MENDELNET 2016 HYDROXYMETHYLFURFURAL IN SYRUPS, DOUGHS AND IN SYRUP´S BISCUITS MARCELA JANDLOVA, JINDRISKA KUCEROVA Department of Food Technology Mendel University in Brno Zemedelska 1, 613 00 Brno CZECH REPUBLIC [email protected] Abstract: Hydroxymethylfurfural (HMF) is formed by dehydration of sugar and Maillard reaction during storage and food processing. The aim was to determine the amount of HMF in selected syrups, honey, cane molasses and sucrose, and then in doughs and biscuits made from these sweeteners. Biscuits were baked at 175 °C, 200 °C and 225 °C. In doughs was measured pH. The lowest specified value of HMF was in biscuits with sucrose, the dough with sucrose had the highest value of pH. Key Words: HMF, bakery products, corn syrup, wheat syrup, date syrup INTRODUCTION HMF is highly reactive crystalline colorless solid, in the air immediately browns, causes brown or yellow-brown color in the product. HMF respectively its reaction products exhibit slightly "fruity odor" (Bacílek and Kamler 2009). HMF is well soluble in water, ethanol, methanol and ethyl acetate, and less soluble in petroleum ether. Absorption maxima of HMF are at 284 nm and 230 nm (Gökmen and Morales 2014). HMF is formed in Maillard reaction and dehydration of sugars during caramelization. Maillard reaction is non-enzymatic browning, which leads to a chemical reaction between a reducing sugar and an amino acid at high temperature. HMF occurs in processed foods. HMF molecule consists of furan ring, a hydroxymethyl group and a carbonyl group (Gökmen and Morales 2014). The main precursors for the formation of HMF are amino acids and sugars, particularly hexoses. HMF is formed in carbohydrate´s foods such as jams, fruit juice concentrates and honeys. Increasing temperature during storage or processing is leading to the faster formation of HMF. It is therefore appropriate to reduce the temperature e.g. using a vacuum residue or biscuits. HMF quantity is increased at lower pH. Therefore, temperature should be optimized during the storage and processing of acidic foods, to reduce the amount of HMF. The amount of HMF is increased with the length of storage and HMF formation is accelerated with low or average moisture (Gökmen and Morales 2014). HMF is formed more in bread, which contains glucose, than sucrose. Also the formation of HMF is higher in more acidic dough and leading to intensive surface browning (Gökmen et al. 2007). Fructose is degrading (to HMF) more rapidly in comparison to sucrose and glucose (Přidal 2013). Hydroxymethylfurfural is probably toxic and mutagenic substance (Velíšek and Hajšlová 2009) In living organisms. HMF is converted to 5-sulfoxymethylfurfural (SMF) which is genotoxic (Capuano and Fogliano 2011). HMF short study on the creation of cancer in the intestinal tract can not be clearly labeled as carcinogenic HMF. Daily dietary intake of HMF is in the hundreds mg/kg, which is much higher than the income of other toxic food substances. Intake of HMF is estimated 4 to 30 mg/person/day. Intake above 350 mg/person /day is possible, e.g. consumption of prune beverages. In experiments it was found that the amount of 80–100 mg of HMF/kg body weight/day showed no adverse effect in experimental animals. Of the mentioned knowledge seems to be the current exposure hydroxymethylfurfural for humans safe (Abraham et al. 2011). HMF is included in U.S. Public Health Service's National Toxicology Program on sheet for Toxicological studies (Rupp 2003). The amount of HMF is recorded only in honeys. According to the Czech Decree no. 76/2003 Coll. the current limit of HMF with floral and honeydew honeys is 40 mg/kg, and for honeys from areas with 577 | Page MENDELNET 2016 tropical climates and their mixtures is 80 mg/kg. The amount of HMF in honey is an indicator of the age of honey, honey heating and poor storage conditions. The requirements for the amount of HMF in honey are incorporated in the legislation of the European Union, and in the Codex Alimentarius (Zappala et al. 2005). The quantity of HMF occurring in honey, is monitored for quality reasons. HMF is highly toxic to bees, wherefore bees must be supplementary feeding sugar of good quality and no overheated honey, in which is higher levels of HMF (Přidal 2013). Bee feeding of hydrolyzed sucrose catalyzed by acid is danger to bees, because it contains HMF. In contrast, the enzymatic hydrolysis of sucrose doesn´t arise HMF (Titěra 2009). HMF quantities in some foods: biscuits from 0.5 to 74.5 mg/kg, white bread 3.4 to 68.8 mg/kg, breakfast cereals from 6.9 to 240.5 mg/kg, honey 10.4–58.8 mg/kg, dried fruit from 25 to 2900 mg/kg, marmalade from 5.5 to 37.7 mg/kg, malt 100 to 6300 mg/kg, instant coffee from 400 to 4100 mg/kg, coffee 100–1900 mg/kg, chicory 200 to 22500 mg/kg, beer from 3.0 to 9.2 mg/l, red wine from 1.0 to 1.3 g/l, balsamic vinegar 316.4 to 35251.3 mg/l (Capuano and Fogliano 2011). The aim was determining the amount of HMF in selected sweeteners, doughs and biscuits. pH test to determine the dependence on the amount of HMF in dough and biscuits and baking temperature dependence of different formation HMF. MATERIAL AND METHODS Sweeteners used: syrups: date, wheat and corn syrup, as well as honey, cane molasses and sucrose. All sweeteners were purchased in the Czech Republic. Dough and biscuits were made at the Department of Food Technology. Used recipe was 40 g of sweetening agent per 100 g of flour. Biscuits were baked at 175 °C, 200 °C and 225 °C. The preparation of sample for the determination of HMF (Zhang et al. 2012): First, solutions Carrez I and Carrez II were prepared. Solution Carrez I: to 15 g K4[Fe (CN)6] · 3H2O (from Lach-Ner, Ltd.) was dissolved in water in 100 mL volumetric flask, and the solution was thoroughly mixed. Carrez II solution: 30 g of ZnSO4 · 7H2O (from Lach-Ner, Ltd.) was dissolved in water in 100 mL volumetric flask. A sample of 1 g was put into a 20 mL centrifuge tube with a cap and 250 µL Carrez I below 250 µL Carrez solution II and 9.5 mL of demineralized water. Centrifuge tube was shaken vigorously on a shaker (450 revolutions/min.) for 5 minutes and subsequently centrifuged for 15 min. at 4000 revolutions/min. and 4 °C. The solution was filtered through a 0.45 µm filter disk, loaded in Eppendorf microtube (1.5 ml) which were sealed and stored in a freezer at -18 °C until determination of HMF by HPLC. The values measured by HPLC were computed in Microsoft Excel 2010 HMF has been measured on high pressure liquid chromatography, HPLC (Agilent 1100 Series) with UV/VIS detection. The column used was an Agilent ZORBAX Eclipse XDB-C18 column size 4.6 x 150 mm, particle size 5 µm, manufactured in the USA. Detection was carried out at a wavelength of 284 nm and 25 °C. Mobile phase of methanol-water (5 : 95; V : V) was used isocratic elution. The mobile phase flow was 1 mL/min, the volume of sample was 10 µL. Data were obtained and processed utilizing Agilent ChemStation software. Standard 5- hydroxymethyl-2-furancarbaldehyd (Merck spol. S r.o.) was used for calibration (0.1–100 µg/mL). Each sample was measured twice, in the case of different results of three to four times with the exclusion of outliers. The pH of doughs was measured by table pH meter HANNA Instruments pH 212. Electrode of pH meter was put in the dough and measured three times. RESULTS AND DISCUSSION The lowest average value of the amount of HMF (Table 1) was found in the sweetener sucrose and the highest measured value was in cane molasses. HMF quantity of the doughs varied from 0–8.06 mg/kg in biscuits from 0.48 to 13.99 mg/kg. Table 1 Average concentration of HMF in sweeteners, doughs and biscuits and pH of doughs 578 | Page MENDELNET 2016 Sweetener pH of dough Concentration of HMF [mg/kg] Sweetener Dough Biscuits Biscuits Biscuits 175°C 200°C 225°C Sucrose 7.19 0.12 0 0.68 0.81 0.48 Honey 6.60 20.24 2.72 9.44 8.96 20.43 Wheat syrup 6.47 4.64 0.68 2.30 2.60 1.90 Corn syrup 6.31 0.47 0 1.67 0.99 3.08 Date syrup 5.79 8.36 1.29 3.50 4.16 6.69 Cane molasses 5.66 41.38 8.06 13.99 10.83 10.39 The amount of HMF and pH were correllated in doughs (R = -0.62), in biscuits (baked at 175 °C R = -0.54; at 200 °C R = -0.53; at 225 °C R = -0.23). Further, the correlation of the HMF and baking temperature for each type of biscuit was estimated separately (with sucrose R = -0.60; honey R = 0.85; wheat syrup R = -0.57, corn syrup R = 0.66, date syrup R = 0.95, cane molasses R = -0.92). Statistical significance has not been found for any of the above mentioned correlation coefficients (R, alpha 0.05). However in the study Vorlová et al. (2006) the amount of HMF in fruit syrups, syrups for preparation of beverages was determined. Average value of HMF's concentration was in fruit syrups 6.6 mg/kg, which is higher than average value for wheat and corn syrup, which we measured.