effects of different smoking methods on the …...iii abstract the effects of different smoking...

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.

iii

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.

v

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.

vii

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.

http://starfsfolk.hi.is/en/simaskra/16421

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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

x

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

xi

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

xii

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

xiii

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


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


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



Figure 15: Salt content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns,





Figure 16: Salt content in hot smoked whole mackerel, smoked in the cabin or Bradley kilns,





xiv

Figure 17: Water activity in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns,





oC). ............................ 40

Figure 18: Water activity in hot smoked whole mackerel, smoked in the cabin or Bradley kilns,





oC). ........................... 41

Figure 19: Total lipid content in hot smoked mackerel fillets, smoked in the cabin or Bradley



air packaging, V for vacuum packaging, B for smoking in the Bradley kiln R to storage at


oC). ............................ 42

Figure 20: Total lipid content of whole smoked mackerel, smoked in the cabin or Bradley kilns,





oC). ............................ 42

Figure 21: TVB-N (N/100 g) in hot smoked mackerel fillets, smoked in the cabin or Bradley





oC). ............................ 43

Figure 22: TVB-N (N/100 g) in hot smoked whole mackerel, smoked in the cabin or Bradley





oC). ............................ 44

Figure 23: TPC (logCFU/g) in hot smoked fillets mackerel, smoked in the cabin or Bradley





oC). ........................... 45

Figure 24: TPC (logCFU/g) in hot smoked whole mackerel, smoked in the cabin or Bradley





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 o

C), 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

xv

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

xvi

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

xvii

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

1

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

2

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

3

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).

4

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).

5

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.

7

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).

8

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

9

(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.

10

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.

11

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).

12

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

Compound Molecular

weight(g/mol)

Boiling

point(0C)

Melting

point(0C)

Solubility in water

(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

Benzo(j)fluoranthene 252 - 166 7.6 ×10 -4

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).

13

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

Acenaphthylene

Anthracene

Benzo(a)anthracene

ACE Questionable

ACEN Questionable

ANT Negative

BaA Positive

Unknown

Unknown

3

2

Benzo(b)fluoranthene

Benzo (g, h,i)perylene

Benzo(a)pyrene

Chrysene

Dibenzo(a,h)anthracene

Fluoranthene

Fluorene

Indeno(1,2,3cd) pyrene

Phenanthrene

Pyrene

Naphthalene

Benzo(k)fluoranthene

BbF Positive

BghiP Positive

BaP Positive

CHRY Positive

DBahA Positive

FLT Positive

FLU Negative

IcdP Positive

PHE Questionable

PYR Questionable

NAPH Positive

BfK Positive

2

3

1

2

2

3

3

2

3

3

2

2

1- Carcinogenic 2- Probably carcinogenic 3- Not classifiable.

Fernández-Sánchez et al., (2004); IARC (2001).

14

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).

15

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.

16

Table 3: Maximum limits of BaP and PAH4 in selected foodstuffs under EU 1327 /2014

Product Levels of BaP

(μg /kg)

Levels of PAH4

(μg/kg)

Cereals for baby foods

Smoked meat and meat products

Smoked fish and fish products

Coconut oil

Fats and oils

Smoked mullusks

Cocoa beans

1.0

2.0

2.0

2.0

2.0

5.0

5.0

1.0

12.0

12.0

20.0

10.0

30.0

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 smokehouse 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).

17

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

type

Levels/μg/kg Country Reference

Beef Smoking BaP 0.15 China Chung et al., 2011

Pork

Pork

Lamb

Chicken

Fish

Fish

Fish

Fish

Sausage

Fish

Fish

Smoking

Smoking

Smoking

Smoking

Smoking

Smoking

Smoking

Smoking

Smoking

Smoking

Smoking

BaP

PHE

BaP

BaP

BaP

PHE

BaP

BaA

BaP

BaP

BaP

0.30 China

9.32 Spain

2.80 Estonia

6.50 Estonia

10.6 Czech Rep

12.37 USA

12 China

60.08 Ghana

30 Czech Rep

73.78 Ghana

˂ 0.5 Iceland

Chung et al., 2011

Lorenzo et al., 2010

Reinik et al., 2007

Reinik et al.,2000

Simko, 1991

Forsberg et al., 2012

Wang et al., 1999

Palm et al., 2012

Suchanová et al., 2008

Essumang et al.,2013

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

18

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.

19

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 o

C 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

20

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

21

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.

22

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

23

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).

25

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

26

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.

27

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.

28

Table 5: Experimental design and sampling groups

Group

abbrev.

Fillet/whole Smoking

equipment

Storage

temperature

Packaging

material

Sampling days

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.

29

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

30

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

31

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.

33

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.

34

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

35

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

36

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


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.

37

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).

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

38

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



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).

39

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




temperature (20°C), and C to storage at chilled conditions (4°C).

40

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




temperature (20 oC), and C to storage at chilled conditions (4

oC).

41

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





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).

42

Figure 19: Total lipid content in hot smoked mackerel fillets, smoked in the cabin or Bradley kilns, and



packaging, V for vacuum packaging, B for smoking in the Bradley kiln R to storage at


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





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

43

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





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

44

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





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).

45

Figure 23: TPC (logCFU/g) in hot smoked fillets mackerel, smoked in the cabin or Bradley kilns, and





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





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

46

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 o

C), 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.

47

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

48

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 o

C), 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.

49

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



50

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

μg/kg

Cabin whole

μg/kg

Bradley

fillets μg/kg

Bradley whole

μg/kg

5-Methylchrysene ˂ 1c

Benzo-c-fluorene 3.05 ± 0.07a

Cyclopenta(c, d)pyrene 6.15 ± 0.64 a

Dibenzo(a, l)pyrene ˂ 1c

Dibenzo(a, h)anthracene ˂ 0.5c

Dibenzo(a, e)pyrene ˂ 1c

Dibenzo(a, h)pyrene ˂ 0.5c

Benzo(a)anthracene 8.85 ± 0.07a

Chrysene 10.75 ± 0.21a

Benzo(a)pyrene 2.8 ± 0.14a

Benzo(b)fluoranthene 3.45± 0.07a

Benzo(k)fluoranthene 1.45 ± 0.07 a

Dibenzo(a,i)pyrene ˂ 1c

Benzo(j)fluoranthene 2.8 ± 0.1a

Benzo(g, h, i)pyrelene 1.0 ± 0.0c

Indeno(1,2,3-cd)pyrene 1.1 ± 0.0 a

Sum of PAH4 25.85 ± 0.07a

˂ 1c

1.6 ± 0.1b

˂ 1 b

˂0.5c

˂ 0.5c

˂ 1c

˂ 1c

1.95 ± 0.21b

2.35 ± 0.07b

1.05 ± 0.07b

1.15 ± 0.07b

˂ 0.5b

˂ 1c

˂ 1b

˂ 1c

˂ 0.5 b

8.1 ± 0.03b

˂ 1c

˂ 1c

˂ 1b

˂ 1c

˂ 0.5c

˂ 1c

˂ 1c

˂ 1c

˂ 1c

˂0.5c

˂ 0.5c

˂ 0.5b

˂ 1c

˂ 0.5b

˂ 1c

˂ 0.5 b

Inaplicable

˂ 0.5c

˂ 1c

˂ 1b

˂ 1c

˂ 0.5c

˂ 1c

˂ 1c

˂ 0.5c

˂ 0.5c

˂ 0.5c

˂ 0.5c

˂ 0.5b

˂ 1c

˂ 0.5b

˂ 0.5c

˂ 0.5 b

Inapplicable

*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

51

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.

53

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

54

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.

55

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