1 DIFRUCTOSE ANHYDRIDES AS QUALITY MARKERS OF HONEY AND 2 COFFEE. 3 A. Montilla1, A.I. Ruiz- Matute2, M.L. Sanz2*, I. Martínez-Castro2, M.D. del Castillo1 4 5 6 7 8 9 1Instituto de Fermentaciones Industriales (C.S.I.C.) 10 2Instituto de Química Orgánica General (C.S.I.C.) 11 c/Juan de la Cierva, 3 28006 Madrid (Spain) 12 13 14 *Telephone : +34 91 562 2900 15 Fax: + 34 91 564 4853 16 Email: [email protected] 17 18 19 20 Abbreviated running head: Difructose anhydrides in honey and coffee 21 22 23 24 25 1 26 ABSTRACT 27 28 Difructose anhydrides (DFAs) are pseudodisaccharides produced by 29 condensation of two fructose molecules by means of caramelization reaction which 30 takes place during heating of sugars or sugar-rich foodstuffs. The aim of this research 31 was to evaluate the feasibility of DFAs as chemical markers of honey authenticity and 32 sugar-roasted torrefacto coffee. DFAs were analysed by gas chromatography coupled to 33 mass spectrometry after conversion to their trimethylsilyl (TMS) derivatives. -D- 34 fructofuranoside-1,2’:2,1’--D-fructofuranoside (DFA7) and -D-fructofuranoside- 35 1,2’:2,1’--D-fructopyranoside (DFA9) can be used as quality markers of honey and 36 coffee. DFA7 and DFA9 were detected in honey added with 5% fructose and sucrose 37 caramels and 15% of glucose caramels. Torrefacto coffees showed DFAs values ranged 38 from 0.195 to 0.570 g/100g whereas only traces were found in natural roasted coffees. 39 Quantities from 0.073 to 0.189 g/100g were measured in blends of natural and 40 torrefacto roasted coffees. A relationship between DFAs content in torrefacto coffees 41 and roasting conditions was observed. In conclusion, this study indicated that DFAs are 42 useful chemical indicators to control honey authenticity and torrefacto coffee roasting. 43 44 45 46 47 KEYWORDS: Difructose anhydrides, caramelization, honey, coffee, authenticity, 48 torrefacto. 49 2 50 INTRODUCTION 51 In recent times, food quality assurance is getting a great importance for 52 consumers, producers and regulatory authorities. Food quality requires the control of 53 nutritional value, sensorial properties, authenticity and safety. Various chemical 54 compounds can be used as quality markers. By definition, good chemical indicators of 55 quality may be considered those natural food components which concentrations are 56 modified during processing such as proteins, fats, or aminoacids; those formed during 57 processing and storage, or those incorporated to foods as contaminants and adulterants 58 (Lee and King, 1996). Carbohydrate breakdown products formed by isomerization, 59 Maillard and caramelization reactions have been widely employed as very good markers 60 of quality in a great number of processed foods (Corzo, Delgado, Troyano, Olano, 1994; 61 Sanz, del Castillo, Corzo, Olano, 2000). 62 Difructose anhydrides (DFAs) are pseudodisaccharides formed during 63 caramelization reaction consisting of two fructose residues and possessing physiological 64 functions (Manley-Harris & Richards, 1997; Saito, Tomita, 2000). This reaction takes 65 place during heating of sugars or food products rich in sugars, and results in a minor 66 volatile fraction and a major proportion of non-volatile components (90-95 %) including 67 DFAs. The relative proportion of DFAs depends on food composition and processing 68 (Defaye, Fernandez, Ratsimba, 2000). DFAs analysis in caramels was first described by 69 Tschiersky and Baltes (1989) and more studies have been conducted to give insight to 70 their chemical structure (Defaye & García-Fernández, 1994; Defaye & García- 71 Fernández, 1995). Methodologies have been settled for analysis of DFAs as suitable 72 tracers of caramelization in food and food additives as caramels, chicory and dehydrated 73 fruits (Defaye et al., 2000). DFA content in caramels prepared from fructose, glucose 74 and sucrose may be determined by gas chromatography-mass spectrometry (GC-MS) of 3 75 their trimethylsilyl (TMS) derivatives (Ratsimba, Fernandez, Defaye, Nigay, Voilley, 76 1999). 77 Honey is a natural food elaborated by honeybees either from nectar of blossoms 78 (nectar honey) or from secretions of living parts of plants or excretions of plant sucking 79 insects on the living part of plants (honeydew honey). Honeydew honeys usually have 80 darker colours than nectar honeys and in many areas are more valued than these ones 81 (Prodolliet & Hischenhuber, 1998). There are evidences of honey frauds by caramels 82 addition to nectar honeys to enhance their colour. Detection of honey adulteration is not 83 always easy and represents a problem to solve. DFA analysis by GC may provide an 84 inexpensive easy adequate approach to be employed for the routine quality control of 85 this food. 86 For coffee companies is very important to check the roasting degree of coffee 87 beans because it strongly affects taste, acidity, body, aroma and flavour. Coffee beans 88 may be roasted following two main ways known as "conventional or natural" without 89 sugar addition and "torrefacto" when sugar is added. The roasting degree in each 90 country is determined by preferences of consumers. In general, North European 91 countries mainly consume light-roasted, American medium-roasted and South European 92 countries dark-roasted coffees; some South American countries, Portugal and Spain 93 mainly consume blends of natural and torrefacto coffees (Maetzu, Andueza, Ibáñez, de 94 Peña, Bello, Cid, 2001). Natural and torrefacto roasted coffee present differences in the 95 volatile fraction perceived by sensorial analysis in the intensities of the nutty, roasty, 96 earthy, burnt and caramel notes (Sanz, Meatzu, Zapelena, Bello, Cid, 2002). Torrefacto 97 roasting also contributes to the brownish colour of the coffee brew (Maetzu et al., 98 2001). 4 99 Very few references related to quality control of torrefacto coffees are available. 100 As it is known, Arabica coffees are more valued by consumers than Robusta coffees 101 because their sensorial properties. Previous studies have observed that torrefacto 102 roasting mask the poor sensorial properties of Robusta coffee (Maetzu et al., 2001) and 103 could be used as a fraud. The influence of torrefacto roasting on the volatile fraction of 104 coffee and its sensorial properties has been described (Sanz et al., 2002); however, 105 studies related to the non-volatile compounds formed by caramelization in this type of 106 coffee have not been conducted yet. Therefore, since caramelization takes place during 107 torrefacto roasting, DFAs may be formed and provide useful information related to the 108 degree of torrefacto roasting and the composition of blends. 109 In our knowledge, DFAs have not been previously determined either in honey or 110 in torrefacto coffee. Consequently, the aim of this research was to find out the feasibility 111 of DFAs as chemical indicators of honey authenticity and control torrefacto roasting. 112 113 MATERIALS AND METHODS 114 Reagents 115 Glucose, fructose, sucrose and phenyl-glucoside were acquired from Sigma 116 (St. Louis, USA). High-purity water was produced in-house using a Milli-Q Synthesis 117 A10 system (Millipore, Bellerica, Mass., USA) and was used throughout. 118 119 Samples 120 Honey samples (n=20): 16 nectar honeys from different botanical origins (citrus, 121 rosemary, heather, acacia, eucalyptus and multifloral) and 4 honeydew honeys were 122 purchased to beekeepers and local markets. 5 123 Coffee samples (n=17): 6 natural (N), 4 torrefacto (T) and 7 coffee blends 124 including 20N/80T (n=1), 30N/70T (n=1) and 50N/50T (n=5), from industrial brands 125 were purchased at local markets. 126 In order to evaluate the influence of roasting on DFAs formation, Arabica coffee 127 beans were roasted under controlled conditions by a coffee company (Café La 128 Mexicana, Rodríguez y Mateus, S.A Madrid) to obtain 3 natural and 2 torrefacto 129 coffees. Natural roasting was carried out at temperatures ranged from 150ºC to 198ºC 130 for 15 min. Torrefacto coffees were obtained adding sugar to Arabica beans during 131 roasting at 203-214ºC for 18 min. In addition, coffee blends (20N/80T (n=1), 30N/70T 132 (n=1) and 50N/50T (n=1)) were produced in the laboratory from commercial coffees 133 having known DFA contents. A comparative analysis of DFAs values obtained for 134 commercial and produced in-house blends was carried out. 135 136 Sample preparation 137 138 Acid caramels: 139 10g of glucose, fructose and sucrose were separately mixed with 0.1g of citric 140 acid and 1mL of water and heated at 160ºC for 150 minutes, 140ºC for 35 min and 141 140ºC for 25 min, respectively. 3mL of additional water were added to each sample at 142 the end of the cooking process and heated for two minutes at the same temperatures. 143 Honey 144 Acacia honey was added with 5, 10 and 15% of caramel. Caramels and honey 145 samples (0.5 g) were diluted to 25mL with 70% methanol, prior to DFAs analysis. 6 146 Coffee extracts 147 Hot water (75ºC, 60 mL) was added to ground coffee (10g), stirred for 5 min and 148 filtered through Whatman no.4 filter paper. 3mL of filtrates were deproteinated and 149 defatted by stirring with 25mL of methanol followed by standing for at least 1h. The 150 clear supernatants obtained were employed for DFAs analysis. 151 152 Analytical methods 153 All analyses were carried out in duplicate. 154 155 Gas chromatography analysis 156 Methanol solutions of caramels (0.5mL), honey (0.5mL) and coffees (2mL) were 157 derivatised, prior to GC analysis. 0.5 mL of 1 mg/mL phenyl--D-glucoside, internal 158 standard, was added to the samples. Afterwards, methanol was evaporated under 159 vacuum at 38-40 C. 2.5% Hydroxylamine chloride in pyridine (350L) was added to 160 the dry samples and kept at 75ºC for 30 min.