Several physical properties of aflatoxin-contaminated pistachio nuts ...

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identification of Aspergillus flavus-infested nuts, at harvest and in ... growth split nuts are less contaminated, and pistachios with sound hulls are almost clean.
Food Additives and Contaminants, November 2005; 22(11): 1144–1153

Several physical properties of aflatoxin-contaminated pistachio nuts: Application of BGY fluorescence for separation of aflatoxin-contaminated nuts

EBRAHIM HADAVI Science and Research Campus of Islamic Azad University, Tehran, Iran (Received 15 April 2005; revised 29 June 2005; accepted 27 July 2005)

Abstract The primary objective was to evaluate and find a proper method for visual identification of aflatoxin-contaminated pistachio nuts. The feasibility of using bright greenish yellow fluorescence (BGYF) in pistachio nut as a discriminating factor for identification of Aspergillus flavus-infested nuts, at harvest and in post-harvest, is investigated. Results show a strong relationship between BGYF and aflatoxin content at harvest. The factors affecting the application of this method in postharvest stages are also discussed. The relationship between inside-brown kernels and aflatoxin presence is confirmed. At harvest, the brown kernels are a subdivision of fluorescent fraction. The share of different pistachios based on hull types (with sound hull, growth split and early-split) in contamination is studied. The early-split nuts are the most contaminated nuts, growth split nuts are less contaminated, and pistachios with sound hulls are almost clean. The effect of inappropriate handling on the percentage of fluorescent nuts is studied. The percentage of visible mould in samples is observed which shows a good relationship with the presence of BGY fluorescence.

Keywords: Pistachio, Aspergillus flavus, sorting, aflatoxin, BGY fluorescence, Kojic acid

Introduction Nowadays the aflatoxin as an established potent carcinogen has become a health hazard for worldwide pistachio consumers. As the consumers are more and more conscious on their health, the mycotoxin problem becomes more significant and consequently preventive and corrective measures become more important. Other pistachio-producing countries have been able to reduce sources of variations because of their industrialized structure, lower spore concentration of contaminating fungi in producing areas, limited number of orchard owners, cultivation of one or two improved cultivars and employment of clonal rootstocks and mechanized and quick harvest and transport and processing. Furthermore, the contaminated nut (especially earlysplit nuts) properties and staining patterns are studied, and based on this, some sorting methods are developed and are applied. In Iran, the contaminating fungi are long-term established, besides numerous local varieties are cultivated commercially

Correspondence: Ebrahim Hadavi. E-mail: [email protected] ISSN 0265–203X print/ISSN 1464–5122 online ß 2005 Taylor & Francis DOI: 10.1080/02652030500306976

and labour-consuming traditional methods still prevail. With this vast variation, definition of a pattern for suspicious nuts is unlikely because not all cultivars’ early-split nuts show similar staining patterns. Moreover, poor post-harvest handling makes staining so abundant which decreases hand-sorting effectiveness for removing highlycontaminated nuts. Because of this situation, development of an efficient method for removing highly-contaminated nuts, which could be applicable for most cultivars, is indispensable. Aflatoxin contamination is highly heterogeneous and concentrated in a few nuts, estimated as one highly-contaminated nut per 10,000 to 1,000,000 pistachios (Schatzki & Pan 1996) and one per 25,000 nuts (Sommer et al. 1986). It is obvious that introducing a screening method for removal of these contaminated nuts could use this distribution pattern as an advantage. We should bear in mind that because of the unique structure of the pistachio nut, which is comprised of two phases of bony white shell and dark kernel, the introduction of sorting

Physical properties of aflatoxin-contaminated pistachio nuts methods based on colour have specific difficulties and errors even though these methods work well for other nuts like peanuts. The relationship between Aspergillus flavusinfection and BGY fluorescence and aflatoxin was first reported in cottonseed (Marsh et al. 1969). The fluorescence was so easily detected as to suggest its use in locating fibres infected with A. flavus prior to harvest. Because of its bright greenish yellow colour under an ultraviolet light it was referred to as the ‘BGY’ fluorescence (BGYF). The BGYF formed readily in living cotton fibres incubated with A. flavus, but fibres of the same kind, which were autoclaved and subsequently incubated with the fungus, showed no BGYF in spite of heavy growth on them, role of a heat-labile factor revealed. Later, it was observed that the capability of causing fluorescence was a characteristic of living cells, which exhibit peroxidase activity. Finally, the evidence suggested that for BGYF to form in a higher plant under the influence of A. flavus, at least three events must occur: A. flavus must infect the plant tissue and grow in it to some degree; kojic acid (which is a known metabolite of A. flavus) must be formed; and the kojic acid must be transformed to one or more BGYF compounds, probably in a peroxidase type of reaction. All the isolates of A. flavus tested caused BGYF. In his study Marsh et al. (1969) concluded that the BGYF serves as a diagnostic marker to locate A. flavus infections in cotton fibre at harvest and later evidence might warrant similar results for other plants under defined conditions. In another study, the structure of the mentioned BGYF compound was characterized as a substance with molecular weight of 282 and by further characterization of the compound with 1H- and 13C-NMR spectroscopic analysis, it was confirmed that it was a dehydrogenated dimer of two kojic acid molecules, linked through the C-6 positions (Zeringue et al. 1999). For corn, it is reported that the largest part of A. flavus strains in the surveyed area were non-toxigenic but all of them produced kojic acid which results in BGYF kernels without aflatoxin (Wicklow 1999). In corn, BGYF has made the basis for the ‘black light test’ to identify aflatoxin contaminated corn lots. A count of one BGYF particle per kg obtained from a given corn sample is an indication that the sample should be tested for aflatoxin by chemical means such as HPLC or ELISA (Shotwell & Hesseltine 1981). In figs, the association of BGYF with decay by only four species including A. flavus in two Aspergillus sections is reported (Doster & Michailides 1998). In the first years after the rise of the aflatoxin problem in Iranian pistachios exported to US, the viability of BGY fluorescence as a screening method for removal of contaminated nuts was evaluated

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(Dickens & Welty 1975). BGYF was observed on the shells of 7% (average) of the nuts in samples taken from 46 aflatoxin-contaminated commercial lots of Iranian pistachio nuts. The fluorescent nuts contained 50% of the aflatoxin in samples. Fluorescent kernels constituted from 4–17% of the total kernels. Except for four samples, the aflatoxin content was higher in fluorescent nuts with an average of 150 mgkg1 compared to 14 mgkg1 in nonfluorescent nuts. Because A. flavus grew only from 21% of the fluorescent nuts and 4% of the BGYF shells, it appeared that fungi other than A. flavus produced BGYF in pistachio shells. Finally, because a direct relation between aflatoxin content and A. flavus and BGYF was not observed, it was concluded that fungi other than A. flavus could create BGYF in pistachio shells. In another study working on pistachios exported by Iran and Turkey, the BGYF soundness for identification of contaminated pistachios was investigated again (Steiner et al. 1992). By putting sideby-side the results with that of Dickens and Welty, because of observed non-specific nature of BGYF as a criterion for aflatoxin screening in pistachios and related variations, it was concluded that ‘‘analysis of nuts with fluorescent shells was not an appropriate means to find all kernels containing high aflatoxin concentrations’’. It was found that ‘‘in pistachio nuts, the aflatoxins were located in brown or brown spotted kernels’’, so the brown colour was introduced as a criterion to find pistachio kernels containing high aflatoxin concentrations. The BGYF under UV-excitation was used to detect aflatoxin in pistachio nuts (McClure & Farsaie 1980). It was found that the ratio of the fluorescence of pistachio at 490 and 420 nm could be a basis for BGYF detection. These two wavelengths were used in an automatic optical sorter for removal of aflatoxin-infected pistachio nuts (Farsaie et al. 1981). However, no report about the viability of this method at commercial level has been published. Many reasons could be assumed for this, first of all is the ceasing of pistachio nut imports from Iran in the same years, and the different nature of the aflatoxin problem in US pistachios which was more easy to sort and accord by international legal limits in those years. This resulted in the elaboration of alternative techniques which focused on extensive work on the physical properties of early-split nuts for removing them as the main source of aflatoxin (Pearson et al. 1994). Based on obtained results, a machine vision system for detection of early-split pistachio nuts was developed (Pearson & Schatzki 1998). Later, the relationship between kernel decay and the type of shell discoloration of early-split nuts was established as well (Doster & Michailides 1999). This relationship was demonstrated in other kinds of nuts

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including growth split and unsplit pistachios along possible mechanisms for each kind of discoloration. Finally, implementing shell characteristics by processors to identify poor quality nuts was suggested. As can be concluded from the above brief review, successful results for aflatoxin detection based on BGYF in cotton and corn are focused at harvest time and in unsuccessful trials for pistachio, the samples are gathered from pistachios exported by Iran and Turkey which are usually poorly handled after harvest. This leads to two main problems; first, as it is mentioned in literature, production of BGY fluorescence is dependent on the presence of peroxidase, which is present in living tissues like pistachio hull. Therefore, we could expect nonBGYF pistachio nuts, which are, contaminated in post-harvest stages including storage and transportation and shipment in case of sufficient water activity, for growth of fungi. These BGYF ‘false-negative’ results can change the result by build-up of high levels of aflatoxin in nuts without BGYF that are referred to in some literature (Steiner et al. 1992); the second problem is BGYF ‘false-positive’ results which are studied here. These are created by inappropriate handling of freshly harvested nuts to the processor, i.e., the transport of fresh nuts in bulk-compacted state and without proper ventilation and time restriction that is common in traditional methods of post-harvest handling in Iran. In this study by control of mentioned factors and collecting samples directly from the orchard and from processors which work under supervision of an experimental HACCP plan in which harvest and transport to processor is done in ventilated state without compaction, the mentioned false results are controlled. As a result, the sorting based on BGYF had promising results. Besides the discolouration of kernel sections is studied and the result complied with that of BGYF.

Materials and methods Samples from orchards Pistachio nuts were collected in the first week of October 2003, from six orchards near Kerman and Rafsanjan. The orchards were cultivated under prevailing conditions of the region and were flood irrigated. The pistachios were collected by selecting random rows and searching all sides of tree for different kinds of nuts based on hull condition, i.e. sound nuts (non-split), growth split and earlysplit nuts in separate rounds. By this procedure of sampling the heterogeneous distribution of contaminated nuts was covered well as practiced earlier in literature. Three local cultivars of

Kalleghoochi, Akbari and Momtaz were included. Those early-split pistachios that were deformed and routinely removed through processing by screening or ‘hand-picked out’ were not collected or excluded later. The samples were stored in a limited ventilation situation, i.e. they were placed in polyethylene bags in room temperature in the shade to resemble improper transport of fresh pistachio, for different periods and then were hulled and dried in a wellventilated glasshouse. All orchard samples were divided into fluorescent and non-fluorescent groups, employing a UV cabin under 360 nm wavelength. Any little or questionable amount of fluorescence was considered positive and included in F group. Then they were halved and their cross sections were observed for presence of brown colour or brown spots and classification was made subsequently. Medical gloves were worn for individual nuts handling. Some sub-samples of F and brown kernels were divided into further subsamples to assess the quantitative or qualitative nature of the feature as well. For samples collected from processing terminals after division based on BGYF, cross section was examined only for two of them from F group, which included brown coloured kernels. All the resulted sub-samples were photographed once prior aflatoxin analysis with a high resolution digital camera for possible further evaluation and are available. The aflatoxin tests were carried out by an independent accredited laboratory based on AOAC Official Method 999.07 (AOAC 2000). For 50 g of test portion 5 g NaCl and 120 ml MeOH and 100 ml hexane is added and blended in high-speed blender for 3 min. The mixture is filtered, 20 ml filtrate is mixed with 130 ml distilled water, 75 ml withdrawn and passed by flow rate of 2–3 ml/min from conditioned immunoaffinity column (EASIEXTRACT, R-Biofarm). The column is washed with 10–20 ml water and dried applying vacuum; 500 ml methanol is passed by gravity, after 1min another 1500 ml methanol is passed; air pressure applied to collect remainder MeOH. For quantification, 200 ml is injected to reverse-phase high-performance liquid Chromatography with post-column derivatization using kobra cell and fluorescence detector. LOD was 0.3 and 1.2 mg/kg for aflatoxin B1 and total aflatoxins respectively. LOQ was 1 and 2 mg/kg for aflatoxin B1 and total aflatoxins respectively. Small samples were homogenized using ultra-turrax prior to extraction. In case the filtered sample was not enough for final quantification of possible higher levels, it is noted in the results that the actual amount is more than the given amount. All the given amounts for aflatoxin concentrations in text are the total amount of aflatoxins in samples,

Physical properties of aflatoxin-contaminated pistachio nuts which in nearly all cases is the same as or near aflatoxin B1 concentration. For correlation coefficient calculation, 0 was assigned for samples, which were not considered contaminated (concentrations below 2 and 4, for aflatoxin B1 and total aflatoxins respectively) and 1 for contaminated samples. For fluorescent and brown coloured kernels, a similar method was applied. The resulted correlation coefficient was evaluated for significance by t-value resulting from the following formula in which r ¼ correlation coefficient, N ¼ number of observations and degrees of freedom ¼ N2. t¼

r sqrt½ð1  r 2 Þ=ðN  2Þ

The samples were coded as follows: The first letter corresponds with the first character of cultivar; the second letter corresponds to orchard location and the proceeding letter(s) signifies type of pistachio collected (i.e. S for sound nuts, ES for earlysplit and GS for growth split nuts respectively). F is used for fluorescent nuts and NF is used for non-fluorescent nuts; the cross-section colour is mentioned separately. Samples from processing terminals Five cumulative samples were collected from terminals that were under the supervision of the Department of Pistachio Affairs in a national experimental HACCP plan for first year, so harvest and transportation practices were controlled to ensure ventilation and avoid compaction, and nuts were hulled during 8 hours after harvest. These are named as follows: .

.

.

.

.

KGSD: Sample from nuts, which were processed, and sun-dried after mechanical drying; TMain: Collected from Sinkers line of flotation tank, after removal of nuts with adhering hull (in Iran these nuts are named ‘‘goo’’); TFloat: Collected from Floaters line of flotation tank after removal of blank nuts; FMain: Collected from Sinkers line of flotation tank, after removal of nuts with adhering hull (goo); HRND: Collected from output of an air-based gravity separator (table) system which substitutes for water floatation tank in some terminals.

Results and discussion The results are shown in Table I. In Table II a summary is given. Unexpectedly high concentrations of aflatoxin were observed in some samples. This could be related to much higher frequencies of Aspergillus moulds (Doster & Michailides 1994b).

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This is possibly due to higher concentration of related fungi spores compared to the US. Pistachio litter and hull are good mediums for A. flavus growth so could increase spore counts and the inoculation rate in Iranian producing areas in which disposal of hulling residues in production fields is a regular practice (Doster & Michailides 1994a, Mahoney & Rodriguez 1996). For example, 17.45 g of pistachio in MHES/HF could contaminate a 723 kg of otherwise clean bulk of pistachio. This situation demands a special consideration for finding applicable methods toward decreasing in-orchard contamination in Iranian producing areas as the first step for a longterm program. As only in three cases trace amounts of G group of aflatoxins was observed, we could conclude that A. flavus is the dominant aflatoxin producer in our samples. Brown coloured kernels Except for two single non-fluorescent and brownkernel nuts which had ND and 1.34 mgkg1 (AAES/ NF, KAES/NF), and a brown spotted sample with 31.51 mgkg1 aflatoxin (KHGS/NF), all the brown coloured group nuts were in the fluorescent portion with large amounts of aflatoxin. As summarized in Table II, at harvest the brown coloured kernels contain more than 98.1% of total aflatoxins and actually are a subdivision of fluorescent nuts. In other words, by removal of fluorescent nuts, the brown kernel part is removed as well, which means the removal of at least 99.2% of aflatoxin in our study. In two samples both from Akbari cultivar (AA, AM), the aflatoxin content in brown kernels is lower even though aflatoxin concentrations in brown kernels still are substantially higher than normal nuts. In addition, as could be drawn from Table I, these samples contain the least aflatoxin content as well, which could be considered a sign for difference among varieties in aflatoxicosis susceptibility and characteristics. These two samples also had three sub-samples (AMES/F-brown kernels with heavy mould, AMES/F-brown kernels and AAES/Fbrown kernels), which their actual concentration was not quantified and contain higher levels than reported here. The possible higher levels of aflatoxin in these samples would raise the share of brown kernels and fluorescent nuts in contamination further. We can conclude that any sign of brown colour or spots in kernels are a good criterion for identification of highly-contaminated nuts that become contaminated either in pre-harvest or postharvest. Based on substantially more contaminations in brown coloured nuts compared with brown-like and brown-spotted nuts, it seems that this factor (browning) has a tendency to be quantitative in nature and probably related to the extent of physical

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Table I. Results of aflatoxin tests of pistachio sub samples separated based on BGY fluorescence and then cross-section colour.

Samplea

Kind of pistachiob

Divided based on BGYF presence - then by kernel’s cross section appearancec

Nuts with visible mould infestation

KG (2)

S

F-Sound Kernels (14) NF-Sound Kernels (584) F-Sound Kernels (34.9) F-Brown-like Kernels (23) F-Brown Kernels with Black Fungal Colony (7) F-Brown Kernels with Green Fungal Colony (8.8) NF-Sound Kernels (100) F-Sound Kernels (2.3) NF-Sound Kernels (571) F-Sound Kernels (43) F-Brown Kernels (7.6) F-Brown Kernels with Heavy Mould (7.6) NF-Sound Kernels (168) NF-Sound Kernels (136) F-Sound Kernels (20.1) F-Brown Kernels (9) NF-Sound Kernels (12) F-Sound Kernels (70.5) F-Brown Kernels (15.9) NF-Sound Kernels (119.5) NF-Brown-Spotted Kernels (9.9) NF-Sound Kernels (85.1) F-Sound Kernels (113.6) F-Brown Kernels (7.34) NF-Sound Kernels (27.3) NF-Brown Kernels (1.3) HF-Sound Kernels (71.5) HF-Brown Kernels (9.1) F-Sound Kernels (76.5) F-Brown-Spotted Kernels (2.5) NF-Sound Kernels (36.2) F-Sound Kernels (5.7) NF-Sound Kernels (91.6) HF-Sound Kernels (56.8) HF-Brown Kernels (12.4) F-Sound Kernels (46.4) F-Brown Kernels (8.8) NF-Sound Kernels (6.3) NF-Brown Kernels (1.4) HF-Sound Kernels (49.3) HF-Brown-Spotted Kernels (5.4) F-Sound Kernels (58.6) F-Brown Kernels (4.8) NF-Sound Kernels (13.5) F-Sound Kernels (65.7) NF-Sound Kernels (92.8) HF-Sound Kernels (124) HF-Brown Kernels (17.45) F-Sound Kernels (15.6) F-Brown-Like Kernels (2.5) NF-Sound Kernels (1.7) HF-Sound Kernels (83.3) HF-Brown Kernels (5.7) F-Sound Kernels (19.5) F-Brown-Like Kernels (1.9) NF-Sound Kernels (1.1)

0% 0% 11% 9% 100% 100% 1% 0% 0% 7% 53% 100% 0% 0% 15% 22% 0% 7% 25% 0% 10% 0% 7% 41% 0% 100% 1% 44% 4% 0% 0% 0% 0% 14% 100% 4% 57% 0% 100% 6% 0% 5% 100% 0% 0% 0% 6% 100% 13% 80% 0% 4% 0% 15% 0% 0%

ES

AM (2)

S ES

KH (24)

S ES

GS

AA (36)

S ES

GS

KA (48)

S ES

GS

MH (72)

S ES

GS

Aflatoxins concentration (mg/kg)d B1

B2

Total

0.88 ND 4.97 21.43 380.96 8801.8 ND 14.52 ND 39.88 682.84 526.47 106.83 0.68 25.57 538.03 ND 19.87 51559 1.27 28.49 ND 121.99 374.13 1.4 ND ND 51.61 58.76 6.04 2.95 ND ND 27.9 61689 5.14 578.55 37.16 1.34 ND 14.73 ND 594.81 235.89 ND ND 124.42 78557 0.5 206.28 ND 0.69 2.78 ND 0.5 3.94

ND ND 0.69 2.83 0.77 513.6 ND 2.33 ND 4.16 2.39 13.02 4.33 ND 1.14 0.69 ND 1.37 5089 ND 3.02 ND ND 1.13 ND ND ND ND 3.51 ND ND ND ND 1.29 5816 ND 1.23 3.59 ND ND 0.94 ND ND 19.26 ND ND 6.21 4342 ND 9.96 ND ND ND ND ND ND

0.88 ND 5.66 24.26 381.73 9315.39 ND 16.85 ND 44.04 685.23 542.81* 111.16 0.68 26.71 538.72 ND 21.24 56648.71 1.27 31.51 ND 121.99 375.26 1.4 ND ND 51.61 62.27 6.04 2.95 ND ND 29.19 67504.77 5.14 579.78 40.75 1.34 ND 15.67 ND 594.81 255.15 ND ND 130.63 82899.79 0.5 216.24 0.84* 0.69 2.78 ND 1.11* 3.94

a The number in bracket shows the duration of inappropriate storage (h); bS ¼ Sound, ES ¼ early split, GS ¼ growth split; c F ¼ Fluorescent, NF ¼ Non-Fluorescent, HF ¼ High-Fluorescent, the number in bracket shows weigh of the sub sample (g); d In no case aflatoxin G1 is detected; *The subsample has low levels of aflatoxin G2; ND ¼ Not Detected.

Physical properties of aflatoxin-contaminated pistachio nuts

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Table II. Contamination distribution in samples collected from orchards between BGYF vs. non-BGYF nuts and natural coloured cross-sections vs. all types of brown-coloured kernels. Total of all samples (calculated from Table I). Weigh percentage

Concentrated aflatoxin in each part

Mean aflatoxin concentration (mg kg1)

BGYF pistachios Non-BGYF pistachios

25% 75%

99.2% 0.8%

2414.99 9.86

Brown coloured kernels Natural coloured kernels

5% 95%

98.1% 1.9%

14157.42 41.73

growth of fungi in intra-cotyledonary space. In an earlier study on kola nuts, it was noted that the inner surfaces of the cotyledons were infected more rapidly than the outer surfaces by A. flavus (Adebajo 2000). Correlation coefficient between the occurrence of all brown coloured types and aflatoxin contamination occurrence was 0.404 which is considered statistically significant ( p  0.01, df ¼ 52). Correlation coefficient between the occurrence of brown coloured kernels (excluding intermediate types) and aflatoxin contamination occurrence was 0.431 which is considered statistically significant as well ( p  0.01). Aflatoxin contamination in nuts with different hull conditions (1) Pistachios with sound hull (unsplit). As could be calculated from Table I, mean concentration of aflatoxin in F and NF portions are 0.58 and 0.059 mgkg1 respectively, that demonstrates a good separation resulting from near ten-fold concentration of aflatoxin in F portion. In none of the non-fluorescent unsplit pistachio samples notable aflatoxin was observed, even after 72 h of storage in unfavourable conditions. In fluorescent portion of sound pistachios, the contamination mean is neglectable and in no case, sound pistachios are contaminated even with peak of 16.85 mgkg1 contamination in AMS/F, which makes up 0.06 mgkg1 in AMS. This pinpoints the protective role of intact pistachio hulls during growth stages in the orchard. Any measure for reduction of any kind of hull damage including splitting could reduce the number of potential contaminated nuts. The fluorescence occurrence in unsplit nuts is relatively low at harvest and reached to maximum of 2.3% (in KGS) even though after storage in unfavourable conditions, BGYF percentage raised up to 41% in MHS but still no aflatoxin was detected. We assume that this could be because of entrance of fungi through slight hull surface damages, which gave proper establishment site to fungus in prolonged non-ventilated circumstance to grow towards and on the surface of the shell, with BGY footprints and inadequate time for progress toward kernel and toxin production. In AMS/F and

KGS/F 16.85 and 0.88 mgkg1 aflatoxin is detected respectively. As these samples were not stored in unfavourable situations, we could conclude that the contamination originated from the orchard. The possible ways and role of insects could be a matter of investigation. It is reported that the vascular system of pistachio nuts could act as a route for the infiltration of kernels with A. flavus and other fungi (Denizel 1992). (2) Growth split (cracked hull) pistachios. This kind of hull split takes place in less than 15 days of harvest and is characterized by ragged brown edges and much wider splits than early splits and random orientation of split on hull (Pearson et al. 1994). In this group, 910447 ng and 4018 ng aflatoxin is present respectively in F and NF portions. Mean concentration of aflatoxin in these portions are 1916.36 and 22 mgkg1 respectively, which demonstrates accumulation of more than 99.5% of aflatoxin in F portion. In growth split pistachios, like sound pistachios, significant increase of F percentage by unfavourable storage is noticed, which is accompanied by an increase of aflatoxin. Earlier harvest, if applicable, can reduce growth-split associated problems to a least possible amount. In Californian orchards, it is estimated that the incidence of decay by aflatoxin-producing fungi in growth split nuts was substantially lower than that of early-split nuts (Doster & Michailides 1994b). In our study, the concentration and variation of aflatoxin in this group is more than expected. It sounds that in Kerman and Rafsanjan circumstances any hull shortage has greater effects compared to other locations, probably because habituation of fungus and improper measures like uncontrolled dispersal of processing resulted in green hulls in the orchards of the production area, which is estimated around 200,000 tons per year by fresh weight. The pistachio hull is a good medium for A. flavus growth and sporulation (Mahoney & Rodriguez 1996). It was reported earlier that the Aspergillus moulds were found at much higher frequencies in Iran compared to California and pistachio litter is introduced having an important role in the infection of nuts by increasing the amount of Aspergillus inoculum in pistachio orchard (Doster & Michailides 1994a).

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(3) Early-split pistachios. As noticed before, this group was the main source for high contaminations in pistachio nuts because the fungus has the most time for establishment and development of contamination when compared to other kinds of split. Like for preceding groups, the substantial part of aflatoxin is in F portion of this group. In F and NF portions 2426507 ng and 18975 ng aflatoxin are present respectively. Mean concentration of aflatoxin in these portions are 4890.6 and 63.03 mgkg1 respectively. This demonstrates accumulation of more than 99.9% of aflatoxin in F portion. In early-split pistachio nuts, like sound and growth split pistachios, significant increase of BGYF percentage by unfavourable storage is noticed. Generally, it is observed that based on aflatoxin content, early-split nuts ranked first, growth split nuts follow them, and sound (unsplit) nuts are near completely clean pistachios. Cross-section of a noncontaminated single pistachio (AAES/NF) in this group which had brown kernel but not fluorescent, was examined under UV light; A fluorescent BGY trail was observed which matched roughly with vascular tissue which proposes possible infection in the orchard by fruit stem defects or sucking insects and subsequent growth of fungus through vascular tissue into the kernel. Penetration of fungus to pistachio kernel through vascular tissue was investigated earlier (Denizel 1992). Samples collected from processing terminals In five samples collected from processing lines, which were divided based on fluorescence occurrence, the orchard results are confirmed (Table III). Mean concentration of aflatoxin in F portion is 158.43 mgkg1 while for NF portion this amount drops to 2.28 mgkg1 of total aflatoxins. In these samples by removing minimum 1.8% and maximum 7.3% with mean of 4.4% of BGYF pistachio nuts at least 51.5% with peak of 100% and average of 82.8%

of aflatoxin is excluded as well. Two samples (KGSD HRND) had higher amounts of aflatoxin in fluorescent portions than mentioned which could increase the contamination share of this portion further. Fluorescence as a discriminating factor for aflatoxin-contaminated pistachio nuts As can be seen in Table I, in all samples except AM, regardless of cultivar or unfavourable storage period, more than 99% of aflatoxin is accumulated in BGYF nuts. Even when considering only early-split nuts of any orchard in Table I (with mean concentration of 2808 mgkg1) we see that except KG and AM which still show a good separation, in all other samples the contamination is reduced below the legal limit of EU countries. These results comply with reports on cotton and corn at harvest and show that in pistachio nuts a similar mechanism for BGYF creation by A. flavus persists. We propose the use of this BGY fluorescence as an identification criterion for A. flavus-contaminated nuts and consequently the majority of aflatoxin-contaminated pistachio nuts at harvest. Correlation coefficient between occurrence of fluorescence and aflatoxin contamination occurrence in all orchard samples was 0.388, which is considered statistically significant ( p  0.01). Provided that the handling of fresh nuts ensures enough ventilation without excessive compaction, this criterion is practicable at the end of the processing line. The results from processing terminals confirm this claim as well. Presumably, if the storage condition secures proper water activity for prevention of fungal growth this principle could be applied after storage as well. Because of its common mechanism this discriminating factor worked well among tested cultivars and is almost definitely applicable on other cultivars as well. In particular, we can suggest it for the large diversity of cultivars cultured in Iran and either it may yield for other tree-nuts as well. Using automated techniques for

Table III. Aflatoxin distribution between fluorescent and non-fluorescent sub-samples that were resulted from separation of samples collected from processing terminal; based on BGYF occurrence on shell. Calculated aflatoxin concentration (mg kg1)

Aflatoxin concentration in sub-samples divided based on BGYF occurrence on shell (mg kg1)

KGSD

9.74

FMain

0.34

HRND

36.51

TMain

1.69

T Float

0.83

550 *ND 8.93 *ND 314.45 *14.78 35.47 *0.84 12.22 *ND

Sample name

*Non-BGYF pistachios. The ‘‘’’ sign indicates that the actual contamination is more than presented value.

Physical properties of aflatoxin-contaminated pistachio nuts detection of BGY-fluorescent nuts like UV-based sorters could enhance the detection efficiency further. It is worth mentioning that the presence of BGY fluorescence does not ascertain aflatoxin contamination, rather it is indicative of A. flavus growth record. It maybe that the fungus has not had enough time for complete establishment toward kernel and toxin production, it is of non-toxigenic strains, or the fungus has given up the site to competitive microorganisms later. Observation of intensive black mould colonies probably A. niger together with BGYF and high aflatoxin B1 concentrations points to A. flavus growth history that possibly has given up the site to A. niger, a more aggressive species, later. Improper handling of freshly-harvested pistachios creates BGYF in uncontaminated nuts. Post-harvest contaminations which are produced by adequate water activity for fungus to germinate and develop after the pistachio nut has been dried, are not characterized by BGY fluorescence, apparently because lack of peroxidase activity to yield BGYF substance from kojic acid produced by A. flavus. These factors reduce the observed correlation between BGYF and contamination in poorly handled nuts. This is probably the source of earlier unsuccessful attempts at using BGYF as a sorting basis for aflatoxin in pistachio nut. Observation of visible mould occurrence The percentage of nuts with any visible fungal growth was observed qualitatively and calculated for every sample (see Table I). As can be seen, consequent data complies with previous results from fluorescence and brown kernels. The correlation coefficient between occurrence of visible mould and aflatoxin concentration was 0.436, which is considered statistically significant ( p  0.01). Fluorescent and brown-kernel nuts with mean fungal growth percentage of 51.7% ranked 1st; Fluorescent and normal kernels are in the next place with mean of 7.93% and finally non-fluorescent and normal nuts had the least amount of fungal growth with a mean of 1.2% (Two single non-fluorescent pistachios with brown section and with fungal growth are not included). This indicates that by removing fluorescent nuts most of the moulded nuts are removed as well. Earlier works indicate that in tests using hull inoculation, A. flavus prefers entering kernels via vascular tissue at nut stem or shell sutures after hull colonizing (Sommer et al. 1976, 1986). In this study in most cases, it was visibly observed that fungus growth was concentrated in the placental region of the kernel where the conducting tissue enters the kernel. This matched with browning of intra-cotyledonary

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space. Generally, we can conclude that in-orchard contaminations probably obey the same rules as inoculation tests, which are, reported earlier, i.e. the fungus gains entry from any kind of splits in hull and grow between hull and shell space until reaching to nut stem and after penetrating via numerous microscopic passages here, the fungus reaches the kernel in the placental region. The fungus possibly follows the least physical restriction with adequate moisture path, which directs it to intra-cotyledonary space, the observed brown colour or spots here, probably may act as an indicator of physical growth and/or restriction pattern of fungus. This can explain why the BGYF is observed on the shell and propose that direct entrance and growth of fungus in the kernel of early-split nuts is an infrequent phenomenon. In another words, the hull split, not the shell split, is the main contributing factor in fungi spore establishment in pistachio nuts. On the other hand, in spotted kernels, the spots were not overlapped with the stem end. These spots may be a sign of controlled fungi by plant defence mechanisms in case of penetration from other than stem end entrance point. These samples show lower levels of aflatoxin compared to brown kernels as is evident in Table I. Fluorescence expansion during unfavourable storage conditions By increasing storage time in unfavourable conditions, the percentage of fluorescent nuts increases rather exponentially with correlation coefficient of 0.94 that is considered statistically significant ( p  0.01). In addition, the intensity of BGYF increases to such an extent that we divided the F portion further to High-Fluorescence (HF) and Fluorescent with the purpose of evaluating the qualitative or quantitative nature of fluorescence. It was observed that any direct relationship between shell fluorescence intensity and contamination levels cannot be formulated. Possibly despite fungus has grown between the hull and the shell and produced BGY fluorescence, either it could not penetrate into the kernel or if penetrated, it had not enough time for toxin production. However, even with standard inoculations, a large variability in aflatoxin production is reported by Mahoney and Rodriguez (1996). Regarding present data, it is concluded that fluorescence is better to be regarded rather qualitative, as done here, to yield better for removal of contaminated nuts. An increase of storage period in unfavourable conditions also resulted to relatively more aflatoxin build-up although the correlation coefficient was lower (0.81) and is considered statistically insignificant. The relative constant percentage of brown

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coloured nuts supports this idea as well. This, perhaps, was modified by working on a single cultivar and certainly changes by more prolonged incubation periods. In an additional sample from a delaying bulkhandled pistachio, which had a bulk-core temperature of 39 C, prior discharge in the processing terminal, in addition to extensive visible staining which is common for this kind of pistachio handling, 21% of nuts were BGYF. This kind of handling can decrease effectiveness of later visual sorting by increase of stained nuts as well. This experiment is comparable to the BGYF percentage range of 4–17% that was reported by Dickens and Welty in Iranian pistachio lots, which were exported to US. Based on present results it is expected that BGYF percentage in Iranian normal maintained, harvested and handled pistachios be around 4%. Late harvest and improper handling could significantly increase this percentage. For other countries with lower spore concentration lower percentages are projected. Here we notice the BGYF percentage in pistachio works as an applicable index for judgement on pistachio-handling quality until hulling. A promising different pattern of lower contamination and brown kernel contamination is noted in Akbari cultivar. Further work may reveal substantial difference between cultivars in aflatoxin production capacitance and lead to breeding and using of cultivars that limit or reduce aflatoxin production by invading fungi. Conclusion BGY fluorescence could be used for identification of most A. flavus-infested pistachio nuts and consequently the substantial majority of aflatoxincontaminated nuts. The BGYF worked well among different cultivars studied. The observed contaminations are very high which first point out the possible high inoculation rate by fungus in the Iranian production area and, second, to the ability of this method for separating highly contaminated nuts. The growth split pistachios showed relatively higher contaminations comparing reports from US. Poor handling of fresh harvested nuts could lead to an increase of BGYF nuts along with visible discoloration, which reduces both the effectiveness of sorting based on BGYF and prevailing hand-sorting. The percentage of BGYF nuts in a lot also could help handlers to assess the pre- and post-harvest handling quality of nuts. Contaminations that take place after rehydration of dried nuts due to improper storage are not characterized by BGYF because lack of enzymatic activity. The browning of intra-cotyledonary space as a proof for aflatoxin presence, which

was earlier raised by Steiner et al., is confirmed in studied cultivars at harvest. It is concluded that hull splits or lesions could provide suitable places for fungus establishment, then it grows toward the kernel via the shell surface and stem end with BGYF footprints.

Acknowledgments The author wishes to acknowledge the authorities of the Iranian Ministry of agriculture in Tehran, Kerman and Rafsanjan, particularly Hamid Fizi and Behrouz Gheibi for help and support and Fariborz Shojaee for special consideration and accuracy during aflatoxin tests.

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Steiner WE, Brunschweiler K, Leimbacher E, Schneider R. 1992. Aflatoxin and fluorescence in brazil nuts and pistachio nuts. Journal of Agriculture and Food Chemistry 40:2453–2457. Wicklow DT. 1999. Influence of Aspergillus Flavus strains on aflatoxin and bright greenish yellow fluorescence of corn kernels. Plant Disease 83:1146–1148. Zeringue HJ Jr, Shih BY, Maskos K, Grimm D. 1999. Identification of the bright-greenish-yellow-fluorescence (BGY-F) compound on cotton lint associated with aflatoxin contamination in cottonseed. Phytochemistry 52:1391–1397.