Chemistry of Food Contaminants and Their Remediation Or Mitigation

Journal of Chemistry

Chemistry of Food Contaminants and Their Remediation or Mitigation

Lead Guest Editor: Alberto Fiore Guest Editors: Photis Papademas, Vlasios Goulas, and Giuseppe Meca Chemistry of Food Contaminants and Their Remediation or Mitigation Journal of Chemistry

Chemistry of Food Contaminants and Their Remediation or Mitigation

This is a special issue published in “Journal of Chemistry.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents

Chemistry of Food Contaminants and Their Remediation or Mitigation Alberto Fiore , Photis Papademas, and Vlasios Goulas Editorial (2 pages), Article ID 2756389, Volume 2019 (2019)

Oxidative Degradation of Amoxicillin in Aqueous Solution by Thermally Activated Persulfate Juanjuan Zhao, Yujiao Sun , Fachao Wu, Minjian Shi, and Xurui Liu Research Article (10 pages), Article ID 2505823, Volume 2019 (2019)

Pollution Abatement of Heavy Metals in Different Conditions by Water Kefir Grains as a Protective Tool against Toxicity Giorgio Volpi , Marco Ginepro, Janeth Tafur-Marinos, and Vincenzo Zelano Research Article (10 pages), Article ID 8763902, Volume 2019 (2019)

Effects of Formulation and Baking Process on Acrylamide Formation in Kolompeh, a Traditional Cookie in Iran Fatemeh Shakeri, Somayeh Shakeri, Sajad Ghasemi, Antonio Dario Troise, and Alberto Fiore Research Article (6 pages), Article ID 1425098, Volume 2019 (2019)

Primary Investigation into the Occurrence of Hydroxymethylfurfural (HMF) in a Range of Smoked Products Laura-Artemis Bouzalakou-Butel, Pantelis Provatidis, Keith Sturrock, and Alberto Fiore Research Article (8 pages), Article ID 5942081, Volume 2018 (2019)

Separation and Enrichment of Omega 3, 6, and 9 Fatty Acids from the By-Products of Vietnamese Basa Fish Processing using Deep Eutectic Solvent Thanh Xuan Le Thi , Hoai Lam Tran , Thanh Son Cu, and Son Lam Ho Research Article (10 pages), Article ID 6276832, Volume 2018 (2019)

Critical Effects of Smoking Parameters on the Levels of Polycyclic Aromatic Hydrocarbons in Traditionally Smoked Fish and Meat Products in Finland Mirja Hokkanen , Ulla Luhtasela, Pirkko Kostamo, Tiina Ritvanen, Kimmo Peltonen, and Marika Jestoi Research Article (14 pages), Article ID 2160958, Volume 2018 (2019) Hindawi Journal of Chemistry Volume 2019, Article ID 2756389, 2 pages https://doi.org/10.1155/2019/2756389

Editorial Chemistry of Food Contaminants and Their Remediation or Mitigation

Alberto Fiore ,1 Photis Papademas,2 and Vlasios Goulas 2

1School of Applied Science, Division of Food and Drink, Abertay University, Dundee, UK 2Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, Limassol, Cyprus

Correspondence should be addressed to Alberto Fiore; a.ﬁ[email protected]

Received 1 July 2019; Accepted 1 July 2019; Published 15 July 2019

Copyright © 2019 Alberto Fiore et al. "is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

"e food industry and research community are particularly metals such as Ca, K, Mg, and Na analysed by ICP. "ey have focused on developing new mitigation strategies to avoid or concluded that water kefir grains could be an efficient ad- alleviate the occurrence of chemical contaminants in the sorber/biosorber of metals on polluted water contaminated food chain. Food chemical contaminants could be present as by heavy metal ions. naturally occurring, entering the food chain as an additive, F. Shakeri and coworkers have evaluated the occurrence by cross-contamination, or formed under different pro- of acrylamide, a potential carcinogenic compound formed cessing conditions. Food contaminants also come from the during the heat processing of starch-rich foods, in Kolompeh phases of food processing, packaging, transportation, and traditional sugary Iranian cookie. "e authors have evaluated storage [1]. "e implications of these contaminants on industrial and household processing conditions, evaluating human health range from mild gastroenteritis to fatal cases different recipes of traditional and industrial processing of of hepatic, renal, and neurological syndromes [2]. "erefore, Kolompeh. In a controlled environment, they have produced regulations are always up to date with legal limits aiming to different Kolompehs: traditional and sugar enriched. "ey guarantee to the consumer the safety of food products. "us, have used the industrial oven and the traditional oven (direct the aim of this special issue was to publish research papers heating) at 273°C to bake the different recipes. "ey have addressing recent advances on the common food contam- concluded that in order to reduce the exposure to acrylamide inants and to offer a deeper insight into food chemistry. to the population, control of the type of raw material and Numerous manuscripts have been evaluated, but only six increasing the automatization level even on traditional food have been successfully published. J. Zhao and coworkers preparation need to be in place to reduce the acrylamide investigated the remediation of antibiotic residues that their exposure. abuse poses a great threat to public health and food security. L.-A. Bouzalakou-Butel and coworkers investigated the In particular, they focused on oxidative degradation of occurrence of hydroxymethylfurfural (HMF), a potential amoxicillin (AMO) in aqueous solution by thermally acti- harmful compound formed in different food products under vated persulfate (TAP). "ey concluded that the TAP was different food processing regimes, in selection of smoked efficient in AMO degradation in aqueous solution under a food (cheese, fish, and meat). "e authors have also analysed pseudo-first-order kinetic model and in drinking water the volatile fraction of the smoked product to correlate with under the experimental condition. Particularly, they sug- HMF. "e authors concluded that, in all the samples ana- gested that TAP could be a valid technology for water re- lysed, HMF was present ranging between 30 and 330 ppb for mediation contaminated by AMO. processed meat. "e highest amount of HMF was found in G. Volpi and coworkers evaluated the use of water kefir the smoked fish, while the cheese samples showed a wide grains as an absorber of heavy metal ions. More specifically, range of HMF concentrations. "e authors discovered a they investigated the role of two different microbial colonies positive correlation between HMF and the measured phe- in the remediation of both heavy metals and mineral water nols in cheese samples, while the correlation for fish and 2 Journal of Chemistry processed meat samples was not significant but indicative. "is finding could lead to other research works between smoking processes employed and the formation of HMF. T. X. Le "i and coworkers investigated the role of deep eutectic solvents (DESs), a new class of ionic liquid analogues, which show promising results in green solvent extraction due to their biodegradability, low toxicity, and easy way of use. "ey optimised the extraction of omega 3, 6, and 9 fatty acids from fish industry waste. "ey concluded that the proposed approach shows an increasing recovery of polyunsaturated fatty acid from 57% to 91% in the raw material (fish waste). "is eco-friendly method shows a potential in the recovery of polyunsaturated fatty form food by-product. Finally, the work of M. Hokkanen and coworkers investigated the four marker polycyclic aromatic hydrocarbons (PAH4) on different traditionally smoked Finnish fish and meat products. PAH4 are classified as human carcinogenic teratogenic, haematological, and immune-toxic compounds; therefore, the exposure to those contaminants is high risk to human health. "eir wide sampling of the Finnish smoked product includes a detailed information on the production process, and the results were able to correlate to the best smoking practice to obtain a safer product; the authors highlighted indirect smoking, and having a distance of at least five meters between the food and smoke source and a smoking time no longer than 5 hours are recommended. In conclusion, the current special issue offers updated information to the readers on the recent advancements regarding food contaminants giving further thoughts with handling of harmful compounds and their mitigation strategy. It is clearly evidenced that the food processing contaminants are a current topic and need further investigation towards their remediation or the mitigation of their formation. Conflicts of Interest "e editors declare that they have no conflicts of interest regarding the publication of this special issue. Acknowledgments "e editors would like to thank all the authors, reviewers, and the editorial team of the Journal of Chemistry for their great support and contribution in making this special issue possible.

Alberto Fiore Photis Papademas Vlasios Goulas References [1] R. Krska, A. Becalski, E. Braekevelt et al., “Challenges and trends in the determination of selected chemical contaminants and allergens in food,” Analytical and Bioanalytical Chemistry, vol. 402, no. 1, pp. 139–162, 2012. [2] I. A. Rather, W. Y. Koh, W. K. Paek, and J. Lim, “"e sources of chemical contaminants in food and their health implications,” Frontiers in Pharmacology, vol. 8, p. 830, 2017. Hindawi Journal of Chemistry Volume 2019, Article ID 2505823, 10 pages https://doi.org/10.1155/2019/2505823

Research Article Oxidative Degradation of Amoxicillin in Aqueous Solution by Thermally Activated Persulfate

Juanjuan Zhao,1,2 Yujiao Sun ,1 Fachao Wu,2 Minjian Shi,2 and Xurui Liu2

1College of Water Sciences, Beijing Normal University, Beijing 100875, China 2Environmental Engineering Department, North China Institute of Science and Technology, Beijing 101601, China

Correspondence should be addressed to Yujiao Sun; [email protected]

Received 27 September 2018; Revised 10 February 2019; Accepted 17 February 2019; Published 12 March 2019

Guest Editor: Giuseppe Meca

Copyright © 2019 Juanjuan Zhao et al. /is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Antibiotic residues and antibiotic resistance genes (ARGs) pose a great threat to public health and food security via the horizontal transfer in the food production chain. Oxidative degradation of amoxicillin (AMO) in aqueous solution by thermally activated persulfate (TAP) was investigated. /e AMO degradation followed a pseudo-first-order kinetic model at all tested conditions. /e pseudo-first-order rate constants of AMO degradation well-fitted the Arrhenius equation when the reaction temperature ranged from 35°C to 60°C, with the apparent activate energy of 126.9 kJ·mol−1. High reaction temperature, high initial persulfate concentration, low pH, high Cl− concentration, and humic acid (HA) concentration increased the AMO degradation efficiency. ·− /e EPR test demonstrated that both ·OH and SO4 were generated in the TAP system, and the radical scavenging test identified ·− that the predominant reactive radical species were SO4 in aqueous solution without adjusting the solution pH. In groundwater and drinking water, AMO degradation suggested that TAP could be a reliable technology for water remediation contaminated by AMO in practice.

1. Introduction contaminants [7]. Persulfate (PS) is usually applied as the precursor of sulfate radicals by reaction equation (1): Antibiotic residues ubiquitously exist in surface water, O O O O O O groundwater, and soil because of overuse and misuse in Activation human and veterinary medicines, leading to the prevalence S S 2 S (1) of antibiotic resistance genes (ARGs) in natural environ- O O O O O O ment [1]. ARGs as a kind of emerging contaminants pose a great threat to public health and food security mainly due to Sulfate radicals could be generated through scission of the persistence in environment and the horizontal transfer in the peroxide bond of persulfate by energy including heat the food production chain [2, 3]. /us, it is urgent to take [8, 9], ultraviolet [5, 10], ultrasound [11], radiolysis [12], and effective measures to eliminate the antibiotics in natural catalyzer [13–16]. Among these methods, TAP is particularly environment in order to reduce the influence of ARGs. attractive for removing organic contaminants because it is a Advanced oxidation processes (AOPs) which could simple and effective method to produce sulfate radicals with produce highly active, oxidizing radicals such as hydroxyl a high reaction stoichiometric efficiency (RSE) [17]. In this radicals, sulfate radicals, and other radicals are promising study, amoxicillin (AMO) belonging to β-lactam antibiotic, techniques to degrade recalcitrant contaminants into which was the top-priority human and veterinary antibiotic, harmless products (CO2 and H2O) [4–6]. Recently, sulfate was chosen as the target contaminant. Currently, AMO was radical-based AOPs has received increasing amount of in- degraded by various physical-chemical processes including terest due to its low cost, high efficiency, and friendly en- the use of zero-valent iron [18, 19] and AOPs such as vironment in degradation and mineralization of recalcitrant Fenton’s reagent [20, 21], photo-Fenton process [22, 23], UV 2 Journal of Chemistry

and UV/H2O2 processes [24], microwave-assisted Fenton’s column (5 μm, 4.6 mm × 250 mm, GL Sciences Inc., Japan). oxidation [25], photocatalytic adsorbents [26], photo- /e mobile phase was made of a mixture of 80% ultrapure catalytic ozonation process [27], and photo-Fenton process water and 20% methanol, and the flow rate was 1.0 mL/min. with Goethite [28]. To the best of our knowledge, this is the /e column temperature was maintained at 40°C. 10 μL of first report on oxidative degradation of AMO by TAP in sample was injected into the HPLC. A FiveEasy plus pH meter aqueous solution. (Mettler Toledo) was applied to test the solution pH. A MS- /e aim of this present study was to investigate TAP for 5000 electron paramagnetic resonance (EPR) instrument the remediation of highly AMO-contaminated water, in- (Freiberg instruments, Germany) was applied to identify cluding the influence factors of the reaction temperature, radical species in the TAP system under the following in- initial PS concentration, pH and Cl−, Ca2+, Mg2+, humic acid strument conditions: modulation amplitude, 1.0 G; modula- (HA), and dissolved oxygen. /e predominant radicals were tion frequency, 100 kHz; sweep width, 100 G; sweep time, − − identified by electron paramagnetic resonance (EPR), and 120 s; and microwave power, 10.00 mW. Cl , NO3 , and 2− AMO degradation in real waters was evaluated, which SO4 were analyzed by the ICS-2100 ion chromatograph + + provides fundamental and practical knowledge to treat (Dionex, USA) with Ionpac AS11 column, and Na , NH4 , AMO-contaminated waters. K+, Mg2+, and Ca2+ were analyzed by the AQ ion chromatograph (/ermo Fisher, USA) using the Ionpac CS12A 2. Materials and Methods column. PS concentration was monitored according to previous literature [29]. Total organic carbon (TOC) was mea- 2.1. Chemicals and Materials. Amoxicillin trihydrate sured by a vario TOC analyzer (Elementar, Germany). (C16H19N3O5S·3H2O, AMO, ≥98.0%), humic acid (FA ≥ 90%), and tert-butyl alcohol ((CH3)3COH, TBA, 3. Results and Discussion ≥99.5%) were obtained from Aladdin Industrial Corpora- tion (Shanghai, China). Sodium persulfate (Na2S2O8, PS, 3.1. Effects of Reaction Temperature on Degradation of AMO. ≥98%) was purchased from /ermo Fisher Scientific (New Reaction temperature is a critical factor that should be Jersey, USA). Methanol (CH3OH, MeOH, HPLC grade) was considered for applying TAP to degrade organic contami- obtained from J. T. Baker (USA). 5,5-Dimethyl-1-pyrrolidine- nants. Effects of the reaction temperature (35–60°C) on N-oxide (DMPO) was acquired from Macklin Biochemical AMO degradation by TAP are shown in Figure 1(a). It could Co. Ltd (Shanghai, China). be seen that AMO oxidative degradation was temperature dependent. /ere was only 22% AMO oxidized by persulfate at 35°C within 330 min. However, obvious degradation was 2.2. Water Samples. Groundwater was collected from a well observed with the increasing temperature. /e removal of ° ° in Nanxin village (40 3′32.9″N, 116 47′50.9″E) (Beijing, AMO was completely achieved by TAP at 55°C within China), and drinking water was taken from Yanjiao town 330 min, and the complete degradation time was decreased ° ° (39 57′18.3″N, 116 48′11.6″E) (Hebei, China). All the water to 90 min at 60°C. samples were filtered through a 0.22 μm membrane and In addition, the AMO degradation well-fitted a pseudo- ° stored at 4 C before further experiments. first-order kinetic model as shown in the following equation: dC − � k C. (2) 2.3. Experimental Procedures. All experiments were per- dt obs formed in 250 mL conical flasks with ground glass stoppers. At the beginning of every experiment, a 10.00 mL·1.010 It can also be written as follows: mmol·L−1 AMO stock solution was diluted to 100.00 mL and C ln � −kobst. (3) heated to the designated temperature for 20 min. /e re- C0 action was started by the addition of 1.00 mL·1.010 mol·L−1 k was the pseudo-first-order rate constants (min−1), PS stock solution; therefore, [AMO]0 was 0.1 mM, and [PS]0 obs was 10 mM. 1.00 mL samples were removed from the conical and it was determined by the plots of ln(C/C0) versus re- flask at each desired time interval and quickly quenched by action time (t), as shown in Figure 1(b). t1/2 was defined as 1.00 mL methanol before analysis. /e initial pH values of equation (4), and kinetic parameters of AMO degradation by −1 TAP at different conditions are shown in Table 1: the solution were adjusted by 1.0 mol·L NaOH and H2SO4 in order to investigate the effects of the initial pH. TBA and ln 2 0.6931 t � � , ( ) MeOH as scavenging agents were applied to identify the 1/2 4 kobs kobs dominating radicals in the TAP system. All the samples were performed in triplicates, and the standard deviations were E ln k � ln A − a , (5) depicted as error bars in figures. /e control experiments obs RT were performed without PS at the same conditions. 15267.38 ln k � 42.21 − . (6) obs T 2.4. Analytical Methods. /e concentration of AMO was analyzed using a high pressure liquid chromatography (LC- kobs in the oxidative degradation by TAP increased 20AT, Shimadzu, Japan) equipped with a WondaSil C18-WR significantly when the reaction temperature raised from Journal of Chemistry 3

1.0 0

0.8 –1

0.6 ) 0 0 –2 C /

0.4 ln ( C /

–3 0.2

0.0 –4 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Time (min) Time (min)

35°C 50°C 35°C 50°C 40°C 55°C 40°C 55°C 45°C 60°C 45°C 60°C

(a) (b) 0.035

0.030 –3

–4 15267.38 0.025 lnk = 42.21 – obs T –5 r = 0.9954 ) obs

–1 0.020 ln k –6 (min 0.015 –7 obs obs k 0.010 0.00300 0.00305 0.00310 0.00315 0.00320 0.00325 1/T

0.005

0.000 35 40 45 50 55 60 Temperature (°C)

(c) Figure 1: Eﬀects of reaction temperature on AMO degradation by TAP (a). Plot of ln(C/C0) versus reaction time t for kobs determination with the pseudo-ﬁrst-order kinetic model (b). Plot of ln kobs versus 1/T for Ea determination with the Arrhenius equation (c). Experimental ° conditions: [AMO]0 � 0.1 mM; [PS]0 �10 mM; T � 35–60 C; reaction time � 330 min.

° ° 35 C to 60 C. Furthermore, ln kobs and 1/T followed degradation efficiency increased with increasing [PS]0 from Arrhenius equation (5), and AMO degradation by TAP was 2 to 20 mM. /ere was about 31, 54, 91, and 100% AMO identified as equation (6) with high correlation coefficient degraded at 330 min in the TAP system when [PS]0 was 2, 5, (r � 0.9942). /e apparent activate energy (Ea) of AMO 10, and 15 mM, respectively. All of AMO was removed at −1 degradation was calculated to be 126.9 kJ·mol according to 20 mM [PS]0 at 210 min. Furthermore, kobs increased line- equation (6), which was comparable to those of other re- arly with increasing [PS]0 according to Figure 2(c), sug- fractory organics, such as ketoprofen (157.02(±8.9) kJ·mol−1) gesting that the AMO degradation rate was in positively −1 −1 [10], triclosan (121 kJ·mol ) [30], naproxen (155 kJ·mol ) proportion to [PS]0. Similar phenomenon observed by Yang [31], ibuprofen (168(±9.5) kJ·mol−1) [9], bisoprolol et al. [33], Chen et al. [34], and Nie et al. [35] was possibly −1 −1 ·− (119.8(±10.8) kJ·mol ) [17], and diuron (166.7 kJ·mol ) due to more SO4 released by high concentration of TAP. [32]. /ese comparable apparent activate energy indicated % RSE defined as the ratio of the concentration of the that the TAP system could be suitable for degrading re- polluters degraded to the PS consumed [15, 31] is a critical fractory organics in wastewater treatment. parameter to evaluate the sustainability of TAP. % RSE values at different [PS]0 values in the TAP system at reaction time of 330 min are shown in Figure 3. /e maximum % RSE 3.2. Effects of Initial PS Concentration on Degradation of value was reached at [PS]0 �10 mM. When [PS]0 increased AMO. PS concentration is also a significant factor that from 2 mM to 10 mM, % RSE increased gradually with the influences AMO degradation by TAP. Effects of [PS]0 on increase of AMO degradation efficiency. When [PS]0 was AMO degradation are shown in Figure 2. AMO degradation from 10 mM to 20 mM, % RSE decreased due to the followed the pseudo-first-order kinetic model, and AMO quenching reactions between radical and radical or radical 4 Journal of Chemistry

Table 1: Kinetic parameters of AMO degradation by TAP at S O 2− + H+ ⟶ HS O − (7) different conditions. 2 8 2 8 −1 − ·− · Operating conditions kobs (min ) t1/2 (min) r HS2O8 ⟶ SO4 + HSO4 (8) [AMO]0 � 0.1 mM, [PS]0 �10 mM ° T � 35 C 0.0007 990.14 0.9886 SO ·− + OH− ⟶ ·OH + SO 2− (9) T � 40°C 0.0013 533.15 0.9924 4 4 T � 45°C 0.0028 247.54 0.9985 T � 50°C 0.0071 97.62 0.9949 /erefore, the reaction mechanisms with contami- T � 55°C 0.0114 60.80 0.9978 nants would vary with the predominant radicals. ° ·− T � 60 C 0.0311 22.29 0.9902 It is reported that SO4 reacts with recalcitrant ° [AMO]0 � 0.1 mM, T � 50 C compounds by electron transfer [37], addition- [PS]0 � 2 mM 0.0013 533.15 0.9894 elimination, and hydrogen atom abstraction [38], [PS]0 � 5 mM 0.0024 288.79 0.9894 while ·OH reacts with recalcitrant compounds by [PS]0 �10 mM 0.0071 97.62 0.9949 addition of C�C double bonds or abstraction of � [PS]0 15 mM 0.0092 75.34 0.9959 hydrogen from the C-H, N-H, or O-H bond [39]. [PS]0 � 20 mM 0.0165 42.01 0.9965 ° (ii) AMO speciation changed with the changing of so- [AMO]0 � 0.1 mM, [PS]0 �10 mM, T � 50 C pH � 2 0.0159 43.59 0.9992 lution pH, as shown in Figure 5. When pH < pKa1 of pH � 5 0.0067 103.45 0.9738 AMO (2.4), an AMO molecule accepts a proton pH � 8 0.0060 115.52 0.9920 forming an ion with a positive charge. When pKa1 pH � 10 0.0058 119.50 0.9991 (2.4) < pH < pKa2 (7.4), AMO exists in the form of − [Cl ]0 �1 mM 0.0058 119.50 0.9968 molecule in aqueous solution. When pKa2 (7.4) − [Cl ]0 �10 mM 0.0082 84.52 0.9825 < pH < pKa3 (9.6), an AMO molecule loses a proton − [Cl ]0 �100 mM 0.0082 84.52 0.9944 forming an ion with a negative charge. When [HA] � 0.1 mg/L 0.0097 71.31 0.9752 0 pH > pKa3 (9.6), an AMO molecule loses two protons � [HA]0 1 mg/L 0.0103 67.16 0.9768 forming an ion with two negative charges. As shown [HA]0 �10 mg/L 0.0103 67.10 0.9836 in Figure 4, the highest kobs was attained at pH 2 due to [HA]0 � 20 mg/L 0.0098 70.72 0.9727 + 2+ the protonation of AMO, resulting in AMO with a [Ca ]0 �1 mM 0.0073 94.95 0.9978 2+ � positive charge which increased the electrostatic at- [Mg ]0 1 mM 0.0074 93.66 0.9948 ·− Groundwater 0.0059 117.47 0.9920 traction to SO4 . With the increase in pH, kobs de- Drinking water 0.0053 130.77 0.9968 creased which could also be attributed by the deprotonation of AMO, resulting in the electrostatic ·− repulsion to SO4 . and PS [5]. /e TOC removal increased with the increasing [PS]0 from 2 mM to 20 mM, as depicted in Figure 3. /is phenomenon might be due to the attacking on AMO and its 3.4. Effects of Matrix in Aqueous Solution on degradation intermediates by the radicals formed at high PS Degradation of AMO concentrations. It can be clearly seen that high TOC removal 3.4.1. Cl− Concentration. /e effects of different anions on and high % RSE were not reached at the same time in AMO degradation were studied. Figure 6 shows the effects of Figure 3. /is was in accordance with the literature reported Cl− concentration. When Cl− concentration was 1 mM, k by Ghauch et al. [5]. obs was slightly lower than that in deionized water, and when − Cl concentration was 10 mM and 100 mM, kobs was a little 3.3. Effects of Solution pH on Degradation of AMO. higher, indicating that higher Cl− concentration could Solution pH is an important factor for TAP to degrade promote AMO oxidative degradation. /is phenomenon contaminants because it could obviously affect radical species, was identical with the effect of Cl− concentration on the contaminant formations, and reaction mechanisms. Effects of degradation of sulfamethazine by TAP [40]. It was probably different pH values on AMO degradation by TAP are shown due to the formation of reactive chlorine species including · ·− in Figure 4. As it can be seen, AMO degradation efficiency Cl , Cl2 , Cl2, and HOCl, which were moderate oxidants increased significantly with decreasing pH, indicating that produced by reactions (10)–(13) [41] and could react with lower pH was favorable to degrade AMO. /is result was AMO molecular to promote AMO degradation efficiency: consistent with the previous reports about sulfate radical- SO ·− + Cl− ⟶ SO 2− + Cl·, based oxidation of fluoroquinolone [36] and triclosan [30]. 4 4 · (10) /is phenomenon could be explained as follows. Cl E0� � � 2.41 V (i) /e predominant radical species were affected by Cl− solution pH. At acid conditions, sulfate radical was the dominating radicals, formed as equations (7) and (8), · − ·− Cl + Cl ⟶ Cl2 , and the reactivity increased with decreasing pH. At ·− (11) basic conditions, hydroxyl radical was the main radical 0 Cl2 E �− � � 2.09 V converted from sulfate radical by using equation (9): Cl Journal of Chemistry 5

1.0 0

0.8 –1

0.6 ) 0 0

C / –2

0.4 ln ( C /

0.2 –3 0.0 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Time (min) Time (min)

2 mM 15 mM 2 mM 15 mM 5 mM 20 mM 5 mM 20 mM 10 mM 10 mM

(a) (b) 0.020

0.020

0.015 k = –0.00123 + 0.00082(AMO) 0.015 ) obs 0 –1 r = 0.9784 0.010 (min ) obs k –1 0.005 0.010 (min 0.000

obs obs 0 5 10 15 20 k (PS)0 (mM) 0.005

0.000 2 5 10 15 20

(PS)0 (mM)

(c) Figure 2: Eﬀects of [PS]0 on AMO degradation by TAP (a). Plot of ln(C/C0) versus reaction time t for kobs determination with the pseudo- ° ﬁrst-order kinetic model (b). Plot of kobs versus [PS]0 (c). Experimental conditions: [AMO]0 � 0.1 mM; [PS]0 � 2–20 mM; T � 50 C; reaction time � 330 min.

2+ 2+ Cl ·− + Cl ·− ⟶ 2Cl− + Cl , with the eﬀects of Ca and Mg on ketoprofen degradation 2 2 2 because Ca2+ and Mg2+ could not activate persulfate [42]: ( ) 0 Cl2 12 E �− � � 1.36 V kobs, with cation − kobs, deionized water� Cl % change in kobs � kobs, deionized water + + − + +, Cl2 H2O ⟶ HOCl Cl H × 100%. HOCl (13) E0� � � 1.48 V (14) Cl−

3.4.3. HA Concentration. Humic acid (HA) is naturally 3.4.2. Ca2+ and Mg2+. Ca2+ and Mg2+ are common cations abundant in groundwater, river water, soils, and sediments, in aqueous matrices which contribute to the water hardness. and understanding the effects of HA on the degradation of Effects of Ca2+ and Mg2+ on AMO degradation by TAP are organic compounds is important to apply TAP to in situ 2+ 2+ shown in Figure 7. % change in kobs of Ca and Mg in chemical remediation (ISCO). /e effects of HA on AMO aqueous solution defined as equation (14) was 2.82% and degradation are investigated and shown in Figure 8. AMO 4.22%, respectively, which indicated that Ca2+ and Mg2+ had degradation efficiency increased gradually with the increase no effect on AMO degradation. /is result was consistent of HA concentration from 0.1 to 10 mg/L, indicating that HA 6 Journal of Chemistry

6 100

5 95 0

4 90 %RSE TOC/TOC 3 85

2 80

2 5 10 15 20

(PS)0 (mM) %RSE

TOC/TOC0 Figure 3: % RSE and TOC/TOC0 at diﬀerent [PS]0 in the TAP system. Experimental conditions: [AMO]0 � 0.1 mM; [PS]0 � 2–20 mM; T � 50°C; reaction time � 330 min.

1.0 0.018

0.015

) 0.012

0.8 –1 0.009 (min

obs 0.006 k 0.6 0.003 0 0.000 25810

C / pH 0.4

0.2

0.0 0 50 100 150 200 250 300 350 Time (min)

pH 2 pH 8 pH 5 pH 10 Figure 4: Effects of pH on AMO degradation by TAP. Solid lines represent the pseudo-first-order kinetic model fits. Insert: changes of kobs ° at different pH values. Experimental conditions: [AMO]0 � 0.1 mM; [PS]0 �10 mM; T � 50 C; reaction time � 330 min.

+ NH 3 NH NH NH 2 2 2 H H H H NH S NH S NH S NH S pK = 2.4 pK = 7.4 pK = 9.6 a1 a2 a3

O N O N O N O N HO HO HO O O O O O

OH OH O O O O O O Figure 5: Changes of AMO speciation by solution pH. could promote AMO degradation efficiency obviously. /is quinone and phenol functional groups in HA. When HA phenomenon was consistent with some previous reports. concentration continuously increased to 20 mg/L, the AMO /e quinone functional groups in HA could efficiently ac- degradation efficiency decreased a little compared with that tivate persulfate to degrade 2,4,4′-trichlorobiphenyl was in 1–10 mg/L HA solution possibly due to the HA’s ·− verified, and the activation of persulfate was induced by the quenching effect to SO4 and ·OH because of the electron- formation of semiquinone radicals [43]. Phenol-activated rich sites in HA [10]. persulfate by the phenoxide form was investigated, and the significant role of phenol in the activation of persulfate was documented by Ahmad [44]. /us, the increasement on 3.4.4. Dissolved Oxygen. In order to investigate the effect of AMO degradation was possibly due to the contribution of dissolved oxygen on AMO degradation by TAP, three Journal of Chemistry 7

1.0 0.010 1.0 0.012

0.008 0.010 ) ) 0.8 –1 0.006 0.8 0.008 –1

(min 0.004 0.006 (min obs k obs

k 0.004 0.6 0.002 0.6 0.002 0 0.000 0 0 mM 1 mM 10 mM 100 mM 0.000 C / C / 0 mg/L 0.1 mg/L 1 mg/L 10 mg/L 20 mg/L 0.4 0.4

0.2 0.2

0.0 0.0 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Time (min) Time (min)

0 mM 10 mM 0 mg/L 10 mg/L

1 mM 100 mM 0.1 mg/L 20 mg/L

1 mg/L Figure 6: Effects of Cl− concentration on AMO degradation by TAP. Solid lines represent the pseudo-first-order kinetic model fits. Figure 8: Effects of HA concentration on AMO degradation by − Insert: changes of kobs at different Cl concentrations. Experimental TAP. Solid lines represent the pseudo-first-order kinetic model fits. � � � ° conditions: [AMO]0 0.1 mM; [PS]0 10 mM; T 50 C; reaction Insert: changes of kobs at different HA concentrations. Experi- � ° time 330 min. mental conditions: [AMO]0 � 0.1 mM; [PS]0 �10 mM; T � 50 C; reaction time � 330 min.

1.0 0.008 1.0 0.008 0.006 )

0.8 –1 0.006

0.004 ) –1 (min 0.8

obs 0.004 k 0.002 (min obs

0.6 k 0.002 0 0.000 0.6 Deionized Ca 1 mM Mg 1 mM 0

C / water 0.000 0.4 123

C / Systems 0.4 0.2 0.2 0.0 0 50 100 150 200 250 300 350 0.0 Time (min) 0 50 100 150 200 250 300 350 Time (min) 2+ Ca 1 mM Mg2+ 1 mM System 1 Deionized water System 2 System 3 Figure 7: Effects of different cations on AMO degradation by TAP. Figure Solid lines represent the pseudo-first-order kinetic model fits. 9: Effects of dissolved oxygen on AMO degradation by TAP. Solid lines represent the pseudo-first-order kinetic model fits. Insert: changes of kobs at different pH values. Experimental conditions: [AMO] � 0.1 mM; [PS] �10 mM; T � 50°C; reaction Insert: changes of kobs at different conditions. Experimental con- 0 0 ° time � 330 min. ditions: [AMO]0 � 0.1 mM; [PS]0 �10 mM; T � 50 C; reaction time � 330 min. systems were designed as follows: System 1 was operated as depicted in Section 2.3 as the control experiment; System 2 effect of dissolved oxygen on AMO degradation efficiency by was conducted in a closed vial and before reaction nitrogen TAP could be neglected. /erefore, AMO degradation by was filled in order to remove oxygen in solution and conical TAP could be carried out in both the oxic condition and flask. System 3 was carried out in an open vial. /e results are anoxic condition. shown in Figure 9. It can be seen that the AMO degradation rate was higher in System 3 than in System 2, suggesting that the oxic condition facilitated the degradation rate of AMO 3.5. Identification of Predominate Radical Species on AMO by TAP. However, at reaction time of 330 min, AMO Degradation. Radical species on AMO degradation were degradation efficiency remained between 88% and 91% tested and identified by EPR. As shown in Figure 10, the under a different dissolve oxygen, demonstrating that the generation of DMPO-OH was obviously demonstrated by its 8 Journal of Chemistry

334 336 338 340 342 Magnetic ﬁeld (mT) Figure 10: EPR spectrum of radical adducts in the TAP system. DMPO-OH, DMPO-SO4. Experimental conditions: ° [DMPO]0 �100 mM; [PS]0 � 75 mM; T � 45 C; reaction time � 20 min.

hyperfine splitting constants (AN � AH � 14.92 G), suggest- 1.0 ing that ·OH existed in the TAP system. DMPO-SO4 was also monitored according to the simulation spectra (AN � c1 c2 0.8 13.97 G, AH � 9.94 G, AH � 1.44 G, and AH � 0.79 G) [45]. /erefore, sulfate radical and hydroxyl radical were reactive oxidative species in the TAP system. 0.6 0 Furthermore, in order to identify the dominating re- 0.008 C / active radicals on AMO degradation by TAP, MeOH (with 0.4 0.006 ) α-hydrogen) and TBA (without α-hydrogen) were chosen as –1 0.004 (min radical scavengers because of the different reaction constants 0.2 obs between alcohol and radicals. /e constant with which k 0.002 ·− 7 −1 −1 MeOH reacts with SO4 (1.1 × 10 M ·s ) is parallel to that 0.000 0.0 Scavenger free TBA MeOH with ·OH (9.7 ×108 M−1·s−1), while the constant with which 0 50 100 150 200 250 300 350 TBA reacts with SO ·− (4.0–9.1 × 105 M−1·s−1) is much lower 4 Time (min) than that with ·OH (3.8–7.6 ×108 M−1·s−1) [46]. /us, ·− MeOH is considered to scavenge both SO4 and ·OH with a MeOH similar rate constant, and TBA is considered to scavenge TBA ·OH efficiently. As depicted in Figure 11, after the addition of Scavenger free MeOH and TBA, the AMO degradation efficiency was Figure 11: Effects of different scavengers on AMO degradation by observed to be approximately 53 and 59%, respectively, TAP. Solid lines represent the pseudo-first-order kinetic model fits. ·− suggesting that SO4 was the dominating radical in Insert: changes of kobs at different scavengers. Experimental con- ° degrading AMO by TAP. ditions: [AMO]0 � 0.1 mM; [PS]0 �10 mM; T � 50 C, [alcohol]0/ [PS]0/[AMO]0 � 30000 :100 :1; reaction time � 330 min. 3.6. Performance of AMO Degradation by TAP in Real Waters. In order to evaluate the feasibility of applying TAP to probably due to the relatively high ionic strength which might degrade AMO under real environmental conditions, hinder the degradation of contaminants in TAP, suggesting groundwater and drinking water were applied (Table 2). that application of TAP for remediation of AMO in AMO degradation by TAP in groundwater and drinking groundwater and drinking water might be efficient. water is shown in Figure 12. It could be seen that AMO degradation in groundwater and drinking water followed 4. Conclusions a pseudo-first-order kinetic model, and kobs in groundwater was slightly higher than that in drinking water. /is In this study, AMO degradation by TAP was effectively result was possibly because higher Cl− concentration achieved in aqueous solution. For any experiment condition, could promote AMO degradation efficiency according to AMO degradation followed a pseudo-first-order kinetic the previous study (as shown in Section 3.4.1). Compared model. /e apparent activate energy of AMO degradation with deionized water, AMO degradation efficiency in was calculated to be 126.9 kJ·mol−1 according to the groundwater and drinking water was a little lower Arrhenius equation ranged from 35°C to 60°C. On increasing Journal of Chemistry 9

Table 2: Characteristics of water samples. − − − 2− − + + + 2+ 2+ Waters pH Cl NO2 NO3 SO4 H2PO4 Na NH4 K Mg Ca Groundwater 7.61 39.7982 n.d. 0.0412 39.6060 n.d. 25.74 0.26 0.66 27.09 65.90 Drinking water 7.98 11.9522 n.d. 1.3448 14.2461 n.d. 71.29 n.d. 1.42 11.00 40.92 n.d., not detected.

1.0 0.008 References

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Research Article Pollution Abatement of Heavy Metals in Different Conditions by Water Kefir Grains as a Protective Tool against Toxicity

Giorgio Volpi , Marco Ginepro, Janeth Tafur-Marinos, and Vincenzo Zelano

Department of Chemistry, University of Turin, Via P. Giuria 7, 10125 Turin, Italy

Correspondence should be addressed to Giorgio Volpi; [email protected]

Received 2 July 2018; Revised 6 December 2018; Accepted 19 December 2018; Published 21 January 2019

Guest Editor: Vlasios Goulas

Copyright © 2019 Giorgio Volpi et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

'is research paper addresses the hypothesis that Water Kefir grains can be used as absorbers of metal ions and reports the first application of the Water Kefir grains as a protective tool against toxicity by heavy metal ions. 'e aim of this study is to evaluate the concentration of heavy metal ions in several Water Kefir solutions during the fermentation process under various conditions. Two colonies of Water Kefir grain were used, and the concentrations of Cd, Co, Cr, Cu, Mn, Ni, Pb, Ba, and Ca were measured in Water Kefir grain solutions at different contact times (0, 24, 48, and 72 hours), different pH values in citric and acetic buffers, and different Water Kefir grains/metal solution ratios, with and without sucrose (5%). Optical emission spectroscopy was used to measure the concentrations of metal ions. Among the tested experimental conditions, the best combination for pollution abatement is sucrose (5%), contact time 24 hours, starting pH � 4.5, acetate buffer, and Kefir grains/metal solution ratio 1 :1. In these conditions, the heavy metal abatement by Water Kefir grains is particularly effective for Cr and Pb (70%) and good for Cu, Ni, and Mn (50%).

1. Introduction Water Kefir beverage is used as a dietary supplement to rebalance the intestinal microflora and as a probiotic sup- Water kefir is an acid, softly alcoholic and fragrant fer- plement [12–20]. mented drink whose fermentation is started with solid Nowadays, investigations on Water Kefir grains are still Water Kefir grains. 'ese Water Kefir grains contain an very incomplete, and most of the scientific research available insoluble polysaccharide and bacteria and yeasts responsible has analysed its biological diversity [1, 10, 21]. 'e structure for the fermentation [1–3]. 'e insoluble Water Kefir grains and the biochemical composition of the Water Kefir grain act as inoculum when added to a mixture of water and sugar polysaccharide has been also studied [22–24]. 'e microbial (sucrose), possibly with extra ingredients such as lemon, diversity of Water Kefir is based on a constant consortium of dried figs, and many others. After 24–48 hours of in- principally lactic and acetic acid bacteria and yeasts; how- cubation, a yellowish fermented drink is obtained; it has a ever, different Water Kefir colonies display different mi- fruity aroma and an acidic, slightly sweet, and slightly al- crobial species [25, 26]. Nevertheless, the fermentation coholic taste [2, 4–9]. conditions, pH modification, and presence and concentra- Water Kefir is accessible worldwide, but the real origin of tion of heavy metals have been poorly reported compared to the grains is still uncertain. It has been proposed that the the vast investigation of the microbial diversity of Water Water Kefir grains came from the Opuntia cactus. “Water Kefir [3, 27–30]. kefir” is the typical name in Western Europe, but also other Water Kefir colonies could interact with heavy metals appellations are in use for this fermented beverage, both physically and chemically due to their structure and depending on the country, such as “African bees,” “Cal- functional groups. ifornia bees,” “Japanese beer seeds,” “ginger beer plants,” In general, the chance of heavy metal contamination in “Tibicos,” “Tibi grains,” “ale nuts,” “balm of Gilead,” food and water is high due to the increasing anthropic “Beb` ees,”´ and “sugary kefir grains” [10, 11]. In general, activities. For these reasons, it is important to define/ 2 Journal of Chemistry establish the chemical quality of water, particularly the and Na in mineral water were determined (Ca � 3.92 mg/L, content of heavy metals, in order to evaluate the possible K � 0.76 mg/L, Mg � 0.76 mg/L, and Na � 2.21 mg/L). human health risk [31–37]. Metals such as zinc, copper, iron, Sodium hydroxide pellets (Reagent grade, Sigma and manganese are essential since they play an important Aldrich), glacial acetic acid (ACS reagent, Sigma Aldrich), role in biological systems, whereas chromium, lead, and and citric acid (ACS reagent, Sigma Aldrich) were used to cadmium are toxic even in traces. 'e essential metals can make buffer solutions as received from commercial suppliers also produce poisonous effects when the intake is excessively without further purification. elevated. As a consequence, there are concentration limits of them that are established for food and water in most countries. 2.3. Labware. 'e risk of contamination was minimized by 'erefore, the focus of this work is to understand if using glassware as little as possible and employing new Water Kefir grains can be used for pollution abatement of plastic (polypropylene) vessels and pipette tips. All labware heavy metal ions at different conditions (contact time, was washed with 10% nitric acid solution and rinsed several starting pH, buffer type, and metal concentration). More- times with deionized water. over, the study deeps the importance of the fermentation process (in sucrose presence) for the adsorption of metal 2.4. Water Kefir Samples. Two colonies of Water Kefir with ions and so identifying the best condition for possible ap- different grains sizes (Figure 1) have been purchased by plication of Water Kefir grains as a protective tool against Kefiring (Kefiring di Fabio Marcolongo, http://www. toxicity. To this end, two Water Kefir grains were tested, and kefiring.com). 'e composition of Water Kefir grains of the concentrations of heavy metals were evaluated in a both Colonies 1 and 2 is Lactobacilli, yeast, lactic cocci acid Water Kefir fermentation process as a function of time (24, bacteria, and Enterococci. Both colonies are used to prepare 48, and 72 h) in the presence or absence of sucrose at dif- the Water Kefir drink to homemade purpose. ferent starting pH values (pH � 3.5, 4.5, and 6.0) in different In order to reproduce the domestic preparation con- buffer types (acetate and citrate). Kefir/metal solution ratios ditions, in laboratory, the Water Kefir grains of Colonies 1 of 1 :1 and 1 :10 were also evaluated. An analytical method and 2 were kept in 1 L commercial mineral water with an originally used to analyse metals in natural water was addition of 0.5 g of citric acid and 100 g of commercial adapted for the determination of Cd, Co, Cr, Cu, Mn, Ni, Pb, sucrose (at 20°C). 'e choice of citric acid is justified by the Ba, Ca, K, Mg, and Na, by ICP-OES (inductively coupled lemon juice addition according to homemade preparation. plasma optical emission spectroscopy) in Water Kefir 'e sucrose-citric acid solution has been replaced every 72 beverages after removing of Water Kefir grains by filtration. hours at 20°C.

2. Materials and Methods 2.5. Sample Preparation and Analysis. 'e water content in the grains was evaluated after 24 hours dehydration in oven 2.1. Apparatus. pH meter Metrohm mod. 713 was used for at 120°C, resulting to be 85% for sample 1 and 84% for pH determination. ICP-OES Optima 7000 DV PerkinElmer sample 2. was used for quantification of metal ions. Measurements 'e samples were prepared as follows: (1) 1 g of Kefir were taken at the following wavelengths: 228.802 nm for Cd, grains was added to 10 mL of mineral water and sucrose 228.616 nm for Co, 267.716 nm for Cr, 324.754 nm for Cu, (5%). (2) 1 g of Kefir grains was added to 10 mL of a metal 257.610 nm for Mn, 231.604 nm for Ni, 220.353 nm for Pb, ion solution (Water Kefir grains/metal solution ratio 1 :10) 455.403 nm for Ba, 317.933 nm for Ca, 766.490 nm for K, prepared with or without sucrose (5%); the initial pH was 285.213 nm for Mg, and 589.592 nm for Na. 1.88, and for varying the pH conditions, a citrate buffer (NaOH 1 M and citric acid 5·10−3 M) was added. 3.5, 4.5, and 6.0 pH values were chosen because of slightly acidic pH of 2.2. Chemical Reagents. Multielement standard solution, homemade Water Kefir drink (pH approx. 3.5–4.0). 'e 1000 mg/L (CertiPUR , Merck KGaA), was used, diluted as buffer concentration was reasonably diluted (5·10−3 M) to necessary, to obtain working® standards acidified with nitric guarantee a proper initial pH without preventing the met- acid (approx. 0.2% wt/v) for calibration curve; high-quality abolic activity of Water Kefir grains (Figure 2). 1 g of Kefir concentrated nitric acid (70%, ACS reagent, Sigma Aldrich) grains was added to 10 mL or 10 g of Kefir grains was added and ultrapure water obtained using a Milli-Q system to 10 mL, 1 :10 or 1 :1 ratios, respectively, prepared with (Millipore, Milford, MA) were used. sucrose (5%) in acetic buffer (NaOH 1 M and acetic acid Solution of metals for Water Kefir colonies was prepared 5·10−3 M). 'e initial pH was 4.5. at different final concentrations of Cd (100 µg/L), Co 'e pH solution was monitored from 24 hours up to 240 (300 µg/L), Cr (1 mg/L), Cu (5 mg/L), Mn (5 mg/L), Ni hours (1) or every 24 hours up to 72 hours for the other (400 µg/L), Pb (200 µg/L), and Ba 1 mg/L (as internal experimental conditions. standard) and acidified with nitric acid (10−2 M). 'e pH After filtration of Water Kefir grains, ICP analysis of the value of the final solution was 1.88. supernatant solution has enabled to evaluate the amount of Commercial mineral water and commercial sucrose biosorbed or bioaccumulated metals. Both heavy metals and were used for Water Kefir tests. 'e contents of Ca, K, Mg, mineral water metals (Ca, K, Mg, and Na) were monitored. Journal of Chemistry 3

(a) (b)

Figure 1: Water Keﬁr grains: Colony 1 (a) and Colony 2 (b).

8.0

7.0

6.0

5.0 pH

4.0

3.0

2.0 0 24487296240 Time (h)

Colony 1 Colony 2

Figure 2: pH trend for Colonies 1 and 2 in mineral commercial water. Conditions: 5% sucrose, initial pH � 7.0, 1 :10 Water Keﬁr/metal solution ratio. 'e results are expressed as the mean of three replications.

'e concentration of metal ions was measured every 24 3. Results and Discussion hours up to 72 hours. An external calibration was performed for quantifying 3.1. pH Trend. 'e Water Kefir is a weakly acidic beverage, each element. 'ree replicates were performed at each but its pH can greatly change depending on the fermentation concentration level, and RSD% values were <2%; therefore, time, the addition of other ingredients, and the amount of the least-squares regression line was utilized for quantifi- sugar. 'e pH trend of a simulated homemade Water Kefir cation, and R2 values of the calibration curves were 0.9900 to solution, that is in commercial mineral water added with 0.9999 depending on the element. sugar, is shown in Figure 2. Each experiment had three independent replications of 'e acid conditions in which Kefir colonies grow were the experiments, and the mean data were used for the simulated with addition of citric acid. A citrate buffer evaluation of results. All of them were expressed with a SD. (5·10−3 M) was used to change the pH of metal solution from 'e data dispersion was evaluated by calculating the stan- 1.88 to 3.50, 4.50, and 6.00. dard error of the mean (SEM) (for standard errors, µg/L Figure 3 shows the trend of pH as a function of time in concentrations, see also SI Tables S1 and S2). presence or absence of sucrose for both colonies. 'e A total of 96 samples were studied. All samples were starting pH was 3.50, 4.50, and 6.00, and Water Kefir grains/ carefully handled to avoid contamination; the appropriate metal solution ratio was 1 :10. quality assurance procedures and precautions were followed Starting from pH � 3.5, 4.5, and 6.0 in presence of to ensure the reliability of results. sucrose, pH decreased in the first 24 hours due to the 4 Journal of Chemistry

4.0 4.0

3.8 3.8

3.5 3.5 3.5 3.5 pH pH

3.3 3.3

3.0 3.0 0 244872 0244872 Time (h) Time (h)

1 1 2 2

(a) (b) 5.5 5.5

5.0 5.0

4.6 4.5 4.5 4.5 pH pH

4.0 4.0

3.5 3.5 0 244872 0244872 Time (h) Time (h)

1 1 2 2

6.5 6.5 6.1 6.0 6.0 6.0

5.5 5.5 pH pH

5.0 5.0

4.5 4.5

4.0 4.0 0244872 0 244872 Time (h) Time (h)

1 1 2 2

(e) (f)

Figure 3: pH trend of Colonies 1 and 2 in absence and presence of sucrose (5%). Conditions: 5·10–3 M citrate buffer, initial pH � 3.5, 4.5, and 6.0, and 1 :10 Water Kefir/metal solution ratio. Left: absence of sucrose. Right: presence of sucrose. n � 3. Journal of Chemistry 5 well-known fermentation process. In the absence of sucrose, (pH � 6.0). Moreover, in relation to the previously published pH remained unchanged or slightly increased. A different studies, two Water Kefir grain/metal solution ratios were trend in presence-absence of sucrose is clearly observable, if experimented: 1 :10 and 1 :1, that is, 1 g·Kefir/10 ml solution the other experimental conditions remain constant. 'e two and 10 g·Kefir/10 ml solution. While a 1 :10 ratio is very Water Kefir colonies show a peculiar and different behaviour similar to the conditions described to prepare home water in the same conditions, probably due to the peculiar size and Kefir beverage (approx. 100 g·Water Kefir grains/1 L·water), form of the Water Kefir grains. the 1 :1 ratio highlights the metal abatement and is most frequently used in the literature studies. Figure 5 shows the trend of Cr concentration as a 3.2. Determination of Metal Ions in the Supernatant Solutions. function of time in the presence of sucrose in citrate and As reported above, the citrate buffer (5·10−3 M) was used to acetate buffers, for both colonies. 'e starting pH was 4.50 change the pH, and the concentration of heavy metals in the and Water Kefir grains/metal solution ratio was 1 :10. As can supernatant solutions was monitored every 24 hours up to be seen, the acetate buffer solution has a more pronounced 72 hours for each different initial pH values. effect on Cr abatement than citrate one. Similar trends were Figure 4 shows the trend of metal concentration (Cr, Ni, obtained also for the other metals (see SI Tables S1 and S2 for Pb, and Cu) as a function of time in presence or absence of other metal ions). 'is result has confirmed what previously sucrose, for both colonies. 'e starting pH was 3.50, 4.50, reported; that is, citrate anion has more complexing power and 6.00, and Water Kefir grains/metal solution ratio was 1 : than acetate anion and so a larger amount of metals is 10. available in solution for absorption/accumulation in acetate 'e concentrations of metal ions did not change sig- buffer. nificantly in the absence of sucrose (for other metal ions) at Two different Kefir/metal solution ratios were studied the three different initial pH values studied. At initial pH � 6, and compared (1 :10 and 1 :1). Figure 6 shows the trend of the concentrations of Ni, Cd, and Cr slightly reduced (lower Cr, Pb, Ni, and Cu concentration as a function of time in the than 20%), while the contents of Co, Pb, Mn, and Cu were presence of sucrose for both colonies. 'e starting pH was markedly decreased (20–75%). Generally, the concentration 4.50 and Water Kefir grains/metal solution ratio was 1 :10 trends at 24–48–72 hours were not susceptible to strong and 1 :1 in acetate buffers (see SI Table S2 for all other metal modification. ions). As can be noted, 1 :1 ratio shows more efficient Also in the presence of sucrose, there were not in- abatement of metals than 1 :10 ratio. A possible explanation teresting modifications in the concentration of metals after is that a saturation effect occurs when the absolute quantity 72 hours (see SI Table S1 for other metal ions). Usually, the of metals increases ten times. content of metals decreased in the first 24 hours and then 'e trends of heavy metal ions abatement as a function returned approximately at the initial value in the next 48 of time are reported in Figure 7 for Pb, Cu, Ni, Cr, and Ca hours. (see SI Table S2 for all other metal ions). 'e starting pH was Comparing the two colonies, it is evident that Colony 1 4.50 and Water Kefir grains/metal solution ratio was 1 :10 adsorbs/accumulates a greater amount of metal ions than and 1 :1 in acetate buffers. Colony 2 at every initial pH value. 'is happens probably For Cr, Pb, and Mn, the difference between 1 :1 and 1 :10 because Water Kefir Colony 1 acidifies the solution more ratio is moderate (10–20%), while for Cu, Ni, Cd, and Co, slowly than Colony 2, so that metal ions are gradually this difference is much more evident (>20%). Likely, the redissolved in the solution. As is common knowledge, metal reason lies in the peculiar Water Kefir colony affinity for ion precipitation-complexation equilibria are influenced by each metal. pH values [38, 39]. In this work, the pH values are modified Figure 8 shows the trend of Ca concentration as a by the Kefir metabolic activity. function of time in the presence of sucrose for both colonies. Moreover, it can be noted that the sucrose presence is 'e starting pH was 4.50 and Water Kefir grains/metal essential to have a microbial activity and, consequently, to solution ratio was 1 :10 and 1 :1 in acetate buffers. As can observe bioaccumulation and/or biosorption phenomena which be seen, the Ca concentration increases in the presence of permit metal ions abatement. However, bioaccumulation/ sucrose instead of decreasing as occurs for heavy metals; biosorption phenomena due to Water Kefir grains activity presumably, this element is replaced by heavy metals during could be reduced in the presence of citrate since, as known, the bioaccumulation/biosorption process. 'is fact is mainly citrate anion easily forms metal complexes in solution. noticeable for Ca because Na, K, and Mg are at lower 'erefore, to evaluate the buffer complexing effect, other concentrations and rarely form coordination complexes. samples were prepared with acetate buffer because acetate anion is a weaker ligand compared to citrate in forming metal 4. Conclusions complexes. Because of the importance of starting pH value and 'e Water Kefir grains are able to retain heavy metal ions sucrose presence, in this second data set, acetate buffer dissolved in aqueous solution. 'eir metabolic activity is (5·10−3 M) was employed at pH � 4.5 with sucrose. 'e influenced by the surrounding conditions: sugar, contact starting pH value was chosen to make a compromise be- time, pH, buffer, Kefir grains/metal solution ratio. tween the physiological pH of Kefir (3.5–4.0) and the pH 'e presence of sucrose is necessary to have a mi- value that shows the most successful metal abatement crobial activity that induces a metal retention in acid 6 Journal of Chemistry

Cr (0% sucrose) Cr (5% sucrose)

1000 1000

900 900 Cr (ppb) 800 Cr (ppb) 800

700 700 0 2448720244872 Time (h) Time (h) 1 pH 3.5 2 pH 4.5 1 pH 3.5 2 pH 4.5 2 pH 3.5 1 pH 6.0 2 pH 3.5 1 pH 6.0 1 pH 4.5 2 pH 6.0 1 pH 4.5 2 pH 6.0

(a) (b) Cu (0% sucrose) Cu (5% sucrose) 6000 6000

5000 5000

4000 4000 Cu (ppb) 3000 Cu (ppb) 3000

2000 2000 0 2448720244872 Time (h) Time (h) 1 pH 3.5 2 pH 4.5 1 pH 3.5 2 pH 4.5 2 pH 3.5 1 pH 6.0 2 pH 3.5 1 pH 6.0 1 pH 4.5 2 pH 6.0 1 pH 4.5 2 pH 6.0

430 430

380 380 Ni (ppb) Ni (ppb)

330 330 02448720244872 Time (h) Time (h) 1 pH 3.5 2 pH 4.5 1 pH 3.5 2 pH 4.5 2 pH 3.5 1 pH 6.0 2 pH 3.5 1 pH 6.0 1 pH 4.5 2 pH 6.0 1 pH 4.5 2 pH 6.0

(e) (f) Pb (0% sucrose) Pb (5% sucrose) 250 250 200 200 150 150 100 100 Pb (ppb) Pb (ppb) 50 50 0 0 0 2448720244872 Time (h) Time (h) 1 pH 3.5 2 pH 4.5 1 pH 3.5 2 pH 4.5 2 pH 3.5 1 pH 6.0 2 pH 3.5 1 pH 6.0 1 pH 4.5 2 pH 6.0 1 pH 4.5 2 pH 6.0

(g) (h)

Figure 4: Cr, Cu, Ni, and Pb concentration as a function of time in absence and presence of sucrose (5%) for Colonies 1 and 2. Conditions: 5·10–3 M citrate buﬀer, initial pH � 3.5, 4.5, and 6.1, and 1 :10 Water Keﬁr/metal solution ratio. Left: absence of sucrose. Right: presence of sucrose. n � 3. Journal of Chemistry 7

1200 Cr

1000

800

600

Cr (ppb) 400

200

0 0244872 Time (h) 1 citrate 2 citrate 1 acetate 2 acetate

Figure 5: Cr concentration as a function of time in citrate buffer (5·10−3 M) and acetate buffer (5·10−3 M) for Colonies 1 and 2. Conditions: 5% sucrose, initial pH � 4.5, and 1 :10 Water Kefir/metal solution ratio. n � 3.

1200 7000 Cr Cu 1000 6000 5000 800 4000 600 3000 Cr (ppb) Cu (ppb) 400 2000

200 1000

0 0 02448720244872 Time (h) Time (h)

(a) (b) 500 250 Ni Pb 400 200

300 150

200 100 Ni (ppb) Pb (ppb)

100 50

0 0 02448720244872 Time (h) Time (h)

Figure 6: Cr, Cu, Ni, and Pb concentration as a function of time in acetate buffer (5·10−3 M) for Colonies 1 and 2 at 1 :10 and 1 :1 Water Kefir/metal solution ratios. Conditions: 5% sucrose and initial pH � 4.5. n � 3. condition. In fact, the reported data show that heavy metal resulting in the best performance of abatement of metals ions are significantly absorbed on the Water Kefir grains after 24 hours. If the initial pH value is too low (3.5), metal surface only in the presence of sucrose, during the met- ions stay in solution; if the initial pH is too high (6.0), metal abolic activity. ions are quickly adsorbed. When the fermentation activity of 'e most appropriate starting pH is 4.5, which was the sample decreases, the pH value turns to acid condition slightly modified by microorganisms during fermentation and, consequently, metals are redissolved. 8 Journal of Chemistry

100 100

80 80

60 60

40 40 % abatement % abatement

20 20 Cr Cu

0 0 0 2448720 244872 Time (h) Time (h)

1 1:10 2 1:10 1 1:10 2 1:10 1 1:1 2 1:1 1 1:1 2 1:1

(a) (b) 100 100

80 80

60 60

40 40 % abatement % abatement

20 20

Ni Pb 0 0 0 2448720 244872 Time (h) Time (h) 1 1:10 2 1:10 1 1:10 2 1:10 1 1:1 2 1:1 1 1:1 2 1:1

Figure 7: % abatement of Cr, Cu, Ni, and Pb as a function of time in acetate buﬀer (5·10−3 M) for Colonies 1 and 2 at 1 :10 and 1 :1 Water Keﬁr/metal solution ratios. Conditions: 5% sucrose and initial pH � 4.5. n � 3.

8000 Ca 7000

6000

5000 Ca (ppb) 4000

3000

2000 0244872 Time (h)

1 1:10 2 1:10 1 1:1 2 1:1

Figure 8: Ca concentration as a function of time in acetate buﬀer (5·10−3 M) for Colonies 1 and 2 at 1 :10 and 1 :1 Water Keﬁr/metal solution ratios. Conditions: 5% sucrose and initial pH � 4.5. n � 3. Journal of Chemistry 9

Precipitation, adsorption, and complexation equilibria sugary kefir fermentation-a review,” Food Microbiology, are controlled by the buffer type besides the pH value, as vol. 66, pp. 86–95, 2017. mentioned above. Complexing anions like citrate should be [2] Y. Karaca, “Production and quality of kefir cultured butter,” avoided because they compete with the biosorption/ Mljekarstvo, vol. 68, no. 1, pp. 64–72, 2018. bioaccumulation phenomena. 'e acetate buffer has negli- [3] D. Laureys, A. Van Jean, J. Dumont, and L. De Vuyst, “In- gible complexing properties. vestigation of the instability and low water kefir grain growth during an industrial water kefir fermentation process,” Ap- 'e 1 :10 ratio brings lower abatement than 1 :1 ratio, plied Microbiology and Biotechnology, vol. 101, no. 7, probably because the metal ion abatement depends also on pp. 2811–2819, 2017. the absolute quantity of metal ions, and in this condition, a [4] A. A. Bengoa, M. G. Llamas, C. Iraporda, M. T. Dueñas, saturation effect occurs on the Water Kefir grains’ surface. A. G. Abraham, and G. L. Garrote, “Impact of growth tem- 'erefore, among the tested experimental conditions, perature on exopolysaccharide production and probiotic the best combination for pollution abatement is sucrose, 24 properties of Lactobacillus paracasei strains isolated from hours, pH � 4.5, acetate buffer, Kefir grains/metal solution kefir grains,” Food Microbiology, vol. 69, pp. 212–218, 2018. ratio 1 :1. In these conditions, the abatement of heavy metals [5] D. Cais-Sokolinska,´ B. Stachowiak, Ł. K. Kaczynski,´ by Water Kefir is particularly effective for Cr and Pb (approx. P. Bierzunska,´ and B. 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Research Article Effects of Formulation and Baking Process on Acrylamide Formation in Kolompeh, a Traditional Cookie in Iran

Fatemeh Shakeri,1 Somayeh Shakeri,2 Sajad Ghasemi,3 Antonio Dario Troise,4 and Alberto Fiore 5

1Research Center of Tropical and Infection Disease, University of Medical Science, Kerman, Iran 2Department of Food Science and Engineering, Yazd Branch, Islamic Azad University, Yazd, Iran 3Department of Food Science, Engineering and Technology, Campus of Agriculture and Natural Resources, University of Tehran, Karaj, Iran 4Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Italy 5Division of Food & Drink School of Science, Engineering and Technology, Abertay University, Dundee DD1 1HG, UK

Correspondence should be addressed to Alberto Fiore; a.ﬁ[email protected]

Received 26 July 2018; Revised 15 November 2018; Accepted 9 December 2018; Published 2 January 2019

Academic Editor: Susana Casal

Copyright © 2019 Fatemeh Shakeri et al. +is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. +ermal treatments and recipes are two critical aspects for the formation of acrylamide at ordinary household cooking conditions and industrial level. Kolompeh is a traditional Iranian cookie, and the aim of this study was to monitor acrylamide formation in four different recipes: traditional sugary Kolompeh (TSK), traditional simple Kolompeh (TSIK), industrial sugary Kolompeh (ISK), and industrial simple Kolompeh (ISIK). Along with the measurement of reducing sugars, moisture, and pH, acrylamide was quantified by gas chromatography mass spectrometry (GC-MS). Results indicated that acrylamide content was 1758, 1048, 888, and 560 μg/kg for TSK, TSIK, ISK, and ISIK, respectively, revealing that the kind of thermal treatment in combination with higher concentrations of reducing sugars were the major driver for acrylamide formation. In particular, acrylamide concentration in TSIK direct heating was 1.87 times higher than industrial indirect heating treatment, highlighting that domestic preparation of Kolompeh required a specific attention as a source of potential toxic molecule formation.

1. Introduction ability to stabilize azomethine ylide, one of the precursors of acrylamide [4, 5]. Polysaccharides as cellulose and pentosans Acrylamide (2-propenamide, C3H5NO) is usually formed are other efficient precursors of acrylamide while the yield in during heat processing of starch-rich foods at temperatures presence of aldehydes or ketones, such as 2-hydroxy-1- higher than 120°C in presence of asparagine. Roasting, butanal and hydroxy acetone, is higher than the one in toasting, baking, frying, broiling, and grilling are the typical presence of glucose [6]. thermal treatments that promote acrylamide formation due Other minor routes of acrylamide formation include 2- to the presence of an acrylic and amide group. Considering propenal and acrylic acid in presence of amino acids with its high reactivity, acrylamide is defined as a potential nitrogen in their side chains such as glutamine, lysine, and carcinogenic, reprotoxic, and neurotoxic contaminant for arginine or a β-proton in the amino acid adjacent to al- humans [1–3]. anine [4, 7, 8]. +e backbone molecules of acrylamide Looking at the chemical aspects, asparagine and re- originate from asparagine, which forms the acrylamide by ducing sugars are the two key precursors of acrylamide direct deamination and decarboxylation, but the reaction formation. In particular, fructose is more reactive than is done inefficiently in the extremely low acid media glucose, as a consequence of the lower melting point and the [8–10]. 2 Journal of Chemistry

+e Maillard reaction (MR) is the main pathway for Kolompeh is a traditional cookie of Kerman and Iran acrylamide formation in foods; however, some minor and it is composed by two parts: the coating consists of pathways may occur also in non-Maillard environments, as wheat flour, butter, milk, sugar, egg, baking powder in the case of 3-aminopropionamide, that avoid the re- (NaHCO3), and bakery yeast, and occasionally spices such quirement of reducing carbonyls [7, 9, 10]. as cinnamon, cardamom, and nutmeg are added [23]. +e Acrylamide formation is common to a wide variety of core is characterized by the presence of butter, walnut, food products [11]. Depending on the kind of thermal almonds, pistachio, and date paste. Along with the treatments and the concentration of precursors, acrylamide common ingredients, Kolompeh cookies can be produced concentration can go up to 5000 μg/kg. In this frame, bakery following two different recipes and two different pro- products and cookies along with potato products, burgers, cesses. On the one hand, recipes can include reducing nuggets, coffee, and cereals are the most relevant source of sugars in the coating, as in the case of sugary Kolompeh, acrylamide. and on the other hand, sugar is not added to the coating Several methods have been developed for the detection mixture (simple Kolompeh). Along with the addition of and measurement of acrylamide in food products, where gas sugar, Kolompeh cookies are produced in ordinary chromatography mass spectrometry (GC-MS) and liquid household cooking conditions by using direct oven chromatography mass spectrometry (LC-MS) are the heating and at the industrial level by using indirect techniques of choice [12]. Recently, rapid methods for de- heating. tection of acrylamide such as color-indicating methods, +is study deals with the effects of the baking process enzyme-linked immunosorbent assay (ELISA) methods, and formulation of Kolompeh on acrylamide formation, supramolecular recognition-based methods, electrochemical aiming at investigating the correlation of acrylamide, re- biosensing methods, and fluorescent sensing methods have ducing sugars, pH, and moisture content. +e final goal is to been published [13–15]. introduce smart strategies to control acrylamide formation Mitigation of acrylamide formation in foods is one of the during ordinary household cooking conditions. most challenging aspects related to the Maillard reaction, as the simultaneous formation of desired and undesired 2. Materials and Methods molecules are not easily separated [11, 15]. In this respect, 13 control of the reaction mechanisms of acrylamide formation 2.1. Materials. Acrylamide and [1,2,3- C3]-acrylamide is one of the prerequisites to reduce acrylamide content in (98%) standards purchased from Sigma (St. Louis, MO) foods and the most effective strategies encompass: storage and Cambridge Isotope Laboratories, Inc. (Andover, MA), and tuning of the thermal treatment (temperature, time, and respectively. Methanol, formic acid, n-hexane were obtained water activity), quality of raw materials with the selection of from Merck (Darmstadt, Germany). Analytical grade so- cultivars with low reducing sugar concentration, blanching dium thiosulfate pentahydrate, hydrobromic acid, hydro- (temperature and time), reduction of the pH, addition of chloric acid, potassium bromide, and bromine (99.8%) were antioxidants, replacement of reducing sugars, and oxidation purchased from Sigma (St. Louis, MO). of free asparagine into aspartic acid by using asparaginase Food ingredients as wheat flour, sugar, and sunflower oil [16, 17]. were purchased from a local market. All chemicals and Other effective strategies can be designed in the pro- solvents were of analytical grade. duction process such as the use of vacuum baking, radio- frequencies, and ohmic heating. In the case of acrylamide formation, heat treatment of foods needs to be optimized to 2.2. Preparation of Kolompeh Cookies. Kolompeh cookies, preserve beneficial outcomes as the formation of flavor, traditional sugary Kolompeh (TSK), and traditional simple texture, color, and microbiological safety and to limit un- Kolompeh (TSIK) were prepared according to the tradi- desired effects. While for industrial production, this may be tional method, and two different samples were produced achieved more effectively/sustainably by consistent fine- according to the following recipe. For TSK samples: 500 g tuning of technological processes, within ordinary house- wheat flour and 100 g sugar were added to 300 g margarine hold cooking conditions much more attention needs to be and 80 mL cow’s milk with 3% fat. Upon careful mixing dedicated to the recipes and to the thermal exchange, as little using a kitchen robot equipped with a hook, 90 g of egg yolks modifications can be implemented during production were added along with 2 g salt and 50 mL of distilled water. processes [18–20]. For TSIK samples, the same recipe was used without the +e relationship between household cooking condi- addition of sugars. Twenty biscuits were prepared for each tions and negative outcomes of MR has been investigated batch and were cooked in triplicate. by several authors. Trevisan and coworkers demonstrated +e dough of all samples was rolled out to discs with that home cooking methods did not affect dietary intake of 7 cm diameter and 2 mm thickness using a manual laminator Maillard reaction products (MRPs) in beef and early and and stored at room temperature. After 30 min, each disc was furosine as a marker of lysine Amadori compounds rounded with 10 g of date paste. Traditional samples (TSK predominated in cooked beef [21]. Similarly, Lamberti and TSIK) were baked in the traditional oven (direct et al. observed domestic boiling-modified milk protein heating) at 273°C, and industrial samples (ISK and ISIK) profile, causing a minor reduction in milk allergenicity were baked in the industrial oven (indirect heating) at 200°C [22]. for 15 min. Journal of Chemistry 3

2.3. Moisture Content and pH. Samples (5 g) were weighed Differences were significant at p ≤ 0.05. +e correlation of accurately into standardized plates and then dried until reducing sugar concentration and moisture content with constant weight in an oven at 105°C overnight. Moisture acrylamide formation was analyzed by the Pearson corre- content was reported as a percentage according to the lation coefficient. standard ISO 6540 :1980 (International Standard Organi- sation, 1980). +e samples were analyzed in triplicate. For 3. Results and Discussion pH determination, finely ground cookies (1 g) were mixed with 100 mL of water and vortexed for 3 min. +e mixture 3.1. Effect of Formulation on Acrylamide Formation. was then held at room temperature for 1 hour to separate Results indicated that acrylamide content in the two dif- solid and liquid phases. After carefully removing the su- ferent recipes treated with direct and indirect heating, pernatant layer, the pH was measured using a CG-837 pH namely, TSK, TSIK, ISK, and ISIK were 1758 ± 4.45, 1048 ± meter (Schott, Mainz, Germany). Analysis was performed in 79.63, 888 ± 12.175, and 560 ± 99.1 μg/kg, respectively triplicate. (Table 1). Results of this study showed that TSK and TSIK had a significantly (p < 0.05) higher level of acrylamide content 2.4. Reducing Sugar. +e nonstoichiometric sugar oxidation than ISK and ISIK (Table 1). +e highest level of acrylamide process in the presence of alkali (Fehling reagents) was used was produced in the TSK sample and the lowest of acryl- for quantitative determination of reducing sugars according amide concentration obtained by the ISIK sample. +e to the method described by Miller [24]. Each sample was concentration of reducing sugar also exhibited a clear re- analyzed in triplicate. lationship with acrylamide formation in a complex environment such as Kolompeh cookies; indeed, TSK samples 2.5. Extraction and Measurement of Acrylamide by Gas with added sugars showed the maximum level of acrylamide. Chromatography Mass Spectrometry (GC-MS). GC-MS +e dough processing was not controlled in the traditional analysis was performed according to Castel and Wei- products that lead to an increase of reducing sugars and Chich [25, 26], using a Varian ion trap Saturn 2200 GC- cookies baked at elevated temperatures in traditional sam- MS (Agilent Technologies, Santa Clara, CA), and separation ples [27]. Increasing reducing sugar concentration and of acrylamide and its labeled internal standard was achieved temperature of baking led to an increase of the MR that leads by using a Varian DB5 column (DB5 30 m × 250 μm × to the increase of acrylamide formation in the traditional 0.25 μm). An aliquot of 10 g sample and 100 μL of d3- sugary Kolompeh. Results reported here were in agreement acrylamide solution (2 µg·mL−1) were carefully mixed with with other cookies’ preparation only for industrial processes 10 mL of water. Samples were mixed for 20 minutes and then with no sugar added, while in presence of sugar and during centrifuged at 4000 rpm at the temperature of 4°C for 10 household production, the concentration was undoubtedly minutes. +e supernatant was filtered using 0.45 μm nylon higher than cookies preparation [28–30]. Looking at the syringe filters and 300 μL brominating reagent (200 g of concentration of reducing sugars as one of the active pre- potassium bromide, 10 ml of hydrobromic acid, and 160 ml cursors of acrylamide formation, results revealed that the of bromine-saturated water were mixed into a 500 ml vol- concentration in traditional and industrial Kolompeh were umetric flask and made up to volume with deionised water) 10.04% and 9.84% (Tables 2 and 3). According to our results, added to 3 mL of the filtered sample. +e mixture was the concentration of reducing sugars had a positive Pearson vortexed and incubated in an ice bath for 1 hour. One drop correlation coefficient (0.964, Table 4) with acrylamide of 1 N sodium thiosulfate was added to each sample to formation. decompose any remaining bromine. Samples were extracted +e moisture content in traditional and industrial with 2 mL ethyl acetate and then centrifuged for 10 minutes, Kolompeh was 8.11 and 11.19% (Tables 2 and 3). Our in- and the organic layer was transferred to autosampler vials vestigations indicated the moisture content of Kolompeh for analysis. Analysis of 2,3-bromopropionamide (2,3-BPA) showed a negative Pearson correlation (−0.987, Table 4) with and the internal standard was performed in the PCI SIM acrylamide formation. +e moisture content and acrylamide mode with the following parameters: injector: 180°C; Xfer formation, as expected, had an inverse relationship. +e line: 180°C; trap: 120°C; ionization mode: CI. +e SIM moisture content in traditional Kolompeh was lower than masses selected for PCI were 152, 150, 106, and 70 for 2,3- industrial Kolompeh, leading to higher acrylamide forma- BPA, while for 2- BP(C13)A, 154, 152, 110, 108, and 73 were tion in traditional Kolompeh than industrial products. +e used. +e quantification ions were 152 and 154 for 2,3-BPA evaporation of water promoted the formation of brown and 2- BP(C13)A, respectively. A calibration curve was compounds and unequivocally increased the reaction rates prepared in the range 200–2000 μg/kg by following the same of MR [31]. High baking temperature and direct heating for brominating procedure described above. traditional samples caused the decrease in moisture content in traditional samples, and the removal of water was one of the key aspects for decarboxylation of Amadori compound 2.6. Statistical Analysis. Acrylamide content in all samples and hence for the formation of acrylamide. It was hy- was analyzed for variance (ANOVA) and subjected to pothesized that acrylamide formation mainly occurred in Duncan’s multiple test by using the SPSS software V. 24. the crust independently from the recipe or the thermal Data are expressed as means of triplicate analyses ± SD. treatments. Previous studies reported similar results: 4 Journal of Chemistry

Table 1: Acrylamide content in Kolompehs (μg/kg). temperature on acrylamide formation, and heating tem- Min Max SD Mean perature and acrylamide formation exhibited an exponential correlation from 160 to 230°C, while prolonging the thermal TSK 1720 1800 4.45 1758a b treatment over 20 min acrylamide concentration decreased TSIK 970 1120 79.63 1048 ISK 790 1200 12.175 888c as a consequence of polymerization and Michael addition ISIK 480 600 99.1 560d reaction. [32] Moreover, the presence of free amino groups Traditional sugary Kolompeh (TSK), traditional simple Kolompeh (TSIK), can influence the elimination of acrylamide as a consequence industrial sugary Kolompeh (ISK), and industrial simple Kolompeh (ISIK). of the Michael addition [35, 36]. Significant differences were determined by ANOVA analysis and Duncan test (p ≤ 0.05). Different letters indicate significant differences. 4. Conclusion

Table +e effects of sugar and baking processes on acrylamide 2: Characters of traditional Kolompeh formation in Kolompeh biscuits were investigated by taking Min Max SD Mean into account two different recipes, with and without sugar Moisture content (%) 6.3 10.1 2.0 8.1 addition, processed in two different conditions that mim- Reducing sugar (%) 9.8 10.3 0.07 10.0 icked the industrial scale and household production pro- pH 5.4 5.9 0.02 5.6 cesses. In both cases, with and without sugars, acrylamide formation was 1.97 and 1.87 times higher in the domestic Table 3: Characters of industrial Kolompeh preparation than industrial process. +e optimization of indirect heating at the industrial level guaranteed an effective Min Max SD Mean control of acrylamide formation, while the addition of re- Moisture content (%) 10.4 11.9 0.2 11.2 ducing sugar for the formation of texture or flavor devel- Reducing sugar (%) 8.5 10.7 0.6 9.8 opment should be kept under control in order to avoid the pH 5.4 5.8 0.01 5.6 formation of potentially toxic compounds. Independently, from the addition of the sugar, household cooking requires Table 4: Correlation coefficient of reducing sugar and moisture particular attention as the direct heating and the higher content with acrylamide formation. temperature are effective in the formation of acrylamide. +e results of the experiments were as expected, and especially in Pearson correlation p value line with the recent findings of Mesias et al., in order to coefficient reduce the exposure to the ACR in the population, a Reducing sugar concentration (%) 0.964∗∗ 0.002 ∗∗ standardization of raw material (limiting the precursors) Moisture content (%) −0.987 0.0001 and increasing the level of automatization will reduce ac- ∗∗ Correlation significant at the 0.01 level. rylamide [37]. moisture, acrylamide, and MR rates were inversely corre- Data Availability lated, and the external crust is the principal situ in which the reaction occurs [27, 32]. Data are present within the article. Our results indicated that the pH did not significantly change (5.60 and 5.63 for the industrial and traditional Conflicts of Interest products). In both industrial and domestic preparation, pH was slightly acid as a consequence of the production of +e authors declare that they have no conflicts of interest. hydrogen atoms during the course of the MR. In this respect, the pH showed a limited impact on the formation of MRPs, Acknowledgments in particular acrylamide. We gratefully appreciate University of Medical Science, Kerman, Iran. +is work has been partially funded by a grant 3.2. Effect of Baking Process on Acrylamide Formation. provided by the council for Research at the Research Center Baking process highly affected acrylamide formation. Direct of Tropical and Infectious Disease, University of Medical heating and elevated temperature led to an increase in ac- Science, Kerman, Iran. We thank Miss Morag Steele, rylamide formation, and the hypothesized protection of the Abertay University, for the English language revision of coating layer was not effective in mitigating the formation of manuscript. potentially toxic molecules. Traditional household baking process increased acrylamide concentration from 888 to 1758 μg/kg in the sugary sample, and acrylamide content References increased from 560 to 1048 μg/kg in the without sugar [1] J. G. F. Hogervorst, P. A. van den Brandt, R. W. L. Godschalk, sample (Table 1). As previously observed, increasing the F.-J. van Schooten, and L. J. Schouten, “+e influence of single temperature of the heat process from 200 to 230°C acryl- nucleotide polymorphisms on the association between dietary amide formation increased 2-3 times [33, 34]. Rydberg and acrylamide intake and endometrial cancer risk,” Scientific coworkers reported similar results for the effect of baking Reports, vol. 6, no. 1, p. 34902, 2016. Journal of Chemistry 5

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Research Article Primary Investigation into the Occurrence of Hydroxymethylfurfural (HMF) in a Range of Smoked Products

Laura-Artemis Bouzalakou-Butel,1 Pantelis Provatidis,1 Keith Sturrock,2 and Alberto Fiore 1

1Division of Food & Drink, School of Science, Engineering & Technology, Abertay University, Dundee DD1 1HG, UK 2Division of Science, School of Science Engineering and Technology, Abertay University, Dundee DD1 1HG, UK

Correspondence should be addressed to Alberto Fiore; a.ﬁ[email protected]

Received 15 June 2018; Revised 25 October 2018; Accepted 11 November 2018; Published 5 December 2018

Academic Editor: Annalisa De Girolamo

Copyright © 2018 Laura-Artemis Bouzalakou-Butel et al. .is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

5-Hydroxymethylfurfural (HMF) is produced in foods through many different pathways. Recently, studies have revealed its potential mutagenic and carcinogenic properties. Determination of HMF was originally used as an indicator of both the extent of thermal processing a food had undergone and food quality. It has been identified in a variety of food products such as bread, breakfast cereals, fruit juices, milk, and honey. In addition to the thermal processes that lead to the formation of HMF during thermal treatment, food smoking also creates conditions that result in the formation of HMF. .is can take place within the food due to the elevated temperatures associated with hot smoking or by the proximity of the products of the pyrolysis of the wood matrix that is used for smoking (cold smoking). .is may lead to further contamination of the product by HMF over and above that associated with the rest of the preparation process. Until now, there have been no studies examining the relation between the smoking procedure and HMF contamination in smoked food. .is study is a primary investigation measuring HMF levels in three categories of smoked food products, cheese, processed meat, and fish, using HPLC-UV. .e amount of HMF found in all three product categories supports our hypothesis that HMF levels are due to both internal pathways during processing and external contamination from the smoke generation matrix (wood) employed. .e results ranged from 1 ppb (metsovone traditional Greek smoked cheese) to 4 ppm (hot-smoked ready-to-eat mackerel). Subsequently for smoked cheese products, a correlation was found between HMF and phenolic compounds generated by the smoking procedures and identified by SPME-GCMS. It was observed that cheese samples that had higher concentrations of HMF were also found to have higher concentrations of syringol and cresols. It is important therefore to understand the smoking procedure’s effect on HMF formation. .is will aid in the development of mitigation strategies to reduce HMF formation while retaining the flavour of the smoked products.

1. Introduction .e burning of wood results in pyrolysis/thermal degradation of its main constituents: cellulose, hemicellulose, Food smoking is one of the oldest techniques used for the and lignin. More than 400 volatile compounds have been preservation and flavouring of food [1]. Nowadays, such found in the smoke of wood, with phenols mainly being processing is used to improve the organoleptic properties of responsible for the smoky flavour [4]. A range of factors have some food by providing unique flavours [2] originating from been found to influence the smoking process. .ese include the smoke applied, usually by burning wood. Smoking can the temperature during the pyrolysis, the wood type also alter the colour of foods and promote dehydration, (hardwood or softwood), the part of the wood (heartwood or which affects the texture and reduces the bacterial growth. sapwood), the moisture concentration of the wood, and the Products that are usually smoked are fish and meat, but these presence of air during the process [5]. Hot smoking and cold techniques are also applied to cheeses and spices [3]. smoking are natural vaporous methods utilising smoke 2 Journal of Chemistry produced during combustion under different conditions. occurs, and HMF is an intermediate by-product under weak .e temperature for hot smoking processes is usually in the alkaline conditions [13]. Subsequently, HMF reacts further range 55°C to 80°C, and the duration is short. On the with nitrogen-containing compounds and polymerises contrary, cold smoking takes place over longer periods and giving nitrogenous polymers known as melanoidins, which can last for days, at temperatures between 15°C to 25°C [2, 4]. are responsible for the brown colouration of the food sur- .e type of polysaccharide present in the wood leads to faces. .e parameters mainly responsible for the quantity particular degradation products. Hemicellulose constitutes and quality of the browning as a consequence of the Maillard 20–35% of wood mass, and it mainly contains hexoses and reaction are the sugar and amino group source, the extent pentoses connected by β-(1,4) bonds. Compounds produced and type of thermal processing, and pH [14]. Caramelisa- by the pyrolysis of hemicellulose are mainly furans and tion, a controlled pyrolysis of sugars, is another non- carboxylic acids, together with aldehydes that give smoked enzymatic browning reaction that occurs due to thermal products their characteristic brown colour. Cellulose is a treatment and can also lead to HMF formation [15]. linear polymer of glucose monomeric units connected by HMF has already been identified and measured in many β-(1,4) bonds that constitute approximately 40–50% of food products such as dried fruits, breads, jams, coffee, softwoods and hardwoods. .e pyrolysis of cellulose, caramel, fruit juices, cookies, breakfast cereals, milk, and depending on the burning temperature, can lead to different honey. A number of influences on its formation have been products such as anhydrous sugars at lower temperatures discovered. Examples are, in cookies, the relationship be- and furans, pyrans, and glycoaldehyde at higher tempera- tween sugars, mainly glucose, and HMF concentration is tures [6]. Lignin, a cross-linked polymer of several phe- linear. For fruit juices, the acidity during hydrolysis was nylpropanoids constitutes 20–25% of wood matrix and found to be an important factor [16–21]. In honey, mea- provides to the smoke phenolic compounds mainly syringol surement of HMF is an indicator of quality age and thermal (2,6-dimethoxy-phenol), eugenol (2-methoxy-4-(prop-2- treatment, and there is a strict regulatory limit. .e Honey en-1-yl)phenol), isoeugenol (2-methoxy-4-(prop-1-en-1- (Scotland) Regulations 2015, for example, place a limit of not yl-phenol), guaiacol (2-methoxyphenol), p-cresol (4-meth- more than 40 mg/kg for all honey except baker’s honey and ylphenol), and phenol. It has been reported that they could not more than 80 mg/kg for honey from tropical regions. In be used as indicators of the temperature and duration of the addition, it has been reported that HMF could be produced smoking process [7]. Phenols and cresols are produced from when bees are fed with sucrose syrup with an acidic flavour further degradation of guaiacols. Syringol levels are higher additive [22]. from the pyrolysis of hardwoods compared to softwoods as Determination of HMF levels in food products has the lignin structure is different between the two types of become important as HMF is metabolised in human body wood with the former containing more syringol [8]. mainly to 5-hydroxymethylfurfuroic acid (HMFA) [23] and Among other components, 5-hydroxymethylfurfural to 5-sulfoxymethylfurfural (SMF) [24, 25]. .e sulphuric (HMF) has been identified in wood smoke [9]. .ere are two group of the SMF can be removed easily, resulting in the main mechanisms of HMF production during the burning of production of an ester with genotoxic and mutagenic effects. the wood, either the dehydration of hexoses under mild .is ester further reacts with DNA, RNA, and proteins acidic conditions or formed as an intermediate in the leading to their damage. Work has been conducted using in Maillard reaction [10]. vitro and in vivo experiments in animals to identify the During pyrolysis, cellulose initially depolymerises extent of the carcinogenic properties of SMF [26–30]. Rats forming several anhydrous sugars, mainly levoglucosan. fed with honey with different concentrations of HMF Different levels of levoglucosan result from different burning showed adverse health effects as the HMF concentration sources. Subsequently, the thermal degradation of levoglu- increased [31]. cosan leads to the 1,6- glycosidic bond cleavage and the In this paper, an initial investigation into HMF presence subsequent formation of glucopyranose by rehydration. in smoked food products was conducted. We also report our .ere are two pathways to produce HMF. .e first pathway attempt to link the extent and the type of smoking by includes the rearrangement reaction of 1,6-anhydro-β-D- comparing the phenolic compounds produced during glucopyranose and the second results from the thermal smoking with the different levels of HMF. degradation of levoglucosan. Additionally, further reactions of HMF lead to the production of furfural and formaldehyde 2. Materials and Methods by dehydroxymethylation of the furan ring. In addition, according to [11], phenol and benzene are produced from 2.1. Materials. .e food samples were purchased from local rearrangement reactions of HMF [12]. retailers in the Dundee area. Formic acid was purchased .e other main route to the formation of HMF is the from Sigma-Aldrich (St. Louis, MO, USA), 5-(hydrox- Maillard reaction. .e Maillard reaction plays an important ymethyl)furfural (HMF) was from Fisher Scientific (98%, role in the taste and appearance of food and occurs during ACROS Organics™, USA), and the Carrez clarification kit cooking. .e Maillard reaction is a complex network of was obtained from Merck (Darmstadt, Germany). nonenzymatic reactions with different pathways. Maillard starts when an amino group from an amino acid or a protein condenses with a reducing sugar leading to Amadori 2.2. Sampling. An appropriate sampling technique was used rearrangement products. At a later stage, 1,2-enolization for each of the different sample types. For the cheeses, Journal of Chemistry 3 an 8 cm3 cube was cut from the body of the cheese and a total run time of 97 min. .e injection temperature was grated. For processed meats, a portion size slice (8 cm × 8 cm 250°C; the injection mode was splitless and the sampling × 2 mm) was used. For fish, the flesh was separated from the time 1 minute. .e ion source temperature was 200°C, in- skin and each treated was separately. Approximately 25 g of terface temperature 250°, solvent cut time 4.5 min, and mass each sample was weighed on an analytical balance (Fish- range 33–495. Finally, the ionization voltage was 70 eV. .e erbrand™ Analytical Balances, UK), freeze-dried (Edwards analytes were identified by comparing their mass spectra Freeze Dryer Micro Modulyo, USA), ground (WARING, with those recorded in NIST 147. commercial blender, Aberdeen), and subsequently stored at −20°C until required. 2.5. Statistical Analysis. For the statistical analysis, XLStat (version 2014.5.03, Addinsoft, NY) was used. Significant 2.3. HMF Analysis. .e method used for HMF de- differences between the samples with a confidence interval of termination was that of Fiore et al. [32] with some modi- 95% were performed by using a Tukey test, and for the fication. Briefly, 0.5 g of the ground sample was placed in a correlation between HMF concentration and the percentage 50 mL centrifuge tube (Conical, Foam Rack, Sterile, Blue of volatiles, principal component analysis (PCA) was used. Cap, PP, Fisherbrand, USA). 9 mL of Ultrapure water (UPW) water (with 0.1% formic acid) was added followed by 3. Results and Discussion 0.5 ml of Carrez I (potassium ferrocyanide) and 0.5 ml Carrez II (zinc acetate). .e mixture was homogenised (IKA 3.1. HMF. .e analysis of HMF using HPLC with diode Dispersers T 18 digital ULTRA-TURRAX , USA) for 1 array detection at 280 nm and 313 nm was performed on minute and then centrifuged for 10 minutes® at 4000 rpm at three different groups of smoked products (cheese, pro- 4°C (Jouan CR3.12 Refrigerated Benchtop Centrifuge, USA). cessed meat, and fish). .ere were clear indications of the .e supernatant was collected into a 50 mL centrifuge tube. presence of HMF in all three categories. .e range of Another 5 mL of UPW was added to the precipitate, and the concentrations varied from 4 ppb (metsovone cheese) up to solution was homogenized and centrifuged as above. .is 3000 ppb (mackerel). .e different nature of the samples last procedure was repeated one more time after collecting (cheese, processed meat, and fish) along with other factors the supernatant. From the combined supernatant layers, such as the type of smoking (hot/cold smoking) or use of 1 mL was transferred to an Eppendorf tube (Fisherbrand, smoke condensates and the type of burning matrix (hard- 1.5 mL) and centrifuged for 10 minutes at 14000 rpm 4°C woods, softwoods, and herbs) are considered to be re- (Eppendorf centrifuge 5417R, Canada). .e supernatant was sponsible for the different concentration levels of HMF that collected and passed through a Minisart 0.2 µm syringe filter were identified. (15 mm) into HPLC vials (10 mm Screw, 2 mL, Phenom- enex) and analysed by HPLC with the equipment which 3.1.1. Occurrence of HMF in Cheese Products. In Figure 1, consisted of a .ermo Scientific (Germany) Ultimate 3000 the mean concentrations and standard deviations of HMF in Pump, Dionex DDA-100 diode array detector, and a Dionex the cheese samples are presented. Samples C1 and C2 had autosampler ASI-100 (San Jose, CA). .e mobile phase was the highest mean concentrations (657 ppb and 475 ppb, methanol: UPW (90 :10) at a flow rate of 0.8 mL/min under respectively), while sample C3 had the lowest mean con- isocratic conditions. .e column employed was the Synergy centration of 4 ppb. For samples C4, C5, and C6, the mean 4 μm Hydro-RP 80 A, 25 × 4.6 cm (Phenomenex, USA). .e concentrations ranged from 48 ppb to 88 ppb. C1, C2, and diode array detector was set to read absorbance at 280 nm C3 (German, Dutch, and Greek cheeses, respectively) had and 313 nm. For the calculation of concentration of HMF, a characteristic brown rinds, the source of which can be at- calibration curve was constructed with the HMF range of 20 tributed to the Maillard reaction or the formation of alde- to 1000 ppb (equation y � 0.0029x and R2 � 0.9951). hydes during thermal decomposition [34]. It is interesting to note that sample C3 (metsovone, Greek) had the lowest 2.4. Volatile Compounds Analysis. For the detection of concentration of HMF. Metsovone is a traditional smoked volatile compounds, the method of Guillen and Errecalde Greek cheese of protected designation of origin (PDO). It is [33] was used with modifications. Briefly, 2.00 g of the dry cold smoked for 1–2 days after maturation with natural sample was placed in a SPME vial. .e instrument used was a smoke from burning grasses, leaves, and herbs of the area. QP2010 HS-SPME-GCMS fitted with an AOC-5000 auto- However, samples C1 and C2 (German and Dutch cheeses) sampler (Shimadzu, Tokyo, Japan). .e SPME fibre are smoked with wood as the burning material. .ese dif- employed was a PDMS/DVB from Supelco (UK) and used as ferences in the smoke generation material may be re- follows: 10 min of incubation at 60°C followed by 30 min sponsible for the observed concentrations of HMF. Sample extraction time and 10 min desorption time. A 30 m, C4 (Austrian cheese) was a processed cheese produced by 0.25 mm id, 0.25 µm film thickness Zebron ZB-wax capillary mixing smoked cheddar and smoked butter, but the final column (Phenomenex, USA) was used with the carrier gas product is not smoked; thus, this different process of pro- (He) set at a total flow of 94 mL/min and a column flow of duction might be the cause of the low level of HMF found. 1.78 ml/min. .e initial column oven temperature was 40°C Finally, samples C5 and C6 are Scottish cheddars from the rising to 60°C at 4°C·min−1 (2 min hold) and then up to same producer (Orkney) single and triple smoked, respec- 190°C at 2°C·min−1 and 230°C at 5°C·min−1 (15 min hold) for tively. .e smoke is produced naturally from wood from the 4 Journal of Chemistry

HMF in smoked cheese 800 D 700

600 C 500

400

300 HMF (PPB) 200 B 100 A, B A, B A 0 C1 C2 C3 C4 C5 C6 Cheese samples

Figure 1: HMF concentration in smoked cheeses (Table 1 for sample details). Significant differences were determined by ANOVA analysis and Tukey test (p ≤ 0.05). Different letters indicate significant differences (n � 3). local area and most of it is hardwood. .e lack of the brown HMF concentration than the cheese and processed meat rind may be indicative of the lower amount of HMF samples. Furthermore, in each sample, the flesh had signifi- compared to samples C1 and C2. Between the two samples cantly higher (p < 0.05) HMF concentration than the skin. For (C5 and C6), the triple smoked (C6) had a slightly higher sample F1 (haddock), the flesh (F1-fl) had a mean concen- concentration of HMF possibly due to the additional tration of 1202 ppb, while the skin (F1-sk) had a significantly smoking. .e difficulty of analysing cheese products could lower mean concentration of 835 ppb. Sample F3 (salmon) be related to the difficulty that Morales and Jimenez-P´ erez´ showed corresponding results with a mean value for the flesh [35] faced when analysing infant milk, which is the com- (F3-fl) of 1326 ppb and for the skin (F3-sk) 1011 ppb. ANOVA plexity of compounds in the heat-treated milk and the fact showed the amount of HMF in the flesh samples of F1-fl and that other compounds were reported to coelute with HMF F3-fl had no significant difference (p > 0.05). .e results for during chromatographic analysis. In addition, the heat sample F2 (mackerel) were significantly higher for the flesh (F2 treatment of milk before it is transformed to cheese may fl) at 2930 ppb, while the skin F2-sk had a value that was not affect the HMF concentration as shown in the work of significantly different from F1-sk and F3-sk. For smoked fish Magarinos et al. [36]. products, hot smoking is the method that is most commonly used, unlike cheeses and processed meats which are generally cold smoked. .ere is a difference in the temperatures of these 3.1.2. Occurrence of HMF in Processed Meat Products. smoking processes (hot smoking is generally carried out at For smoked processed meat, the different mean HMF 55° ° ° ° concentrations and standard deviations are presented in C to 80 C and cold smoking at 15 C to 25 C [2, 4]), and this Figure 2. Previous work has shown that HMF production is may be the explanation for the higher HMF concentration either a consequence of the presence of pentoses contrib- found in fish products. In addition, the burning matrix that uting to the Maillard reaction or from the dephosphoryla- was used for sample F3 was beechwood, a hardwood high in tion of ribose phosphate within the meat. Additionally, it has hexoses and pentoses, and the presence of which leads to the been observed that the level of acidity plays an important production of HMF during thermal treatment. It is also in- role in the extent of HMF production [37]. Sample M3 teresting to note that the smoked mackerel (F2) was a ready- (German smoked peppered salami) had the highest mean to-eat hot-smoked product that has undergone thermal ´ HMF concentration (331 ppb). HMF concentrations found treatment equal to cooking. Perez-Palacios et al. [38] rec- in samples M1 and M2 (oak smoked ham, 32 ppb, and ommended that fried fish should be included in high HMF beechwood smoked cooked ham, 46 ppb, respectively) were concentration food groups and that generally the product not significantly different (p ≤ 0.05) from each other and handling and cooking procedure affects HMF levels. It is were considerably lower when compared with the other postulated that similar processes are at work here. meat samples. Samples M4 (German smoked ham, 169 ppb), M5 (Bavarian smoked ham, 171 ppb), and M6 (Italian smoked prosciutto, 180 ppb) showed no significant differ- 3.2. SPME-GC-MS Analysis and Results ence (p ≤ 0.05) between their mean values. 3.2.1. Major Smoking Process-Related Compounds. Principal component analysis (PCA) was used to find a 3.1.3. Occurrence of HMF in Fish. For smoked fish samples, correlation between the concentration of HMF and some a slightly different sampling method was followed. As the selected phenolic compounds. .e presence and amounts of skin of the selected fish was not edible, each sample was these phenolics are an indication of the degree of smoking divided in flesh and skin and analysed as two individual the foods were exposed to and hence may be linked to HMF samples (Figure 3). Fish products in general showed higher levels found. For this study, the phenolic compounds that Journal of Chemistry 5

Table 1: Sample coding for all smoked products. Smoked cheese C1 C2 C3 C4 C5 C6 German smoked Austrian Orkney single-smoked Orkney triple- Dutch smoked cheese Smoked metsovone cheese smoked cheese mature cheddar smoked cheddar Smoked processed meat M1 M2 M3 M4 M5 M6 Oak smoked Beechwood smoked German peppered German smoked Italian smoked Smoked bavarian ham ham cooked ham smoked salami ham prosciutto Smoked fish F1 F2 F3 Smoked Ready-to-eat smoked Hot-smoked salmon haddock fillets mackerel filets sk: skin; fl: flesh

HMF in smoked processed meat 400.0 C 350.0 300.0 250.0 B 200.0 B B 150.0 HMF (PPB) 100.0 A 50.0 A 0.0 M1 M2 M3 M4 M5 M6 Meat samples

Figure 2: HMF concentration in smoked processed meats (Table 1 for sample details). Significant differences were determined by ANOVA analysis and Tukey test (p ≤ 0.05). Different letters indicate significant differences (n � 3).

HMF in smoked ﬁsh 3500 D 3000 2500 2000 1500 C C C B

HMF (PPB) 1000 A 500 0 F1-sk F1-f l F2-sk F3-f l F3-sk F3-f l Fish samples

Figure 3: HMF concentration in smoked fish Table 1 for sample details). Significant differences were determined by ANOVA analysis and Tukey test (p ≤ 0.05). Different letters indicate significant differences (n � 3). were chosen to explore any potential relationships were and C2 (Dutch smoked cheese), and both of these are syringol, eugenol, isoeugenol, guaiacol, p-cresol, and phenol smoked using wood. When these are compared to C3 because they are the major components identified in wood (metsovone smoked cheese) where the amount of phenolics smoke, and previous studies have linked different wood was high, the product had the most intense smoky smell and types and smoking conditions with different concentration flavour, but the HMF concentration was the lowest among of these phenolic compounds [8]. For the cheese samples the samples analysed. Taking into consideration the smoking (Figure 4), a positive correlation was observed between HMF procedure of this traditional smoked cheese (smoked with and syringol and p-cresol for C1 (smoked German cheese) grasses, leaves, and herbs), it can be noted that while burning 6 Journal of Chemistry

6 Phenol

4 C3 Guaiacols 2

C6 0 C1 C2 Syringol

F2 (19.33%) C5 C4 Cresols –2 Eugenol HMF

–4

–6 –8 –6 –4 –2 0 2 4 6 8 10 F1 (74.11%) (a) 4 4 HMF Eugenol 3 3 Guaiacols F1 2 M3 2 M4 1 Cresols 1 Phenol Syringol Cresols 0 0 Phenol M6 –1 M5 F2 F3 F2 (21.16%) –1 Eugenol M2 F2 (31.84%) –2 HMF Guaiacols –2 M1 –3 Syringol –3 –4 –4 –3 –2 –1 0 1 2 3 4 5 –4 –3 –2 –1 0 1 2 3 4 5 6 F1 (47.36%) F1 (68.16%) (b) (c)

Figure 4: Principal component analysis of all smoked samples (PCA): (a) biplot of smoked cheese (axes F1 and F2: 93.44%); (b) biplot of smoked meat (axes F1 and F2: 68.52%); (c) biplot of smoked fish (axes F1 and F2: 100.00%). herbs can also result to the production of phenolics, they do was analysed with high-pressure liquid chromatography- not consist of cellulose and hemicellulose which are found in coupled diode array detector (HPLC-DAD). .e highest wood and particularly associated with HMF formation [39]. HMF concentration was shown in smoked fish samples, while A correlation between HMF and guaiacols and phenol was cheese samples showed the widest range of results. After also in evidence for some meat samples. M3 (German reviewing scientific data regarding different smoking pro- peppered smoked salami) had the highest concentration of cesses such as the smoking temperature, smoking dura- both HMF and these phenolics. Although fish samples in tion, and wood matrix, the variance of HMF concentrations general had the highest HMF values, some of the phenolics was expected due to a variety of influencing factors (syringol, isoeugenol, and phenol) were found in very low [2, 4, 5, 7, 11, 13]. Traditionally, smoked fish is cooked by a hot concentrations, and so no correlation was found between smoking procedure, whereas cheese and processed meat are HMF and phenolics in the fish samples analysed. Many usually cold smoked at lower temperatures. .ere is a cor- factors seem to influence the amounts of phenolics in the relation between these smoking procedures and the level of smoked products. In previous studies of smoked fish, several HMF found with both cheese and processed meat having major phenolic compounds were found for different lower HMF levels. .e fish sample with the highest HMF smoking processes [40, 41]. Another factor that influences concentration was a ready-to-eat mackerel (F2) with a value of the smoky smell and flavour attributed to phenolic com- approximately 3000 ppb. Also, traditionally smoked metso- pounds are the type of wood, the burning temperature, the vone cheese had the most intense smoky flavour and smell but different smoke generation methods, and the fat content of with the lowest HMF concentration (4 ppb). .e range of the food. Lighter smokiness results from softer hardwoods HMF concentration in smoked processed meat products was that burn rapidly at low temperatures while for harder between 30 and 330 ppb (M1 and M3, respectively). hardwoods, the opposite has been observed [42].

4. Conclusion 4.2. GC and Correlation. .e PCA analysis of the potential correlation between HMF concentration and the amount of 4.1. HMF. .e measurement of HMF concentration in selected phenolic compounds discovered in smoked food smoked cheese, smoked processed meat, and smoked fish products as a function of the smoking procedure was Journal of Chemistry 7 conducted. .e characteristic phenolic compounds dis- [8] A. Hitzel, M. Pohlmann,¨ F. Schwagele,¨ K. Speer, and W. Jira, covered by SPME-GC-MS were syringol, guaiacol, eugenol, “Polycyclic aromatic hydrocarbons (PAH) and phenolic isoeugenol, p-cresol, and phenol. .e investigation into a substances in meat products smoked with different types of potential correlation between phenolics compounds and wood and smoking spices,” Food Chemistry, vol. 139, no. 1–4, HMF was based on previous work that found that the pp. 955–962, 2013. abundance of phenolics (mainly from the pyrolysis of lignin [9] K. Abraham, R. Gürtler,K. Berg, G. 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Research Article Separation and Enrichment of Omega 3, 6, and 9 Fatty Acids from the By-Products of Vietnamese Basa Fish Processing using Deep Eutectic Solvent

Thanh Xuan Le Thi ,1,2 Hoai Lam Tran ,3 Thanh Son Cu,4 and Son Lam Ho4

1Dong ap University, Dong ap, Vietnam 2Graduate University of Sciences and Technology (GUST)–Vietnam Academy of Science and Technology (VAST), Vietnam 3Department of Chemical Technology, Ho Chi Minh City University of Food Industry, Ho Chi Minh, Vietnam 4Institute of Applied Materials Science (IAMS) - VAST, Vietnam

Correspondence should be addressed to anh Xuan Le i; [email protected]

Received 11 June 2018; Revised 9 October 2018; Accepted 25 October 2018; Published 2 December 2018

Guest Editor: Photis Papademas

Copyright © 2018 anh Xuan Le i et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Omega 3, 6, and 9 fatty acids were separated and enriched successfully from the by-products of Vietnamese Basa ﬁsh processing by the deep eutectic solvent. e total amounts of omega fatty acids were about 57% in the raw material, and they were amounted to 91% after the ﬁrst separation by DES. e optimal mass ratio is 20 g methyl ester with 200 g methanol and 15–20 g DES. Moreover, the ionic liquid-DES was successfully synthesized with the molar ratio of choline chloride/urea of 1 :1 and 2 :1. e characteristics of DES were determined and demonstrated by FTIR, TGA, DSC, 1H-NMR, and 13C-NMR analysis methods.

1. Introduction Our studies [2] show that the by-product of Basa-fish processing is 59%. us, there will be about 65–70 thousand e scientific name of Basa fish is Pangasius bocourti, tons of waste each year. Of which, 8.4% are fatty acids, a catfish in the family Pangasiidae of high economic value, equivalent to 546.000–588.000 tons. Omega 3, 6, and 9 fatty raised in many countries around the world. is species is acids of by-product are 57%, meaning that 300.000–335.000 native to the Mekong Delta in Vietnam and to the Chao tons of omega 3, 6, and 9 annually are not used properly for Phraya River basin in ailand [1]. is fish is an important its role. food in the international market. In the Mekong Delta, Omega 3, 6, and 9 play an important role and is nec- catfish provide for essential demands over the country, and essary for the human body. e body cannot self synthesis they are exported to other countries. It is promoting the these omegas which can prevent cardiovascular diseases, development of Vietnamese fisheries industry. Average cancer, lower blood pressure, lower cholesterol, and tri- annual production of Vietnamese Basa-catfish from 2013 to glycerides in blood, brain accretion, and cardiac regulation 2017 is from 1.1 to 1.2 million tons. However, the main part and prevent thrombosis [3, 4]. of exporting fish is fillet meat and the other parts such as e production process of omega 3, 6, and 9 has been head, bone, fat, and skin (total ∼60–65%) are still not fully done by many methods as distillation in high temperature to utilized. e by-products of Basa-fish processing including give the useful compounds [5], crystalline at low temperature viscera, skin, scales, bones, and skeletons are becoming in ethanol, acetone or solvent mixtures [6], the supercritical ° a major problem for industries, causing environmental fluid used as carbon dioxide (CO2) at 31.1 C and 72.9 atm to pollution and the loss of large quantities of high-value achieve the all of fatty acids [7, 8]. Silver-coated chroma- nutrients such as omega 3, 6, and 9 unsaturated fatty acids. tography and high-performance chromatography methods 2 Journal of Chemistry may isolate polyunsaturated fatty acids [9–11], using the Table 1: Parts of catfish details. complex of urea is the method to concentrate polyunsaturated No. Products Weight (g) Percentage (%) fatty acids from waste of poultry and fish [12–15]. 1 Fat 106.01 6.057 Deep eutectic solvents (DESs) are now widely acknowl- 2 Fillet meat 615.00 35.142 edged as a new class of ionic liquid analogues due to their 3 By-products 1028.99 58.800 characteristics and properties that are similar to ionic liquids. 4 Error 0.001 DESs show great promise as green solvents because they have the important properties such as biodegradability, low toxicity, and easy preparation [16]. DES was successfully used to 942.80 g of the extracted solution, the fatty acid was not efficiently extract baicalin from Scutellaria baicalensis Georgi found; i.e., there was not fatty acid in this section. by Wang et al. [17]. In this work, we introduce some initial e obtained fatty acids were esterified by methanol with results when using DES to separate and enrich omega 3, 6, and an FA : methanol ratio of 10 :1. e mixture was heated to ° 9 in the fat of Vietnam’s Basa fish processing waste. 60 C and stirred at 120 rpm for 3 hours. Next, the mixture was cooled down to room temperature. en, the ester 2. Materials and Methods products were separated and washed several times. Finally, Na2SO4 was used to remove water from the methyl ester of 2.1. Materials. e ionic liquid-DES is synthesized from the FA. e final products were labeled “methyl esters.” e choline chloride and urea that were supported by Acros chemical compositions of methyl esters were analyzed by the Organics Company. GC-FID method. A volume 1750 g of Basa fish was processed according to the exporting fish processing method. e average per- 2.3.2. Omega 3, 6, and 9 Separation and Enrichments from centages of the fat, fillet meat, and by-products are shown in Methyl Esters by DES. Methyl ester, methanol, and DES with Table 1. In which, the by-product was dominant (58.8%) and a ratio of 20 (g) : 200 (g) : m (g) were added to a 3-stage vial was used to separate and enrich the omega 3, 6, and 9. equipped with a backwash, heating element, and stirrer at 120 rpm and heated in a temperature of 45°C for 1 hour. e obtained homogeneous mixture was allowed to cool down to 2.2. Synthesis of the Ionic Liquid-DES 4°C and then cooled at 4°C for 8 hours. e resulting mixture 2.2.1. Sample Preparation. e general procedure to syn- was divided into two layers: the upper layer was liquid and thesize ionic liquid-DES based on choline chloride and urea the bottom layer was the crystal solid. e crystal part was [18, 19] as follows: choline chloride and urea are mixed to- washed with cold methanol. e washed methanol was gether at a molar ratio of 1 :1 (and 2 :1) in a flask fitted with added into the liquid part above, then was evaporated methanol in a vacuum evaporator, dried by Na SO , and a heating element and magnetic stirrer. e mixture was 2 4 analyzed the chemical compositions. heated to 60°C for 5 to 7 minutes until it becomes homo- e crystal solid was added with 200 ml H O, boiled, and geneous. Cooling at room temperature to prevent the crys- 2 stirred until they dissolved and formed two layers. e upper tallization occurs. When changing the molar ratio between layer contained the fatty acids. ese FAs were separated and choline chloride and urea to 1 : 2 and performing the same as described above, then cooling the reaction mixture down to then removed water by Na2SO4. Finally, the obtained fatty room temperature, the mixture rapidly crystallizes into solids. acids were analyzed for the chemical compositions by GC/FID method. e lower layer contained methanol, water, and DES. e methanol and water were removed by 2.2.2. DES’s Characteristics. e used analysis methods the evaporation in vacuum. DES rechecked the physical include FTIR on EQUINOX 55 (Bruker), frequency from properties for reusable purpose. In this work, the mass of 400 to 4000 cm−1, KBr template, the NMR technology by DES has been changed from 5 g to 10, 15, 20, 25, and 30 g. Brucker AC 500, and TGA-DSC by LABSYS evo SETARAM equipments. Viscosity is determined by DV III Ultra 2.4. Determination of Fatty Acids. e fatty acids were ob- (Brookfield). Water concentration is determined by tained from the by-products, and the unsaturated fatty acids SCHOTT Instruments TitroLine KF trace. by DES were analyzed by GC-FID method (GC-Agilent 6890N, flame ionization detector (FID), HP-INOWAX × × 2.3. Omega 3, 6, and 9 Separation and Enrichment GC column: 30 m 0.25 mm 0.25 μm, the temperature range of 159–245°C; nitrogen). 2.3.1. Sample Preparation. e by-product was added with 2 liters of water and boiled for 30 minutes. e mixture was 3. Results and Discussion allowed to cool down to 4°C. e mixture was separated into 3.1. Synthesis Ionic Liquid-DES two parts after cooling. e above part is a white grease layer that was separated and weighed 86.10 g. e rest was filtered, 3.2.1. Synthesis DES Base on Choline Chloride and Urea. dried at 45–50°C in vacuum, and weighed 942.80 g. e fatty Both choline chloride and urea are soluble in water. e acid was extracted from both above parts by methanol and melting points of choline chloride and urea were at 302°C n-hexane. In 86.6 g of the grease, the obtained result was and 133°C, respectively. However, their mixture becomes 75.175 g of fatty acid, corresponding to a yield of 87.3%. In a homogeneous solution as increasing the temperature to Journal of Chemistry 3

60–75°C. e products of reaction depend on the mass ratio In addition, two urea molecules can combine to form of choline chloride and urea. Moreover, some by-products biurea [20]: such as ammonium cyanide, NH , HOCN, and biurea can be 3 H N formed at reaction temperature, as below: 2 2 CO H2NCNH C NH2 + NH3 H2N – + T °C H2N CO NCO NH4 O O

H2N (3) (1) erefore, the reaction temperature and mass ratios of Ammonium cyanide is destroyed, and NH3 will be choline chloride and urea must be selected to prevent the formed as follows: formation of by-product. – + NC O NH NC OH + NH 4 3 (1) Choline Chloride : Urea (1 :1) Solution. (2)

CH3 CH3 + [CO(NH ) ] + – 2 2 + – CH N CH CH OH Cl 3 2 2 CH3 N CH CH2 OCN ( ) CH 4 3 CH3

+ NH4Cl + H2O

(2) Choline Chloride : Urea (2 :1) Solution.

CH3 CH3 + [CO(NH ) ] + – 2 2 + – 2 CH3 N CH2 CH2 OH Cl CH3 N CH CH2 OCN

CH3 CH3 (5) CH3 + – + NH4Cl + 2H2O CH3 N CH CH2 Cl

CH3

By-products in both cases are water, ammonium chlo- this mix will melt at low temperature and does not crystallize ride, and ammonium hydroxide. ese can be removed by at room temperature (32–33°C). heating in vacuum at low temperature (<60°C). Anionic NCO− and cation choline combine by weak static bond, so (3) Choline Chloride : Urea (1 : 2) Solution.

CH3 CH3 + [CO(NH ) ] + – 2 2 + – CH N CH 3 2 CH2 OH Cl CH3 N CH CH2 OCN (6) CH3 CH3

+ HO C N + NH4Cl + NH4OH 4 Journal of Chemistry

Table 2: Physical index of DES solution at 30°C. Choline chloride : urea ratio No. Index 1 :1 ratio 2 :1 ratio 1 pH 6.65 5.92 2 Density (g/ml) 1.197 1.167 3 Viscosity (cP) 61.5 57.3 4 Electronic conductivity (mS) 1.64 2.69 5 Water concentration (%) 0.65 0.81

(a)

(b)

(c) Transmittance (%) 1661.19 1607.64 1438.79 952.43 865.14 783.35 629.28 582.83 554.43 505.86 457.68 430.94 418.21 2966.11 1329.19 1281.80 1235.46 1161.79 1135.60 1082.56 1054.91 1004.21 3325.05 3194.28

4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm–1) Figure 1: FTIR of DES solutions were synthesized with the choline chloride/urea molar ratios: a-(1 :1), b-(2 :1), c-(1 : 2).

With 1 : 2 ratio of choline chloride : urea, the by-products as diagram (1) and to form new product with different include by-products in case of 1 :1 ratio and isocyanic acid. electronic conductivities. e conductivity of urea in water is is is the factor which changes products’ comeback initial 0.01 mS, while the conductivity of choline chloride in water state and crystallize at room temperature. is 5.6 mS. When creating new products, the conductivity Based on this initial survey, we choose DES solution with decreases considerably, where a 2 :1 solution has a greater 1 :1 and 2 :1 ratio of choline chloride : urea to continue electrical conductivity, it is explained by the excess choline optimization to determine the best ratio. chloride content, which results in higher conductivity of the solution. is result confirms that choline chloride is chemically related to urea according to the reaction scheme 3.2.2. Physical Properties of DES Solution. Physical prop- (1) and produces a new product with conductivity different erties of DES solution with choline chloride : urea ratio in 1 : from that of the original. 1 and 2 :1 are shown in Table 2. Electronic conductivity of urea in water is 0.01 mS, while 3.2.3. Analysis and Identification electronic conductivity of choline chloride in water is 5.6 mS. After the reaction, electronic conductivity of solution de- (1) FTIR Analysis. Figure 1 shows the FTIR spectra of DES creases clearly. In case of 2 :1 ratio, the higher value of solutions that were synthesized with the molar ratio electronic conductivity can be explained by the remaining of choline chloride/urea: a-(1 :1), b-(2 :1), c-(1 : 2). In quality of choline chloride after reaction. is result con- FTIR of solutions (a) and (b), vibrational frequencies at tributes in confirming the bond of choline chloride and urea 3230–3550 cm−1 and 3445.63 cm−1 can be attributed to the Journal of Chemistry 5

CCU−MeOD−1H 50 5.732 4.810 4.047 4.042 4.036 4.032 4.027 4.022 4.016 3.548 3.542 3.538 3.535 3.529 3.336 3.333 3.330 3.326 3.323 3.253 0 (a)

–50 (b) –100

–150 Weight (%) (c) –200

–250

–300 50 100 150 200 250 300 350 400 450 500 550 600

Temperature (°C) 9 8 7 6 5 4 3 2 1 0 ppm Figure 2: TGA of (a) choline chloride, (b) urea, and (c) choline chloride : urea 1 :1. (a) CCU−MeOD−C13CPD 163.502 69.030 69.008 57.083 54.775 54.743 54.711 49.512 49.342 49.170 49.001 48.831 48.660 48.490 20 Exo 10 (c) 0 –10 –20 (b) –30 –40

Heatflow ( µ V) –50 –60 –70 (a) –80 50 100 150 200 250 300 350 400 450 500 550 600 Temperature (°C) 200 180 160 140 120 100 80 60 40 20 ppm Figure 3: DSC of (a) choline chloride, (b) urea, and (c) choline (b) chloride : urea (1 :1). Figure 4: NMR spectrum of DES (1 :1): 1H NMR (a) and 13C NMR (b). vibration of the hydrogen bond between OH groups with average intensity. e vibrational frequencies at 1054.91 cm−1 positions are the little strong vibration of C-N 3180–3220 cm−1 and 3194.28 cm−1 are the frequencies of in choline chloride. N-H in the amide group of urea. e vibrational frequencies e FTIR spectra of solutions show unclearly because of at 1660–1705 cm−1 and 1661.19 cm−1 are the frequencies of the crystallization. Results of chemical-physical properties C�O in urea. And the frequencies of 1590–1620 cm−1 and and FTIR show that both solutions are the good ionic 1607.64 cm−1 indicate the average vibration of N-H in the liquids. amide group that combines with vibration of C-N in urea. In 2800–3300 cm−1, the longer vibration of N-H bond in (2) TGA and DSC Analysis of DES (1 :1). e clear diﬀerence amide of urea and O-H shows that the amide group of urea between thermal gravity of choline chloride, urea, and bond to O-H of choline. e range from 2700 to 3000 cm−1 product mixes is displayed in Figure 2. In 350–400°C range, and 2966.11 indicates the strong vibration of –NH2. e choline chloride (a) and urea (b) are both completely range from 2280 to 3380 cm−1 is the average vibration of decomposed. But in their mix, at 250°C, there is fast + N-H bond in –NH3 . changing mass including increasing and decreasing mass. Especially, in the range of 2100–2225 cm−1, 2158.95 cm−1 After that, their mix’s mass decreased slowly to 80°C. is contributes to vibration of N≡C-O− (cyanide ionic). Vi- demonstrates that between choline chloride and urea, there bration at 629.28 cm−1 is also the strong vibration of NCO−. is bonding, not to mechanical mixing. In 1150–1210 cm−1, 1161.79 cm−1, C-N bond vibrates with Similar to TGA analysis, DSC result of choline chloride, average intensity. e 1135.60 cm−1, 1082.56 cm−1, and urea, and their products is also clearly diﬀerent (Figure 3). 6 Journal of Chemistry

FID1 B, back signal (C:\VN01PCC00060\2016\04\Fatty acid\06\06-04-2016 2016-04-06 22-25-01\16040571.D)

600 Area: 3853.43

500

400 19.651-C 16:0 (palmitic acid) 23.947-C 18:1 n9 (cis-oleic acid)

pA 300 23.207-C 18:0 (stearic acid)

200 20.333-C 16:1 (palmitoleic acid) Area: 549.665

25.044-C 18:2 n6 ( cis -linoleic acid) Area: 421.115 26.527 - C 18:3 (gamma-linoleic acid) 16.414-C 14:0 (myristic acid) 100 34.970-C 22:6 ( cis -4,7,10,13,16,19-docosahexaenoic acid) 31.960-C 20:5n3 ( cis -5,8,11,14,17-eicosapentaenoic acid) 30.606-C 20:3n3 (cis-11,14,17-eicosatrienoic acid) 17.927-C 15:0 (pentadecanoic acid) 21.298-C 17:0 (margaric acid) 27.612-C 20:1 (eicosenoic acid) 33.621-C 24:0 (lignoceric acid) 30.037-C 20:3n6 ( cis -8,11,14-Eicosatrienoic acid) 30.740-C 20:4 (arachidonic acid)ARA 25.832-C 18:3 (alpha-linolenic acid) ALA 29.074-C 21:0 (heneicosanoic acid) 26.843-C 20:0 (arachidic acid)

23.557-C 18:1 ( trans ) (elaidic acid) Area: 22.9047 17.246-C 14:1(myristoleic acid) 15.091-C 13:0 (tridecanoic acid) 13.863-C 12:0 (lauric acid) Area: 7.76306 0

5 10 15 20 25 30 35 min (a) FID1 B, back signal (C:\VN01PCC00060\2016\04\Fatty acid\06\06-04-2016 2016-04-06 22-25-01\16040569.D)

700

Area: 5646.15

600

500 19.671-C 16:0 (palmitic acid) 23.990-C 18:1 n9 ( cis -oleic acid) 400 pA

300

20.345-C 16:1 (palmitoleic acid) Area: 782.103 23.242-C 18:0 (stearic acid) 200 16.412-C 14:0 (myristic acid) 25.061-C 18:2n6 ( cis -linoleic acid)

Area: 612.57626.540 - C 18:3 (gamma-linoleic acid)

100 31.965-C 20:5n3( cis -5,8,11,14,17-eicosapentaenoic acid) 34.975-C 22:6 ( cis -4,7,10,13,16,19-docosahexaenoic acid) 30.610-C 20:3n3 ( cis -11,14,17-eicosatrienoic acid) 17.925-C 15:0 (pentadecanoic acid) 27.623-C 20:1 (eicosenoic acid) 21.300-C 17:0 (margaric acid) 17.171-C 14:1 (myristoleic acid) 30.043-C 20:3n6 ( cis -8,11,14-Eicosatrienoic acid) 30.744-C 20:4 (arachidonic acid) ARA 33.622-C 24:0 (lignoceric acid) 25.835-C 18:3 (alpha-linolenic acid) ALA 29.081-C 21:0 (heneicosanoic acid) 18.744-C 15:1 ( cis -10-pentadecenoic acid) 34.130-C 24:1 (nervonic acid)

Area:26.858-C 20:0 (arachidic acid) 45.3455 23.603-C 18:1 ( trans ) (elaidic acid) 15.088-C 13:0 (tridecanoic acid) 13.861-C 12:0 (lauric acid) 0

5 10 15 20 25 30 35 min

(b)

Figure 5: Chromatogram of GC/FID analysis of the fatty acids in methyl ester (a) and in methyl ester-DES (b).

° 1 Mix’s crystallization temperature is about 300 C, while shown as follows: (a) H NMR (CDCl3, 500 MHz): 3.2 (1H), choline chloride crystallizes at 200°C and urea crystallizes at 3.3 (5H), 3.5 (5H), 5.4 (7H), 5.8 (1H), 5.7 (1H) and (b) 13C ° 100, 250, and 400 C. NMR (CDCl3, 125 MHz): 58.1, 59.5, 63.0, 63.0, 118, and 163.5.

(3) NMR Analysis of DES (1 :1). Figure 4 shows the NMR 3.2. Determination of Fatty Acid in Samples. e composi- spectrum of DES solution that was synthesized with the tion of the crude materials was determined by GC/FID molar ratio of choline chloride/urea: (1 :1). Results are method (Figure 5(a)) and is shown in Table 3. Journal of Chemistry 7

Table 3: e chemical compositions of fatty acids before separation and enrichment by DES. No. Fatty acids Raw methanol extract (%) Methyl ester (%) 1 Saturated fatty acids 31.37 35.58 2 Unsaturated fatty acid 2.85 3.35 3 Omega 3 2.00 1.84 4 Omega 6 16.44 14.48 5 Omega 9 40.71 40.63 6 Undeﬁned 6.63 4.12 Total 100.00 100.00

Table 4: Classification of saturated fatty acids, unsaturated fatty acids, and omega in methyl ester. Classification Name of compounds Percentage (%) Myristic acid (C14 : 0) 1.96 Palmitic acid (C16 : 0) 26.55 SFA Stearic acid (C18 : 0) 6.78 Arachidic acid (C20 : 0) 0.29 USFA Palmitoleic acid (C16 :1) 3.35 cis-5,8,11,14,17-Eicosapentaenoic acid (C20 : 5n3) 0.42 (EPA) α-Linolenic acid (C18 : 3n3) (ALA) 0.46 OM-3 cis-11,14,17-Eicosatrienoic acid (C20 : 3n3) 0.15 Nervonic acid (C24 :1n9) + cis-4,7,10,13,16,19- 0.63 docosahexaenoic acid (C22 : 6n3) (DHA) Linoleic acid (C18 : 2n6c) (LA) 12.41 c-Linolenic acid (C18 : 3n6) (GLA) 1.05 OM-6 cis-8,11,14-Eicosatrienoic acid (C20 : 3n6) 0.18 Arachidonic acid (C20 : 4n6) (AA) 0.48 Oleic acid (C18 :1n9c) 40.21 OM-9 cis-11-Eicosenoic acid (C20 :1n9) 0.42 Undefined 4.11 Total 100.00

e chemical compositions of methyl esters were not e omega fatty acid compositions were determined by significantly different to one’s before esterification reaction. the GC/FID method (Figure 5(b)) and are shown in Table 7 e total content of omega 3, 6, and 9 in the raw materials is and Figure 7. It is that although the separation efficiency is 57%; 39% are non-omega 3, 6, and 9 fatty acids; 4% are low, the purity of omega 3, 6, and 9 was high. e content of unidentified substances. Omega 3, 6, and 9 and non-omega omega FA was reached from 83% to 91% for the first sep- are identified in Table 4 and Figure 5(a). aration, and the optimal mass of DES was in a range from 15 to 20 g/20 g of methyl esters (Table 7 and Figure 7(a)). Eighty percent FA of methyl esters is combined with 3.3. Separation and Enrichment of Omega 3, 6, and 9 by DES. DES to form the crystal solid, as shown in Section 2.3. In In this work, we use DES with a choline chloride/urea ratio this mixture, there are mainly SFA and UFA. Omega FAs of 1 :1 to separate and enrich omega 3, 6, and 9 because it has were accounted for only 20–30% as shown in Table 8 and a pH of nearly 7, small electronic conductivity (1.64), and Figure 7(b). water low content (0.65%). ese properties will not cause e FA separation process from the crystal solid was also side effects with multiple coupling compounds in the carried out with DES; however, the omega fatty acids were sample. Figure 6 illustrates the process of enriching omega not obtained. is result indicated that DES was not capable from the sample. of separating omega FA from the non-omega FA when the Effectiveness of process delamination during the par- omega FA/non-omega FA ratio was less than 1. ticipation of DES is shown in Table 5. e performance of omega FA separation from the 4. Conclusions liquid layer varies from 12% to 19.5%, depending on the mass of DES (5–30 g per 20 g of methyl ester). It changes e ionic liquid-deep eutectic solvent was successfully insignificantly when increasing the mass of DES. For omega synthesized with the molar ratio of choline chloride/urea of 3, 6, and 9, the separation efficiency is between 17% and 29%, 1 :1 and 2 :1. e characteristics of DES were determined depending on the mass of DES. is performance can reach and demonstrated by FTIR, TGA, DSC, 1H-NMR, and 13C- over 50% for omega 3 and 6 as shown in Table 6. NMR analysis methods. DES’s physical properties were 8 Journal of Chemistry

(a) (b) (c)

(d) (e) (f)

Figure 6: (a) DES and methanol extract; (b) DES, methanol extract, and methyl ester after stirring and heating; (c) mixture after 8 hours of cooling; (d) omega-rich fatty acids in the liquid layer; (e) crystal solid under the microscope; (f) fatty acids separated from crystal solid.

Table 5: e dependence of the liquid/crystal solid ratios on mass of DES. Mass of DES (g) Liquid (g) Crystal solid (g) Loss (g) 5 2.40 17.52 0.08 10 2.87 17.08 0.05 15 3.30 16.60 0.10 20 3.60 16.33 0.07 25 3.82 16.09 0.09 30 3.90 16.00 0.10

Table 6: e efficiency of omega FA separation from the liquid layer. Mass of DES (g)/20 g methyl ester Mass of fatty acids 0 5 10 15 20 25 30 Omega 3, 6, and 9 FA (g) 11.39 1.99 2.42 3.00 3.25 3.30 3.34 Efficiency (%) — 17.47 21.06 26.33 28.53 28.97 29.32 Omega 3 and 6 FA (g) 3.26 1.12 0.89 1.22 1.74 1.21 1.54 Efficiency (%) — 34.00 27.30 37.42 53.37 37.12 47.24

Table 7: Fatty acid compositions of the liquid layer. Mass of DES (g) SFA (%) UFA (%) Omega 3, 6, and 9 (%) Undeﬁned (%) Total (%) 5 11.76 2.90 82.97 2.37 100 10 7.46 3.14 84.35 5.05 100 15 3.30 2.37 91.03 3.30 100 20 2.99 2.64 90.30 4.34 100 25 8.22 2.17 86.60 3.01 100 30 6.66 3.70 85.52 4.12 100 Journal of Chemistry 9

100 100

80 80

60 60

40 40

Percentage of acids (%) 20 20 Percentage of fatty acids (%)

0 0 –5 0 5 10 15 20 25 30 35 –5 0 5 10 15 20 25 30 35 Content of DES (g) Content of DES (g) Omega fatty acids Non-omega fatty acids Non-omega fatty acids Omega fatty acids

(a) (b)

Figure 7: e dependence of omega FA and non-omega FA in the content of DES: (a) in liquid layer and (b) in crystal solid.

Table 8: Fatty acid compositions of crystal solid. Mass of DES (g) SFA (%) UFA (%) Omega 3, 6, and 9 (%) Undefined (%) Total (%) 5 60.45 13.80 25.32 0.43 100 10 62.22 15.07 22.40 0.36 100 15 65.73 15.12 18.57 0.58 100 20 63.75 14.68 20.08 0.49 100 25 55.43 12.41 31.64 0.52 100 30 54.62 14.30 30.51 0.57 100 determined: the electronic conductivity of DES is 1.64 mS References and 2.69 mS and the viscosity is 61.5 cP and 57.3 cP, corresponding to the molar ratio of choline chloride/urea of 1 :1 [1] T. R. Roberts and C. Vidthayanon, “Systematic revision of the and 2 :1. Asian catfish family Pangasiidae, with biological observations DES with a choline chloride/urea ratio of 1 :1 was used to and descriptions of three new species,” Proceedings of Academy of Natural Sciences of Philadelphia, vol. 143, pp. 97–144, 1991. enrich omega 3, 6, and 9 because it has a pH of nearly 7, [2] T. X. Le i, “Survey composition and content of omega-3,6,9, small electronic conductivity (1.64), and water low content extracted from catfish at Mekong delta Vietnam by extraction (0.65%). e results show that the percentage of omega 3, 6, method with the traditional solvents,” Vietnam Journal of and 9 was increased from 57% in raw materials to 91% for Chemistry, vol. 55, no. 5e34, pp. 551–556, 2017. the first separation. e optimal mass ratio is 20 g methyl [3] R. Kapoor and U. Patil, “Importance and production of acetate with 200 g methanol and 15–20 g DES. omega-3 fatty acids from natural sources,” International Food DES was only used to separate and enrich the omega FA Research Journal, vol. 18, pp. 491–497, 2011. when the omega FA/non-omega FA ratio is greater than 1. [4] C. M. Yates, P. C. Calder, and G. Rainger, “Pharmacology and therapeutics of omega-3 polyunsaturated fatty acids in Data Availability chronic inflammatorydisease,” Pharmacology and erapeu- tics, vol. 141, pp. 272–282, 2014. e data used to support the findings of this study are [5] D. Patil and A. Nag, “Production of PUFA concentrates from available from the corresponding author upon request. poultry and fish processing waste,” Journal of American Oil Chemists’ Society, vol. 88, no. 4, pp. 589–593, 2011. [6] L. Vazquez´ and C. C. Akoh, “Enrichment of stearidonic acid Conflicts of Interest in modified soybean oil by low temperature crystallization,” Food Chemistry, vol. 130, no. 1, pp. 147–155, 2012. e authors declare that they have no conflicts of interest. [7] F. Sahena, I. S. M. Zaidul, S. A. Jinap et al., “Fatty acid compositions of fish oil extracted from different parts of Acknowledgments Indian mackerel (Rastrelliger kanagurta) using various techniques of supercritical CO2 extraction,” Food Chemistry, e authors wish to thank the Ministry of Education and vol. 120, no. 3, pp. 879–885, 2010. Training of Vietnam, B2017.SPD.04 Project, for their fi- [8] S. Tang, C. Qin, H. Wang et al., “Study on supercritical ex- nancial support of this work. traction of lipids and enrichment of DHA from oil-rich 10 Journal of Chemistry

microalgae,” Journal of Supercritical Fluids, vol. 57, no. 1, pp. 44–49, 2011. [9] J. Guo, C. Wang, Z. Wu et al., “Purification of essential linoleic acid from pinus armandi franch seed oil by silver-silica gel chromatography column,” in Proceedings of the 4th In- ternational Conference on Bioinformatics and Biomedical Engineering (ICBBE), vol. 1–4, Chengdu, China, June 2010. [10] J. T. Dillon, J. C. Aponte, R. Tarozo, and Y. Huang, “Puri- fication of omega-3 polyunsaturated fatty acids from fish oil using silver-thiolate chromatographic material and high performance liquid chromatography,” Journal of Chroma- tography A, vol. 1312, pp. 18–25, 2013. [11] P. Fagan and C. Wijesundera, “Rapid isolation of omega-3 long-chain polyunsaturated fatty acids using monolithic high performance liquid chromatography columns,” Journal of Separation Science, vol. 36, no. 11, pp. 1743–1752, 2013. [12] M. Wu, H. Ding, S. Wang, and S. Xu, “Optimizing conditions for the purification of linoleic acid from sunflower oil by urea complex fractionation,” Journal of American Oil Chemists’ Society, vol. 85, no. 7, pp. 677–684, 2008. [13] L. Z. Cheong, Z. Guo, Z. Yang et al., “Extraction and enrichment of n-3 polyunsaturatedfatty acids and ethyl esters through reversible π–π complexation with aromatic rings containing ionic liquids,” Journal of Agricultural and Food Chemistry, vol. 59, no. 16, pp. 8961–896, 2011. [14] V. Crexi, M. Monte, and L. A. Pinto, “Polyunsaturated fatty acid concentrates of carp oil: chemical hydrolysis and urea complexation,” Journal of American Oil Chemists’ Society, vol. 89, no. 2, pp. 329–334, 2012. [15] C. Fie, J. Salimon, and M. Said, “Optimisation of urea complexation by Box-Behnken design,” SainsMalays, vol. 39, pp. 795–803, 2010. [16] E. L. Smith, A. P. Abbott, and K. S. Ryder, “Deep eutectic solvents (DESs) and their applications,” Chemical Reviews, vol. 114, no. 21, pp. 11060–11082, 2014. [17] H. Wang, X. Ma, Q. Cheng, X. Xi, and L. Zhang, “Deep eutectic solvent-based microwave-assisted extraction of baicalin from Scutellaria baicalensis Georgi,” Journal of Chem- istry, vol. 2018, Article ID 9579872, 10 pages, 2018. [18] A. Florea, A. Petica, L. Anicai, and T. Visan, “Formation from choline chloride based ionic liquids,” UPB Scientific Bulletin, Series B, vol. 72, no. 2, pp. 115–126, 2010. [19] A. P. Abbott, S. Nandhra, S. Postlethwaite, E. L. Smith, and K. S. Ryder, “Electroless deposition of metallic silver from a choline chloride-based ionic liquid: a study using acoustic impedance spectroscopy, SEM and atomic force microscopy,” Physical Chemistry Chemical Physics, vol. 9, no. 28, pp. 3735–3743, 2007. [20] G. Wiedemann, “Biuret decomposition product of urea,” Justus Liebig’s Annalen der Chemie, vol. 68, no. 3, pp. 323–326, 2006. Hindawi Journal of Chemistry Volume 2018, Article ID 2160958, 14 pages https://doi.org/10.1155/2018/2160958

Research Article Critical Effects of Smoking Parameters on the Levels of Polycyclic Aromatic Hydrocarbons in Traditionally Smoked Fish and Meat Products in Finland

Mirja Hokkanen ,1 Ulla Luhtasela,2 Pirkko Kostamo,2 Tiina Ritvanen,1 Kimmo Peltonen,1 and Marika Jestoi2

1Finnish Food Safety Authority Evira, Research and Laboratory Services Department, Helsinki FI-00790, Finland 2Finnish Food Safety Authority Evira, Food Safety Department, Helsinki FI-00790, Finland

Correspondence should be addressed to Mirja Hokkanen; mirja.hokkanen@evira.ﬁ

Received 8 June 2018; Revised 7 August 2018; Accepted 3 September 2018; Published 10 October 2018

Guest Editor: Alberto Fiore

Copyright © 2018 Mirja Hokkanen et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Eighty fish products and 62 meat products were sampled and analysed in Finland, in the year of 2012 for four marker polycyclic aromatic hydrocarbons (PAH4) with an accredited gas chromatography-tandem mass spectrometry method. In general, the determined PAH4 levels were relatively low and below the maximum levels. ,e mean concentrations of smoked fish samples were 0.7 μg·kg−1 for benzo[a] pyrene and 3.9 μg·kg−1 for the PAH4 sum, whereas in smoked meat samples, mean benzo[a]pyrene and PAH4 sum levels were 2.2 μg·kg−1 and 11 μg·kg−1, respectively. However, PAH4 sum concentrations ranged from not detected to 200 µg·kg−1 particularly among meat products, underlining the importance of controlling the smoking process. In this study, the effect of selected smoking parameters, i.e., smoking technique (direct/indirect), smoking time (less than five hours/more than five hours), smoke generation temperature (optimised/nonoptimised), and the distance (less than five metres/more than five metres) between the food and the smoke source, confirmed the linkage between the smoking factors and the PAH4 levels formed in fish and meat products. As guidance for a safe smoking process, it was demonstrated that an indirect smoking technique, a shorter smoking time, an optimised smoke generation temperature, and a longer distance from the smoke source generated lower PAH concentrations in food products. However, while a shorter smoking time generated lower PAH levels in meat products, the levels in fish products were unexpectedly higher than in those smoked for a longer time. Other factors, such as the smoking type (cold smoking/warm or hot smoking) and the fish size, may have affected this result.

1. Introduction have teratogenic, haematological, and immunotoxic effects, and their concentrations in food should therefore be as low Polycyclic aromatic hydrocarbons (PAHs) consist of a ver- as reasonably achievable (ALARA principle) [6–8]. satile group of organic compounds that have at least two or PAHs are formed during the incomplete combustion of more aromatic rings joint together [1, 2]. ,ey are fat soluble organic matter, and they are widely distributed in the envi- and chemically stable compounds that are classified as ronment via air [6, 7]. Industry, traffic, smoking, forest fires, human carcinogens [1]. Several metabolic pathways may and volcanic eruptions generate PAHs, and humans are result in reactive intermediates inducing mutagenic or consequently mainly exposed by inhalation, skin contact, and carcinogenic processes of PAHs [3]. ,e carcinogenic ca- ingestion [6, 7]. Despite also being environmental contami- pacity varies between them, despite having similar structural nants, PAHs are formed in food processing, such as drying, properties [4]. ,ose with four to six fused rings, such as grilling, roasting, and smoking [6, 7, 9]. For nonsmokers, the benzo[a]pyrene (BaP), are effective carcinogens belonging to diet appears to be the main source of PAH exposure [9, 10]. Group 1 carcinogens according to the International Agency Food smoking is one of the oldest preservation methods for Research on Cancer (IARC) [5]. Additionally, PAHs and is still widely used [11–14]. However, smoking is 2 Journal of Chemistry nowadays mainly used to obtain the desired colour, flavour, fish/fishery products and 62 meat/meat products) throughout aroma, and appearance in the smoked food rather than for Finland from smokehouses and retail shops with their own preservation purposes [14, 15]. Traditional smoking is generally smoking chambers. Among the meat and meat products, performed by the formation of smoke from wood [16, 17]. different types of pork products were the most common Smoke is defined as the result of thermal pyrolysis of wood when (68%) (Figure 1(a)), and for the fish and fish products, either access to oxygen is limited [6]. PAHs and other chemical warm or cold smoked salmon products were predominant compounds occur in smoke particles, which can migrate into the (82%) (Figure 1(b)). Sampling was conducted according food product being smoked [2, 18]. Wood smoke contains to Commission Regulation (EC) No. 333/2007 [24] and a combination of antioxidant and antimicrobial chemicals followed a risk-based sampling plan (e.g., the smoking (e.g., phenols, carboxylic acids, aldehydes, and acetic acids), but technique used and production volumes). Only so-called also some harmful compounds, such as PAHs [12, 17, 19]. PAHs worst-case samples from the consumer perspective, which are potential health hazards associated with smoked foods, in were most likely to contain PAH compounds, were chosen which they typically occur as a complex mixture [6, 13]. In for chemical analysis. ,erefore, liquid-smoked samples, for Finland, smoking with direct and indirect techniques is widely instance, were not included. During the sampling, detailed used in the processing of meat and fish products. For direct information on the production process was recorded. smoking, smoke is generated from an open fire in the same However, incompletely reported smoking parameters were chamber as the smoked product, whereas in indirect smoking, omitted. the smoke is generated in an external chamber separated from the food and the smoke is led to the product from the external 2.2. Chemicals and Reagents. Organic solvents (dichloro- smoke generator [16, 20]. Alongside the smoking technique, the methane, hexane, acetone, cyclohexane, methanol, and type of process (grilling, roasting, smoking, and drying), the toluene) were HPLC or ultra resi-grade and purchased from distance between the food and the smoke source, the process J. T. Baker (Deventer, Netherlands). Ethyl acetate (HPLC time, and temperature impact the formed PAH levels [6]. grade) was obtained from VWR Chemicals (Fontenay-sous- Commission Regulation (EC) No. 1881/2006 specifies Bois, France). Ethanol was provided by Altia Oyj (Rajamaki,¨ the maximum levels (MLs) of BaP in different foodstuffs Finland) and was of Aa grade (≥99.5%). Celite 545 and [21]. In 2008, the European Food Safety Authority (EFSA) florisil (100–200 mesh) used for accelerated solvent extraction concluded that BaP alone is not a suitable indicator for the (ASE 200 , Dionex Corporation) were obtained from Merck occurrence and toxicity of PAHs in foods [3]. ,erefore, ad- (Darmstadt,® Germany) and from Sigma-Aldrich (Steinheim, ditional MLs for the sum of four PAHs (PAH4: BaP, benz[a] Germany), respectively. Supelclean ENVI-Chrom P (6 mL, anthracene (BaA), chrysene (CHR), and benzo[b]fluoranthene 500 mg) solid-phase extraction cartridges were provided (BbFA)) were set in Commission Regulation (EU) No. by Supelco (Bellefonte, PA, USA). ,e analytical standard 835/2011 [22]. ,e MLs for smoked meat and fish products mixture (PAH-Mix 183, 10 µg·mL−1) was purchased from smoked from 1.9.2012 to 31.8.2014 are 5.0 µg·kg−1 for BaP Dr. Ehrenstorfer GmbH (Augsburg, Germany). In addition, and 30.0 µg·kg−1 for the PAH4 sum. ,e MLs applicable the isotopically labelled compounds (13C6 BaA, 13C6 CHR, from 1.9.2014 for smoked meat and fish products are 13C6 BbFA, and 13C4 BaP) containing 100 µg·mL−1 each 2.0 µg·kg−1 for BaP and 12.0 µg·kg−1 for the PAH4 sum [22]. were supplied by LGC Standards GmbH (Wesel, Germany) However, in some Member States, the lower levels set from and used as internal standards. ,e stock solutions of the 1.9.2014 could not realistically be reached for traditionally individual 13C PAHs were prepared and combined in smoked products. ,erefore, Commission Regulation (EU) No. toluene. All the standard solutions were stored at +4°C, 1327/2014, amending Regulation (EC) No. 1881/2006, was set protected from the light. for three years, permitting certain Member States, including Finland, to produce and consume traditionally smoked meat and meat products, as well as fish and fish products, in their territory 2.3. Sample Preparation. PAH4 compounds were deter- that comply with the PAH MLs of 5.0 µg·kg−1 for BaP and mined using a gas chromatography-tandem mass spec- 30.0 µg·kg−1 for the PAH4 sum [23]. In this study, the PAH4 trometry (GC-MS/MS) method accredited according to ISO levels of Finnish smoked fish and meat products were de- 17025. ,e method was based on the publication of Veyrand termined. In addition, particular attention was paid to in- et al. [25], with minor modifications. All samples were vestigating the effect of selected smoking parameters on the packed in aluminium foil or in a sales package and stored at PAH4 levels to develop science-based guidance for a safe +5°C prior to homogenisation in a food mixer. According to smoking procedure. Particularly aimed at small and the manufacturer’s information, the fish or meat skin was medium-sized enterprises (SMEs), guidance will strengthen removed before homogenisation if the skin was not an edible their expertise and thereby prevent and reduce PAH con- part of the product. MLs are only applicable to the edible tamination in traditionally smoked fish and meat products. parts of smoked products. ,e samples were homogenised in a mixer, and approximately 100 g of each sample was taken 2. Materials and Methods to be freeze-dried. Half a gram of the dried sample was spiked with internal standards (13C-labelled PAHs) followed 2.1. Samples. During 2012, the municipal food control au- by the extraction of PAHs using ASE 200 . ,e extraction thorities and the inspection veterinarians of the Finnish cells were filled with celite and florisil and® prewashed with Food Safety Authority Evira collected 142 samples (80 dichloromethane before adding the sample and extracting Journal of Chemistry 3

2% 3% 3% 3% 5% 2% 5%

11% 5%

11%

68%

82% Pork Reindeer Salami Turkey Salmon Herring Sausage Others Whiteﬁsh Vendace Canned ﬁsh Others (a) (b)

Figure 1: Percentage distribution of sampled (a) smoked meat (n � 62) and (b) fish (n � 80) products. with hexane/acetone (50/50, v/v). ,e extract was evapo- Table 1: Precursor and product ions of PAH4 compounds. rated, diluted into cyclohexane, and purified in the solid- phase extraction (SPE) cartridge. ,e SPE cartridge was first Compound and precursor ion (m/z) Product ion (m/z) Benzo[a]pyrene 252.3 226.2, 250.1 conditioned (ethyl acetate, cyclohexane) and washed with 13 cyclohexane/ethanol (70/30, v/v), after which the sample ISTD benzo[a]pyrene C4 256.0 254.1 Benz[a]anthracene 228.2 202.2, 226.2 extract was introduced into the SPE cartridge. ,e PAH 13 ISTD benz[a]anthracene C6 234.0 232.2 compounds were eluted with cyclohexane/ethyl acetate Chrysene 228.2 202.1, 226.1 (40/60, v/v). Finally, the solvent was evaporated with ni- 13 ISTD chrysene C6 234.0 254.1 trogen, and the sample was redissolved in 100 µl toluene and Benzo[b]fluoranthene 252.3 226.0, 250.1 13 transferred to a GC vial. For the GC-MS/MS analysis, one ISTD benzo[b]fluoranthene C6 258.0 256.2 microliter of the final sample extract was injected into the Underlined product ions are used as quantifier ions. GC injector. In the case of canned fish samples, the fish and oil were separated and analysed separately. ,e individual ° −1 ° results were considered when calculating the final result for 15 min, and 14 C·min to 350 C for 5 min. Quantification the canned fish product. was performed by using the internal standard calibration curve with seven standards from 0 to 800 ng·mL−1. ,e data were processed using MassLynx V4.1 software supplied by 2.4. GC-MS/MS Analysis. ,e final detection and quantifi- Waters (Manchester, United Kingdom). cation of the analytes were performed using a gas chromatograph (Agilent, 6890N) coupled to a Micromass Quattro 2.5. Statistical Analysis. ,e statistical analysis was per- Micro GC triple quadrupole analyser (Waters, Micromass). formed using IBM SPSS Statistics, version 25 (SPSS Inc., Analysis was carried out using the Select PAH capillary column Chicago, Illinois). Boxplots were performed to visualise the × × from Agilent J&W (30 m 0.25 mm 0.15 µm). Helium (AGA, distribution of the data sets. Finland) was applied as the carrier gas at the constant flow rate of 1.0 mL·min−1 and argon (AGA, Finland) as the collision gas. ,e injection was done in splitless mode by utilising the 2.6. Method Validation and Quality Control. ,e GC-MS/MS electron ionisation (EI) technique (70 eV). Ion transitions were method complies with the criteria for the official control monitored with the multiple reaction monitoring (MRM) of PAH4 according to Commission Regulation (EU) No. mode, and the two most predominant fragments for each 836/2011 [26]. Method validation was performed for fish, analyte were utilised. Precursor ions and the selected meat, and oil matrices using Commission Decision (EC) product ions are given in Table 1. ,e injector and the No. 657/2002 as a guideline [1]. ,e following parameters transfer line temperature were held at 300°C and the ion were successfully tested: specificity, selectivity, linearity, source at 275°C. ,e GC oven temperature was initially kept repeatability, within-laboratory reproducibility, apparent at 110°C for 0.7 min and then raised at 5°C·min−1 to 180°C, recovery, the limit of detection (LOD), the limit of 3°C·min−1 to 230°C for 7 min, 28°C·min−1 to 280°C for quantification (LOQ), and trueness. ,e proficiency test 4 Journal of Chemistry organised by FAPAS was used to assess the trueness of the Table 2: ,e calculated maximum permitted ion ratio tolerance for method. Smoked meat® test material was analysed for BaP, fish and meat samples. BaA, and BbFA, and our z-scores were −0.7, 0.1, and −0.4, Maximum permitted Maximum permitted respectively. Compound ion ratio tolerance ion ratio tolerance Quality controls of the method were performed both for fish samples for meat samples before and during the sample run. Each GC-MS/MS run was Benzo[a]pyrene 4.0–7.5 4.5–8.4 preceded by a system performance check with the standard Benz[a]anthracene 4.0–6.6 3.9–7.3 solutions containing 0.011 and 0.064 µg·mL−1 of PAH4 Chrysene 3.6–5.9 3.7–6.1 standards (PAH-Mix 183, Dr. Ehrenstorfer GmbH, Augs- Benzo[b]fluoranthene 6.2–12 5.0–9.3 burg, Germany). In addition, the standard solutions were injected at the beginning, in the middle, and at the end of each sample batch. Individual internal standards were used for each PAH4 compound. ,e smoked large fish products (n � 72) contained 79% ,e spiked matrix sample was used to check the func- fillets, 13% pieces, and 8% whole fish samples, whereas all tionality of the whole method by spiking the blank fish or eight small fish samples represented the whole fish. When meat sample with PAH4 standards at the level of 5.0 µg·kg−1 comparing the data between the products of larger fish and analysing in each sample batch. ,e performance criteria species (e.g., salmon and whitefish) and smaller fish set in Commission Regulation (EU) No. 836/2011 consider the (e.g., herring, vendace, and roach), those of smaller fish recovery values 50–120%, the LOD ≤ 0.30 µg·kg−1, and the were found to contain higher median concentrations of LOQ ≤ 0.90 µg·kg−1 for each of the four substances acceptable PAHs (Table 3). ,e minimum and maximum values were [26]. All our performance values were within these limits. approximately at the same level in both large and small According to Commission Decision (EC) No. 657/2002 [1], fish species. the maximum permitted tolerances for relative ion in- Within the framework of the study, four meat product tensities were defined during fish and meat validation and samples were found noncompliant with the legislation, and applied as an identification criteria (Table 2). A chro- in all these cases, direct smoking was used. ,e competent matographic peak which was eluting at the retention time of authorities enforced the operator of these products to a target PAH was identified if qualifier ion was present and modify the smoking process. After modifications, follow-up the ion ratio between quantifier and qualifier was within the samples were taken to ensure the products were compliant acceptable ion ratio. As an example, benzo[a]pyrene in with the legislation before allowing the placement of the a meat sample with ion ratio of 5.3 is illustrated in Figure 2. products on the market. Four canned smoked fish products were analysed, comprising either vendace or roach. ,e final results were 3. Results calculated by combining the relative proportions of PAHs in ,e method used for the determination of PAH4 is the fish and vegetable oil in a can. In all of the cases, the routinely used and is accredited according to ISO 17025 PAH4 contamination was almost five times higher in the [27]. ,e LOD and LOQ of the method were 0.26 µg·kg−1 vegetable oil than in the fish part. A similar oil-fish PAH and 0.87 µg·kg−1 for each PAH4 analyte in fish and meat, ratio (5 :1) was reported by Lawrence and Weber [29] in respectively. Upper bound concentrations were calcu- canned smoked sardine. However, all the canned fish lated, assuming that all values of the individual PAH4 samples were compliant with the legislation. compounds less than the LOQ were equal to the LOQ. Furthermore, the concentrations below the LOD were 3.1. Effect of the Selected Smoking Parameters on the PAH4 calculated as zero. Measurement uncertainty (MU) was Levels. ,e effect of the selected smoking parameters was based on the validation and proficiency test data [28]. studied using smoked fish and meat samples. Additionally, Estimated as expanded uncertainty, MU varied from 16 to smoked salmon fillets (n � 57) were evaluated separately in 27% in fish and from 10 to 22% in meat, depending on the order to eliminate the interfering factors, such as other fish PAH4 compound. For the PAH4 sum, MU was 44% in fish species and effect of the skin. Salmon fillets were all smoked and 38% in meat. without the skin. Regarding the smoking time, more in- In general, the determined PAH4 levels in smoked fish vestigation in fish was needed; thus, cold and warm/hot and meat samples were relatively low and below the pre- smoked salmon fillets as well as large and small fish species vailing MLs. Regarding the smoked fish products, the mean were studied. concentrations were 0.7 µg·kg−1 for BaP and 3.9 µg·kg−1 for the PAH4 sum. For smoked meat products, the mean BaP and PAH4 sum levels were 2.2 µg·kg−1 and 11 µg·kg−1, re- 3.1.1. Smoking Technique. ,e samples were smoked using spectively. It is noteworthy, however, that our results dis- either a direct or an indirect smoking technique. In smoked played wide variation, particularly in meat products, for fish samples, direct smoking technique produced only slightly which the PAH4 sum concentrations ranged from not de- higher BaP and PAH4 sum concentrations than indirect tected to 200 µg·kg−1 (Table 3). Moreover, median PAH smoking. Exceptionally, one sample smoked with indirect levels in pork products were higher than in other meat technique contained highest PAH4 concentrations products. (Figure 3(a)). For smoked salmon fillets, the results Journal of Chemistry 5

14021320 Smooth (Mn, 2×3) F5: MRM of 3 channels, EI+ 252.3 > 250.1 e Benzo[a]pyrene 4.510 + 004 252.3 > 250.1 RT = 32.62 (%)

2 14021320 Smooth (Mn, 2×3) F5: MRM of 3 channels, EI+ 252.3 > 226.2 Benzo[a]pyrene 7.205e + 003 252.3 > 226.2 RT = 32.61 (%)

11 14021320 Smooth (Mn, 2×3) F5: MRM of 3 channels, EI+ ISTD 256 > 254.12 2.479e + 004 Benzo[a]pyrene13C4 256.0 > 254.1 RT = 32.61 (%)

11 31.75 32.00 32.25 32.50 32.75 33.00 33.25 33.50 33.75 34.00 34.25 34.50 34.75 35.00 (min) Figure 2: Multiple reaction monitoring chromatogram of benzo[a]pyrene at the level of 5.1 µg·kg−1 in a meat sample.

Table 3: Median and minimum-maximum concentrations (µg·kg−1) for BaP and PAH4 in products of large and small fish and in pork and other meat products. BaP PAH4 sum Product type Minimum-maximum Minimum-maximum Median (µg·kg−1) Median (µg·kg−1) (µg·kg−1) (µg·kg−1) Large fish products (n � 72) 0 0–4.9 0.3 0–26 Small fish products (n � 8) 0.9 0–3.8 8 0–21 Pork products (n � 42) 0.8 0–40 1.9 0–200 Other meat products (n � 20) 0 0–6.8 0.4 0–38

illustrated direct smoking producing higher PAH levels than between the large and small fish products. All the small fish the indirect smoking (Figure 3(b)). Moreover, direct products were smoked in less than five hours, and there- smoking generated clearly higher PAH levels in smoked fore, the comparison between the large and small fish meat samples than indirect smoking (Figure 3(c)). products was performed in that time range. ,e results show the small fish samples containing higher median PAH levels (Figure 4(d)). In smoked meat samples, the 3.1.2. Smoking Time. ,e effect of smoking time was exam- PAH concentrations were higher for both BaP and ined by dividing the PAH results into two categories according the PAH4 sum when the smoking time exceeded five to whether the smoking process took less or more than five hours (Figure 4(e)). hours. Surprisingly, the shorter smoking time generated higher PAH concentrations in fish samples, particularly observed for the PAH4 sum (Figures 4(a) and 4(b)). ,e comparison be- 3.1.3. Smoke Generation Temperature. ,e temperature tween cold smoked and warm or hot smoked salmon fillets required for smoke generation was defined as optimised if indicated that in cold smoking process, only two samples the thermal field was maintained at 400–600°C during the contained detectable PAH concentrations (Figure 4(c)). In whole process. In a nonoptimised case, the temperature may warm or hot smoked salmon fillets, which were mostly vary during the process, and it is not therefore controlled. As smoked in less than five hours, PAH levels were clearly expected, both for fish and meat samples, lower amounts of higher than in cold smoked products. Moreover, the fish PAHs were formed when the temperature was optimised size may affect the observed PAH levels, which was studied (Figures 5(a)–5(c)). Particularly, as shown in Figure 5(b), 6 Journal of Chemistry

25 ∗

20 ∗ ) –1 15 ∗ ( μ g·kg 10

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0 BaP PAH4 BaP PAH4 Direct smoking (n = 35) Indirect smoking (n = 45) Smoking technique (a) 25

∗ ) 15

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0 BaP PAH4 BaP PAH4 Direct smoking (n = 27) Indirect smoking (n = 30) Smoking technique (b) 200 ∗

150 ) –1 100 ( μ g·kg

50 ∗ ∗ ∗ 0 ∗ ∗ BaP PAH4 BaP PAH4 Direct smoking (n = 23) Indirect smoking (n = 39) Smoking technique (c)

Figure 3: (a) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for ﬁsh products divided according to the smoking technique used. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. (b) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for salmon ﬁllet products divided according to the smoking technique used. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. (c) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for meat products divided according to the smoking technique used. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. Journal of Chemistry 7

20 ) –1 15 ∗ ( μ g·kg 10

5 ∗ ∗ ∗ 0 BaP PAH4 BaP PAH4 Smoking time <5h (n = 54) Smoking time >5h (n = 18) Smoking time (a) 30

20 ∗

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5 ∗ ∗ 0 BaP PAH4 BaP PAH4 Smoking time <5h (n = 36) Smoking time >5h (n = 16) Smoking time (b) Smoking time Smoking time <5h(n =2) >5h(n = 11) <5h(n = 21) >5h(n =2) 25

) 15 –1

( μ g·kg 10

5 ∗

0 ∗ BaP PAH4 BaP PAH4 BaP PAH4 BaP PAH4 Cold smoked salmon fillet (n = 13) Warm or hot smoked salmon fillet (n = 23) Smoking type (c) Figure 4: Continued. 8 Journal of Chemistry

25 ∗ ∗ 20 ∗

) ∗ –1 15 ( μ g·kg 10

5 ∗ ∗ 0 BaP PAH4 BaP PAH4 Large fish (n = 46) Small fish (n =8) Fish size (d)

200 ∗

150 ) –1 100 ( μ g·kg

50 ∗ ∗ ∗ ∗ ∗ 0 BaP PAH4 BaP PAH4 Smoking time <5h (n = 22) Smoking time >5h (n = 22) Smoking time (e)

Figure 4: (a) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for fish products divided according to the smoking time. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 72 samples contained the relevant information. (b) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for salmon fillet products divided according to the smoking time. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 52 samples contained the relevant information. (c) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for cold smoked and warm or hot smoked salmon fillets divided according to the smoking time. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 36 samples contained the relevant information. (d) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for large and small fish products smoked in less than five hours. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 54 samples contained the relevant information. (e) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for meat products divided according to the smoking time. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 44 samples contained the relevant information.

PAH levels were clearly higher for smoked salmon ﬁllets in there were no detectable levels of BaP in meat products the nonoptimised system. when the distance from the smoke source was over ﬁve metres (Figure 6(c)).

3.1.4. Distance between the Food and the Smoke Source. 4. Discussion In this study, the reported length between the food and smoke source was either under or over five metres. ,e Food safety and especially the risk management of PAHs longer distance (over five metres) generated lower concen- must be the first priority in food smoking processes, al- trations of PAHs in both fish and meat products though smoking extends the food storage period and im- (Figures 6(a)–6(c)). ,e comparison between PAH levels proves the flavour, composition, and preservation of the in smoked fish (Figure 6(a)) and salmon fillets (Figure 6(b)) vitamin content [20]. ,e Codex Alimentarius recommends demonstrated that similar results were obtained. Furthermore, optimisation of the smoking process to produce smoked Journal of Chemistry 9

) 15 –1

( μ g·kg 10 ∗ 5 ∗

0 BaP PAH4 BaP PAH4 Temperature optimised (n = 18) Temperature not optimised (n = 17) Temperature optimisation (a) 25

) 15 –1

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5 ∗ ∗ 0 ∗ BaP PAH4 BaP PAH4 Temperature optimised (n = 11) Temperature not optimised (n = 13) Temperature optimisation (b)

200 ∗

150 ) –1 100 ( μ g·kg

50 ∗ ∗ ∗ 0 ∗ BaP PAH4 BaP PAH4 Temperature optimised (n = 15) Temperature not optimised (n = 11) Temperature optimisation (c)

Figure 5: (a) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for ﬁsh products divided according to the temperature optimisation. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 35 samples contained the relevant information. (b) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for salmon ﬁllet products divided according to the temperature optimisation. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 24 samples contained the relevant information. (c) Boxplot diagrams of BaP and PAH4 in µg·kg−1 wet weight for meat products divided according to the temperature optimisation. ,e line in the central box represents the median, the whiskers show the distribution outside, the central quartiles and the circles and stars are outliers. N is the number of samples. 26 samples contained the relevant information. 10 Journal of Chemistry

25 ∗ ∗

20 ∗ ) –1 15 ( μ g·kg 10 ∗ 5 ∗ ∗ ∗ 0 BaP PAH4 BaP PAH4 Distance <5m (n = 60) Distance >5m (n =9) Distance (a) 25

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( μ g·kg 10

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∗ 0 BaP PAH4 BaP PAH4 Distance <5m (n = 42) Distance >5m (n =8) Distance (b)

200 ∗

150 ) –1 100 ∗ ( μ g·kg

50 ∗ ∗

0 ∗ BaP PAH4 BaP PAH4 Distance <5m (n = 38) Distance >5m (n =5) Distance (c)

Figure 6: (a) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for fish products divided according to the distance. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 69 samples contained the relevant information. (b) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for salmon fillet products divided according to the distance. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 50 samples contained the relevant information. (c) Boxplot diagrams of BaP and PAH4 concentrations in µg·kg−1 wet weight for meat products divided according to the distance. ,e line in the central box represents the median, the whiskers show the distribution outside the central quartiles, and the circles and stars are outliers. N is the number of samples. 43 samples contained the relevant information. Journal of Chemistry 11 products that have good microbiological quality and sensory smoking generated higher concentrations of BaP and PAH4 properties with low PAH levels [20]. In order to control the than indirect smoking. ,e European Commission reported PAH levels in smoked products, it is important to identify in 2004 an average BaP concentration of 5.3 µg·kg−1 for the critical parameters. directly smoked fish, whereas for indirectly smoked fish, the Overall, due to the risk-based, i.e., selective sampling in BaP concentrations ranged from 0.1 µg·kg−1 to 2.0 µg·kg−1 this study, the detected PAH4 levels in fish and meat were [36]. ,e direct contact of the product with combustion probably higher than would have been obtained using ob- gases is definitely an important source of PAH contami- jective sampling. ,ey were nevertheless relatively low and nation [3, 6, 15]. In the direct smoking process, the com- comparable to other studies [18, 30, 31], with some ex- bustion temperature is often very high and challenging to ceptions. However, the large variation in PAH levels be- control [16, 18]. ,e replacement of direct smoking by tween the samples demonstrated how variable the smoking indirect smoking may reduce PAH contamination [7, 34]. process can be in Finnish smokehouses. ,e smoking However, 100% of the directly smoked fish products and equipment used are rather self-made than commercial; thus, 82% of the directly smoked meat products were compliant e.g., the length of smoking tube and the model of the filter with the legislation, so the direct technique is applicable may vary from one device to another. In 1999, the levels of 11 when properly controlled. Similar findings were observed in PAHs in different food categories were determined in Swedish meat and fish products that had been traditionally Finland. Among smoked meat products, the highest BaP directly “sauna” smoked, in which the smoked meat concentrations were observed in smoked pork products products contained higher levels of BaP than the smoked fish (5.6–13.2 µg·kg−1), whereas in the smoked fish product products. ,e “sauna” smoking produced increased BaP category, a smoked herring sample contained a sum con- levels in nine meat samples (6.6–36.9 µg·kg−1) and in six fish centration of 11 PAHs of 270 µg·kg−1, but a relatively low samples (8.4–14.4 µg·kg−1) [18]. BaP concentration of 0.7 µg·kg−1 [32]. Due to the different ,e smoking time is one of the critical factors to consider combinations of PAHs analysed, comparison with our re- in the smoking process, and it should be kept as short as sults is challenging, but we observed the same trend of higher possible, taking into account food safety and the product PAH levels in smoked pork products compared to other shelf life, because a prolonged smoking time increases the meat products. ,e data analysis did not reveal any par- PAH exposure of the product [19, 20, 37]. ,is was especially ticular smoking parameter alone to be the reason for the noticeable in the meat sample results, whereas for fish higher PAH levels in pork, but some factors were found samples, a shorter smoking time generated unexpectedly related to long smoking time up to 36 hours, no distance higher PAH concentrations than a longer smoking time. between the food and smoke source or irregular cleaning of Fish results were divided into different groups to be able to the smoking equipment. Altogether, the high variation evaluate them better. Similar findings were done in salmon among the results demonstrated the smoking process either fillets, which in turn were divided into cold smoked and to be controlled or not. Products of herring and also other warm or hot smoked fillets. ,e temperature differs gen- small fish species such as vendace, roach, and mackerel erally from 12 to 25°C in cold smoking, from 25 to 45°C in contained higher PAH levels than those of larger fish species warm smoking, and from 40 to 100°C in hot smoking [13]. (salmon and whitefish). ,is may be attributed to the as- Warm or hot smoking procedure generated more PAHs sumption that herring is often more heavily smoked than than the cold smoking, with some exceptions. Most of the salmon due to the smaller surface area-to-volume ratio cold smoked salmons (85%) were smoked more than five [16, 29]. hours, whereas 91% of the warm or hot smoked products Additionally, small fish species are consumed with the were smoked less than five hours which explains the skin, which contains more PAHs than the other parts of the higher PAH levels in salmon fillets within shorter smoking fish. According to previous studies [18, 33], the skin acts as time. ,e results are in agreement with Danish studies, a barrier to penetration by smoke particles, thereby pre- which indicated hot smoking producing higher PAH venting PAH contamination. ,e uptake of PAHs into the levels than cold smoking [16]. Moreover, all the small fish fish tissue is greater if the skin has been removed before species, such as herring and vendace, were smoked in less smoking. When reaching the product surface, PAHs accu- than five hours with higher median PAH levels compared mulate on the skin surface and may migrate into the un- to larger fish species (e.g., salmon and whitefish). A derlying fatty tissue due to their solubility in fat. ,e water smaller product size may result in the formation of higher activity and fat content have a significant role in the diffusion PAH levels [16]. process [15]. For smoked meat, similar conclusions were ,e most significant parameter influencing the PAH made by Ciecierska and Obiedzinski´ [34], who demon- concentrations in smoked products is the smoke generation strated lower PAH levels in the interior than the exterior of temperature [13]. ,e composition of smoke is dependent the same products. In canned fish products, PAHs can on the temperature, which needs to be regulated to reduce the migrate from the fish to the oil or remain in the fish lipid formation of PAHs [20]. According to Go¨bel [37], PAHs easily [35]. However, the oil mainly acts as a preservative, and form in the temperature range between 660 and 740°C. When many consumers pour away most of it before food con- temperature is increased within the range 400–1000°C, there is sumption [35]. a linear increase in the formation of PAHs in smoke [18, 38]. As expected, the smoking technique had a clear impact Based on our results, smaller amounts of PAHs were formed on the PAH levels. For both meat and fish products, direct when the smoke generation temperature was optimised to 12 Journal of Chemistry

400–600°C than in nonoptimised conditions. ,e difference in Data Availability PAH levels between the optimised and nonoptimised processes was obvious in both fish and meat samples, which emphasises ,e data used to support the findings of this study are the significance of temperature optimisation. Typically, the available from the corresponding author upon request. smoke generation temperature varies between 500 and 800°C and is controlled by the air supply [33]. Conflicts of Interest Besides the temperature, the distance between the food and the smoke source is among the important factors ,e authors declare that there are no conflicts of interest to observe. PAHs are bound to smoke particles, and regarding the publication of this paper. a longer distance from the smoke source to the smoked food may therefore lead to lower PAH levels in the food Acknowledgments [20]. ,e analysed fish and meat samples smoked at a distance over five metres from the smoke source con- We are very grateful for the sampling performed by the tained lower levels of PAHs. In indirect smoking, the municipal food control authorities and the inspection vet- length of the smoking tube varied between 0.2 and erinarians of Evira. We thank Kristian Alho for performing 23 metres. Naturally, all the samples smoked with the the PAH analysis and Dr. Jukka Ranta for the statistical direct technique were placed in the same chamber as the advice. In addition, we would like to thank the whole sample smoke source, and the distance was therefore always pretreatment team. under five metres. It can be expected that PAH levels will then be higher, because, as previously mentioned, direct References smoking leads to higher concentrations of PAHs in the products. To be able to reduce the PAH levels in the final [1] Commission of the European Communities, “Commission decision of 12 August 2002 implementing Council Directive product, an external smoke generator combined with 96/23/EC concerning the performance of analytical methods a long tube could be a good option. and the interpretation of results,” Official Journal of the Evaluating the significance of smoked meat and fish in European Union, vol. L221, pp. 8–36, 2002. the average diets of several European countries, the Scientific [2] P. Simko,ˇ “Determination of polycyclic aromatic hydrocar- Committee on Food (SCF) estimated the intake of BaP from bons in smoked meat products and smoke flavouring food smoked fish and meat to form only a small part of the total additives,” Journal of Chromatography B, vol. 770, no. 1-2, dietary intake [13]. However, in local communities or in pp. 3–18, 2002. certain countries, e.g., in Finland or Sweden [18], where [3] European Food Safety Authority, “Polycyclic aromatic hy- smoked fish and meat traditionally constitute a signifi- drocarbons in food. Scientific opinion of the panel on con- cant proportion of the diet, the intake of BaP and other taminants in the food chain adopted on 9 June 2008,” EFSA Journal, vol. 724, pp. 1–114, 2008. PAH4 compounds from these sources is significantly [4] M.-K. Song, M. Song, H.-S. Choi, Y.-J. Kim, Y.-K. Park, and higher [13]. Regarding the European Commission’s es- J.-C. 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[8] World Health Organization, “Evaluation of certain food 5. Conclusions additives and contaminants in WHO food additive report series,” vol. 28, October 2016, http://www.inchem.org/ Based on our results to obtain safe fish and meat products documents/jecfa/jecmono/v28je18.htm. regarding PAHs in traditional smoking, indirect smoking [9] M. Rose, J. Holland, A. Dowding et al., “Investigation into the with distance more than five meters between food and formation of PAHs in foods prepared in the home to de- smoke source as well as smoking time less than five hours termine the effects of frying, grilling, barbecuing, toasting and and an optimised temperature (without jeopardising mi- roasting,” Food and Chemical Toxicology, vol. 78, pp. 1–9, crobiological food safety) are recommended. 2015. Journal of Chemistry 13

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