EFFECTS OF SPICE AND SPICES BLEND RATIO ON MICRO NUTRIENTS, HEAVY METALS, SHELF LIFE AND SENSORY QUALITY OF SMALL FISH ( Oreochromis niloticus, Pylodictis olivaris and Cyprinus carpio) PRESERVATION, GAMBELLA REGION, ETHIOPIA.

Dagne Tarle Tarse^*1, Aregay Berhe Gebre^*2 and Endale Tesfaye^*3.

*1 and *2, Gambella University, Department of Food Engineering, Ethiopia.

*3, Gambella University, Department of Chemistry, Ethiopia.

Aregay Berhe, Endale Tsfaye, Dagne Tarle Tarse, EFFECTS OF SPICE AND SPICES BLEND RATIO ON MICRO NUTRIENTS, HEAVY METALS, SHELF LIFE AND SENSORY QUALITY OF SMALL FISH ( Oreochromis niloticus, Pylodictis olivaris and Cyprinus carpio) PRESERVATION, GAMBELLA REGION, ETHIOPIA.(2017)SDRP Journal of Food Science & Technology 2(2)

The study was conducted to assess effect of drying methods and pre-treatments on shelf-life, micro-nutrients, Hg level and sensory quality of dried fish. It was conducted in factorial arrangement of 3×2×3 with 3 drying methods (sun, smoke and oven drying), 2 preservatives (blend ratio and red pepper) and 3 small fish (tilapia, cat fish and carp) species laid out in CRD. Fresh fillets were analyzed for Ca, Fe, Zn, Vit-A, level of Hg, sensory and microbial quality. Dried fillets were stored and analyzed for the expected parameters at 1 month interval and for microbial status every 20 days for 60 days. In fresh samples, a high load of aerobic bacteria (AB) of 4.75 log₁₀ cfu/g was observed and mould counts were not detected in all the 3 species of fish and milk sample. The Ca in fresh fillets (1140.16- 1199.70 mg/100g) was equivalent to Ca in fresh milk (1192.33 mg/100g). Regarding freshly dried fillets, high load of AB (5.22-5.38 log₁₀ cfu/g) with no (P<0.05) significant difference was observed in untreated fillets of the 3 species whereas the initial load of moulds were <1.28 log₁₀ cfu/g. After 60 days of storage, the maximum load of AB and moulds were 7.20-7.57 and 6.08-6.95 log₁₀ cfu/g, respectively. Minerals and Hg level of fresh samples were  20.40-34.33 mg/100g of Fe,  4.75-6.77 mg/100g of Zn,  0.07-0.18mg/100g  of vit-A  and 0.01-0.11mg/100g  of Hg.  The micro-nutrients and Hg level after drying were 1497.86‑1561.51mg/100g  of Ca, 26.50‑53.55 mg/100g  of  Fe,  4.85‑11.51 mg/100g of Zn,  0.03‑0.08mg/100g  of  vit‑A and 0.00‑0.11mg/100g  of  Hg.  After the storage of 3 months, levels of Ca, Fe, Zn,  Vit‑A and Hg were 967.49‑1187.84 mg/100g, 25.69‑53.28mg/100g,  4.13‑11.00 mg/100g,  0.01‑0.06mg/100g  and  0.00‑0.11mg/100g  respectively.  Micro‑elements showed slightly reduction in every month of storage time. Overall acceptability of treated and untreated samples reached 5.28 (like slightly) and 4.62 (neither like nor dislike) respectively after 3 months. The total load of AB (7.20 log₁₀ cfu/g) in all untreated samples was the reason that why samples were not allowed for panelists to taste. This was the point of sensory rejection in which the number of microbial load should be below the 10⁷-10⁸ log₁₀ cfu/g EU [26].

Keywords: small fish, drying, milk, minerals, Hg, shelf‑life, sensory quality, Gambella region, Ethiopia.

The contribution of fish to human nutrition and its positive impact on health, has received different emphasis in developed and developing countries, FAO [2006]. Different species of fish had benefits for the world as food for human consumption  Mdegela R. et al., [72]. The global traditional and improved fish processing technologies in aquatic food production had more than double since 1970, with a total of approximately 93.2 million metric tons in 1997 Akinneye J., O. et al., [4]. According to MoA 2012/13 fiscal year, Ethiopian consumption on fish was mainly depending on Tilapia/Koroso/Oreto and Cat fish/Ambaza/aggula because of their access abundant relative to other species. For instance, Koroso accounted for 75% of the fish production followed by Ambaza, with 12% and other types of fish account for 13% in which Carp/Duba was included was reported. Baro-river passing through Gambella region is a good source of small fish. Similarly, Alwero reservoir is also known by its naturally wild fish production in Gambella regional state which is located in Anywaa zone, Abobo District Dagne Tarle et al, [19]. Although fishing was done on a continuous basis by dam or river-side communities and local farmers in Gambella region, it was noticeable that a bumper harvest occurs mostly during dry seasons to ensure supply of fish Eyo, [27]. This was because of the reduced water level of the water body makes easy for harvesting. The period of fish scarcity was often encountered especially during flood and raining seasons, during which fish are in short supply. Thus, it was imperative to process and preserve some of the fish harvested in the period of abundance, so as to ensure an all year round supply. Small fish (Adappe or Dey Rech) that eaten as whole, provide a particularly rich source of Ca, vitamin-A, Fe and Zn in human consumption. Small fish are ‘’milk’’ in most parts of the world where there is no access for livestock milk. This was because the Ca in small fish was equivalent to that in milk Hansen et al [45].

The drying of fish was one of world's oldest known preservation methods that lengthen storage life of dried fish. Fish should be preserved to assure the best possible consumption supply at all time, assure safety of products, and reduce wastes to the barest possible extent Akinneye, J. O. et al [4]. To keep the quality and safety of fish, it was essential to minimize water activity in fish in appreciable quantities in good condition until its use is required FAO, [32]. Environmental contaminants may enter the food chain, accumulate in organisms and affect their survival. Hence, the abundance and quality of commercially important fish species, an important ecosystem service of water bodies, may be at risk Gebremariam Z. and Desta Z.  [38]; Gebremariam Z. & Pearce, J.G. [39]. In light of the previous studies on the metal pollution, elements present in effluents were Cd, Hg, Cr, Pb, Se and As (Gebremariam Z. & Desta Z.  [38]; Gebremariam et al. [39].  Although As is a metalloid and Se a non-metal, we address both together with Cd, Hg, Cr and Pb, and for reasons of brevity, termed as “heavy metals” for the whole group (Gebremariam & Desta [38]; Gebremariam et al. [39]. The potential of these elements for increased human exposure & health risk is troubling; therefore,they were listed as priority pollutants by the US Environmental Protection Agency Mc Kinney and Rogers [71].

Major problems related with small fish processing and drying are loss of quality due to contamination that affects micro-nutrients and shelf-life of small fish in HH consumption (Sablani et al., [81]. Lack of small fish preservation skill/activities are seen for year round supply for perennial alleviation of malnutrition and food insecurity. ‘’Adappe’’ for ‘’Kac’’ or ‘’Dey Rech’’ for ‘’Dak Mithini’’ are also more associated with heavy metals than large fish (Larissa D. et al [61]. Therefore, upgrading the traditional fish processing technology and adoption of preservation methods with specific antioxidants and appropriate storage periods are needed in the region to minimize the dried fish problems. Application of herbs, spices and their essential oils with anti-microbial effects compared to synthetic additives should be practiced. The objectives of this study were: to study the effects of preservation methods and spice and spices blend ratio on micro‑nutrients, heavy metals, shelf-life and sensory quality of small fish preservation. Moreover, to evaluate effect of spice and spices blend ratio on safety, sensory quality and micro‑nutrients including heavy metals of preserved fish. Furthermore, to evaluate the effect of preservation and preservation methods on micro‑nutrients, heavy metals and shelf life of fish.

(a)   Description of the Study Area

Harvesting, processing and preservation of small fish were conducted in Abobo Woreda that located in Gambella Regional State which was at about 777 km in southwest part of Ethiopia from Addis Ababa. It is situated in the lowland of the Baro-Akobo River Basin between latitude 6022’ and 8030’N, and longitudes 33010’and 35050’ E and it covers a total area of about 34,063km2, while the total area of the River Basin is about 75,910 km2 CSA, [17]. The annual rain fall and mean annual temperature in the region are 1,247mm and 34.370C, respectively GPNRS, [43].The rain fall regime is unimodal, referred to as the ‘’Sudan Type’’, occurs in the lowlands along the border with Sudan Coppock [15].The rain fall varies with season, about 60% to 70% occurs during the wet season (i.e.,May to October) & 30 to 40% with dry season (November to April). December, January & February are the driest months; only about less than 2% of annual rainfall occurs in these months over the lowlands of the Region while about 4‑6%occurs over  highlands GPNRS[43].

(b)    Experimental Location

The small fish species were collected from Alwero reservoir, Abobo District in Gambella region where drying, pre-treatments and sensory evaluations were conducted. The analyses for the micro-nutrients of fresh and dried fish samples such as iron, zinc, calcium and preformed vitamin A as retinol including heavy metal (Hg) were conducted using AAS in ‘Jije laboratory’’/ Jije Laboglass P.L.C. Testing Services, Addis Ababa. The total microbial analyses of fresh and dried samples like aerobic bacterial plate counts (aerobic plate counts) and total moulds were conducted in Food microbiology laboratory at four kilo of Addis Ababa University.

(c)    Experimental Materials

Fish: The experimental materials included three small fish species namely, tilapia (Oreochromis niloticus), flat head cat fish (Pylodictis olivaris), and common carp (Cyprinus carpio). After the fish were caught, selections of right quality fish were done based on age, length and type followed by evisceration. Total of 30 kg small fish from the three species with 10 kg of each of the three species were collected from the reservoir.

Ginger (Zingiber officinale) and Garlic (Allium sativum): These were obtained from the local markets at Gambella town. About 12kg of each garlic and ginger was cleaned, washed, chopped and stored in a refrigerator.

Capsicum/Chili (red pepper): This was purchased from the local markets in the same town. A total of 12 kg red pepper was cleaned, chopped and stored in a refrigerator.

Chemicals/Reagents/medias: Buffered pepetone water, Aerobic plate count agar, Sabouraud Dextrose Agar Medium, NaSO4, CuSO4, H2SO4, NaOH, petroleum ether, N-hexane, Methyl, Nitric acid, perchloric acid, HCl, calcium chloride (CaCl2) solution, KMnO4, FeCl3, Phytic acid, phosphoric acid, Hydrogen Cyanide (HCN)&Ammonium hydroxide   ISO [54].

(d)   Experimental Design and Treatment

The experiment of this study was laid out in a factorial arrangement of 3 x 2 x 3 in a completely randomized design (CRD) with three replications. These were three species of small fish (tilapia, cat fish and common carp) with two types of preservatives (blend ratio and red pepper) and three methods of drying (sun, smoke and oven drying). The controls were fresh and dried small fish with no treatment.

(a)   Sample Preparation

Small fish preparation: The process of small fish preparation was carried out immediately after sufficient experimental fish were obtained. After the small fish were collected, evisceration was conducted by cutting the lower belly of the fish and opened to allow for drying as a whole based on the following procedures: The whole small fish cut into steak (cross-sections taken from a dressed fish). Trimmed small fish steak was cut into flat strips of width x thickness x length of cm, according to FAO [32; 31] standard procedures on sizes suitable for domestic use.

Blend ratio preparation: The cleaned garlic and ginger were chopped and minced before being used for the treatment. Fresh blend juice was prepared from the both grinded types of preservatives with  equal ratio by adding 250 ml of distilled water to 1 kg minced blend and press under 4 fold cheese cloth for the treatment based on FAO [32; 31] for traditional meat drying of Qwanta. The dilution of juices then was filtered using 4 fold cheese cloths (muslin cloth). The filtrate of juice was collected in air tight bottles and stored in refrigerator at 4oC until they would used for the treatment Nduagu et al [77].

Red pepper/Chili juice preparation: The clean chili was chopped and minced Salam et al, [83]. The minced chili was then crushed and mixed with 250 ml of water per kilogram to get the diluted chili juice. The dilution of chili juice then was filtered using 4 fold cheese cloths. The filtrate of chili juice dilution was collected in air tight bottles and stored in refrigerator at 4oC until they would used for the treatment Nduagu et al [77].

Pre-drying treatment of fillets: The steaks of small fish samples about (1000 g) of each fish type was submerged in 1000 ml (1:1 w/v) of blend ratio juice or chili juice in flat bowl of 2000 ml capacity Suleiman, [87] and Wilson [97]. The steaks of small whole fish lots were uniformly treated in their respective juice by turning them up and dawn for 10 minutes. The treated samples were dried using sun, smoke or oven. Another 5 kg steaks of each fish type was prepared without treatment to be dried in sun, under smoking or oven as control Wilson and Lawrie [97]. One fourth kg of each of the fish type was stored in deep freeze (-200C) until needed for analyses based on the UK’s recommended storage temperature. Every material used for slicing, treatment and drying activities of the small fish was properly cleaned and washed with cold running water followed by washing with hot water.

(b)   Drying of Small Fish

Sun drying: The sliced samples were loaded on flat tin sheets which were ready for the drying purposes of slices outside in sun. Uniform arrangements of slices were made with no surface contact between the neighboring slices. Each piece of fillet was placed at least about 10-15 cm far from the other for good drying. Drying was conducted with the tin sheets on top of the surface FAO, [32] and [31] and raised about 1 m above the floor allowed air to circulate underneath it. Drying process in sun was conducted until the slices were dried to desired level as determined by physical inspection. The whole set up of drying facility allowed to bring indoors overnight and covered properly. After the slices were dried, they were packed in polypropylene plastic bags and stored at room temperature at Abobo District in Gambella region. The fresh and dried samples were transported to Addis Ababa for micro-mineral and heavy metal absorption including microbial detections.

Smoking: Sliced fillets were sparsely spread into each treated drying tin sheets. Fork was used to scramble half of them and left the rest with the moisture intact. Dried the fillets under smoking and checking to make sure that the smoke was white using physical inspection. Finally the dried samples were packed in polyethylene bags and stored at room temperature to check its shelf-life AOAC [6].

Oven drying: The oven drying was done with the use of thermostat controlled oven of South African model (model 220). The oven drying temperature to dry the small fish samples was adjusted to 125°C for 3 hours based on the recommendations of Food Chemistry Laboratory Manual for food samples Bultosa [12]. Space of 1-1/2 inches on the sides, front, and back of the trays was allowed so that air can circulate all around them in the oven AACC[1]. After the slices were dried, they were stored in packed form in water proof plastics to be stored in ambient condition at the study area  AOAC[6].

(c)   Data Collection

(1)    Determination of element concentrations

The concentrations of Ca, Fe and Zn were determined by flame atomic absorption spectrometry (FAAS) making deuterium lamp the background correction for the detection Desta et al. [27] and Deribe et al. [21]. In addition to this vitamine-A was tested with HPLC. The concentration of Hg was analyzed by cold vapour atomic absorption spectrometry (FIMS 100, PerkinElmer, USA) at a wavelength of 253.7 nm. Working standard solutions for system calibration and control of analytical accuracy was obtained by dilution of stock solutions (1,000 mg l-1, Merck, Germany) using purified water (18 MO cm-1) and analytical grade HNO3 (GFAAS) or HCl (HGAAS, cold vapour AAS). Precision for determination of concentration was better than 10%. Although certified fish reference materials allowing testing of analytical results in a natural matrix was not applied, reliability of measurements was not considered to be significantly restricted, due to the following observations: All samples were completely digested, and matrix effects were reduced due to careful pyrolysis during FAAS. Furthermore, liberation of elements from solution as hydrides or volatile metal (Hg) was also considered to significantly reduce matrix effects.

(2)    Human exposure estimates

Though Ethiopians were traditionally meat eaters, eating habits have been shifting in favor of fish in the areas  and  communities  where  there  was  a  regular  and  sufficient  supply. In those communities, annual fish consumption can exceed 10 kg person-1 FAO [31]. This value (00.19 kg week-1) was used for the calculation of the estimated weekly intake (EWI) of heavy metals through fish from Alwero reservoir. The EWI was calculated by multiplying mean concentrations in fish muscles (edible part, wet weight) with 0.19 kg. Measured dry weight values was converted to wet weight values by dividing the values by factor 5.9, as water content in the sampled fish species.

(3)    Microbial quality

In this study, microbial analyses were done to assess APC and presence of moulds in stored samples: The load test was conducted on fresh fillets as well as on dried ones at the beginning of the experiment. Similar tests were conducted on dried slices of small fish within twenty days up to the storage period of sixty days.

Sampling: In the first microbiological analyses, about 50g of three representative samples of the fresh and freshly dried slices were randomly taken. For the 1^st, 2^nd and 3^rd analyses, about 50g of slices from each of the fish types randomly sampled. Three representative fish products of each fresh in the first analyses and 21 dried samples for each fish types either dried on oven, under smoke or sun will be analyzed before storage. For each of the first, second and third analyses, a total of 63 samples of small fish were microbiologically analyzed. In general, overall 87 samples were microbiologically analyzed in this study.

Preparation of serial dilution: Sampling of the product lots for the microbiological analyses was done by aseptically weighing 25g from each sample type and diluting it with 225 mL of bufferd pepetone water ISO, [54], method 4833 for preparation of 1:10 dilution level. Samples were homogenized for 2 minutes using stomacher after placing in the diluents. Decimal dilutions 10-2, 10-3 up to 10-6 was prepared by transferring 1 mL of the previous dilution (1:10) into test tubes containing 9 ml of 0.1% peptone H2O ISO, [54] method 4833). All dilutions were thoroughly mixed before they were platted. Estimated numbers of colonies per gram of sample were calculated for APC according to Maurine and James [70] with the formula indicated below:

Formula: N = ΣC/ ((1 * n1) + (0.1 * n2)) * V * (d)

C was the sum of colonies on all plates to be counted;

n1 is the no of plates to be counted at the 1st dilution ;

n2 is the no of plates to be counted at the 2nd dilution;

v is the volume applied in each plate;

d is the dilution from which the 1st count obtained.

v  Microbial load count: Aerobic plate count (APC) was conducted by taking samples from the 1:10 dilution levels. Total numbers of moulds were counted by taking scraps from the colony counted under APC.

Aerobic plate count: Aerobic plate (APC) was conducted according to ISO [54] method 4833) using the pour plate technique. The estimation number of colonies per gram of sample was calculated according to Maurine and James [20].

 Counting of moulds: About 10% of typical colonies grown on APC agar was transferred and plated onto Sabouraud Dextrose Agar Medium. The identification and load of total moulds were estimated based on Libby, Maeda et al and Dagmar [65;64] respectively.

(4)     Sensory evaluation

The samples were evaluated using a seven point hedonic scale basis (7= like very much, 6 = like moderately, 5 =like slightly, 4 = neither like nor dislike, 3 = dislike slightly, 2 = dislike moderately and 1 = dislike very much) ES, [25], EU, [26] and Lawless,H.T. and Heymann, H.[63].

(d)    Statistical Analysis

Statistical analyses were conducted on all data collected to test for significance difference among treatment means. Analysis of variance was performed by one-way ANOVA procedures with statistical software (version SAS 9.1) and means were evaluated at the P<0.05 level of significance using fisher’s LSD and Duncan's new multiple range test (Gomez, K.A. and Gomez, A. A. [42].

In Gambella region, particularly in Coastal areas, little children are involved in catching and trading of capture small fish around Alwero dam. Children’s involvement in catching, processing and trading should be used as a real economic empowerment tool to the stakeholders around Alwero dam like other Asian countries where there was a lack of cow milk. A similar trend was observed in a study in Congo, small fish were used by stakeholders those catched by traditional basket trap methods. These fish are mostly for household consumption. The author pointed out these small fish for the high prevalence of malnutrition and suggested building capacity of sustainable aquatic resource management as one of the strategies to improve the current nutritional status of the poor. Fishing activities were an important customary practice for the local small children who spent their time around the coastal part of the reservoir as well as Baro-river to cope with food insecurity, especially during a food scarcity period. Lack of commercialization and increased migrants made it difficult for the locals to engage in fishing, resulting in increasing vulnerability to food insecurity. However, small-scale fisheries and under nutrition cannot be simply connected as a causal relationship because the nutritional status is determined by many factors. Fishing activities are only a small part which can influence some of the key factors which directly and indirectly relate to nutritional status. As the authors pointed out in this study, the high prevalence of under nutrition in fishing communities should be carefully analyzed by identifying their basic and underlying determinants. Nevertheless, small size fish fishing contribute to the mentioned nutritional micro-nutrients in rural poor households who do not have enough income and other assets to sustain themselves from agriculture. The present study was conducted to evaluate the effect of sun drying, smoke drying, oven drying and two types of preservatives (blend ratio and red pepper) juices on micro-nutrients, Hg contents, microbial quality and shelf-life of small tilapia (O. niloticus), flat head cat fish (P. olivaris), and common carp (C. carpio) fish fillets collected in Alwero dam, Abobo woreda, Gambella region.

(a) Microbial Load

Results of this study clearly revealed that microbial growth was increasing through the increasing storage period. Standards guidelines often use much lower bacterial population as indices of acceptability. In a recent European study by consumers, fish was assumed ‘‘not to be in a good enough condition to be stored for long’’ when total load of aerobic plate count were 106 cfu/g EU [26]. Enumeration of aerobic bacteria under aerobic plate counting and total mould estimation were conducted under this study to estimate microbial load both in fresh and dried stored fillets of the common three species of small fish.

(1) Enumerations of microorganisms in small fresh fish: The load of aerobic plate counts and total moulds of the respective fresh fillets of the three small fish species were indicated in Table 2.

Very few load under APC with no significant difference and absence of moulds under total moulds count were found in fresh samples that reported in log 10 cfu/g. The low mean values of APC and none of the total moulds count were observed in the fresh samples were attributed to mostly the degree of safety of the products and dilution levels (10-5 and 10-6) from which samples were used for plating. According to Maurine and James [70],suitable colony counting range is 25 250. When plates of all dilutions have no colonies it is reported as less than 25 colonies estimated count. Existence of few APC counts in fresh samples of milk and small fish species in table 2 may bedue to the handling and cross contaminations through the process of evisceration and filleting the fish. An estimation of the total number of microorganisms: named total load of aerobic plate count and total moulds enumerations of freshly caught fish were laid within (102-106 cfu/g) of an acceptability index in standards, guidelines and specifications EU[26]. Therefore, the results of the total aerobic plate counts and total moulds in present study agreed with this standard acceptability index. Similar results about these micro flora were also observed in reports of Huss et al., [46] and Gram and Dalgaard, [44] in which the number of microbials fitted the standards of load in shelf-life of fish (107-108).

(2) Aerobic plate counts in dried fillets of fish

The total load of aerobic plate counts within the interval of twenty days in sixty days stored products were presented in Table 3. In that the minimum load of aerobic plate counts recorded in sample, the safe the products were illustrated within the table 3. As compared to the load of Aerobic plate counts in the fresh fillets reported in table 3, an increase in aerobic plate counts (APC) was observed in the treated and untreated fillets over the storage periods (0-60th days). The maximum and minimum load of APC of zero day storage were recorded in untreated sundried tilapia and blend ratio treated oven dried cat fish with 5.38 and 4.60 log 10 cfu/g, respectively. After the storage of 20 days, the fillets were with the minimum 4.96 (blend ratio treated oven dried cat fish) and maximum 6.23 (untreated smoke dried cat fish) log10 cfu/g. Similarly, the maximum (6.71) and the minimum (5.53) log10 cfu/g after 40th day storage were recorded in untreated smoke dried cat fish and red pepper treated sun dried carp fish fillets. At the end of the 60th day’s storage the lowest (6.48) and the highest (7.57 log10 cfu/g) APC were recorded in red pepper treated sundried carp and untreated smoke dried cat fish, respectively.

There was significant differences (P<0.05) in load of aerobic bacteria between the untreated and treated dried fish fillets of the three species throughout the storage periods (0-60th days). However, no significant differences (P>0.05) were recorded between most of the blend ratio and red pepper treated fillets of the three species and higher load of bacteria was observed in untreated dried fillets of the respective species throughout the storage period of 60 days. These significant variations between the untreated dried fillets and all treated dried fillets showed that blend ratio and red pepper have inhibitory effects on growth of these microorganisms through drying of the small fish fillets of the three experimental fish species. All the treated fillets of small fish reach at 106 cfu/g after the 40th day storage and it can be concluded that it is not appropriate to stored for such a long period. The untreated sundried and oven dried fillets reach points of sensory rejection at 20th and 40 th days storage period were good evidence among the reasons of this research work that they did not allowed to taste by panelists after the storage of sixty days. This was based on a recent European study by consumers, in which fish was assumed “not to be in a good enough condition to be stored for long” when CMT were 106 cfu/g EU [26]. After the 60th day storage period the load of aerobic bacteria in some samples reached 107-10 8 cfu/g. At the point of sensory rejection, the APC of small fish products could typically be 107-108 cfu/g EU [26].Therefore, this result showed that differently treated fillets have a shelf-life of around sixty days storage period. Nevertheless, standard guidelines and specifications often use much lower CMT as indices of acceptability.

(3) The load of total moulds on dried fillets of small fish stored for sixty days

All of the fillets from the 3 small fish spp had total number of moulds count of >0.72 log10 cfu/g (Table 4) where as for total load of moulds in all the treated freshly dried fillets had with the value of ND. The maximum and minimum load of moulds after the storage of 20 days were recorded in untreated smoke dried tilapia and red pepper treated oven dried cat fish with 4.59 and 2.41 log10 cfu/g respectively. After the storage of 40 days the fillets were with the minimum count of mould of 3.44 (pepper treated oven dried tilapia) and maximum of 5.60 (untreated dried tilapia) log10 cfu/g. Similarly, the highest (6.95) and the lowest moulds (5.45) log10 cfu/g counts after 60th day storage were recorded in untreated dried tilapia and pepper treated oven dried tilapia respectively.

Results revealed that significant (P>0.05) differences were observed in total load of moulds between treated and untreated dried fillets of the three species through out of storage periods of sixty days. No Significant differences (P<0.05) were observed between most of the blend ratio and red pepper treated dried fillets of the three species through the storage period of 60 days. The Significant (P>0.05) variations between the treated and untreated sun, smoke and oven dried fillets showed that, the pre-treatment technology in fish preservation have necessarily inhibitory effects on the number of total moulds counts. The result also showed scrupulous hygienic measures during handling and preparation of spice dilutions before applying them on filleted fillets. All the fillets after the 60th day storage period should not be stored further by taking the increase of the total moulds as warning signals. The fish fillets approached at the point of sensory rejection of 10 7 -108 cfu/g after the storage of 60 days showed that shelf-life of differently treated fillets should be less or equal to sixty days storage period Gram and Dalgaard [44]. Results in present study indicated that samples treated with blend ratio and red pepper juices were microbiologically stable than the control samples as these had longer shelf-life and were not covered by visible moldy mass of mycelium during 60 days of storage within twenty days interval of tests. There were steady increases in mould counts as storage period progressed in all the treatments. However, treated fillets showed lower mould counts as compared to untreated fillets in their respective drying methods. Therefore, combination of preservatives with drying resulted in variations of microbial levels (i.e. moulds). According to Jallow 1995, it was reported as a shelf life of dried fish storage was 3-9 months when stored properly. The differences may be due to the influence of environmental factors. Almost all the recorded results about total moulds before storage were < 1 log10 cfu/g.

The observation of large visible mould mass of mycelium from the first twenty days storage under ambient condition obtained in untreated fillets indicated that effectiveness of blend ratio and red pepper as anti-fungal agents which resulted in extended shelf life of treated fillets. Appropriate treatment with blend ratio and red pepper gave lower load of moulds than untreated sun, smoke and oven dried samples that could extend shelf life of the small dried fish. Due to this, the microbial populations (moulds) for all the treatments observed in this study were within the recommended limits for good quality fish product according to ICSMF[47;48]. Significant (P<0.05) variations in moulds were observed between untreated sundried and the rest dried fillets of experimental fish within 20 days interval until the 60 days storage. The untreated sun, smoke and oven dried fillets approached point of sensory rejection after forty days storage. However, treated fillets were not reached even if after 60 days storage periods. This showed effectiveness of the blend ratio & red pepper extracts were antimycotic agents Magawata & Shina [68] with the load of total molds in appropriate suitable range for consumption with 4.57 to 5.23 log10 cfu/g.

(b) Acceptability of Samples

Sensorial acceptability was conducted based on the 7 hedonic values. These were 7= like very much,6 = like moderately,5 =like slightly,4 = neither like nor dislike,3 = dislike slightly, 2 = dislike moderately and 1 = dislike very much. Results were discussed as follows.

(1) Acceptability of Fresh & Dried Samples before Storage

Sensory evaluation of the fresh samples of the three fish species and the milk samples resulted in scores between 6 (like moderately) and 7 (like very much) in a hedonic scale of 7 points, for all the sensory parameters over the dried fillets (Table 5). This showed that freshness makes a major contribution to the quality of fish or fishery products. Quality is a function of freshness; freshness is essential for quality but is not by itself a quality factor based on these sensorial analyses Zambuchini [63].

Where, LSD= least significant difference, trtmnt=treatment,DM=drying method,CV=coefficient of variation, Spp. = species, T= tilapia, Ct=cat fish, Cr=common carp, C=control samples, Br=blend ratio, Rp=red pepper, SD=sun drying, SmD=smoke drying, OD=oven drying, C=dried control, Ctt=fresh control, Cl=fresh milk sample and the values are mean ±SD in that the mean values followed by the same letter in a column are not significantly different at 5% level of significance.

Significant (P<0.05) differences have been noticed between the treated and untreated samples where as no (P>0.05) variations was observed mostly between the fresh and treated samples of the three species in all the parameters considered. No significant (P>0.05) differences was recorded between the fresh small fish of the given three species and the fresh milk sample in all sensorial parameters. The highest scores were observed for all parameters in fresh samples relating to the dried samples as discussed in Table 5. All the untreated dried fillets of the three fish species showed the lowest scores with no statistical difference in most of the parameters.

The picture of the sensory scores for overall acceptability is almost the same for treated samples as well as untreated samples. The story is almost the same regarding other sensory parameters such as color, odour, taste and texture as reported in Table 5. Generally the scores of the fresh and dry fillets were greater than 4.44 considering all parameters before the storage, i.e. in the liking ranges.

(2) Effect of Treatment Combination on Sensory Score of Dry Fillets after storage of 1 month

Organoleptic attributes of appearance, odor, taste, texture, and general acceptability of the fish samples were evaluated by consumer panelist, selected from among the local people in Gambella at Abobo District in Gambella region. A 7-point hedonic scale was used with 7 for like very much, down to 1 for dislike very much (Lawless and Heymann 1998). For the evaluation, presentation to the panelists about the samples was conducted at area. The sensorial attributes of the tested fillets by the panelists after the one month storage period were illustrated in Table 6 below. The fillets were subjected to different treatments combinations of drying method, preservatives and fish species and subjected to sensory evaluation before storage at ambient condition. The scores of all sensory parameters varied from 3.52 to 6.22 hedonic values. All these scores remained between dislike slightly and light moderately among the seven hedonic values. It revealed that the control samples (untreated sun, smoke and oven dried fillets) received lower panel scores than blend ratio and red pepper juice treated fillets of the three species with regards to color, odor, taste, texture and over all acceptability. There were significant (P<0.05) differences between most of the untreated dried fillets of all fish species and treated fillets for all of the parameters. However, some of the dried samples were with no significance differences (P>0.05) among the untreated and treated dried fillets. Result of the present study showed blend ratio and red pepper enhances the palatability/ flavor for consumers in the sensory evaluation. Similar results were obtained by Sallam et al [82] which was around 4.21 as the minimum value in their research Lawless, and Heymann [63] and Lawrie [64].

(3) Sensory acceptability test results of dried fish fillets after the storage of 2 months.

The results of sensorial analysis of dried fillets after the second month storage period were presented in Table 7. The dried fillets after the second month storage were evaluated by panelists get the values within 3.36 (dislike slightly) to 6.02 (like moderately) hedonic values. Consumers noted significant (P<0.05) differences in color, odor, taste, texture and overall acceptability between the untreated and treated dried fillets of fish. Blend ratio treated smoke dried tilapia and red pepper treated oven dried the same species were with the highest mean (6.00) and (6.02) scores for color respectively, whereas untreated oven dried fillets of the same species had the lowest mean (3.36) score for the attribute taste. There were no significant (P<0.05) variations among the most untreated and treated dried fillets for the individual sensorial parameters. All the sensorial values were in their steady reduction up to the second month relative to values recorded before and one month storage in all of the respective attributes. The results obtained were similar to the report by Ikeme and Bhandary [50] in which treated samples were generally more preferred over the untreated samples. According to Ihuahi et al [49] report, similar result in which the treated samples with preservatives recorded highest acceptability. No differences (P>0.05) were observed between most of untreated dried fillets of three species for the individual attributes: color, odor, texture and overall acceptability. There were steady decreased values in all sensorial parameters through progressive storage periods of 2 months

Panelist found or preferred the flavor of the blend ratio and pepper to the combination of allicinic and sharp effects as well as pungency flavor of the treated fish fillets respectively. The OA scores decreased as storage time increased to 2 months in all the fillets.

(4) SA of Dried Fish Fillets after Storage of three Months

The consumer acceptability of the dried three months stored fish fillets were presented in Table 8. The dried fillets after the three months storage period were evaluated by panelists get the values within 3.04 to 5.98 means from the hedonic values. There was significant variation (P<0.05) in some of the tested parameters.

All sensorial attributes throughout the storage periods were observed in Tables 5 8 in this study. Panelists were not allowed to evaluate taste panel after the third month storage period because of the microbial load tested after forty days storage indices. This was done due to the ethical clearance about the products that given to the consumers based on closer microbial load to the point of sensory rejection of 107-108 cfu/g after forty days storage period EU [26]. The panelists score for the dried fillets of fish on the 90th day storage period is closed to the flavor and degree of rancidity of the product. From the scores, the panelist detected rancid odor almost in all the control fillets (sun, smoke and oven dried). The detection of rancidity in the control sample was as a result of increase in lipid oxidation, and this affected the flavor and overall acceptability of the treated and untreated experimental fish fillets. The overall acceptability scores decreased while the storage time increased in all the fillets of fish, and this agrees with the findings of Idris et al. (2010).

(5) Effect of Storage Time on SA of Dried Fillets

The effect of storage time on sensory acceptability of dried small fish fillets is illustrated in five different figures as follows. Figure-1 presents effect of storage time on sensory acceptability of oven dried small fish whereas figure-2 reports effect of storage time on SA of sundried small fish. Furthermore, figure-3 states the effect of storage time on sensory acceptability of smoke dried small fish. However, figure-4 and 5 revealed that the effect of spice and small dried fish species on storage period of the three months respectively.

Figure-1 Presents Sensory evaluation scores of oven dried fillets of small fish stored for three months. Immediately after drying the sensory scores of color, odor, taste, texture and overall acceptability were between 5 and 7 in a scale of seven. As the storage time increased, the values decreased for all the attributes reaching the minimum but above 4.5 by the end of third month. The reduction values of the attributes as the storage time increased, made the samples were not allowed to taste by panelists at end of the third month based on microbial guidelines. Therefore, most samples were more sensitive to spoilage as they scored the lowest values of all attributes starting from the beginning. However; the samples were still acceptable after the storage of three months except for the taste attribute.

Figure-2 shows the sensory evaluation score of sundried fillets stored for the same duration. The score of the sensory attributes and of the overall acceptability were between 4 and 6 in a scale of seven showing similar reduction as compared to those of the oven dried samples. The scores of all the sensory attributes reduced as the time of storage increased reaching as low as score 4. The values of all attributes showed the lower sensory score starting from the beginning and reduced onwards in sensory acceptability of the sundried fillets as compared to that of the oven dried samples as the storage time increased. In three months of storage the samples were not liked by the sensory panelist.

All the sensory attributes of the samples dried by smoking were the highest at 0 months storage as compare with the previous two drying methods which were scored 6 and above in analyses. Oven drying is the second next to the smoking and sun drying is the third when the three methods of sample drying were compared each other at the time of storage. Similar trends were observed as samples were stored at ambient condition with the rest two methods in storage of smoked dried samples.

The two treatmental spices such as blend ratio of garlic with ginger and red pepper juices had with the highest point of hedonic scores and with more acceptances by panelists than the one which was not treated by spices. Moreover, the reduction in values of sensory attributes, treated samples was very slow as compare to the untreated three months stored samples. Therefore, the blend ratio and red pepper juices had more enhanced even after the storage in sensory acceptances than the dried stored control samples tested with no treatment. It is recommended to use these locally available spices before the preservation of the small fish species around the area of the study.

(6) Effect of Fish Species on Overall Acceptability of Dried Stored Fillets

Figure-5 shows the overall acceptability rate of the dried fillets during the storage periods. The acceptability reduced with increase in storage time for all the three fish species; however species carp exhibited relatively higher score than the other two species while species flat head cat fish received the lowest score around the third month storage time. The two species such as tilapia and flat head cat fish had similar values before and up to the storage of 2 months. The point of SA was steadily in reduction through the storage of the three months as indicated in figure-5 as follows.

All the overall acceptability ratings of the dried fillets of the three fish species remained above 4 which indicated a liking response of the three months storage period. Generally drying can preserve the fillets of the three small fish species to acceptable status by the consumers up to three months storage time.

(c) Effect of drying methods and application of preservatives on micro-nutrients & heavy metals of small fish

‘Food security is a condition when all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life’. This definition includes the nutritional aspect which is described as ‘access to nutritious food to meet their dietary needs’, however, this review uses the term ‘food and nutrition security’ to emphasis the support to access and appropriate utilization of micronutrient-rich foods, including the process through which they are cooked and absorbed in the body, and then used in physiologic functions at individual level. The terms, ‘small fish’ is distinguished in this paper, based on length. The fish under 25 cm at maximum size are basically categorized as small fish, except some fish species which the maximum matured length is over 25 cm but small individuals of these species are most often consumed Roos[79].Small fish (‘’Adappe’’ or ‘’Dey Rech’’) are a good source of Ca, Fe, Zn, and Vit-A in daily human consumption. Moreover, ‘’Adappe’’ or ‘’Dey Rech’’ are milk as it proved in this paper and cited by different authors in different countries. However, major problems related with small fish processing and drying are loss of quality due to contamination that affects micro-nutrients and shelf-life of small fish in HH consumption Sablani et al.[81;82]. Lack of small fish preservation activities are seen for year round supply for perennial alleviation of malnutrition and food insecurity. ‘’Adappe’’ for ‘’Kac’’ or ‘’Dey Rech’’ for ‘’Dak Mithini’’ are also more associated with heavy metals than large fish Larissa D. et al [61]. Therefore, upgrading the traditional fish processing technology and adoption of preservation methods with specific antioxidants and appropriate storage periods are needed in the region to minimize the dried fish problems. Application of herbs, spices and their essential oils with anti-microbial effects compared to synthetic additives should be practiced at the area. Table 6 below summarizes the studied micro-elements and Hg contents in small fresh samples:

The prevalence of osteoporosis in developing countries is low compared to most industrialized countries despite an apparent low Ca intake. It is possible, however, that food surveys have overlooked important Ca sources in developing countries. Small fish eaten with the bones can be a rich source of Ca, even though Ca from bone may be considered unavailable for absorption. In the present study, absorption of Ca from indigenous Bengali small fish was compared with the Ca absorption from milk. As indicated in the table 6 there is no significant (P>0.05) variation between the Ca content in fresh zebu cow milk and all the three species of fresh small fish which positively encourage the hypothesis in this work. Therefore, it ensures the small fish those consumed as a whole are milk especially for children around Gambella region, as there is an access of these products. This was similar result with Larissa D. et al [61]. Milk sample have different results as compared to some of the results of Fe and Vit-A except of that Zn. all small fish species in these results have high calcium content having >800 mg/100g raw, edible parts. This is strongly agreed with the results reported by Roos [79]. The calcium in fish bones has the same bioavailability as milk (Hansen et al.[45]; Larsen et al.[45]. Therefore, small fish consumed with bones are important as a source of calcium, especially in populations with low intakes of milk and milk products. Small fish are rich by these important minerals when eaten as a whole. Bones of small fish are easily consumable, concentrated with Fe in the head and viscera with sufficient source of Vit-A around eyes for human consumption. The presence of heavy metals in the environment is a cause for major concern. Even at low concentration levels, they can cause problems as a result of toxicity and their tendency to permeate the food chain Folandifard R. and Ebrahim A. [36]. The term ‘heavy metals’ refers to metals whose specific gravity is greater than 5 g/cm3 in their standard state Cruz GC.et al. [16]. Fish accumulate metals by ingesting particulate material suspended in water, through food, through the ion-exchange of dissolved metals across lipophilic membranes (e.g., the gills), and through adsorption onto tissue and membrane surfaces Squadrone S. et al [86]. Fish are considered bio-indicators of the quality of ecosystems because of their ability to bio-Concentrate and integrate a contaminant load Taghavi Jeldar H. et al [88]. Heavy metals may enter the human body through the consumption of fish and thereby create a serious health hazard Sen I. et al. [84]. The measurement of heavy metals in fish can be useful to assessing potential health risks to humans associated with the consumption of fish Ahmad AK and Sarah AA [2]. The present study was undertaken to determine the levels of Hg in the three most frequently used fish caught from Alwero dam fresh waters. As a result of this study, consumers and authorities can become better aware of the possible risks of contamination through daily intake of fish. Mercury is a heavy metal therefore it should not exceed the following standards used by different countries because there is no standard hazard element in our country. Table 10: Guidelines of JECFA, MAFF, the European Commission and China’s national regulations regarding heavy metals (mg/kg)

Source: Environmental Health Engineering and Management Journal (2016)

Therefore, the concentration of the heavy metal (Hg) in this study in which the samples were taken in Alwero reservoir was below the standard hazardous load. So, it is strongly advice as expert for the stake holders in Gambella regional state in general and Abobo woreda in particular that the small fish which is rich in Ca and other minerals are safe from this toxic metal.The effect of sun drying, smoke drying, oven drying and two types of preservatives (blend ratio & red pepper juices) on micronutrients of tilapia (O.niloticus),cat fish (P. olivaris), and common carp (C. carpio) fish fillets was as follows.

(1) Effect of Drying on Micronutrient Composition of Small Fish

The micro-nutrient composition of fresh and dried fillets of the three small species of fish namely cat fish, carp and tilapia are presented in Table 11. The Ca contents of the fresh cat fish, fresh carp and tilapia were 1199.70, 1154.65and 1140.16 mg per 100 gm respectively. There was no significant (P>0.05) differences in calcium content between the milk samples and that of fish samples. Therefore, the hypothesis of this paper work is true which status Ca in milk is equivalent to Ca in small fish cited in different countries. For instance, small Bengali fish (397 mg Ca in total) and skimmed milk (377 mg Ca in total). There was no significant difference b/n the two M. Hansen et al [67]. The calcium contents in all the three species of fish samples were higher than the fresh samples as indicated in table 11 with no significant (P>0.05) differences statistically. These were due to the loss of water during drying the samples to the minimum moisture level makes the micronutrient to be concentrated in composition except vitamin-A because vitamins are sensitive due to light and may lost as the samples stay for long time in ambient condition. This is why Significant (P<0.05) variation was observed in vitamin-A content between the fresh and dried samples of the three species of fish. Milk sample was with similar results of all the dried samples of the three species of fish in vitamin-A content.

Carp was with higher in mercury both in fresh and dried form samples with significantly (P<0.05) different as compare to the rest two species. This was due to that carp was mostly been in deeper area of the water body where it lives and stays within mud. However, since mercury is a heavy metal the estimated weekly intake therefore is below the standards used by different countries as indicated in table 10 EHEMJ, [24]. The Hg content of each of the three species different forms (dry and fresh) was similar as indicated in table 11. Milk sample was significantly (P<0.05) different in Hg content from the fish samples except from the samples of cat fish. As In general, there were an increased content of micro-nutrients as the drying methods reduced the moisture content in fish samples to the lower extent. These results were agree with the results of Mohammed Malakootian et al [75] in which their results were laid between the range of 0.065-0.08 mg/100gm.  

(1)   Effect of Drying Methods interaction with the species of fish on Micro-nutrients Composition of Small Fish: Significant (P<0.05) differences in calcium content were observed in smoke dried samples in relation to the rest two methods of drying methods. This may revealed that additional Ca absorption within the smoke was occurred during the smoking of fish samples. The Ca content obtained from the smoke dried (2529-2600mg/100gm) samples were higher than the results reported by Kawarazuka N. [57] (776-1061 mg/100gm) which is almost similar to the results obtained from sun and oven dried samples as illustrated in table 12.

The contents of Fe in this paper were slightly higher values whereas closely the similar zinc (6.6-20.20 mg/100gm) contents were observed as compared with the results of Kawarazuka N. [57]. No Significant (P>0.05) differences were recorded in most of the samples in micro-nutrients between the three species of fish dried by the three methods of drying in interaction of method of drying with the fish species. The content of vitamin-A is lower than the results reported by Kawarazuka N. [57] due to the variation of species of fish and type of water resource. The values of Hg were below the standard requirement that stated as estimated weekly intake in human consumption.

(3)     Effect of Drying Methods interaction with the preservatives on Micro-nutrients Composition of Small Fish

The effect of the interaction between the method of drying and kind of preservatives on micro-nutrients and the given heavy metal will be discussed as follows in table 13.

(4)     Micro-nutrients and heavy metal results of dried fish fillets before storage

All the results of micro-nutrients including heavy metals analyzed from the dried samples before storage were discussed in table 14. Significant (P>0.05) differences in Ca contents between the treated and untreated dried fillets in all the three methods of drying was observed. Furthermore, Significant (P>0.05) differences in Ca contents between the blend ratio and red pepper juice treated smoke dried and samples treated with the similar treatments in other methods of drying was recorded. Therefore, this implication showed that smoke drying had with great influence than other methods of drying. The contents of Fe, Zn and vitamin-A in treated dried fillets of the three species of fish were slightly higher than those of untreated samples in all of the three methods of drying. This is the fact that the composition in combination of the fish samples to with that of preservatives make the values higher than those of untreated samples. moreover, the content of Hg is lower than that of standard maximium refereed in previous table 10.That mean this paper work indicate that the small fish ‘’Adappe’’ for ‘’Kac’’ or ‘’Dey Rech’’ for ‘’Dak Mithini’’ were advisable and free from heavy toxic metals those caught from the Alwero reservoir for human consumption as they dried before they store at ambient condition in Abobo district in Gambella region.

(5)     Micro-nutrients and Hg results of dried fish fillets after the storage of one month

Calcium is necessary to give strength to bones and teeth. When our dietary intake of calcium is greater than our bodies' requirements some of the excess calcium is stored in our bones. When our day-to-day intake of calcium does not meet requirements, the calcium stored in bone becomes available to meet this shortfall. Calcium has other important roles. It is essential for normal clotting of blood and is a vital link in transmission of nerve impulses. It is also an essential element in enzyme regulation, in the secretion of insulin in adults, and in regulation of muscle function. During periods of growth the demand for calcium is greater than usual, although some calcium is incorporated into bone at certain other stages of life. Thus children, adolescents and pregnant and lactating women need additional calcium. Adults continually need to replace calcium that is lost from the body in urine and faeces and to a lesser extent in sweat. The results of Ca values in this research were highly related with the recommended daily dietary intake of Ca in Australia in which the intake in infants (1100mg), Children (1200mg), Adult men (300-500mg), Adult women (700-1200mg), Pregnancy (3rd trimester) (800mg) and lactation stage (800-1000mg) Kawarazuka N. [57]. Therefore, taking consideration the easily available small fish‘’Adappe’’ for ‘’Kac’’ or ‘’Dey Rech’’ for ‘’Dak Mithini’’ in every day meals to enhance the sufficient Ca intake to the body as well as to the health conditions in general. Now take a look the contents of these micro-nutrients and Hg contents after the storage of one month as illustrated in table 15. All the micro-nutrients stated in table 15 showed that slightly reduction in contents due to the moisture absorption at the time of storage at ambient condition of the woreda. However, the content of Hg was varying as indicated in table 15 after the storage of one month.

(6)     Micro-nutrients and heavy metal results of dried fish fillets after the storage of 2 months

Small fish are rich in micronutrients, in particular, vitamin A, Ca, Fe and Zn, as they are consumed as a whole with bones, heads and viscera where most micronutrients are concentrated. These species are commonly consumed by the poor, and thus have a high potential to address micronutrient deficiencies. These minerals were reduced in values from the previous 1st month storage.  Most of the vitamin A in fish is concentrated in eyes and viscera (Roos, 2001), therefore fish species which are eaten as whole are an important source of vitamin A. In Alwero dam, 3 small fish spp, tilapia, flat head cat fish and carp were identified as having appropriate content of vitamin A, >0.02 µg/100g raw edible parts with no (P>0.05) significant variation between all the treated and untreated dried samples after the storage of two months illustrated in table 16. These results of vitamin-A were lower than from results obtained by Roos et al.[79;80] in Cambodia due to species variation and type and variation of water bodies. Vit-A is a fat-soluble vitamin, therefore about 3-5g fat/meal are required to enhance absorption of vit-A Castenmiller & West, [13]. Fish which are rich in vitamin A, cooked with some vegetables with some vegetable oil give an ideal combination to maximize vit-A intake. Unlike other minerals and water-soluble vitamins such as vitamin C, vit-A can be stored in the liver for 3-4 months Olson[78]. Therefore, vitamin A rich fish only consumed in a particular season are still effective to meet the nutritional needs for a longer period. A similar trend with the previous month for the content of Hg was seen after the storage of 2 months.

(7)      Micro-nutrients and Hg results of dried fish fillets after the storage of 3 months

Consequently, the minerals were with steady reduction onwards storage of dried small fish which are eaten with bones that contribute to Ca intake in mg/100gm which is equivalent to Ca in milk in mL. However, all small fish ’’Adappe’’ for ‘’Kac’’ or ‘’Dey Rech’’ for ‘’Dak Mithini’’ spp have high Ca content such as tilapia, cat fish and carp, having minimum of 967.49 mg/100gm after storage of 3 months. The results of Ca values were similar to data reported by Roos [79] having a value of Ca 800 mg/100g of whole edible parts even if after the storage of 3 months. The Ca content of these fish is the same to that of milk & Ca in fish bones has the same bioavailability as milk Hansen et al. [45] & Larsen et al. [45]. Therefore, small fish consumed with bones are important as a source of Ca, especially in populations with low intakes of milk & milk products. Based on this study there are possibilities that small fish of the 3 spp found to have a high content of Fe in its dried edible whole parts, after using traditional drying methods. Although, there were reduction in amount of Fe through storage time to 3 months, the Fe values  after storage of 3 months still high & sufficiently enough as nutritional intake. Serving of the traditional sour soup made with small tilapia (O. niloticus), eaten mostly with pourage & sometimes with rice, the most common meal in the study area. Furthermore, the composition of Fe in fish is different from that in plant-source foods, containing large amounts of haem Fe & high molecular sub‑pool of complex‑bound non‑haem Fe, with a higher bioavailability than non‑haem Fe. Zn content after storage of 3 months was enough in amount to be as source of foods from animals (fish), in micro‑nutrient context as dairy products, while cereals & legumes  contain  inhibitors  of  Zn absorption,such as phytic acid (International Zn‑nutrition consultative group; 2004). 

Because habitual diets of  the poor dominated  by staple foods reduce bioavailability of Zn, little Zn intake is expected from  such diets.Yet, daily Zn requirement in women in 3^rd semester pregnancy & lactating  with low  bioavailability diet is as high as 20 mg/day (WHO/FAO [95].

Children with low zinc bioavailability diet are required 8.3-11.2 mg/ day depend on the body weight. Small fish after the storage of three months eaten as whole are very rich in zinc compare with other animal source foods and large fish species. Zn content after the storage of three months was laid between 4.85-11.51 mg / 100g of small fish commonly consumed by poor people in area. These results were similar to the results reported by Kawarazura N. [57] and Roos [79] with 6.6-20.20 mg / 100g and 20.3 mg / 100g values of Zn respectively.  As requirement is very high, it is difficult to meet unless a significant amount of animal-source foods are taken every day (unlike vitamin A, zinc cannot be stored in human body WHO/FAO [95] and is therefore needed from everyday diet). In this respect, adding small fish, even a small amount to a plant based diet can greatly increase zinc intake and compensate for the low bioavailability induced by the phytic acid of the staple foods. Overall, small fish are rich in micronutrients and consumed frequently in the everyday diets, contribute to the intake of multiple micronutrients from a meal. Therefore, utilizing locally available small fish has the potential as a food-based strategy to enhance micronutrient intakes. These was not only the recommendation of this paper work Gibson et al. [41] also noted that adding small dried fish to plant based diets can enhance the content and bioavailability of iron, zinc and calcium. In Malawi, according to the data calculated from the food composition, serving 24g of the small dried fish consumed whole with bones, in a meal, twice a day, can lead to significant increases in iron, zinc, and calcium intakes to meet the needs of children Gibson et al.[40]. Small fish also have many advantages because they can be available for a long period by processing in peak seasons and storing them for year-round consumption, are more affordable for the poor as they are purchased in small portions, and can also be more evenly divided between household members Thilsted et al .[90]. Similar trends were seen for the contents of Vitamin-A and Hg with the previous months analysis.

(a)      Summary

This study was conducted to assess the effect of drying methods and pre-treatments on shelf-life, micro-nutrients & Hg content & sensory quality of dried fish. The experiment was conducted in factorial arrangement of 3×2×3 with 3 drying methods 2 preservatives treatment & 3 small fish (tilapia, flat head cat fish and carp) sp for food insecurity ‘’Adappe’’ for ‘’Kac’’ or ‘’Dey Rech’’ for ‘’Dak Mithini’’  laid out in Completely Randomized Design (CRD). Fresh fillets were analyzed for their micro-nutritional value (Ca, Fe, Zn & Vitamin-A), content of Hg, sensory (color, odor, taste, texture & OA) and microbiological quality (total APC & total load of moulds). The dried fillets were stored at ambient condition & the samples were analyzed for the micro-nutritional quality, heavy metals & sensory acceptability at 1 month interval & for microbial status every 20 days starting from the end of drying operation. Fresh fillets & fresh milk & untreated dry fillet were used as control. In fresh samples, a high load of AB of 4.75 log10 cfu/g was observed on tilapia, and total mould count was not detected (ND) in all the three species of fish and milk sample. The total load of bacteria in fresh samples were 4.75±0.01, 4.47±0.01, 4.74± 0.27 and 4.51±0.23 log10 cfu/g  in  fresh  tilapia, cat fish, carp & cow milk respectively. Regarding freshly dried fillets, high load of aerobic bacteria (5.22-5.38 log10 cfu/g) with no (P<0.05) Significant difference was observed in untreated fillets of the three species whereas the initial load of moulds was <1.28 log10 cfu/g in all freshly dried fillets. After 60 days of storage, the maximum load of AB and moulds was 7.20-7.57 and 6.08-6.95 log10 cfu/g, respectively, in untreated fish fillets, higher than that in treated (6.48-6.69 and 5.45-5.81 log10 cfu/g) samples respectively for both organisms. The micro-nutrient compositions and the Hg content of the fresh samples were 1140.16-1199.70 mg/100gm of Ca, 20.40-34.33 mg/100gm of Fe, 4.75-6.77 mg/100gm of Zn, 0.07-0.18mg/100gm of vitamin-A and 0.01-0.11mg/100gm  of Hg contents. The Ca in fresh small fish (1140.16- 1199.70 mg/100gm) is equivalent to the Ca content in fresh milk (1192.33 mg/100gm). The content of micro-nutrients and Hg values after drying were laid between 967.85-5415.00 mg/100gm of Ca, 26.50-53.55 mg/100gm of Fe, 4.85-11.51 mg/100gm of Zn, 0.03-0.08mg/100gm of vitamin-A and 0.00‑0.11mg/100gm of Hg before storage. After the storage of 3 months, values of Ca, Fe, Zn Vitamin‑A & Hg were 967.49‑1187.84 mg/100gm,25.69‑53.28  mg/100gm, 4.13‑11.00 mg/100gm, 0.01‑0.06mg/100gm  and 0.00‑0.11mg/100gm, respectively. All the 3 drying methods made an increased in most of the micro-nutrients while reduction in vitamin-A values due to the sensitivity of it to the light. However, the values of micro-elements showed slightly steady reduction in every month through the storage period of 3 months due to the moisture absorption during the time of storage. The overall acceptability of treated & untreated samples reached the hedonic values of 5.28 (like slightly)& 4.62 (neither like nor dislike) respectively after the storage of 3 months. Fish was assumed “not to be in a good enough condition to be stored for long” when CMT were 106 cfu/g (EU, 1995). After the 60th day storage period the load of bacteria in untreated samples reached 107-10 8 cfu/g. this was the point of sensory rejection where the APC of fish products could typically be 107-108 cfu/g (EU, 1995).Therefore, this result showed that differently treated fillets have a shelf-life of less than 90 das storage period. Nevertheless, standard guidelines & specifications often use much lower CMT as indices of acceptability. The total load of bacteria (reached above 7.20 log10 cfu/g) in all untreated samples was the reason that why samples were not allowed for panelists for taste. This was the point of sensory rejection in which the number of microbial load should be below the 10⁷-10⁸ log10 cfu/g based on guide lines of EU [26]. Results in this study showed that micro-nutritional values of dry fish did not change much during the storage period of three months. The highest preferences of consumers were recorded in treated fish fillets than untreated ones. As the storage time of fish fillets increase, there was reduction of the sensory acceptability of the products through storage of 90 days.

(b)     Conclusion

The present study was conducted to examine the effect of drying methods on micro-nutritional composition, heavy metals, shelf-life and sensory quality of three small fish species commonly used in Gambella Regional state, Abobo District. It was to evaluate the effects of preservation methods and spice blend ratio on micro‑nutrients, heavy metals, shelf-life and sensory quality of small fish preservation. Traditional fish processing around Alwero dam in Abobo District produce cheap important and necessary micro-nutrients sources for the poor population. This is the strategic direction to alleviate food insecurity utilizing the small fish ‘’Adappe’’ for ‘’Kac’’ or ‘’Dey Rech’’ for ‘’Dak Mithini’’ were advisable and free from heavy toxic metals those caught from the Alewero dam for human consumption in Abobo district in Gambella regional State, Ethiopia. However, Upgrading the traditional fish processing technology and adoption of sun drying and smoke drying with locally available specific antioxidants and appropriate storage were needed in the region and in the woreda particularly to minimize quality defects related to dried fish. To solve the perennial problem of malnutrition in the area, the high post-harvest loses in fisheries need to be minimized or eliminated. Drying and drying methods did not affect the micro-nutritional values (Ca, Fe, and Zn) with reduction of Vitamin-A and variably change of Hg. Moreover, the application of blend ratio and red pepper juices treatments showed retardation in microbial content of small fish preservation. The numerated microbial loads were low enough due to drying and methods of drying with blend ratio and red pepper juice treatments than untreated fillets. Fresh fillets of the three small fish species had preferable sensory attributes than dried fillets. However, dried fillets were also between the hedonic scale of more than 5 (like slightly) and more than 6 (like moderately) had values with good acceptability having higher shelf-life than untreated fillets up to the end of the third month storage. Therefore, blend ratio and red pepper which are easily available at the place should be used to extend the shelf life of dried fillets. Moreover, important measures need to be taken to train local people in processing, preservation and utilization of local spices in hygienic practices.

1. AACC, 2000.Approved methods of American Association of Cereal Chemists,Inc.,St.P, USA.

2. Ahmad AK, Sarah AA. 2015. Human health risk assessment of Malakootian et al heavy metals in fish species collected from catchments of former tin mining. International Journal of Research Studies in Science, Engineering and Technology 2015; 2(4): 9-21.

3. Ahmed, M., Lorica, M. H. (2002). Improving developing country food security through aquaculture development - lessons from Asia. Food Policy, 27, 125 - 141. 00007-6

   View Article           

4. Akinneye, J. O., Amoo, I. A. and Bakare, O. O. 2010. Effect of drying methods on the chemical composition of three species of fish (Bonga spp., Sardinella spp. and Heterotis niloticus).

5. Al Sayegh Petkov?ek S, Mazej Grudnik Z, Pokorny B. 2012. Heavy metals and arsenic concentrations in ten fish species from the ?alek lakes (Slovenia): assessment of potential human health risk due to fish consumption. Environ Monit Assess 2012; 184(5): 2647-62. PMid:21713497

   View Article      PubMed/NCBI     

6. AOAC, 2000. Official Methods of Analysis of the Association of Official Analytical Chemists (14th Ed.), Washington, p.1193.

7. APHA, 1984. Microbiological examination of foods; parallel food testing in EU (fish, 1995).

8. APHA, 1992. Compendium of methods for the microbiological Examination of foods, 3rd ed., C. Vander dent, and splittstoesser, D, (Eds), APHA, Washington Dc 2: 1264 pp.

9. Askari SaryA, Khodadadi M, Mohammadi M. 2010.Concentration of heavy metal (Cd, Pb, Ni, Hg) in muscle, gill and liver tissues of Barbus xanthopterus in Karoon River. Iranian Scientific Fisheries Journal 2010; 19(4): 97-106.

10. Awira R., Friedman, K., Sanui, S. Kronen, M., Pinca, S., Chapman, L., Magron, F. (2008). Kiribati country report: profiles and results from survey work at Nggela, Marau, Rarumana and Chubilopi. New Caledonia: Pacifific Regional Oceanic and Coastal Fisheries Programme. Nyirenda, D., Husken, S., Kaunda, W. The nutrition fish supplementation on the response to anti retroviral therapy: a literature review. WorldFish Centre.

11. B. Zambuchini.2007/2008. Shelf-life and Molecular Traceability of Fish Products.

12. Bultosa, G. 2005. Food Chemistry Laboratory Manual with adjustable oven drying temperature levels for meat and fish samples, University of Haramaya, Ethiopia.

13. Castenmiller, J.J.M., West, C. E. (1998). Bioavailability and bioconversion of carotenoids. Annual review of nutrition,18, 19 ? 38. PMid:9706217

    View Article      PubMed/NCBI     

14. Conner and Hall, 1994. Shelf-life and quality of fish products.

15. Coppock, D.L. 1994. The Borana plateau of southern Ethiopia: Synthesis of pastoral research, development and change, 1980-1991. International Livestock Center for Africa (ILCA), Addis Ababa, Ethiopia. 299p.

16. Cruz GC, Din Z, Feri CD, Balaoing AM, Marie Gonzales E, Navidad HM.2009.Analysis of toxic heavy metals (arsenic, lead, and mercury) in selected infant formula milk commercially available in the Philippines by AAS. E-International Scientific Research Journal 2009;1(1): 40-51.

17. CSA, 2007. Census conducted by the Central Statistical Agency of Ethiopia (CSA).

18. Dagmar, N. 2013. Lab Manual of Veterinary Microbiology, University of Haramaya,Ethiopia.

19. Dagne Tarle, Mitiku Eshetu, Solomon Abera & Getahun Asebe, 2015. Global Journal of Medical Research: L Nutrition & Food Science: Effect of Drying Methods and Pre- Treatments on Shelf Life and Microbial Quality of Fish (Oreochromis Niloticus, Pylodictis Olivaris and Cyprinus Carpio) Species Commonly used in Gambella Region, Ethiopia.Vol 15 issue 1 version 1.0 year 2015.page 11.

20. Dagne Tarle, Mitiku Guya, Solomon Abera and Getachew Bekele Fereja, 2016. Turkish Journal of Agriculture - Food Science and Technology, 4(1): 22-30, 2016. Effect of Drying Methods and Pre-Treatments on Nutritional Value and Sensory Quality of Fish (Oreochromis niloticus, Pylodictis olivaris and Cyprinus carpio) Species Commonly Used in Gambella Region, Ethiopia.

21. Deribe, E., Rosseland, B. O., Borgstr?m, R., Salbu, B., Gebremariam, Z., Dadebo, E., et al . (2011) . Bioaccumulation of persistent organic pollutants (POPs) in fish species from Lake Koka, Ethiopia: The influence of lipid content and trophic position. Science of the Total Environment, 410?411, 136?145. PMid:21978619

    View Article      PubMed/NCBI     

22. Desta, Z., Borgstr?m, R., R?sseland, B. O., & Gebremariam, Z. (2006). Major difference in mercury concentrations of the African big barb, Labeobarbus intermedius (R.) due to shifts in trophic position. Ecology of Freshwater Fish, 15, 532?543.

    View Article           

23. Durali, M., Uluozlu, O. D., Hasdemir, E., Tuzen, M., Sari H. and Suicmez, M. 2005. Determination of trace metals in seven fish species in lakes in Tokat, Turkey. Food. Chem., 90: 175-179.

    View Article           

24. EHEMJ, 2016. Environmental Health Engineering and Management Journal.

25. ES, 2823, 2006. Ethiopian Standard of quality check.

26. EU, 1995. Estimations of the total number of microorganisms from fresh water fish with acceptability index in standards, guidelines, and specifications.

27. Eyo, A. A.1997.Post harvest losses in the fisheries of Kainji Lake. A consultancy report submitted to Nigerian/German (GTZ)Kainji lake fisheries promotion project, March, pp: 75.

28. Eyo, A. A. 2001. ''Fish processing Technology in the tropics'' publish by National Institute for Freshwater fisheries research (Niffr) PMB 6006, New Bussa, Nigeria104-152pp.

29. Eyo, A. A. 1987. Description of traditional Smoking Kiln.Report to Kainji Lake research.

30. Eyo, A. A. 1986. Significance of fish handling preservation and processing in the development of Nigeria inland fisheries with special reference to Kanji Lake. Fisheries society Nigeria: 3rd annual conference proceedings.

31. FAO, 1981, 1988, 2000, 2006, 2010 and 2011. The state of world fisheries and aquaculctur FAO fisheries and aquaculture dep: food and agricltur organization of the united nation, Rome.

32. FAO, 1990 and 2005. Manual on Simple Methods of Meat Preservation, FAO, User's, guide Rome .

    View Article           

33. FAO, 1998. Responsible Fish Utilisation. FAO technical guidelines for fisheries. No: 7.

34. Farkas, A., Salanki, J. and Specziar, A. 2003. Age-and size-specific patterns of heavy metals in the organs of freshwater fish Abramis brama. Water Res., 37: 959-964. 00447-5

    View Article           

35. Farkas,T.,Csengeri, I., Majors, F. and Olah, J. 1978. Metabolism of FA in fish. II.Biosynthesis of fatty acids in relation to diet in the carp.(Cyprinus carpio L.) .Aquaculture, 14: 57-65. 90140-0

    View Article           

36. Foladifard R, Ebrahimi AA. 2010. Efficiency study of nickel (II) and cadmium (II) biosorption by powder of waste activated sludge from aqueous solutions. Iranian Journal of Health and Environment 2010; 3(4): 419-30. [In Persian].

37. Gandotra,R., Koul,M., Gupta, S. and Sharma, S. 2012. Change in composition and microbial count by low temperature preservation in Fish Muscle Of Labeo Rohita (Ham-Buch). PMCid:PMC3861146

38. Gebremariam, Z., & Desta, Z. (2002). The chemical composition of the effluent from Awassa textile factory and its effects on aquatic biota. Ethiopian Journal of Science, 25, 263?274.

39. Gebremariam, Z., & Pearce, J. G. (2003). Concentrations of heavy metals & related trace elements in some Ethiopian rift-valley lakes &their in-flows. Hydrobiologica, 429, 171?178.

40. Gibson, R. S., Hotz, C. (2001). Dietary diversification/modification strategies to enhance micronutrient content and bioavailability of diets in developing countries, British Journal of Nutrition, 85(2), 159 - 166.

    View Article           

41. Gibson, R. S., Hotz, C., Temple, L., Yeudall, F., Mtitimuni, B., Ferguson, E. (2000). Dietary strategies to combat deficiencies of iron, zinc, and vitamin A in developing countries: development, implementation, monitoring, and evaluation. Food and Nurition Bulletin, 21(2), 219 - 231.

    View Article           

42. Gomez, K.A. and Gomez, A. A. 1984. Statistical procedures for agricultural research. 2nd ed. John Wiley and Sons Inc. New York.

43. GPNRS (Gambella Peoples National Regional State). 2011. Action Plan of Adaptation to Climatic Change. 63p.

44. Gram, L. and Dalgaard, P. 2002. Fish spoilage bacteria - problems and solutions. Curr. Opin. Biotechnology. 13, 262-266. 00309-9

    View Article           

45. Hansen, M., Thilsted, S. H., Sandstrom, B., Kongsbak, K., Larsen, T., Jensen, M., Sorensen, S. (1998). Calcium absorption from small soft-boned fish. Journal of Trace Elements in Medicine and Biology, 12, 148 - 154. 80003-5

    View Article           

46. Huss, H. H., Dalgaard, P. and Mejlholm, O. 1997. Application of an iterative approach for development of a microbial model predicting the shelf-life of packed fish.International Journal of Food Microbiology, 38, 169e179.

47. ICMSF (International Commission on Microbiological Specifications for Foods), 1986. sampling plans for fish and shellfish, In: Microorganisms in Foods. Sampling for Microbiological Analysis: Principles and Scientific Applications, Vol. 2, 2nd Edition. University of Toronto Press, Toronto, Canada.181 196 Pp.

48. ICSNF, 1986 and ICMSF, 1997 and 1998. Microbial quality standards specifications.

49. Ihuahi, J. A., Omojowo, F. S. and Ugoala, A. E. 2005. Effect of spice treatment on the quality of hot-smoke catfish (Clarias gariepinus) National Institute for Freshwater fisheries research NIFFR, In: Fish Processing, Quality control and Package for international markets pp363-36.

50. Ikeme, A.I. and Bhandary, C. S. 2001. Effect of spice treatment on the quality of hot smoked mackerel (Scomber scombrus) presented at the seventh FAO expert consultation in Africa, Senegal. 2001.1-10pp.In Workshop on Seeking Improvement in Fish Technology in West Africa. IDAF Inc. p. 3-15, 293-297.

51. International Zinc Nutrition Consultative Group. (2004). Assessment of the risk of zinc deficiency in populations and options for its control. Food and Nutrition Bulletin, 25(1).

52. ISO, 2002. Method 6579, 4th edition. Microbiology food and animal feeding atuffs-Horizontal method for the detection of Salmonella spp.

53. ISO, 2006. Microbiology food and animal feeding atuffs-Horizontal method for the detection and enumeration of coliforms.No. 4831, 3rd edition.

54. ISO, 2003. Microbiology food and animal feeding, Horizontal method for the enumeration of microorganism-colony counting technique at 30 degree Celsius.No. 4833, 3rd ed.

55. Jallow, A. M. 1995. Contribution of improved chokor oven to artisanal fish smoking in IDAF Technical Report. No: 66.

56. Jiang and Zhou, 2003 and Barbara, Z. 2007. Natural anti oxidative application on fish product.

57. Kawarazuka N. (2010). The contribution of fish intake, aquaculture, and small-scale fisheries to improving nutrition: A literature review. The WorldFish Center Working Paper No.2106. The WorldFish Center, Penang, Malaysia. 44 p. The WorldFish Center, Penang, Malaysia.

58. King and Johnson, 1987. How to made fish drying racks.Natural Resources Institute Technical Leaf let No: 1.

59. Koki IB, Bayero AS, Umar A , Yusuf S. Health risk assessment of heavy metals in water, air, soil and fish. African Journal of Pure and Applied Chemistry 2015; 9(11): 204-10.

    View Article           

60. Larissa Dsikowitzky & Mesfin Mengesha & Elias Dadebo & Carlos Eduardo Veiga de Carvalho & Sven Sindern Received and pulished: 2012. Assessment of heavy metals in water samples and tissues of edible fish species from Awassa and Koka Rift Valley Lakes, Ethiopia.

61. Larsen, T., Thilsted, S. H., Kongsbak, K., Hansen, M. (2000). Whole small fish as a rich calcium source. British Journal of Nutrition, 83, 191 - 196. PMid:10743499

    PubMed/NCBI     

62. Lawless, H.T. and Heymann, H. 1998. Sensory evaluation of food: Princples and practices.

63. Lawrie, R. A. 1981. Meat Science (4th Edition). Percamon Press, New York.

64. Libby,J. A.1975.Meat Hygiene(4thEd).Lea&Fibiger.Washington Square,Philadelphia.Pp 658.

65. M. A., Wahab, M. A., Milstein, A. (2004). Effects of adding different proportions of the small fish punti (puntius sophore) and mola (amblypharyngodon mola) to a polyculture of large carp. Aquaculture Research, 35, 124 - 133.

    View Article           

66. M. Hansen, S.H. Thilsted, B. Sandstr?m, K. Kongsbak, T. Larsen, M. Jensen and S.S. S?rensen: 1998. Journal of Trace Elements in Medicine and Biology: Volume 12, Issue 3, November 1998, Pages 148?154. 80003-5

    View Article           

67. Magawata, I. and Shina, A. A. 2013. Effect of spice treatment on the quality of solar dried, Africa cat fish Clarias Gariepinus (Burchell, 1822) In Sokoto, Nigeria.

68. Mansour, S. A. and Sidky, M. M. 2002. Ecotoxicological studies. 3. Heavy metals contaminating water and fish from fayoum Governorate, Egypt. Food Chem., 78: 15 22. Marine Dry Fishes (Harpodon nehereus, Johnius dussumieri and Lepturacanthus savala). Am. J. Food Technol., 7: 429-436. DOI:10.3923/ajft.2012.429.436.

    View Article           

69. Maurine, L. and James, T. P. 2001. US Food and Drug Administration (FDA).Science and Research Laboratory Method. Bacteriological Analytical Manual (BAM).8th ed.Revision A. PMid:11545342

    PubMed/NCBI     

70. McKinney, J., & Rogers, R. (1992). Metal bioavailability. Environmental Science and Technology, 26, 1299.

    View Article           

71. Mdegela R., Ruttaisire, J., Obua, J. and Okoth, S. 2010. Fishery and Aquaculture cluster proceeding workshop.

72. MoA, 2012. The report done by MOA on the total fish production and productivity.

73. Moeller, A., Spencer, D. M., Richard, F. A. and Shane, S. Q. 2003. Elements in ?sh of malibu creek and malibu lagoon near Los Angeles, California. Marine Pollut. Bull., 46: 424-429. 00466-6

    View Article           

74. Mohammad Malakootian1, Mohammad Seddiq Mortazavi, Abdolkarim Ahmadi. 2016. Heavy metals bioaccumulation in fish of southern Iran and risk assessment of fish consumption.

75. Murano, S. P. 2003. Understanding Food Science and Technology. Thomson Learning Academy Resource Center. Belmont, US.

76. Nduagu, C., Ekefan, E. J. and Nwankiti, A. O. 2008. Effect of Some Crude Plant extracts on Growth of Colletotrichum capsici (Synd) Butler and Bisby causal agent of Papper anthracnose. J. App. Bioscie. 6: 184-190.

77. Olson, J. A. (1996). Biochemistry of vitamin A and carotenoids. In: Sommer, A., West, K.P. Jr. (edt). Vitamin A deficiency: health, survival, and vision (pp. 221 - 250). New York: Oxford University Press.

78. Roos, N. (2001& 2007). Fish consumption and aquaculture in rural Bangladesh: Nutritional contribution and production potential of culturing small indigenous fish species (SIS) in pond polyculture with commonly cultured carps. Ph.D. Thesis, Department of Human Nutrition, The Royal Veterinary and Agricultural University, Denmark.

79. Roos, N., Islam, M., Thilsted, S. H., Ashrafuddin, M., Mursheduzzaman, M., Mohsin, D.M., Shamsuddin, A. B. M. (1999). Culture of mola (amblypharyngodon mola) in polyculture with carps - experience from a field trial in Bangladesh, NAGA WorldFish Centre Quarterly, 22(2), 16 - 19.

80. Sablani, S., Rahman, M. S., Mahgoub, O. and AL-Marzouki, A. 2002. Sun and solar drying of fish sardines. Proceedings of the international drying symposium Vol.c:1662 -1666.

81. Salam, M. A. 2002. Seasonal changes in the biochemical composition of body muscles of a freshwater catfish, Heteropneustes fossilis. Bangladesh J. Life Sci., 14 (1&2): 47-54.

82. Salam, M.A., Alam, N., Nasiruddin, M., Nabi, R. and Howlader, M. Z.H. 1995. Biochemical composition of body muscles and its caloric contents of tawes (Puntiusgonionotus, Bleeker). Bangladesh J. Sci. Res., 13(2): 205-211.

83. Sen I, Shandil A, Shrivastava VS. 2011. Study for determination of heavy metals in fish species of the River Yamuna (Delhi) by inductively coupled plasma-optical emission spectroscopy (ICP-OES). Advances in Applied Science Research 2011; 2(2): 161-6.

84. Spencer, J. and Clifford, A. L. R. 2000. Microbiology. Methods and Protocols, Enzymes in Non aqueous Solvents.

85. Squadrone S, Prearo M, Brizio P, Gavinelli S, Pellegrino M, Scanzio T, et al. 2013. Heavy metals distribution in muscle, liver, kidney and gill of European catfish (Silurus glanis) from Italian Rivers. Chemosphere 2013; 90(2): 358-65.

    View Article           

86. Suleiman, M. N. 2010. Fungitoxic Activity of Neem and Pawpaw Leaves Extracts on Alternaria Solani, Causal Organism of Yam Rots: Adv. Environ. Biol., 4: 159-161.

87. Taghavi Jelodar H, Fazli H, Salman Mahiny A. 2016. Study on heavy metals (chromium, cadmium, cobalt and lead) concentration in three pelagic species of Kilka (Genus Clupeonella) in the southern Caspian Sea. Iranian Journal of Fisheries Sciences 2016; 15(1): 567-74.

88. Thilsted, S. H., Roos, N. (1999). Policy issues on fisheries in relation to food and nutrition security. In Ahmend, M., Delgado, C., Sverdrup-Jensen., Santos, R. (ed.). Fisheries policy research in developing countries: issues, priorities and needs (pp. 61 - 69). Makati City, Philippines, International Center for Living Aquatic Resource Management. PMid:10574542

    PubMed/NCBI     

89. Thilsted, S. H., Roos, N., Hassan, N. (1997). The role of small indigenous fish species in food and nutrition security in Bangladesh. WorldFish Centre Quarterly, July-December (Supplement), 82 - 84.

90. Thilsted, S. H., Sandstrom, B., Kongsbak, K., Larsen, T., Jensen, M., Sorensen, S. (1998). Calcium absorption from small soft-boned fish. Journal of Trace Elements in Medicine and Biology, 12, 148 - 154. millennium development goals, 211 - 226.

91. Tilahun, K., A. Mengistu and S. Mengistu. 2015. The Journal of Agriculture and Natural Resources Sciences. 2(1): 267 280. 2015.Journal homepage: Print ISSN:2345-4377Onlin e ISSN:2383-238X .Seasonal Dynamics in Botanical Composition of the Rangelands of Gambella, Southwestern Ethiopia.

    View Article           

92. Toth,I., Polthast, K., Chicheste, C. O., Mark, E. M. K. and Schweigert, B. S. 1984. Chemical aspects of smoking of meat and fish product, in.(Eds) Advances in food Research , Academic New York PP 87.

93. UNCEF, 2005. Riverside fishing activities in Ethiopia.

94. WHO/FAO. (2004). Vitamin and mineral requirements in human nutrition. Second edition. Geneva: World Health Organization and Food and Agriculture Organization.

95. WHO/WFP/UNICEF. (2006). Preventing and controlling micronutrient deficiencies in populations affected by an emergency. Geneva: Joint statement by the World Health Organization, the World Food Programme and the United Nations Children's Fund.

96. Wilson, N. R. P., 1981. Meat and Meat Products.Applied Science publishers, London. pp 660. PMid:7286460

    PubMed/NCBI     

97. Yohannes YB, Ikenaka Y, Nakayama SM, Saengtienchai A, Watanabe K, Ishizuka M. 2013. Organochlorine pesticides and heavy metals in fish from Lake Awassa, Ethiopia: insights from stable isotope analysis. Chemosphere 2013; 91(6): 857-63.

    View Article           

98. Zhang Z, He L, Li J, Wu Z. 2007. Analysis of heavy metals of muscle and intestine tissue in fish ? in Banan section of Chongqing from Three Gorges Reservoir, China. Polish J Environ Stud 2007; 16(6): 949-958.

99. Zhuang P, Li ZA, McBride MB, Zou B, Wang G. 2013. Health risk assessment for consumption of fish originating from ponds near Dabaoshan mine, South China. Environ Sci Pollut Res Int 2013; 20(8): 5844-54. PMid:23508535

    View Article      PubMed/NCBI     

100. Zugarramurdi, J. A., Parin, M. A. and Lupin, H. M. 1993. Manual on economic engineering applied to fish industry. FAO. Rome. Pp 107 210.iukumar, G.,Abraham, T.J. and Jayachandran, P.1995.Sanitation in fish curing yards of Tuticorin, Tamil Nadu. Fish. Technol. 32 (2).136 -138.