Gymnarchus niloticus (Cuvier, 1829), generally referred to as the African knife fish, is a fish commonly used for ceremonial purposes either as a live fish or smoked fish. Gymnarchus niloticus has oily flesh and a strong, rich flavour. It is relished as a food fish among the fish population in Nigeria due to its good taste and solid flesh. It has the ability to attain a very good weight of up to 15 kg [1], and its good market price is about two times or three times the price of the popular Clarias gariepnus. The fish is eaten fresh or processed and forms a much cherished delicacy that cuts across socio-economic, age, religious, and educational barriers [2]. In Nigeria only a negligible portion of fish is preserved by various processing methods [3]. As Nigeria has intensified efforts in fisheries production, there is evidence that processing has been expanding with product distribution to the remote parts of the country [4].

Fish is one of the foods that needs careful handling [5]. This is because fish spoils after capture due to high tropical temperatures, which accelerate the activities of enzymes, bacteria, and chemical oxidation of fat in the fish. Also, due to poor handling, about 30—50% of fish harvested in Nigeria are wasted [5]. These losses could be minimized by the application of proper handling, processing, and preservation techniques [6]. The purpose of processing and preservation is to get fish to the consumer in good, useable condition; the steps necessary to accomplish this begin before the fish expedition starts and do not end until the fish is eaten or processed into oil, meal, or feed [7]. Soon after the death of a fish, considerable irreversible changes take place and result in fish spoilage, and the resultant effect is the decomposition of the fish. This spoilage is caused by lipid oxidation and microbial proliferation [8].

Lipid oxidation causes a reduction in the nutritional value of the fish; it also impacts offensive odour on the fish, which affects its consumption and ultimately its marketability [9]. Microbial contamination can be dangerous to the health of humans, as reported in Europe (contamination of fruits by E. coli in Europe, especially in Germany). Apart from nutritional loss, the presence of microorganisms in fish can result in massive economic loss. This is very common in developing countries with inadequate and inefficient storage facilities [10]. This lipid oxidation and microbial proliferation can be slowed down or completely stopped by two main methods, which are processing and preservation.

Preservation methods help to keep the fish in a fresh state so that changes in texture, physical appearance, and taste can be minimized. Processing methods usually change the texture, physical appearance, and taste of the fish so that deterioration is minimized or stopped. Akintola et al. [8] reported different types of preservation methods: drying, smoking, canning, salting, freezing, and chilling. These processing methods have their advantages and disadvantages, which dictate their usage in particular parts of the world. Smoking has enjoyed the widest acceptance and usage in Nigeria [11]. In spite of the relative success of smoking, especially in terms of increasing the shelf life and nutritional quality of fish, the volume of post-harvest losses, particularly in sub-Saharan Africa, is quite worrisome. It becomes imperative to put measures in place that prevent lipid oxidation and further microbial proliferation; these measures include the use of synthetic antioxidants during smoking [10]. Synthetic antioxidants like Butylated Hydroxytoluene (BHT) and Butylated Hydroxyanisole (BHA) have been very effective in controlling rancidity [12]. Hence there is a need to work on improving the traditional fish smoking methods in order to prolong the shelf life of smoked fish. This need calls for the introduction of food preservatives and antioxidants, which are very good at prolonging the shelf life of food by either killing microorganisms or controlling their growth on food [13]. With the increase in post-harvest loss, there is a need for measures to remedy the situation. Therefore, use of natural spices during smoking will help reduce post-harvest losses by delaying fish spoilage. The aim of this work is to determine the effectiveness of the natural plant spices (Citrus paradisi, Xylopia aethiopica, and Piper nigrum) on prolonging the shelf life of smoked Gymnarchus niloticus (Curvier 1829).

Study Area

The study area is Otuocha River, found in Umuleri, a major community of the Umuobanam clan in Anambra East Local Government, Anambra State.

Sample Collection

Samples of fresh water Gymnarchus niloticus were purchased from Otuocha market, a town in the east of the Niger River and the headquarters of the Anambra East local government area, Anambra State, Nigeria. The fish samples were identified using key features. The spices grape, black pepper, and Negro pepper were purchased from a local market in Awka (Eke-Awka), Anambra State, Nigeria. The charcoal was also sourced from a local vendor in Eke-Awka market. The smoking kiln used for smoke-drying belongs to the Department of Fisheries and Aquaculture, Nnamdi Azikiwe University, Awka.

Fish Preparation

The fish (Gymnarchus niloticus) was selected, weighed, measured, degutted, and washed. After that, the fish was cut into steaks, washed in clean water, and left to drain in a plastic sieve.

Preparation of the Spices

30g of Negro pepper and black pepper were ground into powder using a kitchen grinding machine, and 500ml of grape juice was extracted and diluted with 500ml of water. The ground black pepper and Negro pepper were mixed with 500 ml of water and heated for 5 minutes and allowed to cool. The fish samples were soaked in the spice extract for 30 minutes, then drained and smoke-dried in a smoking kiln.

Processing Techniques

The drying of the fish was conducted using a charcoal oven. The pre-treated fish samples were arranged on the oven trays and allowed to dry for 24 hours, during which turnover of the fish was done at intervals to achieve a uniformly dried product. The processing of the treatments was done on different days to avoid mixing flavours in the oven. The samples after processing were allowed to cool and packaged in a light polythene bag (figure 1-4). The samples were labelled as follows:

Control (salt): treatment A

Black pepper: Treatment B

Negro pepper: Treatment C

Grape: Treatment D

Storage Technique

The samples were stored for 60 days at room temperature (25°C) wrapped in a light polythene bag and monitored daily in order to determine the effect of the three spices on the shelf life of the smoked fish specimen.

Organoleptic Evaluation

The smoke-dried fish were served to 10 independent panellists comprising male and female students of Nnamdi Azikiwe University, Awka, who were familiar with sensory attributes of fish such as taste, aroma, appearance, and texture. A nine-point (1982) hedonic scale (like extremely=9, like very much=8, like moderately=7, like slightly=6, neither like nor dislike=5, dislike slightly=4, dislike moderately=3, dislike very much=2, dislike extremely=1) was adopted according to ISO to evaluate changes in taste, aroma, appearance, and texture of the fish sample. The samples in the batch were served; batch A (control) was coded A, batch B (black pepper) was coded B, the sample in batch C (Negro pepper) was coded C, and batch D (grape) was coded D.

Bacteria Identification

Sample Collection and Culturing

A small portion of each of the ground fish samples weighing 1 g was transferred aseptically into 9 ml of distilled water in a test tube. Samples were serially diluted 4-fold using 1:10 dilution. 1 ml of the dilution was taken from the 4th test tube into two sterile petri dishes with labelling. Nutrient agar was added into the petri dishes accordingly, mixed, and allowed to get together. The nutrient agar was then incubated at 37°C for 24 hours.

Plate Reading and Bacterial Isolation

After 24-hour incubation, bacterial growth on nutrient agar was observed and counted. Different bacterial colonies were isolated and sub cultured according to their colony morphology. The isolates were characterized and identified based on their colony characteristics and biochemical reactions as follows:

Biochemical Tests

The following biochemical tests were done on the isolate for further identification: Gram staining, catalase, urease, coagulase, citrate, motility, and sugar fermentations.

Gram-Staining Technique

A drop of distilled water was placed on a clean, grease-free glass slide, and a colony of the isolate was picked with a sterilized wire loop and emulsified. The glass slide was passed over the flame four times to heat fix. The smear was flooded with crystal violet for 60 seconds and rinsed with distilled water. Lugol's iodine was added for 60 sec and then immediately decolorized with acetone for 10 sec with distilled water. The smear was counterstained with safranin for 30 sec and rinsed with distilled water. The smear was then allowed to air dry, after which oil immersion was added and viewed under a microscope using a x100 objective lens. Gram-positive results appear purple in colour, while gram-negative results are pink/red in colour.

Catalase Test

This test is used to detect the presence of catalase, an enzyme that catalyzes the release of oxygen from hydrogen peroxide when broken down. One drop of hydrogen peroxide solution was dropped on a clean glass slide, followed by the inoculation of a 24-hour-old culture on the slide. The presence of gas bubbles indicates a positive test, while the absence of gas bubbles indicates a negative reaction.

Methyl Red Test

This test determines whether the organism performs mixed acid fermentation when supplied glucose. Tubes of MR broth were inoculated with a pure culture of the isolates and incubated at 35°C for 4 days. 5 drops of methyl red reagent were added to the culture; a positive test is indicated by red colour formation, while no change denotes negative.

Citrate Utilization Test

Simmon’s citrate agar was prepared in accordance with the manufacturer's manual; 38 g of the media was weighed and dissolved in 100 ml of water. The media was boiled for 15 min and autoclaved for 15 min. The media was allowed to cool to a temperature of about 40°C before pouring it. After pouring, the media was allowed to gel, and isolates were inoculated on it.

Urease Test

Urease agar was prepared in accordance with the manufacturer's manual; 24 g of the media was weighed and dissolved in 100 ml of water. The media was boiled for 15 min and autoclaved for 15 min. The media was allowed to cool to a temperature of about 40°C before pouring it into test tubes. Media was allowed to gel, and isolates were inoculated on it. A colour change was observed after 24 hours.

Coagulase Test

A sterile wire loop was used to pick a colony from an overnight culture and mixed with a normal saline place at the end of a clean glass slide. A drop of blood plasma was added and incubated at 37°C for 1–6 hours. Clumping within 1 to 6 hours (1–6 hrs) indicates a positive reaction (Ogbulie et al., 2014).

Indole Test

Sterile nutrient broth 5 ml was added into a tube and inoculated with the test organism. The tubes were covered with sterile cotton wool and incubated overnight at 37°C. After incubation, 0.5 ml of Kovac reagent was added to the broth. A red ring layer on the surface of the media indicates a positive indole result, and a brown colour indicates a negative indole result (Gaffa and Azoro, 2005).

Fungi Isolation and Identification

Sample Preparation and Culturing

A small portion of each of the ground fish samples, weighing 1 g, was transferred aseptically into 9 ml of distilled water in a test tube. Samples were serially diluted 5-fold using 1:10 dilution. 1 ml of the dilution was taken from the 5th test tube into two sterile petri dishes with labelling. Molten SDA was added into the petri dishes, mixed, and allowed to gel together. The media was left for 5 days at room temperature.

Fungal Isolation and Identification

After the incubation period, cultured plates were examined. The isolates were subcultured and allowed to stay for 3 days. Fungi were then identified as follows:

Macroscopic morphology: the colour, shape, texture, and size of the fungi colonies were observed and compared with a standard fungi atlas.

Microscopic Using Lactophenol Cotton Blue

Two drops of lactophenol cotton blue were placed on a clean grease-free slide. Using a sterile wire loop, a small portion of the fungus was collected and teased in the lactophenol and then covered with a coverslip. The slide was mounted on the microscope stage and was focused with x10 and observed with the x40 objective lens. Under the microscope the type of spores and hyphae in the smear was noted.

Figure 1: Fish Sample Smoked with Salt.

Figure 2: Fish Sample Smoked with Black Pepper.

Figure 3: Fish Sample Smoked with Grape.

Figure 4: Fish Sample Smoked with Negro Pepper.

The mean values of the organoleptic ratings by the panellist are graphically represented in fig 5 (sensory analysis for taste), fig 6 (sensory analysis for aroma), fig 7 (sensory analysis for appearance), and fig 8 (sensory analysis for texture) below.

Figure 5: Sensory Analysis for Taste in G. niloticus Smoked with Different Spices.

Figure 6: Result of Sensory Analysis of Aroma.

Figure 7: Result of Sensory Analysis for Appearance.

Figure 8: Result of Sensory Analysis for Texture.

Table 1 shows the general acceptability of the different spices in taste, aroma, appearance, and colour as rated by the panellist. Table 2 shows the effect of the organic spices on mould infestation of the smoke-dried Gymnarchus niloticus during the time of storage. The sign (+) indicates that the fish sample was positive for mould infestation that particular week, while the sign (-) indicates a negative response to mould. Table 3 shows the fungi isolated and identified from each sample at the 8th week of storage, the frequency with which each fungus occurred in the fish samples, and the percentage occurrence. The sign (+) indicates the fish sample was positive for the identified fungi, while the sign indicates the (-) absence of the identified fungi. Table 4 shows the total fungi count of isolated and identified fungi carried out in the test samples. Table 5 shows the bacteria isolated and identified from each sample at the 8th week of storage, the frequency with which each bacterium occurred in the fish samples, and the percentage occurrence. The sign (+) indicates the fish sample was positive for the identified fungi, while the sign indicates the (-) absence of the identified fungi. Table 6 shows the total plate count of isolated and identified bacteria carried out in the test samples.

Table 1: General Acceptability of Smoked Fish Sample Treated with Different Spices.

Table 2: Effect of Mold Attack on the Smoked Gymarchus niloticus.

Table 3: Fungi Isolation and Identification on the 8th Week of Storage.

Table 4: Fungi Count in Each Treatment.

Table 5: Bacteria Isolation and Identification on the 8th Week of Storage.

Table 6: Total Plate Count of the Isolated Bacteria.

Quality of food is the characteristic of food that is acceptable to the consumer. In references to fish, the characteristics that impact distinctive quality may be described in four different attributes: colour and appearance, flavour (taste and aroma), texture, and nutritional value. According to Okeke et al. [14], the organoleptic qualities (texture, colour, taste, and flavour) of smoked dried fish determine the acceptability to consumers and the market price of the fish. As consumers, these four attributes typically affect us in the order specified above; for example, we evaluate the visual appearance and colour first, followed by the taste, aroma, and texture. With an ever-growing world population and the need to store and transport the food from one place to another where it is needed, food preservation becomes necessary in order to increase its shelf life and maintain its nutritional value; therefore, a good preservation technique must prevent microbial spoilage without affecting its nutritional quality and nutritional value [15].

In this study three organic spices were used to evaluate the effect of the organic spices on the organoleptic qualities and shelf life of hygienically smoke-dried Gymnarchus niloticus. Taste is the sensation produced when substances in the mouth react chemically with the taste receptor cells located on the taste buds in the oral cavity, mostly on the tongue. Our gustatory system allows the perception of different food constituents as alkali metallic salts (salty), acid (sour), sugar (sweet), and bitter compounds [16]. The result of the organoleptic analysis shows higher preferences for sample A (control 7.33±0.21) treated with salt only than for B (black pepper 5.40±0.46), D (grape 5.17±0.6), and C (Negro pepper 3.54±0.46); this, however, agrees with Tairu et al. (2017), whose study found the salt-treated sample tastier than the other samples he analyzed. This is similar to the findings of Abdel et al. [18]; the general acceptability in all the sensory parameters indicated that the spices may have inherent chemical compounds responsible for the pleasant colour, taste, and flavour/aroma in oven-dried Gymnarchus niloticus.

The results from the table show that the salt-treated sample started moulding on the 6th week (42 days) of the experiment, and treatment C (Negro pepper) started moulding at the 7th week (49 days). The occurrence of fungi might be as a result of the difference in chemical composition [19] and the fact that different moulds react differently to the fish samples. However, B and D showed no sign of physical deterioration during the study, but there was a presence of fungal spores. This result is in agreement with Dylag [20], who stated fungal spores in some small quantities are naturally found in both indoor air and outdoor air. Dylag [20] also stated in his work “fungi present in the home and their impact on human health" that mould are the most common, and when they are above the permissible limit, they can cause allergic reactions, conjunctivitis, cough, upper respiratory tract infections, and congestion.

According to Agwuna et al. [21], a microbial count of less than 30 colonies or less than 2.4 x 104 colony-forming units per number for the viable bacteria and yeast in mixed culture is negligible and insignificant in food quality and safety assessment, but values higher than the figure pose a risk to human health. Therefore, Piper guinensis, Xylopia aethiopica, and Citrus paradisi had chemical preservatives that have effective preservation potentials for smoke-dried fish during storage. This antioxidant could be part of the reason for the low microbial count, which made the fish within acceptable limits after 8 weeks of storage, as also recorded by the International Commission on Microbial Safety for Food and for good fish products [22]. This is therefore similar to the findings of Amuneke et al. [23] in their study "Effect of natural preservatives on the organoleptic characteristics and storage stability of smoked Heterotis niloticus," in which Xylopia aethiopica and Piper guinensis were also used.

This new study shows that the three indigenous spices used in this study, Piper guinensis, Citrus paradisi, and Xylophia aethiopicum, had chemical preservative and antioxidant properties. Piper guinensis (uziza) and Citrus paradisi were found to be the most effective in the overall preservation of smoked-dried fish during storage, but there is no significant difference in the acceptability of treatments. All three spices used in the course of this study have good acceptability and can be used for smoking. Hence, the following will be recommended:

• Further study should be carried out on Citrus paradisi extract as a preservative for fresh fish.
• Further studies should be carried out to test higher levels of Citrus paradisi, Piper guineense, and Xylopia aethiopica inclusion to determine the limit they might affect sensory attributes since the levels tested were acceptable.

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