Antioxidant Purple Corn Protein Concentrate from Germinated Andean Purple Corn Seeds

Ecuador Andean purple corn (Zea mays L.) was subjected to a germination process at 15–40 ◦C for 24–168 incubation hours. Purple corn protein concentrates (PCPCs) were obtained by alkaline extraction at pH 8.0 and pH 10.0, followed by an isoelectric precipitation process at pH 4.0, pH 5.0 and pH 6.0. Proteins and phenolic content of PCPCs was calculated. PCPC antioxidant properties were determined by the ferric-reducing antioxidant power (FRAP) in vitro method and by the 2,2-azinobis, 3-ethyl-benzothiazoline-6-sulfonic acid, (ABTS) in vitro method. Andean purple corn seeds were able to germinate under the germination conditions tested in this study. The higher percentage of germination was of 63.33% at 168 h/25 ◦C. The PCPCs protein profile was characterized for the presence of six bands with molecular weights of 14.50 kDa, 20.12 kDa, 25.18 kDa, 41.85 kDa, 59.59 kDa, and 65.87 kDa. Germinated PCPC presented a high TPC content with ranges of 605.71–1820.00 mg gallic acid equivalents (GAE)/g PCPC dry weight (DW), germinated PCPC/72 h/25 ◦C presented a higher value of 1820.00 mg GAE/g PCPC, DW. All germinated PCPCs samples assayed presented strong antioxidant activity when measured by the ABTS and FRAP methods. Germinated PCPC/144 h/35 ◦C presented high antioxidant activity by ABTS with 804.35 μmol Trolox equivalents (TE)/g PCPC DW and germinated PCPC/144 h/30 ◦C presented a high value by the FRAP method, 987.83 μmol TE/g PCPC DW.


Introduction
Corn (Zea mays L) stands at third place in importance of cereal crops after wheat and rice with 1147 million tons of fresh weight of seeds produced in 193 million hectares during 2018 [1]. In Ecuador, corn is one of the main crops in the Andean region. In 2018, more than 365,334 hectares were harvested with a total production of 1,324,147 tons. This represents an average yield of 3.62 t/ha [1]. Corn belongs to the Family Poaceae and Genus Zea [2,3]. There are several types of corn grains with different colors such as yellow, blue, brown, green, and purple. Purple corn, also called purple maize, is a native crop of the Andean region in South America cultivated in Peru, Ecuador, Bolivia, and Argentina [4,5]. Maize is a cereal crop widely spread throughout the world from latitude 580 N to 400 S, from sea level up to more than 3000 m of altitude and in areas with an annual rainfall between 250 mm and 5000 mm [6]. The countries with major corn production in the world are the USA with 30%, China 15%, EU 14%, Brazil 4%, and India 3% [6]. Corn is used for animal and human food for their chemical composition and a high nutritive value. Corn grains have a high starch content (72%), 7-13% protein content, 2.5% of fiber, and 4.8% of oil content. Corn grains have an important content of vitamins and minerals [7][8][9][10][11].
Germination is a biological process that allows plants to use seeds to conserve species. With this process, a series of biological mechanisms are initiated that allow the growth of an embryo. The embryos begin germination with the entry of water and end with the elongation of the embryo axis-terminal. During this complex process, different biological compounds of the seeds undergo catabolism and synthesis processes [12][13][14]. In the early stages, many of the phytocompounds are used for embryo growth. The synthesis of new phytocompounds used for different purposes including protection mechanisms is activated. In addition, during germination, the seeds reduce some components that are considered anti-nutrients (phytic acid and lectins) [15][16][17][18].
At the biotechnological level, sprouts are used to generate germplasm banks. For many years, sprouts have attracted the interest of the food industry for the nutritional value of their components and their biological properties such as the antioxidant activity [19]. Antioxidant compounds are used in the food industry to preserve processed foods against lipid oxidation [20][21][22]. Recent studies have demonstrated their inhibitory effects against certain diseases such as cancer [23,24]. Therefore, there is a considerable interest in the search for natural compounds with antioxidant properties [25][26][27]. Many natural extracts obtained from plants containing mainly polyphenols, flavonoids, carotenes, and anthocyanins have been described in the scientific literature for their high antioxidant activity [28,29].

Plant Material
Andean purple corn seeds (Zea mays L., INIAP-199 bunch of grapes) were collected from 40 corn plants of the crop grown by the Bolivar State University (Guaranda), and the National Institute of Agricultural Research (INIAP), (Quito). The crop was grown in the city of Guaranda, Bolivar in Ecuador at an altitude of 2800 m, south latitude 01 • 34 15 and west longitude 79 • 0 02 . The average annual temperature of the site is 13 • C with 75% humidity. The harvest was carried out by hand once the seeds reached physiological and commercial maturity. The seeds were manually shelled and dried in a drying rack in the open air until reaching a humidity of 14%. Then, they were stored in plastic containers.

Germination of Purple Corn Seeds
Andean purple corn seeds were germinated according to the methodology described by Paucar-Menacho et al. (2016) [39]. One hundred seeds were submerged for disinfection in 0.1% sodium hypochlorite solution (1:5 p/v) for 30 min at room temperature and then washed with distilled water. The seeds were then submerged in distilled water (1:5 p/v) at room temperature for 24 h. Subsequently, the hydrated seeds were introduced to the BINDER KBF 240 (LabReCo, Horsham, PA, USA) constant climate chamber on wet filter paper, with a water circulation system to always keep the seeds moist. Germination was carried out in the dark at the following temperatures with durations of 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. Three replications were made for each germination condition. The percentage of germination was calculated to each treatment of germination. The percentage of germination = (% normal seeds + % anormal seeds + % dead seeds) = 100%. Normal seeds were evaluated as good ridicule, primary roots and secondary roots well grow; hypocotyl well grow; plumule with good development, with leaves well grow and a healthy cotyledon. Abnormal seeds were evaluated as damaged primary root and absence of secondary roots and cotyledons and leaves deformed, necrotic, or damaged by infections.

Purple Corn Protein-Phenolic Concentrate (PCPC) from Germinated Seeds
Germinated and non-germinated Andean purple corn seeds were ground in a Perten 120 laboratory mill (Perten Instruments, Hägersten, Sweden) until a flour (<500 mm) was obtained. Purple corn flour defatted (10 g) was resuspended in 100 mL of Milli-Q water. The pH of the solution was adjusted to pH 8.0 and pH 10.0 with 2.0 M NaOH. The solution obtained was shaken for 60 min and centrifuged at 10,000× g for 60 min at 4 • C. The precipitate obtained was separated and discarded (fiber, ash, and carbohydrates). The pH of the supernatant was adjusted to pH 4.0, pH 5.0 and pH 6.0 with 1 NHCl. The supernatant solutions were centrifuged at 10,000× g for 30 min at 4 • C. The pH of the precipitate obtained was adjusted to pH 7.0 using a 0.5 M NaOH solution. PCPC samples produced were dried by a lyophilization technique and frozen and stored at −80 • C [40]. PCPC protein content was calculated by the Dumas method using a macro elemental analyzer (Elemental Vario Macro Cube, Langenselbold, Germany). The PCPC protein percentage was determined with the equation % of protein = 6.25 × % N, where 6.25 is the conversion factor and N is the percentage of nitrogen determined by the instrument [41].

PCPC Electrophoresis Analysis
PCPC proteins profile was characterized by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method using a mini-protean cell electrophoresis system (Bio-Rad, Hercules, CA, USA). The gels were prepared with a concentration of 12.00% polyacrylamide. The tinting of gels of polyacrylamide was made using a Coomassie Brilliant Blue G-250 solution for 24 h with shacked up. PCPC protein molecular weight was calculated with the help of Gel Documentation Imagen systems (Analytic Jena Tower, Jena Germany). A marker of standard with molecular weights of 10 kDa-200 kDa (Bio-Rad, Hercules, CA, USA) was used [42].

PCPC Quantification of Total Phenolic Content (TPC)
The samples for TPC analysis were extracted in all PCPCs samples. The TPC was extracted using methanol (70%) from lyophilized PCPC and stirred for 5 min, followed by an ultrasound technique for 10 min. The extracts were centrifuged, filtered, and calibrated. An aliquot of the solution was separated and mixed with distilled water, Folin Ciocalteau reagent, and with sodium carbonate (20%). The samples absorbance was measured at 765 nm. TPC quantification was made with a standard calibration curve of gallic acid (GA). The standard curve obtained was (y = 0.0021x + 0.0033, R 2 0.9982). TPC results were expressed as mg gallic acid equivalents GAE/g of PCPC, DW [43].

Assay of Antioxidant Activity by ABTS Method
Germinated PCPCs (200 µL) were mixed with 3800 µL of ABTS solution (composed of 7 mM ABTS solution with 2.45 mM potassium persulfate solution in a 1:1 ratio) and then diluted with phosphate buffer until obtaining an absorbance of 1.1 ± 0.01 a 743 nm. For the determination of the concentrations, a calibration curve was performed with the Trolox standard solution (200 µmol to 1000 µmol). The curve obtained was (y = 0.012x + 0.2089, R 2 = 0.9901). The data were expressed as µmol of trolox equivalents TE/g PCPC, DW [44].

Assay of Antioxidant Activity by the Ferric-Reducing Antioxidant Power (FRAP) Method
Germinated PCPCs (1 mL) were mixed with 2.5 mL of pH 6.6 buffer solution. 5 mL of 1% potassium ferrocyanide solution was added. The sample was incubated in a water bath at 50 • C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid solution, 2.5 mL of distilled water and 0.5 mL of 1% ferric chloride were added. Samples and standards were homogenized in a vortex and rested for 30 min in the dark. Finally, the absorbance of the solutions was measured at 700 nm.
For the determination of the concentrations, a calibration curve was performed with the Trolox standard (200 µmol to 1000 µmol). The curve obtained was (y = 0.0016x + 0.1324, R 2 = 0.9985). The data were expressed as µmol of trolox equivalents TE/g of PCPC, DW [45].

Statistical Analysis
Results were presented as mean ± standard deviation (n = 3). Statistical differences of the samples were evaluated with one-way ANOVA analysis (p < 0.05) followed by the Tukey test. The statistical differences were presented with a different letter.

Purple Corn Flour Proximal Analysis
Proximal analysis of purple corn flour showed that protein content was 8.58 ± 0.07%, fat content 5.73 ± 0.10%, ash content 0.02 ± 0.00%, moisture content 11.70 ± 0.05%, fiber content 2.91 ± 0.26%, and carbohydrates content 71.06 ± 0.16%. Their composition depends on the environmental conditions of the cultivars, temperature, variety, and type of seeds (yellow, white, black, blue, and purple seeds). Trehan et al. (2018) reported the chemical composition of three varieties of white, yellow, and purple corn flours. They reported a fat content for yellow corn flour (2.85-5.23%), white corn flour (2.03-4.95%), and purple corn flour (1.70-4.61%). They reported protein content of yellow, white, and purple corn flour (8.44-8.70%), (8.73-9.54%), and (9.53-9.88%), respectively [46]. Mansilla et al. (2020) reported different genotypes of purple corn cultivars from Argentina with protein contents between 9.48% to 11.50% and fat contents between 6.72% and 8.21% [47]. The results of protein content of purple corn flour reported in this study were lower than those reported by the other researchers, while the fat content was higher.

Germination (%) of Purple Corn Seeds
Andean purple corn (100 seeds) were put in the germination process in the incubator for 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h at 15 • C, 20 • C, 25 • C, 30 • C, 35 • C, and 40 • C. Table 1 showed the germination percentage obtained at different conditions. Germination percentages were obtained with values between 0.00 ± 0.00 and 63.33 ± 7.23. The highest germination percentages were obtained at the temperature of 25 • C with germination values between 42.00% and 63.33%.
The seeds incubated at 15 • C for 24h to 96 h did not showed germination. At 120 h, they presented a 6.00 ± 0.58% of germination. These values increased with a germination time of 168 h, presenting the highest percentage with a value of 63.33%. The increase in % of germination was proportional to the increase in the incubation time of the seeds to germinate. As the germination temperature increases, the germination percentage decreases. The germinated rates obtained at 40 • C varied between 9.33% and 26.00%. These were the lowest values of germination obtained.
The results obtained present significant differences at p < 0.05 when compared to incubation temperature with time of incubation of germination of the Andean purple corn seeds. Govender, Aveling, and Kritzinger (2007) reported percentages of germination of yellow and white varieties from northern KwaZulu-Natal and southern Mozambique with values of 18.70 to 100% depending on the variety. Germination process was made at 25 • C for 7-11 days [8]. Figure 1 showed the registered pictures of the Andean purple corn germinated obtained at different temperatures and different germination times. Figure 1a

PCPCs Protein Profile
Once germinated, the Andean purple corn seeds were used to obtain flour. The flour was used to obtain protein-phenolic concentrates by alkaline extraction (pH 8.0 and pH 10.0) followed by  Results were expressed as mean ± standard deviation (n = 3) and were evaluated by one-way Anova and Turkey test (p < 0.05). Statistical differences were indicated with different letters. Temperatures groups were compared with times groups.

PCPCs Protein Profile
Once germinated, the Andean purple corn seeds were used to obtain flour. The flour was used to obtain protein-phenolic concentrates by alkaline extraction (pH 8.0 and pH 10.0) followed by isoelectric precipitation (pH 4.0, pH 5.0, and pH 6.0). Once the PCPCs were obtained, their protein profile was analyzed using the SDS-APGE technique. Figure 2 showed the PCPC protein profile. Figure 2a

PCPCs Protein Content Quantification
The protein content present in PCPCs obtained from germinated seeds at 72 h, 120 h, and 168 h incubated at 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C was quantified using the Dumas method. Table 2 summarizes the results obtained from the protein quantification analysis. Protein concentration in PCPCs ranges between 9.38% (8.0-6.0 pH (alkaline-Ip)) to 33.56% (10.0-4.0 pH (alkaline-Ip)) these values correspond to PCPC germinated at 25 °C. The highest value was obtained after 168h of incubation. The PCPCs obtained at the two extraction pHs (pH 8

PCPCs Protein Content Quantification
The protein content present in PCPCs obtained from germinated seeds at 72 h, 120 h, and 168 h incubated at 15 • C, 20 • C, 25 • C, 30 • C, 35 • C, and 40 • C was quantified using the Dumas method. Table 2 summarizes the results obtained from the protein quantification analysis. Protein concentration in PCPCs ranges between 9.38% (8.0-6.0 pH (alkaline-Ip)) to 33.56% (10.0-4.0 pH (alkaline-Ip)) these values correspond to PCPC germinated at 25 • C. The highest value was obtained after 168h of incubation. The PCPCs obtained at the two extraction pHs (pH 8.0 and pH 10.0) present similar protein percentage values. Small differences were observed in PCPCs from sprouts 72 h/25 • C, 120 h/20 • C, and 120 h/25 • C. The highest values obtained were PCPC 168 h/25 • C with values of 29.06%, 30.00%, and 33.56%; PCPC 168 h/30 • C presented values of 23.75%, 27.53%, and 29.38%; PCPC 168 h/35 • C presented percentages of 23.80%, 27.60%, and 29.40%. All PCPCs obtained from germinated corn seeds at different times and different temperatures presented protein percentage values higher than the percentage of corn flour from non-germinated corn seeds (8.53%). Statistical analysis indicates significant differences when the time of germination was compared to the pHs alkaline and the isoelectric precipitation at p < 0.05. Corn seed has albumins protein that represent 8.00% of protein, DW. Albumins proteins are soluble in a water solution. Globulins protein represent 9.00% of protein soluble in salts and glutelin proteins represent 40.00% of protein, DW, which are soluble in alkaline solutions [51]. PCPC presented albumins, glutelin and globulin proteins. Normal purple corn seeds were used in this study to get germinated PCPCs. The protein contents reported in this work are in line with values reported in the literature for different zein protein concentrates. Corn protein concentrate are used for industrial applications such as adhesives, biodegradables plastics, coating (edible, moisture-resistant) for food products, cosmetic powders, dietary fibers, textile fibers, microencapsulated pesticides, microspheres, long acting matrix tablet formulations, nutrient delivery system for ruminants, high potency sweeteners, hair fixative, and other industrial purposes [52][53][54][55][56][57][58][59][60][61][62].  [64]. PCPC concentrates from germinated kernels increased the TPC content when compared to the TPC content reported for purple corn flour.

TPC Content of Germinated PCPCs
The isoelectric precipitation pH (pH 4.0, pH 5.0, and pH 6.0) used to precipitate the proteins allows the isolation of a considerable amount of phenolic components. PCPCs continue to maintain a purple coloration which indicated their presence in protein concentrates. Total phenolic can be separated from PCPCs with repeated methanol extractions. Six consecutive extraction processes allowed the isolation of 100.00% of the phenolic components present in the PCPCs from germinated seeds. All PCPCs tested in this work with alkaline extraction and isoelectric precipitation had a high TPC content. Future work on HPLC-MS-MS should be carried out to identify the phenolic components present in the PCPCs and to determine whether there are differences in the phenolic depending on the pH extraction and the pH precipitation.   In the light of the above discussion, we consider that in the protein concentrates obtained from plants, the presence of phenolic in the samples and their possible influence on the biological activities that are evaluated must be considered. For this reason, we decided to name them purple corn protein phenolic concentrates (PCPC).

Germinated PCPCs Antioxidant Activity by ABTS and FRAP Methods
Germinated PCPCs obtained at pH 10.0 of alkaline extraction and pH 4.0 of precipitation, 48-168 h and 20-40 • C incubation temperatures were used to evaluate the antioxidant activity by ABTS and FRAP methods. Table 4 25 µmol TE/g PCPC, DW. In general, higher antioxidant activities were observed for similar behavior at high temperatures and high germination times presenting higher antioxidant activity. Statistical analysis showed significant differences when the groups of temperatures were compared to the incubation times at p < 0.05. Coco and Vinson (2019) reported antioxidant activity of raw kernel 8.73-13.40 mg of catechin/g for nine varieties of corn by the FRAP method. Raw kernel was subjected to in vitro simulation digestion. The antioxidant activity was evaluated by the FRAP method. They found a value of 0.76 mg catechin/g of sample [65]. Yang and Zhai (2010) reported antioxidant activity of total anthocyanins content (TAC) isolated from purple corn kernels using the DPPH, FRAP, and TEAC methods (16.20 mmol and 18.70 mmol of FeSO 4 /g DW). Results are expressed as mean ± standard deviation (n = 3) and were evaluated by one-way Anova and Turkey test (p < 0.05). Statistical differences were indicated with different letters.
Various researchers have reported a relationship between polyphenol and anthocyanin content of purple corn kernels and their antioxidant activities [4]. López-Martinez et al. (2014) described antioxidant activity of three varieties of purple corn using nitric oxide radical scavenging activity and superoxide radical scavenging activity methods. The ethanol extracts showed a strong antioxidant activity in the following order: generic purple > Oaxaca 332 > Veracruz 42 with 62.00%, 48.00%, and 32.00% of NO scavenging activity, respectively [66].
The protocol used in this study to obtain antioxidant PCPC was validated from a lot of seeds of Andean purple corn of the variety (Zea mays L., INIAP-199 bunch of grapes) grown in Guaranda, Ecuador. The proteins and polyphenols content present in seeds can vary with the variety tested and the seeds growing conditions. For this reason, this protocol could be validated with different varieties of corn seeds (purple, white, and yellow corn) grown under different environmental conditions, different irrigation conditions, and different fertilization conditions. The protocol could also be validated on transgenic corn seeds to compare the results.

Conclusions
Andean purple seeds can be germinated in a wide range of temperatures (15-40 • C) and times (24-168 h). Alkaline extraction (pH 8.0 and pH 10) followed by the isoelectric precipitation (pH 4.0, pH 5.0, and pH 6.0) is a good method to obtain protein phenolic corn concentrate (PCPC). A higher concentration of protein and TPC can be obtained in PCPC compared with non-germinated corn kernel flours. The germination process of Andean purple corn kernels can be used to obtain PCPC with biological activities, such as antioxidant activity, that can increase the nutritional value and quality of this food product. The germinated purple corn PCPCs have a high content of phenolic components that are responsible for the antioxidant activity. The protocol used to obtain antioxidant PCPCs in this study is a preliminary protocol that must be tested in different varieties of corn seeds grown under different environmental conditions. This protocol needs to consider the influence of the dormancy stages of the seeds.