Aceh Journal of Animal Science

The increasing use of synthetic antimicrobials has created a situation leading to a pond ecological imbalance and enrichment of multiple multi-resistant pathogenic microorganisms. This study was conducted to investigate the impact of spices and natural preservatives using a mixture of Onion Bulb (OB), Holy Basil (HB) and Turmeric Rhizome (TR) on shelf–life, flesh and microbial quality of smoked Clarias gariepinus during 56 days storage. Spices were added at the point of processing with five treatments of 8 fresh C. gariepinus (1.2-2kg) were distributed into experimental containers; the control (without Onion Bulb, Holy Basil and Turmeric Rhizome), (TR + OB) 2 , (TR+ HB) 3 , (OB +HB) 4 , (TR + OB + HB) 5 and the experiment were carried out in triplicates. Clarias gariepinus were smoked in a smoking kiln at 40 º C-60ºC and 120-150 º C for 6 and 18 hours respectively. Proximate composition, biochemical parameters, organoleptic assessment, and microbial analysis were carried out using standard methods. The result showed that the crude protein of C. gariepinus was higher in the treated groups compared to the control. Also, the result shows that the biochemical parameters, organoleptic assessment and the microbial loads in smoked C. gariepinus were reduced in the treated groups than in the control at 1 day, 28 days and 56 days storage respectively. The results indicated that using onion bulb, holy basil, and turmeric rhizome in combination may be useful in improving the shelf life, consumer acceptability of smoked C. gariepinus and preventing bacteria pathogens in smoked C. gariepinus.


Introduction
Aquaculture is the fastest growing foodproducing sector and developed as an important component of food security (Ekelemu et al., 2021;Bello, 2014). Fish is one of the cheapest and most promising sources of animal protein with 93.7% digestion by people who consume fish as animal protein (Bello, 2014). Fish is more susceptible to tissue decomposition, development of rancidity, and microbial spoilage as soon as they leave water/caught (Kiin-Kabari, 2011). Food spoilage affects the texture, nutritional value and flavour of the food making the food unacceptable for consumption (Yoganathan, 2020). Bio-preservation of food systems has recently increased globally due to the economic lost, deterioration and poisoning of food products by food pathogens which is often responsible for the depreciation in quality and safety (Ekelemu et al., 2021). Food preservation and safety measures can be used to prevent the growth and activity of food-borne pathogens in foods to acceptable levels (Yoganathan, 2020). Food preservation enhance food quality by inhibiting spoilage in food by increasing nutritional value, texture, flesh quality and flavor (Wakoli et al., 2014). The uncontrolled and repeated use of chemicals such as Butylhydroxyanisole (BHT) in aquaculture to preserve fish for a long time has been incriminated carcinogenic, considering the negative effects on human health, current foodborne disease management tend to concentrate on environmentalfriendly, preventive methods such as the use of natural products that have antimicrobial and antioxidant properties (Bello et al., 2013). Natural Olusola & Martins antioxidant does not have harmful effects and can helps to improve human health compared to the synthetic antioxidants (Bober et al., 2018;Campos et al., 2015).
Spices and herbs that possess antimicrobial activity against foodborne pathogens and food spoilage bacteria, they have been used for the preservation of foods and beverages primarily due to their phytochemical content (Alex and Eagappan, 2017;Embuscado, 2015). However, when spices are added to food, they increase the shelf-life preventing food loss due to their antioxidant and antimicrobial benefits (Ekelemu et al., (2021;Amuneke et al. (2020). The antioxidant and antimicrobial potential of spices such as onion bulb, holy basil and turmeric rhizome among others have been investigated and found to be effective (Olusola, 2021). But information on the combination of spices or herbs as a bio-preservative agent has not been well elucidated. This study was investigated at assessing the synergy of onion bulb, holy basil and turmeric rhizome on the flesh quality and shelf-life of African catfish C. gariepinus under ambient storage for 8 weeks. African catfish is one of the popular freshwater fish in the world (Marimuthu et al., 2017;Ayoola and Bamiro, 2017), and this species has been cultured intensively and extensively in Nigeria (Adeniyi et al., 2021).

Collection of plant materials and identification
Spices such as onion bulb, turmeric rhizome, and holy basil were purchased from Oja -Oba market, Okitipupa, Ondo State, Nigeria and were identified at the Department of Biological Sciences, Olusegun Agagu University of Science and Technology, Okitipupa. Preparation of onion bulb, turmeric rhizome, and holy basil 4500 g of fresh onion bulb, turmeric rhizome, and holy basil was ground with a fabricated hammer mill and 3500 g of water extract were poured into a 45 litres experimental plastic bowls until required for use.

Collection of experimental fish and processing of experimental sample
One hundred and twenty C. gariepinus (1.2 -2 kg) were purchased from a farm in Akure, Nigeria and packed in oxygenated bags transported to the Department of Fisheries and Aquaculture Technology Teaching and Research Farm, Olusegun Agagu University of Science and Technology, Okitipupa. The C. gariepinus were killed, dissected with a sterile knife and aseptically eviscerated, washed and rinsed in distilled water and placed on a basket to allow the water to drain. The fish was dressed with bamboo stick and were soaked in the water extracts of equal proportion of (TR + OB), (TR+ HB), (OB +HB) and (TR + OB + HB) for 2 hours. The control was without a mixture of these spices. The fish was cold-smoked between 40ºC-60 º C for 6 hours and hot smoked between 120 º C-150 º C for 18 hours. The fish were allowed to cool and stored in perforated experimental plastic containers Fish storage The smoked fish was kept in 15 plastic containers with 8 fish per replicate for 56 days at the Fisheries and Aquaculture Technology Laboratory, Olusegun Agagu University of Science and Technology, Okitipupa at 25 º C.

Microbiological analysis
One gram (1 g) of each sample was obtained aseptically from the smoked C. gariepinus and analyzed for the total viable counts and Enterobacteriacea counts using plate count agar by spread plate technique. They were separately macerated and put into the sterile capped bottle containing sterilized peptone water (10 ml). The dilutions of 10 4 were spread on plate count agar and incubated at 37°C for 24 hours. The colonies were counted for total viable counts and Enterobacteriaceae counts and the count was recorded as log10 Colony Forming Unit, CFU/g (AOAC, 2005).

Organoleptic assessment
The subjective evaluation of the quality of smoked fish quality were done at 1 day, 28 days and 56 days storage on parameters such as taste, flavour, odour, texture, appearance and overall acceptability. This was carried out by experienced ten (10) trained panelists using the hedonic scale of 1-7. The panels awarded score to control, (TR + OB)2, (TR+ HB)3, (OB +HB)4, and (TR + OB + HB)5. Treatment with less than 2 was recorded unfit. The mean values of these parameters were recorded and calculated.

pH determination
The pH of each sample (treatment) was measured at 1 day, 28 days and 56 days storage. Fish sample (5 g) from each treatment was weighed and homogenized in 30 ml of sterile distilled water, autoclaving at 121 º C for 15 minutes, and the sample was allowed to cool for 1 hour before the reading was taken and recorded.

Analytical method
The quality of C. gariepinus (1) from each treatment was assessed for their proximate composition using the methods of the Association of Official Analytical Chemists (AOAC, 2005) at 1 day, 28 days and 56 days storage.

Statistical analysis
The values of proximate composition, biochemical composition, organoleptic assessment, microbial loads and pH obtained from this study were subjected to descriptive statistics and a oneway analysis of variance (ANOVA) using SPSS (Statistical Package for Social Science 2006 version 20.0). Duncan's multiple range tests was used to compare the differences among individual means at P= 0.05.

Proximate composition of smoked C. gariepinus treated with the combinations of turmeric, rhizome, onion bulb and holy basil
The results revealed a decrease in the value of crude protein, ash and ether extract as the period of storage increased while the increase in the value of moisture and the nitrogen-free extract was obtained as the period of storage increased. The treated groups had better values than the control (Table 1).

Biochemical composition of C. gariepinus treated with a combination of turmeric, onion bulb and holy basil
The values of Total Volatile Base (TVB), Free Fatty Acid (FFA), Peroxide Value (PV) and Thiobarbituric Acid Level (TBA) recorded at 1 day and 56 days of storage were higher in the control when compared with the treated groups, the values decrease as the storage period increases (Table 2).

Organoleptic assessment of smoked C. gariepinus in a combination of turmeric, holy basil and onion bulb
The values recorded in odour, flavour, texture, taste, appearance and overall acceptability were better in the treated groups compared to the control. The highest values of overall acceptability were obtained in (TR + OB)2 (9.2; 8.2) at 1 day and 28 days after smoking respectively while (OB + HB)4 recorded highest overall acceptability (6.0) at 56 days and the least in the control (3.0) ( Table 3).

Microbial analysis of smoked C. gariepinus treated with a combination of turmeric, holy basil and onion bulb
The result of the microbial load revealed higher values of Enterobacteriaceae and total viable counts in the control when compared to the treated groups, the values increased as the storage period increased. There were significant differences (P<0.05) among the treatments at 1 day, 28 days and 56 days (Table   4).

pH determination of smoked C gariepinus treated with a combination of turmeric, onion bulb and holy basil
The values of the pH decreased as the week of the storage increased the value obtained in the control and the treated groups were relatively similar and there was no significant difference (P >0.05) among the treatments at 1 day and 28 days but, there were significant differences (P<0.05) at 56 days among the treatment groups (Table 5).

Discussion
The results of the proximate composition of the fish revealed that catfish treated with a combination of spices (onion bulb, holy basil and turmeric rhizome) had a higher percentage of protein than the control, the crude protein and fat were better in the treated groups at 1 day and 56 days compared to the control. The crude protein and fat were recorded highest in the mixture of turmeric rhizome and holy basil (43.54±0.01; 3.56±0.01) and the least in the cont rol (40. 59± 0.01 ; 3.56 ±0.04) respectively at 56 days. There was a significant difference (P< 0. 05) among the treatments.
The quality loss of proximate composition was higher in the control fish during storage. The values of crude protein and lipid were decreased with the increase of the storage period while the values of moisture content were increased with the storage period. Moisture content is one of the principal determinants in analyzing the quality of dried food products. It has been reported that the lower the moisture content, the higher the quality of the products. This result was similar to the report of Abdul and Aktar (2011) who reported better crude protein and lipid in fish treated with spices compared to the control. Also, Hassan et al. (2013); Abdul and Aktar, (2011) reported an increase in the moisture content as the length of storage increased and a decrease in crude protein, and lipid as the storage period increased which was in accord with the present findings.
The results of biochemical composition revealed that Total Volatile Base (TVB), Free Fatty Acid (FFA), Peroxide Value (PV) and Thiobarbituric Acid Level (TBA) were decreased in the treated groups compared to the control. However, the values increased as the period of storage increased. The treated groups had better values of PV, TVB, FFA, and TBA at the 56 days of storage and they were significant difference (P< 0.05) among the treatments. A combination of onion bulb and holy basil had better values of PV and FFA and a mixture of turmeric rhizome and onion bulb had best values in TVB and TBA at 56 days of storage. This is because these natural spices possessed phenolic compounds (phenolic acids, polyphenols and flavonoids) which helps to prevent oxidative rancidity and prolong the shelf life of the fish in the treated groups. These results were in accord with the report of Yoganathan (2020) who reported that application of spices inhibits the free-radical mediated damages like lipid oxidation thus preventing oxidative rancidity. Also, the present study supports the report of Abdul and Aktar (20011);and Farid et al. (2014) who reported that the nutritional value of dried fish deteriorates with the increase in storage period.
The result of a mixture of these natural spices treated smoked fish had a better quality than the control fish when assessed by sensory and nutritional quality. The values of odour, flavour, texture, taste, appearance and overall acceptability decrease as the period of storage increases was better in the treated groups compared to the control. The overall acceptability was obtained in the mixture of onion bulb and holy basil (6.0) against the least value obtained in the control (3.0) at 56 days of storage. This study shows that inclusion of a mixture of onion bulb, holy basil and turmeric rhizome gave a better acceptability of preserving fish products as they sustained longer shelf life, reduced bacterial and enzymatic activities and a better sensory score than that of control fish. This study supports the report of Alex and Eagappan, (2017) and Akter et al. (2013) who reported that dried fish treated with spices and herbs had better shelf life and flesh quality than the control. Also, Yoganathan (2020) reported that a better shelf life and flesh quality was obtained in brine salted dried fish than the control which was similar to this present study.
Alex and Eagappan, (2017); Taniya and Kannan, (2016); Kumolu -Johnson et al. (2015) reported that application of herbs and species that possessed antioxidant and anti-bacterial properties helps to prevent the microbial growth and increasing the shelf life of the product. The results show that the Enterobacteriaceae counts and total viable counts were higher in the control than in the treated groups, the values of Enterobacteriaceae counts and total viable counts increased as the period of storage increased. The increase in moisture content of all the groups could lead to the proliferation of microorganisms, thereby resulting in the increase in the microbial loads in all the treatments over time. This was in agreement with Ekelemu et al. (2021) who reported that microbial loads increased with the duration of storage. The combination of onion bulb and holy basil recorded the lowest Enterobacteriaceae counts and total viable counts at 1 day and 56 days of storage. There was a significant difference (P< 0.05) among the treatments. This study was in concordance with the report of Taniya and Kannan, (2016) who reported higher bacterial counts as the storage period increases in all the treatments with a reduction in microbial count in treated groups compared to the control.
The result revealed a variation in the values of pH obtained among the treatments, there was no significant difference (P > 0.05) at 1day storage among the treatments, these values decrease as the storage period increased and there was a significant difference (P < 0.05) among the treatments. This study aligned with the report of Olusola, (2021) who reported a decrease in values of pH as the period of storage increased in smoked C. gariepinus. Olusola & Martins The result of this present study shows that a mixture of spices would be helpful in enhancing the shelf-life, and consumer acceptability of smoked Clarias gariepinus and reducing post-harvest losses of fish due to the antimicrobial and antioxidant activity of their natural constituents. The mixture of onion bulb, holy basil and turmeric rhizome could have a synergistic effect and possibly influence the prevention of food spoilage microorganisms.