Novel approach for the use of Ozone in fumigation of white maize grains (Zea mays L.) for the prevention of aflatoxins

Abstract

In Africa and especially in Mozambique the family farming sector cultivates more than 90% of agricultural land and is characterized by a subsistence orientation. The family farm averages less than 4 hectares, with post-harvest losses estimated at around 30%. One of the causes being the development of fungi and mycotoxins (1).

Mycotoxins are dangerous contaminants that endanger the health of humans and animals. The incidence of these mycotoxins is high in foods in many parts of Africa. The development of appropriate control methods to reduce this incidence is a pending task since there is no point in developing methods that cannot be later replicated in the field. Given the scope and complexity of the problem, it is necessary to develop an economical and practical method that can be used by smallholder farmers.

Of all the methods found in the literature, the use of ozone to combat aflatoxins seems to be one of the least explored. This may be because not so long ago, ozone production equipment’s were very expensive and had a very low ozone concentration production. Today relatively inexpensive equipment with high ozone production yields can be found in the market. What makes this method a potential option for the control of aflatoxins.

All the studies reviewed regarding the use of ozone to control pests or reduce mycotoxin concentration have been conducted to demonstrate its efficacy. That is, they measure the reduction of a known initial contamination in the product. From these studies it is concluded that at the laboratory level, ozone is an effective means to reduce the concentration of mycotoxins, molds and some pests in the products. However, no studies were found on how to use ozone gas in a practical way in family farming, as a preventive treatment for mold contamination and its toxins in grains.

Given the need to understand if it is possible and practical to use ozone gas to fumigate grain in storage as it is often stored by smallholder farmers in Africa the following was evaluated. The feasibility to carry out a fumigation with ozone gas using the blanket method and the capacity of ozone to prevent the appearance of aflatoxins in the product. For this, the results of the treated samples were compared against the untreated control sample.

The experimental results show that the presence of aflatoxins was not detected in any of the treated samples, and they were only detected in the control samples after 6 months. It was observed that an increase in the treatment time reduces the concentration of fungi from the field in the initial product. In the present work it is concluded that it is possible to carry out ozone fumigation using the blanket method. That ozone has a protective effect on the treated samples, preventing the appearance of aflatoxins and that ozone gas does not affect the sensory characteristics of the product in terms of color, smell, and appearance.

?Keywords: Ozone fumigation, ozonization, aflatoxins, mycotoxins.

Introduction

Mozambique is a developing country that, until the early 1990s, suffered from a prolonged civil war. It is one of the poorest countries in Africa, and the livelihood of more than 80% of its population depends on agriculture. Most farmers are subsistence smallholders, farming less than three hectares (1). The productivity of maize, the main staple food crop, has an average yield of 1.0 t/ha.(2). It is also a country with a high incidence of aflatoxins, especially in crops such as maize and peanuts.

Ozonation is the process by which stored products are exposed to ozone gas to terminate or reduce unwanted biological activity. This is how ozonation is presented as a potential method to control pest infestation, the development of molds and mycotoxins. Following a review of the scientific research on ozonation, it was found that, at least in some cases, ozonation appears to be very effective against infestation; that the effects reported in most cases are not causing any damage to the crops; and that the concentration of mold and their respective mycotoxins in the treated products is significantly reduced. (3,4)

Now, how are these lab test results transferred to storage facilities?

At laboratory level, the tests consisted of placing the product in a sealed environment and passing a stream of ozone gas through the product. This is not possible in practice since the product is usually bagged and stacked in warehouses awaiting distribution and sale, so the only viable options to treat the product with ozone would be to open the bags and pass the product through a machine that doses the ozone and then re-bag the product again or carry out the treatment using fumigation sheets to cover the entire product and injecting the ozone gas inside for a certain time. It is this second option which was evaluated.

So, to determine if ozone gas can prevent the development of mycotoxins, especially aflatoxin B1 in maize, using the blanket fumigation method, the following was experimentally evaluated: the reduction of fungal load from the field after ozone treatment, the effects of ozone on product moisture and sensory characteristics and finally the development of aflatoxins was monitored in the product for 9 months. Three treatment times were assessed and compared with a control sample.

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1)???Background

A grain production system consists of several stages. One of these stages is the harvest in which the opportune moment is sought, generally determined by the moisture content of the grain. Subsequently, in the post-harvest, the processes of reception, storage and conservation of the grains are carried out until they are commercialized. Internationally, postharvest losses in stored grains average 5%, however, in underdeveloped countries these percentages increase up to 30% (5). Among the main causes that generate post-harvest losses is the presence of biotic agents such as insects, fungi, rodents and even birds.

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1.1 Molds

Molds are organisms that lack chlorophyll, provided with a thallus, generally filamentous and branched, through which they absorb the organic nutritional principles of the environment. They are of very varied size and preferentially asexual reproduction (by spores); They live as parasites or on decomposing organic matter or parasites of plants or animals.

The mold that can potentially produce mycotoxins and that commonly attack maize both in the field and in storage belong to the genera Aspergillus, Fusarium y Penicillium (6,7); being able to develop in grains with relative humidity contents between 65 to 90% (5). The growth of these fungi and the contamination of food products with mycotoxins are a consequence of the interaction between the mold, the host, and the environment. According to García and Heredia, 2006 cited by (8) the interaction of these factors determines the infestation and colonization of the substrate, as well as the type and quantity of mycotoxins produced.

Today these molds and their toxins are already a serious problem which could worsen with the effects of climate change. It is expected that with an increase in temperature, the areas where these fungi and their toxins can develop will increase. Additionally, in climates where these fungi already develop, it is likely that if drought conditions become more frequent, they could stimulate greater contamination by aflatoxins. (9). Finally, in countries with very cold climates, it is not expected to change, because temperatures would not reach optimal levels for its development and production of toxins.

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1.2 Mycotoxins.

Mycotoxins are secondary metabolites that have harmful effects on human and animal consumers. Mycotoxin contamination in grains is generally an additive process; can start in the field, increase during harvest and drying operations, and continue to accumulate during storage (10–14). Grain water content and temperature are critical factors affecting mycotoxin production (10,13,15). Many molds can produce more than one mycotoxin and quite a few mycotoxins are produced by more than one species of mold. Often more than one mycotoxin is found within the same substrate (13,14,16,17). The production of mycotoxins is null or very low with water activities (aw) below 0.85; however, the growth of toxigenic fungi can occur in water activity ranges from 0.7 to 0.85. (10,18)

Aflatoxins are the most studied and controlled mycotoxins. These are produced by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus (10,13,17,19). Toxicologically, they are considered powerful toxins, related to the genesis of cancer, mutations, and multiple alterations in fetal development (10). Animal experiments have shown that aflatoxins can produce acute and chronic toxicity. Acute effects include hepatic necrosis, nephritis, and pulmonary congestion. Chronic effects include cell damage, carcinogenicity, teratogenicity, and mutagenicity in animals. (10,13,14,19).

Fig. 1. Chemical structure of the four main aflatoxins (10)

Pure aflatoxins are crystalline, tasteless, odorless, and colorless (10). Chemically, aflatoxins are derivatives of difuranocoumarin produced by the polyketide pathway. (10). They are stable in foods and resistant to degradation under normal cooking procedures. (20). There are four main aflatoxins, B1, B2, G1 and G2, with the letters referring to the color of their fluorescence under ultraviolet light (blue or green) and the number indicating the relative migration distance in a thin layer of the chromatographic plate. (19). There are many other less common aflatoxins. Aflatoxin B1 is the most potent known natural carcinogen (19,21) and, in general, if the term aflatoxin is used in the singular, the author refers to aflatoxin B1.

Cereals are probably the most important source of mycotoxin intake, as they are susceptible to fungal attack either in the field or during storage. The production of aflatoxins is favored both by factors that occur in the field and in storage (22).

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1.3 Mycotoxins control methods

Control methods can be divided into strategies designed to reduce the growth of toxigenic fungi and strategies for detoxification of contaminated products. Additionally, the methods can also be divided into pre- and post-harvest strategies. Some methods have been found to significantly reduce the incidence of mycotoxins. However, the complete eradication of a mycotoxin contamination is not achievable at the moment. (23)

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1.3.1 In the field

Rain at or near harvest means unacceptable concentrations of aflatoxins in many products in hot regions (9). In the field, aflatoxin production increases with water stress, high temperatures and damage to the host plant caused by insects belonging to the genera Heliothis and Spodoptera (24). These insects damage plant tissue, thus creating entry portals for the mold. However, aflatoxin can occur in the absence of an apparent injury. Once the temperature conditions, drought stress and inoculum levels are reached, A. flavus can enter the plant through various portals. This variability has made it difficult to develop control measures.

Various field control measures are currently being used or explored, including modification of cultural practices; development of resistant crops using molecular and proteomic techniques; competitive by exclusion using strains that do not produce aflatoxins and development of field treatments that would block aflatoxin production.

However, as mentioned in Devreese et al.(22) It has been found that the most important strategy in the field is the application of Good Agricultural Practices (GAP). These include crop rotation, land preparation or tillage, the correct use of chemicals, integrated pest management and correct irrigation, among others.

These parameters are controllable however the environmental conditions are not. Relative humidity and temperature are factors of great incidence in the development of mold and the production of toxins. Therefore, the planting and harvesting of crops must be carefully planned in order to avoid adverse environmental conditions that favor the development of molds.

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1.3.2 Postharvest control.

In storage, the following conditions are decisive in the increase of aflatoxin synthesis in maize grain: high temperature and humidity, aeration and primary inoculum from the field. However, according to Klinch M. 2007 cited by (8) this synthesis can be controlled by keeping the available moisture at levels lower than those that will allow the growth of the mold.

As mentioned in Schrodter, 2004 cited by (22) Postharvest storage conditions are key in preventing the development of molds and the production of toxins. Prevention strategies include humidity and temperature control, good storage practices, pest control, and the removal of damaged or infected grains. For example, grains should be stored with humidity below 15% to avoid hot spots of high humidity that are favorable for the development of molds.

Within the detoxification strategies, these must destroy or inactivate mycotoxins, generating non-toxic by-products, without affecting the nutritional value, physical characteristics and technical properties of the products. Some of the detoxification processes include radiation, oxidation, reduction, ammonization, alkalization, acidification, deamination, and ozonation. (13). Many of these processes are not permitted by the European Union because the chemical transformation could result in other toxic substances, failing to comply with the requirement of not generating toxic by-products.

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1.3.3 Ozonisation.

Ozone (O3) is an allotropic form of oxygen (O2), that is, it is made up of the same atoms, but they are combined differently. The difference is the presence of three oxygen atoms, while "common oxygen" has only two. Ozone is then enriched oxygen (O3). Ozone is a gaseous compound that is naturally present in the atmosphere and formed because of high-energy ultraviolet radiation. (25,26).

Ozone gas is a powerful oxidant, which can be successfully used to control stored product pests, mold and mycotoxins. (3,4,26–32). The United States Food and Drug Administration (FDA) has recognized the application of ozone as a safe substance (GRAS) for use as an oxidizing agent in food processing (26). When applied directly to cereal grains, ozone (O3) and hydroxyl radicals (OH) generated in the process can react with mycotoxins, promoting their degradation to lower molecular weight products, thus eliminating, or reducing their biological activity in terms of toxicity (33). The effectiveness of ozone in mycotoxin decontamination depends on several factors, such as ozone concentration, exposure time, moisture content and food temperature (26,29,31). Using different conditions, some recent studies have shown that it is possible to obtain a high reduction of aflatoxin levels and microbiological contamination in cereal products. However, more studies should be carried out to know the potential of ozone to reduce mycotoxins and microorganisms in a wider variety of cereal products, since the contamination of these foods is a relevant problem in terms of health and economy. Some of the studies on the use of Ozone in the control of insects, mold and their mycotoxins are detailed below.

McKenzie, K.S. 1997 (29) investigate the degradation and detoxification of mycotoxins in the presence of Ozone. Where equimolar solutions of various mycotoxins were treated with ozone at 2, 10 and 20% by weight of Ozone for a period of 5 minutes and analyzed by HPLC. The results reported a rapid degradation of Aflatoxins B1 and G1 with 2% ozone while aflatoxins B2 and G2 were more resistant to oxidation and required higher levels of ozone (20%).

Méndez, F 2003 (32) evaluated the flux characteristics of ozone through a grain less porous than maize such as wheat, and the effects of prolonged exposure to a high concentration of ozone (50 ppm) on grain quality. He found that in a first phase the ozone degraded rapidly as it moved through the grain and a second phase in which the ozone moved freely through the grain with little degradation. He found that increasing the ozone flow velocity from 0.02 to 0.04 m/s facilitated deeper penetration in the first phase and that treatment of grains with 50 ppm ozone for 30 days had no deleterious effects on grain quality characteristics for end users.

Wang, S 2010 (31) compare the effectiveness of three treatments with ozone. A dry method, wet method and aqueous method on aflatoxin B1 and other mycotoxins in cereals. His results showed that the wet method of fumigation with ozone is the one that best degrades mycotoxins.

Bonjour, E.L. 2011 (30) and Lemic D. et. al. 2019 (27) studied the efficacy of ozone gas fumigation in controlling insects in wheat stored in silos. Bonjour found that a dose of 70 ppm of ozone for 3 days eliminates 100% of the larvae and adults of the species Sitophilus oryzae and Tribolium castaneum. For the case of Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus surinamesis, 100% mortality was not achieved. Concluding that ozone has the potential to control some of the insects that attack wheat in storage. On the other hand, Lemic reported the ability of ozone to reduce the speed of insects and their level of activity. This reduces the chance of insects escaping from treated objects. The results of this research suggest that ozone has the potential to become a realistic option for suppressing harmful insects in storage systems, either alone or as a complement to other control methods.

De Brito, J 2018 (28) concluded that ozonation reduces both the total count of mold, as well as the incidence rate of Aspergillus spp and Penicillium spp in maize grains exposed to ozone gas at a concentration of 2.14 mg/L for a period of 50 hours. They confirmed that ozone gas has a fungicidal effect and can be used in the control of genera Aspergillus and Penicillium in maize grains.

Porto, Y. 2019 (33) reported a reduction of three logarithmic cycles for the count of colony forming units of molds and a reduction of the level of contamination of aflatoxins of up to 57% for maize grits. The parameters of the experiment were an ozone concentration of 20 to 60 mg/L, an exposure time of 120 to 480 min, and a mass of 1 to 5 kg.

Finally, Daneshniya M. et al 2019 (34) studied the effect of ozone on microbiological properties in rice flour. He studied the effect of ozone concentration at three concentration levels of 10, 20 and 30 g/h and exposure time of 15 and 30 minutes. Non-ozonized samples were introduced as control samples. After the ozonation process, the samples were examined twice for 2 months at room temperature. The experimental results suggested that the microorganisms "Staphylococcus aureus" and "Coliform bacteria" were detectable and counted only in the control sample; and negative results were observed in samples treated with ozone at different concentrations and ozonation process times.

Despite the above, the information on the use of gaseous ozone as a mycotoxin prevention agent and for the preservation of cereals outside the laboratory is still very limited. Also, it has not been evaluated if it can be used by blanket fumigation which is the common practice in sub-Saharan Africa.

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2)???Problem definition

In Mozambique, as in many developing countries, where agriculture is a significant contributor to the Gross Domestic Product (GDP), mycotoxins are present in the value chains of many crops, such as corn and peanuts (12). Agriculture constitutes 24% of GDP and 80% of the population depends on it as their main source of income (35). Some of the most commonly grown crops in Mozambique and widely consumed by the population are maize, cassava and peanuts, which are easily contaminated with mycotoxins.

Exposure to mycotoxins and their association with health issues can contribute to a significant public health burden. This is especially significant in less developed countries. The health effects of exposure can be divided according to concentration, acute or chronic. In cases of acute aflatoxicosis, the individual has been exposed to moderate to high levels of aflatoxins. Acute aflatoxicosis is characterized by symptoms such as nausea, diarrhea, abdominal pain, fever, anorexia, drowsiness, acute liver damage, coma, and ultimately death (10,12–14,19). In cases of chronic aflatoxicosis, the individual has been exposed to low or moderate levels of aflatoxins for prolonged periods of time. Chronic aflatoxicosis is characterized by symptoms such as liver cancer, chronic hepatitis, jaundice, hepatomegaly, gallbladder inflammation, growth retardation in children, cirrhosis, and fatty liver. (10,12–14,19).

On the other hand, economic losses related to mold spoilage and mycotoxin contamination impede access to urban and international markets, contributing to rural poverty, constraining economic growth and exacerbating gender inequality. In Africa, the restrictions derived from this problem limit personal, social and national opportunities for development. Mycotoxin contamination is a serious obstacle to many of the Sustainable Development Goals.

We can say that mycotoxins are neglected despite being a major public health problem and their control is underfunded and not considered a priority by many African governments. (12,15,16). As with any other food safety challenge, the mycotoxin problem requires comprehensive strategic initiatives that reduce the challenges regarding health, trade, income, and food security.

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3)???Justification

Famine is one of humanity's most difficult problems today. Millions of tons of food are lost annually in the post-harvest stage due to molds and mycotoxins (5,8,14,36). Aflatoxin contamination is a growing problem for trade, food safety and health issue in sub-Saharan Africa, where smallholder farmers do not have the resources to protect their crops (37,38).

According to researchers from the International Institute of Tropical Agriculture (IITA), exposure to mycotoxins is an obstacle to the health and well-being of the population of Africa, where high levels of aflatoxin contamination have been confirmed. Many smallholder farmers are unable to prevent contamination during the production and storage of their crops because they lack cost-effective methods to control it (39).

Smallholder farmers in Africa resort to traditional storage methods and the use of pesticides to prevent weevils. But these methods are not pest-proof, and so much of the stored crop is lost when it is needed most. They also lack drying equipment, and most keep their maize and peanut crops in the fields to sun dry. Sometimes they are stored before they are completely dry, which makes them vulnerable to aflatoxins (36,39).

Sub-Saharan Africa loses more than 450 million dollars annually in the trade of maize and peanuts, among other crops, due to aflatoxins (39). Many people, without knowing it, consume contaminated food, with the consequent expense for public health, in a region whose health centers are saturated.

In Kenya, considered the country most affected by aflatoxins in East Africa, around 200 people died after eating contaminated maize between 2004 and 2006 (40). Approximately two million bags of maize were declared unfit for human consumption due to high aflatoxin levels in 2010. The Gambia, Malawi, Mozambique, Senegal and Zambia, once net exporters of groundnuts, lost lucrative groundnut markets to the European Union, the United States and South Africa due to aflatoxins in their products (39).

To overcome poverty, it is necessary not only to combat food insecurity, but also to address food safety. Because if a food is not safe, it is not food. Being potent carcinogens, aflatoxins are clearly a food safety concern. Against this, there is a very high rate of cases that could be prevented. This project evaluated the possibility of preventing the development of molds and mycotoxins in maize by fumigating the recently harvested product with ozone gas. In order to reduce the initial field load and thereby reduce the possibility of mycotoxin development. If this works, it could become an economical, ecological and safe technique to prevent the development of mycotoxins.

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4)???Materials and methods

4.1 Maize

In July 2021, ten tons of white maize (Zea mays L) were purchased in Malema district, Nampula province in northern Mozambique. In Mozambique, the maize harvest in the north is from April to July (41). The province of Nampula was selected due to the high incidence of aflatoxins in maize in that area mentioned by Cambaza, et al 2018 (12). The purchase was made just after the harvest from smallholder farmers in the area. The product was purchased in virgin woven polypropylene (PP) bags with a net content of fifty kilograms each. A total of two hundred sacks of maize were purchased to carry out the experiment.

The technical requirements requested for the purchase of the product were that the maize grains have a moisture content of less than 15%, grains damaged by pests less than 4%, dirt less than 0.1%, free of pests and that the product has been recently harvested.

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4.2 Ozone generator

There are several methods for ozone production. These can be through the distillation of water, the hydrolysis of water, the use of ultraviolet light or the most widely used method, the corona effect, where ozone is produced from atmospheric oxygen.

In this method the corona cell is composed of the anode, the cathode and a dielectric barrier material. The most commonly used dielectric materials are ceramic, glass and quartz. Glass is usually found in older generators. Glass has the disadvantage of being less consistent in its composition and therefore is currently the least used in ozone generators. Quartz is the most widely used as it offers a consistent dielectric with high heat resistance. On the other hand, deposits do not form on quartz as easily as on ceramic, and quartz is easier to clean if necessary. Finally, quartz can be shaped in many ways, giving manufacturers greater flexibility in crown design. With respect to the materials that make up the anode and the cathode, the most common are stainless steel, aluminum and titanium. Stainless steel is the most common material in the construction of ozone generators due to its high resistance to ozone and low cost, the only drawback being the fact that it is not a good conductor of heat. On the other hand, aluminum is a great conductor of heat, its drawback being that it is not very resistant to some by-products of ozone. Titanium is used as anode material in some ozone generators offering a great compromise between heat transfer and material resistance. (25).

For the experiment, the model YT-015-10A ozone generator from the company Guangzhou Jiahuan appliance technology Co. Ltd of China was chosen (Fig 2). The ozone generation principle consists of the corona effect method with a titanium electrode and a quartz tube as dielectric barrier material. This equipment was chosen due to its low cost compared to the ozone yields that it can produce. In addition, the equipment has a built-in oxygen concentrator.

The technology for oxygen concentration consists of pressure swing adsorption. This concentrates the oxygen by passing the air through a fixed bed of zeolite at low pressure (3 to 6 bar), until almost all the nitrogen present in the air is adsorbed. Along with nitrogen, water vapor, carbon monoxide and carbon dioxide are also adsorbed. Obtaining at the outlet oxygen concentrates of 90 to 95%. The oxygen concentrator has a production capacity of 5 liters of oxygen per minute.

Ozone is obtained by passing the oxygen current obtained from the concentrator between two electrodes subjected to a high alternating potential. To prevent the formation of an electric arc, a dielectric barrier material of uniform thickness is placed between the two electrodes, creating an equipotential surface. The corona effect is due to the accumulation of high potential electrical charges on the conductors. When this accumulation of electrical charges reaches saturation, the surrounding air becomes slightly conductive and the electrical charges escape, producing a characteristic sound and emitting light. This causes the oxygen molecule (O2) in the air to break down into two oxygen atoms (O1) which join another oxygen molecule (O2) to form ozone (O3) and is then released into the environment. (19,25).

The energy absorbed by an oxygen molecule breaks it down into two oxygen atoms.

O2 + Energy -> O + O?

Each of these atoms binds to an oxygen molecule to form an ozone molecule.?

O + O2 -> O3

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4.3 Ozone

Ozone was discovered in 1840 by Sch?nbein during his observations on the electrolysis of water which produced an odor at the anode identical to that of an electric arc (Sch?nbein, 1838-1840). Sch?nbein proposed that this smell was due to a different chemical substance, which he called "ozone" (25).

Ozone is a bluish-colored gas with a boiling point of -112°C. at atmospheric pressure (26,42). Ozone can partially dissolve in water and the solubility of ozone is thirteen times that of oxygen (25,26,42,43). The oxidation potential is 2.07 volts which shows that ozone is a powerful oxidant, in fact it is one of the strongest oxidants available for water treatment.

Ozone is quite unstable in aqueous solutions; its average life in water is 20 minutes. In air, ozone has an average life of 12 hours, which makes it impossible to store (42,43).

It has been used as a disinfecting agent in the production of drinking water in France since the beginning of the 20th century. The potential utility of ozone for the food industry lies in the fact that ozone is 52% stronger than chlorine and has been shown to be effective on a much broader spectrum of microorganisms than chlorine and other disinfectants (26). Complementing the effectiveness is the fact that ozone, unlike other disinfectants, leaves no chemical residue and degrades to molecular oxygen by natural reaction or degradation. (26). The fact that ozone has a relatively short life is both an advantage and a disadvantage.

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4.4 Fumigation sheets

Successful fumigation requires that a concentration of the fumigant be maintained within the fumigation chamber during the exposure period (44). The demand for fumigation sheets is generally so low that it is difficult to find a variety of materials from which to choose. Therefore, it is necessary to select the most suitable material among those available.

A fumigation sheet is very different from a conventional tarp or waterproof cover. Basically, a fumigation sheet is required to retain the fumigant, which allows the dispersion of the gas at a sufficient concentration and retains the gas for a sufficient time to carry out the treatment (44). The combination of characteristics required in a fumigation sheet to be used for ozone fumigation is low permeability to ozone, durability, flexibility, and chemical resistance to ozone.

Ozone is highly corrosive, although many different types of plastics have properties that make them suitable for use in an ozone environment, many other plastics are highly vulnerable to degradation when exposed to ozone. (45).

Table 1. Characteristics of plastic materials

(Adapted from Friendship R. 1989 and Marco A. et al 2012)

Note: Ratings for plastic range from A for excellent to D for poor.

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Common plastic and rubber materials have a C rating for chemical resistance to ozone. These are not recommended for constant exposure as softening, loss of strength and swelling may occur if used in a continuous ozone environment. These include acetal (Delrin), neoprene, and polypropylene. These materials that are not safe for ozone fumigation should be avoided (26,45).

Plastics, rubbers, and elastomers that have a "poor" D rating should not be used under any circumstances in applications where ozone is present. Class D materials include all nylon materials and all natural rubbers. Poorly rated materials are severely affected by ozone exposure, leading to severe cracking, cracking and corrosion (26,45).

The fumigation blankets selected and used were made of 250-micron polyvinyl chloride (PVC), because they are the ones with the best chemical resistance to ozone, in addition to their good permeability and low cost.

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4.5 Ozone detector

The ozone detector in air from Guangzhou Jiahuan Appliance Technology Co., Ltd of China was used for the experiment. The detection principle is that of electrochemical theory with a measurement range of 0 to 100 parts per million and an accuracy of ≤ ± 2% F.S. (Full Scale).

The electrochemical theory technology is based on the fact that many toxic gases are very reactive and under suitable conditions they produce electrons that are measured by the electrochemical sensor. This consists of a micro-reactor, where in the presence of ozone it reacts and generates a very low electric current, but measurable with a micro-ammeter. The amount of ozone present is related to the amount of current produced (46).

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4.6 Location and design

Maize was transported in 50 kg bags to one of the World Food Program warehouses in the city of Matola, Maputo province. The ten tons (200 bags) were separated into four blocks of two and a half tons. The product was placed on plastic pallets, 20 cm from the concrete floor and half a meter from the wall and between blocks.

The configuration used was thirteen bags (5-3-5) for the base and second row while the third and fourth rows were placed with only twelve bags (5-2-5), leaving a hole in the center of the block in the third and fourth row. This was done with the aim of improving the exposure of the bags to the gas.

Fig. 2. Bags configuration

4.7 Process

Once the four blocks were assembled in the warehouse, samples were taken from each block and an additional sample made up of samples from the 4 blocks.

A PVC hose from the ozone generator was placed in the center of the block. The dimensions of the hose were 3 meters long by 15 mm thick and 5/8” in diameter. The outlet of the hose was in the upper part of the block to take advantage of the fact that ozone is denser than air and due to gravity, it will tend to go down. Then the first block was covered with the fumigation sheet and the bases were sealed using bags filled with sand. Finally, the oxygen concentrator was turned on first and a minute later the ozone generator was turned on. The equipment was regulated for an ozone production of 10 grams per hour. The equipment was left operating for a total of 22 hours for the first block. The process was repeated for a total of 44 hours for the second block and a total of 66 hours for the third block.

At the end of each ozone treatment, samples were taken from each treated block for analysis. The analysis performed on the samples were: moisture, total mold, total aflatoxins, aflatoxin B1, B2, G1 and G2, color, odor, and appearance. New samples were taken from the four blocks (3 with treatment and 1 without treatment) three months after treatment and new analysis′ were performed. This was repeated 6 and 9 months after the initial treatment.

The temperature and humidity of the warehouse were controlled, and the product was regularly inspected to verify the absence of pests.

The experimental unit used is shown in figure 3.

Fig. 3. Scheme of the experimental unit

?The four blocks were left in the WFP warehouse for a period of 9 months, during which samples were taken for analysis every 3 months.

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4.8 Ozone concentration calculations

The following formula was used to calculate the theoretical concentration under the fumigation blankets:

Concentration (gr/m3) = O3 production (gr/hr) x time (hr) / volume (m3)

Source: Holger Claus, Ozone generation by ultraviolet lamps, 2021 (43)

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The ozone generator produces 10 gr/hr of ozone, the volume under the blanket is 4.8 m3 (2m x 2.4m x 1m), the time of each treatment were 22, 44 and 66 hours and knowing that 1 gram ozone/m3 equals 0.0467% (or 1% = 21.4 g/m3) (43) then:

The theoretical ozone concentration reached was 2.1% for the 22-hour treatment, 4.2% for the 44-hour treatment, and 6.4% for the 66-hour treatment.

Note that this would imply that none of the ozone degrades over time and that perfect mixing of the ozone under the blanket is unlikely. In reality, ozone is heavier than air, so without air circulation it would tend to sink. It is also to be expected that without air circulation, the ozone concentration near the hose outlet will be higher than average and that as the gas penetrates the product, it reacts.

During the experiment, the concentrations of ozone under the blanket were measured at the end of each treatment and a high concentration was confirmed, above the detection range of the equipment (100 ppm) near the outlet of the hose, while the points farthest from the concentration reached levels of 6 ppm for the 22-hour treatment, 21 ppm for the 44-hour treatment, and 76 ppm for the 66-hour treatment. This confirms the slow passage of ozone through the product.

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4.9 Treatments costs

In the case of ozone fumigation, we can see that the main cost is the energy consumption of the ozone generator. In our case, the electrical energy consumption of the ozone generator used is 0.8 kWh and the average price for businesses in Mozambique is 0.068 USD per kWh, while the average price in the world for businesses is 0.125 USD per kWh (47). Table 2 shows the energy costs in US dollars for each treatment time, both in Mozambique and the world average.

Table 2. Electricity costs

Note: The costs were calculated at the time of the experiment.

On the other hand, the cost of phosphine fumigation in Mozambique is around 1.28 USD per metric ton not including the cost of dislocation of the equipment. Knowing that the average maize productivity of the subsistence farmer in Mozambique is 1 ton per hectare and that they have an average of 3 hectares, then the average production is 3 tons per farmer. The average cost of phosphine fumigation would be 3.84 USD per smallholder farmer.

Another option that has been gaining strength to combat aflatoxins is the use of biological control products that use Aspergillus strains that are not capable of producing aflatoxins, also known as atoxigenic. These products are used prior to flowering and protect the product as a means of biological control of the development of aflatoxins. The estimated cost of using these products to treat one hectare varies between 7 and 12 USD. Which gives a total cost for a farmer with 3 hectares of between 21 to 36 USD only in the cost of the product (16).

Table 3 compares the basic costs of the three options evaluated for the treatment of a production of 3 tons of maize or 3 hectares of crops.

?Table 3. Comparative costs of fumigation with ozone, phosphine and biological control

?For small subsistence farmers, the most economical option is ozone fumigation, the difference in cost compared to phosphine fumigation being only 6.5%. With respect to the biological control product, the difference in cost is 5 to 7 times greater.

Regarding the safety of products, the use of ozone gas is safer than phosphine gas, mainly due to the high toxicity of phosphine. Although in both cases training is required to carry out the fumigation, the security measures are less in the use of ozone. Biological control products are considered safe for health.

For the elimination of insects, phosphine gas is more efficient than ozone since phosphine eliminates almost 100% of the insects present in the product (if the fumigation is well carried out) while with ozone the effectiveness depends on the type of insect and of the stage of life. However, phosphine does not degrade mycotoxins and it is in this sense that ozone fumigation is more efficient. Biological control products do not kill insects and only prevent the development of aflatoxins in the product.

Regarding the environment, we can say that ozone fumigation and biological control products do not leave any toxic residue in the process. Over time, ozone gas decomposes into oxygen that remains in the atmosphere, while phosphine leaves toxic residues from the fumigation process that must be carefully discarded and that affect the environment.

?

4.10 Sampling

Aflatoxins are not evenly distributed in agricultural products; therefore, it is not surprising that sampling is the largest source of variation in aflatoxin testing (48). There are various approaches that can be taken to minimize variation, but yet there is no global agreement on aflatoxin sampling protocols for any crop. (49).

The need to obtain a representative sample deserves particular consideration as an incorrect sampling plan can greatly affect the reliability of measured mycotoxin levels (49). The sample size can significantly reduce the sampling error. Sampling is one of the most important steps contributing to analytical variability due to the inhomogeneous nature of aflatoxin distribution in food and feed (50).

Due to this, the samples were taken by adding all the bags that make up the different sub-lots (treatment times) up to a total of 10 Kg of sample for each sub-lot.

?

4.11 Laboratory analysis

Intertek, the only company in Mozambique accredited with ISO 17025, was hired for the laboratory analysis. The requested analysis were for Aflatoxin B1, B2, G1, G2, Totals, humidity, molds and appearance (color, smell). Intertek is a service provider in the area of total quality assurance for industries around the world. The services they provide are auditing, inspection, laboratory analysis and certification.

Regarding the ISO 17025 standard, it is a standard oriented to the evaluation of conformity. It contains the general requirements for the competence of testing and calibration laboratories. The ISO 17025 standard was developed to guide laboratories in quality management and technical requirements for proper operation. This standard complies with the technical requirements of ISO 9000. Therefore, any organization that complies with the requirements of ISO 17025 also complies with the requirements of ISO 9000. (51).

While the requirements of ISO 9000 are generic and can be applied to all types of organizations, the requirements of ISO 17025 are specific to testing and calibration laboratories. The standard addresses issues such as: the technical competence of personnel, the ethical conduct of personnel, the use of well-defined tests and calibration procedures, participation in proficiency tests, and the contents of test reports and certificates.

The main objective of ISO 17025 is to ensure technical competence and reliability of analytical results. For them, it uses both management requirements and technical requirements that affect the improvement of the quality of the work carried out in the laboratories. (51).

These requirements are used as tools for the dissemination of collective knowledge, which facilitates the integration of personnel, provides flexibility in adapting to changes in the environment and allows problems to be detected for early resolution.

?

4.12 Aflatoxins.

For the analysis of aflatoxins, the method used is based on the ISO 16050 standard - Determination of aflatoxin B1, and total content of aflatoxins B1, B2, G1 and G2 in cereals, nuts and derived products ─ Method by high performance liquid chromatography (HPLC). The equipment used for aflatoxin testing was the Perkin Elmer Ultus A10 HPLC.

?The principle of the method is based on extracting the test sample with a mixture of methanol and water. Filter the sample extract, dilute with water and apply to an affinity column containing specific antibodies for aflatoxins B1, B2, G1 and G2. Isolate aflatoxins, purify and concentrate on column, followed by separation of antibodies with methanol. Quantify aflatoxins by reverse-phase high-performance liquid chromatography (HPLC) with fluorescence detection and post-column derivation.

?

4.13 Moisture.

For moisture analysis, the method used is based on the ISO 6540:2021 standard Determination of moisture content in ground grains and whole grains. The principle of the method is based on extracting all the moisture contained in the sample without altering the chemical composition of the sample, in particular oxidation and loss of volatile organic substances. The equipment used for moisture testing was the Labotech EcoTherm oven.

?

4.14 Mold count.

For the mold count, the method used is based on the ISO 7954 standard. To find out the mold load of a food, the usual technique of counting on agar plates is used from the series of decimal dilutions of the product. Then, from the aerobic incubation of the plates at 25 degrees centigrade for 5-7 days, the number of molds per gram or per milliliter of the sample is calculated from the number of colonies obtained on the plates chosen at dilution levels to give a meaningful result.

The technique consists of suspending the mixture in a ratio of 1/9 in tryptone soy broth. The initial dilution of the sample (1:10) is kept for 2 hours at room temperature. Then, starting from this dilution, serial decimal dilutions (1:100, 1:1,000, 1:10,000, etc.) are made in tryptone water, 0.01% Tween 80 solution, or Ringer's solution.

The seeding of the counting plates is carried out in mass, and it is important to homogeneously disperse the sample in the counting medium to obtain correct values. Next step it is left to solidify on a horizontal surface at room temperature.

Then it is incubated in a culture oven at 25 °C for 5-7 days. Passed this time frame, the grown colonies are counted, although the readings begin on the third day to observe the formation of invasive aerial mycelia. The count is established from the number of colonies that appeared in a single dilution, specifically the dilution that provides an average value of fungal colonies between 0 and 30 (0 to 50). The number of colonies counted is multiplied by the plate dilution factor to obtain the total count of mold colony-forming units per gram or milliliter of food.

?

4.15 Sensorial analysis

Sensory testing of the maize kernels involved the use of the human senses in the objective evaluation of the product. Characteristics such as appearance, odor, and color were analyzed by trained raters to assess product quality.

The sensory tests were carried out by a sensory panel with the following requirements:

??????Neutral attitude towards the product to be tested

??????Linguistic competence

??????No allergies or intolerances to the test product

??????Good senses (without olfactory, visual disorders, etc.)

For the sensory test, the samples were prepared under the following criteria:

??????The differences between the samples should be as small as possible, that is, all evaluators, if possible, ?should receive the same samples.

??????All samples must be at room temperature.

??????The sample should be transferred to a neutral container.

??????Samples are neutrally coded prior to testing to remain anonymous.

??????The samples are presented in neutral containers; these must have the same shape and color for all testers.

For the analysis of the samples, the triangle test was carried out, which is a scientific method in which three products are presented to a test panel. Two of the products are identical, and one is different from the others. All 3 products are blind tested to see if the tester can detect a sensory difference. Tables are used for the evaluation, which allows judging the distinctiveness of the samples.

?

4.16 Water activity (Aw)

For the calculation of the water activity in corn, the sorption isotherms of maize calculated by Mu?oz R. 1992 (52).

Table 4. Water activity values in maize grains

?

Fig 4. Equilibrium isotherm for maize between 5oC and 70oC. Maize equilibrium moisture. Modified Henderson Model (52)

Equilibrium Humidity (%bh)

????????????????????????Equilibrium Relative Humidity (%) Aw

4.17 Drop test.

To verify the resistance of woven polypropylene to the effects of ozone. At the end of the treatments, a drop test was carried out based on the ISO 7965-2 1993 standard for thermo-flexible plastic bags (53) and adapted by the World Food Program. The test consisted of evaluating 12 bags, three for each treatment block and three for the control sample. For this evaluation, the bags were dropped from a height of 1.2 meters from the side of the upper and lower seams and two drops from the front and back of the bag from a height of 1.6 meters. At the end, the bags were checked in search of product loss or damage to the container.?

?

4.18 Temperature and humidity measurement in warehouse

During the experiment, the temperature and humidity in the warehouse were recorded. For the measurement, the Sper Scientific No. 800254C temperature and humidity sensor was used, which has a temperature range of -10 oC to 60 oC with a resolution of 0.1 oC and a humidity range of 0 to 95% with a resolution of 1%. Temperature and humidity were recorded daily at 8 am, noon and 5 pm.

The monthly averages of the temperature and humidity measurements in the Maputo warehouse during the experiment are shown in table 4. As can be seen, the relative humidity remained between 72 and 81% while the temperature ranged between 13.1 oC and 31.4 oC with an average range between 20 and 26.1 oC.

Table 5. Record of temperature and humidity in Maputo warehouse

?

5)???Results

5.1 Aflatoxins B1 analysis

The results of aflatoxin B1 in the corn samples for the analysis at 6 and 9 months are shown in table 6. As can be seen, the aflatoxin levels were negative for all treatments and only reported in the control sample both at month 6 and 9 after treatment. Aflatoxin B1 was also analyzed before and after treatment and at 3 months, being undetectable in all cases. In the case of Aflatoxins B2, G1 and G2, it was not detectable in all cases.

Table 6. Aflatoxin B1 values in parts per billion (ppb)

?ND: Not Detectable (<1 mg/kg)

?

Since the data was non-parametric and since it involved more than 2 samples, the Moods median test was performed for samples from different populations with a significance level of 0.05. In addition, the results were only compared after 6 months, which is when the presence of aflatoxin B1 was detected in the samples.?????????????????????????????????????????????????????????????????????

Table 7. Resume:

Table 8. Mood test:

XLSTAT 2022.2.1.1308 - Mood test - Microsoft Excel 16.61507

Interpretation:

H0: The median levels of aflatoxin B1 from month 6 are the same.

Ha: At least one median aflatoxin B1 level from month 6 is different.

Since the value calculated for P-value (0.019) is less than the level of significance alpha = 0.05, the null hypothesis H0 is rejected, and the alternative hypothesis is accepted with 95% confidence.

?

5.2 Mold count

The results of mold in the maize samples are shown in table 9. As can be seen, the levels of mold were decreasing for both the control and the products treated with ozone. A drastic reduction of the fungal load is also observed at the beginning of the product treated for 66 hours.

Table 9. Molds mean values

Note: values are expressed as means

?

5.3 Product moisture and water activity calculation

The results of the moisture value in the maize samples treated with ozone are shown in table 10. As can be seen, the moisture levels vary between 13.14% and 13.99%, finding the highest moisture levels at month 6 of the experiment.

Table 10. Average moisture values

Note: values are expressed as means

A normality test was carried out, finding that the data for humidity value has a normal behavior. Given this, the One way Anova statistical analysis was performed to compare the mean moisture levels between the three treatments and the control. The results found at a confidence level of 95%, the P-value was 0.94587407.

H0 = The mean moisture level in the control = mean moisture level in T1 = mean moisture level in T2 = mean moisture level in T3.

Ha = The mean moisture level in the control ≠ mean moisture level in T1 ≠ mean moisture level in T2 ≠ mean moisture level in T3.

As P-value > 0.05, the null hypothesis H0 is accepted, and the alternative hypothesis is rejected with 95% confidence, that there is no difference in the mean moisture values between the control and the treatments.

Having the humidity of the maize on a wet basis, the maximum temperatures in storage and the sorption isotherms of the maize, the maximum water activity in the maize was calculated, which are shown in table 11.

Table 11. Calculated maximum water activity values for maize

?

5.4 Sensory characteristics of the product????????

The results of the sensory characteristics in the maize samples are shown in tables 12, 13 and 14. As can be seen, the sensory characteristics of the product were the same for all treatments, including the control sample, and remained the same throughout the experiment.

Table 12. Maize color results

?Table 13. Maize odor results

?Table 14. Maize appearance

?

5.5 Drop test???

The results of the maize sack drop test are shown in Table 15. As can be seen, no sacks were damaged during the drop tests.

Table 15. Drop test results

?

6)???Discussion

The objective of this work can be divided into two parts. The first part consists of evaluating the feasibility of carrying out ozone fumigation using fumigation blankets, both from a practical and economic point of view. The second part consists of evaluating whether ozone fumigation prevents the appearance of aflatoxins in maize. For this, 3 samples of 2.5 tons of maize each, were treated with ozone for 22 hours, 44 hours and 66 hours, using fumigation sheets. The results of aflatoxins, mold count, moisture and sensory evaluation of the treated samples were then compared against a control sample. These analysis were performed after fumigation, and every 3 months up to 9 months after fumigation. The result was that none of the treated samples reported any aflatoxins and only aflatoxin B1 was found in the control samples at 6 and 9 months.

There are two possible explanations for these results. The first explanation is that the ozone treatment eliminated most of the Aflatoxin producing molds protecting the product and preventing the appearance of aflatoxins. The second explanation is that aflatoxins appeared only in the control sample because it was the sample with the highest water activity during month six (0.711), while in the treated products the water activity only reached 0.705. This increase in water activity due to climate conditions could have stimulated the activity of the present microorganisms creating some pockets in the product with an even higher water activity that the Aflatoxin producing molds took advantage and produce the toxin. However, this second explanation is unlikely because both the treated samples and the control reached a water activity higher than 0.7, the limit reported for the development of aflatoxins by Gimeno (2002) and Dhanasekaran et al (2011).

Several studies have confirmed the oxidative power of ozone to reduce the microbial load and degrade mycotoxins. The reduction in the concentration of mold from the field by ozone was corroborated in this study, as well as the fact that ozone does not affect the sensory characteristics of the product. Contrary to what was reported by Wang, S et al (2010) who reported a reduction in moisture content from 15.56 to 12.86 in maize after ozone treatment, the moisture values of the treated product in our case do not differ statistically from the control sample.

This study was the first to evaluate the possibility of using ozone under the blanket method as a preventive treatment for aflatoxins. During the last few years there has been great interest in finding viable methods for the aflatoxin problem. The use of ozone gas as a method of prevention and treatment of agricultural products prone to contamination with aflatoxins is promising. The study's findings offer a new economic and viable perspective for the prevention of aflatoxin development for the smallholder farmer in Africa. More research is required to evaluate doses or treatment times based on various amounts of product.

Although the blanket fumigation technique does not facilitate a uniform distribution of the ozone gas due to the almost zero air circulation, the ozone gas was distributed throughout the entire fumigation volume. Future research could evaluate ways to standardize the concentration of ozone gas under the fumigation sheet.

The study has one main limitation and that is that the storage conditions of the experiment are not the same as those of the smallholder farmer in Africa. WFP's storage conditions are superior in quality to the conditions in which small farmers normally store the product. Future research could evaluate the product under the same storage conditions as of the smallholder farmer.?????????

?

7)???Conclusions

The conclusions reached in this work are detailed below:

???Ozone gas has a protective effect against the appearance of aflatoxins on the treated product, however, it is necessary to do more tests to determine the times for different amounts of product and the effects on the different ways of accommodating the product in the stacks in the warehouse.

???The 250 micron PVC blankets allow Ozone fumigation to be carried out for products packed in woven polypropylene bags with good gas retention inside the fumigation volume. However, it is necessary to evaluate ways to uniform the distribution of the gas under the fumigation sheet. This would reduce the required fumigation times.

???This blanket fumigation method could also be used to treat products contaminated by aflatoxins. This would require testing the time required for the degradation of aflatoxins for different tonnages.

???The ozone fumigation time required to have a reduction of 1 logarithmic cycle in the concentration of mold in 2.5 tons of maize with equipment that produces ozone at a rate of 10 gr/hr in an approximate volume of 4.8 m3 was 66 hours.

? The ozone gas does not affect the sensory characteristics of smell, color or appearance of the treated maize grain for up to 66 hours, nor does it damage the woven polypropylene bags.

? Ozone fumigation under the blanket method is an economical and viable technique to replicate in family farming in Africa.

?

?

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