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Title: Antioxidant Activity of Peptide Fractions from Chickpea Globulin Obtained by Pulsed Ultrasound Pretreatment
Authors: María Fernanda González-Osuna, Wilfrido Torres-Arreola, Enrique Márquez-Ríos, Francisco Javier Wong Corral, Eugenia Lugo-Cervantes, José Carlos Rodríguez-Figueroa, Guillermina García-Sánchez, Josafat Marina Ezquerra-Brauer, Herlinda Soto-Valdez, Alejandro Castillo, Carmen Lizette Del-Toro-Sánchez *
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Horticulturae 2023, 9, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/horticulturae
Article 1
Antioxidant Activity of Peptide Fractions from Chickpea Glob-
2ulin Obtained by Pulsed Ultrasound Pretreatment
3María Fernanda González-Osuna 1, Wilfrido Torres-Arreola 1, Enrique Márquez-Ríos 1, Francisco Javier Wong-Co- 4 rral 1, Eugenia Lugo-Cervantes 2, José Carlos Rodríguez-Figueroa 3, Guillermina García-Sánchez 4, Josafat Marina 5 Ezquerra-Brauer 1, Herlinda Soto-Valdez 5, Alejandro Castillo 6, and Carmen Lizette Del-Toro-Sánchez 1,* 6
1 Departamento de Investigación y Posgrado en Alimentos, Universidad de Sonora. Encinas y Rosales s/n, 7 83000, Hermosillo, Sonora, Mexico; fernandagonzalezosuna@gmail.com (M.F.G.-O.); wilfrido.torres@uni- 8 son.mx (W.T.-A.); enrique.marquez@unison.mx (E.M.-R.); francisco.wong@unison.mx (F.J.W.-C.); josafat.ez- 9 querra@unison.mx (J.M.E.-B.); carmen.deltoro@unison.mx (C.L.D.-T.-S.) 10
2 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), A.C., Unidad 11 de Tecnología Alimentaria, Zapopan, México; elugo@ciatej.mx (E.L.-C.) 12
3 Departamento de Ingeniería Química y Metalurgia, Universidad de Sonora. Encinas y Rosales s/n, 83000, 13
Hermosillo, Sonora, Mexico; jose.rodriguez@unison.mx (J.C.R.-F.) 14
4 Coordinación de Tecnología de Alimentos de Origen Animal, Centro de Investigación en Alimentación y 15 Desarrollo A.C. (CIAD, A.C.), 83304, Hermosillo, Sonora, Mexico; guilleg@ciad.mx (G.G.-S.) 16
5 Coordinación de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y 17 Desarrollo A.C. (CIAD, A.C.), 83304, Hermosillo, Sonora, Mexico; hsoto@ciad.mx (H.S.-V.) 18
6 Animal Science Department, Texas A&M University, College Station, TX 77843-2471, USA; alejandro.cas- 19
tillo@ag.tamu.edu (A.C.) 20
* Correspondence: carmen.deltoro@unison.mx 21
Abstract: Protein hydrolysates and peptides can show biological activities. Pulsed ultrasound im- 22 prove bioactivities. Among matrices from which protein hydrolysates can be obtain, chickpea is an 23 excellent source. To evaluate the effect of pulsed ultrasound to obtain chickpea peptide fractions, 24 we applied ultrasound pretreatment on globulin concentrate and measured it properties. This doc- 25 ument presents the antioxidant activity of chickpea protein concentrates and the improve in antiox- 26 idant activity of protein hydrolysates and peptide fractions due to ultrasound pretreatment. The 27 electrophoretic profile, amino acid profile, and antimicrobial activity of hydrolysates were also de- 28 termined. Two hydrolysates had the highest antioxidant activity: HGb (91.44% ABTS inhibition, 29 73.04% hemolysis inhibition and 5185.57 µ mol TE /g dried sample in FRAP assay) and HGb-20 30 (48.25% ABTS inhibition, 100% hemolysis inhibition and 2188.53 µ mol TE /g dried sample in FRAP 31 assay). Peptide fractions inhibited 100% of hemolysis on human erythrocytes. The hydrolysates 32 from chickpea proteins obtained with savinase have antioxidant activity by SET and HAT mecha- 33 nisms. The application of obtained compounds for development of functional foods or for food 34
preservation should be considered. 35
Keywords: chickpea protein hydrolysates; globulin; ultrasound pretreatment; antioxidant peptide 36
fractions 37
38
1. Introduction 39
During past decades, the noticeable increase in the prevalence of non-communicable 40 diseases such as cerebrovascular and cardiovascular diseases, cancer, and diabetes has 41 been one of the biggest concerns for world population [1,2]. On the other hand, food loss 42 and waste are major problems for food safety, the economy, and the environment [3]. For 43 those reasons, research has focused on identifying bioactive compounds with multiple 44 biological activities, that could be used for health or food preservation matters [4]. Among 45 active compounds that could be employed for this purpose, peptides and protein 46 Citation: To be added by editorial
staff during production.
Academic Editor: Firstname Last- name
Received: date Revised: date Accepted: date Published: date
Copyright: © 2023 by the authors.
Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/).
hydrolysates are to be considered given their multifunctional activities like antioxidant, 47 antihypertensive, antimicrobial, and anti-inflammatory, among others [4,5]. These bioac- 48 tive molecules have gained interest in the food industry, more specifically, the ones with 49 antioxidant and antimicrobial activities [6-8]. Antioxidant protein hydrolysates reduce or 50 prevent oxidation whereas antimicrobial protein hydrolysates insert into membranes of 51 microorganisms, disrupting them, resulting in bacterial killing [8,9]. 52 Identifying protein sources from which protein hydrolysates and peptides can be ob- 53 tained is an important matter [10]. Several studies indicate that protein hydrolysates and 54 peptides derived from legumes have different bioactive properties [11-13]. In that sense, 55 chickpea peptides have shown antioxidant capacity and antimicrobial capacity [14-17]. 56 The biological activities were attributed to presence of basic, acidic, and hydrophobic 57
amino acids. 58
Protein hydrolysates and bioactive peptides are inactive within the parent protein, 59 therefore must be isolated by processes such as fermentation, gastrointestinal digestion, 60 and enzymatic hydrolysis [9]. In addition, the use of pretreatments enhances the biological 61 activity of obtained compounds [10]. Pulsed ultrasound prior to enzymatic hydrolysis or 62 ultrasound-assisted hydrolysis generates increases in the bioactivity of the hydrolysates 63 and peptides obtained [18,19]. Increments in biological activity result from the cavitation 64 phenomenon, produced by acoustic waves during the application of ultrasound, where 65 the microbubbles generated cause conformational changes in the proteins. With hydro- 66 phobic regions exposed, efficiency of the enzymes increases, allowing the obtaining of 67 peptides and protein hydrolysates with more favorable amino acid composition [20-23]. 68 Ultrasound treatment on chickpea proteins has been used recently, with a focus on 69 improving functional properties such as emulsification, foaming, and gel properties, of 70 chickpea protein isolates [24-26]. To improve antioxidant activity, ultrasound pretreat- 71 ment has been used as part of the extraction technique, enhancing phenolic compounds 72 extraction from chickpea seeds [27,28]. As far as we know, there are no studies where 73 pulsed ultrasound pretreatment is used to obtain antioxidant peptides and protein hy- 74
drolysates from chickpea. 75
To evaluate the effect of pulsed ultrasound to obtain chickpea peptide fractions, we 76 applied ultrasound pretreatment on globulin concentrate and measured it properties. This 77 document presents the antioxidant activity of chickpea protein concentrates and the im- 78 prove in antioxidant activity of protein hydrolysates and peptide fractions due to ultra- 79 sound pretreatment. The electrophoretic profile, amino acid profile, and antimicrobial ac- 80
tivity of hydrolysates were also determined. 81
2. Materials and Methods 82
2.1. Materials 83
Chickpea grains (Cicer arietinum L.) of kabuli type, variety Blanco Sinaloa harvested 84 in 2017 were obtained from “El Compa” plantations in Hermosillo, Sonora, Mexico 85 (28.596062 N, -111.468929 W) and were provided by Grupo Aliansa (Sonora, Mexico). 86 Savinase (EC 3.4.21.62) obtained from Bacillus sp. was purchased from Sigma-Aldrich (St. 87 Louis, MO, USA). All reagents and chemicals used were of analytical or HPLC grade and 88 were also acquired from Sigma-Aldrich unless otherwise specified. 89
2.2. Preparation of Chickpea Flour 90
Chickpea flour was obtained according to the methods by Tovar-Pérez et al. [29] and 91 Tontul et al. [30], with modifications. Five kilograms of raw chickpea seeds were lyophi- 92 lized. Lyophilized chickpea seeds were ground in a Thomas-Wiley mill (Thomas Scien- 93 tific, New Jersey, USA) and flour was sieved through a 2 mm mesh. The resulting flour 94 was milled in a Braun food processor (Gillette Commercial, Massachusetts, USA) and 95 passed through a 35-mesh sieve and 0.425 mm pore size. Flour was defatted by extracting 96 twice with petroleum ether (1:5 w/v) for 24 h. Chloroform was used twice (1:5 w/v) for 24 97
Horticulturae 2023, 9, x FOR PEER REVIEW 3 of 17
h for remotion of alkaloids. Acetone was used twice (1:3 w/v) for 24 h for removal of pol- 98 yphenols [31]. Flour was evaporated to dryness and stored at -18 ° C until use. 99
2.3. Preparation of Chickpea Protein Concentrates 100
Extraction of soluble proteins from chickpea flour was performed according to the 101 methods by Voigt et al. [32] and Tovar-Pérez et al. [29], with modifications. Chickpea flour 102 was extracted successively with 10 mmol/L Tris-HCl (pH 7.5 containing 2 mmol/L EDTA), 103 0.5 mol/L NaCl (containing 2 mmol/L EDTA and 10 mmol/L Tris-HCl pH 7.5) and 0.1 104 mol/L NaOH, to obtain albumin (Al), globulin (Gb) and glutelin (Gt) concentrates, respec- 105 tively. Suspensions (1:2 w/v) were stirred for 5 min and centrifuged at 9,000 rpm for 80 106 min at 4°C. Each supernatant was dialyzed (molecular weight 12 kDa cutoff) against de- 107 ionized water for 48 h at room temperature, with a change every 8 h. The contents of the 108 dialysis tubes were centrifuged, lyophilized, and stored at -18 ° C. Total protein concen- 109 tration in chickpea protein extracts was determined as total nitrogen multiplied by 6.25, 110
using micro Kjeldahl [33,34]. 111
2.4. Preparation of Chickpea Protein Hydrolysates 112
Chickpea hydrolysates were obtained according to the methods by Garcia-Mora et al. 113 [13,33], with some modifications. Freeze-dried chickpea protein concentrates were sus- 114 pended in deionized water (2% w/v), equilibrated at 40°C. The pH value was adjusted to 115 8 using 0.1 M NaOH. Enzymatic proteolysis employing savinase was carried out at an 116 enzyme-substrate ratio (E/S) of 0.1 U/mg of soluble protein at 40°C, for 2 h and pH 8. The 117 reaction was finalized by heating samples at 80 ° C for 15 min. Hydrolysates were centri- 118 fuged at 9,000 rpm at 10°C for 20 min, lyophilized, and stored until use, being labeled as 119 albumin hydrolysate (HAl), globulin hydrolysate (HGb) and glutelin hydrolysate (HGt). 120 Soluble protein concentration in hydrolysates was determined according to the method 121 by Bradford [35]. Bovine serum albumin (BSA) was used as standard at a concentration 122
range from 0 to 1 mg/mL. 123
2.5. Antioxidant Activity of Protein Concentrates and Protein Hydrolysates 124
2.5.1. ABTS•+ Scavenging Activity 125
ABTS assay was carried out according to the method described by Re et al. [36]. 19.3 126 mg of ABTS (2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) 127 were dissolved in 5 mL of water to prepare the cationic radical ABTS. 88 µ L of potassium 128 persulfate (0.0378 g/mL) were added to ABTS·+ solution and the mixture was incubated 129 in the dark for 16 h. 1 mL of the ABTS radical was added to 88 mL of water, and the 130 absorbance was adjusted to 0.7 ± 0.02 at 734 nm. The samples (20 μL) were mixed with the 131 ABTS radical solution (270 μL) and incubated for 30 min, and the absorbance was read 132 after 30 min at 734 nm using a microplate reader (Thermo Fisher Scientific Inc. Multiskan 133 GO, New York, USA). The results were expressed as the scavenging activity percentage 134 of Abs734 (RSA %). Additionally, for protein hydrolysates the 50%-inhibitory concentra- 135 tion (IC50) was expressed as the μg/mL of hydrolysate that scavenged half of ABTS•+ rad- 136
ical. 137
2.5.2. Evaluation of the Protective Effect on Human Erythrocytes 138 The protective effect in human erythrocytes was evaluated according to the method 139 by Hernández-Ruiz et al. [37]. In this assay, erythrocytes hemolysis is induced with AAPH 140 (2,2'-Azobis(2-amidinopropane) dihydrochloride) radical. were washed three times with 141 phosphate-buffered saline (PBS) at pH 7.4. A suspension of erythrocytes was prepared 142 with PBS (2%). A mixture of erythrocytes (100 μL), sample (100 μL) and AAPH radical 143 (100 μL) was prepared and incubated at 37°C under stirring at 30 rpm, in darkness, for 3 144 h. Afterwards, 1 mL of PBS was added to the mixture and centrifuged at 1500 rpm for 10 145 min. The absorbance of the supernatant was measured at 540 nm using a microplate 146
reader (Thermo Fisher Scientific Inc. Multiskan GO, New York, USA). The results were 147 expressed as a percentage of hemolysis inhibition (PHI) compared to a similar reaction 148 without sample, and the value was calculated using the following equation: 149
PHI (%)=AHI-AS
AHI x 100 (1)
where AHI was defined as absorbance of hemolysis induced by AAPH at 540 nm while 150
AS was defined as absorbance of the sample at 540 nm. 151
152
2.5.3. Ferric Reducing Antioxidant Power (FRAP) 153
The FRAP assay, in which ferric ion (Fe+3) is reduced to ferrous ion (Fe+2), was deter- 154 mined using the method by Benzie and Strain [38], with modifications. The FRAP reagent 155 was prepared in a 1:1:10 ratio of 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) in 40 mM 156 HCl, 20 mM FeCl3·6H2O and 0.3 M sodium acetate buffer (pH 3.6), respectively. The sam- 157 ples (20 µ L) were mixed with the FRAP reagent (280 µ L) and incubated for 40 min. The 158 absorbance was read at 638 nm (Thermo Fisher Scientific Inc. Multiskan GO, New York, 159 USA). A standard curve was made with Trolox, with concentrations ranging from 0 to 200 160 µ mol. Results were expressed as micromoles of Trolox equivalents per gram of dried sam- 161
ple (µ mol TE/g dried sample). 162
2.6. Ultrasound Pretreatment on Globulin Concentrate 163
As shown in Table 2, HGb showed greater antioxidant activity among the different 164 hydrolysates. Furthermore, being globulin one of the predominant protein fractions, Gb 165 was chosen to perform pulsed ultrasound pretreatment according to the method by Wang 166 et al. [18], with modifications. 100 mg of freeze-dried globulin concentrate were dissolved 167 in 20 mL of distilled water in a glass beaker, to obtain protein solutions with substrate 168 concentration of 5 g/L. Each sample was treated in a probe-type sonicator Branson Digital 169 Sonifier SFX 550 (Branson Ultrasonics Corporation, Connecticut, USA) with a sonotrode 170 with a 12.7 mm diameter tip. The samples were treated with 3 conditions: 10, 20 and 30 171 min, at powers of 33, 77 and 119 W, respectively, with pulsed on-time and off-time of 2 172 and 2 s. The beaker was surrounded by an ice bath to keep the sample at room tempera- 173 ture during the ultrasound treatment. The control consisted of a globulin concentrate 174 without ultrasound pretreatment. Immediately after the application of ultrasound pre- 175 treatment, hydrolysis with savinase was carried out according to the conditions described 176 above. The soluble protein concentration and the antioxidant activity of the hydrolysates 177
obtained was determined as previously described. 178
Samples were coded as globulin concentrate (Gb) or globulin hydrolysate (HGb), fol- 179 lowed by time of pulsed ultrasound pretreatment (10, 20, and 30). For example, Gb-20 is 180 the globulin concentrate pretreated with pulsed ultrasound in conditions of 20 minutes, 181 77 W; and HGb-10 hydrolysates are those produced from globulin concentrate pretreated 182 with pulsed ultrasound in conditions of 10 minutes, 33 W and hydrolyzed by savinase. 183 184
2.7. Ultrafiltration 185
Hydrolysates with the highest antioxidant activity were used to obtain peptide frac- 186 tions by ultrafiltration, according to the method by Tovar-Pérez et al. [29], with some mod- 187 ifications. The Millipore Amicon® model 8200 agitation cell (EMD Millipore Corporation, 188 Massachusetts, USA) and ultrafiltration membranes with 10 and 3 kDa molecular weight 189 cutoff were used, obtaining 3 peptide fractions for each hydrolysate (molecular weight > 190 10, 10-3 and < 3 kDa). Subsequently, the antioxidant activity of the peptide fractions was 191
determined as described above. 192
193
2.8. Electrophoretic Profile (SDS-PAGE) 194
Horticulturae 2023, 9, x FOR PEER REVIEW 5 of 17
The profiles of chickpea proteins and hydrolysates were analyzed in SDS-PAGE, with 195 12% resolution gel and 4% stacking gel [39]. 20 μL of sample were loaded in the gel and 196 protein separation was performed at 100 V. Gels were stained with Coomassie Brilliant 197 Blue R-250 and destained with a methanol:water:acetic acid solution (500:400:100 mL). 198 Same conditions were used to analyze the profile of globulin hydrolysates obtained with 199 pulsed ultrasound pretreatment. The molecular weight of the protein concentrates and 200 hydrolysates was determined by comparison with a wide range molecular weight marker. 201
2.9. Amino Acid Profile 202
The amino acid composition of the hydrolysates with the highest antioxidant activity 203 (HGb and HGb-20) was determined by reverse-phase high-performance liquid chroma- 204 tography (RP-HPLC) using a Hewlett-Packard 1200 series HPLC system (Hewlett-Pack- 205 ard Co., Waldbronn, Germany) according to the method by Vázquez-Ortiz et al. [40]. The 206 samples were hydrolyzed in sealed tubes using 6 M HCl in an evaporator at 150 °C for 12 207 h. The hydrolyzed samples were resuspended on sodium citrate buffer solution (pH 2.2), 208 and derivatized with O-phthalaldehyde (OPA) for determination of primary amino acids. 209 Chromatographic separation was carried out using a C18 column (4.6 mm ID × 100 mm; 210 Agilent Technologies, Inc., Palo Alto, CA, USA), and the integrations were calculated us- 211 ing ChemStation software (Agilent Technologies Inc., California, USA). Fluorescence 212 emission was monitored continuously at 330 and 418 nm. The results were expressed as 213
amino acid residues. 214
215
2.10. Antimicrobial Activity of Protein Hydrolysates 216
2.10.1 Bacterial Strains and Growth Conditions 217
Staphylococcus aureus (ATCC 65384) and Salmonella enterica subsp. enterica ser. Typhi- 218 murium (ATCC 14028) were employed in the experiments. Strains were maintained in 219 tryptic soy broth (TSB) containing glycerol (20%) at 4 °C until use. A loopful of bacteria 220 was transferred to 3 mL of TSB and incubated at 37 °C overnight. A loopful of culture was 221 then transferred to mannitol salt agar or bismuth sulfite agar, and cultures were grown at 222 37 °C until reaching the desired number of colony-forming units per mL (CFU/mL) for 223
use in inhibitory assays [41]. 224
2.10.2. Plate Preparation and Analysis 225
Antimicrobial activity was evaluated by the method described by Griffin et al. [42] 226 with some modifications. Mueller-Hinton agar was prepared and different concentrations 227 of HGb and HGb-20 (10, 5, 1 mg/mL) were added to 15 mL aliquots of molten, tempered 228 agar to give final concentrations of 2.0% v/v. The agar-samples solutions were vortexed 229
for 15 seconds and immediately poured into Petri dishes. 230
The plates were inoculated by pipetting 10 µ L of a freshly prepared bacterial suspen- 231 sion (104 CFU/mL) onto the agar surface. One agar plate containing the test organism in 232 the absence of the test material was used as a positive control with each experiment. Plates 233 were set up in duplicate and the entire method was repeated twice. After inoculation the 234 plates were left to stand for 30 min, then incubated for 24 h at 37 °C. After incubation the 235 plates were examined for the presence or absence of growth. The MIC was recorded as 236 the concentration of test material where at least five out of six readings showed no visible 237
growth or only isolated colonies (≤ 20). 238
239
2.11. Statistical Analysis 240
Experimental data were subjected to analysis of variance (ANOVA) followed by 241 Tukey test at a 0.95 (p<0.05) confidence level. Results are presented as mean with standard 242 deviation. All trials were performed in triplicate. Statistical analysis was carried out using 243 JPM software, version 8.0 (SAS Institute Inc.; North Carolina, USA). The graphics were 244
made with SigmaPlot software, version 10.0 (Systat Software Inc., Illinois, USA). Descrip- 245 tive statistics was applied for the electrophoretic analysis, FTIR and RMN data. 246
3. Results and Discussion 247
3.1. Chickpea Protein Concentrates 248
Three protein concentrates were obtained from chickpea flour and their protein con- 249 tent was determined, as shown in Table 1. The amount of concentrate obtained varied 250 from 1.93 to 3.51 g/100 g of flour. Glutelin and globulin concentrates showed higher yield 251 than albumin, with significant difference between glutelin and albumin. Several studies 252 with chickpea seed have reported a higher globulin and glutelin content compared to al- 253 bumin content, as occurred in this study. Liu, Hung and Bennett [43] reported a content 254 of 28.5% albumin, lower than 43.5% globulin. Singh and Jambunathan [44] reported a con- 255 tent of 56.6% and 18.1% of globulin and glutelin, higher than albumin content (12%). This 256 suggests that there may be variations in the yield of the protein concentrates obtained 257 from chickpea, those differences being attributed to factors such as seed variety, fraction- 258
ation technique and storage conditions [29,43]. 259
260 Table 1. Protein content of protein concentrates obtained from chickpea seed. 261
Protein concentrate Quantity of protein (g/100 g flour*)
Protein content (g/100 g concentrate)
Al 1.93 ± 0.26 b 82.73 ± 1.19 a
Gb 2.93 ± 0.64 ab 82.25 ± 4.77 a
Gt 3.51 ± 0.56 a 85.23 ± 6.58 a
Values are shown as mean ± standard deviation of triplicate determinations. Means with different superscripts, in the same column, are significantly different (p < 0.05).
*Defatted flour, without alkaloids and phenols.
262 The protein content of the concentrates was determined from 82.73 to 85.23 g/100 g 263 of concentrate and were not significantly different (Table 1). The results in this study are 264 similar to those described in literature, where values of 83.57 ± 0.22 to 84.8 ± 0.3 g/100 g of 265 chickpea globulin concentrate are reported [45,46]. In consequence, the process of obtain- 266 ing protein hydrolysates was favored by high protein content of the concentrates. 267 268
3.2. Antioxidant Activity of Protein Compounds 269
3.2.1. ABTS•+ Radical Scavenging Activity 270 Table 2 shows the antioxidant activity of chickpea protein concentrates and hydrol- 271 ysates as determined by the ABTS assay. The protein concentrates displayed values from 272 10.68 to 37.07% ABTS inhibition, with significant differences between them, being higher 273 for glutelin concentrate. For hydrolysates, the ABTS inhibition ranges were from 87.75 to 274 92.37%, being higher for albumin hydrolysate, the difference between hydrolysates being 275 significant. Evangelho, Vanier, Pinto, De Berrios, Guerra Dias and Rosa Zavareze [47] re- 276 ported 68% ABTS inhibition for bean globulin hydrolysates obtained using alcalase. Ngoh 277 and Gan [48]. determined a 53.33% ABTS inhibition for bean protein hydrolysates obtained 278 with protamex. The ABTS inhibition values determined in the present study were mark- 279 edly higher than those described above, the difference may be attributed to the enzyme 280 used for the hydrolysis process. Savinase allowed the obtaining of lentil protein hydroly- 281 sates with higher activity antioxidant than those obtained with alcalase or protamex [33]. 282 The IC50 values determined for ABTS assay ranged from 1.54 to 2.12, being lower for 283 albumin (1.54 ± 0.06) and globulin (1.56 ± 0.17) hydrolysates than for glutelin hydrolysate 284 (2.12 ±0 .02) (p < 0.05). Other legume hydrolysates, such as mung bean (Vigna radiata) and 285 poroto (Erythrina edulis), exhibited higher IC50 values, 54.65 and 84.14 µ g/mL respectively, 286
Horticulturae 2023, 9, x FOR PEER REVIEW 7 of 17
therefore having lower antioxidant activity that the three chickpea hydrolysates deter- 287 mined in this study [49,50]. Differences in the IC50 value may be due to the enzyme used, 288 given that the studies described above alcalase was used. As mentioned previously, the 289 use of savinase allows the production of hydrolysates with greater antioxidant activity 290
than those obtained with alcalase [33]. 291
Table 2. Antioxidant activity of protein concentrates obtained from chickpea seed. 292 Concentrate ABTS inhibition (%) Hemolysis inhibition (%) FRAP (µmol TE/g d s)
Protein Hydrolysate Protein Hydrolysate Protein Hydrolysate Al 10.68 ± 1.53 cB 92.37 ± 0.12 aA 80.38 ± 2.84 aA 58.76 ±8.24 bB 128.07 ± 13.06 bB 1624.41 ± 131.92 bA Gb 33.10 ± 0.76 bB 91.44± 0.55 bA 67.38 ± 2.40 bB 73.04 ± 3.10 aA 644.27 ± 150.28 aB 5185.57 ± 698.59 aA Gt 37.07 ± 0.68 aB 87.75 ± 0.66 cA 64.89 ± 2.99 bB 77.40 ± 6.27 aA 446.07 ± 45.89 aB 1323.83 ± 178.18 bA Values are shown as mean ± standard deviation of triplicate determinations. Means with different lowercase superscripts, in the same column, are significantly different (p < 0.05). Means with different capital letter superscripts between columns, for each anti- oxidant assay, are significantly different (p < 0.05). Concentrations of proteins and hydrolysates were 200 µ g/mL.
3.2.2. Evaluation of the Protective Effect on Human Erythrocytes 293 The results of the protective effect on human erythrocytes of chickpea protein con- 294 centrates and hydrolysates are presented in Table 2. In this assay, the AAPH radical causes 295 lysis of the cell membranes of erythrocytes but if the AAPH radical is reduced by proton 296 donation from an antioxidant compound, hemolysis is inhibited. The ability of com- 297 pounds to inhibit the AAPH radical, preventing hemolysis, is measured. The chickpea 298 protein concentrates showed values from 64.89 to 80.38% hemolysis inhibition, with sig- 299 nificant differences between them, being higher for albumin. For hydrolysates, hemolysis 300 inhibition ranged from 58.76 to 77.40%, being higher for glutelin and globulin hydroly- 301 sates, without significant differences between them. The increase in the antioxidant capac- 302 ity of globulin and glutelin hydrolysates, in comparison to the concentrates from which 303 they were obtained, is attributed to the increase of the exposure of the side chains of the 304 amino acids present in the hydrolysates, which can act by donating protons to stabilize 305 the AAPH radical and stop the chain reaction of the radical, thus preventing damage to 306
erythrocytes [51-53]. 307
Most studies of protective effect in human erythrocytes are for compounds such as 308 phenols, ginsenosides and flavonoids [37,51-52]. In the matter of compounds of protein 309 nature, Zheng, Dong, Su, Zhao and Zhao [54] studied the protective effect in human eryth- 310 rocytes of different dipeptides against the effects caused by AAPH-induced oxidation, 311 finding hemolysis of 10.64, 3.61, 23.12 and 20.30% for Tyr-Gly, Trp-Gly, Cys-Gly and Met- 312 Gly, respectively, results close to those determined for chickpea hydrolysates in the pre- 313 sent study. Zhan, Wang, Liu, Guo, Gong, Hao, et al. [55] evaluated the protective effect of 314 globulin hydrolysates of sachi seeds (Plukenetia volubilis) on erythrocytes, with hemolysis 315 inhibitions of < 25% at 500 µ g/mL of hydrolysates and > 80% at 2000 µ g/mL of hydroly- 316 sates, concentrations far superior to the ones evaluated in this study (200 µ g/mL). 317
3.2.3. Ferric Reducing Antioxidant Power (FRAP) 318
The results of the FRAP assay for chickpea protein concentrates and their hydroly- 319 sates are shown in Table 2. The concentrates showed a FRAP value from 128.07 to 644.27 320 µ mol TE/g dried sample, being higher for globulin. The hydrolysates showed a high re- 321 ducing power, with FRAP values from 1323.83 to 5185.57 µ mol TE/g dried sample, with 322 significant differences between them, being greater for globulin hydrolysate. Santos Agui- 323 lar, Soares de Castro y Sato [56] obtained rice protein hydrolysates using a Bacillus lichen- 324 iformis protease and alcalase, determining values of 18.78 and 19.31 µ mol TE/g sample for 325 FRAP assay, respectively. Piñuel, Vilcacundo, Boeri, Barrio, Morales, Pinto, et al. [57] ob- 326 tained a bean protein concentrate, with a FRAP value of 95.80 µ mol TE/g sample, while 327
the hydrolysate obtained after an in vitro gastrointestinal digestion showed 225.77 µ mol 328
TE/g sample for FRAP assay. 329
The chickpea hydrolysates obtained in this study, specially HGb, are antioxidant 330 compounds with great electron donor capacity, as explained by the amino acids found in 331 abundance in the globulin fraction. A high Asp content was determined in the globulin 332 hydrolysate (HGb, Table 3). Asp is a negatively charged amino acid that possesses electron 333 excess, those electrons can be donated, reducing the ferric ion-TPTZ complex to ferrous 334 ion-TPTZ, mechanism evaluated in this test [38]. It is known that acidic amino acids can 335 interact with transition metals through their charged residues avoiding oxidative pro- 336
cesses [2]. 337
338 3.3. Antioxidant Activity of Ultrasound Pretreated Globulin Hydrolysates 339 Figure 1 presents the antioxidant activity of globulin concentrate and globulin hy- 340 drolysate pretreated with pulsed ultrasound determined by three assays. Pulsed ultra- 341 sound pretreatment affected the antioxidant capacity of the protein concentrates and hy- 342 drolysates in the assays that evaluate SET mechanism, such as ABTS and FRAP. A reduc- 343 tion in the percentage of ABTS inhibition for globulin concentrate with three pulsed ultra- 344 sound pretreatments (<20.9%) and for globulin hydrolysates with ultrasound pretreatment 345 (48.25 to 72.85% ABTS inhibition) compared to the globulin concentrate and hydrolysate 346 without ultrasound pretreatment was observed (Figure 1a). 347
Gb Gb-10 Gb-20 Gb-30 HGb HGb-10 HGb-20 HGb-30
inhibition (%)
0 20 40 60 80
100 ABTS
Hemolysis
y y
y
y y
x x x a
b
c
d e
f g
h
a
348
X Data
Gb Gb-10 Gb-20 Gb-30 HGb HGb-10 HGb-20 HGb-30
FRAP value µmol TE/g dried sample
0 1000 2000 3000 4000 5000
6000 a
d d
b bc
cd d d
b
349 Figure 1. Antioxidant activity of globulin concentrates, and hydrolysates pretreated with pulsed ul- 350 trasound. (a) ABTS and Hemolysis inhibition assays; (b) FRAP assay. Results are plotted as mean ± 351 standard deviation of triplicate determinations. Different letters in the same parameter are signifi- 352 cantly different (p < 0.05). Concentrations of proteins and hydrolysates were 200 µ g/mL. 353
Horticulturae 2023, 9, x FOR PEER REVIEW 9 of 17
The same behavior was shown in the FRAP assay, globulin hydrolysate has a greater 354 reducing power than the hydrolysates pretreated with pulsed ultrasound (1134.34 to 355 2188.53 µ mol TE/g dried sample for FRAP value). For FRAP assay, there were no signifi- 356 cant differences between globulin concentrate and the globulin concentrates pretreated 357 with pulsed ultrasound (Figure 1b). Although there was a decrease in the FRAP value for 358 the hydrolysates obtained with ultrasound pretreatment, the results are notoriously supe- 359 rior to that reported for rice and bean hydrolysates, whose FRAP value were determined 360 of 19.31 and 225.77 µ mol TE /g of sample, respectively [56,57]. Xia, Zhai, Huang, Liang, 361 Yang, Song, et al. [58] reported the use of ultrasound pretreatment to obtain antioxidant 362 hydrolysates from microalgae protein (Dunaliella salina), where the use of ultrasound gen- 363 erated a decrease in antioxidant activity (evaluated as % DPPH inhibition) within 30 364 minutes, as happened in the present study in the ABTS and FRAP assays. Misir and Koral 365 [59] reported a higher percentage of ABTS inhibition for rainbow trout protein hydrolysate 366 obtain by conventional hydrolysis compared to that obtained with an ultrasound-assisted 367
hydrolysis. 368
Figure 1a shows that ultrasound pretreatment favored greatly in the protective effect 369 on erythrocytes assay, since 100% erythrocyte hemolysis inhibition was achieved for glob- 370 ulin hydrolysates obtained with pretreatments with ultrasound, percentage notoriously 371 higher than the one achieved by globulin hydrolysate without ultrasound (73.04%). In ad- 372 dition, there was an increase in the inhibition of hemolysis of the globulin concentrate pre- 373 treated with ultrasound (70.47 to 76.39%). Authors have reported that the use of pulsed 374 ultrasound pretreatment increased antioxidant activity in DPPH inhibition assay and hy- 375 droxyl radical inhibition assay [23,60]. The results observed in the different carried out 376 assays indicate that the use of ultrasound pretreatment favors obtaining hydrolysates with 377 proton donation capacity. In the amino acid profile for HGb-20 (Table 3) a high content of 378 Arg was found, a basic amino acid that has the capacity to act as a proton donor [61]. A 379 high content of Ala and Gly was also found. These hydrophobic amino acids confer anti- 380 oxidant capacity to hydrolysates by increasing their solubility in related substrates. These 381 amino acids could be exposed by the effect of ultrasound pretreatment, causing that hy- 382 drolysates rich in these amino acids were obtained, which could facilitate the union of 383 globulin hydrolysates with the erythrocyte lipid membranes, donating protons to the 384
AAPH radical, inhibiting their harmful effect [2,22]. 385
3.4. Antioxidant Activity of Peptide Fractions 386
Figure 2 presents the results of antioxidant activity evaluation of three peptide frac- 387 tions of the globulin hydrolysates with the highest antioxidant activity (HGb and HGb- 388 20). In the ABTS assay, a remarked decrease in the percentage of inhibition by the peptide 389 fractions of HGb (7.76 to 13.40%) and HGb-20 (3.06 to 6.61%) was observed, compared to 390 the hydrolysates from which they came (Figure 2a). Similar behavior occurred in the FRAP 391 assay, where the peptide fractions of HGb showed FRAP values from 226.08 to 550.62 µ mol 392 TE/g dried sample, while the peptide fractions of HGb-20 had FRAP values from 226.08 to 393 372.02 µ mol TE/g dried sample, significantly different to the FRAP value of the hydroly- 394 sates HGb and HGb-20 (Figure 2b). Ngoh and Gan [48] reported the same behavior for 395 peptide fractions of bean hydrolysates, where the hydrolysate had a higher percentage of 396 inhibition of the ABTS radical (53.3%), compared to that found in peptide fractions of same 397 molecular weight of the ones of this study (19.79, 2.61 and 42.18%). 398 In the protective effect on erythrocytes assay (Figure 2c), favorable results were 399 found. Peptide fractions obtained from HGb showed from 86.68 to 100% hemolysis inhibi- 400 tion, significantly greater than hydrolysate (73.04%). Low molecular weight peptides, be- 401 ing a short chain of amino acid residues, have smaller steric hindrance compared to hy- 402 drolysates, allowing the interaction between hydrophobic and hydrophilic amino acids 403 with free radicals, neutralizing them [58]. The peptide fractions of HGb-20 maintained the 404 ability to inhibit hemolysis, with values from 99 to 100%. Pulsed ultrasound pretreatment 405
favored obtaining peptide fractions with hydrophobic amino acids, which facilitated the 406 interaction between peptide fractions and erythrocyte membranes [22]. The peptide frac- 407 tions obtained from the hydrolysates HGb and HGb-20 showed antioxidant capacity, 408 whose mechanism of action is HAT, mechanism measured by the performed assay [52]. 409
X Data
HGb HGb-20
ABTS inhibition (%)
0 20 40 60 80 100
Hydrolysate
> 10 kDa 10-3 kDa
< 3 kDa a
b
c cd de
ef ef f
a
410
X Data
HGb HGb-20
FRAP value µmol TE/g dried sample
0 1000 2000 3000 4000 5000
6000 Hydrolysate
> 10 kDa 10-3 kDa
< 3 kDa a
b
c c
c c c c
b
411
X Data
HGb HGb-20
Hemolysis inhibition (%)
0 20 40 60 80
100 Hydrolysate
> 10 kDa 10-3 kDa
< 3 kDa
a bc a a
ab c d e
c
412 Figure 2. Antioxidant activity of globulin hydrolysates and peptide fractions. (a) ABTS assay; (b) 413 FRAP assay; (c) Hemolysis inhibition assay. Results are plotted as mean ± standard deviation of trip- 414 licate determinations. Bars with different letters are significantly different (p < 0.05). Concentrations 415
of hydrolysates and peptide fractions were 200 µ g/mL. 416
Horticulturae 2023, 9, x FOR PEER REVIEW 11 of 17
3.5. Electrophoretic Profiles 417
Figure 3a shows the electrophoretic profile of chickpea proteins and hydrolysates 418 obtained in this research. Different electrophoretic profiles were observed for albumin, 419 globulin and glutelin concentrates (Figure 3a; lanes B, C, and D). For albumin, bands with 420 molecular weights from 97 to 21 kDa were observed, with three main bands with molecu- 421 lar weights of approximately 97, 48 and 40 kDa (Figure 3a, lane B). Papalamprou, Doxas- 422 takis, Biliaderis and Kiosseoglou [62] reported bands with molecular weights of 87, 31.4 423 and 23.9 kDa, corresponding to the chickpea albumin fraction. Chang [63] reported protein 424 bands with molecular weights of 93.6, 51, 41.7 and 38.9 kDa for chickpea albumin analyzed 425 on a 12% SDS-PAGE gel. Electrophoretic profiles determined in previous studies were like 426 to the one obtained in the present study. For globulin, a banding pattern from 97 to 21 kDa 427 was observed, with three main bands with molecular weights of approximately 70, 60 and 428 50 kDa (Figure 3a, lane C). Chang, Alli, Molina, Konishi and Boye [64] identified bands 429 with molecular weights of 70.2, 50.7 and 35 kDa, corresponding to vicilin subunits, bands 430 with molecular weights of 40.6 and 39.5 kDa, corresponding to legumin α-subunits and 431 bands with molecular weights of 23.5 and 22.5 kDa, corresponding to legumin β-subunits. 432 The bands of 70 and 50 kDa identified for the globulin concentrate in this study would 433 correspond to the chickpea globulin vicilin fraction, while the bands with weights between 434 30 and 21 kDa would correspond to legumin β-subunits, and the 40 kDa band would cor- 435 respond to legumin α-subunit. Legumin and vicilin are the main groups of proteins from 436 chickpea globulin. For glutelin concentrate, an electrophoretic profile with bands from 60 437 to 25 kDa was observed, with the main protein bands around molecular weights of 55 and 438 35 kDa (Figure 3a, lane D). Chang [63] reported molecular weights of 51, 38.1 and 26.2 kDa 439 for chickpea glutelin subunits. The results of this study coincide with previously reported 440 bibliography, since the most intense protein bands for chickpea glutelin are in the range of 441
55-50 and 40-35 kDa. 442
443
Figure 3. (a) SDS-PAGE profile of chickpea protein concentrates and hydrolysates. Lane A: molecular 444 weight marker; lane B: Al; lane C: Gb; lane D: Gt; lane E: HAl; lane F: HGb; lane G: HGt; (b). SDS- 445 PAGE profile of globulin concentrates, and hydrolysates pretreated with pulsed ultrasound. Lane A: 446 molecular weight marker; lane B: Gb; lane C: Gb-10; lane D: Gb-20; lane E: Gb-30; lane F: HGb; lane 447
G: HGb-10; lane H: HGb-20; lane I: HGb-30. 448
449 Degradation of protein subunits by the effect of enzymatic hydrolysis with savinase 450 was appreciated, with presence of bands with molecular weights from 30 to 21 kDa for 451 albumin hydrolysates (Figure 3a, lane E), and bands with molecular weights from 45 to 21 452 kDa for globulin and glutelin hydrolysates (Figure 3a, lanes F and G).In the study by Gar- 453 cia-Mora, Peñas, Frias and Martínez-Villaluenga [33] the use of savinase caused a complete 454 degradation of the lentil protein subunits in a hydrolysis time of two hours, forming 455
smaller fragments, as occurred in this study. 456
The electrophoretic profile of globulin concentrates and globulin hydrolysates with 457 ultrasound pretreatment obtained in this study are observed in Figure 3b. No differences 458 were found between the banding pattern of globulin concentrate without ultrasound pre- 459 treatment and the globulin concentrates pretreated with ultrasound (Figure 3b; lanes B, C, 460 D and E). Results are in accordance with the study of Wang, Wang, Li, Bai, Li and Xu [26], 461 where there was no difference in bands between a chickpea protein isolate with and with- 462 out ultrasonic treatment. Ultrasound treatment causes changes on tertiary structure of pro- 463
teins but does not on secondary structure [23]. 464
Difference in the electrophoretic profile of globulin hydrolysates with ultrasound 465 pretreatment (Figure 3b; lanes G, H, and I) in comparison to the control hydrolysate lies in 466 the disappearance of the 45 kDa band (indicated by an arrow in Figure 3b; lane F). The 467 degradation of the 45 kDa band could be attributed to ultrasound pretreatment, since ul- 468 trasound modifies the structure of proteins, which facilitate bonding between protease and 469 the substrate, increasing the hydrolysis efficiency [18,20]. 470
3.6. Amino Acid Composition 471
The amino acid contents of the two hydrolysates with the highest antioxidant activ- 472 ity, HGb and HGb-20, are presented in Table 3. Both hydrolysates had a high content of 473 Asp, Arg, Ser, Thr, Gly and Ala, without significant differences between treatments. Like 474 this study, Misir and Koral [59] found not differences between the amino acid content of 475 rainbow trout by-products hydrolysates obtained with ultrasound-assisted hydrolysis and 476 those obtained with conventional hydrolysis, using alcalase in both hydrolysis process. 477 These results showed that the amino acid content depends on the protein source and does 478 not depend on the ultrasound pretreatment prior to hydrolysis. 479 Table 3. Amino acid profile (residues/1000 amino acid residues) of globulin hydrolysates. 480
Amino acid HGb HGb-20
Aspartic acid 119 106
Glutamic acid 18 15
Histidine 37 39
Serine 147 144
Arginine 96 99
Glycine 113 114
Threonine 169 174
Alanine 105 106
Tyrosine 25 27
Methionine 7 8
Valine 18 22
Phenylalanine 33 31
Isoleucine 7 9
Leucine 48 43
Lysine 58 64
Total 1000 1000
Values are mean of triplicate determinations, with standard deviation less than 5%.
Paredes-López, Ordorica-Falomir and Olivares-Vázquez [45] reported high contents 481 of Asp, Glu, Arg, Lys, Ser and Leu for chickpea globulin. Liu, Hung and Bennett [43] de- 482 termined Asp, Glu, Arg and Leu as the predominant amino acids in a chickpea globulin 483 concentrate. Results of the present study coincide with those described above, finding 484 abundance of acidic, basic, and hydrophobic amino acids to which the antioxidant activity 485 of globulin hydrolysates can be attributed. Hydrophobic amino acids such as Gly and Ala 486 increase the solubility of protein hydrolysates in lipids, due to their nature, causing the 487
Horticulturae 2023, 9, x FOR PEER REVIEW 13 of 17
interaction with the target organs through hydrophobic interactions with the lipid bilayer 488 of cells, promoting the neutralization of free radicals. In turn, Asp can neutralize free rad- 489 icals due to its excess of electrons [2,22]. The basic amino acid Arg acts as a proton donor 490 given its guanidine group [16]. The globulin hydrolysates of the present study showed SET 491 and HAT mechanisms, according to the antioxidant activity assays performed. 492
3.7 Antimicrobial Activity of Globulin Hydrolysates 493
Given that protein hydrolysates could display multiple bioactivities, the antimicro- 494 bial activity of globulin hydrolysates with the highest antioxidant activity (HGb and HGb- 495 20) was evaluated. Results are shown in Table 4. A descending trend for CFU of Salmonella 496 enterica was observed when incrementing HGb concentration, with value of 38 ± 2.08 CFU 497 for 10 mg/mL, significantly different to other concentrations of the same hydrolysate. By 498 contrast, the lower value of CFU (35 ± 3.05) for HGb-20 was with 5 mg/mL of said hydrol- 499 ysate. For Staphylococcus aureus, the descending trend occurred when incrementing HGb- 500 20, with value of 48 ± 2.64 CFU for 10 mg/mL, with significant differences to other concen- 501 trations of the same hydrolysate. On the other hand, the lower value of CFU (58 ± 4) for 502
HGb was at 5 mg/mL of the hydrolysate. 503
Table 4. Evaluation of the antimicrobial activity of globulin hydrolysates. 504 Concentration
(mg/mL)
Salmonella enterica (CFU)
Staphylococcus aureus (CFU)
HGb HGb-20 HGb HGb-20
0 76 ± 6.93 aA 76 ± 6.93 aA 109 ± 8.50 aA 109 ± 8.50 aA 1 73 ± 3 aA 68 ± 2.89 aA 79 ± 1.41 bA 65 ± 2.83 bB 5 59 ± 2.3 bA 35 ± 3.05 cB 58 ± 4 cA 65 ± 4.16 bA 10 38 ± 2.08 cB 56 ± 2.08 bA 66 ± 2.81 cA 48 ± 2.64 cB Values are shown as mean ± standard deviation of triplicate determinations. Means with different lowercase superscripts, in the same column, are significantly different (p <
0.05). Means with different capital letter superscripts between columns, for each micro- organism, are significantly different (p < 0.05).
505 MIC values were not determined given that more than 20 UFC were counted in all 506 cases. Even though, in this preliminary assay the inhibition of a Gram+ microorganism by 507 HGb-20 and the inhibition of a Gram- microorganism by HGb was observed, implying 508 differences in the composition of the amino acids present in the peptide sequences of each 509 type of hydrolysate, which could be attributed to differences in the affinity between the 510
hydrolysates and the evaluated microorganisms. 511
Amino acids present in the HGb sequences would have higher interaction with the 512 lipopolysaccharides of the outer membrane of Gram-negative bacteria compared to the 513 amino acids present in the HGb-20 sequences, causing the disintegration of microorgan- 514 isms [7]. On the other hand, the higher affinity of the amino acids present in the HGb-20 515 sequences with the peptidoglycan cell wall of the Gram-positive bacteria could be because 516 of the ultrasound pretreatment carried out in obtaining the globulin hydrolysates [65]. 517 However, it is necessary to carry out more studies to determine the antimicrobial activity 518
of globulin protein hydrolysates. 519
4. Conclusions 520
The hydrolysates from chickpea proteins obtained with savinase have antioxidant 521 activity, displaying a high reducing power, especially those obtained from globulin by 522 conventional hydrolysis process. The application of pulsed ultrasound pretreatment to 523 chickpea globulin allows the production of hydrolysates and peptide fractions with a high 524 protective effect in human erythrocytes, due to the exposure of hydrophobic amino acids, 525
abundant in chickpea globulin, improving the proton donation mechanism of hydroly- 526 sates and peptide fractions obtained from globulin. Globulin hydrolysates have different 527 interaction affinity to bacteria owing to the use or absence of ultrasound treatment prior 528
enzymatic hydrolysis. 529
As previously stated, protein hydrolysates display multiple biological activities 530 given their amino acidic composition, being antioxidant and antimicrobial activities of 531 great relevance in health and food industry. The compounds identified in this study could 532 be used as ingredients in the elaboration of functional foods, being necessary to evaluate 533 their potential beneficial effect in vivo assays. Moreover, protein hydrolysates could be 534 used for preservation of food matrices, being applied directly to food products, or being 535 added in films and coatings made with biopolymers. In this manner, the effect of the ap- 536 plication of chickpea protein hydrolysates on food preservation should be evaluated. 537 538 Author Contributions: Conceptualization, A.C. and E.L.-C.; methodology, J.M.E.-B. and H.S.-V.; 539 validation,., W.T.-A. and E.M.-R; formal analysis, M.F.G.-O. and G.G.-S.; investigation, J.C.R.-F.; re- 540 sources C.L.D.-T.-S., data curation, F.J.W.-C.; writing—original draft preparation, M.F.G.-O.; super- 541 vision, C.L.D.-T.-S.. All authors have read and agreed to the published version of the manuscript. 542 Funding: This research was funded by Universidad de Sonora, grant number USO313007398. 543 Institutional Review Board Statement: The study was carried out in accordance with the Declara- 544 tion of Helsinki of 1975. The clinical laboratory is accredited by ISO-IEC 17.025 (NMX-EC-17025) 545 and ISO 15.189 prepared by the technical committee ISO/TC 212 (Clinical Laboratory Testing and 546 In vitro Diagnostic Systems) taking as reference the ISO/IEC 17.025 and ISO 9001 standards. 547 Informed Consent Statement: Informed consent was obtained from all subjects involved in the 548 study. Informed consent was for blood donation. Written informed consent has been obtained from 549
the patients to publish this paper. 550
551 Data Availability Statement: The original contributions data presented in this research are included 552 in the article; further inquiries can be directed to the corresponding authors. 553 Acknowledgments: María Fernanda González Osuna acknowledges the Consejo Nacional de Cien- 554
cia y Tecnología (CONACyT, Mexico) for a Ph.D. scholarship. 555
Conflicts of Interest: The authors declare no conflict of interest. 556 557
