Microbial Safety and Quality of Smoked Clarias gariepinus (Burchell, 1822) on Different Concentration of Preservatives during Ambient Storage  

A. O. Isaac , S. E. Olusola , O. A. Oyelese
Department of Wildlife and Fisheries Management, University of Ibadan, Nigeria
Author    Correspondence author
International Journal of Aquaculture, 2014, Vol. 4, No. 21   doi: 10.5376/ija.2014.04.0021
Received: 28 Mar., 2014    Accepted: 21 May, 2014    Published: 24 Jun., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Isaac A.O., Olusola S.E., and Oyelese O.A., 2014, Microbial safety and quality of smoked Clarias gariepinus (Burchell, 1822) on different concentration of preservatives during ambient storage, International Journal of Aquaculture, Vol.4, No.21: 123-130 (doi: 10.5376/ija.2014.04.0021)

Abstract

This experiment was conducted to assess the microbial safety and quality of smoked Clarias gariepinus using different concentrations of pepper and salt spice mixture as preservatives. Ninety catfish, Clarias gariepinus (1,000 – 1,250g) were allocated to five treatments and replicated twice. The prepared weight to weight spice mixture of pepper 50% and salt 50% was neatly rubbed on the Clarias gariepinus using different concentrations after degutting: E1= 0%, E2= 5%, E3 =10%, E4= 15%, E5 = 20%. The Clarias gariepinus were cold smoked in an oven between 40℃ to 65℃ for 6hrs and hot smoked between 120-150℃ for 18hrs. This study were carried out for 18 weeks were proximate composition, organoleptic assessment, Total Viable Count (TVC), biochemical parameters such as Peroxide Value (PV), Thiobarbituric Acid (TBA), Free Fatty Acid (FFA) and Total Volatile Base (TVB) were investigated. Data were analyzed using descriptive statistics and ANOVA at p = 0.05. Results showed that crude protein of Clarias gariepinus were better in treated groups compared to the control and increased with increasing level of pepper and salt spices at 18weeks. Also, better organoleptic assessment was recorded in treated groups compared to the control, best assessment was recorded in 20% spice mixture (6.72,5.80,4.91,4.51) compared to the control (6.49, 4.91, 4.07, 3.03) after 24hours, 6, 12 and 18 weeks respectively and were significantly different (p <0. 05) from the control. Total viable count from the study revealed that bacterial loads in smoked Clarias gariepinus were more affected by pepper and salt spice mixture than the control after 24hours and 18 weeks and this were significantly different ( p <0. 05) than control. The PV, TBA, FFA and TVB were lower in the treated groups compared to the control after 24 hours, 6, 12 and 18 weeks. These results indicated that using pepper and salt spice mixture may be useful in improving the shelf life and consumer acceptability of smoked Clarias gariepinus and it was concluded that 20% spice mixture would positively influence shelf life, reduce and prevent infections in smoked Clarias gariepinus.

Keywords
Clarias gariepinus; Preservatives; Microbial safety; Organoleptic assessment; Proximate composition
Introduction
Catfish, (C. gariepinus) accounts for about 80% of aquaculture production in Nigeria (FDF, 2003). Catfish are a lean and highly nutritious fish that contain high amounts of vitamins, proteins, minerals, and little or no saturated fat, and is low in carbohydrates. However, catfish like any other fish species, could result in significant economic loss due to its perishable nature, if adequate preservation techniques are not adopted (Clucas and Ward, 1996). Fish and fisheries products are among the most perishable commodities worldwide mainly due to microbial spoilage. About one-third of the world’s food production is lost annually as a result of microbial spoilage (Omojowo et al., 2010).
Smoking is the process through which volatiles from thermal combustion of wood penetrate meat or fish flesh (Simko, 1991). Hard curing by salting and smoking permits lengthy preservation by removing moisture, which is essential for bacteriological and enzymatic spoilage (Antonia da Silva, 2002). Various food preservation techniques have been utilized to improve the microbial safety and extend the shelf life of fish in general including freezing, chemical preservation, salting, and smoking (Nickelson et al., 2001). The quality of smoked product is dependent on several factors including the quality of the fish at the time of smoking, and the nature of wood and type of smoking procedure employed (Antonia da Silva, 2002). Consumers are rediscovering the good taste of smoked seafood, including smoked catfish. To satisfy the consumer demand, it is necessary to produce good quality and safe smoked seafood products. The objectives of this study were to evaluate the effect of different concentrations of salt and pepper spice mixture on the microbial, physical, organoleptic and nutritional quality of smoked catfish during 18-week storage at room temperature.
1 Results
1.1 Proximate composition of smoked C. gariepinus treated with salt and pepper spice mixture
There was a decrease in the protein level in the final proximate composition (18 weeks) when compared with the initial (after 24 hours smoking). Although, the values were significantly different (p <0.05) among the treatments. The highest crude protein was recorded in treatment 5 while the least was in control. Also, high moisture content was recorded in all the treatments at 18 weeks compared to the values obtained after 24 hours smoking (Table 1).

 

 

Table 1 Proximate composition of C. gariepinus after 24 hours smoking and at the end of the experiment (18 weeks)

 

1.2 Biochemical composition of C. gariepinus
The biochemical composition: Peroxide value (PV), Thiobarbituric acid (TBA mg/kg); Free fatty acid (FFA mgKOH/g) and Total Volatile Base (TVB mg/100g) showed an increase in all the sample determined, increased in values were observed as the storage period increased; the highest values was recorded in the control sample ( 2.39, 0.39, 7.27, 6.16; 4.31, 0.62, 8.93, 12.65; 13.35, 2.25, 14.86, 21.33; 32.00 3.15 28.85, 44.14) for 24 h after smoking, 6, 12 and 18 weeks respectively while the least in 20% spiced mixture of pepper and salt (2.05, 0.2, 3.89, 4.57; 3.58, 0.31, 2.44, 5.09; 4.13, 0.44, 4.80, 6.29; 10.91, 0.95, 6.19, 12.91) respectively (Table 2)

 

 

Table 2 Biochemical composition and microbial analysis of smoked C. gariepinus with different concentration of preservatives

 

1.3 Organoleptic assessment of smoked C. gariepinus with different concentration of salt and pepper spice mixture
The organoleptic assessment of C. gariepinus 24 hours after smoking compared to the values recorded at 6, 12 and 18 weeks indicated a decrease in appearance, quality, texture, odour, flavour and taste (overall acceptability) as the storage period increased. The highest overall acceptability was obtained in 20 % spice mixture of salt and pepper (6.72, 5.80, 4.91, 4.15) at 24 h after smoking, 6, 12 and 18 weeks respectively while the least in control (6.49, 4.91,4.07, 3.03) at 24 h after smoking, 6, 12 and 18 weeks respectively (Table 3).

 

 

Table 3 Overall acceptability of smoked C. gariepinuswith different concentration of salt and pepper spice mixture

 

1.4 Microbial analysis of smoked C. gariepinus with different concentration of salt and pepper spice mixture
Increasing values were recorded in total viable count among the treatments with highest values in control 2.91 log10 cfu/g; 53.31 log10 cfu/g) at 24 h after smoking and 18 weeks respectively and the least in 20% spice mixture of salt and pepper (2.26 log10 cfu/g; 8.13 log10 cfu/g) at 24 h after smoking and 18 weeks respectively (Table 4).

 

 

Table 4 Total viable count of smoked C. gariepinus with different concentration of salt and pepper spice mixture

 

1.5 Probable bacteria in smoked C. gariepinus
The bacterial isolates obtained at 18 weeks were identified as Staphylococcus aureus, Clostridium welchii, Proteus morganii, Escherichia coli and Bacillus subtilis. The characterization of the isolates is as shown in Table 5.

 

 

Table 5 Probable identification of bacterial spices present in smoked C. gariepinus with different salt and pepper concentrations at 18 weeks

 

2 Discussion
In assessing the quality of a fish the consumer may consider its freshness, the safety of the fish in terms of the microbial load and the presence or absence of pathogenic organisms as well as the palatability of the fish. The moisture content of the fish subjected to different treatments decreased after smoking. This decrease was due to loss of water during smoking but, the values increase in all treatments after 18 weeks. This may be due to environmental factors that brought increases in moisture content of fish. This is similar to Fakunle et al. (2009). The moisture content of the dried catfish is of a great importance during storage, the result of this study showed that the moisture content was still at a safe level (9.59 -12.82%) which is within the recommended safe moisture content of dried fish (8 to 13%) as reported by World Health Organization.
There was a general increase in the crude protein content of the fish after smoking (24 hours), this might be due an increase in the dry matter content per unit of weight following sample dehydration during smoking and reduction in the moisture contents after the smoking before autolysis becomes pronounced. Decreases in values of crude protein were observed in all the treatment at 18 weeks. This might be as a result of long period of storage. Although higher crude protein was observed in treated group (58.39 – 63.24) compared to the control (55.79) and these values differs significantly (p< 0.05) among the treatments. This agrees with the findings of Puwastien et al. (1999), and Tao and Linchun (2008) who recorded increased in crude protein in treated groups compared to the control. This increase may be due to the preservatives effects which slow down autolysis in the fish muscles and consequently slow down the protein break down. However, this result shows that storage time may cause a decrease in the protein content of smoked catfish which is similar with the report of Ufodike and Obureke (1989) where there was decrease in crude protein of preserved Oreochromis niloticus.
The fat content of smoked C. gariepinus increased significantly at 18 weeks compared to the values obtained after 24 hours after smoking, this might be due to loss of moisture and an increase in the dry matter content per unit of weight following sample dehydration. The values of crude fibre showed a decrease in all treatments at the end of the experiment. There was a general increase in ash content of the smoked C. gariepinus at 18 weeks and this may be attributed to the salt uptake during smoking as reported by FAO (1992). Also, this finding is similar to the work of Oetterer et al., (2003).
Measurement of peroxides value is used to estimate the degree of rancidity and the result of the peroxide value revealed that the values increased as the storage period increased and the treated groups recorded lower values of peroxides value compared to the control and there were significant differences (p<0.05) among the treatments. Also, results obtained from the study showed that the values decreased as the concentration of salt and pepper spice mixture increased. A similar result was observed by Antonia da Silva et al, (2008). The results of Thiobarbituric acid revealed that the values increased as the weeks of storage increased and the treated groups recorded lower values of Thiobarbituric acid compared to the control and there were significant differences (p<0.05) among the treatments. This might be as a result of anti-oxidant properties present in pepper and antimicrobial properties present in salt (Ravishankar and Juneja, 2000).
The result of free fatty acid in this present study revealed that the values were higher at 24 hours after smoking and the values decreased as the weeks of storage increased. Although the study showed lower values at 6 weeks and increase in values at 12 and 18 weeks, the values recorded in the treated groups were better than the control. There were significant differences (p <0.05) among the treatments. The values of free fatty acid recorded in all the treatments decreased as the concentration of salt and pepper spice mixture increased. This might be as a result of the mechanism of action of pepper and salt spice mixture. The result of total volatile base revealed that the values increased as the weeks of storage increased and the treated groups recorded lower values of total volatile base compared to the control and there were significant differences (p<0.05) among the treatments. Also, the values decreased as the concentration of salt and pepper spice mixture increased. The results of total volatile base shows that the values obtained were still within the limit of acceptability of fish which is 30-mg N per 100g (Eyo, 2001).
The aim of bacteriological test is to determine qualitatively and quantitatively the microbial flora that is harboured by the skin, gut, gills and tissue of fish. The results of the study revealed lower microbial loads at 24 hours after smoking with highest value (2.91 log10 cfu/g) of total viable count recorded in control and the least in 20% salt and pepper spice mixture (2.26 log10 cfu/g). At 18 weeks, significant increase in values of total viable count were recorded with highest value in control (53.31 log10 cfu/g) and least (8.13 log10 cfu/g) in 20% salt and pepper spice mixture. The reason for this might be as a result of increase in moisture content of all the treatments at 18 weeks, high pH and temperature that might tend to encourage the growth of microorganisms. These values decreased as the concentration of salt and pepper inclusion increased. There were significant differences (p<0.05) among the treatments, the treated groups recorded decrease in microbial loads compared to the control which could be attributed to the antimicrobial properties present in salt (Ravishankar and Juneja, 2000) and anti oxidant properties in pepper (Deni, 1996). Antimicrobial action suppresses growth for extended periods of time, assuring a longer shelf life and increased product safety
Also, isolation, identification and characterization of microorganisms were done at the end of the experiment to ascertain the presence of food borne pathogens present in the fish, the results revealed the presence of Staphylococcus aureus, Clostridium welchii, Proteus morganii, E. coli and Bacillus subtilis from smoked C. gariepinus at 18 weeks and the effects on the fish which can create serious public health hazards (ICMSF, 1986)
Organoleptic assessment is very important in quality assessment and the quality of the smoked fish (both treated and untreated) was evaluated immediately after smoking (24 hours), 6, 12 and 18 weeks of storage. The assessment was based on taste, flavour, texture, appearance and odour (overall acceptability). The fish flesh overall score was given to both control and treated groups (5 – 20% salt and pepper spice mixture) using a hedonic scale of 1-7, fish scoring less than 2 being regarded as unacceptable. The overall acceptability of the smoked C. gariepinus were higher after 24 hours smoking than value on 6, 12 and 18 weeks, the acceptability decrease as weeks of storage increased and increased as the concentration of salt and pepper spice mixture increased, they were significantly different (p <0.05) among the treatments and this results show that 20% salt and pepper spice mixture were more acceptable (4.15) compared to the control (3.03) at 18 weeks. The result of organoleptic assessment shows that salt and pepper spice mixture retards the activities of bacteria, enzymes and chemicals in fish. This is due to the movement of water out of bacteria cell that is more than into the cell and this result is in agreement with the report of Omojowo et al. (2010).
3 Conclusion
This study demonstrated the effect of smoking, salt and pepper spicing on proximate composition, biochemical parameters, microbial loads and organoleptic assessment of smoked C. gariepinus during 18 weeks ambient storage. Using pepper and salt spice mixture may be useful in improving the shelf life and consumer acceptability of smoked C. gariepinus and it was concluded that 20% spice mixture would positively influence shelf life, reduce and prevent pathogens in smoked C. gariepinus.
4 Materials and Methods
4.1 Sample collection and treatment
Ninety (90) C. gariepinus (1000-1250g) was purchased from the Research and Teaching Farm of the Department of Aquaculture and Fisheries Management, University of Ibadan, Nigeria. The fish were acclimatized for two weeks before the commencement of the experiment. The fish were killed by severing the spinal cord with a sterile knife and aseptically eviscerated, washed and rinsed in distilled water and placed on a white tray. The prepared weight to weight spice mixture of pepper 50% and salt 50% was neatly rubbed on the C.gariepinus with different concentrations after degutting: E1= 0%, E2= 5%, E3 =10%, E4= 15%, E5 = 20%. The C.gariepinus were bent, hooked together with palm-tree material and were cold smoked in an oven between 40℃ to 65℃ for 6 hours and hot smoked between 120-150℃ for 18 hours for proper drying. The C.gariepinus were removed after 18 hours, allowed to cool and placed in perforated plastic containers. Each treatment was separately placed in a plastic container in the office for further assessment.
4.2 Isolation of microorganism/counts
Five grammes (5g) representative sample was obtained aseptically from the loin muscle of the smoked catfish, Clarias gariepinus and were separately macerated and put into sterile capped test tube containing sterilized peptone water and homogenized (Shalaby et al., 2006). Serial dilution was carried out and 1ml each from 10-1 to 10-6 dilution factors were dispensed into Petri dishes that were appropriately labelled and molten sterile medium (MacConky agar for total viable count) was poured aseptically into each Petri dish. The plates were swirled gently for even distribution of inocula and allowed to set /gel and then incubated at 37℃ for 24-48 hours. The organisms grew into visible different colonies after 24 hours. Visible colonies were counted and recorded as Total viable count, the result were expressed in cfu/g.
4.3 Biochemical assessment
Total Volatile Base (TVB), Free fatty acid (FFA), peroxide value (PV) and Thiobarbituric acid level (TBA) were determined.
4.4 Free fatty acid determination (FFA)
10g of the sample (melted fat) was mixed with neutral solvent and titrated with aqueous 0.1M Sodium hydroxide and shaken constantly until a pink colour which persisted for 15 seconds was obtained. The values were calculated as;
FFA = Titration value (mL) × weight of Oleic acid / Weight of the sample used (Pearson, 1968).
4.5 Total volatile base value (TVB)
The total volatile base values were determined using Conway micro-diffusion method (Conway, 1968). The values were calculated as;
TVB = (ml of HCl)/(ml of NaOH) = (mlA- mlB × f × 96 ×14 × 100)/(2 × 25 ×100).
4.6 Determination of peroxide value (PV)
The PV was determined in duplicate using titrimetric method as described by AOAC, (2005). 30 ml glacial acetic acid-chloroform solution (3:2 v/v) was added to 5g of extracted fat and swirled. Potassium iodide (0.5ml of KI solution) was added to react with the peroxides and iodine was liberated. After 1 min., 30 ml of water was added. Then the solution was titrated with 0.1 N sodium thiosulfate (Na2S2O3) using 0.5ml of 1% starch as indicator, until the blue colour disappeared. The peroxide value was calculated by multiplying ml of Na2S2O3 by normality and by 1000 then divided by grams of catfish sample.
4.7 Determination of thiobarbituric acid level (TBA)
Oxidative stability of smoked catfish was measured by Thiobarbituric acid reactive substance (TBA-RS) following the procedure Cd19-90 (AOCS, 1996). Results were expressed as mg malonaldehyde /Kg dry weight. A 5g sample of fat from the belly flap of the fish was combined with distilled water and thoroughly mixed. After addition of 2.5 ml of 4 N HCl, Dow antifoam A, and boiling beads, the sample was distilled and the first 50 ml collected. An aliquot of the distillate (5 ml) was combined with 5 ml of TBA reagent and heated in a boiling water bath for 35 min. Absorbance of the solution was determined at 530 nm, using a slope of TEP (1,1,3,3-tetraethoxypropene, Sigma Chem. Co., St. Louis, MO) standard curve, which was derived to correlate with the expected range of values. Absorbance readings were calculated as µ moles TBARS/kg and then converted to milligrams of malonaldehyde (or TBARS) per kilogram of sample. The TBARS was calculated by the following formula:
TBARS = (T*V1*1000) / (V2*W)
Where: TBARS value in µ moles/kg fish
T = µ moles malonaldehyde (TEP) equivalent to absorbance of sample as determined from the standard curve; V1= volume (ml) of distillate collected, usually 50ml;
V2= volume (ml) of distillate aliquot withdrawn for analysis, usually 5ml;
W= weight of fish added to distil, usually 5g.
4.8 Organoleptic assessment
Sensory evaluation was carried out according to the method of Clucas and Ward, (1996). Evaluation was done at 24 hours after smoking, 6, 12 and 18 weeks on taste, flavour, texture, appearance and overall acceptability. The mean overall acceptability was calculated and recorded.
4.9 Analytical methods
Proximate composition of the smoked C.gariepinus was analyzed 24 hours after smoking and 18 weeks. One fish from each treatment were taken 24 hours after smoking and 18 weeks and analyzed for their proximate composition according to the methods of Association of Official Analytical Chemists {A.O.A.C, (2005)}
4.10 Statistical analysis
Proximate composition, organoleptic assessment, microbial loads and biochemical parameters that resulted from the experiment were subjected to one- way analysis of variance (ANOVA) using SPSS (Statistical Package for Social Science 2006 version 15.0). Duncan multiple range test was used to compared differences among individual means.
Authors’ contributions
This work was carried out by three authors; AOI, SEO and OAO. Author AOI conceived the idea; AOI, SEO and OAO designed the study and wrote the protocol, author OAO supervised the study. Author AOI performed the experiment and author SEO performed the statistical analysis as well as managed the literature searches. Also, Author SEO wrote the first draft of the manuscript. All authors read and approved the final manuscript.
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