Saudi Journal of Biological Sciences (2011) xxx, xxxxxx

King Saud University

Saudi Journal of Biological Sciences

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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

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.
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* 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

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

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Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005

2 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

Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005

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) 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

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).

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.

Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005

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,

Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005

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. 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.

Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005

6 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-

S. Al-Sohaibani et al. 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 Biomass 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 E. cardamom Acetone 80 60 40 20 10 Control Chloroform 80 60 40 20 10 Control 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 ND ND ND 0.35 10 1.72 30 ND ND ND ND 0.21 05 2.13 25 Aatoxin production ND 20 0 45 10 60 10 110 20 280 10 10 20 10 50 10 80 10 120 20 270 20 ND ND ND ND ND 170 ND ND ND ND NA 250 A. niger ML01 Biomass 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 ND ND ND ND ND 2.43 08 ND ND ND ND 0.18 10 2.45 05 Aatoxin production NA 10 0 40 10 50 10 80 20 110 20 ND ND 10 0 30 10 70 10 100 30 ND ND ND ND ND 110 10 ND ND ND ND NA 110 20

S. aromaticum

Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005

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

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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Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005

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Please cite this article in press as: Al-Sohaibani, S. et al. Xerophilic aatoxigenic black tea fungi and their inhibition by Elettaria cardamomum and Syzygium aromaticum extracts. Saudi Journal of Biological Sciences (2011), doi:10.1016/j.sjbs.2011.06.005