Ohmic Heating Nixtamalization Modifies Maize Starch and Affects the Structural and Physicochemical Characteristics of Instant Masa Flours ^†

Abstract

The objective of this study was to examine the changes in starch processed under various ohmic heating (OH) conditions in relation to the characteristics of nixtamalized maize. Ground and dehydrated nixtamalized doughs (masas) were analyzed. Samples were prepared using both OH and traditional nixtamalization methods for comparison. The OH process variables included cooking temperature (85 and 90 °C), heating time (0, 5, and 10 min), and voltage (120 and 130 V). Starch modifications were assessed through viscosity measurements, differential scanning calorimetry (DSC), X-ray diffraction, and scanning electron microscopy (SEM). The results showed that viscosity in OH-treated samples was influenced by both thermal conditions (time and temperature) and the electric field (at 130 V), due to gelatinization and electroporation, evidenced by starch granule damage in SEM. DSC and X-ray diffraction revealed gelatinization and a loss of crystalline structures, along with new interactions between starch components that stabilized the system and reduced peak viscosity in the OH masa flours.

1. Introduction

Historically, the preponderance of maize in the diets of Mexico and Central America is considered a consequence of nixtamalization, a technique that increased iron, niacin, lysine, and tryptophan bio-availabilities, and improved the palatability of maize products [1]. In this process, grains are cooked in an excess of alkaline solution, steeped for several hours. During these stages, the solution penetrates and degrades the pericarp, so that water and calcium ions diffuse into the grain, leading to softening of the inner structures and changes in the starch granules [2]. Afterwards, the soaked grains (nixtamal) are washed and separated from the cooking liquid (nejayote), which is discarded. Finally, nixtamal is ground to produce a dough or “masa”, which is the starting point of a variety of products such as tortillas, atole, tamales, tex-mex style taco shells, chips, and snacks [3]. Masa is also versatile in that it can be used fresh or dehydrated and ground to obtain flours, which can be reconstituted with water to form the aforementioned dishes. According to the Official Mexican Standard NOM-F46-S 1980, instant masa flours, or nixtamalized maize flour, are defined as “the product obtained from the grinding of healthy, clean, previously nixtamalized and dehydrated corn grains (Zea mays)”.

Traditional nixtamalization was designed for maize processing at a household level, but it is now used, virtually unchanged, in semi and industrial operations. This has brought to light several problems of the traditional process when scaled up, including energy inefficiency during cooking, loss of nutrients, and production of significant amounts (~2.9 m³ of nejayote per Mg of processed maize) of untreated wastewater [4]. These issues have prompted the modification of the traditional process and the development of new technologies to make nixtamalization more sustainable. Such ecological processes include the use of different additives (enzymes or various salts) to reduce the impact of nixtamalization effluents while using conventional equipment. Other alternatives imply the application of new mechanical/thermo-mechanical technologies like extrusion, low-shear laminar transport, and high energy milling, or different heating mechanisms such as ultrasound, infrared, ohmic heating, or microwaves (for a review see: [5]).

With regards to ohmic heating (OH) assisted nixtamalization, it is a novel approach that consists of the flow of an electrical current (alternating) through a semi-solid mixture of raw ground grains and an alkaline solution. The mixture’s resistance limits the electrical flow, causing internal heating and cooking as a consequence [5]. This use of the OH technology seems to have great potential to replace traditional nixtamalization at an industrial scale since cooking time is reduced, no wastewater is produced, and electrical energy is utilized efficiently [6,7]. Additionally, the nutritional profile of masa is enhanced since whole grains can be used and the pericarp is preserved, which increases fiber and phytochemical contents [8,9].

Experimental studies on OH nixtamalized products claimed that masa, instant masa flours, and tortillas produced by this method have similar quality attributes to their traditionally or commercially made counterparts [6,7]. Flours prepared by continuous OH, however, produced softer tortillas with increased water retention, a lower tendency toward retrogradation, lower viscosity, and a higher process output. These studies also showed that controlling moisture and temperature played a significant role in reducing the presence of undesirable nixtamalization outcomes (e.g., higher moisture and temperature tended to interfere with tortilla making ability), and these parameters were related to starch modifications that occurred during OH assisted nixtamalization, but the studies did not address the topic any further.

Structural changes in starch during nixtamalization are related to swelling of the granules and physicochemical processes such as gelatinization, annealing, and the loss of crystallinity. These modifications are responsible for the rheological properties of masa and, ultimately, for the texture of the products made from it [2]. It is important to perform structural studies on starch to gain a better understanding of the processes that lead to desirable or undesirable nixtamalized product characteristics. In this context, the use of techniques like scanning electron microscopy, differential scanning calorimetry (DSC), and X-ray diffraction, for example, has proven to be excellent tools to gain knowledge on the modifications of starch during traditional, ultrasound assisted, and extrusion nixtamalization processes used to produce masa, flours, or tortillas, but they have not been used to the same extent in the study of the OH process [2,5]. This type of study is particularly relevant for process variables that are inherent to OH, such as voltage, field strength, conductivity, frequency, power, and resistance, for example [10,11]. Effects such as hydrolysis and the formation of micropores on starch granules, attributable to the application of electric fields and OH during processing, have been reported for foods and isolated starches, such as rice, cassava, potato, and corn [11,12,13,14,15,16]. These starch modifications affect the electrical resistance and conductivity of the medium, changing the efficiency of the process, and are said to produce pre- and gelatinized starch with special properties, such as improved solubility, viscosity, and swelling power, for example. Recently, Flores Garcia et al. [17] studied the effects of electrical fields during OH treatments of maize native starch, finding that both thermal and non-thermal phenomena such as electrolysis occurred, and it was the latter that resulted in increased resistant starch. To our knowledge, no full-length publications have studied the effects of OH assisted nixtamalization on whole maize and how they relate to starch modifications [18].

Therefore, the aim of this study was to monitor the starch-related changes in rheological, thermal, and morphological characteristics of instant masa flour during OH nixtamalization under different temperature (85 and 90 °C), heating time (0, 5, and 10 min), and voltage (120 and 130 V) conditions.

2. Materials and Methods

2.1. Biological Material

Mature maize (Zea mays L.) grains, obtained from the 2016 harvest in Sinaloa, Mexico, were used to make instant masa flours. Prior to processing, the grains were manually cleaned from debris using a metallic sieve (5 mm).

2.2. Production of Instant Masa Flours

2.2.1. Traditional Nixtamalization Process

Grains were mixed with a 1.3% Ca(OH)₂ solution using a ratio of 2 L solution/kg of maize. The mixture was first cooked in an aluminum steamer at 90 °C for 25 min and then steeped for 12 h. Afterwards, the cooking liquid was separated from the nixtamal, and the latter was washed with clean water to remove excess lime and pericarp residues. Nixtamal was then ground in a stone mill (FUMASA, mod. US-25) to obtain masa. Finally, masa was dehydrated at 270 °C using a flash type dryer (Cinvestav-AV, M2000, Queretaro, Mexico) to a moisture of 8% and ground in a hammer mill (Pulvex S.A. de C.V., Mexico City, Mexico, model 200) using a 0.5 mm mesh size. Nixtamal instant flour samples were placed in polyethylene containers and stored at 4 °C until analysis.

2.2.2. Nixtamalization Process Using Ohmic Heating

Maize grains were ground in a disc mill (Nixtamatic, Mexico) to a mean particle size of 1410 μm and then mixed with lime in a ratio of 3 g Ca(OH)₂/kg maize. The dry mixture was conditioned for 10 min with enough water (50 °C) to reach a moisture of 55% in a commercial mixer (Kitchen Aid model K45SS; St. Joseph, MI, USA). The maize–lime–water mixtures were then cooked in a batch ohmic heating cell described previously by Gaytán-Martínez et al. [6]. A factorial design (2 × 2 × 3) was used to apply the cooking conditions to the raw mixtures in the OH nixtamalization process, with the following factors: voltage (120 and 130 V), temperature (85 and 90 °C), and holding time (0, 5, and 10 min). The latter factor referred to the duration of isothermal or constant temperature heating once processing temperature was reached at a given voltage.

The sample was placed in the cooking cell, and a thermocouple (type T) was inserted at the center of the heating equipment. The desired temperature and voltage were set prior to starting to heat. Voltage was applied to each sample, with a power of 1 W/g, calculated considering the electrical properties of each maize and lime water mixture, using Equation (1).

$$\mathrm{P}={\mathrm{V}}^2/\mathrm{R}$$

(1)

where P is the power (W); V is the voltage applied across the electrodes, and R is the electrical resistance (Ω). To monitor the process, a LabView (National Instruments, Austin, TX, USA, 2008) computer program was developed to record the voltage, current, and temperature. With these data, the electrical conductivity (EC) of the uncooked mixtures was estimated over the cooking period with the following equation,

$$\mathrm{E}\mathrm{C}=\mathrm{L}/\mathrm{A}\mathrm{R}$$

(2)

where L is the gap between the electrodes (m), A is the cross-sectional area of the ohmic heater (m²), and R is the electrical resistance (Ω). The EC was plotted by the computer program, and the conductigrams for each treatment are presented in the results. Once the samples were cooked, they were flash dried and pulverized to produce suitable instant masa flours and finally stored in the manner described above.

2.3. Characterization of Starch Changes on Instant Flours

2.3.1. Scanning Electronic Microscopy (SEM)

SEM images of the nixtamal and OH flours were obtained to assess the morphological changes in maize starch granules after the different nixtamalization processes [19]. Flour samples were analyzed on a scanning electron microscope (JEOL, JSM-6060 LV, Tokyo, Japan) in low vacuum mode, with the electron beam set between 5 and 15 kV. The sample was placed on a sample holder with double-sided tape attached and was coated with gold [20]. The Nixtamal sample micrograph was obtained according to the protocol described above on a JEOL, JSM-6060 LA (Tokyo, Japan).

2.3.2. Viscosity Profile Analysis

Viscosity in maize flours depends on the degree of starch gelatinization in the sample due to the type of process applied (traditional or OH nixtamalization). Viscosity profiles are used to study the physical and structural transformations of heat and moisture treated starch as it transitions from a suspension into a gel [19]. The viscosity curve was obtained in a rheometer (Anton Paar, MCR-101, Graz, Austria) using the AACC 61-02.01 method as modified by Ménera-López et al. [7]. Samples of the instant flours (3 g) were adjusted to 14% moisture, and distilled water was added to keep the sample weight constant during the test. The rotating paddles were first maintained at 50 °C for 2 min to stabilize temperature and ensure particles were dispersed homogeneously and then heated up to 92 °C at a rate of 5.6 °C/min and held constant for 5 min. The samples were finally cooled down to 50 °C at the same rate. Maximum, minimum, and final viscosities (all in cP) were obtained from the profile. Setback viscosity (total setback) was calculated as the difference between final and minimum viscosities.

2.3.3. X-Ray Diffraction Analysis

To assess the structural changes in maize starch that occurred as a consequence of the nixtamalization process, raw maize, OH, and traditional instant flours were studied using X-ray diffraction. This method allows us to monitor the order–disorder transformation of starch elicited by gelatinization via the changes in sample polarization [20]. The technique was performed on a diffractometer (Ultima IV, Rigaku Americas, Austin, TX, USA) with a detector D/tex ultra. Samples were adjusted to 7% moisture and ground into a fine powder (250 μm sieve). The X-ray diffraction patterns were obtained at 35 kV and 15 mA, with a CuK_α radiation wavelength of 0.15406 nm, and from 5 to 70° on a 2θ scale with a step size of 0.02°.

2.3.4. Thermal Properties

DSC measurements were carried out to study the endothermic transitions that occurred in maize starch as it gelatinized during nixtamalization processes to produce instant flours. Thermal analysis of the flours was performed in a differential scanning calorimeter (DSCI STAR System, Mettler Toledo, Greifensee, Switzerland) in the manner described by [20]. Flour samples (3 mg, ground to 250 μm particle size) and 7 mg of distilled water were placed in an aluminum crucible and sealed tightly. The sample was subjected to ramp heating from 30 to 110 °C at a 10 °C/min heating rate. Gelatinization parameters: onset (T_o), end (T_e), and peak (T_p) temperatures, and enthalpy (ΔH_gel) were obtained from the thermogram. Temperature range (ΔT) was calculated as T_e-T_o. Starch gelatinization percentage or degree (%SG) was estimated in relation to the enthalpy of unprocessed maize (ΔH_m) using Equation (3).

$$\%\mathrm{S}\mathrm{G}=(1-({∆\mathrm{H}}_{\mathrm{g}\mathrm{e}\mathrm{l}}-{∆\mathrm{H}}_{\mathrm{m}}\left)\right)\times 100$$

(3)

2.3.5. Statistical Treatment

The temperature and viscosity data obtained from the thermal and rheological characterization of instant masa flours produced with the OH processes were analyzed using the General Linear Model on Minitab 20 (Minitab Inc., State College, PA, USA) to assess the effect of the different factors: voltage (2 levels), temperature (2 levels), and time (3 levels). When significant effects were found, means were compared using the Tukey test. Additionally, a Dunnett test was performed to establish if the samples produced by OH treatments were different from those produced using the traditional nixtamalization process (Nixtamal flour). For ANOVA effects and comparisons between means, significance was set at p ≤ 0.05.

3. Results and Discussion

3.1. Electrical Conductivity

The changes in EC of OH maize–lime–water systems as the different nixtamalization treatments were applied can be seen in Figure 1. In general, systems showed a sharp increase in EC during the first 50 s of OH and then a brief reduction after temperature surpassed 70 °C. A second, smaller peak could be observed around 100 s, particularly in the more severe OH treatments as they approached process temperatures (85 or 90 °C), followed by a final reduction and relative stabilization of EC as the period of constant temperature (isothermal heating or holding time for 5 or 10 min) was reached.

EC is one of the factors that determine the efficiency of the OH process, and for cereal–water systems, the conductigrams are closely related to starch gelatinization [11,12]. EC first peak occurred concurrently with temperature rise in the sample, until around maize starch gelatinization temperature (65–67 °C), due to thermal boosting of particle movement [12,21]. The subsequent small rise in EC observed in the conductigrams could be associated with a decrease in apparent viscosity as gelatinization proceeded and temperature increased (Figure 1), perhaps due to molecular alignment and electrical disruption of some granule remnants under low shear conditions [12,22].

The ionic strength of the maize–lime–water mixtures could also contribute to the rapid increase in EC and to a faster overall heating rate. Voltage also affects the heating rate via the increase in the applied power at higher voltages (Equation (2)). In the present study, the heating rates of the OH samples were 16 °C∙min⁻¹ and 19 °C∙min⁻¹ for 120 V and 130 V, respectively, which correspond to the short time it took the samples to reach the process temperatures of 85 and 90 °C, approximately 70–95 s. As heating progressed, further changes of starch, now forming a hydrogel, would increase the resistance of the mixture, causing the observed decrease in EC. This could be caused by a reduction in water activity and of the surface or paths available for particle motion, but also increased viscosity of the matrix [12,21,23]. Due to the rapid heating, particularly at 130 V, it could be expected that all possible starch modifications related to gelatinization had already occurred soon after process temperature was reached because of the electrical field strengths applied (Figure 1) [17]. But, as the isothermal heating period rose to 5 and 10 min and the samples cooked further, starch seemed to reach its final level of gelatinization, and EC increased slightly during that period, which could be attributed to the temperature applied [11].

3.2. Changes in Microstructure

Figure 2 presents the micrographs of the samples, focusing on the starch granules. Flours treated at 120 V and 85 °C still possessed recognizable native maize starch structures, characterized by their polyhedral and spherical shapes (Figure 2a). At this voltage, the granules showed signs of disruption and aggregation that became more prominent as holding time (Figure 2a–c) and temperature (Figure 2d–f) increased. One of the signs of early granule disruption caused by OH was the appearance of indentations (holes) in the swollen surface, which is consistent with previous observations regarding pregelatinized starches [24]. Loss of granular structure and pores was also visible in native maize starch treated with OH at different electric field strengths [17].

The granule disruption and agglomeration were more visible in treatments at 130 V (Figure 2g–i) and comparable to the most intense treatments at 120 V. At this high voltage, no granular structure remained after the process temperature was reached (Figure 2g,j) and was a consequence of the rapid heating rate caused by the electrical field strength. At 5 and 10 min treatments, the structures appeared to be more distorted or cooked, suggesting that further changes occurred in the matrix due to holding time (Figure 2h,i,k,l). In this context, SEM showed that the starch disruption induced by the electric fields during OH nixtamalization treatments was continuous and irreversible, which has also been reported in the modification of starches via pulsed electric fields [14]. The Nixtamal sample consisted mostly of aggregated and even collapsed granules, likely caused by the combination of thermal and mechanical effects that occur during the wet-milling stage [25]. The disruption in this sample was significant, but it appeared less profound than in the higher voltage and/or higher temperature/time samples, as some recognizable structures were still visible, similar to the 120 V samples cooked for 0 or 5 min.

In conjunction with EC observations, the micrographs seem to also indicate that, at short holding times, surface disruption and partial gelatinization were more affected by voltage, while at longer times (10 min isothermal), temperature played a larger part in the disruption/agglomeration of the inner granule structures. It could be speculated that during OH nixtamalization, the holes produced by the electric field eroded the surface of starch granules early in the process (Figure 2). As a result, they would be more permeable for water diffusion, so swelling and initial lixiviation of starch components would increase [13,26]. These electrical changes have also been reported to increase water solubility and water absorption of maize starch [17]. In these conditions, a larger percentage of current would pass through the maize–water–lime mixture, causing a greater level of internal heat generation, increasing the temperature quickly [12]. Consequently, as temperature is held constant, thermal disruption would increase, swollen granules would further gelatinize or break down, and a higher degree of cooking will be achieved in the flours [11].

3.3. Thermal and Rheological Properties

3.3.1. Thermal Parameters

Thermal parameters obtained from the calorimetry analysis are listed in

Table 1. Thermal and rheological parameters of instant flours made by two nixtamalization methods, Ohmic heating and traditional.

Process	Treatment			Viscosity		Thermal Parameters
	Voltage (V)	Temperature (°C)	Time (min)	Maximum (cP)	Setback (cP)	To (°C)	Tp (°C)	Te (°C)	ΔT (°C)	ΔHgel (J∙g−1)	%SG
Ohmic Heating	120	85	0	2241 ab	2789 a	65.83 d	72.45 d	79.84 bc,A	14.01 ab,A	3.97 ab,A	48.44 ab,A
			5	2381 a	2772 ab	66.14 cd	72.63 cd	80.83 abc	14.69 a,A	4.66 a,A	39.49 b,A
			10	2114 abc	2555 abc	66.91 abc	73.21 bdc	81.35 ab	14.44 ab,A	4.21 ab,A	45.39 ab,A
		90	0	2179 abc	2622 abc	66.73 abc	73.3 abcd	81.19 ab	14.46 ab,A	3.74 ab,A	51.49 ab,A
			5	1903 abc	2418 abc	66.81 abc	73.72 ab	81.08 ab	14.27 ab,A	3.02 ab,A	60.76 ab,A
			10	1699 c	2190 c	67.12 ab	73.87 ab	81.26 ab	14.15 ab,A	2.78 b	63.94 a
	130	85	0	1882 abc	2330 abc	66.67 bcd	73.04 bcd	80.4 abc,A	13.73 ab,A	3.69 ab,A	52.14 ab,A
			5	1961 abc	2416 abc	66.17 cd	73.22 bcd	81.15 ab	14.98 a,A	3.28 ab,A	57.39 ab,A
			10	1774 bc	2254 abc	66.65 bcd	73.22 bcd	79.01 c,A	12.36 b,A	3.17 ab,A	58.88 ab,A
		90	0	1861 abc	2381 abc	67.36 ab	73.81 ab	80.88 abc	13.53 ab,A	3.48 ab,A	54.86 ab,A
			5	1748 bc	2274 abc	66.72 abcd	73.46 abc	80.91 a	14.19 ab,A	3.02 ab,A	60.76 ab,A
			10	1730 bc	2217 bc	67.56 a	74.21 a	82.3 a	14.74 a,A	4.38 ab,A	43.19 ab,A
Traditional	Nixtamal instant flour (control)			4887 A	4281 A	65.16 A	70.98 A	79.08 A	13.91 A	4.26 A	44.70 A
Standard Error of the Mean				104	110	0.159	0.186	0.387	0.422	0.325	4.22

Means that share a lowercase letter are not significantly different (Tukey test, p < 0.05). Means that share an “A” are not significantly different from the control (Dunnet test, p < 0.05). Gelatinization onset (T_o), end (T_e), and peak (T_p) temperatures. ΔH_gel, gelatinization enthalpy with respect to raw maize. Temperature range of gelatinization, ΔT = T_e − T_o. %SG, starch gelatinization percentage.

. The Oh processing factors (voltage, time, and temperature) had significant effects on T_o and T_p (p ≤ 0.017), and most interactions between the factors were non-significant, except voltage*time for T_o. Temperature affected T_e, ΔH_gel, and %SG (p ≤ 0.045), while ΔT was mainly affected by the interaction between all three factors. In fact, cooking temperature and/or its interactions with time and/or voltage were the factors that accounted for most of the variance observed in all thermal parameters. In general, OH samples cooked at higher voltages and/or times showed higher gelatinization onset, peak, and end temperature values, apart from the treatment at 130 V-85 °C-10 min, which presented the lowest T_e (Table 1). In most treatments, ΔT tended to increase from 0 to 5 min, stabilizing or decreasing after that (Table 1). At higher voltages and temperatures, ΔH_gel was generally lower, while %SG was higher. The exception in these parameters was the treatment at 130 V-90 °C-10 min, where ΔT and ΔH_gel presented some of the highest values in the group, while %SG was among the lowest (Table 1).

Regarding the comparison with the control, i.e., Nixtamal flour, gelatinization onset and peak temperature values of OH instant flours were significantly higher than those of the control (p ≤ 0.001), with the exception of the T_o of the mildest OH treatment (120 V-85 °C-0 min). Regarding ΔT and T_e, most of the OH treatments were not significantly different from Nixtamal flours (Table 1) but tended to be higher. The starch gelatinization of samples (as ΔH_gel and %SG) in OH flours was not statistically different than in Nixtamal flours (Table 1).

In Figure 3, the DSC curves of OH flours are presented. They showed a typical single endothermic peak of maize starch undergoing gelatinization but keeping some degree of internal order in the granules [19,27]. Also, size and shape variations in the thermograms across the processing conditions were observable (Figure 3). Comparing the two voltages applied, it appeared that the samples processed at 120 V had less gelatinized starch than the samples cooked at 130 V, so they required more energy to elicit phase transition during DSC. At 120 V, increasing holding time at the studied temperatures appeared to reduce the amount of crystalline order of some of the granules, so the peaks were less sharp (Figure 3b) or seemingly unchanged from one treatment to another (Figure 3a). The Nixtamal flour sample behaved closely to OH samples that were processed at higher voltages or longer times, suggesting a similar change in the crystallinity and gelatinization of starch granules in those samples (Figure 3e).

The presence of additional small peaks can be seen in the OH thermograms of Figure 3a–c, corresponding to samples 120 V-85 °C-10 min, 120 V-90 °C-10 min, and 130 V-85 °C-0 min, respectively. In Figure 3a, a small peak around T_o could indicate a minor thermal event related to the presence of free fatty acids (whose melting temperatures are around 66–68 °C) that could be entrapped by amylose-fatty acid complexes formed during OH nixtamalization [28]. In Figure 3b,c, the additional peaks were found after T_e and could also be attributed to the melting of amylose or amylopectin containing complexes and/or small retrograded fractions of differing thermal stability [17,29]. Although not as clearly visible as the peaks of Figure 3b or c, most thermograms showed incipient thermal events after T_e, between 85 and 110 °C which could point towards similar melting or dissociation incidents [20]. Finally, in Figure 3c, a peak forming a “shoulder” in the main endothermic peak could point out that the starch population was more heterogeneous than in other samples, so the shoulder may represent the gelatinization of a more crystalline group of granules, perhaps less affected by the nixtamalization process [30,31].

The differences in transition temperatures (T_o and T_p) indicated that a larger amount of ordered structures are present in the samples nixtamalized by OH when compared to the traditional method, even if the %SG or ΔH_gel of both processes were similar (Table 1). This could be due to the milling of nixtamal after cooking, which causes additional thermo-mechanical stress to starch granules, leading to more gelatinization, a reduction in the number of whole starch granules, and probably a more disorganized structure, which could also be observed in their thermogram [25,32]. Fast and homogeneous temperature increase during OH could also contribute to higher T_o and T_p by gelatinizing the least stable granules quickly while increasing gelatinization constraints for the more stable granules [30,31]. OH flours tended to have broader ΔT than Nixtamal flours, and this also points to the existence of slightly higher crystallite heterogeneity, and thus different stabilities [33], in OH processed starch granules.

In this study, increasing voltage, temperature, and time reduced the enthalpy of gelatinization. Due to its fast nature, the period of isothermal heating in OH nixtamalization is important since it replaces the cooking-steeping stage of the traditional process, where most starch changes occur and ultimately affect the product quality [2]. Previous research has shown that the application of stronger electric fields enhances heating rate, water diffusion, and subsequent starch gelatinization, particularly during extended isothermal heating periods [13,17]. This would imply that, at higher temperature and holding time levels, treatments at 130 V would have achieved higher gelatinization degrees than those of treatments at 120 V. This effect could be due to electrically induced alteration in the structure of starch granules lowering their thermal stability and causing partial granular dissolution, which could be seen in Figure 2, in a similar way to the observations in high-pressure damaged starch granules [30]. This damage could enhance solid loss in the crystalline and molecular regions of the granules during the first moments of OH isothermal heating at a given temperature but would not cause gelatinization of the complete granule population, corresponding to previous observations of systems under limited hydration [31].

The results of this study indicated that increasing holding time also increased T_o and T_p. At the most severe treatment (130 V, 90 °C and 10 min), it was observed that %SG was lower, but transition temperatures were high (Table 1), and the curves in Figure 2d show evidence of starch reorganization or increased order, as the increased sharpness of the peaks [34]. In this context, more intense alkaline heat treatment could create new charged interactions between starch components, stabilizing the matrix and further reducing water availability for gelatinization of the remaining stable granules [31]. The interaction of these effects would increase the overall gelatinization energy requirements (ΔH_gel) in the most severe OH treatment.

Interestingly, the %SG OH samples also showed variation across the temperature, time, and voltage conditions, from 39.5 to 63.9%, suggesting that these treatments could achieve a wide range of starch gelatinization degrees. Previous OH nixtamalization studies have stated that this process provides the manufacturer with the ability to control the degree of gelatinization of maize starch within a narrow set of conditions, namely, temperature and moisture [6,7]. This study has demonstrated that voltage and holding time also influence the degree of maize starch gelatinization during OH nixtamalization. In conjunction, the results of these studies highlight an opportunity to optimize the process in terms of energy use and reduction in processing time without compromising product quality but also offer the possibility to explore this heating alternative and transform starch in a manner that is more efficient than conventional heat and moisture processes.

3.3.2. Viscosity Profile

The values of maximum and setback/retrogradation viscosities of control and OH flours are listed in Table 1, while Figure 4 shows the viscosity profiles of flours produced by OH. As can be seen in Table 1, both parameters in OH flours were lower than in Nixtamal flours (p < 0.05). OH flours showed maximum viscosity values between 1730 and 2381 cP and between 2189 and 2789 cP for retrogradation or setback viscosity, while nixtamal flours were at least twice as viscous.

These results could be related to the inherent differences between the two processes. OH process can be considered milder than traditional nixtamalization due to the overall short processing time and low mechanical stress; therefore, even though enough swollen granules gelatinized and even broke down completely (see the changes in Figure 1), granule remnants and chain associations forming an amorphous network were still present [11,22,34]. This would result in OH flours having a lower peak viscosity and better water absorption capacity than Nixtamal samples, which are subjected to lower thermal damage but high shear during milling [6,25]. This could be beneficial in terms of the rehydration of the OH flours to obtain a smooth and uniform masa that can be shaped into tortillas or other products. Setback viscosity represents a measure of the rapid retrograde tendencies of amylose molecules during cooling and the changes in final viscosity caused by the re-association of starch chains [19]. The formation of amylose–amylose and amylose–amylopectin complexes during gelatinization would be reflected in the increase in final viscosity observed in all the pasting profiles [17]. In this context, lower maximum and setback viscosities in OH flours could indicate that intermolecular hydrogen bonds were formed between amylose and pericarp fiber components such as arabinoxylans, or even other starch fractions, preventing excessive leaching [35]. These bonds will contribute to producing a more stable gel matrix in OH samples than in Nixtamal flours, thus, final and setback viscosities would be higher in the latter as they are more prone to retrogradation (Figure 3 and Table 1). In terms of the properties of the flour, setback values of instant Nixtamal flours tend to be somewhat high (see, for example: [36], who reported values above 3000 cP), which helps form cohesive masas (doughs) since the presence of retrograded fractions contributes to structure; however, excessive setback viscosity (over 4200 cP in the same study) could lead to masas with an undesirable adhesiveness and poor handling [36]. Lower setback viscosities, as the ones observed in the studied OH flours, have been found in previous studies and were related to the production of softer and more pliable masas as well as desirable quality attributes in the resulting tortillas when compared to the traditional process, namely softness and the prevention of hardening or staling after reheating or storing [6,7,37].

In terms of the OH process parameters, it can be seen in Table 1 that a continuous reduction in maximum and setback viscosity appeared as treatment intensity increased in terms of voltage (p ≤ 0.001, 130 V > 120 V), temperature (p ≤ 0.014, 90 °C > 85 °C), and time (p ≤ 0.02, 10 min > 0 min). These results imply that as conditions become more thermally intense, at higher voltages, there were fewer intact starch granules available to develop viscosity in the OH samples prior to analysis [19,25]. This combined effect of the studied factors can also be appreciated in the lowering peak viscosity of the profiles presented in Figure 4 for the more intense OH treatments.

This, along with the thermal and SEM results presented above, seemed to indicate that the gelatinization of starch under OH was affected by non-thermal effects due to the application of an electrical field for a prolonged time [11,17]. Electrical effects in OH-treated starch are due to the low frequency of the applied treatments (generally between 50 and 60 Hz) which lead to the accumulation of charges in the membranes of the granules and the reorganization of their structure [38]. This would explain the erosion of the granule surface, or “pores”, observed in the micrographs, in a phenomenon called electroporation or electropermeabilization, which is known to enhance trans-membrane water and solute diffusion in vegetable tissues [13,39]. This phenomenon would alter the structure of starch granules, making them more susceptible to water absorption, swelling, and thermal damage, causing partial granular dissolution and increasing amylose leaching [17]. However, as mentioned above, gelatinization degree would not be as pronounced as in traditional nixtamalization due to the low shear of the medium keeping these lixiviates in a continuous gel matrix with low viscosity. Accordingly, previous studies by Gaytán-Martínez et al. [6] and Ménera-López et al. [7] have hypothesized that electroporation contributed to the starch granule damage, swelling, and gelatinization that occur during OH nixtamalization.

3.3.3. X-Ray Diffraction

The diffractograms of the studied treatments can be seen in Figure 5. They show peaks at 2θ values of 15, 17,18, and 23° (Figure 5: 200, 031, 211, and 231, respectively), which are characteristic of orthorhombic crystalline A-type starches, which are common for cereals such as maize [32,40]. The samples also showed small peaks at 2θ values of 10.05, 11.2, and 26.3° (Figure 5: 020, 101, and 051, respectively), which have also been found in other A-type starches [40]. Flores-Garcia et al. [17] reported the presence of V-type peaks in OH-treated maize starch at 7°, 13°, and 20°, and the authors of said study suggested that the intensity of V-type peaks was associated with the non-thermal effects of OH. In our study, only the peak at 20° (Figure 4: 040) was found. This could be attributed to the more complex nature of the whole maize samples compared to pure starch, as it includes lipids, proteins, and gums.

Traditional nixtamalization is known to change the width and sharpness of these peaks when compared to raw maize due to gelatinization and partial disruption of the crystalline structure, but the basic organization of the polymers remains largely unchanged [41]. We observed this behavior in the nixtamal and OH flours with respect to raw maize, in terms of how defined the peaks were and in the preservation of the characteristic diffraction A-type patterns, but both types of processing increased the intensity of the diffractograms (Figure 5). The preservation of the A-type pattern suggests that the overall chemical structure of starch was not altered by alkaline cooking.

Regarding starch in OH flours specifically, increasing voltage and heating duration generally increased the diffraction pattern intensity, and their peaks were also more intense than those of nixtamal samples (Figure 5). As cooking time increased, peak intensity of samples heated at 90 °C (Figure 5c,d) was not as pronounced as in samples cooked at 85 °C (Figure 5a,b). These differences could have been caused by the different degrees of crystallinity loss in the starch, achieved by the processing conditions during OH. At a molecular level, the combination of electrical effects and thermal effects at higher voltage and temperature probably caused damage in the outer layer of the starch granules, inducing leaching of amylopectin [17,42]. This would contribute to the disruption of amylopectin crystallites as gelatinization occurred, reflected in the reduction in enthalpy at the high voltage and high temperature treatments [33,43].

Although from a crystallographic point of view, the presence of one plane is not sufficient to prove the existence of a crystalline structure associated with the diffraction pattern of V-type starch [20], the formation of amylose complexes could still occur without amounting to actual recrystallization. It was observed that the peak corresponding to the formation of amylose–lipid complexes (2θ, 20°) increased its intensity with nixtamalization treatments, particularly in OH (Figure 5). These changes have been attributed to the reorganization of the amorphous region of the starch granule and the formation of new associations between leached amylose and naturally occurring maize internal lipids during cooking (and annealing if present) and/or the transformation of less ordered (Type I) into more ordered ones (Type II) amylose–lipid complexes [41]. The interaction of amylose with lipids likely occurred quicker in OH than in traditional nixtamalization due to electroporation. The formation of amylose–lipid complexes is known to increase the stability of the helices in the starch system, affecting the functionality of starch, which could be another factor accounting for the lower viscosity of OH samples [20]. Similar observations regarding the formation of amylose–lipid (V-type polymorph) structures have been reported for the thermo-alkaline treatment of maize grains of intermediate hardness, as well as in the production of other nixtamalized products such as tamales [41,44].

The improved formation of such complexes during OH nixtamalization has the additional implication of obtaining products with enhanced content of resistant starch, an important fiber component. In this regard, Flores García et al. [17] postulated that the intensity of the electric field in OH could increase the resistant starch fraction by non-thermal mechanisms, such as electrolysis, which would add to the thermal effects observed in traditional nixtamalization. Water electrolysis produces free radicals, such as -OH groups, which can interact with the components of starch. Additionally, the electrolysis of amylose and amylopectin chains can break the α-(1,4) bonds and oxidize the hydroxyl (-OH) groups. Oxidation produces carbonyl (C=O) and carboxyl (COOH) groups that hinder enzymatic digestion, thus increasing resistant starch. Recently, Dominguez-Hernandez et al. [9] reported that OH nixtamalized instant flours produced at 120 V and 85 °C contained almost twice the dietary fiber of traditionally processed flours and therefore had better nutraceutical properties than nixtamal flours.

4. Conclusions

The rheological properties and thermal transition temperatures of instant masa flours produced by OH were different from those obtained by the traditional method, at comparable levels of gelatinization.

Morphology of the OH flours, thermal properties, as well as X-ray diffraction patterns indicated that during processing, gelatinization and electroporation phenomena occur in a low mechanical stress setting, which enhances gelatinization but allows sufficient starch granule remnants, crystalline structures, and chain associations to remain and form a stable matrix with low viscosity and lower retrogradation tendencies. The proposed scenario seems to indicate that the desirable properties of the studied instant flours are a consequence of simultaneous thermal and non-thermal effects occurring in maize starch treated with OH.

Viscosity values showed that a range of gelatinization degrees in OH flours can be obtained, depending on the intensity of the process. Additionally, OH promotes the formation of resistant starch to a greater extent than traditional nixtamalization due to the electrolysis of the water and starch molecules. In conjunction, these results indicate there is an opportunity to control and optimize the process within a small set of cooking temperature, time, and applied voltage conditions to obtain nixtamalized products with enhanced functional properties derived from the modification of starch.