Effect of Nixtamalization on Aflatoxins, Functional and Acceptability of Maize from Mulindi, Rwanda

Rwanda relies highly on maize for human food as well as for animal feed and industrial uses. However, high temperature and wet climate conditions in Rwanda favor the growth of fungi that may lead to mycotoxin contamination in maize and subsequent degradation of its quality. Therefore, this study aimed to assess the effect of nixtamalization on the aflatoxins, the functional and acceptability of maize grains from Mulindi, Rwanda. The maize samples were collected from the Mulindi market in Rwanda; representative samples were collected by the guidelines for sampling food for mycotoxin determination. Subsequently, the treated maize samples were dry-milled to produce fine flour, and the flour obtained was analyzed for swelling capacity, water absorption capacity, water solubility, acceptability, and aflatoxins, which were quantified by the HPLC method. The HPLC results showed that the highest total aflatoxin level in maize grains was 44.99 ppb. Cooking maize in water and calcium hydroxide of 1% resulted in a reduction of aflatoxin with degradation percentages of 25% and 88%, respectively. However, in all treatments, aflatoxins B₁ and B₂ were less reduced compared to aflatoxins G₁ and G₂. Cooking maize in pure water showed the lowest reduction of aflatoxin compared to the other treatments. However, cooking maize in water with 1% calcium hydroxide decreased aflatoxin to a higher percentage. Cooking maize in lime enhanced the swelling capacity, water absorption capacity, and water solubility of maize, as well as its acceptability. However, the nixtamalization process was observed to reduce aflatoxins levels in maize and improve the functional properties of maize, and therefore, its application could be encouraged for safe products in the market.

Introduction

Aflatoxin is a type of mycotoxin produced by molds, mainly Aspergillus parasiticus and Aspergillus flavus. Based on their fluorescence under UV light, there are four major types of aflatoxin named B₁, B₂, G₁, and G₂, and their two metabolites M₁ and M₂. However, B₁ is known to be the most harmful mycotoxin among others [1]. Several factors are reported to encourage Aflatoxin contamination; these include temperature and humidity [2]. The other mentioned factors are cropping damage due to insect activities, heavy rain during harvesting, inappropriate drying of crops before storage, and improper storage facilities [3].

The average temperature in Rwanda ranges between 14 °C and 30 °C, while the relative humidity is between 71% and 79%, and these conditions favor fungi growth [4]. In addition, abiotic and biotic stresses result in the proliferation of toxigenic fungi, which produce mycotoxins in maize during its production stages. Therefore, controlled agriculture practices and food processing techniques are needed to prevent fungal growth in the consumed commodities. Osuret and others [5] reported that poor crop handling leads to aflatoxin contamination of foods.

The ingestion of mycotoxin-contaminated food leads to chronic and acute effects, including edema, impairment of the function of macrophages, and decreased antibody response to vaccines. For instance, in Kenya, an outbreak caused by higher consumption of aflatoxin-contaminated maize was reported, which resulted in 317 aflatoxicosis cases and the death of 125 persons [6]. Aflatoxins should be reduced and detoxified by biological, chemical, and/or physical methods from the contaminated foodstuff. Some of these methods have their limitations. However, it is recommended that the treated products should be free from the chemicals used, and their nutritional value should not be compromised.

Rwanda as a country is facing a challenge of aflatoxin in maize due to poor storage and drying facilities combined with high rainfall. Application of nixtamalization, a process of cooking maize in lime solution at boiling temperature and steeping in a plastic container for 24 hours, has been found to reduce the level of aflatoxin in maize [7]. In addition, cooking maize grains in lime solution has many benefits over unprocessed grains for food preparation, such as improved nutritional value, aroma, flavour, and reduced mycotoxins. It has also been found to increase the bioavailability of vitamin B3 niacin, which reduces the risk of pellagra disease and increases calcium intake due to its absorption by the kernels during the steeping process [8]. Little information is available on the study of the effects of nixtamalization treatment on aflatoxin and the functional and acceptability status of nixtamalized maize from Rwanda.

This study aimed at examining the effect of nixtamalization on aflatoxin levels, functional properties, and acceptability of maize from the Mulindi market in Rwanda. The findings from this study are expected to contribute to the solution for aflatoxin decontamination and improvement of grain functional and sensory quality.

Materials and Methods

Materials Sampling and Preparation

The maize grain samples were purchased from the Mulindi open air market, Kicukiro District in Rwanda. Random sampling was done from different vendors from where a representative sample of 20 kilograms was drawn. The aggregation sample was mixed, the representative sample was taken according to Whitaker guidelines for sampling food for mycotoxin analysis [9], and put in amber paper bags. The maize samples were stored in an airtight plastic container at room temperature (25 °C–27 °C) prior to laboratory analysis. Lime [Ca (OH)₂], chemicals (Methanol, TFA, Phosphate-Buffer Saline), and aflatoxin standards were purchased from Sigma-Aldrich in Kenya. During laboratory analysis, the sample was divided into portions; the first maize grain sample portion was analyzed for total aflatoxin, the second was boiled (normal cooking) in water, and the final sample was subjected to a nixtamalization process (alkaline cooking). Subsequently, both samples were dry-milled to produce fine flour. The maize flour, after alkaline cooking, was analyzed for aflatoxins, swelling capacity, water solubility, and water absorption capacity. The quantification of aflatoxins was undertaken according to the AOAC Official Method 2005:08 [10], and their identification was confirmed by HPLC with a fluorescence detector; this method offers good sensitivity and precision coupled with ease of automation. However, HPLC is expensive in initial capital investment and requires skilled and experienced staff to operate and maintain equipment.

Preparation of Untreated Maize Flour (control)

The maize grain sample was mixed with an automatic mixer to obtain a homogeneous sample. About 500 g of maize were milled with an electric miller (Model: CM 200, RetschGmbH -42781, Haan, Germany) and sieved to get fine flour. The flour was packaged in an amber paper bag and stored at a refrigeration temperature of 4 °C for two days before laboratory analysis.

Preparation of Flour from Maize Grain Cooked in Water

Maize grain sample was weighed to 500 g, then boiled in 1.5 L of water, followed by steeping in a plastic container for 24 hours. The grains were removed out of the water, well-drained, and oven-dried at 60 °C for 90 minutes. Then, the maize sample was milled and sieved with a 0.25 mm mesh screen, and the flour obtained was packed in an amber paper bag and stored at a refrigeration temperature of 4 °C for subsequent laboratory analysis.

Preparations of Flour from Maize Cooked in an Alkaline Solution

A sample of 500 g maize grains was boiled in 1.5 L of water with 5 g of 1% Ca (OH)₂ at a temperature of 92 °C for 30–45 minutes. After 24 hours of steeping, maize grains were washed with normal water, with the aim of removing pericarps and the remaining lime to form nixtamal. The nixtamal was oven-dried at 60 °C for 90 minutes and milled with a disc miller (Model: CM 200/Retsch GmbH/42781, Haan, Germany) into flour. Subsequently, nixtamalized maize flour was stored at a refrigeration temperature of 4 °C for laboratory analysis. Non-nixtamalized maize flours were treated in a similar manner.

Aflatoxin Extraction and Determination

Both untreated and treated maize flour were subjected to aflatoxin analysis using the Association of Official Analytical Chemists standard method (2005:08) [10]. Specifically, 5 g per each sample and the control were weighed in a test tube. Then, they were mixed with 20 ml and 70% methanol for 2 hours using magnetic stirring and centrifuged at 3000 rpm for 15 minutes. 9 ml of sample extract was put in a salinized test tube and evaporated to almost dryness (<0.5) with a nitrogen evaporator (Model: 1041/Gesellschaft fur/Germany). The dried sample was reconstituted with Phosphate-buffered saline buffer to 10 ml). The mixture was filtered with a membrane filter of 0.2 µm, put in an immunoaffinity column cleanup on a vacuum manifold, and a drop-off storage solution of 1 ml of the diluted extract was transferred into the column during aflatoxin clean-up. The immunoaffinity column was washed with 2 ml × 10 ml water, rinsed dilution test tube and container also with the first 10 ml of water dropped through the column by gravity. Aflatoxins were eluted from the column with 3 ml × 0.5 ml of methanol to the salinized test tube, and methanol was stood in the column to make sure all aflatoxins were free from the column. During derivatization with trifluoro acetic acid (TFA), the elute was evaporated to dryness with a nitrogen stream, and 200 µL trifluoro acetic acid was added, closed the cap, and mixed with a vortex blinder for 1min and incubated for 30 minutes at room temperature. The sample mixture was diluted to 500 µL with acetonitrile-water solution (30:70) mixed for 1 minute and filtered through a 0.2 µm membrane filter. The aflatoxins were analyzed with HPLC (Shimadzu/Japan, equipped with a fluorescence detector RF 20A/Autosampler SIL30AC) and a fluorescence detector on the same day. Aflatoxins were analyzed as their TFA derivatives and they were eluted in the following order: G₁, B₁, G₂, and B₂. However, aflatoxins were identified according to their retention time and quantified with their external standard curves.

Analysis of Functional Properties

Swelling Capacity

Swelling capacity was determined using the method described by Oloyo and others with some minor modifications [11]. Specifically, one gram of maize flour sample was weighed and mixed with 10 ml of distilled water in a centrifuge tube and heated in the water bath at a temperature of 80 °C for 30 minutes. The sample mixture was shaken continuously during heating. After heating, the suspension was centrifuged at 1000 rpm for 15 minutes and the supernatant was decanted, and the weight of the paste was recorded. Thus, the swelling capacity was determined using (1):

(1)% Swelling capacity=AB×100where A stands for the weight of the paste in grams and B stands for the weight of the dry maize flour in grams.

Water Absorption Capacity

Water absorption capacity was determined following the method of Sefa-Dedeh et al. [7]. Five grams of maize flour were weighed and mixed with 30 ml of distilled water at 70 °C. The mixture was stirred and allowed to stand for 30 minutes and then centrifuged (Model Max. drehzahl/Srotor/spanning/220v, Germany) at 3000 rpm for 15 minutes. The increase in weight was noted by weighing the paste. The water absorption capacity was expressed as a percentage of the initial sample weight.

Solubility

Water solubility was determined using the method described by [12]. A maize flour of 0.5 g was weighed, mixed with 10 ml distilled water, and heated in a water bath (Model: LSB-015S; DATHAN LABTECH CO, LTD/ KOREA) at a temperature of 60 °C for 30 min without mixing. After centrifuging the sample at 1600 rpm for 10 minutes, 5 ml of the supernatant was dried in a hot air oven, weighed, and calculated using (2):

(2)% solubility=NM×2×100where N stands for the weight of the soluble starch in grams and M stands for the weight of the sample (dry basis) in grams.

Effect of Lime Cooking (nixtamalization) on the Sensory Properties of Maize Flour

Maize flour porridges were prepared using both nixtamalized and non-nixtamalized/raw flours, separately. The porridge cooking method practiced in households was simulated. The cooked porridges were subjected to a sensory evaluation test using 30 randomly selected persons who were familiar with maize porridge as untrained panelists. The untrained panelists were 14 males and 16 females aged between 25 and 32 years old, and they were presented with coded samples and were requested to indicate their liking for each product on odor, taste, texture, appearance, after tase and overall acceptance using a ranking method based on 7-point hedonic scale.

Statistical Analysis

Data were analyzed using Statistical Package for Social Sciences (SPSS) version 25. The significance differences were determined using the Turkey test at p ≤ 0.05. The comparison of the mean differences was done by the analysis of variance (ANOVA).

Results and Discussion

Quantification of Aflatoxin Levels in Maize Grain

The concentration of total aflatoxins was quantified in the maize grain sold at Mulindi market, Rwanda. The aflatoxin B₁ levels in the maze samples varied from 35.98 ± 3.59 and 26.43 ± 3.53 ppb for RMF and CMF, respectively; this was slightly high compared to the recommended limits. The findings correlate to another research done on aflatoxin B₁ contaminated flour samples from different markets of Kigali city, which indicated that 40% of maize flour samples from these markets were contaminated with aflatoxin B₁ at the level (15.6 ppb) which is higher than the 5 ppb limit set by East African Community countries (EAC) [13]. According to the Kenyan Bureau of Standards, the postharvest practices involved in maize from the field to the market and handling by processors may increase mycotoxin proliferation [6]. Limited knowledge on the proliferation of moulds by farmers has been found to be a cause for the increase in the levels of aflatoxin contamination in addition to the unavailability of proper postharvest infrastructure [14]. Therefore, in this study, the higher levels of aflatoxin may be attributed to the dearth of information on aflatoxin contamination in maize and infrastructures which are at the development stage in Rwanda. Moreover, researchers have indicated that aflatoxin contamination is highly due to environmental conditions [15].

Effect of Nixtamalization on Aflatoxin’s Levels in Maize Flour

The results on the effect of nixtamalization on the aflatoxin levels in maize flour are presented in Table I. Three different maize flour samples (raw/uncooked, boiled, and nixtamalized) were analyzed for aflatoxin using an HPLC method. The analysis revealed the levels of the different aflatoxin fractions in the maize samples used. The RMF showed a significantly higher levels of the different aflatoxin fractions compared to the other treatments. There was no significant difference between RF, CMF, and NMF samples on the levels of AFG1 and AFG2. However, NMF showed the lowest significant level for AFB1, AFB2 and total aflatoxin than the other treatments (Table I). In all treatments, aflatoxins B₁ and B₂ were less reduced compared with aflatoxins G₁ and G₂. This indicates that aflatoxins B₁ and B₂ showed higher resistance to alkaline cooking than aflatoxins G₁ and G₂. These results correlate to those by Méndez-Albores et al. [16], who reported 90% degradation of total aflatoxin by nixtamalization. This is supported by another finding on the reduction of aflatoxin by nixtamalization process in maize kernels intended for tortilla production, indicating that aflatoxins G₁, and G₂ are more reduced during the nixtamalization process than aflatoxins B₁ and B₂ with the average reduction of 93%, 90%, 98%, and 97%, respectively [17]. However, the reduction of aflatoxin by lime cooking depends on the type of aflatoxin.

Table I. Effect of Different Treatments on the Concentration of Aflatoxin Contents in Maize Samples

Maize treatments	AFG1 ppb	AFG2 ppb	AFB1 ppb	AFB2 ppb	Total Aflatoxin ppb
RMF	1.96 ± 0.74 b	0.57 ± 0.50 a	35.98 ± 3.59c	7.65 ± 6.95c	44.99 ± 2.79 c
RF	0.44 ± 0.31 a	0.28 ± 0.24 a	30.88 ± 1.29bc	4.35 ± 0.27b	34.81 ± 1.51 b
CMF	0.29 ± 0.21 a	0.59 ± 0.51 a	26.43 ± 3.53 b	6.95 ± 1.78bc	33.81 ± 5.04 b
NMF	0.42 ± 0.20 a	0.84 ± 0.12 a	3.58 ± 0.65 a	0.65 ± 0.33 a	5.26 ± 0.39 a
	103.10	67.66	54.22	61.21	52.66
p value	0.004	0.411	<0.0001	0.000	<0.0001

Méndez-Albores and his collaborators also found that cooking, prolonged steeping, and washing maize facilitate the reduction of aflatoxin [16]. Therefore, the slight decrease in aflatoxin observed in the flour from maize cooked in normal water may be due to the prolonged steeping and washing after cooking maize, whereas the decrease of aflatoxin observed in flour from the nixtamalized maize partly due to cooking and steeping maize kernels in calcium hydroxide solution which facilitated the removal of pericarps. This observation agrees with the study that reported that steeping and cooking maize in lime reduced aflatoxin by 50% [18]. Moreover, aflatoxin detoxification by dehulling method has been proposed by many researchers in the field.

Nixtamalization process reduced aflatoxin (44.99 ppb to 5.26 ppb) with a degradation of 88%, which is lower than 10 ppb of the total aflatoxin limit set by African countries, whereas cooking maize in pure water showed a slight reduction (44.99 ppb to 33.81 ppb) with a degradation of 25% which is higher than the regulatory limit for human consumption. Cooking aflatoxin-contaminated maize in lime solution and prolonged steeping is a better way of detoxification of maize-based foods, especially in countries where maize is considered a staple food for humans.

Effect of Nixtamalization on the Functional Properties of Maize Flour

The results for swelling capacity, water absorption capacity, and solubility are presented in Table II.

Table II. Functional Properties of Maize Flour

Maize treatments	Swelling capacity g/g	WAC g/g	Solubility %
NMF	5.53 ± 0.19 b	2.72 ± 0.21 a	0.87 ± 0.07a
CMF	5.23 ± 0.11 ab	3.39 ± 0.01b	0.75 ± 0.21a
RMF	5.08 ± 0.19 a	2.53 ± 0.04a	1.23 ± 0.25a
Coefficient ofvariation	4.63	14.03	28.58
p values	0.038	0.000	0.052

Swelling Capacity

The swelling capacity values ranged from 5.08 to 5.53 g/g, with flour from untreated maize having the lowest value, followed by flour from maize cooked in water, and lastly that from maize cooked in lime (nixtamalized) having the highest value. Both treatments significantly (p < 0.05) increased the swelling capacity of the flour with lime cooking, showing the highest increase. This could be attributed to both the properties of starch and the effect of heating [19]. A study by Xie et al. reported that the swelling capacity of starch in cereal foods is related to the starch gelatinization property and is considered as the amylopectin property, its value decreasing linearly with amylose content [20]. It has been reported in several cereal starches that a high proportion of long chains of amylopectin leads to an increase in the swelling properties of the sample. Furthermore, heating an aqueous suspension of starch granules in excess water leads to hydration and disruption of structures due to the breakage of hydrogen bonds, and consequently, water molecules become linked through hydrogen bonding to the hydroxyl group on amylopectin and amylose, causing an increase in swelling [21].

Water Absorption Capacity (WAC)

Water absorption capacity is the ability of the product to incorporate water. On the one hand, water absorption capacity facilitates the digestion of foods, while on the other hand, high water activity could cause food spoilage by microorganisms [22]. The water absorption capacity ranged between 2.53–3.39 g/g, with flour from untreated maize having the lowest value and flour from maize cooked in pure water (without lime) having the highest value. Apart from the untreated flour sample, other treatments significantly (p < 0.05) increased the water absorption capacity, with the flour from maize cooked in water having the highest increase compared to flour from nixtamalized maize (Table II). This is, however, contrary to the report by Sefa-Dedeh et al. [7] showed a reverse trend of increased WAC of flour from maize cooked in lime solution (1%) while that of cooked in normal water decreased.

The different trends observed in the water absorption capacity of nixtamalized maize flour could be due to the gelatinization of starch as well as Ca⁺² and Ca (OH)⁺- starch interaction and the saturation of starch hydroxyl sites in the maize sample, which may lead to the decreased water absorption capacity observed [7]. The effect of lime on water absorption capacity has also been studied by Zazueta-Morales et al. [23], who reported that maize cooked in lime solution absorbs more water than maize cooked in normal water. The low value of water absorption capacity indicates the compactness of the starch polymer, while the high value is attributed to the loss of its structure due to the thermal treatment [24]. The maize was thermally treated at a temperature of 92 °C for 35–45 minutes. Therefore, the water absorption capacity could have been affected by the cooking temperature [25]. Other changes that occur due to heating may also influence the WAC. It has been reported that temperatures above the gelatinization of starch (approximately 60 °C) may result in protein denaturation and starch gelatinization [26]. Furthermore, the high water absorption capacity of the cooked sample may be due to the presence of more hydrophilic carbohydrates like soluble sugars with good water-holding capacity [27].

Solubility

Water solubility depends on the characteristics of starch, which can be calculated as the percentage of dry matter in the supernatant. The solubility ranged between 0.75% to 1.23%, with the flour from untreated maize having the highest value and flour from cooked maize having the lowest value. However, there was no significant difference (p < 0.05) in the solubility between the treatments. The results indicated that cooking maize reduced the solubility of flour, which could be attributed to the effect of thermal processing of unfolding protein and exposing its hydrophobic ends, hence reducing the solubility [28]. In addition, the solubility could be due to the amount of amylose leaching; thus, the higher the amount of amylose leaching, the higher the solubility [29]. On the other hand, the decrease in solubility of the flour from cooked maize may be due to the low content of amylose.

Effect of Lime Cooking (Nixtamalization) on the Sensory Properties of Maize Flour

The results of the sensory evaluation are presented in Table III. Except for appearance, there was no significant difference in the organoleptic scores between the nixtamalized and non-nixtamalized maize porridge. The appearance had the highest discrimination. The organoleptic scores showed that the participants could distinguish the flours (NFP and NNFP), indicating that although the formulations were not significantly different, the organoleptic scores were different in the respective organoleptic parameters. The product appearance did not influence the consumer’s taste and odour, which were not significantly different. However, the overall acceptability and after-taste did not show any significant differences. There were no differences between the texture of the products and the rest of the organoleptic scores. The NNFP resulted in relatively high scores for odour, taste, texture, and overall acceptability, although the aftertaste was also well received compared to the NFP, whose appearance was the most appealing.

Table III. Sensory Evaluation of Nixtamalized and Non-Nixtamalized Maize Flour Porridge

Products	Organoleptic parameter
	Appearance	Odor	Taste	Texture	After taste	Overall acceptability
NNFP	5.30 ± 1.21 a	5.78 ± 1.01 a	5.53 ± 1.07a	5.50 ± 1.38 a	5.57 ± 1.33a	5.87 ± 1.25 a
NFP	6.00 ± 0.87 b	5.43 ± 1.57 a	5.27 ± 1.31a	5.27 ± 1.34 a	5.17 ± 1.26a	5.80 ± 0.89 a
p values	0.013	0.331	0.392	0.509	0.237	0.813

According to the scores of the nixtamalized and non-nixtamalized maize porridge evaluated, only the appearance of the nixtamalized product was preferred compared to other organoleptic properties. The findings are in close agreement with those of Fernández-Muñoz et al. [30], which reported that lime concentrations of >2% may produce a nixtamalized product with organoleptic characteristics unsuitable for human consumption. This indicates that the good appearance observed in the nixtamalized flour porridge could be due to the use of low amounts of lime for the nixtamalization process. Furthermore, the removal of the pericarp improved the appearance of the nixtamal and, subsequently, the nixtamalized product (porridge). A study of tortilla production from maize involving an alkaline cooking process reported that a high concentration of lime results in a yellowish product even when it is from white maize kernels [31]. On the contrary, in this study, the maize porridge cooked from non-nixtamalized (raw) flour appeared yellowish, probably because it was milled without the removal of the pericarp, whereas the porridge cooked from nixtamalized maize flour appeared white because the maize was milled after dehulling. Cooking and prolonged steeping influence starch gelatinization and imbibition of the lime solution, softening the pericarp (removed during washing), unlike in the untreated maize grains, thereby influencing the appearance of the lime-cooked maize. According to Serna-Saldivar et al. [31], the color of nixtamalized maize grains changes from white to yellow depending on the type of maize and the concentration of lime used for treatment.

Conclusion

This study revealed that aflatoxin contamination remains a threat to maize consumers in Rwanda due to the high level of aflatoxin exposure from maize. The high level of maize contamination by toxigenic fungi is possibly associated with a lack of awareness by farmers and market vendors on aflatoxins, inadequate storage facilities, and inappropriate storage conditions. In this study, aflatoxin B₁ was predominantly high among other aflatoxins (B₂, G₁, and G₂), nixtamalization reduced the aflatoxins to the allowable level of below 10 ppb for total aflatoxins. Nixtamalization process was better than cooking maize in ordinary water, although both processes were effective in aflatoxin degradation. However, although mycotoxin can be controlled by nixtamalization process through degradation and modification of chemical compounds to be less toxic, maize processors should prevent mycotoxin from maize before processing and use grain that is free from mycotoxins to ensure the safety of maize-based products.

The nixtamalization process affected the functional properties of the maize flour. Mainly, flour from nixtamalized maize had increased swelling capacity, which ranged from 5.08 (control) to 5.53 g/g, and water absorption capacity, which ranged from 2.53 (control) to 2.72 g/g. However, solubility did not have a significant change. Alkaline cooking also enhanced the organoleptic properties of maize products, especially the most appealing appearance. Therefore, the nixtamalization process can be employed in the improvement of functional quality and mycotoxin reduction in maize-based foods, as well as the improvement of the organoleptic quality that influences the acceptability of food by consumers.