Effects of different smoking methods on the formation of polycyclic aromatic hydrocarbons (PAHs) and physicochemical quality in smoked Atlantic mackerel products.
Benjamin Aidoo
Master Thesis in Environment and Natural Resources
June 2017
Master Thesis in Environment and Natural Resources
Effects of different smoking methods on the formation of polycyclic aromatic hydrocarbons (PAHs) and physiochemical quality in smoked Atlantic mackerel products
Benjamin Aidoo
Supervisors: María Guðjónsdóttir & Sigurjón Arason
60 ECTS
Faculty of Food Science and Nutrition School of Health Sciences Reykjavik, June 2017.
Abstract The effects of different smoking methods and temperatures on the formation of PAHs in smoked mackerel was studied. The research also aimed at finding out how different smoking methods, storage temperature and packaging materials affect the stability of hot smoked mackerel by analyzing their physicochemical properties. Analysis of water and lipid content, free fatty acids (FFA), total plate counts (TPC), water activity, total volatile basic nitrogen (TVB-N), pH, peroxide values (PV), and thiobarbituric acid reactive substances (TBARS) were performed.
Furthermore, the study indicated a stable total lipid content in the smoked fillets and whole mackerel during storage at chilled temperatures (0-4°C). The formation of free fatty acids (FFA), PV and TBARS in the smoked whole mackerel was more delayed than in the fillets during storage. Vacuum packaging also reduced the formation of TVB-N in smoked fillets, in comparison to the air packaging.
The study showed that the level of chrysene was 10.75 ± 0.21 μg/kg and < 1 μg/kg in the cabin and Bradley smoked fillets, respectively. A significant difference was observed between the cabin smoked fillets and the cabin smoked whole fish (p = 0.0005). Benzo(a)pyrene, which is the main indicator for the presence of PAHs in food was observed to be 2.8 ± 0.14 μg/kg and < 0.5 μg/kg in the cabin and Bradley smoked fillets, respectively, whereas in the cabin and Bradley smoked whole fish it was 1.05 ± 0.07 μg/kg and < 0.5 μg/kg, respectively.
Furthermore, the sum of the four PAHs (i.e. chrysene, benzo(b)fluoranthene, benzo(a)pyrene, and benzo(a)anthracene), which are the main concerns of the EU regulation, was 25.85 μg/kg and 8.1 μg/kg in the cabin smoked fillets and whole mackerel, respectively. The results indicated that the cabin-smoked fillets contained benzo(a)pyrene and PAH4 levels that are above the acceptable limit of 2 μg/kg and 12 μg/kg respectively per the EU No. 1327/14 regulation. However, the cabin smoked and the Bradley smoked whole mackerel are safe for human consumption since the levels of benzo(a)pyrene and PAH4 fall below the acceptable limits.
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Samantekt Rannsökuð voru áhrif mismunandi reykingaraðferðar og hitastigs á myndun fjölhringja kolvetni (Polycyclic aromatic hydrocarbons, PAH) í reyktum makríl. Einnig voru áhrif mismunandi reykingaraðferða, geymsluhitastig og pökkunarefna á stöðugleika heitreyktra makrílafurðir kannaðar, með því að fylgjast með eðlis- og efnaeiginleikum þeirra. Greiningar á vatns-, fitu- og saltinnihaldi,vatnsvirkni, sýrustig, fríum fitusýrum, oxunarafurðum (Peroxide values PV og thiobarbituric acid reactive substances, TBARS) og hvarfgjörnum köfnunarefnissamböndum (Total volatile base nitrogen, TVB-N), ásamt greiningum á heildarfjölda örvera voru framkvæmdar.
Rannsóknin sýndi að fituinnihald reyktu makrílafurðanna var stöðugt í gegnum geymslutímabilið ef notast var við kældar aðstæður (0-4°C). Myndun fría fitusýra og oxunarafleiðanna var hærri í flökunum en í heilum fiski. Einnig mátti hægja á myndun TVB- N ef flökin voru geymd við lofttæmdar aðstæður í stað loftumbúða.
Rannsóknin sýndi ennfremur að magn PAH efnisins chrysene náði styrknum 10.75 ± 0.21 μg/kg í flökum sem reykt voru í s.k. klefareykofni (cabin smoker), en voru < 1 μg/kg í flökum sem reykt voru í Bradley reykofninum Einnig mátti sjá marktækan mun á PAH gildum hvort um var að ræða reykingu á heilum fiski eða flökum þegar klefareykofninn var notaður (p = 0.0005). Benzo(a)pyrene, sem er notaður sem vísir um tilvist PAH í matvælum mældist sem 2.8 ± 0.14 μg/kg í klefareyktu flökunum, en < 0.5 μg/kg í flökum sem reykt voru í Bradley reykofninum. Sambærileg gildi í heila fiskinum voru hins vegar 1.05 ± 0.07 μg/kg í klefareykofninum og < 0.5 μg/kg í heila makrílnum úr Bradley reykofninum.
Summa PAH efnanna 4, sem EU reglugerðir einblína á (þ.e. chrysene, benzo(b)fluoranthene, benzo(a)pyrene og benzo(a)anthracene), mældist 25.85 μg/kg í reyktum makríl flökum og 8.1 μg/kg í heilreyktum makríl þegar klefareykofninn var notaður. Þetta bendir til þess að reyking með klefaofninum leiði til of hárrar myndunar á PAH efnunum fjórum, að benzo(a)pyrene meðtöldu í reyktu flökunum, en ásættanleg mörk fyrir þessi efni skv. EU reglugerð nr. 1327/14 eru 12 μg/kg fyrir PAH4 og 2 μg/kg fyrir benzo(a)pyrene. Hins vegar má telja að heilreykti makríllin sé öruggur til mannlegrar neyslu þar sem innihald benzo(a)pyrens og PAH4 voru innan æskilegra marka.
v vi Acknowledgement (and funding) My heartful gratitude goes to Professor Brynhildur Davíðsdóttir of the Faculty of Economics and Faculty of Life and Environmental Sciences for helping and guiding me through the realization of my dream. Also, many thanks go to Bjargey Anna Guðbrandsdóttir, Programme Coordinator, Environment and Natural Resources for helping in choosing good courses throughout my studies. I would also like to express a special gratitude to the project partners Síldarvinnslan and University of Iceland Research fund for providing the fish and funds respectively for this project.
This dream would not have materialize without the emmense support, encouragement, criticisms and comments from my supervisors, Professor Sigurjon Arason, Professor María Guðjónsdóttir and Dr. Magnea Karlsdottir, of Faculty of Food Science and Nutrition, University of Iceland and Matis – Icelandic Food and Biotech R&D respectively. I cannot thank you enough for this gesture. I would like to express my sincere gratitude to Professor María Guðjónsdóttir for her endless sacrifices, encouragement and positive comments that made this work a better one.
Special thanks also go to Dr. Paulina Romotowska, Hildur Inga Sveinsdóttir and Dang Thi Thu Huong who took me through an in-service training on the entire project and again to my project partners, Mrs. Eunice Konadu Asamoah of Department of Marine Fisheries Sciences, University of Ghana and Mr. Osvaldo da Costa, a good teamwork you exhibited.
Finally, I would to thank my family and friends, who have supported me in diverse ways to reach this far. Your confidence, motivation and encouragement during this work is highly appreciated and it would not have been possible without you.
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Table of content
Abstract ...... iii Samantekt ...... v Acknowledgement (and funding) ...... vii Table of content ...... ix List of tables ...... xii List of figures ...... xiii Abbreviations ...... xvi 1 INTRODUCTION ...... 1 1.1 Objective of the study ...... 5
2 LITERATURE REVIEW ...... 7 2.1 Polycyclic Aromatic Hydrocarbons (PAHs) ...... 7 2.2 Classifications and structures of Polycyclic Aromatic Hydrocarbons ...... 7 2.2.1 Classification by molecular weight ...... 7 2.2.2 Classification by number of rings ...... 8 2.2.3 Classification by ring arrangement ...... 8 2.3 Polycyclic Aromatic Hydrocarbons of special concern ...... 8 2.3.1 Benzo(a)pyrene ...... 9 2.3.2 Benzo(a)anthracene ...... 9 2.3.3 Chrysene ...... 10 2.3.4 Benzo(b)fluoranthene ...... 10 2.4 Physicochemical Properties of Polycyclic Aromatic Hydrocarbons ...... 10 2.4.1 Vapor pressure ...... 11 2.4.2 Solubility ...... 11 2.4.3 Boiling point ...... 12 2.5 Carcinogenicity of Polycyclic Aromatic Hydrocarbons ...... 12 2.6 Reactions of Polycyclic Aromatic Hydrocarbons ...... 14 2.6.1Reactions of Benzo(a)pyrene with Deoxyribonucleic acid (DNA) ...... 14 2.7 Toxicological Effect of Polycyclic Aromatic Hydrocarbons ...... 15 2.8 Regulations of Dietary exposure of Polycyclic Aromatic Hydrocarbons ...... 15 2.9 Dietary exposure of Polycyclic Aromatic Hydrocarbons ...... 16 2.10 Alternative sources and distribution of Polycyclic Aromatic Hydrocarbons ...... 17 2.11 Contributing factors for Polycyclic Aromatic Hydrocarbons formation in food ...... 19 2.11.1 Preservation methods ...... 19 2.11.1.1 Smoking ...... 19 2.11.1.2 Smoking equipment ...... 20
ix 2.12.2 Effect of chemical composition of the product ...... 20 2.11.3 Type of fuel used ...... 21 2.11.4 Temperature ...... 21 2.12 Other methods of fish preservation ...... 22 2.12.1 Salting ...... 22 2.1.2 Cooling and freezing ...... 22
3 METHODS AND MATERIALS ...... 25 3.1 Materials ...... 25 3.1.1 Raw Material ...... 25 3.1.2 Smoking equipment ...... 25 3.1.3 Chemicals ...... 26 3.2 Pre-trial ...... 26 3.3 The main experiment ...... 27 3.3 .Physicochemical methods...... 29 3.3.1 Temperature measurements ...... 29 3.3.2 Water activity ...... 29 3.3.3 pH measurements ...... 29 3.3.4 Water content ...... 29 3.3.5 Salt content ...... 29 3.3.6 Lipid content ...... 29 3.3.7 Peroxide Value (Primary Oxidation Product) ...... 30 3.3.8 Thiobarbituric Acid Reactive Substances (TBARS) ...... 30 3.3.9 Free Fatty Acids (FFA)...... 30 3.3.10 Total Volatile Basic Nitrogen (TVB-N) ...... 31 3.3.11 Total aerobic plate count (TPC) measurements ...... 31 3.3.12 Polycyclic Aromatic Hydrocarbon Analysis ...... 31 3.4 Statistical analysis...... 31
4 RESULTS AND DISCUSSIONS ...... 33 4.1 Pre-trial ...... 33 4.2 The main experiment ...... 33 4.2.1 Temperature measurement during the main experiment smoking process ...... 33 4.3 Physicochemical analysis ...... 34 4.3.1 Characteristics of the raw material (Atlantic mackerel) ...... 34 4.3.2 pH measurements ...... 35 4.3.3 Water content ...... 37 4.3.4 Salt content ...... 38 4.3.5 Water activity ...... 40
x 4.3.6 Lipid content ...... 41 4.3.7 TVB-N Analysis ...... 42 4.3.8 Microbial Analysis ...... 44 4.3.9 Free Fatty Acids (FFA)...... 45 4.3.10 Lipid oxidation ...... 47 4.3.11 Polycyclic Aromatic Hydrocarbons (PAH) Analysis ...... 50
5 CONCLUSION ...... 53 6 FUTURE PERSPECTIVES ...... 53 REFERENCES ...... 55
xi List of tables Table 1: Physico-chemical properties of common Polycyclic Aromatic Hydrocarbons ...... 12
Table 2: Carcinogenic and mutagenic properties of Polycyclic Aromatic Hydrocarbons ...... 13
Table 3: Maximum limits of BaP and PAH4 in selected foodstuffs under EU 1327 /2014 ...... 16
Table 4: Comparative study on PAH levels from different foodstuffs ...... 17
Table 5: Experimental design and sampling groups ...... 28
Table 6: The chemical composition of the mackerel before smoking ...... 34
Table 7: Levels of Polycyclic Aromatic Hydrocarbons in smoked mackerel ...... 50
xii List of figures Figure 1: The chorkor smoker ...... 3 Figure 2: Atlantic mackerel (Scomber scombrus)...... 4 Figure 3: Structures of some Polycyclic Aromatic Hydrocarbons (adapted from Phillips, 1999)...... 7 Figure 4: Keta-annealated and peri-fused Polycyclic Aromatic Hydrocarbon (adapted from ATSDR, 1995)...... 8 Figure 5: Alternate and non-alternate Polycyclic Aromatic Hydrocarbon (adapted from ATSDR, 1995)...... 8 Figure 6: Activation of BaP leading to DNA lesions (adapted from Stansbury et al., 1994). .... 14 Figure 7: Smoking equipment used for the study ...... 25 Figure 8: Experimental design for the smoking with the two different kilns...... 28 Figure 9: Temperature profiles within the cabin and Bradley smoking kilns as measured on the top and bottom racks of the kilns...... 33 Figure 10: Temperature profile inside the whole muscle from the Bradley and cabin kilns ...... 34 Figure 11: Comparison of pH of the hot smoked mackerel fillets from the cabin and Bradley smokers, stored at either 4 °C or 20 °C for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C)...... 36 Figure 12: Comparison of pH of hot smoked whole mackerel, smoked in the cabin and Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 37 Figure 13: Water content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 37 Figure 14: Water content in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C)...... 38 Figure 15: Salt content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C)...... 39 Figure 16: Salt content in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C)...... 39
xiii Figure 17: Water activity in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 40 Figure 18: Water activity in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 41 Figure 19: Total lipid content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 42 Figure 20: Total lipid content of whole smoked mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 42 Figure 21: TVB-N (N/100 g) in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 43 Figure 22: TVB-N (N/100 g) in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 44 Figure 23: TPC (logCFU/g) in hot smoked fillets mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 45 Figure 24: TPC (logCFU/g) in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 45 Figure 25: Free fatty acids (FFA; g FFA/100g samples) in smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 46 Figure 26: Free fatty acids (g FFA/100g samples) in whole smoked mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 47
xiv Figure 27: Comparison of the development of lipid hydro peroxide (PV; µmol/g samples) and thiobarbituric acid reactive substances (TBARS; µmol/g samples) in smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 o C or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)...... 48 Figure 28: Lipid hydroperoxide (PV; µmol/g samples) and thiobarbituric acid reactive substances (TBARS; µmol/g samples) in whole smoked mackerel smoked in the cabin or Bradley kilns, and stored at either 4 o C or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C)...... 49
xv Abbreviations ATSDR Agency for Toxic Substances and Disease Registry CAC Codex Alimentarius Commission CONTAM Contaminants in Food Chain CSIR Council for Scientific and Industrial Research DNA Deoxyribonucleic acid EC European Commission EFSA European Food Safety Authority EPA Environmental Protection Agency EU European Union FAC Fillets air chilled FAO Food and Agricultural Organization FAR Fillets air room FBVC Fillets Bradley vacuum packed FFA Free Fatty Acids FVC Fillets vacuum chilled FVR Fillets vacuum room GDP Gross Domestic Products GHS Ghana Health Service HMW High molecular weight IARC International Agency for Research on Cancer ICMSF International Commission on Microbial Specifications for Foods MMW Medium molecular weight MOFAD Ministry of Food and Agriculture PAH Polycyclic Aromatic Hydrocarbons PUFA Polyunsaturated fatty acids PV Peroxide value RASFF Rapid Alert System for Food and Feed RNA Ribonucleic acid SCF Scientific Committee on Food TBA Thiobarbituric acid TBARS Thiobarbituric reactive substances
xvi TCA Trichloroacetic acid TCDC Technical Cooperation in Developing Countries TEP Tetraethoxypropane TVB-N Total volatile basic nitrogen UNDP United Nations Development Program WAC Whole air chilled WAR Whole air room WBVC Whole Bradley vacuum chilled WHC Water Holding Capacity WHO World Health Organization WVC Whole vacuum chilled WVR Whole vacuum room
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xviii 1 INTRODUCTION
In recent years, human activities have contributed to the rise in the levels of contaminants in the environment. Although some of these contaminants occur naturally, most of them are generated through daily human activities. Food contamination usually occurs through environmental sources, industrial food processing and food preparation methods such as grilling and smoking (WHO, 2006; SCF, 2002). The method of cooking, preservation and storage contributes to the introduction of substantial amounts of contaminants such as polycyclic aromatic hydrocarbons (PAHs) into foods (Chen & Chen, 2001). Smoking of food products increases the shelf life of the food by reducing the effects of dehydration, inhibit anti-microbial activities and anti-oxidants that arise from the smoke (Alcicek & Atar, 2010). However, food processing methods such as smoking and grilling, which involve the use of direct flames at high temperatures, generate genotoxic PAHs (Jagerstad & Skog, 2005). During industrial smoking, heating, and drying processes, which involve combustion further exposes the foods to PAHs from the fuel used to generate the smoke (Guillen et al., 1997). Furthermore, PAHs can be generated from fats within the foods through thermal degradation or polymerization (Chen et al., 2005; Phillips, 1999).
Polycyclic Aromatic Hydrocarbons (PAHs) are group of compounds which consist of fused aromatic benzene rings. PAHs are produced when carbon compounds burn incompletely in air, for instant in tobacco and charbroiled meat (European Food and Scientific Authority, 2005; Suchanova et al., 2008). Polycyclic Aromatic Hydrocarbons (PAHs) can also be formed by pyrolysis of organic matter, as well as during industrial processes, such as burning of wood to produce charcoal (Scientific Committee on Food (SCF), 2002; WHO, 2006). PAHs arise mainly from sources such as direct release of fossil oil and its products, or by combustion of fossil fuels, wood and other organic substances (Readman et al., 2002; Yunker et al., 2002). PAHs have thus been observed in air, water, food, soil and sediments (Tao et al., 2004; Nizzetto et al., 2008).
PAH formation is also common during cooking processes. A Codex Alimentarius Commission formed by the Food and Agricultural Organization and the World Health Organization reported that PAHs formation depends on certain factors, such as the applied cooking method, the fat content in the food, the temperature, duration of the smoking, distance between food and heat source and the fuel used for cooking or smoking (Joint FAO/WHO CAC, 2008). In addition, the EU Regulation 1881/2006, which was amended in 2014 as EU Regulation 1327/2014 seems to be more concerned regarding the maximum levels of PAHs in smoked dietary products. Dietary daily exposure of PAHs in the Dutch market has been estimated to be between 5 and 17 μg/day from cereals, fish and meat (De Vos et al., 1990). In New Zealand, a high daily intake of PAHs through consumption of cereals, vegetables, fruits, and oven-baked pizza and meat, resulting in a daily intake level of 1.2 μg/day was found (Thomson, et al., 1996). Also, Menzie et al. (1992), found that the dietary intake of PAHs was 3.15 μg/day, which represents 96% of the total daily PAH exposure from fruits, vegetables and grilled meat. PAHs in grilled foods have been found to
1 range from 0 to 130 mg/kg (Farhadian et al., 2010). However, the European Food Safety Authority (EFSA, 2008) reported that, the average levels of dietary exposure to genotoxic and carcinogenic PAHs from smoked foodstuffs are 1.73 mg/day. Many of the PAHs pose immunotoxic, genotoxic, mutagenic and carcinogenic effects to humans as well as the environment (Phillips, 1999; Castillo et al., 2004). Some of the meat and fish products found in the Ghanaian market are prepared over an open flame with uncontrolled heat and with fuel sources made from wood sawdust or hot plates with cooking oil. PAHs are thus introduced into the food through its preparation with extremely high-temperature methods, and consuming such food products puts the consumers at risk and may be harmful to their health.
Several studies have been performed on dietary exposure of PAHs to humans (Alomirah et al., 2011; Reinik et al., 2007; Orecchio & Papuzza, 2008; Forsberg et al., 2012). Grilled and smoked foods, especially fish, contain high concentrations of PAHs (Guillen et al., 1997; Phillips, 1999). The levels of PAHs accumulated in these foods depends on the amount of smoke generated and the type of wood (Essumang et al, 2013; Maga, 1988; Varlet et al., 2007). However, very few such studies have been done on the effect of different smoking methods on PAH formation during the smoking of fish.
Fish serves as a major protein source in many diets. Apart from proving adequate food resources, the fishing industry employs about 10% of the Ghanaian population, which generates about US$ 1 billion total revenues annually (Ministry of Food and Agriculture, MOFAD, 2016). Thus, in Ghana, the fish industry is of great economic importance because about 4.5% of the country’s Gross Domestic Products (GDP) come from the export of fish (MOFAD, 2016). Also, most Ghanaian diets contain fish which provide protein source intake of about an average per capita consumption of 23.7kg annually (Anon, 2011).
Ghana has very similar fish species as Iceland, such as the anchovies, sardines, tuna, mackerel and salmon, which are classified as fatty fishes. With the lean fish and fatty fish is most common in Ghanaian. The most exploited species includes chub mackerel, anchovies and sea breams. The catch capacity from these species amounts to about 70 to 80 % of the total marine fish catch capacity per annum (Armador et al., 2006). The catch is processed in several ways by smoking, salting, drying, and deep frying to prolong the shelf life of the fish. However, the most widely used method of fish preservation in Ghana is smoking due to the smoky flavor and the long storage time of the fish before spoiling. In Ghana, almost all the fish species caught from the freshwater, aquatic and domestic marine are smoked and it is estimated to be applied to 70 to 80 % of the fish catch (Nunoo et al., 2015). The smoked products losses their quality due to poor handling thus affecting market value. This post-catch loss is estimated to be 40 % due to poor packaging, storage, transportation and during smoking (Aryee & Oduro, 2013).
Previously very primitive traditional smokers like the cylindrical or rectangular ovens made of mud or metals were used for smoking of fish in Ghana until 1969 when the Food and Agriculture Organization of the United Nations (FAO) and the Food Research Institute of the
2 Council for Scientific and Industrial Research (CSIR), introduced the ‘Chorkor smoker’ (Figure 1). These primitive ovens produced a lot of smoke, less fish could be smoked, longer time to smoke, less quality products, less energy efficient and exposes the fish processors to diseases.
Figure 1: The chorkor smoker
The traditional kilns were criticized because of the poor quality of the product and inefficient fuel used leading to a low market value (United Nations Development Program on Technical Cooperation in Developing Countries, UNDP/TCDC, 2001). The fuel smoker used in the chorkor smoker produced a lot of smoke making the product less valuable and exposing the women to diseases. Thus, there is a need for the introduction of a safer and more efficient technical ways of smoking even with the existence of the ‘Chorkor smoker’ which is moderate in terms of energy consumption efficiency and its production of smoke (Okyere- Nyarko et al, 2015). Recently, modern technologies of smoking kilns, such as the Bradley and cabin smokers, have been developed and these are more energy efficient and produce less smoke. Thus, products of higher quality and greater market value may be produced. These new smoking kilns also have their limitations, such as the fuel used and the temperature at which this equipment are operated. Another limitation of these methods is the high temperature of the heat source, which can lead to deposition of high levels of polycyclic aromatic hydrocarbons (PAHs) in the smoked product, thus posing an increased health risk for humans who consume it. The Cancer Control of Ghana Health Service reported that 16,600 cancer cases are estimated in Ghana and the prevalence rate of 109.5 cases per 100,000 persons. Another report by Breast Care International indicated that breast cancer cases among Ghanaian women are due to the levels of PAHs in Ghanaian diets (GHS, 2011).
3 The risk posed by PAHs, such as benzo(a)anthracene, benzo(a)pyrene, chrysene and benzo(b)fluoranthene, has been of great concern recently. These four PAHs are common in most foods and are thus often referred to as PAH4. Most of the studies done in Ghana on smoking of PAHs in fish involved the use of species including chub mackerel (Scomber japonicas), cat fish (Chrysichthys auratus), Sardines/herrings (Sardinella aurita). These species are chosen for these types of studies because of the fatty levels and also their availability in the markets.
The Atlantic mackerel, Scomber scombrus L., is a fast swimming, pelagic schooling species found in the Atlantic Ocean (Figure 2). Atlantic mackerel is a fatty fish belonging to the scombridae family. The Atlantic mackerel normally lives in deep waters during winter but move to the shores during spring. The feeding patterns of the mackerel depends on the season, which results in variation in the fatty and water content (Romotowska et al., 2016). In Icelandic waters, mackerel spawning and travelling period is in the summer (June– September), which is a heavy feeding season and during this period the mackerel enters Icelandic fishing waters. The lipid content in the mackerel muscle increases during this period, resulting in changes in the fatty acid composition due to the heavy feeding
(Astthorsson et al., 2012; Jansen et al., 2012). Mackerel has high vitamin B12, as well as omega-3 polyunsaturated fatty acids (PUFAs), which when consumed have shown to reduce the effects from coronary artery disease, as well as depression (Delagado-Lista et al., 2012; Perica & Delas, 2011). Mackerel thus serves as a good source of phosphatidylserine, which plays a key role in cell cycle signaling. However, higher amounts of PUFAs makes the fatty fish more unstable during processing and storage as a result of degradation and oxidation of the lipids (Saeed & Howell, 2002). The demand for the mackerel has become low despite its numerous economic importance. As such, the mackerel that is underutilized ends ups as being used for animal feeds or other products (FAO, 2007).
Figure 2: Atlantic mackerel (Scomber scombrus).
4 1.1 Objective of the study The objectives of the present study were to determine how different smoking methods and temperatures affect the formation of PAHs in hot smoked Atlantic mackerel (Scomber scombrus). The research also aimed at studying how different storage temperatures and packaging materials affected the quality and stability of hot smoked mackerel by analyzing their physicochemical and microbiological properties during storage. This was performed by analysis of water and fat content, free fatty acids (FFA), water activity, total volatile basic nitrogen (TVB-N), pH, peroxide value (PV) and thiobarbituric acid reactive substances (TBARS), as well as total plate counts (TPC). The specific objectives of the study were to: Identify and compare the levels of PAH4 in hot smoked mackerel from the cabin and Bradley smoking kilns. Evaluate the storage life of hot smoked mackerel products stored at 0 - 4°C and 15- 20°C using analysis of physicochemical properties. Compare the physicochemical characteristics of the smoked mackerel from the cabin and Bradley smoking kilns.
5
2 LITERATURE REVIEW
2.1 Polycyclic Aromatic Hydrocarbons (PAHs) Polycyclic Aromatic Hydrocarbons (PAHs) are a group of environmental organic compounds which contain two or more condensed aromatic rings made of carbon and hydrogen atoms (Suchanová et al.,2008). Humans are exposed to PAHs through various routes, such as cigarette smoking, by skin contact, or through food ingestion. The European Food Safety Authority reports that the most common medium of PAHs exposure is through food (EFSA, 2005). PAHs can be found in both raw and processed foods but their formation are highly dependent on the applied cooking methods.
2.2 Classifications and structures of Polycyclic Aromatic Hydrocarbons The structures of PAHs are arranged such that they can be linear, angular or clustered. The structure of a few common PAHs can be seen in Figure 3. The branched PAHs are more stable and less reactive due to the presence of one electron in the non-bonding orbital. PAHs may be grouped based on how they are formed, their molecular weights, and number of rings or how the rings are arranged.
Anthracene Phenanthrene Benzo(a)anthracene
Chrysene Pyrene Fluoranthene
Benzo(a)pyrene fluorene Benzo(k)fluoranthene
Indeno(123-cd) pyrene Acenaphthene Naphthalene Figure 3: Structures of some Polycyclic Aromatic Hydrocarbons (adapted from Phillips, 1999).
2.2.1 Classification by molecular weight PAHs may be classified based on their molecular weight. Those which have two-three ring are grouped under low molecular weights (LMW) and generally have a molecular weight ranging from 152 g/mol to 178 g/mol. Examples includes acenaphthene, fluorene, anthracene, acenaphthylene, and phenanthrene. PAHs, which have four rings, are grouped under medium molecular weights (MMW) and have molecular weight of 184 g/mol to 202 g/mol, and examples include fluoranthene and pyrene. High molecular weight PAHs (HMW) are PAHs which contain five to seven rings with a molecular weight of 208 g/mol to 278 g/mol. Examples include benzo(a)anthracene, benzo(b)fluoranthene, chrysene, and benzo(a)pyrene (ATSDR, 1995).
7 2.2.2 Classification by number of rings PAHs may also be classified by the number of rings and the PAHs within each classification have similar chemical structures. PAHs are then classified under two, three, four and five rings system etc. The two-ring PAHs have only one member, naphthalene, whereas the three– ring homologs include acenaphthene, fluorene, phenanthrene, and anthracene as examples. The four-ring group includes fluoranthene, pyrene, benzo(a)anthracene, chrysene etc. PAHs with five rings include cyclopenta(c,d)pyrene, dibenzo(a,h)anthracene, and PAHs that have six rings include benzo(g,h,i)perylene.
2.2.3 Classification by ring arrangement PAHs are also often classified based on how their rings are arranged. The ring arrangement can be either keta-annealated or peri-fused. With the keta-annealated arrangement the rings are fused linearly or in a straight line (Figure 4). An example of compounds classified under this group are anthracene, naphthacene, and pentacene. In the peri-fused PAHs the rings are arranged in an angular shape. Examples are phenanthrene and benzo(a)anthracene.
Anthracene Phenanthrene Figure 4: Keta-annealated and peri-fused Polycyclic Aromatic Hydrocarbon (adapted from ATSDR, 1995).
PAHs can also be classified into alternant (e.g. benzo(a)pyrene, chrysene) and non- alternant (e.g. fluoranthene, benzo(k)fluoranthene). This distinction is based on the electron density distributed around the molecule. The alternant PAHs have an equally distributed electron density, whereas the no alternant PAHs have two different molecules due to an uneven distribution of the electron density from one portion of the molecule to another (Figure 5).
Benzo(a)pyrene – alternant PAH Fluoranthene – non-alternant PAH Figure 5: Alternate and non-alternate Polycyclic Aromatic Hydrocarbon (adapted from ATSDR, 1995).
2.3 Polycyclic Aromatic Hydrocarbons of special concern The most prevalent PAHs found in food exist in the gaseous or in a particulate phase. About 47% of the total polycyclic aromatic hydrocarbons (PAHs) found in the environment exist as gases (Snook et al., 1976). Most of the four-ring PAHs with a molecular weight of 184 g/mol to 202 g/mol, e.g. phenanthrene and pyrene, have been found to exist in the gas phase, whereas PAHs with five-six rings, such as benzo(a)pyrene generally are particulates and gaseous. Many PAHs exist in the environment, and the US Environmental Protection Agency
8 (USEPA) has recognize sixteen (16) of them which have a potential of posing carcinogenicity (Cloarec et al., 2002). However, the European Food Safety Authority (EFSA) panel on contaminants in the Food Chain (CONTAM Panel) identified 15 PAHs which are mostly found in food (EFSA, 2008). Moreover, the European Union Scientific Committee on Food therefore also identified 8 PAHs that are capable of posing carcinogenicity through dietary exposure, which agrees with the results obtained by EPA and the CONTAM panel. These PAHs are benzo(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, chrysene, indeno(1,2,3-cd)pyrene, benzo(g,h,i)perylene, and dibenzo(a,h)anthracene. Out of these, four of them have been commonly observed in food. These four benzo(a) anthracene, benzo(a)pyrene, benzo(b)fluoranthene and chrysene. Therefore, the CONTAM panel has focused mostly on these PAH4 and concluded that benzo(a)pyrene is the most common PAH observed in food. However, it should not be the only marker used to determine the presence of PAHs in food but rather the sum of all the four PAHs (EFSA, 2011).
2.3.1 Benzo(a)pyrene Benzo(a)pyrene (BaP) is produced from incomplete combustion of fuels at temperatures between 300°C and 600°C. The molecular formula is C20H12 and it has a boiling point of 310 °C. BaP is primarily in the particulate state. Benzo(a)pyrene has a vapor pressure of 5.0×10–7 mm Hg, and a low aqueous solubility of 0.0038 mg/L, suggesting that it exist in the non- aqueous phase. Benzo(a)pyrene is very harmful due to its ability to form carcinogenic and mutagenic metabolites, such as (+)-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, which intercalate into DNA, and thus interferes with transcription. The presence of BaP is moderately persistent in the environment. It is usually bound in the soil so it does not leach easily into the ground water, although there has been detection of some BaP in ground water. Benzo(a)pyrene in contact with water will adsorb strongly to sediments and particulate matter. When such PAHs are adhered strongly to the sediments it becomes more difficult to be broken down by microbes or reactive chemicals except by sunlight, which can degrade it (Juhasz & Naidu, 2000).
2.3.2 Benzo(a)anthracene Benzo(a)anthracene is an atmospheric contaminant generated around power plants and mostly around highways. It is classified as high molecular weight due to the presence of four benzene rings in its structure. The structure contains an angular shaped benzene ring and is considered as a perifused PAH. It has the chemical formula C18H12. Benzo(a)anthracene exist as a gas and binds to particulates in the atmosphere and can only be removed by ozonolysis reactions mechanism. The half-life is less than a day, but depends on the nature of the particulate matter it is adhered to (U.S. EPA, 1994). Tobacco smoke, automobile exhaust, coal tar and petroleum asphalt are sources of benzo(a)anthracene in the environment, while when it comes to food it is mainly found in roasted coffee, broiled charcoal, and barbecued or smoked meats.
9 2.3.3 Chrysene Chrysene consists of four benzene rings. Chrysene is a white crystalline solid, which is produced from coal tar. It has a very strong blue fluorescence, which is yellowish due to the traces of yellow-orange isomer tetracene impurities. Although it is denser than water, chrysene is insoluble in water. It is usually used in the industry to join electrical parts or to preserve wood. Some evidence shows that chrysene can cause cancer in laboratory animals when contaminated with more strongly carcinogenic compounds such as BaP.
2.3.4 Benzo(b)fluoranthene Benzo(b)fluoranthene, B(b)F is a colorless solid, which is insoluble in most solvents. Impure benzo(f)fluoranthene exists as an off-white powder. It has a molecular formula of C20H12. It is produced from incomplete combustion of hydrocarbons, coal, exhausts of cars, and in cigarette smoke. Benzo(b)fluoranthene has a vapor pressure of 5.0×10–7 mm Hg at 25°C, which indicates that it can exist in both the vapor and particulate phases in the atmosphere.
2.4 Physicochemical Properties of Polycyclic Aromatic Hydrocarbons Most PAHs has a “bay-region” and a “k-region” epoxide, which give the PAHs their characteristic reactions. The bay regions are associated with PAHs with 4 and 5 carbon atoms, and the k-region is found in the 9th and 10th oleinic aromatic double bonds, and has high electron density. The differences in the epoxide region accounts for the variation in the physical and chemical characteristics of the PAHs (Ferrarese et al., 2008). Due to the double bonds the k-region epoxides, PAHs pose more carcinogenic activities in the high molecular weight (HMW), since they adhere strongly to soils, making them highly resistant to microbial degradation. On the other hand, PAH gets into food through the smoke that is produced during the cooking process and sometimes the fats that drips on the hot heat source and vaporizes on the surface of the food. However, the bay-region in the low molecular weight PAHs makes them more soluble, volatile, and easily degradable. The physicochemical properties of vapor pressure, solubility, melting, and boiling points of some selected PAHs are summarized in Table 1.
10 2.4.1 Vapor pressure PAHs have low vapor pressures and are thus easily adhered to particulate matter in the atmosphere. Their vapor pressures are inversely proportional to the number of rings they contain, and as such, decrease with increasing molecular size. PAHs with rings between two to four rings vaporize easily and exist as gases. The lower molecular weight PAHs are not easily adhered to particulate matter and are therefore free to move in the atmosphere (Ministry of the Environment, 1997). Some PAHs found in the vapor phases can absorb onto particulate matter (Ravindra et al., 2008). The vapor pressures of PAHs vary due to differences in molecular weight, thus causing the PAHs to distribute in different concentrations in the vapor and the absorbed phases (Abdel-Shafy & Mansour, 2016).
2.4.2 Solubility PAHs are highly lipophilic and non-polar due to their aromatic ring structure. The occurrence of PAHs in the environment and food are mainly influenced by physiochemical factors that allows their absorption and distribution. The relative solubility of PAHs in water and organic solvents are determined by the ability of the PAH to be distributed and transported in the environment (Table 1). Most PAHs are thus insoluble in water but soluble in organic solvents (Choi et al., 2010). The solubility of PAHs in water decreases as the molecular mass increases (Johnsen et al., 2005; Ferrarese et al., 2008), and depends largely on the number of rings it contains. PAHs containing five or more rings are less soluble in water than those with fewer rings. The PAHs with two or three rings, dissolve readily in water, thus becoming more available for organisms to consume because they are easily broken down easily and readily taken up by aquatic organisms and thus enter the food chain (Choi et al., 2010; Johnsen et al., 2005; Mackay & Callcott, 1998). Most of these PAHs exist in the solid states attached to food, soils, and sediments, and sometimes in water and dissolve in any oily substance in the water. The larger PAH members are also poorly soluble in organic solvents as well as lipids, and PAHs stabilize when accumulated in fats and oils, making them more difficult to dissolve (Arrebola et al., 2006).
11 2.4.3 Boiling point PAHs are colorless, white to pale yellow-green solids at room temperature. All completely unsaturated PAHs are solid at room temperature and are often attached to particulate matter (Aronstein et al., 1993). They have relatively high melting and boiling points (Table 1). Most of the PAHs exist in the particulate phases, which are produced in wood smoke. Table 1: Physico-chemical properties of common Polycyclic Aromatic Hydrocarbons Molecular Boiling Melting Solubility in water Compound weight(g/mol) point(0C) point(0C) (mg/L) Acenaphthene 154 96 95 3.8 Acenaphthylene 154 65 92 3.9 Anthracene 178 342 218 4.5 ×10 -2 Benzo(a)anthracene 228 400 158 1.1 ×10 -2 Benzo(a)pyrene 158 310 179 3.8 ×10 -3 Benzo(b)fluoranthene 252 - 168 1.5 ×10 -3 Benzo(k)fluoranthene 252 480 216 7.6 ×10 -4 Benzo (g, h, i) pyrelene 276 330 273 2.6×10 -4 Chrysene 228 448 256 2.0 ×10 -3 Fluoranthene 202 - 11 2.6 ×10 -1 Fluorene 166 295 116 1.9 Indeno(1,2,3-c,d)pyrene 276 330 164 6.2 ×10 -2 Phenanthrene 178 340 100 1.1 Pyrene 202 393 156 1.3 ×10 -1 Dibenzo(a,h)anthracene 278 - 262 6.0 ×10 -4 -4 Benzo(j)fluoranthene 252 - 166 7.6 ×10 Pissinatti & de Souza (2017)
2.5 Carcinogenicity of Polycyclic Aromatic Hydrocarbons
PAHs have several carcinogenic effects due to the activities that they are involved in when they enter the body of the organism. The reactive bay regions of the PAHs contain metabolites such as epoxides and dihydrodiols, which can bind to the DNA of the organism when ingested. This causes cancer, tumors, mutations and developmental deformations due to changes in the DNA arrangements and replications during cell division. Exposure of high levels of PAHs for long periods of time results in the development of lung cancer, skin cancer and stomach cancer from inhalation, ingestion and skin contact, respectively. PAHs have a low acute toxicity, and it is therefore difficult to evaluate the effects of individual PAHs to the total carcinogenicity of complex PAH mixtures in humans. The International Agency for Research on Cancer (IARC) (1987), and EPA (1994) evaluated PAHs and labelled them as having carcinogenic, and mutagenic effects. The EPA and IARC proposed some selected carcinogenic PAHs activities, which are of great concern (Fernandez-Sánchez et al., 2004).
12 The biological activity of the PAHs depends on the structure of the PAH in question, and thus each different isomer of the PAHs exhibit different carcinogenic activity. The number of rings also influence the level of toxicity of the PAHs. Studies have shown that high molecular weight PAHs pose more threats to organisms and humans than those of lower molecular weight PAHs (Ledesma et al., 2014; Chen & Chen, 2005; Iwegbue et al., 2015; Orecchio et al., 2009). The presence of benzo(a)pyrene-diol- epoxide activates the tumor suppression ability in certain cells, leading to cancer (Pfeifer, 2002). Alomirah et al. (2011) found the cancer risk of benzo(a)pyrene in children and adults after consuming grilled vegetables, chicken and smoked fish. It was concluded that the adults were more at risk from the higher consumption rate as compared to children at a ratio of 1: 3 (children 2.63 and adult 9.3 μg /kg /day). Essumang et al. (2012) found carcinogenic risk of ingesting benzo(a)pyrene, chrysene, benzo(a)anthracene and indenol(1,2,3-cd)pyrene in smoked sardines to be in a 1:3 adult to child ratio (children 1.6 and adult 6.1 μg /kg /day) respectively.
High consumption of smoked salmon prepared by the Native American fish method has been found to pose carcinogenicity due to the higher levels of PAHs produced from this smoking method (Forsberg et al., 2012). The fillets were exposed to direct smoke from smoldering wood for several hours for 2-3 days. The study revealed that consuming such smoked fish could result in an elevated cancer risk because of the high levels of PAHs formed (Forsberg et al., 2012). Table 2 summarizes the risk of genotoxicity of and carcinogenicity of the 15 PAHs compounds. Table 2: Carcinogenic and mutagenic properties of Polycyclic Aromatic Hydrocarbons Name of compound Abbreviation Genotoxity IARC Classification Acenaphthene ACE Questionable Unknown Acenaphthylene ACEN Questionable Unknown Anthracene ANT Negative 3 Benzo(a)anthracene BaA Positive 2 Benzo(b)fluoranthene BbF Positive 2 Benzo (g, h,i)perylene BghiP Positive 3 Benzo(a)pyrene BaP Positive 1 Chrysene CHRY Positive 2 Dibenzo(a,h)anthracene DBahA Positive 2 Fluoranthene FLT Positive 3 Fluorene FLU Negative 3 Indeno(1,2,3cd) pyrene IcdP Positive 2 Phenanthrene PHE Questionable 3 Pyrene PYR Questionable 3 Naphthalene NAPH Positive 2 Benzo(k)fluoranthene BfK Positive 2 1- Carcinogenic 2- Probably carcinogenic 3- Not classifiable. Fernández-Sánchez et al., (2004); IARC (2001).
13 2.6 Reactions of Polycyclic Aromatic Hydrocarbons
2.6.1Reactions of Benzo(a)pyrene with Deoxyribonucleic acid (DNA) PAHs metabolics result in the formation of products such as quinones, phenols, epoxide intermediates and dihydrodiol (ATSDR, 1995). During the process, the carcinogenic compound formed is converted into carcinogen which binds covalently to information carrying molecules such as the deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) resulting in the formation of carcinogens (Figure 6). The process takes place in three enzymatic reactions resulting in the formation of the final product. A BaP diol epoxide is formed which suppresses the tumors’ ability resulting in the formation of cancer. The BaP diol epoxide metabolite formed reacts with and binds to DNA, forming mutations, which may result in cancer (Denissenko et al., 1996). PAHs enter internal adipose tissues and are stored in the organs. The PAHs are then activated by specific enzyme by epoxidation which is conjugated with glutathione. The epoxides that are non-conjugated with glutathione are then converted into phenols and diols. The PAHs metabolites are non-polar and as such are difficult to be excreted through feces and urine.
Figure 6: Activation of BaP leading to DNA lesions (adapted from Stansbury et al., 1994).
14 2.7 Toxicological Effect of Polycyclic Aromatic Hydrocarbons The toxicity of PAHs in aquatic organisms are mostly due to the metabolism and photo- oxidation in the presence of ultraviolet light. Organisms that are exposed to PAHs are likely to have different adverse effects, such as immunotoxicity, genotoxicity, mutagenicity and carcinogenicity (Philips, 1999). Low molecular weight PAHs pose more toxic effects than heavy PAHs (Wenzl et al., 2006; Ferraresa et al., 2008). Mice exposed to high levels of benzo(a)pyrene during pregnancy result in an increase in formation of micronuclei during early stages of gestation thus causing deformities in the DNA (Wang et al., 1990). There is furthermore an increased risk of decrease in body weight of the offspring and birth defects if the mice were exposed to benzo(a)pyrene. Benzo(a)pyrene poses toxic threats to cells, tissues of animals, and even the development of the immune systems and abnormalities during birth (Essumang et al., 2013). Induction of skin cancer in laboratory animals exposed to PAHs develops adverse skin deformities (ATSDR, 1995). People with normal skin and pre-existing skin conditions may be at risk of developing adverse dermal effects of rashes and cancer when exposed to benzo(a)pyrene, anthracene, benzo(a)anthracene (ATSDR, 1995). Women exposed to high levels of PAHs may be at increased risk of reproductive dysfunction (Perera, 2003). Benzo(a)pyrene exposure may reduce fertility and the ability to bear children due to placenta dysfunction (Huel et al., 1992). Exposure to PAHs results in the adverse development of liver and skin diseases in humans (ATSDR, 1995).
2.8 Regulations of Dietary exposure of Polycyclic Aromatic Hydrocarbons The occurrence of PAHs in the environment has called for regulations to reduce its impact on the environment and human health. Th US Environmental Protection Agency (EPA) has developed a standard criterion, which seeks to protect carcinogens effects on humans through PAH exposure. In 2008, the European Food Safety Authority (EFSA) reported the oral carcinogenic effects of PAHs particularly focus on BaP. A limit was set to form indicator basis for PAHs analysis in food (EFSA, 2008). The regulation was based on the route of dietary exposure and it was concluded that the methods of cooking are a possible route of exposure of PAHs to humans. Therefore, the Scientific Committee on Food (SCF) amended the limits for PAH in food in 2011. The maximum level for BaP in most foods was set to 2 μg/kg. The new acceptable maximum levels of BaP in food are detailed in the Commission Regulation (Regulation, EU 835/2011), amending the previous regulation of No 1881/2006. Under the new regulation, emphasis was placed on PAH4, due to their predominant dietary exposure. The regulation stipulated that as from 2014 the maximum levels for the sum of PAH4 should not exceed 12 μg/kg, whilst that of BaP alone was set to a maximum of 2 μg/kg. The maximum levels for BaP and most carcinogenic PAH4 are shown in the table 3.
15 Table 3: Maximum limits of BaP and PAH4 in selected foodstuffs under EU 1327 /2014 Product Levels of BaP Levels of PAH4 (μg /kg) (μg/kg) Cereals for baby foods 1.0 1.0 Smoked meat and meat products 2.0 12.0 Smoked fish and fish products 2.0 12.0 Coconut oil 2.0 20.0 Fats and oils 2.0 10.0 Smoked mullusks 5.0 30.0 Cocoa beans 5.0 30.0 (EFSA, 2014)
2.9 Dietary exposure of Polycyclic Aromatic Hydrocarbons Many thermally processed and uncooked food products, such as bread, oils, cereals, smoked meat and fish, has been found to contain some amounts of PAHs (Phillips, 1999; Gomaa et al., 1993). The concentrations of PAHs in uncooked foods depends mainly on the source of the foods. For example, fruits and vegetables grown around heavily traffic roads has been found to contain benzo(a)pyrene, chrysene and dibenzo(a,h)anthracene (Larsson, 1984).
Several studies have been done to determine the exposure of diet in human. Analysis of PAHs in diet in the UK revealed that the major route of exposure was through cereals with only a fraction coming from fruits, vegetables and sugar. Out of a total daily consumption of 1.46 kg of food and beverages, the daily exposure was found to be 3.7 μg/kg (Dennis, 1984). A similar study done in the Dutch also reported an average daily intake of between 5 and 17 μg/kg. It was concluded that cereal products are the major source of PAHs whereas fruits and vegetables also contribute but to less extent. Another study done on Italian cereals, vegetables, fruits, milk and meat revealed that these food stuffs contributes to high levels of PAHs especially oven-baked pizza and barbecued meat (Lodovica, 1995).
During cooking of food, particularly meat over open flame, there is a possibility of PAHs formation. If direct contact of the meat to the flame occurs, pyrolysis of the fats in the meat can also leads to PAHs being formed, which then deposit on the meat. This can also happen when the fats drips into the flame, and thus generate PAHs. In Sweden, 19 samples of smoked fish and smoked fish products from a commercial smoke- house revealed a BaP concentration of higher 1 μg /kg (Larsson, 1982). Cooking food using charcoal is also a way for PAHs to get into the food. Charred foods contain PAHs, but their level depend on the proximity of the heat source to the meat, as well as the fat content of the food (Howard & Fazio, 1980). A study on PAHs in smoked mackerel revealed that the combined level of benzo(a)pyrene), benzo(b)fluoranthene and chrysene was 6.9 μg/kg during open fire smoking. The levels of BaP were < 0.5 μg/kg and 1.1 μg/kg in the kiln cabin and open fire respectively (Odoi, 2014). In Egypt, studies on the sources and daily intake of PAHs was analyzed using smoked fish. The results showed that the smoked fish contained phenanthrene, fluorene, acenaphthylene, and acenaphthene at mean concentrations of 122, 111, 80.4, and 76 μg/kg, respectively, with naphthalene being the most common PAH (Abdel-Shafy & Mansour, 2016).
16 In China, analysis on PAHs revealed that smoked lamb contained high levels of BaP and it was attributed to the fat content, the type of wood used for the smoking and the heating temperature (Chen & Chen, 2001). This highest level was due to the smaller distance of the food to the source of heat (charcoal). Commercially smoked meat products from the Italian market (e.g. sausages pitina) were found to contain BaP at a concentration as low as 0.1 μg/kg but this level increased when the smoking was done with the meat placed close to the fire (Purcaro, 2009). A study on barbecued meat was furthermore tested to determine the level of PAH4 from the Danish market. The results showed BaP levels of 63 μg/kg, and a total level of 195 μg/kg for PAH4, respectively (Duedahl – Olesen et al., 2006). However, in Germany with 22 samples of smoked meat products showed BaP level of 0.15 µg/kg though the samples were prone to contain PAHs because of their blackened surface and smoky odor (Jira et al., 2008). Similar study done in the US showed the presence of phenanthrene as high as 12 μg/kg (Forsberg et al, 2012) when the fish was smoked for three days. Table 4 shows a comparative study done on PAHs from different foodstuffs. Table 4: Comparative study on PAH levels from different foodstuffs Food Method PAH Levels/μg/kg Country Reference type Beef Smoking BaP 0.15 China Chung et al., 2011 Pork Smoking BaP 0.30 China Chung et al., 2011 Pork Smoking PHE 9.32 Spain Lorenzo et al., 2010 Lamb Smoking BaP 2.80 Estonia Reinik et al., 2007 Chicken Smoking BaP 6.50 Estonia Reinik et al.,2000 Fish Smoking BaP 10.6 Czech Rep Simko, 1991 Fish Smoking PHE 12.37 USA Forsberg et al., 2012 Fish Smoking BaP 12 China Wang et al., 1999 Fish Smoking BaA 60.08 Ghana Palm et al., 2012 Sausage Smoking BaP 30 Czech Rep Suchanová et al., 2008 Fish Smoking BaP 73.78 Ghana Essumang et al.,2013 Fish Smoking BaP ˂ 0.5 Iceland Odoi, 2014 *Levels marked with bold exceeded the maximum limit of PAH levels from EU 1327/14
2.10 Alternative sources and distribution of Polycyclic Aromatic Hydrocarbons Polycyclic Aromatic Hydrocarbons are chemicals which comprises of different compounds aiding many biological activities in carcinogenic and mutagenic processes (RASFF report, 2007). Origination of PAHs can be environmental sources (natural and anthropogenic), food processing in the industry (e.g. smoking, heating and drying) and cooking methods (e.g. grilling, frying and roasting) (EFSA, 2008; Fromberg et al., 2007; Guillen & Sopelana, 2003), as mentioned before. Industrial oil burning as a source is identified using the markers, fluoranthene, pyrene and chrysene. Burning oil produces produces high levels of volatile PAHs, and low levels of high molecular weight PAHs (Ravindra et al., 2006).
Polycyclic Aromatic Hydrocarbons can also be found in the air when mixed with dust. Higher levels of PAHs are generated at workplaces from processes such as coal tar production, aluminum production, smokehouse operations, municipal trash incineration and
17 asphalt production (Woodard & Snedeker, 2001). Human exposure to PAHs is common through the air and drinking water but it is noted that the highest exposure is through food (Suchanova et al., 2008). The occurrence of PAHs in effusive and explosive rocks is due to volcanic and volcanic hydrothermal activities in geothermally active regions, such as Iceland (Geptner et al., 1999a; 1999b). This also results in the deposition of volcanic ash in the atmosphere. The distribution of PAHs is higher and diverse due to the alteration of the hydrothermal rocks and minerals (Geptner et al., 2003). Also, studies have shown that the accumulation of hydrocarbons in Skjálfandi area due to the emanation of gas from the Öxarfjörđur geothermal field (Ólafsson et al., 1993). Polycyclic Aromatic Hydrocarbons can also be found in tobacco smoke. Humans are exposed to PAHs through cigarette smoking. The main route of exposure of PAHs for smokers is through cigarette smoking while non-smokers are exposed through food. Daily PAH exposure from smoking increases by 30-fold daily (ESFA, 2008). The concentration level of carcinogenic PAHs through smoking is estimated at 5μg/day (Menzie et al., 1992). The effects of non-smokers to PAHs is associated with their exposure to tobacco smoke in the environment. About 50% of attributed risk for bladder cancer is associated with cigarette smoking (Maclure et al., 1989). Humans can also be exposed to PAHs through indoor air. During food preparation, unvented space heaters produces smoke from indoor combustion contribute to human exposure to PAHs (Lioy & Greenberg, 1990).
Soil can also be contaminated with PAHs in areas like coal burning sites, wood preserving facilities using creosote, and sewage treatment plants. Soils that are found around vehicular fallouts contains higher levels of PAHs compared to urban areas. Essumang et al. (2006) found a concentration of various types of PAHs ranging from 3,500 μg/kg to 111,200 μg/kg with the highest concentration being from acenaphthene in the soil of Ghana. Heavy traffic and aerial deposition from forest fires and lightening could also lead to high concentrations of PAHs in the soil (Man et al., 2013). Soils which contains high levels of PAHs tend to have lower organic matter activities making it highly toxic (Shrestha et al., 2015). PAHs concentration in the soil may arise from human activities including discarding open burnt crop residues, land clearing and providing nutrients for next growth cycle (Lemieux et al., 2004).
PAHs can also get into water through different ways such as agricultural activities, mining and run off. Water can become contaminated with PAHs from runoff in urban areas, waste water from certain industries (e.g. creosote manufacturing, aluminum smelting), or from PAHs carried in the air. The World Health Organization (WHO) gives guidelines for PAH standard in drinking water in which the maximum concentration of benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, benzo(g,h,i)perylene should be 0.2 µg/l (WHO, 2004). The European Commission (EC) directive on water for human consumption intends that water should be monitored based on microbiological, chemical of health concern and indicator parameters.
18 2.11 Contributing factors for Polycyclic Aromatic Hydrocarbons formation in food
2.11.1 Preservation methods PAHs are generated through many preservation and cooking methods such as smoking, roasting, frying and grilling. In raw foods, PAHs are often generated through atmospheric deposition, unlike in cooked foods where it is attributed to the method of preparation, such as grilling, barbecuing and smoking (Lyons, 1997).
2.11.1.1 Smoking
Smoking is one of the oldest methods used to preserve meat and fish (Stolyhwo & Sikorski, 2005; Swastawati et al., 2000). Furthermore, the smoke produced from the burning wood enhances the aroma and taste in the food. Smoking gives flavor and rich brown color in fish thus enhancing the sensory quality of the product (Kolodziejska et al. 2002). The wood smoke releases chemicals such as formaldehyde and alcohols, which can preserve the food (Horner, 1997). During cooking the smoke contaminates the food because of incomplete combustion of the fuel. The amount of smoke depends on the fuel used, the smoking flavor agent and temperature. The smoke that is produced is made of particulates and gases. Modern controlled kilns can be used to control the formation of smoke and can produce high quality products (Ciecierska & Obiedzinski, 2007). In today’s smoking, the smoked product has high water content, and a low salt content to meet the consumers taste (Kolodziejski et al., 2002). To increase storage stability a drying step is often added in the fish smoking processing to remove moisture, which increases the storage stability and affects the texture of the final product.
Basically, there are two main methods of smoking; hot smoking, which is also referred to as traditional smoking, and cold smoking. The traditional smoking methods involves salting the fish before smoking, and wood is the most common fuel used for this type of smoking. Normally the hot smoking occurs at temperatures between 70 °C to 80 °C, which results in cooking of the fish. During cold smoking the temperatures are kept below 30 °C (Arason et al., 2014). Cold smoking of fish involves the use of flavors and curing with smoke. The fish is placed in an unheated chamber and smoke is pumped through the chamber for about 12-24 hours. Usually chips and sawdust are used to provide the heat so that the aromatics of the wood oils can be released unto the fish.
During hot smoking, the process goes through three stages (i.e. drying, smoking and partial cooking) and the temperature of the smoke ranges from 40°C to 100 °C. The core temperature of the product being smoked typically reaches 85 °C at the end of smoking (Andrzej & Zdzislaw, 2005). Each stage of the smoking process has an impact of the shelf life of the fish, flavor and texture of the final product. The first step is pre-drying at a temperature between 30 oC to 40 o C, followed by smoking or partial cooking at a temperature between 40 to 60 o C, which gives the fish a characteristic color and odor. The final stage is the actual smoking and cooking stage where the fish is subjected to a temperature between 70 oC to 100 oC (Kenneth & Hilderbrand, 1992). The initial temperature of the smoke house is
19 generally kept at 30°C, and the air inlets are kept open to about half or up to three quarters to facilitate an even air circulation. The fish is then dried after smoking in an oven. The drying process depends on the level of dryness required to reach the desired texture and water activity, thereby inhibiting microbiological and enzymatic activities in the product. The water activity in the smoked product must be less than 0.85 to keep the product stable at room temperature (Arason et al., 2014).
2.11.1.2 Smoking equipment
Modern smoking methods, involves the use of smoke generators, where the food is separated from the smoke inside the chamber (Joint FAO/WHO Codex Alimentarius Commission, April, 2008). Exposing the fish or meat products directly to the smoke could result in higher concentration of PAHs compared to indirect exposure methods (Roda et al., 1999). Some traditional smoking methods involve exposing the fish directly to smoke from smoldering wood for several hours up to 2 or 3 days (Forsberg et al, 2012). However, in most developing countries, especially Africa, smoking of food products is done in traditional ovens or drums, such as altona smokers, banda smokers, or the chorkor smoking kilns (Olatunji et al., 2015; Palm et al., 2011). Generally, the smoking technique involves either hanging the food or placing it on racks in a chamber designed to contain the smoke. In commercial smokehouses, steam pipes are often used to supplement the heat of a natural sawdust fire or hardwood fire. The smoking fuel used, straws and wood chips, burn partially in the smokehouses generating PAHs. The smoke that is generated from painted wood and corrugated cardboards produces more smoke, which also contains PAHs (Esposito et al., 2015). Hot smoking can result in a higher level of PAHs than cold smoking (Joint FAO/WHO CAC, 2008; Kleter, 2004) due to the higher temperatures obtained.
2.11.2 Effect of chemical composition of the product Food products that have a high amount of fat are more susceptible to PAHs formation than lean products (Phillips, 1999; Kazerouni et al., 2001). PAHs accumulate in fats and oils and binds together making them more stable (Arrebola et al., 2006). Lipid and fatty acid composition vary greatly between species, and even within the same species, which may be affected by catching ground, season and feed availability (Romotowska et al., 2016). The relative lipid content in mackerel ranges between 4% and 24% during the winter and summer respectively because of seasonal variations in feeding (Leu, et al., 1981). The lipid content of the fish may also change when the fish is processed or stored under certain conditions for later processing. During fish processing and storage, e.g. smoking, there is likelihood of deterioration of vitamins and lipids, which reduces the nutritional value of the product.
PAH concentrations in fish or meat being processed are thus influenced by the amount of fat present (Kendirci et al., 2014; Stumpe- Viksna et al., 2008; Djinovic et al., 2008). Furthermore, if the fat drips from the product upon heating into the heat source it will generate more smoke. Several studies have shown that the levels of the PAHs are a result of the pyrolysis of fat that dripped from the meat on the heat source during the grilling process (Chen & Chen, 2001; Chung et al., 2011; Lee et al., 2016). Formation of carcinogenic PAHs
20 associated with fat content can thus be reduced during grilling if the melting fat does not encounter the heat source (Farhadian et al., 2011). Also, the cooking method used influenced the formation of PAHs. Grilling and barbecuing draws out more fats from food product than smoking, but a short smoking time and lower temperature reduces the levels of PAHs formed compared to grilling (Chen & Lin, 1997).
The lipid content in fatty fish with high polyunsaturated fatty acids (PUFA) can change during processing making it more susceptible to hydrolysis and oxidation during storage (Richards, 2000; Aubourg, 2005). Fatty acids hydrolyze in the fish muscle and cause degradation during storage. The fatty acids composition and lipid content affects the oxidation of the lipid. This is a result of the formation of the oxygen species and oxidative compounds facilitating the metabolic processes in the stored fish (Richards, 2002). The unsaturation of the fatty acids in relation to the lipid content is affected by the amount of feed source available and the temperature variation of the ocean (Bandara et al., 2001; Celik, 2008). Higher ocean temperatures decrease the rate of fatty acids saturation (Orban et al., 2011).
2.11.3 Type of fuel used The type of fuel that is used in the preparation and processing of food items generates PAHs. Most of these fuels especially wood, burns to produce smoke when there is insufficient supply of oxygen. Most often hardwood, saw dust, and other materials are used as fuel for during the smoking (Kleter, 2004). These materials burn to produce a lot of smoke which hitherto comes into contact with the food to contaminate it. The high concentrations of PAHs in the grilled meat samples could also be attributed to the type of wood used for the charcoals (Palm et al., 2011). Kakareka et al. (2005) realized a high concentration of BaP, the most carcinogenic PAH to ranging from 55,000 to 968,000 μg/kg was due to the heavy soot on the skin of the meat. Smoking fish for a longer time using hardwood as fuel has the potential of producing high levels of PAHs and such fish is not safe for human consumption (Essumang et al., 2013).
2.11.4 Temperature PAH formation is greatly influenced by temperature. The levels of PAHs that accumulate in smoked foods are affected by the type of smoke generator, the type of fuel used, the smoking duration and combustion temperature, as mentioned before. The temperature at which smoke is formed affects the duration of the smoking process. Thermal decomposition of wood at high temperatures has the potential of generating high levels of PAHs. McGrath et al. (2007) found that the temperature for smoke production from wood fires ranges between 500 and 550 o C, whereas an increase in temperature from 400 to 1000 0 C could result in the formation of very high levels of PAHs (Larsson, 1982). The required temperature for PAH formation can also depend on the type of compounds being burnt. Ledesma et al. (2014) reported that when tertiary tar compounds are burnt at a temperature above 750 0C PAHs are formed. The amount of smoke that is formed inside the oven is dependent on the smoke generator which in turn influence the PAH formation.
21 2.12 Other methods of fish preservation Fish spoil fast and as such different methods are used to reduce spoilage. Storing fish at low temperatures inhibits bacterial growth, thus minimizing spoilage. High temperatures at tropical regions facilitates bacterial growth to initiate flesh fish spoilage. Fish decomposition usually occurs from the enzymatic reactions (Sikorski & Kolakowska, 1994), lipid oxidation (Romotowska et al., 2016), and microbial activities (Hansen et al., 1995) in the fish.
2.12.1 Salting Salting of fish is used to prolong the shelf life by lowering the water activity (Swastawati et al., 2000). Salting involves the use of sodium chloride (NaCl) as a preservative, which penetrates the tissues, thus limiting the bacterial growth and may cause inactivation of the enzymes. The salt can cause the fish muscle to swell or shrink depending on the concentration (Gudjónsdóttir et al., 2013). The amount of salt that is used in the salting influences the distribution of water and salt in the muscle (Gudjónsdóttir et al., 2016). A higher salt content also has the tendency to lower the initial freezing point of the food (Chen, 1985) and affects the water holding capacity (WHC) (Bocker et al., 2008).
Light salting is recommended prior to smoking since it may help to increases the moisture content of the product and reduce drip (Thorarinsdottir et al, 2002). Light salting is done either by injection of the fish with salt, or by immersion of the fish in brine resulting in a salt concentration of up to 2% (Gudjónsdóttir et al, 2010). Brine injection has been found to increase the rate of drip loss due to the high tendency of puncturing the muscle cells making it more porous. It makes the muscle more susceptible to water draining from the muscle and thus reducing the water holding capacity i the salting is not performed at optimal conditions and injection settings (Gudjónsdóttir et al, 2010).
2.12.2 Cooling and freezing Freezing has been one of the oldest methods used for preserving fish (Karlsdottir et al., 2014). Lowering the temperature is an effective way to reduce the enzyme and bacterial actions which causes spoilage, and thus cooling and freezing slows down food deterioration (Aubourg et al., 2005; Saeed & Howell, 2002). When the fish is stored at chilled temperatures the quality of the fish is it ensured, especially regarding reduced gaping, peritoneum deterioration, and viscera rapture and bruises, and reduces enzyme activities (Aubourg, 2001). Before freezing of fish cold temperatures are preferred to prevent microbial spoilage. The preferred cooling temperatures for mackerel on board fishing vessels, prior to freezing is between -1 0C to -1.8 0C (Margeirsson et al., 2012). An optimum temperature of 0 to 2 ºC enables fish to become stable since the water in the muscles does not freeze which can cause aggregation and destruction of muscle proteins (Lauzon et al., 2009). The freezing method and frozen storage conditions help stabilizing the nutritional and sensory attributes of the fish products (Erickson, 1997). Large crystals formed inside the fish muscle during slow freezing have furthermore the tendency of rupturing the fish tissues to
22 cause drip during freezing. The drips become channel for soluble protein, vitamins and minerals to leach out giving the fish to have undesirable appearance.
In fatty fishes, frozen storage can also lead to lipid oxidation and hydrolysis, reducing the shell life of the product (Aubourg, 1999; Aubourg & Gallardo, 2005). Lipid oxidation occurring during frozen storage involves the formation of various unstable compounds in the muscle, especially hydroperoxides as primary oxidation products, and thiobarbituric acid reactive substances (TBARS) and other secondary oxidation products (Aubourg, 1999).
23
3 METHODS AND MATERIALS
3.1 Materials
3.1.1 Raw Material Atlantic mackerel used in the study was provided by the Síldarvinnslan Fish Processing Company, located in Neskaupsstaður, Iceland. The fish was caught in the Atlantic Ocean, South East of Iceland on August, 2016. After landing, the fish were frozen in blocks and transported to the MATIS laboratory in Reykjavík. The fish were then thawed, cleaned and divided into two groups. One group was de-headed and filleted, while the remaining group was left as whole.
3.1.2 Smoking equipment The smoking equipment used for the study were the open drum, cabin smoking kiln and a Bradley smoker. The open drum was made of a cylindrical drum with an open top and with a perforated base. The fish were placed on racks, which were placed over the open top within the drum. The fire source was beneath the wire trays and could be controlled, thus resulting in lower chances of charring the fish. The cabin smoker was constructed by MATIS in collaboration with the United Nations University, Fisheries Training Programme (UNU-FTP) as a prototype of a refined smoking unit used in Tanzania. The cabin smoker was made of a wooden frame, and has a metal drum beneath where the wood is placed to provide the heat. A long metal tube is joined from the drum to the top of the cabin to serve as a chimney, so that the heat can pass through and distribute the smoke through the smoking unit. A small metal plate is attached to the hole, which regulates the heat entering the chamber and ensures an even circulation of hot air through the smoking chamber. A removable wooden frame with a wire mesh is fixed inside the chamber, on which the fish are placed during the smoking process. The Bradley smoker had a polished stainless steel interior and an epoxy steel exterior, with temperature, time, and smoke controls (Figure 7).
Open fire drum Bradley smoker Cabin smoker Figure 7: Smoking equipment used for the study
25 3.1.3 Chemicals All of chemicals that will be used for the project will of analytical grade and bought from Sigma-Aldrich Company, Taufkirchen, Germany.
3.2 Pre-trial A pre-trial was performed to determine the required brine concentrations for an optimal salt concentration in the final product, the needed time for smoking, to choose appropriate smoking methods, and which smoking kilns to use in the main experiment. Fish fillets from two frozen blocks were left at room temperature overnight to thaw completely. With the aim of obtaining a salt concentration of 4% in the fish muscle of the final product, two different brine concentrations of 8% and 12% were tested during brining prior to the smoking. The fillets were brined for 90 minutes in either salt concentration in a ratio of 2:1 (brine: fish) at kept in the cooler at 0-4°C. After the brining, the fish were placed on plastic racks and left overnight to drain in the cooler at 0-4°C. The open fire drum, the Bradley and cabin smoking kilns were used for the pre-trial. Birch and beech wood, and hickey wood was used as fuels in the open fire drum and Bradley, respectively, and were obtained from Húsasmiðjan Reykjavik, Iceland, whereas the dung used in the cabin smoker was obtained from a farm in Borgarfell, Iceland. The different smoking kilns were set up and pre-heated for 60 minutes. The fish were then placed on wire mesh trays and inserted into the smoking kilns. Two temperature loggers were used to follow the temperature profile in the fish and on the tray to measure the internal temperatures of the smoking kiln and fish muscle, respectively. The temperature of the fish muscle was measured every 10 minutes until the desired temperature of 70 °C was attained. The fish were then taken out of the smoking kilns and dried at 50 °C for 2 hours in a convection oven (Convotherm, HUD 1010, Germany). The fish were then air packed and kept at room temperature while the remaining half was vacuumed parked and kept at 0-4°C.
26 3.3 The main experiment To obtain the desired 3% salt concentration in the samples, both fillets and whole mackerel were placed in a brine with 8% salt concentration and kept at 0-4°C for 45 and 60 minutes, respectively. The fish were then placed at on drying racks at 0-4°C and kept overnight to drain the excess water. The smoke in the cabin kiln smoking chamber was generated with birch wood chunks and beech wood chips, while the hickory wood was used to generate heat in the Bradley. Both smoking kilns were heated up for 30 minutes before the fish were placed inside. The smoking was performed and the temperature inside the oven and the muscle of the fish were monitored in 1 minute intervals until the required temperature in the cabin was reached. The fillets and whole fish were taken out of the smoker and then dried for 2 and 3 hours respectively in the convection oven (Convotherm, HUD 1010, Germany) at 50 °C.
After the smoking and drying the samples were packed in plastic bags, either under vacuum conditions or air packaging, and placed at either 0-4°C or 15-20°C. The samples were then grouped and identified per the smoking method, packaging material and storage temperature. The samples were identified as follows: Fillets smoked in the cabin smoker were labelled with the initial ‘F’ only, whereas the whole fish smoked in the cabin smoker was identified by ‘W’. The notations ‘FB’ and ‘WB’ indicates fillets and whole fish, respectively, which was smoked in the Bradley smoker. The sample groups and experimental design are summarized in Table 5 and Figure 8, respectively.
Analysis of total volatile base nitrogen (TVB-N), salt content and water content were performed at the chemical laboratory after 5, 7, 10, 18, 25, 30 and 35 days of storage at either 0 - 4°C or 15 - 20 °C as indicated in Table 5. Also, the other chemical analysis like Peroxide value (PV), Thiobarbituric acid reactive substances (TBARS), and Free fatty acids (FFA), were performed in each of the respective storage days. PAH analysis was on the other hand only performed on day 5 of storage, to assess the effect of the smoking methods on PAH formation.
27 Table 5: Experimental design and sampling groups Group Fillet/whole Smoking Storage Packaging Sampling days abbrev. equipment temperature material FAC Fillet Cabin 0 - 4°C Air 5,10,18 FAR Fillet Cabin 15-20 °C Air 5,7,10 FVR Fillet Cabin 15-20 °C Vacuum 5,10,18 FVC Fillet Cabin 0 - 4°C Vacuum 5,15,25,35 FBVC Fillet Bradley 0 - 4°C Vacuum 5,15,25,35 WAC Whole Cabin 0 - 4°C Air 5,10,18 WAR Whole Cabin 15-20 °C Air 5,7,10 WVR Whole Cabin 15-20 °C Vacuum 5,10,18 WVC Whole Cabin 0 - 4°C Vacuum 5,15,25,35 WBVC Whole Bradley 0 - 4°C Vacuum 5,15,25,35
Figure 8: Experimental design for the smoking with the two different kilns.
28 3.3 Physicochemical methods
3.3.1 Temperature measurements The temperatures inside the cabin and Bradley smoking kilns as well as in the fish muscle were monitored using iButton data loggers (DS 1922E, California, USA) every 10 minute interval, by placing temperature loggers at 3 different positions (i.e. inside the fish muscle, on the top rack, and bottom rack) for the entire smoking period. During the 1 minute interval for monitoring the temperature inside the fish and smoking kilns the time of smoking was also monitored.
3.3.2 Water activity
The water activity (aw) of the sample was determined using the Aqua Lab water activity meter (4TE, Washington, USA). About 2 g of the fish muscle were placed in meter and the temperature and water activity was read.
3.3.3 pH measurements The pH of the sample was determined using a pH electrode (SE 104 – Mettler Toledo Knick, Berlin, Germany) which was connected to a portable pH meter (Portames 913 pH, Knick, Berlin, Germany). The electrode was inserted into the fish muscle and the measurement read when the pH stabilized.
3.3.4 Water content The water content of the mackerel samples was determined by measuring the weight difference by drying 5 g of the minced samples at 104 oC 1 oC for 4 hours (ISO, 1999). Results were calculated as g water/100 g sample.
3.3.5 Salt content The salt content in the fish sample was determined with the titration method described in AOAC (2000). 5g of the sample were weighed and placed in extraction bottles. 200 mL of deionized water was added to the sample and shaken for about 50 minutes. 20 mL of nitric acid were then added to 20 mL of the supernatant and titrated with silver nitrate. The salt content in the water phase (Z-value) for each sample was then calculated as: Equation 1 where: msalt is amount of the salt and mwater is the amount of water in the sample.
3.3.6 Lipid content The lipid was extracted using the method by Bligh & Dyer (1959) with adaptations. 25 g of the grounded sample were weighed into a 250 mL centrifuge bottles. 25 mL of chloroform and 50 mL of methanol were added and homogenized for 2 minutes. 25 mL of chloroform were added to the mixture and homogenized again for 1 minute before 25 mL of 0.88% KCl
29 were added and mixed for 1 minute. The mixture was then centrifuged for 20 minutes at 2500 rpm at 4 o C. The lower chloroform phase containing the fat was extracted using a glass pipette. The chloroform phase content was then filtrated on a glass microfiber filter under suction. The suction flask content was poured into a 50 mL volumetric flask and filled with chloroform to the mark. The lipid content was determined gravimetrically and the results were expressed as g lipid/ 100 g of the sample.
3.3.7 Peroxide Value (Primary Oxidation Product) Lipid hydroperoxides (PV) were determined with a modified version of the ferric thiocyanate method (Shantha & Decker, 1994). 5g of the sample were weighed into a 50 mL tube and 10 mL ice-cold chloroform: methanol (1:1) solution were added. The mixture was homogenized for 10 seconds, and 5 mL of 0.5 M Sodium chloride were then added and homogenized for 30 seconds. The mixture was then centrifuged at 5100 rpm for 5 minutes (TJ-25 Centrifuge, Beckmann Coulter, U.S.A.). Approximately 5 mL of the bottom layer were collected and transferred into a 25 mL tube, using a transfer pipette. 50 μL of the lower layer of the extracts and 900 μL of a chloroform: methanol solution were placed into Eppendorf tubes, followed by 5 μL of ammonium thiocyanate and ferrous chloride solution (1:1), which was then vortexed. The samples were allowed to stand at room temperature for 10 minutes. 100 μL was placed in polypropylene microplate (Eppendorf, microplate 96/F-PP) and the absorption read at 500 nm (Tecan Sunrise, Austria). A standard curve was prepared using cumene hydroperoxides. The results were expressed as μmol lipid hydroperoxides per g of the sample.
3.3.8 Thiobarbituric Acid Reactive Substances (TBARS) TBARS were determined with a modified method of Lemon (1975). 5 g of the sample were weighed into a 50 mL tube and 5 mL of TCA solution were added into the test tube and the solution was homogenized for 10 seconds. 5 mL of the TCA solution was then added and homogenized again for another 10 seconds. The mixture was then centrifuged at a speed of 5100 rpm for 20 minutes at 4 o C (Beckman Counter TJ-25, Rotor TS-5.1-500). 5 mL of the supernatant were then collected by filtration using Whatman 4 filter paper. 900 μL of TBA were placed in 1.5 mL Eppendorf tubes along with 100 μL of the supernatant, and the solution was then vortex for 10 seconds. Small holes were then made on the cap of the Eppendorf tubes with needles to allow space for expansion during heating in the water bath. The Eppendorf tubes were placed in the water bath at 95 o C for 40 minutes and later allowed to cool down on ice. 200 μL of the supernatant were placed in the micro plate and the absoption was read at 530 nm (Tecan Sunrise, Man-nedorf, Switzerland). The TBARS results were expressed as μmol malondialdehyde diethyl acetal per g of sample.
3.3.9 Free Fatty Acids (FFA) The free fatty acid (FFA) content was determined using the method of Lowry & Tinesley (1976), with a modification as described by Bernardez et al. (2005). 1 mL of the lipid extracted (Bligh & Dyer, 1959) was pipetted into bottles and evaporated using the nitrogen
30 jet set at 55 °C for 10 minutes. After cooling down, 3 mL of cyclohexane and 1 mL of cupric acetate–pyridine reagent were accurately added and vortexed for approximately 40 seconds. After centrifugation at 2000 rpm for 10 minute at 4 o C ((Beckman Counter TJ-25, Rotor TS- 5.1-500), the absorption of the upper layer was read at 710 nm (UV- 1800 spectrophotometer; Shimadzu, Kyoto, Japan) The FFA concentration in the sample was calculated as μmol oleic acid, based on a standard curve spanning a 2-14 μmol range. The results were expressed as grams FFA per 100 g of total lipids.
3.3.10 Total Volatile Basic Nitrogen (TVB-N) The total volatile base nitrogen (TVB-N) was determined using the method described by Malle & Poumeyrol (1989). The TVB-N was measured by steam distillation (Struer TVN distillatory, STRUERS, Copenhagen, Denmark) and titration. 7.5% aqueous trichloroacetic acid solution was used to extract the TV-N from the fish muscle. The distilled TVB-N was the collected in boric acid solution and titrated with sulphuric acid solution. The TVB-N (mg N/100 g) was then calculated according to the equation: Equation 2 where: a is the volume of sulphuric acid in mL, and b is the normality of sulphuric acid (0.0340 N) and 14 is the molecular weight of nitrogen.
3.3.11 Total aerobic plate count (TPC) measurements The microbial quality of the raw and smoked mackerel was determined using the total aerobic plate counts (TPC). 20 g of the fillets and whole were weighed in stomacher bags and mixed with 180 mL of maximum recovery diluent (MRD). The mixture was homogenized for 2 minutes in a Waring laboratory blender and serially diluted up to 109 and inoculated in growth media in petri dishes. 1 mL of 1/10 dilutions was transferred using a pipette to the Petri plates, and melted iron agar at 45 °C was poured on the plates for the mixture to solidify. The plates were covered with a thin layer of iron agar and then incubated at 22 °C for 48 hours. All microbiological analyses were conducted in duplicate and data were expressed as a logarithm of the number of colony-forming units (log cfu/g).
3.3.12 Polycyclic Aromatic Hydrocarbon Analysis 50 g of the samples were minced and kept in a small glass jars with screw caps and sent to the Eurofins WEJ contaminant laboratory for the analysis. The skin, head and bones were removed from the samples leaving only the edible parts for analysis.
3.4 Statistical analysis The mean values and standard deviations from all analytical methods were plotted separately against storage time using Microsoft excel (2016). A t-test using Microsoft Excel (2016) (Microsoft Inc., Redmond, Washington, USA) was performed to see if the samples were significantly different. The significance level was defined at 0.05 for all statistical tests.
31
32 4 RESULTS AND DISCUSSIONS
4.1 Pre-trial The fillets that were brined in the 8 % NaCl solution for 90 minutes had a salt content of 1.5 % after the smoking, whereas the fillets brined in the 12 % solution had 2% salt concentration after the brining, resulting in a dominating salty taste. It was concluded that the time for brining (90 minutes) was too long, so the time should be reduced to 45 minutes for the fillets, and 60 minutes for the whole fish to get an optimum salt content in the main experiment. Also, the temperature in the open drum reached 92 oC after 50 minutes, and the fish was thoroughly smoked. However, a lot of smoke was produced from the wood used during the open fire smoking, making it difficult to control the fire. The dung was therefore not recommended as a fuel for the smoking, but wood was favored instead. The fish muscle smoked in the cabin reached a high temperature of 71 oC after 120 minutes, while the temperature in the Bradley only reached 58 oC after 80 minutes in the fish muscle. Nonetheless, the cabin and Bradley were the recommended smoking kilns for the main trial.
4.2 The main experiment
4.2.1 Temperature measurement during the main experiment smoking process The temperature profile measured from the cabin and Bradley as well as inside the muscle (both fillets and whole) are shown in Figure 9. There were fluctuations in the temperature both the smoking chamber and inside the fish muscle. The temperature at the top rack in the cabin smoker was higher and reached 142 oC after 27 minutes of smoking, whereas the bottom rack recorded a temperature of 120 oC after 64 minutes. In the Bradley, on the other hand, the highest temperature recorded was 107 oC in the bottom rack, whereas the top rack recorded a temperature of 55 oC after 210 minutes of smoking.
Figure 9: Temperature profiles within the cabin and Bradley smoking kilns as measured on the top and bottom racks of the kilns.
33 The temperature measurements in the fillets smoked in the Bradley and cabins smokers are illustrated in figure 10. The highest temperature in the fillet muscle in the cabin was observed as 70 0C at 23 minutes. The time taken for the fish to be well smoked was 106 minutes in the cabin kiln, whereas in the Bradley the internal temperature of the fillet muscle reached 51 0C after 196 minutes, indicating insufficient cooking of the fillets when using the Bradley smoker. The highest internal temperature in the whole fish from the Bradley was 44 0C after 196 minutes, whereas in the cabin the temperature reached 68 0C after 120 minutes.
Figure 10: Temperature profile inside the whole muscle from the Bradley and cabin kilns
4.3 Physicochemical analysis The shelf life of the products during storage was assessed by sensory evaluation performed by United Nations University, Fisheries Training Programme fellow Eunice Konadu Asamoah. The results of the sensory evaluation were thoroughly reported in her final report and will not be discussed in further detail in the current manuscript.
4.3.1 Characteristics of the raw material (Atlantic mackerel) The chemical composition of the thawed raw mackerel was analyzed (Table 6). Table 6: The chemical composition of the mackerel before smoking Product Chemical components Water content 59.5 ± 0.8 % Lipid content 21.8± 1.5% Salt content 0.3 ± 0.0% Total volatile basic nitrogen 18.4± 0.7 N/100g FFA 1.5 0.7 g FFA/100 g pH 6.76 0.21 Peroxide value (PV) 0.72 0.5 μmol/g muscle TBARS 0.25 0.00 μmol/g muscle
34 The quality of the fish depends on certain biological factors (size of the fish, total lipid content and fatty acid composition etc.) and environmental factors (geographical location, available feeding, geographical location, season of catch) (Huss, 1995). The diets of mackerel have shown to affect the stability of the product during processing and storage (Arason et al., 2015). The lipid content in the present study was 21.8 ± 1.5% in the raw material. A similar lipid content (21.6 ± 0.6%) was observed in mackerel caught in end of July in North-East Iceland (Romotowska et al., 2016b). However, a different lipid content was observed in fish caught in 2012, which was high (29.7 ± 7.2%), compared to fish caught in 2013 (20.3 ± 4.5%). In general terms, these variations in lipid content were influenced by the feeding conditions, ocean temperature, feed availability and competition for feed (Aubourg et al., 2004; Romotowska et al., 2016a). A water content of 59.5 ± 0.8 % was observed in the raw material, which is in good agreement with the study of Romotowska et al. (2016a), which stated a water content of 59.5 ± 3.7 % and 55.0 ± 3.7 % in mackerel caught in North-East Iceland in 2013 and 2012, respectively. The water content differed between years in the Romotowska study be due to changes in ocean temperature, the size of the stock of the fish and increase competition in feed (Nøttestad et al., 2012, 2013). Furthermore, high levels of PUFAs in fatty fish affects the stability of the raw material during processing and storage because of lipid oxidation. The FFA differs from findings by Romotowska et al. (2016a) who observed 1.8 FFA/100 g and 0.7 FFA/100 g in mackerel caught during ending July 2012 and 2013, respectively. Different primary oxidation (lipid hydroperoxide) were also observed in mackerel caught from North -East of Iceland at the end of July 2012 and middle of July 2013 respectively. A higher peroxide value (0.72 0.5 μmol/g) was observed in the present study compared to results from Romotowska et al. (2016a) at 0.09 μmol/g in mackerel caught in ending July 2012 and 0.22 μmol/g from middle July 2013. However, a higher secondary oxidation was observed in at a level of 0.2 μmol/g and 0.7 μmol/g in mackerel caught at beginning August 2012 and 2013 respectively from eastern part of Iceland. A lower TBARS (0.25 0.00 μmol/g) was observed in the present study compared to the results from Romotowska et al (2016) study. These differences in the chemical composition of the raw materials could be attributed to different season, year, feed available, ocean temperature and the location in which the fish were caught.
4.3.2 pH measurements
The pH in the raw material was higher (6.76 0.21) compared to the smoked products (Figure 11). This is in good agreement with the studies of Leroi & Joffraud (2000a) and Doe (1998) who reported that a lower pH in smoked products could be attributed to the lower moisture content and salting. A general increase in pH was observed in the fillets from day 5 to day 25. However, the pH in the fillets kept in air packaging and stored at 20 o C (FAR) was significantly higher at day 10 of storage than the fillets kept at other conditions (chilled and/or vacuum packed). This increase in pH during storage may be due to the microbial degradation and development of TVB-N formation, causing spoilage in the smoked products. This agrees with Reddy et al.(2000), who stated that the pH in smoked products increased because of the development of volatile basic components, such as ammonia, and
35 trimethylamine causing fish spoilage. Furthermore, Richards & Hultin (2000), stated that an increase in pH ay delay rancidity and the development of TBARS in rainbow trout. On the other hand, the vacuum-packed fillets from the cabin smoker, stored at room temperature (FVR), decreased through storage. These differences in pH due to packaging material and storage conditions are likely due the presence of increased microbial spoilage in the air packed samples, when stored at the higher storage temperature. The pH of fillets stored at 4 o C from both smoking equipment increased with storage time. However, the fillets from the cabin (FVC) had a significantly higher pH (p = 0.047) than fillets from the Bradley (FBVC) although all were stored at the same temperature (4°C).
Figure 11: Comparison of pH of the hot smoked mackerel fillets from the cabin and Bradley smokers, stored at either 4 °C or 20 °C for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C). The pH in the whole smoked mackerel stored at 20 o C (WVR) was significantly lower than WAC (p = 0.05), and the pH in the whole mackerel from the Bradley was significantly higher (p = 0.03) after smoking (on day 5) compared to the other treatments (Figure 12). However, these pH differences seemed to stable during the storage. The observed differences in the pH on day 5 are therefore mainly attributed to the different smoking methods (cabin and Bradley), although some effect of the storage conditions and packaging material may not be excluded.
36
Figure 12: Comparison of pH of hot smoked whole mackerel, smoked in the cabin and Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
4.3.3 Water content
The raw material had a higher water content (59.15 0.75) compared to the fillets after smoking in all groups (Figure 13). The smoking process probably decreased the water content and the drying process could also have caused some dehydration of the samples. A significant increase in water content was observed in the chilled and vacuum packed groups through storage, but not in the samples stored in air packaging and/or room temperature conditions (FAC, FAR and FVR). The small differences seen in these groups between sampling days are believed to be primarily due to individual variation. However, no significant difference in water content was observed between the different smoking methods in the fillets.
Figure 13: Water content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC)
The water content in the whole mackerel decreased compared to the raw material during smoking (Figure 14), with the WVC and WAR significantly different on day 5. Opposed to the fillets, the water content in the WBVC and WVC decreased slightly with
37 storage up until day 25, where an increase in water content was observed again. The difference could be due to oxidative degradation of the muscle as a similar trend was observed in the TBARS developed in the Bradley smoked whole mackerel. However, there were no significant changes in the water content during storage for the other treatments.
Figure 14: Water content in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C).
4.3.4 Salt content A significant difference was observed in the salt content between the raw material and the smoked samples after 5 days of storage (Figure 15). The salt content in the raw material (0.3 0.00%) was lower than in the smoked products. This could be due to the partial dehydration obtained during the smoking processes and changes in the wet weight of the fillets. This finding agrees with the results reported by Goulas et al. (2005), who observed an increase in 2.1 – 2.2 % salt content in smoked chub mackerel. A slight decline in the salt content was observed in the air packed fillets (FAC) after day 5, from an average of 2.25 0.07% to 1.8 0.00% at day 18. The difference was statistically significant (p = 0.01). Although the salt content was generally stable during storage (Figure 15), and there were no significant differences in the salt content between the groups in the smoked fillets (p > 0.05).
38
Figure 15: Salt content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C). The overall average salt content in the Bradley smoked whole mackerel was higher (0.8 0.09%) than in the raw material (0.30 0.00%). The resulting Z-value of the muscle after smoking was 1.3%. The salt content stayed very stable throughout storage, and there were no significant differences between the groups (Figure 16). The salt content in the whole mackerel was though significantly lower than in the fillets, indicating that the skin is an affective barrier to salt uptake during brining (Gallart-Jornet, 2007).
Figure 16: Salt content in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C).
39 4.3.5 Water activity The combining preservative effect of the salt, smoke, and heat caused the water activity to decrease in the smoked products, compared to the raw material. Generally, lowering the water activity hinders bacterial growth in the fish (Hildebrand, 1992). However, the water activity (aw) in the present study only decreased from 1.00 0.00 in the raw material to 0.98 0.00 in the smoked products (Figure 17). This water activity was not low enough to ensure a stable product at room temperature (Arason et al., 2014). A decrease in the water activity was observed in FBVC and FVR during the first two weeks of storage, while FAR fluctuated in water activity. These fluctuations in FAR are believed to be mostly due to individual differences between fishes during sampling. No significant effect was observed due to the packaging or storage methods, nor due to the smoking methods. The water activity for the fillets, which were vacuum packed and stored at 4 o C (FVC), was stable during the whole storage period.
Figure 17: Water activity in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
40 On the other hand, the water activity in the whole mackerel stored at chilled temperatures decreased at day 5, especially the Bradley smoked mackerel compared to the raw material (Figure 18). However, there was no significant differences between the groups. A slightly increasing trend was observed in the water activity in the air packed sample during storage but the difference was not significant (p > 0.05).
Figure 18: Water activity in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
4.3.6 Lipid content A higher lipid content was observed in the smoked fillets on day 5 compared to the raw material (21.8± 1.5%), indicating the effect of drying and water loss during the smoking process (Figure 19). This agrees with observations from Espea et al. (2002), who obtained a similar result during smoking of salmon. The lipid content in the smoked fillets then decreased with storage time, until day 25. This could be because of formation of free fatty acids because of enzymatic activities in the fish muscle. A slight increase was observed in the lipid content within FBVC and FVC after day 25, but it was only significant in the fillets smoked in the Bradley (FBVC). Furthermore, the lipid content was not significantly influenced by the different smoking methods in agreement with the observations of Alcicek & Atar (2010).
41
Figure 19: Total lipid content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC). The lipid content in the whole smoked mackerel was higher than in the raw material, in agreement with the results of the fillets (Figure 20). However, the analysis showed a decrease in the lipid content in the vacuum-packed samples stored at 4 o C (WBVC and WVC) during the first two weeks of storage. However, after that no significant changes were observed.
Figure 20: Total lipid content of whole smoked mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
4.3.7 TVB-N Analysis The total TVB-N is a parameter used to determine the quality of fish products. TVB-N is influenced by microbacterial activities in the fish resulting in the production of dimethylamine, ammonia and other volatile nitrogenous compounds which cause spoilage in seafood (Huss, 1995). In the present study, the TVB-N in the smoked fillets was higher in all the groups compared to the raw material (18.4 ± 0.7 N/100 g) (Figure 21). This agrees with the findings of Alcicek & Atar (2010), who stated that smoking resulted in an increase in the
42 level of TVB-N of rainbow trout. The results are, furthermore, in agreement with the findings from Bennour et al. (1991), who concluded that the initial TVB-N in smoked samples were significantly higher than the non-smoked samples. It is, however, likely that partial dehydration of the smoked samples is contributing to this difference as well. After 5 days of storage, the TVB-N increased in the room temperature stored fillets, and was significantly higher in FVR and FAR (p < 0.05) compared to the other groups. The highest TVB-N was recorded after 18 days of storage in fillets which had been vacuum packed and stored at room temperature 20 oC (FVR = 51.2 4.2 N/100g). No significant changes observed in TVB-N between FVC and FBVC indicated that there was no significant effect of the smoking methods. However, the vacuum packaging seemed to delay the formation of TVB-N in the fillets, in comparison to the air packaging, especially when used in combination with chilled conditions, but no significant TVB-N formation was observed during the storage duration for these groups.
Figure 21: TVB-N (N/100 g) in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC). The changes in TVB-N for the whole smoked mackerel during storage are shown in Figure 22. The TVB-N in the smoked products was significantly higher (p < 0.05) in all the smoked products at day 5 compared to the raw material (18.4± 0.7 N/100 g). The increase in TVB-N for WAR and WVR after day 5 was very high compared to the other groups. Although showing a similar trend as the TVB-N formation in the fillets, the TVB-N formation was significantly higher in the whole mackerel. There was a steady increase in the TVB-N for the whole, air packed mackerel stored at 4 o C (WAC) during storage. However, no significant increase in the TVB-N was observed in the vacuum-packed samples stored at 4 o C (i.e. WBVC, WVC and WAC were stable). The highest TVB-N was recorded in WAR on day 10 (88.2 12.7 N/100g), which was significantly higher than both WAC (p = 0.025) and WVR (p = 0.039), indicating that both temperature and packaging solutions had a significant effect on the storage stability with regards to TVB-N formation. No difference was though observed between the smoking methods with regards to TVB-N formation in the whole mackerel. The results show clearly that the packaging material and temperature influences the level of TVB-N in the samples, as mentioned before. This was in agreement with results from
43 Goulas & Kontominas (2005), who reported that TVB-N production in smoked mackerel could increase as a result of partial dehydration. The vacuum packed smoked products had a lower TVB-N since restricted access to air lead to a decrease in the deamination capacity of the micro-organisms which cause spoilage, leading to a lower production of volatile compounds (Rodrigues et al., 2016). Fairly constant TVB-N values were obtained throughout the storage time in both fillets and whole mackerel when stored at cold temperatures. This could be associated with the deposition of antimicrobial smoke constituents (e.g. formaldehyde, phenols), higher water content, and to the effect of the increased salt level. Various maximum limits have been set as acceptable TVB-N levels in foods, dependent on different processing conditions, specific treatments, and products. The acceptable limit in seafood set by the EU (EEU, 1995) is 35 mg N/100g of muscle, while other acceptability levels have been reported by different authors; e.g. 35 – 40 mg N/ 100 g (Connell, 1990), 25- 30 mg N/100 g (Lopez – Caballero et al., 2000) 20-25 mg N/100 g (Kim et al., 2002). If the EU limit is applied the TVB-N results in the current study compiles well with the point of rejection of the samples, based on the sensory evaluation (Asamoah, 2017).
Figure 22: TVB-N (N/100 g) in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
4.3.8 Microbial Analysis The TPC in the raw material was lower than the smoked fillets (Figure 23). A significantly higher TPC was observed in FVR compared to the raw material (p = 0.00), FVC (p = 0.02), and FAC (p = 0.003). At day 5 a significant difference in TPC was observed between the Bradley and the cabin smoked fillets (p=0.001). Also, the TPC of the air packed fillets stored at room temperature (FAR) was lower compared to counts of those stored at chilled temperatures, (FAC) (p = 0.001).
44
Figure 23: TPC (logCFU/g) in hot smoked fillets mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC). The total plate count for smoked whole products were higher than the raw material (Figure 24). On day 5, the TPC in WAR was significantly higher than in WAC (p = 0.02), WVC (p = 0.04), and WBVC (p = 0.009). These variations could be due to the different packaging materials and storage temperatures. The highest TPC was observed in WAR at day 10 (9.1 0.1 log CFU/g), which thus exceeded the maximum limit of 7 log CFU/g.
Figure 24: TPC (logCFU/g) in hot smoked whole mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
4.3.9 Free Fatty Acids (FFA)
The FFA in the raw material (1.5 0.7 g FFA/100 g) was lower compared to the smoked products (Figure 25). An increase was observed in the FFA development in all the samples, and the fillets stored at 20 oC (FAR) increased very sharply (5.4 0.7 g FFA/100 g) with storage. The sharp increase in the FFA in the FAR shows that there was a significant difference between storage treatments, and that air packaging and storage at room
45 temperature triggered the formation of FFA due to increased enzymatic activities. This results agrees with a similar study performed by Huss (1995) on the free fatty acids formation in herring stored at different temperatures. Similar observations were also obtained by Aubourg et al. (1997) who observed a similar trend in the FFA development in sardine fillets stored at cold temperatures, as well as in the study of Hwang & Regenstein (1993), who observed an increase in FFA content in minced white mackerel muscle stored at 2 – 37 o C. Surprisingly, a decrease in FFA was observed in FAC after day 10, while no significant changes were observed in the FFA of FVC and FBVC up until day 25 of storage. The FFA in the Bradley smoked fillets decreased after that, while those of the cabin smoked fillets remained stable. The FFA in the cabin smoked fillets were higher than in the Bradley smoked fillets at day 5 (p = 0.05), and again at the end of storage (day 35). However, no significant differences in FFA could be attributed to the different smoking methods used.
Figure 25: Free fatty acids (FFA; g FFA/100g samples) in smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
In general, there was an increase in the FFA in all whole smoked mackerel treatments compared to the raw material (Figure 26). The highest FFA development occurred in WBVC (3.81 0.57 g FFA/100 g) at day 25 compared to other groups, especially WVC, which was relatively stable throughout the storage. This indicated that the different smoking methods had a significant effect on the FFA production in the smoked whole mackerel. The FFA in WBVC was significantly higher (p = 0.02) than WAR at day 5. This difference between smoking methods, may be due to the lower temperature obtained inside the fish muscle during the smoking in the Bradley. This aided enzymatic activity in the fish in the Bradley smoked whole fish. A higher FFA development was generally observed in the fillets than in the whole mackerel (2.78 0.19 g FFA/100 g), indicating that the increased exposure of the muscle to air in the fillets influenced the FFA formation. Furthermore, the smoked mackerel that was stored at room temperatures were more prone to lipid hydrolysis during the storage period. This indicated that lipid deterioration was affected by temperature, which aids enzymatic activities, causing the product to spoil faster.
46
Figure 26: Free fatty acids (g FFA/100g samples) in whole smoked mackerel, smoked in the cabin or Bradley kilns, and stored at either 4 oC or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC).
4.3.10 Lipid oxidation The rate of lipid oxidation was assessed in the muscle by analysis of the primary and secondary oxidation products of peroxide value (PV) and thiobartituric acid reactive substances (TBARS), respectively. The peroxide value in the raw material (0.72 0.5 μmol/g muscle) was higher compared to the smoked products (Figure 27). A significantly higher PV (p = 0.174) was observed for the cabin smoked, vacuum-packed fillets stored at 20 oC (FVR) at day 5 compared to the Bradley smoked fillets stored at the same conditions. However, no significant difference was observed between the smoking methods through the remains of the storage duration. It is possible that the antioxidants from the wood smoke influenced the lipid oxidation. This phenomenon is explained by Horner (1997), who stated that during the hot smoking process, phenolic antioxidants substances are deposited on the fish, which delays autoxidation of the unsaturated lipids. The air packed fillets were slightly higher in PV values compared to the vacuum-packed fillets, in agreement with the observations of Adeyemi et al. (2013), who also observed an increasing peroxide value in smoked mackerel during storage at room temperature. This may be caused by the higher access of air to the lipids in the muscle, which lead to increased rancidity in the product. However, this difference between air and vacuum packed samples was not significantly different at all sampling points. The TBARS in the raw material was relatively higher than in the smoked products (0.25 0.00), in agreement with the lower PV in the smoked fillets. This indicated that the smoking is an effective storage method to decrease degradation due to oxidation, possibly due to the lowering of the water content in the muscle during smoking. The fillets, which were vacuum packed and kept at 4 oC (FBVC and FVC) had lower TBARS values compared to the air packed samples. Thus, the presence of air in the packaging material facilitated the easy development of TBARS. The increase in PV for the vacuum packed and chilled fillets (FBVC and FVC) observed at the end of the studied storage period, indicated that the primary oxidation peak had not yet been reached in these groups. This agrees with the fact that no
47 formation of the secondary product, TBARS was observed in the FBVC and FVC fillets, under these storage conditions.
Figure 27: Comparison of the development of lipid hydro peroxide (PV; µmol/g samples) and thiobarbituric acid reactive substances (TBARS; µmol/g samples) in smoked mackerel fillets, smoked in the cabin or Bradley kilns, and stored at either 4 o C or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20 oC), and C to storage at chilled conditions (4 oC). For the whole mackerel, a general increase in the PV was observed in the samples, although it was faster in the air stored samples (Figure 28). Although the PV fluctuated during the storage period, the PV formation in the WBVC and WVC took their maximum values on day 25, followed by the decomposition of this primary oxidation product. This agrees with the non-existent formation of TBARS in these groups during the storage period. The results from this study are in excellent agreement with the study of Bugueno et al. (2003) where no changes in TBARS values were observed in vacuum packed smoked brined salmon until after 25 days of cold storage. Furthermore, according to Huss (1995), lower storage temperature reduces the lipid degradation with time. However, the increase in TBARS in the air packed samples, and those stored at room temperature, indicate that both primary and secondary oxidation was occurring in the whole smoked mackerel during air packed storage.
48
Figure 28: Lipid hydroperoxide (PV; µmol/g samples) and thiobarbituric acid reactive substances (TBARS; µmol/g samples) in whole smoked mackerel smoked in the cabin or Bradley kilns, and stored at either 4 o C or 20 oC for up to 35 days. The letter F refers to fillets, A to air packaging, V for vacuum packaging, B for smoking in the Bradley kiln, R to storage at room temperature (20°C), and C to storage at chilled conditions (4°C).
49 4.3.11 Polycyclic Aromatic Hydrocarbons (PAH) Analysis The levels of the PAHs in the smoked samples are shown in Table 7. Chrysene showed the highest PAH level with a concentration of 10.75 ± 0.21 μg/kg and < 1 μg/kg in the cabin and Bradley smoked fillets, respectively. This was followed by benzo(a)anthracene, at a level of 8.85 ± 0.07 μg/kg in the cabin smoked fillets. However, a significant difference was observed between the cabin smoked fillets and the cabin smoked whole fish (p = 0.0005). The level of chrysene in the fillet was as high as 10.75 ± 0.21 μg/kg, while the whole mackerel contained 2.35 ±0.07 μg/kg, indicating a significantly higher (p = 0.0004) formation of chrysene in the fillets. Significantly higher levels of benzo(b)fluoranthene (p = 0.0009), benzo(a)anthracene (p = 0.004) were also observed in the cabin smoked fillets, compared to those smoked in the Bradley. Benzo(a)pyrene, which is the main indicator for the presence of PAHs in food was observed to be 2.8 ± 0.14 μg/kg and < 0.5 μg/kg in the cabin and Bradley smoked fillets, respectively, whereas in the whole smoked mackerel it was 1.05 ± 0.07 μg/kg. Furthermore, the sum of the four PAHs (i.e. chrysene, benzo(b)fluoranthene, benzo(a)pyrene, and benzo(a)anthracene), which are the main concerns of the EU regulation, was 25.85 μg/kg and 8.1 μg/kg. in the cabin smoked fillets and whole fish, respectively. Table 7: Levels of Polycyclic Aromatic Hydrocarbons in smoked mackerel Type of PAH Cabin fillets level Cabin whole Bradley Bradley whole μg/kg μg/kg fillets μ g/kg μg/kg 5-Methylchrysene ˂ 1c ˂ 1c ˂ 1 c ˂ 0.5 c Benzo-c-fluorene 3.05 ± 0.07a 1.6 ± 0.1 b ˂ 1 c ˂ 1 c Cyclopenta(c, d)pyrene 6.15 ± 0.64 a ˂ 1 b ˂ 1 b ˂ 1 b Dibenzo(a, l)pyrene ˂ 1c ˂0.5c ˂ 1 c ˂ 1 c Dibenzo(a, h)anthracene ˂ 0.5c ˂ 0.5 c ˂ 0.5 c ˂ 0.5 c Dibenzo(a, e)pyrene ˂ 1c ˂ 1c ˂ 1 c ˂ 1 c Dibenzo(a, h)pyrene ˂ 0.5c ˂ 1 c ˂ 1 c ˂ 1 c Benzo(a)anthracene 8.85 ± 0.07a 1.95 ± 0.21b ˂ 1c ˂ 0.5c Chrysene 10.75 ± 0.21a 2.35 ± 0.07b ˂ 1c ˂ 0.5c Benzo(a)pyrene 2.8 ± 0.14a 1.05 ± 0.07b ˂0.5c ˂ 0.5c Benzo(b)fluoranthene 3.45± 0.07a 1.15 ± 0.07b ˂ 0.5 c ˂ 0.5 c Benzo(k)fluoranthene 1.45 ± 0.07 a ˂ 0.5b ˂ 0.5b ˂ 0.5b Dibenzo(a,i)pyrene ˂ 1c ˂ 1c ˂ 1 c ˂ 1c Benzo(j)fluoranthene 2.8 ± 0.1a ˂ 1b ˂ 0.5b ˂ 0.5b Benzo(g, h, i)pyrelene 1.0 ± 0.0c ˂ 1c ˂ 1 c ˂ 0.5 c Indeno(1,2,3-cd)pyrene 1.1 ± 0.0 a ˂ 0.5 b ˂ 0.5 b ˂ 0.5 b Sum of PAH4 25.85 ± 0.07a 8.1 ± 0 .0 3 b Inaplicable Inap plicable *Different subscript letters (a, b, c) indicate a significant difference (p < 0.05) within each type of PAH between the different products (fillets or whole fish, smoked in either the cabin or Bradley kilns).
As stated earlier, the actual levels of PAHs in smoked foods depend on several variables in the smoking process, including type of smoke generator, combustion temperature, and degree of smoking (Larsson, 1982; Moret et al., 1997). In the present study, the levels of PAHs were higher in the cabin smoked fillets than in the Bradley due to the
50 difference in the temperature in the oven, and the smoke generator. The PAHs levels in the Bradley smoked samples were generally below the detection threshold levels, indicating that the PAH generation was largely affected by the type of smoke generator. This can be partly explained by the fact that the cabin generated more smoke than the Bradley, but this could be attributed to the kind of wood that was used during the smoking. This agrees with Moret et al. (1997) and Larson (1982), who concluded that smoke generated by thermal pyrolysis of wood could result in high levels of PAHs due to limited access to oxygen. This observation is also in agreement with the findings by Essumang et al. (2013), who observed differences in PAHs levels in mackerel, tuna, and sardines, when using either acacia, sugarcane bagasse or mangroves as fuels for the smoking. The acacia, a hardwood with a high lignin content, produced a lot of smoke, which resulted in higher levels of PAHs compared to the other types of wood. Furthermore, the higher levels of PAHs in the cabin could also be a result of the higher temperature obtained, while an appropriate cooking temperature was not reached using the Bradley. During the smoking of the fillets in the cabin there was too much wind which caused so much wood to be used for the smoking and ashes were deposited on the fish after smoking. Again, the wood chunks were wet due to the poor weather conditions and this resulted in a lot of smoke generated in the smoking chamber. This made monitoring very difficult and as such some of the fish were burnt so it be possible that the samples selected for the PAHs analysis were burnt and had a lot of smoke on them. The duration of smoking may also influence the levels of PAHs generation. Chen & Lin (1997) stated that a shorter smoking time and lower temperature would reduce the formation of PAHs. It took approximately 106 minutes and 210 minutes for the fish to reach their maximum temperature in the cabin and Bradley, respectively in the current study. Duedahl-Olesen et al. (2010) and Varlet et al. (2006) reported that the time used for smoke curing had a significant impact on the levels of PAHs. Also, a similar trend was observed by Essumang et al. (2013) in mackerel, tuna, and sardine, each smoked for 120 minutes and 240 minutes. However, in the current study lower PAH levels were obtained using the Bradley, although the smoking during was longer. This may be explained by the fact that the PAHs were converted to volatile ones and released into the surroundings instead of to the fish. This same observation was found by Essumang et al., (2013) in their work. This may also indicate that the smoking temperature is a more dominating variable with respect to formation of PAHs in mackerel fillets, compared to the smoking duration. Lower levels of PAHs in the whole smoked mackerel may also be attributed to the presence of the skin, which served as a highly affective barrier to the passage of smoke into the flesh. This confirms the findings from Gobel(2006) that the uptake of PAHs into the flesh of skinless fillets is higher than when the skin in on. Also, the lipid content in the fillets was slightly higher than in the whole fish, which could lead to increased formation of the PAHs. A higher lipid content increases the risk of melting of the lipids, which may have dripped into the fire during the smoking process, resulting in higher levels of PAHs. This agrees with the suggestions of Gobel (2006), which stated that to minimize the formation of PAHs the melted fat should be reduced from encountering the smoldering wood. This agrees with the observations of Chen & Lin (1997), stating that PAHs are formed at high temperatures, so direct contact of meat with the cooking flame during charcoal grilling at high temperatures should be avoided to reduce the formation of PAHs.
51
52 5 CONCLUSION
The study showed a lower salt content in the whole mackerel than in the fillets indicating that the skin served as a barrier for salt uptake in agreement with earlier studies. Also, a stable salt content was observed in smoked mackerel during storage. The water activity in the vacuum packed fillets and whole mackerel were stable during storage. However, in order to prolong the shelf life of smoked fish a lower water activity than reached in the current study is needed to keep the product stable at room temperature.
The study also indicated a stable lipid content in the smoked mackerel fillets and whole mackerel stored at chilled temperatures. However, the fillets were prone to FFA formation, possibly due to increased exposure of air to the muscle. Furthermore, the study showed high stability of the muscle lipids (as assessed by primary and secondary oxidation products, and free fatty acids) when stored at cold temperatures, and especially when combined with vacuum packaging. However, high enzymatic activity and oxidation was observed at room temperatures leading to an increase in the formation of free fatty acids. Vacuum packaging also seemed to reduce the formation of TVB-N in smoked fillets, in comparison to the air packaging, especially when the products are stored at chilled conditions.
The cabin smoker was recommended as the smoking equipment for smoking fish since it provides the required temperature needed to inhibit the enzymatic activities in the fish for some time before the fish muscle starts to spoil. However, using the cabin smoker resulted in PAH4 and BaP levels above the acceptable 12 μg/kg and 2 μg/kg in the fillets, while benzo(a)pyrene in the cabin smoked whole mackerel was below the acceptable limit. Mackerel should therefore be smoked whole, since the skin seems to act as a barrier to the PAH formation and accumulation in the edible muscle. On the other hand, the benzo(a)pyrene and PAH4 levels in the Bradley smoked mackerel fillets and whole were very low due to the lower temperature obtained. However, the use of the Bradley smoker was not optimal since the products were not stable in terms of lipid stability and oxidation causing the product to spoil faster.
Wood turned out to be the best fuel for the smoking of the fish but the temperature should be regulated as well as the duration of smoking so that the levels of PAHs formed will be minimal. Furthermore, to minimize the formation of PAHs, the melted fat be should not encounter the smoldering wood. Also, since PAHs are formed at high temperatures, direct contact of the smoke to the fish during the smoking should be avoided to reduce the degree of PAHs formed.
6 FUTURE PERSPECTIVES
Further studies are needed on how formation of polycyclic hydrocarbons occurs with the use of the cabin smoker in local smoke houses in Ghana, in comparison with the traditional ovens
53 that are currently being used. This could give a deeper insight and further understanding for the government to invest in new and healthier technologies for fish processing and management. This will further boost the fishing industry in Ghana. It is also recommended that more sample replicates will be used during further studies, to ensure that stronger conclusions can be made, and variations in the results due to the differences in the individual samples can be minimized.
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