Antimicrobial, antiviral and antioxidant activities of “água-mel” from Portugal

Food and Chemical Toxicology 56 (2013) 136–144

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Antimicrobial, antiviral and antioxidant activities of ‘‘água-mel’’ from Portugal Maria G. Miguel a,⇑, Leonor Faleiro b, Maria D. Antunes a, Smail Aazza a, Joana Duarte b, Ana R. Silvério b a b

Universidade do Algarve, IBB-Centro de Biotecnologia Vegetal, Faculdade de Ciências e Tecnologia, Edif. 8, Campus de Gambelas, 8005-139 Faro, Portugal Universidade do Algarve, IBB-Centro de Biomedicina Molecular e Estrutural, Faculdade de Ciências e Tecnologia, Edif. 8, Campus de Gambelas, 8005-139 Faro, Portugal

a r t i c l e

i n f o

Article history: Received 24 November 2012 Accepted 4 February 2013 Available online 17 February 2013 Keywords: ‘‘Água-mel’’ Phenols Melanoidins Biological properties

a b s t r a c t ‘‘Água-mel’’ is a honey-based product produced in Portugal for ancient times. Several attributes have been reported to ‘‘água-mel’’ particularly in the alleviation of simple symptoms of upper respiratory tract. Samples of ‘‘água-mel’’ from diverse beekeepers from different regions of Portugal were studied in what concerns antimicrobial, antioxidant and antiviral properties. The amounts of phenol and brown pigment were also evaluated and correlated with the antioxidant activities. A great variability on the levels of these compounds was found among samples which were responsible for the variability detected also on the antioxidant activities, independent on the method used. Generally, antioxidant activity correlated better with brown pigments’ amount than with phenols’ content. The antimicrobial activity found for ‘‘água-mel’’ samples confirm the virtues reported by popular findings. In addition, this work also reveals the antiviral properties of ‘‘água-mel’’ evidenced by a decrease on the infectivity of the Qb bacteriophage. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction A non-enzymatic reaction which occurs during baking, roasting, broiling, and frying of food is responsible for the formation of flavour compounds and brown pigments. For example, heat treatment of food is used extensively to increase the palatability of food. The brown pigments have a relative high impact on the quality of foods, because the colour is a key factor in consumer acceptance. This brown colour is attributed to the reaction between reducing carbohydrates and amino acids or proteins, known as Maillard reaction. During this reaction, there is the formation of a complex mix of diverse components of different molecular weights, including aldehydes, ketones, dicarbonyls, acryl amides, heterocyclic amines (all of them contributing to flavour), melanoidins and advanced glycation endproducts (AGEs) (both being polymeric products formed at the advanced steps of Maillard reaction) (Wang et al., 2011). Melanoidins are coloured compounds reported as possessing antioxidant activity and other biological properties. According to a recent review made by Wang et al. (2011), melanoidins present capacity for scavenging free radicals and chelating metal ions in in vitro assays. In vivo studies have revealed that a food melanoidin containing pyrrolinone reductonyl-lysine present in bread crust, caraffa malt,

Abbreviations: AAPH, 2,20 -Azobis-2-methyl-propanimidamide, dihydrochloride; ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; AGEs, glycation endproducts; AU, absorption units; AUC, area under the curve; FL, fluorescein; ORAC, oxygen radical activity capacity; PFU, plaque-forming unit. ⇑ Corresponding author. Tel.: +351 289800900; fax: +351 289818419. E-mail address: [email protected] (M.G. Miguel). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.02.007

or pronyl bovine serum albumin decreased oxidative stress and thiobarbituric acid reactive substances levels, and increased tocopherol in the plasma of rats (Somoza et al., 2005). Antimicrobial activity has been also described for melanoidins and they can act as bacteriostatic or bactericidal, depending on the concentrations. At low concentrations, melanoidins exert a bacteriostatic activity by the chelation of iron from the culture medium and at high concentrations they are bactericidal by chelating magnesium ions from the outer membrane, leading to a destabilization of the inner membrane with the consequent release of intracellular molecules (Rufian-Henares and de la Cueva, 2009). Honey is a source of high concentrations of reducing sugars (glucose and fructose), also possessing free aminoacids and proteins. The presence of these components is responsible for the non-enzymatic browning commonly observed during prolonged storage of honey, as well as when it is submitted to heating. The formation of these brown pigments generally coincides with an increase of antioxidant and antimicrobial activities (Brudzynski and Kim, 2011, 2011a,b; Turkmen et al., 2006a,b). The high molecular weight melanoidins were already recognised by Brudzynski and Miotto (2011a,b) as being the main components responsible for radical-scavenging ability of Canadian honeys. A honey-based product known as ‘‘água-mel’’ is produced in Portugal for ancient times. This product also produced in Sardinia region (Italy) (Spano et al., 2008) is obtained from honeycombs which still possessing residues of honey are crumbled and dipped into warm water for separating waxes which are removed carefully from the top. The remaining mixture (water, honey, pollen and some propolis) is submitted to heat, until obtaining a brown,

M.G. Miguel et al. / Food and Chemical Toxicology 56 (2013) 136–144

honey-like product, called ‘‘abbamele’’. In Portugal the production of ‘‘água-mel’’ follows practically the same steps reported for ‘‘abbamele’’ from Italy. In the majority of cases, there is no control of temperature during the production process only depending on the producer tradition. Nevertheless and more recently, some beekeepers start to register the temperature and °Brix throughout processing. According to these beekeepers, the heat treatment at 100–108 °C is done until 70–77°Brix is achieved. In Portugal, Figueira and Cavaco (2012) reported for the first time the changes in physical and chemical parameters (viscosity, total soluble solids and Hunter colour parameters L, a, b, chroma and hue angle) of that product during the production process, and Miguel et al. (in press) reported the chemical characterisation and microbiological quality of ‘‘água-mel’’ produced by several beekeepers from Algarve and Alentejo. ‘‘Abbamele’’ from Sardinia region possessed antioxidant activity (Jerkovic´ et al., 2011). Portuguese people uses ‘‘água-mel’’ since ancient times, as sweetener in cakes, tea, and, of great importance, as natural medicine on the alleviation of simple symptoms of upper respiratory tract. The evaluation of the antimicrobial, antiviral and antioxidant activities of ‘‘água-mel’’ is the main objective of this work. The correlation between antioxidant activity and the phenolic content of ‘‘água-mel’’, its colour and the Maillard reaction like products, such as melanoidins, formed during the ‘‘água-mel’’ production, is also intended to evaluate in the present work. For this purpose, several samples of ‘‘água-mel’’ from various beekeepers and obtained in diverse years were evaluated.

2. Methods

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2.3. Total content of polyphenols and flavonoids Polyphenol content was spectrophotometrically determined as described by Floris et al. (1994) and also reported for the quantification of phenols in ‘‘abbamele’’ from Sardinia (Spano et al., 2008). This method is based on Folin Ciocaletu’s reagent but modified to prevent interference by reducing sugars. Data are expressed as mg of caffeic acid equivalents/g of ‘‘água-mel’’ (CAEs). The same fractions used for the quantification of total phenols were used in the quantitative evaluation of total flavonoids. The method used was that reported by Kong et al. (2012). Data are expressed as mg quercetin equivalents/g of ‘‘águamel’’ (QEs).

2.4. Capacity for scavenging 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) The determination of ABTS radical scavenging was carried out as reported by Miguel (2010). Briefly, the ABTS radical was generated by the reaction of (7 mM) ABTS aqueous solution with K2S2O8 (2.45 mM) in the dark for 16 h and adjusting the absorbance at 734 nm to 0.7 at room temperature. Samples (25 ll) were added to 275 ll ABTS, and the absorbance at 734 nm was read after 6 min. Several concentrations of samples were made and the percentage inhibition calculated from the formula: [(A0  A1/A0)  100] was plotted against sample concentration and IC50 was determined (concentration of sample able to scavenge 50% of ABTS free radical). A0 is the absorbance of negative control (blank sample containing the same amount of solvent and ABTS solution); A1 is the absorbance of the sample.

2.5. Chelating metal ions The degree of chelating of ferrous ions by ‘‘água-mel’’ was evaluated according to Miguel et al. (2010). Briefly, samples were incubated with 0.05 ml of FeCl24H2O (2 mM). The addition of 0.2 ml of 5 mM ferrozine initiated the reaction, and after 10 min, the absorbance at 562 nm was measured. An untreated sample served as the control. The percentage of chelating ability was determined according to the following formula: [(A0  A1)/A0  100], in which A0 is the absorbance of the control and A1 the absorbance of ‘‘água-mel’’ sample. The values of IC50 were determined as reported above.

2.1. Samples Samples of ‘‘água-mel’’ were given by the following producers; through the beekeepers Association ‘‘Associação dos Apicultores do Sudoeste Alentejano e Costa Vicentina’’ (AASACV), Portugal: – – – – – – – – – – – – – – – – – – –

1A/2010: Producer 1A/year of production 2010. 1A/2011: Producer 1A/year of production 2011. 2A/2011: Producer 2A/year of production 2011. 1B/2008: Producer 1B/year of production 2008. 1B/2010: Producer 1B/year of production 2010. 1B/2011: Producer 1B/year of production 2011. 2B/2011: Producer 2B/year of production 2011. 1C/2011: Producer 1C/year of production 2011. 1D/2011: Producer 1D/year of production 2011. 1E/2011: Producer 1E/year of production 2011. 1F/2011: Producer 1F/year of production 2011. 1H/2011: Producer 1H/year of production 2011. 1I/2011: Producer 1I/year of production 2011. 1J/2011: Producer 1J/year of production 2011. 1K/2011: Producer 1K/year of production 2011. 1L/2011: Producer 1L/year of production 2011. 1M/2011: Producer 1M/year of production 2011. 1N/2011: Producer 1N/year of production 2011 1O/2011: Producer 1O/year of production 2011.

Samples were kept at room temperature and flasks were opened at aseptic conditions, in a laminar flow chamber (BIOHAZARD, Bio II A, Telstar, Madrid, Spain). For each sample of ‘‘água-mel’’, the producers provided 3 bottles, which served as replicates. For each bottle, three determinations were done. Thus, data are the mean of nine determinations (n = 9).

2.2. Analyses of água-mel colour and melanoidin content ‘‘Água-mel’’ colour was determined spectrophotometrically (Shimadzu 160-UV spectrophotometer) by measuring net absorbances at (A560–A720). Melanoidin content was estimated based on the browning index (net absorbance at A450–A720) (Brudzynski and Miotto (2011a). In both cases, the measurements were conducted using a 1 cm path length, quartz cell, being the results expressed as absorption units (AU).

2.6. Oxygen radical activity capacity (ORAC) The ORAC method with fluorescein (FL) as the fluorescent probe was previously described by Ou et al. (2001). This assay is based on the capacity of antioxidants in a sample to quench peroxyl radicals generated from the thermal decomposition of AAPH (2,20 -Azobis-2-methyl-propanimidamide, dihydrochloride). A mass of 0.414 g AAPH was dissolved in 10 ml phosphate buffer 75 mM (pH 7.4) and was kept in an ice bath. The solution of fluorescein (0.00419 mM) was prepared in phosphate buffer and kept in the dark at 4 °C. A new concentration (8.16  105 mM) was made before reaction. Trolox standard 0.02 M was prepared in phosphate buffer and diluted to 50, 25, 12.5, and 6.25 lM. In each well of plate, 150 ll of fluorescein working solution and 25 ll of sample, blank (milliQ water), or standard were placed. The plate was covered with a lid and incubated in the preheated (37 °C) BioTek Synergy™ 4 Hybrid Microplate Reader for 10 min. Twenty-five microlitre AAPH solution were added to each well. The Microplate was shaken for 10 s and fluorescence was read every minute for 2 h at excitation of 485 nm and emission of 527 nm. ORAC values are calculated according to Cao and Prior (1999). Briefly, the net area under the curve (AUC) of the standards and samples was calculated. The standard curve is obtained by plotting Trolox concentrations against the average net AUC of the two measurements for each concentration. Final ORAC values are calculated using the regression equation between Trolox concentration and the net AUC and are expressed as lmol Trolox/g ‘‘água-mel’’.

2.7. Nitric oxide scavenging capacity The nitric oxide (NO) scavenging activity of samples was measured according to the method described by Ho et al. (2010). In this method 50 ll of serially diluted sample of ‘‘água-mel’’ were added to 50 ll of 10 mM sodium nitroprusside in phosphate buffer saline (PBS) into a 96-well plate and the plate was incubated at room temperature for 90 min. Finally, an equal volume of Griess reagent was added to each well and the absorbance was read at 546 nm. Several concentrations of samples were made and the percentage inhibition calculated from the formula: [1  (Asample  Asample blank)/(Acontrol  Acontrol blank)]  100, where (Asample  Asample blank) is the difference in the absorbance of a sample, with or without 10 mM sodium nitroprusside, and (Acontrol  Acontrol blank) is the difference in the absorbance of the PBS control, with or without 10 mM sodium nitroprusside. Percentage inhibition was plotted against sample concentration and IC50 was determined (concentration of sample able to scavenge 50% of NO free radical).

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2.8. Lipoxygenase inhibition activity

2.11. Statistical analysis

Lipoxygenase is known to catalyse the oxidation of unsatured fatty acids containing 1–4 diene structures. The conversion of linoleic acid to 13-hydroperoxy linoleic acid was followed spectrophotometrically by appearance of conjugate diene at 234 nm on a UV/visible spectrophotometer, according to that described by some authors (Frum and Viljoen, 2006). The reaction was initiated by the addition of 3 ll lipoxygenase solution (0.054 g in 1 ml borate buffer 0.005%, Tween 0.1 M, pH 9) to 937 ll borate buffer, 10 ll sample and 50 ll linoleic acid (0.001 M). The absorbance was read every minute for 5 min. The percentage inhibition of the enzyme was calculated and the IC50 were determined.

The significant differences between the antimicrobial, antioxidant activities, phenols, colour and melanoidins of the different água-mel samples were determined by ANOVA through the SPSS 18.0 program (Inc., Chicago IL., USA) and using the Tukey post hoc test. Correlations between phenol content, melanoidins and antioxidant activity were achieved by Pearson correlation coefficient (r) at a significance level of 99% (P < 0.01).

3. Results and discussion

2.9. Antimicrobial activity

3.1. Polyphenol content

For the determination of the antimicrobial activity of the ‘‘água-mel’’, samples 1B/2010; 1B/2011; 1F/21011; 1H/2011 were used. The antimicrobial activity of those samples was tested against Escherichia coli DSM 1072, Salmonella enterica subspecies enterica serovar Typhimurium ATCC 14028; Enterobacter aerogenes DSM 30053, Staphylococcus aureus CFSA2; S. aureus ATCC 6538, Methicillin-resistant S. aureus (MRSA) 6, MRSA 12, Listeria monocytogenes EGD, L. monocytogenes C882, Enterobacter faecalis NCTC 775, Saccharomyces cerevisae DSM 70449, Candida albicans ATCC 90028. The determination of antimicrobial activity of ‘‘água-mel’’ samples was done by using a microdilution method according to EUCAST (2000) at concentrations of 20%, 30%, 40%, and 50% (w/v) in Brain Heart Infusion Broth (BHI) for bacteria and in Yeast Malt Broth (YM) for yeasts. The microbial cells were recovered from 80 °C by growing either in BHI agar or YM as appropriate and incubated at 37 °C the bacterial cultures and at 25 °C the yeasts cultures. Previous to each assay the microbial cells were grown in fresh culture media and an exponential culture was obtained. From this culture the inoculum was prepared by suspending the microbial cells in BHI broth supplemented with ‘‘água-mel’’ at appropriate concentration and a volume of 20 ll of this microbial suspension was used to inoculate a microplate well with 180 ll of the same medium. The microplate was incubated at 37 °C during 15 h for bacterial and C. albicans cultures. S. cerevisae was grown at 25 °C during 20 h. The microbial growth was followed by measurement of the absorbance at 600 nm (A600nm). At end of the growth experiment the viability of the cells was evaluated by plating 30 ll of the culture in each well in BHI agar or YM agar. The antibiotic chloramphenicol (30 lg/ml) and the antifungal agent cycloheximide (10 lg/ml) were used as control. The inoculation of BHI broth supplemented with the antibiotic/antifungal agent was done as described above. The percentage of inhibition of microbial growth and percentage of growth was determined according to Patton et al. (2006). Three independent experiments were done.

Polyphenol content and antioxidant activities of ‘‘água-mel’’ samples through the capacity for scavenging free radicals or inhibiting lipoxygenase are presented in Table 1. Polyphenol concentrations were much lower than those reported for ‘‘abbamele’’ samples from Sardinia (Jerkovic´ et al., 2011). These highest amounts of total phenols are attributed to the interferences of reducing sugars in the method based on Folin Ciocalteu’s reagent. In the present work, a modified method of the Folin Ciocalteu’s reagent was used for determining polyphenol content. In this method a previous extraction of sugars was done before the quantification of polyphenols, according to that reported by Floris et al. (1994). Following this procedure, the total phenols found are substantially inferior being closer to those reported by Spano et al. (2008) for ‘‘abbamele’’ from Sardinia. The levels of polyphenols in ‘‘água-mel’’ samples were greatly dependent on the producer and year of production as well (Table 1). Sample 1B/2011 presented the highest levels of polyphenols in contrast to those of 1E/2011, 1B/2008 and 1C/2011. The highest and the lowest values of polyphenol contents were found for ‘‘água-mel’’ samples produced by the same producer but in different years (1B/2008 and 1B/2011). Two main factors may have contributed to this variability: storage period of ‘‘água-mel’’ and floral origin of the honey from which ‘‘água-mel’’ was obtained. The last hypothesis was already proposed by Spano et al. (2008) for ‘‘abbamele’’ samples from Italy. The amount of polyphenols (1.323 ± 0.088) present in the sample 1B/2011, which presented the highest concentration, was even higher than reported in the literature (Al et al., 2009; Ferreira et al., 2009) for honeydew honey that is known to have the highest levels of polyphenols. Nevertheless, the remaining samples had lower concentrations of total phenols as the samples of ‘‘abbamele’’ from Sardinia (Italy) using the same method of quantification (Spano et al., 2008). As reported for polyphenols, the amounts of flavonoids present in ‘‘água-mel’’ samples were also greatly dependent on the producer and year of production as well (Table 1). They also were within the ranges found for honey from diverse botanical origin of Italy and northeastern Portugal (Pichichero et al., 2009; Estevinho et al., 2012). These authors showed the influence of botanical origin of honey on the flavonoid content.

2.10. Antiviral activity The antiviral activity was determined by following the effect of ‘‘água-mel’’ samples on the infectivity of the enterobacteria phage Qb DSM13768. The genome of the enterobacteria phage Qb is constituted by a single stranded, linear positivesense RNA molecule, which is encapsulated in an isosahedral protein capsid of about 24–26 nm of diameter and is the type specie of the genus Allolevivirus of the Leviviridae family (International Committee on Taxonomy of Viruses). The enterobacteria phage was replicated in E. coli DSM 5210 for 4–6 h at 37 °C. The phage culture was centrifuged at 2500g during 10 min. The supernatant was filtered (mixed cellulose esters membrane of 0.45 lm of pore, Pall Corporation, USA). The filtrate was centrifuged at 15,000g during 15 min and resuspended in BHI with 25% (v/v) glycerol to obtain the enterobacteria phage stock suspension, which concentration was 109–1012 PFU/ml. The bacteriophage infectivity was determined by using a microplate method adapted from the described by McLaughlin (2007) and confirmed by a standard overlay agar assay (Fisher et al., 2009; Vo et al., 2009). Solutions of ´ ’’água-mel’’ at 30%, 40%, and 50% (w/v) in BHI broth were inoculated with 109 PFU/ml of the Qb bacteriophage and incubated at 37 °C. The number of PFU/ml was determined after 24 h and 48 h of contact of the bacteriophage with the ‘‘água-mel’’ sample. The microplate method included the distribution of the E. coli DSM 5210 culture in exponential phase (O.D600 nm of about 0.25–0.30) into wells (180 ll per well) and addition of 20 ll of the phage suspension in ‘‘água-mel’’ at different concentrations. The last row of the microplate was inoculated with the bacterial culture and the appropriate concentration of ‘‘água-mel’’ without phage (in the same proportion as for phage suspension) to control the activity of ‘‘água-mel’’ against the host bacterium. Bacterial cell lysis was followed by optical densities readings in a microplate reader (Tecan Infinite M200, Tecan) during 3 h. Following the determination of which dilutions showed lysis the number of PFU/ml was determined by the plaque assay that by mixing 100 ll of the E. coli culture with 100 ll of the diluted phage suspension and 3 ml of melted soft agar (0.75% [w/v] agar) were added to each host/phage suspension and homogenised thoroughly. This mixture was transferred to a Petri dish containing solid agar medium (1.5% [w/v] agar). The inoculated plates were incubated at 37 °C. The number of PFU/ml was determined after overnight incubation, in triplicate for each ‘‘água-mel’’ concentration. Three independent experiments were done.

3.2. Antioxidant activity ‘‘Água-mel’’ samples having polyphenols should have antioxidant activity, because such components are able to scavenge free radicals and chelate metal ions which could catalyse the lipid peroxidation (Bravo, 1998). They also inhibit lipid peroxidation by acting as chain-breaking peroxyl-radical scavengers (Miguel, 2010). Our results show such capacity of ‘‘água-mel’’ samples measured through diverse methods. Nevertheless for each assay, a great variability of antioxidant abilities was found. For example, six samples revealed to be significantly better as chelating agents: 1A/2011, 1J/ 2011, 1B/2011, 2A/2011 and 1B/2010, in contrast to that of 1N/ 2011, the worst one (Table 1). In what concerns the capacity for scavenging free radicals, 2A/2011 had the lowest IC50 values, that

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M.G. Miguel et al. / Food and Chemical Toxicology 56 (2013) 136–144 Table 1 Phenol and flavonoid contents and antioxidant activities of ‘‘água-mel’’ samples obtained from diverse beekeepers of Alentejo and Algarve (Portugal). Samples 1A/2010 1A/2011 2A/2011 1B/2008 1B/2010 1B/2011 2B/2011 1C/2011 1D/2011 1E/2011 1F/2011 1H/2011 1I/2011 1 J/2011 1 K/2011 1L/2011 1 M/2011 1 N/2011 1O/2011

Phenol content1 de

0.512 ± 0.086 1.122 ± 0.086ab 1.183 ± 0.086ab 0.332 ± 0.086e 0.536 ± 0.086de 1.323 ± 0.086a 0.977 ± 0.086bc 0.386 ± 0.086e 0.510 ± 0.086de 0.310 ± 0.086e 0.428 ± 0.086e 0.787 ± 0.086cd 0.505 ± 0.086de 1.021 ± 0.086bc 0.789 ± 0.086cd 0.568 ± 0.086de 0.791 ± 0.086cd 0.503 ± 0.086de 0.903 ± 0.086bc

Flavonoid content2 de

0.184 ± 0.027 0.341 ± 0.027b 0.327 ± 0.027b 0.143 ± 0.027de 0.185 ± 0.027de 0.444 ± 0.027a 0.281 ± 0.027bc 0.127 ± 0.027de 0.161 ± 0.027de 0.094 ± 0.027e 0.094 ± 0.027e 0.149 ± 0.027de 0.121 ± 0.027de 0.346 ± 0.027b 0.202 ± 0.027cd 0.085 ± 0.027e 0.182 ± 0.027de 0.145 ± 0.027de 0.185 ± 0.027de

Chelating activity3 g

1.035 ± 0.132 0.258 ± 0.132h 0.414 ± 0.132h 1.072 ± 0.132g 0.521 ± 0.132h 0.413 ± 0.132h 1.192 ± 0.132fg 3.236 ± 0.132c 1.093 ± 0.132fg 3.368 ± 0.132c 3.134 ± 0.132cd 2.206 ± 0.132e 4.282 ± 0.132b 0.280 ± 0.132h 1.516 ± 0.132f 4.374 ± 0.132b 2.772 ± 0.132d 7.762 ± 0.132a 1.462 ± 0.132fg

ABTS3

ORAC4 gh

0.665 ± 0.042 0.660 ± 0.042gh 0.295 ± 0.042j 2.892 ± 0.042b 0.663 ± 0.042gh 0.263 ± 0.042j 0.592 ± 0.042hi 1.165 ± 0.042f 2.000 ± 0.042d 2.877 ± 0.042b 2.220 ± 0.042c 0.787 ± 0.042g 3.027 ± 0.042a 0.517 ± 0.042i 1.067 ± 0.042f 2.270 ± 0.042c 1.318 ± 0.042e 2.960 ± 0.042ab 2.197 ± 0.042c

Lipoxygenase3 cd

200.233 ± 27.024 295.533 ± 27.024ab 379.867 ± 27.024a 137.133 ± 27.024def 163.633 ± 27.024de 318.433 ± 27.024ab 302.067 ± 27.024ab 75.800 ± 27.024efg 81.967 ± 27.024efg 30.300 ± 27.024g 64.167 ± 27.024fg 120.367 ± 27.024defg 120.367 ± 27.024defg 269.733 ± 27.024bc 165.933 ± 27.024de 83.467 ± 27.024efg 118.033 ± 27.024defg 59.033 ± 27.024fg 166.400 ± 27.024de

NO3 fg

4.153 ± 0.316 2.327 ± 0.316h 1.743 ± 0.316hi 8.137 ± 0.316c 7.027 ± 0.316d 1.163 ± 0.316i 1.717 ± 0.316hi 6.923 ± 0.316d 2.393 ± 0.316h 17.073 ± 0.316a 4.940 ± 0.316ef 4.317 ± 0.316fg 7.607 ± 0.316cd 3.730 ± 0.316g 5.480 ± 0.316e 7.840 ± 0.316cd 8.460 ± 0.316c 12.660 ± 0.316b 3.933 ± 0.316fg

8.343 ± 1.684efg 4.277 ± 1.684fg 4.137 ± 1.684fg 9.593 ± 1.684ef 7.513 ± 1.684efg 2.603 ± 1.684g 5.867 ± 1.684fg 8.620 ± 1.684ef 7.630 ± 1.684efg 15.843 ± 1.684cd 16.327 ± 1.684cd 16.467 ± 1.684cd 20.883 ± 1.684c 11.8000 ± 1.684de 17.607 ± 1.684c 39.047 ± 1.684a 29.777 ± 1.684b 37.860 ± 1.684a 16.583 ± 1.684cd

Values in the same column followed by the same letter are not significant different (p < 0.01) by the Tukey’s multiple range test. 1 (mg of caffeic acid equivalents/g of ‘‘água-mel’’ ± standard error, n = 9). 2 (mg of quercetin equivalents/g of ‘‘água-mel’’ ± standard error, n = 9). 3 IC50 (mg/ml ± standard error, n = 9). 4 Trolox Equivalent (lmol/g ± standard error, n = 9).

is, the best sample for scavenging ABTS. Concerning the capacity for scavenging peroxyl radicals (measured through the ORAC method), this sample was also the most effective because it had the most elevated value of trolox equivalent. For scavenging free ABTS radicals and peroxyl radicals, 1I/2011 and 1E/2011 were the less effective, respectively (Table 1). Nitric oxide (NO) is a mediator of physiological processes such as smooth muscle relaxation, neuronal signalling, inhibition of platelet aggregation and regulation of cell mediated toxicity. Low concentrations of NO are sufficient to affect these beneficial functions, nevertheless high concentrations is implicated in having a role in the pathogenesis of vasodilatation, non-specific host defence, ischemic stroke, septic shock and acute and chronic inflammation. However, during infections and inflammations, formation of NO is elevated and may be the responsible for some undesired deleterious effects (Jagetia et al., 2004; Kumar et al., 2008). However, in the aerobic conditions, the NO molecule is very unstable and reacts with the oxygen to produce NO2, N2O4, N3O4, the stable products nitrate and nitrite. NO is also able to react with superoxide originating peroxynitrite. All of these products formed are genotoxic (Jagetia et al., 2004). Therefore, compounds able to scavenge that reactive nitrogen substance may be useful because such prevents the formation of other products deleterious for the human health. Sample 1B/2011 was the best for scavenging NO radicals in contrast to those of 1L/2011 and 1N/2011, which possessed the highest values of IC50 (Table 1). The lipoxygenase assay has been used as an indication of the anti- inflammatory and antioxidant activities because lipoxygenase catalyse reactions on arachidonic acid that generate metabolites important to the mediation of inflammatory responses after the oxidation of this acid to hydroperoxyeicosatetraenoic acids (HPETE) and hydroxyeicosatetraenoic acids (HETE) (Akula and Odhav, 2008). Lipoxygenase also catalyse the oxidation of other unsaturated fatty acids containing 1–4-dienes. For example, the conversion of linoleic acid to 13-hydroperoxy linoleic acid is generally followed spectrophotometrically by the appearance of a conjugate diene which can be measured at 234 nm, constituting a method generally used for the evaluation of anti-lipoxygenase activity, that is, anti-inflammatory activity (Akula and Odhav, 2008). Concerning this ability, all samples possessed the capacity for inhibiting

Table 2 Colour and melanoidin content of ‘‘água-mel’’ samples obtained from diverse beekeepers of Alentejo and Algarve (Portugal). Samples 1A/2010 1A/2011 2A/2011 1B/2008 1B/2010 1B/2011 2B/2011 1C/2011 1D/2011 1E/2011 1F/2011 1H/2011 1I/2011 1 J/2011 1 K/2011 1L/2011 1 M/2011 1 N/2011 1O/2011

‘‘Água-mel’’ colour (A560–720)a e

0.209 ± 0.008 0.434 ± 0.008b 0.430 ± 0.008b 0.205 ± 0.008e 0.234 ± 0.008d 0.510 ± 0.008a 0.357 ± 0.008c 0.155 ± 0.008f 0.161 ± 0.008f 0.040 ± 0.008h 0.091 ± 0.008g 0.158 ± 0.008f 0.091 ± 0.008g 0.252 ± 0.008d 0.143 ± 0.008f 0.060 ± 0.008h 0.107 ± 0.008g 0.090 ± 0.008g 0.163 ± 0.008f

Melanoidin content (A450–A720)a 1.258 ± 0.040ef 2.474 ± 0.040b 2.849 ± 0.040a 1.203 ± 0.040f 1.359 ± 0.040e 2.934 ± 0.040a 2.176 ± 0.040c 0.738 ± 0.040i 1.197 ± 0.040f 0.233 ± 0.040l 0.547 ± 0.040j 0.811 ± 0.040hi 0.600 ± 0.040j 1.479 ± 0.040d 0.873 ± 0.040h 0.381 ± 0.040k 0.557 ± 0.040j 0.420 ± 0.040k 1.065 ± 0.040g

Values in the same column followed by the same letter are not significant different (p < 0.01) by the Tukey’s multiple range test. a Absorption units (AU).

lipoxygenase, nevertheless with different strengths because the sample 1B/2011 was the most active in contrast to that of 1E/ 2011 (Table 1) with IC50 of 1.163 and 17.073 mg/ml, respectively. The Pearson correlation coefficients (r) showed a strong relationship between the phenol content and peroxyl scavenging ability of samples measured through ORAC method (r = 0.794, p < 0.01, n = 57), and a negative correlation between phenols’content and IC50 values for ABTS, lipoxygenase, chelating and NO which r values found were: r = 0.675 (p < 0.01), r = 0.649 (p < 0.01), r = 0.502 (p < 0.01) and r = 0.321 (p < 0.05), respectively. Stronger correlation between the flavonoid content and IC50 values of chelating activity and NO scavenging (r = 0.601 and r = 0.530; p < 0.01, respectively) than phenol content and these activities was found. In buckwheat honey, a dark-coloured honey, Zhou et al. (2012) attributed the weak chelating activity of their samples to the low

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A

B

Fig. 1. Positive correlation between ORAC, expressed as Trolox equivalent (TE), and phenol content (A); positive correlation between ORAC, expressed as Trolox equivalent (TE), and melanoidin content measured at A450 nm (B).

A

B

Fig. 2. Negative correlation between NO scavenging ability, expressed as IC50, and phenol content (A); negative correlation between NO scavenging ability, expressed as IC50, and melanoidin content measured at A450 nm (B).

A

B

Fig. 3. Negative correlation between chelating ability, expressed as IC50, and phenol content (A); negative correlation between chelating ability, expressed as IC50, and melanoidin content measured at A450 nm (B).

content of flavonoids because the presence of o-diphenolic groups in the 3,4-dihydroxy position in ring B and the ketol structure, 4oxo, 3-OH or 4-oxo, 5-OH in the C ring of the flavonols are essential to chelate metal ions. The inverse correlations found are due to the fact that the results are expressed in IC50, this meaning that the lower these values the higher the antioxidant activities will be.

3.3. Colour, Maillard reaction products/melanoidin content and UV absorbing compounds of ‘‘água-mel’’ from diverse producers The degree of browning measured through the absorbance of samples between 420 and 450 nm is used to assess the extent to

which the Maillard reaction occurs in foods and, therefore, may constitute one way to evaluate melanoidins’ content since these constitute the late-stage of the Maillard reaction products (Borrelli et al., 2002; Martins et al., 2001). On the other hand, the natural colour of honeys is assessed on the net absorbance of A560–A720 (Bruddzynski and Kim, 2011).

3.3.1. Colour of ‘‘água-mel’’ A great variability was found on the colour of ‘‘água-mel’’ samples, ranging the values from a minimal of 0.040 for 1E/2011 to 0.510 for 1B/2011 (Table 2). The lowest value found in ‘‘águamel’’ from the producer 1E/2011 is even lower than those obtained for pumpkin and blueberry honeys from Canada (Brudzynski and

Table 3 Antibacterial activity of ‘‘água-mel’’ samples from Alentejo and Algarve (Portugal). Sample

% (w/v)

Microorganisms E. aerogenes DSM 30053

1B/2010

1B/2011

1H/2011

Antibiotic* Sample

1B/2010

1B/2011

1F/2011

1H/2011

99.57 ± 4.47 98.72 ± 0.11 98.92 ± 0.35 97.89 ± 0.18 98.30 ± 0.17 97.16 ± 0.60 97.30 ± 1.40 91.06 ± 2.26 104.08 ± 1.24b 84.10 ± 2.12 99.35 ± 0.42 102.12 ± 1.57 11.04 ± 1.85 92.73 ± 0.97 99.61 ± 1.52 102.72 ± 4.29 98.73 ± 0.18

E. faecalis NCTC 775

E. coli DSM 1072

S. typhimurium ATCC 14028

L. monocytogenes EGD

L. monocytogenes C882

S. aureus CFSA2

S. aureus ATCC 6538

77.66 ± 1.71 98.11 ± 1.04 97.79 ± 0.53 97.52 ± 0.16 92.18 ± 3.01 94.20 ± 1.62 96.47 ± 5.54 82.91 ± 8.03 70.26 ± 6.61 93.06 ± 4.28 101.45 ± 2.40 105.80 ± 1.04 78.79 ± 0.28 98.33 ± 0.77 99.16 ± 1.29 100.82 ± 6.12 97.72 ± 0.97

74.83 ± 0.81 92.17 ± 0.65 91.85 ± 0.68 91.02 ± 0.11 81.29 ± 0.30 78.93 ± 1.23 84.50 ± 6.54 84.97 ± 1.86 69.81 ± 1.16 90.84 ± 1.66 90.30 ± 1.49 99.06 ± 3.46 76.09 ± 0.55 93.60 ± 1.21 93.38 ± 0.41 92.66 ± 2.11 97.69 ± 0.77

81.29 ± 1.19 90.92 ± 1.16 93.51 ± 0.51 91.73 ± 0.25 82.57 ± 0.44 81.46 ± 2.95 84.67 ± 1.96 87.04 ± 1.53 78.41 ± 3.77 96.86 ± 1.39 99.84 ± 1.73 99.94 ± 3.41 83.04 ± 1.77 92.88 ± 0.55 93.09 ± 0.28 93.03 ± 4.64 97.72 ± 0.97

83.63 ± 1.45 99.05 ± 1.26 97.28 ± 0.55 95.60 ± 0.56 94.92 ± 0.57 94.62 ± 1.21 96.03 ± 13.91 74.07 ± 2.74 58.56 ± 6.18 92.14 ± 0.79 98.16 ± 0.36 118.49 ± 6.69 91.37 ± 2.92 98.28 ± 0.33 102.77 ± 8.75 111.86 ± 7.55 97.26 ± 1.21

86.50 ± 0.81 99.01 ± 0.56 96.00 ± 1.20 94.62 ± 0.62 93.80 ± 0.45 95.27 ± 0.23 92.38 ± 0.80 70.60 ± 1.07 60.07 ± 0.94 92.61 ± 1.42 97.22 ± 2.14 97.52 ± 2.06 94.83 ± 1.71 96.85 ± 0.05 98.03 ± 1.13 101.00 ± 4.41 96.90 ± 0.74

86.61 ± 2.13 96.08 ± 0.34 96.60 ± 1.18 97.77 ± 1.56 95.09 ± 0.69 96.89 ± 0.27 97.54 ± 1.25 100.21 ± 0.32 54.38 ± 1.37 92.54 ± 0.87 97.20 ± 1.19 97.14 ± 1.17 90.31 ± 0.79 97.47 ± 0.71 98.42 ± 0.66 103.67 ± 6.07 97.51 ± 0.29

ND ND ND ND 93.16 ± 0.76 86.78 ± 2.23 83.47 ± 1.59 75.61 ± 1.44 ND ND ND ND 53.19 ± 0.94 87.19 ± 0.81 99.68 ± 2.20 100.96 ± 9.39 97.46 ± 0.24

% (w/v)

20 30 40 50 20 30 40 50 20 30 40 50 20 30 40 50

Antibiotica

Microorganisms MRSA 6

MRSA 12

53.97 ± 11.27 96.56 ± 0.35 97.63 ± 0.71 97.28 ± 0.99 97.32 ± 0.48 97.68 ± 0.15 98.64 ± 0.19 100.35 ± 1.33 33.12 ± 3.93 71.15 ± 12.15 92.40 ± 4.05 97.49 ± 1.53 52.43 ± 1.29 86.18 ± 1.56 98.38 ± 0.21 103.29 ± 1.08 98.06 ± 0.30

ND ND ND ND 96.23 ± 0.56 91.14 ± 0.41 91.07 ± 2.55 81.62 ± 1.14 ND ND ND ND 54.89 ± 3.40 87.14 ± 2.51 98.36 ± 1.43 93.77 ± 7.01 97.70 ± 0.15

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1F/2011

20 30 40 50 20 30 40 50 20 30 40 50 20 30 40 50

b

Data are the mean of three independent experiments ± standard deviation. ND-not determined. ND-not determined. a The antibiotic used was chloramphenicol at 30 lg/ml. b (Italicised values) – The percentage of growth promotion is indicated.

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Table 4 Antifungal activity of ‘‘água-mel’’ samples from Alentejo and Algarve (Portugal).

Data are the mean of three independent experiments ± standard deviation. a The antifungal was cycloheximide at 10 lg/ml. b (Italicised values) – The percentage of growth promotion is indicated.

Kim, 2011). The colour in honey is attributed to the presence and concentration of compounds possessing double bonds which absorb light in visible range (400–700 nm), such as polyphenols, flavonoids, terpenes, carotenoids and Maillard reaction products. The Pearson correlation coefficients found between colour and total phenols were r = 0.769, p < 0.01 (n = 57) and between colour and total flavonoids were r = 0.880, p < 0.01 (n = 57). The contribution of melanoidins on the colour of ‘‘água-mel’’ samples were also confirmed because a direct correlation between A560–720 and A450– A720 was found (r = 0.990, p < 0.01, n = 57), revealing that the

involvement of melanoidin on the colour of ‘‘água-mel’’ is greater than the contribution of polyphenols or flavonoids. 3.3.2. Maillard reaction products/melanoidin content All samples of ‘‘água-mel’’ absorbed at A450nm but to different degree (Table 2). Samples 2A/2011 and 1B/2011 had the highest values (2.849 and 2.934, respectively) in contrast to that of 1E/ 2011 (0.233). This high melanoidin content detected in those samples is related with the best activities detected for the same samples (Table 1). The lowest activity found for 1E/2011 is also related with the lowest content of melanoidins. Studies concerning the effect of prolonged heating on antioxidant activity and colour of honey, some authors (Turkmen et al., 2006a; Turkmen et al., 2006b) showed that increase in treatment temperature favoured both antioxidant activity and brown pigment formation. Other authors studying the effect of heating honey (at 121 °C for 30 min) on the formation of brown pigments they concluded that they were dependent on the honey variety. Heating light and medium-coloured honeys there was a promotion of the formation of brown pigments detected through the increase in net absorbance at A450 nm and heating dark honeys, the net absorbance detected at this wavelength decreased despite of a remarkable darkening of honey. Nevertheless the authors also detected in these samples the precipitation of some brown coloured components, that is, heating honeys contribute to the formation of brown pigments abut it also may affect the solubility of these pigments, depending on the type of honey (Brudzynski and Miotto, 2011). The differences detected in our samples may be therefore attributed to the type of honeys used by the beekeepers and/or to the time and temperature used during the production of ‘‘água-mel’’. A strong correlation between melanoidin content and antioxidant activities was found. The Pearson correlation coefficients (r)

Fig. 4. Antiviral activity of ‘‘água-mel’’ samples against the Qb bacteriophage in BHI supplemented with 30%, 40% and 50% of ‘‘água-mel’’. The number of virus plaques was determined after 24 and 48 h of inoculation and compared with the initial virus number. Results are indicated as Log reduction of virus plaques. Data are the mean ± SD of three independent experiments. For 24 and 48 h of sampling the significant difference of the concentrations of ‘‘àgua-mel’’ is indicated: (A) p < 0.05 and (B) p < 0.01; (C) p < 0.001.

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between the melanoidins’ content and peroxyl scavenging ability, melanoidins’ content and ability for scavenging free ABTS radicals, melanoidins’ content and capacity for scavenging NO, melanoidins’ content and capacity for chelating metal ions, and melanoidins’ content and capacity for inhibiting lipoxygenase were r = 0.902, r = 0.702, r = 0.710, r = 0.698, r = 0.724 (p < 0.01), respectively. In addition, the Pearson correlation coefficients (r) were higher between the melanoidins’ content and the antioxidant activities than phenol content and antioxidant activity, particularly between melanoidins’ content and capacity for scavenging peroxyl radicals; between melanoidin content and capacity for scavenging NO radicals and between melanoidin content and capacity for chelating metal ions (Figs. 1–3). Such data allow hypothesising that melanoidins contribute more for the antioxidant activity of samples of ‘‘água-mel’’ than phenols, at least in what concerns the capacity for scavenging peroxyl and NO radicals and ability for chelating metal ions. On the basis of the chemical reactions involved in the assays of antioxidant activity, the highest correlation found between phenol/ melanoidin content and ORAC method which mechanism is based on hydrogen atom transfer reaction, our results may reveal that the ability of the phenols and melanoidins present in ‘‘água-mel’’ for preventing oxidation may preferentially occur through this type of reaction. In addition, melanoidins are better scavengers of NO radicals than phenols.

3.4. Antimicrobial activity The antimicrobial activity of the four selected ‘‘água-mel’’ samples is summarised in Table 3. The group of Gram-negative bacteria; E. aerogenes, E. coli and S. typhimurium were susceptible to all tested ‘‘água-mel’’ samples. However the growth of E. aerogenes was promoted when the ‘‘água-mel’’ samples 1B/2010 and 1F/ 2011 were used at 20% (w/v) (Table 3). Above 20% (w/v) all samples showed a percentage of growth inhibition higher than 80% (w/v). The tested Gram-positive bacteria, including the MRSA strains, were susceptible to the examined ‘‘água-mel’’ samples. Nevertheless the 1B/2011 sample at 20% (w/v) achieved the highest values of inhibition for all bacteria varying between 92.18 ± 3.01% of growth inhibition for E. faecalis and 98.30 ± 0.17% for E. aerogenes. This high inhibitory activity may be associated to its highest phenol and melanoidin content (Tables 1 and 2). It is interesting to note that 1F/2011 and 1H/2011 samples at 50% (w/v) caused bacterial cell lysis (percentage of growth inhibition is higher than 100%). On the other hand the sample 1B/ 2011, at this same concentration, the percentage of growth inhibition of E. faecalis, L. monocytogenes EGD and C882 strains was affected (Table 3). This observation may be linked to the presence of a protective substance that at 50% (w/v) reaches an amount sufficient to decrease the impact of the antimicrobial components of ‘‘água-mel’’ against these two Gram-positive bacteria. No significant differences (p > 0.05) were observed between the percentage of growth inhibition of ‘‘água-mel’’ samples and the antibiotic for all tested bacteria. The antifungal activity was determined against the yeasts S. cerevisae and C. albicans. The results are summarised in Table 4. Both yeasts were susceptible to the tested ‘‘água-mel‘‘ samples. However, from all tested microorganisms the S. cerevisae strain was the less susceptible (p < 0.05) and its growth was promoted by 1B/2010 and 1H/2011 at 20% (w/v). This high resistance may be associated with its high resistance to high osmolality and its less susceptibility to honey has been reported (Gomes et al., 2010). The percentage of growth inhibition of ‘‘água-mel’’ samples and the antifugal agent (cyclohexamide) was similar (p < 0.05).

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3.5. Antiviral activity The antiviral activity of ‘‘água-mel’’ samples was determined against the enterobacteria phage Qb and the results are indicated as the Log reduction on the formation of virus plaques in BHI supplemented with 30%, 40%, and 50% (w/v) of ‘‘água-mel’’. The results are depicted in Fig. 4. The highest antiviral activity is observed after 48 h of inoculation. The ‘‘água-mel’’ samples 1B/2011 and 1H/2011 were the most active (p < 0.05) against the virus achieving 8 Log of reduction after 48 h of inoculation and the sample 1F/2011 was the least active (p < 0.05). Qb phage is a F-specific RNA bacteriophage and this type of phage due to its high correlation with those of enteric viruses have been used to evaluate the antiviral action of several chemical agents either in Europe or in the USA and Japan (Casteel et al., 2008; Fisher et al., 2009; Havelaar et al., 1993; Meschke and Sobsey, 2003; Shang et al., 2007; Shirasaki et al., 2009; Vo et al., 2009). The antiviral activity of honey has been reported (Al-Waili, 2004; Shahzad and Cohrs, 2012; Zeina et al., 1996). However, its mode of action still remains to be elucidated. The composition of ‘‘água-mel’’ is similar to honey except the presence of hydrogen peroxide that due to the heat treatment will disappear (Molan, 1992). Hence its antiviral activity will certainly result from the components with documented action against virus, as polyphenols and propolis (Amoros et al., 1992; Vynograd et al., 2000). The higher antiviral activity was found for samples 1B/ 2011 and 1H/2011, the samples that had the highest polyphenol content; 1.323 ± 0.086 and 0.787 ± 0.086, respectively (Table 1). In our study the ‘‘água-mel’’ samples caused a decrease on the infectivity of the Qb bacteriophage suggesting its potential use to combat enteric viruses. 4. Conclusions Samples of ‘‘água-mel’’ from diverse beekeepers of Portugal had antioxidant, antimicrobial and antiviral activities which may be attributed to the phenol and brown pigments (melanoidins) formed during the production. A strong correlation was found between phenol and antioxidant activity independent on the assay performed, nevertheless more evident between melanoidin content and antioxidant activity. A great variability on the phenol content and antioxidant activity was found, which may suggest variability on the mode of production and/or type of honey used by beekeepers. This heterogeneity may indicate a need for conducting the production of ‘‘água-mel’’ under controlled conditions if beekeepers intend to see their product valorised and accepted in the food market. Conflict of Interest The authors declare that there are no conflicts of interest Acknowledgements The authors wish to thank the Ministério da Agricultura, Mar, Ambiente e Ordenamento do Território (Portugal) through the Programa Apícola Nacional 2011–2013, Medida 6A, for its financial support of this research. The authors are grateful to Adrian Rochefort for his technical assistance on microbiological assays. References Akula, U.S., Odhav, B., 2008. In vitro 5-lipoxygenase inhibition of polyphenolic antioxidants from undomesticated plants of South Africa. J. Med. Plants Res. 2, 207–212. Al, M., Daniel, D., Moise, A., Bobis, O., Laslo, S., Bogdanov, 2009. Physico-chemical and bioactive properties of different floral origin honeys from Romania. Food Chem. 112, 863–867.

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