Gordana R. Dimić

Effects of Onion (Allium cepa L.) and Garlic (Allium sativum L.) Essential Oils on the Aspergillus versicolor Growth and Sterigmatocystin Production

In the present study the effects of individual and combined essential oils (EOs) extracted from onion (Allium cepa L.) bulb and garlic (Allium sativum L.) clove on the growth of Aspergillus versicolor and sterigmatocystin (STC) production were investigated. The EOs obtained by hydrodistillation were analyzed by GC/MS. Twenty one compounds were identified in onion EO. The major components were: dimethyl-trisulfide (16.64%), methyl-propyl-trisulfide (14.21%), dietil-1,2,4-tritiolan (3R,5S-, 3S,5S- and 3R,5R- isomers) (13.71%), methyl-(1-propenyl)-disulfide (13.14%), and methyl-(1-propenyl)-trisulfide (13.02%). The major components of garlic EO were diallyl-trisulfide (33.55%), and diallyl-disulfide (28.05%). The mycelial growth and the STC production were recorded after 7, 14, and 21 d of the A. versicolor growth in Yeast extract sucrose (YES) broth containing different EOs concentrations. Compared to the garlic EO, the onion EO showed a stronger inhibitory effect on the A. versicolor mycelial growth and STC production. After a 21-d incubation of fungi 0.05 and 0.11 μg/mL of onion EO and 0.11 μg/mL of garlic EO completely inhibited the A. versicolor mycelial growth and mycotoxins biosynthesis. The combination of EOs of onion (75%) and garlic (25%) had a synergistic effect on growth inhibition of A. versicolor and STC production.

Cultivation of Polyporus squamosus for pectinase production in aqueous two-phase system containing sugar beet extraction waste.

Cultivation of the fungus Polyporus squamosus for pectinase production was studied in a polyethylene glycol/crude dextran aqueous two-phase system, with sugar beet extraction waste as pectin source. Fungal growth was restricted to the bottom phase and the amounts of biomass and exo-pectinase activity produced were superior to in homogeneous cultivation. The partition coefficients of endo-pectinase and exo-pectinase were 4.26 and 2.78, respectively. The top phase yields in the single extraction step were about 90% for both pectinases.

Antifungal activities of basil ( Ocimum basilicum L.) extract on Fusarium species

The basil extract composition was determined by the GC-MS method and 38 different components were identified. The major components of the basil extract were estragol (86.72%), trans-α-bergamotene (2.91%), eucalyptol (2.67%), trans-ocimene (1.04%), linalool (0.72%), methyl-eugenol (0.71%), etc. The antifungal potential of the basil extract was tested against Fusarium oxysporum, F. proliferatum, F. subglutinans , and F. verticillioides isolated from cakes, using the agar plate method. Extract concentrations of 0.35 and 0.70% (v/v) significantly inhibited the growth of F. proliferatum (33.37 and 44.30%, respectively) and F. subglutinans (24.74 and 29.27%, respectively) whereas other investigated Fusarium species exhibited much lower sensitivity. The basil extract completely inhibited the growth of investigated Fusarium spp. at the concentration of 1.50% (v/v). Higher concentrations (0.35 and 0.70% (v/v)) reduced growth of aerial mycelium in all tested species. Strong medium pigmentation in the case of F. proliferatum and F. verticillioides was observed. The microscopic examination of the samples confirmed the presence of hyphae deformations with a frequent occurrence of fragmentations, thickenings and diminished sporulation. In addition to the basic, sensory, role the extract of basil has in the food product, it exerted significant antifungal properties, depending on its concentration. Key words : Basil (Ocimum basilicum L.) extract, components, antifungal activity, Fusarium spp.

Antifungal activity of oregano (Origanum vulgare L.) extract on the growth of Fusarium and Penicillium species isolated from food

The effect of the oregano extract (Origanum vulgare L.) on the growth of Fusarium and Penicillium species isolated from cakes and ready-for-use fresh salads from different kinds of vegetables was investigated. The contents of the active component of extract were identified by GC-MS and they include: carvacrol (34.2%), carvone (18.5%), p-cimene (8.05%), thymol (3.74%). The oregano extract showed the ability to reduce mould growth at all applied concentrations. Stronger inhibitory effect on the growth of Penicillium species, contrary to Fusarium, was determined. At extract concentration of 2.50 mL/100 mL, growth of P. aurantiogriseum, P. glabrum and P. brevicompactum was completely inhibited during 14 days of incubation. At the same concentration, growth of Fusarium proliferatum was inhibited by 81.71%, F. oxysporum by 85.84%, F. verticillioides by 86.50%, P. chrysogenum by 86.2% and F. subglutinans by 88.85%.

Investigation of toxigenic potential of fungal species by the use of simple screening method.

Potential for the biosynthesis of aflatoxin B1 (AFLB1), ochratoxin A (OTA), diacetoxyscirpenol (DAS), T-2 toxin (T2), and zearalenone (ZON) was investigated in different fungal species belonging to the genera: Aspergillus, Fusarium and Penicillium. The majority of investigated isolates originated from cereal grains, crushed oil soybean seed and fodder mixtures. The simple screening method developed by Filtenborg et al. (1983) was applied with few modifications concerning the type of the medium and cultivation temperature. In order to optimise the biosynthetic conditions for different mycotoxins, the following control cultures, known as mycotin producers were used: OTA - A. ochraceus CBS 108.08, DAS - F. semitectum (SL-B i SL-C), T2 - F. sporotrichioides (ITM-391, M-1-1, R-2301) and ZON - F. graminearum (GZ-LES). The fungi were cultivated on the standard medium (YESA - 2% yeast extract, 15% sucrose and 2% agar, pH 6.5), three modifications of the basic medium (YESAZn - the standard medium supplemented with 0.23 mg/l ZnSO4 x 5 H2O; PPSA - the medium in which yeast extract was replaced with peptone-1; PPSAZn - the medium in which yeast extract was replaced with peptone-1 and supplemented with 0.23 mg/l ZnSO4 x 5 H2O), and the potato-dextrose agar (PDA). The earlier biosynthesis of tested mycotoxins was recorded under the following cultivation conditions of fungal species: AFLB1 - after 14 days on PDA at 27±1°C, OTA - after 10 days on YESA and YESAZn at 27±1°C, DAS - after 10 days on PPSA and PPSAZn at 27±1°C, T2 - after 7 days on PPSAZn and PPSA at room temperature (20-24°C), and ZON - after 1 week on YESA and YESAZn at room temperature (21-24°C).

Antifungal activity of the basil (Ocimmum basilicum L.) extract on Penicillium aurantiogriseum, P. glabrum, P. chrysogenum, and P. brevicompactum

This study was aimed at investigating the antifungal potential of basil (Ocimmum basilicum L.) extract against toxin-producing Penicillium spp. (P. aurantiogriseum, P. glabrum, P. chrysogenum, and P. brevicompactum) isolated from food. The basil extract composition was determined by the GC-MS method. The major component identified in the extract was estragole (86.72%). The determination of the antifungal activity of basil extract on Penicillium spp. was performed using the agar plate method. Basil extract reduced the growth of Penicillium spp. at all applied concentration levels (0.16, 0.35, 0.70, and 1.50 mL/100mL) with the colony growth inhibition from 3.6 (for P. glabrum) to 100% (for P. chrysogenum). The highest sensitivity showed P. chrysogenum, where the growth was completely inhibited at the basil extract concentration of 1.50 mL/100mL. The growth of other Penicillium spp. was partially inhibited with the colony growth inhibition of 63.4 % (P. brevicompactum), 67.5% (P. aurantiogriseum), and 71.7% (P. glabrum). Higher concentrations (0.70 and 1.50 mL/100mL) reduced the growth of the aerial mycelium of all tested Penicillium species. In addition, at the same extract concentrations, the examination of microscopic preparation showed the deformation of hyphae with the frequent occurrence of fragmentations and thickenings, occurrence of irregular vesicle, frequently without metulae and phialides, enlarged metulae. The results obtained in this investigation point to the possibility of using basil extract for the antifungal food protection.

Fungi and mycotoxins: Food contaminants

The growth of fungi on food causes physical and chemical changes which, further affect negatively the sensory and nutritive quality of food. Species from genera: Aspergillus, Penicillium, Fusarium, Alternariа, Cladosporium, Mucor, Rhizopus, Eurotium and Emericella are usually found. Some of them are potentially dangerous for humans and animals, due to possible synthesis and excretion of toxic secondary metabolites - mycotoxins into the food. Their toxic syndroms in animals and humans are known as mycotoxicoses. The pathologic changes can be observed in parenhimatic organs, and in bones and central nervous system also. Specific conditions are necessary for mycotoxin producing fungi to synthetize sufficient quantities of these compounds for demonstration of biologic effects. The main biochemical paths in the formation of mycotoxins include the polyketide (aflatoxins, sterigmatocystin, zearalenone, citrinine, patulin), terpenic (trichothecenes), aminoacid (glicotoxins, ergotamines, sporidesmin, malformin C), and carbonic acids path (rubratoxins). Aflatoxins are the most toxigenic metabolites of fungi, produced mostly by Aspergillus flavus and A. parasiticus species. Aflatoxins appear more frequently in food in the tropic and subtropic regions, while the food in Europe is more exposed to also very toxic ochratoxin A producing fungi (A. ochraceus and some Penicillium species). The agricultural products can be contaminated by fungi both before and after the harvest. The primary mycotoxicoses in humans are the result of direct intake of vegetable products contaminated by mycotoxins, while the secondary mycotoxicoses are caused by products of animal origin. The risk of the presence of fungi and mycotoxin in food is increasing, having in mind that some of them are highly thermoresistent, and the temperatures of usual food sterilization is not sufficient for their termination. The paper presents the review of most important mycotoxins, their biologic effects, the condition of their synthesis, occurence in food, permitted tolerant intake, as well as the possibility of their degradation. [Projekat Ministarstva nauke Republike Srbije, br. TR 31017]