In Vitro Activity of Neem (Azadirachta indica) Oil on Growth and Ochratoxin A Production by
5. Materials and Methods 1. Fungal Strains
SixAspergillus carbonariusstrains were evaluated as follows: two reference strains (FRR5690 and A2034) from the CSIRO Collection Centre, Australia and fourA. carbonariusstrains (RCG1, RCG2, RCG3 and RCG4) isolated from dry grapes in Argentina [48]; all of them were OTA producers on yeast extract sucrose (YES, HiMedia Laboratories Pvt. Ltd., Mumbai, India) medium (2% yeast extract, 15% sucrose).
5.2. Culture Medium
Commercial neem oil (Base Fértil Agrícola, Cravinhos, SP, Brazil) was used in this study. According to the manufacturer’s certificate of analysis, neem oil was extracted from seeds and contained 0.12%
p/p of azadirachtin (=1200 ppm). Neem oil was added to the Czapek yeast extract agar (CYA, HiMedia Laboratories Pvt. Ltd., Mumbai, India) at final concentrations of 0.1%, 0.3%, 0.5% or 1.0% (v/v) at 45◦C.
Plates containing CYA media without neem oil were used as control.
5.3. Inoculation and Incubation Conditions
Spore suspensions of the sixA. carbonariusstrains were obtained by scraping the surface of a 7-day-old colony cultured in 2% malt extract agar (MEA, HiMedia Laboratories Pvt. Ltd., Mumbai, India) and transferring the conidia to a tube containing 10 mL of sterile distilled water supplemented with 0.1% Tween 20. The solution was homogenized and read in a spectrophotometer (530 nm) to obtain a transmittance between 80% and 82%, which corresponds to 1–5×106colony forming units per milliliter (CFU/mL). Then the Petri plates were needle-inoculated centrally with 10μL of the spore suspension. The plates were incubated at 25◦C±2 for a maximum of two weeks.
5.4. Growth Assessment
Two perpendicular diameters of the growing colonies were measured daily until the colony reached the edge of the plate or for a maximum of two weeks. The percentage inhibition of diameter growth (PIDG) values were determined according to the equation as below:
PIDG(%) = Diameter of sample −Diameter of control Diameter of control × 100
Growth rate (mm day−1) was calculated by linear regression of colony diameter against time for each strain at each set of conditions tested, and the time at which the line intercepted the x-axis was used to calculate the lag phase (h) in relation to isolate and essential oil. In all cases, the experiments were carried out with three replicates per treatment. The growth of fungal cultures containing different concentrations of neem oil was compared with that of the control culture that was grown with no EO.
5.5. Ochratoxin A Extraction from Culture
Ochratoxin A production was analyzed after 2, 7 and 10 days of incubation. The methodology proposed by Bragulat, Abarca and Cabañes [49] with some modifications was used. On each sampling occasion, three agar plugs were removed from different points of the colony and extracted with 1 mL of methanol. The mixture was centrifuged at 14,000 rpm for 10 min. The solutions were filtered (syringe filters, 17 mm, 0.45μm, nylon membranes), evaporated to dryness, and re-dissolved in 200μL of mobile phase (acetonitrile:water:acetic acid, 57:41:2) and the extracts injected into the high-performance liquid chromatography system.
5.6. OTA Detection and Quantification
The OTA production was detected and quantified by reversed phase in a high performance liquid chromatography system Hewlett Packard Serie 1100 (HP/Agilent, Santa Clara, CA, USA) with fluorescence detection (λexc330 nm;λem460 nm) using a C18column (Supelcosil™LC-ABZ, 150×4.6 mm, 5μm particle size), connected to a precolumn (Supelguard™LC-ABZ, 20×4.6 mm, 5μm particle size). The mobile phase was pumped at 1.0 mL/min. The injection volume was 100μL and the retention time was around 4±1 min. The detection limit of the analyses was 1 ng/g [50].
5.7. Statistical Analyses
Statistical analyses were conducted using PROC GLM in SAS program (SAS Institute Inc., Cary, NC, USA). The differences between growth inhibition percentage, lag phase and OTA production at different concentration levels of neem oil by sixAspergillus carbonariusstrains at 2, 7 and 10 days were analyzed statistically by analyses of variance (ANOVA). The statistical models used in ANOVA considered the effects of the dependent variables and “strains” as a covariate within the different concentration levels of neem oil. The independence of the covariate was formally checked. Means were compared by Fisher’s LSD test to determine the influence of the neem oil on the ecophysiology of the strains assayed [51]. Orthogonal polynomial contrasts were used to determine linear, quadratic and cubic responses to neem oil. The OTA production data contained some results of “not detected” (ND).
That is, the OTA concentration was not detected above the detection limit (DL) of the used method.
The actual concentration represented by ND is some value below the DL, however, the analytical method cannot determine whether the ND is truly zero or some unquantifiable value between zero and the DL. For this situation we used the substitution method, replacing the ND with the DL value (1 ng/g). In the cases of “not growth” (NG) results, we performed the substitution with zero.
Author Contributions:Investigation, M.P.R., A.L.A. andÁ.A.d.O.; formal analysis, K.M.K.; project administration, C.A.d.R.R. and K.M.K.; supervision, L.R.C. and M.I.d.A.; writing—original draft, M.P.R. and A.L.A.;
writing—review and editing, M.P.R., A.L.A.,Á.A.d.O., L.A.S., G.L.B., L.A.M.K. and K.M.K.
Funding: The authors would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais (PRPq-UFMG), Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto (SECYT-UNRC) and Fondo para la Investigación Científica y Tecnológica (FONCYT-PICTO).
Conflicts of Interest:The authors declare no conflicts of interest.
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