Título da Dissertação: Antifungal and antiaflatoxigenic marjoram essential oil activity on barley and its effect on craft beer.

Orientador: Prof. Dr. Miguel Machinski Junior

Data da Defesa: 07/03/2018



INTRODUCTION. Mycotoxins are natural secondary metabolites of filamentous fungi without being biochemically important for fungal development. Among the mycotoxins, aflatoxinsare one of the most concern because of health and economic impact, and frequently contaminates commodities such as cereals, peanuts, milk, dairy products, beer and dried fruits and vegetables.Since cereals are one of the main ingredients of beer, it is necessary to consider the aflatoxin contamination in this beverage, because mycotoxins can be transmitted from  contaminated grains into beer. The reduction of aflatoxins contamination on beer can be achieved through controlling fungal contamination of malt and
barley. Essential oil has been studied as a natural, eco-friendly and safe fungicide. Among the several essential oils used as fungicides, marjoram essential oil (Origanum majorana – MEO) has been proved to be non-toxic, non-irritant, non-sensitizing antimicrobial, antioxidant and antiinflammatory.
AIMS. The objective of this study was to assess the antifungal and antiaflatoxigenic activity of MEO on A. flavus, in vitro and in situ on barley. Considering the strong aroma of MEO, it was also evaluated its effect on the beer’s sensory characteristics.
MATERIAL AND METHODS. Marjoram essential oil (MEO) was purchased from Ferquima Ind. e Com. Ltda (Vargem Grande Paulista, Brazil) and it was characterized by gas chromatography-mass spectrometry (GC–MS) with an automatic injector. A. flavus(AF42) was obtained from Toxicology Laboratory collection (Department of Health Basic Sciences, State University of Maringa, Brazil), an aflatoxin B1 and B2 (AFB1 and AFB2) producer. Determination of minimum inhibitory concentration (MIC) was performed according to the standard method M38-A preconized by the Clinical and Laboratory Standards Institute, with adaptations for macrodilution in broth for filamentous fungi. Mycelial growthwas evaluated measuring the diameter of growing colonies inoculated in PDA containing different concentration of MEO. Through the scanning electron microscopy, it was possible to determine the morphological characteristics of A. flavus by Quanta 250 Scanning Electron Microscope. To determine the wet weight of the mycelia and ergosterol content, fungi was grown in liquid medium Yeast Extract Sucrose and MEO in the same concentrations used for mycelial growth. After the incubation period, the mycelial biomass was weighted and used for the wet weight of mycelium and extraction of ergosterol content. The filtered was used for aflatoxins determination. Ergosterol determination was performed by high-performance liquid chromatography (HPLC) Finnigan Surveyor Plus with a UV/VIS Finnigan Surveyor detection system at a wavelength of 282 nm. Aflatoxin analyses were performed by a FinniganSurveyor Plus HPLC system, coupled to a Finnigan Surveyor fluorescence detection system; excitation and detection wavelengths were 365 nm and 430 nm, respectively. To evaluate the antiaflatoxigenic MEO effect on barley, Barley grains donated by Agrária cooperative (Guarapuava, PR, Brazil) were hydrated until they reached a desirable aw (0.95). Six different treatments were performed: fungal control; negative control; positive control with Derosal Plus®; and 3 treatments with MEO at 1.56, 3.12 and 12.5 μL/mL. Each treatment had 200g of rehydrated barley grain, and all of them, except negative control, were pulverized with spore solution. From each treatment, 50g of barley was taken out to extract aflatoxin, and aflatoxin analyses were carried out by a Finnigan Surveyor Plus HPLC system, coupled to a Finnigan Surveyor fluorescence detection system; excitation and detection wavelengths were 365 nm and 430 nm, respectively. In order to evaluate the effect of MEO on beer, it was brewed two Saison Ale, 5% alcohol by volume, using the same process, bitterness hops and yeast (Fermentis US-05). One of them had the malt (a mix of Pilsen and Viena malt 3:1 w/w) pulverized with 3.12 μL/mL MEO solution.The other one, without MEO solution, was used as a
control. After that, a triangular test was taken with 50 subjects to detect if the presence of MEO interferes in the overall sensory attributes.
RESULTS AND DISCUSSION. The main components of MEO are Terpinen-4- ol (22.02%) and Terpineol, cis-β (20.07%), showing a good antifungal activity with MIC at 3.12. There was a significant growth inhibition at 1.56, 3.12 and 6.25 μL/mL of MEO with the percentages of 24.85, 91.04 and 99.26%, respectively. Concentrations above 12.5 μL/mL of MEO and nystatin inhibited 100% growth of A. flavus. However, there was no inhibition at concentrations below 0.78 μL/mL of MEO. Regarding the morphological characteristics, the morphology structure changed according to the concentration of essential oil: the abnormality increased on high concentrations. The principal antifungal mechanism of EOs is disruption of ergosterol production. Therefore, the results obtained in this study suggest that MEO acted on the cell membrane, showing a dose-dependent decrease in ergosterol and mycelial wet weight on increasing concentration of EO. However, the dose-dependent relation for both is up to the concentration 0.78 μL/mL of MEO. A possible reason for the increase of ergosterol content and mycelial weight is that at low fungicide concentrations, the fungicide creates some stress condition and the fungus acts defensively as a response. The results showed that MEO inhibited aflatoxins production. A
complete inhibition of AFB1 and AFB2 were achieved with treatments higher than 3.12 μL/mL. When MEO is applied in barley grains, there was a significant reduction of aflatoxin in all MEO concentration, but the concentration of 1.56 μL/mL was the most efficient. The main limitation using essential oils on food is due to the strong flavor they impart to foods.Our sensory evaluation showed that there was no significant difference (p<0.05) between malted barley treated with MEO and control
(without essential oil).
CONCLUSIONS. The present study demonstrated that Origanum majorana essential oil presents significant antifungal and antiaflatoxigenic activity against A. flavus. Furthermore, MEO can be used as a natural fungicide for barley used for brewing, because MEO did not change beer sensory attributes.
Key words: antifungal; aflatoxins; marjoram essential oil; A. flavus; barley; craft beer.

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