B. Cie?lak
　　Department of Food Biotechnology, Microbiology and Evaluation, Warsaw University of Life Sciences, Warsaw 02-776, Poland
　　Received: April 14, 2011 / Published: July 20, 2011.
　　Abstract: Bread is still the most important food in Polish households. Staropolski and Pytlowy are two typical Polish breads, prepared with wheat and rye flour. The aim of this study was to examine the occurrence of volatile contaminants such as furan and its derivatives, and present a volatile profile of the most common furan derivatives in the described products. To measure concentration of chemical compounds such as furan and furan derivatives, fast and effective solid-phase micro-extraction/gas chromatography/mass spectrometry (SPME/GC/MS) methods were adapted. This study showed the differences in furan occurrence between Pytlowy and Staropolski, however, the greater differences were observed between different times and temperatures of baking rather than between the bread types. Clearly, the higher concentrations were found in products prepared during the longest baking time period of 50 min, and temperatures of 230 °C and 240 °C. The highest furan level was detected in Staropolski 28.89 ±1.16 μg/kg, baked for 50 min at 230 °C in bread crust. The volatile fraction of bread crust was composed of 5-9 furan derivatives while crumb layer contained only furan-2-pentyl. It was concluded that furan is present in almost each type of bread and percentage of furan derivatives in all volatile fractions ranged from 0.5 % to 60.6% in crust of examined bars.
　　Key words: Furan, furan derivatives, bakery products, bread, SPME/GC/MS.
　　 1. Introduction
　　Preparation of Staropolski and Pytlowy typical Polish breads involves three steps: dough mixing(flours, water, salt and sourdough typical for this kind of product); fermentation and preparing of bread bar; volume increase in the oven and baking. The most important for the occurrence of volatile compounds are two stages: fermentation and baking. Fermentation processes are associated with microorganism metabolism and in consequence provide products of carbohydrate decomposition. More complicated chemical reactions involve thermally-induced bakery processes: gelatinization of starch, denaturation of proteins, transformation of raw material to light, porous and readily digestible product. Two typical substances are specific for bread browning: hydroxymethylfurfural (HMF) and furosine [1]. Both compounds are furan derivatives and belong to bread volatile fraction. An analytical method used to measure their occurrence is HPLC. In headspace of bread there are other volatile furan derivatives with high volatility.
　　Furan is known as highly volatile, heterocyclic compound present in thermally processed food. It was considered as a possible human carcinogen, classified by IARC (International Agency for Research on Cancer) as 2B group member [2]. Cis-2-buten-1,4-dial was identified as a main furan metabolite, which bound cellular peptide fraction and nucleotides [3, 4].
　　The occurrence and formation of furan and its derivatives were reviewed [5] in different kinds of food at the end of 70th. First described furan formation pathways were decomposition of carbohydrates, caramelization, and Maillard’s reactions. Furan
　　 derivatives were also formed as a result of carbohydrate decomposition and Maillard’s reactions, in cases furan derivatives such as furfural were furan precursors. Furan could form from ascorbic acid in model systems[6] and during oxidation of fatty acids [7].
　　Production of some food products includes addition of food flavouring substances. Twenty-five substances considered by EFSA Flavouring Group [8] as food additives are structurally related to the group of 25 furfuryl and furan derivatives. Some of them occurred naturally in food during processing, some were added to final product [8].
　　For the analysis of furan and its derivatives in different food matrices many authors used automated headspace analysis coupled with GC-MS [6, 9-11]. Another very popular method, SPME combined with GC/MS, was also widely used [12, 13].
　　Browning is a chemical reaction that produces proper yellow-gold colour of bread crust during bread baking. Maillard’s reactions, caramelization and decomposition of carbohydrates are very intensive in the browning process. Those reactions are temperature-, pH-, moisture content-dependent, and are also influenced by the presence of metal ions and inner sugar structure [14]. Time and temperature of baking and crust appearance of a bread bar are the main indicators of properly processed bread.
　　Furan level in bread products varied depending on bread type, ingredients and stage of toasting [15]. In a whole bread bar furan amount was in the range between n.d. to 29 μg/kg. In the crust the amount of furan was higher and ranged from 24 μg/kg to 193 μg/kg.
　　Bread as a popular food product is prepared in different countries according to different, local recipes, with various ingredients. Pytlowy and Staropolski are Polish breads, prepared with two types of flours: rye and wheat. The difference is the percentage of flour: Staropolski contains 50%/50% of each kind of flour, Pytlowy contains 83% rye flour to 17% wheat flour(Table 1). Both breads are baked in special form and have a dry crusty dark crust.
　　Table 1 Proportion of dough ingredients per 10 kg.
　　


　　The aim of this study was to measure furan levels in typical Polish breads and analyse furan derivatives formed in the crumb and crust layers of these breads. Furan level was measured according to the description of US FDA [9], furan and its derivatives was calculated in relation to internal standard (1,2-dichlorobenzene).
　　 2. Material and Methods
　　2.1 Reagents and Sample
　　Furan (99.9%), furan-d4 (> 98 atom%), were supplied from Sigma-Aldrich Poland. 1,2-dichlorobenzene (99%) was supplied from POCH Poland.
　　Two typical Polish breads were baked in a Warsaw local bakery. The composition of both breads is presented in Table 1.
　　The thermal process was performed at three different times: 30 min, 40 min and 50 min, and at three different temperatures: 220 °C, 230 °C and 240 °C.
　　Procedures for preparing dough were typical for each product. The procedures were presented on the official pages of Polish Government-Ministry of Agriculture and Rural Development [16].
　　After baking samples were cooled and then placed in special 40 mL sealed vials. Crumb and crust were stored separately at -18 °C, until further analysis, but not longer than 7 days.
　　2.2 Method
　　The method used was a modification of the US FDA[9] method. Cooled samples were stored for minimum 2 h in a refrigerator (4 °C). Then 10 g of sample, 10 mL of water (Milipore 18,2 m?), 0.5 g NaCl, 100 μL of working solution of furan-d4 (5 ng/μL) were added by a syringe, and 1 μL of internal standard (0.01% 1,2-dichlorobenzene in methanol) were added by a syringe to 10 g of each sample. The vial was immediately sealed, and vigorously shaken for 2 min. For furan quantitation usually two ranges of a four and three-point calibration were used, corresponding to 0, 10, 20, 50, 100, 150 ng of furan per vial. For furan derivative percentage point calculation: to relative peak areas were calculated by dividing the peak area of the internal standard. To increase the clarity of figures presented data were standardized to 0 mean and 1 standard deviation.
　　All the analysis was performed in triplicate. The calculations were performed in Statistica 8.0. 2.3 Volatile Compounds Analysis
　　Extraction of volatile compounds was performed at 30 °C by means of SPME (CAR/PDMS fiber). Desorption was for 1 min at 220 °C in the injector of gas chromatograph coupled with mass spectrometer(QP2010, Shimadzu) equipped with ZB-WAX plus capillary column (30.0 m length, 0.25 mm internal diameter, 0.25 μm film thickness, Phenomenex). Injection was in splitless mode at 220 °C. Helium flow was 0.67 mL/min. Oven temperature program was as follows: 40 °C for 2 min, increased at 7 °C/min to 220 °C (2 min). Interface temperature was 220 °C. Ion source temperature was 220 °C. Mass acquisition range was from 35 to 650 m/z. Furan derivatives compounds identification was based on comparison with reference spectra from the libraries (Wiley7N2 and NIST147).
　　Table 2 Furan content in crust/crumb layer of examined bread.
　　


　　Furan content was measured in bread produced in a local bakery, all baked bars were of standard value and acceptable for consumers. Table 2 presents results of furan analysis in separated Pytlowy and Staropolski bread parts as crumb and crust. As it is shown in Table 3 the crust layer contained higher amounts of furan than crumb, ranging from 0 to 28.89 ± 1.16 μg/kg.
　　The level of furan was very different in each type of sample, and an increase in temperature did not correspond to an increase in furan content in each case. Staropolski seemed to be a more stable source of furan than Pytlowy. This suggests that the type of flour used played a role, especially in availability of substrates for furan formation. Wheat flour gave a relatively stable furan increase in response to temperature and time of baking.
　　All furan derivatives were measured using the same analytical procedure as furan, so that the comparison of percentage share in headspace volatile fraction was possible.
　　There were significant differences in the level and composition of furan derivatives between crust and crumb in both breads. The crust layer was the richest in furan derivatives.
　　In the crumb layer there was only furan, 2-pentylwas present. The level of this furan derivative was usually below 1% in volatile fraction. The profiles of furan derivatives composition percentage share are presented in Figs. 1-3 and Table 3.
　　In bread crumb the temperature and the time of baking process seemed to have a comparable effect on the formation of furan and its derivatives.
　　Table 3 Comparison of furan derivatives [%] in examined bread crumb.
　　


　　


　　Fig. 1 Furan and its derivatives in the crumb layer of examined breads-30 min baking time.
　　


　　Fig. 2 Furan and its derivatives in the crumb layer of examined breads-40 min baking time.
　　Profiles of furan and its derivatives in bread crumb were similar for different baking times and bread types.
　　The crust layer of both Pytlowy was similar to Staropolski was richer in furan derivatives than bread crumb. Results of identification and percentage point calculation are presented in Table 4.
　　


　　Fig. 3 Furan and its derivatives in the crumb layer of examined breads-50 min baking time.
　　Table 4 Comparison of furan derivatives [%] in examined bread crust.
　　


　　The comparison Pytlowy and Staropolski showed that the number of furan derivatives including furan was greater in Pytlowy, where nine furan derivatives were identified in three baking conditions: 230 °C/40 min, 230 °C/50 min and 240 °C/50 min. All furan derivatives found in bread crust are described in Table 4. Furan, 2-pentyl- that formed in bread crumb was also present in the crust layer, but the amount was almost fifty times higher than in crumb. Figs. 4-6 show percentage share of furan derivatives including furan in headspace volatile fraction. The dominant compound was 2-furancarboxyaldehyde. Over 25% of 2-furancarboxyaldehyde was measured in Pytlowy crust prepared at 230 °C/50 min and 240 °C/30 min. The second prevalent derivative in products baked for 30 min was furan-2-pentyl, which dominated in Pytlowy crust at the lowest baking temperature of 220 °C.
　　


　　Fig. 4 Furan and its derivatives in the crust of examined breads-30 min baking time.
　　


　　Fig. 5 Furan and its derivatives in the crust of examined breads-40 min baking time.
　　


　　Fig. 6 Furan and its derivatives in the crust of examined breads-50 min baking time.
　　In the crust baked for 40 min the dominant furan derivative was 2-furancarboxyaldehyde, however, the second most abundant one was not so evident, 2-furanmethanol seemed to be typical in products baked at 240 °C.
　　The richest volatile fraction was found in bread crust baked for 50 min, but the dominant compound, 2-furancarboxyaldehyde, reached the maximum value of over 36% at 230 °C. A profile for breads crust baked for 50 min implicated that prolonged baking time had a significant influence on quality and quantity of furan derivatives composition in the volatile fraction.
　　In crumb headspace of examined breads was identified, while in crust a maximum of eight compounds with a furan ring were present. Furan amount was lower than the amount of other furan derivatives such as HMF or 2-furancarboxyladehyde. This could be the result of different pathways of formation of these substances in the same food matrix. Sometimes 2-furancarboxyladehyde was presented as a furan precursor.
　　Furan 2-pentyl was a derivative that disappeared in more drastic conditions of thermally processed bread.
　　Fig. 7 presents the percentage sum of every furan compounds identified during the analysis of Polish breads. The highest value was detected for Pytlowy crust-60.6% and the lowest value for Pytlowy crumb-0.6%. The bread volatile fraction contained higher amounts of furan compounds when the temperature conditions were more drastic during baking, while, the lowest amounts were found in crumb baked at relatively low temperature, with relatively high water content.
　　Differences in furan levels between the crust and crumb layers were a result of different temperature inside the bread bar, where temperature could be in the range of 105 °C to 110 °C [17]. Assessed the percentage share of water in crust layer at 5% points was measured [18]. Differences in the processing temperature between crust and crumb could cause changes in water content, pH and chemical matrix in the same bread bar.
　　There was indicated that furan content of bread also depended on its shape or, on the surface-to-volume ratio. This probably means the smaller the bread the higher the respective furan content. In case of Pytlowy and Staropolski the shape and described above ratio were the same, but because bread was baked in special bakery forms only the upper crust layer was the main source of furan. As a consequence, total amount of furan were lower than in products baked without forms. Another important aspect is that the relationship between shape and surface-to-volume ratio was the same for all samples.
　　The furan richest product measured [15] was“farmer baguette”-193 μg/kg, with the ingredients listed as: wheat flour, sunflower oil and barley malt flour. Those compounds were a rich source of carbohydrates and fatty acids. In contrast, Pytlowy and Staropolski did not content any additional sources of carbohydrates and fatty acids responsible for furan formation and consequently exhibit lower furan values.
　　In the study [19] the highest furan concentration was measured in cake baked for 6 min, while HMF in the same amount was measured after 8 min of baking. This result indicated different pathways of formation of these compounds. HMF identified in some publications as a furan precursor in bread accumulated at a slower rate than 2-furancarboxyaldehyde, which could be associated with a chosen analytical procedure favoring highly volatile substances.
　　A comparison of furan and furan derivatives in crumb and crust of Polish breads indicated that different conditions of baking time and temperature induced different quantity and quality of furan compounds in the same product.
　　2,5-dimethyl-3(2H) furanone 4-hydroxy known as DMHF was described as a sour aroma compound found in bread, especially in wheat bread. There was relation[20] that DMHF levels of 6.0 mg/kg and 6.7 mg/kg in bread depended on the amount of yeast supplemented dough and was dominant in compounds of aroma. In Pytlowy and Staropolski DMHF was not identified.
　　Large number of furan compounds in bread headspace fraction should be confirmed in future investigations.
　　


　　Fig. 7 Sum [%] of furan and its derivatives in volatile fraction of examined breads.
　　In conclusion, furan content was higher in crust than in crumb in the same bread product.
　　Flour type as well as temperature and time of baking could determine furan and furan derivatives levels.
　　In Pytlowy and Staropolski breads volatile compounds fraction contained furan derivatives in the range one-for crumb to nine for crust.
　　Total amount of volatile furan derivatives including furan could represent over 60% of all volatiles, so these compounds could play a significant role to human health.
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