Next Article in Journal
Micro RNA-124a Regulates Lipolysis via Adipose Triglyceride Lipase and Comparative Gene Identification 58
Next Article in Special Issue
The Role of Food Peptides in Lipid Metabolism during Dyslipidemia and Associated Health Conditions
Previous Article in Journal
Genome-Wide Identification and Evolution of HECT Genes in Soybean
Previous Article in Special Issue
Major Peptides from Amaranth (Amaranthus cruentus) Protein Inhibit HMG-CoA Reductase Activity
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 16
Issue 4
10.3390/ijms16048536
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Characterization of Peptides Found in Unprocessed and Extruded Amaranth (Amaranthus hypochondriacus) Pepsin/Pancreatin Hydrolysates

by
Alvaro Montoya-Rodríguez
1,2,
Jorge Milán-Carrillo
1,
Cuauhtémoc Reyes-Moreno
1 and
Elvira González De Mejía
2,*
1
North West Regional Program in Biotechnology, University of Sinaloa, FCQB-UAS, AP 1354, CP 80000 Culiacán, Sinaloa, Mexico
2
Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 228 ERML, MC-051, 1201 West Gregory Drive, Urbana, IL 61801, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2015, 16(4), 8536-8554; https://doi.org/10.3390/ijms16048536
Submission received: 15 September 2014 / Revised: 25 September 2014 / Accepted: 2 April 2015 / Published: 16 April 2015
(This article belongs to the Special Issue Bioactive Proteins and Peptides Derived from Food)
Browse Figures
Versions Notes

Abstract

:
The objectives of this study were to characterize peptides found in unprocessed amaranth hydrolysates (UAH) and extruded amaranth hydrolysates (EAH) and to determine the effect of the hydrolysis time on the profile of peptides produced. Amaranth grain was extruded in a single screw extruder at 125 °C of extrusion temperature and 130 rpm of screw speed. Unprocessed and extruded amaranth flour were hydrolyzed with pepsin/pancreatin enzymes following a kinetic at 10, 25, 60, 90, 120 and 180 min for each enzyme. After 180 min of pepsin hydrolysis, aliquots were taken at each time during pancreatin hydrolysis to characterize the hydrolysates by MALDI-TOF/MS-MS. Molecular masses (MM) (527, 567, 802, 984, 1295, 1545, 2034 and 2064 Da) of peptides appeared consistently during hydrolysis, showing high intensity at 10 min (2064 Da), 120 min (802 Da) and 180 min (567 Da) in UAH. EAH showed high intensity at 10 min (2034 Da) and 120 min (984, 1295 and 1545 Da). Extrusion produced more peptides with MM lower than 1000 Da immediately after 10 min of hydrolysis. Hydrolysis time impacted on the peptide profile, as longer the time lower the MM in both amaranth hydrolysates. Sequences obtained were analyzed for their biological activity at BIOPEP, showing important inhibitory activities related to chronic diseases. These peptides could be used as a food ingredient/supplement in a healthy diet to prevent the risk to develop chronic diseases.

Graphical Abstract

1. Introduction

Amaranth (Amaranthus hypochondriacus) is a native grain of Mexico, where it has been used since pre-Columbian civilizations by Aztecs and Mayas [1,2,3]. The importance of amaranth has resurged in the past 20 years because of its agricultural features, since it is a fast growing cultivar with tolerance to drought conditions. In addition, amaranth can grow in poor soils where common crops cannot grow. Likewise, amaranth has important nutritional and nutraceutical properties [4,5,6]. Amaranth grain has excellent chemical composition with high concentration of proteins (13%–19%), which is higher than in cereals [7,8,9,10]. Globulins, albumins and glutelins are the principal proteins in amaranth [11]. These proteins have great nutritional quality and value, with excellent balance of essential amino acids such as lysine and sulfur-containing amino acids, which are deficient in traditional cereals and legumes, respectively [12,13,14]. The quality of amaranth proteins is comparable to the optimum protein reference pattern in the human diet, almost reaching the requirements according to FAO/WHO [15,16]. It also presents high digestibility (90% of the proteins are digested) [17]. These characteristics suggest that amaranth could be used in a mixture or combination with cereals to improve the quality of the protein and have a better nutritive food [18]. The combination of amaranth and maize flour in a ratio 50:50 almost reaches the 100 score on the nutritional scale. The limiting amino acids in amaranth seed are leucine, isoleucine and valine. However, they are not a serious problem, since they are in excess in most common grains [12]. In addition, amaranth does not contain gluten which is suitable for people with celiac disease [19]. Due to the high protein concentration, amaranth could be a source of bioactive peptides. Bioactive peptides are inactive within the parent protein; however, with enzymatic digestion or food processing, peptides can act as physiological modulators of metabolism releasing these bioactive peptides [20]. Extrusion is a high temperature-short-time food processing technology that has been used to get precooked flours with high nutritional value [21] and also it shows a positive effect on antioxidant capacity [22]. Some studies of amaranth have reported the presence of peptides with biological activities such as anti-hypertensive, anti-oxidative, anti-thrombotic, among others [11,23,24]. Our group has evaluated the biological activity of the peptides present in the hydrolysates from unprocessed amaranth (UAH) and extruded amaranth (EAH) [25,26]. We found that peptides present on these hydrolysates have anti-inflammatory and anti-atherosclerotic potential. However, the specific characteristics of these peptides were not reported.
Hence, the objectives of this study were to perform an extensive characterization of peptides found in UAH and EAH, and to determine the effect of the hydrolysis time on the profile of the peptides produced. For the first time, this manuscript provides novel information on the time-course of the effect of gastrointestinal enzymes to produce peptides from amaranth proteins with potential biological activities throughout hydrolysis. Peptides were characterized by mass spectrometry and their presence in amaranth proteins was identified. This complete characterization of peptides during simulated gastrointestinal digestion of amaranth proteins is unique since it has not been provided in previous publications.

2. Results and Discussion

2.1. Pepsin and Pancreatin Hydrolysates

Unprocessed and extruded amaranth flour hydrolysates were obtained after 360 min of hydrolysis; 180 min with pepsin and 180 min with pancreatin. After 360 min of hydrolysis, the freeze dried material was obtained and the mass yield (weight of the hydrolysate in grams/weight of original material in grams) was 31% w/w.
The protein electrophoretic profile of UAH gave a distinct protein pattern, characteristic of amaranth proteins [27]. For instance, the 28 kDa band corresponded to amaranth albumins that are water soluble proteins, while 7S and 11S globulin fractions corresponded to proteins of molecular masses 33 and 57 kDa, respectively. The extrusion process changed the protein profile of the amaranth flour showing barely detectable amaranth albumins and 7S and 11S globulins. Extrusion also led to the production of smaller molecular mass proteins and peptides. As expected, the degree of hydrolysis increased in relation to digestion time. Maximum degree of hydrolysis was reached after pancreatin digestion at 360 min for both flours, UAH and EAH. After 180 min of pepsin hydrolysis, most large molecular mass proteins were converted to smaller proteins of molecular mass less than 37 kDa, while pancreatin digestion led to transformation of large molecular mass proteins leading to the production of peptides with molecular mass less than 10 kDa [28].

2.2. MALDI-TOF Hydrolysates Characterization

The unprocessed amaranth hydrolysates characterized by MALDI-TOF are shown in Table 1 and Table 2. Table 1 shows the hydrolysates 10, 25 and 60 min after hydrolysis. The MM of these peptides was between 520 and 3580 Da for these three hydrolysate times. More peptides were present after 10 and 60 min of hydrolysis than after 25 min of hydrolysis. However, the hydrolysate at 25 and 60 min showed peptides with lower MM than the hydrolysate at 10 min. Table 2 shows the hydrolysates at 90, 120 and 180 min after pancreatin hydrolysis. Peptides from 511 to 3900 Da appeared at 90, 120 and 180 min. However, peptides longer than 1200 Da were more abundant only at 120 min.
Table 3 and Table 4 show the extruded amaranth hydrolysates characterized by MALDI-TOF. Table 3 shows the hydrolysates at 10, 25 and 60 min after pancreatin hydrolysis. Peptides with MM between 511 and 4100 Da were present at these hydrolysis times. However, peptides longer than 2100 Da appear with a weak intensity. Table 4 shows the hydrolysates at 90, 120 and 180 min after pancreatin hydrolysis. Peptides from 511 to 3800 Da appear at 90, 120 and 180 min. However, peptides longer than 2100 Da appear with weak intensity and were only present at 120 min.
The hydrolysis time impacted on the MM of the peptides in both flours (Table 5). More peptides with low MM appeared after 90 min of hydrolysis in both flours. However, in the extruded amaranth hydrolysates, peptides with low MM appeared immediately after 10 min of hydrolysis. This could be explained by the extrusion process since higher levels of free amino acids were present and the fact that during protein enzymatic hydrolysis amino acids were released [28,29]; extrusion could cause protein hydrolysis, producing small peptides during the process. In addition, the extrusion process produced partial denaturation of the proteins, being the proteins more available for the enzyme action. The extrusion process showed more peptides with lower MM at the end of the hydrolysis and also more peptides with biologically active sequences.
MM of 527, 567, 802, 984, 1295, 1545, 2034 and 2064 Da appeared consistently in the whole hydrolysis time in both flours. MALDI-TOF analysis showed high intensity peaks of MM at 10 min (2064 Da), 120 min (802 Da) and 180 min (567 Da) in UAH, while EAH showed high intensity at 10 min (2034 Da) and 120 min (984, 1295 and 1545 Da). In addition, most of these peptides showed a high percent of area under the curve in the MALDI-TOF spectrum analysis. Due to the fact that these peptides appeared consistently throughout hydrolysis, the time where each one showed the highest intensity was selected to be analyzed by MS-MS to know their sequence.

2.3. Characterization of UAH and EAH Amaranth Peptides by MS-MS

After MALDI-TOF analysis, peaks with the highest intensity were selected to be fragmented by MS-MS to know their peptide sequence. Figure 1 shows the peaks with the highest intensity found in UAH. Figure 1A shows the MALDI-TOF spectrum for the peptide with MM of 2064 Da and Figure 1B shows the MS-MS spectrum for the same peptide. After the MS-MS fragmentation, analysis of the spectrum shows that the potential sequence of the peptide was CAPYYLERWYRRKLF (2064 Da). Likewise, Figure 1C shows the MALDI-TOF spectrum for the peptide with MM of 802 Da and the MS-MS fragmentation was shown in Figure 1D, with a sequence for the peptide EGDAZPGE (802 Da). Figure 1E shows the MALDI-TOF spectrum for the peptide with MM of 567 Da and in Figure 1F the MS-MS spectrum of the peptide sequence with MM of 567 Da, GTFNE.
Ijms 16 08536 g001 1024
Figure 1. MALDI-TOF analyses of unprocessed amaranth hydrolysates after 10 min (A); 120 min (C) and 180 min (E) with pancreatin. The peaks with molecular mass of 2064 (B); 802 (D) and 567 (F) were analyzed by MS-MS. These peaks were present along the hydrolysis times and showed the highest intensity at these hydrolysis times.
Figure 1. MALDI-TOF analyses of unprocessed amaranth hydrolysates after 10 min (A); 120 min (C) and 180 min (E) with pancreatin. The peaks with molecular mass of 2064 (B); 802 (D) and 567 (F) were analyzed by MS-MS. These peaks were present along the hydrolysis times and showed the highest intensity at these hydrolysis times.
Ijms 16 08536 g001
Table
Table 1. Characterization by MALDI-TOF of peptides from unprocessed amaranth pancreatin hydrolysates at 10, 25 and 60 min.
Table 1. Characterization by MALDI-TOF of peptides from unprocessed amaranth pancreatin hydrolysates at 10, 25 and 60 min.
MM (Da)10 RA25 RA60 RA
IntensityArea% of AreaIntensityArea% of AreaIntensityArea% of Area
520---853325203.47---
52227966980.9716,06150446.95---
52738479381.31539613741.8923,05347125.65
543------461710191.22
563---629017742.44681614331.72
56728058261.1511,59832004.4113,10727973.35
571---360710511.4538817950.95
613---329810001.3835157740.93
616---422213571.87489711171.34
617---10,31032564.4812,61430453.65
61928719201.28350511321.56470410781.29
620---390513921.92540915771.89
62129119111.2710,78834134.7013,87134774.17
623------376311521.38
625---409814351.98585414701.76
802258510051.40349015412.1231089711.16
821------25607710.92
825---395717292.38500815911.91
829---311815612.15429414081.69
931425119072.66451122593.11307310651.28
984------267010941.31
102818927541.05------
104514057201.00------
1060195210571.47302018032.48---
1160199411011.53185012171.68---
1234------248714241.71
1295213013931.94385128503.93351721072.52
1435------166812921.55
1545334327033.77280825653.53143311341.36
184599025153.51---136931233.74
1899---112415032.07---
2014231628253.9491211661.6182110401.25
20346795880912.28366946286.37464753846.45
2050126025593.57---129222892.74
2056------80313581.63
206422,15725,39835.40980913,09218.0316,88320,85924.99
2071------327838544.62
2076304840235.61239732034.41---
2086168420132.81---281833844.05
2106179024043.35135618732.58188820632.47
26095779711.35------
27834917321.02------
2889114219522.7258212991.79---
3027138026123.6496723673.263316560.79
3267------3957850.94
3581------58313691.64
MM = Molecular mass; Da = Daltons; RA = Unprocessed flour with pancreatin; 10, 25, 60 = Minutes of hydrolysis with pancreatin after 180 with pepsin. Italics indicate the mass of the peptides with the highest intensity or % area.
Table
Table 2. Characterization by MALDI-TOF of peptides from unprocessed amaranth pancreatin hydrolysates at 90, 120 and 180 min.
Table 2. Characterization by MALDI-TOF of peptides from unprocessed amaranth pancreatin hydrolysates at 90, 120 and 180 min.
MM (Da)90 RA120 RA180 RA
IntensityArea% of AreaIntensityArea% of AreaIntensityArea% of Area
511---445611470.97---
51839429111.86------
52010,31121624.42791817871.51---
52216,11334076.9712,11529172.47---
52728,308581911.9030,90168635.8117,70841867.01
543688721514.40678517171.4535789521.59
54434027651.57------
551348317013.48---313916012.68
563812216393.35605513481.14641816412.75
56715,28531496.44129026612.2519,12728494.77
57146528881.8238157620.6534388751.47
60537028551.75---24296891.15
61340869231.89---31139501.59
61435599421.93------
616583313742.81393611921.01427512542.10
61714,66634797.12934125552.16982027504.61
619572913012.66421312791.08331110551.77
620643418423.7712,15934052.88369511511.93
62116,89841228.43---10,01930525.11
623446713942.85---26968561.43
625694517243.53456914561.23369312982.17
642516311962.45------
802---847430292.57364611912.00
82132659571.96------
825653319864.06484316161.37371014032.35
829558217483.58417814471.23282212272.06
931---554625092.1222938711.46
984---865145143.82797434945.85
1028------324112712.13
1029------298813442.25
1100---385221771.8419719911.66
1103------15927321.23
1152------16228701.46
1161------16749181.54
1182------180210411.74
1236206111682.39409421551.83---
1295204512762.61572239773.37564135035.87
1436---291421601.83---
1545------191615682.63
1845---288061935.24---
2034163817643.61479155464.7084910401.74
2046---106416931.43---
2050---247646153.91---
2056---142826732.26---
20645467605212.3817,25025,78221.847614941915.78
2076141215923.26264434732.94---
2086137414142.89613870245.95---
2102---130615101.28---
21068389441.93206023892.02---
2118---146315561.32---
326793616703.4287716971.4487318123.04
3585------1544110.69
3910---2785980.511433770.63
3931---3026530.55---
MM = Molecular mass; Da = Daltons; RA = Unprocessed flour with pancreatin; 90, 120, 180 = Minutes of hydrolysis with pancreatin after 180 with pepsin. Italics indicate the mass of the peptide with the highest intensity or % area.
Table
Table 3. Characterization by MALDI-TOF of peptides from extruded amaranth pancreatin hydrolysates at 10, 25 and 60 min.
Table 3. Characterization by MALDI-TOF of peptides from extruded amaranth pancreatin hydrolysates at 10, 25 and 60 min.
MM (Da)10 EA25 EA60 EA
IntensityArea% of AreaIntensityArea% of AreaIntensityArea% of area
511402312001.18------
52744,86483488.1832,29475465.7032,76571958.73
543961923632.32806118531.40733316562.01
563533113031.2810,37125281.91621313561.64
567823320542.0110,79746523.5112,11127453.33
57124066460.63606214161.0743509491.15
61327056380.63561813661.0331968411.02
61634778530.84714918231.38476512411.51
617863822472.2017,48447593.6010,56528633.47
61928809080.89646818331.38371911721.42
620423310651.04711823871.80459710781.31
621940825912.5419,36154294.1012,25434054.13
62325378150.80532317071.29363811201.36
625386111021.08839525451.92528416131.96
802------691830913.75
821---403713251.00---
825356212891.26713025051.89526517932.17
829285310681.05578421961.66438416582.01
931------424722362.71
984------343119382.35
1029175110671.05------
1058---285323281.76---
1152------216115641.90
1175------228712441.51
1295---273722531.70301023172.81
1510-------17192.08
1545---638367055.07152038374.65
2014---103615681.18376064887.87
2034161824962.45679696067.26440214,64917.77
2064401063996.2715,95125,29519.1110,24836544.43
2076105517641.73386453004.00268121792.64
2106---213729532.23155533594.07
3267---3066040.46118417492.12
3581---56112200.9255517492.12
4046111913,32213.0649062464.72---
405981311,55511.3329237042.80---
4080170622,88722.4436156434.26---
4098---28041093.10---
41059631403313.76------
4121---56989446.76---
MM = Molecular mass; Da = Daltons; EA = Extruded flour with pancreatin; 10, 25, 60 = Minutes of hydrolysis with pancreatin after 180 min with pepsin. Italics indicate the mass of the peptide with the highest intensity or % area.
Table
Table 4. Characterization by MALDI-TOF of peptides from extruded amaranth pancreatin hydrolysates at 90, 120 and 180 min.
Table 4. Characterization by MALDI-TOF of peptides from extruded amaranth pancreatin hydrolysates at 90, 120 and 180 min.
MM (Da)90 EA120 EA180 EA
IntensityArea% of AreaIntensityArea% of AreaIntensityArea% of Area
511343110351.28404716721.44288912252.14
52733,33368728.4935,2831166410.0727,734679311.85
54310,12721562.6710,82427982.41562914662.56
551424213121.62------
563645713941.72649418931.63482413572.37
56711,13124473.0211,45431502.72849122273.89
57132046340.7832229730.84---
61335808541.0631478930.7724927311.28
616408212501.55456311561.0029239261.62
61712,15730443.76968227522.37885625014.36
619430310771.3336709950.8628399201.61
620525014381.78430013731.1832959981.74
62113,40134854.3110,85931882.75872625674.48
623349411061.37316510010.8624807841.37
625531514391.78429314021.21341011211.96
663---332411160.96---
802765033184.10686028902.49734234095.95
825453215231.88428316511.42299912482.18
829424814911.84348115141.31231310771.88
931517123132.86345017011.47371519773.45
984653229783.68933146834.04927151839.04
1013---280616861.45---
1030------191210811.89
1058---252616531.43---
1100---317918411.59305518203.18
1152286417172.12415626072.25274118793.28
1175208710391.28252316381.41---
1182242213941.72442328982.50376926174.57
1295340323042.85789059015.09349330485.32
1510171814891.84158216171.40---
1545330429873.6910,22810,3108.90259730215.27
1716120914351.77------
2034330843715.402113357022.03---
2064850612,65615.6414,57525,5232.33326150528.81
2076431062027.67189827031.69---
2106270037064.58126619641.88---
32672064340.5488221810.6885922863.99
3581---2847850.46---
3799---167536100---
MM = Molecular mass; DA = Daltons; EA = Extruded flour with pancreatin; 90, 120, 180 = Minutes of hydrolysis with pancreatin after 180 min with pepsin. Italics indicate the mass of the peptides with the highest intensity or % area.
Table
Table 5. Effect of hydrolysis time and extrusion process on peptide profile of amaranth proteins. Higher occurrence of peptides with low molecular mass appears after 90 min of hydrolysis.
Table 5. Effect of hydrolysis time and extrusion process on peptide profile of amaranth proteins. Higher occurrence of peptides with low molecular mass appears after 90 min of hydrolysis.
Hydrolysis Time (min)
-UNPROCESSED (%AUC)EXTRUDED (%AUC)
MM (Da)1025609012018010256090120180
0–100010.0448.2637.5572.7535.7457.6327.9334.6646.0250.8941.8263.70
1000–200014.2813.6910.883.8614.1120.501.058.5312.9515.2826.0223.50
2000–300072.0434.7948.2018.4347.6517.5210.4533.7936.7833.2929.138.81
>30003.643.263.374.992.504.3660.5823.024.220.543.023.99
MM: Molecular mass; Da: Daltons; AUC: Area under curve; min: Minutes.
The peaks with highest intensity present in EAH are shown in Figure 2 and Figure 3. Figure 2A,C show the MALDI-TOF spectrum for the peptides with MM of 527 and 2034 Da, respectively. While in Figure 2B,D their MS-MS spectra are shown, respectively. The sequence for these MM were RSHK (527 Da) and NRPWWWHPGGGGGGGGLGAGT (2034 Da). Figure 3 shows the MALDI-TOF and MS-MS spectrum of 984 Da (3A and 3B, respectively), 1295 Da (3C and 3D, respectively) and 1545 Da (3E and 3F, respectively). The sequences of these 3 peptides were HGSEPFGPR, RPRYPWRYT and RDGPFPWPWYSH, respectively. All of these peptides, found in both flours, unprocessed and extruded, are hydrophobic and most of them (except EGDAZPGE and GTFNE) present positive net charge [25].
It is important to mention that, based on the MS-MS identification of some of the peptide sequences, amino acid sequences present in proteins such as 11S globulin seed storage protein, polyamine oxidase, glucosyltransferase, among others, were found.
Silva-Sánchez et al. [11] and Velarde-Salcedo et al. [30] also found peptides derived from the hydrolysis of amaranth (Amaranthus hypochondriacus L.) proteins by MS-MS analysis.
Ijms 16 08536 g002 1024
Figure 2. MALDI-TOF analyses of extruded amaranth hydrolysates after 10 min (A) and 25 min (C) with pancreatin. The peaks with molecular mass of 527 (B) and 2034 (D) were analyzed by MS-MS. These peaks were present along the hydrolysis times and showed the highest intensity at these hydrolysis times.
Figure 2. MALDI-TOF analyses of extruded amaranth hydrolysates after 10 min (A) and 25 min (C) with pancreatin. The peaks with molecular mass of 527 (B) and 2034 (D) were analyzed by MS-MS. These peaks were present along the hydrolysis times and showed the highest intensity at these hydrolysis times.
Ijms 16 08536 g002
Peptide sequences in amaranth have been associated with potential biological activities such as antioxidant capacity; inhibitors of dipeptidyl peptidase IV (DPP-IV) important in the management of diabetes; angiotensin converting enzyme inhibitor; and antithrombotic activity [31]. Montoya-Rodriguez et al. [25,26] reported that extruded amaranth hydrolysates showed potential anti-inflammatory and anti-atherosclerotic activity in vitro, probably attributed to the production of bioactive peptides during the extrusion process. The degree of hydrolysis had a differential effect on the antioxidant capacity of UAH and EAH [25,26]. For instance, in UAH maximum antioxidant capacity was reached after 120 min of pepsin digestion while maximum antioxidant capacity was reached after 25 min of pancreatin digestion. This can be attributed to different antioxidant characteristics of the peptides generated during hydrolysis; different peptides have different antioxidant capacity dictated by their amino acid composition. For instance, the most reactive amino acids are sulfur-containing Met and Cys, aromatic Trp, Phe, and His, attributed to its imidazole ring. Most of the molecular masses of the peptides ranged from 500 to 1800 kDa showed high values of antioxidant capacity. Peptides with similar molecular masses were found in this study. Aside from the degree of hydrolysis, extrusion affected the antioxidant capacity of the hydrolysates. Pepsin hydrolysates from UAH had higher antioxidant capacity than hydrolysates from EAH. Extrusion can also lead to interactions between proteins and other macromolecules such as starch and lipids. Amaranth grain has a high starch content (60%–70%); which makes the protein less available as an antioxidant. However, when pancreatin was used, those interactions could have been hydrolyzed freeing the generated peptides hence the observed increase in antioxidant capacity after pancreatin hydrolysis in EAH; pancreatin is a mixture of enzymes including amylases, lipases and proteases, leading to almost complete hydrolysis and production of small molecules. More research is needed to determine the structural changes of amaranth protein-carbohydrates interactions under the extrusion conditions used in this study.
Ijms 16 08536 g003 1024
Figure 3. MALDI-TOF analyses of extruded amaranth hydrolysates after 120 min (A,C,E) with pancreatin. The peaks with molecular mass of 984 (B); 1295 (D); and 1545 (F) were analyzed by MS-MS. These peaks were present along the hydrolysis and showed the highest intensity at these hydrolysis times.
Figure 3. MALDI-TOF analyses of extruded amaranth hydrolysates after 120 min (A,C,E) with pancreatin. The peaks with molecular mass of 984 (B); 1295 (D); and 1545 (F) were analyzed by MS-MS. These peaks were present along the hydrolysis and showed the highest intensity at these hydrolysis times.
Ijms 16 08536 g003
Tovar-Pérez et al. [32] reported peptides from amaranth hydrolysates with ACE-inhibitory activity. Vecchi and Añon [33] reported that peptides present in the globulin 11S of the amaranth showed ACE-inhibitory activity. Maldonado-Cervantes et al. [34] reported that the amaranth lunasin-like peptide, obtained from fragmented amaranth proteins, showed potential anti-cancer activity. Enzymatic hydrolysis or food processing could be used to obtain peptides with potential biological activity; however, the method used will depend on several factors such as the bioactive peptide of interest to be released and intended use of the peptide. Although the complete mechanism of absorption and the bioavailability of specific peptides are still under investigation, there is enough evidence to conclude that food bioactive peptides are bioavailable and can be absorbed into the body [35].
The significance and importance of this study was that it provides new information on the kinetics of hydrolysis of amaranth proteins, exposed to gastrointestinal enzymes, to produce peptide sequences with potential biological activities.

3. Experimental Section

3.1. Materials

The amaranth (Amaranthus hypochondriacus) grain was purchased at a local market in Temoac, Morelos, Mexico. The amaranth grains were packed in lots of 1 kg and storage in refrigeration (5–10 °C) until their use.

3.2. Extrusion Process

The extruded amaranth flours were obtained according to procedure recommended by Milán-Carrillo et al. [22] The amaranth grains (1 kg lots) were milled and mixed with lime (0.21 g Ca(OH)2/100 g amaranth) and conditioned with purified water to reach a moisture content of 28%. Each lot was packed in a polyethylene bag and stored at 4 °C for 8 h. Before extrusion, the flours were tempered at 25 °C for 4 h. A single screw laboratory extruder Model 20 DN (CW Brabender Instruments, Inc., South Hackensack, NJ, USA) with a 19 mm screw-diameter; length to diameter 20:1; nominal compression ratio 2:1; and die opening of 3 mm was used. The inner barrel was grooved to ensure zero slip at the wall. The temperature in the barrel was the same for the three zones and the end zone was cooled by air. A third zone, at the die barrel, was also electrically heated but not cooled by air. The feed rate was 30 rpm. Extrusion temperature (ET) was defined as temperature at the die end of the barrel. Extrusion operation conditions were: ET, 125 °C and screw speed (SS) of 130 rpm. After extrusion the extrudates were cooled, equilibrated at environmental conditions (25 °C, RH = 65%), milled (UD Cyclone Sample Mill, UD Corp, Boulder, CO, USA) to pass through an 80-US mesh (0.180 mm) screen, packed in plastic bags, and stored at 4 °C.

3.3. Preparation of Amaranth Protein Hydrolysates

In vitro simulated digestion of amaranth protein was performed following the procedure by Megías et al. [36] with several modifications. Briefly, unprocessed and extruded amaranth flours were suspended in water (1:20 w/v) and a sequential enzyme digestion was carried out with pepsin (EC 3.4.23.1, 662 units/mg; enzyme/substrate, 1:20 (w/w); pH 2.0) and pancreatin (8× USP; enzyme/substrate, 1:20 (w/w); pH 7.5) at 37 °C for 3 h each. The hydrolysis with pepsin was stopped increasing the pH until 7.5 to start the hydrolysis with pancreatin, which was stopped by heating at 75 °C for 20 min, and the resulting hydrolysates were centrifuged at 20,000× g for 15 min at 4 °C. Aliquots of 50 mL were taken at 10, 25, 60, 90, 120 and 180 min during both hydrolysis, with pepsin and pancreatin. The total hydrolysis time was 360 min (180 min with pepsin followed by 180 min with pancreatin). Once the hydrolysates were desalted, they were freeze dried in a Labconco (Kansas, MO, USA) Freeze Dryer 4.5.

3.4. Peptide Characterization

For sample preparation, 1 mg of amaranth hydrolysates (UAH and EAH) were dissolved in 1 mL of deionized water and analyzed by using an UltrafleXtreme MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a frequency tripled Nd:YAG solid state laser using the FlexControl 1.4 software package (Bruker Daltonics). MS/MS analysis of each ion of interest, the peptide with the highest intensity under the curve, was performed at 500 Hz in LIFT mode using a randomized raster, summed and saved for analysis. Data processing was performed using the FlexAnalysis 3.4 software package (Bruker Daltonics, Bremen, Germany) and Biotools 3.2 (Bruker Daltonics, Bremen, Germany). The percentage of each peptide was calculated based on the area under the curve method. The total area for each sample was calculated by taking the sum of all the areas under the curve. Then the area under the curve for each peptide identified was divided by the total area and the percentage was calculated. Potential biological activities of the peptides were obtained using BIOPEP tool (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep, accessed on 18 August 2014). Protein sequences were confirmed using BLAST® tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

4. Conclusions

The hydrolysis time impacted the peptide profile in both hydrolysates, from unprocessed and extruded flours. The extrusion process had a higher impact on the peptide profile, producing more peptides with lower MM and biological activity. The in vitro digestion with pepsin and pancreatin produced peptides with biological activity, which represented the simulated digestion and demonstrated that peptides could be formed during real digestion. In addition, the extrusion process was a good alternative as pre-treatment because it could break-down food proteins and made them more available to enzyme action. In summary, unprocessed and extruded amaranth flours are sources of peptides with potential biological activity, such as ACE-inhibitor and DPP-IV inhibitor, which are related with the prevention of important chronic diseases. Amaranth flours, unprocessed and extruded, as well as the peptides found in both flours, could be used as an ingredient or food supplement in a healthy diet to prevent the risk to develop chronic diseases.

Acknowledgments

Alvaro Montoya-Rodríguez was supported by a CONACYT Fellowship. This work was supported by a research grant from the University Autonomous of Sinaloa, Culiacán, Mexico in collaboration with the University of Illinois.

Authors Contributions

Jorge Milán-Carrillo and Cuauhtémoc Reyes-Moreno proposed the project. Alvaro Montoya-Rodríguez developed and wrote the manuscript. Elvira González de Mejía provided scientific guidance throughout the research, suggested, revised and edited the manuscript. All authors read and approved the manuscript.

Abbreviations

ACE-Inhibitor
Angiotensin converting enzyme-inhibitor
DPP-IV-Inhibitor
Dipeptidyl peptidase IV inhibitor
EAH
Extruded amaranth hydrolysate
ET
Extrusion temperature
FAO
Food and agriculture organization
MALDI-TOF
Matrix-assisted laser desorption/ionization- time-of-flight mass spectrometer
MM
Molecular mass
MS-MS
Tandem mass spectrometry
RH
Relative humidity
rpm
Revolutions per minute
SS
Screw speed
UAH
Unprocessed amaranth hydrolysate
USP
Enzyme units (United States Pharmacopeia)
WHO
World health organization

Conflicts of Interest

The authors have declared no conflict of interest.

References

  1. Milán-Carrillo, J.; Montoya-Rodríguez, A.; Reyes-Moreno, C. High-antioxidant capacity beverages based on extruded and roasted amaranth (Amaranthus hypochondriacus) flour. In Hispanic Foods: Chemistry and Bioactive Compounds, 1st ed.; Tunick, M.H., González de Mejía, E., Eds.; American Chemical Society: Washington, DC, USA, 2012; Volume 1109, pp. 199–216. [Google Scholar]
  2. Pavlik, V. The revival of amaranth as a third-millennium food. Neuroendocrinol. Lett. 2012, 33, 3–7. [Google Scholar] [PubMed]
  3. Zapotoczny, P.; Markowski, M.; Majewska, K.; Ratajski, A.; Konopko, H. Effect of temperature on the physical, functional, and mechanical characteristics of hot-air-puffed amaranth seeds. J. Food Eng. 2006, 76, 469–476. [Google Scholar] [CrossRef]
  4. Gallegos-Tintoré, S.; Chel-Guerrero, L.; Corzo-Ríos, L.J.; Martínez-Ayala, A.L. Péptidos con actividadantioxidante de proteínas vegetales. In Bioactividad de Péptidos Derivados de Proteínas Alimentarias; Segura-Campos, M., Chel-Guerrero, L., Betancur-Ancona, D., Eds.; OmniaScience: Terrassa, Spain, 2013; Volume 4, pp. 111–122. [Google Scholar]
  5. Khandaker, L.; Masum-Akond, A.S.M.G.; Ali, M.B.; Oba, S. Biomass yield and accumulations of bioactive compounds in red amaranth (Amaranthus tricolor L.) grown under different colored shade polyethylene in spring season. Sci. Hortic. 2010, 123, 289–294. [Google Scholar]
  6. Avanza, M.V.; Puppo, M.C.; Añón, M.C. Structural characterization of amaranth protein gels. J. Food Sci. 2005, 70, 223–229. [Google Scholar] [CrossRef]
  7. Alvarez-Jubete, L.; Wijngaard, H.; Arendt, E.K.; Gallagher, E. Polyphenol composition and in vitro antioxidant activity of amaranth, quinoa buckwheat and wheat as affected by sprouting and baking. Food Chem. 2010, 119, 770–778. [Google Scholar] [CrossRef]
  8. Ferreira, T.A.; Gómez-Áreas, J.A. Calcium bioavailability of raw and extruded amaranth grains. Cienc. Tecnol. Aliment. 2010, 30, 532–538. [Google Scholar] [CrossRef] [Green Version]
  9. Repo-Carrasco-Valencia, R.; Hellstrom, J.K.; Pihlava, J.M.; Mattila, P.H. Flavonoids and other phenolic compounds in andean indigenous grains: Quinoa (Chenopodium quinoa), kaniwa (Chenopodium pallidicaule) and kiwicha (Amaranthus caudatus). Food Chem. 2010, 120, 128–133. [Google Scholar] [CrossRef]
  10. Aguilar, E.G.; Peiretti, E.G.; Uñates, M.A.; Marchevsky, E.J.; Escudero, N.L.; Camiña, J.M. Amaranth seed varieties: A chemometric approach. Food Measure 2013, 7, 199–206. [Google Scholar] [CrossRef]
  11. Silva-Sánchez, C.; Barba de la Rosa, A.P.; León-Galván, M.F.; De Lumen, B.O.; de León-Rodríguez, A.; González de Mejía, E. Bioactive peptides in amaranth (Amaranthus hypochondriacus) seed. J. Agric. Food Chem. 2008, 56, 1233–1240. [Google Scholar] [CrossRef] [PubMed]
  12. Grobelnik-Mlakar, S.; Turinek, M.; Jakop, M.; Bavec, M.; Bavec, F. Nutrition value and use of grain amaranth: potential future application in bread making. Agricultura 2009, 6, 43–53. [Google Scholar]
  13. Morales de León, J.; Camacho, M.E.; Bourges, H. Aminoacid composition of some Mexican foods. Arch. Latinoam. Nutr. 2005, 55, 172–186. [Google Scholar] [PubMed]
  14. Awasthi, C.P.; Kumar, A.; Singh, N.; Thakur, R. Biochemical composition of grain amaranth genotypes of himachal pradesh. Indian J. Agric. Biochem. 2011, 24, 141–144. [Google Scholar]
  15. Rastogi, A.; Shukla, S. Amaranth: A new millennium crop of nutraceutical values. Crit. Rev. Food Sci. 2013, 53, 109–125. [Google Scholar] [CrossRef]
  16. Shevkani, K.; Singh, N.; Chand-Rana, J.; Kaur, A. Relationship between physicochemical and functional properties of amaranth (Amaranthus hypochondriacus) protein isolates. Int. J. Food Sci. Technol. 2014, 49, 541–550. [Google Scholar] [CrossRef]
  17. Grobelnik-Mlakar, S.; Turinek, M.; Jakop, M.; Bavec, M.; Bavec, F. Grain amaranth as an alternative and perspective crop in temperate climate. J. Geogr. 2010, 5, 135–146. [Google Scholar]
  18. Gorinstein, S.; Pawelzik, E.; Delgado-Licon, E.; Haruenkit, R.; Weisz, M.; Trakhtenberg, S. Characterization of pseudocereal and cereal proteins by protein and amino acid analyses. J. Sci. Food Agric. 2002, 82, 886–891. [Google Scholar] [CrossRef]
  19. Alvarez-Jubete, L.; Arendt, E.K.; Gallagher, E. Nutritive value of pseudocereals and their increasing use as functional gluten free ingredients. Food Sci. Technol. 2010, 21, 106–113. [Google Scholar] [CrossRef]
  20. Pihlanto-Leppälä, A.L.; Koskinen, P.; Piilola, K.; Tupasela, T.; Korhonen, H. Angiotensin I-converting enzyme inhibitory properties of whey protein digests: Concentration and characterization of active peptides. J. Dairy Res. 2000, 67, 53–64. [Google Scholar] [CrossRef] [PubMed]
  21. Milán-Carrillo, J.; Gutiérrez-Dorado, R.; Perales-Sánchez, J.X.K.; Cuevas-Rodríguez, E.O.; Ramírez-Wong, B.; Reyes-Moreno, C. The optimization of the extrusion process when using maize flour with a modified amino acid profile for making tortillas. Int. J. Food Sci. Technol. 2006, 41, 727–736. [Google Scholar] [CrossRef]
  22. Milán-Carrillo, J.; Montoya-Rodríguez, A.; Gutiérrez-Dorado, R.; Perales-Sánchez, X.; Reyes-Moreno, C. Optimization of extrusion process for producing high antioxidant instant amaranth (Amaranthus hypochondriacus L.) flour using response surface methodology. Appl. Math. 2012, 3, 1516–1525. [Google Scholar]
  23. Barba de la Rosa, A.P.; Fomsgaard, I.S.; Bente, L.; Mortensen, A.G.; Olvera-Martínez, L.; Silva-Sánchez, C.; Mendoza-Herrera, A.; González-Castañeda, J.; de León-Rodríguez, A. Amaranth (Amaranthus hypochondriacus) as an alternative crop for sustainable food production: Phenolic acids and flavonoids with potential impact on its nutraceutical quality. J. Cereal Sci. 2009, 49, 117–121. [Google Scholar] [CrossRef]
  24. Caselato-Sousa, V.M.; Amaya-Farfán, J. State of knowledge on amaranth grain: A comprehensive review. J. Food Sci. 2012, 77, 93–104. [Google Scholar] [CrossRef]
  25. Montoya-Rodríguez, A.; Milán-Carrillo, J.; Dia, V.P.; Reyes-Moreno, C.; González de Mejía, E. Pepsin-pancreatin protein hydrolysates from extruded amaranth inhibit markers of atherosclerosis in LPS-induced THP-1 macrophages-like human cells by reducing expression of proteins in LOX-1 signaling pathway. Proteome Sci. 2014, 12, 30–42. [Google Scholar] [CrossRef] [PubMed]
  26. Montoya-Rodríguez, A.; González de Mejía, E.; Dia, V.P.; Reyes-Moreno, C.; Milán-Carrillo, J. Extrusion improved the anti-inflammatory effect of amaranth (Amaranthus hypochondriacus) hydrolysates in LPS-induced human THP-1 macrophage-like and mouse RAW 264.7 macrophages by preventing activation of NF-κB signaling. Mol. Nutr. Food Res. 2014, 58, 1028–1041. [Google Scholar]
  27. Benítez, R.; Ibarz, A.; Jordi-Pagan, J. Protein hydrolysates: Processes and applications. Acta Bioquim. Clin. Latinoam. 2008, 42, 227–236. [Google Scholar]
  28. Chun, C.; Haifeng, Z.; Mouming, Z.; Hua, C. Effects of extrusion treatment on enzymatic hydrolysis properties of wheat gluten. J. Food Process Eng. 2008, 34, 187–203. [Google Scholar]
  29. Sui, X.; Jiang, L.; Wang, C.; Li, Y. The comparison of amino acid content of the hydrolysate under extrusion pretreatment with non-extrusion pretreatment. Appl. Mech. Mater. 2011, 66–68, 702–708. [Google Scholar]
  30. Velarde-Salcedo, A.J.; Barrera-Pacheco, A.; Lara-González, S.; Montero-Morán, G.M.; Díaz-Gois, A.; González de Mejia, E.; Barba de la Rosa, A.P. In vitro inhibition of dipeptidyl peptidase IV by peptides derived from the hydrolysis of amaranth (Amaranthus hypochondriacus L.) proteins. Food Chem. 2013, 136, 758–764. [Google Scholar] [CrossRef]
  31. Dzubia, M.; Dzubia, B. In silico analysis of bioactive peptides. In Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals; Yoshinori, M., Eunice, L.C., Bo, J., Eds.; Blackwell Publishing Ltd. and Institute of Food Technologists: Chicago, IL, USA, 2010; Volume 22, pp. 325–340. [Google Scholar]
  32. Tovar-Pérez, E.G.; Guerrero-Legarreta, I.; Farrés-González, A.; Soriano-Santos, J. Angiotensin I-converting enzyme inhibitory peptide fractions from albumin 1 and globulin as obtained of amaranth grain. Food Chem. 2009, 116, 437–444. [Google Scholar] [CrossRef]
  33. Vecchi, B.; Añón, M.C. ACE inhibitory tetrapeptides from Amaranthus hypochondriacus 11s globulin. Phytochemistry 2009, 70, 864–870. [Google Scholar] [CrossRef] [PubMed]
  34. Maldonado-Cervantes, E.; Jeong, H.J.; León-Galván, F.; Barrera-Pacheco, A.; de León-Rodríguez, A.; González de Mejía, E.; de Lumen, B.O.; Barba de La Rosa, A.P. Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells. Peptides 2010, 3, 11635–1642. [Google Scholar]
  35. González de Mejía, E.; Martínez-Villaluenga, C.; Fernández, D.; Urado, D.; Sato, K. Bioavailability and safety of food peptides. In Food proteins and Peptides: Chemistry, Functionality Interactions, and Commercialization; Hettiarachchy, N.S., Sato, K., Kannan, A., Eds.; Taylor & Francis: Boca Raton, FL, USA, 2012; pp. 297–330. [Google Scholar]
  36. Megías, C.; Yust, M.; Pedroche, J.; Lquari, H.; Girón-Calle, J.; Alaiz, M. Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates. J. Agric. Food Chem. 2009, 52, 1928–1932. [Google Scholar] [CrossRef]

Share and Cite

MDPI and ACS Style

Montoya-Rodríguez, A.; Milán-Carrillo, J.; Reyes-Moreno, C.; De Mejía, E.G. Characterization of Peptides Found in Unprocessed and Extruded Amaranth (Amaranthus hypochondriacus) Pepsin/Pancreatin Hydrolysates. Int. J. Mol. Sci. 2015, 16, 8536-8554. https://doi.org/10.3390/ijms16048536

AMA Style

Montoya-Rodríguez A, Milán-Carrillo J, Reyes-Moreno C, De Mejía EG. Characterization of Peptides Found in Unprocessed and Extruded Amaranth (Amaranthus hypochondriacus) Pepsin/Pancreatin Hydrolysates. International Journal of Molecular Sciences. 2015; 16(4):8536-8554. https://doi.org/10.3390/ijms16048536

Chicago/Turabian Style

Montoya-Rodríguez, Alvaro, Jorge Milán-Carrillo, Cuauhtémoc Reyes-Moreno, and Elvira González De Mejía. 2015. "Characterization of Peptides Found in Unprocessed and Extruded Amaranth (Amaranthus hypochondriacus) Pepsin/Pancreatin Hydrolysates" International Journal of Molecular Sciences 16, no. 4: 8536-8554. https://doi.org/10.3390/ijms16048536

Article Metrics

Back to TopTop