Yash Pal Sharma & Geeta Sumbali. Department of Botany, University of Jammu, Jammu 180 006 (J&K), India. Received 23 February 1999; accepted 6 July ...
Mycopathologia 148: 103–107, 1999. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Incidence of aflatoxin producing strains and aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga Mill.) from the Indian State of Jammu and Kashmir Yash Pal Sharma & Geeta Sumbali Department of Botany, University of Jammu, Jammu 180 006 (J&K), India Received 23 February 1999; accepted 6 July 2000
Abstract An investigation was undertaken to obtain data on the occurrence of aflatoxins and the aflatoxin producing potential of Aspergillus flavus strains isolated from dry fruit slices of quinces produced in jammu and Kashmir, India. A total of 147 A. flavus isolates recovered from dr fruit slices were grown in liquid rice flour medium and screened for the production of various aflatoxins by thin layer chromatography. The results showed that 23.14% of the tested isolates were aflatoxigenic, producing aflatoxins B1 and B2 in varying amounts. Aflatoxins G1 and G2 were not detected. All 25 of the investigated market samples were also found to be aflatoxin B1 positive and the level of contamination ranged from 96 to 8164 µg/kg of the dry fruit which is quite high in comparison to the permissible level of 30 ppb. As per these results biochemical composition of dry fruit slices of quinces, along with climatic conditions seem to be very favourable for aflatoxin production by the toxigenic A. flavus strains. Therefore, monitoring of aflatoxins in dry fruit slices of quinces is recommended for this region. Key words: Aflatoxigenic, Aspergillus flavus, dry fruit slices, quince.
Introduction Aflatoxins are a group of structurally related secondary mold metabolites produced in various agricultural commodities by some strains of Aspergillus flavus, A. parasitcus and rarely by A. nomius [1]. They are reported to be hepatotoxic, carcinogenic, teratogenic and mutagenic [2, 3]. In view of these health risks, aflatoxin prone agricultural commodities are subjected to stringent regulations in most of the countries and thus have a significant impact on the international economics [4]. From India, the first few reports of aflatoxicosis came from western states [5–7] and later on aflatoxin contamination in foods and feeds was also reported from Bihar [8–11], Andhra Pradesh [12, 13], Gujarat [14], Uttar Pradesh [15, 16], Tamil Nadu [17], Punjab [18] and Karnataka [19]. However, food and feed samples from other Indian states also need to be monitored quickly and efficiently.
The Indian Government under 57A of Prevention of Food Adulteration Rules, 1955, has imposed the maximum permissible level of 30 ppb for aflatoxin B1 in all foods prone to aflatoxin contamination [20]. The incidence and level of aflatoxin contamination in foods and feeds is variable in different Indian states and is largely determined by the local environmental conditions, agronomic practices, storage structures, processing and transport facilities. In all these respects, north Indian state of Jammu and Kashmir was considered to provide a favourable situation for toxigenic mold proliferation but literature shows little investigation of foodstuffs from this state [21]. No work has been done so far to assess aflatoxin contamination in dry fruit slices of quinces (Cydonia oblonga). In view of this, the aim of our present investigation was to verify whether dry fruit slices of quinces (Cydonia oblonga), which are valued in tis state for its religious, nutritional and commercial significance, contain aflatoxins.
104 We examined this possibility and, in this communication report our results on the aflatoxin production by 147 Aspergillus flavus strains isolated from the mycoflora present on the surface of dry fruit slices of quinces. An evaluation of the aflatoxin levels in the samples taken from main points of production, storage and distribution all over the state was also made.
Materials and methods Isolation of Aspergillus flavus from dry fruit slices of quinces In the present study, 147 isolates of A. flavus were recovered from the samples by employing both the direct plating of fruit slices on agar medium and by surface washing method in which aliquots of samplewater suspension were poured in sterilised petri plates and then plated with medium. Isolation medium included Czapek’s Dox agar (CDA) supplemented with rose bengal (0.20 g/L) and streptomycin sulphate (0.06 g/L) as antibacterial agent. The antibiotic was added just before pouring the petri plates which were then incubated at 28 ◦ C for 8 days. Analysis of aflatoxins in A. flavus cultures All the isolates were grown statically in 250 mL Erlenmeyer flasks with 100 mL of autoclaved rice flour liquid medium (rice flour, 40 g/L; sucrose 30 g/L; yeast extract 2 g/L) and incubated for 10 days at 208 ◦ C. After the incubation period, contents of the flasks were filtered through Whatman #1 filter paper and the filtrate was extracted thrice with chloroform (Total volume 50 mL). The organic phases were combined, evaporated to dryness (37 ◦ C) and finally dissolved in 1 mL of chloroform for analysis of aflatoxins. In the present investigation, thin layer chromatography (TLC) was used for analysis as it is simple, fast and low cost method that could reliably be used as an alternative to more expensive methods [22]. Analysis for aflatoxins in samples Samples were obtained from 25 different market stocks kept in various storage structure (gunny bags, nylon bags, polyethylene bags, wooden boxes). From each stock, dry fruit slices of quinces were taken at various points as composite sample and used for analysis.
Aflatoxins were determined in samples by previously described procedure [23]. Each of the 25 composite samples of dry fruit slices of quinces were taken, finely ground in a Rico Model Deluxe grinder by intermittent grinding at slow speed for 2 minutes and then at high speed for another 2 minutes. A 50 g portion of the finely ground sample was stirred with 250 mL of methanol : water (60 : 40 v/v) on a mechanical shaker for 30 minutes. This solution was filtered through whatman #1 filter paper and 125 mL of the filterate was taken in a separating funnel and shaken vigorously with 50 mL of n-hexane for 2 minutes. Lower methanol layer was then extracted with 50 mL chloroform. The chloroform layer was taken in a conical flask containing 5 g cupric carbonate, shaken and then filtered through whatman #42 filter paper having a bed of anhydrous sodium sulphate. Cupric carbonate was again washed with 25 mL of chloroform and filtered through anhydrous sodium sulphate bed. The two chloroform extracts were pooled and evaporated until nearly dry. The residue was dissolved in 1 mL of chloroform and analysed by TLC. Known aliquots of the extracts obtained from ‘in vitro’ cultures and market samples were spotted on silica gel coated and activated TLC plates. Developing solvent system consisted of toluene : isoamylalcohol : methanol (90 : 32 : 2 v/v). Developed plates were allowed to dry in the dark and examined under longwave (366 mm) UV light. In addition to their emission of blue and green fluoresence, B and G toxins were identified by using standards placed side by side with the extract on the chromatoplates. Confirmatory tests, when necessary, were performed according to Stack and Pohland [24]. Quantitative estimation of aflatoxins was done spectrophotometrically [25] using ‘UVIKON’ model 922 spectrophotometer. Silica gel of each spot was scraped and collected individually in clean dry centrifuge tubes. For extraction, 5 ml of cold methanol (Spectroscopy Grade) was added to each tube and centrifuged at 3000 r.p.m. for 5 minutes. Aflatoxin content was determined by recording the ultra violet absorption spectrum of the methanolic solution in a spectrophotometer at 360 nm for AF B1 and AFG1 and 362 nm for AFB2 and AFG2 . The amount of aflatoxin content in the sample was calculated according to the following formula:
Aflatoxin concentration:
D × M × 106 mg/mL E × 1 × 1000
105 Table 1. Production of aflatoxins by Aspergillus flavus isolates from dry fruit slices of quinces Number of isolates screened
Number of isolates producing: No Only B1 aflatoxins aflatoxins
147 Production range (µg/L) Means ± standard deviation
113
19 99–3730 1069 ± 1030.7
Only B2 aflatoxins
Both B1 and B2 aflatoxins
3 243–599 426 ± 178.8
12 553–3857∗ ; 71–3054∗∗ 2027 ± 1111.2∗ ; 1593 ± 1138.3∗∗
Figures marked ∗ and ∗∗ indicated aflatoxins B1 and BB2 , respectively.
where D is the optical density; M is the molecular weight of aflatoxin; E is the molar extinction coefficient; and I is the path length (2 cm cell was used).
Results and discussion In the present investigation, 113 out of 147 A. flavus isolates from dry fruit slices of quinces did not produce aflatoxins in media (Table 1). There are many reports that some A. flavus isolates do not produce aflatoxins and the percentage of these atoxigenic A. flavus isolates varies depending upon the substrate [26–28]. Among the confirmed toxigenic isolates, 35.3% produces both aflatoxin B1 and B2 , 55.9% produced only aflatoxin B1 , whereas, 8.8% elaborated only aflatoxin B2 . Lack of aflatoxin B1 production by some isolates and exclusive production of aflatoxin B2 could be explained by total conversion of aflatoxin B1 to B2 [26]. Few other workers have also reported exclusive production of aflatoxin B2 by A. flavus strains [29, 30]. It was also noted that among the toxigenic isolated producing both aflatoxins B1 and B2 , majority of the isolates produced aflatoxin B1 in greater amount than aflatoxin B2 (Table 1). This observation clearly depicts that A. flavus isolates may even differ in their capacity to interconvert the aflatoxins. Similar observation has been recorded earlier by Heathcote and Hibbert [31]. However, none of the tested A. flavus isolates produced the G aflatoxins [28]. It was found that even under similar physical and nutritional factors, both toxigenic and atoxigenic isolates of A. flavus varied in their sclerotia producing ability. Among the toxigenic isolates, eight produced abundant small sclerotia (
