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Food Chemistry 153 (2014) 3543

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Food Chemistry
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Physicochemical and biochemical properties of honeys from arid regions


Hosam M. Habib , Fatima T. Al Meqbali, Hina Kamal, Usama D. Souka, Wissam H. Ibrahim
Department of Nutrition and Health, College of Food and Agriculture, United Arab Emirates University, P.O. Box 15551, Al Ain, United Arab Emirates

a r t i c l e

i n f o

a b s t r a c t
This study was conducted to evaluate the quality of 11 honeys from arid regions for rst time, and compare it with 5 different honeys from non-arid regions. Mean values obtained for physicochemical parameters were: pH 4.76 0.55; 17.32 1.8% moisture; 80.95 1.60 Brix sugar; 69.05 4.41% total sugar; 413.81 178.48 lS cm1 electrical conductivity; 17.58 7.68 meq/kg free acidity; 11.05 3.18 meq/kg lactonic acidity; 28.63 9.6 meq/kg total acidity; 12.66 20.39 mg/kg HMF; 0.58 0.03 water activity; and 0.98 0.62 colour intensity. Potassium was the major mineral (1760.54 685.24 mg/kg). All the samples showed considerable signicant variations with reference to their physicochemical and biochemical properties, moreover, the total free amino acids and total carotenoids were 61.13 63.16 mg/100 g and 4.07 10.05 lg/100 g respectively. Acrylamide was detected only in one sample at 2.39 0.22 lg/kg. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 14 July 2013 Received in revised form 3 December 2013 Accepted 9 December 2013 Available online 14 December 2013 Keywords: Honey Colour characteristics Physicochemical Biochemical properties Arid regions

1. Introduction Honey is the natural sweet product produced by Apis mellifera bees from nectar of plants (nectar honey), from secretions of livings parts of plants or excretions of plant-sucking insects of the living part of plants (honeydew honey) (Silva, Videira, Monteiro, Valento, & Andrade, 2009). This natural complex foodstuff is produced in almost every country and largely used as food source. Honey cannot be considered a complete food by human nutritional standards, but it offers potential as a dietary supplement (Silva et al., 2009). Honey mainly contains simple sugars or monosaccharides [of which fructose and glucose are the main components (65%)] and approximately 18% water, (Silva et al., 2009). Proteins, avour and aroma, phenolic compounds (phenolic acids and avonoids), free amino acids, organics acids, vitamins and minerals constitute minor components of honeys (Silva et al., 2009). Honey commercially available varies greatly in quality all over the world. This is largely assessed on the basis of colour, avour and density. Honey composition is inuenced by the plant species, climate, environmental conditions and the contribution of the beekeeper. In general, honey is either monooral or multioral depending on the source of the plant (Andrade et al., 1999; Anklam, 1998; Azeredo, Azeredo, Souza, & Dutra, 2003; Gonzalez-Miret, Terrab, Hernanz, Fernandez-Recamales, & Heredia, 2005). Several types of honeys are produced in arid regions. However, the information available on their chemical and physical properties is limited. Also there has been no particular research to determine

their essential physical, chemical and biological properties such as, mineral, free amino acids, sugar and carotenoid proles in different varieties of honey produced in arid regions. Therefore, the current study was conducted to assess the physicochemical properties composition and biochemical properties of honey samples from arid regions for the rst time, as well as comparing it with different non-arid regions honey samples. 2. Materials and methods 2.1. Materials All of the chemicals and reagents used were of analytical grade, sucrose, fructose, glucose, maltose, formic acid, acrylamide, methanol, phosphoric acid, acetonitrile, bovine serum albumin, hexane, acetone, lutein, cryptoxanthine, zeaxanthine, lycopene, a-carotene, b-carotene, c-carotene and ethyl acetate were purchased from Sigma (St. Louis, MO, USA). FolinCiocalteus phenol reagent, HCl, FeSO4_7H2O, HMF, and standard solutions (1000 mg/l): [aluminum (Al), arsenic (As), sulphur (S), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), phosphorus (P), lead (Pb), vanadium (V), zinc (Zn), calcium (Ca), potassium (K), sodium (Na), magnesium (Mg), and strontium (Sr)], were obtained from Merck (Darmstadt, Germany). AccQ_Tag kit was purchased from (Waters, Miliford, MA, USA). 2.1.1. Honey samples The present study was performed on eleven reputed commercial honey brands from arid regions (8 monooral and 3 heterooral) and ve from non-arid regions (3 monooral and 2

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E-mail address: hosamh@uaeu.ac.ae (H.M. Habib). 0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.12.048

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H.M. Habib et al. / Food Chemistry 153 (2014) 3543

heterooral) (Table 1). Fresh honey samples weighing 2501 kg, packed and sealed in glass bottles, were purchased from a local market, and some samples provided directly by local UAE beekeepers, and stored at 4 C. The samples were diluted 10 time using deionized water and were kept at 80 C and analysed at the earliest in such a way that none of the samples exceeded the storage period beyond 6 months. The honey samples were thawed at ambient temperature before the analyses were performed. 2.2. Methods 2.2.1. Physicochemical analysis 2.2.1.1. Water content, RI, Brix. Water content was determined by refractometry, measuring the refractive index (RI) according to AOAC Methods (AOAC 969.38B, 2003), using a standard model Abbe type refractometer at 20 C. Water content (%) was then obtained from the Chataway table. 2.2.1.2. Water activity. Water activity of liquid honey samples was measured using an Rotronic Hygrolab (Rotronic Instrument Corp. Hauppauge NY, USA) at 20 C. (Acquarone, Buera, & Elizalde, 2007). 2.2.1.3. Acidity (free, lactone, and total). Free, lactone, and total acidity were determined as follows by the titrimetric method (AOAC 962.19, 2003): 10 g honey samples were dissolved in 75 ml, CO2-free water in a 250 ml beaker. The electrode of the pH meter (Mettler Toledo Delta 320) was immersed in the solution, stirred with a magnetic stirrer and titrated with 0.05 N NaOH to pH 8.5 (free acidity). Then the addition was stopped; immediately 10 ml of 0.05 N NaOH were added and without delay back-titrated with 0.05 N HCl to pH 8.30 (lactone acidity). Total acidity resulted from adding free plus lactone acidities. The results were expressed as milliequivalents/kg (meq/kg). 2.2.1.4. pH determination. pH measurements were performed potentiometricaly a 20 C using a pH-meter Sartorius Professional Meter PP-15 (Sartorius Ag, Goettingen, Germany) in Honey samples diluted with freshly deionized water, ranging from 10% to 100% (w/v) (Silva et al., 2009).

2.2.1.5. Redox potential (Eh). Millivolt measurements were performed at 20 C using a pH-meter cyber scan pH 6000 (Eutech instruments, Nijkerk, Netherlands). Honey samples were diluted with freshly deionized water, ranging from 10% to 100% (w/v) (Dimin s, Kuka, Kuka, & Cakste, 2006). 2.2.1.6. Ash. Ash was indirectly determined using the measured electrical conductivity and applying the following equation: X1 = (X2 0.143)/1.743, were: X1 = ash value; X2 = electrical conductivity in lS/cm at 20 C (Piazza, Accorti, & Persano Oddo, 1991). 2.2.1.7. Electrical conductivity. Electrical conductivity was measured at 20 C in solutions of honey samples in deionized water with specic electrical conductivity lS/cm1 using a conductivity meter WTW 1970i (Werksttten, GmbH, Germany), (Silva et al., 2009). 2.2.1.8. Colour intensity. The net absorbance of the honey samples was determined by the method of Beretta, Granata, Ferrero, Orioli, & Facino, 2005. The honey samples were diluted to 50% (w/v) with warm (4550 C) milli Q water and the solution was ltered through a 0.45 lm lter. There was a complete absence of coarse particles in the honey solutions as all the commercial samples were non-crystalline liquid honeys. The absorbance was measured using a spectrophotometer at 450 and 720 nm and the difference in absorbance was expressed as mAU. 2.2.1.9. Colour. Visual colour was measured using Hunter colorimeter model ColorFlex (Hunter Associates Laboratory, Reston, VA, USA) in terms of L (lightness), a (redness and greenness) and b (yellowness and blueness). The instrument (45/0 geometry, 10observer) was calibrated with a standard black and white tile followed by measurement of each honey samples (Beretta et al., 2005). 2.2.2. Biochemical analysis 2.2.2.1. Sugar prole analysis. Honey (1 g) was dissolved in 10 ml acetonitrile: water (1:1) solution. The mixture was homogenised with constant shaking for at least 30 min. Then the samples were

Table 1 Classication of honey types and regional sources. Sample Type of honey Family Botanical name Common name Ghaf Wild jujube Wild mountain Wild jujube Wild mountain Wild jujube Wild mountain Marya herbal Wild mountain Herbs Mountain herbal Local name Region Sensory characteristics (colour, consistency) Light amber, less viscous Slightly dark amber, very less viscous Light amber, less viscous Light amber, less viscous Slightly dark amber, viscous Dark amber, very viscous Light amber, viscous Dark amber, viscous Light amber, viscous Slightly dark amber, less viscous Light amber, viscous

Arid regions H1 Monooral H2 Monooral H3 Monooral H4 H5 H6 H7 H8 H9 H10 H11 Monooral Monooral Monooral Monooral Monooral Heterooral Heterooral Heterooral

Fabaceae Rhamnaccae Fabaceae Rhamnaccae Fabaceae Rhamnaccae Fabaceae Fabaceae

Prosopis juliora Ziziphus spina-csisti Acacia tortilis Ziziphus spina-csisti Acacia tortilis Ziziphus spina-csisti Acacia tortilis Acacia tortilis

Ghaf honey Alain sider Ras ul Khaima Samar Oman sider Oman samer Garden sider Doany samer Ashab marya samer Ashab gablaya Ashab Ashab gbalya

UAE UAE UAE Oman Oman Yemen Yemen Yemen UAE Omani Yemen

Non arid H12 H13 H14 H15 H16

regions Monooral Monooral Monooral Heterooral Heterooral

Rhamnaccae Rhamnaccae Myrtaceae

Ziziphus spina-csisti Ziziphus spina-csisti Leptospermum scoparium

Wild jujube Wild jujube Manuka Black forest Black forest

Pakistan sider Kashmir sider Manuka El ghabat el sawda El ghaba el sawda

Pakistan Kashmir New Zealand KSA(Imported) Germany

Light amber, viscous Light amber, less viscous Light amber, very ne granulated, medium solid Dark amber, less viscous Dark amber, less viscous

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ltered through a 0.45 lm syringe lter. 1 ml of the concentrated ltrate was then diluted with acetonitrile: water (1:1) solution to a nal volume of 25 ml. This mixture was then ltered again through the 0.45 lm syringe lter into vials and set for injection in the Waters ACQUITY UPLC with ELSD. The injection volume was 10 ll, and separation was carried out on an ACQUITY UPLC BEH Amide Column C18 (2.1 150 mm, 1.7 lm) maintained at 35 C with a run time of 20 min. The mobile phase A was 80/20 methanol/H2O with 0.2% triethylamine (TEA), and mobile phase B was 30/70 methanol/H2O with 0.2% triethylamine (TEA) with a ow rate of 0.17 ml/min, according to (Waters, ACQUITY UPLC BEH Amide, Application Notebook, 2009). 2.2.2.2. Acrylamide. The procedure of sample preparation involved an automated extraction method using accelerated solvent extraction unit ASE 350 (DIONEX, CA, USA). Honey samples (5 g) were extracted for 20 min using 10 mM formic acid in 34 ml cells of the ASE 350. The conditions for the ASE extraction unit were set at 70 C, 1500 psi, with heat up time of 5 min and static time of 4 min, followed by 3 static cycles, ush volume 60%, and a purge time (N2) of 120 s (DIONEX, CA, USA, application note 409). Quantication of acrylamide in the above extracted honey samples was performed on a UPLCMS/MS with the electrospray positive ionisation (ESI+). In detail, an ACQUITY UPLC quaternary pump system equipped with the micro vacuum degasser, thermostated autosampler and thermostated column compartment (Waters, Milford, MA, USA) was coupled with a Micromass Quattro Ultima triple-quadrupole mass spectrometer from Micromass Company Inc (Manchester, UK). The analyte elution (injection volume 10 ll) was carried out on a UPLC BEH C18 column (50 mm length, 2.1 mm i.d., 1.7 lm particle size) (Waters, Milford, MA, USA) maintained at 25 C with a run time of 3 min. The mobile phase was 10% methanol/0.1% formic acid in water with a ow rate of 0.2 ml/min. The conditions used for electrospray source were as follows: capillary voltage, 3.5 kV; cone voltage, 50 V; source temperature, 100 C; desolvation gas temperature, 350 C; desolvation gas ow, 400 L/ h nitrogen; cone gas ow, 45 L/h nitrogen; argon collision gas pressure to 3 103 mbar for MS/MS, which gave a highest acrylamide response in this study. The collision energy (CE) was optimised for each multiple reaction monitored (MRM) transition (Zhang, Jiao, Cai, Zhang, & Ren, 2007). 2.2.2.3. Hydroxymethylfurfural (HMF). Honey (1 g) was dissolved in 10 ml acetonitrile: water (1:1) solution. The mixture was homogenised with constant shaking, for 10 min. This mixture was then ltered again through the 0.45 lm syringe lter into vials and set for injection in the Waters ACQUITY UPLC with TUV. The injection volume was 10 ll, and separation was carried out on an ACQUITY UPLC BEH Amide Column C18 (2.1 100 mm, 1.7 lm) maintained at 22 C with a run time of 5 min. The mobile phase was 0.01 N Phosphoric acid (86%) and acetonitrile (14%) with a ow rate of 0.2 ml/min (Chinnici, Masino, & Antonelli, 2003). 2.2.2.4. Minerals. Honey samples (1 g) were submitted to sequential digestion using CEM Mars 5 microwave digestion system. After the digestion was completed, ultra-pure water was added up to a nal volume of 50 mL. The mineral components selected to be quantied in the digested honey samples were: Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, P, Pb, Zn, Ca, K, Na, Mg, S and Sr. The concentration of these elements in the digested samples was determined using inductively coupled plasma (Varian ICP-OES model 710-ES), (Lachman et al., 2007). 2.2.2.5. Free amino acids. Free amino acids, histidine (His.), serine (Ser.), arginine (Arg.), glycine (Gly.), aspartic acid (Asp.), glutamic acid (Glu.), theronine (Thr.), alanine (Ala.), proline (Pro.), lysine

(lys.), tyrosine (Tyr.), methionine (Met.), valine (Val.), isoleucine (LIc.), leucine (Leu.), phenylalanine (Phe.) and systine (Sys.), were determined as described by Liming, Jinhui, Xiaofeng, Yi, and Jing (2009). 2.2.2.6. Total protein. The total protein content was determined by Lowrys method of protein estimation which is based on the formation of a copperprotein complex and the reduction of phosphomolybdate and phosphotungstate present in FolinCiocalteau reagent to hetero polymolybdenum blue and tungsten blue, respectively. Bovine serum albumin (BSA), (0100 lg/ml) was used as a standard for preparing the calibration curve (Saxena, Gautam, & Sharma, 2010). 2.2.2.7. Carotenoids. Honey (5 g) was dissolved in 45 ml hexane: acetone (60:40) solution. The mixture was homogenised with constant shaking, for at least 10 min with addition of 5 ml water. The mixture was then ltered through a Whatman 150 mm, 42 lter paper and the ltrate was collected and dried under N2. After complete drying the residue was re-dissolved in 2 ml of the mobile phase containing acetonitrile: methanol: ethyl acetate (730:200:70 ml) then ltered through 0.45 lm syringe lter into vials and set for injection in the Waters ACQUITY UPLC with TUV. The injection volume was 10 ll, and separation was carried out on an ACQUITY UPLC BEH Amide Column C18 (2.1 50 mm, 1.7 lm) maintained at 30 C with a run time of 10 min. The mobile phase was ran at a ow rate of 0.2 ml/min. ACQUITY UPLC, PDA detector (450 nm) was used. lutein, cryptoxanthine, zeaxanthine, lycopene, a-carotene, b-carotene, c-carotene were used as a standards for preparing the calibration curve (Chauveau-Duriot, Doreau, Noziere, & Graulet, 2010). 2.2.3. Statistical analysis All analytical determinations were performed in triplicate. Statistical analysis was performed using SPSS for windows (version 19; SPSS Inc., Chicago, IL, USA). The differences of mean values among samples varieties was determined using one-way analysis of variance (ANOVA) followed by Tukey. 3. Results and discussion 3.1. Physicochemical analysis 3.1.1. Water content, RI, Brix Honey moisture content depends on the environmental condition and manipulation by beekeepers at the harvest period, and it can vary from season to season and from year to year (Acquarone et al., 2007). The moisture content (%) in the investigated samples ranged from 13.63% to 20.60% (Table 2), which are within the allowed parameters (620%) according to the international regulations of quality (Codex Alimentarius, 2001). In our samples, the values were similar to those previously reported for different kinds of honey whose corresponding values ranged from 18.7% to 21.8% (Manresa, 2005). Higher moisture content could lead to undesirable honey fermentation during storage caused by the action of osmotolerant yeasts resulting in the formation of ethyl alcohol and carbon dioxide; the alcohol can be further oxidised to acetic acid and water resulting in a sour taste (Chirife, Zamora, & Motto, 2006). Brix (directly related with sugar content) may be a reliable index of adulteration. The analysed samples presented Brix without signicant differences, ranging from 79.0 to 84.10 (average = 80.95 1.60) (Table 2), which are similar to those from others geographical locations (Silva et al., 2009. The Brix results obtained in the current study suggest that the honey samples used in the present study are most likely unadulterated. Similarly, RI

38 Table 2 Physicochemical and biochemical properties of honeys.


pH H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 4.61 0.02ef 5.27 0.00 k 5.08 0.00j 4.24 0.01b 4.44 0.01d 6.33 0.02 m 4.65 0.03 fg 4.68 0.02 g 4.74 0.01 h 4.65 0.01efg 4.61 0.01e 4.95 0.01i 5.32 0.01 l 4.22 0.00b 4.35 0.01c 3.99 0.02a L H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 9.96 0.12i 7.60 0.33 fg 7.85 0.09 g 7.27 0.10ef 6.19 0.09bc 13.77 0 .24j 5.03 0.04a 6.83 0.04de 9.10 0.32 h 7.22 0.23ef 6.40 0.07 cd 8.84 0.03 h 10.17 0.06i 8.88 0.08 h 5.74 0.17b 35.49 0.17 k mV 142.73 0.40i 102.97 0.15b 114.47 0.42c 160.67 0.59k 150.07 0.12j -16.00 0.00a 139.93 0.25 h 135.40 0.40f 133.40 0.30e 137.70 0.26 g 140.03 0.15 h 121.33 0.21d 298.47 0.25o 162.20 0.17 m 154.93 0.15 l 176.53 0.42n a 0.69 0.10bc 2.19 0.42a 0.23 0.63b 0.25 0.16b 1.91 0.37 cd 3.11 0.95d 0.21 0.17b 0.42 0.16b 1.73 0.53a 1.99 1.00 cd 2.54 0.35d 0.67 0.28bc 0.66 0.26bc 0.21 0.36b 0.55 0.33bc 14.50 0.31e Free acidity (meq/kg) 23.82 0.47def 15.23 0.25bc 17.03 0.13c 16.33 0.25bc 25.55 0.08f 3.86 0.10a 4.28 0.30a 15.98 2.10bc 23.31 0.93de 23.29 0.08dc 14.86 0.62b 15.75 0.08bc 16.99 0.15c 28.45 0.89 g 25.34 0.62ef 21.81 0.05d b 11.80 0.06gh 7.50 0.21de 7.37 0.14d 8.00 0.30e 6.64 0.16c 16.02 0.13i 4.93 0.04a 6.05 0.13b 9.50 0.20f 7.51 0.33de 5.02 0.20a 10.26 0.19 g 10.39 0.03 g 7.57 0.17de 5.93 0.06b 36.54 0.17 h

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Lactonic acidity (meq/kg) 13.65 0.10dcd 7.15 0.25a 7.97 0.15a 6.15 0.51a 15.01 0.12d 14.79 0.65d 6.60 0.30a 10.66 0.25b 12.51 2.16bc 14.15 0.25 cd 17.31 1.01e 10.56 0.13b 13.86 0.13 cd 12.24 0.30bc 10.89 0.26b 13.99 0.08 cd Colour intensity (mAU) 273.90 2.40a 1700.20 4.60j 1238.65 1.05 h 689.30 0.00cde 679.85 3.35cde 653.35 38.27 cd 638.30 2.00c 976.90 47.86 g 746.80 16.40def 835.30 25.80f 2874.80 100.20 k 976.55 3.65 g 617.10 11.70bc 756.60 2.90ef 1562.75 30.35i 489.10 1.50b

Total acidity (meq/kg) 37.48 0.50 g 22.39 0.11c 25.00 0.15de 22.48 0.71 cd 40.57 0.18 h 18.66 0.55b 10.88 0.58a 26.64 2.33e 35.82 1.24 g 37.44 0.23 g 32.17 1.28f 26.31 0.09e 30.85 0.03f 40.69 0.62 h 36.22 0.88 g 35.80 0.13 g RI 1.4879 0.00a 1.4901 0.00a 1.4849 0.00a 1.4889 0.00a 1.4980 0.00a 1.5026 0.00a 1.4915 0.00a 1.4980 0.00a 1.4949 0.00a 1.4962 0.00a 1.4884 0.00a 1.4937 0.00a 1.4955 0.00a 1.4927 0.00a 1.4945 0.00a 1.4919 0.00a

HMF (mg/kg) 4.45 0.05d 1.08 0.05ab 0.26 0.05ab 0.17 0.01a 79.26 0.87 h 1.16 0.05b 15.33 0.27e 5.36 0.46d 0.75 0.02ab 4.81 0.13d 28.70 0.30f 2.47 0.03c 0.98 0.07ab 15.58 0.52e 4.95 0.06d 37.22 0.22 g Brix (%) 79.00 0.00a 79.80 0.00a 77.90 0.00a 79.50 0.00a 82.80 0.00a 84.10 0.00a 80.50 0.00a 83.00 0.00a 81.70 0.00a 82.00 0.00a 79.20 0.00a 81.20 0.00a 81.80 0.00a 80.90 0.00a 81.40 0.00a 80.40 0.00a

Acrylamide (lg/kg) 2.39 0.22 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

Ash (%) 0.248 0.000 h 0.077 0.000b 0.114 0.001e 0.096 0.000c 0.116 0.000e 0.316 0.000 l 0.077 0.003b 0.104 0.001d 0.244 0.000 g 0.268 0.000j 0.115 0.000e 0.285 0.000 k 0.262 0.002i 0.075 0.000b 0.196 0.000f 0.066 0.000a Water activity (wa) 0.63 0.00 k 0.60 0.00 h 0.62 0.00j 0.56 0.00d 0.53 0.00b 0.52 0.00a 0.64 0.00 l 0.55 0.00c 0.57 0.00 g 0.56 0.00ef 0.62 0.00j 0.57 0.00f 0.57 0.00 g 0.55 0.00c 0.56 0.00e 0.60 0.00i

Total protein (mg/ 100 g) 345.20 1.15b 402.33 1.93d 453.74 7.08 g 355.55 0.87c 578.87 1.25i 456.06 4.17 g 204.84 3.02a 350.49 2.84bc 399.79 1.31d 420.39 2.81e 348.52 0.68bc 405.54 1.32d 435.81 4.01f 347.79 1.11bc 475.14 4.52 h 427.70 1.23ef

Moisture (%) 19.40 0.00 l 18.60 0.00j 20.60 0.00 m 19.00 0.00 k 15.40 0.00c 13.63 0.29a 18.00 0.00i 14.80 0.00b 16.60 0.00ef 16.20 0.00d 19.20 0.00kl 17.00 0.00 g 16.40 0.00de 17.50 0.00 h 16.80 0.00 fg 18.00 0.00i

Electrical conductivity (lS cm1) 570.67 0.58 h 274.67 0.58b 338.67 1.15e 307.33 0.58c 341.67 0.58e 690.67 0.58 l 273.37 5.74b 320.67 1.15d 564.67 0.58 g 605.67 0.58j 340.67 0.58e 636.67 0.58 k 596.33 3.21i 270.33 0.58b 480.33 0.58f 254.67 0.58a

Honey SD. Different letters in a column denote signicant differences, P < 0.05.

values did not show any signicant differences among the tested samples, as presented in Table 2. 3.1.2. Water activity The water activity of the honey samples varied from 0.52 to 0.64, with an average of 0.58 0.03 as presented in Table 2. Our results are quite similar to those of Greek honeys for which the wa values ranged from 0.53 to 0.67 (Lazaridou, Biliaderis, Bacandritsos, & Sabatini, 2004). The water activity is an important factor, which governs the food stability by preventing or limiting microbial growth. The osmotolerant yeasts are able to grow at a minimal water activity of 0.6 (Chirife et al., 2006). The honey sample H6, which had the lowest moisture content was 13.63 0.29%, had the lowest water activity as well. 3.1.3. Acidity (free, lactone, and total) Honey acidity is due to the presence of organic acids, mainly gluconic acid, in equilibrium with their corresponding lactones or internal esters, and to inorganic ions, such as phosphate, sulphate and chloride (Terrab, Recalames, Hernanz, & Heredia, 2004). Table. 2 illustrate the free acidity, and lactonic acidity is considered as the acidity reserve when the honey becomes alkaline, while the total acidity is the sum of free and lactonic acidities. Table 2 illustrates that the total acidity observed in the current study for different honey samples were acceptable (below 50 meq/kg) (Codex Alimentarius, 2001), indicating the absence of undesirable fermen-

tation. Lactonic acidity ranged from 6.15 0.51 to 17.31 1.01 meq/kg (average = 11.72 3.31 meq/kg). Total acidity varied between 10.88 0.58 and 40.69 meq/kg, with a mean value of 29.96 8.44 meq/kg. The results obtained for acidity were in agreement with data reported from other geographical locations (Terrab, Dez, & Heredia, 2002; Terrab et al., 2004). The variation of total acidity has been attributed to harvest season. 3.1.4. pH The pH is a parameter that is correlated with honey storage and with microorganism growth that could change the texture and honey stability (Feas, Pires, Iglesias, & Estevinho, 2010). The pH limit is not described in EU Directive 2001/101/EC, however, honey pH should be low to avoid microbiological contamination. The pH values for studied honey samples averaged at 4.76 0.55, and the range was between 3.99 0.02 and 6.33 0.02 (Table 2), which were acceptable values and comparable with those obtained in other works (Corbella & Cozzolino, 2006; Feas, Pires, Estevinho, Iglesias, & Araujo, 2010; Gomes, Dias, Moreira, Rodrigues, & Estevinho, 2010). 3.1.5. Redox potential (Eh) Table 2 shows the Eh values of honey samples. The range was between 16.00 0.00 and 298.47 0.25 mV, with average of 141.93 56.31 mV. H6 had the lowest redox potential, while H13 had the highest. Several substances found in honey participate in

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oxidationreduction processes. The oxidationreduction potential of honey partially indicates the characteristics of honey and physicochemical processes in it during storage and treatment. According to oxidationreduction potential, honey can be divided into different kinds. Every such kind of honey has its characteristic oxidationreduction potential (Dimin s et al., 2006). The redox potential may also be an interesting indicator of the antioxidant efciency of food. 3.1.6. Ash Ash content is a parameter used for the determination of the botanical origin (oral, mix or honeydew). The results found (0.066% to 0.316%) (Table 2) are within the limit allowed for honeys (0.6%), indicating clearness of honey samples and possibly lack of adulterations with molasses. 3.1.7. Electrical conductivity The electrical conductivity of honey is closely related to the concentration of mineral salts, organic acids and proteins. This parameter shows great variability according to the oral origin and it is important for the differentiation of honeys of different oral origins. The results obtained for the honey samples under study varied between 154.67 0.58 and 690.67 0.58 lS cm1 (average = 429.19 152.78 lS cm1). These values are below the maximum limit indicated by the international regulations of quality (Codex Alimentarius, 2001) for nectar honey (800 lS cm1), as shown in Table 2. 3.1.8. The colour intensity The colour intensity (ABS450) is supposed to be related to pigments (carotenoids, avonoids, etc.), which are also known to have antioxidant properties (Frankel, Robinson, & Berenbaum, 1998). The ABS450 values for the samples ranged from 524 to 1678 mAU (Table 2). The reported ABS450 values for some Italian and Slovenian honeys are in the range of 253413 mAU and 70495 mAU, respectively (Beretta et al., 2005; Bertoncelj, Dobersek, Jamnik, & Golob, 2007). 3.1.9. Colour The colour characteristics are presented in Table 2, which summarizes the means, standard deviations and ranges of the parameters L, a and b, obtained with the Hunter colorimeter for honey samples. The colour of the honeys was light to dark amber, with reddish or green tinge. H16 and H6 had the highest average values of parameter L that indicates lightness, 35.49 0.17 and 13.77 0.24, respectively, while H7 and H15 had the lowest average values of parameter L, 5.03 0.04 and 5.74 0.17 respectively. Honey samples analysed had red, yellow and green components: green components (negative a values) for H9, H7, H2, H3, H14. On the other hand, H16 and H6 also had the highest average values of parameter b value at 36.54 0.17 and 16.02 0.13, respectively. In general the parameters L, a and b of the honey samples analysed had signicant variations among the colour parameter. 3.2. Biochemical analysis 3.2.1. HMF HMF was investigated among the honey samples to determine their quality. All samples analysed showed HMF values within the parameters allowed according to the international regulations of quality (Codex Alimentarius, 2001) for honeys of declared origin from regions with a tropical or arid climate (lower than 80 mg/kg). HMF content is widely recognised as a parameter of honey sample freshness, because it is absent in fresh honeys and tends to increase during processing and/or aging of the product. Several factors inuence HMF levels, such as temperature and time of heating,

storage conditions, pH and oral source, so it provides an indication of overheating and storage in poor conditions (Fallico, Arena, Verzera, & Zappala, 2006). The data in Table 2 showed that H5 had the highest HMF values (79.26 0.87 mg/kg) while H4 had the lowest values (0.17 0.01 mg/kg).

3.2.2. Acrylamide Acrylamide is formed from reducing sugars and asparagine in the Maillard reaction in a complex mechanism. Table 2 presents the acrylamide in honey samples. All samples did not had acrylamide except sample H1 which had very low amount of acrylamide 2.39 0.22 lg/kg honey. 3.2.3. Minerals Mean contents of each mineral found in the 16 honeys expressed in mg/kg fresh weight are shown in Table 3. Some minerals were found in considerable amounts, and others in very small amounts or not detected at all. The mineral content is an important index of a possible environmental contamination when heavy metals are detected. On the other hand, the major mineral content contribution [potassium (K), calcium (Ca), sodium (Na), magnesium (Mg) and iron (Fe)] can provide also the nutritional value of honey products and can be considered a potential indicator of geographical origin of honey (Silva et al., 2009). The range of potassium content was 86.00 1.262690.29 49.96 mg/kg, independent of geographical origins; however, the lowest potassium content was found in H7. On the other hand, H2, showed the highest potassium contents. The mineral composition of sodium (6.76 0.06531.77 1.37 mg/kg), calcium (7.87 0.09 183.90 1.28 mg/kg), magnesium (2.28 0.1292.99 1.21 mg/ kg), phosphorus (8.99 0.10264.18 1.48), sulphur (6.97 0.10 333.73 2.11), iron (1.15 0.04110.79 1.32 mg/kg), manganese (below <0.01 0.0010.31 0.24), zinc (0.30 0.026.73 0.15) and copper (0.26 0.011.91 0.01) were minority macro- and micro-elements compared with potassium concentration in honey samples. Nevertheless, the highest sodium and calcium contents were obtained for H1, while H7 had the lowest value. The determination of potassium, sodium, magnesium, calcium, phosphorus, sulphur and iron contents in honey is useful to determine the nutritional value of some characteristic honey samples and to establish a botanical origin differentiation. Nevertheless, the mineral content is not yet a quality parameter of the EU Directive (Codex Alimentarius, 2001). The total mineral contents of heavy metals in the honey samples ranged from 1.24 0.03 to 13.93 0.08, <0.010 to 3.96 0.44, 0.06 0.00 to 0.74 0.01, <0.01 0.00 to 13.31, <0.02 0.00 to 1.93 0.05, and 0.00 to 7.78 0.02 mg/kg for Al, Sr, Cd, Cr, Mo, and Ni, respectively. While the readings for Pb, Co, and As, were all below 0.01 mg/kg. These values can be compared to the amount of heavy metals reported in New Zealand honey (Vanhanen, Emmertz, & Savage, 2011). Overall, the amount of heavy metals was comparable with other honey types analysed elsewhere in the world.

3.2.4. Total protein content Total protein contents are shown in Table 2. The highest values of total protein was found in H5 (578.87 1.25 mg/100 g of honey) while the lowest values were found for H7(204.84 3.02 mg/100 g of honey). Honey protein level is dependent on the type of ora: since it is variable, protein content in honeys can be attributed to the presence of enzymes introduced by bees themselves, and others derived from the nectar. Similar protein values in honey were previously reported by Azeredo et al. (2003) and Escuredo, Miguez, Fernandez-Gonzalez, and Seijo (2013).

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H.M. Habib et al. / Food Chemistry 153 (2014) 3543

Table 3 Mineral contents of honeys samples are expressed as mg/kg honey.


K H1 H2 H3 H4 H5 H6 H7 H8 1415.53 4.83c 2690.29 49.96j 2476.46 525.51ij 2044.14 9.82fgh 914.03 10.39b 2502.40 19.86ij 86.00 1.26a 1715.36 7.96cdef Na 531.77 1.37 m 99.27 1.75f 88.59 0.75e 144.94 2.50i 112.92 1.37 h 56.79 0.05d 6.76 0.06a 146.57 0.97i Ca 183.90 1.28 h 71.93 4.38 cd 72.56 3.79 cd 157.05 6.46 g 118.60 0.31f 77.68 0.54 cd 7.87 0.09a 110.69 0.58ef Mg 70.37 0.23 h 28.01 1.75b 32.76 2.08c 75.30 1.84i 57.25 0.28g 39.69 0.46e 2.28 0.12a 90.45 0.33j P 100.20 0.35f 38.28 1.07b 46.63 0.68 cd 110.74 3.28 g 113.73 1.33 g 39.95 0.23b 8.99 0.10a 264.18 1.48 k Fe 6.45 0.05f 3.06 0.10bcd 1.97 0.10ab 2.82 0.27bc 6.60 0.06f 2.81 0.05bc 1.15 0.04a 3.02 0.02bcd S Mn Zn 1.05 0.02b 1.33 0.09bc 1.50 0.03c 3.70 0.18 g 6.65 0.25i 1.55 0.02c 0.30 0.02a 4.16 0.07 h Cu 0.34 0.02ab 0.40 0.02bc 0.28 0.05a 0.49 0.07de 0.58 0.02f 0.44 0.00 cd 0.26 0.01a 0.70 0.02 g 0.39 0.01bc 1.91 0.01j 0.56 0.01ef 0.39 0.02bc 0.49 0.03de 0.44 0.04 cd 1.58 0.03i 1.15 0.01 h As <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a

115.11 0.97f 0.33 0.01b 52.75 2.13b 0.01 0.00a 57.55 0.39bc 0.22 0.01b 170.92 4.26 h 0.23 0.04b 202.45 2.44j 0.40 0.00b 53.18 0.50b 0.30 0.00b 6.97 0.10a <0.01 0.00a 333.73 2.11 l 0.39 0.00b

H9 2026.24 6.10efgh 107.43 0.39 g H10 2205.65 38.81ghi 20.54 0.24b H11 1800.19 18.28cdfg 171.68 2.03 k H12 2333.63 2.87hij 58.62 0.26d H13 1923.97 21.91defg 158.31 1.88j H14 1547.21 71.97 cd 195.99 2.72 l H15 1637.47 23.82cde H16 850.02 2.12b Al H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 3.07 0.08e 3.73 0.27 g 2.16 0.04b 1.96 0.14b 2.72 0.04 cd 2.17 0.05b 1.24 0.03a 2.11 0.03b 2.46 0.06c 13.93 0.08 k 5.62 0.12i 3.09 0.06e 3.42 0.06f 4.83 0.05h 2.84 0.04de 7.77 0.06j 34.10 0.61c 32.50 0.40c Sr

76.67 0.25 cd 37.86 0.41de 49.35 0.77d 66.78 0.84c 92.99 1.21j 192.97 1.64j 248.20 7.14i 160.65 0.46 l 92.74 0.21e 76.74 1.32 cd 81.60 0.36d 161.99 12.26 g 100.96 1.88e 49.25 0.47b Cd 0.55 0.09gh 0.21 0.02bc 0.22 0.00bc 0.27 0.02 cd 0.38 0.01ef 0.41 0.04ef 0.14 0.00ab 0.06 0.00a 0.46 0.02 fg 0.74 0.01i 0.17 0.00bc 0.64 0.05 hi 0.65 0.02hi 0.34 0.01de 0.27 0.06 cd 0.46 0.00 fg 35.18 0.76 cd 44.17 0.75c 43.65 0.32f 47.06 1.13 cd 58.90 3.28 g 121.28 0.11 h 110.05 0.50 k 26.93 0.15b Cr 1.09 0.01c <0.01 0.00a <0.01 0.00a <0.01 0.00a 0.55 0.01b <0.01 0.00a <0.01 0.00a <0.01 0.00a 0.08 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a 13.31 0.25d 162.45 0.33i 50.13 0.51d Mo

3.58 0.02cde 72.57 0.99d 4.86 0.04e 159.34 2.15 g 110.79 1.32 h 321.68 0.90 k 3.19 0.08bcd 59.45 2.18c 2.92 0.07bc 78.05 0.35d 2.42 0.07abc 180.73 0.63i 4.27 1.05de 63.26 0.06 g Ni 0.10 0.02b 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.41 0.04c 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.49 0.04d 0.13 0.02b 0.00 0.00a 0.00 0.00a 0.00 0.00a 0.00 0.00a 7.78 0.02e 107.38 2.45e 52.09 0.74b Pb

0.34 0.01b 6.73 0.15i 8.46 0.10e 3.66 0.02 g 1.41 0.01c 6.64 0.02i 0.37 0.01b 1.49 0.04c 0.25 0.00b 3.58 0.02 g 0.27 0.01b 2.81 0.05e 10.31 0.24f 3.29 0.05f 4.74 0.06d 2.04 0.02d Co <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a

2.19 0.01 g 0.96 0.07bc 1.20 0.07 cd 3.96 0.44 h 1.68 0.00ef 0.76 0.01b <0.00 0.00a 1.70 0.02ef 1.12 0.01bcd 0.18 0.00a 2.36 0.05 g 0.86 0.05bc 1.43 0.01de 1.81 0.16f 0.26 0.00a 0.19 0.00a

<0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a <0.02 0.00a 1.93 0.05b

<0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a <0.01 0.00a

Honey SD. Different letters in a column denote signicant differences, P < 0.05.

3.2.5. Sugar The monosaccharaides glucose and fructose are the major constituents of honey. Fructose is always the most important sugar quantitatively followed by glucose. In the current study, fructose was higher than glucose in all the honey samples analysed. Fructose and glucose contents of honey samples ranged from

32.26 0.07 g/100 g to 42.42 0.10 g/100 g and 27.78 0.10 g/ 100 g to 32.35 0.04 g/100 g respectively. Sucrose, maltose and rafnose were the minor sugars in honey as shown in Table 4; the ranges were 0.56 0.431.66 0.01, 0.31 0.022.09 0.09 and 0.0010 0.0010.0079 0.0032, respectively. Overall the total sugar ranged from 61.21 1.09 to 77.49 0.63. All the honeys

Table 4 Sugar of honeys samples are expressed as percentage. Fructose Sugar (%) H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 37.16 0.16f 33.26 0.05bc 32.84 0.09b 33.14 0.14bc 42.42 0.10j 36.44 0.05de 33.45 0.05c 39.28 0.27 h 36.82 0.07ef 39.87 0.09i 32.26 0.07a 38.32 0.03 g 38.95 0.06 h 39.21 0.58 h 39.28 0.06 h 35.95 0.06d Glucose 27.78 0.10a 31.72 0.03c 31.41 0.09c 31.61 0.05c 31.64 0.04c 28.59 1.16ab 32.01 0.10c 32.25 0.72c 29.28 0.06b 32.35 0.04c 27.93 0.07a 31.75 0.43c 31.65 0.05c 28.61 0.52ab 29.37 0.06b 29.45 0.05b Sucrose 0.98 0.02b ND ND 0.88 0.01ab 1.60 0.02c 0.72 0.00ab 0.92 0.02b 1.66 0.01c 0.56 0.43a ND ND 0.91 0.01b 0.87 0.01ab ND 1.40 0.01c ND Maltose 0.48 0.03ab 0.31 0.02a 0.50 0.02ab 0.52 0.03ab 0.59 0.04ab 0.98 0.14abc 0.38 0.05a 4.29 0.02d 1.72 1.46bc 0.44 0.02ab 1.02 0.96abc 2.09 0.06c 2.09 0.09c 1.01 0.04abc 0.48 0.02ab 1.40 0.04abc Rafnose 0.0065 0.0005ab 0.0035 0.0009ab 0.0047 0.0005ab 0.0064 0.0005ab 0.0050 0.0020ab 0.0038 0.0013ab 0.0053 0.0023ab 0.0020 0.0022ab 0.0070 0.0076ab 0.0010 0.0018ab 0.0079 0.0032b ND ND ND 0.0047 0.0005ab ND Total sugar 66.41 0.22bcd 65.29 0.09bc 64.75 0.09b 66.15 0.12bc 76.25 0.11 h 66.74 1.04bcd 66.77 0.16 cd 77.49 0.63 h 68.39 1.72de 72.65 0.10 g 61.21 1.09a 73.06 0.51 g 73.56 0.11 g 68.82 0.92ef 70.54 0.08f 66.80 0.14 cd F/G ratios 1.34 0.00e 1.05 0.00a 1.05 0.01a 1.05 0.00a 1.34 0.00e 1.28 0.05d 1.04 0.00a 1.22 0.03c 1.26 0.01cd 1.23 0.00 cd 1.15 0.00b 1.21 0.02bc 1.23 0.00 cd 1.37 0.02e 1.34 0.00e 1.22 0.00c

Honey SD. Different letters in a column denote signicant differences, P < 0.05. ND, not detected.

H.M. Habib et al. / Food Chemistry 153 (2014) 3543 Table 5 Carotenoids contents of honeys samples are expressed as lg/100 g honey. Lutein Carotenoids H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 (lg/100 honey) 0.0707 0.0026ab 0.0740 0.0105b 0.3186 0.0128f 0.1842 0.0064 cd 0.0136 0.0029a ND 0.3025 0.0182ef 0.4980 0.0471 g 0.0410 0.0010ab 0.4271 0.0252 g 0.4959 0.0225 g 0.1545 0.0438c 0.0768 0.0061b 0.2813 0.0055ef 0.1593 0.0234c 0.2473 0.0475de Cryptoxanthine 0.1558 0.0041c 0.0781 0.0071ab 0.0519 0.0063a 0.0615 0.0038a ND 0.2263 0.0269d 0.0917 0.0020b 0.2752 0.0180e ND 0.2321 0.0032d 0.2209 0.0137d ND ND 0.0727 0.0095 ab ND ND Zeaxanthine 38.6644 1.4230c 0.3082 0.0224a 0.3216 0.0254a 0.2777 0.0208a 0.3644 0.0212a 0.7667 0.0654a 0.1020 0.0191a 0.1463 0.0216a 0.2688 0.0379a 17.3199 0.3182b 0.0796 0.0098a 0.1987 0.0205a 0.3501 0.0288a 0.5330 0.0445a 0.1572 0.0285a ND b-Carotene ND ND 0.0165 0.0019c 0.0151 0.0005bc 0.0820 0.0087e ND 0.0324 0.0056d 0.0964 0.0049f ND 0.0173 0.0022c 0.0820 0.0012e ND ND 0.0177 0.0017c 0.0067 0.0012ab 0.0113 0.0008bc

41

c-Carotene
ND ND ND ND ND ND ND ND ND 0.0787 0.0092d ND 0.0066 0.0003ab 0.0148 0.0025c ND ND 0.0100 0.0016bc

Total carotenoids 38.89 1.42c 0.46 0.04a 0.71 0.05a 0.54 0.02a 0.46 0.02a 0.99 0.09a 0.53 0.04a 1.02 0.04a 0.31 0.04a 18.08 0.32b 0.88 0.03a 0.36 0.05a 0.44 0.03a 0.91 0.06a 0.32 0.05a 0.27 0.05a

Honey SD. Different letters in a column denote signicant differences, P < 0.05. ND, not detected.

Table 6 Free amino acids of honeys samples are expressed as lg/100 g honey.
His. Ser. Arg. 168.13 6.28 g 33.55 0.33b 52.50 2.17c 297.79 3.78 h 2.34 0.19a 81.25 1.25d 132.54 4.45e 720.58 6.03j 437.09 1.49i 75.38 0.90d 147.38 3.24f 29.13 0.25b 1084.25 10.82 l 1.96 0.64a ND 943.96 7.77 k Gly. 135.29 5.15c 2194.42 6.56 k 1415.42 3.82j 919.88 57.52 g 735.42 15.02e 1118.83 9.12 h 1368.04 6.50j 361.13 2.74d ND 785.83 6.41f 2196.58 10.22 k 3.70 0.19a ND 1268.75 7.60i ND 85.63 2.84b Met. ND 188.84 1.44j 85.33 0.80 g 3.63 0.13a 3.75 0.13a 10.38 0.76c 95.00 1.25 h 175.92 0.50i ND 9.42 0.19bc 23.33 1.91d ND 46.67 2.93f 35.83 1.91e 5.96 0.31ab ND Asp. ND 650.67 1.28i 11.58 0.80b 62.34 1.58f ND 30.00 1.25c 285.42 1.91 h ND ND 38.42 0.31d 57.50 1.25e 1.77 0.10a ND 111.29 1.32 g ND ND Val. ND 55.46 0.71b ND ND ND ND ND ND ND 65.42 1.91c ND ND 102.00 0.76d ND ND 45.00 1.25a Glu. 31.55 5.27bc 62.00 0.78e 105.00 1.25 g 0.46 0.07a ND 5.00 0.13a 37.17 1.39c 269.79 5.61i ND 29.92 0.19b 50.00 1.25d 29.92 0.19b 81.09 0.32f 121.25 1.25 h ND 61.67 0.85e Llc. ND 46.13 0.13 h 3.71 0.07bc 4.25 0.13c ND 1.21 0.07a 15.50 0.13f 149.67 0.80 k ND 25.88 0.38 g 11.63 0.76e 59.50 3.28j ND 7.54 0.19d 55.96 0.89i 16.50 0.13f Thr. ND 52.92 0.72e 93.75 1.25 g 0.62 0.01a 2.46 0.07a 11.96 1.56bc 48.71 2.39e 261.25 1.25 h ND 10.46 0.19b 18.33 0.72c 3.67 0.19a 0.55 0.07a 80.42 1.91f 0.95 0.01a 26.54 7.63d Leu. ND 1890.83 14.81j 335.42 1.91i 24.29 0.69b ND 36.63 0.76c 49.17 0.81d 253.71 0.19 h ND 111.71 1.48f 97.92 1.91e 3.75 0.13a ND 209.58 2.60 g ND 17.46 0.15b Ala. 45.00 1.25e 310.83 1.91 k 244.58 7.32i 11.92 0.90bc 6.21 0.19a 21.58 0.80 cd 294.25 4.98j 476.75 6.31 m ND 62.46 1.31f 120.83 4.39 h 11.29 1.19b 24.21 0.81d 85.00 2.50 g 22.96 0.14d 346.21 3.81 l Pro. 2031.92 7.51a 7918.75 6.96n 7247.08 6.17 m 5224.17 26.94j 5629.17 19.09 k 4943.33 64.11i 2606.75 20.07c 2490.96 15.99b 2681.58 5.65c 3021.67 18.55e 4070.00 65.53 h 3595.83 26.02f 6821.79 3.94 l 5609.58 15.63 k 3695.42 64.84g 2892.42 6.57d Total FAA (mg/100 g) 2.83 0.02a 15.14 0.04n 11.38 0.02 m 6.67 0.09 g 7.40 0.03i 7.44 0.08i 5.49 0.04f 8.62 0.02j 4.30 0.01d 5.04 0.02e 8.49 0.06j 3.74 0.03b 11.21 0.04 l 8.94 0.03 k 4.14 0.07c 6.93 0.10 h

Free amino acids (lg/100 g honey) H1 419.11 1.94c ND H2 ND ND H3 ND 500.94 1.24d H4 ND ND H5 ND 359.92 1.13c H6 ND 360.37 0.76c H7 ND ND H8 1762.50 8.50 g 410.83 1.08c H9 H10 H11 H12 H13 H14 H15 H16 632.30 1.13d ND ND 3.49 0.16a 792.50 2.29e 26.08 0.15b ND 1739.29 2.15f Lys. Free amino acids (lg/100 g honey) H1 ND H2 1734.92 14.54j H3 1285.83 1.91i H4 126.33 1.26a H5 658.75 3.31d H6 813.25 2.05e H7 558.50 3.77c H8 1009.21 3.09 g H9 H10 H11 H12 H13 H14 H15 H16 546.50 5.85c 800.46 1.02e 937.50 12.50f ND 2227.79 19.76 k 1162.08 1.91 h 360.00 2.50b 655.59 6.12d ND ND 756.83 3.56e ND ND 218.67 1.26b ND 103.08 89.27a Tyr.

ND ND ND ND 1.21 0.07a ND ND 272.29 2.49c ND 1.51 0.02a ND ND 26.00 0.38b ND ND ND

Honey SD. Different letters in a column denote signicant differences, P < 0.05. ND, not detected.

presented a value of glucose plus fructose higher than 60 g/100 g, which is the value required for all the kinds of honey according to the international regulations of quality (Codex Alimentarius, 2001). Fructose/Glucose (F/G) ratio has been recommended to

evaluate honey granulation, because glucose is less water soluble than fructose. The proportion of fructose to glucose depends largely on the nectar source (Anklam, 1998). Most investigators reported an F/G average ratio around 1.2, which coincides with

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our data for honeys according to the international regulations of quality (Codex Alimentarius, 2001). 3.2.6. Carotenoids The levels of different carotenoids are shown in Table 5. Lutein, cryptoxanthine, zeaxanthine, b-carotene and c-carotene were quantied in all honey samples, and results ranged from ND to 0.4980 0.0471, ND to 0.2752 0.0180, ND to 38.6644 1.4230, ND to 0.0964 0.0049, ND to 0.0787 0.0092 lg/100 g honey, respectively. In general, the highest amount of carotenoids were found in the honey from arid regions, and some honey values were not within the quantication limit of the method. In Portuguese honeys, Ferreira, Aires, Barreira, & Estevinho, 2009 reported carotenoid content in the range from 8.64 0.06 mg/kg to 9.49 0.15 mg/kg. These values are clearly much higher than the values found in the present study. In this case it must be taken into account that honey composition is rather variable and depends not only on its oral source, but also on the geographical zone, as well as on seasonal and environmental factors, which may be responsible for the detected differences. H1 from arid regions mono oral had the highest amount of total carotenoids 38.89 1.42 followed by H10 from arid regions heater oral 18.08 0.32. 3.2.7. Free amino acids Free amino acid contents are shown in Table 6. H8 mono oral honey from arid regions had the highest level of His. (1762.50 8.5 lg/100 g), while H11 hetero oral honey from arid regions had the highest level of Ser. (756.83 3.56 lg/100 g). H13 mono oral from non-arid regions had the highest level of Arg. (1084.25 10.82 lg/100 g). H11 and H2 from arid regions had the highest levels of Gly., 2196.58 10.22, 2194.42 6.56 lg/ 100 g, respectively. H2, also from arid regions, had the highest levels of Asp. (650.67 1.28 lg/100 g). H8, a mono oral honey from arid regions, exhibited the highest level of Glu. (269.79 5.61 lg/ 100 g). H3, a mono oral honey from arid regions had the highest level of Thr. (93.75 1.25 lg/100 g), while H16, a hetero oral honey from non-arid regions, had the highest level of Ala. (346.21 3.81 lg/100 g). In addition, Pro. was found in all honey samples in quite large amount, ranging from 2031.92 7.51 to 7918.75 6.96 lg/100 g. H2, a mono oral honey from arid regions had the highest level of Proline. Lys. results showed that the highest level was found in H2, a mono oral honey from arid regions (1734.92 14.54 lg/100 g). On the other hand, H8, a mono oral honey from arid regions, showed the highest level of Tyr. (272.29 2.49 lg/100 g), while H2, another mono oral from arid regions, had the highest values of Met. (188.84 1.44 lg/100 g). Val. was detected only in some honey samples, and H13 had the highest value (102.00 0.76 lg/100 g). LIc. showed the highest value in H8 mono oral from arid regions (149.67 0.80 lg/100 g), moreover, Leu. had the highest value in H2 mono oral from arid regions (890.83 14.81 lg/100 g). In general, total free amino acids were higher in mono oral honey samples from arid regions. H2 had the highest value (15.14 0.02 mg/100 g). In addition, Sys. and Phe. were not detected in all honey samples under investigation. To the best of our knowledge, this is the rst time free amino acids have ever been analysed in honey. 4. Conclusion Since characterisation of honey from arid regions is not yet available, this study provides a preliminary but comprehensive and detailed evaluation of physicochemical and biochemical properties composition of some of these honeys. In conclusion, our approach, using all the parameters previously mentioned, provides an entrance to further research with a larger number of samples

in order to improve the interest and knowledge about this honey and its quality proles.

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