Food Research International 33 (2000) 833838

www.elsevier.com/locate/foodres

Hydroxymethylfurfural and methylfurfural content of selected bakery products

nez , Bele n Garca-Villanova , Eduardo Guerra-Herna ndez * Antonio Ramrez-Jime
n y Bromatologa, Facultad de Farmacia, Universidad de Granada, Departamento de Nutricio Campus Universitario de Cartuja, 18012 Granada, Spain Received 27 January 2000; accepted 3 April 2000

Abstract Hydroxymethylfurfural (HMF), methylfurfural (MF) and colour (100-L*) were determined in nine common varieties of selected bakery products from Spain. Identical sample preparation and HPLC conditions were used to determine HMF in all bakery products. Recovery of HMF was 93.3%. The HMF values ranged from 4.1 to 151.2 mg/kg. The colour index (100-L*) ranged from 23.1 to 42.9. No linear correlation was found between HMF and colour. MF was determined by gas-liquid chromatography. The precision was 12%. The MF values ranged from 1.6 to 11.5 mg/kg. Fried products showed higher values than baked products. Linear correlation was obtained (r2=0.875) between HMF and MF for baked products. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Bakery; HMF; MF; Colour

1. Introduction In Spain, bakery speciality products constitute 18% of the total consumption of products manufactured mainly with cereals (Saura-Calixto & Gon i, 1993). The products included in the present study, such as croissants, doughnuts, etc., represent an important group of this type of product. They are made with baked or fried fermented dough. The basic ingredients are our, fat, sugar, eggs, milk, yeast, avour and additives. The manufacturing process involves three steps: dough-mixing, dough fermentation and baking or frying (Humanes Carrasco, 1993). The chemical reactions involved in this process are essentially the Maillard reaction and caramelization. The Maillard reaction is favoured in foods with a high protein and carbohydrate content and an intermediate moisture content at temperatures above 50 C and at a pH of 47 (Kroh, 1994), producing changes in colour (melanoidins), avour (aldehydes and ketones), functional properties and nutritional value (blocking or
* Corresponding author: Tel.: +34-958-243867; fax: +34-958243869. ndez). E-mail address: ejguerra@ugr.es (E. Guerra-Herna

destruction of lysine) (O'Brien & Morrisey, 1989; Reineccius, 1990). Caramelization (degradation of sugars) needs more drastic conditions (temperatures >120 C, pH<3 or pH>9 and low Aw) (Kroh). Water content distribution and temperature play an important role in developing the sensory characteristics of these products. During baking, the water content on the surface of the loaf becomes lower than in the middle and this, combined with the high temperature, is one of the factors that makes the crust dierent from the crumb (Thorvaldsson & Kjjo ledbrand, 1998). Hydroxymethylfurfural (HMF) is an intermediate product in the Maillard reaction (Berg & Van Boekel, nez-Pe rez, 1997) and is 1994; Morales, Romero & Jime also formed from the degradation of sugars at high temperatures (Kroh, 1994). This indicator has been analysed in commercial baby and breakfast cereals (Garca-Villa ndez, Martinez-Gomez & Montilla, nova, Guerra-Herna ndez, Garca-Villanova & Montilla1993; Guerra-Herna mez, 1992; and has been used to control pasta drying Go (Resmini, Pellegrino, Pagani & De Noni, 1993), baby ndez-Artigas, Guerra-Herna ncereal processing (Ferna dez & Garca-Villanova, 1999) and sliced bread toasting nez, 1998). Brown pigment formation is (Ramrez-Jime desirable during bread-making.

0963-9969/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0963-9969(00)00102-2

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nez et al. / Food Research International 33 (2000) 833838 A. Ramirez-Jime

Furfural and Methylfurfural are also furanic compounds formed in non-enzymatic browning reaction during thermic treatments. They have been determined in fruit juices (Sun, Severson & Kays, 1993), wines (Guerra ndez, Montilla & Garca-Villanova, 1990), beer Herna (Harayama, Hayase & Kato, 1994), coee (Kanjahn, Jarms & Maier, 1996) and infant formula (Albala-Hurtado, Veciana-Nogues, Izquierdo-Pulido & Vidal-Carou, 1997). In model systems with gluten, starch and glucose heated at 150 C for 1 h under atmosphere-simulating aerobic and anaerobic baking conditions, furanic compounds were identied, especially furfural and methylfurfural, among other avour substances (Berry & Gramshaw, 1988; Izzo & Ho, 1993). The brown colour index in solid products has been applied to study the modelling of bread-crust browning kinetics during baking (Zanoni, Peri & Bruno, 1995), the toasting of wheat nez, 1998; Rychlik & Grosch, 1996) bread (Ramrez-Jime and the twin-screw extrusion of corn and soy products (Konstance et al., 1998). The objectives of the present study were to develop a method of methylfurfural determination in bakery products and to determine the HMF and 100-L* (colour index) values of an important group of selected bakery products. 2. Materials and methods 2.1. Samples Nine common bakery speciality products from Spain were studied. Information on their ingredients, formulation and processing conditions was provided by the company that manufactured them. The dough formula used by the company for each product, identied here by a letter, was as follows. Products A, B, C and D: baking our (5 kg), water (2 l), previously fermented dough (1 kg), baker's yeast (500 g), lard (1 kg), sucrose (1 kg), liquid egg (500 ml), salt (20 g), additives, plus margarine (1.5 kg) for product C; product E: doughnut our (our, vegetable fat, salt, sucrose, starch, preservatives and natural avours) (5 kg), water (2.2 l) and baker's yeast (450 g); products F and G: baking our (5 kg), sunower oil (3 l), water (2 l), previously fermented dough bread (1 kg), baker's yeast (500 g), sucrose (500 g), raisins (500 g), anise seed (150 g), sesame seed (100 g), salt (150 g) and dough-conditioning agents (50 g); product H: baking our (5 kg), water (2.7 l), lard (4 kg), sucrose (2 kg), previously fermented dough bread (500 g), baker's yeast (200 g) and salt (100 g); product I: baking our (5 kg), sunower oil (2.5 liters), water (2 l), liquid egg (2.5 l), sucrose (3.2 kg), leavening agent (175 g), powder milk (175 g), lemon essence. The fat used for fried products (D and E) was a mixture of palm fat and soy oil. The description of samples is as follows:

. Product A: weight 25 g, oval and smooth form; fermentation 3035 C/30 min; baking 220 C/810 min, moisture 13.6%. . Product B: weight 60 g, oval form with central ssure; fermentation 3035 C/4560 min; baking 220 C/ 1012 min, moisture 19.8%. . Product C (croissant): weight 60 g, crescent form; fermentation 3035 C/60 min; baking 180200 C/ 1820 min, moisture 16.8%. . Product D: weight 50 g, cylindrical form; fermentation 3035 C/25 min; frying 160 C/15 s, moisture 21.1%. . Product E (doughnuts): Weight 60g, ring-shaped form; fermentation 30-35 C/30 min; frying 160 C/ 3040 s, moisture 25.2%. . Product F: the fermented dough is varnished with liquid egg before baking. Weight 250 g, stick form; fermentation 3035 C/30 min; baking 200 C/30 min, moisture 18.8%. . Product G: the fermented dough is varnished with liquid egg and sucrose is added on the top before baking. Weight 250 g, round at form; fermentation 3035 C/30 min; baking 200 C/30 min, moisture 20.5%. . Product H: sucrose is added on the top before baking. Weight 100 g, square form; fermentation 30 35 C/60 min; baking 220 C/1012 min, moisture 19%. . Product I (``biscuits''): weight (1000g), rectangular form. No fermented product, baked 180 C/90 min, moisture 14%. All the samples were stored at 50 C. Before analysis the products were thawed at room temperature for 3060 min according to the sample size. The sample was then divided into very small parts and left at room temperature overnight to reduce the water content. The samples were then ground and passed through a 500 mm sieve. 2.2. Colour determination The colour of samples was measured using the CIE L* a* b* colour system, where L* is lightness, a* is redness and b* is yellowness. The instrument used was a reectance spectrophotometer Elrepho 2000 (Datacolor S.A., Spain). The colorimetric parameters L*, a*, b* were referred to illuminant D65 and the instrument was calibrated using a BaSO4 standard. The samples were lyophilised prior to the analysis. Duplicate analyses were performed on the ground samples. 2.3. HMF determination 2.3.1. Reagents Analytical reagent grade chemicals were used. The claried solution was composed of 15% potassium

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ferrocyanide (w/v) (Merck, Darmstadt, Germany) (Carrez I) and 30% zinc acetate (w/v) (Merck, Darmstadt, Germany) (Carrez II). A standard stock solution containing 200 mg/l 5-(hydroxymethyl)furfural (Merck, Darmstadt, Germany) was used to prepare the working standard solutions (0.020.5 mg/l). 2.3.2. Apparatus The liquid chromatographic system used in this study consisted of a Konic model 500A (Barcelona, Spain) with a 20 ml injection loop chromatograph, a Spherisorb S5 ODS2 (25040 mm i.d.) column (Sugelabor, Madrid, Spain), a UV Konic detector model 200 UVIS (Reno, NE) set at 284 nm and Hewlett Packard integrator model 3394A (Avondale, PA). The mobile phase was water acetonitrile (95:5) (Panreac, Barcelona, Spain). 2.3.3. Procedure The HMF determination was performed following the method of Garca-Villanova et al. (1993). The ground sample (0.4 g) was weighed into a 10 ml centrifuge tube to which 7 ml of deionised water were then added. The centrifuge tube was shaken vigorously for 1 min and the sample was then centrifuged for 10 min at 5000 rpm. The same procedure was followed twice more. The supernatants were claried with 0.5 ml each of Carrez I and II solutions. The resulting mixture was centrifuged for 10 min at 5000 rpm. The solution was diluted to a total volume of 25 ml with deionized water. A 2 ml aliquot of this solution was ltered through an 0.2 mm disk lter before injection. 2.3.4. Chromatographic conditions Twenty ml of ltered solution were separated in a reversed-phase C18. The ow rate was 1 ml/min. The HMF was completely separated out in 8 min and the run time was 15 min. The external standard method was used for the calibration. The HMF solution concentrations and the height of the peak obtained were considered as the variables to obtain the linear regression equation. The concentration ranges were 0.020.5 mg/l. The linear regression equation used was (n=7) Y=292.17X 0.27, where Y is the peak height and X is the HMF concentration. The correlation coecient was 0.9999. Duplicate samples were analysed. 2.4. 5-Methylfurfural determination 2.4.1. Reagents Analytical reagent chemicals were used. The claried solution was composed of 15% potassium ferrocyanide (w/v) (Merck, Darmstadt, Germany) (Carrez I) and 30% zinc acetate (w/v) (Merck, Darmstadt, Germany) (Carrez II). A standard stock solution containing 500 mg/l 5methylfurfural (Merck, Darmstadt, Germany) was used

to prepare the working standard solutions (10500 mg/l). The internal standard was a solution of borneol (DGF, Granada, Spain) (15 mg/l borneol in dichloromethane). 2.4.2. Apparatus Separation and determination of 5-methylfurfural was performed using Perkin-Elmer capillary gas chromatograph model Sigma 3B with ame ionisation detector (FID) (Norwalk, CT) and Perkin-Elmer data station model Sigma 15 (Norwalk, CT). 2.4.3. Procedure Two g of ground sample was weighed into a 50 ml centrifuge tube to which 25 ml of deionized water were then added. The tube was shaken vigorously for 1 min and the sample was then centrifuged for 10 min at 5000 rpm. The procedure was then repeated. The combined supernatants were claried with 0.5 ml each of Carrez I and II solutions and centrifuged for 10 min at 5000 rpm. The supernatant was extracted twice with 50 ml of dichloromethane and concentrated by the Kuderna Danish system to 2 ml and then passed under a nitrogen stream to remove the solvent. The residue was dissolved with 0.2 ml of internal standard solution. A volume of 1 ml of extract was injected into a methyl silicone capillary column (C-382 25QC2/SGL-1) (25 m 0.25 mm i.d., 0.25 mm lm thickness) (Sugelabor, Madrid, Spain). The GC setting was: injector and detector temperature 250 C; temperature/time program: start at 40 C (5 min), increasing 35 C/min up to 100 C, then at 100 C for 10 min and increasing 35 C/min up to 180 C, then at 180 C for 5 min. The ow rate of the carrier gas (nitrogen) was 1.5 ml/min. The gas chromatography system had a split ratio 1:20. The 5-methylfurfural was completely separated out in 7 min and the run time was 20 min. The internal standard method was applied for the calibration. The calibration of the chromatograph was carried out by the addition of 5-methylfurfural standard solutions on sample H, in which no 5-methylfurfural was detected, increasing quantities of methylfurfural standard within the range expected in the samples. The 5methylfurfural/borneol area and concentrations were considered as the variables to obtain the linear regression equation. The range of 5-methylfurfural assayed was between 10 and 500 mg/l and the samples were within this range. The linear regression equation used was (n=6), Y=0.01233+9.3988 103X where Y is the ratio of area methylfurfural/borneol and X is the mg/l 5methylfurfural concentration. The correlation coecient was r2=0.9999. All the extracts were duplicated and injected twice. 2.5. Moisture determination Moisture was obtained by gravimetric determination (AOAC, 1990).

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2.6. Statistical analysis Analysis of linear regression test used a Sigma package supplied by Horus Hardware S.A., Madrid, Spain. 3. Results and discussion Fig. 1 depicts the HPLC chromatograms for the HMF standard and the HMF from sample A. The same method and chromatographic conditions were applied to all the dierent samples with no need for modications. The identity and purity of the HMF was conrmed by diodearray. Recovery was determined by the standard addition procedure. HMF standard was added to sample E. Six addition levels were used between 17.4 and 154.9 mg/ kg. The recovery ranged between 90.1 and 97.8 (Table 1 ) and the mean value was 93.33.06%. Study of the precision of HMF method in similar products (breads) with low and high HMF concentrations showed coecients of variation of 1.57 and 2.60%, nez, 1998). respectively (Ramrez-Jime Table 2 shows the HMF contents. The values ranged from 4.1 mg/kg (product F) to 151.2 mg/kg (product A). Products A, B and C were made with the same

dough and fermentation time and baking conditions proportional to their weight. However, wide HMF differences were found between product A (151.2 mg/kg) versus products B (41.4 mg/kg) and C (39.9 mg/kg). The addition of pasteurised liquid egg on the surface of product A could partly account for this result. Products E and D showed values of 10.7 and 17.1 mg/ kg, respectively. Both products were fried rather then baked; the frying time was between 15 and 35 s. Products F and G are made with similar dough, weight, fermentation and baking characteristics. The HMF value was 4.1 mg/kg for product F and 14.2 mg/ kg for product G. The dierent crust/crumb proportion of the two products may explain this dierence. Studies on HMF values of derivative cereals have also shown variability. They ranged between 0.4 and 65.5 mg/ ndez-Artigas et al., 1999; kg for infant cereals (Ferna ndez et al., 1992), 3.7 and 193.3 mg/kg for Guerra-Herna breakfast cereals (Garca-Villanova et al., 1993), 2.2 and nez, 1998) and 0.08 87.7 mg/kg for breads (Ramrez-Jime and 7 mg/kg for pasta drying (Acquistucci & Bassotti, 1992; Resmini et al., 1993). The colour index (100-L*) for the bakery speciality products in the present study ranged between 23.1 and 42.9 (Table 2). The reproducibility of the refractometer colour method in bread products has been reported to nez, 1998). be 0.30% (CV) (Ramrez-Jime
Table 1 HMF recovery of product E Added 17.4 34.8 69.7 92.9 123.9 154.9
a b c

Totala 28.1 45.5 80.4 103.6 134.6 165.6

Detectedb 26.3 42.1 76.7 94.3 131.7 149.2

Recovered %c 93.6 92.5 95.4 91.0 97.8 90.1

The product E contained 10.7 mg/kg HMF. Mean of three samples with two injections per sample. MeanS.D. 93.33.06.

Table 2 HMF, MF and colour in bakery speciality products Product A B C D E F G H I Fig. 1. HPLC and GLC chromatograms of (A) HMF standard, (B) HMF from sample A, (C) MF standard and (D) MF from sample D.
a b

HMFa 151.2 41.4 39.9 17.1 10.7 4.1 14.2 9.5 15.6

MFa 6.5 3.9 3.9 6.2 11.9 n.d.b 1.8 n.d. 1.6

100-L* 34.4 28.8 37.6 40.5 32.6 35.0 27.0 23.1 42.9

mg/kg of dry matter. n.d., not detected.

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The 100-L* parameter was considered by Resmini et al. (1993) and Fernandez-Artigas et al. (1999) as a browning index in the control of pasta-drying and ourtoasting. No linear correlation was found between HMF and 100-L* (r2=0.004). The variability of the ingredients used to manufacture these products, and their contribution to the colour may explain the lack of correlation between thermic treatment and colour. However, this phenomenon was not observed in bread products. The common breads showed a linear correlation of r2=0.9023. nez, 1998). (Ramrez-Jime The analyses of other furanic compounds (furfural and methylfurfural) showed quantiable methylfurfural values and traces of furfural. The GLC chromatograms of the methylfurfural standard and the methylfurfural from sample D are depicted in Fig. 1. The precision of the methylfurfural study was tested on sample D and the coecient of variation was 12%. Table 2 shows the methylfurfural content. The values ranged from 1.6 to 11.9 mg/kg. Methylfurfural was not detected in samples F and H. The fried products (D and E) showed higher values than the baked products. The baked product A had the highest value of HMF and also a high value of methylfurfural. The fried products were heated at 170 C for 1530 s and the baked products at 200 C for 1015 min. The time and temperature conditions of baked products could favour the loss of methylfurfural due to volatility. No methylfurfural was detected in a study of nez, 1998). Fura wide variety of breads (Ramrez-Jime fural and methylfurfural were the main avour components in model systems of gluten, starch and glucose heated at 150 C for 1 h (Berry & Gramshaw, 1988; Izzo et al., 1993). In the present study, a linear correlation was found between HMF and MF in baked products (r2=0.875) but no correlation was obtained (r2=0.088) when fried products were considered. In conclusion, the method developed for methylfurfural determination proved adequate in this setting. A wide range of HMF and 100-L* values may be determined in selected bakery products. The furanic compounds (HMF and MF) show correlation in these products. Acknowledgements The authors would like to thank ``Alcampo'' market for providing the samples and Richard Davies for assisting with the translation.

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