Uticaj ekstrakata žačina na rast plesni i biosintezu mikotoksina

Abstract

Cilj istraživanja doktorske disertacije bio je da se ispita pojedinačni i sinergistički uticaj ekstrakata kima (Carum carvi L.), bosiljka (Ocimum basilicum L.), origana (Origanum vulgare L.) i etarskih ulja crnog (Allium cepa L. kultivar Kupusinski jabučar) i belog luka (Allium sativum L. kultivar Bosut) na rast plesni izolovanih iz prehrambenih proizvoda, kao i njihov uticaj na biosintezu mikotoksina. Ukupan broj plesni u uzorcima salata od povrća spremnih za konzumiranje kretao se od 10,0 do 5,5´102 cfu/g, u uzorcima poslastičarskih proizvoda do 6,1´102 cfu/g i u proizvodima od mesa do 60,0 cfu/g. Najveći broj plesni izolovan je na DG18 podlozi (1,53´102 cfu/g), a najmanji na MY50G (42,0 cfu/g). U ukupnoj mikopopulaciji svih ispitivanih uzoraka dominirale su vrste rodova Penicillium (39,07%), Cladosporium (23,40%) i Aspergillus (20,42%). Vrste iz rodova Alternaria, Fusarium i Eurotium su bile zastupljene sa 5,85%, 4,97% i 2,76%. Dominantne vrste u ukupnoj mikopopulaciji bile su C. cladosporioides (21,63%), A. niger (16,0%) i P. aurantiogriseum (11,81%). Dominirali su potencijalni producenti ohratoksina A (31,89%), proizvoñači fumonizina (4,74%), moniliformina (1,43%) i sterigmatocistina (1,54%). Izolati A. versicolor su biosintetisali sterigmatocistin u koncentracijama od 56,3 i 109,2 ng/mL. Ostale potencijalne toksin-produkujuće vrste nisu pokazale sposobnost produkcije mikotoksina. Mikotoksikološkim ispitivanjem hrane u dva uzorka salata spremnih za konzumiranje (kupus beli rezani i FIT salata) utvrñen je sadržaj sterigmatocistina u koncentracijama od 3,5 i 5,5 mg/kg. Kao glavna komponenta u ekstraktu kima odreñen je karvon (43,98%), u ekstraktu bosiljka estragol (metil kavikol) (86,72%), a u ekstraktu origana karvakrol (34,20%) i karvon (18,05%). Najveći deo etarskog ulja crnog luka činili su: dimetil-trisulfid, metil-propil-trisulfid, dimetiltetrasulfid, dietil-1,2,4-tritiolan, metil-(1-propenil)-trisulfid, metil-(1-propenil)-disulfid. Dialildisulfid, dialil-trisulfid, metil-alil-trisulfid i metil-alil-disulfid su glavne komponente koje su odreñene u etarskom ulju belog luka. Koncentracija od 0,35 mL/100 mL ekstrakta kima je bila fungicidna (MFC) prema C. cladosporioides, dok je 0,70 mL/100 mL potpuno inhibirala rast A. carbonarius, A. wentii, E. nidulans, Eurotium spp., C. cladosporioides, P. glabrum, P. brevicompactum, F. subglutinans i F. verticillioides. Na rast P. chrysogenum i P. aurantiogriseum ista koncentracija bila je inhibitorna (MIC). Najslabije delovanje ovaj ekstrakt ispoljio prema A. niger, A. versicolor, F. oxysporum i F. proliferatum. Primena ekstrakta bosiljka u koncentraciji od 0,70 mL/100 mL pokazala je fungicidno delovanje na C. cladosporioides. Koncentracija od 1,50 mL/100 mL potpuno je inhibirala rast A. wentii, A. versicolor, E. nidulans, E. herbariorum, E. chevalieri, E. rubrum, P. chrysogenum i Fusarim spp. Ekstrakt bosiljka je najslabije delovao prema A. niger, A. carbonarius, P. aurantiogriseum, E. amstelodami, P. glabrum i P. brevicompactum. Ekstrakt origana je pokazao najslabije ihibitorno delovanje na rast ispitivanih plesni. Primena ekstrakta u koncentraciji od 1,50 mL/100 mL je bila fungicidna prema E. rubrum. Koncentracija od 2,50 mL/100 mL je pokazala fungicidno delovanje na E. rubrum, E. herbariorum, A. wentii, C. cladosporioides i P. aurantiogriseum, a inhibitorno prema E. nidulans, E. chevalieri, E. amstelodami, P. glabrum i P. brevicompactum. Ovaj ekstrakt je najslabije delovao na A. niger, A. carbonarius, F. proliferatum, F. subglutinans i P. chrysogenum. Etarsko ulje crnog luka pokazalo je signifikantno jače antifungalno delovanje na ispitivane plesni u odnosu na etarsko ulje belog luka. Koncentracija od 14,0 mL/100 mL ulja belog luka fungicidno je delovala prema E. rubrum, E. chevalieri i C. cladosporioides, dok je ulje crnog luka na ovoj koncentraciji pokazalo fungicidni efekat i na E. herbariorum i E. amstelodami. Za ostale plesni fungicidna koncentracija iznosila je 28,0 mL/100 mL, osim za A. niger i P. aurantiogriseum. Neke od ispitivanih smeša bosiljka i kima, bosiljka i origana, origana i kima i etarskih ulja lukova pokazale su sinergističko delovanje na inhibiciju rasta A. wentii, E. herbariorum, F. verticilllioides i P. aurantiogriseum sa FICindex od 0,63 do 0,97. Začinski ekstrakti i etarska ulja lukova su pored ograničavanja rasta kolonija plesni uzrokovali i promene u makro i mikromorfologiji. Potpuna inhibicija biosinteze sterigmatocistina i rasta A. versicolor postignuta je pri koncentraciji od 0,20 mL/100 mL ekstrakta kima i origana u periodu od 21 dana. Na ovoj koncentraciji ekstrakt bosiljka je inhibirao biosintezu sterigmatocistina za 88,73% i rast plesni za 52,56%. Smeša koja je sadržavala 75% ekstrakta kima i 25% ekstrakta bosiljka potpuno je inhibirala rast plesni i biosintezu sterigmatocistina u YES bujonu tokom 21 dana inkubiranja. Pojedinačne koncentracije etarskih ulja crnog i belog luka od 5,0 i 10,0 mL/100 mL i u smeši sa 1,50 mL/100 mL etarskog ulja crnog luka i 0,50 mL/100 mL etarskog ulja belog luka bile su potrebne za potpunu inhibiciju rasta A. versicolor i biosintezu sterigmatocistina. Dodatak smeše ekstrakata kima i bosiljka (0,35 mL/100 mL ekstrakta kima + 0,70 mL/100 mL ekstrakta bosiljka) u svež kupus rezanac uticao je na smanjenje inicijalne kontaminacije plesnima za 93,9%, uz pojavu intenzivnijeg, ali prihvatljivog mirisa i neznatne promene boje. Definisani matematički model za komparaciju uticaja ekstrakata i etarskih ulja na rast plesni može se primenjivati u formiranju matrica inhibicije i optimizaciji vremena i koncentracije antifungalnih agenasa. Dobijena saznanja o antifungalnom delovanju ekstrakata začina i etarskih ulja lukova mogu biti značajna u poboljšanju antifungalne zaštite namirnica, smanjenju biosinteze mikotoksina i ukupnim smanjenju šteta izazvanih delovanjem plesni.

The aim of this PhD thesis was to study the individual and synergistic effects of extracts of caraway (Carum carvi L.), basil (Ocimum basilicum L.), oregano (Origanum vulgare L.) and the essential oils of onion (Allium cepa L. cultivar Kupusinski jabučar) and garlic (Allium sativum L. cultivar Bosut) on the growth of moulds isolated from food products. The study also focused on the impact of extracts and the essential oils on mycotoxins biosynthesis. The total number of moulds detected in samples of vegetable salads “ready for use” ranged from 10.0 to 5.5´102 cfu/g. In cake and pastries, as well as, meat samples, the number reached 6.1´102 cfu/g and 60.0 cfu/g, respectively. The highest number of mould colonies was isolated in DG18 medium (1.53 ´ 102cfu/g) and the lowest in MY50G medium (42.0 cfu/g). The species of the genera Penicillium (39.07%), Cladosporium (23.40%) and Aspergillus (20.42%) prevailed in the entire mycopopulation of all tested samples. Species of the genera Alternaria, Fusarium and Eurotium were represented with 5.85%, 4.97% and 2.76%, respectively, while C. cladosporioides (21.63%), A. niger (16.0%) and P. aurantiogriseum (11.81%) were the most dominant species in the entire mycopopulation. Potential producers of ochratoxin A (31.89%) accounted for the largest share of the isolated mycopopulation. The share of producers of fumonisin, moniliformin and sterigmatocystin amounted to 4.74%, 1.43% and 1.54%, respectively. Sterigmatocystin was biosynthesised in the concentration of 56.3 ng/mL and 109.2 ng/mL by both isolates of A. versicolor, while other potential toxin producers did not show the ability of mycotoxin production. Mycotoxicological investigation showed the sterigmatocystin content in two samples of vegetable salads "ready for use" (shredded white cabbage and FIT salad - carrot, lettuce and red chicory) in concentrations of 3.5 mg/kg and 5.5 mg/kg, respectively. The major component in the extract of caraway was carvon with a share of 43.98%. The basil extract contained estragol (methyl cavicol) in the highest percentage (86.72%), while carvacrol (34.20%) and carvon (18.05%) were major components of the oregano extract. As for the essential oil of onion, dimethyl trisulphide, methyl propyl trisulphide, dimethyl tetrasulfid, diethyl-1, 2, 4-tritiolan, methyl-(1-propenyl)-trisulphide, and methyl-(1-propenyl) – disulfide constituted the largest share. The major components isolated in garlic essential oil were diallyl disulfide, diallyl-trisulphide, allyl methyl trisulphide and allyl methyl disulfide. The concentration of the caraway extract of 0.35 mL/100mL exhibited fungicidal effect (MFC) on C. cladosporioides, while the concentration of 0.70 mL/100mL completely inhibited the growth of A. carbonarius, A. wentii, E. nidulans, Eurotium spp., C. cladosporioides, P. glabrum, P. brevicopmactum, F. subglutinans and F. verticillioides. The same concentration showed the inhibitory effect (MIC) on the growth of P. chrysogenum and P. aurantiogriseum. The poorest effect of the caraway extract was expressed on the growth of A. niger, A. versicolor, F. oxysporum and F. proliferatum. The basil extract application in the concentration of 0.70 mL/100mL showed fungicidal effects (MFC) on the growth of C. cladosporioides. The concentration of 1.50 mL/100mL completely inhibited (MFC) the growth of A. wentii, A. versicolor, E. nidulans, E. herbariorum, E. chevalierii, E. rubrum, P. chrysogenum and Fusarim spp. The poorest effect of the basil extract was exhibited on A. niger, A. carbonarius, P. aurantiogriseum, E. amstelodami, P. glabrum and P. brevicompactum. The oregano extract showed the weakest growth inhibition influence on all of the tested moulds. The application of this extract in the concentration of 1.50 mL/100mL was fungicidal (MFC) to E. rubrum. The concentration of 2.50 mL/100mL showed fungicidal effects (MFC) on the growth of E. rubrum, E. herbariorum, A. wentii, C. cladosporioides and P. aurantiogriseum and inhibitory effects (MIC) on E. nidulans, E. chevalieri, E. amstelodami, P. glabrum and P. brevicompactum. The weakest effect of this extract was expressed on the growth of A. niger, A. carbonarius, F. proliferatum, F. subglutinans and P. chrysogenum. Onion essential oil showed a significantly stronger antifungal effect on the tested moulds in comparison to garlic essential oil. While the concentration of 14.0 mL/100mL of garlic oil had a fungicidal effect on E. rubrum, E. chevalieri and C. cladosporioides, the same concentration of onion oil was also fungicidal to E. herbariorum and E. amstelodami. With an exception of A. niger and P. aurantiogriseum, the concentration that showed a fungicidal effect on the remaining moulds equalled 28.0 mL/100mL. Some of the tested mixtures of basil with caraway, basil with oregano, oregano with caraway, and essential oils of onion and garlic, showed a synergistic effect on the growth inhibition of A. wentii, E. herbariorum, F. verticilllioides and P. aurantiogriseum with the FIC index ranging from 0.63 to 0.97. Apart from the inhibitory effect on the mould colony growth, the spices extracts and the essential oils of onion and garlic also caused changes in the macro- and micro- morphology of the moulds. Complete inhibition of the growth of A. versicolor and sterigmatocystin biosynthesis was achieved at a concentration of 0.20 mL/100mL of the extract of caraway and oregano in the period of 21 days. At this concentration the basil extract delayed the sterigmatocystin biosynthesis by 88.73% while the mould growth was inhibited by 52.56%. Mixtures containing 75% of the caraway extract and 25% of the basil extract completely inhibited the mould growth and sterigmatocystin biosynthesis in YES broth during 21 days of incubation. The concentrations of 5.0 m L/100mL (onion essential oil) and 10.0 m L/100mL (garlic essential oil) applied in a mixture containing 1.50 mL/100mL of onion and 0.50 mL/100mL of garlic essential oil were necessary for a complete inhibition of the growth of A. versicolor and sterigmatocystin biosynthesis. The addition of the mixture of caraway and basil extracts (0.35 mL/100mL of caraway + 0.70 mL/100mL of basil) to fresh shredded cabbage influenced the reduction of initial mould contamination by 93.9%. This was accompanied by the occurrence of acceptable more intense flavour and slight discoloration. The defined mathematical model for comparing the effects of extracts and essential oils on the growth of moulds can be applied in establishing inhibition matrices and optimisation of the time and the concentration of antifungal agents. The obtained results on the antifungal effects of the spices extracts and onion and garlic essential oils can be beneficial for improving the antifungal protection of food and reducing the mycotoxin biosynthesis as well as the overall damage caused by the action of moulds.