Studies on the quality of fresh water and marine fish sold in and around Greater Hyderabad Municipal Corporation

Introduction

Fish constitute an essential source of first class protein, essential fatty acids, vitamins, mineral elements and other micronutrients. Growing awareness of healthy diet is enabling increased consumer acceptance and demand for fish and fishery products. India is one among the largest fish-producing countries of world with contribution of fisheries to food security, employment, economic prosperity and trade. Despite its importance, fish spoil much more rapidly than many other food items derived from animal protein source, due to its high moisture content, relatively high water activity, slightly alkaline near neutral pH, presence of abundant endogenous enzymes and nutrient-rich muscle that furnish ample conditions suitable for deteriorative biochemical and microbiological processes after catching. Therefore the factor of freshness in fish quality is of paramount important. Degradation processes include the breakdown of lipids (oxidation) and proteins (autolysis, degradation) by endogenous enzymes and the spoilage by microorganisms that grow on fish meat post-mortem. These factors have the tendency of deteriorating the sensorial properties, nutritional value and hence market life and food safety. Contamination of fish occurs at various stages in the supply chain such as landing, transportation, processing, handling, icing, storage and retail display. Maintaining good sanitation practices and appropriate temperature management helps limiting the microbial spoilage and the growth of food-borne pathogens. Key food-borne microorganisms that are often implicated in fish and fishery products include Escherichia coli, Salmonella spp., Staphylococcus aureus, Vibrio spp., Aeromonas spp., Pseudomonas spp., Clostridium spp. And Shigella spp. The presence of these organisms in fish may be the indicator of unsanitary food handling practices, Public health risks. Psychochemical parameters including pH, water holding capacity, E.R.V, (extract released volume), T.B.A (thiobarbituric acid), peroxide value and FFA (free fatty acids) indicate fresh quality, protein functional properties, oxidation of lipids and thus deteriorate. Microbiological parameters which helps in determining hygienic and microbiological quality included: Total viable bacterial count, total coliform count, faecal The Greater Hyderabad Municipal Corporation has fish from various freshwater and marine environments to meet consumer demand in a densely populated area of millions of individuals. Variations in the handling and storage procedure coliform count, yeast and mould count.res practiced in retail markets may have the potential to affect its quality and safety attributes. So this study was conducted to analyze the physicochemical and microbiological quality of certain commercially available freshwater and marine fish fillets available in the fish markets of Greater Hyderabad Municipal Corporation, Telangana state and also the incidence and load of some important foodborne pathogens.

Materials and methods

Sample collection

A total of eight fish varieties were selected for the study, comprising four freshwater species catla, rohu, tilapia, and murrel and four marine species mackerel, tuna, pomfret, and hilsa. Samples were collected from retail fish markets located within the Greater Hyderabad Municipal Corporation, including Gudimalkapur, Budwel, and Ramnagar.

Determination of freshness and physicochemical quality

Freshness of the samples was studied using an EU freshness grading scale developed by the European Union, EU (1996) [1] . Samples were graded using organoleptic qualities into three grades of freshness, namely: Extra-E (good quality); A-Good; B-Satisfactory; and-Unsuitable.

The physicochemical quality of the samples

The physicochemical quality indices were determined using pH, WHC, ERV and the level of lipid oxidation of the fish. Water holding capacity (WHC), extract release volume (ERV) were determined using standards methods according to FSSAI Manual (2012) [2] , a level which has been established to indicate optimum shelf life, The TBA level was determined according to Witte et al. (1970) [3] , expressed as mg malondialdehyde (MDA)/kg of fish. The peroxide value (PV) and free fatty acids (FFA) were estimated using standard methods described in FSSAI and Koniecko.

Microbiological quality determination

Standard methods of WGU (2014) were used for the estimation of total viable count (TVC), total coliform count (TCC), faecal coliform count (FCC) and Total Yeast & Mould count (TYMC). Fish samples were screened for presence and loads of selected food borne pathogens like E. coli, Salmonella spp., Clostridium spp., S. aureus, Vibrio spp., Aeromonas spp., Pseudomonas spp and Shigella spp.

Isolation of pathogenic organisms

Selective Isolation of Staphylococcus aureus. Escherichia coli., Salmonella spp., Clostridium spp., Vibrio spp., Shigella spp., Aeromonas spp. And Pseudomonas spp was performed from the fish samples by BAM (1998) [4] . Staphylococcus aureus spp; The inoculam were plated on Voges Johnson agar and incubated at 37C overnight for the appearance of black colonies. These colonies are typical of S.aureus and result was recorded as log CFU/g. Escherichia coli The inocula (10-fold dilution) were spread on Levine's EMB agar and incubated at 37C. Bluish-purple colonies with green metallic shine were designated as E. coli; Salmonella spp. The samples were first pre-enriched in buffered peptone water, enriched in tetrationhate broth and sub-plated on XLD agar. Presumptive Salmonella colony are usually pink with or without black center; Clostridium spp. The sample were applied on SPS selective medium and incubated at 37C under anaerobic condition to obtain Black cotton-wool type colony (Clostridium spp); Vibrio spp; The samples were directly enriched in Alkaline peptone water, then plated on TCBS agar and characteristic large yellow and green colonies were selected for further biochemical characterization ;Shigella spp. The selected samples were pre-enriched in Shigella broth then directly inoculated on Salmonella-Shigella agar. Colourless colony was picked; Aeromonas spp. The enrichment of Samples was performed in Tryptic Soy Broth. The diluted inoculam were spread on A.hydrophila and P.aeruginosa Selective medium A.hydrophila and P.aeruginosa selective media. Translucent colonies of A.hydrophila and blue-green colonies of P.aeruginosa were presumptively considered positive.

Identification and characterisation of isolates

All the presumptives isolates obtained on the selective media were identified on the basis of morphology, Gram reaction and bio-chemical characteristics by referring to the schemes of Cowan and Steel (1993) [5] . The following bio-chemical tests viz. Methyl red, Voges-Proskauer (VP), indole, citrate utilization, nitrate reduction, oxidase and catalase, motility tests and 3% NaCl tolerance test were conducted to confirm the isolates and additional testes like coagulase and DNase test was perform for confirmation of S. aureus, Congo red binding and haemlysis on 5% sheep blood agar was performed to confirm E. coli.

Results

Freshness assessment

Freshness assessment showed clear differences between freshwater and marine fish. All murrel and tilapia samples were classified as Egrade (100%), indicating excellent freshness. Catla samples comprised 83.33% E-grade and 16.66% A-grade, whereas rohu showed 66.66% E-grade and 33.33% A-grade. Marine fish were predominantly classified as A-and B-grade. Mackerel, tuna, and pomfret each showed 50% A-grade and 50% Bgrade samples. Hilsa showed 66.66% A-grade and 33.33% B-grade samples. No marine fish sample was classified as E-grade. Freshness loss is primarily associated with endogenous biochemical changes in fish muscle and generally precedes microbial spoilage (Ehira et al., 1987) [6] . The comparatively lower freshness grades observed in marine fish may be associated with differences in handling, transportation, storage duration, and temperature conditions. Prolonged holding and high ambient temperatures are known to accelerate fish quality deterioration. Freshwater fish showed higher pH values than marine fish. The mean pH of freshwater fish ranged from 6.65 ± 0.04 to 6.84 ± 0.03, while marine fish ranged from 5.84 ± 0.04 to 6.37 ± 0.03. Among freshwater species, murrel recorded the highest pH (6.84 ± 0.03), followed by rohu (6.74), catla (6.72), and tilapia (6.65). Among marine fish, hilsa showed the highest pH (6.37 ± 0.03), followed by pomfret (6.27), mackerel (5.98), and tuna (5.84 ± 0.04). The observed differences may be associated with species, muscle composition, season, diet, physical activity, and stress during capture, and other physiological factors. The values obtained were generally comparable with earlier reports.

Water-holding capacity

Water-holding capacity was significantly higher in freshwater fish than marine fish. Freshwater fish showed WHC values ranging from 74.79 ± 0.01% to 75.33 ± 0.13%, while marine fish ranged from 70.74 ± 0.01% to 70.82 ± 0.02%. Murrel recorded the highest WHC (75.33 ± 0.13%) among all species, whereas tuna recorded the lowest (70.74 ± 0.01%). Among marine fish, hilsa had the highest WHC (70.82%), followed by pomfret (70.80%), mackerel (70.79%), and tuna (70.74%).

Reduced WHC may be associated with changes in myofibrillar protein functionality, which plays an important role in water retention. Loss of WHC may also contribute to changes in fish texture during deterioration.

Extract release volume

ERV, which can be used as an indicator of incipient spoilage, was higher in freshwater fish than marine fish. Freshwater fish showed values ranging from 28.90 ± 0.

Thiobarbituric acid value

TBA values were higher in marine fish than freshwater fish.

Freshwater fish recorded values ranging from 0.94 ± 0.05 to 1.01 ± 0.01 mg MDA/kg, whereas marine fish showed values from 1.05 ± 0.01 to 1.55 ± 0.15 mg MDA/kg. Among freshwater fish, catla and murrel showed the lowest values at approximately 0.94 mg MDA/kg, followed by rohu (0.98) and tilapia (1.01). Among marine fish, pomfret showed the highest TBA value (1.55 ± 0.15 mg MDA/kg), followed by hilsa (1.26 ± 0.13), tuna (1.24 ± 0.07), and mackerel (1.05 ± 0.01). The higher TBA values in marine fish indicate greater lipid oxidation compared with freshwater fish.

Peroxide value and free fatty acids

Peroxide value was not detected in any of the freshwater fish samples.

In marine fish, PV ranged from 1.37 ± 0.00 to 3.58 ± 0.06 mg%, with the highest value in tuna (3.58 ± 0.06), followed by mackerel (3.47 ± 0.06), pomfret (2.80 ± 0.00), and hilsa (1.37 ± 0.00). Similarly, FFA were not detected in freshwater fish, whereas marine fish showed measurable levels. Tuna recorded the highest FFA value (3.19%), followed by mackerel (2.51 ± 0.10%), pomfret (2.16 ± 0.01%), and hilsa (1.26 ± 0.13%). The presence of PV and FFA in marine fish indicates greater lipid degradation and oxidation compared with freshwater fish.

Table 3. Results of Chemical Parameters of fish sample

Microbiological quality

Total viable count

The TVC was higher in freshwater fish than marine fish. Freshwater fish showed counts ranging from 6.45 to 7.10 Log CFU/g, while marine fish ranged from 5.73 to 6.13 Log CFU/g. Among freshwater species, murrel recorded the highest TVC (7.10 Log CFU/g), followed by catla (6.99), rohu (6.76), and tilapia (6.45). Among marine fish, tuna showed the highest count (6.13 Log CFU/g), followed by mackerel (5.91), hilsa (5.73), and pomfret. The higher bacterial load in freshwater fish indicates greater microbial contamination and may be related to source water quality, handling, transportation, and retail conditions. Log CFU/g, while marine fish ranged from 2.36-2.51 Log CFU/g. Yeasts and moulds contribute to deterioration and decomposition of fish and may also produce metabolites associated with food spoilage and health concerns.

Table 4. results of microbial assessment of fish

Total and faecal coliform counts

Incidence of pathogenic organisms

Escherichia coli: E. Coli occurred much higher frequency in freshwater fish (83.33%) than marine fish (50% The incidence of Pseudomonas spp was higher in the case of marine fishes, where Hilsa has the highest incidence (66.66 %), it was followed by Mackerel, Tuna, and Pomfret (50 % each). In fresh water fishes, the incidence in Murrel was 33.33 %, in Catla, Rohu and Tilapia was 16.66 %. Shigella spp. Was identified in 16.66 % samples of Catla, tilapia, mackerel and pomfret, whereas it was not observed in rohu, murrel, tuna and hilsa. Presence of these bacteria suggests that contamination might occurred due to harvesting, handling, processing and storage. Pathogenic organism loads Loads of individual pathogenic bacteria showed marked difference between fresh and marine water fishes; loads for E.coli recorded was between 5.47-5.84 Log CFU/g for freshwater fishes and 3.69-4.77 Log CFU/g for marine fishes, more loads were found in the former which is in accord with the incidence. Loads for Salmonella spp recorded was between 0-2.47 Log CFU/g. No Salmonella spp detected in Murrel, Mackerel, Pomfret. Whereas in the positive samples, the loads varied between 2.00 and 2.47 Log CFU/g. The loads for Clostridium spp recorded was between 1.00-2.32 Log CFU/g and 1.00-2.30 Log CFU/g for fresh and marine water fish respectively. For Staphylococcus aureus higher loads was observed for marine fish (5.30-5.90 Log CFU/g) compared to freshwater fish (4.69-4.87 Log CFU/g). Loads of Vibrio spp recorded was 2.00-2.47 Log CFU/g and 3.00-3.47 Log CFU/g for fresh and marine water fish. Also loads of Aeromonas spp was significantly higher for marine fish (5.47-5.84 Log CFU/g) compare to fresh water fish (2.47-2.77 Log CFU/g).

Loads of Pseudomonas spp was between 2.47-3.69 Log CFU/g for fresh and 5.60-5.84 Log CFU/g for marine fish, and that of Shigella spp ranged between 2.00-2.47 Log CFU/g for positive samples; the bacteria was absent from rohu, murrel, tuna and hilsa.

Table 6. Result of microbial count of pathogenic organisms

Discussion

Freshness serves as the primary indicator of fish quality, which diminishes mainly due to biochemical changes before microbial decay sets in. Within the scope of this study, all samples of murrel and tilapia were classified as excellent (E-grade) on the freshness scale.

Freshness among rohu and catla was predominantly excellent, whereas the majority of marine species fell into the acceptable to good (A-and B-grade) range, suggesting lower freshness compared to freshwater samples. Elevated ambient temperatures and extended holding periods likely contribute to this decline in freshness. The pH, water-holding capacity (WHC) and extract release volume (ERV) of freshwater fish were significantly higher than that of marine fish. Fresh samples of freshwater fish displayed pH values ranging from 6.65-6.84 with highest pH value in murrel (6.84). Similarly, WHC and ERV values of freshwater fish (29.73 mL highest in murrel) indicate better physical quality compared to marine fish (pH 5.84-6.37) but these can vary with factors such as fish species, seasons, diet, activity level, level of stress, and body muscle characteristics. Marine fish indicated greater degree of chemical spoilage. TBA value in marine fish ranges from 1.05 to 1.55 mg MDA/ kg with highest in pomfret (1.55 mg MDA/kg), whereas in freshwater fish it was within range 0.94 to 1.01 mg MDA/ kg and highest in murrel. Fresh samples of freshwater fish recorded no values for peroxide value and FFA which were within range in marine fish from 3.58% (tuna to 0.81%) and 3.19% (tuna to 0.6%) respectively, whereas tuna registered the highest values for PV and FFA Showing higher lipid oxidation and hydrolytic he degradation in marine species. Microbiologically, marine fish had low count of TVC Coiform counts and faecal coliform with respect to that of freshwater fish. Lowest TVC was (7.1log CFU/g) observed for murrel (fresh water fish). Fresh water species of Fish species have relatively high counts on Total coliform and faecal coliformas compared to marine species. These can result from contaminated water sources, handling, processing, transportation and storage. Freshness levels of marine species were generally found to be lower than fresh water species which may be caused by local environmental, physiological and biological factors (Osborne, 1985). Fresh and chilled samples of Fish showed poor hygiene practices and possible spoilage in terms of high aerobic count and coliform presence. Total Aerobic counts (TAC) of all samples were high. Total coliform (TC) counts also were high in some fresh and chilled marine fish samples. However fresh water species were more dominated in case of presence of coliform and faecal coliform as expected because of being found in stagnant polluted water body. Total yeast and mold counts were in a higher range in marine fish compared to freshwater fish. Among the selected pathogenicity E.coli was more found in freshwater species (83.33% in case of Tilapia), Whereas in case of Marine species Vibrio were found (83.33%), Aeromonas and Pseudomonas (66.66%), Where as in case of Catla and Rohu the counts were highest for S. Aureus,(50% in each). Total count for Clostridium in Rohu and Catla (50% in each case) while Shigella sp. (0% to 16.66%).

Among pathogenicity E.coli counts were high in freshwater species (5.47-5.84log CFU/g) as compare to Marine species, while Aureus, Vibrio, Aeromonas and Pseudomonas counts were significantly high in case of Marine fish species. Based on this work, freshwater species showed best fresh but greater counts of Total coliform, faecal coliform etc. where as amrineries indicated lower freshness with greater amount of lipid deterioration. Thus, it is important to utilize ice packing, appropriate handling, transportation and rapid marketing methods for maintaining overall quality of fish and to avoid consumer's risk.

Conclusion

The present investigation recorded significant differences in physicochemical and microbiological quality of the freshwater and marine fish sold in GHMC. Freshwater fish with the exception of catla, Rohu revealed fairly good status of sensory freshness, pH, WHC, ERV than marine fishes. All of the murrel and tilapia samples are E-grade, most of the Catla and Rohu samples are also E-grade as assessed by sensory.

Marine fish had comparatively lower pH, WHC and ERV, but revealed a significantly higher TBA value, PV and FFA than freshwater. Lower values of PV and FFA in freshwater fish may suggest for less lipid breakdown. Microbiological survey of freshwater fishes reported higher value of TVC, TCC and FCC where, marine fish samples had a comparatively larger yeast and mold count. Several pertinent foodborne microorganisms have been isolated from the samples of both marine and freshwater fish. E.coli were most frequently isolated from fresh water, being maximum in tilapias; Vibrio, Aeromonas and Pseudomonas are significantly more prevalent and present in higher quantities in marine fish samples. Most fish samples possess generally good freshness and physicochemical quality for consumption but presence of several indicator microorganisms and foodborne pathogens may reflect the concern for hygiene. Good hygiene, careful handling, sufficient amount of icing, and maintained controlled temperature at storage and during transport, use of potable water and constant attention toward microbiological scrutiny are necessary to preserve quality of fish and to ensure safety. Hence it can be suggested to follow good fish handled and good retail practice throughout.

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