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Electron beam irradiation – An emerging technology for fungal

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Chapter · January 2008

DOI: 10.13140/2.1.1818.4960

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 Novel Techniques and Ideas in Mycology

13
Electron beam irradiation – An emerging
technology for fungal decontamination of
food and agricultural commodities

Kandikere R. Sridhar* and Rajeev Bhat

Microbiology and Biotechnology, Department of Biosciences, Mangalore University,

Mangalagangotri 574 199, Mangalore, Karnataka, India (*[email protected])

Sridhar, K.R. and Bhat, R. (2008) Electron beam irradiation – An emerging technology for
fungal decontamination of food and agricultural commodities. In: Novel Techniques and Ideas
in Mycology (eds. K.R. Sridhar, F. Bärlocher, & K.d. Hyde). Fungal Diversity Research Series
20: 271-303

Electron Beam Irradiation (EBI) is a fast, effective and environmentally safe method of
sterilization and fungal decontamination of a variety of commodities. EBI allows
decontamination of many food and agricultural commodities (e.g. food, feed, fruits, vegetables,
mushrooms, diary products), economically-valued plantation produce (e.g. vanilla, coffee, tea,
spices, honey) and traditional herbal products, which usually suffer from fungal contamination
and mycotoxin interference during post harvest processes. EBI technology can also be
implemented to decontaminate and preserve the traditional and oriental foods (e.g. pickles,
marinated foods, seaweeds, parboiled rice). Compost and wastewaters can be effectively treated
through EBI to inactivate pathogenic fungi. In view of the proposed banning of several
fumigants (e.g. sulphur fumes, ethylene dioxide, ethylene oxide) which are detrimental to
environmental and human health ,EBI will be a safe alternative. As EBI uses less energy than
conventional techniques, it may replace dry sterilization techniques in future.

Key words: Electron beam irradiation, agricultural produce, plantation produce, food
commodities, herbal products, post harvest technology, compost, wastewater

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 Novel Techniques and Ideas in Mycology

Introduction

Providing safe and adequate food supplies for mankind has been a
challenge since time immemorial. The Food and Agriculture Organization
(FAO) of the United Nations has estimated the annual loss of the world’s food
supply to be about 25% due to microbial contamination, improper handling and
storage. Increasing concerns by consumers over food losses due to infestation,
microbial contamination and food-borne diseases has stimulated worldwide
interest in the prevention of fungal contaminants of food and agricultural
commodities. Stringent laws imposed by the European Union (EU) and several
countries on the quality and safety of imported materials have encouraged the
introduction of non-conventional alternatives for food preservation due to ban
of use of chemical fumigants (e.g. ozone depleting chemicals: methyl bromide,
ethylene dioxide) in developed (in 2005) and in developing (in 2015) countries
(Anon, 1995, 1996; UNEP, 1995) have opened up possibilities of
commercializing food irradiation on a large scale. Irradiation as a technique of
preservation has been extensively employed for decontamination, disinfestation
and shelf life improvement of food and agricultural products prone to rapid
deterioration. Over the last six decades, the physical and chemical changes
induced by absorption of ionizing radiation in food and agricultural
commodities have been debated at national and international forums. The
objective of the present chapter is to highlight the technology, importance and
novelty of employing electron beam irradiation (EBI) to prevent fungal
contamination and thus enhance the quality of food and agricultural
commodities.

The background

The foundation for food irradiation research was laid by the discovery of
X-rays by the German scientist Wilhelm Conrad Roentgen in 1895. In 1896, a
French physicist, A.H. Becquerel, discovered the emission of radiation from
naturally occurring radioactive materials. This was followed by Misch’s
proposal in Germany to employ ionizing radiations to prevent food spoilage.
Some attempts have been made to employ radiation to treat fungal diseases of
scalp and foot. In 1905, the United States and United Kingdom issued patents to
J. Appleby and A.J. Banks for the efficacy of using ionizing radiations to kill
food-borne pathogens. Subsequently, a French patent was issued to O. Wust for
employ ionizing radiation (X-rays) for bacterial decontamination in packed
foods in 1930. The Food Additive Amendment of the Federal Food, Drug and
Cosmetic Act (1958) defined irradiation sources as a ‘food additive’ rather than
as a preservation process, which delayed the commercialization of food
irradiation. However, an upsurge in the use of radiation technology for food

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 Novel Techniques and Ideas in Mycology

preservation occurred in 1980, when the Joint Expert Committee on Irradiation

(FAO/WHO/IAEA) declared that ‘irradiation of food up to an overall dose of 10
kGy presents no toxicological hazard with no nutritional or microbial problems’
(WHO, 1981). A series of conventions of experts were convened by
WHO/FAO/IAEA (1994, 1997, 1999) and concluded that food irradiation
employed to achieve the desired technological target does not pose health risks
and irradiated food is safe for consumption. Radiation processing of food has
been cleared for commercial use for at least one food product in 35 countries. At
present, irradiated foods are commercially available in 28 developing and
developed countries (IAEA, 1995; Loaharanu, 1996). Food and agricultural
produce need to be processed to enhance the shelf life and prevent spoilage
caused by insect pests, parasites and microbes. In some instances, prevention of
sprouting (e.g. onion, potato) and early ripening (e.g. fruits) is necessary to
minimize insect sprays and fumigants. Irradiation needs to be considered along
with conventional methods of food preservation like freezing, canning and
drying to prevent spoilage of perishable food products during storage. Table 1
deals with the use of gamma irradiation of food and commercial applications.

Table 1. Gamma irradiation of food and its commercial applications

Product Purpose Dose (kGy)

Potato, onion, garlic, Sprout inhibition, reduction of 0.03-0.15
ginger post harvest loss
Strawberries Control mould infection 2-3
Mushrooms Shelf life extension 1.5-2.5
Spices, dried herbs, Hygienization by reducing 6-14 (maximum, 30
vegetable microflora kGy)
Dry enzyme Microbial disinfection 10
preparations

Radiation processing

Technology. Radiation processing involves precise exposure of food and

agricultural commodities to ionizing radiations such as gamma rays (cobalt-60
and caesium-137) or machine generated X-rays (5 Mev) and high-energy
electrons (8-10 Mev). Ionizing radiation in EBI technology is provided through
electrons, while mainly by photons in X-rays and gamma rays. The amount
provided by the source is known as dose and measured in kilo Grays (kGy) (1
kGy = 1,000 kJ or MR; 1 MR = 1,000,000 ergs/gram). Depending on the
technical specifications, radiation processing is broadly classified into three
categories:
1. Low dose (<1 kGy), mainly used for sprout inhibition of onions,
potatoes, ginger, garlic and shallots and for disinfestation of stored

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cereals, grains, dry fruits and spices. A low dose is also employed to
delay ripening in fruits.
2. Medium dose (1-10 kGy), for hygienisation of whole spices, spice
powders, spice mixtures and for elimination of spoilage microbes in
fruits and seafoods. This range of dose is employed for shelf life
improvement during long-term storage.
3. High dose (10-45 kGy), necessary to make foods sterile, wherein no
refrigeration is required. Some spices recommended for export has been
given clearance for this dose range. This dose is used to produce sterile
foods in hospital diets for patients with compromised immune systems.
This dose also employed for foods used by astronauts during space
flight.

Packed foods or agricultural commodities are allowed to pass through a

radiation chamber on conveyor belts in such as way that they will not come in
direct contact with radioactive materials. Nutritionists are of the opinion that
radiation processing produces no greater nutritional loss than other food
processing methods like cooking or canning. Nutrient losses can be minimized
by irradiation of foods in anoxic conditions or while freezing. Table 2 compares
the characteristic features of gamma irradiation with EBI.

Table 2. Comparison between gamma irradiation and EBI technology*

Gamma irradiation (Co-60) Electron beam irradiation

Well established technology New technology
Permanent source of radiation emission Radiation emission only on operation
state
Simple to operate Operating skills necessary
Limited options of operations Numerous options of operation
No mass radiation penetrates deep into Small mass, radiation penetration
materials limited
Low dose rate (absorbed dose per unit Required dose rate is higher
time)
required

* The only disadvantage faced by EB irradiators is the requirement of a steady and

continuous power supply, which may be a constraint in the third world countries

Electron accelerators. Following the success of laboratory studies

employing gamma rays from radioisotopes (Co-60 and Cs-137), a remarkable
development in accelerator technologies was seen in the late sixties. These
technologies provided a new source of radiation. The technological efficiency of
machine-generated radiation is comparable with gamma rays. In 1933, van de
Graff proposed the first electron accelerator, wherein a stream of electrons from
a heated cathode was accelerated through an evacuated tube between cathode
and anode with an energy input of 4 Mev. Cleleand (1959) developed a more

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compact and cost-effective accelerator, which could be used for food

irradiation. A high-energy source of electrons, ‘Dynamitron’ had the capacity to
convert low voltage AC-power to high voltage DC-power using a radio
frequency oscillator. Varieties of machines and accelerators differ in the
generation of voltage as well as in potential difference (Ramler, 1982). In the
last two decades, a technological revolution developed reliable accelerators
having high power at a low manufacturing cost. The available accelerators for
radiation processing with varying beam power of 10-200 kw include: LINAC,
RHODOTRONS, CYCLOTRONS, MICROTRONS and DYNAMITRONS.
Apart from food and agricultural sectors, accelerators are useful in
several industries: chemical and petrochemical industries (for cross-linking, de-
polymerization, grafting and polymerization of polyethylene, polypropylene,
lubricants), coating and additives (curing, grafting, polymerization of adhesive
tapes, wood/plastic composites) and health and pharmaceutical industries (for
sterilization of ethical drugs, ointments, powders and medical disposables). In
medicine, accelerators are being extensively used for radiotherapy, nuclear
medical applications, radiobiology and synchrotron radiation techniques.
Accelerators are useful in monitoring and control of pollution in the
environment. In pilot studies in Japan and Germany, they were used to eliminate
sulphurdioxide and nitrogen oxides from flame gases of thermal plants and steel
industries. Accelerators are also employed in disinfection sewage sludge,
allowing their safe disposal.

EBI technology. The EBI technology offers a fast, effective and

environmentally safe alternative to fumigation. A standard source of electricity
(AC) is sufficient to generate electrons for irradiation. The EBI allows non-
nuclear and accelerated generation of radiation, is available when required and
the machine can be switched ‘on’ or ‘off’, as necessary. Machine sources
employed provide constant output without radioactive decay and waste
generation normally associated with isotope sources. Energy of the beam and
amount of radiation can be manipulated to suit the size of packages to be
irradiated. The penetration depth can be predicted and the product can be
irradiated from one or from both sides. Machine sources are economical and
useful for high throughput and high dose applications. Radiation shielding is
required only when the machine is on. No radioactive materials need to be
transported, which minimizes the risks of occupation hazards. There is no
change in the radiation dose due to decay of the source. Thus, the EBI
technology is safe, cost effective and could be installed in places like airports or
harbors for quarantine treatments of imported commodities.

Irradiation to destroy microbes

A number of objectives have to be fulfilled for decontamination or

elimination of moulds in food and agricultural commodities to meet the

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international standards. The energy from electrons or X-rays has to target and
inactivate fungal nucleic acids. This damage occurs directly as a result of
electron and photon interactions with DNA and RNA and indirectly through the
radiolytic products of water, which further react with nucleic acids.
Microorganisms with large genomes are usually more susceptible to radiation
than those of smaller genomes. When ionizing radiation reaches microbes, its
high energy breaks chemical bonds in molecules that are vital for cell growth
and integrity, and this results in microbial death. The cellular destruction caused
by disruption of the genetic material is the principal effect of radiation (Murano,
1995), and enables destruction of insects, inactivation of parasites, delay in
ripening and prevention of sprouting.
Table 3 summarizes the history of disease outbreaks by fungi that played
a significant role in human life. Treating foods with ionizing radiations raises
some important queries concerning microbial safety:
1. Can irradiation produce mutated microorganisms and possibly more
virulent pathogens? The Food and Drug Administration (FDA) does not
consider radiation-induced mutation a concern with respect to increased
virulence or heat resistance, since no evidence has been reported to show
such effects.
2. Can irradiation reduce the numbers of relatively harmless spoilage
microorganisms while allowing pathogens to grow undetected without
competition? In fact, radiation has been shown to reduce the virulence of
any surviving pathogens (Farkas, 1989). The EBI technology has been
shown to be more effective on fungal spores as D-10 value (the minimal
dose required to kill an organism) is usually higher in gamma irradiation
(Blank and Corrigan, 1995).

Table 3. History of outbreaks of diseases due to fungal pathogens in food and

agricultural commodities

Disease Fungus Year and country

Late blight of potato Phytophthora infestans 1840-1847 (Ireland,
Europe, USA,
Canada)
Rust of coffee Hemieilia vastatrix 1867 (Sri Lanka)
Downy mildew of Grapes Plasmopara viticola 1878-1882 (wine
industries in France)
Chestnut blight Endothia parasitica 1904 (USA)
Red rot of sugarcane Physalospora 1938-1939 (Java)
tucumanensis
Helminthosporium disease Helminthosporium oryzae 1942 (India)
of rice (Great Bengal
Famine)
Wheat Rust Puccinia spp. 1946-1947 (India)
Corn leaf blight Helminthosporium maydis 1969-1970 (USA)

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 Novel Techniques and Ideas in Mycology

Current status of EBI technology

The effect of gamma rays and EBI on the fungal contaminants

(osmophilic moulds, Aspergillus flavus and coliforms) in black pepper,
turmeric, rosemary and coriander has been studied by Ito and Islam (1994). A
dose of 4 kGy eliminated or reduced to below detectable limit most of the
contaminant microflora. Migdał et al. (1995) used the industrial linac facility
(electron irradiator: ELEKTRONIKA 10-10) with 10 Mev strength on spices
and vegetables (dry parsley leaf, parsley root, carrot, celery leaf and root, red
beet and dry mushrooms) and found that 5 kGy was sufficient to reduce the
initial microbial load by a factor of 105. The EB (400 Gy) was employed by
Hayashi et al. (1998) and found effective to inactivate the insect pests of cut
flowers. Soft electrons energies (300 or <300 Kev) was used by Hayashi (1998)
for surface decontamination of pulses, grains, spices and dehydrated vegetables
and reduced the microbial load to <10 cfu/g with minimal quality deterioration.
In 1999, Hayashi used electrons at an accelerator voltage of 170-180 Kev to
reduce the microbial contaminants of radish and alfalfa seeds to below
detectable levels. Soft electrons up to 7.5 kGy were employed to reduce the
microbes in soybeans (Todoriki et al. 2002). This dose was sufficient to make
high temperature (120°C) sterilization of soymilk unnecessary and resulted in
improved gelatinization property.
Studies on the influence of EBI on mechanical features and microbial
contamination of plastic cups, polystyrene and polypropylene cups used in dairy
products have been carried out by Tacker et al. (2002). The efficacy of EBI (4-5
kGy) in decontamination of naturally occurring moulds and yeasts has been
tested with deliberately contaminated cups (Alternaria sp.). No naturally
occurring moulds or yeasts were detected after EBI, but decontamination of
inoculated cups required a dose of ≥7.5 kGy. Moreira et al. (2002) showed a 2-
log cycle decrease of microorganisms on the surface of apples exposed to EBI
(1.2-4 kGy). Mittendorfer et al. (2002) reported that EBI (5-7 kGy) effectively
inactivated yeasts, molds (Penicillium and Aspergillus) and their spores in food
packaging materials (yogurt cups, containers, bottles and seal caps) before
shipment. Palekar et al. (2004) showed that EBI effectively reduced the total
aerobic microbial counts and extended the shelf life of water-washed fruits and
chlorine-washed fruits (sliced cantaloupe). Kikuchi et al. (2003) compared the
efficacy of EBI (<60 keV and 300 keV) to decontaminate soybean varieties
(Enrei and Vinton) and showed that 26 kGy was required for achieving
complete sterilization. Except for the delay in germination, no changes were
induced by EBI in qualitative parameters such as lipid oxidation, lipoxygenase
activity and radical scavenging activity compared to gamma rays. EB irradiated
wheat product retained the quality without any signs of spoilage by
contaminating moulds, yeasts and pathogenic bacteria until the packaging
material was intact (Placek et al. 2004).

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Food commodities and EBI

Food, feed and silage. Risks posed by consumption of contaminated

foods are high. Commonly consumed food grains, fruits and beverages
worldwide include rice, wheat, barley, sorghum, rye, sugar (sugar cane and
sugar beets), grapes, spices, cocoa, coffee, wine and beer. Through improper
processing, drying and storage all can become prone to fungal contamination.
Under favourable conditions (humidity, moisture, water activity and
temperature), fungi produce several toxic metabolites (mycotoxins), which
present severe health hazards. Mycotoxins are known for teratogenic, mutagenic
and carcinogenic effects in livestock and humans. For instance, peanuts harbour
at least 24 different taxa of fungi. Aspergillus flavus is often dominant and can
produce high levels of aflatoxins (Costantini, 1993). Aflatoxins and fumonisins
have also been recorded in high concentrations in corn (Anon, 2003). Patulin is
a heat-resistant mycotoxin produced by Penicillium, Aspergillus and
Byssochylamys on apples. Whole apples or pasteurized/non-pasteurized apple
juice consumed are known to be susceptible to patulin contamination. Table 4
summarizes some common mycotoxins occurring on food products and their
effect. Contamination of mycotoxins in food can be prevented by creating
unfavourable conditions for mould growth (e.g. alteration of temperature,
moisture, oxygen, heat), however, due to ease in handling EBI, can be definitely
advantageous.
The efficacy of EBI treatment in combination with other methods to
prevent the growth of moulds will be of immense value. Due to its nutritional
versatility, seafoods form an important part of the normal diet of coastal
dwellers. As they are highly perishable, gamma irradiation has been commonly
employed. EBI may also serve as an effective means of decontamination and
preservation of fresh sea foods. In most coastal regions, sun dried sea foods
(fishes, squid, squilla, rayfish, prawns) are popular. Available information on
fungal contaminants of these products is meager, but as an example, salted fish
commonly harbors the fungus Polypaecilum pisce. Depending on the processing
and storage practice of dried sea foods, mould contamination may be common.

Table 4. Common food products, contaminating mycotoxins and their biological

effects

Food product Mycotoxin Effect Fungus

Corn Fumonisin, Cancer-causing Fusarium
aflatoxin/zearalenone, effects, esophageal verticillioides
ochratoxin cancer/ estrogenic,
kidney-related
problems

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 Novel Techniques and Ideas in Mycology

Peanuts, corn, nuts, Aflatoxin (B1, B2, G1, carcinogens Aspergillus

grains, milk G2) aflatoxin M1 flavus, A.
parasiticus
Apples Patulin Brain and lung Penicillium
hemorrhage, expansum, P.
carcinogen patulum,
Aspergillus sp.,
Byssochylamys
sp.
Grains, (wheat, Deoxynivalenol Vomiting Fusarium
corn, barley, rye). (DON) (vomitoxin) graminearum
Cereals (corn, Ochratoxin Carcinogenic Aspergillus sp.,
barley, wheat, oats), Penicillium sp.
edible animal
tissues, milk

Numerous reports are available globally on contamination of feeds and

forages (silage) with fungi and their spores. These problems are more common
in tropical countries with hot climates. The use of additives is expensive for
small-scale farmers. Animal feed and fodder are an important source of
hazardous microbes. Feed-borne fungal contamination and aflatoxins affect the
livestock as well as human health through animal products (e.g. milk, eggs,
meat, offal; Bastianelli and Le Bas, 2000). Aflatoxin is known to accumulate in
feeds in hot and humid conditions in groundnuts and oilseed meal and its
presence in tiny amounts can diminish performance and cause hepatic toxemia
in commercial animals. Thus, prevention of fungal contamination and storage
conditions has to be improved. Additives (e.g. coccidiostatics, antibiotics) are
used to control contamination from coccidiosis, but excessive dosage
contributes to microbial resistance. Mycotoxin contamination of fodder is due to
contamination in the field (harvest and storage) and mutualistic endophytic
fungi (Chhabra and Singh, 2005). Aspergillus flavus, A. ochraceus and A.
parasiticus proliferate in fodder in tropical warm humid conditions, while
Penicillium expansum and P. verrucosum predominate in temperate conditions.
Aflatoxin ranges between 0 and 4,200 µg/kg in animal feeds (e.g. groundnut
cake, maize, wheat, rice bran and wheat bran, cotton seed cake). About 85% of
maize samples were contaminated with aflatoxin B1 (8-68 µg/kg) and fumonisin
(160-2,590 µg/kg). In northern Vietnam, aflatoxin B1 (9-96 µg/kg) and
fumonisin B1 (271-3,447 µg/kg) were found in feed-grade maize (Placinta et
al., 1999). The endophytic fungus, Neotyphodium coenophialum produce
ergopeptine alkaloid and ergovaline in perennial tall fescue and N. lolii produce
indole isoprenoid lolitrem alkaloid and lolitrem B in perennial ryegrass.
Ergopeptine alkaloid and ergovaline reduce the growth, reproductive
performance and milk yield in cattle, while indole isoprenoid lolitrem alkaloid
and lolitrem B induce neurological effects in ruminants.
Lupin stubble is a valuable fodder for sheep in Australia. The infection
of lupin stems, pods and seeds with Phomopsis leptostromiformis causes the
production of phomopsins, which results in liver damage, photosensitization and
reduced reproductive potential in sheep (D’Mello and MacDonald, 1998). Table

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 Novel Techniques and Ideas in Mycology

5 deals with the origin and impacts of major mycotoxins in feeds and forages.
The relative impact of individual toxins in feeds distinctly differs
geographically.

Table 5. Origin and effects of major mycotoxins in feeds and forages (Partial source:
D’Mello, 2000; Chhabra and Singh, 2005)

Mycotoxin Fungal species Source Effects

Aflatoxins Aspergillus flavus, Peanut meal, Inhibition of protein
A. parasiticus cottonseed and palm synthesis, lipogenesis
kernel cake, maize, and glycogenesis, Cause
compound feeds of cancer
Cyclopiazonic Aspergillus flavus Oilseed meals and Weight decrease,
acid compound feeds mortality, lesions on
proventriculus, gizzard,
ulcers in proventriculus,
affect liver, spleen
Ochratoxin A Aspergillus ochraceus, Barley grains, wheat Inhibition of protein
Penicillium grains synthesis, glycogenesis
viridicatum, P.
cyclopium
Citrinin Penicillium citrinum, Corn, Rice, Cereal Diuresis and necropsy-
P. expansum grains lesions in kidney
Deoxynivalenol Fusarium culmorum, Cereal grains Reduced feed intake,
F. graminearum vomitoxin
Sterigmatocystin Aspergillus versicolor, Cereals (rye, wheat Hepatotoxic,
A. nidulans and barley) hepatocarcinogenic
Zearalenone Fusarium culmorum Cereal grains Hyperoestrogenic,
F. graminearum, reproductive
F. sporotrichioides abnormalities
Fumonisin, Fusarium moniliforme Cereal grains, maize Pulmonary oedema,
moniliformin, kernels leukoencephalomalacia,
fusaric acid human esophageal cancer
Lolitrem Neotyphodium lolii Grasses Gastrointestinal smooth
alkaloids muscles
Ergot alkaloids Claviceps purpurea Cereal grains Ergotsim, convulsive and
sensory neurological
disorders,
vasoconstriction,
gangrene of vascular
system, neuroendocrine
control of anterior
pituitary

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Silages also harbour yeasts such as Candida, Saccharomyces and

Torulopsis. Currently, no cost-effective methods are available to detoxify
contaminated feeds and forages. Deactivation of fungal toxins has been tried
and deoxynivalenol (DON) and zeraleone was reduced in grains by cleaning and
washing with water or mild sodium carbonate solution (Charmley et al., 1994).
Milling contaminated wheat also reduced mycotoxins in flour by between 15-
100% (Tanaka et al., 1986). Solar drying (6-14 hr) (Gowda et al., 2003) and UV
irradiation of contaminated feeds reduced the mycotoxins (Chhabra and Singh,
2005). Currently, ammoniation (treatment with ammonium hydroxide or
gaseous ammonia at high temperatures and pressure) is the method of choice for
detoxification of aflatoxin-contaminated oilseeds used as animal feed. Applying
organic acids prior to storage prevents mould contamination in high moisture
feeds and grains. Mixtures of organic acids: Lupro-Mix® NC (propionic acid,
38%; formic acid, 34%; ammonia 8% and water 20%), Lupro-Grain®
(propionic acid, 92%; ammonia, 4%; propanediol, 4%) and sodium bentonite
are commercially available preservatives. To preserve the quality of silage, EBI
to replace chemicals will be economical and a better choice to prevent mould
contamination.

Fruits and vegetables. Consumption of fruits and vegetables has

increased rapidly worldwide due to the status conferred on them.
Epidemiological studies have shown that consumption of fruits, vegetables and
derived food products have health benefits and protect against chronic diseases
including cardio- and cerebrovascular diseases and cancer (Ames et al., 1993;
Hertog et al., 1997). Growing consumption of minimally processed vegetables
for nutritional benefits has raised questions about their safety. Fungal diseases
during post-harvest storage are well known. Often the fungal invasion begins
when the fresh produce is kept in an uncontrolled environment or invasion
occurs through surface cuts or damages of the produce. Fungal contamination
depends on the produce, as well as on the climatic region. Rhizopus spp. are
common and cause considerable loss of fruits and vegetables. Raw sprouted
seeds consumed as nutritional supplement are prone to be contaminated by
toxigenic moulds as they are wet and nutritionally rich. Table 6 gives a brief
overview of the common fungal contaminants of fresh produce and their effects.
Fresh-cut cantaloupe in modified atmospheric packages (oxygen, 4%;
carbon dioxide, 10%) was irradiated at EBI (0.5, and 1.0 kGy) by Boynton et al.
(2006). The irradiated samples had a lower and more stable rate of respiration
than non-irradiated samples for about 20 days. Total plate counts were
significantly higher in non-irradiated samples. The color and texture of samples
were stable and showed good sensory properties at 1 kGy with highest
sweetness and flavor intensity and lowest in off-flavor after 17 (±3) days
storage. The low-dose EBI of fresh-cut cantaloupe combined with modified
atmospheric packaging promises to extend shelf life. Similarly, the impact of
EBI on packaged fresh blueberries at greater than 1.0 kGy on the quality has
been evaluated by Moreno et al. (2007). The berries were stored (temperature,
5°C; RH, 70.4%) for 14 days and tested at 0, 3, 7 and 14 days for

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physicochemical, textural, microstructural, and sensory properties. Irradiation at

doses higher than 1.1 kGy significantly affected the texture of berries (they
became soft and less acceptable throughout storage). The dose, 3.2 kGy
influenced the color of berries at the end of storage. In terms of overall quality,
texture and aroma, berries exposed to 3.2 kGy were unacceptable based on
sensory evaluation. The dose of irradiation did not influence the density, pH,
water activity, moisture, acidity and juiciness of berries. The EBI of blueberries
at 1.6 kGy was found to be feasible for decontamination as well as to maintain
the overall quality attributes of blueberries.

Table 6. Common storage fungi on fresh produce and their effect

Produce Fungus Effect

Stone fruits, pome Sclerotinia spp. Brown rot
fruits
Banana Gleosprorium musarum Rot
Citrus fruits Penicillium spp., Blue mould or green
Sclerotinia sclerotiarum mould, watery soft disease
Grapes Penicillium italicum Blue mould rot
Papaya Colletotrichum gloeosporioides Anthracnose
Musk melons Cephalosporium roseum, Pink mould rot, blue
Penicillium spp. mould rot
Apple and pears Aspergillus niger, Botrytis cinerea, Black mould rot, gray
Penicillium expansum mould rot, soft rot
Potato tubers Fusarium caeruleum and Rot, silver scurf
Helminthosporium spp.
Apricot Sclerotinia fructicola Rot
Onions Botrytis spp., Aspergillus niger, Neck rot, blue mould rot,
Penicillium sp. discoloration
Carrots Sclerotinia sp., Botrytis spp. Rot
Celery Sclerotinia sclerotiorum Pink rot
Lettuce Sclerotinia sclerotiorum Wilting, rot
Cucurbits Pythium aphaniderantum Fruit rot
Ginger Pellicularia spp., Fusarium spp. Rhizome rot
Potato Alternaria solani Early blight
Areca nut Phtophthora arecae Areca rot

Altekruse and Swerdlow (1996) document drastic changes worldwide in

dietary habits, including higher per capita consumption of fresh or minimally
processed fruits and vegetables, including salads. The presence of cut surface
and damaged tissues makes minimally processed product prone to microbial
contamination and decreased shelf life (Nguyen and Carlin, 1994). Most
literature on minimally processed products indicates the presence of potential
bacterial pathogens like Listeria monocytogenes, Yersinia entercolitica and E.
coli (Beaufort et al., 1992; Nguyen and Carlin, 1994; Archer and Kvenberg,
1988), but little information is available on fungal contamination. Safety of
ready to eat fruits and vegetables depends primarily on refrigeration and

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hygienic practices, which are quite difficult to ensure at large scales.

Overchilling and refrigeration may lead to cold injuries favouring fungal
contamination (e.g. Alternaria rot), as observed in tomatoes, squash and peppers
(Anon, 1992). Sometimes, post harvest treatment involves the use of fungicides
like borax mixture, sodium carbonate, boric acid (2%), sulphur dust, Thiram,
Maneb and sodium dimethyl dithiocarbonate (emulsion wax). Sodium
hypochlorite (liquid laundry bleach) is one of the most readily available of these
sanitizers. Excessive use of hypochlorite can result in off flavors, tissue damage
and change the surface pH of the produce, which may encourage microbial
growth. Considerable consumer resistance to chemically synthesized additives
and antibiotics in preservation has made it mandatory to use natural
preservatives possessing antimicrobial properties. Even the use of modified
atmospheric packaging (MAP) is not foolproof in preventing fungal
contamination leading to development of off flavors.
Hoogerwerf et al. (2002) have shown that high oxygen modified
atmosphere inhibits the growth of food-associated mould like Rhizophus
stolonifer, Botrytis cinerea and Penicillium discolor. The EBI at low lethal
doses may be employed in combination with natural additives or atmospheric
packaging to ensure fungal decontamination for safety and shelf life
improvement.

Mushrooms. Mushrooms are cultivated on the surface of solid substrates

like composted straw, cotton wastes, wood logs, saw dust, rice bran and rice
straw. Some substrates are used for mushroom cultivation without pretreatment
(e.g. Japanese mushroom or Shiitake, Lentinus edodes grown on wood logs). In
other instances, the raw materials are steam-treated or autoclaved before
mushroom cultivation. However, due to the sheer bulk of the raw material, it is
likely that some heat-resistant fungal spores will survive. A series of fungal
pathogens have also been identified while growing edible mushrooms (e.g.
Aspergillus fumigatus, Aspergillus spp., Humicola insolens, Mucor spp.,
Mycogone sp., Penicillium spp., Rhizomucor pusillus, Torula thermophile,
Verticillium sp.). Other fungi isolated belong to Trichoderma and Chaetomium
(Fermor et al., 1985). Substrates often harbour “weed” fungi leading to major
losses. Fungal viruses have also been reported from edible mushrooms
(Agaricus bisporus) (Atkey, 1985). Common methods to control weed fungi in
raw material during mushroom cultivation include the use of chemicals and
fungicides. The extensive use of fungicides has led for the development of
resistant fungal spores (Elliot, 1985). Harvested mushrooms also undergo
deterioration by fungal contaminants during storage in spite of packing and
refrigeration. EBI is a promising approach for the decontamination of raw
materials for mushroom cultivation, and to prevent fungal growth during and
after production of mushrooms.

Bakery products. Most popular bakery foods become contaminated with

various toxigenic moulds during different stages of preparation. The most
common contaminants are Aspergillus spp., Penicillium spp. and Rhizopus spp.

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Aspergillus flavus and A. ochraceus have been recovered from flours used for
bread and other bakery products (Pitt and Hocking, 1985; Christensen, 1987;
Pitt and Hocking, 1985; Filtenborg et al., 1996; Visconti and Bottalico, 1983).
Similarly, Penicillium verrucosum contamination has been reported in cakes
(Williams, 1990) and carcinogenic aflatoxins are also evident in bakery
products (Pohland and Wood, 1987). Chocolates, despite low water content, can
become contaminated with Chrysosporium spp. (e.g. C. farinicola) leading to
quality deterioration. Similarly, jams are known to have Eurotium spp.,
Penicillium coryophilium and Wallemia sebi, while Zygosaccharomyces rouxii
is common on honey (moldbacteria.com/myblog/2005_07_01_moldbacteria_archive.html;
Brysch-Herzberg, 2004; Lugauskas and Stakéniené, 2002).
Fungal contaminants are usually prevented by exposing the product to
infrared, microwaves and by the addition of fungal inhibitors (e.g. ethanol,
propionic acid, sorbic acid, acetic acid) (Legan, 1993). Permissible limits of
chemical preservatives in bakery products have been reduced in Europe by EU
Directive 95/2/CE (Anonymous, 1995). Are conventional methods of
preservation still effective under these restrictions? The effects of some of the
commonly used preservatives (e.g. calcium propionate) on conidial germination
have been tested with fungi contaminating bakery products. Tests revealed that
the use of sub-optimal salt concentrations will be ineffective (Lavermicocca et
al., 2000). Recently, Guynot et al. (2005) have isolated the most common fungi
that spoil bakery products (Eurotium spp., Aspergillus spp. and Penicillium spp.)
and studied their responses to 20 essential oils (e.g. cinnamon leaf, rosemary,
thyme, bay and clove essential oils). The findings underlined the potential of
essential plant oils as an alternative to preserve bakery products. A drawback is
that oils produce off flavors or alter the original aroma of the product on long-
term storage. Char et al. (2005) have worked on the growth of xerotolerant
moulds in milk jam (a typical Argentine sweet-spread prepared from milk) and
studied the influence of water activity, pH and addition of potassium sorbate
(1,000 ppm) on the growth of Eurotium chevalieri, Aspergillus fumigatus and
Penicillium brevicompactum. Certain combinations of these factors were found
to assure inhibition of fungal growth during a critical storage period. However,
this method needs further refinement to preserve the bakery products. Again,
EBI promises to be an effective alternative for the decontamination of bakery
products.

Dairy products. Post-manufacture contamination of sweets produced

from milk and milk-based products is common (Soomro et al., 2003; FRI
Newsletter, 2004; capemaycountygov.net/cit-e-access/webpage.cfm?TID=5&TPID=5041).
Dairy scientists have concentrated on health risks associated with bacterial
contamination from Listeria, Legionella or Salmonella and ignored risks posed
by yeasts and other fungi. The problem becomes more serious when the product
has to be stored for extended periods. Although yeast and mould counts may
vary depending on the hygienic environments and storage conditions employed,
in a majority of cases fungal contamination seems almost inevitable and
relatively harmless, common moulds (Aspergillus, Penicillium, Mucor,

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Rhizopus, Geotrichum, Syncephalastrum, Cladosporium) (Malhotra and Prasad,

1999; Dhand et al., 2001) and highly toxigenic moulds (Aspergillus flavus, A.
parasiticus, A. niger, A. fumigatus, A. oryzae, A. ochraceus, A. terreus,
Penicillium expansum and P. viridicatum) (Mor and Singh, 2000; Naresh
Kumar et al., 1996).
The most common preservatives to prevent spoilage are potassium
sorbate, benzoic acid and propionic acid. In view of increasing consumer
resistance, a search for alternatives is inevitable. Although bacteriocins are
recommended as natural preservatives (e.g. nicin, subtilin), their use is still in its
infancy. A combination of bactericins and EBI to preserve dairy products looks
promising.
Fungi are involved in the ripening of a variety of cheeses (Roquefort,
Gorgonzola, Brie and Camembert). Even though these moulds are safe for
consumption, they may be associated with harmful bacteria (Listeria, Brucella,
Salmonella and E. coli) and contamination by other moulds (e.g. Penicillium
discolor). Current method to prevent mould growth on cheese includes plastic
wrap and refrigeration. Alternatively, EBI technology may prove to be more
effective in preserving cheese at room temperature for extended periods or
refrigeration of cheese after EBI might be appropriate.

Brewing. Barley, an important raw material for the production of beer,

harbours many types of bacteria, toxigenic moulds, and heat-resistant yeasts.
The moist warm conditions of malting favour rapid growth by these
microorganisms. Standard specifications for brewing quality have been outlined
by the Analysis Committee of the European Brewery Convention (1984). Some
of the common moulds isolated from barley before harvest, during storage and
during malting belong to Fusarium, Rhizopus and Aspergillus. Although most
of these moulds or their spores are killed by boiling, a ‘mouldy flavor’ may
persist in the final product leading to rejection of consignments. Presence of
these moulds may cause ‘gushing’ (uncontrollable overfoaming of the beer
when opened from the can or bottle). Even the addition of the enzyme ‘papain’
as chill-proof agent may be insufficient for complete decontamination of the
container. Experiments with contaminated grains (Ochratoxin A by Aspergillus
ochraceus) showed a reduction in mycotoxin after proper brewing (Gjersten et
al., 1973). Some non-beneficial strains of wild yeasts have also been isolated in
large amounts during beer production and fermentation. Barnett et al. (1983)
and Gilliland (1971) discussed these contaminants in detail. Several non-
alcoholic beverages (e.g. soymilk, ginger beer, Soborodo and Kunu-Zaki) have
been reported to harbour a wide range of toxigenic moulds (Aspergillus,
Candida, Penicillium, Trichoderma) leading to quality deterioration as well as
consumer rejection (Osuntogun and Aboaba, 2004). Use of EBI might prevent
nonspecific fungal interference.

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Post harvest problems and EBI

Cocoa. Cocoa is an important plant product necessary for manufacturing

cocoa powder and butter, chocolates and various beverages. The Draft
regulations by the EU has stated that ochratoxin A levels in cocoa bean and
powder imports must not exceed 2 ppb, while finished cocoa products
(chocolate) must not contain more than 1 ppb (Harrison, 2000; Probert, 2003).
There is a growing concern about fungal contamination rates in cocoa and its
products at different stages of processing and storage. Toxigenic moulds can
contaminate cocoa beans during fermentation, drying, transport and storage and
presence of ochratoxin A is also known in cocoa. As cured beans are highly
hygroscopic, they actively absorb moisture. Successful storage of cured cocoa
beans depends on low moisture content (≤8%, w/w). Some of the common
storage moulds isolated during cocoa bean fermentation are Aspergillus flavus,
A. fumigatus, A. niger, A. tamarii, Penicillium and Mucor spp. Usually
decontamination and disinfestation of cocoa beans is achieved by fumigation
with methyl bromide or phosphine or a mixture of 98% carbondioxide and 2%
phosphine (CMAAC, 1999). Most of these chemicals are now banned or have
ozone-depleting property. Methods to replace fumigation include high/low
temperatures treatments, atmospheric storage (elimination of oxygen/flushing of
carbon dioxide), impregnating jute bags with neem oil, gamma irradiation and
biological control agents (e.g. Bacillus thuringiensis) (Bullington, 1998). Even
though cocoa beans are protected during storage by their shell, chemical
fumigation likely penetrates to the edible portion. Recent observations indicate
that ochratoxin A accumulates in shells and industrial-shelling methods could
prevent the toxin in final cocoa products (Amézqueta et al. 2005). Whether such
methods are practicable in tropics and developing countries prone to high mould
contamination is questionable. Treating cocoa beans by EBI may be a better
choice than the current methods in processing and storage of cocoa beans.

Vanilla. Vanilla is one of the most valuable flavoring agents in the food
industry ($4,000/kg) (Muheim, and Lerch, 1999). Green vanilla beans harvested
are devoid of flavor, but acquire it during post-harvest processing (curing)
lasting 2-6 months (Ranadive, 1994). The main objective of curing the beans is
to develop flavor in vanilla (enhancing enzymatic hydrolysis of flavor
precursors to vanillin). Subsequently, drying cured beans helps in extraction,
enhances and preserves the flavor compounds generated (Adedeji et al., 1993).
Curing of vanilla beans comprises four major steps i.e. killing, sweating, drying
and conditioning.
The current methods of killing beans are based on the ancient Mexican
method (wilting the beans in the sun until beans became brown; Balls and
Arana, 1941). Contemporary methods include sun drying, oven drying, hot
water exposure and scratching, or freezing (Childers et al., 1959, Ranadive,
1994). After killing, beans are allowed to sweat at high humidity and high
temperature (45-65ºC) for 7 to 10 days, which allows the characteristic vanilla
flavor, aroma and color to develop. During sweating, moisture is allowed to

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escape to prevent spoilage by microorganisms. After sweating, beans become

brown and develop a characteristic flavor and aroma. Traditional drying to
terminate enzyme activity lowers the moisture content of these beans from
about 60-70% to 25-30%. This protects against microbial spoilage of beans
(Ranadive, 1994).
The drying of beans to attain desired size and to preserve the flavor as
well as the vanillin content is one of the most difficult steps in curing. A wide
variety of physical methods (e.g. sonification, blending) are used to prevent
microbial contamination (Lindahl and Bakken, 1995). After curing, the beans
are graded as top quality (long, fleshy, supple, dark brown to black, oily
appearance, strongly aromatic, free from physical injuries and with 30%
moisture) and low quality beans (hard, dry, thin, brown to reddish brown and
with poor aroma). Four to five bundles of cured beans are wrapped in butter
paper or polypropylene bags, sealed and stored in airtight
wooden/aluminium/styrofoam boxes.
Vanilla beans may be prone for fungal contamination at various steps
following their harvest. As post-harvest, processing and storage practices are by
and large traditional in most regions, fungal contaminants or mycotoxins in
finished product cannot be ruled out, but until recently no detailed reports were
available on fungal contamination or mycotoxins in finished vanilla and
possible implications for human health. At each step of curing (killing, sweating
and drying), EBI technology may become be useful to minimize the risk of
fungal contamination or invasion However, the specific dose of EBI required at
different steps without impinging on the flavor and aroma of vanilla will have to
be standardized. EBI of the final product of vanilla will be successful in
eradicating fungal contaminants and assure quality and improved shelf life.

Copra. Copra (dried kernel) of coconut (Cocos nucifera) is highly

valued as a source of edible oil and cake. The residue after oil extraction is used
as livestock feed. The main copra exporting countries are the Philippines, Papua
New Guinea, Vanuatu, Mozambique, Malaysia, the Pacific Islands, India and
Sri Lanka. Harvesting copra is tedious. The ripe coconuts are allowed to dry,
split, deshelled and the kernel is removed and sun dried. The lack of appropriate
post-harvest technology causes significant losses during harvest, seasoning,
drying and storage. Improper open-air drying, high moisture content,
inappropriate storage practices and delayed transport facilitate microbial
invasion. Such inadequacies resulted in declining exports of copra, oil and cake
(Rouziere, 1994; Corbett, et al., 1937; FAO/WHO, 2004). Different moulds
(Rhizopus sp., Aspergillus niger, Aspergillus flavus and Penicillium glaucum)
are involved in the deterioration of copra and its products. Philip et al. (1981)
investigated the fungal contaminants of copra (Botryodiplodia theobromae,
Mucor hiemalis, Curvularia senegalensis and Rhizopus oryzae) and their
impacts on degradation of amino acids. Head et al. (1999) studied the impact of
processing of copra by different methods (sun drying, drying in a kukum dryer,
smoke drying), followed by storage (2, 4 and 8 weeks) on aflatoxin
contamination (<20 µg/kg). Clearly, copra is highly susceptible to fungal

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invasion resulting in deteriorating quality. As there is no information on the use

of natural preservatives or chemicals for safe storage purposes, it is imperative
to investigate new methods to prevent fungal invasion. This may include EBI.

Coffee and tea. Coffee and tea are the most common non-alcoholic
beverages (vonl.com/chips/slbt.htm). Coffee is obtained by roasting beans of
Coffea arabica (Arabica) and Coffea canephora (Robusta) varieties. The quality
of coffee bean is profoundly influenced by the processing methods during post
harvest (wet or dry) and storage. In wet processing, the ripe berries are pulped,
fermented, washed and sun dried prior to dehulling. In dry processing, ripened
(occasionally unripened) berries are directly sun-dried, dehulled and stored
(Clifford; 1985, Barel and Jacquet, 1994 Avallme et al., 2001). As the processed
coffee beans are highly hygroscopic, they absorb moisture during improper
storage, which encourages fungal contamination. In many coffee-growing
regions, fumigants (which are either banned or likely to be) are employed
commercially in order to control storage pests or microbial contaminants. A
great diversity in fungal contaminants has been reported from processed coffee
beans. Some of the commonly isolated moulds belonged to Aspergillus (A.
niger, A. ochraceus, A. flavus, A. carbonarius, A. wentii and A. versicolor),
Penicillium and Cladosporium (Mislive et al., 1983; Micco et al., 1989,
Pohland 1992; Taniwaki et al., 2003). Levi et al., (1980) has reviewed the
occurrence of mycotoxins in coffee and indicated the presence of aflatoxins,
ochratoxin A and sterigmatocystin. The most studied toxin among these is
ochratoxin A, which is present in green coffee at a high concentration (Micco et
al., 1989; Frank, 1999; Romani et al., 2000; Leoni et al., 2001). A wet physical
method has been suggested to limit the risks of ochratoxin A production in
green coffee (Suarez-Quitroz et al., 2005). However, recontamination by
toxigenic moulds can occur during long-term storage. Some mycotoxins in
green coffee beans (mainly ochratoxin A) have been shown to be destroyed or
reduced to below detection limits during roasting (Micco et al., 1989; Blanc et
al., 1998). EBI might be a suitable alternative to replace conventional
techniques. The dried green coffee beans, prior to storage can be subjected to
the maximum permissible limits of radiation treatments for effective
decontamination. If the sterility of green coffee beans can be achieved at low
dose, artificial inoculation by beneficial microbes or moulds capable of
decaffeinating may help avoid current chemical decaffeination methods.

Spirulina and green coffee. Spirulina platensis is known for antioxidant

activity, which is very important in human health. NemtŃnu et al. (2006)
attempted biological decontamination of S. platensis and ground green beans of
Arabica coffee through EBI (5-40 kGy). The non-irradiated coffee was
dominated by Aspergillus niger and A. ochraceus. Intact and ground green
coffee beans were devoid of fungal and bacterial load at 5 and 10 kGy
respectively, while S. platensis lost bacterial and fungal contaminants at 10 and
40 kGy. Even at 40 kGy, S. platensis retained antioxidant activity of up to

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45.9%. Similarly, the rheological and spectrophotometric analyses did not

reveal modifications of irradiated green coffee irradiated at 40 kGy.
Tea (Camellia sinensis L.) has been consumed for its taste and health-
promoting benefits. Tea has rich antioxidant, antipyretic, anti-inflammatory and
antimicrobial properties. Its consumption is known to reduce the risk of cancer,
lowers the risk of heart disease and stroke (Cheng et al., 2005; Chen et al.,
2005, Chanadiri et al., 2005; Wirth and Hakenberg; 2005, Raza and John, 2005,
Siddiqui et al., 2005; Augustniak et al., 2005). After harvest (plucking of tender
leaves), the black tea undergoes a series of processing like withering,
maceration, fermentation, firing, rolling drying and grading. Moulds and
bacteria have the opportunity to grow during processing (Sanderson 1983).
Sivaplan (1982) reported that tea is strongly hygroscopic and easily absorbs
moisture from the atmosphere during or after processing. Reports are not
available on health risks caused by consumption of tea due to mould
contamination possibly due to low moisture (3-4 %) in dried leaves. The
International Food and Agricultural Organisation has established a standard on
black tea, stating that it should be packed at a moisture <4%. (FAO, 1995)
Some studies have been performed on the mycoflora of tea leaves of four
popular brands in Muscat (Elshafie et al., 1999). Common fungal species were
Aspergillus flavus, A.niger, Penicillium spp. and Paecilomyces spp. None of the
A. flavus isolates screened were toxigenic. Mould contamination of regular and
herbal teas has been documented (Kuminsky et al., 1996; Le Bars, 1988).
Martins et al., (2001) studied the natural occurrence of fumonisin (FB1 and
FB2) in black tea obtained from supermarkets in Lisbon (Portugal). Their
samples contained fumonisin B1 (FB1) at levels ranging from 80-280 µg/kg.
Similarly, Omurtag and Yazicioglu (2004) measured the potential levels of FB1
and FB2 in several herbal teas consumed regularly in Turkey (FB1, 86.9±8.4%;
FB2, 102±6.8%). Microbial contamination of bags with herbal tea and brewed
tea were tested by Wilson et al. (2004) and demonstrated the potential risk of
nosocomial infection.
The FAO (1995) cautioned against moisture absorption and deterioration
of quality of tea by mould attack particularly during shipment to other countries.
Tea processing does not rely on any chemicals to control microbial
contamination. This may be due to the fact that tea contains large amounts of
antimicrobial polyphenolic compounds (Chung and Murdock, 1991). Gangneux
et al. (2004) examined the efficiency of disinfection procedures such as boiling
and microwave heating to eradicate the spores of Aspergillus fumigatus. Thus,
risks of mould contamination and their toxins in tea during processing, storage
and transport cannot be ruled out and it warrants a safe and foolproof method of
decontamination, which may include EBI technology.

Cashew. Cashew (Anacardium occidentale L.) is a small to medium

sized, fast growing, tropical tree, which has been cultivated extensively for its
economically important nutritional and industrial products (cashew apple, nuts
and nut shell liquid). Almost all tropical countries cultivate this crop with
variations in size and taste. The nuts appear as an appendage on the end of a

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fleshy edible bright yellow or red peduncle (fruit or cashew apple). The seeds
are dehulled, which involves cleaning, roasting, pre-grading and drying (open
sun drying or furnace drying followed by peeling grading and packaging). After
obtaining the nuts, they are dried to attain appropriate moisture (4-6%) and
packed in polythene or jute bags. The usual packaging employed for export of
kernels is in airtight tins (capacity, 25 lb), which involves substituting air with
carbon dioxide.
As many other agricultural products, cashew nuts are hygroscopic
(Pixton, 1967). Adebajo and Diyaol (2003) isolated many fungi from cashew
nuts (Aspergillus niger, A. restrictus, A. flavus, A.fumigatus, A. ochraceus, A.
tamarii, Pencicllium citrinum, P. digitatum, Rhizopus nigricans, Mucor pusillus
and Syncephalastrum sp.). However, other reports indicate that cashew nuts are
devoid of mycotoxins (Abdel-gawad and Zohri, 1993). Cashew nuts are mostly
consumed raw or after mild roasting. If any contaminant moulds are present,
there is a risk of food poisoning. Although mould contamination may not be
apparent upon visual examination, fungal contaminants may still be present as
cashew nuts are highly nutritious. Low doses of EBI may suffice to
decontaminate fungi without affecting the overall taste of cashew nuts.

Spices. Spices are highly valued products and provide a major part of
foreign exchange for producer countries. Most spices are rich in antioxidants
and anti-microbials, and as a result, their use as natural preservatives in food
industries has increased. Common spices include: black pepper, cardamom,
cumin, fenugreek, saffron, garlic, fennel, turmeric, ginger and chilli. The
moisture in spices is usually low, which largely protects them against microbial
spoilage. Inadequate drying and storage are primarily responsible for fungal
contamination. Drying in most the producer countries is primarily by spreading
them on floors. Spices harbour some microbes capable of causing spoilage or
diseases (IAEA, 1992). The most commonly isolated toxigenic fungi include
Aspergillus flavus and others belonging to Alternaria, Penicillium, Fusarium
and Mucor. Currently, chemical fumigants (e.g. ethylene oxide) and thermal
treatment with steam are most commonly used to disinfect spices. Fumigants
are less effective and traces of harmful toxic residues may be retained, while
thermal treatment may alter the aroma and flavor of spices. Stringent quality
specification has been introduced in some of the leading importing countries
like US, Germany, UK, Netherlands, France and Japan to ensure high quality
and safety. The roles played by FDA, American Spice Trade Association
(ASTA) in USA and provision under the Law on Food and Consumer Goods
(LFCG) (August 1974) in Germany is worth referring to. Based on these
regulations, if the defect level exceeds permissible limits, legal action can be
enforced or the exporting country can be blacklisted (e.g. toxin limits:
aflatoxins, 4 ppb; AFB1, 2 ppb). Processing spices by gamma irradiation (10-14
kGy) has been well established and is an approved technology to improve the
quality in many European countries, US and India. Gamma irradiation has been
successfully implemented and studied for quality amelioration of spices (Farkas,
1998; Chatterjee et al., 1998; Vajdi and Pereira, 1973; Mahfouz-Al-Bachir,

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2005; Kuruppu et al., 1988). Improving the quality of spices without impinging
on the original quality and flavor is a critical task and thus the application of
EBI can play a significant role in preserving and enhancing the commercial
value of spices and their blends.

Honey. Bee honey, normally contaminated with a variety of saprophytic

(Saccharomyces, Schizosaccharomyces and Torula) as well as pathogenic
microbes (aerobic Bacillus, anaerobic Clostridium spores, and spores and small
fragments of moulds; Migdał et al., 2000). Osmophilic yeasts in honey initiate
fermentation, clostridia pose a risk of alimentary infection (primarily in infants
due to their underdeveloped immune system), surgical applications in injury and
in ophthalmology. The EBI of honey at 10 kGy decreased the aerobic and
anaerobic bacteria and fungi by up to 99%, did not affect nutritional properties
of honey (e.g. color, taste, acidity, diastase activity) and increased the antibiotic
index of different types of honey (1.8 to 2.6) (Migdał et al., 2000).

Herbal products. There is an upsurge in the popularity of herbal

medicinal products like Ayurveda, Traditional Chinese Medicine (TCM).
Nearly 70-80% of the world population, primarily in the third world relies on
such non-conventional medicines (Akerele, 1993). These medicines are
administered orally, and contaminants may pose health risks. These include
presence of heavy metals, pesticides, radioactive compounds and
misidentification of raw materials.
In addition, microbial contamination is a major concern. It can be traced
to the collection, preparation and preservation of the drug. In most instances,
crude drugs are stored in warehouses under warm, humid conditions. High
humidity favours the growth of yeasts and moulds. Insect infestation may
further stimulate microbial contamination of the herbal raw material. Fungi such
as Penicillium, Aspergillus, Rhizopus, Mucor, Cladosporium and
Aureobasidium spp. have been isolated from herbal drugs (Hitokoto et al.,
1978). In some studies toxigenic fungi (e.g. Aspergillus flavus, A. candidus, A.
niger, A. luchuensis, A. ochraceus, A. nidulans, Fusarium moniliforme, F.
oxysporum, Chaetomium, Alternaria alternata, Penicillium citrinum) were
isolated from raw plant materials used for drug preparation (Ayres et al., 1980;
Azeez and Youssef, 1991; Hitokoto et al., 1978; Misra, 1981; Roy et al., 1987).
Presence of mycotoxins in medicinal plants has also been documented
(Chourasia, 1990; Rani and Singh, 1990; Roy and Chourasia, 1990).
Fumigants are used to decontaminate the raw materials used for herbal
medicines (e.g. sulphur fumes, ethylene dioxide, ethylene oxide). Most of these
chemicals have been banned due to environmental and health hazards. There are
a few reports where gamma irradiation has been employed to decontaminate the
raw materials (Damle et al., 1988; Karmazin et al., 1988, 1990; Venskutonis et
al., 1996). EBI at standardized dose might prove efficient in decontamination of
raw materials and finished herbal drugs or products.

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Compost, waste waters and EBI

Agricultural residues contain high levels of lignocellulose, and their

enzymatic hydrolysis is slow due to compositional heterogeneity and structural
complexities. Physical or chemical treatments of lignocellulosic materials to
modify their chemical structure for enhanced degradation are expensive. EBI
may be an effective alternative for partial degradation of lignocellulosic wastes
prior to bioconversion by fungi. For instance, the white-rot fungus
(Phanerochaete chrysosporium) and other filamentous fungi (Humicola sp.,
Phialophora sp., Trichoderma reesi) are capable of degrading lignin and
cellulose. There are possibilities of enhancement of such transformation on
treating agricultural wastes by EBI prior to fungal treatment. During the
composting process, various members of macro-fauna (earthworms, millipedes,
mites, beetles and worms) usually invade and make compost ready to use.
Beneficial mycoflora species can also be inoculated at later stages (e.g.
ectomycorrhizae, AM fungi, Trichoderma sp.) before the compost is applied to
crops. However, the use of compost harboring fungal pathogens can be
dangerous. One of the classical examples is the case of organic farming in
coffee plantations. Aspergillus ochraceus is a potential nephrotoxin producing
fungus, which has frequently been isolated from coffee beans. During on-farm
composting of coffee wastes the cycle of A. ochraceus may complete itself and
the compost becomes contaminated. EBI technology may effectively sterilize
finished composts and eliminate dangerous pathogens. Such treated composts
are ready for inoculation with bioprotectants (e.g. herbicidal, insecticidal,
parasitic, entomo-pathogenic, nemato-pathogenic fungi) or macrofauna (e.g.
earthworms, millipedes). Incorporating fungal bioprotectants into compost after
EBI has incredible potential for controlling dispersal of plant pests and
pathogens.
Domestic and industrial wastewaters possess a variety of pollutants and
fungal pathogens, which pose problems during biological treatment. The EBI at
1 kGy, enhanced the biodegradability of textile wastewater (BOD5/COD) and
facilitated activated sludge process (Kim et al., 2007). The refractory organic
compounds were transformed into more easily biodegradable low molecular
compounds (e.g. oxalic acid, formic acid). With EBI, even at a short hydraulic
retention time of the activated sludge, high organic removal efficiencies were
achieved with good COD and BOD5 removal efficiencies. The textile
wastewater was effectively treated by integrating EBI and an activated sludge
process, which reduces the cost of biological treatment. Pre-irradiation of
activated wastewater sludge with EBI at 8 kGy effectively suppressed foaming
(Čuba et al., 2003). Similarly, EBI was also effective in destroying aromatic
organic compounds in industrial effluents (Duarte et al., 2000).

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Outlook

EBI is an effective method for mould decontamination and sterilization.

In future, it may replace conventional dry sterilization techniques. High dose
EBI sterilization may allow energy saving compared to the conventional
techniques. Soil remediation employs a variety of methods (physicochemical,
thermal and biological) to reduce or eliminate pollutants. EBI technology might
allow the initial elimination of microbes followed by re-inoculation of desired
microbes, to scavenge the pollutants through processes such as bioventing,
bioslurry, land treatment and land farming. Solid state fermentation is an
efficient technique to produce a variety of microbial secondary metabolites (e.g.
organic acids, antibiotics, steroids). Pre-sterilization of substrate is necessary
before inoculating the desired fungus. Sterilization can be achieved through EBI
at appropriate doses. It is necessary to investigate whether EBI at sub-lethal
doses enhances the production and extractability of fungal secondary
metabolites. EBI technology can also be implemented in the decontamination
and preservation of traditional and oriental foods (e.g. pickles, marinated foods,
edible seaweeds, parboiled rice). There is an increasing popularity to use plant-
based environment friendly biodegradable food packaging materials instead of
plastics and other synthetic polymers. Commonly used plant materials include
leaves (banana, areca, grass, palms), shoots (bamboo), sugarcane wastes (straw)
and cornstarch. However, there is insufficient information on the microbial
contaminants of these raw materials or finished products. These biodegradable
packaging materials may be used more safely after decontamination by EBI.
Surface disinfection of plant materials (tissues and seeds) has been achieved by
many chemicals (e.g. mercuric chloride, ethanol, sodium hypochlorite,
chloramine-T, sulphuric acid). Depending on the material, EBI (single or
double side) can be employed with environmentally friendly, low energy
electrons. Similarly, economically important goods (e.g. textile, leather, books)
can be disinfected through EBI. The EBI will become a leading technology
destruction of microbes of hospital wastes and in medical waste management.
This technology will also facilitate quarantine procedures between geographic
regions. On suspicion of transfer of biological weapons (e.g. pathogenic
moulds), EBI technology may play a major role in national security. To inform
the consumers, a logo named ‘Radura’ is usually displayed on the cover of
irradiated products. It includes the dates, year and batches, when the irradiation
was executed (Fig. 1). Radiation processing can be an essential part of an
overall Hazard Analysis Critical Control Point (HACCP) program for the
processing of food and agricultural commodities. However, Good
Manufacturing Practices (GMP) and Good Agricultural Practices (GAP) may
provide additional safety to consumers. The technology may be of great benefit
to food processors, distributors and agriculturalists in the near future. Although
irradiation is not presently permitted for organic foods, it may occupy a
prominent place in decontamination of microbes including fungi once its
advantages are fully realized. The current chapter has outlined several areas of
applied mycology where EBI technology might be beneficial by facilitating

293
 Novel Techniques and Ideas in Mycology

decontamination. It is still in its infancy, and implementation on pilot scale may

provide data on where it will fulfill its promise.

Fig. 1. Radura logo to indicate that the product has been ‘Electron Beam Irradiated’

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