Carry-Over of Aflatoxins to Fig Molasses from Contaminated Dried Figs

Abstract

The carry-over of aflatoxins to fig molasses produced by using two different processing techniques from contaminated dried figs (> 1000 ppb of total aflatoxins) were examined by using a HPLC technique. The effects of extraction, bleaching and concentration steps on the reduction of aflatoxin levels were also investigated. The reductions in total aflatoxin levels in fig molasses produced by using the two techniques were detected to be 62 and 38%, respectively. Extraction, bleaching and concentration steps were observed to cause 22% reduction in total aflatoxin levels.

Keywords:

• Aflatoxins
• Figs
• Molasses

INTRODUCTION

Aflatoxins, potent carcinogenic, mutagenic, and teratogenic metabolites produced by the fungal species Aspergillus flavus and Aspergillus parasiticus, can contaminate human foods and animal feeds. Such contamination is the result of invasion by molds before and during harvest, or because of improper storage of agricultural commodities. The major agricultural commodities susceptible to aflatoxin contamination are corn, peanuts, figs, tree nuts, rice, dried fruits, pumpkin seeds, and cottonseeds.[1–3] With increasing knowledge and awareness of aflatoxin as a potent source of health hazards to both man and farm animals, a great deal of effort has been made to completely eliminate the toxin or reduce its content in foods and feedstuffs to significantly lower levels.[2] Several physical (heat, irradiation, UV, and visible light),[4–7] chemical (chlorine, hydrogen peroxide, ozone, bisulphite, ammonia, alkalis, acids)[8–10] and biological methods[11–14] for detoxification of aflatoxins have been reported, but they are not applicable to foods because of safety aspects.

By the mid 1970s there had been some recalls of dried figs in the USA because of aflatoxin contamination[15] and it has been reported that fig fruits can accumulate aflatoxin comparable to those found in peanuts and cereal grains. Aspergillus flavus was shown to be a vigorous pathogen to ripe fig fruits[16] and fig was found to have the highest potential for aflatoxin formation considering the uncooked fig fruits.[17] In 1985, a laboratory of a food retail chain reported high aflatoxin contamination in the dried figs imported to Switzerland. With the recognition of figs as a high risk commodity for aflatoxins, a simple and rapid sorting procedure in which BGY (bright greenish-yellow) fluorescence under ultraviolet (UV) radiation (365 nm) is used [18] This technique is widely used in the fig manufacturing industry as a physical sorting procedure. A number of studies have been reported to give promising results for degradation of aflatoxins in dried figs by using sodium bisulphite or sulphur dioxide alone or in combination with other chemicals [19–21] However, the toxicological aspects of aflatoxin degradation by using sodium bisulphite and other chemicals are still not relevant for fig industry. Thus, sorting under UV lamps seems to be the most suitable method for efficiently cleaning dried figs from aflatoxins on industrial scale. The contaminated figs sorted out under UV lamps are disposed as toxic wastes and this leads to important economic losses in fig industry. The aim of the present study was to investigate the possibility of aflatoxin decontamination at different steps while producing molasses from these figs. For this purpose, fig molasses was produced from contaminated figs by using two different processing techniques and the carry-over of aflatoxins was observed.

MATERIALS AND METHODS

Dried Fig Samples

The aflatoxin contaminated dried figs screened under UV light (365 nm) as suggested in the Swiss method[18] were supplied by Silver Gida A. S. The initial aflatoxin levels were detected to be different during molasses production by the two different techniques that were applied.

Aflatoxin Standard Solutions

Aflatoxins B₁, B₂, G₁, and G₂ were supplied from Sigma Chemical Company. A mixed standard solution in toluene:acetonitrile (98:2) containing a total of 8.5 μg ml⁻¹ (2.86 μg B₁ and G₁, 1.43 μg B₂ and G₂) was prepared as a primary standard to be used in aflatoxin analysis.

Sampling

A batch of 5 kg aflatoxin contaminated fig samples were obtained as the laboratory sample. 600 g sub samples obtained by using the quartering technique as suggested in Pearson[22] were used to produce molasses from figs (300 g) and to determine aflatoxin contents of the raw material (300 g). The same sampling procedure was applied in two different processing techniques used during molasses production.

Production of Fig Molasses

The flow chart for production of fig molasses by two different processing techniques is shown in the Figure 1. In the processing technique (I), 300 g contaminated dried fig samples were washed by using tap water to remove the filth, sand particles and pesticide residues. The solid-liquid extractions with three steps were applied to obtain fig extract. In each extraction step, 100 g of dried figs were treated with water (1:1.5 solid-liquid ratio) for 90 minutes at 75°C in a shaking water bath (shaking: 300 speed/min). The extraction continued until the dry matter content of the solution was 5°Brix and the solid particles were removed from the extract by using a thin cotton cloth. In the bleaching step, the extract was treated with earth (94% CaCO₃) to neutralize and clarify the solution. The solution was filtered through coarse filter paper and concentrated under vacuum in a rotary evaporator (Bibby Rotary Vacuum Evaporator RE 100) at 90°C to 76°Brix. The concentrate was pasteurised in an autoclave at 82°C for 20 minutes.

Figure 1 Production of fig molasses by two different processing techniques (I);[27] (II).[28]

In the processing technique (II), the fig samples were washed by using tap water as described in technique (I). In the solid-liquid extraction step, 125 g of dried figs were treated with water (1:1.5 solid-liquid ratio) for 30 minutes at 75°C shaking water bath (shaking: 300 speed/min). The extraction continued until the dry matter content of the solution was 5°Brix and solid particles were removed from the extract by using a thin cotton cloth. The extract was treated with 2% enzyme solution (Ultrazyme 100, Schweizerische ferment AG, Basel) and the mixture was treated with the 5% gelatine solution (commercial type gelatine, Rousselot LTD, London) for clarification. In the filtration step, the solution was filtered through coarse filter paper and concentrated under vacuum in a rotary evaporator as described in the processing technique (I).

HPLC Analysis

The aflatoxin extraction and purification steps of the analysis were performed as suggested in Steiner et al.[18] A reversed phase high-pressure liquid chromatographic system (Hewlett Packard Series 1050) involving a fluorescence detector (Waters 470 Scanning fluorescence detector, Em: 432 nm, Ex: 365 nm) was used for quantification of the aflatoxin levels. The aflatoxins (B₁, B₂, G₁, and G₂) were separated by using C₁₈ μ-Bondapak column (3.9 × 300 mm) at an elution rate of 0.7 ml min⁻¹ and pressure between 85.3–111.8 × 10⁵ Pa. The elution solvent consisted of acetonitrile:methanol:water (15:15:70 v/v/v) as described by Supelco.[23] Aflatoxin standards were obtained from Sigma Chemical Co. and a solution in toluene:acetonitrile (98:2) containing a total of 8.5 μg ml⁻¹ (2.86 μg AFB₁ and AFG₁, 1.43 μg AFB₂ and AFG₂) was used.

RESULTS AND DISCUSSION

The aflatoxin contents of figs and the fig molasses produced by two different processing techniques are given on Table 1. The total aflatoxin levels of dried figs used in both techniques were significantly high (<1000 ppb) with aflatoxin B₁ being the highest among the other aflatoxins. It can be also observed that the total levels of aflatoxins used in technique (II) were almost four times higher than those used in technique (I), thus the molasses obtained from these figs involved the aflatoxins at significant levels. It can be observed from Table 1 that by using technique (I) the lowest carry-over was detected in aflatoxin B₁ and a reduction of 62% was obtained in total aflatoxins, while only 38% of reduction in total amount of aflatoxins was obtained by using technique (II). This result may be due the high amount of aflatoxin levels (< 4000 ppb) present in the figs used for production of fig molasses, as well as the inefficiency of the production stages used in techniques (II). The effects of various stages in technique (I) such as extraction, bleaching and concentration on aflatoxin levels were also investigated. In order to determine the efficiency of these steps, the aflatoxins remained in the residue after the extraction step and in the bleaching earth were determined. The difference between the aflatoxin levels before and after the concentration step were also calculated (Table 2). As it can be observed from Table 2, the contribution of the mentioned stages to the reductions of total aflatoxin contents were not as high as expected, and the data obtained was not sufficient to explain the total 62% reduction obtained by application of this technique. In a study conducted on aflatoxin removal from pistachio nuts, it was detected that 38–100% reduction in aflatoxin B1 was obtained by treatment with natrolite (a natural zeolite).[24] The addition of zeolites to bleaching earth in technique I can be efficient in adsorbing and removal of aflatoxins during production of fig molasses. However, the effects of this procedure on the sensory quality of the molasses should also be investigated.

Table 1 Aflatoxin levels of dried figs and fig molasses and carry-over values

	Technique I			Technique II
Aflatoxins	Figs (μg/kg)	Fig molasses (μg/kg)	Carry-over of aflatoxins from fig to molasses (%)	Figs(μg/kg)	Fig molasses (μg/kg)	Carry-over of aflatoxins from fig to molasses (%)
B1	969 ± 334	342 ± 58	35	2026 ± 352	1383.7 ± 359	68
B2	0.3 ± 0.02	0.2 ± 0.1	67	248.8 ± 61	155.3 ± 53	62
G1	46 ± 11	21 ± 10	46	1790 ± 196	1006.2 ± 129	56
G2	96 ± 26	55 ± 20	57	231.3 ± 38	105.3 ± 24	46
Total	1111.3 ± 78	418.2 ± 30	38	4296 ± 356	2650.5 ± 168	62

Table 1 The reductions in total aflatoxin levels during extraction, bleaching and concentration steps as applied in technique I

Stage	Reduction in total aflatoxin level (%)
Extraction	7
Bleaching	5
Concentration	10
Total	22

CONCLUSION

The results of the present study indicated that the manufacture of molasses from contaminated figs using the processing technique (I) has significant (p < 0.05) contribution to the reductions of aflatoxin levels in the final product. Since the figs used in the study were highly contaminated, it was only possible to reduce this level to 418.2 ppb. This level was significantly high in comparison with the maximum permitted level (10 ppb (total aflatoxins) as stated in the regulations.[25,[26] Thus, technique I seemed to be promising in case of manufacturing molasses from dried figs involving total aflatoxin levels lower than 60 ppb. Further studies for increasing the effects of bleaching and filtration steps on aflatoxin reduction by using different bleaching agents and filtration materials are also recommended.