Abstract: Effect of pre-chilling on the sensory, microbiological and biochemical quality of silver pomfret (Pampus argenteus) stored in a standardized combination of dry ice and wet ice at the ratio of 1:0.2:0.5 (wt/wt/wt) was studied. Silver pomfret were chilled in ice till their core temperature reached to 0°C before their storage in the combination of dry ice and wet ice. The pre-chilled fish stored in the combination were sensorially acceptable up to 27 h without ricing, whereas the fish was acceptable only up to 18 h when they were stored without pre-chilling. Total bacterial count, psychrophilic count, H2S producers and lactics count was 107, 106, 105 and 103 cfu g-1, respectively at the end of storage period in the pre-chilled fish. Moraxella constituted 50% of the total bacterial flora in raw pomfret and became 2%, when they were pre-chilled with ice. Moraxella was also found to be the dominant (29%) flora in pre-chilled stored pomfret, whereas Flavobacterium was dominant in control pack. TMA-N and TVB-N did not exceed the limit of acceptability. Hypoxanthine (Hx) and Free Fatty Acids (FFA) content were 7.94 mg/100 g and 19.84% as oleic acid, respectively at the end of the storage period.
INTRODUCTION
The rate of deterioration in fish is highly temperature dependent and can be inhibited by the use of low storage temperature (Sivertsvik et al., 2002). Chilling generally slows down the deterioration of seafood. The prevalent method of retarding spoilage of seafood in India, as well as in other tropical countries, is storage in ice (Surendran et al., 1989). The most common chilling medium for preserving fresh fish is ice. However, the quantity of crushed ice required for chilling fresh fish is quite substantial which is at least 1:1 ratio (wt/wt) and sometimes is even higher with tropical conditions (Lima dos Santos et al., 1981). When the atmospheric surrounding of the product is modified to reduce oxygen concentration, the shelf life is increased considerably due to the reduction in the rate of chemical oxidation by oxygen as well as in the growth of aerobic microorganisms (Stiles, 1991).
Carbon dioxide is the most important gas used in modified atmospheric packaging of fish. Because of its bacteriostatic and fungistatic effect, it inhibits the growth of many spoilage bacteria (Sivertsvik et al., 2002). Carbon dioxide enriched atmospheres have been increasingly used in the seafood trade during the last few years (De la Hoz et al., 2000). Dry ice has gained popularity in India for the rapid transportation of fresh fish by air by reducing the temperature rapidly as well as modifying the packing environment with CO2. It acts as coolant in the present trends of shipping of fresh seafood (Schoemaker, 1990). Recently, dry ice is often mixed with wet ice to save shipping weight, cost and extend the cooling energy of wet ice. Several exporters use dry ice blindly in combination with wet ice without any scientific basis for transportation of fresh fish in chilled condition. It has been reported by us that the fish, Emperor breams (Lethrinus miniatus) stored in a combination of dry ice and ice at the ratio of 1:0.2:0.5 (wt/wt/wt) were superior when compared to their storage in wet ice only (Jeyasekaran et al., 2004a). However, there is no clear report on the effect of pre-chilling on the quality of fish stored in dry ice and wet ice. Since silver pomret (Pampus argenteus) is one of the commercially important fish varieties that is chilled and exported from India and the knowledge of specific spoilage organisms of different fish from various aquatic environments of tropical region under different packaging condition is still limited (Sivertsvik et al., 2002), the present study was undertaken to investigate the effect of pre-chilling on the quality of silver pomfret (P. argenteus) stored in the standardized combination of dry ice and wet ice in relation to their shelf life.
Materials and Methods
Raw Material
Silver pomfret (Pampus argenteus) were procured from fish landing
center of Tuticorin, India, which is situated in the same campus where our laboratory
is located. Time interval between harvesting and the arrival of fish at the
landing center was 6 to 7 h and during this period they were iced. They were
immediately brought to the laboratory in insulated containers. They had an average
length of 24 cm and weight of 195 g. Whole fish were beheaded, gutted and washed
in tap water. They were divided into 2 lots of 15 kg each. First lot was chilled
with wet ice till the core temperature of fish reached to 0°C, which took
40 min. Pre-chilled fish were packed with a standardized combination of dry
ice (Thermosafe Dry ice Machine, USA) and wet ice (Ziegra Flake ice Maker, Germany)
at the ratio 1: 0.2: 0.5 (wt/wt/wt) of fish to dry ice to wet ice (Jeyasekaran
et al., 2006). Second lot was packed with the same combination of dry
ice and wet ice without pre-chilling, which served as control. Gloves were worn
during handling of ice and fish. Care was taken to avoid direct contact of fish
with wet ice and dry ice. In order to avoid direct contact between the ice and
fish, dry ice was wrapped in kraft paper pouches and wet ice in polythene bags.
Fish was not re-iced during the entire process of study. Packages were wrapped
in polythene bags (Polypropylene, 200 gauge), placed in conical shaped styrofoam
boxes and sealed airtight with cellophane tape. The boxes were stored at room
temperature (33±1°C). One pack from each lot was periodically opened
and analyzed in triplicate for sensory, bacteriological, biochemical and physical
quality until sensory rejection. This study was conducted during the months
of August - October 2005 in the Fish Quality Control Laboratory of Department
of Fish Processing Technology, Fisheries College and Research Institute, Tuticorin,
India.
Sensory Evaluation
Sensory characteristics and overall acceptability of silver pomfret (P.
argenteus) were assessed by a panel of six experienced Faculty of Fisheries
College and Research Institute on the basis of ten point scale on each sampling.
Sensory characteristics studied included general appearance (inclusive of color),
odor and texture of fish. Scale employed for evaluating sensory quality of chilled
silver pomfret was developed based on the guidelines given by Lima dos Santos
et al. (1981) and is given in Table 1. The scores were
given in the decreasing order scale with 10-9 for excellent, 8-7 for good, 6-5
for fair and acceptable, 4-3 for poor and 2-1 for very poor. The mean of the
scores given by the panel represented the overall sensory quality (Huss, 1988).
A score of 3 to 4 constituted unacceptable and shelf life failure.
Bacteriological Quality Analysis
Bacteriological analysis carried out in this study included total bacterial
count, total psychrophiles, total lactics, total H2S producers, total
coliforms and total anaerobic sulphite reducers. Fish muscle (25 g) was homogenized
using 225 mL physiological saline (0.85% NaCl) and serial decimal dilutions
of each homogenate were carried out with the same diluent for the respective
bacteriological analysis (APHA, 2001).
| Table 1: | Scoring system for evaluating sensory quality of pre-chilled
silver pomfret (Pampus argenteus) stored in a combination of dry
ice and wet ice |
Appropriate dilutions were spread plated onto Trypticase Soya Agar (TSA) for the enumeration of total bacterial count and total psychrophilic count. The plates were incubated at 37°C for 24 h for the enumeration of total bacterial count, whereas they were incubated under refrigerated condition (5°C) for 7 days for the enumeration of psychrophiles. The colonies from mesophilic plates (having 30 to 300 colonies) at each sampling were isolated and identified by various biochemical tests (Le Chevallier et al., 1980; Balows et al., 1992). Double-layer pour plate technique was followed for the enumeration of total lactics using deMan Rogosa Sharpe (MRS) lactobacillus agar (Surendran et al., 2003). Pour plate technique was followed for the enumeration of H2S producers using peptone iron agar. Inoculated plates were incubated at the room temperature for 48 h. After appropriate incubation, the number of colonies developed on the plates were counted and expressed as colony forming units per gram (cfu g-1). Most Probable Number (MPN) technique was followed for the enumeration of total coliforms and total anaerobic sulphite reducers using lauryl sulphate tryptose broth and Differential Reinforced Clostridial Medium (DRCM), respectively. The tubes were incubated at 37°C for 24 h for the enumeration of total coliforms. The tubes showing acid and gas production were counted as positive and expressed as MPN count g-1. The inoculated tubes were incubated in a water bath at 37°C for 4 days for anaerobic sulphite reducers. The tubes exhibiting black precipitate were counted as positive and expressed as MPN count g-1.
Biochemical Quality Analysis
Biochemical quality indices studied included Total Volatile Base Nitrogen
(TVB-N), trimethylamine nitrogen (TMA-N), hypoxanthine (Hx) and Free Fatty Acid
(FFA). TVB-N and TMA-N contents were determined by the Conway micro-diffusion
method (Cobb et al., 1973). Hx content was estimated using the method
of Burt (1976). Free fatty acid content was determined by the method described
by Pearson (1968).
Physical Quality Analysis
Physical parameters studied were pH, temperature and gas composition. pH
was determined by using pH meter (Digisun Electronics Digital pH Meter 707,
India) by taking 10 g of homogenized sample in 100 mL distilled water. Changes
in temperature of packages and storage room were recorded by using Ultrafreezer
temperature probe (Consort Model T 852, Belgium). Gas composition of packages
and storage environment was measured by gas analyzer (PBI Dansensor CheckMate
9900, Denmark).
Statistical Analysis
Analysis of variance (ANOVA) was performed by using statistical package
(SPSS 10.0) to examine whether any significant difference exists between different
treatments, with respect to the different fish quality characteristics at 5%
level.
Results and Discussion
Sensory Quality
Changes in the sensory scores of pre-chilled pomfret stored in a combination
of dry ice and wet ice are given in Fig. 1. Raw fish exhibited
fresh seaweedy odor, bluish-white translucent appearance and very firm texture
with an overall sensory score of 9.8. Initial characteristic features were maintained
until 12 h of storage in pre-chilled package, whereas fish in control pack retained
the initial characters only up to 1 h of storage. On 15 h, pre-chilled fish
lost its fresh seaweedy odor, but appeared as shiny white with firm texture,
whereas fish in control pack exhibited slight ammoniacal odor. Even on 21 h,
fish in pre-chilled package exhibited shiny appearance, but the skin became
very slight yellowish in color, whereas in control pack, the fish were sensorially
unacceptable with strong fecal odor and complete loss of texture with a score
of 3.9. On 27 h, fish slightly lost its texture, meat color became slight red
and exhibited ammoniacal odor. Huss et al. (1997) reported that the release
of off-odors might be due to chemical changes in fish muscle. The fish became
sensorially unacceptable on 30 h with strong ammoniacal odor and complete loss
of texture. The changes in the sensory score between the treatments were found
to be significant (p<0.05).
| Fig. 1: | Changes in the sensory score of pre-chilled silver pomfret
(Pampus argenteus) stored in a combination of dry ice and wet ice |
It has been earlier reported that combination of dry ice and wet ice at this ratio (1:0.2:0.5) extended the storage life of seerfish (Scomberomorus commersonii) by about 60% when compared to ice alone (Sasi et al., 2003).
Bacteriological Quality
Changes in total bacterial count of pre-chilled pomfret stored in a combination
of dry ice and wet ice are given in Fig. 2. The initial total
bacterial count was found to be 105 cfu g-1, which was
maintained when the fish were chilled with ice before packing. On 1 h of storage,
total bacterial count reduced by a log, which then increased only after 18 h
in pre-chilled pack. But, in control pack, the initial count reduced by a log
only on 6 h and the same was maintained up to 12 h of storage. The changes in
total bacterial counts between the two treatments were statistically significant
(p<0.05). Earlier workers also reported that the application of CO2
gaseous environment and exposure to low temperature inhibited the bacterial
growth (Clark and Lentz, 1969; Jay, 1987). On further storage, total bacterial
count increased continuously and reached a count of 107 cfu g-1
at the end of storage period. Papadopoulos et al. (2003) also observed
that a mesophilic count of 107 cfu g-1 was the maximum
level for the acceptability of marine fish.
Bacterial flora associated with raw fish, chilled fish, pre-chilled and dry ice cum wet ice stored fish and control pack fish stored in a combination of dry ice and wet ice are shown in Fig. 3. Fresh fish carried a bacterial flora comprising of Moraxella, Micrococcus, Alcaligenes, Pseudomonas and Corynebacterium. Moraxella constituted 50% of the total bacterial flora. Shift in bacterial flora was observed when the fish were chilled with ice before packing. Corynebacterium constituted about 31% of the flora and Aeromonas and Escherichia coli newly appeared in chilled fish in addition to the flora already observed in the raw fish except Pseudomonas. Moraxella (29%) again dominated when the pre-chilled fish were stored in a combination of dry ice and wet ice. It was followed by Pseudomonas, Aeromonas, Alcaligenes, Micrococcus, Escherichia coli, Flavobacterium, Corynebacterium and Plesiomonas. Pseudomonas and H2S producing bacterial population were found to be specific spoilage bacteria in fish from tropical waters (Gram and Huss, 1996). However, Lindsay et al. (1986) reported that dominant spoilage flora such as Pseudomonas and Shewanella in fish stored under aerobic refrigerated conditions were effectively inhibited by atmosphere enriched with 20% CO2 or more and this might be the reason, Pseudomonas constituted only 17% of the flora in the present study. Flavobacterium (36%) was dominated in control pack fish followed by Acinetobacter, Pseudomonas, Alcaligenes, Bacillus, Staphylococcus, Moraxella, Micrococcus and Vibrio.
Changes in total psychrophiles count of pre-chilled pomfret stored in a combination of dry ice and wet ice are given in Fig. 4. Fresh fish exhibited a psychrophilic population of 104 cfu g-1, which maintained when the fish were chilled with ice before packing and also on the 1 h in control pack.
| Fig. 2: | Changes in total bacterial count of pre-chilled silver pomfret
(Pampus argenteus) stored in a combination of dry ice and wet ice |
| Fig. 3: | Bacterial flora associated with raw, chilled, fish in pre-chilled
and control pack stored in a combination of dry ice and wet ice |
| Fig. 4: | Changes in total psychrophilic count of pre-chilled silver
pomfret (Pampus argenteus) stored in a combination of dry ice and
wet ice |
In pre-chilled pack, psychrophilic population reduced by a log on the 1 h and further reduced by another log on the 6 h and the same was maintained until the 12 h of storage. The population continuously increased from 15 h onwards and reached to 106 cfu g-1 at the end of storage period. Similar psychrophilic count of 106 cfu g-1 was also observed by Jeyasekaran et al. (2006) when tuna (Euthynnus affinis) chunks packed in a combination of dry ice and wet ice. However, in the control pack, the initial population reduced by a log on the 6 h, which maintained up to 12 h. On further storage, the population increased and reached to 105 cfu g-1. Changes in psychrophilic count showed significant difference (p<0.05) between the treatments.
Changes in total lactics count of pre-chilled pomfret stored in a combination of dry ice and wet ice are given in Fig. 5. Fresh fish had a total lactics count of 102 cfu g-1, which maintained until the 21 h of storage. Similar initial lactics count was also found in our earlier studies in tuna (Euthynnus affinis) chunks stored in the combined package of ice (Jeyasekaran et al., 2006).
| Fig. 5: | Changes in total lactics count of pre-chilled silver pomfret
(Pampus argenteus) stored in a combination of dry ice and wet ice |
| Fig. 6: | Changes in total H2S producers count of pre-chilled
silver pomfret (Pampus argenteus) stored in a combination of dry
ice and wet ice |
On the 24 h, lactics count increased by a log and the same was maintained until the end of storage period. Jay (1987) reported that most of the lactic acid bacteria could grow in the pH range of 4.0 to 4.5. Since the pH of silver pomfret varied from 6.23 to 6.95 in the present study, the growth of lactics was not high. In the case of control pack, initial count increased by a log on the 1 h and further to 105 cfu g-1 on the 12 h, which maintained till the end of storage period. The changes in total lactics count between the treatments were statistically significant (p<0.05).
Changes in total H2S producers count of pre-chilled pomfret stored in a combination of dry ice and wet ice are given in Fig. 6. Initial H2S producers count was 102 cfu g-1, which maintained till the 18 and 6 h in pre-chilled and control pack, respectively. Huss (1988) suggested that CO2 has an inhibitory effect on H2S producing bacteria and their count was comparatively lower than the total bacterial load, which supported the findings of the present investigation. On further storage, H2S producers increased by a log at each sampling period from 21 h onwards to reach 105 cfu g-1 at the end of storage period in pre-chilled fish, whereas in control pack the initial count increased to 104 cfu g-1 on the 12 h, which maintained up to 15 h and reached 105 cfu g-1 at the end of storage period. Significant difference (p<0.05) in H2S producers count was observed between the treatments.
Changes in total coliforms and anaerobic sulphite reducers count of pre-chilled pomfret stored in a combination of dry ice and wet ice are presented in Table 2. Initial coliform count was found to be 13 MPN g-1, which reduced to 8.0 MPN g-1 when the fish chilled before packing. In pre-chilled pack, the coliforms population became zero on the 1 h, which maintained up to 6 h, whereas in control pack, the count became zero only on the 6 h of storage.
| Table 2: | Changes in total coliforms and total anaerobic sulphite reducers
of pre-chilled silver pomfret (Pampus argenteus) stored in a combination
of dry ice and wet ice |
| DC-Discontinued | |
Ramachandran et al. (1990) found the presence of coliforms particularly E. coli in immediately iced Chinese herring, Hilsa toli at a level of 260 MPN g-1; however, it did not survive after 2 days of storage. Coliforms reappeared on the 12 h in both the packs and on further storage, the count increased gradually to 345 MPN g-1 in pre-chilled pack and 1600 MPN g-1 in control pack at the end of storage period. The initial total anaerobic sulphite reducers were 0.9 MPN g-1. The same level of anaerobic sulphite reducers count was also initially observed by Jeyasekaran et al. (2004b) in tuna (Euthynnus affinis). On the 1 h of storage, the count increased to 4.5 MPN g-1 and then to 9.5 MPN g-1 on the 6 h of storage in both the packages. This might be due to the displacement of oxygen by carbon dioxide inside the package. On further storage, fish in control pack exhibited higher count of 140 MPN g-1. But, count in pre-chilled pack became zero on the 15 h onwards to 24 h of storage.
Biochemical Quality
Changes in TVB-N content of pre-chilled pomfret stored in a combination
of dry ice and wet ice are given in Fig. 7. Fresh fish had
a TVB-N content of 5.19 mg%, which decreased after chilling and further to 3.79
mg% during storage on 1 h, whereas in control pack, TVB-N content increased
to 12.45 mg% on the 1 h. On further storage, TVB-N content was found to be in
an increasing trend in both the packs and exceeded the acceptable limit in control
pack at the final storage period. Significant difference (p<0.05) was observed
in TVB-N contents between the treatments. Similar increasing trend was also
reported in our earlier studies in emperor breams (Jeyasekaran et al.,
2004a). Leblanc and Leblanc (1992) also found that haddock (Melanogrammus
aeglefinus) fillets super-chilled with CO2 snow exhibited lower
TVB-N content than iced fillets.
Changes in TMA-N content of pre-chilled pomfret stored in a combination of dry ice and wet ice are given in Fig. 8. Initial TMA-N content of fresh fish was 2.72 mg%, which decreased after chilling with wet ice and further reduced to 1.32 and 2.50 mg% on the 1 h of storage in pre-chilled and control pack, respectively. On further storage, TMA-N contents were found to increase gradually, but did not exceed the acceptable limit in pre-chilled pack at the final storage period, which might be due to the effect of CO2 on TMO reducing bacteria (Sasi et al., 2003). Changes in the TMA-N contents between the treatments are statistically significant (p<0.05). It has also been reported that the rejection limit for TVB-N and TMA-N content in fish varied with species and processing condition (Huss, 1988; Dalgaard, 2000).
Changes in hypoxanthine (Hx) content of pre-chilled pomfret stored in a combination of dry ice and wet ice are given in Fig. 9. Fresh fish had an initial hypoxanthine content of 3.26 mg/100 g, which decreased to 2.90 mg/100 g after chilling and further reduced to 2.48 mg/100 g on the 1 h in pre-chilled pack, whereas the content was 3.40 mg/100 g in control pack. Hypoxanthine content was in increasing trend during further storage and reached to 7.94 and 8.88 mg/100 g in pre-chilled and control pack, respectively at the end of storage period. The changes in hypoxanthine content between the treatments were found to be significant (p<0.05).
| Fig. 7: | Changes in TVB-N content of pre-chilled silver pomfret (Pampus
argenteus) stored in a combination of dry ice and wet ice |
| Fig. 8: | Changes in TMA-N content of pre-chilled silver pomfret (Pampus
argenteus) stored in a combination of dry ice and wet ice |
| Fig. 9: | Changes in hypoxanthine content of pre-chilled silver pomfret
(Pampus argenteus) stored in a combination of dry ice and wet ice |
Our earlier study on lethrinus (Lethrinus miniatus) stored in a combination of dry ice and ice at the ratio of 1:0.2:0.5 showed that hypoxanthine level reached to 8.20 mg/100 g at the end of storage period (Jeyasekaran et al., 2004a), which was almost similar to that of the present study.
Changes in Free Fatty Acid (FFA) content of pre-chilled pomfret stored in a combination of dry ice and wet ice are given in Fig. 10. Initial FFA content of fresh fish was 23.28% as oleic acid, which was in decreasing trend till the 12 and 6 h in pre-chilled and control pack, respectively.
| Fig. 10: | Changes in free fatty acid content of pre-chilled silver pomfret
(Pampus argenteus) stored in a combination of dry ice and wet ice |
| Fig. 11: | Temperature profiles of pre-chilled silver pomfret (Pampus
argenteus) stored in a combination of dry ice and wet ice |
On the 15 and 12 h onwards, the content increased and reached to 19.84 and 21.58% as oleic acid in pre-chilled and control pack, respectively at the end of storage period and their changes were statistically significant (p<0.05). Jeyasekaran et al. (2004a) also observed a FFA level of 31.7% as oleic acid in dry ice and wet ice stored lethrinus (Lethrinus miniatus) at the end of storage period.
Temperature profiles of pre-chilled pomfret stored in a combination of dry ice and wet ice are given in Fig. 11. Temperature is one of the important factors influencing the growth of bacteria. After chilling with wet ice, immediately after packaging, the temperature recorded was 3.1°C. At that time, room temperature was 33.4°C. Low temperature of 1.3°C was observed at the 3 h of storage in pre-chilled pack and at that time control pack exhibited a temperature of 3.2°C. After that, temperature gradually increased in both the packages throughout the storage period. The results of present study confirmed the fact that the rate of deterioration spoilage is highly temperature dependent (Sivertsvik et al., 2002).
Changes in gas composition of pre-chilled pomfret stored in a combination of dry ice and wet ice are presented in Table 3. The atmospheric gas composition during this study was oxygen at 19.9%, carbon dioxide 0.5% and nitrogen 79.7%. On 1 h after packing, both the packages had a carbon dioxide level of 100%, which was due to the sublimation of dry ice inside the package. On further storage, the carbon dioxide level decreased slowly and simultaneously the oxygen and nitrogen levels increased due to the occupation of atmospheric air. Earlier studies reported that the longer shelf life obtained in dry ice packed fish might be due to high content of CO2 in such packages (Dhananjaya and Stroud, 1994; Randell et al., 1999).
| Table 3: | Changes in gas composition of pre-chilled silver pomfret (Pampus
argenteus) stored in a combination of dry ice and wet ice |
| *DC-Discontinued | |
Conclusions
Based on the sensory, bacteriological, biochemical and physical quality analyses, the present investigation concluded that chilling before packaging in dry ice and wet ice extended the shelf life of silver pomfret (Pampus argenteus) by 50% and also resulted in better quality. Hence, the chilled fish exporters may adopt pre-chilling as a prerequisite for exporting fish in dry ice and wet ice to maintain the quality as well as increase the shelf life, in addition to considerable reduction in airfreight.
Acknowledgements
Authors are grateful to the Indian Council of Agricultural Research, Government of India, New Delhi for financial assistance. We thank the Dean, Fisheries College and Research Institute, Tamilnadu Veterinary and Animal Sciences University, Tuticorin for facilities and encouragement.