LOGARITHMIC AND PERCENTAGE REDUCTIONS OF BACTERIAL ISOLATES IN GALATEA PARADOXA TREATED WITH CITRUS AURANTIFOLIA AND NACL: PUBLIC HEALTH IMPLICATION AND NUTRITIONAL ASSESSMENT

Shell fishes constitute a vital source of food for humans due to its high nutritional values. Bacteriological and nutritional assessments of Galatea paradoxa treated with Citrus aurantifolia and NaCl were determined using bacteriological and analytical protocols. The results revealed a reduction from 4.845 to 2.301 Log CFU/g in Total Heterotrophic Bacterial Counts (THBC) in G. paradoxa treated with 10% NaCl for 5 mins. The G. paradoxa treated with 7.5 % NaCl for 5 min had a reduction in Total Coliform Counts (TCC) ranging from 3.903 to 2.398 Log CFU/g, while Total Faecal Coliform Counts (TFC) in G. paradoxa treated with 5 % and 10 % for 10 min reduced by 99.99 %. There was 53.46% THBC reduction in G. paradoxa treated with 10% C. aurantifolia for 5 min; THBC in G. paradoxa treated with 10% C. aurantifolia for 10 min reduced by 79.36 %; THBC in G. paradoxa treated with 10 % equal concentrations of NaCl and C. aurantifolia decreased by 99.99 % within 10 min, while TCC in G. paradoxa treated with 7.5 % equal concentrations of NaCl and C. aurantifolia within 10 min of exposure had 99.99 % decrease. The predominant survived bacterial genera in treated samples were Bacillus, Vibrio and Micrococcus. There was insignificant difference (p ≥ 0.05) between the nutritional compositions of treated and untreated samples. This study showed that G. paradoxa could be treated with C. aurantifolia and NaCl so as to avert possible foodborne diseases associated with consumption of this aquatic food.


INTRODUCTION
An increase in population worldwide has resulted in a substantial increase in consumption of aquatic food such as Galatea paradoxa (Samya and Mohammed, 2006). Galatea paradoxa (Born 1778) are freshwater clams, bivalve and filter feeding mollusc that belong to order 'Veneroidea'; superfamily 'Tellinoidea' and family 'Donacidae' (Moses, 1990). These aquatic animals, without vertebral column, have two hinged calcareous shells that aid its protection and are endemic in West African countries such as Ghana, Nigeria and Cameroun (Etim and Brey, 1994;Villalobs and Elguezabel, 2001). The high nutritional values of shellfishes have triggered its increased consumption (Ekanem and Adegoke, 1995;Simopoulos, 2003). Galatea paradoxa constitute a vital source of food for humans due to its high protein content, low cholesterol content, significant amounts of omega-3-fatty acids (Ekpenyong et al., 2013), vitamins, iron, potassium, zinc, copper, manganese and selenium (Davies and Jamabo, 2016).
The G. paradoxa are a suitable bio-indicator of environmental pollution (Chiesa et al., 2018;Okon et al., 2020). They can accumulate human pathogenic organisms from sewage contaminated waters and also accumulate toxins in its soft tissues through feeding on toxic phytoplankton (Gram et al., 2002). The ingestion of contaminated soft tissues of G. paradoxa by humans may result in food-borne related diseases (Hathal et al., 2005). The soft tissue of G. paradoxa is consumed after frying, smoking, roasting, steaming or cooking (King, 2000;Villalobs and Elguezabel, 2001) and G. paradoxa also serves as a means of livelihood some dwellers in parts of Southern Nigeria.
Sodium chloride (NaCl) is one of the most extensively used food additives as a preservative, enhancing the flavour and enzymatic activities responsible for organoleptic parameters (Cheng et al., 2003;Silva et al., 2003), and improving water adsorption which aids in inhibiting growth of spoilage and pathogenic organisms (Lawrence et al., 2003;Man, 2007). The reduction of water activity due to addition of salt and presence of ions exerts osmotic pressure effects on the microorganisms, thus, increase the shelf life of the processed food (Anbalagan et al., 2014). The inadequacy of NaCl as a sole preservative in ready-to-eat and other food products has necessitated its combination with other preservation processes such as addition of chemicals, drying and osmotic dehydration.
The Food Safety and Inspection Service of the United States Department of Agriculture has approved the use of lime (Citrus aurantifolia) juice as a natural antimicrobial agent having been recognized as safe for its application in food (Skrivanova et al., 2011). The juice of C. aurantifolia has been reported to reduce the growth of some bacteria in family Enterobacteriaceae and its antibacterial activities has attributed to its low pH that can penetrate the bacterial membranes (Davidson and Taylor, 2007;Bradley et al., 2011). However, there is a need to evaluate natural occurring organic compound that can be applied as post-harvest treatment to G. paradoxa. Consequently, this study determined the effect of citrus aurantifolia and NaCl singly and in combination on bacterial loads and nutritional quality of G. paradoxa.

MATERIALS AND METHODS Collection of Samples
Freshly harvested G. paradoxa were obtained from two major markets (Itam and Akpan Andem markets) in Uyo using sterile wide-mouth plastic containers and were transported to the Microbiology Laboratory, University of Uyo. The G. paradoxa was identified and confirmed by a Fish Taxonomist in the Department of Fisheries and Aquaculture, University of Uyo. Galatea paradoxa were extensively washed with sterile distilled water and rinsed with normal saline to remove all extraneous materials before shucking. The edible part was aseptically removed as described by APHA (1998) and was transferred into sterile containers for bacteriological and nutritional analyses. Bacteriological Analysis of Samples Ten (10) grams of each fleshy blended parts of G. paradoxa was aseptically suspended into 90 ml sterile distilled water, vigorously shaken to dislodge adhered bacteria and ten-fold serial dilutions were made to obtain dilutions 10 -1 to 10 -3 . One (1) mL of aliquot was pour-plated in triplicate onto each plate of Nutrient Agar (NA), MacConkey Agar (MCA), Eosine Methylene Blue Agar (EMB) and the plates were aerobically incubated at 37°C for 24 hr. After incubation, colonies on plates were counted and multiplied by the dilution to obtain the Total Heterotrophic Bacterial Counts (THBC), Total Coliform Counts (TCC) and Total Faecal Coliform Counts (TFC), respectively. The discrete colonies were sub-cultured onto freshly prepared NA plates and aerobically incubated at 37°C for 24 hr. The pure cultures of isolates were streaked onto NA slant, incubated at 37°C and stored in a refrigerator at 4 °C for characterization and identification. All isolates were Gram stained and subjected to convectional biochemical tests (Holt et al., 1994).

Effect of C. aurantifolia and NaCl on the Bacterial Loads of G. paradoxa
The C. aurantifolia juice was extracted using the method of Ndelekwute and Enyenihi (2017). Fleshy part of G. paradoxa was suspended into sterile conical flasks containing varied concentrations (2.5 %, 5.0 %, 7.5 % and 10 %) of NaCl and C. aurantifolia, respectively. The contents of sterile conical flasks were allowed to stand for 5 and 10 min, respectively. Thereafter, 10g of each fleshy part was blended, separately suspended into 90 ml sterile distilled water, vigorously shaken to dislodge adhered bacteria and dilutions were made to obtain 10 -1 and 10 -3 . One (1) ml of the aliquot was pour-plated in triplicate onto each plate of NA, MCA, EMB and incubated aerobically at 37 °C for 24 hr. The same procedure was carried out for combination of NaCl and C. aurantifolia at ratio of 1:1 (vol/vol). The same procedures were carried on control (G. paradoxa untreated with NaCl and C. aurantifolia). After incubation, the bacterial counts were recorded and mean, standard deviations were appropriately calculated.

Proximate Analysis of G. paradoxa treated with NaCl and Citrus aurantifolia
The moisture, lipid and ash contents of fleshy part of G. paradoxa samples were carried out using the methods of AOAC (2005). The fibre and protein contents were obtained by Kjeldahl's procedure and subsequently converted to crude protein by multiplying the values obtained with a protein conversion factor of 6.25. The energy content was calculated as follows: Energy Kcal 100g = (crude lipid x 8) + (crude protein x 2) + (CHO x 4), where CHO was carbohydrate contents of G. paradoxa. All determinations were done in triplicates and values obtained were expressed as mean ± standard deviation.

Statistical Analysis
The Statistical Package for Social Sciences (IBM SPSS Version 22.0) was used for data analysis. The significant difference (p ≤ 0.05) between the nutritional compositions of G. paradoxa treated with NaCl / Citrus aurantifolia and the untreated G. paradoxa were determined using one-way Analysis of Variance (ANOVA).

RESULTS
The logarithmic and percentage reductions of bacterial loads in G. paradoxa treated with NaCl are presented in Table 1. The results revealed a THBC reduction in G. paradoxa treated with 10% NaCl for 5 mins from 4.845 to 2.301 Log CFU/g, while THBC in G. paradoxa samples treated with 10% NaCl for 5 mins decreased from 4.845 to 1.146 Log CFU/g. The G. paradoxa treated with 7.5 % NaCl for 5 min had a TCC reduction ranging from 3.903 to 2.398 Log CFU/g; G. paradoxa treated with 7.5 % at 10 min had a reduction in TCC (3.903 to 1.041 Log CFU/g), while TFC in G. paradoxa treated with 5 % and 10 % for 10 min reduced by 99.99 % (Table 1) The logarithmic and percentage reductions of bacterial loads in G. paradoxa treated with C. aurantifolia are presented in Table 2. The results showed 53.46% THBC reduction in G. paradoxa treated with 10% C. aurantifolia for 5 min, while THBC in G. paradoxa treated with 10% C. aurantifolia for 10 min reduced by 79.36 %. G. paradoxa treated with 7.5 % C. aurantifolia for both 5 min and 10 min did not have TCC, respectively; while G. paradoxa treated with 7.5 % C. aurantifolia for 5 min had no TFC ( Table 2).
The logarithmic and percentage reductions of bacterial loads in G. paradoxa treated with combination of NaCl and C. aurantifolia are presented in Table 3. The THBC in G. paradoxa sample treated with equal concentrations (10 %) of NaCl and C. aurantifolia decreased by 99.99 % within 10 min of exposure, while TCC and TFC in G. paradoxa treated with equal concentrations (vol / vol) of 7.5 % (NaCl and C. aurantifolia) within 10 min of exposure had 99.99 % decrease ( Table 3).
The trends of occurrence of bacterial isolates from G. paradoxa treated with NaCl and C. aurantifolia singly and in combination are shown in Table 4. The results revealed that B. subtilis, S. aureus, M. varians, E. coli, K. pneumoniae, V. cholerae and P. aeruginosa survived in G. paradoxa treated with 5 % of NaCl or C. aurantifolia within 5 min of exposure. Although, B. subtilis were predominant in all the treated samples, but combination of 10% equal volume (vol / vol) of C. aurantifolia and NaCl killed the bacterial isolate.
The comparative analyses of proximate compositions of G. paradoxa treated with NaCl and C. aurantifolia singly and in combination are presented in Table 5. The highest moisture content of 67 ± 1 % was obtained from sample Cd (control), followed by sample Cb (G. paradoxa treated with 10 % C. aurantifolia) with 62 ± 1 %, while the lowest moisture content of 50 ± 2 % was obtained from sample Cc (G. paradoxa treated with equal volume of 10 % NaCl and 10 % C. aurantifolia). The ash content was highest in sample Cd with 3.950 ± 0.01% and lowest in sample Cc with 3.75 ± 0.03%. Samples Cc and Ca had the lowest fibre (0.023 ± 0.02 %) and protein (32.10 ± 0.25 %) content respectively, while the highest fibre (0.045 ± 0.01 %) and protein (33.229 ± 0.10 %) content was obtained in sample Cd.. The crude lipid was highest in sample Ca with 1.360 ± 0.02 % and was lowest in sample Cc (1.290 ± 0.001%). The total carbohydrate was highest in sample Ca (62.700 ± 0.11%) and was lowest in sample Cc (58.606 ± 0.20 %). There was no statistically significant difference (p > 0.05) between the nutritional compositions of G. paradoxa treated with NaCl / Citrus aurantifolia and the untreated G. paradoxa (Table 5).

DISCUSSION
Even though shellfishes, G. paradoxa, are substantially nutritious and have become an increasingly significant source of inexpensive proteins and other nutrients essential for maintenance of healthy body of a large section of world population, nevertheless, shellfishes harbour some pathogenic microorganisms attributable to poor hygienic conditions of the water bodies from where they are obtained (Adebayo-tayo et al., 2006;Oranusi et al. 2018).
In our study, a high THBC, TCC and FCC were obtained from G. paradoxa and these high bacterial loads substantiated the results of Ekanem and Adegoke (1995) and Oranusi et al. (2018) who discretely observed unacceptable bacterial loads in shell fishes. The high bacterial loads from G. paradoxa not treated with NaCl and C. aurantifolia in our study conform with the findings of Antai (1998) and Tonbarapagha et al. (2018) who obtained high microbial loads in shell fishes, but these findings contradicted the report of Udoh et al. (2017) who reported low bacterial loads from G. paradoxa in Cross River, Nigeria. Our findings showed that bacterial loads from G. paradoxa in Uyo, Akwa Ibom State, exceeded the acceptable limits for shellfishes as specified by FDA (1991) and ICMSF (2005). The high bacterial loads in the shell fishes, G. paradoxa, could be attributed to poor handling and processing in the markets (Odu et al., 2010;Akinjogunla et al., 2011).
In this study, logarithmic and percentage reductions in bacterial loads (THBC, TCC and TFC) in G. paradoxa treated with different concentrations of NaCl and C. aurantifolia singly or in combination for ≥ 10 mins were obtained. The decrease in logarithmic and percentage reductions of bacterial loads in G. paradoxa treated with different concentrations (5 to 10%) of NaCl agrees with Soyiri et al. (2008) and Anbalagan et al. (2014) who reported that NaCl concentrations between 7.5 and 10 % eliminated all pathogenic bacteria from shell fishes. A medium containing 10 % NaCl has been reported as unfavourable medium for proliferation of pathogenic microorganisms (Onyeagba and Isu, 2006;Anbalagan et al. 2014). Similarly, studies have showed that NaCl removed water from food products by osmosis and as NaCl content in food increased its water content also decreased, thus, leading to plasmolysis of cell walls of pathogenic micro-organisms (Anbalagan et al. 2014;Orjimelukwe et al. 2017). This study revealed 79.36 % THBC reduction in G. paradoxa treated with 10% C. aurantifolia for 10 min. The reduction in bacterial loads to an acceptable level for human consumption in our study corroborated the findings of Rodrigues et al. (2000) and similarly agrees with the results of Mata et al. (1994) who reported extinction of some bacterial isolates in acidic medium containing lime (C. aurantifolia) juice.
The eleven bacterial genera obtained from G paradoxa not treated with NaCl and C. aurantifolia were Staphylococcus, Micrococcus, Salmonella, Streptococcus, Escherichia, Klebsiella, Bacillus, Enterobacter, Vibrio, Enterococcus and Pseudomonas. The isolation of S. pyogenes, V. cholerae, E. coli, S. enterica and S. aureus in our study substantiated the reports of Udoh et al. (2017) who obtained these bacterial isolates from G paradoxa in Cross River, Nigeria.
Our findings revealed 33.229 % protein content in untreated G. paradoxa (control) and this value was lower than 47.0 % obtained by Ehigiator and Akise (2016) in Delta State, but our findings agrees with Ivon and Eyo (2018) who reported 32.10 % protein content in G. paradoxa from Calabar River, Nigeria. The moisture content in G. paradoxa was high in this study and this was in conformity with Zhu and Bai (2007). The high moisture content in shellfish has been attributed to the quantity of water absorbed into their cells from the external environment (Davies and Jamabo, 2016). A high ash content was discretely obtained from the untreated and G. paradoxa treated with NaCl and C. aurantifolia, and this concurs with results of Adeleke and Odedeji (2010). The ash content greater than 0.5 % has been reported as an indication of good mineral content in food (Adeleke and Odedeji, 2010).

CONCLUSION
This study showed that G. paradoxa, harboured some pathogenic bacteria of public interest and its treatment with equal concentration (vol/vol) of 10 % C. aurantifolia and NaCl singly or in combination for 10 min holding time will avert possible foodborne diseases associated with consumption of this aquatic food.