LWT - Food Science and Technology 44 (2011) 1922e1930

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LWT - Food Science and Technology

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Physico chemical and bioactive properties of honeys from Northwestern Argentina

Mara Ins Isla a, b, *, Ana Craig a, Roxana Ordoez a, b, Catiana Zampini a, b, Jorge Sayago a, b, Enrique Bedascarrasbure c, Alejandro Alvarez c, Virginia Salomn c, Luis Maldonado c
a b c

Facultad de Bioqumica, Qumica y Farmacia, Facultad de Ciencias Naturales e IML, Universidad Nacional de Tucumn, San Miguel de Tucumn, Argentina INQUINOA (Instituto de Qumica del Noroeste Argentino), CONICET, Universidad Nacional de Tucumn, Ayacucho 461, 4000 e San Miguel de Tucumn, Argentina Estacin Experimental Agropecuaria Famaill, Instituto Nacional de Tecnologa Agropecuaria, Ruta provincial 301, km 32, Famaill, Tucumn, Argentina

a r t i c l e i n f o
Article history: Received 22 September 2010 Received in revised form 2 April 2011 Accepted 11 April 2011 Keywords: Argentinean honeys Antioxidant activity Antibacterial activity

a b s t r a c t
Honey is used for its nutritional and functional properties. The Argentinean Northwest is a region with a growing potential for honey production, but up to now, few physicochemical and biological studies have been carried out. The aim of this study is to characterize monooral (Prosopis sp and Citrus lemon) and multioral honey samples from the Argentinean Northwest from a physicochemical and functional standpoint. The results showed that the honeys had good properties of stability and freshness. The highest content of avonoid and phenolic compounds correspond to multioral honeys. A positive correlation was observed between colour intensity and avonoid or phenolic compounds content (R2 0.98 and R2 0.92, respectively). The avonoids, chrysin and pinocembrin were present in all samples analyzed, while hesperidin and hesperetin were numerically more important in lemon honey (>1 mg/kg), providing a valuable marker of botanical origin. The highest antioxidant activity against ABTS radical cation was detected in the darkest honey samples. All tested honeys showed antibacterial activity with MIC values between 0.10 and 0.25 g/mL on Gram-positive and Gram-negative antibiotic resistant bacteria. Neither pH nor osmolarity affected bacterial growth. The phenolic compounds and hydrogen peroxide were responsible for antimicrobial activity by bioautographic assays. The antioxidant and antimicrobial properties found in honeys from the Argentinean Northwest make them products of high added value and excellent quality. 2011 Elsevier Ltd. All rights reserved.

1. Introduction Traditionally, honey has been considered to have therapeutic properties since ancient times (Molan, 1992). It has been well established that the factors responsible for the antimicrobial activity are pH, sugar content, hydrogen peroxide levels and the presence of some phytochemicals, mainly phenolic compounds including phenolic acids and avonoids (Al et al., 2009; Allen, Molan, & Reid, 1991; Alandejani, Marsan, Ferris, Slinger, & Chan, 2009; Molan, 1992; Weston, 2000; Weston, Brocklebank, & Lu,

* Corresponding author. INQUINOA (CONICET), Universidad Nacional de Tucumn, Ayacucho 461, 4000 e San Miguel de Tucumn, Argentina. fax: 54 381 424 8169. E-mail address: misla@tucbbs.com.ar (M.I. Isla). 0023-6438/$ e see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.04.003

2000). It has been demonstrated that the presence of avonoids may contribute to the antioxidant effects observed in some honeys (Al-Mamary, Al-Meeri, & Al-Habori, 2002; Aljadi & Kamaruddin, 2004; Kk et al., 2007). Some studies have shown that avonoids inhibit auto-oxidation reactions and have a scavenging effect on free radicals by different mechanisms (Cos et al., 1998). Argentina is the second largest producer after China and accounts for 20% of the world production, with an average of 350 106 kg per year (Bedascarrasbure & Maldonado, 1995; Sabio & de los Santos, 2005). The Argentinean Northwest (ANW), due to its climate and vegetation, is a region with a growing potential for honey production, especially monooral honey, but there are few physicochemical and biological studies of these honeys. In this work, we characterized ANW honey samples from the physicochemical and functional standpoint (antioxidant and antibacterial activities).

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2. Materials and methods 2.1. Honey samples Thirteen honey samples were obtained from the ANW provinces of Tucumn, Santiago del Estero, Jujuy and Salta: a) monooral samples: algarrobo honey (Prosopis nigra) MASE 101; citrus honeys (Citrus lemon): MLT 201 to MLT 209 and MCMJ 301. b) multioral samples: MHT 290 and MMS 401. All samples were collected directly from beekeepers in the 2006e2008 period and stored in the dark at 4  C. 2.2. Chemicals Methanol, water and acetic acid (HPLC grade) were purchased from, Sintorgan (Argentina). Sucrose, AlCl3, NaOH, Na2CO3, gallic acid and Folin-Ciocalteau reagent were purchased from Merck (Germany). Sigma Aldrich and Fluka provided 2, 2-diphenyl-1picrylhydrazyl-hydrate p.a. (DPPH) and 2, 20 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), gallic acid, quercetin, hesperidin, hesperetin, pinocembrin and chrysin. Amberlite XAD-2 was purchased from Supelco (Bellefonte, USA). The antimicrobial agents were supplied by Sigma Chemical Co (USA) and Laboratorio Britania S.A. (Argentina). All chemicals and reagents were of analytical grade. 2.3. Physicochemical analysis 2.3.1. Moisture Water was determined from the refractive index of the honey by reference to a table derived from a formula developed by Wedmore, 1955. 2.3.2. Colour Honey colour was determined by measuring the light transmittance of bubbles and debris free honey samples in a colorimeter (Hanna C221, Hungary) using glycerol as standard. The results were expressed in mm Pfund (IRAM 15941-2, 1997). 2.3.3. pH and free Acidity The honey samples were dissolved in CO2 free water, the pH measured with a pHmeter (Altronix, Argentina) and the solution titrated with 0.05 mol/L sodium hydroxide solution up to pH 8.3 to obtain the free acidity (AOAC, 1990). 2.3.4. Electrical conductivity Honey electrical conductivity was determined with a conductimeter (Consort C830, Belgium) on a 200 g/L aqueous solution at 20  C (IRAM 15945, 1997). 2.3.5. Hydroxymethylfurfural Hydroxymethylfurfural (HMF) content was determined by UV absorbance at 284 nm. The difference between the absorbance of a clear aqueous honey solution (claried with Carrez I and II solutions) and the same solution after addition of bisulphite was determined to prevent other components from interfering. The HMF content was calculated after subtracting background absorbance at 336 nm (White, 1979a, b). 2.3.6. Reducing sugars and apparent sucrose Reducing sugar content was determined by the method of Fehling-Causse-Bonnans (IRAM 15934, 1995). Apparent sucrose

was calculated as the difference between total reducing sugars and reducing sugars. Honey samples were treated with HCl concentrated and then neutralized with 6 mol/L NaOH for total reducing sugars determination. 2.4. Flavonoids extraction Each sample was diluted with ve parts of water acidied with concentrated HCl, pH 2. The homogenate was then extracted with hexane and the aqueous fraction was mixed with Amberlite XAD-2 resin (0.84e0.25 nm). The resin slurry was packed into a glass column (20 2 cm). Polar compounds, like carbohydrates, were eluted with water acidied with HCl, pH 2, and subsequently with distilled water. Hydrophobic compounds, like avonoids, attached to the resin surface, were eluted with methanol. The methanolic extract was evaporated at 40  C and the residue was resuspended in distilled water (5 mL). Three successive extractions with ethyl ether (5 mL) were made and the fractions were combined and then evaporated. The residue containing mainly phenolic compounds were resuspended in methanol and analyzed by HPLC (Ferreres, Ginert & Toms-Barbern, 1994). Extracts were conserved at 20  C in the dark until use. 2.4.1. Phenolic composition by HPLC Phenolic compounds were detected by HPLC Gilson (USA) using an RP-C18 column (Phenomenex column size 4.6 250 mm; particle size 5 mm). It was eluted using a solvent gradient: water-acetic acid; 99:1, mL/mL (A) and methanol (B), starting with 100 mL/L of B (0e15 min), until reaching 900 mL/L(15e60 min), remaining at 900 mL/L B (60e65 min) and then decreasing to 100 mL/L of B (65e75 min). The solvent ow was 1 mL/min and run temperature of 25  C. Compounds detection was carried out at 290 nm and the identication by UV-spectra and co-chromatography with standard compounds (hesperidin, hesperetin, pinocembrin and chrysin). 2.4.2. Total phenolic content Total phenolic content was determined by the FolineCiocalteau reactive (Singleton, Orthofer, & Lamuela-Raventos, 1999). A honey solution (1 g/L) was mixed with 0.2 mL of 2 mol equi/L FolineCiocalteau reagent (Fluka, Switzerland) and 0.8 mL of sodium carbonate solution (159 g/L). The reaction mixture was heated at 50  C for 5 min in a water bath. Absorbance was measured at 765 nm after cooling. Results were expressed as mg gallic acid equivalents/g of honey (mg GAE/g of honey) using a calibration curve over the range of 5e50 mg gallic acid. 2.4.3. Estimation of total avonoids A method described by Popova, Silici, Kaftanoglu, and Bankova (2005) was used for total avonoids determination. Briey, 0.5 mL AlCl3 (20 g/100 mL) was added to 0.5 mL of honey samples. After 1 h at room temperature, absorbance was measured at 420 nm. Total avonoid contents were expressed as mg quercetin equivalents/g of honey (mg QE/g of honey), using a calibration curve over the range of 5e30 mg quercetin. 2.5. Antioxidant activity 2.5.1. DPPH free radical scavenging activity Antioxidant activity was determined for all samples using the method described by Ordoez, Gmez, Vattuone, and Isla (2006) based on DPPH scavenging. The discoloration grade of DPPH solution (1.5 mL of 300 mmol/L in 96 ethanol) with different phenolic compounds extracted of honey samples or dilutions of honey was recorded at 515 nm after 15 min. Methanol (HPLC grade) was used for honey sample preparation. Quercetin and butylated

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hydroxy-toluene (BHT) were used as positive controls (1e50 mg/mL). The antioxidant activity was expressed as a percentage of radical scavenging activity (RSA%). SC50 values (sample concentration required to scavenge 50% of DPPH free radicals) were calculated. 2.5.2. ABTS free radical scavenging activity The antioxidant capacity assay was carried out by the improved ABTS method as described by Re et al. (1999). ABTS radical cation (ABTS) was produced by reaction between an ABTS stock solution with 2.45 mmol/L potassium persulfate (nal concentration) and allowing the mixture to stand in the dark at room temperature (23  C) for 12e16 h before use. ABTS solution (1 mL; absorbance of 0.7 0.02 at 734 nm) was added to each preparation and mixed thoroughly. The reactive mixture was allowed to stand at room temperature and the absorbance reading was taken at 30  C exactly 1 min after initial mixing. Thereon, it was taken for 6 min at 1 min intervals. Quercetin and butylated hydroxy-toluene (BHT) were used as positive controls (1e10 mg/mL). Results were expressed in terms of percentage of residual scavenging activity (%RSA). SC50 values (sample concentration required to scavenge 50% of DPPH free radicals) were calculated. 2.5.3. Autographic assay The components of the different extracts (15 and 30 mg of total phenolic compounds) were separated by TLC (Kieselgel 60 F254 0.2 mm, Merck) using chloroform: ethyl acetate (4.4:0.6, mL/mL) as development solvents. The separated components were vizualized with ABTS after the TLC plates were dry. A volume of 3 mL of soft medium (agar 9 g/L) containing 1 mL ABTS solution was distributed on each TLC plate. After solidication, the plates were incubated at room temperature for 0, 0.5 and 1 min in the dark. The antioxidant activity appeared as clear spots against a dark greenblue background (Zampini, Ordoez, & Isla, 2010). The separated components were also visualized under ultraviolet light (254 and 365 nm, UV Lamp Model UVGL-58 Mineralight Lamp, USA) followed by spraying with 10 g/L methanolic solution of diphenylboric acid aminoethyl ester, NP reagent (Wagner, Bladt, & Zgainsky, 1984) and were compare the retention factor (Rf) values (ratio of the distance travelled by the compound to the distance travelled by the solvent).

The cell number in CAMHB was estimated using a serial dilution technique according to the recommendations of the CLSI 2006 (Clinical Laboratory Standards Institute) for each assay. 2.6.2. Minimal inhibitory concentration (MIC) MIC values of Argentinean honeys against the test organisms were determined by serial agar macrodilution method (CLSI, 2006). The same volume (1 mL) of dilutions of each honey was added to 9 mL of MHA (Meller-Hinton agar) medium. After cooling, the plates were inoculated in spots with 2 mL of each bacterial cell suspension (5 104 CFU) and incubated aerobically for 16e20 h at 35  C. A growth control of each tested strain was included. Controls of distilled water were carried out. MIC100 was dened as the lowest concentration of honey at which no colony was observed after incubation. MIC values were also determined for different commercial antibiotics. Resistance was dened for each case according to CLSI (2006): levooxacine (Lvx, MIC ! 8 mg/mL), piperacillin/tazobactam (Tzp, ! 128 mg/mL), imipenem (Ipm, MIC > 16 mg/mL), meropenem (Mem, MIC > 16 mg/mL), ceftriaxone (Cro, MIC > 128 mg/mL), cefotaxime (Ctx, MIC > 128 mg/mL), ceftacidime (Caz, MIC > 32 mg/mL), cefuroxime (Cxm, MIC ! 32 mg/mL), cefepime (Fep, MIC ! 32 mg/mL), amikacin (Amk, MIC > 16 mg/mL) and ampicillin/sulbactam (Sam, MIC ! 32 mg/mL) for Gram-negative bacteria and oxacillin (Oxa, MIC > 16 mg/mL), streptomycin (Str, MIC > 300 mg/mL), ampicillin (Amp, MIC > 64 mg/mL), methicillin (Met, MIC > 16 mg/mL), gentamycin (Gen, MIC > 100 mg/mL) and vancomycin (Van, MIC > 6 mg/mL) for Gram-positive bacteria. 2.6.3. Determination of factors responsible for antimicrobial activity 2.6.3.1. Effect of pH. The pH effect on microbial growth was determined adjusting the culture medium pH between 4 and 6, using sodium citrate buffer (0.1 mol/L). 2.6.3.2. Effect of osmolarity. Different commercial sugar solutions such as fructose, glucose and sucrose were diluted and added to the culture medium (9 mL of MHA) to obtain the same sugar concentration that was determined for each honey sample previously. The mixture was homogenized and poured into Petri dishes. 2.6.3.3. Effect of hydrogen peroxide. Different dilutions of honey samples (1 mL) were treated with 100 ml of a 20 g/L peroxidase solution in potassium phosphate buffer 0.1 mol/L, pH7 and added to the culture medium. Corresponding controls were performed without peroxidase. After cooling, the plates were inoculated in spots with 2 mL of each bacterial cell suspension (5 104 CFU) and incubated aerobically for 16e20 h at 35  C in all experiments. A growth control of each tested strain was included. Controls of distilled water were carried out. 2.6.3.4. Effect of phenolic compounds. Honey samples or phenolic compounds obtained from honey samples (75 mg) were seeded on TLC plates in dot or separated by TLC according to described in 2.5.3.Then, were practiced the bioautographic assay, plates were covered with 3 mL of soft medium (BHI with 6 g/L agar) containing 105 colony forming units (CFU) of S. aureus (F7) or P. aeruginosa (F305), incubated at 35  C for 16e20 h and sprayed with a 2.5 mg/mL MTT solution (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) in PBS (10 mmol/L sodium phosphate buffer, pH 7, with 0.15 mol/L NaCl). Plates were incubated in the dark at 35  C for 1 h for colour development (Nieva Moreno, Isla, Cudmani, Vattuone, & Sampietro, 1999). The separated components were also visualized under ultraviolet light and NP reagent and the Rf values were compared with the Rf values of commercial avonoids.

2.6. Antimicrobial assays 2.6.1. Culture media and microbial identication Clinical isolates of Staphylococcus aureus (F7, F19, F22), Enterococcus faecalis (F203, F208, F223), Escherichia coli (F301), Morganella morganii (F339), and Pseudomonas aeruginosa (F305) were obtained from clinical samples from Hospital Dr. Nicols Avellaneda, San Miguel de Tucumn, Tucumn, Argentina. The following reference strains were included in the study: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 35218, and Klebsiella pneumoniae ATCC 700603. The strains were identied by the use of biochemical proles according to the recommendations of the Manual of Clinical Microbiology (Murray, Baron, Pfaller, Tenover, & Yolken, 1999). All organisms were maintained in brain-heart infusion (BHI medium) containing 300 mL/L glycerol at 20  C. Before testing, the suspensions were transferred to trypticase soy agar supplemented with 50 g/L sheep blood (Difco) and aerobically grown overnight at 35  C. Individual colonies were isolated and suspended in 5 mL of 9 g/L NaCl solution. The inocula were prepared by adjusting the turbidity of the suspension to match the 0.5 McFarland standard and diluted in CAMHB (cation e adjusted MellereHinton broth) in order to achieve the adequate inoculum in each case.

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2.7. Statistical analysis Analyses were made in triplicate, and the data are presented as mean standard deviation. GraphPad Prism 5.0 software was used to perform analysis of correlation between two variants by Pearson test with the level of signicance set at p < 0.05 and of variance (ANOVA) with Tukey posttest at a condence level of 95%. 3. Results and discussion The study was conducted in 13 honey samples from beehives of different phytogeographical regions of the Argentinean Northwest. 3.1. Physico-chemical properties The data obtained are presented in Table 1 and show the good quality of the honey samples studied. 3.1.1. Moisture content Water content is a good criterion to establish honey quality; high moisture content can produce honey fermentation during storage, resulting in the formation of ethyl alcohol and carbon dioxide. The alcohol can further be oxidized into acetic acid and water with the ensuing sour taste (Chirife, Zamora, & Motto, 2006). Honey moisture content depends on various factors such as harvesting season, degree of maturity reached in the hive and climatic factors (Finola, Lasagno, & Marioli, 2007). Values between 14.1 g/100 g and 18.8 g/100 g were obtained. All tested honeys had moisture contents below 20 g/100 g, which is the maximum prescribed limit as per the Codex standard for honey (Codex Alimentarius, 2001). 3.1.2. Color The colour characteristics are presented in Table 1. Based on the USDA classication, the ANW honeys studied can be regarded as extra white (colour of the samples >8 and 17 mmPfund) and white (>17 and 34 mmPfund). Sample MCMJ 301 was considered as extra light amber (>34 and 50) and MMS 401 (>114 mmPfund) as dark amber. The colour intensity was supposed to be related to pigments (carotenoids, avonoids) that are also known to have antioxidant properties. 3.1.3. pH All the honeys analyzed were acidic. Their pH values ranged from 3.18 to 4.10 (Table 1). In general, honey is acidic in nature regardless of geographical origin. The pH values of Brazilian and Spanish honeys have been found to vary between 3.49 and 4.53, 3.10 and 4.05, respectively (Azeredo, Azeredo, de Souza, & Dutra, 2003).

3.1.4. Free acidity Acidity affects the avor and aroma of honey and is due to the presence of organic acids, particularly gluconic, pyruvic, malic and citric, in equilibrium with lactones or esters and inorganic ions (Echingo & Takenaka, 1974). Table 1 shows the acidity values obtained for all samples. None of the honeys exceeded the limit allowed by the Codex Alimentarius (Codex Alimentarius, 2001) and Standard Mercosur (MERCOSUR Technical Regulation of Identity and Honey Quality, 1994) (40 and 50 meq/kg respectively), indicating the absence of undesirable fermentation. In addition, the average value 25.13 meq/ kg may be taken as an indicator of honey freshness. Sample MASE 101, from Santiago del Estero (9.9 meq/kg) had the lowest value, while the highest corresponded to multioral honeys MMS 401 from Salta (33.4 meq/kg) and MCMJ 301 from Jujuy (36.8 meq/kg). All lemon honey from Tucumn showed relatively homogeneous values between 21.4 and 28.0 meq/kg while citrus honey from Spain ranged from 9.20 to 29.20 meq/kg obtained by Serrano, Villarejo, Espejo, and Jodral (2004). 3.1.5. Electrical conductivity Table 1 shows that sample MASE 101 from Santiago del Estero (0.684 mS/cm) had the highest value followed by MMS 401 from Salta (0.466 mS/cm). The lemon and multioral honeys from Tucumn showed lower values ranging from 0.121 to 0.223 mS/cm. None of the samples exceeded the maximum allowed by the Codex Alimentarius of 0.8 mS/cm (Codex Alimentarius, 2001). The results are consistent with other studies that indicate that citrus honeys have low levels of conductivity (Corbella & Cozzolino, 2006; Serrano et al., 2004; Terrab, Gonzalez, Diez, & Heredia, 2003). 3.1.6. HMF Over-heating of honey samples during processing or storage for very long periods could lead to the conversion of sugars to HMF. A low level of HMF is an indicator of the freshness of honey. In all samples the HMF content was found to be lower than the values recommended by the Codex Alimentarius (Codex Alimentarius, 2001) and by Standard Mercosur (MERCOSUR Technical Regulation of Identity and Honey Quality, 1994) (40 mg/kg). The HMF values were between 4 and 26.28 mg/kg (Table 1). 3.1.7. Reducing Sugar and apparent sucrose Sugars represent the main components of any type of honey. The reducing sugar content in the honey samples ranged from 67.7 to 73.5 g/100 g and the apparent sucrose from 0.4 to 5.6 g/100 g (Table 1). The data indicated that the majority of soluble sugars in honey samples are reducing sugars. A higher apparent sucrose content observed in MLT 204 could be attributed to an early harvest of honey,

Table 1 Physicochemical parameters of Argentinean Northwest honey samples (mean standard deviation, n 3). Sample number 101 201 202 203 204 205 206 207 208 209 301 290 401 H (g/100 g) 15.2 16.9 16.5 16.4 16.8 17.0 16.9 16.9 16.8 16.9 17.6 14.1 18.8 0.1 0.2 0.2 0.1 0.1 0.2 0.1 0.2 0.2 0.1 0.1 0.1 0.0 Colour (mmPfund) 32 12 17 16 18 22 16 21 15 10 96 40 126 12 4 6 5 6 5 4 7 6 3 11 2 14 pH 4.10 3.37 3.36 3,37 3.24 3.21 3.25 3.22 3.27 3.18 3.56 3.43 3.62 0.04 0.06 0.03 0.14 0.19 0.05 0.14 0.19 0.08 0.17 0.11 0.01 0.15 FA (meq/kg) 9.9 21.4 24.3 26.3 27.1 26.7 26.8 27.2 28.0 22.7 36.8 16.1 33.4 0.7 0.5 0.6 0.5 0.2 0.6 0.6 0.9 0.5 0.5 0.3 0.9 0.5 EC (mS/cm) 0.684 0.186 0.223 0.178 0.213 0.199 0.205 0.180 0.195 0.121 0.324 0.194 0.466 0.007 0.002 0.004 0.006 0.007 0.008 0.004 0.004 0.007 0.006 0.006 0.002 0.003 HMF (mg/kg) 13.1a 2.1 24.7 3.7 24.5 4.7 21.0 5.1 25.6 5.7 26.3 3.2 25.2 2.7 26.3 6.0 25.9 4.3 9.5a 2.3 49.0 2.2 4.0a 1.3 20.3 4.6 RS (g/100g) 71.5 2.2 70.8 2.5 70.8 1.9 68.9 2.4 69.1 2.2 69.8 1.9 69.9 1.8 67.7 3.3 70.7 4.4 69.7 1.3 73.5 1.3 68.6 2.3 69.3 1.8 AS (g/100 g) 1.6 2.0 0.4 2.7 5.6 3.1 2.8 3.7 1.1 3.9 4.3 2.6 0.7 0.5 0.5 0.0 0.4 0.1 0.3 0.4 0.1 0.3 0.2 0.1 0.5 0.1

H, Moisture; FA, Free acidity; EC, Electrical conductivity; HMF, Hydroxymethylfurfural; RS, Reducing sugars; AS, Apparent sucrose. a determined in fresh samples.

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Fig. 1. Phenolic compounds (-), as mg GAE/g honey, and avonoids ( ), as mg QE/g honey, content in analyzed honey samples.

wherein sucrose from nectar has not been fully transformed into glucose and fructose by bees (Guler, Bakan, Nisbet, & Yavuz, 2007). All other samples showed sucrose levels below 5 g/100 g, prescribed maximum limit by Codex Alimentarius. 3.2. Total phenolic and avonoid content Total phenolic content of ANW honeys ranged from 187.30 to 1073.21 mg GAE/g. The highest values were obtained for the

multioral honeys MCMJ 301 and MMS 401 (1073.21 and 875.75 mg GAE/g, respectively), approximately three times higher than those obtained for samples of monooral lemon honeys. The concentration and type of polyphenolic substances in honey is variable and depends on the oral origin (Kk et al., 2007). Fig. 1 shows the phenolic and avonoid recovery for each analyzed honey. A positive correlation was observed between colour and avonoid or phenolic compounds content (R2 0.98 and R2 0.92, respectively). Multioral honeys (MHT290 and MMS 401) have the highest avonoid content and darkest colours (Fig. 2). Hesperedin, hesperetin, pinocembrin and chrysin contents of ANW honeys are shown in Table 2. These results are the rst report on honey from Northwest Argentine (Tucuman, Salta and Jujuy). In general, the range of total phenolics in honey obtained from ANW was coincident with that determined in multi and monooral honey from other country (from 325.9 to 1003.9 mg GAE/kg of honey), (Meda, Lamien, Romito, Millogo, & Nacoulma, 2005; Vit, Gutirrez, Titera, Vendar, & Rodrguez-Malaver, 2008).

3.3. Biological activities 3.3.1. Antioxidant activities A positive signicant correlation between honey colour and ABTS scavenging activity was observed (R2 0.73). Thus, the Argentinean honeys with more colour and electrical conductivity

Fig. 2. Reverse-phase HPLC of honey samples number MHT 290 (A) and MMS 401 (B).

M.I. Isla et al. / LWT - Food Science and Technology 44 (2011) 1922e1930 Table 2 Hesperidin, hesperetin, pinocembrin and chrysin content in methanolic extracts of unioral and multioral honeys from Argentinean Northwest (mean standard deviation, n 3). Sample Hesperidin mg/kg 101 201 202 203 204 205 206 207 208 209 301 290 401 nd 2.62 4.38 6.04 3.95 4.23 5.52 3.97 6.35 3.87 3.49 0.60 0.27 0.20 0.35 0.51 0.28 0.23 0.48 0.30 0.55 0.30 0.16 0.04 0.01 0.35 1.83 0.21 1.00 1.50 0.29 nd nd 1.16 0.15 0.37 0.31 0.54 0.01 0.12 0.01 0.14 0.10 0.02 0.05 0.05 0.02 0.45 0.15 0.40 0.37 0.25 0.34 0.66 0.04 1.45 1.05 0.00 0.02 0.01 0.02 0.01 0.03 0.03 0.02 0.02 0.03 0.03 0.11 0.09 0.10 0.09 0.01 0.36 0.31 0.30 0.13 0.14 0.04 0.22 0.08 0.36 0.62 0.01 0.01 0.00 0.02 0.03 0.01 0.01 0.01 0.00 0.02 0.01 0.03 0.04 Hesperetin Pinocembrin Chrysin

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0.09 0.01 0.03 0.02 0.04

nd: not detected.

were also those with higher free radical scavenging capacity. There was a signicant correlation between the antiradical activity and phenolic compounds content of ANW honeys (R2ABTS 0.63). The highest antioxidant activity was exhibited by MMS 401 with SC50 values of 10 and 2.73 mg/mL for DPPH and ABTS, respectively (Fig. 3A). All honey samples showed a concentration-dependent pattern. The order of ABTS antioxidant activity for honey extracts was as follows: MMS 401 > MASE 101 > MCMJ 301 > lemon honey with SC50 values of 2.73, 3.32, 3.62, and 3.94 mg/mL respectively (Fig. 3A). The SC50 values of ANW honey were lower than the SC50 value of BHT SC50ABTS 5:0 0:1 mg=mL At the same phenolic compounds concentration, the free radical scavenging percentage was different, depending on the honey sample. These results indicate that the quality, not the quantity, of polyphenols is the major determinant of the antioxidant capacity of honey and that the chemical composition of the honey sample was dependent on its oral origin. In all samples analyzed by autographic assay there were at least ve zones with antioxidant activity that would correspond to phenolic compounds according to the Rf values (Fig. 3B).

Fig. 3. A. Scavenging activity of honey samples on DPPH(-) and ABTS ( ) radicals at a phenolic compounds concentration of 5 mg/mL. Samples MASE 101 correspond to Prosopis Honey, MLT 201 to MLT 209 to Lemon Honey, MCMJ 301 to Citrus and Monte Honey and MMS 401 to Monte Chaqueo Honey. B. Autographic assay: 15 and 30 mg of phenolic compounds extracted from honey samples MASE 101 (I) and MMS 401 (II) were developed in F254 silica gel plates using chloroform: ethyl acetate (4.4:0.6 mL/mL as solvent). Then, the plates were covered with 3 mL of soft medium (agar 9 g/L) containing 1 mL ABTS. Arrows indicate the Rf values of compounds with antioxidant activity.

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Table 3 Minimal inhibitory concentration (MIC) of ANW honey against Gram-positive and Gram-negative sensitive and antibiotic resistant bacteria. Bacterial strains 101 Gram (D) S. aureus F7 F19 F22 ATCC29213 E. faecalis F203 F208 F223 ATCC29212 Gram (L) E. coli F301 ATCC25922 ATCC35218 P. aeruginosa F305 K. pneumoniae ATCC700603 M. morganii F339 MIC (g/mL) 201 202 203 204 205 206 207 208 209 290 301 401 Phenotype of clinical isolate

0.20 0.10 0.20 0.10 0.20 0.20 0.20 0.20

0.25 0.15 0.15 0.15 0.25 0.25 0.25 0.25

0.20 0.15 0.15 0.15 0.20 0.20 0.20 0.20

e e e e e e e e

e 0.15 0.15 0.15 e e e e

0.20 0.10 0.20 0.15 e 0.20 e e

0.25 0.10 0.20 0.10 e e e e

0.20 0.10 0.20 0.10 0.25 0.25 0.25 0.25

e 0.15 0.25 0.15 e e e e

e 0.20 0.25 0.20 e e 0.25 e

0.20 0.05 0.10 0.10 0.20 0.20 0.20 0.20

0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20

0.20 0.05 0.15 0.10 0.20 0.20 0.15 0.20

Metr OxarGenr Mets OxasGensVans Metr OxarGensVans Control strain Genr Strr Vans Amps Genr Strr Vans Ampr Genr StrsVans Amps Control strain

0.10 0.15 0.20 0.10 0.20 0.15

0.15 0.15 0.15 0.15 0.20 0.15

0.15 0.15 0.20 0.15 0.20 0.15

e 0.20 0.25 0.15 e e

0.15 0.15 e 0.10 e 0.15

0.15 0.15 0.15 0.10 e 0.15

0.10 0.15 0.20 0.10 e 0.15

0.10 0.15 0.20 0.10 0.25 0.10

0.15 0.15 0.20 0.10 e 0.15

e 0.20 0.20 0.15 0.25 0.20

0.10 0.15 0.10 0.10 0.15 0.10

0.20 0.15 0.20 0.10 0.20 0.20

0.10 0.10 0.10 0.10 0.15 0.10

Lvxr Cror CTxr Cxmr SamrMemr Control strain Control strain Lvxr Cror CTxr Cazr Ipmr Amkr Memr Tzpr Control strain Lvxr Cros CTxr Cazr Ipmr Amkr Memr Tzpr

() indicates that bacterial growth was not observed until a concentration of 0.25 g/mL. All experiments were carried out in triplicate, rResistant, ssusceptible; vancomycin (Van), ampicilin (Amp), gentamycin (Gen), streptomycin (Str), methicillin (Met), oxacillin (Oxa),levooxacine (Lvx), piperacillin/tazobactam (Tzp), imipenem (Ipm), ceftriaxone, (Cro), cefotaxime (Ctx), ceftacidime (Caz), cefuroxime (Cxm), cefepime (Fep), amikacine (Amk), ampicillin/sulbactam (Sam) and meropenem (Mem).

Fig. 4. A) Effect of H2O2 on bacterial growth. Line 1: S. aureus (ATCC 29213, F22, F19, F7), Line 2: E. faecalis (ATCC 29212, F223, F208, F203). Line 3: P. aeruginosa (F305), E. coli (ATCC 35218, ATCC 25922, F301). Line 4: K. pneumoniae (ATCC 700603), M. morganii (F339). A1). Honey MASE 101 (25 g/100 mL) without peroxidase; A2). Honey MASE 101 (25 g/100 mL) with peroxidase B) Bioautographic assay: the comparison of honey MASE 101 (I) and MMS 401 (II) in silica gel F254 plates were made. B1) Phenolic compounds extracted (75 mg) were loaded on TLC in dot. B2) Phenolic compounds extracted (15e30 mg) were loaded and developed using chloroform: ethyl acetate (4.4:0.6 mL/mL as solvent I, II and III) The plates were then covered with 3 mL of medium containing 6 g/L BHI agar and 105 CFU/mL of S. aureus (F7). The plates were incubated at 35  C for 16e20 h and revealed with MTT. The arrows indicate the microbial growth inhibition zones whose Rf values match those of phenolic compounds (avonoids). The Rf value of one antibacterial compound (0.51e0.53) was coincident with Rf value of Pinocembrin. III) The plates were visualized with UV-NP reagent.

M.I. Isla et al. / LWT - Food Science and Technology 44 (2011) 1922e1930

1929

3.3.2. Antimicrobial activity In this work, antibiotic resistant Gram-positive and Gramnegative bacteria isolated from skin lesions (S. aureus, E. faecalis, P. aeruginosa, E. coli, M. morganii, K. pneumoniae) were used. In our work conditions, both Gram-negative and Gram-positive bacteria were susceptible to different samples of ANW honey (Table 3) in contrast with other reports (Basualdo, Sgroy, Finola, & Marioli, 2007; Estevinho, Pereira, Moreira, Dias, & Pereira, 2008; Socha, Juszczak, Pietrzyk, & Fortuna, 2009; Taormina, Niemira, & Beuchat, 2001) that showed that Gram-negative, (P. aeruginosa and E. coli), are more resistant than Gram-positive bacteria. Prosopis and multioral honeys inhibited the growth of both Gram-positive and Gramnegative microorganisms while lemon honeys were less active. Honey has multiple components (polyphenolics, sugars, acidity and hydrogen peroxide) which contribute to its antimicrobial activity (Lee, Churey, & Worobo, 2008; Molan, 1992). For this reason, the effect of polyphenols, sugars (glucose, fructose and sucrose), acidity and hydrogen peroxide on the growth bacterial was evaluated. According with the obtained results, neither sugars nor pH were responsible for the antibacterial activity demonstrated in ANW honey. Based on the results of peroxidase tests and phenolic effect, the antibiotic activity of ANW honey against S. aureus was partially attributed to the production of hydrogen peroxide and the presence of phenolic compounds (Fig. 4). In order to determine whether the antibacterial activity required the presence of phytocomplex or isolated compounds, chromatographic runs of ANW honey ethanol extracts were made. Bioautographic analysis revealed the presence of at least 5 antibacterial bands (Rf values of 0.15, 0.32, 0.41, 0.53 and 0.70) against S. aureus in MSAE101 and 4 components (Rf values of 0.07, 0.26, 0.51 and 0.84) active in MMS 401. Most of the antibacterial bands correspond to Rf values of avonoid compounds detected by NP reagent (Fig. 4) and the Rf value of one antibacterial component (0.51e0.53) are coincident with Rf value of Pinocembrin. The antioxidant and antibacterial properties of honey may be mainly due to its phenol content as evidenced by the signicant correlation observed between the antioxidant and antibacterial activities with phenolic content (R2 0.73). All the analyzed Argentinean honey samples, principally multioral and Prosopis, demonstrated antioxidant and antimicrobial activities. Hence, ANW honey can be used as food with therapeutic potential as antioxidant and as topical administration antibacterial agent against multi-resistant bacteria. Acknowledgements This research was supported by grants from Consejo de Investigacin de la Universidad Nacional de Tucumn, Argentina (CIUNT26D-430), Consejo Nacional de Investigaciones Cienticas y Tcnicas (CONICET-PIP-704) and Programa Nacional Apcola del INTA (PROAPI) y rea Estratgica de Tecnologa de Alimentos (AETA). References
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