Sugarcane, Saccharum officinarum
, is a tropical tall perennial grass cultivated in several countries of the world. In Africa, sugarcane is the second most cultivated crop after cassava, where Egypt maintains the second position after South Africa [1
]. In Egypt, around 97% of the total sugarcane production, 16 million tons in 2014, is cultivated in the upper part of the country [2
]. From the economical point of view, sugarcane is an important cash crop beside cotton and a major contributor of income and employment for farmers. The plant has a high sucrose and low fiber content and is used mainly for raw sugar and molasses production (brownish-black viscous syrup known as black honey in Egypt), in addition to the grass left over or bagasse, which is used as an animal feed supplement or fertilizer. Furthermore, several secondary industries such as vinegar, alcohol, chipboard, paper, some chemicals, plastics, paints, fiber, insecticides and detergents are based on sugarcane and its wastes [2
]. It has been estimated that 80% of the world’s sugar comes from sugarcane [7
], where Brazil and India are the largest producers worldwide [4
]. The annual consumption of sugar in Egypt in 2010 was estimated to be 34 kg per capita [7
During the harvesting time, chewing raw sugarcane is a common practice. In addition, sugarcane juice is considered the most popular fresh juice in Egypt, with cane juice shops spreading through all the Egyptian cities. Indian and Pakistani people share the same habit with Egyptians regarding chewing raw sugarcane and consumption of juice [9
]. Apart from its sweet taste and being a source of energy and minerals, sugarcane juice consumption, in traditional medicine, helps in the treatment of many diseases such as jaundice, kidney stones, urogenital tract infections, and in lowering blood pressure, and healing dermal wounds; it is also reported as a natural antioxidant under various experimental conditions [10
According to the Food and Agriculture Organization, 25% of the world’s crops are contaminated by fungal toxic metabolites [12
]. Sugarcane is a suitable host for many saprophytic fungi, especially the aflatoxigenic ones that belong to the Aspergillus
]. Products of secondary fungal metabolism, some of them being toxic and thus termed mycotoxins, can be formed either in the field and/or during storage. The most significant mycotoxins in terms of food and feed safety are aflatoxins, ochratoxin A, fumonisins, zearalenone, trichothecenes, and ergot alkaloids that have carcinogenic, mutagenic, teratogenic, cytotoxic, neurotoxic, nephrotoxic, estrogenic, dermotoxic, and immunotoxic effects [12
Ingestion of contaminated food is the principal route for human exposure to mycotoxins [17
]. One of the most important aspects in the risk analysis of food contaminants is to determine the degree of human exposure [18
]. In the case of mycotoxins, this exposure is generally assessed by taking into account data on mycotoxin occurrence in foodstuffs as well as data on the dietary consumption of the concerned population [19
], although analytical methods for determination of individual exposure by analysis of biofluids have recently been developed [21
There are some methods used to assess dietary consumption, generally known as market basket, 24 h dietary recall and food record methods, food frequency methods or dietary history [25
]. The degree of exposure is measured in terms of probable daily intake (PDI) per unit of body weight, and is generally expressed in ng/kg of body weight (b.w.) per day. Afterwards, the PDI value is compared with the tolerable daily intake (TDI) which is determined by certain toxicological studies for risk analysis. Several exposure and risk assessment studies for mycotoxins in different food commodities were carried out during the last few years [25
]; nevertheless, no exposure assessment for mycotoxins from contaminated sugarcane grass and juice has been conducted yet.
The growth of toxicogenic fungi in a sugarcane crop field was documented in numerous studies [13
]. However, these previous studies discussed only the isolation of different fungal species from the plant and the prevalence of their mycotoxins after inoculation of the isolated fungi in culture media without determining the natural (co-)occurrence of the produced mycotoxins. Ahmed et al. (2010) screened the contamination of sugarcane juice sold in Pakistan with several mycoflora including Aspergillus flavus
, A. fumigatus
, and A. niger
], while Hariprasad et al. (2015) investigated the natural aflatoxin uptake by sugarcane from contaminated soil and its persistence in sugarcane juice and jiggery (the natural sweetener made by concentrating the sugarcane juice) using thin layer chromatography and ELISA [34
]. To the best of the authors’ knowledge, no reports have been published for the natural occurrence of multiple mycotoxins in cane grass and juice. Moreover, no regulations in Egypt or other countries for mycotoxins in this commodity have been established so far. Therefore, it was worthwhile to perform this survey to screen for a wide range of (toxic) fungal metabolites in sugarcane grass and juice sold in Assiut City, Egypt, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an estimation of the seasonal human exposure to mycotoxins from sugarcane juice in order to evaluate the possible health risks.
The sugarcane crop and its by-products have a great agro-industrial and economic value. Upper Egypt governorates are the main home to sugarcane cultivation and its industry in Egypt, particularly in the governorates of Sohag, Qena and Aswan [36
]. The choice of Assiut City, not a major state for sugarcane cultivation in Egypt, was based on the availability of the samples and collection of data by the authors.
During harvest time, cutting of the sugarcane stalk disrupts the physiology of the plant and acts as a portal of entry for pathogenic bacteria and fungi. Some of these microorganisms, under elevated temperatures and high humidity, use the sugar as a source of energy for growth and produce different metabolic by-products that, on one hand, may cause processing problems in the mill and refinery and, on the other hand, can be toxic to animals and humans [33
]. Red rot, smut, wilt and sett rot are the most important fungal diseases affecting sugarcane agriculture and causing remarkable economic losses [38
]. This necessitates a detailed investigation of the probable effect of mycotoxins on the sugar industry and the other by-products such as alcohol as one of the main products derived from sugarcane.
In the light of the aforementioned results, the level of AFB1
in sugarcane was higher than the highest maximum level regulated by the EU (12 μg/kg for unprocessed almonds, pistachios, and apricot kernels) [41
] and Egyptian standards for peanuts intended for human consumption (5 μg/kg) [42
]. AFs in sugarcane crops can be derived from two sources, either through the natural AF uptake from contaminated soil in the field [34
] or from fungal attack to the outer fiber layer, especially after insect invasion or other parasites pre- or post-harvest [43
]. Also, it is worth mentioning that chewing raw sugarcane is a common practice, especially during the harvesting time. However, no regulations for AFs in fruit or crop juices have been set. Recently, Hariprasad et al. (2015) detected higher concentrations of AFB1
in sugarcane juice, 0.5–6.5 μg/kg, collected from local vendors in India [34
]. In a previous study, AFB1
was detected in mango (0.03–0.72 μg/L), guava (0.04–0.20 μg/L), apple (0.03–0.07 μg/L) and orange (0.006–0.07 μg/L) juices collected from local markets of six governorates in Egypt. The maximum concentration of AFG1
in mango, orange, guava and apple nectar was 0.08, 0.21, 0.34, and 0.55 μg/L, respectively [46
]. The detection of multiple mycotoxins in different Egyptian canned fruits juices and beverages was also investigated by Abdel-Sater et al. (2001) using thin layer chromatography. Total AFs (AFB1
) in apple beverages ranged from 20–30 μg/L and AFB1
in guava juice was at a concentration of 12 μg/L [47
Two hypotheses for sugarcane juice contamination with the detected metabolites including AFs were developed; (1) contaminated sugarcane stems used for juice production; (2) contamination of the instruments used in juicing. A reported case in China showed that 3-NPA was the main causative agent of acute poisoning after consumption of moldy sugarcanes [48
]. Moreover, 88 persons died out of the 884 exposed to moldy sugarcane during the period of 1972 until 1988 [49
]. In animals the systemic administration of 3-NPA is suspected to cause Huntington's disease–like symptoms [50
]. In a survey from Argentina in 2011 and 2014, the maximum concentration in native grass for grazing cattle ranged from 28.8–120 μg/kg [51
] which is lower than the present report; however, higher concentrations were detected by Ezekiel et al. (2012) in poultry feed from Nigeria (up to 947 μg/kg) [52
]. Therefore, the toxic effect of 3-NPA may affect humans through chewing raw sugarcane grass while it may affect animals through feeding on grass left over or on the bagasse of sugarcane.
Sugarcane juice consumption is a daily habit in Egypt, continuing throughout the year. However, the consumption rate greatly increases during the summer months. A common approach to estimate mycotoxin exposure is generally obtained through the combination of contamination results with the consumption data. So far, to the best of the authors’ knowledge, there are no available data on dietary exposure to mycotoxins from sugarcane juice and/or juice consumption in Egypt or anywhere else of the world.
There are some methods used to assess dietary intake such as the food frequency questionnaire. This approach was adopted to assess the dietary exposure to different mycotoxins by several previous studies in Brazil, Spain, Japan, Malaysia [25
]. In this study, the juice frequency questionnaire (online and paper based) was chosen. The participants, 91 males and 90 females, were of different ages (18–65 years old). The difference in PDI values between males and females is related to the juice intake rates. The reason may be connected with the nature of Egyptian society where males are more likely to be in contact with streets and juice shops. Also, the difference between winter and summer is due to climatic factors such as temperature.
Extensive AF exposure studies with estimation of PDI values have been reported, although little is available in the literature concerning the AFB1
contamination in juices or similar beverages [46
]. In Egypt, the PDI values of AFB1
from contaminated corn-based snacks for adults ranged from 0.42 to 11.30 ng/kg b.w./day; however, the consumption data (50 g/day) were estimated by the authors based on the content packaging of the commercial product and PDI values were estimated with the mean value of positive samples [55
]. In another study from Cairo using the same foodstuffs, the probable daily intake of aflatoxin B1
was 3.69 ng/kg b.w./day, although the value was calculated with the mean of the positive samples as well [56
]. The same author in another survey, El-Sawi in 2006, estimated the PDI of AFB1
(0.097 ng/kg b.w./day) from corn using the mean values of contaminated samples in the calculations [57
]. The mean PDI values of AFB1
were assessed worldwide: Korea (1.19 and 5.79 ng/kg b.w./day from rice) [27
], Malaysia (24.3 to 34.0 ng/kg b.w./day and for total AFs was 28.81 to 58.02 ng/kg b.w./day from 236 individual food composites consisting of 38 different types of foods) [53
], Lebanon (0.63–0.66 ng/kg b.w./day from Lebanese diets) [58
], Japan (0.003 to 0.004 ng/kg b.w./day, from different 24 foods categories including peanuts) [32
], and Brazil (2.3 to 4.1 ng/kg b.w./day of AFs from peanuts for high consumers) [28
Consequently, the estimated PDI levels of AFB1 and AG1 for the Egyptian population from sugarcane juice consumption in both seasons may contribute to a public health implication due to the fact that even low levels of AFs contamination, which might fall with the permissible limits, can lead to haptic cancer in the long run, in addition to the inevitable exposure to AFs from other contaminated food commodities. Also, serious health implications can be expected in case of high rates of juice consumption.
The study aimed to detect multiple mycotoxins occurring naturally in the sugarcane crop and juice for the first time. Indeed, the present work will open the door for further studies on the occurrence of more mycotoxins in this important economic crop. Furthermore, the presence of these metabolites either in their present (parent) form or in a modified form in the secondary products, which are based on sugarcane products such as raw sugar, vinegar, alcohol, chipboard, paper, some chemicals, plastics, paints, fiber, insecticides and detergents and molasses, cannot be excluded.
The study sheds light on the non-negligible doses of aflatoxins through the consumption of juice in Upper Egypt, especially in the summer. However, the survey was limited to 181 persons in Assiut City. Also, it should be noted that chewing raw sugarcane grass is a common practice during the harvesting time which indicates an additional source of mycotoxin exposure. These first data for exposure warrant further larger-scale multi-mycotoxin-based studies aimed at providing a comprehensive assessment in other Egyptian cities as the consumption may vary among different social groups within Egypt. Due to the seasonal fluctuation of the fungal species [33
] and the difference in consumption rates of juice as shown in the present survey, follow-up detection studies in summer and in other cities of the country are urgently needed to provide better insights regarding the number of contaminating mycotoxins and their quantities in both grass and juice.
It is highly recommended that a study for risk assessment in different foodstuffs be performed to estimate the contribution of sugarcane juice in comparison with other foods and beverages in the Egyptian diet. This report will assist the other countries where sugarcane grass cultivation and juice consumption are common to assess the occurrence of different mycotoxins in these commodities and the consequent possible health risks. Another important point needs to be mentioned herein regarding the contribution of grass left over from sugarcane in animal feed, as it is a very cheap supplement and a rich source of fiber. A detailed investigation must be conducted to detect the contaminating mycotoxins and the possibility of co-exposure in producing farm animals.
In order to limit the fungal attack and the potential production of toxic metabolites, appropriate pre-harvest precautions should be applied. Considering the juice, it should be produced in a hygienic media after a proper cleaning of the grass, especially the outer external cortex of the plant, as well as the wringers or the machine used for juicing, which can be a source of contamination.