Gamma radiation against toxigenic fungi in food, medicinal and aromatic herbs

S. Aquino1 1UNINOVE –Health Departament. Universidade Nove de Julho. Av. Dr. Adolpho Pinto,109. CEP: 01156-050 - São Paulo, SP - Brazil

Practices of harvesting, handling and production may cause additional contamination and microbial growth. In fact, bacteria and fungi occur naturally as a microflora of fruits, vegetables and herbs. Microbial purity is an important aspect of wholesome products, considering that medicinal plants are often collected from wild sources. These products are sold in open-air markets, and most toxigenic moulds grow very well in this environment when exposed for long periods in open markets, without protective packaging, proper temperature maintenance, and moisture control. The presence of toxigenic moulds represents a potential risk of mycotoxin contamination. Mycotoxins are secondary metabolites produced by different fungi genera as Aspergillus, Fusarium and Penicillium. Among the mycotoxins, aflatoxin B1 (AFB1) is the one most commonly found in food and is considered to be the most toxic compound, with the liver representing the main target organ and this mycotoxin is produced by Aspergillus flavus and Aspergillus parasiticus. In addition to its hepatotoxic action, AFB1 is also highly mutagenic, carcinogenic, and probably teratogenic to animals and the International Agency for Research on Cancer (IARC) has classified AFB1 as a group 1 human carcinogen. The regulatory recommendations determine the detection of aflatoxins in food or medicinal plants, which presence, even in small amounts, can be hazardous to health. The treatment using ionizing radiation to the decontamination of fresh fruits and vegetables to eliminate pests from imported agricultural commodities was reported in many studies. Irradiation has been used to preserve foods and this method inactivates microorganisms that decompose foods, particularly bacteria, moulds and yeasts. This treatment also destroys pathogenic organisms, including worms and insects, which degrade the quality of stored foods. Ionizing radiation has been widely recognized as a method of decontamination of foodstuffs and medicinal plants.

Keywords: Ionizing; radiation; fungi; food; medicinal plants; mycotoxins

1. Introduction

Fungi can contaminate foods from cultivation to harvest, during transportation and storage, and in various production phases, whenever the fungus is under favorable conditions of temperature and humidity. The contamination of the materials, taken directly from nature, depends on the available surface, so that flowers and leaves contain about 100 times more contamination than fruits and seeds. The effects of fungal invasion include a reduced germination potential, development of visible moldiness, discoloration, unpleasant odor, loss of dry matter, heating, chemical and nutritional changes, loss of quality, and production of mycotoxins [1-4]. The toxigenic moulds most frequently isolated from foods or grains are Aspergillus, Penicillium and Fusarium. In storage conditions, Aspergillus and Penicillium are predominant and the Fusarium spp. is an important plant pathogen. Aflatoxins (AFB1, AFB2, AFG1 and AFG2) are mycotoxins produced by Aspergillus flavus (AFB1 and AFB2 producer) and A. parasiticus (AFB1, AFB2, AFG1 and AFG2 producer). These species are commonly recognized in grains as maize or peanuts. Aflatoxin B1 is most toxic of the group followed in decreasing toxicity by AFG1, AFB2 and AFG2. Aflatoxins are recognized in some species as responsible for toxic signs and lesions, reduced growth, immunosuppression and liver cancer [5]. Among a hundred of secondary metabolites the Fusarium species produce simultaneously fumonisins, zearalenone, trichothecenes and fusaric acids. Fumonisins are mycotoxins that cause human esophageal cancer and human neural tube defects. Other mycotoxin produced by Fusarium spp. called zearalenone (ZEA) was already detected in food and feeds and it is implicated in hyperestrogenic syndromes in humans [5, 6]. The International Agency for Research on Cancer [7] has classified the toxins derived from Fusarium spp. (including fumonisins) as possibly carcinogenic to humans. Many different products consumed by humans may be contaminated by a various mycotoxins, as an example is Penicillium expansum that produces a mycotoxin known as patulin in contaminated apples and derived juices. Considering the high worldwide use of herbal products as alternative medicines, it is necessary to set standards for toxigenic moulds in crude herbal drugs in order to reduce the risks for consumers. In 1998, the World Health Organization published a document about quality control methods for medicinal plant materials and some suggestions regarding general limits for contaminants were included [8, 9]. The general limit (colony-forming units per gram) to untreated plant materials harvested under acceptable hygienic conditions is the maximum of 105 cfu g -1 (mould propagules). For plant materials that have been or that have been used as topical dosage forms, the maximum limit to yeasts and moulds are 104 cfu g -1. Finally, to plant materials for internal use (yeasts and moulds) the maximum is 103 cfu g -1. The WHO document also states that any material of plant origin should be tested for the presence of aflatoxins. Aquino et al. [10] studied 80 samples of medicinal plants and the results

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demonstrated that 93.7% of samples were contaminated and 75% of them were above the limit established by the WHO document (total count for molds of 103

cfu g -1). The microbiological control of food and herbs is the mainly concern of many countries around the world. It is also a concern of the pharmaceutical and food industry to follow drug and food laws. As consumption soars, the responsibility of regulatory agencies and manufacturers to ensure product safety to consumers increases [10]. However, methods for decontamination are restricted. The use of ethylene oxide in food has been forbidden within countries of the European Union or it has been used as a decontaminant method to medical and hospital products. Physical methods of separation, thermal inactivation, solvents extraction, grain drying, controlled atmosphere storage, adsorption from solution, microbial inactivation and fermentation are described to prevent the fungal growth or avoid the mycotoxins [11, 12]. One of methods applied to avoid fungi is the treatment by ionizing radiation.

1.1 Ionizing radiation applications

The Food and Agriculture Organization of the United Nations (FAO) estimated that 25% of all food products are wasted after harvest worldwide [13]. According to Forsythe [14], most economic losses of foods are due to infestation with insects, fungal contamination and premature germination. The use of ionizing radiation to control the microbial growth in foods or applied in medicine has been investigated since the late 19th century. Ionizing radiation has been widely recognized as a method of decontamination of foodstuffs. Exposing food to radiation treatment delays spoilage and improves safety by eliminating or reducing pathogenic microorganisms. The treatment by gamma radiation is based in an electromagnetic radiation characterized by high frequency waves (X and gamma rays). Gamma radiation is a very high-energy that carries enough energy to remove an electron from an atom or molecule [15]. Food preservation using radiation is principally achieved using a gamma source such as cobalt-60 source (60Co) or electrons, generated by high-energy electron beam accelerators. Electron beams and gamma rays differ greatly in their ability to penetrate matter. Generally gamma rays exhibit higher penetration into food compared to electron beams [16]. Blank and Corrigan [17] evaluated the resistance of microorganisms to electron beam radiation and compare results to those obtained by gamma radiation. The D10 values for all conidiospores were higher when gamma treatment was applied. The electron beam treatment was significantly more effective in only one-half to two-thirds of the organisms evaluated. The application of electron beam treatment, although lacking the penetration of 60Co. The advantage of gamma radiation is the high penetrability and uniformity of the dose that is expressed (according to International System Units) by Gray (Gy) that represents absorbed radiation dose of ionizing radiation, which permits to treat products of different sizes and shapes. The old unit of absorbed dose was rad (radiation absorbed dose). One Gy is equivalent to 100 rads [15, 18]. Irradiation has been used to preserve foods and to produce foods free of pathogenic microorganisms and is therefore an important tool for the control of food contaminating microorganisms. The treatment by gamma radiation contributes to the quality of stored foods, to reduce economic losses (resulting from food deterioration) and also to food safety, thus favoring the acceptance of products exported by developing countries [19- 22]. In some countries, the treatment using ionizing irradiation may be forbidden or require special registration procedure. This method is applied in United States to the decontamination of fresh fruits and vegetables to eliminate pests from imported agricultural commodities [9, 23]. The Joint FAO/ IAEA/WHO Expert Committee for the evaluation of toxicological, nutritional, chemical and physical aspects of foods treated with ionizing radiation concluded that foods treated with doses of up to 10 kGy (kilogray) are safe and nutritionally adequate as long as they are produced according to good manufacturing practices [13]. Regarding aromatic herbs or powdered spices, it has been reported that exposure to gamma irradiation in the dose range from 6.0 to 10.0 kGy is adequate to sterilize pepper, cardamom, nutmeg, cinnamon, fennel and turmeric without causing significant chemical or sensory alterations [24, 25]. Aziz et al. [26] concluded that doses of up to 10 kGy are highly effective in microbial decontamination and have no adverse effects on the nutritional quality of cereal grains. Aquino et al. [10] reported that almost 100% of samples of medicinal plants (Peumus boldus, Camellia sinensis, Cassia angustifolia and Maytenus ilicifolia) collected from open-air markets and drugstores in Brazil were contaminated with fungi. In this study, the dose of 10 kGy was the dose required for complete elimination of fungal contamination in these materials and the sterilized conditions were kept after 30 days, in all packed samples, in contrast to control samples (nonirradiated).

1.2 Gamma radiation effects on fungal growth

The susceptibility of microorganisms and/or their spores to gamma radiation has been well established. The ionizing radiation produces chemical changes on substrate that inactivate microorganisms. Many applications are realized also to reduce the microorganism number and consequently eliminate the risks of a poisoning disease. The energy of ionizing radiation affects directly the microbial DNA molecules, causing the damage on fungal or bacterial cells. Other effect of

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radiation (known as the indirect effect) is the interaction of energy with water molecules present on substrate or food, producing free radicals and ions that attack the microorganism DNA, killing the microbes [15, 27-29]. The lethal dose of radiation can vary according to the organism and comparing with vegetative form of bacteria, fungi are more resistant to ionizing radiation doses. In general, the vegetative forms of bacteria are more sensible to radiation than fungi (Table 1). The effectiveness of the treatment is dependent on several factors including the composition of the food, the number and type of microorganisms and the dose applied [30]. According to Chirinos et al. [31] a dose of 1.08 kGy would be sufficient to reduce E. coli O157:H7 in the hamburgers submitted to gamma radiation in 4 log cycles. In another experiment using chicken meat, the dose recommended in order to control Salmonella spp. was 3.8 kGy [32].

Table 1. Lethal doses of ionizing radiation (kGy)

Organisms Dose (kGy)

Humans 0.0056-0.0075

Insects 22-93

Virus 10-40

Yeasts 4-11

Moulds 1.3-11

Escherichia coli (Gram negative) 1.0-2.3

Salmonella spp. (Gram negative) 3.7-4.8

Bacillus subtilis (spores) 12-18

Clostridium botulinum (A) (spores) 19-37

Staphylococcus aureus (Gram positive) 1.4-7

Adapted from Frazier [33] Aziz and Moussa [34] investigated the effects of gamma radiation on the fungal micoflora of fruits stored at refrigeration temperatures (below 10 ºC) and observed a progressive reduction of fungal contamination in samples treated with 1.5 and 3.5 kGy. Ladaniya et al. [35], who exposed three citrus species to low gamma radiation doses (0.25, 0.5, 1.0 and 1.5 kGy) and then stored them at 6–7 ºC for 75–90 days, showed that the dose of 1.5 kGy was not sufficient to completely control fungi, but only delayed fungal growth and, thus, increased the shelf-life of the fruits. The authors reported that the fungal flora in the different fruit samples are sensitive to gamma radiation, and observed a completely inhibited at 5 kGy radiation dose. Rowley and Brynjolfsson [36] reported that radiation resistance of any particular fungus is influenced by many factors as the availability of water in suspending medium. This may be attributed to the indirect effect of primary water free radicals (OHº, Hº, e-) resulting from water radiolysis which are, certainly, much more in saline solution than in dried corn [37]. It is possible to conclude that microbial resistance to gamma radiation depends of many factors as an individual sensibility or the compounds of substrate. For example, in solutions or a substrate with high amount of free water, the dose will be more effective because of indirect effects of radiolysis and in an opposite condition, such as a dry or powdered substrate, the chosen dose should be high. Yeasts are more resistant than filamentous fungi. Application of different doses of electron beam and gamma irradiation (0, 2.5, 5, 7.5, 10, 15 and 30 kGy) for decontamination of Lotus seeds, revealed a significant dose-dependent decrease in the fungal contaminants [38]. However, the authors observed that contaminant yeasts survived up to 10 kGy dose, which were completely eliminated at 15 kGy. Application of medium doses of gamma irradiation in the range of 0.5 and up to 3 kGy to both buffered saline solution and to corn caused a slight decrease in the count of isolated Fusarium verticillioides while a 5 kGy dose caused a dramatic reduction in fungal count. From the calculated D10 value it is obvious that the fungal spores were more radiorresistant in corn than in saline solution [37]. Hammad and El-Baza [39] reported that the D10 value of Aspergillus flavus was 0.43 and 0.5 kGy in buffered saline solution and in smoked herrings, respectively.

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In addition, corn as a suspending medium may contain certain compounds that act as protective agents (these compounds are known as scavengers) which give protection against irradiation damage of the fungal spores. Schubert [40] mentioned that scavengers may react with free radicals liberated from water radiolysis hence protecting or reducing the radiation damage to the cell normally attacked by these radicals, which explain the high doses (between 10 and 15 kGy) recommended for complete decontamination when yeasts are present in the substrate. During the yeasts fermentation is produced lactic acid, acetic acid and alcohol that act as scavengers, giving a protective effect to yeasts against the free radicals formed by irradiation. High doses of gamma radiation are applied to fungal control, as demonstrated by Aziz and Abd El-Aal [41] that showed the complete elimination of toxigenic moulds in coffee beans and food commodities with doses from 5 to 10 kGy. Aziz and coworkers [42], investigating 84 medicinal plants from different localities of Cairo in Egypt, revealed that the effective dose for the elimination of fungi and actynomicets was 5 kGy for all examined medicinal plants. Braghini et al. [43] evaluated the effects of a range of gamma radiation doses on the growth of Alternaria alternata in artificially inoculated cereal samples (sun-flower, corn, wheat and rice). The authors reported that fungal contamination decreased with increasing gamma radiation dose. The water activity (Aw) was the same (0.98) before and after irradiation in all the substrates. A comparison between the control group (0 kGy) and the groups irradiated with 2, 5 and 10 kGy showed a decrease of fungal counting at 2 and 5 kGy but a complete absence of growth was observed at 10 kGy for all the four substrates. Ferreira-Castro et al. [44], who studied the effects of gamma radiation on corn samples artificially contaminated with Fusarium verticillioides, observed fungal growth in 80% of the samples irradiated to 5 kGy and a complete decontamination at 10 kGy. Aziz et al. [42] demonstrated that there is a correlation between radioresistance of A. flavus spores and the percentage of total lipid of mycelium. Aquino et al. [45] demonstrated a higher resistance of the Aspergillus flavus to gamma radiation, which showed no growth after exposure to 10 kGy. According to Salama et al. [46] fungi are more resistant to radiation due to the natural radioprotective agents present in mycelia. The variation in gamma radiation resistance in filamentous fungus strains can be explained by multiple factors. The cell walls of some fungi contain appreciable fractions of lipids (up to 20%) as in the case of some Aspergillus species. It was observed in guarana samples (powdered and grains) that the fungal contamination was reduced in 85% to the acceptable limit, using the dose of 5 kGy, but 20% of the genus Cladosporium and Rhizopus and 10% of Penicillium were recovered from samples irradiated with the dose of 5 kGy. Meanwhile, 10 kGy was the dose required for complete elimination of fungal contamination as demonstrated in Fig. 1 [47]. Some investigators postulated that filamentous fungi produce numerous metabolites, such as alcohols, acids, enzymes, pigments, polysaccharides, and steroids, as well as some complex compounds, such as ergotinine, and antibiotics, including penicillin, notatin, flavicin, and fumigacin. In addition, intracellular fungal components (sulfhydric compounds, pigments, aminoacids, proteins and fatty acids) have been reported to be responsible for radioresistance of fungi [42]. Melanin is a black pigment (a polymer) that protects organisms against UV rays and ionizing radiation. This pigment has also been associated with fungal radioresistance, especially among dematiaceous fungi. Aquino [10], analyzing medicinal plant samples, demonstrated a higher resistance of Phoma spp. to a radiation dose of 5 kGy. Other studies have also shown a higher resistance of dematiaceous fungi (Alternaria alternata, Cladosporium cladosporioides, Curvularia lunata, etc.) to gamma radiation [28].

Fig.1The effect of gamma radiation doses on Rhizopus spp. in a medium culture. Many studies have demonstrated a high resistance of some species of Alternaria spp. strains when exposed to elevated levels of radiation. This genus was tolerant to doses of up to 4 kGy because this fungus produces melanin, accumulated inside the mycelium. Analysis of the fungal microbiota in soil samples collected around the Chernobyl reactor (which contained radionuclides with a long half-life) revealed a predominance of pigmented fungi, including A. alternata. This fact led to the use of A. alternata strains isolated from the radioisotope-contaminated environment around reactor nº 4 of the Chernobyl nuclear power plant as a model for the genetic study of resistance to gamma radiation [48-51].

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1.3 Effects of gamma radiation on mycotoxins

The presence of water has an important role in the destruction of aflatoxin by gamma energy, since radiolysis of water leads to the formation of highly reactive free radicals. These radicals can readily attack AFB1 at the terminal furan ring, giving products of lower biological activity [15]. The mutagenic activity of AFB1 in an aqueous solution (5 g µL-1 water) was reduced by 34%, 44%, 74% and 100% after exposure to gamma rays at 2.5; 5; 10 and 20 kGy, respectively (19). Addition of 1 mL of 5% hydrogen peroxide to an aqueous AFB1 solution (50 µg/mL) resulted in 37-100% degradation of the toxin at a dose of only 2 kGy [19]. There are a number of conflicting reports that show different results in the increase, decrease or even unaffected the production of mycotoxins after irradiation of fungi under various laboratory conditions. Many studies showed that the fungal strain, condition of sporage, humidity and irradiation dose affect mould growth and toxin production [34, 52-54]. The effect of irradiation on the aflatoxin content of food and feed was previously shown by Aziz and Moussa [34] that reported the degradation of AFB1, observed in plum stored at refrigeration and irradiated at 3.5 kGy, decreasing of 380-500 µg/ kg to 20 µg/ kg. The authors treated fruits with different gamma radiation doses and observed a progressive decrease in fungal count and mycotoxin levels (penicillic acid, patulin, cyclopiazonic acid, citrinin, ochratoxin A and aflatoxin) at doses of 1.5 and 3.5 kGy. The authors reported also that no mycotoxins were detected in fruits treated with 5 kGy. Aquino et al. [45] studied the influence of Aw on the aflatoxins reduction in maize samples, using doses of 2 and 5 kGy. The reduction of AFB1 was 68.9% and AFB2 was 97.6%, of total aflatoxins amount in samples, submitted at the dose of 2 kGy. The reduction of AFB1 and AFB2 in samples irradiated with 5 kGy was 46% and 94%, respectively. The higher sensitivity of AFB1 and AFB2 in samples irradiated with 2 kGy compared to 5 kGy may be explained by higher Aw at 2 kGy (0.91) compared to 5 kGy (0.88) and a concomitantly higher gamma energy which may result in an increased formation of highly reactive free radicals (the radiolytic products of radiolysis of water) that are formed by broken molecules of water. In this same work, it was demonstrated that high dose of 10 kGy associated with a high Aw (0.94) resulted in no detectable levels of AFB1 and AFB2. These data showed that with the increase of Aw the effect of gamma radiation is more effective to control aflatoxins in a substrate. Braghini et al. [55] studied the effects of different gamma radiation doses on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seeds samples, inoculated with Alternaria alternata spores and irradiated with 2, 5 and 7 kGy. The Aw of all samples was adjusted to 0.98. This work revealed a decrease in toxin levels that was proportional to the radiation dose used. The production of AOH and AME was higher in the control group when compared to the irradiated groups. The percent reduction of AOH and AME was 99% to both toxins in the groups irradiated with 5 and 7 kGy. There are resistant mycotoxins, as fumonisins. The reduction in fumonisins levels after irradiation was observed by Ferreira- Castro et al. [44] using corn samples artificially contaminated with Fusarium verticillioides. The dose applied in the experiment was 5 and 10 kGy, with a reduction of fumonisin amount of 21% and 62.5%, respectively. In this work it was demonstrated that the dose of 10 kGy was not sufficient for a complete elimination of fumonisins, even the Aw of the analyzed samples ranged between 0.83 and 0.86. The results of this study using 5 kGy are similar with the data described by Visconti et al. [56] that showed that 15 kGy sterilized the naturally contaminated corn flour effectively, but reduced fumonisin content only in approximately 20% of total amount. Aziz et al. [42] found that wheat flour irradiated with 6 kGy eliminates fungi, but as much as 8 kGy was needed for complete degradation of the fumonisin. Molecules of fumonisins are very stable, and their destruction is likely to be difficult. Fumonisins also have relatively high thermo and light stability. Ordinary cooking does not reduce concentrations of this toxin substantially. A significant decrease of fumonisins concentrations can be expected only in 15 min at temperatures above 218 ºC [7, 58-60]. Removal of fumonisins from maize and maize-based products by means of chemical reactions is an object of many studies. Ammoniation was tested as a means of fumonisin removal, but the results were not always satisfactory [57].

2. Mycotoxins and inoculum size effect

The size of the inoculum affects toxin formation, as demonstrated with A. parasiticus and A. flavus [61, 62]. A medium inoculated with a larger number of aflatoxigenic fungi spores will develop less aflatoxin than a medium inoculated with a smaller number of spores. The toxin production is apparently suppressed when the number of inoculated spores per unit volume of substrate exceeds a certain level [63]. The same effect was observed when barley was fumigated with phosphine or methyl bromide. Obviously, the reduction in the competing flora, either by irradiation or by fumigation, enhanced mycotoxin production [64, 65]. Applegate and Chipley [66] exposed Aspergillus ochraceus to low doses of gamma radiation using 10, 25, 50, 100, and 150 krad (or 0.1, 0.25, 0.5, 1.0, 1.5 kGy) of 60Co irradiation resulted in an increase in the ochratoxin A producing potential of cultures developing from irradiated spores as compared to nonirradiated controls. The authors compared the ochratoxin A production after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures

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developing on both media. The most increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with low doses as 10, 25, or 50 krad (0.1, 0.25, 0.50 kGy). This study revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50 and 100 krad (0.1, 0.25, 0.5 and 1.0 kGy) when grown on synthetic media. Cultures derived from spores having been exposed to low doses of 10, 25 and 50 krad (0.1, 0.25 and 0.5 kGy), produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Exposures to high doses of 400 or 600 krad (4 or 6 kGy) resulted in complete inhibition of spore germination and, consequently, no ochratoxin production was detected. In the same work, it was observed that, of the two substrates used (wheat and synthetic) the quantities of ochratoxin A produced were significantly higher in the natural substrate than synthetic media, using low doses. Ferreira-Castro et al. [44] observed the same effect of gamma radiation on Fusarium verticilioides inoculum. The unirradiated samples (control group) showed the amount of 6.4 mg/kg to FB1 and 0.5 mg/kg of FB2. The concentrations in the samples irradiated with a low dose of 2 kGy (12.3 mg/kg for FB1 and 0.8 mg/kg for FB2) were higher comparing to the control samples, which may be attributed to the size of the inoculum (fungal burden). The amount of fumonisins found in the irradiated samples proved that applying low doses, the energy was not enough to eliminate completely the fungal contamination and the reduction of inoculum produced more toxins. The authors reported also that the frequency of fungi viable cells after irradiation with 2 kGy was 36% (the control samples showed 72% of viability). The irradiation decreased inoculum by three log cycles and increased the concentrations of fumonisins almost two-fold as compared with the control group. A similar reduction was reported by different studies that showed a decrease in the number of spores by approximately four log cycles, either by simple dilution or by irradiation and caused a two-fold increase in the toxin production by A. parasiticus [61] and up to a 12-fold increase in the toxin production by A. flavus [62]. Because of this effect on inoculum size, the dose chosen to fungal decontamination in natural products is the high dose that eliminates completely the mycobiota in samples, to avoid the increase of mycotoxins if a small inoculum survives. In the literature, many studies showed that doses above 10 kGy is enough to this objective, but depends of substrate conditions (constituents, temperature, Aw, etc.) and it is necessary testing the samples 7 to 10 days after the treatment to sure that no mycotoxins are not formed in irradiated food or herbs.

3. Chemical and Aw changes on irradiated food and herbs

Many reviews have summarized the nutritional adequacy of irradiated foods. They clearly demonstrate that irradiation results in minimal, if at all noticeable, changes in the taste, provided that the optimal dose for each type of food is not exceeded [30]. In general, irradiation to the recommended doses changes the chemical composition of foods very little. According to Diehl [30, 15], at doses below 1 kGy, nutritional losses are considered to be insignificant, and none of the chemical changes found in irradiated foods is harmful, dangerous or even lying outside of the limits normally observed [67, 68]. Calucci et al. [69] reported increased free radical content and ascorbic acid losses in nine species and aromatic herb samples submitted to gamma ray irradiation at a dose of 10 kGy, according to commercial practices. Migdal [70] and Owczarczyk [71] have shown that the content of biologically active substances in nine investigated herbal raw materials did not change in a significant degree after irradiation (10 kGy) nor did the pharmacological activity of 10 phytopreparations (also irradiated with 10 kGy). Aquino et al. [47] analyzed the irradiated samples of powdered guarana with the doses of 5 and 10 kGy and comparing with nonirradiated samples intending to determine changes in the chemical structure of the substances contents. The analysis of samples submitted to gamma radiation showed no significative chemical differences among them, considering the spots of extracts observed under iodine steams and ultraviolet light, after chromatography, using in parallel control samples. Peregrino and Leitão [72] studied the effects of gamma radiation on compounds of Mikania glomerata Sprengel (a Brazilian medicinal plant), popularly known as "Guaco”. The results indicated that the treatment of Mikania glomerata leaves with gamma rays in the assayed doses of 3.5 and 5 kGy did not affect the chromatographical profiles of fluid extracts and tinctures prepared from them. Extracts from the same plant (untreated) were used as standards. In fact, the results obtained showed that there was an increase in the coumarin content in the extracts obtained from irradiated plants. The increase in the coumarin content which was found with this procedure can be considered as beneficial, since this is one of the active constituents of this plant. A similar result using high doses was observed by Koseki et al. [73] that reported that powdered and dehydrated herbs irradiated with doses of 10, 20 and 30 kGy showed the identical therapeutical action of non-irradiated samples of Rosmarinus officinalis Linné, Nasturtium officinale R. Br, Cynara scolymus Linné and Ocimum basilicum Linné. In the extracts of medicinal plants (Peumus boldus, Camellia sinensis, Maytenus ilicifolia and Cassia angustifolia) treated using a 60Co gamma ray source with doses of 5 and 10 kGy, the phytochemistry analysis of control samples and irradiated samples showed no difference among them, as demonstrated by thin layer chromatography using the hexanic

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and ethanolic extracts. The irradiation process did not modify the compounds of these medicinal plants studied, keeping intact the phytochemistry profile of all analyzed medicinal samples [10]. Water activity (Aw) is defined as the ratio of the vapor pressure of water in a material (p) to the vapor pressure of pure water (po) at the same temperature. The Aw of a food is the ratio between the vapor pressure of the food itself, when in a completely undisturbed balance with the surrounding air media, and the vapor pressure of distilled water under identical conditions. The Aw of 0.80 it means that the vapor pressure is 80% of pure water. The Aw increases with temperature and the moisture condition of a product can be measured as the equilibrium relative humidity (ERH), expressed in percentage or as the Aw expressed as a decimal, considering that in the pure water the Aw value is 1.0. Water activity is equal to equilibrium relative humidity divided by 100: (Aw = ERH/100) where ERH is the Equilibrium Relative Humidity (%) [74]. The Aw of substrate is affected by gamma radiation because of indirect effect (water radiolysis). Aquino et al. [47, 10] analyzed the Aw of different substrates as medicinal plants (Peumus boldus, Camellia sinensis, Maytenus ilicifolia, and Cassia angustifolia) and guarana (Paullinia cupana). In all samples treated by gamma radiation it was observed a constant decrease of Aw levels ( 0.01 to 0.05) comparing with control values. The data are showed in Tables 2 and 3.

Table 2. Average of Aw values of twenty samples powdered and grains of guarana.

Dose Samples Powdered Grains

Control (0 kGy) 0.46 0.51 5 kGy 0.44 0.49

10 kGy 0.41 0.47

Adapted from Aquino et al. (2007)

Table 3. Average of Aw values of twenty samples of medicinal plants.

Medicinal plant Control (0 kGy) 5 kGy 10 kGy C. angustifolia 0.58 0.55 0.53 P. Boldus 0.55 0.53 0.51 M. ilicifolia 0.51 0.50 0.49 C. sinensis 0.53 0.52 0.50

Adapted from Aquino et al. (2010)

Most foods have an Aw value above 0.95 and that will provide sufficient moisture to support the growth of bacteria, yeasts, and mold. The amount of available moisture can be reduced to a point which will inhibit the growth of the organisms [74]. The decrease of Aw caused by gamma radiation confers a protective result against toxigenic molds, since the minimum Aw permitting Aspergillus spp. germination and growth ranged from 0.80 to 0.82, according to Pitt and Miscamble [75]. Hunter [76] proposed the value of 0.87 as the minimum required for aflatoxin production. A level of Aw above 0.87 is necessary for Fusarium spp. growth, while value above 0.90 is essential for fumonisin production [77].

4. Conclusion

The process of gamma radiation treatment is an important tool to the fungal control in foods and medicinal plants. In some cases, these products are consumed as a natural or raw presentation. Applying high doses (above 10 kGy) fungal burden is eliminated, keeping the quality of product. In despite of this technology to be applied to reduce the risk of fungal growth and mycotoxins forming, as a mechanism of prevention in the food and herbs contamination, it is necessary to have a monitoring Program of Good Manufacturing Produce (GMP) to avoid the fungal contamination during manufacturing process, storage and exposure of products on the market.

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