Amylolytic enzyme potential of corn kernels subjected to different germination processes

Potencial enzimático amilolítico de grãos de milho submetidos a diferentes processos de germinação

Abstract

Corn is one of Brazil's main agricultural commodities and has high industrial potential, mainly due to its starch, the main reserve substance. Malting, a process involving controlled germination followed by drying, triggers a series of biochemical reactions that promote the activation of hydrolytic enzymes, especially amylases, responsible for the mobilization of starch into simple sugars. This study aimed to investigate the amylolytic enzyme potential of corn kernels subjected to different germination processes, with and without prior maceration, over 120 hours. Physiological viability, water content, enzymatic activity, and total starch, amylose, and amylopectin contents were evaluated. The results showed that the grains had high initial physiological quality, with more than 80% normal seedlings. Prior maceration anticipated root protrusion and promoted greater enzyme activity in the ^{first hours, reaching 2,173.49 mg}_AR g_malt^{−1 in 84 hours, while in grains germinated without maceration, the activity increased progressively, reaching a maximum 1,759.44 mg}_AR g_malt^{−1, followed by a slight decrease after 108 hours. A gradual} reduction in starch, amylose, and amylopectin contents was observed in both treatments, confirming the action of amylolytic enzymes in the degradation of energy reserves. Both processes, without maceration and with prior maceration, were efficient, but suggest different applications depending on their purpose. Corn malt obtained without maceration can be considered for bakery products and food formulations, while malt subjected to prior maceration, due to its higher enzymatic activity and, consequently, higher content of fermentable sugars, is more suitable for the production of fermented beverages and first-generation ethanol. Thus, corn malt stands out as a technological, versatile, and sustainable alternative for use in functional foods, baking, fermented beverages, and biofuels.

Keywords:
starch; enzymatic activity; amylase enzyme; maceration; corn malt

Resumo

O milho é uma das principais commodities agrícolas do Brasil e apresenta elevado potencial de aproveitamento industrial, sobretudo em função do seu amido, principal substância de reserva. A maltagem, processo que envolve a germinação controlada seguida de secagem, desencadeia uma série de reações bioquímicas que promovem a ativação de enzimas hidrolíticas, em especial as amilases, responsáveis pela mobilização do amido em açúcares simples. Este estudo teve como objetivo investigar o potencial enzimático amilolítico de grãos de milho submetidos a diferentes processos de germinação, com e sem maceração prévia, ao longo de 120 horas. Foram avaliados a viabilidade fisiológica, o teor de água, a atividade enzimática e os teores de amido total, amilose e amilopectina. Os resultados demonstraram que os grãos apresentaram elevada qualidade fisiológica inicial, com mais de 80% de plântulas normais. A maceração prévia antecipou a protrusão radicular e promoveu maior atividade enzimática nas primeiras horas, atingindo 2173,49 mg_AR g_malte⁻¹ em 84 horas, enquanto nos grãos germinados sem maceração a atividade foi progressiva e sustentada, alcançando 1759,44 mg_AR g_malte⁻¹ em 108 horas. Foi observada a redução gradual dos teores de amido, amilose e amilopectina em ambos os tratamentos, confirmando a ação das enzimas amilolíticas na degradação das reservas energéticas. Os dois processos, sem maceração e com maceração prévia, foram eficientes, porém sugerem aplicação distinta conforme o seu objetivo. O malte de milho obtido sem maceração pode ser considerado para produtos de panificação e formulações alimentícias, enquanto o malte submetido à maceração prévia, por apresentar maior atividade enzimática e, consequentemente, maior teor de açúcares fermentescíveis, sendo mais adequado à produção de bebidas fermentadas e etanol de primeira geração. Dessa maneira, o malte de milho destaca-se como uma alternativa tecnológica, versátil e sustentável para aplicação em alimentos funcionais, panificação, bebidas fermentadas e biocombustíveis.

Palavras-chave:
amido; atividade enzimática; enzima amilase; maceração; malte de milho

1. Introduction

Brazil ranks third among the world's largest corn (Zea mays L.) producers, behind only China and the United States (USDA, 2024). In the 2023/24 harvest, which corresponds to the period from February to December 2024, the country it exported 34.92 million tons (Brasil, 2025a). The Midwest region of Brazil is the main producer of this commodity in the country, accounting for more than 50% of national production, with the state of Mato Grosso leading in productivity (Chaves et al., 2022; Silva et al., 2023).

Corn grain is rich in nutrients and is composed on average of 72% carbohydrates, 10% protein, 5% fat, as well as fiber, sugars, minerals, and vitamins, which are distributed in four physical structures: endosperm, germ, pericarp, and tip (Ma et al., 2020; Oliveira et al., 2022). This composition may vary depending on genetics, type of fertilization, and environmental conditions of cultivation (Anderson and Almeida, 2019; Oliveira et al., 2022). Approximately 98% the starch, a polysaccharide formed by the macromolecules amylose and amylopectin, is located in the endosperm (Oliveira et al., 2022).

Starch stands out in different industrial sectors, such as the food industry, where it acts as a stabilizer, thickener, gelling agent, and bulking agent; in the pharmaceutical industry, as an excipient and vehicle in formulations; and in the biofuel industry, where starch it is converted into ethanol through fermentation processes (Miranda et al., 2021; Chaves et al., 2022).

In recent years, the use of malted corn has gained prominence as a functional and sustainable alternative in the industry. Malt is obtained from the germination of grains, followed by drying, a process in which several biochemical and physiological reactions occur (Lemmens et al., 2019). Among these reactions, water absorption, increased respiratory activity, and the synthesis or activation of hydrolytic enzymes, such as amylases, proteases, and lipases, which act in the mobilization of grain reserve compounds, stand out (Tuan et al., 2019).

The corn malting process contributes to improving starch digestibility, in addition to improving the texture, palatability, and nutritional value of foods (Oliveira, et al., 2022). Sprouted corn is rich in simple sugars, which facilitate the action of fermentative microorganisms, and is used in fermentation processes mainly for the production of functional beverages and beers (Wu and Xu, 2019). In addition, malt enzymes improve the sensory quality of baked goods, intensifying their color, flavor, and aroma (Wu and Xu, 2019).

The resulting enzymatic activation of malting increases the versatility of corn, making it a promising raw material for the biofuel industry, especially in ethanol production (Fasim et al., 2021). Amylase enzymes are classified as α-amylases (EC 3.2.1.1; 4-α-D-glucan glucanohydrolase) and β-amylases (EC 3.2.1.2, 4-α-D-glucan maltohydrolase), which hydrolyze α-1,4 bonds in the glycosidic molecules of amylose and amylopectin in starch, generating fermentable sugars, such as glucose and maltose, which will later be converted into ethanol by yeasts during alcoholic fermentation (Mondal et al., 2022).

Enzymes can be obtained from microorganisms (fungal or bacterial), animals, and plants (Monteiro and Silva, 2009), and the corn malting process has been characterized as an economical and efficient form of processing, capable of promoting the production of amylolytic enzymes (Oliveira et al., 2022), which may be an alternative to add value to this cereal.

Thus, the objective of this study was to investigate the potential of amylolytic enzymes obtained from malted corn kernels under different conditions of time and germination processes.

2. Materials and Methods

2.1. Materials

Corn ears of the Pioneer 3707 cultivar were manually collected at the Mantra farm located in the rural area of Vera municipality (-12.349019798317757, -55.265258861196735), Mato Grosso, Brazil. The samples were transported to the Laboratory of Agroindustrial Projects and Processes at the Federal University of Mato Grosso (UFMT – Sinop). At the laboratory, the grains were manually removed from the ears, and the initial water content was determined gravimetrically (AOAC, 2000). The remaining grains were stored at a temperature of -20 °C for further analysis.

2.2. Grain viability

Three tests were performed to assess grain viability: germination, accelerated aging, and electrical conductivity.

2.2.1. Germination test

The germination test was used to verify the development of the embryo's essential structures. For this test, four replicates were performed each containing 50 corn kernels, which were distributed on Germitest paper (28 × 38 cm, Germilab) previously moistened with water in a volume three times the weight of the dry paper. These were then folded into rolls and kept at 25 °C for seven days in a Biochemical Oxygen Demand (BOD) incubator (Model Series EI08, Caltech). On the fourth and seventh days, normal, abnormal, and dead seedlings were counted, and the results were expressed as a percentage of normal seedlings (Brasil, 2025b; Krzyzanowski et al., 2020).

2.2.2. Accelerated aging test

The accelerated aging test was used to assess the degree of tolerance of the grains to high relative humidity and temperature. Three hundred seventy-five corn grains were separated and distributed on screens suspended inside 11 × 11 × 3.5 cm (Mylabor) Gerbox-type plastic boxes containing 40 mL of distilled water and kept in a BOD-type incubator (Model Series EI08, Caltech) at 41 °C for 72 hours. After this period, the grains were subjected to the germination test as described in the previous test (2.2.1.) (Krzyzanowski et al., 2020).

2.2.3. Electrical conductivity test

The electrical conductivity test was used to analyze the integrity of the cell membrane system through the leaching of electrolytes in the soaking solution. Four replicates were performed, each containing 50 corn kernels, which were placed in plastic cups and weighed on an analytical balance (Model M254Ai, Weblabor). Next, 75 mL of distilled water was added and kept in a BOD incubator oven (Model Series EI08, Caltech) at 25 °C for 24 hours. After this period, the electrical conductivity of the immersion solution was read using a conductivity meter (Model pH Smart, Linelab). The results were expressed in μS cm^-1 (Krzyzanowski et al., 2020).

2.3. Controlled germination

The viable grains underwent controlled germination for subsequent extraction of amylolytic enzymes, determination of enzyme activity, and quantification of total starch, amylose, and amylopectin.

The corn grain samples were germinated using two different methods: some of the grains were germinated directly, without undergoing the maceration process, while others were macerated before germination.

Germination without maceration or direct germination: 2,500 dry corn kernels were distributed on Germitest paper (28 × 38 cm, Germilab), previously moistened with water in a volume three times the weight of the dry paper. Next, 50 rolls were made, each containing 50 grains, and these were kept in a chamber at 20 °C for 120 hours.

Germination after maceration: before germination, the corn kernels underwent maceration. The grains were washed with 2.5% sodium hypochlorite to prevent contamination by microorganisms, and then soaked in distilled water at a ratio of 1:3 (w/v). Every 12 hours, the water was completely removed and the grains were left to rest for one hour, in order to promote aeration before the water was replaced. After 60 hours, the grains were removed from the water to determine their water content and then taken for germination. The 2,500 macerated corn grains were distributed on 28 x 38 cm Germitest paper (Germilab), previously moistened with water in a volume three times the weight of the dry paper. Next, 50 rolls were made, containing 50 grains each, and these were kept in a chamber at 20 °C for 120 hours. In both methods, five rolls were removed every 12 hours to count the primary roots and seedlings, determine the water content, and dry the germinated grains.

2.3.1. Primary roots and normal seedlings

Primary roots are the first roots to emerge from the seed during germination, originating from the embryo's radicle, and normal seedlings have basic structures such as roots, stems, and leaves.

In each germination roll, 50 corn grains were observed with the naked eye to count the number of grains with primary roots and normal seedlings. Then, the radicles and aerial parts were removed for subsequent drying of the grains.

2.3.2. Water content in germinated grains

To determine the water content of the germinated grains, five corn grains were removed from each germination roll, weighed in porcelain crucibles, and dried in an air convection oven (Model SL- 102, Solab) at 105 °C for 24 hours. After this period, the grains were weighed again to calculate the loss of mass due to water evaporation, according to the AOAC 934.01 (AOAC, 2000) method.

The water content was calculated using Equation 1.

$$CW=(M_{H2O}/M_S)x100$$(¹⁾

where: CW = Water content (%); M_H2O = Mass of water (g); M_S = Mass of sample (g).

2.3.3. Drying of germinated grains (Malt)

The grains were dried in a climatic chamber (Model BTL-433, BTX-475, Espec) at 54 °C for 8 to 10 hours (Biazus et al., 2006) to interrupt the germination process, obtaining the malt. The malt was then crushed and stored under vacuum -20 °C until the extraction of amylolytic enzymes, determination of enzymatic activity, and quantification of total starch, amylose, and amylopectin.

2.3.4. Enzymatic extraction for amylases

The extraction of amylolytic enzymes was carried out in an ice bath to minimize enzymatic activity loss. The extraction of amylolytic enzymes was carried out in an ice bath to minimize enzymatic activity loss. For this procedure, 1 g of germinated grain was weighed into a 25 mL beaker, followed by the addition of 3 mL of a 0.001 mol L⁻¹ hydrochloric acid (HCl) solution at pH 3.0. The mixture was then homogenized using a hand mixer (Model SQ-4264, Só Qualidade) until a uniform milky suspension was obtained. The solution was then placed in a 2 mL Eppendorf tube and centrifuged at 12,000 g min⁻¹ at 4 °C for 10 minutes in a refrigerated centrifuge (Model SL-706, Solab). The supernatant was removed with a micropipette transferred to another Eppendorf tube, and stored at -20 °C for subsequent determination of enzyme activity.

2.3.5. Enzymatic activity

Enzymatic activity was determined by measuring reducing sugars using the 3,5- dinitrosalicylic acid (DNS) method (Miller, 1959). For this process, 250 μL of 1% starch solution in Tris (hydroxymethyl) aminomethane buffer with HCl pH 7.0 was pipetted into a 15 mL test tube, 250 μL of enzyme extract was added, then the test tube was placed in a water bath (Model TE-056MAG, Tecnal) at 50 °C for 5 minutes. After this period, the test tube was removed from the water bath, 500 μL of DNS solution was added, and it was homogenized in a vortex mixer (Model K45-2810, Kasvi), then heated in boiling water for 5 minutes. After cooling, the tube was kept in an ice bath for 5 minutes and 5 mL of sodium and potassium double tartrate solution (KNaC₄H₄O₆) was added. The reading was performed spectrophotometrically (Model V-5000, Metash) at a wavelength of 540 nm, and the results were expressed in mg of reducing sugars produced by starch degradation per gram of malt (mg_AR g_malte^-1).

2.3.6. Total starch, amylose, and amylopectin contents

The total starch content was determined using the iodine-starch spectrophotometric method (Model UV-vis/NIR Cary 5000, Agilent) at a wavelength of 590 nm (Humphreys and Kelly, 1961) , while the method of Williams et al. (1970) was used to determine the amylose and amylopectin content at a wavelength of 605 nm.

The amount of amylopectin was calculated by the difference between the total starch content and the amylose content (Equation 2).

$$\%amylopectin=\%starch-\%amylose$$ (2)

2.4. Statistical analysis

The corn grain vigor analysis data were submitted to descriptive statistical analysis. The remaining variables were subjected to a normality test followed by analysis of variance (ANOVA), considering both time and treatment as fixed factors. When significant differences were detected (p < 0.05), means were compared using Tukey’s test. The level of significance adopted was 5%, with confidence intervals of 95%. All statistical procedures were performed using R software, version 4.4.3 (R Development Core Team, 2024).

3. Results

The results obtained refer to the initial characterization of the grains, the evaluation of physiological viability, as well as the count of primary roots and normal seedlings, water content, enzymatic activity, and total starch, amylose, and amylopectin contents under different germination conditions.

Immediately after harvest, the corn grains had a water content of 12.47 ± 0.32%. Regarding physiological viability, 87.60 ± 2.97% of normal seedlings were observed in the germination test, 82.00 ± 3.16% in the accelerated aging test, and an electrical conductivity of 8.3 ± 1.31 μS cm⁻¹.

In the primary roots and normal seedlings count, corn kernels germinated without maceration showed progressive primary root emergence, with values close to zero in the first 48 hours and reaching 100% after 84 hours, a period during which the formation of normal seedlings had not yet occurred (Figure 1a). In the process of germinated corn after maceration, primary root emergence began as early as 12 hours after germination, and normal seedling formation began at 60 hours (Figure 1b).

Figure 1
(a) Primary root emergence (%) and normal seedling formation (%) in the process of corn germinated without maceration; (b) corn germinated after maceration, over 120 h.

The hydration of corn kernels subjected to germination without maceration occurred gradually and continuously throughout the process, reaching a water content of 40.41 ± 0.48% after 120 hours. In the maceration process, the grains remained immersed in water for up to 60 hours, during which a significant increase (p < 0.05) in water content was observed in the first 12 hours (30.93 ± 1.19%), reaching 39.19 ± 0.75% at the end of maceration. Only after this period were the grains subjected to germination and then evaluated as germinated grains after maceration. In this treatment, the water content continued to increase, reaching 45.29 ± 0.80% at the end of 120 hours (Table 1).

Table 1
Water content (%) of corn kernels under different treatment conditions and germination times.

Germination time (hours)	Corn germinated without maceration (%)	Macerated corn (%)	Corn germinated after maceration (%)
0	12.47 ± 0.32 a	12.47 ± 0.32 a	N.A.
12	24.25 ± 1.20 b	30.93 ± 1.19 c	39.84 ± 0.51 fg
24	27.21 ± 0.48 b	33.91 ± 0.46 d	40.57 ± 0.80 fgh
36	31.43 ± 1.47 c	36.39 ± 0.81de	40.40 ± 1.03 fg
48	32.09 ± 0.43 c	37.58 ± 0.65 e	40.41 ± 1.44 fg
60	33.13 ± 0.28 c	39.19 ± 0.75 fg	41.24 ± 0.99 fgh
72	33.38 ± 1.19 cd	N.A.	41.84 ± 1.76 ghi
84	36.45 ± 2.54 de	N.A.	42.73 ± 1.48 ghij
96	38.50 ± 1.51 ef	N.A.	43.51 ± 1.60 hij
108	41.41 ± 1.35 fgh	N.A.	44.72 ± 1.74 ij
120	40.41 ± 0.48 fg	N.A.	45.29 ± 0.80 j

• Mean ± absolute standard deviation; letters in the same column and row that do not differ from each other according to Tukey's test with p < 0.05; N.A. = not applicable. Macerated corn: water content during soaking (0-60 h); Corn germinated after maceration: water content during germination after soaking.

The enzymatic activity of corn malt increased progressively with germination time and at all times analyzed. In addition, prior maceration of the grains resulted in higher enzymatic activity compared to direct germination. These differences were statistically significant (p < 0.05) as indicated ^{by Tukey's test. In the first 12 hours of germination, enzyme activity was 200.72 ± 40.72 mg}_AR g_malt^{-1 in unmacerated grains, while macerated grains showed 500.93 ± 43.71 mg}_AR g_malt^{-1, indicating} an increase of approximately 2.5 times due to maceration. Maximum activity was observed after 108 hours for unmacerated grains (1,759.44 ± 52.64 ^mg_AR g_malt^-1) and at 84 hours for macerated grains (2,173.49 ± 44. ^mg_AR g_malt^-1) (Table 2).

Table 2
Enzymatic activity (mgAR gmalt^-1 ) of corn grain under different germination treatment conditions.

Germination time (hours)	Germinated corn without maceration	Germinated corn after maceration
12	200.72 ± 40.72 a	500.93 ± 43.71 e
24	372.48 ± 67.70 c	859.93 ± 15.41 b
36	642.58 ± 25.08 e	1,434.85 ± 23.14 d
48	680.72 ± 29.33 e	1,490.47 ± 43.97 df
60	982.66 ± 34.00 g	1,537.21 ± 26.81 f
72	1,334.20 ± 27.30 j	1,762.02 ± 30.35 hi
84	1,537.79 ± 18.43 f	2,173.49 ± 44.02 k
96	1,534.63 ± 29.93 f	1,733.34 ± 41.30 hi
108	1,759.44 ± 52.64 hi	1,814.20 ± 44.57 h
120	1,549.83 ± 61.38 f	1,723.88 ± 17.58 i

• Mean ± absolute standard deviation; letters in the same column and row that are not different from each other according to Tukey's test with p < 0.05.

During germination, the total starch, amylose, and amylopectin contents varied over time, with differences between the germination processes without maceration and germination after maceration, as shown in Table 3.

Table 3
Starch, amylose, and amylopectin contents (%) of corn kernels under different germination treatment conditions.

Germinated Corn Without Maceration				Germinated Corn After Maceration
Time (h)	Starch (%)	Amylose (%)	Amylopectin (%)	Starch (%)	Amylose (%)	Amylopectin (%)
0	78.45 ± 1.62 a	18.61 ± 0.26 a	59.84 ± 1.51 a	78.45 ± 1.62 a	18.61 ± 0.26 a	59.84 ± 1.51 a
12	77.54 ± 0.13 ac	18.17 ± 0.37 ac	59.37 ± 0.29 ac	68.89 ± 0.38 b	16.09 ± 0.32 b	52.80 ± 0.46 b
24	76.57 ± 1.18 ac	17.87 ± 0.16 c	58.70 ± 1.33 ac	65.54 ± 0.19 d	15.59 ± 0.28 bd	49.94 ± 0.14 d
36	75.33 ± 0.44 cf	16.99 ± 0.09 f	58.34 ± 0.40 ac	64.13 ± 0.23 de	14.92 ± 0.35 e	49.21 ± 0.38 d
48	73.38 ± 0.26 fh	16.08 ± 0.10 b	57.29 ± 0.17 ce	57.40 ± 0.68 g	13.82 ± 0.05 g	43.58 ± 0.74 f
60	71.82 ± 0.67 h	15.73 ± 0.05 b	56.09 ± 0.62 e	52.53 ± 0.94 i	12.59 ± 0.14 hj	40.23 ± 0.45 g
72	68.27 ± 0.58 bj	15.09 ± 0.14 de	53.18 ± 0.67 b	51.19 ± 0.40 i	10.96 ± 0.05 j	39.94 ± 1.00 g
84	66.15 ± 0.76 dj	13.83 ± 0.19 g	52.32 ± 0.61 b j	45.91 ± 0.81 k	10.18 ± 0.14 k	35.73 ± 0.69 h
96	64.24 ± 0.27 de	12.76 ± 0.27 h	51.48 ± 0.24 bdi	44.53 ± 0.54 kl	09.43 ± 0.05 l	35.10 ± 0.49 h
108	62.91 ± 0.22 em	11.98 ± 0.16 i	50.93 ± 0.34 bdi	44.04 ± 0.77 kl	08.32 ± 0.14 m	35.72 ± 0.84 h
120	61.23 ± 0.30 m	10.99 ± 0.14 j	50.24 ± 0.44 dj	43.45 ± 0.75 l	07.50 ± 0.18 n	35.94 ± 0.90 h

• Means followed by the same lowercase letter in the column and row within each variable (starch, amylose, or amylopectin) do not differ from each other according to Tukey's test at 5% probability (p < 0.05). Values are presented as mean ± absolute standard deviation.

The starch content in germinated corn without maceration showed average values ranging from 78.45 ± 1.62% to 61.23 ± 0.30% at 120 hours. In the treatment with prior maceration, the starch content decreased significantly (p < 0.05) to 43.45 ± 0.75% after 120 hours of germination. The amylose content in germinated corn without maceration had an initial average of 18.61 ± 0.26%, progressively decreasing to 10.99 ± 0.14% at 120 hours. In grains germinated after maceration, the values ranged from 18.61 ± 0.26% at 0 h to 7.51 ± 0.18% at 120 hours. The amylopectin content started in both treatments at 59.84 ± 1.51% at 0 hours. For corn germinated without maceration, this content decreased to 50.24 ± 0.44% at 120 hours, while in grains germinated after maceration, the final value was 35.94 ± 0.90%, with both reductions being statistically significant (p < 0.05) according to Tukey's test.

^{Thus, it was observed that as enzyme activity (mg}_AR g_malt^{-1) increases, starch content (%)} decreases in both treatments with (Figure 2a) and without maceration (Figure 2b).

Figure 2
(a) Relationship between enzyme activity (mg_AR g_malte^-1) and starch content (%) in the corn process germinated without maceration; (b) corn germinated after maceration over 120 h.

4. Discussion

The corn grain had an initial water content of less than 13%, which is considered ideal for maintaining physiological quality during storage (Zambiasi et al., 2020). Water contents below this limit are essential to reduce tissue metabolic activity, preventing microbiological deterioration and contributing to the preservation of grain viability over time (Dubal et al., 2022). The high percentage of normal seedlings observed in the germination (87.60%) and accelerated aging (82.00%) tests indicated that the analyzed grains presented physiological quality under stress conditions, according to the established criteria for corn seeds (Krzyzanowski et al., 2020).

The reduced electrical conductivity (8.3 μS cm⁻¹) suggests good cell membrane integrity, a condition associated with minimal solute leaching and the absence of relevant physiological damage (Srii and Nagarajappa, 2024). Similar results were obtained by Ponte et al. (2025), who associated higher electrical conductivity values with cell membrane rupture and reduced vigor in popcorn seeds. Thus, the grains evaluated proved to be physiologically viable and proceeded to controlled germination.

Visible germination, characterized by the protrusion of the primary root, normally occurs when the grains reach between 30 and 40% water content (Oliveira et al., 2022). Both the water content and the physical characteristics of the grain structures showed different behaviors among the evaluated treatments. Figure 1a and Table 1 show that grains germinated without maceration presented late root emergence, which is associated with a slower hydration process, occurring gradually and continuously over 120 hours. This tends to delay metabolic reactivation in intact grains due the greater physical resistance by external tissues, such as the pericarp and aleurone (Wellmann et al., 2023). On the other hand, grains that underwent prior maceration (Figure 1b and Table 1) had their external structures softened by immersion in water, which reduces resistance to water entry and anticipates the physiological events associated with germination (Wellmann et al., 2023).

In the initial stage of germination, water is the limiting factor for maintaining embryo multiplication and expansion (Kerbauy, 2019), since rapid hydration favors the activation of embryo metabolism and the mobilization of reserves, directly reflecting on germination performance (El-Maarouf-Bouteau, 2022). As shown in Table 1, it was observed that the water content tends to stabilize at a certain point in both grains subjected to direct germination and those germinated after maceration. This period is known as the stationary phase and corresponds to the stage in which metabolic processes intensify, with greater demand for energy and degradation of reserve substances (Kerbauy, 2019). It is important to note that, in addition to adequate soaking, oxygen availability is essential for efficient germination (Oliveira et al., 2022). During maceration, although the water content reached about 30% in the first 12 hours, root protrusion only occurred after the onset of controlled germination, when the grains were no longer immersed in water, since immersion reduces the availability of oxygen to the embryo. This behavior shows that, even with a water content adequate for metabolic activation, low oxygen availability under immersion conditions temporarily limits embryonic development (Vieira and Carvalho, 2023).

The higher enzymatic activity for macerated grains compared to unmacerated grains indicated that maceration not only accelerated the onset of germination but also brought maximum enzyme production forward. On the other hand, in unmacerated grains, high enzymatic activation was maintained for a longer period. Maximum production corresponds to the point at which amylolytic enzymes reach their highest efficiency in starch degradation, which is expected behavior during germination, as it coincides with the moment of highest metabolic demand of the seedling (Munõz- Llandes et al., 2023).

Enzymatic activity is directly associated with water content, since adequate grain hydration promotes metabolic activation and stimulates the embryo to synthesize and release the hormone gibberellin (GA), which acts on the cells of the aleurone layer to produce hydrolytic enzymes, especially amylases (Hedden, 2025). These results indicated that both processes were efficient, but each has advantages depending on the desired technological application. Germination with prior maceration, by providing rapid starch mobilization and anticipation of maximum enzymatic activity, may be more suitable for processes that require high concentrations of fermentable sugars in a shorter time, such as in the production of fermented beverages (beers, distillates) and first-generation ethanol (Gopinath, et al., 2017). Germination without maceration, on the other hand, by maintaining enzymatic activity for longer, may be interesting in applications that require prolonged enzymatic action, such as in the production of bakery products and food formulations aimed at digestibility and nutritional enrichment (Monteiro and Silva, 2009; Gopinath, et al., 2017). Farias et al. (2009) reported maximum enzymatic activity of 2,538.38 mg_AR g_malte⁻¹ for amylases in corn germinated for five days with prior maceration. Similarly, other authors who also adopted maceration before germination described a progressive increase in amylase activity (Liu et al., 2024; Sharma and Gujral, 2020).

Total starch, amylose, and amylopectin contents (Table 3) gradually decreased throughout the germination process. This behavior confirms that the amylolytic enzymes produced during germination are metabolically active, promoting the conversion of these reserve polysaccharide to simple sugars, which are essential to meet the high energy demand of the growing embryo (Liu et al., 2024). In a similar study, Ma et al. (2020) reported significant reductions of up to 52.5%, 46.5%, and 54.2% in total starch, amylose, and amylopectin contents, respectively, in grains germinated for up to five days. Liu et al. (2024) also observed a 61.2% reduction in total starch content after 72 hours of germination, reinforcing the importance of amylolytic enzymes in mobilizing energy reserves. The study demonstrated that germinated corn kernels have high enzymatic potential, with the ability to efficiently mobilize starch reserves through the action of amylolytic enzymes, which can add value to this cereal widely cultivated in Brazil.

5. Final Considerations

The germination processes of corn grains, with and without prior maceration, demonstrated high amylolytic enzymatic efficiency, confirming their capacity to effectively mobilize starch reserves. Owing to its simplicity and ease of implementation, malted corn represents a versatile and sustainable technological alternative suitable for various industrial sectors, depending on the intended application. Beyond adding value to one of the most widely cultivated cereals in Brazil, the use of corn malt may contribute to reducing dependence reliance on traditional raw materials, enhance the competitiveness of domestic production, and open new opportunities for use in functional foods, baking, fermented beverages, and biofuels.

Acknowledgements

The authors would like to thank the Federal University of Mato Grosso (UFMT), Sinop Campus, for its institutional support, as well as the Center for Innovation and Technology in Bioenergy and Sustainability (CIT/UFMT-Sinop) for its technical and scientific support. They also thank the Electrochemical and Polymer Materials Laboratory at the Federal University of Uberlândia (UFU), Pontal Campus, for its collaboration and infrastructure. Finally, they thank the company Biomassa Giacomelli & Filhos for donating the corn samples used in this study.

• Data Availability Statement
  The data supporting the findings of this study are available from the corresponding author upon reasonable request. The datasets are not publicly available due to institutional restrictions but may be shared upon justified demand.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request. The datasets are not publicly available due to institutional restrictions but may be shared upon justified demand.

Publication Dates

• Publication in this collection
  20 Mar 2026
• Date of issue
  2026

History

• Received
  21 Oct 2025
• Accepted
  22 Jan 2026