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Growth inhibition of some Eurotium and Aspergillus species with spice extracts

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Growth inhibition of some Eurotium and Aspergillus species with spice extracts

Abstract

The antifungal activity of spice extracts obtained from caraway seed, garlic and origanum was tested against antifungal activity of Eurotium herbariorum E. amstelodami, Aspergillus flavus and A. sydowii. Caraway seed extract has been proved to possess the highest inhibitory effect on all investigated mould species. The concentrations of caraway extract, sufficient to inhibit the growth completely were: 0,5% for E. herbariorum, E. amstelodami and A. sydowii, and 1% for A. flavus; of garlic, 1% for Eurotium spp. and 2% for A. sydowii, and of origanum, 1% for E. herbariorum and 2% for E. amstelodami. The results of colonies diameter measuring showed that garlic and origanum extracts have no significant suppressing ability on micellar growth of A. flavus, while garlic was more efficient in other test cultures.
Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci., Matica Srpska Novi Sad,
¥ 113, 63—70, 2007
UDC 635.7:632.4
Gordana R. Dimiã
Sunåica D. Kociã-Tanackov
Dragana Karaliã
Tehnološki fakultet, Bulevar Cara Lazara 1, Novi Sad, Serbia
GROWTH INHIBITION OF SOME EUROTIUM AND
ASPERGILLUS SPECIES WITH SPICE EXTRACTS
ABSTRACT: The antifungal activity of spice extracts obtained from caraway seed,
garlic and origanum was tested against antifungal activity of Eurotium herbariorum, E. am-
stelodami, Aspergillus flavus and A. sydowii. Caraway seed extract has been proved to pos-
sess the highest inhibitory effect on all investigated mould species. The concentrations of
caraway extract, sufficient to inhibite the growth completely were: 0,5% for E. herbario-
rum, E. amstelodami and A. sydowii, and 1% for A. flavus; of garlic, 1% for Eurotium spp.
and 2% for A. sydowii, and of origanum, 1% for E. herbariorum and 2% for E. amsteloda-
mi. The results of colonies diameter measuring showed that garlic and origanum extracts
have no significant supressing ability on micellar growth of A. flavus, while garlic was mo-
re efficient in other test cultures.
KEY WORDS: spice extracts, antifungal activity, moulds
INTRODUCTION
Natural antimicrobic agents extracted from plants are very interesting, and
their activity was investigated by many researches. In food production, their
importance is increasing, as being a possible choice for food protection from
microorganisms. In that way, the use of synthetic preservers would decrease
( A r o r a and K a u r, 1999; N i e l s e n and R i o s, 2000; S m i t h - P a l
-
m e r et al., 2001; L o p e z - M a l o et al., 2002; M o r e i r a et al., 2005).
Most preservers are believed to have a bad influence on human health.
Some kinds of spice herbs, besides their use as flavour ingredients of
food, possess antimicrobial properties. Mustard, sage, rosemary, and thyme are
outstanding in their expressive antimicrobial activity. Essential oils, e.g. their
active components (eugenol, carvacrol, thymol, cinnamaldehyde, allyn, limone
-
ne, geraniol, etc.) are the antimicrobial agents of spices (S a v i ã and D a n o n,
1982; M o n t v ille and Matthews, 2005).
Numerous species of moulds cause food deterioration, however, some
species are producers of different toxic substances. Aflatoxins (AF) and steri
-
63
gmatocystine (ST) are toxic secondary metabolites of some Aspergillus and
Eurotium species, which are important contaminants and potential causes of
spoilage of intermediate and low-moisture content food (P i t t and H o c k i n g,
1985; G u y e n o t et al., 2003; D i m i ã et al., 2005).
Antifungal activity of three kinds of spices against toxigenic moulds, E.
herbariorum, E. amstelodami and A. sydowii, potential producers of sterigma
-
tocystine and aflatoxigenic A. flavus, was investigated in this work. These mo
-
ulds are related since aflatoxins and sterigmatocystine have a common precur
-
sor in their biosynthesis, and sterigmatocystine can be converted into aflatoxin
B1 (K e l l e r et al., 1997; D u r a koviã and Durakoviã, 2003).
MATERIALS AND METHODS
Isolates of moulds
Moulds including E. herbariorum, E. amstelodami, A. flavus and A.
sydowii, isolated from different spices, were used as test microorganisms. The
cultures used in this experiment were cultivated on slant Saboraud maltose
agar (SMA) for 7 days, at 25°C.
Spices
Extracts of spice herbs: caraway seeds, garlic, and origanum were sup-
plied by “Etol Celje", Slovenija.
Medium
SMA medium was used to follow the growth inhibition of moulds. The
medium was poured into 250 ml Erlenmayer flasks and autoclaved at 121°C,
for 15 min. After sterilization, 0; 0,07; 0,1; 0,5; 1 and 2% concentrations of
spice extracts were added into flasks.
Antifungal investigations
After the addition of spice extracts, equal portions of SMA medium were
poured into sterile Petri plates. Each concentration was prepared in triplicate.
Inoculation was performed by transfering conidia into the center of agar medi
-
um, as a point. Mould growth inhibition was estimated measuring the diameter
of colonies every day during the incubation at 25°C, for 7 days.
64
RESULTS
Inhibitory effect of three kinds of spice extracts on two Eurotium, and
two Aspergillus species was investigated. Lower concentration of caraway seed
extract (0,07 and 0,1%) affected all four toxigenic moulds, during 7 days of
incubation at 25°C (Table 1). The activity against E. herbariorum was higher
(47 and 76,5%), while the growth inhibition of A. flavus was insignificant (5,1
and 18,6%). Compared to the control, the effect on E. amstelodami and A.
sydowii was similar. Caraway seed extract inhibited completely the growth of
E. herbariourum, E. amstelodami and A. sydowii, in concentration of 0,5%.
The same concentration inhibited partially the growth of A. flavus (88,1%).
Total growth inhibition was achieved with 1% concentration.
Tab. 1 Antifungal activity of caraway extract on the growth of moulds
Conc. (%)
Micelial growth inhibition (%)
E. herbariorum E. amstelodami A. flavus A. sydowii
0.07 47 22.2 5.1 25
0.1 76.5 50 18.6 39.3
0.5 100 100 88.1 100
1 100 100 100 100
2 100 100 100 100
No inhibitory effect of garlic extract (0,07 and 0,1%) (Table 2) on A. fla-
vus was found. On the contrary, 0,07% of garlic extract insignificantly stimu-
lates the growth of this mould. As shown in Table 2, 0,1% at garlic extract
decreased the growth of E. herbariorum by 41,4%, E. amstelodami by 33,3%
and A. sydowii by 10,7%. The concentration of 0,5% was not sufficiently effi-
cient to prevent significantly the growth of the investigated moulds, A. flavus
especially. Garlic extract inhibited completely the growth of both Eurotium
species in concentration of 1%, insignificantly (32,2%) of A. flavus, and
partially (85,7%) of A. sydowii. A. sydowii was completely inhibited at 2% le
-
vel of garlic extract, but A. flavus was not (42,4%).
Tab. 2 Antifungal activity of garlic extract on the growth of moulds
Conc. (%)
Micelial growth inhibition (%)
E. herbariorum E. amstelodami A. flavus A. sydowii
0.07 11.7 27.8 0* 7.1
0.1 41.4 33.3 0 10.7
0.5 52.9 61.1 5.1 42.8
1 100 100 32.2 85.7
2 100 100 42.4 100
* stimulation of micelial growth
Neither Eurotium, nor Aspergillus species showed sensitivity to 0,07%
extract of origanum (Table 3). Also, no inhibition activity against A. sydowii at
low concentrations (0,07 and 0,1%) was found, moreover, 0,07% extract even
65
favoured the micellar growth. Origanum extract inhibited completely inhibited
the growth of E. herbariorum and E. amstelodami at higher concentrations (1
and 2%), and almost totally (96,4%) the growth of A. sydowii at 2% level. No
particular effect was exposed on A. flavus.
Tab. 3 Antifungal activity of origanum extract on the growth of moulds
Conc. (%)
Micelial growth inhibition (%)
E. herbariorum E. amstelodami A. flavus A. sydowii
0.07 0 0 0 0*
0.1 11.8 5.5 1.7 0
0.5 47 33.3 10.2 10.7
1 100 66.7 18.6 46.4
2 100 100 49.1 96.4
* stimulation of micelial growth
The effect of caraway seed extract on germination and growth rate of E.
herbariorum, E. amstelodami, A. flavus and A. sydowii during seven days of
incubation, is presented in Figure 1, having in mind that caraway expressed
the strongest inhibition effect in our experiments.
66
Fig. 1. Inhibition of E. herbariorum, E. amstelodami, A. flavus and A. sydowii by
caraway extract
The decrease of growth rate with increased content of caraway in the agar
medium was more expressed in E. herbariorum, than in other mould species,
both at the beginning and at the end of incubation. The beginning of germina
-
tion, compared to the control, was delayed for three (0,07%) and six days
(0,1%). In all caraway treatments, where E. amstelodami was growing, the rate
of colonies spreading was higher, and the germination was inhibited only at
concentration of 0,1%, in two days. The observation for A. sydowii growth
was similar. At lower levels of caraway extract (0,07 and 0,1%), the growth of
A. flavus was the same as in the control. The diameters of colonies differed
only in 0,1 cm. Although the beginning of the growth was not affected by
0,07 and 0,1% of caraway, difference in growth rate was observed later. This
difference was practically at the same level, till the end of the experiment. In
case of 0,5% concentration, the colonies of A. flavus became visible only the
fifth day after the inoculation of agar plates.
Sporulation of all moulds decreased with the increase of content of ca
-
raway, garlic and origanum extracts. The highest concentrations where growth
of moulds was evident, caused greatest changes in appearance of colonies
compared to the control.
DISCUSSION
These experiments have shown that caraway seed, garlic and origanum
have antifungal activity against the tested toxigenic Eurotium and Aspergillus
species. Caraway in concentrations higher than 0,5%, efficiently prevents the
growth of moulds. Eurotium spp. exhibited higher sensibility. E. herbariorum
was the most sensitive to caraway extract. Garlic extract is also a significant
antifungal agent for the same kinds, however, for A. sydowii, hit was not so
efficient as a caraway seed extract. The tested organisms were less sensitive to
origanum, compared with garlic. Spice extracts had the lowest effect on A. fla-
vus.
Garlic and origanum have some inhibitory effects, however, neither of
them is significantly efficient in growth reduction of A. flavus. Neilsen and
R i o s (2000) investigated some spices and found garlic to be efficient in
growth inhibition of Emericella fibuliger, Penicillium commune, P. corylo
-
philum, P. roqueforti and P. solitum, however, the inhibition of A. flavus
growth was very poor, similar to origanum. In general, the inhibitory potential
of origanum for these species was week, whereas vanilla had no effect at all.
Caraway is the spice which has high inhibitory effect on growth of mo
-
ulds. This effect can be compared to clove (M a l b r o u k and E l - S h a y e b,
1980). This activity can be attributed to the presence of carvon and limonene,
present in caraway seed essential oil in significant quantities (50 to 85%, and
20 to 30%), respectively (S a v i ã and D a n o n, 1982).
It was found that the essential oils of caraway, clove, onion, and garlic
may affect preventively the synthesis of sterigmatocystin and aflatoxin (H i
-
tokoto et al., 1980; H a s a n and M a h m o u d, 1993). The combined ef
-
fect of 16 spices and different water activities (0,80 to 0,90 a
w
)onEurotium
67
spp. (E. amstelodami , E. herbariorum, E. repens and E. rubrum) was tested,
and the results have shown a complete inhibitory effect of lemongrass, clove,
cinammon, bay and thyme at all a
w
values (G u y e n o t et al., 2003). The
inhibitory effect of garlic was stated many times (M e i - C h i n and W e n
-
- S h e n, 1998; Y i n and T s a o, 1999; B e n k e b l i a, 2004).
The essential origanum oil is rich in timol and carvakrol, and this spice is
a very strong growth inhibitor of A. niger (B a r a t t a et al., 1998) and A.
ochraceus, procuder of ochratoxin A (B a s ilico and Basilico, 1999).
Özcan (1998) found that origanum extract, besides thyme and savory (out
of 31 tested ones), inhibits completely the growth A. parasiticus, in 2% con
-
centration during incubation at 30°C, for 10 days.
The spice extracts of caraway seed, garlic and origanum, investigated in
this work, caused morphological changes besides growth inhibition of coloni
-
es, and this points to the possible changes on a cell level. The investigation of
cell ultrastructures of A. niger, after exposure to the effect of thyme essential
oil, showed damages of cell wall, cellular membrane and organellas (R a s s o
-
l i et al., 2006).
CONCLUSION
Having in mind the obtained results, the investigated spice extracts can be
used to limit or prevent the development of harmful moulds in food (preser-
ved, or fresh stored food), as additives, surface prevention, or use in products
packed under modified atmosphere. A number of active components of spices
and their derivates are being further investigated.
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INHIBICIJA RASTA NEKIH EUROTIUM I ASPERGILLUS VRSTA
EKSTRAKTIMA ZAÅINA
Gordana R. Dimiã, Sunåica D. Kociã-Tanackov, Dragana Karaliã
Tehnološki fakultet, Bulevar Cara Lazara 1, Novi Sad, Srbija
Rezime
Ekstrakti zaåina dobijeni iz kima, belog luka i origana bili su ispitani
na antifungalni aktivitet prema Eurotium herbariorum, E. amstelodami, Aspergil
-
lus flavus i A. sydowii. Ekstrakt kima imao je najjaåi inhibitorni efekat i to na
69
sve tri vrste plesni. Koncentracije ekstrakta kima dovoqne da kompletno in
-
hibiraju rast bile su 0,5% za E. herbariorum, E. amstelodami i A. sydowii i1%za
A. flavus; za beli luk 1% za Eurotium spp. i 2% za A. sydowii i za origano 1% za
E. herbariorum i2%zaE. amstelodami. Rezultati merewa dijametra kolonija su
pokazali da ekstrakt belog luka i origana ne supresuju znaåajno micelarni rast
A. flavus, dok je beli luk bio efikasniji prema ostalim test-kulturama.
70
... However, aflatoxin accumulation in the poorly stored maize This is in addition to the expected microbial competition within the microhabitat as evidenced in the low fungal counts for control samples. This fact is further supported by a similar study which showed that garlic, oregano and caraway seeds contained certain compounds in their extracts that inhibited the growth of some Eurotium and Aspergillus species (Dimic et al., 2007). Furthermore, our findings are strengthened by the reports of Kiin-Kabari et al. ...
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The antifungal effects of essential oils from Thymus eriocalyx and Thymus x-porlock were studied with special reference to the mechanism of inhibition of Aspergillus niger growth at ultrastructural level. Minimal inhibitory (MIC), minimal fungicidal (MFC) concentrations, and fungicidal kinetics of the oils were determined. Transmission electron microscopy (TEM) of A. niger exposed to MIC levels of the oils showed irreversible damage to cell wall, cell membrane and cellular organelles. The oils analyzed by GC and GC/MS led to identification of 18 and 19 components in T. eriocalyx and T. x-porlock oils respectively. The results are compared and discussed with the data in literature. It is concluded that the essential oils could be safely used as preservatives.
The effects of black pepper, cinnamon, peppermint, cumin, ginger and clove on growth and aflatoxin formation of Aspergillus flavus were studied in rice powdercorn steep (RC) medium. The effects of the first five spices were judged to be inhibition of aflatoxin formation rather than of mycelial growth. Clove completely inhibited both mycelial growth and aflatoxin formation at a concentration above 0.1%. No aflatoxin was produced when cumin and mint levels of 5% and 10% were used. Black pepper and ginger levels of 10% decreased aflatoxin formation by 100%. Higher concentrations of cinnamon, mint, cumin and ginger stimulated mycelial growth.
Article
Antifungal activity and minimal fungicidal concentration (MFC) of extracts of garlic, bakeri garlic, Chinese leek, Chinese chive, scallion, onion bulb and shallot bulb against Aspergillus niger, A. flavus and A. fumigatus were examined. These Allium plants possessed antifungal activity, with garlic showing the lowest MFC. With the exception of scallion, the inhibitory effect of Allium plants against three Aspergillus species decreased with increasing incubation and heating temperature (P < 0.05). Acetic acid treatments of the extracts increased the inhibitory effect for all plants against three fungi (P < 0.05), and there was no significant difference in this effect among the three pH (2, 4, 6) treatments (P > 0.05) investigated. Acetic acid, at pH = 4, plus heat treatments of the extracts resulted in a greater inhibitory effect for all Allium plants against the three fungi than heat treatment alone (P < 0.05). Treatments of the extracts with NaCl, at concentrations of 0.2 and 0.4 M, did not affect the inhibitory effect of the plant extracts. The combination of acetic acid plus Allium plants was indicated to be an effective way to inhibit fungal growth.
Article
The effect of modified atmosphere packaging (MAP) on the most important spoilage fungi of bread was investigated. Penicillium commune, P. roqueforti, Aspergillus flavus and Endomyces fibuliger were able to grow at oxygen levels down to 0.03%, while the chalk mould E. fibuliger was capable of growing even in the presence of an oxygen absorber. High levels of carbon dioxide retarded growth but not completely. As an alternative to MAP active packaging (AP) using volatile essential oils (EO) and oleoresins (OL) from spices and herbs were tested against a range of fungi commonly found on bread. Concentrations of 1, 10 or 100 microl EO or OL were added to a filter paper placed in the lid of a Petri dish inoculated with one of the test fungi. The Petri dish was sealed hermetically to avoid the exchange of gases. Mustard essential oil showed the strongest effect. Cinnamon, garlic and clove also had high activity, while oregano oleoresin only inhibited growth weakly. Vanilla showed no inhibitory effect towards the tested microorganisms at the applied concentrations. A. flavus was more resistant than the other microorganisms while P. roqueforti was the most sensitive. Mustard essential oil was investigated in greater detail. The minimal inhibitory concentration (MIC) for the active component, allyl isothiocyanate (AITC), was determined for the same species and an additional three moulds and one yeast. MIC values ranged from 1.8 to 3.5 microg/ml gas phase. Results showed that whether AITC was fungistatic or fungicidal depended on its concentration, and the concentration of spores. When the gas phase contained at least 3.5 microg/ml, AITC was fungicidal to all tested fungi. Results of sensory evaluation showed, that hot-dog bread was more sensitive to AITC than rye bread. The minimal recognisable concentration of AITC was 2.4 microg/ml gas phase for rye bread and between 1.8 and 3.5 microg/ml gas phase for hot-dog bread. These findings showed that the required shelf-life of rye bread could be achieved by active packaging with AITC. Active packaging of hot-dog bread, may nevertheless require the additional effect of other preserving factors to avoid off-flavour formation
Article
The effects of selected concentrations of antimicrobials from natural (vanillin, thymol, eugenol, carvacrol or citral) or synthetic (potassium sorbate or sodium benzoate) origin on Aspergillus flavus lag time inoculated in laboratory media formulated at water activity (a(w)) 0.99 and pH 4.5 or 3.5, were evaluated. Time to detect a colony with a diameter > 0.5 mm was determined. Mold response was modeled using the Fermi function. Antimicrobial minimal inhibitory concentration (MIC) was defined as the minimal required inhibiting mold growth for 2 months. Fermi function successfully captured A. flavus dose-response curves to the tested antimicrobials with a highly satisfactory fit. Fermi equation coefficients, Pc and k, were used to compare antimicrobials and assess the effect of pH. Important differences in Pc and k were observed among antimicrobials, being natural antimicrobials less pH dependent than synthetic antimicrobials. A large Pc value represents a small antimicrobial effect on A. flavus lag time; thus, high concentrations are needed to delay growth. A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate (at pH 3.5), and sodium benzoate (at pH 3.5) than to vanillin or citral. MICs varied from 200 ppm of sodium bcnzoate at pH 3.5 to 1800 ppm of citral at both evaluated pHs.
Article
To investigate the volatile fractions of 16 essential oils for activity against the more common fungi causing spoilage of bakery products, Eurotium amstelodami, E. herbariorum, E. repens, E. rubrum, Aspergillus flavus, A. niger and Penicillium corylophilum. The study applied 50 microl of pure essential oils in a sterilized filter paper, were carried out at pH 6 and at different water activity levels (0.80-0.90). First, a wheat flour based agar medium was used, where cinnamon leaf, clove, bay, lemongrass and thyme essential oils where found to totally inhibit all microorganisms tested. These five essential oils were then tested in sponge cake analogues, but the antifungal activity detected was much more limited. Five essential oils showed potential antifungal capacity against all species tested, over a wide range of water availability. Their activity, however, seems to be substrate-dependent. More research is needed to make them work in real bakery products, as in the preliminary study limited effectiveness was found. The potential of the cinnamon leaf, clove, bay, lemongrass and thyme essential oils against species belonging to Eurotium, Aspergillus and Penicillium genus has been demonstrated.
Koncentracije ekstrakta kima dovoqne da kompletno inhibiraju rast bile su 0,5% za E. herbariorum, E. amstelodami i A. sydowii i 1% za A. flavus; za beli luk 1% za Eurotium spp. i 2% za A. sydowii i za origano 1% za E. herbariorum i 2% za E. amstelodami
  • Tri Vrste Plesni
tri vrste plesni. Koncentracije ekstrakta kima dovoqne da kompletno inhibiraju rast bile su 0,5% za E. herbariorum, E. amstelodami i A. sydowii i 1% za A. flavus; za beli luk 1% za Eurotium spp. i 2% za A. sydowii i za origano 1% za E. herbariorum i 2% za E. amstelodami. Rezultati merewa dijametra kolonija su pokazali da ekstrakt belog luka i origana ne supresuju znaåajno micelarni rast A. flavus, dok je beli luk bio efikasniji prema ostalim test-kulturama.