Influence of processing in the prevalence of polycyclic aromatic hydrocarbons in a Portuguese traditional meat product

Food and Chemical Toxicology 49 (2011) 1340–1345

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Influence of processing in the prevalence of polycyclic aromatic hydrocarbons in a Portuguese traditional meat product L.C. Roseiro ⇑, A. Gomes, C. Santos Instituto Nacional dos Recursos Biológicos, I.P. L-INIA, Unidade de Investigação de Tecnologia Alimentar, Estrada do Paço do Lumiar, Campus do IAPMEI (Edifício S), 1649-038 Lisboa, Portugal

a r t i c l e

i n f o

Article history: Received 16 February 2011 Accepted 12 March 2011 Available online 17 March 2011 Keywords: Polycyclic aromatic hydrocarbons Dry-fermented meat sausage HPLC Smoking process

a b s t r a c t The concentration of 16 polycyclic aromatic hydrocarbons (PAHs) was determined in traditional dry/fermented sausage along distinct stages of processing under two different technological procedures (traditional and modified processes). The influence of product’s position in the smoking room, on the variation of contaminants and in their migration dynamics from the outer into the inner part, was also followed up. Raw material mixtures presented expressive total PAH values, 106.17 lg kg 1 in wet samples and 244.34 lg kg 1 in dry mater (DM), expressing the frequent fire woods occurred in the regions pigs were extensively reared. Traditional processing produced a higher (p < 0.01) total PAH levels comparatively to modified/industrial procedures, with mean values reaching 3237.10 and 1702.85 lg kg 1 DM, respectively. Both, raw materials and final products, showed PAH profiles with light compounds representing about 99.0% of the total PAHs, mostly accounted by those having two rings (naphthalene–27.5%) or three rings (acenaphtene–16.9%; fluorene–27.1%; phenanthrene–19.5% and anthracene–3.9%). The benzo[a]pyrene (BaP) accumulated in traditional and modified processed products never surpassed the limit of 5 lg kg 1 established by the EU legislation. PAHs in products hanged in bars closer to heating/smoking source speed up their transfer from the surface/outer portion to the inner part of the product. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants (Mottier et al., 2000), mostly formed by the incomplete combustion of organic matter (Danyi et al., 2009), making the human exposure practically unavoidable. However, several studies confirmed that diet is the major source of human contamination to PAHs (Falcó et al., 2003; Ibánez et al., 2005; Jira, 2004; Martí-Cid et al., 2008; Martorell et al., 2010; Phillips, 1999; Stolyhwo and Sikorski, 2005). Smoking, one of the oldest processing methods applied in food preservation by the action of anti-microbial constituents such as phenols, is still widely used in Portuguese traditional dryfermented sausages production, also to obtain peculiar sensorial characteristics (phenol derivatives, carbonyls, organic acids, among others). The smoke generated from wood combustion under low oxygen environment, contains considerable amounts of PAHs (Maga, 1988), some of them having carcinogenic and mutagenic properties (Janoszka et al., 2004; Yoon et al., 2007). The composition and concentration of PAH profiles contaminating smoked meat products depend on multiple factors, with the type of wood,

⇑ Corresponding author. Tel.: +351 217127107; fax: +351 217127162. E-mail address: [email protected] (L.C. Roseiro). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.017

its moisture content as well as the temperature associated to smoke generation having maximum effects (Garcia-Falcón and Simal-Gándara, 2005; Guillén et al., 2000; Maga, 1988). Among PAHs, the benzo[a]pyrene (BaP) concentration has received particular attention due to its higher contribution to overall burden of cancer in humans, being used as a marker for the occurrence and effect of carcinogenic PAHs in food (Rey-Salgueiro et al., 2009). According to EU legislation, a limit of 5.0 lg kg 1 for smoked meat products has been agreed (Regulation (EC) No. 1881/2006 of 19 December 2006). The goals of the present study were to investigate the PAHs profile and concentrations immediately after drying/smoking stage in a Portuguese traditional meat product ‘‘Chouriço Grosso’’, processed by two different technologies. PAHs distribution and deposition rate in products located at different positions in the smoking room were also assessed in order to clarify the variation observed. 2. Materials and methods 2.1. Preparation of ‘‘Chouriço grosso’’ Coarsely trimmed boned hams and shoulders (75–85%) and belly or back fat (25–15%) from ‘‘alentejano’’ cross breed pig carcasses were used in the formulation, after mincing to about 2  2 cm. Those two raw materials were then mixed for about 5 min and, in the mean time, added of NaCl (3.2% w/w), paprika paste (4% w/w), raw garlic paste (2% w/w) and tap water (6.4% w/w). The mixture was stuffed

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L.C. Roseiro et al. / Food and Chemical Toxicology 49 (2011) 1340–1345 into pig natural casings (20–25 cm long and 5–7 cm in diameter). The same batch of ‘‘Chouriço Grosso’’ (150 kg) was further processed according to traditional (T) and modified (M) practices. Under the traditional processing, raw products stayed continuously in the traditional smoking room under discontinuous smoking regime for the next 40 days. Differently, those produced under the modified processing procedure were first held in a controlled drying room for 12 days, without smoking, being then transferred to the same traditional smoking room until an aW value of about 0.88 was reached. For determining the effect of smoking/drying practices on PAHs content, duplicates of ‘‘Chouriço Grosso’’ from batches processed under both technologies were analyzed. After processing, final products were immediately frozen ( 80 °C), packaged under vacuum in Cryovac BB4L plastic bags (Grace SA, Barcelona, Spain – permeability to oxygen: 30 cm3/m2/day/bar at 23 °C and 0% of relative humidity) and stored protected from the light until analysis. 2.2. Smoking room characteristics and procedures The smoking room, within the traditional style, had a natural opening ventilator on the top and a smoke generating furnace underneath, with a size around 3.5  3.5  4.0 m and an overall capacity of about 1000 kg. Products stayed hanged on six trolleys with four height levels, each having seven rows of sausages. Smoke production from ‘‘Quercus ilex’’ wood combustion was operated about 180 cm below, on a iron device having a kind of protecting roof, to prevent the fat rendering from the products to fall down in the burning wood, avoiding the risk of a fire and reducing the formation of carcinogenic PAH associated to the incomplete combustion of carbon and hydrogen in fat (Larsson et al., 1983). Under such conditions, the smoke stream flow pattern is deeply affected in its way up, spreading around and determining its temperature and composition as well, since it does not reach the first level of products directly. No forced ventilation was applied, with the smoke flow rate inside the room being greatly determined by the outer weather conditions and the opening size of the door existing at the ground level.

dibenzo[a,h]anthracene – DhA, benzo[g,h,i]perylene – BgP, indeno[1,2,3-cd]pyrene – IcP) was determined from the outer (including the casing) and inner portions by HPLC according to Simko et al. (1993). Thirty grams of homogenized samples were saponified with a mixture of potassium hydroxide, methanol and water for 3 h under reflux. After the addition of 100 ml of methanol and water mixture (8:2, v/v), the PAHs were extracted four times with 50 mL of n-hexane. The extracts were combined and 100 mL of 10% (p/v) Na2WO4 water solution was added. The extract was dried over anhydrous Na2SO4 and evaporated to a volume of 1 ml using a rotary vacuum evaporator. The concentrated extract was applied in a Florisil column (Sep-Pak Plus Florisil, Waters, MA, USA) and eluted with 165 mL of n-hexane. After evaporation to dryness, the residue was dissolved in 3 mL of methanol and filtered through a Acrodisc membrane 25 mm GHP, GF 0.45 lm (Waters, Milford, MA). PAHs were separated using a HPLC system consisting of an Alliance Separation Module 2695, a Multi k Fluorescence detector 2475 and a Dual k UV/VIS detector 2487 (Waters, Milford, MA). Separation was performed on a reverse phase PAH C18, S-5 lm; 250  3.0 mm (Waters, Germany), using a gradient elution program with a mixture of acetonitrile and water which started at 20% acetonitrile for 1 min, reaching 56% in 5 min and 100% in 30 min at a flow rate of 0.5 mL min 1. The eluted compounds were revealed with two programmable detectors (UV/VIS and fluorescence) connected in series. Detection parameters are shown in Table 1. The PAHs quantification in different samples was made in duplicates for each assay condition and carried out through the external standard method. The sum of crysene, benzo[a]pyrene, benzo[a]anthracene and benzo[b]fluoranthene (PAH4), adopted as a suitable indicator for the occurrence and toxicity of PAHs in food (EFSA Journal, 2008) was calculated. The PAHs content was expressed as lg kg 1 in dry matter (DM) of sample. DM was measured by drying the samples at 103 ± 2 °C to constant weight (ISO 1442, 1997).

2.5. Statistical analysis 2.3. Reagents and standards n-Hexane, methanol and potassium hydroxide used in this study were of analytical grade and acetonitrile was of HPLC grade, all obtained from Panreac (Barcelona, Spain). Water was purified with a Milli-Q System (Millipore, Bedford). PAH standard mixture of 16 PAHs (EPA 610 Polynuclear Aromatic Hydrocarbons Mixture) from Supelco (Bellefonet, PA, USA) was used.

The influence of processing technology (T) and time (t) on data was evaluated by ANOVA using the software Statistica 6.0 (StatSoft Inc., 2001). The Tukey post hoc test was used for comparison of mean values, with differences being considered significant at p < 0.05.

3. Results and discussion 2.4. PAHs analysis The quantification of 16 PAH (naphthalene – NAP, acenaphthylene – ACY, acenaphtene – ACE, fluorene – FLR, phenanthrene – PHE, anthracene – ANT, fluoranthene – FLT, pyrene – PYR, benzo[a]anthracene – BaA, chrysene – CHR, benzo[b]fluoranthene – BbF, benzo[k]fluoranthene – BkF, benzo[a]pyrene – BaP, Table 1 Detection parameters of different PAHs. UV detector

ACY, IcP NAP, ACE, FLR, PHE ANT, FLT, PYR, BaA, CHR, BbF, BkF, BaP DahA BgP

254 – –

Fluorescence detector k Ex (nm)

k Em (nm)

– 260 260

– 366 430

ACY – acenaphthylene, IcP – indeno[1,2,3-cd]pyrene, NAP – naphthalene, ACE – acenaphtene, FLR – fluorene, PHE – phenanthrene, ANT – anthracene, FLT – fluoranthene, PYR – pyrene, BaA – benzo[a]anthracene, CHR – chrysene, BbF – benzo[b]fluoranthene, BkF – benzo[k]fluoranthene, BaP – benzo[a]pyrene, DhA – dibenzo[a,h]anthracene, BgP – benzo[g,h,i]perylene.

Table 2 PAHs composition (lg kg

1

PAHs have been found in water, air, soil and also in vegetation/ pastures around, originated from diverse sources such as engine exhausts, fire woods and other combustion types (Bartos et al., 2009; Zhang et al., 2009). So, contamination of food from animal origin with PAHs is then a realistic matter, attending to their permanent formation in the environment (Crepineau et al., 2003; Garcia-Falcón et al., 2006; Rey-Salgueiro et al., 2004). Concentrations found in animal tissues will depend on different factors, including the PAH type, the route of administration and the presence or absence on inducers and inhibitors of hydrocarbon metabolism within the organism (Ciganek and Neca, 2006; Wenzl et al., 2006). The PAHs contamination profile of raw materials mixture used in sub-batches T and M (Table 2), which will express the contribution from environment and animal feed in pork obtained from extensively reared autochthonous pigs, is similar to that found in smoked processed meat sausages, but with compounds having, obviously, lower concentrations (total amount of 244.34 lg kg 1 in DM). Among the 16 PAH analyzed only 12 compounds were detected, with acenaphthylene, dibenzo[a,h]anthracene, benzo[g,h,i] perylene and indeno[1,2,3-c,d]pyrene being always absent. Such

DM) in the raw materials used in manufacture of dry-cured fermented sausages.

Raw material

NAP

ACY

ACE

FLR

FEN

ANT

FLT

PIR

BaA

CHR

BbF

BkF

BaP

DbA

BgP

IcP

PAH total

Without seasoning With seasoning SE p

67.20 81.77 1.98

nd nd – –

41.24 46.95 2.09 ns

66.18 53.53 1.06

47.53 46.54 0.33 ns

9.52 9.14 0.05

5.72 5.72 0.03 ns

4.50 4.67 0.01

0.79 0.80 0.05 ns

0.60 0.71 0.03 ns

0.55 0.50 0.04 ns

0.26 0.27 0.01 ns

0.26 0.26 0.01 ns

nd nd – –

nd nd – –

nd nd – –

244.34 250.83 5.46 ns

⁄

⁄

⁄

⁄⁄

NAP – naphthalene, ACY – acenaphthylene, ACE – acenaphtene, FLR – fluorene, PHE – phenanthrene, ANT – anthracene, FLT – fluoranthene, PYR – pyrene, BaA – benzo[a]anthracene, CHR – chrysene, BbF – benzo[b]fluoranthene, BkF – benzo[k]fluoranthene, BaP – benzo[a]pyrene, DhA – dibenzo[a,h]anthracene, BgP – benzo[g,h,i]perylene, BgP – benzo[g,h,i]perylene, IcP – indeno[1,2,3-cd]pyrene, nd – not detected, SE – standard error. In same column, means with different letters are significantly different. ns = not significant. ⁄P < 0.05, ⁄⁄P < 0.01.

⁄ ⁄⁄⁄

ns ns ⁄⁄ ⁄

⁄⁄

40

30

22

0 14

Raw material Modified Traditional Modified Traditional Modified Traditional Modified Traditional SE Technology (T) Time (t) Tt

NAP – naphthalene, ACY – acenaphthylene, ACE – acenaphtene, FLR – fluorene, PHE – phenanthrene, ANT – anthracene, FLT – fluoranthene, PYR – pyrene, BaA – benzo[a]anthracene, CHR – chrysene, BbF – benzo[b]fluoranthene, BkF – benzo[k]fluoranthene, BaP – benzo[a]pyrene, DhA – dibenzo[a,h]anthracene, BgP – benzo[g,h,i]perylene, BgP – benzo[g,h,i]perylene, IcP – indeno[1,2,3-cd]pyrene, nd – not detected, SE – standard error, nd – not detected. In same column, means with different letters are significantly different. ns = not significant. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

⁄

⁄⁄

⁄⁄

ns

ns

ns

ns

⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄ ⁄⁄⁄

⁄⁄⁄ ⁄⁄⁄

⁄⁄

⁄⁄⁄

⁄⁄ ⁄⁄

⁄⁄ ⁄⁄⁄

⁄⁄

⁄⁄

⁄⁄⁄

⁄⁄

⁄⁄⁄ ⁄⁄ ⁄⁄⁄

⁄⁄⁄

⁄⁄

⁄⁄

⁄⁄

IcP

nd nd nd nd nd nd nd nd nd – – – – nd nd nd nd nd nd nd nd nd – – – –

BgP DbA

nd nd nd nd nd nd nd nd nd – – – – 0.26f 0.57ef 0.68def 0.98cdef 1.66cde 1.89cd 3.33ab 2.17bc 3.53a 0.22

BaP BkF BbF

0.55bc 0.68bc 0.41bc 1.50b 0.01c 0.67bc 2.83a 1.25bc 3.48a 0.23

CHR

0.60b 2.14b 2.13b 3.22b 20.28a 4.10b 9.69ab 9.89ab 19.48a 2.41 0.79 1.09 3.13 3.68 15.44 4.24 12.65 10.79 17.98 2.44

BaA PYR

4.50 7.81 17.58 20.14 101.64 33.44 118.56 78.89 154.42 19.26 5.72 12.32 41.12 31.86 207.62 63.21 187.57 150.79 299.17 37.60

FLT ANT

9.52 12.15 46.47 30.23 186.91 71.17 198.13 157.41 297.32 41.93

PHE

47.53 65.22 179.73 140.62 708.89 274.05 774.66 628.90 1078.50 128.56

FLR

66.18 92.55 239.35 51.53 801.01 344.57 713.60 484.98 877.21 148.46

ACE

41.24b 31.16b 40.97b 29.99b 119.58b 63.75b 301.48a 110.87b 284.55a 27.58

ACY

nd nd nd nd nd nd nd nd nd – – – –

NAP

67.20b 44.59b 20.50b 15.39b 88.50ab 63.63b 42.08b 65.98b 199.52a 22.88 ns

Time (days) Process

DM) in the dry-cured fermented sausages manufactured by traditional and traditional modified process. 1

Table 3 PAHs composition (lg kg

0.26f 0.52def 0.34ef 0.70cde 0.55def 0.77cd 1.56b 0.93c 1.94a 0.06

PAH total

L.C. Roseiro et al. / Food and Chemical Toxicology 49 (2011) 1340–1345

244.34e 270.80e 592.40de 329.85e 2252.09abc 925.48cde 2366.14ab 1702.85bcd 3237.10a 428.23

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profile must be associated with the wood used in smoking/drying operation, which is considered to have a significant influence on the contamination level (Garcia-Falcón and Simal-Gándara, 2005; Toth and Potthast, 1984; Viksna et al., 2008a). The concentration in raw materials for the distinct compounds was inversely related to their molecular weight, with light members representing 99.4% of the total PAHs, mostly from those having two rings (NAP – 27.5%) or three rings (ACE – 16.9%; FLR – 27.1%; PHE – 19.5% and ANT – 3.9%). These results are consistent with those reported by Ciganek and Neca (2006) and Mottier et al. (2000), but comparatively, the concentrations in wet products detected by these authors were considerably lower than ours (6.55 vs 29.2; 0.34 vs 17.92; 0.98 vs 28.76; 6.95 vs 20.65 and 0.76 vs 4.13 lg kg 1, respectively). Such higher contamination level may be related to the frequent fire woods occurred during the last decade, affecting the ecosystem supporting the free-range feeding of pigs (Garcia-Falcón et al., 2006). Among toxic compounds, BaA, CHR, BbF, BkF and BaP reached mean amounts of 0.34, 0.26, 0.24, 0.11 and 0.11 lg kg 1 DM, respectively, showing a variation range similar to that indicated by the research teams referred before. The results also fit well with those presented in EFSA Journal (2008) regarding this type of products. The addition of seasonings (red paprika and garlic pastes) and curing salts, in the amounts used at the present study, did not affect considerably the initial PAHs concentration in raw materials mixture but, apparently, they increased slightly the initial PAHs concentration observed in raw materials mixture, due basically to the increased concentration of NAP, PHE, ANT, FLT, CHR and NAP, ACE and PYR, respectively. Attending to the compositional (meat/fat) variation of raw materials mixture, these trends seem irrelevant. The PAHs composition and content in the manufactured dry-cured fermented sausages, randomly picked up at distinct processing stages from the traditional (T) and the modified (M) sub-batches, are shown in Table 3, with total and most individual compounds concentrations differing significantly between both technologies. Despite the general impact in the results from the products holding time in the smoking/drying house, no significant effect resulted from both factors interaction was verified in FLR, FEN, ANT, FLT, PYR, and BaA. Due to the delay in the entrance of

Table 4 Variation in the contamination degree (lg kg analyzed within the same sub-batch. Modified process

NAP ACY ACE FLR PHE ANT FLT PYR BaA CHR BbF BkF BaP DBA BgP IcP

1

DM) observed among final products

Traditional process

Min

Max

SD

Min

Max

SD

17.70 nd 50.22 169.98 411.76 104.65 107.74 57.22 8.01 7.12 0.72 0.68 1.58 nd nd nd

93.60 nd 202.61 791.14 908.28 238.31 229.89 118.45 14.63 12.84 2.72 1.51 2.90 nd nd nd

28.14 nd 61.33 248.01 205.34 54.34 51.45 26.37 2.92 2.50 0.67 0.29 0.57 nd nd nd

45.87 nd 187.00 355.76 530.81 131.33 141.91 79.63 8.00 5.01 0.63 0.72 1.20 nd nd nd

420.64 nd 452.91 1715.10 1725.33 654.46 539.03 243.55 32.87 38.11 7.42 3.49 6.57 nd nd nd

132.44 nd 107.97 565.74 475.89 178.64 144.53 68.42 9.93 12.44 3.00 1.30 2.27 nd nd nd

NAP – naphthalene, ACY – acenaphthylene, ACE – acenaphtene, FLR – fluorene, PHE – phenanthrene, ANT – anthracene, FLT – fluoranthene, PYR – pyrene, BaA – benzo[a]anthracene, CHR – chrysene, BbF – benzo[b]fluoranthene, BkF – benzo[k]fluoranthene, BaP – benzo[a]pyrene, DhA – dibenzo[a,h]anthracene, BgP – benzo[g,h,i]perylene, IcP – indeno[1,2,3-cd]pyrene.

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L.C. Roseiro et al. / Food and Chemical Toxicology 49 (2011) 1340–1345 Table 5 PAH content (lg kg 1 DM) in dry fermented sausages placed into distint vertical plans into the smoking space. PAHs

Superior

Intermediate

Inferior

SE

NAP ACY ACE FLR PHE ANT FLT PYR BaA CHR BbF BkF BaP DBA BgP IcP Total PAH4

87.09b nd 120.17b 460.35a 532.57b 121.80b 130.63b 75.24b 13.32b 57.66 4.17 2.18a 3.01a nd nd nd 1608.19a 78.16

35.93b nd 116.12a 215.81b 545.97a 131.23a 161.56a 85.03a 17.72a 26.14 1.55 0.87ab 2.25a nd nd nd 1517.88a 47.66

155.04a nd 100.86c 15.92c 37.94c 5.31c 18.85c 15.44c 1.61c 12.34 1.06 0.4b 0.89b nd nd nd 365.66b 15.90

2.16 – 1.41 3.62 5.32 2.25 2.76 2.04 0.34 8.45 0.77 0.31 0.19 – – – 21.79 9.50

p ⁄⁄⁄

– ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄

ns ns ⁄ ⁄⁄

– – – ⁄⁄⁄ ⁄

NAP – naphthalene, ACY – acenaphthylene, ACE – acenaphtene, FLR – fluorene, PHE – phenanthrene, ANT – anthracene, FLT – fluoranthene, PYR – pyrene, BaA – benzo[a]anthracene, CHR – chrysene, BbF – benzo[b]fluoranthene, BkF – benzo[k]fluoranthene, BaP – benzo[a]pyrene, DhA – dibenzo[a,h]anthracene, BgP – benzo[g,h,i]perylene, BgP – benzo[g,h,i]perylene, IcP – indeno[1,2,3-cd]pyrene, nd – not detected, SE – standard error. In same column, means with different letters are significantly different. ns = not significant. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄ P < 0.001.

M products in the smoking house, as well as to the lower smoking intensity and associated temperature (approximately half of the drying weight loss was already accomplished), they appeared at the end of processing less contaminated (p < 0.05) than T counterparts (1702.85 vs 3237.10 lg kg 1 in DM). Those compounds with lower molecular weight were again the major representatives in both products profiles, mainly PHE (37% and 33% in T and M products, respectively) and fluoranthrene (29% and 27% in T and M products, respectively). Without any specific order, ANT, FLT, ACE and PYR, were the next more concentrated PAHs, between 10% and 6% in both processing styles, which gave origin just to small serial modifications between tested smoking/drying technologies. In

relation to PAH4, this contamination indicator accounted almost the same to the overall contamination in both products (1.42% and 1.37%, in M and T products, respectively). The BaP accumulated in products never surpassed the limit of 5 lg kg 1 established by the EU legislation for this kind of products. The high variation in the contamination degree observed among products within the same sub-batch (Table 4), may be related with the uneven smoke flow distribution observed under this smoking system and also to the variation observed in the fat content among the products (Perelló et al., 2009; Roseiro et al., 2008) due to PAHs lipophylic character. Detected minimum (1477.93 lg kg 1 DM – T products; 923.30 lg kg 1 DM – M products) and maximum (5250.47 lg kg 1 DM – T products; 2518.04 lg kg 1 DM – M products) mean values of total PAHs, could also be associated to changes occurring in the smoke composition along the smoking operation, due to variations in the combustion temperature. The contribution from the heavy compounds to such variation is much visible in T products. To identify the source of that variation, the effect associated to the disposition of sausages into the smoking room at distinct vertical plans was investigated (Table 5). The results showed a lower PAHs accumulation in those products placed at the inferior plan (365.66 lg kg 1) when compared with the products at intermediate (1517.88 lg kg 1) and superior (1608.17 lg kg 1) levels. Such trend may be determined by the smoke flow distribution, emerging from the reflection achieved with the device placed above the burning wood, which tend to spread it toward the lateral walls of the compartment. Consequently, the smoke concentrates upwards, circulating then around the sausages. Viksna et al. (2008b) also found out differences in the levels and distribution pattern of BaP in products processed at different points and plans at the smoking house. The profile pattern also changed between products placed in the lower plans and those hanged at intermediate and upper bars, which not differed. In the later groups of products, the five most concentrated compounds were by decreasing order PHE, FLR, FLT, ANT and ACE whereas in former one this list was NAP, ACE, PHE, FLT and FLR. Another different pattern observed among sausages placed in distinct vertical plans in the smoking room concerns to the penetration rate of PAHs from the surface toward the inner portion of

Table 6 Distribution of PAHs (lg kg 1 DM) in the products (outer and inner portions) placed in different vertical plans. PAHs

Position in the smoking house Superior

NAP ACY ACE FLR PHE ANT FLT PYR BaA CHR BbF BkF BaP DBA BgP IcP Total

Statistic Intermediate

Inferior

SE

Outer

Inner

Outer

Inner

Outer

Inner

70.51a nd 305.51a 1348.92a 1586.28a 365.89a 388.11a 219.37a 40.79a 179.49a 12.70a 6.76a 9.31a nd nd nd 4533.65a

94.89 nd 32.95b 42.21b 36.71b 6.93b 9.46b 7.41b 0.39b 0.33ab 0.15ab 0.03b 0.04a nd nd nd 231.48b

78.24b nd 333.12c 641.35c 1679.30c 406.14c 495.77c 256.40c 52.14c 80.84b 4.74b 2.44b 6.91b nd nd nd 4037.38c

95.30b nd 94.74c 19.35c 55.02c 25.24c 22.38c 18.48c 0.32c 0.40b 0.52b 0.24b 0.23b nd nd nd 332.23c

163.53bc nd 103.73c 16.35c 40.64c 5.76c 7.62c 5.79c 0.19c 7.63b 0.90b 0.06b 0.16b nd nd nd 352.38c

151.04c nd 99.51d 15.73c 36.66c 5.09c 24.13c 19.98c 2.28c 14.56b 1.13b 0.56b 1.24b nd nd nd 371.91c

3.66 – 4.00 5.95 10.42 3.70 4.53 3.23 0.81 18.55 1.71 0.67 0.43 – – – 39.52

Position

Portion

PP

⁄⁄⁄

⁄

⁄⁄

–

–

–

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄

⁄⁄

⁄⁄

⁄

⁄⁄

⁄

⁄

⁄⁄

⁄⁄

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

– – –

– – –

– – –

⁄⁄⁄

⁄⁄⁄

⁄⁄⁄

NAP – naphthalene, ACY– acenaphthylene, ACE – acenaphtene, FLR – fluorene, PHE – phenanthrene, ANT – anthracene, FLT – fluoranthene, PYR – pyrene, BaA – benzo[a]anthracene, CHR – chrysene, BbF – benzo[b]fluoranthene, BkF – benzo[k]fluoranthene, BaP – benzo[a]pyrene, DhA – dibenzo[a,h]anthracene, BgP – benzo[g,h,i]perylene. BgP – benzo[g,h,i]perylene. IcP – indeno[1,2,3–cd]pyrene. nd – not detected. SE – Standard error. In same column, means with different letters are significantly different. ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.

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products (Table 6). The highest concentration of PAHs is supposed to be achieved on meat products outer portion, soon after the end of the smoking operation. Investigations regarding the PAHs distribution in meat products body showed that, coincidently, near 99% of compounds were in the outer portion (3–5 mm deep, representing approximately 30% of overall product weight) (Jira et al., 2006; Roseiro et al., 2008). However, PAHs also penetrate into the inner product during processing and storage, stabilizing their concentration after some time (Girard, 1991). Afterwards, a decrease in PAHs content is expectable, caused by light decomposition and interaction with other components of the formulation (Dennis et al., 1984; Simko, 1991). In the present study, the penetration rate depended on the distance the products were from the heating source. While in those placed at the upper and intermediate plans, the PAHs are mostly accumulated on the surface and in the outer portion (3 mm deep), in sausages placed close to the furnace, the PAHs concentration is quite equilibrated all over the sausage body or even higher in the inner portion in relation to the heavier compounds (Table 6). The higher maximum temperatures attained by the later products comparatively to counterparts, could speed up and increase the fat rendering, which would work as fluid channels facilitating the transference of PAHs to the inner section of the product. Such hypothesis is also confirmed when the comparison between upper and intermediate samples is made, with the former sausages, which were submitted to slightly higher environmental temperatures, also denoting higher concentrations in the center part of the product for most of PAHs. Measurements based on a greater number of trials and products are needed to confirm this trend. 4. Conclusions The relative contribution to the total contamination level of compounds integrating the PAH4 group was similar in both product types, which means that the slightly lower mean temperature at smoking in the modified procedure did not affect considerably the smoke PAHs profile. Under both processing styles, BaP never exceed the limit (5.0 lg kg 1) established by EU legislation for this kind of products. Sausages location at the smoking room had an important influence in the contamination level and penetration rate of PAHs due to the flow patterns of the smoke reaches the products surface and evolution of their internal temperature. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements The authors would like to thank Salsicharia Estremocense, SA the support given in meat products processing. This research was funded by Fundação para a Ciência e Tecnologia (ERA-FOOD/ 0001/2008-RISKFOODCONT). References Bartos, T., Xupr, P., Klánová, J., Holoubek, I., 2009. Which compounds contributed most to elevated airborne exposure and corresponding health risks in the Western Balkans? Environment International 35, 1066–1071. Ciganek, M., Neca, J., 2006. Polycyclic aromatic hydrocarbons in porcine and bovine organs and tissues. Veterinarni Medicina 51, 239–247. Crepineau, C., Rychen, G., Feidt, C., LeRoux, Y., Lichtfouse, E., Laurent, F., 2003. Contamination of pastures by polycyclic aromatic hydrocarbons in a vicinity of a highway. Journal of Agricultural and Food Chemistry 51, 4841–4845. Danyi, S., Bose, F., Brasseur, C., Schneider, Y.J., Larondelle, Y., Pussemier, L., 2009. Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements

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