MATERIALS AND METHODS

Collection of samples: Fresh water mola (Amblypharyngodon mola) fishes were purchased from a local market of Savar, Bangladesh in the year of 2010. All the samples were aseptically packed in pre-sterilized polythene bags. The polythene bags were sealed air tightly after collecting the samples. The samples were brought out immediately in chilled condition (in insulating foam box with ice) to laboratory for microbiological analysis.

Sample processing and microbiological analysis: Collected fishes were thawed by keeping at room temperature and distributed in separate five small sterile polythene bags. Each bag contained 10 g fish taken from different parts of the fish. These samples were then macerated by using a blender machine under aseptic conditions. To determine the single effect of storage at freezing temperature (-20°C), samples (non-irradiated) were kept at -20°C for 6 months and microbial counts were determined each month interval. Again, to investigate the single effect of gamma radiation, samples were treated with different doses of gamma radiation (2.5, 5.0, 7.5, 10.0 kGy) at a dose rate of 12.5 kGy h^-1 by Co⁶⁰ gamma radiation source (50,000 Ci) at the Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka, Bangladesh followed by microbial counts determination. Finally, to observe the effect of combination of these two treatments, gamma radiated samples were kept at -20°C for 6 months and microbial counts were determined every month.

For microbiological analysis, Total Viable Bacterial Count (TVBC) was done by the standard spread plate method according to the APHA (1984). Total Coliform Count (TCC), Total Faecal Coliform Count (TFC), Total Staphylococcal Count (TSC) and Total Aeromonas Count (TAC) were done in the same way using McConkey agar medium, mFc agar medium, Staphylococcus agar medium and Starch ampicillin agar medium (Akinjogunla et al., 2011; Mahin et al., 2011), respectively. All the viable counts were the average of at least three independent experiments. Bacterial isolates were then identified according to the Bergey’s manual of determinative bacteriology (Buchanan and Gibbons, 1974) and manual for the identification of medical bacteria (Barrow and Feltham, 1993).

Malt Yeast Glucose (MYG) chloramphenicol agar was used for fungal count and their isolation. The plates were incubated at 28°C and counts were recorded after 5 days of growth. Viable fungal counts were the average of at least three independent experiments.

Determination of moisture content: Fish sample (4 g) was taken in the crucible and dried at 105°C for 3 h. Then it was taken at desiccators until it reached at room temperature and weight of the sample was taken. The process was continued until constant weight was obtained. The loss of moisture from the sample was then determined and expressed in percentage as follows:

Determination of protein content: The total nitrogen was determined by the Micro-Kjeldahl procedure (Miller and Houghton, 1945).

RESULTS

Determination of microbial load associated with the fish sample: First the initial microbial load in non-irradiated samples was determined. The Total Viable Bacterial Counts (TVBC) were varied from 8.8x10⁷ cfu g^-1 in sample 5 to 1.27x10⁸ cfu g^-1 in sample 1, with the average of 9.7x10⁷ cfu g^-1 (Table 1). The Total Staphylococcal Counts (TSC) were varied from 2.7x10⁶ cfu g^-1 in sample 5 to 4.2x10⁶ cfu g^-1 in sample 1, with the average of 3.6x10⁶ cfu g^-1.

Table 1:	Initial microbial load of fresh water mola fish samples
TVBC: Total viable bacterial count, TSC: Total staphylococcal count, TCC: Total coliform count, TFCC: Total faecal coliform count, TAC: Total aeromonas count and TFC: Total fungi count

The Total Coliform Counts (TCC) were varied from 4.8x10⁵ cfu g^-1 in sample 4 to 7.3x10⁵ cfu g^-1 in sample 1, with the average of 6.4x10⁵ cfu g^-1. The Total Faecal Coliform Counts (TFCC) were varied from 9.0x10⁴ cfu g^-1 in sample 3 to 1.03x10⁵ cfu g^-1 in sample 2, with the average of 9.7x10⁴ cfu g^-1. The Total Aeromonas Counts (TAC) were varied from 2.7x10⁵ cfu g^-1 in sample 4 to 5.0x10⁵ cfu g^-1 in sample 1, with the average of 4.0x10⁵ cfu g^-1. The Total Fungal Counts (TFC) were varied from 1.6x10³ cfu g^-1 in sample 4 to 3.5x10³ cfu g^-1 in sample 1, with the average of 2.3x10³ cfu g^-1 (Table 1).

Effect of refrigeration, low temperature (-20°C) and their combination treatment during storage period on the associated microorganisms: The average initial Total Viable Bacterial Count (TVBC) of mola fish samples was 9.7x10⁷ cfu g^-1. Non-irradiated samples stored at -20°C showed only negligible change (9.7x10⁷ to 5.6 x10⁶) after 6 months storage (Fig. 1a). Both of the radiation dose 2.5 and 5.0 kGy reduced TVBC count by 3 logarithmic cycle. However, when these radiation treated samples were stored at low temperature, TVBC counts were further reduced by 1 (1.3x10⁴ to 1.3x10³) and 3 (1.1x10⁴ to 6.7x10¹) logarithm cycle respectively after 6 months. Radiation dose 7.5 kGy or more completely eliminated TVBC in the samples without low temperature treatment and when these samples were kept at low temperature, no bacterial growth was found until the end of the study period (Fig. 1a).

The initial average Total Staphylococcal Count (TSC) of non irradiated mola fishes (3.6x10⁶ cfu g^-1) was reduced 2 logarithmic cycle (3.6x10⁶ to 4.2x10⁴ cfu g^-1) after low temperature storage for 6 months (Fig. 1b). Radiation dose of 2.5 and 5.0 reduced the TSC count by 1 and 2 logarithm cycle, respectively without low temperature treatment. However, when both of these radiation treated samples were kept at -20°C, no TSC was found after 3 months storage. Like TVBC, single effect of 7.5 kGy or more completely eliminate TSC counts in the fish samples without any requirement of low temperature treatment (Fig. 1b).

A very negligible change (6.40x10⁵ to 5.2x10⁵ cfu g^-1) in Total Coliform Count (TCC) was found when non-irradiated samples were stored for 6 months in -20°C (Fig. 1c). No viable coliform was found when the samples were treated with irradiation dose 2.5 kGy or more. TCC was not detected even after 6 months of these irradiated samples during their storage at low temperature (Fig. 1c).

In case of Total Faecal Coliform Count (TFCC), the average initial count (9.7x10⁴ cfu g^-1) of non-irradiated fish samples was not significantly changed when the samples were kept at -20°C for 6 months (Fig. 1d). However, the faecal coliforms were completely eliminated with a very low radiation dose of 2.5 kGy. No faecal coliform was detected in any of the irradiated samples throughout the storage period at -20°C (Fig. 1d).

Non-irradiated fish samples also showed only negligible change in Total Aeromonas Count (TAC). This count was not detected when the samples were treated with radiation dose 2.5 kGy or more just after irradiation and even after 6 months of storage at low temperature (Fig. 1e).

Unlike to the other cases, no Total Fungal Count (TFC) was detected in the non-irradiated fish samples after 2 months of storage at -20°C (Fig. 1f). In case of irradiated samples, radiation does of 2.5 kGy or more was enough from total elimination of fungal count. Fungal count was also absent in the irradiated samples kept at low temperature even up to the end of the studied storage period stored (Fig. 1f).

Identification of bacterial isolates from the fresh and stored fish samples: Morphological and biochemical characteristics of the bacteria isolated from fresh mola fish samples revealed that among 76 bacterial isolates, 16 (21.06%) were Staphylococcus aureus, 11 (14.47%) were Micrococcus varians, 9 (11.82%) were Aeromonas hydrophila, 8 (10.53%) were Escherichia coli, 7 (9.21%) were Pseudomonas aeruginosa, 6 (7.89%) were Klebsiella ozaenae, 6 (7.89%) were Bacillus subtilis, 6 (7.89%) were Bacillus megaterium, 4 (5.26%) were Klebsiella edwardsii and 3 (3.95%) were Micrococcus radiodurans (Table 2).

Fig. 1(a-f):

Effect of combined treatment of low temperature and irradiation on (a) Total Viable Bacterial Count (TVBC), (b) Total Staphylococcal Count (TSC), (c) Total Coliform Count (TCC), (d) Total Faecal Coliform count (TFCC), (e) Total Aeromonas Count (TAC) and (f) Total Fungi Count (TFC) of mola fish samples. Different microbial counts after treatment with 0 kGy (closed diamonds), 2.5 kGy (closed squares), 5.0 kGy (closed triangles), 7.5 kGy (open squares) and 10 kGy (open circle) and stored at -20°C up to 6 months of storage are shown

Table 2:	Distribution of isolated bacteria (n = 76) in fresh mola fish

Fig. 2 (a-b):	Changes in moisture (a) and protein (b) contents in the radiation treated fish samples stored at -20°C

However, after 6 months of storage Klebsiella edwardsii and Micrococcus radiodurans were not found in the stored fish samples (data not shown).

Physicochemical analysis of irradiated and non-irradiated fish samples: The initial moisture content of the non-irradiated, 5.0 and 10 kGy irradiated samples were 80.75, 80.63 and 80.38%, respectively (Fig. 2a). After six months of storage this was lowered to 80.18, 80.03 and 80.01%, respectively. Alternatively, the initial protein content of the non-irradiated, 5.0 and 10 kGy irradiated samples were 21.89, 21.65 and 21.47%, respectively (Fig. 2b). After six months of storage this was lowered to 17.87, 17.53 and 17.76%, respectively.

DISCUSSION

In the present investigation, a total of five samples of fresh water mola fish were analyzed for microbial content. The total viable bacterial counts of the sample were varied from 8.8x10⁷ to 1.27x10⁸ cfu g^-1. This may be due to indigenous microbial contaminants, water source, processing or handling and selling condition of the fish sample. Similar results have been reported by Cho et al. (1992). World Health Organization (WHO) documented the primary contamination occurred up to the limit of harvesting, whereas, secondary contamination began at the start of processing as a result of contact with workers and processing equipments. Rashid et al. (1996) studied about microorganisms associated with different types of frozen fresh water fish sample and reported the variable counts in the frozen fish which is almost similar to the present findings.

The total staphylococcal counts were varied from 2.7x10⁶ to 4.2x10⁶ cfu g^-1 in the fresh water mola fish. Presence of Staphylococcus spp. suggests that there was higher level of environmental contaminants and its presence indicates the possible risks of food poisoning as found by Mhlambi et al. (2010).

Total coliform and faecal coliform counts in samples were varied from 4.8x10⁵ to 7.3x10⁵ cfu g^-1 and 9.0x10⁴ to 1.03x10⁵ cfu g^-1, respectively. Presence of coliform and faecal coliform in food is linked with the practice of inadequate hygienic measure, mishandling, improper storage and use of dirty water during marketing and all unhygienic condition of the shops. Munce (1980) and Rashid et al. (1996) also delineated the presence of coliform and faecal coliform in fish samples due to poor hygienic measure during their processing and storage.

The total Aeromonas counts were varied from 2.7x10⁵ to 5.0x10⁵ cfu g^-1. Similar result has been reported by Cho et al. (1992). Aeromonas is dangerous because it can cause food borne diarrhea (Janda and Abbott, 1999; Wadstrom and Ljungh, 1991). Total fungi count was varied from 1.6x10³ to 3.5x10³ cfu g^-1 in the fish samples. Presence of fungi in raw and fish samples has also been reported by Rashid et al. (1996). The presence of Bacillus megaterium, Bacillus subtilis, Micrococcus varians, Escherichia coli, Pseudomonas aerugenosa, Klebsiella ozaenae, Klebsiella edwardsii, Micrococcus radiodurans in fresh water mola fish was also supported by Islam et al. (2001) and Rashid et al. (1996). However, their presence is alarming since the bacterial species are the possible source of food poisoning (WHO, 1992).

Total viable bacterial counts, total coliform counts and total faecal coliform counts were reduced one log in all studied samples after six months of storage. Our previous study on mackerel fish (Scomberomorus guttatus) preservation also showed the similar result (Hossain et al., 2008). Microorganisms maintained at freezing or sub-freezing temperature may be considered dormant and they perform no detectable metabolic activity. Here the initial freezing kills a fraction of the population, where the survivors may remain viable for long period (Pelczar and Reid, 1986). Total Aeromonas were gradually decreased and fungi were totally eliminated in all samples during storage. Slow freezing is more detrimental than quick freezing because of the formation of large ice crystal which disrupts cell membranes as well as brings out solute of the cell. Thus freezing causes the death of the bacterial cell (Fung, 1987). The bacterial genus those survived after six months of storage such as Escherichia, Pseudomonas, Bacillus, Micrococcus-all are more resistant to freezing than other microorganisms (Canada et al., 1964; Trakulchang and Kraft, 1977).

In response to various radiation doses total viable bacterial count was reduced three logs at 2.5 and 5.0 kGy of irradiation doses and no viable count was found when treated with the radiation dose of 7.5 kGy. Total staphylococcal count reduced to one to two logs in all studied samples at 2.5 and 5.0 kGy of irradiation doses and no staphylococcal count was found above these radiation doses. Total coliform, total faecal coliform, total Aeromonas and total fungus were eliminated after irradiated at 2.5 kGy.

This study reveals that microorganisms resistant to freezing may remain associated with the frozen fish samples and can exist during frozen storage since only the fungi were eliminated without need of radiation treatment (Fig. 1f). So, only cold storage or low temperature preservation is not enough for reduction of total bacterial count and controlling bacterial pathogens in the fish samples. Again, only radiation treatment of 7.5 kGy or more was required for complete elimination of TVBC and TSC. But when combination of these treatments was used it was found more detrimental (Fig. 1a, b). Thus, the combination method offers a promising approach for storage life extension of fish at freezing temperature. However, microorganisms associated with raw beef and beef kebab were more sensitive to the radiation treatment as we reported in our previous study (Mahin et al., 2011). Only single effect of 2 kGy gamma radiation was enough for total elimination of TCC, TFCC and TFC in these food samples. No TSC was detected in these samples when treated with 2 kGy radiation dose and kept at -20°C for 2 months. These differences indicated that fish is more vulnerable to microbial spoilage than raw or processed beef. Gomez-Guillen and Montero (2007) also reported that fish is a highly perishable food and its freshness loses much more quickly than in the case of mammal and avian meat.

CONCLUSION

The study clearly indicated that the combination treatment of irradiation and freezing temperature is more powerful than the single treatment of either radiation or freezing for microbial decontamination and long term preservation of a local fish of Bangladesh like mola. Thus, the method can be applied for large scale preservation of mola and any other local fish of Bangladesh for long time without any significant loss of moisture and protein values.