Detection of mycobiota, aflatoxigenic and ochratoxigenic genes, and cytotoxic ability in spices

Abstract Spices are portions of plants because their properties are used as colorants, preservatives, or medicine. The employments of spices have been known since long time, and the interest in the capability of spices is astounding because of the chemical compounds contained in spices. The molds grow on a variety of different crops and foodstuffs including spices often under warm and humid conditions. The mycobiota of five spice species were surveyed. Forty‐six fungal species were obtained. Aspergillus flavus and A. niger were the prevalent species recorded. The aflatoxins (AFs) and ochratoxins (OTs) were detected in some samples and isolates. Cumin had the highest concentration of AFs 8.2 ppb, while ginger had a considerable occurrence of OTs 6.7 ppb. A. flavus obtained from ginger recorded the maximum concentration of AFs 7.5 ppb, and A. niger from turmeric was the highest producer for OTs 3.6 ppb. omt‐A and Aopks genes were detected in all tested A. flavus isolates and two out of four A. niger isolates. One of the important properties of spices is cancer etiology and prevention. Ginger and sage were the highest cytotoxic against four human tumor cell lines.


| INTRODUC TI ON
Spices are characterized as any dried, aromatic vegetables or plant materials in the entire, crushed, or ground forms. They incorporate all the parts of herbaceous plants aside from the leaf, which is viewed as an herb (Billing & Sherman, 1998). Spices are utilized in little concentrations for their preservative and medicinal properties (Sethi, Dutta, Gupta, & Gupta, 2013) and also to enhance or change the taste of food (Al-Mofleh, 2010). In particular, spices have been employed since antiquated circumstances with a view to prevent food spoilage and deterioration and to increase the validity of food (Shan, Cai, Brooks, & Corke, 2007). Investigations have presumed that spices might be extremely helpful since bacteria can develop resistance to conventional anti-infection agents (Gull et al., 2012).
Spices are getting polluted by molds before the drying process during the treatment (Stankovic, Comic, & Kocic, 2006). In addition, the awful states of capacity, ventilation, and high moisture rate are prompt reasons for spoilage of spices by pathogenic microorganisms (Omafuvbe & Kolawole, 2004).
Aflatoxins and ochratoxins A are mycotoxins delivered by few contagious types of the Aspergillus and Penicillium genera (Frisvad, Thrane, & Samson, 2007). Mycotoxins induce dangerous consequences in living organisms since they exhibit carcinogenic, nephrotoxic, hepatotoxic, and immunotoxic effects (Cao et al., 2013).
Aflatoxin and ochratoxin are examined in a variety of dietary products such as spices (Zinedine et al., 2007). Temperature and humidity are considered as the principal factors that control the production of mycotoxin (Griessler, Rodrigues, Handl, & Hofstetter, 2010). The International Agency for Research on Cancer (IARC, 2002) has ordered the naturally occurring blends of aflatoxins as human carcinogens (group 1) and ochratoxin A as potential carcinogen to human (group 2B).
Molecular techniques have been employed as robust methods for detection and identification of fungi (Hassan, Gaber, & El-Hallous, 2014). Consequently, rapid strategies such as polymerase chain reaction (PCR) have been utilized directly for testing foodborne pathogens and for confirmation and genotyping of food microbes. The basic feature of PCR is that organisms are not in need to be cultured, at least not for lengthy periods prior to their detection.
Moreover, PCR-based methods that target DNA offer a good way for rapid diagnosis because of their outstanding specificity and sensitivity (Bandyopadhyay et al., 2016), particularly in developing species-specific primers using multicopy sequences (Awuor et al., 2016).
In the present study, we have evaluated the mycobiota of five types of spices, determination of total AFs and OTs in the most common isolates of A. flavus and A. niger and also in some samples which contaminated by them, detection of AFs and OTs biosynthesis genes in the same isolates also comparison the genetic diversity by using ISSR-PCR.

| Collection of spice samples and mycological isolation
Fifty specimens of spices (10 samples for each) were gathered from different markets in two governorates: Sohag and Qena in Upper Egypt. Each sample was reserved in a sterile polyethylene pack and transferred to the mycological laboratory for fungal examination.
The dilution-plate technique was applied for the quantitative enumeration of contaminated spices, as mentioned by Christensen (1963). A known weight of tested spices (2 g of each of cumin and black cumin, and 0.3 g of turmeric, sage, and ginger samples) was mixed aseptically in 50 ml of sterile distilled water and shaken vigorously. One ml of the proper dilution was poured in sterilized Petri dishes. Fifteen ml of melted DRBC agar media g/L (peptone, 5; dextrose, 10; KH 2 PO 2 , 1; MgSO 4 ·7H 2 O, 0.5; dichloran, 0.002; agar, 15), rose bengal (0.025) and chloramphenicol (0.1) were added as bacteriostatic agents (Baylis, 2003), cooled to 45°C, and poured on the sample suspension in Petri plates which were turned to convey the suspension. Triplicates were read, and the cultures were incubated at 28°C for 7 days. The creating colonies of fungi were isolated, identified, and counted.

| Determination of total AFs and OTs producing potential of Aspergillus flavus and A. niger isolates and also in the samples
The total AFs and OTs producing ability of the isolates were examined The natural occurrence of total aflatoxins and ochratoxins in five samples (one of each type that highly contaminated) was determined by using a slightly modified method based on the Association of Official Analytic Chemists (AOAC) method (Trucksess et al., 1991).
Methanol:water (80:20) solvent (100 ml) and 5 g NaCl were added to 100 g of ground each sample, and the mixture was shacked in a blender at maximum speed for 3 min. Filtration process occurred through fluted filter paper (Whatman 2V; Whatman plc), and the filtrate was diluted (1:4) with water and refiltered through glass-fiber filter paper. Two milliliters of the glass-fiber filtrate was placed on an AFs or OTs Test RWB SR Column (VICAM) and allowed to elute at 1-2 drops/s. The columns were washed two times with 5 ml water, and aflatoxin or ochratoxins were eluted from the column with 1 ml high-performance liquid chromatography (HPLC)-grade methanol. A bromine developer (1 ml) to the methanol extract was added, and the total AFs or OTs concentrations were read in a recalibrated VICAMSeries-4 fluorometer set at 360 nm excitation and 450 nm emissions (Lewis et al., 2005).

| Molecular detection of AFs and OTs biosynthesis genes
All the molecular steps were employed by Gene analyzer 3121 in Scientific Research Center, Biotechnology and Genetic Engineering Unit, Taif University, KSA.

| ISSR analysis
Primers utilized in ISSR analysis were obtained from Amersham Pharmacia Biotech. Following the experiments for optimization of component concentrations, PCR amplification of random primers was carried out in 25 μl volume containing 1 μl (20 ng) of genomic DNA, 12.5 μl of Go Taq ® Green Master Mix, Promega, USA, 1 μl of primer (20 p.mol), and deionized distilled water (up to a total volume of 25 μl) as described by Hassan et al. (2014). For DNA amplification, the C1000TM Thermo Cycler Bio-Rad, Germany, was programmed under the conditions involving denaturation at 94°C for 5 min; 40 cycles of denaturation at 94°C for 30 s, primer annealing at 35°C for 1.5 min, primer extension at 72°C for 2.5 min, and final extension step at 72°C for 7 min. Amplified DNA products were analyzed by electrophoresis in 1.5% agarose gel run in TBE. The gels were stained with ethidium bromide (5 μg/ml). 100 bp DNA ladder (Solis Bio-Dyne ® ) was applied as a standard. DNA was visualized by UV illumination and then photographed by a Bio-Rad Gel Doc 2000 device.

| Data analysis
The amplification results of ISSR-PCR were recorded for the presence as "1" or absence as "0" and missing data as "9." The genetic associations between isolates were evaluated by calculating Jaccard's similarity coefficient for pairwise comparisons depending on the proportion of shared bands produced by the primers. The dissimilarity matrix was generated using of neighbor joined technique, and consequently, the dendrogram was reconstructed. The computations were achieved utilizing the NTSYS-computer program version 2.01 (Rohlf, 2000). For principal component analysis, Jaccard's similarity matrix was amplified.

| Cytotoxic effect on human cell lines
All the cytotoxic effect was carried out in Department of Pharmacognosy, National Research Centre El-Tahrir Str., Dokki, Giza 12622, Egypt.

| Preparation of extracts
Ten g of each powdered spice (ginger, cumin, turmeric, sage, and black cumin) was macerated with 50 ml hexane 80% at room temperature for 3 days. The stock solutions were prepared by adding 1ml dimethylsulfoxide (DMSO) to the residue after evaporation.

| Cell lines
The spice extracts were tested against the following cell lines:

| MTT assay
Cell viability was assessed by the established mitochondrial reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan. All the succeeding steps were completed under sterilized conditions using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME, USA). Cells were suspended in RPMI 1640 medium (for HCT116, HePG 2, MCF7, and PC3), 1% antibiotic-antimycotic combination (containing potassium penicillin 10,000 U/ml, streptomycin sulfate 10,000 µg/ml, and amphotericin B 25 µg/ml), and 1% lglutamine at 37°C with 5% CO 2 . Cells were batch-cultured for 10 days and then seeded at a concentration of 10 × 10 3 cells/well in a fresh complete growth medium in 96-well microtiter plastic plates at 37°C for 24 hr below 5% CO 2 using a water jacketed carbon dioxide incubator (Sheldon, TC2323). The media were aspirated, a fresh medium (without serum) was added, and cells were incubated either alone (negative control) or with different concentrations of the sample to give a final concentration of 100-50-25-12.5-6.25-3.125-0.78 and 1.56 ug/ml. After incubation for 48 hr, the medium was re-aspirated, and 40 ul MTT salt (2.5 μg/ml) was supplemented to each cavity followed by incubation for another four hours at 37°C below 5% CO 2 . For the purpose of dissolving the formed crystals and stopping the reaction, 200 μl of 10% sodium dodecyl sulfate (SDS) in deionized water was supplemented to each cavity and subsequent incubation at

| Mycobiota contamination of spices
The mycobiota of five kinds of spices marketed in Upper Egypt was investigated in this study. Sage samples were found to be more contaminated than other samples (34,562.46 CFU/g of Sage) as illustrated in Table 1. Studies on the fungal contamination of sage are almost completely absent in the previous literature.
Eighteen genera comprised of 41 species, and 5 species varieties were obtained from 50 samples of spices that were bought from the markets in Upper Egypt, 2015. The highly isolated fungi in terms of abundance and frequency, which were recovered from the samples of spices, were as follows: Aspergillus sp. (71.32% and 100%), Eurotium chevalieri (11.76% and 40%), Emericella nidulans var. lata (11% and 22%), Penicillium sp. (4.45% and 66%), and sterile mycelia (1.55% and 34%). The remaining genera were obtained in low or moderate frequencies that formed 9.41% collectively.

| Total AFs and OTs producing potentials of A. flavus and A. niger isolates
Eight isolates of A. flavus and A. niger isolated from the spice samples were subjected to estimate their AFs and OTs producing potentials.
The results are summarized in Table 2. The tested isolates had the abilities to produce total AFs and OTs with the exception of two isolates of A. niger from black cumin and sage samples which exhibited no detectable ochratoxin production. The range of AFs produced by A. flavus was 2.2-7.5 ppb. The levels of OTs produced by A. niger were 3.4 and 3.6 ppb.

| Natural incidence of total AFs and OTs in spice samples
Five samples out of 50 (which had the highest count of A. flavus and A. niger) were naturally contaminated with AFs and OTs. The concentration of the AFs in the spice samples ranged from 1.9 to 8.2 ppb with the highest amount found in cumin sample. Likewise, the reading of the OTs ranged from 4.1 to 6.7 ppb, and the highest reading was recorded in ginger sample, as shown in Table 3.

| Molecular characterization of AFs and OTs biosynthesis genes
The eight Aspergillus isolates identified genes clustered within a 70 kb DNA region in the chromosome that are comprised in the production of AFs and whose DNA sequences have been previously published (Criseo et al., 2008). The polymerase chain reaction (PCR) was applied using two sets of primer for different genes involved in aflatoxin and ochratoxin biosynthetic pathways. The bands of the fragments of omt-A and Aopks genes can be visualized at 320 and 549 bp, respectively (Figure 1). The DNA banding patterns of the examined aflatoxigenic and ochratoxigenic isolates were different.
All aflatoxigenic A. flavus (i.e., isolates, no. 31, 32, 33, and 34) showed DNA fragments that corresponded to the presence of genes. In addition, two of the four A. niger isolates (no. 35 and 36) showed the investigated ochratoxin gene.

| ISSR-PCR analysis, genetic distances, and the cluster dendrogram
The genomic diversity of Aspergillus spp. was investigated by ISSR markers. Results of ISSR-PCR are illustrated in Figure 3. ISSR-PCR reactions were completed with eight Aspergillus isolates. Six different ISSR-PCR markers yielded 84 distinct bands of which 57 bands were polymorphic (68%) and only 27 bands were monomorphic (32%). The number of bands for individual ISSR primers varied from 10 bands for ISSR-16 to 19 bands for ISSR-28. The highest polymorphism was recorded for ISSR-19 and the lowest for ISSR-28. Moreover, the band size of ISSR-8 ranged from 220 to 2,000 bp ( Figure 2).
The genetic similarity and intraspecies differentiation as well as the dendrogram constructed using neighbor-joint method based on Jaccard's similarity coefficients ranging from 0.00 to 1.14 ( Figure 3) were used here. According to the corresponding data, the dendrogram analysis showed that there is genetic distance among native Aspergillus sp. They were grouped into two large clusters: The first cluster consisted of A. flavus-31, A. flavus-32, A. flavus-33, and A. flavus-34, and the second main cluster included the four A. niger only.
The result found that genetic distance among native Aspergillus sp.
was comparatively low between each species and high within each species in general. The smallest genetic distance (0.00) was estimated between A. niger-35 and A. niger-38 (Figure 3).

| Cytotoxic potential of spice extracts
In this experiment, the cytotoxic capability of five extracts of spices against four human tumor cell lines (HCT116, HePG 2, MCF7, and PC3) was probed using the MTT assay (Table 4)

| D ISCUSS I ON
The species A. flavus and A. niger obtained in this study are described as mycobiota contaminated of spices due to the presence of fungi at preharvest or postharvest depend on storage temperature, seed moisture content, relative humidity, and fungal species. Gherbawy, Shebany, Hussein, and Maghraby (2015) found that crushed chili samples were the preferred mediums for fungal growth  (Erami et al., 2007). El-Hamaky, Atef, Yazeed, and Refai (2016) found that a single fragment of about 549 was produced with two positive ochratoxigenic A. niger isolates.  (Akimoto, Lizuka, Kanemastu, Yoshida, & Takenaga, 2015).

| CON CLUS ION
The mycological analysis revealed that sage samples were highly contaminated and that the most prevalent fungi were Aspergillus sp. Cumin had the highest reading of AFs, and ginger was the highest for OTs. All the tested isolates of Aspergillus flavus and A. niger were positive for AFs and OTs production except two isolates of A. niger that exhibited no detectable OTs, omt-A gene was found in all A. flavus isolates, and Aopks genes were detected in 2 out of four isolates of A. niger. The most cytotoxic activity was determined in ginger and sage. Therefore, our findings reveal that the dryness of the spices after harvesting is the main factor to avoid the development of fungi during storage and open up the possibility that natural compound found in these spices may be used to develop new treatment modality for cancer. However, further investigation is needed to determine the mechanism for its action in carcinoma.

ACK N OWLED G M ENTS
This work was supported by South Valley University, Qena, Egypt.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L R E V I E W
All experimental protocols and procedures were reviewed and approved by the Department of Pharmacognosy, National Research Centre El-Tahrir Str., Dokki, Giza 12622, Egypt.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.