Maize Grain Stored in Hermetic Bags : Effect of Moisture and Pest Infestation on Grain Quality

Kenya Agricultural and Livestock Research Organisation (KALRO), Food Crops Research Institute, PO Box 14733-00800, Nairobi, Kenya Kenyatta University (KU), Department of Zoological Sciences, PO Box 43844-00100, Nairobi, Kenya International Maize and Wheat Improvement Centre (CIMMYT), PO Box 1041-00621, Nairobi, Kenya International Centre of Insect Physiology and Ecology (ICIPE), ILRI Campus, PO Box 5689, Addis Ababa, Ethiopia


Introduction
Maize (Zea mays L.) is an important staple food in sub-Saharan Africa but is also used for industrial purposes and animal feed worldwide [1].In the tropics, in locations where there are two annual maize harvests, it is not uncommon for one harvest to coincide with the start of the rainy season.As nearly all smallholder farmers rely on sun-drying prior to grain storage, this poses a serious threat of grain quality deterioration [2].If the crop is stored while still moist and warm, there may be rapid spoilage due to mould growth and insect pests [3].Due to the demand for high quality and safe food by the consumers, there is a need to maintain and protect maize grain from insect damage and fungal infection [4].
Important physical factors that may a ect the quality of stored grain are moisture content; temperature; the type of storage structure used; and the gaseous environment, particularly levels of carbon dioxide and oxygen [5].All these factors may have interactive e ects on mould growth and insect pests [6,7], evolution of carbon dioxide [8], and grain colour and weight [9].e optimum temperature for the development of storage moulds and insects is generally around 30 °C.Requirements for moisture are more wide ranging, with most moulds failing to germinate below 80% relative humidity but insect needing a grain moisture content of at least 10% to 12% [10], which is typically in equilibrium with around 50% to 70% relative humidity.When grain with higher moistures (>14%) is stored under warm conditions, mould growth may result in serious quality changes [3], and insect attack may reduce quality and cause weight losses.Simple and low cost storage facilities that isolate grains from unfavourable environmental conditions are therefore required by farmers.
e main insect pests of stored maize include the larger grain borer Prostephanus truncatus (horn) (Coleoptera: Bostrichidae) and maize weevil Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) [11].For the past 35 years, sub-Saharan Africa has witnessed the outbreak and spread of larger grain borer (LGB) in 20 countries [12].e impact of insect infestation of stored grain is usually expressed as percentage weight loss [13].Losses as high as 36% have been documented on stored maize in Benin [14] and 35% in Kenya [15].e losses are attributed to the use of ineffective storage structures such as cribs, jute, and polypropylene bags for storing grains [16].ese losses affect the food security and income of the 90% of the Kenyan population that relies on the crop as staple food [17] besides contributing to increased food prices due to removal of part of the supply from the market [18].
A promising technology to store grains safely is airtight (hermetic) storage (self-regulated modified atmosphere).
e technology was developed in response to concern about the adverse effects of pesticide residues in grains and environment [19].e respiration of the grains, insects, and moulds within hermetic stores result in depletion of oxygen and increase of carbon dioxide [20,21].Under such conditions, fungal growth may be inhibited [22], and when the oxygen level falls to 10%, insect activity is reduced and insect will die if subjected to less than 2% oxygen for periods in excess of 14 days.Consequently, hermetic storage can be used to maintain grain quality without the need for pesticide application [23].e rate at which oxygen is reduced and carbon dioxide generated is a function of both grain moisture content and the ambient temperature; the rate is low at temperatures below 20 °C [24].
Storage systems based on the hermetic principle include airtight bags, bunkers, and silos.While hermetic metal silos [25] and polythene (plastic) bags [26] have been developed, their use for storage of grains by smallholder farmers is still limited.ere is also scant information on the storability of maize grain at the range of moisture contents that are typical in tropical storage systems.e key to successful hermetic storage is air-tightness and control of moisture content.e present study was undertaken to investigate quality change of maize stored in hermetic bags subject to infestation by Sitophilus zeamais and a variety of initial grain moisture content level.e parameters monitored were moisture content, grain discolouration, insect density, weight loss, and oxygen and carbon dioxide levels.

Preparation of Maize
Grain.Hybrid maize (H614) was cleaned to remove mouldy and broken grains and foreign materials and sun-dried to 12% moisture content.Samples of grain at three further moisture contents 14, 16, and 18% were prepared by adding predetermined amount of distilled water then mixing thoroughly in plastic bags [3,27].e amount of water added was calculated as follows: Q � A ((b−a)/(100−a)), where A � weight of grain, b � desired moisture content, and a � initial grain moisture content.e bags were then kept in a cold room for two weeks.Every other day, the bags were retrieved, grains mixed, and returned to the cold room.Prior to the conditioning of the grains, four grain samples were taken and tested for moisture content using a Foss Infratec ™ 1241 grain analyzer (Sweden)   and weight as the baseline.
e moisture content of the maize grain in the 12, 14, 16, and 18% categories was close to the desired ones: 12.4 ± 0.0, 14.3 ± 0.1, 16.2 ± 0.2, and 18.2 ± 0.2% (wet basis), respectively, while the quality of the grains was 1.2 ± 0.2% discoloured at the start of the storage period.

Test Storage Bags.
e storage bags tested were Super-Grain IV-R ™ bag (74 cm wide and 64 cm length) of 25 kg holding capacity (supplied by GrainPro Inc.) and openweave polypropylene bags of the same capacity.e SuperGrain IV-R ™ bag is tougher than the earlier version of the SuperGrain bag and has an extremely low oxygen permeability (<4 cc/m 2 /day) in which oxygen is reduced from 20% to 8% in 14 days for P. truncatus under controlled conditions of 27 ± 1 °C and 75 ± 1% relative humidity [28].
As recommended by the manufacturer, each SuperGrain IV-R ™ bag was placed inside a woven polypropylene bag to provide support and handling convenience.e open ends of SuperGrain IV-R ™ bags were cut to fit the height di- mension of these polypropylene outer bags.

Experimental
Procedure.e bags were each filled with 20 kg maize grains and then "seeded" with 20 live adult maize weevil (1 beetle/kg grain).
e barn had an LGB population and were interested to see whether this pest would penetrate from the outside into the bags (entry holes) while S. zeamais on the inside potentially made emergence holes on the grains.e bags were closed according to manufacturer's instructions: the entrapped air was squeezed out and then secured tightly with rubber straps.e grains were not inoculated with fungi since maize usually harbours sufficient fungal spores that fungal growth will occur naturally if environmental conditions are conducive.e experiment consisted of eight treatments with four replications: (1) SuperGrain IV-R ™ bag + grains at 12% moisture content; (2) SuperGrain IV-R ™ bag + grains at 14% moisture content; (3) SuperGrain IV-R ™ bag + grains at 16% moisture content; (4) SuperGrain IV-R ™ bag + grains at 18% moisture content; (5)  ® portable oxygen/carbon dioxide analyzer (Pac Check ® 325, Mocon Inc, USA).Measurement of carbon dioxide in polypropylene bags was not considered worthwhile as their open weave would allow free exchange of gas and so it was not expected to accumulate carbon dioxide.
ese measurements were taken every seven days and their means at the end of each storage period calculated.
e bags were stored in a completely randomised factorial design in an open side barn at ambient conditions.e ambient mean monthly temperatures and relative humidity in the barn during the trial were 26 °C (range 23-30 °C) and 54% (range 46-58%), respectively.e highest and lowest mean temperatures were recorded in March/April and June/July, respectively.For relative humidity, the highest records were in April and lowest records in September.Grain bags were sampled destructively after 60, 120, and 180 days storage.
At each sampling interval, bags were unsealed, and about 350 g grains were drawn using a 6-slot probe from the centre and four cardinal points.e samples were put in labelled plastic bags and taken to the laboratory for grain damage analysis.A set of 4.75 mm and 1.0 mm aperture size sieves (Endecott Ltd, UK) were used to separate grains and flour from the insects.Record of adult insects was taken according to species.After sieving, the grains were tested for moisture content using the Foss Infratec ™ 1241 grain analyzer.Half of the grain samples obtained using riffle divider were analysed for damage.e grains were sorted into undamaged, damaged, and discoloured fractions.e number of grains and the weight of each fraction were recorded.
e grains were weighed on a precision electronic scale (to two decimal points).Discoloured grains were expressed as a percentage of the total number of grains.A grain was regarded discoloured when its surface was darkened.Grain weight loss was determined by count and weight method [29]: where W u � weight of undamaged grain; N u � number of undamaged grain; W d � weight of damaged grain; and N d � number of damaged grain.e full contents of each bag were also sieved and all insects present counted according to the species.e insect counts were added to those obtained from the 350 g samples to give a total number of insects present.e total number of adult insects per bag were divided by the initial grain weight (20 kg) and recorded as number of insects per kilogram.

Statistical Analysis
e number of insects was transformed to log 10 (count + 1), while percent discoloured grain, weight loss, and oxygen and carbon dioxide data were square root (√x) transformed in order to stabilize the variances.e transformed data were first analysed using one-way repeated measures ANOVA (SPSS version 20, IBM Corporation 2011) to compare grain moisture content, insect numbers, percent discoloured grain weight loss, and oxygen and carbon dioxide at each storage time among the treatment as the response variable and treatment as the main effect.Storage time represented the repeated factor.Afterwards and separately, each response variable was analyzed using general linear model procedure of GenStat Release 12.1 (VSN International Ltd 2009), with treatment as main effect.Significant differences between the means were separated by the Student-Newman-Keuls (SNK) test at P < 0.05.However, for ease of understanding, the untransformed means are presented.
e treatment effects explained the significant differences observed.SuperGrain IV-R ™ bag maintained moisture content of the grains constantly throughout the storage period (Table 1).e equivalent grains stored in polypropylene bags showed a decrease in grain moisture content over the same storage period, with the final contents ranging from 11.2 to 11.6% after 180 days storage (Table 1).e presence of fermented smell in moist grains at 18% initial moisture content stored in the hermetic bag was observed.ere were differences in the percentage grain discolouration with treatments (F 7,24 � 191.23;P < 0.0001; coefficient of variation � 0.98) and storage period (F 2,48 � 88.56; P < 0.0001; coefficient of variation � 0.79).e interaction (P < 0.0001; coefficient of variation � 0.88) was also significant.e significant differences observed in grain discolouration were explained by the treatment effects.e discoloured grains were 4, 6, and 12 times more in the grains stored in hermetic bags at 14, 16, and 18 than grains with 12% initial moisture content at the end of the experiment, respectively (Table 2).For the grains stored in polypropylene bags, the percentage discolouration reduced with storage period.
e grain darkening and its associated off-odour were indicative of spoilage and changes in the quality of the grains.

Infestation by Sitophilus zeamais and Prostephanus truncatus.
Although the grain was deliberately seeded with S. zeamais, infestation by P. truncatus occurred naturally from a residual population in the barn, thus both insect species were followed during the study.e number of adult S. zeamais differed significantly with treatment (F 7,24 � 197.46;P < 0.0001; coefficient of variation � 0.98) and storage period (F 2,48 � 83.15; P < 0.0001; coefficient of variation � 0.78).
e interaction was significant (P < 0.002; coefficient of variation � 0.65).e significant differences observed were explained by the treatment effects.e population increased marginally 180 days after storage (7-60 adults/kg grain) in SuperGrain IV-R ™ bags compared to equivalent grains (894-1273 adults/kg grain) stored in polypropylene bags (Table 3).SuperGrain IV-R ™ bag with moist grains (18% moisture content) recorded the lowest insect density (7 adults/kg grain) while equivalent grains in polypropylene bags had the highest insects (1273 adults/kg grain) (Table 3).Very few S. zeamais survived in moist grains stored in SuperGrain IV-R ™ bags, and the population was almost the same throughout the storage duration.

Grain Weight Loss.
At the onset of the storage, the weight loss of the grains was 1.2 ± 0.2%.ere were significant mean differences in percentage weight loss with treatment (F 7,24 � 4804.14;P < 0.0001; coefficient of var-iation�0.99)and storage period (F 2,48 � 5521.98;P < 0.0001; coefficient of variation � 0.99).
e interaction was also significant (P < 0.0001; coefficient of variation � 0.99).e significant differences observed were explained by the interaction effects.e results showed significantly increasing trend in grain weight loss over the storage period and treatment.e mean percentage weight loss increased from 1.9 to 20.9 (Table 5) after 180 days of storage.Grain at 12% moisture content stored in the SuperGrain IV-R ™ bags recorded the lowest (1.2%) while grains at 16% moisture content stored in polypropylene bags incurred highest (42.6%) weight loss (Table 5).Overall, irrespective of initial grain moisture content, SuperGrain IV-R ™ bags prevented and maintained almost similar grain weight loss (≤1%) compared to equivalent grains in polypropylene bags over 120 days after storage.As grains in the polypropylene bags lost moisture during the storage period, significantly higher losses were observed (Table 5).By the end of storage period, grains at 12% initial moisture content stored in SuperGrain IV-R ™ bags suffered least weight loss (1.2%), translating to 96.6% weight loss reduction, compared to the equivalent grains in polypropylene bags.e carbon dioxide levels recorded in the grains at different initial grain moisture contents stored in the SuperGrain IV-R ™ bag affected the insect population observed in the bags.e buildup of carbon dioxide varied significantly with the treatment (F 3,12 � 64.11; P < 0.0001; coefficient of variation � 0.94) and the storage period (F 3,36 � 199.43;P < 0.0001; coefficient of variation � 0.94).e interaction between these two factors was also significant (F 9,36 � 104.03;P < 0.0001; coefficient of variation � 0.96).e significant differences observed were explained by the interaction effects.During the first 60 days of storage, carbon dioxide levels increased, reaching a value of 15.7% for grains stored at 12% moisture content and thereafter declined marginally when SuperGrain IV-R ™ bags became perforated by P. truncatus (Table 6).Overall, grains stored at 16 and 18% moisture contents recorded lowest (8.8%) and highest (15.6%) carbon dioxide levels (Table 6).Journal of Food Quality

Discussion
5.1.Moisture Content and Grain Discolouration.Moisture content and temperature are among the most critical factors that affect the quality of grains during storage.e normal harvesting moisture content for most maize farmers in the tropics, particularly in Africa, is 18-20%.e ears are usually put into drying cribs to reduce the moisture content to around 14%, shelled and bagged.Maize is therefore stored while still relatively with high moisture content.e combined effect of warm temperatures and high moisture content results in accelerated grain deterioration and promotes growth of insects and fungi [30].To maintain good quality maize during storage, grains must be protected from changes in moisture content and growth of insects and microorganisms such as fungi.is study demonstrated that moisture of maize grains stored in SuperGrain IV-R ™ bag did not change over the whole storage period, indicating lack of exchanges between the SuperGrain IV-R ™ bag and the outside environment.is suggests that the grains would not dry in the bag if not dried to safe moisture content level before storing.e finding confirms earlier study that showed the moisture content of grains stored in the hermetic bags remains unchanged during storage [31].In contrast, because of permeability of polypropylene bags, grains lost moisture in response to ambient relative humidity.Among factors that influence insect infestation in grain storage ecosystem are water, temperature, and air [32,33], and thus insect damage increases during storage.
e most common mould damage shows grain darkening symptoms.Safe grain storage is assessed by, among other factors, fungal growth, moisture content, and storage period.Infection of maize by storage fungi results in grain discolouration.Grain discolouration is a key factor for assessing the visual quality and market value.Kenya's maize standards and specifications KS01-143 ensure acceptable grain quality classification for producers, processors, and consumers.e standards which are harmonised with regional and international standards compare well with the South African and US corn specifications [34].To facilitate marketing, maize grain is classified into four different grades, based on several factors.For the discoloured grains, the amount varies from 2% for grade 1 (K1) to 6% for grade 4 (K4).ese grades attract different market prices.National Cereals and Produce Board (NCPB) whose core business constitutes commercial grain trading sells 90 kg bag of premier maize grain (grade 2) for human consumption at Ksh.2300 (USD 23) and Ksh.1400 (USD 14) for grade 4 for animal feed processing. is study showed that insignificant discolouration (2% up from 1.2% at the onset of the study) was only found with grains at 12% moisture content stored in SuperGrain IV-R ™ bags 180 days after storage.e results are in agreement with [35] findings on storage studies on pinto beans under different moisture contents and temperature regimes.In contrast, grain discolouration in polypropylene bags reduced with storage period because discoloured grains that showed signs of insect damage (holes and/or tunnelling) were classified as damaged.From health view, consumption of grains with higher percentage of discolouration is a potential risk due to mycotoxin contamination such as aflatoxin produced by the moulds.Cases of aflatoxin poisoning are high in Kenya which is exacerbated by untested maize flour from local posho mills in the rural areas.It would be risky to store moist grains (14-18% moisture content) in hermetic bags due to higher grain discolouration that may result in loss of market price premium and because of associated health hazard due to fungal growth that produce mycotoxins if the grains enter the food chain.However, grain quality before storage will determine whether the grain will maintain quality during longer storage period.As grain moisture level greatly influences the type of moulds that infect and grow on grains, moisture content must therefore be reduced by drying to levels below that at which moulds grow (13%) within the storage environment under tropical conditions.

Sitophilus zeamais and Prostephanus truncatus
Population.A significant reduction of S. zeamais and P. truncatus population when maize grains were stored in SuperGrain IV-R ™ bags was demonstrated in this study.e prevailing mean ambient temperature (26 °C) and relative humidity (54%) during the study were within the range for the development of both insect species.However, the population growth in the SuperGrain IV-R ™ bags was low.e reduction could be attributed to carbon dioxide levels achieved within SuperGrain IV-R ™ bags when properly tied.
For effective control of storage pests, [36] showed that carbon dioxide level above 40% is required.In [37], it is reported that mortality of immature and adult insects rapidly increased when exposed to carbon dioxide levels of 7-19% and defined the level that would kill 95% Tribolium castaneum adult population as 20% in five days.Overall, carbon dioxide levels in the present study were maintained Means within a column followed by the same letter are not significantly different (SNK test, P > 0.05).SGB IV-R ™ : SuperGrain IV-R ™ bag.
6 Journal of Food Quality within 8-16% range, and the observed reduction in insect population probably could be attributed to high temperature (23-30 °C) and lower relative humidity (46-58%) that enhanced insect metabolism and accelerated carbon dioxide toxicity.In the hermetic storage system, insect development is delayed and fecundity altered [31].e reduction in insect population observed is consistent with the findings reported in [31] when maize was stored in PICS bags.PICS and SuperGrain IV-R ™ bags are type of multilayer coextruded tougher plastics with low permeability of gas and good water barrier properties.e population growth of S. zeamais was higher than P. truncatus during the entire storage duration in polypropylene bags.Since P. truncatus was not artificially introduced into the grains, interspecific competition with S. zeamais probably affected its population increase [38].

Grain Weight
Loss. e effectiveness of grain storage is greatly influenced by storage period and weight loss during storage duration.e weight loss levels observed in the bags are attributed to insect population in the bags.Maize grains stored in SuperGrain bags IV-R ™ maintained very low weight loss (1-3%) compared to the equivalent grains in polypropylene bags (35-43%) at 180 days after storage.e weight loss levels were much lower than 13% which has been reported by DeGroote et al. [39].e observed difference probably could be ascribed to differences in the evaluated SuperGrain bag types.In the present study, the used SuperGrain IV-R ™ bag is an improved version of the one reported by [39].e new SuperGrain IV-R ™ bag is char- acterised by ultra-low oxygen permeability, greater toughness, and perforation resistance while retaining the original 0.078 mm thickness [28].Higher losses were recorded in the grains stored in polypropylene bags.SuperGrain IV-R ™ bags kept grains at 12% moisture content within the acceptable low weight loss level (1.2%).Similar observations have been reported under high levels of artificial infestation in the maize grain at similar moisture content stored in the SuperGrain bag [39] and PICS bag [31].When the market supply cannot satisfy the demand, grains at 12% moisture content stored in SuperGrain IV-R ™ bags would be sold at the highest price because of very low percentage of discoloured grains and weight loss thus contributing to improved livelihood income of the smallholder farmers.

Carbon Dioxide
Levels.SuperGrain bag IV-R ™ is a hermetic bag, and its mode of action in protecting the grain against insect pests is through composition of atmospheric gases rich in carbon dioxide and low in oxygen within the storage enclosure that suppress the ability of pests to develop and reproduce [40].Insects die when the oxygen level in the air is reduced below 3% [20] and feeding activity stops when the level falls below 4% [41].Although the carbon dioxide level in moist grains was higher, it did not exceed 20%.e results obtained from this study confirm the study in [3] which showed that the generation of carbon dioxide in grains at 14 and 16% moisture content under hermetic storage conditions did not exceed 20%.e apparent lack of hermetic conditions could have been caused by P. truncatus perforation of the bags, which probably facilitated gaseous exchange during storage between the outside environment and the inside of the bags.e holes were made by this pest from the outside source in the barn as it was not infested with the grains at the onset of the experiment.Perforation of the hermetic bags has been reported by Gracía-Lara et al. [28] when evaluating the effectiveness of SuperGrain IV-R ™ bags and Kukom et al. [31] PICS bags for protection against insect pests in stored maize.In other studies in Niger, Callosobruchus maculatus Fabricius (Coleoptera: Bruchidae) was found to perforate PICS bags which are used for cowpea storage [42].e other factor could be that the respiration of the grains, insects, and microorganism did not greatly contribute to the evolution of carbon dioxide.

Conclusion
e study confirms the effectiveness of SuperGrain IV-R ™ bag as a storage method.Grains should be sorted and sieved to remove debris and broken grains to limit sources and development of insect pests and moulds.e initial grain moisture content remained unchanged, while in polypropylene bags, it reduced.Moist grains must be dried to lower level before storage for a longer period to avoid spoilage due to moulds and grain discolouration.e respiration of grains and insects in the bags did not contribute greatly to the evolution of carbon dioxide due to holes made in the bags by P. truncatus.is study was only carried out at one site; therefore, further study under cool, hot, and humid conditions is needed to confirm the findings.

Table 1 :
Mean (±SE) percent grain moisture content after 60, 120, and 180 days of storage.Means within a column followed by the same letter are not significantly different (SNK test, P > 0.05).
Means within a column followed by the same letter are not significantly different (SNK test, P > 0.05).

Table 4 :
Mean (±SE) number of adult Prostephanus truncatus per kilogram of grain after 60, 120, and 180 days of storage.
Means within a column followed by the same letter are not significantly different (SNK test, P > 0.05).
Means within a column followed by the same letter are not significantly different (SNK test, P > 0.05).

Table 3 :
Mean (±SE) number of adult Sitophilus zeamais per kilogram of grain after 60, 120, and 180 days of storage.Means within a column followed by the same letter are not significantly different (SNK test, P > 0.05).