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Influence of Traditional Smoking on Content Polycyclic Aromatic Hydrocarbons in Dry Fermented Sausages with Collagen and Natural Casings

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Influence of Traditional Smoking on Content Polycyclic Aromatic Hydrocarbons in Dry Fermented Sausages with Collagen and Natural Casings INFLUENCE OF TRADITIONAL SMOKING ON CONTENT POLYCYCLIC AROMATIC HYDROCARBONS IN DRY FERMENTED SAUSAGES WITH COLLAGEN AND NATURAL CASINGS Škaljac Snežana1, Petrović Ljiljana1, Tasić Tatjana2, Ikonić Predrag2, Jokanović Marija1, Tomović Vladimir1, Džinić Natalija1, Šojić Branislav1, Tjapkin Ana1 and Škrbić Biljana1 1 Faculty of Technology, University of Novi Sad, Novi Sad, Serbia 2 Institute for Food Technology, University of Novi Sad, Novi Sad, Serbia Abstract – The concentrations of 13 polycyclic antimicrobial constituents such as phenols. It is aromatic hydrocarbons from Environmental still widely used in Serbian traditional dry Protection Agency list (EPA PAHs) were examined fermented sausages production (Petrovská in two groups of fermented dry sausages (Petrovská klobása) to obtain peculiar sensorial klobása) smoked on traditional way. The sausage characteristics due to phenol derivatives, carbonyls, mixture from the first group of samples was stuffed in the natural casings (N), while the second was organic acids. Wood smoke contains a large stuffed in collagen casings (C). Analyses of PAHs number of polycyclic aromatic hydrocarbons were determined using gas chromatography with (PAH) and their alkylated derivatives [4]. They mass spectrometry. Content of pyrene, anthracene, may be regarded as potentially genotoxic and acenaphthylene, fluorene and phenanthrene was carcinogenic to humans [5] due to a metabolic significantly (P<0.05) lower in sausage with collagen activation in mammalian cell to dioepoxides casings (2.7; 12.8; 10.1; 11.5; 19.1, µg/kg causing errors in DNA replication and mutation, respectively) than in the sausage with natural which initiates the carcinogenic process [6, 7]. casings (8.3; 16.0; 34.9; 38.0; 39.9 µg/kg The United States Environmental Protection respectively). Other examined PAHs were below the Agency (7US-EPA PAHs) has classified seven limit of detection in all the samples. According to the results the maximum acceptable content established PAHs (benzo[a]pyrene, benzo[a]anthracene, by the EU of 5µg/kg for BaP in smoked meat chrysene, benzo[b]fluoranthene, benzo[k] products was not exceeded in any samples of fluoranthene, dibenzo[a,h]anthracene and indeno Petrovská klobása. Results obtained in this study [1,2,3-c,d] pyrene) as Group B2, probable human indicated that total content of 13 EPA PAHs was carcinogens [7]. In turn, the International Agency significantly (P<0.05) lower in sausage with collagen for Research on Cancer (6IARC PAHs) casings (56.2µg/kg) than in the sausage with natural established that benzo[a]anthracene and casings (137.1µg/kg). benzo[a]pyrene are probable human carcinogens, while benzo [b]fluoranthene, benzo[k]fluoranthene, Key Words – Petrovská klobása, Traditional dibenzo[a,h]anthracene and indeno[1,2,3-c,d] smoking, US-EPA PAHs pyrene are possible human carcinogens [8]. Among PAHs, the benzo[a]pyrene (BaP) is used I. INTRODUCTION as a marker for the occurrence and effect of carcinogenic PAHs in food. According to EU Petrovská klobása, which is traditionally produced legislation, a limit of 5.0µg/kg for smoked meat in Bački Petrovac, is fermented dry sausage that is products has been agreed [9]. highly valued product in Serbia and in neighboring countries. It is produced in the traditional way A number of factors related to the smoking without additives and starter cultures [1, 2]. This process affect the composition of smoke as well as product is protected in Serbia with designation of the PAHs uptake in the products, with the origin (PDO) according to Serbian legislation combustion temperature is critical [10]. Also, because of its specific and distinctive quality [3]. deposition and penetration of smoke components Smoking is one of the oldest processing methods into smoked food depends on presence of barriers applied in food preservation by the action of 59th International Congress of Meat Science and Technology, 18-23rd August 2013, Izmir, Turkey such as casings (e.g. natural, collagen casings, B. Method cellulose-peelable casings) [11, 12]. The samples were extracted and cleaned up Thus, the main objective of this study was to using the QuEChERS method (quick easy cheap determine the content of 13 EPA priority PAHs effective rugged and safe): 6 g of ground (acenaphthylene, fluorene, phenanthrene, homogenized sample was placed in a 50 ml anthracene, pyrene, benzo[a]anthracene, chrysene, centrifuge tube, 12 ml of water and 15 ml of benzo [b] fluoranthene, benzo [k] flouranthene, acetonitrile was added and the tube was benzo[a]pyrene, dibenzo[a,h]anthracene, benzo vigorously shaken, followed by the addition of [g,h,i]perylene, and indeno[1,2,3-cd]pyrene) in dry salts (6 g of MgSO4 and 1.5 g of NaCl) and fermented sausage Petrovská klobása smoked in agitation for another 5 minutes. The tube was the traditional (direct smoking) way. The specific then centrifuged at 3000 rpm for 15 minutes aim was to verify can the collagen and natural (Eppendorf 5430 centrifuge); 8 ml of sausages casings affect the levels of PAHs in the supernatant was transferred to a 15 ml tube Petrovská klobása. containing 900 mg of MgSO4, 150 mg of PSA and 150mg of C18 (Restek Q373). The tube was II. MATERIALS AND METHODS shaken for 5 minutes and then centrifuged for 15 min at 3000 rpm. 6 ml of supernatant was A. Material transferred to a glass vial, evaporated to dryness in a stream of nitrogen, reconstituted in 0.2 ml of The materials used in this study were samples of acetone and analyzed. the dry fermented sausages (Petrovská klobása). The basic formulation of sausage mixture The analysis of PAHs was carried out on a GC (Petrovská klobása) was: lean pork meat (85%) 6890N gas chromatograph coupled to a MS and solid fat pork tissue of the back and neck 5975 mass spectrometer (Agilent, Palo Alto, CA, (15%). Other ingredients added per kg of pork USA). The separation of PAHs was performed meat and fat were: red hot spice pepper powder, on Agilent HP-5 MS column (30 m x 0,25 mm x salt, garlic, cumin, sugar, and then the mixture 0,25 µm), injection temperature was 250 °C, of sausages were mixed manually. Sausage injection volume 5 µL (splitless). Helium with a mixture was immediately stuffed in natural flow of 2.6 mL/min was used as carrier gas. The casings – N (pig colon 400-500 mm long and following temperature programme was applied: 45-55 mm in diameter) collagen casings - C isothermal at 70 °C for 2 min, at 25 °C/min to (500 mm long and about 55 mm in diameter). 150 °C, at 3 °C/min to 200 °C, and at 8 °C/min Production the mixture of sausages and smoking to 280 °C. The identification of PAHs was were realised in a registered craft object in Bački performed by mass spectrometry with quadruple Petrovac. Smoking was done on traditional way. mass filter, working in the electron impact (EI) In the smokehouse the smoke was produced by positive ion mode. The temperatures of the the combustion of sweet cherry wood, with the source and the transfer line were 230°C and distance of 3 m between fire and sausages, 280 °C, respectively. Temperature of the which came in direct contact with the smoke quadruple was 150 °C. Acquired data were (direct smoking). The temperature and humidity analyzed using Extracted Ion Chromatogram in the smokehouse were not controlled, but were (EIC) from the total ion chromatogram (TIC) in under the influence of outdoor conditions, i.e. SIM and SCAN mode. temperature was from 1.8 to 16.6°C, and the humidity was from 54.4 to 95.5%. The full Detection limits of the PAHs were 0.6µg/g for smoking process lasted about 10 days with pause. benzo[g,h,i]perylene, 0.5µg/g for dibenzo[a,h] Samples for the analyses were taken at the end anthracene, 0.4µg/g for anthracene, pyrene, of drying process at 60th day. Samples were chrysene and indeno[1,2,3-cd]pyrene and homogenized and stored in dark at – 20 °C until 0.3µg/g for all other PAHs. analyzed. All determinations were made in three samples from each batch in duplicate. 59th International Congress of Meat Science and Technology, 18-23rd August 2013, Izmir, Turkey One way, Post-hoc (Duncan test) was performed New scientific opinion adopted by the European using the software package Statistica 9.1 for Food Safety Authority concluded that BaP alone is Windows, Stat Soft, Tulsa, Oklahoma, USA. not a suitable indicator for the occurrence and Differences were considered significant at toxicity of PAHs in food and that the eight P<0.05. specified PAHs (PAH8) including BaP, BaA, BbF, BkF BgP, CHR, DhA and IcP, for which oral III. RESULTS AND DISCUSSION carcinogenicity data are available, and/or a subgroup of these, PAH4, including BaP, BaA, Content of thirteen EPA polycyclic aromatic BbF and CHR are more suitable markers [18]. The hydrocarbons in traditional dry fermented sausage concentrations of priority PAH8, PAH4, 7US-EPA Petrovská klobása was determined using GS-MS PAHs and 6IARC PAHs were below the limit of method for analysis. The results of content 13 US- detection in all samples of sausages at the end of EPA priority PAHs in dry fermented sausage with drying. Petrovská klobása with natural pig colon (N) and collagen (C) at the end of drying period are given Table 1 Content of polycyclic aromatic hydrocarbons in Table1. (µg/kg) in dry fermented sausage with natural (N) and collagen (C) casings at end of drying period Content of pyrene, anthracene, acenaphthylene, Type of casings Polycyclic aromatic hydrocarbons fluorene and phenanthrene was significantly (µg/kg) Natural Collagen (P<0.05) lower in sausage with collagen casings N C b a (2.7; 12.8; 10.1; 11.5; 19.1, respectively) than in Acenaphthylene Acy 34.9 10.1 b a the sausage with natural casings (8.3; 16.0; 34.9; Fluorene Fln 38.0 11.5 b a 38.0; 39.9, respectively).
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