Antimicrobial activity of yerba mate (Ilex paraguariensis St. Hil.) against food pathogens
Actividad antimicrobiana de la yerba mate (Ilex paraguariensis St. Hil.) contra patógenos alimentarios
José G. Prado Martin
, Ernani Porto, Severino M. de Alencar, Eduardo M. da Glória, Cristina B. Corrêa, Ingridy S. Ribeiro Cabral
Corresponding author
Departamento de Agroindústria, Alimentos e Nutrição, Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ), Universidade de São Paulo, Piracicaba, São Paulo, Brasil
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Prado Martin, Ernani Porto, Severino M. de Alencar, Eduardo M. da Glória, Cristina B. Corrêa, Ingridy S. Ribeiro Cabral" "autores" => array:6 [ 0 => array:4 [ "nombre" => "José G." "apellidos" => "Prado Martin" "email" => array:1 [ 0 => "gui.pmartin@gmail.com" ] "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] 1 => array:2 [ "nombre" => "Ernani" "apellidos" => "Porto" ] 2 => array:2 [ "nombre" => "Severino M." "apellidos" => "de Alencar" ] 3 => array:2 [ "nombre" => "Eduardo M." "apellidos" => "da Glória" ] 4 => array:2 [ "nombre" => "Cristina B." "apellidos" => "Corrêa" ] 5 => array:2 [ "nombre" => "Ingridy S." "apellidos" => "Ribeiro Cabral" ] ] "afiliaciones" => array:1 [ 0 => array:2 [ "entidad" => "Departamento de Agroindústria, Alimentos e Nutrição, Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ), Universidade de São Paulo, Piracicaba, São Paulo, Brasil" "identificador" => "aff0005" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "*" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Actividad antimicrobiana de la yerba mate (<span class="elsevierStyleItalic">Ilex paraguariensis</span> St. Hil.) contra patógenos alimentarios" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2570 "Ancho" => 1881 "Tamanyo" => 282111 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Growth curves of a) <span class="elsevierStyleItalic">S. aureus</span>, b) <span class="elsevierStyleItalic">L. monocytogenes</span> and c) <span class="elsevierStyleItalic">S.</span> Enteritidis under action of yerba mate ethanol extract different pH values. PC = positive control (chlorhexidine 0.12%).</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall">In recent decades, research has shown the potential exploitation of plant products as a bioactive compound source for industrial interest. Leaves, stems and flowers may present biological activities<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a>. In addition, the study of products used in infusions such as teas and beverages has gained increasing prominence<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a>. Among these products, yerba mate stands out, being a product widely used by South American populations both as a source of caffeine in place of or in addition to tea and coffee; also as a therapeutic agent due to its known pharmacological properties such as antioxidant, anti-inflammatory, antitumor, and weight reducing activities<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a>.</p><p id="par0010" class="elsevierStylePara elsevierViewall">In the last years, different investigations have revealed the antimicrobial potential of yerba mate, whose spectrum of activity includes gram-positive and gram-negative bacteria and fungi<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a><span class="elsevierStyleSup">,</span><a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a>. These bioactivities are strictly related to the presence of different classes of compounds, mainly phenolics, whose main representatives in yerba mate are gallic, syringic, caffeic, ferulic and ρ-coumaric acids<a class="elsevierStyleCrossRef" href="#bib0055"><span class="elsevierStyleSup">11</span></a>. However, there are few studies about the influence of pH conditions on the activity of these compounds in crude extracts; these data are extremely important, since this variable is vital for antimicrobial effectiveness.</p><p id="par0015" class="elsevierStylePara elsevierViewall">This study evaluated the antimicrobial activity against food pathogens of the methanolic and ethanolic extracts of yerba mate (<span class="elsevierStyleItalic">Ilex paraguariensis</span> St. Hil.) used to prepare the typical hot mate-based beverage <span class="elsevierStyleItalic">chimarrão</span> and its relation to the content of phenolic compounds. The composition of the extract with the highest antimicrobial activity and phenolic contents was determined by gas chromatography with mass spectrometry (GC-MS) and its antimicrobial activity was evaluated under different pH conditions.</p><p id="par0020" class="elsevierStylePara elsevierViewall">Yerba mate was acquired from the local trade in Campinas, São Paulo, Brazil. The extracts were obtained by percolation. The sample (1:8 w/v) was extracted in hydroethanolic (40:60) and hydromethanolic (30:70) solutions (Synth<span class="elsevierStyleSup">®</span>, Diadema, Brazil) and maintained under refrigeration for 96<span class="elsevierStyleHsp" style=""></span>h, with filtering using qualitative filter paper 12.5<span class="elsevierStyleHsp" style=""></span>μm (Qualy<span class="elsevierStyleSup">®</span>) every 24<span class="elsevierStyleHsp" style=""></span>h. The extract was evaporated in a rotary evaporator at 45<span class="elsevierStyleHsp" style=""></span>°C (Tecnal<span class="elsevierStyleSup">®</span>). Then, the final extract was freeze-dried (Liotop<span class="elsevierStyleSup">®</span>L101) and kept under refrigeration. For the tests, the extracts were dissolved in tryptic soy broth (TSB) (Difco<span class="elsevierStyleSup">®</span>, Franklin Lakes, USA).</p><p id="par0025" class="elsevierStylePara elsevierViewall">Antimicrobial activity from the strain collection of the Laboratório de Higiene e Laticínios (ESALQ/USP), was evaluated against <span class="elsevierStyleItalic">Staphylococcus aureus</span> ATCC 25923, <span class="elsevierStyleItalic">Listeria monocytogenes</span> ATCC 07644, <span class="elsevierStyleItalic">Salmonella</span> Enteritidis ATCC 13076 and <span class="elsevierStyleItalic">Escherichia coli</span> ATCC 25922. All antimicrobial tests were performed in triplicate. Antimicrobial screening was performed by agar diffusion<a class="elsevierStyleCrossRef" href="#bib0030"><span class="elsevierStyleSup">6</span></a>. Two hundred microliters of standardized inoculum (1x10<span class="elsevierStyleSup">8</span><span class="elsevierStyleHsp" style=""></span>CFU/ml) of each organism were transferred to 200<span class="elsevierStyleHsp" style=""></span>ml of TSB plus 0.7% bacteriological agar (final population of 1.5 x10<span class="elsevierStyleSup">5</span><span class="elsevierStyleHsp" style=""></span>CFU/ml). Seventy milliliters of this preparation were transferred to Petri dishes, in which 8<span class="elsevierStyleHsp" style=""></span>mm diameter wells were produced by vacuum pump and 40<span class="elsevierStyleHsp" style=""></span>μl of extracts were distributed (100<span class="elsevierStyleHsp" style=""></span>mg/ml). Negative (40<span class="elsevierStyleHsp" style=""></span>μl of TSB) and positive control (40<span class="elsevierStyleHsp" style=""></span>μl of chlorhexidine 0.12% v/v) were tested.</p><p id="par0030" class="elsevierStylePara elsevierViewall">For MIC determination, the macrobroth dilution method was performed in 96-well microplate<a class="elsevierStyleCrossRef" href="#bib0015"><span class="elsevierStyleSup">3</span></a>. The extract concentrations were obtained by 2-fold serial dilution in the microplate, ranging from 25<span class="elsevierStyleHsp" style=""></span>mg/ml to 0.78<span class="elsevierStyleHsp" style=""></span>mg/ml after the addition of inoculated TSB (1-2 x 10<span class="elsevierStyleSup">5</span><span class="elsevierStyleHsp" style=""></span>CFU/ml). The final volume for each well was 200<span class="elsevierStyleHsp" style=""></span>μl. Positive (200<span class="elsevierStyleHsp" style=""></span>μl of TSB added of 0.12% chlorhexidine v/v) and negative control (200<span class="elsevierStyleHsp" style=""></span>μl of sterile TSB) were tested. Two hundred microliters of sterile TSB were used for broth sterility control. After incubation (35<span class="elsevierStyleHsp" style=""></span>°C/24<span class="elsevierStyleHsp" style=""></span>h), all wells received 30<span class="elsevierStyleHsp" style=""></span>μl of resazurin (0.01% w/v) (Sigma-Aldrich<span class="elsevierStyleSup">®</span>, St. Louis, USA) in order to detect bacterial growth in the wells. Any evidence of color change was considered to be bacterial growth. For MBC determination, 10<span class="elsevierStyleHsp" style=""></span>μl of broth were removed from the wells considered inhibitory and sown in tryptic soy agar (TSA) (35<span class="elsevierStyleHsp" style=""></span>°C/24<span class="elsevierStyleHsp" style=""></span>h). MBC was considered as the lowest concentration at which no growth of colonies on the culture medium surface was observed.</p><p id="par0035" class="elsevierStylePara elsevierViewall">The effectiveness of the yerba mate extract showing the best antimicrobial activity was evaluated at pH 6, 7 and 8 using 96-well microplates<a class="elsevierStyleCrossRef" href="#bib0045"><span class="elsevierStyleSup">9</span></a>. All wells received 100<span class="elsevierStyleHsp" style=""></span>μl of sterile TSB. One hundred μl of extracts were added into the first row of each column and then 2-fold serial dilution was performed (final concentrations ranging from 25<span class="elsevierStyleHsp" style=""></span>mg/ml to 0.78<span class="elsevierStyleHsp" style=""></span>mg/ml), after adding 100<span class="elsevierStyleHsp" style=""></span>μl of inoculated broth (1-2 x 10<span class="elsevierStyleSup">5</span><span class="elsevierStyleHsp" style=""></span>CFU/ml). Control groups: 200<span class="elsevierStyleHsp" style=""></span>μl of inoculated TSB (negative), 200<span class="elsevierStyleHsp" style=""></span>μl of inoculated TSB added of chlorhexidine 0.12% v/v (positive) and 200<span class="elsevierStyleHsp" style=""></span>μl of sterile TSB plus extract (white). The microplates<a name="p95"></a> were incubated in a multilabel plate reader (VictorTMX3, PerkinElmer<span class="elsevierStyleSup">®</span>) (35<span class="elsevierStyleHsp" style=""></span>°C/18<span class="elsevierStyleHsp" style=""></span>h) with absorbance readings performed at 1 hour intervals (600<span class="elsevierStyleHsp" style=""></span>nm).</p><p id="par0040" class="elsevierStylePara elsevierViewall">The total phenolic content was determined with the Folin-Ciocalteau reagent<a class="elsevierStyleCrossRef" href="#bib0065"><span class="elsevierStyleSup">13</span></a>. Five hundred microliters of extracts (10<span class="elsevierStyleHsp" style=""></span>mg/ml) were mixed with 2.5<span class="elsevierStyleHsp" style=""></span>ml of the Folin- Ciocalteau reagent (1:10) and 2.0<span class="elsevierStyleHsp" style=""></span>ml of sodium carbonate solution (Na<span class="elsevierStyleInf">2</span>CO<span class="elsevierStyleInf">3</span>) (4% w/v) (Dinâmica<span class="elsevierStyleSup">®</span>, Diadema, Brazil). After 2 hours of incubation, the absorbance reading was performed at 740<span class="elsevierStyleHsp" style=""></span>nm in visible light spectrophotometer (Femto<span class="elsevierStyleSup">®</span> Plus). The total phenolic content was expressed as gallic acid equivalent (GAE) in g per kg of sample from the gallic acid standard curve. For the gallic acid, the curve was established by plotting concentration (ranging from 2.5<span class="elsevierStyleHsp" style=""></span>μg/ml to 50<span class="elsevierStyleHsp" style=""></span>μg/ml) <span class="elsevierStyleItalic">versus</span> absorbance (nm) (<span class="elsevierStyleItalic">y<span class="elsevierStyleHsp" style=""></span></span>=<span class="elsevierStyleHsp" style=""></span>42.71<span class="elsevierStyleItalic">x</span> + 0.3187; R<span class="elsevierStyleSup">2</span><span class="elsevierStyleHsp" style=""></span>=<span class="elsevierStyleHsp" style=""></span>0.9997, where <span class="elsevierStyleItalic">y</span> is the absorbance and <span class="elsevierStyleItalic">x</span> is the concentration).</p><p id="par0045" class="elsevierStylePara elsevierViewall">The most active extract was submitted to GC-MS analysis (Shimadzu<span class="elsevierStyleSup">®</span> GC-2010 coupled to Shimadzu<span class="elsevierStyleSup">®</span> QP 2010 Plus). The sample purification was performed by the solid phase extraction technique (SPE). Then, the extracts were derivatized adding 100<span class="elsevierStyleHsp" style=""></span>μl of N-methyl-N-(trimethylsilyl)- trifluoroacetamide (MSTFA) reagent (Sigma-Aldrich<span class="elsevierStyleSup">®</span>, St. Louis, USA). The derivatization product was diluted in hexane and the supernatant was transferred to a vial. The chromatographic analysis was carried out as follows: separation in capillary column RTX5MS (30<span class="elsevierStyleHsp" style=""></span>m × 0.25<span class="elsevierStyleHsp" style=""></span>mm × 0.25<span class="elsevierStyleHsp" style=""></span>μm), injection temperature of 280<span class="elsevierStyleHsp" style=""></span>°C and initial volume of 0.5<span class="elsevierStyleHsp" style=""></span>μl in “splitless” mode, and detection at 280<span class="elsevierStyleHsp" style=""></span>°C operating in “scanning” mode (m/z 40-800). The chromatographic conditions were: initial temperature of 80 °C (1<span class="elsevierStyleHsp" style=""></span>min), heating to 250<span class="elsevierStyleHsp" style=""></span>°C (20<span class="elsevierStyleHsp" style=""></span>°C/min) (1<span class="elsevierStyleHsp" style=""></span>min), 300<span class="elsevierStyleHsp" style=""></span>°C (6<span class="elsevierStyleHsp" style=""></span>°C/min) (5<span class="elsevierStyleHsp" style=""></span>min), 310<span class="elsevierStyleHsp" style=""></span>°C (15<span class="elsevierStyleHsp" style=""></span>°C/min) (10<span class="elsevierStyleHsp" style=""></span>min) and 320<span class="elsevierStyleHsp" style=""></span>°C (20<span class="elsevierStyleHsp" style=""></span>°C/min) (10<span class="elsevierStyleHsp" style=""></span>min). Integration was performed using the LabSolutions-CGMS software and the identification of the compounds of interest was performed by comparison of data obtained from GC-MS and the Wiley 8 Library (supplied with the equipment).</p><p id="par0050" class="elsevierStylePara elsevierViewall">The statistical analysis was performed using the Statistical Analysis System software (SAS 2002). The Tukey's test (0.5% probability) was used to compare means.</p><p id="par0055" class="elsevierStylePara elsevierViewall">The methanolic and ethanolic extracts of yerba mate inhibited all evaluated microorganisms, except <span class="elsevierStyleItalic">E. coli</span> (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). The ethanolic extract produced the largest inhibition zones and the lowest MIC on <span class="elsevierStyleItalic">S. aureus</span> and <span class="elsevierStyleItalic">S.</span> Enteritidis; for <span class="elsevierStyleItalic">L. monocytogenes</span>, the values were similar. Furthermore, it showed the lowest MBC on <span class="elsevierStyleItalic">S. aureus</span>, which demonstrates its high <span class="elsevierStyleItalic">in vitro</span> bactericidal activity.</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0060" class="elsevierStylePara elsevierViewall">The study by De Biasi <span class="elsevierStyleItalic">et al.</span><a class="elsevierStyleCrossRef" href="#bib0025"><span class="elsevierStyleSup">5</span></a> found inhibitory activity in the ethanolic extract of yerba mate against several microorganisms and only <span class="elsevierStyleItalic">E. coli</span> was not inhibited, agreeing with our results. However, Vaquero <span class="elsevierStyleItalic">et al.</span><a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a> reported the antimicrobial potential of yerba mate infusion against <span class="elsevierStyleItalic">E. coli</span>, which was the most inhibited microorganism among all evaluated ones. Cogo <span class="elsevierStyleItalic">et al.</span><a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a> reported antimicrobial activity of ethanolic extracts of yerba mate leaves against <span class="elsevierStyleItalic">Helicobacter pylori</span> in MIC as low as those found in this study. These findings reinforce the importance of yerba mate as a research source for new natural antimicrobials.<a name="p96"></a><a name="p97"></a></p><p id="par0065" class="elsevierStylePara elsevierViewall">The ethanolic extract showed the highest total phenolic content. Significant differences were observed between the ethanolic and methanolic extracts; ethanol 60% (v/v) (193.9<span class="elsevierStyleHsp" style=""></span>g GAE/kg) was the solvent which best extracted these compounds in comparison to methanol 70% (v/v) (173.0<span class="elsevierStyleHsp" style=""></span>g GAE/kg). Relating this data to the antimicrobial activity, it could be inferred that the phenolic contents of the extracts are directly related to their antimicrobial potential.</p><p id="par0070" class="elsevierStylePara elsevierViewall">For presenting the best antimicrobial activity, the ethanolic extract of yerba mate was selected for GC-MS analysis. Its components in greatest abundance were 3-O-caffeoylquinic acid (34.02%), 5-O-caffeoylquinic acid (28.45%), 4-O-caffeoylquinic acid (20.83%) and caffeic acid (2.99%) (<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>). Caffeoylquinic acids were the major phenolic compounds and their presence has been reported in previous studies<a class="elsevierStyleCrossRef" href="#bib0055"><span class="elsevierStyleSup">11</span></a>. These compounds have several important biological activities such as antioxidant and antiviral activities<a class="elsevierStyleCrossRef" href="#bib0050"><span class="elsevierStyleSup">10</span></a>. Detected in a lower amount, caffeic acid has been reported in plant extracts as having antimicrobial activity against gram-positive and gramnegative bacteria and fungi<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">7</span></a>.</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><p id="par0075" class="elsevierStylePara elsevierViewall">The effect of pH on the antimicrobial activity was assessed by growth curve analysis for <span class="elsevierStyleItalic">S. aureus, L. monocytogenes</span> and <span class="elsevierStyleItalic">S.</span> Enteritidis, in MIC values at neutral pH (<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>). The extract inhibited the growth of <span class="elsevierStyleItalic">S. aureus</span> at pH 7 and 8, which was not observed at pH 6, being unable to inhibit the growth of this microorganism (<a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a>). This behavior was also observed for <span class="elsevierStyleItalic">L. monocytogenes</span>, and the extract was ineffective only at pH 6. The growth curve of <span class="elsevierStyleItalic">S.</span> Enteritidis demonstrated a slight tendency to growth at pH 8; the extract was not able to inhibit bacterial growth at pH 6, as in other cases.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0080" class="elsevierStylePara elsevierViewall">Gutierrez et al.<a class="elsevierStyleCrossRef" href="#bib0045"><span class="elsevierStyleSup">9</span></a> showed a decrease of antilisterial activity of natural products at pH 6 in comparison to neutral pH, which is in agreement with our results; however, a considerable increase in the lag phase was observed at pH 5. Wen et al<span class="elsevierStyleItalic">.</span><a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">15</span></a> reported an increase in antilisterial activity of phenolic acids with decreasing pH; in the case of chlorogenic acid, better antibacterial activity was observed at a higher pH. According to the authors, this discrepancy may be related to changes in the ionization state and proportion of dissociated molecules at different pH values, resulting in decreased activity of chlorogenic acid at a lower pH.</p><p id="par0085" class="elsevierStylePara elsevierViewall">Since the values of dissociation constants (<span class="elsevierStyleItalic">pK</span>) of phenolic acids are generally higher than 8, the concentration of phenoxide ions is low in solutions with pH lower than 7<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a>. The chlorogenic acid has a <span class="elsevierStyleItalic">pK</span> value of ≈ 8.5<a class="elsevierStyleCrossRef" href="#bib0060"><span class="elsevierStyleSup">12</span></a>. If the antimicrobial activity is related to the concentration of phenoxide ions, the higher the pH of the medium, the more pronounced it would be. The increased antimicrobial activity probably results from the ability of negatively charged phenoxide ions to change the electrochemical balance of the bacterial microenvironment, facilitating cell death<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a>.</p><p id="par0090" class="elsevierStylePara elsevierViewall">According to our results, it could be concluded that yerba mate presents antimicrobial activity against food pathogens. The ethanolic extract was more effective than the methanolic extract in inhibiting <span class="elsevierStyleItalic">S. aureus, L. monocytogenes</span> and <span class="elsevierStyleItalic">S.</span> Enteritidis. The antimicrobial activity observed seems to be related to the presence of compounds derived from chlorogenic acid of known biological activities. The ethanolic extract was active at neutral and at pH 8. Yerba mate is thus a potential source for the extraction of antimicrobial compounds for use by the food industry as a natural preservative in foods and beverages.</p><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conflicts of interest</span><p id="par0095" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:7 [ 0 => array:2 [ "identificador" => "xres272609" "titulo" => "Abstract" ] 1 => array:2 [ "identificador" => "xpalclavsec254679" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "xres272608" "titulo" => "Resumen" ] 3 => array:2 [ "identificador" => "xpalclavsec254680" "titulo" => "Palabras clave" ] 4 => array:2 [ "identificador" => "sec0005" "titulo" => "Conflicts of interest" ] 5 => array:2 [ "identificador" => "xack62358" "titulo" => "Acknowledgements" ] 6 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2012-08-21" "fechaAceptado" => "2013-02-28" "PalabrasClave" => array:2 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec254679" "palabras" => array:5 [ 0 => "Yerba mate" 1 => "Antimicrobial activity" 2 => "<span class="elsevierStyleItalic">Staphylococcus aureus</span>" 3 => "<span class="elsevierStyleItalic">Listeria monocytogenes</span>" 4 => "<span class="elsevierStyleItalic">Salmonella</span> Enteritidis" ] ] ] "es" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Palabras clave" "identificador" => "xpalclavsec254680" "palabras" => array:5 [ 0 => "Yerba mate" 1 => "Actividad antimicrobiana" 2 => "<span class="elsevierStyleItalic">Staphylococcus aureus</span>" 3 => "<span class="elsevierStyleItalic">Listeria monocytogenes</span>" 4 => "<span class="elsevierStyleItalic">Salmonella</span> Enteritidis" ] ] ] ] "tieneResumen" => true "resumen" => array:2 [ "en" => array:2 [ "titulo" => "Abstract" "resumen" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Yerba mate (<span class="elsevierStyleItalic">Ilex paraguariensis</span> St. Hil.) has been studied for its important biological activities mainly attributed to phenolic compounds. This study evaluated the antimicrobial activity of methanolic and ethanolic extracts of yerba mate against food pathogens, such as <span class="elsevierStyleItalic">Staphylococcus aureus</span>, <span class="elsevierStyleItalic">Listeria monocytogenes</span>, <span class="elsevierStyleItalic">Salmonella</span> Enteritidis and <span class="elsevierStyleItalic">Escherichia coli</span> through minimum inhibitory (MIC) and bactericidal (MBC) concentrations, in addition to the determination of chemical composition by gas chromatography with mass spectrometry (GC-MS) and phenolic content. The most effective extract had its activity evaluated under different pH conditions by growth curve analysis. All microorganisms except <span class="elsevierStyleItalic">E. coli</span> were inhibited. The ethanolic extract showed the lowest MIC/MBC (0.78/0.78<span class="elsevierStyleHsp" style=""></span>mg/ml), the highest phenolic content (193.9<span class="elsevierStyleHsp" style=""></span>g.GAE/kg) and the presence of chlorogenic acid derivatives, especially 3-O-caffeoylquinic and caffeic acid. This extract was able to inhibit microbial growth at pH 7 and 8.</p>" ] "es" => array:2 [ "titulo" => "Resumen" "resumen" => "<p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">La actividad biológica de la yerba mate (<span class="elsevierStyleItalic">Ilex paraguariensis</span> St. Hil.) ya ha sido descrita. Dicha actividad generalmente se ha asociado a la presencia de compuestos fenólicos. Este estudio evaluó la actividad antimicrobiana de los extractos etanólicos y metanólicos de la yerba mate contra patógenos alimentarios como <span class="elsevierStyleItalic">Staphylococcus aureus</span>, <span class="elsevierStyleItalic">Listeria</span><a name="p94"></a><span class="elsevierStyleItalic">monocytogenes, Salmonella</span> Enteritidis y <span class="elsevierStyleItalic">Escherichia coli</span> mediante la determinación de la concentración inhibitoria (CIM) y bactericida mínima (CBM). También se efectuó el análisis de la composición química por cromatografía gaseosa-espectrometría de masas (CG-EM) y se determinó el contenido de compuestos fenólicos. El extracto con mayor capacidad inhibitoria se evaluó en diferentes condiciones de pH, por análisis de curvas de crecimiento. Todos los microorganismos fueron inhibidos, excepto <span class="elsevierStyleItalic">E. coli</span>. El extracto etanólico mostró la menor CIM/CBM (0,78/0,78<span class="elsevierStyleHsp" style=""></span>mg/ml), el más alto contenido de fenólicos totales (193,9<span class="elsevierStyleHsp" style=""></span>g.EAG/kg) y la presencia de derivados clorogénicos, principalmente ácido 3-O-cafeoilquínico y cafeico. Este extracto fue capaz de inhibir el crecimiento microbiano a pH 7 y 8.</p>" ] ] "multimedia" => array:3 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 2570 "Ancho" => 1881 "Tamanyo" => 282111 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Growth curves of a) <span class="elsevierStyleItalic">S. aureus</span>, b) <span class="elsevierStyleItalic">L. monocytogenes</span> and c) <span class="elsevierStyleItalic">S.</span> Enteritidis under action of yerba mate ethanol extract different pH values. PC = positive control (chlorhexidine 0.12%).</p>" ] ] 1 => array:7 [ "identificador" => "tbl0005" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "leyenda" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">MIC: minimum inhibitory concentration (mg/ml); MBC: minimum bactericidal concentration (mg/ml); M: methanolic extract; E: ethanolic extract; Averages of triplicates ± standard deviation; -: No inhibition</p>" "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="2" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">S. aureus</span></td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="2" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">L. monocytogenes</span></td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="2" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">S. Enteritidis</span></td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " colspan="2" align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">E. coli</span></td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black"> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">M \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">E \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">M \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">E \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">M \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">E \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">M \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">E \t\t\t\t\t\t\n \t\t\t\t</td></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Inhibition zones (mm) \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">21.67±0.57 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">23.67±0.57 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">10.00±0.00 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">10.33±0.58 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">10.00±0.00 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">13.67±0.57 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">- \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">- \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">MIC \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1.56 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.78 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">6.25 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">- \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">- \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">MBC \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.78 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.13 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">6.25 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">6.25 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">- \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">- \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab391965.png" ] ] ] ] "descripcion" => array:1 [ "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Antibacterial activity of methanolic and ethanolic extracts of yerba mate</p>" ] ] 2 => array:7 [ "identificador" => "tbl0010" "etiqueta" => "Table 2" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:3 [ "leyenda" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">RT: retention time (min).</p>" "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Compounds<a class="elsevierStyleCrossRef" href="#tblfn0005"><span class="elsevierStyleSup">a</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">RT \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Percentage of relative area<a class="elsevierStyleCrossRef" href="#tblfn0010"><span class="elsevierStyleSup">b</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" style="border-bottom: 2px solid black">Ion (m/z, abundance between parenthesis) \t\t\t\t\t\t\n \t\t\t\t</td></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Caffeic acid \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">11.00 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">2.99 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">219 (100), 396 (95), 73 (72), 381 (24), 191 (12), 45 (13); 401 (M+) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">5-O-caffeoylquinic acid \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">19.99 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">28.45 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">345 (100), 73 (94), 255 (58), 307 (41), 147 (21), 397 (15); 786 (M+) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">4-O-caffeoylquinic acid \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">20.64 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">20.83 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">307 (100), 73 (72), 255 (46), 219 (15), 489 (15), 147 (12); 786 (M+) \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3-O-caffeoylquinic acid \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">20.93 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="center" valign="\n \t\t\t\t\ttop\n \t\t\t\t">34.02 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">307 (100), 73 (76), 345 (68), 255 (30), 447 (21), 147 (17); 771 (M+) \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab391966.png" ] ] ] "notaPie" => array:2 [ 0 => array:3 [ "identificador" => "tblfn0005" "etiqueta" => "a" "nota" => "<p class="elsevierStyleNotepara" id="npar0005">All compounds with similarity percentage > 80%.</p>" ] 1 => array:3 [ "identificador" => "tblfn0010" "etiqueta" => "b" "nota" => "<p class="elsevierStyleNotepara" id="npar0010">Peak area in relation to total percentage of peak areas.</p>" ] ] ] "descripcion" => array:1 [ "en" => "<p 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| 2018 July | 10 | 2 | 12 |
| 2018 June | 11 | 2 | 13 |
| 2018 May | 18 | 1 | 19 |
| 2018 April | 8 | 1 | 9 |
| 2018 March | 13 | 0 | 13 |
| 2018 February | 8 | 2 | 10 |
| 2018 January | 10 | 1 | 11 |
| 2017 December | 6 | 0 | 6 |
| 2017 November | 10 | 0 | 10 |
| 2017 October | 6 | 1 | 7 |
| 2017 September | 12 | 3 | 15 |
| 2017 August | 6 | 2 | 8 |
| 2017 July | 6 | 4 | 10 |
| 2017 June | 8 | 17 | 25 |
| 2017 May | 11 | 13 | 24 |
| 2017 April | 13 | 11 | 24 |
| 2017 March | 22 | 21 | 43 |
| 2017 February | 11 | 4 | 15 |
| 2017 January | 11 | 2 | 13 |
| 2016 December | 21 | 6 | 27 |
| 2016 November | 20 | 5 | 25 |
| 2016 October | 27 | 7 | 34 |
| 2016 September | 39 | 16 | 55 |
| 2016 August | 21 | 3 | 24 |
| 2016 July | 34 | 13 | 47 |
| 2016 June | 48 | 33 | 81 |
| 2016 May | 35 | 20 | 55 |
| 2016 April | 28 | 18 | 46 |
| 2016 March | 41 | 17 | 58 |
| 2016 February | 45 | 25 | 70 |
| 2016 January | 27 | 26 | 53 |
| 2015 December | 34 | 20 | 54 |
| 2015 November | 34 | 19 | 53 |
| 2015 October | 70 | 28 | 98 |
| 2015 September | 33 | 16 | 49 |
| 2015 August | 52 | 34 | 86 |
| 2015 July | 38 | 23 | 61 |
| 2015 June | 33 | 29 | 62 |
| 2015 May | 50 | 38 | 88 |
| 2015 April | 33 | 28 | 61 |
| 2015 March | 45 | 9 | 54 |
| 2015 February | 40 | 12 | 52 |
| 2015 January | 44 | 13 | 57 |
| 2014 December | 64 | 14 | 78 |
| 2014 November | 64 | 12 | 76 |
| 2014 October | 56 | 10 | 66 |
| 2014 September | 51 | 8 | 59 |
| 2014 August | 46 | 14 | 60 |
| 2014 July | 71 | 11 | 82 |
| 2014 June | 70 | 10 | 80 |
| 2014 May | 36 | 9 | 45 |
| 2014 April | 34 | 12 | 46 |
| 2014 March | 36 | 14 | 50 |
| 2014 February | 35 | 18 | 53 |
| 2014 January | 27 | 16 | 43 |
| 2013 December | 30 | 12 | 42 |
| 2013 November | 47 | 17 | 64 |
| 2013 October | 61 | 16 | 77 |
| 2013 September | 68 | 22 | 90 |
| 2013 August | 78 | 28 | 106 |
| 2013 July | 36 | 18 | 54 |
| 2013 March | 26 | 0 | 26 |
Show all
