacterial counts in
the range of about 5 x 104 to 5 x 106 have been re-
ported for pond-reared shrimps (18). Following irradia-
tion and 20 d of refrigerated storage, only a small in-
crease in total counts was observed in prawns tested
either shelled or unshelled. Analysis of shelled prawns
may serve as an index for the microbiological quality of
prawns to be used in further processing. Lactobacillus sp.
classified as atypical streptobacteria, constituted the pre-
dominant microbial flora recovered from the irradiated
prawns on plate count agar. Comparison of their D value
(59 krad) with data published on the radiation resistance
of various bacteria. (6,77) indicates that the strains of
Lactobacillus isolated in this study rank among moder-
ately resistant nonsporing bacterial species.
At 8 d of iced storage, a radiation dose of 230 krad
resulted in increased TVBN values as compared with
either 145 krad or control samples. Taste panel evalua-
tions indicated an increase in mushiness in irradiated and
nonirradiated prawn tails during storage. The mushiness
scores may be explained by hepatopancreatic activity,
while the increased TVBN values and increased mushi-
ness observed in prawns that received 230 krad could
JOURNAL OF FOOD PROTE(
145
have been due to enhanced radiolytic activity and con-
sequent proteolysis (7,76). During the first 14 d of refrig-
erated storage, highest TVBN values were found in sam-
ples treated with 230 krad. Between 14 and 29 d of stor-
age the higher irradiation dose (230 krad) resulted in the
lowest TVBN values. This may be related to lower
counts of proteolytic bacteria than in controls. From day
14 onward, changes in TVBN and counts of proteolytic
bacteria followed a similar pattern. No differences in
mushiness were found between treatments from day 14
onward. Counts of proteolytic bacteria were performed on
the basis of the assumption that textural changes in mus-
teins. A delay in the increase of bacterial counts was ob-
served in irradiated samples as compared with control
samples. No differences in mushiness were found be-
tween irradiated and nonirradiated samples in the second
half of the storage period. Therefore, apparently there is
no connection between the mushiness phenomenon and
the increase in bacterial counts. Preceding work had also
indicated that no correlation existed between development
of mushiness and growth of proteolytic bacteria (7).
Biochemical and histological studies are in progress to
elucidate whether there is a linkage between the proteoly-
tic activity from the hepatopancreas and the onset of
mushiness in the prawn muscle.
ACKNOWLEDGMENT
The authors are grateful to H. Weisslowicz and C. Navrot for techni-
cal assistance. This work was supported in part by a grant from the
United States-Israel Binational Agricultural Research and Development
(BARD) Fund.
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', VOL. 49, FEBRUARY 1986
 TOXICOLOGICAL SAFETY OF BACILLUS PULLULANASE
in the dogs given 1.5 g of pullulanase/kg/d were a toxic
reaction due to the consumption of enzyme or were a
consequence of the high content of ash in the test mate-
rial and, hence, of no importance to human consumption.
Accepting as safe a dose level of 1.5 g of pullulanase/kg/
d for the dog study would increase the safety margin
from approximately 900 to 2700.
Based on these estimates on the margin of safety and
the fact that the adverse reactions seen in these studies
were mild and probably due to the high content of ash
in the test material, the use of Promozyme in the produc-
tion of syrups and beer as described should be considered
safe for the consumer. Further, the studies performed also
indicate that no occupational health precautions different
from those generally taken in the manufacturing and use
of enzyme preparations are needed. Hence, the intended
production and use of Promozyme should be regarded as
safe for both consumers and plant workers.
Angel, et al., con't.jromp. 145
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