LWT - Food Science and Technology 65 (2016) 333e340

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LWT - Food Science and Technology

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Antibacterial and antioxidant activity of honeys from the state of Rio

Grande do Sul, Brazil
Francine Manhago Bueno-Costa a, *, Rui Carlos Zambiazi a, Bruna Wendt Bohmer a,

bio Clasen Chaves a, Wladimir Padilha da Silva a, Jerri Teixeira Zanusso b, Iara Dutra c
Fa
a
Department of Food Science and Technology, Universidade Federal de Pelotas (UFPel), Pelotas 96010-900, Brazil
b
Department of Animal Science, Universidade Federal de Pelotas, Pelotas 96010-900, Brazil
c
Apiary Padre Assis, Santiago 97700-000, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Honey contains compounds with antioxidant and antibacterial capacities, such as phenolic compounds
Received 3 January 2015 and carotenoids. Current analysis evaluates the antioxidant and antibacterial activity and determines the
Received in revised form phenolic compounds and carotenoids content in 24 honey samples from the state of Rio Grande do Sul,
13 June 2015
Brazil. Total content of phenolic compounds (from 11.37 to 54.01mgGAE100 g
1), avonoids (from 2.97
Accepted 6 August 2015
Available online 11 August 2015
to 10.46 mgQE100 g
1), phenolic acids (from 0 to 65.47mgCAE100 g
1) and carotenoids (from 0.56 to
6.19 mg b-caroteneKg
1) were found, and the antioxidant activities measured by ABTS (between 8.24
and 111.48 mgtrolox100 g
1) and by DPPH (between 2.48 and 17.21 mgQEA100 g
1) differed signi-
Keywords:
Honey
cantly. All samples showed antibacterial activity, where the H5 and H23 showed the best results (MIC:
Bioactive compounds 10 mg mL
1) against the four pathogenic bacteria tested. A signicant and positive correlations between
Antioxidant capacity the carotenoids and phenolic compounds content was found; likewise, between antioxidant activity
Antibacterial activity (ABTS) and carotenoids content and between color and avonoids content.
2015 Elsevier Ltd. All rights reserved.

1. Introduction (Vandamme et al., 2013) activities.

Honey is the product of beekeeping that has great market po-
tential. Honey contains more than 200 compounds comprising
approximately 38% fructose, 31% glucose, 10% other sugar types, 18%
water and 3% of other compounds. However, precisely the great
mixture of compounds in these 3% is the product's greatest feature,
with special reference to phenolic and carotenoids compounds
(Alvarez-Suarez et al., 2010).
Honey as one of the most complete food for humans, due to its
therapeutic (Blasa, Candiracci, Accorsi, Piacentini, & Piatti, 2007),
antioxidant (Lachman, Orsak, Hejtmankova, & Kovarova, 2010;
Meda, Lamien, Romito, Millogo, & Nacoulma, 2005), antimicrobial
(Escuredo, Silva, Valent~ ao, Seijo, & Andrade, 2012; Vandamme,
Heyneman, Hoeksema, Verbelen, & Monstrey, 2013), antitumoral
(Jaganathan, Mazumdar, Mondhe, & Mandal, 2011), anti-
inammatory (Van Den Berg et al., 2008), antiviral (Watanabe,
Rahmasari, Matsunaga, & Kobayashi, 2014) and antiulcer
* Corresponding author.
E-mail addresses: francinembueno@yahoo.com.br, francinembcosta@gmail.com
(F.M. Bueno-Costa).
Most studies on the effects of honey are concentrated on the
activities of bioactive compounds, especially phenolic compounds,
in the human organism. The most relevant are those widely
distributed in nature, including the phenolic acids and avonoids
(Vermerris & Nicholson, 2006).
Carotenoids were found in small concentrations in the dark
honey (10 mg b-caroteneKg
1) but they were not found in light
colored honey. This fact reveals the effect that carotenoids (Alvarez-
Suarez et al., 2010; Ferreira, Aires, Barreira, & Estevinho, 2009;
Tomasik, 2004) and phenolic compounds have in the honey color
(Estevinho, Prereira, Moreira, Dias, & Pereira, 2008).
Honeys therapeutic activities are mainly related to its antioxi-
dant and antimicrobial properties (Martos, Ferreres, & Tomas-
Barberan, 2000). The antimicrobial activity of honey has been
showed in a recent study with 60 types of honey from different
botanic origins against 16 clinically-derived pathogenic bacteria
(Voidarou et al., 2011).
Several authors also studied the correlations between color and
antioxidant and antibacterial activities with content of the bioac-
tive compounds of honey (Beretta, Granata, Ferrero, Orioli, &
Facino, 2005; Bertoncelj, Dobersek, Jamnik, & Golob, 2007;

http://dx.doi.org/10.1016/j.lwt.2015.08.018
0023-6438/ 2015 Elsevier Ltd. All rights reserved.
334 F.M. Bueno-Costa et al. / LWT - Food Science and Technology 65 (2016) 333e340

Canadanovic-Brunet et al., 2014).
Data on these properties in Brazilian honeys are limited,
therefore, it is currently very important to determine parameters in
honey samples from the state of Rio Grande do Sul, Brazil, especially
due to the productive and economic relevance on the Brazilian
honey market. Current analysis assessed the antibacterial and
antioxidant activities and the content of bioactive compounds in 24
honey samples.
homogenized and were sonicated for 10 min (45  C) until the
complete dissolution of the sugar crystals.
2.2.1. Color
Samples were diluted in water (1:1; w/v) and the absorbance
was measured at 635 nm. Results were calculated
(PFund 38.70 371.39*ABS) (Ferreira et al., 2009) and expressed
in millimeters on a Pfund scale (Fell, 1978).

2. Materials and methods 2.2.2. Bioactive compounds and antioxidant activity

2.1. Materials
2.1.1. Samples
Honeys from entirely randomized different botanic origins were
analyzed (Table 1). Honeys were provided by beekeepers and
collected from several regions in the state of RS, Brazil. The samples
were from the following regions: west (H1, H2, H3, H4, H5, H10,
H11, H14), metropolitan (H6, H16, H19, H20, H21, H22, H23),
southwest (H7, H12, H13), southeast (H8, H9, H17, H18), northeast
(H15) and northwest (H24).
Samples were acquired between January and November 2013
and kept in sterilized dark polyethylene asks under refrigeration
(5  C).
2.1.2. Standards and reagents
All chemical products were of the highest analytic degree.
Quercetin, caffeic acid, gallic acid, DPPH and ABTS were supplied by
SigmaeAldrich (St. Louis, USA), methanol, ciprooxacin and Folin
Ciocalteu 2 N solution were obtained from Merck (Darmstadt,
Germany) and tryptone soya agar, tryptone soya broth and bacte-
riologic peptone were obtained from Oxoid, Basingstone, Hamp-
shire, UK. Water was puried by an Ultra Purication System (Mega
Purity).
2.2. Methods
Prior to all measurements, the samples were previously
2.2.2.1. Total content of phenolic compounds (TCPC). Honey solution
(100 mg mL
1) was previously homogenized and ltered through
quantitative lter, 500 mL of honey solution was added to 2.5 mL of
Folin Ciocalteu (0.2 N). After 5 min, 2 mL of sodium carbonate so-
lution (Na2CO3-75 g L
1) was added and incubated for 2 h in the
dark. The absorbance was measured at 760 nm in a spectropho-
tometer (Singleton, Orthofer, & Lamuela-Raventos, 1999). Standard
curve was dened by known concentrations of gallic acid, ranging
between 0 and 200 mg L
1 (R20.9923) and results expressed in
milligrams of gallic acid equivalents (mgGAE100 g
1).
2.2.2.2. Total content of avonoids (TCF). Honey solution
(100 mg mL
1) was prepared with methanol 50% and previously
homogenized and ltered through quantitative lter, 5 mL of honey
solution was mixed with 5 mL of AlCl3 (2%) in methanol. The
mixture was homogenized and allowed to stand for 30 min. The
absorbance was measured at 415 nm (Arvouet-Grand, Vennat,
Pourrat, & Legret, 1994). Standard curve was dened by known
concentrations of quercetin, between 0 and 40 mg L
1 (R20.9989)
and expressed in milligrams of quercetin equivalents
(mgQE100 g
1).
2.2.2.3. Total content of phenolic acids (TCPA). Phenolic acids were
determined according to the method of Mazza, Fukumoto,
Delaquis, Girard, and Ewert (1999) with some modications.
Appropriate aliquots of solutions prepared with honey (250 mL;
100 mg mL
1 in 50% ethanol solution), 250 mL of acidied ethanol
solution (0.1% HCl in 95% ethanol) and 4.55 mL of solution 2%HCl

Table 1
Honey samples with respective collection data: municipality, botanic origin and color.

Samples Town Botanic origin Pfund scalea Color

H1 Nova Esperana Wild 89 Amber

H2 Unistalda Wild 74 Light amber
H3 Mata Wild 150 Dark amber
H4 Bom Respiro Eucalyptus 116 Dark amber
H5 Cacequi Wild 65 Light amber
H6 Ivoti Wild 123 Dark amber
H7 Bage
Eucalyptus 75 Light amber
H8 Pelotas Eucalyptus 74 Light amber
H9 Pedras Altas Wild 150 Dark amber
H10 ~o Pedro do Sul
Sa Wild 98 Amber
H11 Santiago Wild 89 Amber
H12 Livramento Wild 79 Light amber
H13 ~o Francisco de Assis
Sa Wild 90 Amber
H14 Jaguari Wild 122 Dark amber
H15 Farroupilha Wild 84 Light amber
H16 Nova Petropolis Wild 149 Dark amber
H17 Rio Grande Wild 90 Amber
H18 Cerrito Alegre Wild 122 Dark amber
H19 Camaqua ~ Wild 84 Light amber
H20 Santo Anto ^nio da Patrulha Wild 149 Dark amber
H21 Taquara Eucalyptus 70 Light amber
H22 Gramado Wild 74 Light amber
H23 ^s Coroas
Tre Wild 67 Light amber
H24 Teuto ^nia Eucalyptus 103 Amber
a
In millimeters.
 F.M. Bueno-Costa et al. / LWT - Food Science and Technology 65 (2016) 333e340
were transferred to a 10 mL volumetric ask. Was homogenized
and allowed to stand for 15 min and absorbance was determined at
320 nm. Standard curve was dened by caffeic acid, between 0 and
0.8ug.ml
1 (R20.9992) and results expressed in milligrams of caffeic
acid equivalents (CAEmg100 g
1).
2.2.2.4. Total content of carotenoids (TCC). Honey solution prepared
with 5 g of honey and dissolved in 5 mL of distilled water. After this
solution was mixed with 45 mL of hexane-acetone solution (6: 4),
homogenized for 1 min and allowed to stand in the dark for 30 min.
The absorbance was determined at 450 nm (Ferreira et al., 2009).
Standard curve was dened by compound concentration, between
0.015 and 0.6 ug ml
1 (R20.9966) and expressed in milligram of b-
carotene equivalents (mgb-caroteneKg
1).
2.2.2.5. Antioxidant activity (assay with ABTS and DPPH).
Antioxidant activity described by Re et al. (1999), where the ABTS
assay was done using the same honey solution of phenolic acids
protocol. In reaction was used 100 mL of honey solution and 3900 mL
ABTS solution (0.700 0.05 nm), homogenized and allowed to
stand for 6 min. The absorbance was measured at 734 nm and re-
sults expressed in Trolox equivalents (mgTE100 g
1) with curve
between 0.05 and 0.25 mg ml
1 (R20.9799).
The measure of antioxidant activity was monitored according to
a method reported before (Velazquez, Tournier, Mordujovich de
Buschiazzo, Saavedra, & Schinella, 2003). The DPPH assay was
done by using 750 mL of honey solution that was mixed with 1.5 mL
of DPPH solution in methanol (0.02 mg mL
1). The mixture was
homogenized for 30 min at room temperature and then the
absorbance was determined at 517 nm. Amount of antioxidant was
determined by standard curve of quercetin (0 and 6.25 mg L
1)
(R20.9936) and results expressed in quercetin equivalents antioxi-
dant (mgQEA100 g
1).
2.2.3. Antibacterial activity (MIC)
Bacterial cultures were retrieved from the bacteria collection of
the Laboratory of Food Microbiology, Federal University of Pelotas.
Standard strains of four different bacterial species were analyzed, of
which two were gram negative, Shiguella dysenteriae (ATCC13313)
and Salmonella typhimurium (ATCC14028), and two were gram
positive, Staphylococcus aureus (ATCC25923) and Bacillus cereus
(ATCC1778). Strains were maintained in tryptone soya agar (TSA) at
4  C. Innocuous were prepared in TSA for 24 h at 37  C. Cell sus-
pensions were diluted in peptone water 0.1% by McFarland scale till
the concentration of 106 colony-forming units by milliliters (UFC/
mL). Minimum inhibitory concentration (MIC) of honey extracts
was determined with adapted method by Taveira et al. (2010)
employing serial dilution with the use of sterilized microdilution
plates from 96 pools. The following honey concentrations were
tested for MIC identication: 400, 350, 300, 250, 200, 150, 100, 50,
25 and 10 mg mL
1 in ultra pure water. Further, 90 mL of Trytone
Soya Broth (TSB), 10 mL of suspension with 106 CFU/mL and 100 mL
of each honey solution were added to each pool for MIC. Negative
control consisted by replacing sample by water, whereas the anti-
biotic ciprooxacin (mg.mL
1) was the positive control. Plates with
microdilution were incubated during 24 h at 37  C. Turbidity was
then assessed by a plate reader (Elisa Plate Analyser, Robonik). MIC
for bacteria was determined as the lowest concentration of honey
extracts capable of inhibiting bacteria growth.
2.2.4. Statistical analysis
All analytic determinations were carried out in triplicate and the
data were expressed as means. Analysis of variance followed by
Tukey's test and differences between means at the 95% condence
level were considered statistically signicant (SAS, 1999).
335
Correlations were obtained by Pearson's correlations between co-
lor, TCPC, TCF, TCPA, TCC, ABTS, DPPH and antibacterial activities.
3. Results and discussion
3.1. Color
The possible seven color classes (pfund scale) only three were
found in the studied honeys (Table 1), light amber (41.7%), amber
(25%) and amber (33.3%). Moreti, Sodre
, and Marchini (2006) also
found that the light amber color in 44.5% of 346 honeys from six
Brazilian states (Bahia, Tocantins, Piau, Ceara
, Minas Gerais and
Santa Catarina). Probably the dark tone found in Brazilian honey is
due to wild origin of most honey sold in the country. In Brazil there
is a wide variety of vegetation, which favors the dominance of wild
honey, as well as low professional beekeeping that does not con-
tributes to the appearance of a different product such as, for
example, orange ower honey, monooral honey light tone.
Light-colored honeys have a delicate taste, whereas dark ones
have stronger one (Bogdanov, Ruoff, & Persano Oddo, 2004). Light
honeys, such as the honey from the Harenna Forest in Ethiopia,
showed between 34 and 85 mm (Pfund scale), they are grouped
between extra light and light amber, and sold at rather high prices
(Belay, Solomon, Bultossa, Adgaba, & Melaku, 2015). However, dark
tone honeys are more appreciated in Germany, Austria and
Switzerland (Bogdanov et al., 2004).
3.2. Total content of phenolic compounds (TCPC)
Data shows that TCPC of wild honey (H1) differed signicantly
and it was higher when compared to the other samples. The TCPC
content of the wild honey H22 was lower when compared to the
content of the other samples. In the case of geographical regions
(Fig. 1), the honeys of the western region (H1, H2, H3, H4, H5, H14)
showed the highest contents of phenolic compounds, especially the
samples H1 (111.37 mgGAE100 g
1), H2 (105.62mgGAE100 g
1)
H3 (100.15mgGAE100 g
1) and H4 (87.83mgGAE100 g
1). All
samples were collected at the same time of year (summer) and
were certainly the ideal ripeness. Despite these honeys are classi-
ed as wild, apiaries were located in areas close to some specic
blooms, as Baccharis trimera, Bimucronata mimosa, Schinus ter-
ebinthifoliu and eucalyptus.
TCPC in honey samples ranged between 61.16 and 111.37
mgGAE100 g
1 which were similar to contents related by
Escuredo et al. (2012) and Habib, AlMeqbali, Kamal, Souka, and
Ibrahim (2014). The Manuka honey (Leptospermum scoparium)
has been thoroughly analyzed with regard to its antibacterial
properties, mainly attributed to phenolic compounds and methyl-
glyoxal (Chan et al., 2013; Stephens et al., 2010). The authors
assessed the contents of the phenolic compounds in Manuka
honeys with up to one year storage, and found similar to contents
reported in current analysis, varying between 43.12 and
193.39 mg100 g
1.
Most light amber honey (except H2) had lower content of
phenols (H5, H7, H8, H12, H16, H19, H21, H22, H23). This was an
expected result, it is known that the color reects the content of
pigments present in honey, so light amber honey must have lower
levels of total phenols than amber and dark amber.
3.3. Total content of avonoids (TCF)
TCF in honey samples varied between 2.98 and 10.46
mgQE100 g
1 (Fig. 1), with contents higher than those reported by
Martos et al. (2000) (2.0 and 2.5 mgQE100 g
1) in honey samples
from southern Australia. However, they were lower than those
336 F.M. Bueno-Costa et al. / LWT - Food Science and Technology 65 (2016) 333e340
Fig. 1. Total rate of phenolic compounds (mgGAE100 g
1), avonoids (mgQE100 g
1) and phenolic acids (mgCAE100 g
1), in types of honey sold in the state of Rio Grande do Sul,
Brazil.
registered by Habib et al. (2014), they reported contents of avo-
noids of 109.49 mg catechin100 g
1.
Several authors characterize honey according to its botanic and
geographical region, especially when it is related to avonoid
contents and even to specic avonoids (Iurlina, Saiz, Fritz, &
Manrique, 2009).
Meda et al. (2005) compared avonoids from honeys from
Burkina Faso (Africa), and they veried that the highest content was
8.35 mgQE100 g
1, similar to sample H9 (10.46 mg QE100 g
1).
The TCPC approximately 10% of the average results were avonoids,
ranging from 5% (H7) 17% (H9). TCF also varied according to the
color, honey amber light had on average 5.49 mgQE100 g
1, amber
7.20 mgQE100 g
1 and dark amber 7.66 mgQE100 g
1, as
observed in TCPC, avonoids contents were lower in samples of
light amber color.
3.4. Total contents of phenolic acids (TCPA)
Analyzed types of honey contained total contents of phenolic
acids which ranged between 0.49 and 65.47 mg100 g
1 (Fig. 1).
Samples H22 and H20 showed the highest contents of phenolic
acid, whereas samples H14 had the lowest content. On average,
phenolic acids were present in 30% of the TCPC, which contained
only 1% (H14) and 64% (H20). The percentage of TCPA in relation to
TCPC was higher in the metropolitan region, where on average 50%
of phenolic compounds found in honey that region were phenolic
acids. These variations of phenolic acids and avonoids results
when compared to the total content of phenolic compounds can be
explained by the spectrophotometric method employed is not a
specic method, it detects all the phenolic groups present in the
extract, including protein and ascorbic acid (Naczk & Shahidi,
2004).
It was observed that all samples had more phenolic acids than
avonoids. Coloration of honey ranged between 65 and 150 mm
(PFund scale). Since the scale varies from 0 to 150 mm, most
samples showed a darkish color. The results agree with Bogdanov
et al. (2004), which relate that dark-colored honeys have a high
rate of phenolic acids and low avonoid contents.
3.5. Total content of carotenoids (TCC)
TCC in honeys ranged between 0.56 and 6.19 mg b-
caroteneKg
1, similar contents to those reported by Alvarez-
Suarez et al. (2010), which obtained contents ranging between
1.17 and 5.57 mg b-caroteneKg
1 in honey samples from Cuba.
Boussaid et al. (2014) also analyzed TCC in six monooral honeys of
Tunisia (mint, horehound, eucalyptus, thyme, rosemary and or-
ange) and reported higher contents in orange honey (4.72 mgb-
caroteneKg
1).
Samples of wild (H6 and H10) and eucalyptus (H21) (Fig. 2)
honey failed to provide carotenoid contents within the limit of the
methods detection. Carotenoid may be related to honey coloration.
Honeys which normally have an extra light color (between 9 and
17 mm Pfund) have low carotenoid rates (Alvarez-Suarez et al.,
2010; Ferreira et al., 2009). This relation was not observed in cur-
rent analysis since only slight color variability (light amber; amber;
dark amber) were observed. In contrast, the light amber honey
(H19) showed the highest TCC, demonstrating that although nor-
mally dark honeys have higher amount of carotenoids (H3, H4 and
H18), these compounds may also occur in signicant amounts in
light amber honey.
3.6. Antioxidant activities (DPPH and ABTS)
The activity of the radical (DPPH) varied signicantly among
most samples of honey (Fig. 3). The highest antioxidant activity
(17.21 mgAEQ100 g
1) was observed in the honey sample of
eucalyptus (H24), whereas the lowest activities (2.48 and 2.62
mgEAQ100 g
1) were observed in H13 and H21. H24 also con-
tained high TCF, low TCPC H13 and H21 TCPA and was not detected
by the method employed carotenoids. Meda et al. (2005) deter-
mined antioxidant activities in types of honey in Burkina Faso
(Africa) and registered average rates 12.94 mgQEA100 g
1, or
rather, lower than those in current study. On the other hand, Noor,
Sarfraz, Ali, and Shahid (2014) related honey with 2.85e39.86
mgQEA100 g
1 from different places in Pakistan.
The antioxidant capacity of honey and of its components is a
very useful parameter to correlate phytochemical determinations
(Alvarez-Suarez et al., 2010).
On average eucalyptus honey (9.98 mgQEA100 g
1) showed a
higher antioxidant activity rate that those wild honey (7.63
mgQEA100 g
1). On the other hand, the reverse occurred with
ABTS analysis, where the average content of eucalyptus honey
(52.43 mgAET100 g
1) were lower than those obtained in the wild
 F.M. Bueno-Costa et al. / LWT - Food Science and Technology 65 (2016) 333e340 337

Fig. 2. Total rate of carotenoids in types of honey sold in the state of Rio Grande do Sul, Brazil. Given in mg b-caroteneKg
1 standard deviation. *Different letters in the column
differ by Tukey's (p < 0.05). nd (not detected by the method).

Fig. 3. Antioxidant activity of honeys sold in the state of Rio Grande do Sul, Brazil. Antioxidant activity with DPPH, mgEAQ (equivalent antioxidant quercetin)100 g
1
honey standard deviation. *Different letters in the column differ by Tukey's test (p < 0.05).

honey (60.17 mgAET100 g
1). Honeys antioxidant activity ranged B. cereus.

between 8.24 and 111.48 mgAET100 g
1, by ABTS method (Fig. 4),
which was approximately 7 times higher than the activity found by
DPPH method. Lachman et al. (2010) also compared antioxidant
activity determined by the two methods and always found higher
rates by the ABTS method. The aim of these two determinations
was to detect which one would be the most appropriate in the
evaluation of antioxidant activity in honey. It was found that the
ABTS assay was more appropriate due to the highest correlations
(Table 3) with the content of all phytochemical analysis (phenols,
avonoids, phenolic acids, carotenoids).
3.7. Antibacterial activity (MIC)
Similar to current assay, Escuredo et al. (2012) analyzed anti-
bacterial activity by MIC in 23 honeys from Galicia, against gram
negative and gram positive bacteria and reported a greater inhib-
itory activity against B. cereus. Other authors (Alvarez-Suarez et al.,
2010; Escuredo et al., 2012) also registered that gram positive
bacteria were more sensitive to the honeys antibacterial activities
than gram negative ones.
The current results also were corroborated by Voidarou et al.
(2011). Samples from Cacequi (H5) and Tre ^s Coroas (H23) showed
the best results (MIC:10 mg ml
1) against the four pathogenic
bacteria under analysis. The same samples also showed the best
results with regard to antioxidant activities, both of light amber
color and showed intermediate compounds of phytochemicals.

All types of honey showed antibacterial activity against the four

bacteria tested (Table 2). The antibacterial activity was more 3.8. Correlation matrix
effective with gram negative bacteria than with gram positive ones.
Consequently, more efcient results occurred against S. aureus and There was a positive correlation between TCF and TCC, between
338 F.M. Bueno-Costa et al. / LWT - Food Science and Technology 65 (2016) 333e340

Fig. 4. Antioxidant activity of honey types sold in the state of Rio Grande do Sul, Brazil. Antioxidant activity with radical ABTS, mgTE (trolox equivalent)100 g
1 honey standard
deviation. *Different letters in the column differ by Tukey's test (p < 0.05).

Table 2
Antibacterial activity in honeys in the state of Rio Grande do Sul, Brazil.

Samples Antimicrobial activity (mg mL
1)a

Shiguella dysenteriae Salmonella typhimurium Staphylococcus aureus Bacillus cereus

H1 100 50 10 10
H2 100 50 10 10
H3 50 10 25 200
H4 100 300 300 25
H5 10 10 10 10
H6 25 25 50 25
H7 350 350 50 10
H8 10 25 10 10
H9 150 350 250 150
H10 300 300 250 100
H11 200 300 200 50
H12 150 150 10 10
H13 10 25 10 10
H14 50 100 10 10
H15 50 10 10 10
H16 25 25 10 10
H17 10 25 10 10
H18 25 100 10 50
H19 10 25 10 10
H20 50 300 10 10
H21 100 250 10 10
H22 50 350 50 10
H23 10 10 10 10
H24 300 400 150 200
a
Antimicrobial activity by MIC technique, given in mg of honey/mL of ultra pure water required to inhibit the group of the corresponding strain.

antioxidant activity by ABTS and TCC, between TCPA and color; and
between antibacterial activity (against B. cereus) with TCF (Table 3).
There was also positive correlation between TCPC with antioxidant
activity (ABTS) and color. Even if this correlation is not signicant, it
can not be ignored because it is an important result. For differently
than previously thought, TCPC best correlated with ABTS than
DPPH. In the set of all analyzes the H20 sample contained the
highest levels of antioxidant activity (ABTS), phenols and phenolic
acids and had dark amber color. Moreover, H20 did not contain the
best antibacterial activity, demonstrating that other compounds
(for example hydrogen peroxide) may be more related to the in-
hibition of bacterial than the total content of phenolic compounds.
Several correlations were observed with the antibacterial ac-
tivity. The bacterium shigella had positive correlations with bac-
teria salmonella, staphylococcus and bacillus. Staphylococcus was
also positively correlated with the others bacterias, whereas Ba-
cillus and Salmonella had only correlations with shigella and
staphylococcus.
Different methods are employed to verify in vitro antioxidant
activities in certain foods. When ABTS technique, which is capable
of detecting several compounds (hydrophilic and lipophilic), is
applied, there is a better chance for correlations, such as occurred
with the total content of carotenoids (TCC). Alvarez-Suarez et al.
(2010) also reported correlations between TCF and color, where
 F.M. Bueno-Costa et al. / LWT - Food Science and Technology 65 (2016) 333e340 339

Table 3
Pearson's co-relation coefcient for phenolic compounds, avonoids, antioxidant activity, antibacterial activity and color in honey samples in the state of Rio Grande do Sul,
Brazil.a

TCPC TCF TCPA TCC DPPH ABTS C AASD AAST AASA AABC

TCPC 0.1889 0.0337 0.3843* 0.0322 0.3050 0.3074 0.0577 0.289
0.0344 0.2029
TCF
0.2185 0.0398
0.0019 0.1094 0.5586*
0.0699
0.1153 0.1473 0.4424*
TCPA
0.2245
0.0824 0.2144
0.2257 0.2407 0.0437 0.1492 0.2575
TCC 0.0587 0.6297**
0.0231
0.1206 0.3258
0.1203
0.0778
DPPH 0.0727 0.3056
0.2247
0.1851 0.0825 0.2562
ABTS 0.1185 0.0059
0.1113 0.1455 0.0597
C
0.0585 0.1332 0.2947 0.5203
AASD 0.7347** 0.5580* 0.4208*
AAST 0.6613* 0.3459
AASA 0.4740*
AABC

Pearson's co-relation between total content of phenolic compounds (TCPC), total content of avonoids (TCF), total content of phenolic acids (TCPA), total content of carotenoids
(TCC), antioxidant activity with DPPH (DPPH), antioxidant activity with ABTS (ABTS), color (C), antimicrobial activity in Shiguella dysenteria (AASD), Salmonella typhimurium
(AAST), Staphylococcus aureus (AASA) and Bacillus cereus (AABC).
*
Signicant at p  0,05 ** signicant at p  0.001.
c (Color).
a
95% condence interval.

the highest TCF were detected in amber colored honeys.
Overall, the honey samples showed small differences in the
results, however, the exception occurred in the H20 sample. This
sample comes from an association of Santo Antonio da Patrulha
beekeepers. Therefore, it is a marketed product that meets the
quality and safety requirements in a food processor industry. A
planned apiary, handling and proper processing make all the dif-
ference in the quality of honey. Perhaps for these factors, the H20
sample amber dark color showed the best results of phenolic
compounds and antioxidant activity (ABTS). Nevertheless, H20 did
not contain the best antibacterial activity, demonstrating that
addition of phenolic compounds and carotenoids other properties
(for example, osmolality, acidity and hydrogen peroxide) may be
related to bacterial inhibition.
4. Conclusions
Current analysis showed that honey samples from regions of Rio
Grande do Sul, Brazil, revealed only slight variability in the total
bioactive compounds content. The phenolic compounds were the
bioactive compounds with the largest amounts, mostly due to the
phenolic acids. Carotenoids were detected in small concentrations
at the samples, but enough to contribute their antioxidant activity
(ABTS). Antimicrobial activity, especially with gram positive mi-
croorganisms, staphylococcus and baccilus, suggests that honeys
under analysis may have a relevant role as antibacterial natural
products to decrease the effects of bacterial infections and
contribute for better food.
Acknowledgments

Pozzatto, for his help in
The authors are grateful to Mr. Adi Jose
the acquisition of samples and incentives towards current research.
We thank the CAPES, CNPQ, FAPERGS, PPGCTA for nancial support.
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