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Original article
Effects of temperature, water activity and incubation time on fungal growth and aflatoxin B1 production by toxinogenic Aspergillus flavus isolates on sorghum seeds
Efectos de la temperatura, la actividad de agua y el tiempo de incubación en el crecimiento fúngico y la producción de aflatoxina B1 por aislados toxicogénicos de Aspergillus flavus en sorgo
Amani Lahouara,b, Sonia Marinb, Ana Crespo-Sempereb, Salem Saïda, Vicente Sanchisb,
Corresponding author
vsanchis@tecal.udl.cat

Corresponding author.
a Laboratory of Biochemistry, Unity of Mycotoxicology, Faculty of Medicine of Sousse, University of Sousse, Sousse, Tunisia
b Food Technology Department, University of Lleida, XaRTA-UTPV, Agrotecnico center, Lleida, Spain
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Aflatoxins are a group of toxic chemical compounds that are characterized by their immunotoxic&#44; mutagenic and carcinogenic effects<a class="elsevierStyleCrossRef" href="#bib0340"><span class="elsevierStyleSup">17</span></a>&#46; Aflatoxins are classified as group I carcinogens by the International Agency for Research on Cancer<a class="elsevierStyleCrossRef" href="#bib0520"><span class="elsevierStyleSup">38</span></a>&#46; Among them&#44; aflatoxin B1 &#40;AFB1&#41; is the most frequent and most potent toxin&#46; <span class="elsevierStyleItalic">A&#46; flavus</span> and to a lesser extent <span class="elsevierStyleItalic">Aspergillus parasiticus</span> are among the most widely studied fungal species&#44; as a result of their ability to produce aflatoxins and their potential to persist pre- and post-harvest as a pathogen and saprophyte in the food supply&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">The growth of molds and the accumulation of mycotoxins in food and feedstuff are influenced by multiple variables&#44; such as water activity &#40;a<span class="elsevierStyleInf">w</span>&#41;&#44; temperature&#44; pH&#44; atmosphere composition&#44; substrate&#44; interaction among species&#44; and time<a class="elsevierStyleCrossRefs" href="#bib0390"><span class="elsevierStyleSup">3&#44;37</span></a>&#46; Generally&#44; relative humidity and temperature are considered to be the most critical factors during drying and storage<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">14</span></a>&#46; Various physical and chemical methods have been recommended to reduce mycotoxins&#44; but only a few have been accepted for practical use&#46; Thus&#44; the prevention of fungal growth and mycotoxin production represent key steps in risk management&#46; Many kinetic models have been developed and used to model the growth of toxigenic molds in various food substrates&#46; The effect of biotic and abiotic factors on growth and aflatoxin production have been widely studied using mathematical modeling<a class="elsevierStyleCrossRefs" href="#bib0420"><span class="elsevierStyleSup">4&#44;6&#44;14&#44;16&#44;18&#44;20&#44;28&#44;39</span></a>&#59; however&#44; no studies exist concerning their growth on sorghum seeds&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Sorghum &#40;<span class="elsevierStyleItalic">Sorghum bicolor</span> &#40;L&#46;&#41; Moench&#41; is a drought-resistant crop and an important food resource in terms of nutritional and economic values&#44; especially in semi-arid environments<a class="elsevierStyleCrossRef" href="#bib0430"><span class="elsevierStyleSup">7</span></a>&#46; Sorghum grains can be colonized by several fungal genera during the panicle and grain development stages<a class="elsevierStyleCrossRefs" href="#bib0475"><span class="elsevierStyleSup">21&#44;22</span></a>&#46; Several species of <span class="elsevierStyleItalic">Aspergillus</span>&#44; <span class="elsevierStyleItalic">Alternaria</span>&#44; <span class="elsevierStyleItalic">Fusarium</span>&#44; <span class="elsevierStyleItalic">Cladosporium</span>&#44; <span class="elsevierStyleItalic">Curvularia</span> and <span class="elsevierStyleItalic">Penicillium</span> are among the most prevalent grain mold pathogens in sorghum<a class="elsevierStyleCrossRef" href="#bib0410"><span class="elsevierStyleSup">34</span></a>&#46; Moreover&#44; a number of fungal isolates from this cereal primarily belonging to the genera <span class="elsevierStyleItalic">Fusarium</span> &#40;<span class="elsevierStyleItalic">F&#46; verticillioides</span>&#44; <span class="elsevierStyleItalic">F&#46; graminearum</span>&#44; <span class="elsevierStyleItalic">F&#46; equiseti</span>&#41;&#44; <span class="elsevierStyleItalic">Alternaria</span> &#40;<span class="elsevierStyleItalic">A&#46; alternata</span>&#41;&#44; <span class="elsevierStyleItalic">Aspergillus</span> &#40;<span class="elsevierStyleItalic">A&#46; flavus</span>&#41;&#44; and <span class="elsevierStyleItalic">Penicillium</span> &#40;<span class="elsevierStyleItalic">P&#46; funiculosum</span>&#41; have been reported to produce mycotoxins<a class="elsevierStyleCrossRefs" href="#bib0500"><span class="elsevierStyleSup">27&#44;30&#44;36</span></a>&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">The Tunisian climate is characterized by high temperature and humidity levels that seem to stimulate toxigenic mold growth and mycotoxin production&#46; The Tunisian population consumes large amounts of cereals and cereal-based products&#44; such as wheat&#44; barley and sorghum&#46; The occurrence of aflatoxins in commercialized sorghum in Tunisia was reported for the first time in 1977<a class="elsevierStyleCrossRef" href="#bib0415"><span class="elsevierStyleSup">2</span></a>&#44; and several studies confirmed aflatoxin contamination in Tunisian sorghum<a class="elsevierStyleCrossRefs" href="#bib0290"><span class="elsevierStyleSup">1&#44;8&#44;9&#44;10&#44;19</span></a>&#46; According to Oueslati <span class="elsevierStyleItalic">et al</span>&#46;<a class="elsevierStyleCrossRef" href="#bib0400"><span class="elsevierStyleSup">10</span></a>&#44; AFB1-contaminated cereals represent a real problem for Tunisian consumers&#46; Therefore&#44; it is important to develop prevention and control strategies to minimize aflatoxin contamination in sorghum&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">The objectives of the present work were to determine&#58; &#40;i&#41; the effects of the interactions of water activity&#44; temperature and incubation time on fungal growth and aflatoxin B1 production by Tunisian <span class="elsevierStyleItalic">Aspergillus flavus</span> isolates cultured on sorghum seeds and &#40;ii&#41; to identify the a<span class="elsevierStyleInf">w</span> and temperature limiting conditions for aflatoxin B1 production&#46; The Baranyi model<a class="elsevierStyleCrossRef" href="#bib0415"><span class="elsevierStyleSup">2</span></a> was applied to predict the growth of aflatoxigenic isolates of <span class="elsevierStyleItalic">A&#46; flavus</span> on sorghum seeds as a function of water activity and temperature&#46; Elucidating the ecology and physiology of <span class="elsevierStyleItalic">A&#46; flavus</span> will provide tools to manage AFB1 in sorghum&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Fungal isolates</span><p id="par0030" class="elsevierStylePara elsevierViewall">Three isolates of <span class="elsevierStyleItalic">A&#46; flavus</span> &#40;8&#44; 10 and 14&#41; were used in this study&#46; The three isolates were obtained from sorghum samples collected from the retail market of the Sahel region in Tunisia in 2011 and were kept in the Food Technology department collection of the University of Lleida&#46; All isolates were previously found to produce AFB1 when cultivated in Czapeck Yeast Extract Agar &#40;CYA&#41; and sorghum seeds&#46; The identification of the <span class="elsevierStyleItalic">A&#46; flavus</span> isolates was done microscopically<a class="elsevierStyleCrossRef" href="#bib0325"><span class="elsevierStyleSup">29</span></a>&#46; Molecular characterization of the isolates was also performed by PCR amplification using the primer pair AfAflT-F and AfAflT-R<a class="elsevierStyleCrossRef" href="#bib0365"><span class="elsevierStyleSup">35</span></a>&#46; Two DNA extracts of <span class="elsevierStyleItalic">A&#46; flavus</span> &#91;CECT &#40;Spanish Type Culture Collection&#41; 2695 and <span class="elsevierStyleItalic">A&#46; parasiticus</span> CECT 2681&#93; were used as references&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Experimental design and statistical analysis</span><p id="par0035" class="elsevierStylePara elsevierViewall">A full factorial design of three temperatures &#40;15&#44; 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>six water activities &#40;a<span class="elsevierStyleInf">w</span>&#41; &#40;0&#46;85&#44; 0&#46;88&#44; 0&#46;91&#44; 0&#46;94&#44; 0&#46;97 and 0&#46;99&#41;<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>three isolates &#40;8&#44; 10 and 14&#41; was used&#46; Colony diameters and AFB1 production after 7&#44; 14&#44; 21 and 28<span class="elsevierStyleHsp" style=""></span>d were the dependent variables&#46; Three replicates were included for each treatment&#46; The effects of temperature and a<span class="elsevierStyleInf">w</span> on mycelial growth and AFB1 production after 7&#44; 14&#44; 21 and 28<span class="elsevierStyleHsp" style=""></span>d were statistically analyzed by analysis of variance &#40;ANOVA&#41; using Statgraphics Plus 5&#46;1 &#40;Manugistics&#44; Inc&#46;&#44; MD&#44; USA&#41;&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Preparation of sorghum</span><p id="par0040" class="elsevierStylePara elsevierViewall">The study was performed <span class="elsevierStyleItalic">in vitro</span> on sorghum grains&#46; To determine the growth and aflatoxin patterns on sorghum grains&#44; different amounts of water &#40;calculated from a moisture absorption curve for the sorghum batch used&#41; were added to subsamples of 100<span class="elsevierStyleHsp" style=""></span>g of grain in flasks&#46; The sorghum was allowed to equilibrate at 4<span class="elsevierStyleHsp" style=""></span>&#176;C for 48<span class="elsevierStyleHsp" style=""></span>h with periodic shaking&#46; Once sealed&#44; the flasks were autoclaved for 20<span class="elsevierStyleHsp" style=""></span>min at 121<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; and then single layers of grain were carefully placed in 9<span class="elsevierStyleHsp" style=""></span>cm sterile plastic Petri dishes on a flow bench&#46; The final a<span class="elsevierStyleInf">w</span> levels were 0&#46;85&#44; 0&#46;88&#44; 0&#46;91&#44; 0&#46;94&#44; 0&#46;97 and 0&#46;99&#46; All a<span class="elsevierStyleInf">w</span> values were verified with an AquaLab 3 &#40;Decagon Devices&#44; Inc&#46;&#44; WA&#44; USA&#41; with an accuracy of &#177;0&#46;003 before&#44; during and at the end of the experiment&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Inocula preparation and incubation</span><p id="par0045" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">A&#46; flavus</span> isolates were sub-cultured on potato dextrose agar &#40;PDA&#41; plates and incubated at 25<span class="elsevierStyleHsp" style=""></span>&#176;C for 7<span class="elsevierStyleHsp" style=""></span>d to enable significant sporulation&#46; After incubation&#44; a sterile inoculation loop was used to remove the conidia from the PDA plates&#46; The conidia were suspended in an aqueous solution of 0&#46;05<span class="elsevierStyleHsp" style=""></span>&#37; &#40;w&#47;v&#41; Tween 80&#46; After homogenizing&#44; the spore concentrations were determined using the Thoma cell counting chamber&#46; The suspensions were diluted to adjust the final concentration to vary between 1 and 5<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>10<span class="elsevierStyleSup">5</span> spores&#47;ml&#46; Triplicate plates with single layers of sorghum for each water activity and temperature combination were inoculated centrally with a needlepoint load and incubated for 28<span class="elsevierStyleHsp" style=""></span>d&#46; Previous experiments showed that the number of spores inoculated through this technique was 10&#8211;100 spores on each inoculation point&#46; The incubation temperatures for all three isolates were 15&#44; 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; and the a<span class="elsevierStyleInf">w</span> values were 0&#46;85&#44; 0&#46;88&#44; 0&#46;91&#44; 0&#46;94&#44; 0&#46;97 and 0&#46;99&#46; Plates with the same temperature and a<span class="elsevierStyleInf">w</span> level were stacked in plastic chambers together with 250<span class="elsevierStyleHsp" style=""></span>ml of glycerol&#47;water solution of the same a<span class="elsevierStyleInf">w</span> to maintain the relative humidity equilibrium in the chambers as constant as possible&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Growth measurement and growth rate calculation</span><p id="par0050" class="elsevierStylePara elsevierViewall">The diameter of each growing colony was determined by measurement of two right-angled rays of the colony&#46; Measurements were recorded daily during the 28<span class="elsevierStyleHsp" style=""></span>d of growth&#46; The growth rate &#40;<span class="elsevierStyleItalic">&#956;</span>&#41; &#40;mm&#47;d&#41; and growth time &#40;<span class="elsevierStyleItalic">&#955;</span>&#41; &#40;d&#41; were evaluated for each isolate at each different combination of a<span class="elsevierStyleInf">w</span> and temperature by plotting the colony diameter extension &#40;mm&#41; against time &#40;d&#41; and fitting the Baranyi<a class="elsevierStyleCrossRef" href="#bib0415"><span class="elsevierStyleSup">2</span></a> model using Statgraphics Plus&#46; This model represents the three growth phases &#40;lag&#44; linear&#44; and asymptotic phase&#41;&#46; The proposed primary model has the general form of&#58;<elsevierMultimedia ident="eq0005"></elsevierMultimedia><elsevierMultimedia ident="eq0010"></elsevierMultimedia></p><p id="par0170" class="elsevierStylePara elsevierViewall">Where <span class="elsevierStyleItalic">y</span> is the colony diameter &#40;or radius&#41;&#44; <span class="elsevierStyleItalic">y</span><span class="elsevierStyleInf">0</span> is the initial colony diameter &#40;or radius&#44; usually zero&#41;&#44; <span class="elsevierStyleItalic">y</span><span class="elsevierStyleInf">max</span> is the maximum colony diameter &#40;or radius&#41; attained &#40;asymptotic value&#41;&#44; <span class="elsevierStyleItalic">&#956;</span><span class="elsevierStyleInf">max</span> is the maximum specific growth rate &#40;defined as the slope of the growth curve at the point of inflexion&#41;&#44; <span class="elsevierStyleItalic">&#955;</span> is the lag period &#40;<span class="elsevierStyleItalic">i&#46;e&#46;</span> the intersection of the line defining the maximum specific growth rate with the <span class="elsevierStyleItalic">x</span>-axis&#41;&#44; and <span class="elsevierStyleItalic">t</span> is the time&#46;</p></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Aflatoxin B1 production and quantification</span><p id="par0055" class="elsevierStylePara elsevierViewall">AFB<span class="elsevierStyleInf">1</span> production was determined after 7 and 14<span class="elsevierStyleHsp" style=""></span>d of incubation under the optimal growth conditions &#91;at 25<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;0&#46;97 and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41; and at 37<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;0&#46;94&#44; 0&#46;97&#44; and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#93; and after 21 and 28<span class="elsevierStyleHsp" style=""></span>d of incubation under suitable but not optimal conditions &#91;at 37<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#44; at 25<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;0&#46;91 and 0&#46;94<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41; and at 15<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#93; &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; AFB<span class="elsevierStyleInf">1</span> was extracted from the sorghum samples &#40;20<span class="elsevierStyleHsp" style=""></span>g&#41; with 100<span class="elsevierStyleHsp" style=""></span>ml of 4<span class="elsevierStyleHsp" style=""></span>&#37; acetonitrile aqueous solution containing potassium chloride &#40;9&#58;1&#41;&#46; The mixture was adjusted to a pH of 1&#46;5 with undiluted hydrochloric acid&#44; shaken for 20<span class="elsevierStyleHsp" style=""></span>min and filtered through a Whatman paper&#46; The filtrate was defatted with 100<span class="elsevierStyleHsp" style=""></span>ml of hexane&#46; The solution was shaken for 10<span class="elsevierStyleHsp" style=""></span>min&#46; After separation&#44; the upper phase &#40;hexane&#41; was discarded&#46; This step was repeated with 50<span class="elsevierStyleHsp" style=""></span>ml of hexane&#46; The lower phase was extracted with 50<span class="elsevierStyleHsp" style=""></span>ml of chloroform and 50<span class="elsevierStyleHsp" style=""></span>ml of deionized water&#44; and the solution was shaken for 10<span class="elsevierStyleHsp" style=""></span>min&#46; After separation&#44; the lower phase &#40;chloroform&#41; was collected&#46; The upper phase was re-extracted twice with 25<span class="elsevierStyleHsp" style=""></span>ml of chloroform and 25<span class="elsevierStyleHsp" style=""></span>ml of deionized water&#46; The chloroform extracts were evaporated to near dryness using a rotary evaporator at 40<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; AFB1 was resuspended in 2<span class="elsevierStyleHsp" style=""></span>ml of methanol and evaporated to dryness under nitrogen&#46; Finally&#44; the sample was analyzed with HPLC&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0060" class="elsevierStylePara elsevierViewall">The determination of AFB1 was performed using a Waters &#40;Milford&#44; MA&#44; USA&#41; chromatograph equipped with a reverse phase C<span class="elsevierStyleInf">18</span> silica gel column &#40;Water Spherisorb 3<span class="elsevierStyleHsp" style=""></span>&#956;m ODS2 4&#46;6<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>150<span class="elsevierStyleHsp" style=""></span>mm&#44; Milford&#44; MA&#44; USA&#41;&#44; followed by fluorescence detection &#40;<span class="elsevierStyleItalic">&#955;</span><span class="elsevierStyleInf">exc</span> 362<span class="elsevierStyleHsp" style=""></span>nm&#59; <span class="elsevierStyleItalic">&#955;</span><span class="elsevierStyleInf">em</span> 425<span class="elsevierStyleHsp" style=""></span>nm&#41; &#40;Waters 2475 Florescence Detector&#44; Waters&#44; Milford&#44; MA&#44; USA&#41;&#46; The mobile phase consisted of water&#8211;acetonitrile&#8211;methanol &#40;700&#58;170&#58;170&#41; with a flow rate of 1<span class="elsevierStyleHsp" style=""></span>ml&#47;min&#46; The detection limit &#40;LOD&#41; of the method was 0&#46;075<span class="elsevierStyleHsp" style=""></span>ng&#47;g&#46; The injection volume was 10<span class="elsevierStyleHsp" style=""></span>&#956;l&#44; and the retention time was 8<span class="elsevierStyleHsp" style=""></span>min&#46; Post-column derivatization was achieved using a photochemical reactor for enhanced detection &#40;PHRED&#41; &#40;LCTech UVE&#44; Dorfen&#44; Germany&#41;&#46;</p></span></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Results and discussion</span><p id="par0065" class="elsevierStylePara elsevierViewall">Previous works noted that data obtained regarding mycotoxin production on culture media could not be directly extrapolated to the natural substrates<a class="elsevierStyleCrossRef" href="#bib0530"><span class="elsevierStyleSup">40</span></a>&#46; Thus&#44; the present study was performed directly on sorghum grains&#46;</p><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Effects of water activity and temperature on mycelial growth</span><p id="par0070" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a> shows an example of the observed colony diameters for <span class="elsevierStyleItalic">A&#46; flavus</span> isolates 8&#44; 10 and 14 at two water activities &#40;0&#46;90 and 0&#46;97&#41; and their respective growth adjusted to the Barany model&#46; Under optimum growth conditions&#44; the colony diameters increased linearly until the colonies reached the edge of the Petri dishes&#46; Under limiting growth conditions&#44; a clear lag time was observed prior to growth &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; The Baranyi model was used to determine two growth parameters&#58; the lag phases &#40;d&#41; and the growth rates &#40;mm&#47;d&#41;&#46; The percentage of variance explained by the model &#40;<span class="elsevierStyleItalic">R</span><span class="elsevierStyleSup">2</span> coefficient&#41; obtained for the different treatments &#40;combinations&#41; varied between 85&#46;85<span class="elsevierStyleHsp" style=""></span>&#37; and 99&#46;83<span class="elsevierStyleHsp" style=""></span>&#37;&#44; with 31 combinations showing no growth and only two combinations exhibiting 54<span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>90<span class="elsevierStyleHsp" style=""></span>&#37;&#46; The low MSEs &#40;0&#46;017&#8211;0&#46;673&#41; and the high <span class="elsevierStyleItalic">R</span><span class="elsevierStyleSup">2</span> values &#40;85&#46;85&#8211;99&#46;83<span class="elsevierStyleHsp" style=""></span>&#37;&#41; indicated that the evaluated model could sufficiently determine the growth parameters&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0075" class="elsevierStylePara elsevierViewall">Analysis of variance for the growth rate showed that the single factors a<span class="elsevierStyleInf">w</span> and temperature and the interactions isolate<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>temperature&#44; a<span class="elsevierStyleInf">w</span><span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>temperature and isolate<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span><span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>temperature had significant influences on the growth rate &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; On average&#44; the three isolates behaved similarly&#44; as noted by Mohale et al&#46;<a class="elsevierStyleCrossRef" href="#bib0490"><span class="elsevierStyleSup">24</span></a> with atoxigenic and toxigenic strains of <span class="elsevierStyleItalic">A&#46; flavus</span>&#46; The combined effects of a<span class="elsevierStyleInf">w</span> and temperature on the growth rates &#40;mm&#47;d&#41; of the three isolates tested are shown in <a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#46; The maximum growth rates achieved were 16&#46;29&#44; 14&#46;91 and 14&#46;22<span class="elsevierStyleHsp" style=""></span>mm&#47;d by isolates 14&#44; 8 and 10&#44; respectively&#44; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w&#46;</span> In this study&#44; the optimum temperature for growth was 37<span class="elsevierStyleHsp" style=""></span>&#176;C for all a<span class="elsevierStyleInf">w</span> levels tested &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>&#41;&#46; Additionally&#44; 25<span class="elsevierStyleHsp" style=""></span>&#176;C was a very favorable temperature and yielded high growth rates&#46; Similar results for <span class="elsevierStyleItalic">A&#46; flavus</span> isolates were found by Horn<a class="elsevierStyleCrossRef" href="#bib0485"><span class="elsevierStyleSup">23</span></a>&#46; The extent of seed colonization by <span class="elsevierStyleItalic">Aspergillus</span> section <span class="elsevierStyleItalic">Flavi</span> grew with the increase of water activity and temperature to the optimal conditions of a<span class="elsevierStyleInf">w</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;96 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Samapundo et al<span class="elsevierStyleItalic">&#46;</span><a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">33</span></a> showed that <span class="elsevierStyleItalic">A&#46; flavus</span> had optimum growth on corn at 30<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Schindler et al<span class="elsevierStyleItalic">&#46;</span><a class="elsevierStyleCrossRef" href="#bib0505"><span class="elsevierStyleSup">28</span></a> reported that growth of <span class="elsevierStyleItalic">A&#46; flavus</span> was most favored in wort agar between 29 and 35<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Temperatures between 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C were not tested in our study&#59; however&#44; the mycelial extension of Tunisian <span class="elsevierStyleItalic">A&#46; flavus</span> isolates was significantly higher at 37<span class="elsevierStyleHsp" style=""></span>&#176;C than at 25<span class="elsevierStyleHsp" style=""></span>&#176;C &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; <a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a> showed that very short lag phases were encountered at high temperatures &#40;25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41; and a<span class="elsevierStyleInf">w</span> &#40;0&#46;97 and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; The shorter lag phase period was observed for isolates 10 and 8 at 25<span class="elsevierStyleHsp" style=""></span>&#176;C and for isolate 14 at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Samapundo et al&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">33</span></a> showed that an increase in temperature from 30 to 37<span class="elsevierStyleHsp" style=""></span>&#176;C resulted in a decrease in the colony growth rate and an increase in the lag phase&#46;</p><p id="par0080" class="elsevierStylePara elsevierViewall">No growth was detected at 15<span class="elsevierStyleHsp" style=""></span>&#176;C at any of the a<span class="elsevierStyleInf">w</span> conditions tested for isolates 10 and 14&#59; however&#44; isolate 8 grew at 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; The minimum growth rate observed was 0&#46;9<span class="elsevierStyleHsp" style=""></span>mm&#47;d&#44; which was calculated for isolate 14 at 37<span class="elsevierStyleHsp" style=""></span>&#176;C and 0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; No growth was detected for isolates 10 and 14 at 25<span class="elsevierStyleHsp" style=""></span>&#176;C and 0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; However&#44; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C all isolates grew at 0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; No growth at 0&#46;85 and 0&#46;88<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> was detected after 28<span class="elsevierStyleHsp" style=""></span>d at all tested temperatures&#46; However&#44; Pitt and Miscamble<a class="elsevierStyleCrossRef" href="#bib0440"><span class="elsevierStyleSup">11</span></a> reported that the minimum water activity for growth of <span class="elsevierStyleItalic">A&#46; flavus</span> was between 0&#46;80 and 0&#46;83&#46; The discrepancy in these results may be due to differences in the media used and the availability of nutrients&#46; Some components of the pericarp layer of the grain&#44; such as tannins and high levels of phenol-based pigments&#44; and some antifungal proteins can affect fungal growth<a class="elsevierStyleCrossRef" href="#bib0440"><span class="elsevierStyleSup">11</span></a>&#46; Similar studies have also indicated that the availability of nutrients may affect the chances of growth at marginal a<span class="elsevierStyleInf">w</span> values<a class="elsevierStyleCrossRef" href="#bib0450"><span class="elsevierStyleSup">13</span></a>&#46;</p><p id="par0085" class="elsevierStylePara elsevierViewall">Maximum growth rates were observed for the three isolates at 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C at 0&#46;97 and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; Additionally&#44; the <span class="elsevierStyleItalic">A&#46; flavus</span> isolates assayed grew faster at 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> than at 0&#46;97<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> at 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; with the exception of isolate 14&#44; where the optimum water activity for growth was 0&#46;97<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> at 25<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Similar results were reported by Marin et al&#46;<a class="elsevierStyleCrossRef" href="#bib0465"><span class="elsevierStyleSup">18</span></a> for <span class="elsevierStyleItalic">A&#46; flavus</span> grown on maize extract agar&#44; where the optimal a<span class="elsevierStyleInf">w</span> for growth was 0&#46;994&#46; However&#44; the optimum water activity for growth in some studies was somewhat different from the conditions demonstrated by our data&#46; For instance&#44; Horn<a class="elsevierStyleCrossRef" href="#bib0485"><span class="elsevierStyleSup">23</span></a> found that the optimum water activity for growth was 0&#46;96<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; These differences can be attributed to intraspecific and regional variability among isolates&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Effects of water activity and temperature on aflatoxin B1 production</span><p id="par0090" class="elsevierStylePara elsevierViewall">AFB1 was the only metabolite considered in the data analysis because AFB2 was rarely detected and only in trace amounts whereas aflatoxins G1 and G2 were never detected&#46; AFB1 has been reported to be the major AFB in natural substrates such as sorghum<a class="elsevierStyleCrossRefs" href="#bib0290"><span class="elsevierStyleSup">1&#44;9&#44;10</span></a>&#46; A similar situation was reported by Giorni et al<a class="elsevierStyleCrossRef" href="#bib0535"><span class="elsevierStyleSup">41</span></a>&#46;</p><p id="par0095" class="elsevierStylePara elsevierViewall">Analysis of variance of the effects of a<span class="elsevierStyleInf">w</span> and temperature on AFB1 production by the three <span class="elsevierStyleItalic">A&#46; flavus</span> isolates revealed that the single factors a<span class="elsevierStyleInf">w</span> and temperature and the interactions temperature<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>isolates&#44; a<span class="elsevierStyleInf">w</span><span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>incubation time and temperature<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span>incubation time had a significant effect on AFB1 production after 7 and 14<span class="elsevierStyleHsp" style=""></span>d of growth on sorghum seeds &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; After 21 and 28<span class="elsevierStyleHsp" style=""></span>d&#44; only the single factors a<span class="elsevierStyleInf">w</span> and temperature and the interaction between water activity and isolates significantly affected AFB1 accumulation &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">The maximum accumulation of AFB1 &#40;2&#46;26<span class="elsevierStyleHsp" style=""></span>ppb&#41; was observed for isolate 8<span class="elsevierStyleHsp" style=""></span>at 37<span class="elsevierStyleHsp" style=""></span>&#176;C and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> after 7<span class="elsevierStyleHsp" style=""></span>d of incubation &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46; Generally&#44; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C and 25<span class="elsevierStyleHsp" style=""></span>&#176;C AFB1 accumulation grew with increasing water activities &#40;0&#46;97 and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; No significant differences were found between 0&#46;99 and 0&#46;97<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; at 37<span class="elsevierStyleHsp" style=""></span>&#176;C after 7<span class="elsevierStyleHsp" style=""></span>d of incubation&#46; At 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; significantly higher amounts of AFB1 &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; were produced by the three <span class="elsevierStyleItalic">A&#46; flavus</span> isolates at 0&#46;97 and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; However&#44; Montani et al&#46;<a class="elsevierStyleCrossRef" href="#bib0510"><span class="elsevierStyleSup">32</span></a> found that the most favorable a<span class="elsevierStyleInf">w</span> for aflatoxin production was 0&#46;90<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> at all incubation times studied&#46; In this study&#44; the only isolate that produced AFB1 at 0&#46;94 was isolate 8&#44; which produced 0&#46;23<span class="elsevierStyleHsp" style=""></span>ng&#47;g at 37<span class="elsevierStyleHsp" style=""></span>&#176;C after 7<span class="elsevierStyleHsp" style=""></span>d of incubation and 0&#46;105<span class="elsevierStyleHsp" style=""></span>ng&#47;g at 25<span class="elsevierStyleHsp" style=""></span>&#176;C after 21<span class="elsevierStyleHsp" style=""></span>d&#46; To determine the minimum a<span class="elsevierStyleInf">w</span> that allowed aflatoxin production&#44; we tested a<span class="elsevierStyleInf">w</span> values of 0&#46;85&#44; 0&#46;88 and 0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; AFB1 was not detected at 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; whereas growth occurred at 0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; AFB1 was detected under a narrower range of water activity than the range that allowed growth &#40;0&#46;94 and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; Consequently&#44; it may be possible to avoid aflatoxin accumulation from these strains by storing the commodities at &#8220;safe&#8221; water activity levels &#40;&#8804;0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; At 15<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; AFB1 was not detected &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; Hunter<a class="elsevierStyleCrossRef" href="#bib0370"><span class="elsevierStyleSup">31</span></a> proposed 0&#46;84<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> as the minimal value for aflatoxin production in corn&#44; and Astoreca <span class="elsevierStyleItalic">et al&#46;</span>&#44;<a class="elsevierStyleCrossRef" href="#bib0350"><span class="elsevierStyleSup">26</span></a> proposed 0&#46;83 as the minimum value in corn extract medium&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0105" class="elsevierStylePara elsevierViewall">Several researchers reported a different behavior of the <span class="elsevierStyleItalic">A&#46; flavus</span> isolates regarding temperature&#44; with optimum values varying from 25 to 30<span class="elsevierStyleHsp" style=""></span>&#176;C regardless of the media<a class="elsevierStyleCrossRefs" href="#bib0425"><span class="elsevierStyleSup">5&#44;12&#44;25</span></a>&#46; For instance&#44; Schindler <span class="elsevierStyleItalic">et al&#46;</span><a class="elsevierStyleCrossRef" href="#bib0505"><span class="elsevierStyleSup">28</span></a>&#44; reported that maximal production of aflatoxins on wort media occurred at 24<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; whereas the optimal temperature for AFB1 production was 35<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">The incubation period that allowed higher AFB1 accumulation depended on the temperature&#44; water activity and isolate tested&#46; At the optimal temperature &#40;37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41; and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#44; all isolates required 7<span class="elsevierStyleHsp" style=""></span>d to reach maximum AFB1 production&#59; no AFB1 production was detected after 14<span class="elsevierStyleHsp" style=""></span>d under the same conditions&#46; At water activities above 0&#46;94<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#44; AFB1 accumulated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C was lower after 14<span class="elsevierStyleHsp" style=""></span>d of incubation on sorghum grains than after 7<span class="elsevierStyleHsp" style=""></span>d &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;0001&#41;&#46; The results showed that the optimal a<span class="elsevierStyleInf">w</span> for AFB1 accumulation at 25<span class="elsevierStyleHsp" style=""></span>&#176;C was 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; At 25<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; the amounts of AFB1 detected at 0&#46;97 and 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> after 14<span class="elsevierStyleHsp" style=""></span>d of incubation increased compared to the amounts produced after 7<span class="elsevierStyleHsp" style=""></span>d of incubation&#44; with the exception of isolate 8 at 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#44; whose AFB1 accumulation was higher after 7<span class="elsevierStyleHsp" style=""></span>d of incubation &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;001&#41;&#46; AFB1 decrease following the peak of maximum production was observed under the optimal conditions &#40;37<span class="elsevierStyleHsp" style=""></span>&#176;C and 0&#46;97&#8211;0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; The decrease in AFB1 concentration may be explained by its degradation caused by the mold itself&#46; The detection of AFB1 after 21<span class="elsevierStyleHsp" style=""></span>d and 28<span class="elsevierStyleHsp" style=""></span>d of incubation showed that only isolate 8 produced AFB1 at 0&#46;94<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> and 37<span class="elsevierStyleHsp" style=""></span>&#176;C and 25<span class="elsevierStyleHsp" style=""></span>&#176;C after 7 and 21<span class="elsevierStyleHsp" style=""></span>d&#44; respectively&#46; <span class="elsevierStyleItalic">A&#46; flavus</span> species comprise a high number of isolates with certain genetic variability&#44; which explains why the expression of genes involved in the aflatoxin metabolic pathway is dependent on the environmental conditions&#46;</p><p id="par0115" class="elsevierStylePara elsevierViewall">In our study&#44; maximum AFB1 production could not be linked to a specific time point after inoculation&#44; but overall 7<span class="elsevierStyleHsp" style=""></span>d of incubation was generally sufficient for <span class="elsevierStyleItalic">A&#46; flavus</span> to achieve important AFB1 accumulation under the optimal conditions for AFB1 production &#40;37<span class="elsevierStyleHsp" style=""></span>&#176;C and 0&#46;97&#8211;0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; Nevertheless&#44; the optimum incubation period and temperature required for maximum AFB1 production varied&#44; probably due to the set of conditions tested&#44; the nature and composition of the substrate and intraspecific and regional variability among isolates&#46;</p><p id="par0120" class="elsevierStylePara elsevierViewall">In this study&#44; maximal growth was correlated with maximal aflatoxin production &#40;0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;&#46; However&#44; other studies have shown that good growth may occur without any production of aflatoxins&#46; For example&#44; Rabie and Smalley<a class="elsevierStyleCrossRef" href="#bib0455"><span class="elsevierStyleSup">15</span></a> demonstrated that the maximal production of aflatoxins occurred at 24<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; but the maximal growth of <span class="elsevierStyleItalic">A&#46; flavus</span> isolates occurred at 29 and 35<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><p id="par0125" class="elsevierStylePara elsevierViewall">This is the first study concerning the effect of water activity and temperature on <span class="elsevierStyleItalic">A&#46; flavus</span> growth and AFB1 production on sorghum grains&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">The optimal conditions for mycelial growth and AFB1 production were 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; There was neither <span class="elsevierStyleItalic">A&#46; flavus</span> growth nor AFB1 production at 0&#46;85 and 0&#46;88<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> at all temperatures investigated&#46; Consequently&#44; aflatoxin accumulation could be avoided by storing sorghum at low water activity levels &#40;&#8804;0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; Our results showed that <span class="elsevierStyleItalic">A&#46; flavus</span> isolated from Tunisian sorghum was able to grow in a wide range of water activities &#40;0&#46;91&#8211;0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41; and temperatures &#40;15&#8211;37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;&#44; however&#44; the production of AFB1 occurred at a narrower range of water activities &#40;0&#46;94&#8211;0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41; and temperatures &#40;25&#8211;37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;&#46; Within the range of a<span class="elsevierStyleInf">w</span> evaluated in this study&#44; 0&#46;94 was the limiting level for AFB1 production&#46; The investigation of trends for growth and AFB1 production at different temperatures and water activities in sorghum seeds can provide important information concerning the contamination of sorghum by AFB1&#46; Our study contributes to the understanding of the ecology and physiology of Tunisian <span class="elsevierStyleItalic">A&#46; flavus</span> isolates&#46;</p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Ethical disclosures</span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Protection of human and animal subjects</span><p id="par0135" class="elsevierStylePara elsevierViewall">The authors declare that no experiments were performed on humans or animals for this investigation&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Confidentiality of data</span><p id="par0140" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article&#46;</p></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Right to privacy and informed consent</span><p id="par0145" class="elsevierStylePara elsevierViewall">The authors declare that no patient data appears in this article&#46;</p></span></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Conflict of interest</span><p id="par0150" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest&#46;</p></span></span>"
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        1 => array:2 [
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          "titulo" => "Keywords"
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          "titulo" => "Palabras clave"
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        4 => array:2 [
          "identificador" => "sec0005"
          "titulo" => "Introduction"
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        5 => array:3 [
          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
          "secciones" => array:6 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Fungal isolates"
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              "titulo" => "Experimental design and statistical analysis"
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              "identificador" => "sec0025"
              "titulo" => "Preparation of sorghum"
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            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Inocula preparation and incubation"
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            4 => array:2 [
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              "titulo" => "Growth measurement and growth rate calculation"
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              "titulo" => "Aflatoxin B1 production and quantification"
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          "titulo" => "Results and discussion"
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              "titulo" => "Effects of water activity and temperature on mycelial growth"
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              "titulo" => "Effects of water activity and temperature on aflatoxin B1 production"
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          "titulo" => "Ethical disclosures"
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              "titulo" => "Protection of human and animal subjects"
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              "titulo" => "Confidentiality of data"
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              "titulo" => "Right to privacy and informed consent"
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          "identificador" => "xack209311"
          "titulo" => "Acknowledgments"
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          "titulo" => "References"
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    "fechaRecibido" => "2015-05-23"
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            0 => "Water activity"
            1 => "Incubation time"
            2 => "Temperature"
            3 => "<span class="elsevierStyleItalic">Aspergillus flavus</span>"
            4 => "Aflatoxin B1"
            5 => "Fungal growth"
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            0 => "Actividad de agua"
            1 => "Tiempo de incubaci&#243;n"
            2 => "Temperatura"
            3 => "<span class="elsevierStyleItalic">Aspergillus flavus</span>"
            4 => "Aflatoxina B1"
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        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Sorghum&#44; which is consumed in Tunisia as human food&#44; suffers from severe colonization by several toxigenic fungi and contamination by mycotoxins&#46; The Tunisian climate is characterized by high temperature and humidity that stimulates mold proliferation and mycotoxin accumulation in foodstuffs&#46; This study investigated the effects of temperature &#40;15&#44; 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;&#44; water activity &#40;a<span class="elsevierStyleInf">w</span>&#44; between 0&#46;85 and 0&#46;99&#41; and incubation time &#40;7&#44; 14&#44; 21 and 28<span class="elsevierStyleHsp" style=""></span>d&#41; on fungal growth and aflatoxin B1 &#40;AFB1&#41; production by three <span class="elsevierStyleItalic">Aspergillus flavus</span> isolates &#40;8&#44; 10 and 14&#41; inoculated on sorghum grains&#46; The Baranyi model was applied to identify the limits of growth and mycotoxin production&#46; Maximum diameter growth rates were observed at 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span> at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for two of the isolates&#46; The minimum a<span class="elsevierStyleInf">w</span> needed for mycelial growth was 0&#46;91 at 25 and 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; At 15<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; only isolate 8 grew at 0&#46;99<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#46; Aflatoxin B1 accumulation could be avoided by storing sorghum at low water activity levels &#40;&#8804;0&#46;91<span class="elsevierStyleHsp" style=""></span>a<span class="elsevierStyleInf">w</span>&#41;&#46; Aflatoxin production was not observed at 15<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; This is the first work on the effects of water activity and temperature on <span class="elsevierStyleItalic">A&#46; flavus</span> growth and AFB1 production by <span class="elsevierStyleItalic">A&#46; flavus</span> isolates on sorghum grains&#46;</p></span>"
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        "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">El sorgo&#44; que se consume en T&#250;nez como alimento humano&#44; puede sufrir la colonizaci&#243;n severa de varios hongos toxicog&#233;nicos&#44; con la consiguiente bioacumulaci&#243;n de micotoxinas&#46; Adem&#225;s&#44; el clima de T&#250;nez&#44; caracterizado por las altas temperaturas y humedad&#44; estimula el crecimiento f&#250;ngico y la acumulaci&#243;n de micotoxinas en los productos alimenticios&#46; Este estudio investig&#243; los efectos de la temperatura &#40;15&#44; 25 y 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#41;&#44; la actividad de agua &#40;a<span class="elsevierStyleInf">w</span>&#41; &#40;entre 0&#44;85 y 0&#44;99&#41; y el tiempo de incubaci&#243;n &#40;7&#44; 14&#44; 21 y 28 d&#237;as&#41; sobre el crecimiento y la producci&#243;n de aflatoxina B1 &#40;AFB1&#41; de 3 aislados de <span class="elsevierStyleItalic">Aspergillus flavus</span> &#40;designados como 8&#44; 10 y 14&#41; que se inocularon sobre granos de sorgo&#46; El modelo Baranyi se aplic&#243; para identificar los l&#237;mites del crecimiento y la producci&#243;n de micotoxinas&#46; Las tasas m&#225;ximas de crecimiento para 2 de los aislados se observaron en la combinaci&#243;n 0&#44;99 a<span class="elsevierStyleInf">w</span> y 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; La a<span class="elsevierStyleInf">w</span> m&#237;nima necesaria para el crecimiento del micelio fue de 0&#44;91 a 25<span class="elsevierStyleHsp" style=""></span>&#176;C y 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; A 15<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; solo el aislado 8 creci&#243; a 0&#44;99 a<span class="elsevierStyleInf">w</span>&#44; pero fue incapaz de producir la aflatoxina B1&#46; Es posible evitar la acumulaci&#243;n de aflatoxina B1 en el sorgo almacen&#225;ndolo a baja actividad de agua &#40;&#8804; 0&#44;91 a<span class="elsevierStyleInf">w</span>&#41;&#46; Este es el primer trabajo que ha estudiado el efecto de la actividad del agua y la temperatura sobre el crecimiento de aislados de <span class="elsevierStyleItalic">A&#46; flavus</span> y su producci&#243;n de aflatoxina B1 en granos de sorgo&#46;</p></span>"
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es en pt

¿Es usted profesional sanitario apto para prescribir o dispensar medicamentos?

Are you a health professional able to prescribe or dispense drugs?

Você é um profissional de saúde habilitado a prescrever ou dispensar medicamentos