Technical noTes Journal of Agricultural and Marine Sciences 2021, 26(1): 53–56 DOI: 10.24200/jams.vol26iss1pp53-56 Reveived 11 May 2020 Accepted 30 Nov 2020 Microbiological Assessment of Locally Dried Fish in Oman: Technical Note Aaisha K. Al-Saadia, Abdulrahim M. Al-Ismaili a,*, Mohammed Al-Ruzeikib, Ismail M. Al-Bulushib Abdulrahim M. Al-Ismailia,*( ) abdrahim@squ.edu.om; abdrahim@ hotmail.co.uk, aDepartment of Soils, Water and Agricultural Engineer- ing, Sultan Qaboos University, 123, Al-Khoud, Sultanate of Oman, bDe- partment of Food Science and Nutrition, Sultan Qaboos University, 123, Al-Khoud, Sultanate of Oman Introduction Fish is the main foodstuff around many countries in the world because of its high protein content and nutritional value. However, in the hot climates, fish perishes quickly and it is common to increase their shelf life using different techniques such as drying, smoking and salting (Sultana et al., 2010). In the Gulf region, Oman is the largest fish producer (FAO, 2019) and it has more than 150 kinds of fish and crustaceans (Belwal et al., 2015). Most of the fish in Oman are sold fresh (93%) and the remaining 7% is sold after being dried, salted or smoked (FAO, 2019). In 2018, the total fish production of Oman was about 553 thousand tons which was 59% more than the production in the previous year (MAF, 2018). In the same year, fish export constituted 44% of the total production (about 245 thousand tons). The traditional drying process of fish causes the loss of 30-40% of dried fish due to eating by dogs, birds, cats, and rats, especially during winter when drying takes about a week (Sablani et al., 2002). Indeed, this factor causes a reduction on the revenue of dried fish, which also suffers from the contamination by sand particles. It was reported that sand contamination causes high concentration of ash in the dried fish (Al Ghabshi et al., 2012). In addition, sun-dried fish may become unhealthy التقييم امليكروبيلوجي لألمساك اجملففة حملًيا يف عمان: مالحظة فنية عائشة الساعدي1، عبد الرحيم اإلمساعيلي1،*، حممد الرزيقي2، إمساعيل البلوشي2 Abstract. The presence of microorganisms is one of the major factors affecting the quality of dried fish. In this project, a number of analytical tests were used to verify the presence of biochemical contamination in dried fish, as well as to propose an engineering solution to re- duce the incidence of these contaminations. Two types of fish samples (anchovy and shark) were collected from two local markets (Buraimi and Seeb). The analyzed parameters includ- ed Total Fungal Count (TFC), Total Viable Count (TVC) and enumeration of Escherichia coli (E. coli), Staphylococcus aureus and Coliforms species in dried fish. The results indicated that the anchovy collected from Seeb market had the highest TVC and TFC (TVC= 5.59×105 cfu/ ml, TFC= 3.60×104 cfu/ml) compared to all other samples. It could be recommended to dry fish under more hygienic conditions using solar drier instead of direct drying on beach sands. Keywords: Solar drying, fish drying, fish contamination, dried fish, microbial assessment الدراســة هــذه ويف اجملففــة، األمســاك جــودة علــى تؤثــر الــي الرئيســية العوامــل أهــم أحــد الدقيقــة احليــة الكائنــات وجــود يعتــر املســتخلص: هندســي حــل اقــراح مت وكذلــك حمليــاً، اجملففــة األمســاك يف البيلوجيــة امللــواثت وجــود مــن للتحقــق التحليليــة األختبــارات مــن عــدد اســتخدام مت حمليــا )تســمى والقــرش األنشــوجة ومهــا األمســاك مــن لنوعــن عينــات مجــع مت فقــد ذلــك أجــل ومــن امللــواثت، هــذه وجــود تقليــل علــى يعمــل والعــدد )TFC( للفطــرايت الكلــي العــدد التاليــة: العوامــل وجــود حتليــل مت وقــد والســيب، الرميــي ومهــا حمليــن ســوقن مــن العــوال( و القاشــع اجملففــة. األمســاك يف الكوليفــورم وبكتــراي Staphylococcus aureus بكتــراي و E-coli بكتــراي وتعــداد )TVC( احليــوي الكلــي الــي األنشــوجة يف (g3.60×104 cfu/ml ) احليــوي الكلــي والعــدد (g5.59×105 cfu/ml ) للفطــرايت الكلــي العــدد أن النتائــج أظهــرت وقــد مت مجعهــا مــن ســوق الســيب كان أكثــر منهــا يف العينــات األخــرى. وبنــاء علــى وجــود هــذه امللــواثت يف األمســاك اجملففــة فإننــا نوصــي بتجفيــف الشــاطىء. رمــال علــى املباشــر التجفيــف مــن بــدال الشمســية اجملففــات اســتخدام علــى ونشــجع أفضــل، صحيــة ظــروف يف األمســاك الكلمات املفتاحية: التجفيف الشمسي ، جتفيف األمساك ، تلوث األمساك ، األمساك اجملففة ، التقييم امليكروبيلوجي. 54 SQU Journal of Agricultural and Marine Sciences, 2021, Volume 26, Issue 1 Microbiological Assessment of Locally Dried Fish in Oman: Technical Note when it is attached by insects and larva. It was found that some fishermen apply insecticides to avoid this kind of attacks (Bala & Mondol, 2001). The presence of microorganisms affects the quality of dried fish. Some studies showed that bacteria, molds, and yeasts are potential microorganisms that usually contaminate dried fish (Akwuobu et al., 2019). Therefore, it is important to determinate the microbiological qual- ity of dried fish. The aim of this study was to verify the presence of microbiological contamination in dried fish collected from selected local markets; and to propose an engineering solution to reduce these contaminations. Methodology Two common types of traditionally dried fish in Oman were collected namely; anchovy and shark (locally called Owal) from two different places; Buraimi and Seeb fish markets. The anchovy samples were collected from packaged and unpackaged conditions from the two plac- es. About 500 g was purchased from each place. They were placed in zipped bags and transported to the Food Microbiology laboratory at the College of Agricultural and Marine Sciences, SQU for analysis. Media Preparation All media were prepared according to Al Bulushi (2017) and each test was prepared using the media as men- tioned in Table 1. After preparing the media for each test, it was mixed with distilled water. Then, each mix- ture was poured in a bottle and autoclaved at 121ºC for 2-2.5 h. After autoclaving, 15 ml of the agar media was placed in Petri dishes and cooled at room temperature to solidify the media (Al Bulushi, 2017). Total Fungal Count and Total Viable Count From each sample, 25 g was placed in a sterile stomach- er bag, and 225 ml of maximum recovery diluent was added in the stomacher bag (Interscience/France) to achieve 1/10 (10-1) dilution. Then, 10-2 and 10-3 dilutions were prepared. For plating, 0.1 ml (100 µl) was trans- ferred from each tube of the 10-1, 10-2, and 10-3 dilution mixtures to duplicate plates of the Potato Dextrose Agar (PDA) plate aseptically. Then, the mixture was spread using alcohol flamed spreading from the highest dilution to the lowest dilution (i.e. 10-3 to 10-1). All plates were in- cubated aerobically at an ambient temperature of nearly 23ºC for 3-5 days. After that, the colonies on plates were counted and reported (cfu/g) using Equation 1. For Total Viable Count, all procedures were the same as Total Fungal Count except the differences with the type of agar used (SPCA), temperature (35ºC) and peri- od of incubation of the plates (48 h). Enumeration of Bacteria The samples were stored in a freezer at -40ºC until used for the microbial analysis. Then, the samples were ana- lyzed for 3 types of bacteria; E. coli, staphylococcus au- reus and coliforms as total colony-forming units (cfu/ml) using different agar media, as explained below. Enumeration of E. coli, Staphylococcus aureus and Coliforms For E. coli, about 50 g of fish sample was taken, cut into small pieces and mixed together. Then, a stomacher bag was placed in a beaker and only 25 g of the mixed pieces were placed in the bag. After that, 225 ml of diluent was added to the stomacher bag and blended for 1 min to achieve a homogenized mixture at 10-1 dilution. Then, dilutions of 10-2 and 10-3 were prepared. For plating, 0.1 ml from each tube of the 10-1, 10-2, and 10-3 diluents were transferred to duplicate plates of Tryptone Bile X-Glucuronide Agar (TBXA) plate asepti- cally. Then, the mixture was spread using alcohol flamed spreading from the highest dilution to the lowest dilution (i.e. 10-3 to 10-1). Finally, incubation was done for all plates aerobically at 35ºC for 24 h and only blue or green colo- nies were counted and reported (cfu/g) using Equation 1. For staphylococcus aureus, the diluents were pre- pared as explained earlier for the enumeration of E. coli in dried fish. For plating, molten Baird-Parker agar were used instead of molten TBX Agar. Finally, black colonies were counted and reported (cfu/g) using Equation 1. For coliform species, the diluents were prepared as explained earlier for the enumeration of E. coli in dried fish. For plating, 1 ml from each diluent was transferred to sterile petri dishes. For each dilution, two plates were used. Over a period of 15 min, approximately 12 ml of molten VRB Agar at 44-47ºC was added. The me- dium with the inoculum was carefully mixed by hori- zontal movements (pour plate method) and the medi- um was allowed to cool down. Finally, the dishes were incubated for 24±2 h at 35ºC and only pink colonies were counted and reported (cfu/g) using Equation 1. Moisture Content and Water Activity The moisture content was determined by oven-drying the samples at 105oC for 24 h (Ullah et al. 2016). The wa- ter activity (aw, unit less) of the sample was determined using a water activity meter. The correlation of moisture content and water activity with the analyzed microbial counts was studied using STATA 13.0 (StataCorp, USA) at α = 0.05. 55Technical notes K. Al-Saadi, M. Al-Ismaili, Al-Ruzeiki, M. Al-Bulushi Results and Discussion Microbial Contamination The anchovy collected from Seeb market had the highest TVC and TFC (5.59×105, 3.60×104 cfu/ml, respectively) as compared to all other samples (Table 1). In addition, the presence of 3 species of bacteria; E. coli, staphylo- coccus aureus, and coliforms was detected in anchovy samples more than in shark samples as collected from the two markets. Previous studies reported that anchovy had more contamination than the allowable levels (Aliya et al., 2018; Kumar et al., 2017). It was observed that the moisture content and water activity of the shark from Buraimi market were the highest among all samples (30.37% and 0.74, respectively). In the case of dried fish storage, Bala and Mondol (2001) stated that microbial growth was inhibited when the moisture content was reduced to 25% and particularly mold growth could be avoided when the moisture content was not more than 15%. However, this was not the case in this study, as re- ported in Table 2, which could be related to the types of contaminations present in the samples used in this study. Although, the moisture content of anchovy was lower than 15%, large values of TVC and TFC were observed. The detected microbes in the analyzed samples could have come from several sources, namely, unhygienic handling of fish during drying and storage, and birds and animals’ droppings. In addition, long time open- sun drying under high humidity environments enhances the growth of microorganisms (Sablani et al., 2002). In general, major sources of contamination include pollut- ed coastal water and soil, dust and unhealthy treatment practices (Ginigaddarage et al., 2018). Placing the fish directly on the sand is more susceptible to the contam- ination by dust, insects, and sand (Bremner, 2002). In addition, the stability of microbial growth in dried fish depends on how much moisture they contain during processing and storage stages (Logesh et al., 2012). Rain and humid conditions hamper sun-drying by increas- ing the moisture content of dried fish and made them susceptible to blowfly larvae attacks (Kleih et al., 2003). Techniques to Avoid the Contamination The presence of microbes in dried fish presents a health hazard that needs to be prevented. To avoid the contam- ination of sand, dirt, and animals, the process of drying has to be done quickly using drying shelves, which can help to keep dried fish away from these sources of con- tamination (Ward & Beyens, 2015). Solar dryers, like greenhouse tunnel dryers and forced convective solar dryers can also help to avoid physical and atmospher- ic-driven contamination and they can accelerate the dry- ing process (Sahu et al., 2016; Seerangurayar et al., 2019). These types of solar dryers are highly recommended as they protect dried fish from many sources of contami- nation like rain, humid environment, dust, insects and animals as well as increase the drying rate. Conclusion In this article, we verified the presence of microbiolog- ical contamination in two types of dried fish (anchovy and shark) available in local markets (Seeb and Buraimi). The major issue that affects the quality and safety of dried fish is the contamination caused by bacteria and fungi. Results from this study evidenced the presence of fungi and three species of bacteria; E. coli, staphylococcus au- Table 1. The media used for each biological test. Tests Media Manufacturer/country Total Fungal Count Potato Dextrose Agar (PDA) Sigma-Aldrich/USA Total Viable Count Standard Plate Count Agar (SPCA) Sigma-Aldrich/USA The diluent Maximum Recovery Diluent Sigma-Aldrich/USA Enumeration of E. coli Tryptone Bile X-Glucuronide Agar (TBXA) Sigma-Aldrich/USA Enumeration of Staphylococcus aureus Baird Parker Agar (BPA) Sigma-Aldrich/USA Enumeration of Coliforms species Violet Red Bile Agar (VRBA) Sigma-Aldrich/USA Table 2. Results obtained from different tests. Fish Type (Market) Xm (%) aw TVC=APC (cfu/ml) TFC (cfu/ml) E. coli (cfu/ml) S.aureus (cfu/ml) Coliforms (cfu/ml) Shark (Seeb) 23.66 0.64 1.05×103 2.50×102 0 6.96×103 0 Shark (Buraimi) 30.37 0.74 1.05×104 2.00×103 0 1.33×104 0 Anchovy (Seeb) 14.54 0.69 5.59×105 3.60×104 4.77×102 1.64×106 1.78×103 Anchovy (Buraimi) 14.15 0.65 1.29×105 3.70×103 6.00×102 2.27×105 2.82×102 56 SQU Journal of Agricultural and Marine Sciences, 2021, Volume 26, Issue 1 Microbiological Assessment of Locally Dried Fish in Oman: Technical Note reus and coliforms. The microbial contamination in an- chovy samples was more than in shark samples collected from the two local markets. To avoid the contamination mentioned above, we recommend using solar dryers for fish drying instead of open-sun drying, as they protect the dried fish from different types of contamination and speed up the drying process. References Akwuobu CA, Antiev WS, Ofukwu RA. (2019). 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