J Food Sci Technol

DOI 10.1007/s13197-016-2428-0

ORIGINAL ARTICLE

Effects of heat and pH treatments and in vitro digestion

on the biological activity of protein hydrolysates of Amaranthus
hypochondriacus L. grain
J. López-Sánchez1 • E. Ponce-Alquicira1 • R. Pedroza-Islas2 • A. de la Peña-Dı́az3 •

J. Soriano-Santos1

Revised: 22 November 2016 / Accepted: 30 November 2016

Ó Association of Food Scientists & Technologists (India) 2016

Abstract The aim of this work was to assess the effects of proteolytic activities of T and C. The hydrolysates kept at
temperature (T), time (t) and pH treatments and an in vitro 100 °C for 1 h at pH 4.0, showed a significant increase in
digestion on the stability of the angiotensin I-converting- bioactivity. Conversely, a treatment at pH 7.0 showed no
enzyme-inhibitory activity (ACEIA) and antithrombotic significant difference (p \ 0.05) in the hydrolysates
activity (ATA; assessed as inhibition of platelet aggrega- bioactivities after their digestion. Thus, biological activity
tion) of selected protein hydrolysates of amaranth named of hydrolysates may be preserved or enhanced, depending
Alb1H103 and GloH88 and GluH24 with dipeptidyl pep- on their processing conditions.
tidase IV inhibitory activity (DPPIVIA). Heat treatment
(40–100 °C) for 1 h showed no significant differences Keywords Amaranth  Hydrolysate  ACE-inhibition 
among ACEIA, DPPIVIA and ATA of the heated hydro- DPP IV inhibition  Antithrombotic activity
lysates at pH 4 and 7. There was no statistically significant
loss of any bioactivity under heat treatment for 3 h at pH
4.0. Alb1H103 and GluH24 maintained the inhibitory Introduction
activity of ACE and ATA at pH 7.0 for 3 h, whereas
GloH88 maintained ACEIA and ATA for 2.0 h at pH 7.0. Currently, in order to manufacture third generation
The pH effect on hydrolysates bioactivity was assessed in nutraceuticals (N) and functional foods (FF), bioactive
the range of 2.0–12.0. This was negligible on ACEIA, ATA compounds must be added. It is essential that these com-
and DPPIVIA. The in vitro digestion was performed using pounds maintain their biological activity (bio-functional-
pepsin, trypsin (T) and a-chymotrypsin (C). A previous ity) under the processing conditions used during their
treatment of hydrolysates with pepsin improved the manufacturing. Techno-functional properties, such as
foaming and emulsification, of proteins and their hydro-
lysates are associated with bio-functionality. The loss of
& J. Soriano-Santos stability of bioactivity (bio-functionality) must also be
[email protected] assessed, and all the more so when severe treatment con-
1
Departamento de Biotecnologı́a, Universidad Autónoma
ditions, such as sterilization, grilling and frying, are per-
Metropolitana, Campus Iztapalapa, Av. San Rafael Atlixco formed in order to maintain the expected physiological
No. 186, Col. Vicentina, Ap. P. 55-535, Deleg. Iztapalapa, benefit of a food.
09340 Mexico City, Mexico The effect of the gastrointestinal digestion on those
2
Departamento de Ingenierı́a y Ciencias Quı́micas, compounds must also be taken into account so that their
Universidad Iberoamericana, Prolongación Paseo de la bioavailability and bioactivity can occur in the different
Reforma 880, Lomas de Santa Fe, 01219 Mexico City,
target organs. The stability of the bioactive compounds in
Mexico
3
the gastrointestinal tract may impact the final outcome. For
Departamento de Farmacologı́a, Facultad de Medicina,
example, the antihypertensive and antithrombotic activities
Universidad Nacional Autónoma de México, Circuito
Escolar, Ciudad Universitaria, Coyoacán, (ATA) of peptide fractions of a commercial fermented milk
04510 Mexico City, Mexico product, made with Lactobacillus casei Shirota and

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 J Food Sci Technol

Streptococcus thermophilus, were maintained during stor- interested in the development of N and FF as these may reduce
age. However, the same milk product, after an in vitro the undesirable side effects caused by synthetic drugs (Rao
partial digestion, lost most of its bioactivity and thus its et al. 2012). Thus, in this study there was an assessment of the
effectiveness as FF (Domı́nguez-González et al. 2014). On effect of different treatment conditions of T, t and pH, and an
the other hand, amaranth protein hydrolysates with angio- in vitro digestion on the stability of the bioactivity of hydro-
tensin I-converting-enzyme-inhibitory activity (ACEIA) and lysates of storage proteins as obtained of A. hypochondriacus
dipeptidyl peptidase IV inhibitory activity (DPPIVIA) L. grain, prior to an animal model assessment.
remained constant after in vitro or in vivo digestion (Fritz
et al. 2011; Soriano-Santos et al. 2015). As a result, the
stability of bioactive compounds in foods and foodstuffs Materials and methods
must be evaluated throughout the different steps of pro-
duction and consumption. Such studies, however, are diffi- Amaranth grain flour and protein hydrolysates
cult to execute and should initially be carried out using an
in vitro model system. These would have to consider not Amaranthus hypochondriacus grain cultivar Revancha
only the interactions of the biological compounds with the obtained from INIFAP-Campus Montecillo, México was
molecular structure of the food matrix as there may be other used in this research. The whole grain was milled using a
compounds with which they can react, but also other factors Udy mill (Udy Corporation Fort Collins, CO, USA) until a
such as temperature (T), time (t) of treatment, pH, ionic 60-mesh screen defatted flour was obtained. Then albumin
strength and so forth. Unfortunately, in vitro and in vivo 1 (Alb1) and globulin (Glo) were extracted from defatted
research on the stability of bioactivity of biological com- flour according to the method described by Tovar-Pérez
pounds, under several processing conditions, is still scarce. et al. (2009). Briefly, defatted flour (50 g) was mixed with
Lately there has been a great deal of research on the 300 ml 0.04 Na2SO4 containing 20 mM b-mercaptoethanol
bioactivity of peptides present in protein hydrolysates as the and stirred for 30 min, centrifuged for 20 min at 13,000g;
former offer various physiological benefits such as antioxi- the supernatant was mixed with 50, 70 and 100% (sat.)
dant, immunomodulating, ACEIA, ATA, DPPIVIA, opioid, (NH4)2SO4. Alb 1 was separated from Glob when super-
anticancer, etc. (Dexter and Middelberg 2008). Particularly, natant was dialysed for 24 h against distilled water. These
storage proteins of amaranth grain, in their primary struc- crude proteins were lyophilised and stored at 5 °C.
ture, contain peptides with ACEIA (Fritz et al. 2011; Sori- The residue, after Alb1 and Glo were obtained, was treated
ano-Santos and Escalona-Buendı́a 2015), DPPIVIA with 70% ethanol to discard prolamins. Thus glutelins (Glu)
(Montoya-Rodriguez et al. 2015; Soriano-Santos et al. 2015; were extracted following the method reported by Barba de la
Velarde-Salcedo et al. 2013) and antithrombotic activity Rosa et al. (2010), using 0.1 M Tris buffer at pH 8.0, in a
(Sabione et al. 2015), among others. These bioactivities residue/buffer ratio 1:10. Protein content was assessed by the
would be highly relevant in the making of N and FF. Kjeldahl method (AOAC 2010). Protein extracts were lyo-
Considerable further research has been conducted by philized and stored at 5 °C until alcalase hydrolysis.
Shevkani and Singh (2015) and Shevkani et al. (2014a, b) on Alcalase hydrolysis of proteins was carried out follow-
several novel commodities derived from amaranth grain and ing the method reported by Tovar-Pérez et al. (2009) with
their use in the design of N and FF. Chemical composition, some modifications: 0.5 M phosphate buffer (pH 7.4) was
physicochemical and techno-functional features of proteins, added to a 5 mg/ml of protein solution. The solution was
lipids and carbohydrates occurring in amaranth grain flour incubated for 5 min at 50 °C; then 2.4 UA/ml of alcalase
have been assessed. Even though there has been some work solution in 0.5 M phosphate buffer was added to each test
on the physicochemical and surfactant properties of these tube to reach a final ratio E/S = 0.8 UA/g protein. The
foodstuffs, their bio-functionality still needs to be studied. reaction, at the appropriate time was stopped by adding
Moreover, the chemical composition of foodstuffs derived 100 ll phenylmethylsufonyl fluoride in ethanol (2 mg/ml).
of amaranth and its influence on techno-functional properties Then several amaranth grain protein hydrolysates were
can also affect the bio-functionality. Therefore, this issue obtained at different conditions.
should prompt researchers to assess the relationship between The degree of hydrolysis (DH) was conducted according
bioactivity and the process used to obtain commodities to the method reported by Condés et al. (2009). Free amino
(Soriano-Santos and Escalona-Buendı́a 2015). groups, released by alcalase hydrolysis, were assessed by
Hypertension, type 2 diabetes mellitus and thrombosis are their reaction with 2,4,6-trinitrobenzenesulfonic acid
common diseases for a considerable number of the elderly (TNBS). L-Leucine was used as a standard. The DH was
throughout the world and they also increase the risk of having calculated as reported by the previous authors.
coronary diseases which could eventually be prevented with Molecular weight characterization of amaranth grain
the intake of N and FF. Therefore, consumers may be storage protein hydrolysates was carried out in agreement

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with the method used by Tovar-Pérez et al. (2009), using a control conditions for platelet aggregation experiments were
molecular exclusion column Sephadex G-15 (1.4 9 29 cm; just platelets induced with 5 lM ADP and the average
Pharmacia, Uppsala, Sweden) and a Pharmacia LKB FPLC control aggregation was taken as 100%.
System (Uppsala, Sweden) for peptides separation. 200 ll
of hydrolyzed proteins (15 mg/ml) disolved in 32.5 Stability of bioactive peptides as influenced
mMK2HPO4–2.6 mM KH2PO4, pH 7.5, which contained by physical and chemical factors
0.4 M NaCl and 20 mM 2-mercaptoethanol, were injected
and eluted with the same buffer at 0.2 ml/min. Absorbance Peptide fractions of hydrolysates (80 g/l) were separately
at 214 nm was monitored and 0.5 ml fractions were col- adjusted to pH 4.0 and 7.0 with buffer solution and heated
lected. An ultra-low range molecular weight marker (40, 60, 80, 100 and 120 °C) for 1 h. The pH effect on
(Sigma-Aldrich, St. Louis, MO, USA), containing triose bioactivity of hydrolysates was assessed using various
phosphate isomerase 26.6 kDa; myoglobin 17 kDa; a-lac- buffer solutions at different pH values (1.0, 2.0, 3.0, 4.0,
talbumin 14.2 kDa; aprotinin 6.5 kDa; insulin 3.5 kDa; 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0), which were
bradykinin 1.06 kDa, was used. All experiments through- heated at 100 °C for 1 h. To investigate the effect of the
out this study were performed in triplicate. duration of heat treatment, peptide solutions were adjusted
to pH 4.0 or 7.0 and incubated at 100 °C for 1.0, 2.0, 3.0, or
Measurement of biological activity of hydrolysates 4.0 h. Unheated peptide fractions were used as control (Wu
et al. 2014).
The ACEIA was measured by the method of Hayakari et al.
(1978), adapted by Tovar-Pérez et al. (2009). The deter- Biostability of peptide fractions on an in vitro
mination of DPPIVIA was performed as reported by gastrointestinal digestion
Kojima et al. (1980), adapted by Soriano-Santos et al.
(2015). The % ACE or % DPPIV inhibition was defined as A simulation of human gastrointestinal digestion of peptide
the percentage of ACE or DPPIV activity inhibited by a fractions was performed in vitro by sequential digestion
given concentration of peptide fraction of hydrolysate using pepsin, trypsin and a-chymotrypsin, according to the
obtained from amaranth grain storage proteins (Alb1, Glo method reported by Rui (2012). Briefly: Freeze dried
or Glu). Captopril and the peptide Diprotin A, which peptide fractions were resolubilised in glycine buffer
inhibit ACE and DPPIV activities, were used as control in (0.01 M, pH 2.0) at 1.5% (w/v). Pepsin digestion was
order to compare the protein hydrolysates IC50 values. carried out with E/S = 1/100 (w/w) at 37 °C for 1.5 h.
The antithrombotic activity was measured as the inhi- Then the digestion was followed adding trypsin and a-
bition of platelet aggregation following the method chymotrypsin with E/S 1/150 (w/w) at 37 °C for 2.5 h,
described by González et al. (2010) with some modifica- maintaining the pH at 6.5 with 1 M NaOH by means of a
tions. Briefly: blood was collected from healthy donors, by pH-stat apparatus (MeterLab PHM290, Radiometer-
venipuncture employing 0.129 mmol/l trisodium citrate as Copenhagen, Denmark). The digestion of the sample was
anticoagulant at the blood bank of the National Institute of stopped by heating it in boiling water for 10 min.
Cardiology ‘‘Ignacio Chávez’’. Platelet-rich (PRP) and
platelet-poor plasma (PPP) were obtained as described by Statistical analysis
de la Peña et al. (1993). The samples were centrifuged at
14009g for 4.5 min at 20–24 °C. PRP was carefully Data were analyzed by means by the analysis of variance
withdrawn and pooled. The platelet count was adjusted to (ANOVA) and Tukey multiple comparison test was used to
2 9 103 cells/ml. The PPP was obtained by a second determine the significant differences between means
centrifugation at 4009g for 15 min. (p \ 0.05).
The assays were carried out within 2 h after the blood had
been drawn, in a two channel lumi-aggregometer (Model
560 CA and accompanying software Model 810 AGGRO/ Results and discussion
LINK Chrono-log, Havertown, PA, USA). A 450 ll quan-
tity of PRP was used for each assay. Then 50 ll of the Bioactivity of amaranth protein hydrolysates
peptide fraction of Mr = 12.6 kDa from Alb1H24 or the
peptide fraction Mr = 5.44 kDa from GlobH24 dissolved in Hydrolysis conditions such as T, t, E/S and % of bioactivity
dimethyl sulfoxide (DMSO) was added. The platelet inhibition have previously been reported and evaluated
aggregation was induced with 5 lM ADP, and the response elsewhere by the authors. Table 1 shows the experimental
was recorded over 3 min and estimated in arbitrary units. conditions that were used in order to obtain the different
Light absorption was corrected with PPP and DMSO. The selected peptide fractions from hydrolysates of storage

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Table 1 Experimental conditions used to obtain peptide fractions of hydrolysates (H) of albumin 1, globulin and glutelin (Alb1H, GloH and
GluH, respectively) as obtained of amaranth grain
Hydrolysate Time E/S DH (%) Bioactivity Molecular weight Aminoacid length Peptide fraction
(h) (AU/g protein) (IC50 lg/ml) (kDa) (number of aa) code

Alb1H 10.3 0.77 42.1 ± 0.4 ACEIA = 925 ± 2.2 0.55 4–5 Alb1H103
ATA = 431.1 ± 0.01
GloH 8.8 0.78 16.3 ± 1.7 ACEIA = 1391 ± 1.4 0.40 3–4 GloH88
ATA = 12.8 ± 0.06
GluH 24.0 0.8 75.0 ± 0.5 DPPIVIA = 120 ± 0.6 0.45 3–4 GluH24
Controls
Captopril ACEIA = 6.90 gg/ml
Diprotin A DPPIVIA = 8.43
Buame ATA = 400 lM
Data are the mean ± standard deviation of three replicates
These fractions exhibited angiotensin I-converting-enzyme-inhibitory activity (ACEIA), antithrombotic activity (ATA) and dipeptidyl peptidase
IV inhibitory activity (DPPIVIA). The degree of hydrolysis (DH) was reached at a specific time and alcalase/protein (E/S) ratio. The IC50 values
of peptide fractions can be compared to those of the controls

proteins of amaranth grain which exhibited ACE inhibitory partial thromboplastin time test, prothrombin time test and
activity: Alb1H103, GloH88 and DPPIV (GluH24). The thrombine time test. They found that albumin (3.8 mg/ml)
peptide fraction Alb1H103 was a hydrolysate that has and globulin (2.5 mg/ml) hydrolysates improved the
peptides made up of 4–5 aminoacids (0.55 kDa) with antithrombotic activity whereas glutelin hydrolysate
foaming capacity and inhibitory activity of platelet aggre- exhibited the highest antithrombotic activity. Nevertheless
gation. Conversely, GloH88 has peptides made up of 3–4 it was difficult to compare the concentrations of peptide
aminoacids (0.40 kDa) with emulsifying capacity (Sori- fractions of amaranth protein hydrolysates with
ano-Santos and Escalona-Buendı́a 2015) and also inhibi- antithrombotic activity in the study by Sabione et al. (2015)
tory activity of platelet aggregation. In this work, ATA of and present because the trials evaluated different coagu-
former peptide fractions was observed assessing the inhi- lation factors. In spite of this, Alb1H103
bitory activity of platelet aggregation triggered by ADP. (431.1 ± 0.01 mg/ml) and GloH88 (12.8 ± 0.06 mg/ml)
Blood coagulation is a complex process that involves the had similar antiplatelet activity of the estrogen Buame
confluence of numerous factors and phenomena and con- (400 lM; Flores-Garcia et al. 2012). However, unlike the
sists of three overlapping phases that maintain the integrity study by Sabione et al. (2015), no inhibition of platelet
of the circulatory system: vascular phase, platelet phase, aggregation with GluH24 was observed. The peptide
and coagulate phase. The platelet phase, also called pri- fraction GluH24 is a hydrolysate that has peptides made up
mary hemostasis, implies the adhesion, activation and of 3–4 aminoacids (0.45 kDa). Such fraction displayed
aggregation of the platelets. The coagulation phase, or DPPIVIA and can control postprandial glycemia in strep-
secondary hemostasis, involved the participation of the tozotocin-induced diabetic mice (Soriano-Santos et al.
coagulation factors, many of which were proteases syn- 2015). Table 1 also shows the IC50 value of all hydro-
thesised in the liver as zymogens that, in sequential order, lysates. These values were larger than those observed in the
lead to the clot formation. This coagulation cascade con- controls because the bioactivities were assessed in the
sists of two triggering pathways, the intrinsic and the corresponding peptide fraction. Moreover, there was nei-
extrinsic one, both converging in the common pathway. In ther isolation nor purification of any single peptide in this
order to evaluate the effect of processing conditions on the work. It is known that the more purified a peptide is, the
stability of the potential antithrombotic activity of more bioactivity this will exhibit (Li et al. 2004).
Alb1H103 and GloH88, a simple method was performed,
one that focuses specifically on primary hemostasis. Buame Effect of heat treatment on peptide bioactivity
(N-(3-hydroxy-1,3,5(10)-estratrien-17b-yl)butylamine), a
synthetic estrogen which is a dose-dependant inhibitor on In a previous work, it was reported that the peptide frac-
platelet aggregation, induced by ADP, was used as a con- tions Alb1H103 and GloH88 of amaranth protein hydro-
trol. The method used to assess antithrombotic activity was lysates have foaming and emulsifying capacities, whereas
different from the one reported by Sabione et al. (2015), GluH24 only displays DPPIVIA (Soriano-Santos and
which was done using clotting tests such as activated Escalona-Buendı́a 2015; Soriano-Santos et al. 2015).

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Alb1H103 and GloH88 might be considered as functional change after heating at different temperatures (40–100 °C)
ingredients because of their techno- and bio-functional at pH 7.0 after 2 h of treatment, while heat treatment for
properties (surfactant properties as well as their ACEIA 3.0 h or 4.0 h slightly decreased ACEIA (p \ 0.05). Those
and ATA). Heat treatment is the most important method in authors claimed that the relationship between peptide
food processing. Consequently, bioactivity assurance of structure-bioactivity, was unclear. Heat causes protein
these ingredients should be maintained during thermal denaturation and aggregation during temperature changes
processing for a certain time. Figure 1a, a0 , a00 , b, b0 and b00 from 60 to 90 °C, which may cause that high molecular
shows the effect of heat treatment (40–120 °C) for 1 h, on mass peptides form clusters and, as a result, may hinder
the bioactivities of hydrolysates, at the two more frequent binding ability of the enzyme. Thus, bioactivity may
pH of foods, roughly 4 and 7. There were no significant diminish or be lost (Bloom et al. 2015). In agreement with
differences among ACEIA, DPPIVIA and ATA of the research by Wu et al. (2014), this work suggested that
heated hydrolysates at pH 4 and 7 and those of unheated hydrolysates with ACEIA could be stable in the heat and
hydrolysates (p \ 0.05). This indicates that peptides pre- time treatments of food processes carried out in tempera-
sent in acid or weak alkaline food environment could resist tures ranging from 60 to 100 °C and also for short periods of
thermal processing from 40 to 100 °C. However, at 120 °C time. In a study by Hwang (2010) peptides derived from
treatment, the correspondent bioactivity begins to decrease tuna cooking juice proteins retained ACEIA. They exhib-
when compared with that of the unheated hydrolysate ited acceptable stability after various temperatures, pH
(p \ 0.05). Furthermore, heating time is also a major factor levels and pressure treatments. In fact, they found that
that may affect the peptide bioactivity present in the severe thermal processing at 100 °C for 30 min produced
hydrolysate. Once it was confirmed that the hydrolysate minor changes in peptides. Similarly, Wu and Ding (2002)
treatment at 100 °C kept the bio-functional activity, the observed that soy-protein-derived peptides with ACEIA
effect of time on heat treatment was evaluated. With regard also displayed stability at 100 °C for 30 min. Fu et al.
to the unheated hydrolysate, there was no statistically sig- (2015) studied the effect of different temperatures on the
nificant loss of any bioactivity under heat treatment for 3 h ACEIA of collagen and found that this kept its inhibitory
at pH 4.0 (Fig. 2a, a0 , a00 ). Figure 2b, b0 and b00 on the other capacity at relatively low temperatures (20–60 °C). Con-
hand, exhibits that Alb1H103 and GluH24 maintained the versely, after heating for 2 h at 100 °C, they noted a slight
inhibitory activity of ACE and ATA at pH 7.0 for 3 h. When reduction of the inhibitory activity. Based on the previous
compared to the unheated hydrolysates, there was no sta- data, it would seem that peptides with ACEIA are more
tistically significant loss of bioactivities in GloH88 under stable in a range of temperature (20–100 °C), at least for
heat treatments just for 2.0 h (p \ 0.05) at pH 7.0. Tem- 1 h. However, the loss of stability of bioactivity would have
perature and time may be crucial factors that may lead to to be also assessed under severe treatment conditions, such
loss of bioactivity. Wu et al. (2014) found that the ACEIA as sterilization, grilling and frying, in order to assure that the
of bovine casein-derived peptides showed no significant expected physiological benefit of a food is received.

Fig. 1 IC50 value as a result of the effect of heat treatment DPPIVIA. Values are expressed as mean ± SD. Bars that have the
(40–120 °C). a, a0 and a00 at pH 4.0 and b, b0 and b00 at pH 7.0 for same superscript in a column do not differ significantly (p \ 0.05)
1 h of selected amaranth peptide fractions on their ACEIA, ATA and

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Fig. 2 IC50 value as a result of the effect of time treatment (1–4 h) of and DPPIVIA. Values are expressed as mean ± SD. Bars that have
the selected amaranth peptide fractions heated previously at 100 °C a, the same superscript in a column do not differ significantly (p \ 0.05)
a0 and a00 at pH 4.0 and b, b0 and b00 at pH 7.0 on their ACEIA, ATA

Effect of pH on the peptide bioactivity 2014). pH modification can also prompt physico-chemical
changes of proteins such as denaturation, intermolecular
pH effect was assessed on a range of 2.0–12.0 of inhibitory reactions, reaction with sugars, solubility and, thus, cause
activity of ACE, ATA and DPPIV of Alb1H103, GloH88 and crosslinking, lysine damage, non specific cleavage of pep-
GluH24, respectively. A T = 100 °C was selected for 1 h for tides and damaged amino acids prone to pH changes. A fur-
this assay, which showed that there was no loss of stability of ther explanation holds that pH changes can affect bioactivity
bioactivity, so any change on it would have to be attributed to on account of alterations of one or more aminoacids (Hwang
the influence of pH treatment. Changes of bioactivity of 2010). For example, acid treatments damage glutamine and
different peptide fractions of amaranth protein hydrolysates asparagine, and alkaline treatments not only destroy cysteine,
are shown in Fig. 3a–c. Bioactivity depended largely on the serine and threonine, but also produce lysinoalanine and D-
solubility of the hydrolysate. In general, as solubility amino acids (Kristinsson and Hultin 2003). There are few
increases, IC50 value of the corresponding bioactivity foodstuffs at acid pH and the treatment at pH over 7.0 is
decreases. Conversely, as solubility decreases, IC50 value of scarce. Therefore, in this work, it was observed that pH did
the corresponding bioactivity increases. As for Alb1H103, not have a considerable effect on ACEIA, DPPIVIA and
IC50 values of ACEIA and ATA were roughly constant within ATA. Similarly, a purified hendeca-peptide as obtained of
the pH range of 5.0–7.0; the lowest inhibitory activity, for algae protein waste was subjected to incubation at pH 2–10
both bioactivities, was observed at alkaline pH (8.0–12.0). and temperature 40–100 °C for 1 h and measured for
IC50 value of GloH88, for ACEIA and ATA was kept con- ACEIA. The peptide completely retained its ACEIA, indi-
stant, similar to that of the control, within the pH range of cating that the purified peptide was both pH and heat-
2.0–8.0. At pH 9.0–12.0, the IC50 values of these hydrolysates stable (Sheih et al. 2009). The impact of pH (2.0–10.0) on the
increased. GluH24 yielded a very good solubility at acid or ACEIA of collagen hydrolysates afforded a significant
alkaline pH. Therefore, within the pH ranges of 2.0–4.0 and reduction of ACEIA at strong alkaline conditions (pH 10.0).
7.0–9.0, the DPPIVIA was constant and similar to that of the Similarly, in agreement with our research, collagen peptides
control. Within the pH range of 10.0–12.0, the highest inhi- exhibited good resistance against the acidic and weak alkaline
bitory activity of GluH24 on DPPIV activity was observed. conditions and heat treatment (Fu et al. 2015).
However, at pH 5.0 and 6.0, the DPPIVIA of GluH24
decreased as the IC50 values increased. This may be ascribed Stability of bioactivity of hydrolysates on an in vitro
to the fact that a heat treatment at those extreme pH ranges gastrointestinal digestion
can damage some amino acids of the inhibitory peptides.
Denaturation and even hydrolysis of some peptides can also There are more reports about peptides, as isolated from dif-
affect the inhibitory activity triggered by the pH (Wu et al. ferent natural sources, capable of inhibiting ACE than

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Fig. 3 IC50 value as a result of

the effect of pH (2–12) of the
selected amaranth peptide
fractions heated previously at
100 °C for 1 h on their ACEIA,
ATA and DPPIVIA. Values are
expressed as mean ± SD. Bars
that have the same superscript in
a column do not differ
significantly (p \ 0.05)

studies about peptides capable of inhibiting DPPIV and amaranth protein hydrolysates once they have undergone
antithrombotic activities. By the same token, there are few gastrointestinal digestion. Therefore a simulation of human
examples of biopeptides with ACEIA, such as IPP and VPP, gastrointestinal digestion was performed in vitro using
derived from milk protein, that have been added to com- pepsin, trypsin (T) and chymotrypsin (C). Because T and C
mercial foodstuffs. In addition, conclusive data with respect are serin proteases, the activity of the residual peptide, called
to their effectiveness on hypertension is scarce not only amaranth trypsin/subtilisin inhibitor (ATSI), present in
because there are still insufficient case reports when these protein hydrolysates of amaranth grain, was also assessed.
tripeptides are incorporated in daily diet plans, but also ATSI inhibitory activity was evaluated following the pro-
because functional food regulations are still limited in sev- tocol by Valdes-Rodriguez et al. (1993). Alb1H103 with a %
eral countries (Madureira et al. 2010). To the best of our HD = 42.1 ± 0.4 had a % InhATSI = 35.4 ± 0.6, whereas
knowledge, no reports on commercial products added with GloH88 with a % HD = 16.3 ± 1.7 displayed a higher
peptides that inhibit DPPIV and antithrombotic activities are inhibitory activity of serin proteases
known. One of the aims of the present work was to evaluate (%InhATSI = 46.4 ± 0.8). This is relevant in that authors
the bioactivity of the peptide fractions as obtained of like Hejgaard et al. (1994) observed that a previous treatment

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Fig. 4 Influence of a previous

pH treatment (4 or 7) of peptide
fractions (Alb1H103; GloH88
and GluH24) on ACEIA, ATA
and DPPIVIA, followed by an
in vitro gastrointestinal
digestion. Bars that have the
same superscript in a column do
not differ significantly
(p \ 0.05)

of protein extracts of amaranth with pepsin, containing for GluH24 after the enzymatic digestion. This improvement
ATSI, improved the proteolytic activities of T and C. Simi- of inhibitory activity of the corresponding bioactivity may be
larly, Fig. 4a, a0 , a00 shows that a previous pH treatment at 4.0 due to the degree of hydrolysis of hydrolysates of Alb1H103
or 7.0 (T = 100 °C; t = 1 h) of Alb1H103 and GloH88 and and GloH88 and GluH24 (% DH = 42.1 ± 0.4; 16.3 ± 1.7
GluH24 influenced the bioactivity after a digestion with T and 75.0 ± 0.5, respectively).
and C. It was found that, in general, when the hydrolysates Furthermore, at pH 4.0 ATSI hydrolysis can be pro-
were kept at 100 °C for 1 h at pH 4.0, the corresponding moted because of pepsin activity. Once ATSI is hydrol-
bioactivity resulted in a significant increase (p \ 0.05) as the ysed, no inhibition of serin proteases (T and C) can be
IC50 (mg/ml) values of ACE decreased from 925 ± 2.2 to observed, so these enzymes continue hydrolysing peptides,
622 ± 0.1 and from 1391 ± 1.4 to 895 ± 0.8 for Alb1H103 thus yielding new peptides with different bioactivities. The
and GloH88, respectively. The same effect was obtained resulting biopeptides contained in the hydrolysates can be
when ATA was evaluated as their IC50 (mg/ml) values pro- hydrolysed by T and C because these enzymes can cleave
duced a significant decrease (p \ 0.05) from 431.1 ± 0.01 specific peptide bonds, namely, pepsin breaks peptides
to 226 ± 0.02 and from 12.8 ± 0.06 to 9.4 ± 0.03 for bonds in Tyr, Phe and Leu residues; trypsin in Arg and Lys
Alb1H103 and GloH88, respectively. DPPIVIA decreased residues; chymotrypsin in Trp, Phe, Tyr, Met and Leu
(p \ 0.05) their IC50 (mg/ml) from 120 ± 0.6 to 75 ± 0.2 residues. Huang et al. (2012) observed that two peptides

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(PGVGGPLGPIGPCYE and CAYQWQRPVDRIR), digested by pepsin and pancreatin and afforded an elevated
derived from tuna cooking juice hydrolysates with DPPIV content of free amino acids. The ACEIA of collagen pep-
inhibitory potential, increased significantly upon simulated tides remained stable after an in vitro digestion by gastric
gastrointestinal digestion. This can be attributed to the fact proteases. Thus, the collagen peptides may endure diges-
that new and smaller peptides were formed to increase the tion considerably through the gastrointestinal tract or they
content of DPPIV inhibitory peptides. During the simulated may be partially degraded into smaller peptides whilst
gastrointestinal digestion, the peptides were truncated into retaining their activity. Collagen peptides display potent
smaller fragments, some of which increased the DPPIV ACEIA after an in vitro digestion, possibly due to post-
inhibitory potential. Huang et al. (2012) suggest that the translational hydroxylation of proline in collagen (Fu et al.
smaller peptides may have greater intestinal permeability 2015).
than the original ones. They also found that the DPPIV
inhibitory potential of other peptides isolated from tuna
cooking juice hydrolysates was retained or improved. Conclusion
In contrast, a treatment at pH 7.0 of Alb1H103, GloH88
and GluH24 (T = 100 °C; t = 1 h) does not facilitate Alb1H103 and GloH88, as obtained of amaranth grain,
ATSI hydrolysis by pepsin and, consequently, the digestion exhibited angiotensin I-converting-enzyme-inhibitory
with T and C can be inhibited by ATSI in such a way that activity (ACEIA) and Antithrombotic activity (ATA), and
the peptides of each bioactivity do not undergo a further were stable under a heat treatment from 40 to 100 °C at pH
hydrolysis. Therefore no significant difference (p \ 0.05) 4 and 7 for 3 h, except for GloH88 whose time lapse, at pH
was observed in the bioactivities of the hydrolysates after 7.0, was shorter (2 h). ATA was evaluated as inhibition of
their digestion. In order to prevent the intestinal digestion platelet aggregation. GluH24, with dipeptidyl peptidase IV
from altering the chemical structure of biopeptides, it has inhibitory activity (DPPIVIA), was also stable in the
been suggested that the hydrolysates be obtained by using aforementioned conditions. The pH effect on hydrolysates
only digestive proteolytic enzymes. However, alcalase is bioactivity did not have a considerable effect as long as
one the most sought-after enzymes to hydrolyse proteins peptides were soluble. With regard to the biological
from different sources. Orsini et al. (2011) found that activity of protein hydrolysates, this may be preserved or
biopeptides as obtained of amaranth grain globulin, in an enhanced depending on their processing conditions.
in vitro gastrointestinal digest simulation test, cannot be Therefore, the results suggest that these hydrolysates may
cleaved by the digestive proteolytic enzymes. To the best be considered as biofunctional ingredients to manufacture
of our knowledge, there is not much research on the pro- third generation nutraceuticals and functional foods.
teolytic stability of ACEIA peptides with gastrointestinal
enzymes. Tavares et al. (2011) found that a modest Acknowledgements We are grateful to Prof. Abraham Avendaño-
Martı́nez for proofreading and translating the manuscript.
decrease of ACEIA of peptides as obtained of C. cardun-
culus, in which IC50 = 336.3 ± 34.9 lg/ml was reduced
to a IC50 = 253.6 ± 30.7 lg/ml, when the digestion was
over. Conversely, Akilliioglu and Karakaya (2009) noted References
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