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Saudi Journal of Biological Sciences (2011) 18, 387394

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Xerophilic aatoxigenic black tea fungi

and their inhibition by Elettaria cardamomum
and Syzygium aromaticum extracts
Saleh Al-Sohaibani a, K. Murugan
a
b
c

a,*

, G. Lakshimi b, K. Anandraj

b,c

Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
P.G. and Research Department of Microbiology, K.S.R. College of Arts and Science, Tiruchengodu 637 209, Tamilnadu, India
Shanmuga Industries Arts and Science College, Tiruvannamalai 606 601, Tamilnadu, India

Received 22 May 2011; revised 26 June 2011; accepted 27 June 2011

Available online 2 July 2011

KEYWORDS
Aatoxin;
Black tea;
Elettaria cardamomum;
Syzygium aromaticum;
Xerophile

Abstract Black tea is consumed worldwide and is believed to play a role in cancer prevention.
Xerophilic aatoxigenic fungi are highly hazardous contaminants of tea since they are associated
with tea quality impairment and human health risk. The present study reports isolation of such
xerophilic and aatoxigenic fungi associated with marketed tea. Twenty different tea samples
collected from the local markets of Tamilnadu, India were investigated for fungal contamination.
The results indicated contamination by 0.38% Aspergillus avus. Other common contaminant fungi
including Penicillium spp. (0.30%), Pacelomyces spp. (0.14%), and Mucor spp. (0.19%) were also
isolated. Amongst the fungi isolated Aspergillus niger ML01 and A. avus ML02 were found to
be xerophilic aatoxigenic mycoora. Phylogenetic analysis based on 28S rRNA revealed their close
ancestry. The chloroform and acetone extracts of spices Elettaria cardamomum and Syzygium aromaticum exhibited antifungal inhibitory activity on growth and toxin elaboration of both these xerophilic tea contaminants A. niger ML01 and A. avus ML02. The results advocate the use of these
spices plant or their extracts as novel antimicrobials which may add preservation and avour in
marketed tea.
2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +966 146 75822; fax: +966 145 75833.
E-mail address: murutan@gmail.com (K. Murugan).
1319-562X 2011 King Saud University. Production and hosting by
Elsevier B.V. All rights reserved.
Peer review under responsibility of King Saud University.
doi:10.1016/j.sjbs.2011.06.005

Production and hosting by Elsevier

1. Introduction
People become aware of possible health benets of natural
products. Tea and herbal infusions are hot drinks which are
consumed as daily drinks or for medicinal purposes (Monbaliu
et al., 2010). Tea, the worldwide tonic is one of the most sought
after beverages and its world production reached over 4.73
million tonnes in the year 2008 (FAO, 2010). It has been studied extensively for its health benets, including cancer prevention. Epidemiological studies, however, have not yielded
conclusive results on the cancer-preventive effect of tea

Author's personal copy

388
consumption in humans, possibly owing to different confounding factors (Yang et al., 2009).
Very little information is available on the mycotoxin associated mycoora of tea and the associated quality problems.
Black tea from the dried leaves of the tea plant Camellia
sinensis, processing involves a series of steps before the nal
product is ready. It involves withering, rolling, fermentation
and ring. Fungi have been isolated from all stages of tea processing and manufacturing and recontamination of the nal
product after ring may also occur during sorting and packaging (Elshae et al., 1999). Microbiological contamination of
tea during processing and manufacturing may facilitate their
contamination with mycotoxins. Various studies on black
tea, herbal tea and puerh tea already reported the presence
of Aspergillus, Penicillium, Paecilomyces, Cladosporium, Alternaria, Mucor, Fusarium, Rhizopus, Absidia and Trichoderma.
Among them Aspergillus, Penicillium, Fusarium and Alternaria
species are known as toxigenic producing mycotoxins (Monbaliu et al., 2010). It was observed that tea gets affected by mycotoxigenic and other xerotolerant organisms mostly during the
storage period (Rezacova and Kubatova, 2005). A cup of tea
prepared from contaminated tea leaves may contain microbial
metabolites besides leaf extract some of which might constitute
health hazards to the millions of people who enjoy drinking tea
(Elshae et al., 1999) and hence their contamination deteriorates the tea quality.
This best known fungal secondary metabolite affecting
food crops are aatoxins and Aspergillus avus, Aspergillus
niger and Aspergillus parasiticus are known to produce them.
Aatoxins are toxigenic, carcinogenic, mutagenic and teratogenic in various animal species. Their high level exposure leads
to an acute necrosis, cirrhosis, and carcinoma of the liver
exhibited by haemorrhage, acute liver damage, oedema, alteration in digestion and absorption and/or metabolism of nutrients (Groopman et al., 1999). Aatoxigenic fungal spoilage of
tea is a big drawback to the tea marketability as well as a
threat to the life of consumers (Rezacova and Kubatova,
2005). Restrictions imposed by the food industry and regulatory agencies have led to renewed interest in searching for
alternative food additives, as natural antimicrobial compounds, particularly those derived from plants. A large portion
of traditional medicinal plants of India are found exploited in
food industries since their puried products exhibit antibacterial and antifungal activity besides avour enhancement. One
amongst them is spices which are widely used in combination
with foods. The antimicrobial activities of spices and their
essential oils, their preservative action are well recognized
(Guynot et al., 2003). These spices are used in food preparation throughout the world for the ultimate reason to help
cleanse foods of pathogens and thereby contribute to the
health, longevity and reproductive success of people who nd
their avours enjoyable (Billing and Sherman, 1998). They are
added to tea due to their unique avour.
Syzygium aromaticum (L.) Merr. & Perry. (Family
Myrtaceae), commonly known as clove, is a well known food
avour for exotic food preparations and a popular remedy for
dental, respiratory disorders, headache and sore throat in
traditional medicines of Australia, and Asian countries
(Mishra and Singh, 2008). Eugenol, a main aroma constituent
of its buds was reported to have antifungal activity (Martini
et al., 1996). Because of its antiseptic and antibiotic properties,
clove is frequently used to treat toothache and as an ingredient

S. Al-Sohaibani et al.
in popular toothpastes and mouth fresheners in India (Banerjee et al., 2006). It is also believed to be a stimulant against
digestive disorders and diarrhoea (Chaieb et al., 2007). Recently its therapeutic potential against re-emerging infectious disease giardiasis has also been proved (Machado
et al., 2011).
Elettaria cardamomum (Family Zingiberaceae), the cardamom popularly known as Queen of Spices is widely used for
culinary purposes and traditionally for treating various gastrointestinal, cardiovascular and neuronal disorders. Its powdered seed is frequently prescribed in the treatment of
gastrointestinal disorders and is used as stomachic, resolvent,
retentive, digestive, antiemetic and carminative. It is regularly
used in the treatment of constipation, colic, diarrhoea, dyspepsia, vomiting, headache, epilepsy, cardiovascular diseases
(Khan and Rahman, 1992; Duke et al., 2002), acid peptic disorders, gastritis, ulcer (Jamal et al., 2006). As a spice, cardamom is used in cuisine for curry, coffee, cakes, bread, and
avouring sweet dishes and drinks. They are also used as a avouring component in alcoholic and non-alcoholic beverages,
frozen desserts, candies, baked goods, puddings, condiments,
relishes, gravies, meat, and meat products (El Malti et al.,
2007). They are known as sources of anti-microbial agents acting on dental caries and periodontal disease associated oral
bacteria (Cai and Wu, 1996), on Gram positive and Gram negative bacteria (El Malti et al., 2007).
Due to the harmful effect of aatoxins on human and animal health and their consequence in international food trade,
the aatoxigenic fungal contamination of food has received
worldwide attention. Many food additives, preservatives and
chemicals are found to have effective inhibitory activity
(Bluma and Etcheverry, 2008), but they failed to satisfy the
consumers. Attempts made to harness the natural antimicrobials for food preservation receives increasing attention nowadays due to consumer awareness and a growing concern of
microbial resistance toward conventional preservatives (Moreira et al., 2005). Hence now the challenge is to isolate, stabilize
and incorporate natural antimicrobials into foods without adversely affecting sensory, nutritional and safety characteristics
(Beuchat and Montville, 1989).
Marketed tea often passes through quality assurance tests,
further contamination and growth most likely to occur during
storage after all regulatory conditions have been satised.
Though this contamination of tea is recognized by scientists,
it does not appear high on the priority list for regulatory
authorities in the same way as for coffee, cereals, nuts and a
host of other food products. Further the threats associated
with storage fungi, mostly xerophilic ones have not been well
established in black tea. In this context the present study deals
with determination of the tea aatoxigenic fungal contamination, molecular characterisation and evaluates the GRAS (generally recognised as safe) spices activity on their reduction.

2. Materials and methods

2.1. Sample collection and quality assessment
Twenty different popular marketed brands were chosen and
three pockets of each brand having different production and
expiry dates were collected by random sampling, transferred

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Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum
to Microbiological laboratory, KSR College, Tiruchengode
and were stored at room temperature till further analysis.
The total ash, water soluble ash, alkalinity of water soluble
ash, acid insoluble ash, water extract, and crude bre content
of all samples previously oven-dried at 103 2 C were determined (AOAC, 2000) according to the Prevention of Food
Adulteration Act (PFA, 2006) operative in India. The heavy
metal content of the samples was also determined by atomic
absorption spectrophotometer as described (Seenivasan
et al., 2008).
2.2. Determination of fungal contamination
Aatoxigenic fungal contamination of different brands of marketed black tea samples and their ability to produce toxin was
determined by collecting samples from retail markets in and
around Erode, Tamilnadu, India. Direct inoculation method
was used for the qualitative determination of fungi. 0.5 g from
each sample was taken and equally placed onto ve Petri
dishes containing streptomycin incorporated corn meal agar
(HiMedia, Mumbai, India). The sample inoculated Petri dishes
were incubated at room temperature (24 2 C) and examined daily for seven days. Fungi developed on or around the
tea fragments were counted and recorded. The percentages
of contamination was calculated by dividing the number of
contaminated tea fragments by the total number of tea fragments for each Petri dish multiplied by 100 (Elshae et al.,
1999). The fungal isolates were presumptively identied as described elsewhere (Alexopoules et al., 1996)

389

10 ml liquid nitrogen. From this the DNA was extracted and

puried as described (Cappa and Cocconcelli, 2001).

2.5. PCR amplication and sequencing of 18S rRNA gene

The extracted DNA of the fungal isolates was subjected to
amplication of gene coding for the ribosomal subunit (28S
rRNA). The primers used for amplication were designed
using Gene sher primer designing tool based on 28S rRNA
sequences deposited GenBank Sequence Database of the
National Centre for Biotechnology Information (http://www.
ncbi.nlm.nih.gov) of A. avus and A. niger. The A. avus primers used were forward 5-TCAGTAACGGCGAGTGA-3 and
reverse 3-CATTGCACCCCTGGCTA-5. The primers forward
5-AACCGGGATTGCCTCA-3 and reverse 3-CATTGCACTCCCGGCTA-5 were used for A. niger amplication. Biorad
iCycler iQ real time PCR system, Silicon valley, California was
used for amplication. The reaction was run for 35 cycles of
denaturation at 94 C for 30 s, annealing at 60 C for 30 s,
and extension at 72 C for 2 min. An initial denaturation at
94 C for 30 s and a nal extension at 72 C for 3 min were also
carried out. The amplication products were veried by electrophoresis in 1.2% w/v agarose gel and viewed under UV illumination. The sequencing of obtained 28S rDNA was carried
at Rajiv Gandhi Centre for Biotechnology, Trivandrum, India
using the instrument ABI 3730, Amersham Biosciences, United Kingdom. The obtained DNA sequences were deposited
in NCBI GenBank (Accession numbers EU179522 and
EU182589).

2.3. Screening of xerophilic aatoxin producing Aspergillus

The presumptively identied Aspergillus isolates were screened
for aatoxin production using A. avus/A. parasiticus agar
(AFPA) containing yeast extract 20 g; peptone 10 g; Ferric
ammonium chloride 0.5 g; Agar 20 g per litre (Pitt et al.,
1983). Chloramphenicol (100 mg/l) was also incorporated
for bacterial growth inhibition. Colonies showing bright orange yellow on the reverse, an indicative of aatoxin production were selected for further study. All the selected colonies
were grown on Malt Extract Yeast Extract 50% Glucose
Agar (MY50G) having malt extract 10.0 g; yeast extract
2.5 g; agar 10.0 g and glucose 500.0 g for determining their
xerophilic nature. All the inoculated plates were incubated
at 25 C for 3 weeks and routinely examined for fungal
growth (Roberts and Greenwood, 2002). The colonies grown
on MY50G were conrmed for their aatoxin production
using yeast extract agar (Hara et al., 1974) containing yeast
extract 2 g; sucrose 20 g. After incubation at 25 C for 3 days,
the colonies were exposed to UV (365 nm) and observed for
uorescence. These isolates were identied using phenotypic
and molecular methods (Alexopoules et al., 1996; Samson
et al., 2007).
2.4. Isolation of genomic DNA
Genomic DNA from selected fungal isolates was extracted
using a biomass obtained from 50 ml yeast extract culture
broth cultivated at 30 C. The culture broth was ltered with
Whatman No. 4 lter paper. The obtained mycelial growth
was taken in a mortar and pestle and ground nely adding

2.6. Determination of phylogenetic relationship

The genetic similarity between acquired Aspergillus DNA
sequences were determined using BLAST analysis as described
previously (Tatusova and Madden, 1999). The evolutionary
relationships amongst the isolates were determined by the
analysis and comparison of DNA sequences comparing the
28S rRNA sequences of fungal isolates. The DNA sequences
were aligned rst using CLUSTAL W that calculates a crude
similarity measure between all pairs of sequences by using a
fast and approximate alignment algorithm described by
Wilbur and Lipman (1983) and then determined the order of
sequences to be aligned in the nal multiple alignment. The
resulting distances were used to calculate a phylogenetic guide
tree which uses pair wise sequence distance calculation to perform multiple sequence alignment. The guide tree is calculated
with the MEGA 4 method (Saitou and Nei, 1987; Tamura
et al., 2007).
2.7. Collection of spices and extraction of their bioactive
compound
The sun dried unopened ower buds of S. aromaticum and
dried fruits of E. cardamomum were collected from Kerala
(India), and were authenticated. The samples were coarsely
ground. Ten grams powder was taken and packed using ordinary lter paper. The chloroform and acetone solvents were
used for the extraction of bioactive compounds using a soxhlet
extractor.

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390

S. Al-Sohaibani et al.

Figure 1a

The phylogentic tree of Aspergillus niger ML01 obtained by neighbour-joining method from 28S rDNA sequences.

2.8. Determinations of plant extract inhibitory activity on

growth and aatoxin production
The fungal growth inhibition and antiaatoxigenic activity of
the spice extracts were assessed by monitoring mycelial growth
and aatoxin production as described (Kumar et al., 2008).

The toxigenic fungal isolates spore suspensions were prepared

in an aqueous solution of 0.1% Tween 80 from cultures grown
previously in potato dextrose agar. The inocula was adjusted
to approximately 106 conidia per millilitre determined by a
haemocytometer. 1 ml (106 spores/ml) inoculums were inoculated into 25 ml SMKY broth medium containing sucrose,

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Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum
200 g; MgSO47H2O, 0.5 g; KNO3, 0.3 g; yeast extract, 7.0 g
per litre and pH adjusted to 5.6 0.2 (Ranjan and Sinha,
1991) taken in 100 ml Erlenmeyer ask. Different concentrations (2080 ll) of acetone and chloroform extracts of both
S. aromaticum and E. cardamomum plants mixed and the asks
were incubated on orbital shaker at 150 rpm at 27 2 C for
6 days. The control sets were kept similar to the treatment
group without plant extracts. After growth, the mycelium
was separated by the ltering culture media through Whatman
No. 1 lter paper and dried at 100 C to a constant weight for
biomass determination.
The ltrate was extracted with 20 ml chloroform in a separating funnel. After separation chloroform extract was passed
through anhydrous sodium sulphate kept in Whatman lter
paper No. 42. The extract was evaporated till dryness on water
bath at 70 C. The aatoxin content of the culture ltrate dry
residue was determined by modied Romers thin layer chromatography method (Trucksess and Pohland, 2000). The dry
residue was dissolved in 1 ml chloroform and 50 ll taken from
that was spotted on precoated plates of Silica gel G60 of
dimensions 10 10 cm. The plate is then developed in solvents,
chloroform:acetone (9:1) in one direction and toluene:ethyl
acetate:formic acid (5:4:1) in the second direction perpendicular to the rst. Plates were removed; air dried and examined
visually under UV light at 365 nm and the amount of aatoxin
was quantied by comparing aatoxin (Sigma Aldrich) standard previously prepared with benzene and acetonitrile
solution.

391

2.9. Statistical analyses

Correlations between Aspergillus sp. biomass production and
its aatoxin production were determined using t-test and Levenes test for equality of sample variances and the signicances were determined at 95% condence level.
2.10. GCMS analysis of S. aromaticum acetone extract
By using GCMS the bioactive components of the highly
antiaatoxigenic acetone extract of clove was analysed. Compounds were separated by Gas chromatography and structure
of the components was identied by MS Spectrophotometer.
GCMS analyses were carried out in a PerkinElmer Clarus
500 coupled to a Clarus 500 Mass Spectrometer mass detector
under electron impact ionisation (70 eV). The interface temperature was 250 C and the MS scan range was 40450 atomic
mass units (AMU). The chromatographic column for the analysis was capillary column Elite-5 ms (5%Phenyl 95% dimethylpolysiloxane) size 250 lm ID 0.25 u 30 m. The
stepped temperature programme was as follows: held at
60 C for 2 min. Then the temperature was raised from 60 to
250 C at the rate of 6 C/min, and held for 5 min. The total
run time was 25 min. Helium was used as carrier gas and injected 1 ml/min. Peaks were identied by computer searches
in commercial reference Wiley NIST library.

Figure 1b The cladogram of phylogentic tree for Aspergillus avus ML02 obtained by neighbour-joining method from 28S rDNA
sequences.

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392

S. Al-Sohaibani et al.

3. Results
The collected tea samples when analysed for their prescribed
quality revealed their compliance to the limit prescribed under
the PFA Act. The results of organoleptic analysis of tea samples indicated that all the samples had characteristic avour
and taste. The mean values of quality parameters viz. total
ash (6 0.7), water soluble ash (48 11), alkalinity of water
soluble ash (1.7 4), acid insoluble ash (0.7 2), water extract (44 3), crude bre(12 5) obtained in percentage indicated that all samples conrm to the prescribed standards. The
heavy metal analysis indicated higher values for copper
(22 8 mg/kg), chromium (7 5 mg/kg) and cadmium
(0.08 4 mg/kg) which were within the PFA permissible limit.
Mycoora contamination of the Indian black tea brands
sold in the southern part of India showed considerable variation in terms of average percentages, and was mostly dominated by Aspergillus group. A. niger contamination in the
different batches ranged between 0.43% and 28.54% with an
average of 8.3% for all the brands. The fungal isolates recovered from tea samples were puried and identied by conventional methods. It was observed that A. avus percentage
contamination ranged between 0.09% and 0.87% with an average 0.38. The common contaminants Penicillium spp. (0.30%),
Pacelomyces spp. (0.14%), and Mucor spp. (0.19%) along with
other unidentied members (0.07) were also isolated from most
of the brands. Among the suspected aatoxigenic Aspergillus
spp., three A. avus and one A. niger were tested for their ability
to release aatoxins on AFPA, produced colonies showing
bright orange yellow on the reverse indicated aatoxin produc-

tion. When all the aatoxin producing isolates were grown on

the special low aw MY50G medium, only two isolates (A. niger
ML01 and A. avus ML02) were able to form colonies indicating their potential of growing under low aw conditions.
The amplicons of gene sequences encoding 28S rRNA obtained by PCR amplication had 414 bp for A. niger ML01
and 423 bp for A. avus ML02 and the sequences were
assigned accession numbers (EU179522 and EU182589) upon
submission to GenBank (http://www.ncbi.nih.gov.). Their evolutionary similarity with other reference fungal isolates rRNA
gene sequences deposited in the GenBank databases was determined. The phylogenetic analysis of the gene sequences (Figs.
1a and 1b) of 28S rRNA showed the consensus tree for these
species indicating their close ancestry.
The antifungal activity of the chloroform and acetone
extracts of E. cardamomum and S. aromaticum on growth and
toxin elaboration of xerophilic tea contaminant A. niger and
A. avus strains is presented in Table 1. It was noted that both
the acetone and chloroform extracts of E. cardamomum and S.
aromaticum had growth inhibitory as well as intense antiaatoxigenic activity against the tested fungal isolates. The acetone
extract of S. aromaticum entirely arrested the aatoxin production by both the toxigenic strains where as E. cardamomum
extracts showed moderate activity. The GCMS analysis of
highly active clove (S. aromaticum) acetone extract revealed
the presence of a number of bioactive compounds like
2-methoxy-4-prop-2-enylphenol (eugenol) 68%, eugenil acetate
17%,
8-methylene-4,11,11-(trimethyl)bicyclo(7.2.0)undec-4ene (caryophyllene) 12%, phenol, 2-methoxy-4-(1-propenyl)-.
(Isoeugnol) 1%, (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-

Table 1 In vitro antifungal activity of S. aromaticum and E. cardamomum extracts on values (mean SD) of biomass and aatoxin
production of A. avus ML01 and A. niger ML02. The t-test and Levenes test for equality of sample variances at p < 0.05%.
S. No.

Name of the plant

Extract concentration

Extract inhibition on the aatoxigenic organisms

A. avus ML02

A. niger ML01

Biomass

Aatoxin production

Biomass

Aatoxin production

1
2
3
4
5
6
7
8
9
10
11
12

E. cardamom

Acetone 80
60
40
20
10
Control
Chloroform 80
60
40
20
10
Control

ND
0.72 15
1.14 26
1.74 20
1.85 32
2.30 15
0.52 10
0.80 05
1.21 07
1.54 10
1.85 05
2.21 30

ND
20 0
45 10
60 10
110 20
280 10
10
20 10
50 10
80 10
120 20
270 20

0.61 05
0.85 08
1.10 10
1.24 06
1.72 05
2.45 05
ND
ND
0.81 05
1.08 06
1.45 12
2.41 10

NA
10 0
40 10
50 10
80 20
110 20
ND
ND
10 0
30 10
70 10
100 30

13
14
15
16
17
18
19
20
21
22
23
24

S. aromaticum

Acetone 80
60
40
20
10
Control
Chloroform 80
60
40
20
10
Control

ND
ND
ND
ND
0.35 10
1.72 30
ND
ND
ND
ND
0.21 05
2.13 25

ND
ND
ND
ND
ND
170
ND
ND
ND
ND
NA
250

ND
ND
ND
ND
ND
2.43 08
ND
ND
ND
ND
0.18 10
2.45 05

ND
ND
ND
ND
ND
110 10
ND
ND
ND
ND
NA
110 20

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Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum
1,4,8-triene (-humulene) 1.5%, n-hentriacontane 0.4%, n-nonacosane 0.01% including some other peaks (0.1%) justifying
their bioactive potential.
4. Discussion
Healthy eating and drinking habits are considered to protect
from a variety of diseases, including cancer. Though the popular beverage tea is believed to prevent cancer, the recently
conducted epidemiologic data based meta analytical study
results are insufcient to conclude their role (Sun et al.,
2006). When compared, black tea contains much lower concentrations health benet providing polyphenol catechins than
green tea (Wu and Yu, 2006). Hence the fungal toxin threat
associated with black tea cannot be neglected like other stored
products. Though the analysed black tea samples showed
extensive variation in the heavy metals content, they are within
the PFA permissible limit (Seenivasan et al., 2008). The other
contaminant directly related to health that deserves attention is
the natural toxin, the mycotoxins. The processed dry tea leaves
may carry potential health risk bacteria and fungi due to postprocessing handling and storage contamination (Mishra et al.,
2006). The results of this study revealed the presence of mycoora in marketed black tea sold in the southern part of India
and it corroborates with those reports from Poland (Halweg
and Podsiadlo, 1992), Sultanate of Oman (Elshae et al.,
1999) and Czech Republic (Rezacova and Kubatova, 2005).
Also the presence of Aspergillus fumigatus, A. niger, Penicillium sp., Pacelomyces sp., Mucor sp. and some others were frequently encountered in all those studies. All these fungi would
have been common contaminants and might have entered into
the nal products through different sources. Fungal reservoirs
can be humans, soil, dust, raw materials, drains, equipment
surfaces and ventilation ducts in the industrial processing of
food (Scholte et al., 2002). The presence of fungal strains in
tea leaves and the presence of aatoxin have also been reported
(Elshae et al., 1999; Mishra et al., 2006). Dutta et al. (2008)
enumerated a total of 34 fungal species from air, phyllosphere
and soil samples of tea factory atmosphere which included
some toxin producing fungi like A. niger, A. avus, A. fumigatus and Fusarium lactus. The presence of potentially toxigenic
moulds such as A. avus, A. niger and Penicillium spp. in tea or
on any other food product indicates the potential for mycotoxin contamination. If these fungi nd suitable conditions
for their growth in tea they will produce mycotoxins and constitute a health hazard in tea (Elshae et al., 1999).
Fungi grow over a wide range of environmental conditions
and grow on stored grain ourish most rapidly between 25 and
35 C. This indicates that fungal growth is particularly fast in
the tropics. As fungi grow, they respire, produce heat and free
water, often raise the moisture content and temperature, and
accelerate the rate of spoilage. Many important storage fungi
are xerophilic including the deuteromycotina A. avus and
A. niger (Farrell et al., 2007). The MY50G media used in this
study for xerophilic fungal isolation is a reasonable choice
since the supplementation with glucose lowers the aw value lesser than 0.89 (Pitt and Hocking, 1997). A. niger and other
members of section Nigri are difcult to classify and identify
because they are morphologically very similar. Physiology, secondary metabolites and DNA sequencing can help to differentiate between the species (Mogensen et al., 2009). The sequence

393

comparison of these isolates not only revealed their identity

but also close similarity with their anamorphic forms.
Now-a-days, interest in the use of natural antimicrobials is
growing, especially those from herbs, plants, and spices (or
their components), which are traditional ingredients or avour
enhancers (Lopez-Malo et al., 2005). The aatoxigenic fungal
strains, capable of xerophilic growth, have an inherent threat
of toxin elaboration upon storage warrants incorporation of
efcient fungicidal/toxic agent without affecting consumer
preference. The antimicrobial properties of the spices have
been known and used for centuries. Also their essential oils
have long been recognized as having good fungitoxic activity
(Bluma et al., 2008). When compared, the extracts of clove
completely inhibited the aatoxigenic fungal growth and toxin
production. The cardamom extracts also exhibited signicant
inhibition. Hence the extracts of clove might be safely used
both as a preservative fungistatic stuff and a avouring agent
in tea. The preservative and antifungal potential of
S. aromaticum extracts and their essential oils against the
moulds (Nielsen and Rios, 2000), rice storage fungi Fusarium
culmorum, Penicillium islandicum, A. niger and Aspergillus
candidus (Magro et al., 2010) and common bakery products
spoilage fungi Eurotium, Aspergillus and Penicillium (Guynot
et al., 2003) have already been proved. The present study indicated the preservative potential and management of storage
fungi on tea put up for sale in tropical region. Our results
advocate that these spices are used alone or in conjunction
with other substances to reduce the risk of xerophilic aatoxigenic tea fungi. It may ensure their safety in addition to adding the consumer demanding avour. They could also be used
for the preparation of avoured tea if their organoleptic and
optimal concentrations are determined. Already the S. aromaticum extracts major component eugenol found use as a preservative in a number of stored food products. The accomplished
results of both the plants on xerophilic fungi indicate that they
can stay as novel antimicrobials in tea over the nonbiodegradable and side effects associated with synthetic preservatives.
Hence these spices known to possess intrinsic avour and other
health-promoting properties can also be used to contain the
toxigenic fungi in tea with societal acceptability.

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