Determination of Fatty Acid Profile and 3-Monochloropropane-1,2-diol (3-MCPD) Levels in Bakery Products

NESciences, 2019, 4(2): 114-124 -RESEARCH ARTICLE- Determination of Fatty Acid Profile and 3-Monochloropropane-1,2-diol (3-MCPD) Levels in Bakery Products Şana Sungur*, Ender Azak Hatay Mustafa Kemal University, Science and Letters Faculty, Department of Chemistry, Hatay, Turkey. Abstract Thermally processed foods and refined oils are the most significant sources of 3monochloropropane-1,2-diol (3-MCPD) fatty acid esters. The International Agency for Research on Cancer (IARC) classified 3-MCPD as a “possible human carcinogen (group 2B)” and the UK Food Advisory Committee has recommended reducing its level to minimum in foods. In this study, firstly the fatty acid contents of many foods such as cakes, biscuits, waffles, chocolates, cookies products consumed frequently in daily life were determined by GC-MS. The main fatty acids were determined as palmitic acid and stearic acid. The amounts of 3-MCPD esters were found to be between 0.06 and 0.60 mg kg-1, and the amounts of glycidyl esters were found to be between 0.07 and 8.80 mg kg-1. Keywords: Fatty acid, 3-monochloropropane-1,2-diol (3-MCPD), bakery products, GC-MS. Article history: Received 22 February 2019, Accepted 04 April 2019, Available online 16 May 2019 Introduction 3-monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant formed by heat as a reaction product of triacylglycerols, phospholipids or glycerol and hydrochloric acid in fat-based or fat-containing foods. It occurs in foods in its free (diol) form as well as in the bound esterified (with fatty acids) forms. Depending on the type of food it may occur as a free substance, in the form of an ester with fatty acids or in both forms (Svejkovska et al., 2004). * Corresponding Author: Şana Sungur, e-mail: Natural and Engineering Sciences 115 Toxicological animal studies have shown that the main target organ for 3-MCPD toxicity is the kidney, with chronic oral exposure resulting in nephropathy and tubular hyperplasia and adenomas (JECFA, 2002). The International Agency for Research on Cancer has classified 3MCPD as a “possible human carcinogen (group 2B)” (IARC, 2012). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established for the free compound a provisional maximum tolerable daily intake of 2 µg/kg b.w. Recent studies showed that fatty acid esters of 3-MCPD, i.e., bound 3-MCPD, were found in various foodstuffs such as pickled olives, roasted and green coffee, crisp bread, soda crackers, potato crisps and French fries, salami, certain types of ham, smoked and pickled fish, as well as some cheeses. 3-MCPD has been shown to release under certain processing conditions, such as enzymatic hydrolysis by bakery grade lipase during the baking process (FAO/WHO, 2007). A number of studies reporting levels of 3-MCPD esters in different food commodities have been published in the literature. High levels of 3-MCPD esters have been reported in edible refined plant oils and fats; especially palm oil, for which levels up to 10 mg kg -1 (Weiβhaar, 2011). Coffee creamers, cream aerosols and bouillon cubes presented concentrations in ranges of 130–730, 50– 730 and 380–670 µg kg−1, respectively (Karsulinová et al., 2007). In fried potato products, the amount of 3-MCPD esters was 27–64 µg kg−1 in pre-fried French fries, 100–258 µg kg−1 in fried French fries and 98–2201 µg kg−1 in potato crisps (Ilko et al., 2011). Moreover, levels between 62 and 588 µg kg−1 were reported in infant formula (Zelinková et al. 2009). Samaras et al. (2016) were detected 250 µg kg−1 3-MCPD in waffle samples while 3-MCPD levels were found in the range of 29 – 470 ng g-1 in cookies by Becalski et al. (2015). The aim of the study is to determine fatty acid profile and 3-MCPD levels in bakery products sold in Turkish markets. Materials and Methods Reagents and standards 3-monochloropropane-1,2-diol (98%, 3-MCPD) and d5-3-MCPD (98%) were purchased from Sigma Aldrich (Gillingham, UK). All chemicals were obtained from Merck (Darmstadt, Germany). All chemicals used were of analytical grade and were at least 99.5 % pure. Total fat and fatty acid analysis Total fat (TF) was determined gravimetrically by extraction with petroleum ether at 65–80 ºC (Official Methods 960.39) (AOAC, 1999). The extracted lipid residue was dried at 40 ºC under a stream of nitrogen. For the preparation of fatty acid methyl esters (FAME), a cold method with hexane and 2N KOH in methanol (Bannon et al., 1982) was used. FAME were quantified using a gas chromatograph (Shimatzu QP-2010, Shimadzu Corporation, Kyoto, Japan) fitted with a capillary column Rt-2560 (fused silica), (100 m x 0.25mm id) and flame ionization detector. The oven was held at an initial temperature of 60 ºC for 2 min, then increased to 220 ººC at the rate of 3 ºC per minute, the end temperature of 220 ºC held for 12 minute. The entire time of analysis was 67 minute (TSE 4664 EN ISO 5508). Natural and Engineering Sciences 116 Determination of 3-MCPD and glycidyl ester contents 3-MCPD and glycidyl ester were determined according to the DGF method C-VI 18 (10) which is based on alkaline trans esterification. One analysis was comprised of two assays (A and B). For each assay, about 100 mg of sample was weighed in a tube. After adding 250 µL of methyl tertbutyl ether (MTBE) and 100 µL internal standard solutions, the sample was shaken vigorously. For the saponification of MCPD and glycidyl esters, 350 µL of a methanol/NaOH solution was added. The sample was shaken slowly for 10 minutes. The assay A reaction was quenched with 600 µL of acidic NaCl solution while a chlorine free NaBr solution was used for assay B to avoid the formation of additional MCPD from glycidol. The following preparation steps were similar for both assays. After the addition of 600 μL hexane, the sample was vigorously shaken and incubated for 10 minutes. The sample was again shaken vigorously and the organic hexane layer was dispensed to waste. This step was repeated twice to remove matrix. Free 3-MCPD was extracted by 600 μL MTBE/EtAc (3/2 v/v). The extract was collected in a new 2 mL vial pre-filled with sodium sulfate as drying agent. After adding 30 μL of phenylboronic acid the sample was evaporated to dryness. The phenylboronic acid derivates were redissolved in isooctane and transferred to a new vial with μ-vial insert ready for injection. The fact that phenylboronic acid was not very soluble in isooctane helps reduce the amount of derivatization agent injected. The evaporation step was therefore used both to increase the sensitivity of the analysis and also to remove excess phenylboronic acid in order to protect the MSD. Quantification was performed in Agilent 6890 system equipped with Rxi-17 sil ms (Restek) column (30 m x 0.25 mm x 0.25 µm) (Agilent Technologies, USA). Analysis conditions: MPS: 3 μL injection volume PTV: baffled liner, deactivated solvent vent 40°C (0 min); 12°C/s; 300°C (5 min) Pneumatics: He, constant flow = 1 mL/min Oven: 50°C (2 min (...truncated)