Preparation of corn (Zea mays) and sorghum (Sorghum bicolor) proteins and protein hydrolysates and their antihypertensive activities Luis Fernando Núñez Cartagena Escuela Agrícola Panamericana, Zamorano Honduras November, 2019 i ZAMORANO FOOD SCIENCE AND TECHNOLOGY MAJOR Preparation of corn (Zea mays) and sorghum (Sorghum bicolor) proteins and protein hydrolysates and their antihypertensive activities Special graduation project presented as partial requirement to obtain the Food Science and Technology Bachelor Degree. Presented by Luis Fernando Núñez Cartagena Zamorano, Honduras November, 2019 iii Preparation of corn (Zea mays) and sorghum (Sorghum bicolor) proteins and protein hydrolysates and their antihypertensive activities Luis Fernando Núñez Cartagena Abstract. The most important group of protein in cereals is prolamine, storage protein in cereal grains that contain biopeptides, which can prevent non-transmissible diseases such as hypertension. The objective of the present study was to determine the composition of sorghum, white and yellow corn proteins, as well as to prepare protein hydrolysates in order to evaluate their antihypertensive activities. Protein extraction using the alkaline method in combination with combined ultrasound reached 65.7, 63.5 and 48.1% for white corn, yellow corn and white sorghum, respectively. A comparison was made between corn and sorghum proteins; it was found that there are no significant differences between treatments (P > 0.05), because with the concentration of 1% of protein incubated for 3 hours reached approximately 24% of degree of hydrolysis. However, the hydrolysis time obtained a significant difference (P < 0.05) until reaching a saturation point at 60 minutes and 90 minutes for sorghum and corn, respectively. Corn and sorghum proteins, and their hydrolysates at the point of saturation, were subjected to the angiotensin-converting enzyme inhibition test. The best treatments obtained results of 45.4 and 43.8%, for yellow corn and white corn, respectively. The least effective treatment was white sorghum with 30.5%. It is recommended to use purification and separation methods to differentiate the biopeptides released during hydrolysis. Key words: Alcalase, angiotensin converting enzyme, inhibitors, peptides. Resumen. El grupo de proteínas de mayor importancia en cereales es la prolamina, proteína de almacenamiento en los cereales, en ella se encuentran los biopeptidos que pueden prevenir enfermedades no transmisibles como la hipertensión. El objetivo del presente estudio fue determinar la composición de proteínas de sorgo, maíz blanco y amarillo, así como preparar hidrolizados de proteínas con el fin de evaluar sus actividades antihipertensivas. La extracción de proteína mediante el método alcalino en combinación con ultrasonido alcanzó el 65.7, 63.5 y 48.1% para maíz blanco, maíz amarrillo y sorgo blanco, respectivamente. Se realizó una comparación entre proteínas de maíz y sorgo; se encontró que no hay diferencia significativa entre los tratamientos (P > 0.05), debido a que con la concentración del 1% de proteína incubada por 3 horas alcanzó aproximadamente 24% de grado de hidrolisis. Sin embargo, el tiempo de hidrolisis obtuvo una diferencia significativa (P < 0.05) hasta alcanzar un punto de saturación a 60 minutos y 90 minutos para el sorgo y maíz, respectivamente. Las proteínas de maíz y sorgo, y sus hidrolizados en el punto de saturación fueron sometidos a la prueba de inhibición de la enzima convertidora de angiotensina. Los mejores tratamientos obtuvieron resultados de 45.4 y 43.8%, para el maíz amarrillo y maíz blanco, respectivamente. El tratamiento menos efectivo fue el sorgo blanco con 30.5%. Se recomienda usar métodos de purificación y separación para diferenciar los biopeptidos liberados durante la hidrólisis. Palabras clave: Alcalasa, enzima convertidora de angiotensina, inhibidores, péptidos. iv TABLE OF CONTENTS Cover page ............................................................................................................. i Signature Page ....................................................................................................... ii Abstract ................................................................................................................. iii Table of Contents .................................................................................................. iv List of Tables and Appendices .............................................................................. v 1. INTRODUCTION .............................................................................................. 1 2. MATERIALS AND METHODS ....................................................................... 3 3. RESULTS AND DISCUSSION ......................................................................... 7 4. CONCLUSIONS ................................................................................................. 12 5. RECOMMENDATIONS ................................................................................... 13 6. REFERENCES ................................................................................................... 14 7. APPENDICES..................................................................................................... 18 v LIST OF TABLES AND APPENDICES Tables Page 1. Degree of hydrolysis depending the treatment and time. ......................................... 5 2. ACE inhibitory activities of the treatments in the higher degree of hydrolysis ....... 6 3. Moisture, lipid and protein contents of corns and sorghum flours. .......................... 7 4. Comparison between protein content in flour and protein extraction ...................... 8 5. Degree of Hydrolysis (%) of different corns and sorghum ...................................... 9 6. Angiotensin-converting enzyme (ACE) Inhibitory activities of protein and protein hydrolysates from corns and sorghum ...................................................................... 10 Appendices Page 1. Degree of hydrolysis of yellow corn, white corn and sorghum protein hydrolysate at different times.. ..................................................................................................... 18 2. Angiotensin-converting enzyme Inhibitory activities of protein and protein hydro- lysates from corns and sorghum .............................................................................. 18 1 1. INTRODUCTION Cereals are the dominant crops in world agriculture, with a total of 2,702 million tons being harvested globally in 2017, comprising 1,031 million tons of maize, 759 million tons of rice, 744 million tons of wheat and 60 million tons of sorghum (FAO 2017). They are the major source of calories and protein to the diets of humans and livestock. The reasons for the success of cereals include their adaptability, high yields, and ease of harvest and storage (Lafiandra et al. 2014). The protein of corn has some specific characteristics, such as most abundant amino acids are Glutamic acid, Leucine, Proline, Alanine, Phenylalanine, and Aspartic acid. However, its properties of low water solubility and deficiency in essential amino acids such as Lysine, Histidine, and Tryptophan considerably limit its nutritional quality and the direct application in food ingredient (Zhu et al. 2018). On the other hand, the digestibility of sorghum proteins, especially after cooking, is lower than cereals like wheat and maize. The kafirins of sorghum are resistant to peptidase due to the formation of intramolecular disulfide bonds; this is why it has low digestibility. In varieties rich in tannins, the complexation of the kafirins with phenolic compounds can reduce the protein digestibility up to 50% (De Morais et al. 2015). Since the emergence of Gluten Related Disorders (GRD) resulting from the ingestion of gluten, a mixture of prolamin proteins found mostly in wheat, gluten-free (GF) goods have become more and more popular (Hadjivassiliou et al. 2016; Pellegrini and Agostoni 2015; Balakireva and Zamyatnin 2016). As gluten-free crops, sorghum and corn have gained more interest to be used in gluten-free food products (Ferreira et al. 2016; Emendack et al. 2018). The corn and sorghum proteins are also excellent source of bioactive peptides that have health effects in chronic disease prevention, particularly in regard to diabetes, cardiovascular disease, and cancer (Cavazos and Gonzalez 2013; Zhu et al. 2018; De Morais et al. 2015). The method to extract the protein for the study was alkaline at pH 11, as described previously by De Mesa et al. (2010) with modifications in addition of sonication. The enzyme to hydrolyze, extrated protein for corns and sorghum was Alcalase. The main precursor of the biopeptides is the enzymatic protein hydrolysis consisting of the cleavage of protein molecules into small peptides of various sizes, and eventually amino acids (Nielsen et al. 2001). Alcalase was shown to produce corn gluten meal (CGM) hydrolysates with higher degree of hydrolysis (DH) and higher ACE inhibitory activity 2 compared to other enzymes (Yang et al. 2007). Although peptide fractions with lower molecular sizes were shown to have higher antihypertensive effects (Vasudeva et al. 2006; Yang et al. 2007; Kim et al. 2001). Alcalase has been used for hydrolysis of Sorghum protein, 19% of DH present the higher ACE inhibitory activity with Thr-Lue-Ser (Wu et al. 2015). Hypertension is the single most important risk factor for stroke, the top 2 global cause of death in 2016 (World Heart Federation 2017; WHO 2018). About 75 million American adults (29%) have high blood pressure – one in every three American adults (CDC 2016). The racial disparity in hypertension and hypertension-related outcomes has been recognized for decades with black race with greater risks than Caucasians (Lackland 2015). Angiotensin-converting enzyme (ACE) cleaves at carboxyl terminus of His-Leu of angiotensin I resulting in the producing of angiotensin II, a potent vasopressor, and releases Phe-Arg and Ser-Pro from the carboxyl terminus of bradykinin, a vasodepressor. ACE causes hypertension by interrupting the balance between rennin-angiotensin-aldosterone system (RAS) and kallikrein kinin system (KKS). Thus, inhibition of ACE presents important pharmacological and nutraceutical values in blood pressure control and the prevention of hypertension related complications (Soffer 1976; Natesh et al. 2003; Wang et al. 2015). To overcome the side effects of chemically synthesized drugs, a lot of efforts have been made to discover and apply the natural compounds that are capable of controlling blood pressure (De Leo et al. 2009). Some bioactive peptides (BP) exhibiting ACE inhibitory activities have been isolated from food protein hydrolysates, such as Proline-Serine- Glycine-Glntamine-Tyrosine-Tyrosine (Suh et al. 1999), Alanine-Tyrosine (Yang et al. 2007), and Methionine-Isoleucine/Leucine-Proline-Proline (Wang et al. 2015) from corn, and Threonine-Leucine-Serine (Wu et al. 2015) from sorghum. It was found that dipeptides and tripeptides were able to remain higher potency of bioactivity after adsorption intact through the gastrointestinal tracts than larger peptides (De Leo et al. 2009). The objectives of this study were:  To prepare corn and sorghum meals.  To prepare protein hydrolysates using Alcalase enzyme.  To evaluate proteins and protein hydrolysates for their antihypertensive activities. 3 2. MATERIALS AND METHODS Protein content (AOAC 920.87). Samples were digested using 500 mg of sample, ½ of catalyst tablet (Selenium reaction mixture) and 5 ml of sulfuric acid. In the digestion tubes, the samples were heated for 1.5 hours and shaked for 30 minutes. After this, the digested samples were cooled to room temperature. Kjeldahl method was used to determinate total protein using N×6.25 as conversion factor. Moisture content (AOAC 925.10). Three (3) grams of sample was weighed and heated for five hours 105℃ The dehydrated sample was weighed after cooling down to room temperature. The amount of moisture was determined as the difference between the three grams of sample and the dehydrated sample. Lipid content (AACC 30-25). Five (5) grams of sample was weighed. The sample was transferred to the extractor and extracted with petroleum ether for 5 hours at a condensation rate of 5 to 6 drops/seconds. The petroleum ether was removed from collection flask. The deffated samples were weighed. The amount of fat is the different between the sample weight and the weight from the deffated sample. Preparation of corn and sorghum protein (CP and SP). The corn and sorghum flours were passed through 60 MESH to obtain uniform particle sizes, and defatted using flour: hexane (1:4 ratio) at ambient temperature (25 ℃). The flours were dispersed in deionized (DI) water (1:9 ratio), the pH was adjusted to 11.0 with sodium hydroxide (NaOH 1N) and sonicated for 30 min to disrupt the cell walls. To prevent the overheating of the sample, the beaker containing the sample was placed in an ice and cold- water bath during sonication. Dispersions were stirred for 3 hours followed by a centrifugation at 10,000 g for 30 min at 20 ℃ using a centrifuge (model J2-21, Beckman, Fullerton, Calif., U.S.A.) to extract the protein. The residue was re-extracted two more times to obtain the residual protein. The supernatants were combined and the pH adjusted to 4.5 (isoelectric pH) with hydrochloridic acid (HCl 1N), to isoelectrically precipitate the protein, and then centrifuged at 10,000 g for 20 min at 4 ℃ to obtain the protein isolate. After this, the precipitate was reconstituted in water adjusted to pH 7.0, freeze-dried, and stored at 5 ℃. 4 Preparation of corns (CPH) and sorghum protein hydrolysates (SPH) by Alcalase. Corn protein (CP) or sorghum protein (SP) were prepared in a dispersion (1% in DI water) adjusted to pH 7.0. Based on previous studies, Alcalase (0.5 AU/g protein) was used to obtain CPHs and SPHs. After adding the Alcalase, the samples were incubated at 60 ℃ at varying times (0, 30, 60, 90, 120 and 180 min) in a shaking water bath at 120 rpm (Grant Model OLS200, Cambridge, England). Alcalase was inactivated at 85 ℃ for 5 min and cooled at ambient temperature, then stored at 5 ℃. Degree of hydrolysis of CPH and SPH. o-phthaldialdehyde (OPA) method as described by Nielsen et al. (2001) was used to determine the degree of hydrolysis (DH) with slight modifications. The OPA reagent solution was prepared with 250 mg of sodium-dodecyl sulfate (SDS) and 9.525 g disodium tetraborate decahydrate in 155 ml water; 200 mg OPA in 5 ml ethanol; both solutions were mixed, added with 220 mg dithiothreitol and stirred to a clear solution. A volumetric flask was used to complete 250 ml with DI water. Samples were diluted 5 times to do the spectrophotometric readings. 400 µl of the diluted hydrolysate solutions were added into the OPA reagent (3 ml) and mixed. Absorbance at 340 nm (UV-1601 spectrophotometer, Shimadzu, Kyoto, Japan) was taken exactly 2 min after the addition of the OPA reagent. Blank and standard solutions were prepared using 400 µl of DI water and a serine solution (0.01%), respectively. DH was calculated using the following equations: (Serine-NH2)= SampleOD-BlankOD StandardOD -BlankOD × (0.9516 × V × 100) (X × P) [1] Where, V is sample volume (in liter); X is sample weight (in gram); P is protein content (in %) of the sample, and Serine–NH2 is in meq Serine-NH2/g protein. h= (Serine-NH2)- β α [2] Where,  and  values are estimated to be 1.00 and 0.40, respectively, and h is the number of hydrolyzed bonds. The h total values are estimated to be 9.2 and 7.9 for corn and sorghum, respectively. DH= h htot × 100 [3] Determination of ACE-I inhibitory activity. Inhibitory antihypertensive activity was a modified method of Horax et al. (2017). The mixture of 50 µl CPH or SPH [10 mg hydrolysate/ml in 0.1 M phosphate buffer (PB) (pH 8.3)], 50 µl ACE-I solution [125 mU/ml in 0.1 M PB (pH 8.3)], and 150 µl hippuryl-L- histidyl-L-leucine (HHL) solution [12.5 mM of HHL in PB (pH 8.3) containing 0.5 M 5 NaCl] were incubated at 37 ℃ for 1 hour. 250 µl of 1 N HCL were used to stop the reaction. Negative control was used with the following conditions: 1 N of phosphate buffer was added before the addition of the enzyme. The hippuric acid (HA) liberated from HHL by ACE-I activity was extracted with 1.0 ml of ethyl acetate and centrifuged. The solution of ethyl acetate extract (0.7 ml) was evaporated to drynesss at 90 ℃ in a water bath, then to dissolve the residue, 1 ml of DI water was added. The amount of HA liberated was measured spectrophotometrically at 228 nm. The percentage (%) of inhibition was calculated as follows: [1- ( A – B C – B )] ×100 [4] Where A is absorbance in the presence of hydrolysate; B is absorbance of negative control; C is absorbance in the absence of hydrolysate. Statistical analysis. A statistical analysis was performed using the program SAS, Version 9.4® to find if there were significant differences between corns and sorghum proteins, as well as ACE inhibitory activities. Experimental design. A completely randomized design (CRD) was used, with a factorial arrangement 3 × 7, as shown in Table 1 and 3 × 2, as shown in Table 2 for degree of hydrolysis and ACE-I inhibitory activity, respectively. The separation of means was performed using Duncan and LSMEANS. Table 1. Degree of hydrolysis depending the treatment and time. Time (min) Yellow Corn White Corn White Sorghum Degree of hydrolysis (%) 0 TRT1 TRT8 TRT15 30 TRT2 TRT9 TRT16 60 TRT3 TRT10 TRT17 90 TRT4 TRT11 TRT18 120 TRT5 TRT12 TRT19 150 TRT6 TRT13 TRT20 180 TRT7 TRT14 TRT21 6 Table 2. Angiotensin-converting enzyme (ACE) inhibitory activities of the treatments in the higher degree of hydrolysis. Treatments Time (min) DH ACE inhibitory rate Yellow Corn 0 TRT1 TRT1 Yellow Corn 90 TRT4 TRT4 White Corn 0 TRT8 TRT8 White Corn 90 TRT11 TRT11 White Sorghum 0 TRT15 TRT15 White Sorghum 60 TRT17 TRT17 Location of the study. The analyzes of antihypertensive activities of corns and sorghum were done in a laboratory of protein functionality in the University of Arkansas, USA. 7 3. RESULTS AND DISCUSSION Proximal composition of flours. Table 3 shows that white sorghum had the highest amount of protein with 9.8% in comparison to corns that had 5.7 - 6.0%. As previously shown in others researched grains, protein content range is between 7 - 18% and 6 - 12% for sorghum and corn, respectively, depending on many factors such as the variety of the crop, variation in cultivation practices (e.g. fertilization), as well as environmental conditions (Bean et al. 2011; Williams 1981). For moisture, the highest was obtained for is the white sorghum with 12.3%, but still lower than 15% of moisture as established by the FAO (Codex Alimentarius 2019). In the other hand, lipids in corns were higher with 6.4 and 6.6% and sorghum 5.5%; similar results were reported in previous study (Gwirtz & Garcia 2013; Stefoska 2015). Table 3. Moisture, lipid and protein contents of corns and sorghum flours.1 Flour Moisture Content (%) Lipid content (%) Protein content (%) Yellow Corn 11.7 ± 0.4b 6.4 ± 0.1a 5.7 ± 0.8b White Corn 11.0 ± 0.1b 6.6 ± 0.4a 6.0 ± 0.9b White Sorghum 12.3 ± 0.3a 5.5 ± 0.3b 9.8 ± 0.3a CV (%) 1.26 5.12 8.45 1Values are presented as means ± standard deviation of three replication analysis. Protein content was determined on dry basis. a-b: Different letters for each column represent statistical differences among treatments (P < 0.05). CV: Coefficient of variation. Preparation of corn and sorghum protein (CP and SP). In Table 4, it is shown that about 63 - 65% of protein was obtained from both corns and these results are consistent with previous research of corn protein extraction, which reported extractions ranging 60 - 71%. On the other hand, the lower protein recovery was obtained for white sorghum with 48%. Tapia-Hernández et al. (2019) reported similar results 35.33 - 46.51% of protein recovery in sorghum. Higher amounts of protein, in the range of 66%. The proteins in corn and sorghum are soluble in selected solvents due to their types of proteins, such as: albumins (water), globulins (salt) and glutelin (alkali), zein and kafirin (alcohol). Zein and kafirins are hydrophobic storage proteins in grains that form 45 - 50% 8 and 50 - 60% of the total protein, respectively. Also, prolamins are soluble in pH 11 or above (Shukla and Cheryan 2001; Kamath et al. 2007). Kafirin presents the most hydrophobic trend of the prolamins (Sun et al. 2016). That is why the protein extraction is lower than corns. Table 4. Comparison between protein content in flour and protein extraction.1 Flour Protein content (%) Extraction Protein content (%) White Sorghum 9.8 ± 0.3aX WSP2 48.1 ± 3.4bY White Corn 6.0 ± 0.9bX WCP3 65.7 ± 5.3aY Yellow Corn 5.7 ± 0.8bX YCP4 63.5 ± 4.7aY P value <0.0001 C.V. (%) 10.14 R2 0.991 1Values are presented as means ± standard deviation of three replication analysis. Protein content was determined on dry basis. 2White sorghum protein (WSP), 3white corn protein (WCP) and 4yellow corn protein (WCP). a-c: Different letters in the same column represent statistical differences among treatments (P < 0.05). X-Y: Different letters in the same row represent statistical differences within the same treatment (P < 0.05). CV: Coefficient of variation. In plants, the cell wall consists mainly of cellulose microfibrils embedded in a matrix of hemicelluloses, pectins, proteins and phenolics (Santiago et al. 2013). Proteins represent 10% of the extracellular matrix mass (Komatsu and Yanagawa 2012). The main purpose of ultrasound is to extract the protein in the cell wall. The mechanism of sonication involves high power of ultrasound to cause disruption of cell walls (Tang et al. 2002) and the breaking of covalent bonds (Singh et al. 1990). However, disruption of cell walls has a limited effect in protein extraction. Degree of hydrolysis of CPH and SPH. The proteins previous extracted were subjected to hydrolysys, the process was conducted using a Alcalase enzyme concentration of 0.5 AU/g of protein. Degree of hydrolysis (DH) is the percentage of the number of peptide bonds broken among the total number of peptide bonds in the substrate during protein hydrolysis (Wu et al. 2015). Previous studies reported that ultrasonic bubble cavitation causes crushing, which results in large mechanical shearing forces, which degrade protein structure and open up hydrophilic groups. This opening of structure increases the protein solubility and allows the protease to bind more easily with the protein substrate, which increases the efficiency of hydrolysis (Kadam et al. 2015). As observed in Table 5, the DH increased as the time of incubation was increased, reaching a plateau at 90 min for both white (WCPH) and yellow corn protein hydrolysates (YCPH), 9 and 60 min for white sorghum protein hydrolysate (WSPH). All the treatments presented similar degree of hydrolysis, around 24%. There was significant difference between times (P < 0.05). Table 5. Degree of Hydrolysis (%) of different corns and sorghum.1 Time (min) Yellow Corn White Corn White Sorghum Degree of hydrolysis (%) 0 5.68 ± 0.2dY 5.51 ± 0.4dY 6.60 ± 0.4dX 30 21.55 ± 0.5bX 21.10 ± 0.9bX 22.05 ± 0.9bX 60 22.41 ± 0.7bcY 22.54 ± 1.4bcY 24.51 ± 1.1aX 90 24.07 ± 0.5aX 24.85 ± 0.4aX 24.01 ± 1.1aX 120 23.28 ± 0.7abX 24.17 ± 0.7abX 23.77 ± 0.7aX 150 23.92 ± 1.1aX 23.85 ± 0.8abX 23.80 ± 0.8aX 180 23.60 ± 0.6aX 24.21 ± 0.7abX 24.50 ± 0.6aX P value <0.0001 C.V. (%) 4.31 R2 0.997 1Values are presented as means ± standard deviation of three replication analysis. a-d: Different letters in the same column represent statistical differences among treatments (P < 0.05). X-Y: Different letters in the same row represent statistical differences within treatments (P < 0.05). CV: Coefficient of variation. As shown in table 5, the DH rapidly increased in the first 30 min and then showed a slow increase from 30 to 60 min, or 90 min depending on the treatment. The reduction of the speed in the degree of hydrolysis it could be related with a reduction of substrate in the solution. The analysis of the degree of hydrolysis for yellow corn (YCPH), white corn (WCPH) and sorghum (WSPH). No significant differences were found (P > 0.05) for any of the treatments. WSPH showed similar results to WCPH and YCPH corns, but the difference in the time was because the sorghum protein has less peptide bonds, as previously discussed. These results are consistent with previous studies for sorghum and corn, where it was reported that the concentration of protein was hydrolyzed and this is why the DH did not increase after 90 min (Yang et al. 2007; Wu et al. 2015). Determination of ACE-I inhibitory activity. The samples with the highest DH (the time point where the DH just reached the plateau) and the lowest DH were subjected to the evaluation of ACE inhibitory activities. The highest inhibitory activities were 45.4, 43.5 and 30.5% for YCPH, WCPH, and WSPH, 10 respectively. These results suggest that extensive hydrolysis may release many low- molecular weight peptides and results in high ACE inhibition. This seems to be consistent with previous reported results (Bao et al. 2017; Aluko 2018). These inhibitory activities were obtained with the highest DH for each treatment. When the DH was low, the ACE inhibition rate was also lowered to 5.68, 5.51, and 6.60% for yellow corn, white corn, and white sorghum shown as controls, respectively. As expected, lower degree of hydrolysis had less inhibitory activity, due the limited release of low- molecular weight peptides. (Table 6) Table 6. Angiotensin-converting enzyme (ACE) inhibitory activities of protein and protein hydrolysates from corns and sorghum.1 Treatments 2Initial time 3Final time Degree of Hydrolysis (%) YCPH4 5.68 ± 0.2aY 24.07 ± 0.7aX WCPH5 5.51 ± 0.4aY 24.85 ± 0.4aX WSPH6 6.60 ± 0.4aY 24.51 ± 1.1aX ACE inhibitory rate (%) YCPH 5.03 ± 0.4aY 45.43 ± 1.9aX WCPH 5.27 ± 0.3aY 43.82 ± 1.7aX WSPH 5.95 ± 0.3aY 30.51 ± 2.3bX P value <0.0001 C.V. (%) 3.82 R2 0.998253 1Values are presented as means ± standard deviation of three replication analysis. 2Inicial time is 0 min for YCPH, WCPH and WSPH. 3Final time is 90 min for YCPH and WCPH and 60 min for WSPH. 4Yellow corn protein hydrolysate (YCPH), 5white corn protein hydrolysate (WCP) and 6White sorghum protein hydrolysate (WSPH). a-b: Different letters in the same column represent statistical differences among treatments (P < 0.05). X-Y: Different letters in the same row represent statistical differences within treatments (P < 0.05). These results were obtained using the same concentration of hydrolysate protein at 10 mg/ml. CV: Coefficient of variation. Lower reduction of ACE activity in this study could be related with the low protein extraction rate (De Mesa 2010; Shukla and Cheryan 2001). Several studies reported that bioactives fragments are located in the prolamins, in this specific case, zein and kafirin (Kamath et al. 2007; Zhou et al. 2012; Yano et al. 1996). As previously shown, in other studies it was reported that hydrolyzed protein from corn (CPH) and sorghum (SPH) with a DH between 17 - 19% exhibited higher ACE inhibitory activity (Kamath et al. 2007; Yang et al. 2007; Wu et al. 2015). As previous reported for Kamath et al. 2007 and Yang et al 2007 ACE rate of inhibition is 7 - 46% and 13 - 83% for sorghum and corn, respectively. These inhibition rate values could be beneficial to the state of health of hypertensive individuals. Huang et al. (2011) achieved a reduction of 26.57 11 mmHg in systolic blood pressure to hypertensive rats in four weeks. The reduction in ACE activity could be due to the biopeptides reported in previous studies. For example, hexapeptide [Pro-Ser-Gly-Gln-Tyr-Tyr] and a dipeptide [Ala-Tyr] obtained from corn protein, and from sorghum the tripeptide [Thr–Leu–Ser] (Huang et al. 2011; Wu et al. 2015). Kamath et al. (2007), reported that peptides obtained by enzymatic hydrolysis with Alcalase inhibited the (ACE) enzyme activity by competitively binding with the substrate for the active sites and uncompetitive inhibitors. Also, it was observed that antihypertensive peptides from zein (proline containing) were not susceptible to proteolysis by enzymes of the digestive tract such as chymotrypsin, trypsin or pepsin. The previous author used the same methodology and enzyme so is expected that results obtained was similarities whit the results of this study. 12 4. CONCLUSIONS  The highest amount of protein extract was obtained from white corn and yellow corn, while the least amount of protein extracted was obtained from white sorghum.  The degree of hydrolysis increased during incubation time with Alcalase reaching a plateau after 60 min for sorghum and 90 min for yellow and white corn.  Treatments with a higher degree of hydrolysis showed higher ACE inhibitory activities. Yellow and white corn exhibited higher inhibitory efficiency than white sorghum. 13 5. RECOMMENDATIONS  Evaluate another method of protein extraction in order to reach higher protein concentrations.  Use a method of purification and fractionation to differentiate the biopeptides that were released. 14 6. REFERENCES AACC, American Association for Cereal Chemistry. 2000. Crude Fat in Wheat, Corn, and Soy Flour, Feeds, and Mixed Feeds. AACC Method 30-25.01. ISBN 978-1-891127- 68-2 Aluko RE. 2018. Food protein-derived peptides: Production, isolation, and purification. In: Proteins in Food Processing. 2 ed. Woodhead Publishing. p. 389–412. doi:10.1016/b978-0-08-100722-8.00016-4 AOAC, Association of Official Agricultural Chemist. 2011. Official methods analysis. Method AOAC 920.87 and 925.10. Ed. Washington D.C, United Stated. Balakireva A, Zamyatnin A. 2016. Properties of gluten intolerance: Gluten structure, evolution, pathogenicity and detoxification capabilities. Nutrients. 8(10):644. doi: 10.3390/nu8100644. Bao Z, Zhao Y, Wang X, and Chi YJ. 2017. Effects of degree of hydrolysis (DH) on the functional properties of egg yolk hydrolysate with alcalase. Journal of Food Science and Technology. 54(3):669–678. doi:10.1007/s13197-017-2504-0. Bean S, Loerger B, Smith B, and Blackwell D. 2011. Sorghum protein structure and chemistry: implications for nutrition and functionality. American Chemical Society. 7:131–147. doi:10.1021/bk-2011-1089.ch007 Codex Alimentarius. 2019. Standard for Durum wheat semolina and Durum wheat flour. International food standards. CXS 178-1991. Cavazos A and Gonzalez E. 2013. Identification of bioactive peptides from cereal storage proteins and their potential role in prevention of chronic diseases. Comprehensive reviews in food science and food safety. 12:364-380. https://doi.org/10.1111/1541- 4337.12017 CDC, Centers for Disease Control and Prevention. 2016. High blood pressure frequently asked questions [Internet]. Accessed April 11, 2019. https://www.cdc.gov/bloodpressure/faqs.htm De Leo F, Panarese S, Gallerani R and Ceci LR. 2009. Angiotensin converting enzyme (ACE) inhibitory peptides: production and implementation of functional food. Current pharmaceutical design. 15(31):3622-3643. doi:10.2174/138161209789271834 De Mesa NJ, Alavi S, & Bean SR. 2010. Sorghum Proteins: The Concentration, Isolation, Modification, and Food Applications of Kafirins. Journal of Food Science. 75(5):90- 104. doi:10.1111/j.1750-3841.2010.01623.x 15 De Morais L, Silva S, Duarte HS and Pinheiro-Sant'Ana HM. 2015. Sorghum (Sorghum bicolor L.): Nutrients, bioactive compounds, and potential impact on human health, Critical Reviews in Food Science and Nutrition. 57(2):372-390. doi:10.1080/10408398 Emendack Y, Burke J, Bean S, Wilson J, Hayes C and Laza H. 2018. Composition, functional components, and physical characteristics of grain from staygreen and senescent sorghum lines grown under variable water availability. Cereal Chemistry. 95(5):634-645. doi:10.1002/cche.10077 FAO, Food and Agriculture Organization of the United Nations. 2017. Crop productions [Internet]. Accessed April 11, 2019. FAOSTATS. http://www.fao.org/faostat/en/#data Ferreira SMR, De Mello AP, Dos Anjos MDCR, Krüger CCH, Azoubel PM and de Oliveira Alves MA. 2016. Utilization of sorghum, rice, corn flours with potato starch for the preparation of gluten-free pasta. Food chemistry. 191:147-151. doi:10.1016/j.foodchem.2015.04.085 Gwirtz JA, Garcia-Casal MN. 2013. Processing maize flour and corn meal food products. Annals of the New York Academy of Sciences. doi: 1312:75. 10.1111/nyas.12299 Hadjivassiliou M, Sanders DS and Aeschlimann D. 2016. The neuroimmunology of gluten intolerance. In Neuro-Immuno-Gastroenterology. Springer, Cham. p. 263-285. doi: 10.1007/978-3-319-28609-9_15 Horax R, Vallecios MS, Hettiarachchy N, Osorio LF and Chen P. 2017. Solubility, functional properties, ACE‐I inhibitory and DPPH scavenging activities of Alcalase hydrolysed soy protein hydrolysates. International Journal of Food Science and Technology. 52(1):196-204. doi:10.1111/ijfs.13267 Huang WH, Sun J, He H, Dong HW and Li JT. 2011. Antihypertensive effect of corn peptides, produced by a continuous production in enzymatic membrane reactor, in spontaneously hypertensive rats. Food Chemistry. 128(4):968-973. doi: 10.1016/j.foodchem.2011.03.127 Kadam SU, Tiwari BK, Álvarez C, O'Donnell CP. 2015. Ultrasound applications for the extraction, identification and delivery of food proteins and bioactive peptides. Trends in Food Science and Technology. 46(1):60-67. doi:10.1016/j.tifs.2015.07.012 Kamath V, Niketh S, Chandrashekar, Rajini PS. 2007. Chymotryptic hydrolysates ofa- kafirin, the storage protein of sorghum (Sorghum bicolor) exhibited angiotensin converting enzyme inhibitory activity. Food Chemistry. 100:306-311. doi:10.1016/j.foodchem.2005.10.004 Kim SK, Byun HG, Park PJ, Shahidi F. 2001. Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate. Journal of Agricultural and Food Chemistry. 49(6):2992-2997. doi:10.1021/jf001119u Komatsu S, Yanagawa Y. 2013. Cell wall proteomics of crops. Frontiers in Plant Science. 4(17):1-9. doi:10.3389/fpls.2013. 16 Lackland DT. 2015. Racial Differences in Hypertension: Implications for High Blood Pressure Management. The American Journal of the Medical Sciences. 348(2):135– 138. doi:10.1097/maj.0000000000000308 Lafiandra D, Riccardi G, Shewry P. 2014. Improving cereal grain carbohydrates for diet and health. Journal of Cereal Science. 59:312-326. doi: 10.1016/j.jcs.2014.01.001 Natesh R, Schwager SL, Sturrock ED and Acharya KR. 2003. Crystal structure of the human angiotensin-converting enzyme–lisinopril complex. Nature. 421(6922):551. doi:10.1038/nature01370. Nielsen PM, Petersen D and Dambmann C. 2001. Improved method for determining food protein degree of hydrolysis. Journal of food science. 66(5):642-646. doi:10.1111/j.1365-2621.2001.tb04614.x Pellegrini N and Agostoni C. 2015. Nutritional aspects of gluten‐free products. Journal of the Science of Food and Agriculture. 95(12):2380-2385. https://doi.org/10.1002/jsfa.7101 Santiago R, Barros-Rios J, Malvar RA. 2013 Impact of cell wall composition on maize resistance to pests and diseases. Int. J. Mol. Sci. 14:6960–6980. doi: 10.3390/ijms14046960. Shukla R, Cheryan M. 2001. Zein: the industrial protein from corn. Industrial Crops and Products. 13 (3):171-192. doi:10.1016/S0926-6690(00)00064-9 Singh NK, Donovan GR, Batey IL and MacRitchie F. 1990. Use of sonication and size- exclusion high-performance liquid chromatography in the study of wheat flour proteins. I. Dissolution of total proteins in the absence of reducing agents. Cereal Chem. 67(2):150-161. Soffer RL. 1976. Angiotensin-converting enzyme and the regulation of vasoactive peptides. Annual review of biochemistry. 45(1):73-94. doi: 10.1146/annurev.bi.45.070176.000445 Stefoska A, Beck J, Johnson K, Tapsell C. 2015. Sorghum: an underutilized cereal whole grain with the potential to assist in the prevention of chronic disease. Food Reviews International. 31(4):401-437. Sun C, Dai L, He X, Liu F, Yuan F and Gao Y. 2016. Effect of heat treatment on physical, structural, thermal and morphological characteristics of zein in ethanol-water solution. Food hydrolloids. 58:11-19. doi: 10.1016/j.foodhyd.2016.02.014 Suh HJ, Whang JH and Lee H. 1999. A peptide from corn gluten hydrolysate that is inhibitory toward angiotensin I converting enzyme. Biotechnology Letters. 21(12):1055-1058. https://doi.org/10.1023/A:1005688627350 Tang S, Hettiarachchy NS, Shellhammer TH. 2002. Protein extraction from heat-stabilized defatted rice bran. Physical processing and enzyme treatments. Journal of Agricultural and Food Chemistry. 50(25):7444-7448. doi:10.1021/jf025771w Tapia-Hernández JA, Del-Toro-Sánchez CL, Cinco-Moroyoqui FJ, Juárez-Onofre JE, Ruiz-Cruz S, Carvajal-Millan E, Rodríguez-Felix F. 2019. Prolamins from Cereal By-products: Classification, Extraction, Characterization and its Applications in 17 Micro- and Nanofabrication. Trends in Food Science and Technology. 90:111-132 doi:10.1016/j.tifs.2019.06.005 Vasudeva K, Sajeeda N, Chandrashekar A and Rajini PS. 2006. Chymotryptic hydrolysates of a-kafirin, the storage protein of sorghum (Sorghum bicolor) exhibited angiotensin converting enzyme inhibitory activity. Food Chemistry. 100(1):306-311. doi:10.1016/j.foodchem.2005.10.004 Wang Y, Chen H, Wang X, Li S, Chen Z, Wang J and Liu W. 2015. Isolation and identification of a novel peptide from zein with antioxidant and antihypertensive activities. Food and function. 6(12):3799-3806. doi: 10.1039/c5fo00815h WHO, World Health Organization. 2018. The top 10 causes of death [Internet]. Accessed April 11, 2019. https://www.who.int/news-room/fact-sheets/detail/the-top-10- causes-of-death Williams RJ, & Rao KN. 1981. A Review of Sorghum Grain Moulds. Tropical Pest Management. 27(2):200–211. doi:10.1080/09670878109413652 World Heart Federation. 2017. Stroke and hypertension. Accessed April 11, 2019. https://www.world-heart-federation.org/resources/stroke-and-hypertension Wu Q, Du J, Jia J and Kuang C. 2015. Production of ACE inhibitory peptides from sweet sorghum grain protein using alcalase: Hydrolysis kinetic, purification and molecular docking study. Food chemistry. 199:140-149. doi: 10.1016/j.foodchem.2015.12.012 Yang Y, Tao G, Liu P and Liu JIA. 2007. Peptide with angiotensin I-converting enzyme inhibitory activity from hydrolyzed corn gluten meal. Journal of agricultural and food chemistry. 55(19):7891-7895. doi: 10.1021/jf0705670 Yano S, Suzuki K, Funatsu G. 1996. Isolation from α-zein of thermolysin peptides with angiotensin I-converting enzyme inhibitory activity. Bioscience Biotechnology and Biochemistry. 60(4):661-663. doi: 10.1271/bbb.60.661 Zhou K, Sun S and Canning C. 2012. Production and functional characterization of antioxidative hydrolysates from corn protein via enzymatic hydrolysis and ultrafiltration. Journal of food chemistry. 135:1192-1197. doi: 10.1016/j.foodchem.2012.05.063 Zhu B, He H, Hou T. 2018. A comprehensive review of corn protein‐derived bioactive peptides: production, characterization, bioactivities, and transport pathways. Comprehensive Reviews in Food Science and Food Safety. 18:329-345. doi:10.1111/1541-4337.12411 18 7. APPENDICES Appendix 1. Degree of hydrolysis of yellow corn, white corn and sorghum protein hydrolysate at different times. Appendix 2. ACE Inhibitory activities of protein and protein hydrolysates from corns and sorghum. 0 5 10 15 20 25 30 0 30 60 90 120 150 180 D H ( % ) Minutes YCPH WCPH WSPH 0 5 10 15 20 25 30 35 40 45 50 YC Control WC Control WS Control YCPH WCPH WSPH A C E i n h ib it o ry r at e (% ) Treatments ACE Inhibitory Rate (%) DH Cover page Cover Abstract Table of contents Introduction Material and methods Results and discussion Conclusions Recommendations References Appendices