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Brazilian Archives of Biology and Technology

Print version ISSN 1516-8913

Braz. arch. biol. technol. vol.53 no.6 Curitiba Nov./Dec. 2010 



Efficacy of modified atmosphere packaging to control Sitophilus spp. in organic maize grain



Marcelo De CarliI; Bruna BresolinI; Caciano Pelayo Zapata NoreñaI,*; Irineu LoriniII; Adriano BrandelliI

IInstituto de Ciência e Tecnologia de Alimentos; Universidade Federal do Rio Grande do Sul; Av. Bento Gonçalves, 9500; 91501-970; Porto Alegre - RS - Brasil
IIEmpresa Brasileira de Pesquisa Agropecuária; Rodovia BR 285; Km 174; 99001-970; Passo Fundo - RS - Brasil




The effect of modified atmosphere packaging on the mortality of Sitophilus spp. in organic maize grain was studied. Maize grains were packed with five different atmospheres consisting of 20% O2, or 20, 40, 60, 80% CO2 with remaining N2. The packages were stored for 30 days at 26°C. CO2 and O2 concentrations were monitored inside the packages during the storage. The moisture content, titratable acidity and pH were analysed on the first and thirtieth day. At 5, 15 and 30 days of storage no significant differences were observed in the percentages of dead insects when CO2 was used. The results of progeny indicated that from the fifth day the number of emerging insects was low at 20, 60 and 80% CO2. Complete inhibition of the insects was achieved with 30 days of exposure in CO2 atmospheres.

Key words: Carbon dioxide; Modified atmosphere; Insect mortality.


O efeito da embalagem em atmosferas modificadas sobre os insetos Sitophilus spp. nos grãos de milho orgânico foi estudado. Os grãos de milho foram empacotados em cinco atmosferas iniciais diferentes de 20% de O2, ou 20, 40, 60, 80% de CO2 com balanço de N2. As embalagens foram armazenadas por 30 dias a 26ºC. As concentrações de CO2 e O2 foram monitoradas dentro das embalagens durante o armazenamento. As análises de teor de umidade, acidez e pH foram também realizados no primeiro e trigésimo dia. Os resultados mostraram que no quinto, décimo quinto e trigésimo dia de estocagem não foram encontrados diferenças significativas nas porcentagens de insetos mortos quando CO2 foi usado. Os resultados relativos à progênie indicaram que no quinto dia o número de insetos emergentes foi menor a 20, 60 e 80% CO2. A completa inibição dos insetos foi atingida com 30 dias de exposição em atmosferas de CO2.




The explosion of human population in developing countries is creating an unprecedented demand for greater production of food grains (Moreno-Martinez et al., 2000). The postharvest losses due to infestation by insect pests have required the use of diverse procedures for the control of the arthropods (Gbaye and Odeyemi, 2005). Sitophillus spp. is a major pest of stored maize grain in Brazil and its control must be made efficiently to reduce the loss in grain.

In the recent past, the preservation of cereals and other durable agricultural products during the storage has relied heavily upon the insecticides to control the storage pests. However, the present trend is towards alternative non-toxic control methods that pose no threat to the health of operator or consumer, and which are environmentally friendly (Donahaye et al., 1996). Developing countries have resorted primarily to the use of insecticides (Moreno-Martinez et al., 2000) and most of the contact insecticides have been restricted for use on food materials (Shunmugam et al., 2005). Along with preventive practices, the farmers rely mostly on chemical control of stored products pests. However, this can be potentially harmful to human health in the form of chemical residues (Shunmugam et al., 2005).

The consumers today expect the food products that are pesticide free or with lower levels of residues (Conyers and Bell, 2007). In addition, pressure from the international consumer groups to reduce the chemicals used in food products makes a non-chemical insect control practice highly desirable (Mitcham et al., 2006). It is demanding to develop the alternative methods that are economically feasible and ecologically adjusted to control the storage grain insects and fungi (Moreno-Martinez et al., 2000).

Aeration systems to cool the bulk grains and thereby prevent insect development are being widely used even in warm climates but are only applicable for the bulk grains. Other non-residual treatments such as mechanical impact, irradiation, biological control or heating are suitable for high-quality commodities or niche situations (Donahaye, 2000). CO2 has considerable attraction as a means of disinfecting foodstuff in general and stored durable products in particular (Annis and Morton, 1997). This technology can be used in hermetic storage. The use of controlled atmospheres using either N2 or CO2 closely depends on well-sealed storage structures, which are rarely available in rigid silos, but easily obtainable with flexible plastic liners (Donahaye, 2000). Donahaye and Navarro (2000) reported the use of modified atmospheres (MA) as a non-toxic and environmentally benign alternative to fumigation for the control of insects in stored products. MA has been used in the agricultural and food industries for the protection and preservation of raw materials from harvest to the finished product (Conyers and Bell, 2007).

Disinfestation of stored grain using MA involves the alteration of the natural storage gases such as CO2, O2 and N2 to render the atmosphere in the stores lethal to pests (Jayas and Jeyamkondan, 2002). As a method for insect control in bulk commodities, MA systems increased CO2 or decreased O2 atmospheres, or a combination of both (Bell, 2000; Donahaye and Navarro, 2000). MA may adversely affect the arthropods both through creation of a hypoxic (low O2) environment, as well as from direct physiological effects of the gases (Held et al., 2001).

The maize is an agricultural product widely distributed in Brazil, and is commercialized natural form or with minimal processing to reduce the cost (Biazus et al., 2009). High value commodities, such as organic maize grain may be adapted for MA in flexible plastic containers for transporting overseas to extend its shelf life and to kill the insects. The aim of this study was to evaluate the effect of the atmosphere modified packaging on the mortality of Sitophilus spp. in organic maize grain.




The organic maize grains (variety Fundacep 35) were supplied by Coperfamília (Tenente Portela, Rio Grande do Sul State, Brazil). The samples were cleaned, dried and kept in the fridge at 8°C.


Unsexed adults of Sitophilus spp. were obtained from the Laboratory of Brazilian Agricultural Research Corporation (Embrapa, Passo Fundo, Brazil). In order to obtain an insect population of know age, 550 adult weevils (both sexes) were transferred to 2 l plastic jar containing 1kg of maize grain. These were then covered with a lid of metal mesh. This procedure was repeated four times. The jars were kept at 26°C, 55% RH, in a 12-12 h light and dark photoperiod. After 15 days, all the insects were removed and the maize grains were again kept in the same condition for 56 days. After this period, the adults which emerged from these cultures were used in the experiments.

Experimental procedures

Fifty insects were placed in a plastic flask containing 250 g of maize grains and kept for 40 days as described above. After this period, all the contents (insect and maize) were transferred to the bags with barrier properties (Sealed Air – CRYOVAC). The air was evacuated and a food grade gas mixture (White Martins, Porto Alegre, Brazil) was introduced into the package before heat-sealing using a vacuum sealer (Selovac, model 200). The experiments were conducted for various modified atmosphere packaging: 20% (v/v) O2, 20, 40, 60, 80% (v/v) CO2 with remaining N2 and 30 days exposure at 26ºC.

After the exposure, the packages were opened and all the materials were placed on a table in order to register the number of insects. Dead insects were recognized by their lack of movement despite a range of stimuli (prodding, subjecting to small breeze and leaving undisturbed). Special care was taken to ensure that death had really occurred (Annis and Morton, 1997). If they moved, they were scored as live. Recording of the dead insects were realized at 1, 2, 3, 4, 5, 15 and 30 days exposure to modified atmosphere packaging at 26°C.

After registering the number of dead insects and removing the live ones, the grains were returned to the jars and kept again at 26°C, 55% RH for 45 days to observe the possible emergence of insects. After this period, the emergence of adult insects from any progeny was recorded.

Headspace gas composition

The CO2 and O2 concentrations were measured using a gas analyzer (MOCON, model Pac Check 65). To avoid the modifications in the headspace gas composition due to gas sampling, each package was used only for a single determination of the headspace gas composition (Del Nobile et al., 2006). The O2 and CO2 concentrations in the headspace of packages filled with the maize grains and the insects were measured after 1, 2, 3, 4, 5, 15 and 30 days exposure to modified atmosphere packaging at 26°C.

Moisture, titratable acidity and pH

The moisture content, titratable acidity and pH values were determined after one and 30 days exposure of the grains and insects in AM packaging according to AOAC methods 925.10, 942.15 and 943.02, respectively (AOAC, 1990).

Statistical analysis

A 5x7 factorial design was planned in order to study the influence of CO2 concentration and storage period on the mortality insects. Tukey's test was used to determine the statistically significant differences between the treatment means at p=0.05. The experiments were repeated three times. Statistical analysis was completed using the SAS software (SAS Institute Inc., Cary, N.C. USA).



Physico-chemical analysis

The maize grain with moisture content of 10.43% (m.c.), 3.52 mg NaOH/100g of acidity and pH of 6.25 was used to the test. The results of moisture content, acidity titratable and pH are summarized in Table 1. The moisture content significantly increased (p<0.05) with exposure time at 20% O2, 20 and 40% CO2, but not when 60 or 80% CO2 was used (p>0.05). The highest moisture content on the thirtieth day was observed at 40% CO2.

Titratable acidity was almost constant in the time studied, except at 20 and 80% CO2 (Table 1). In addiction, on the thirtieth day, no differences (p>0.05) were found among the different atmospheres tested. No significant changes in the pH values were observed at 20, 40, 60 and 80% CO2 within the exposure time. In addition, on the thirtieth day, no significant changes in the pH values were found at different atmospheres.

Headspace atmosphere

The evolution of internal CO2 and O2 concentrations with exposure time can be observed in Figures 1 and 2 respectively. At 20% O2, internal levels of CO2 increased while O2 concentrations rapidly decreased. After the fifth day, the levels of CO2 started to decrease. When CO2 was used, the internal CO2 concentrations slightly decreased from the day 3 (Fig. 1). After the fifteenth day, CO2 achieved a steady-state condition. The initial internal O2 concentration was around 0.8% O2, consequence to vacuum produced before inputting CO2 into the package. On the first day the O2 concentration decreased to 0.14%. However, O2 levels slightly increased after the fifth day. On the thirtieth day, the O2 levels significantly increased (p<0.05), except at 20% CO2. This could be due to the package used, which was a O2 low permeability film.





Effect on adult insects

The percentages of mortality under different MA conditions are presented in the Table 2. At 20% O2, the mortality from the second day was significantly lower when compared with any CO2 concentration. During all the exposure times studied at 20% O2, the percentages of dead insects were lesser than 9%. When CO2 was used, from the fifth day, the higher percentages of insect mortalities were obtained and no differences (p>0.05) in the percentages of dead insects were observed at 5, 15 and 30 days of exposure.



Effect on progeny

It was observed that under 20% O2, the emergence of new insects was not repressed in the periods less than 30 days (Fig. 3). The highest amount of adult emergence from the progeny was observed at 20% CO2 and two days of exposure. The insect emergences began to decrease from the fifth day, being the number of live insects lower than three using 20, 60 and 80% CO2. On the fifteenth day, there were less than one insect at 20, 40, 60 and 80% CO2. On the thirtieth day, no live insects were found at 20, 40, 60 and 80% CO2 (Fig. 3).




In the experiments conducted in the absence of insects, no significant changes were found in the moisture content between the first and the thirtieth day. Moreno-Martinez et al. (2000) found that the moisture content of the maize grain stored under hermetic conditions remained almost the same as the initial values. When the tests were conducted in the presence of insects, a significant increase (p<0.05) of moisture content at 20% O2, 20 and 40% CO2 was observed. The increase in the moisture content in the grains was usually a result the metabolic activity of insects. Caneppele et al. (2003) found that the infestation of stored corn with the adults of S. zeamais elevated the seed moisture content, which favored the proliferation of molds by the ninetieth day. The grain stored with high moisture content is subjected to high losses caused by the attack of the insects and fungi (Vásquez-Castro et al., 2008).

However, at 60 and 80% CO2 changes in moisture were not significant (p > 0.05). This behavior was due to the diminished respiration rates of both the insects and grains when they were in modified atmosphere or in low O2 concentrations. In addition, low levels of moisture also decreases the respiratory rate of maize grain.

No significant changes in pH were observed when the atmosphere with 20 CO2 was used, as well as to acidity at 40 and 60% CO2. Rehman et al. (2002) reported that there was no change in pH and titrable acidity of maize grains kept at 10°C for six months storage. However, they observed a decrease in pH and increase in acidity after 30 days of storage at 25 and 45°C.

It was observed that in the atmosphere with 20% O2, there was a high consumption of O2 in the first five days, whereas CO2 increased to 18.1%. This behavior was associated with the respiration of insect, grain and microorganisms in the package, a physiological process that occurred involving the O2 and CO2. Insects are the main consumers of O2, followed by fungi and grains. Thus, insects and fungi deplete the O2, creating a most unfavorable atmosphere for themselves (Moreno-Martinez et al., 2000).

When the experiments were conducted with CO2, the O2 concentration achieved was lower than 0.5% until the fifteenth day. After this period, O2 level increased significantly to values up to 2%, except for 20% CO2. ANOVA revealed that CO2 concentration (p<0.05), time of exposure (p<0.05), and their interaction (p<0.05) were significant factors affecting the CO2 concentration. Figure 1 depicted that the CO2 concentration decreased with the time and initial levels of CO2. On the contrary, the concentration of O2 was independent of initial concentration of CO2 and their concentration began to increase slowly after the fiftieth day (Fig. 1).

The consumption and production of O2 and CO2 by vegetable tissues are metabolically interrelated, making difficult to study the effect of one gas independent the other. The uncontrolled nature of the atmosphere in the package and the time interval required to reach the equilibrium are additional factors that complicate the interpretation of the results (Saltveit, 2003). Besides, there is the transfer of gases through the packaging, leading to an atmosphere richer in CO2 and poorer in O2 (Fonseca et al., 2002).

The mortality observed for Sitophilus spp. when high CO2 concentrations were used could be associated with either O2 depletion or CO2 toxicity or the interaction of both (Annis and Morton, 1997). However, the natural permeability of cellular membranes appeared to be an important protection mechanism for the survival of arthropods under low O2 or high CO2 atmospheres (Mitcham et al., 2006). Hoback and Stanley (2001) described that arthropods were hypoxia-tolerant organisms, although their mortality increased with the lower O2 concentrations. Mitcham et al. (2006) also showed that in the case of using controlled atmosphere, the insecticidal conditions generally contained concentration higher than 20% CO2 and/or lower than 1% O2, depending on the temperature, with the remaining of the atmosphere composed of N2 gas.

ANOVA showed that time, CO2 concentration, and their interaction (p<0.05) were significant factors affecting Sitophilus mortality, indicating that these two factors acted together on the mortality of insects. Similar behavior was observed by Annis and Morton (1997), who reported that the use of time-concentration relationship was not directly applicable to explain the effects of CO2 on S. oryzae because of the interdependent effects of lowered O2 and increased CO2 concentrations. In addition, there was a particular time interval for each species after that an irreversible damage was done to their metabolism (Conyers and Bell, 1996).

In this work, it was shown that after five days of exposure in any initial CO2 concentration studied, the number of insects diminished drastically. In addition to the effect of the concentrations of CO2 and O2 and time, there are other factors that may influence the effect of these atmospheres on the arthropods, such as temperature, grain moisture, insect species and life stage (Pearson and Sorenson, 1970; Mitcham et al., 2006).

In the experiments developed to evaluate the effect on progeny, total inhibition was achieved at 30 days using CO2 atmospheres. This time corresponded to O2 concentrations lower than 2%. Conyers and Bell (2007) mentioned that an increase of CO2 to 10 or 20%, reducing O2 to 5% was sufficient to eliminate the emergence of Sitophilus granarius at 20ºC, but a few individuals emerged at 25ºC. In addition, S. granarius exposed to atmospheres containing 1 to 1.6% O2 with a balance of N2 at 20°C and 70% RH were killed within seven days if the atmosphere also contained 10% or more CO2 (Krishnamurthy et al., 1986). Other studies have shown that the minimum concentration of CO2 useful for the complete disinfestation of S. oryzae was lower than 40% but substantially above 20% CO2 (Annis and Morton, 1997). Arthropod mortality is generally greater in response to elevated CO2 atmospheres when compared with low O2 atmospheres (Mitcham et al., 2006).

This study showed the possibility of using MA packaging in order to protect the maize grains produced organically against insect attack. It was observed that the relationship between the initial CO2 concentration and time was important to achieve the higher percentages of mortality for adult insects. Atmospheres containing higher than 20% CO2 and lower than 2% O2 and higher than five days of exposure were necessary to eliminate most adult insects. However a period of 30 days was necessary to achieve the total inhibition of the progeny.



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Received: January 08, 2009; Revised: October 05, 2009; Accepted: August18, 2010



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