ArticlePDF Available

Abstract and Figures

Canary seed (Phalaris canariensis) is used to feed birds but it has been recently considered a promising cereal with nutraceutical potential for humans. The aim of this work was to analyze the protein fractions from canary seed flour and from milk substitutes (prepared by soaking the seeds in water 12 and 24 h), and to evaluate antioxidant and antihypertensive capacity of peptides obtained after in vitro digestion. Prolamins were the major protein fraction, followed by glutelins. After digestion, albumins and prolamins fractions from milks presented higher levels of peptides than flour, globulins showed more peptides in flour and glutelins were found in similar concentrations in all samples; 24 h milk prolamins had the highest concentration of peptides. Purification by high performance liquid chromatography (HPLC), sequencing of peptides, in vitro antioxidant ABTS (2,2'-azino-bis, 3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays, and antihypertensive capacity (angiotensin converting enzyme (ACE) assay), indicated that peptides from canary seed prolamins were the most efficient compounds with antioxidant and antihypertensive activity. Canary seeds may be considered an accessible and cheap source to prepare milk substitutes with high contents of bioactive peptides with remarkable functional properties to promote better human health and healthy ageing.
This content is subject to copyright. Terms and conditions apply.
Antioxidant and Antihypertensive Potential of Protein
Fractions from Flour and Milk Substitutes from Canary
Seeds (Phalaris canariensis L.)
María Elena Valverde
&Domancar Orona-Tamayo
&Blanca Nieto-Rendón
Octavio Paredes-López
#Springer Science+Business Media New York 2016
Abstract Canary seed (Phalaris canariensis)isusedtofeed
birds but it has been recently considered a promising cereal
with nutraceutical potential for humans. The aim of this work
was to analyze the protein fractions fromcanary seed flourand
from milk substitutes (prepared by soaking the seeds in water
12 and 24 h), and to evaluate antioxidant and antihypertensive
capacity of peptides obtained after in vitro digestion.
Prolamins were the major protein fraction, followed by
glutelins. After digestion, albumins and prolamins fractions
from milks presented higher levels of peptides than flour,
globulins showed more peptides in flour and glutelins were
found in similar concentrations in all samples; 24 h milk pro-
lamins had the highest concentration of peptides. Purification
by high performance liquid chromatography (HPLC), se-
quencing of peptides, in vitro antioxidant ABTS (2,2-azino-
bis, 3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-
diphenyl-1-picrylhydrazyl) assays, and antihypertensive ca-
pacity (angiotensin converting enzyme (ACE) assay), indicat-
ed that peptides from canary seed prolamins were the most
efficient compounds with antioxidant and antihypertensive
activity. Canary seeds may be considered an accessible and
cheap source to prepare milk substitutes with high contents of
bioactive peptides with remarkable functional properties to
promote better human health and healthy ageing.
Keywords Nutraceuticals .Antihypertensive .Antioxidants .
Canary seed proteins .Bioactive peptides
Phalaris canariensis L., commonly known as canary seed, is
an annual cereal crop originating in the Mediterranean region;
moreover, it is mainly cultivated in Argentina and Canada.
Now, it has been considered a promising cereal for human
consumption due to its excellent nutritional and nutraceutical
value, as well as for food industrial purposes [1].
Robinson [2,3] was the first to report the food potential of
canary seed; he also found no toxic characteristics for human
consumption. Abdel-Aal et al. [46] described that canary
seed has 1924 % protein, 59 % crude fat, 68 % total dietary
fiber, 2 % ash, and 5561 % starch. Putnam et al. [7,8]re-
ported a high mineral content in the seeds, and Abdel-Aal
et al. [5] found that they have some B complex vitamins, such
as thiamine, niacin and riboflavin. The oil is highly unsaturat-
ed, including linoleic (55 %), oleic (29 %), and linolenic
(2.5 %) acids; it is worth mentionining that the seed has shown
excellent antioxidant capacity [4]. P. canariesis seed also con-
tains high levels of protein compared with other cereal grains,
the proportion of prolamins and glutelins fractions is 78 %;
they are rich in cysteine, tryptophan, and phenylalanine, but
deficient in lysine and threonine [4,9]. Boye et al. [10]
reported the absence of gluten in canary seed, thus it can
be included in gluten-free diets for people with celiac
disease; besides, no cross-reaction was detected using
ELISA between canary seed proteins and almond, hazelnut,
mustard, peanut, sesame, soybean and walnut proteins.
The potential benefits of canary seeds in human health
has been widely used in folk medicine. They have shown
anti-hypertensive, anti-diabetic and anti-inflammatory effects,
Electronic supplementary material The online version of this article
(doi:10.1007/s11130-016-0584-z) contains supplementary material,
which is available to authorized users.
*María Elena Valverde
Centro de Investigación y de Estudios Avanzados de Instituto
Politécnico Nacional, Km. 9.6 Libramiento Norte Carretera
Irapuato-León, CP 36821 Irapuato, Guanajuato, Mexico
Plant Foods Hum Nutr
DOI 10.1007/s11130-016-0584-z
blood lipid and cholesterol lowering capacity, and have been
also described as remedy for kidney, pancreas, bladder dis-
eases, and obesity [914]. Their use in human health is in-
creasing due to their antihypertensive effects, inhibition of
urinary tract infections, arteriosclerosis, AIDS, gout, rheuma-
tism, edema, gastritis and stomach ulcer; also provide muscle
tone and prevent cardiovascular disorders [15].
P. canariensis is commonly used as a remedy known
Bcanary seed milk substitutes or milk^[15], which is prepared
by soaking the seeds in water for a period of 12 or 24 h then
the seeds are ground with clean water. It has been usually
described as nutraceutical agent for human health; however,
no scientific information has been reported regarding these
seed milks. The aim of this study was to analyze the protein
fractions from canary seed flour, and milk obtained by a tra-
ditional process, in order to determine their antioxidant and
antihypertensive potential.
Materials and Methods
Sample Preparation Canary seed was provided by local pro-
ducers (Irapuato, Gto., Mexico). Seeds were ground in a mill
(Brinkmann Retsch, USA) and passed through an 80 mesh to
obtain the flour. For the milk substitutes, 5 g of seeds were
soaked in 250 mL of distilled water for 12 and 24 h at room
temperature. The soaking water was removed and the seeds
were milled in an Osterizer blender with 250 mL fresh dis-
tilled water. The mixture was percolated through a sieve and it
was lyophilized (FreeZone, 2.5, Labconco) and stored at
20 °C, until use.
Extraction of Protein Fractions Proteins from flour and
milks were fractionated as reported by Sandoval-Oliveros
and Paredes-López [16]; the protein concentration was
determined with a Pierce BCA protein assay kit
(Thermo Scientific, USA).
Polyacrylamide Gel Electrophoresis The first electrophoret-
ic separation of protein and peptides was performed by 13 and
14 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
For two-dimensional gel electrophoresis (2DPAGE) was
used a 2D cleanup kit (GE Life Sciences, Sweden) following
manufacturers instructions. Proteins were loaded on 7 cm
IPG dry strip and were focused according to manufacturers
instructions (GE Life Sciences, Sweden).
In Vitro Digestion of Protein Fractions A sequential enzyme
digestion was carried out following Wang et al. [17] procedure.
ACE Assay The inhibitory effects of canary seed peptides
against ACE were determined according to studies carried
out in our laboratory by Luna-Suárez et al. [18] and Orona-
Tamayo et al. [19].
ABTS Radical-Scavenging Activity This assay was per-
formed according to the method by Zhou et al. [20], with
modifications by Orona-Tamayo et al. [19].
DPPH Radical-Scavenging Activity This assay was mea-
sured according to Martínez-Cruz and Paredes-López [21]
procedure, modified by Orona-Tamayo et al. [19].
Peptides Separation by HPLC The fractionation of flour
prolamins was performed following the method described by
Orsini-Delgado et al. [22] using a preparative C-18 HPLC
column on a HPLC System (Waters, Milford, MA). The main
portion was collected at 9.85 min and then freeze-dried and
stored at 70 °C until use.
Peptide Identification by Liquid Chromatography-Mass
Spectrometry (LC-MS) Analysis Freeze-dried flour prola-
min peptides were applied in a LC-MS system according to
Meneses et al. [23] procedure. The MS spectra data were
collected in positive ion mode and subjected to identification
with Proteome Discoverer 1.4 and BLAST. The functional
prediction of prolamin bioactive peptides was analyzed using
BIOPEP database [24].
Statistical Analysis Experiments were performed with four
repetitions and results were expressed as means ± standard
deviation and analyzed using global LSD (least significant
difference) and univariate analysis of variance (ANOVA).
Statistical analyses were carried out using Statistical Package
for the Social Sciences 17.0 (SPSS Inc., Chicago, IL).
Results and Discussion
Protein Fractions The major protein fraction in canary seeds
was prolamins (Table 1,Online supplementary material):
54 % in flour, 46.3 % in 12 h milk and 38.1 % in 24 h milk.
Tabl e 1 Angiotensin converting enzyme -inhibitory activity (IC
Milk substitutes
Fractions/Samples Flour 12 h 24 h
Albumins 504.8 ± 82
450.1 ± 85
500.5 ± 94
Globulins 442.8 ± 68
384.1 ± 62
483.6 ± 89
Prolamins 217.4 ± 18
276.4 ± 21
279.8 ± 13
Glutelins 348.9 ± 45
396.7 ± 80
518.6 ± 69
Different letters in the same row indicate significant differences (P0.05)
Plant Foods Hum Nutr
Prolamins are the main protein in maize, wheat, barley and
rye; they are determinant for kernel texture in the mature seed,
an essential characteristic of cereal grains [25]. The second
most abundant fraction was glutelins: 26.6 % in flour,
19.7 % in 12 h milk and 12.2 % in 24 h milk. The amount
of globulins was very similar in flour and milks, but albumins
showed significant variations: 4.8 % in flour, 17.3 % in 12 h
milk and 32.8 % in 24 h milk. The protein fraction values were
relatively similar to some previous studies in canary seed flour
[4,9]. The protein concentration changed in 12 and 24 h milk
samples compared with flour; it may be due to the digestion or
degration ofproteins when soaking the seeds in water; further-
more, prolamins were the main fraction in all samples ana-
lyzed (Table 1,Online supplementary material).
Electrophoretic Pattern of Protein Fractions Except for
globulins, there were no significant differences in the electro-
phoretic pattern in flour and milk samples. Albumins present-
ed diffused bands of low molecular weight (20 to 100 kDa)
and clear bands between 35 to 45 kDa; milks showed one
band of around 100 kDa (Fig. 1a). Globulins had bands
around 12 to 20 kDa , and others more clear around 40 to
60 kDa; 12 and 24 h milks exhibited a band of 60 kDa and
an interestingly reduction was observed in a single band
around 37 kDa. Prolamins showed two bands of 10 and
22 kDa approximately; profile which is similar to oat, maize,
rice and barley [25]. An apparent proteolysis in the main band
was found only in 24 h milk. In glutelins, some bands were
located between 25 to 35 kDa and 40 to 60 kDa; a dense band
of approximately 40 kDa appeared in 24 h milks, which may
result by a degradation effect (Fig. 1a). Our results from flour
were similar to those found by Estrada-Salas et al. [9]; but
when canary seeds were soaked in water for milk preparation,
the imbibition period induces proteolysis of some proteins and
electrophoretic pattern changes, which promote the release of
peptides. Up to our knowledge this is the first time that 2D
PAGE electrophoresis is reported in canary seeds. All the
fractions were well separated by SDS-PAGE and 2DPAG E
and some similarities in electrophoretic patterns in all fractions
were observed (data no shown). By SDS-PAGE, prolamins
were detected mainly between 15 and 20 kDa and one band
around 50 kDa; by 2DPAGE, the range of isoelectric point
was rather wide [3,10]. As indicated above, profiles were
similar between flour and milk substitutes; but it was clear a
protein degradation in milks, especially at 24 h (Fig.1b).
Canary Seed Proteins Digestion A large variety of benefits
appear when proteins are digested in the gastrointestinal tract
and thus bioactive peptides are released from food. Canary
seed proteins have been digested more easily under sequential
digestion [26]; our digestion method appears to be appropriate
because after 6 h of in vitro digestion all protein fractions were
completely degraded (Fig. 1a, Online supplementary
material). Estrada-Salas et al. [9] reported similar results in
flour. Albumins and prolamins from milks released more pep-
tides than the same protein fraction from flour, and prolamins
from 12 h milk presented the highest peptides concentration,
Fig. 1 SDS and 2DPAGE from canary seeds protein fractions from
flour and milk subtitutes. Mmolecular marker, Fflour, 12 12 h milk,
24 24 h milk, Ip isoelectric point
Fig. 2 Free radical scavenging activity from canary seeds flour and milk
subtitutes. Fflour, 12 12 h milk, 24 24 h milk
Plant Foods Hum Nutr
followedby12and24halbumins(Fig.1b, Online
supplementary material). Flour globulins released more pep-
tides than the same fraction in milk samples; however,
glutelins showed statistically (p0.05) the same proportion
of peptides in all samples. These results indicate that prola-
mins and albumins generate more peptides after digestion,
which suggest their susceptibility to proteolysis (Fig. 1b,
Online data). Interestingly, when the canary seed is soaked
in water a natural pre-digestion occurs, thus facilitating the
release of peptides from the protein fractions.
ACE Activity of Canary Seed Peptides Food peptides can
be considered an effective and safe strategy against high blood
pressure [27]; peptides from flour and milks were evaluated
for their antihypertensive action. Prolamins peptides showed
more inhibition than peptides from the other fractions, espe-
cially flour with an IC
of 217.4 μg/mL; moreover, it was not
statistically (p0.05) different to globulins and glutelins from
12 h milk and glutelins from flour (Table 1). No effect was
found against angiotensin activity when soaking canary seeds
for 24 h, with exception of prolamins; however, soaking for
12 h considerably increased their activity. Estrada-Salas et al.
[9]reported an IC
value of 332 μg/mL for canary seeds
flour peptides, similar to other seeds and less effective to
our results. Passos et al. [14] reported that the aqueous
extract of canary seeds reduced systolic blood pressure
in hypertensive Wistar rats; the interruption of the treatment
caused a rise in the blood pressure. They reported no negative
Tabl e 2 Peptides identified by
LC-MS in canary seed prolamin
fractions from 12 h milk
Peptide Molecular weight (Da) Peptide-sequences ID Activity function
LSLGT 664.36 GT 7612 ACE inhibitor
LG 7619 ACE inhibitor
SL 7619 Dipeptidyl-peptidase IV inhibitor
TDQPAG 703.38 AG 7600 ACE inhibitor
PA 3179 Dipeptidyl-peptidase IV inhibitor
QP 8532 Dipeptidyl-peptidase IV inhibitor
AG 8760 Dipeptidyl-peptidase IV inhibitor
DQ 8768 Dipeptidyl-peptidase IV inhibitor
TD 8898 Dipeptidyl-peptidase IV inhibitor
QQLQT 797.42 LQ 7831 ACE inhibitor
QL 8874 Dipeptidyl-peptidase IV inhibitor
QQ 8876 Dipeptidyl-peptidase IV inhibitor
QT 8878 Dipeptidyl-peptidase IV inhibitor
FEPLQLA 926.5 PL 7513 ACE inhibitor
LA 7585 ACE inhibitor
LQ 7831 ACE inhibitor
LA 3175 Dipeptidyl-peptidase IV inhibitor
EP 8529 Dipeptidyl-peptidase IV inhibitor
PL 8638 Dipeptidyl-peptidase IV inhibitor
QL 8874 Dipeptidyl-peptidase IV inhibitor
KPQLYQPF 1019.52 LY 3381 ACE inhibitor
KP 7810 ACE inhibitor
PQ 7837 ACE inhibitor
LY 7872 Antioxidant
KP 8218 Antioxidant
KP 8519 Dipeptidyl-peptidase IV inhibitor
QP 8532 Dipeptidyl-peptidase IV inhibitor
PF 8854 Dipeptidyl-peptidase IV inhibitor
PQ 8861 Dipeptidyl-peptidase IV inhibitor
QL 8874 Dipeptidyl-peptidase IV inhibitor
YQ 8943 Dipeptidyl-peptidase IV inhibitor
Bioactive peptides were obtained from BIOPEP database ( [24]
Plant Foods Hum Nutr
side effects in renal function, diuresis or Na
excretion. Our
results showed that proteins from canary seed are a good
source of peptides inhibitors of ACE, mainly prolamins.
Antioxidant Activity of Canary Seed Peptides The ABTS
assay showed different inhibition patterns (Fig. 2a). Albumins
and prolamins peptides of 12 h milk showed the best IC
values, 133.2 and 181.6 μg/mL, respectively (p0.05); also,
albumins and glutelins of 24 h milk showed high IC
176.0 and 213.7 μg/mL, respectively (p0.05). Canary seed
flour presented the lowest IC
values in all protein fractions,
except for prolamins (Fig. 2a). These results clearly demon-
strate that the protein undergoes hydrolysis by soaking the
seeds in water, thus milk samples showed more antioxidant
potential than the flour in all protein fractions. Prolamins pep-
tides, in all cases, were the most efficient samples to inhibit the
DPPH activity (p0.05) comprising the lowest IC
(Fig. 2b).
The 24 h milk showed the best antiradical IC
value (89.7 μg/
mL) followedby flour and 12 h milk (104.2 and 114.1 μg/mL,
respectively). Albumins and globulins peptides showed a sim-
ilar trend against DPPH; milks were more efficient than the
seed flour. Glutelins peptides presented similar IC
flour 248.5, 12 h milk 254, 24 h milk 253.2. In general, 24 h
milk tended to show better inhibition against DPPH in
almost all fractions (albumins: 144.7; globulins: 147.6;
prolamins: 89.7 μg/mL), except for glutelins. Albumins
and globulins from milks showed more antioxidant capacity
than the flour, whereas prolamins and glutelins did not follow
the same trend.
Peptides Separation and Identification HPLC chromato-
gram of prolamins peptides presented a main peak with a
retention time of 9.85 min (Fig. 2,Online supplementary
material); this peak was collected and peptides were analyzed
by LC-MS. Five peptides were identified in a molecular
weight range between 664 and 1019 Da (Table 2). Databank
revealed that some peptides had inhibitory activities against
dipeptidyl-peptidase IV (DPPIV) and ACE. In most cases,
dipeptides encrypted in KPQLYQPF, FEPLQLA and
TDQPAG sequences showed the highest proportion of
DPPIV (dipeptidyl-peptidase), ACE inhibitors and antioxi-
dants, respectively. Dipeptides LA and PA displayed an
inhibition of DPPIV activity [28]. It has been demonstrated
that these peptides of canary seeds showed 43.5 % of inhibi-
tion against DPPIV [9]. Both peptides were found in our se-
quences and can interact between DPPIV mainly via hydro-
phobic interactions. Peptides with GT, LG, LY, LA, LQ, AG,
PL, KP and PQ were associated to antihypertensive functions
with the potential to inhibit ACE [29]; these sequences
were found in canary seed prolamins. We also found two
dipeptides (LY and KP) that have been previously reported
with antioxidant activity [27]. Capriotti et al. [30] reported
peptides with ACE inhibitory activity and antioxidant capacity
in soybean flour and soy milk. Our results suggest that the
prolamins fraction of canary seeds may be considered an ex-
cellent source of peptides with inhibitory capacity against
DPPIV and ACE, as well as antioxidant.
Up to our knowledge, this is the first study for the determina-
tion of protein fractions and their nutraceutical potential of
canary seed milks. Prolamins were the most abundant fraction
in canary seed flour and milk substitutes. Peptides from canary
seed flour and milk substitutes showed a high potential as
antioxidants and ACE inhibitors; particularly prolamins frac-
tion from milk substitutes showed the strongest antihyperten-
sive capacity. It is worth mentioning that canary seed milk is
easy, cheap and fast to obtain. In brief, P. canariensis seeds
showed high nutraceutical levels with the high potential to
impact positively human health. Therefore, canary seed is a
promising cereal for human consumption in the years to come.
Acknowledgments We acknowledge partial support from Consejo
Nacional de Ciencia y Tecnología, México to carry out this study. The
authors wish to thank Talía Hernández Pérez for technical assistance,
CINVESTAV-Irapuato, México.
Compliance with Ethical Standards
Conflict of Interest The authors declare no conflict of interest. This
article does not contain any studies with human or animals subjects.
1. Cogliatti M (2012) Canary seed crop. Sci Agric 1:7588
2. Robinson RG (1978) Chemical composition and potential uses of
annual canary grass. Agron J 70:797800
3. Robinson RG (1979) Annual canary grass: a potential food grain
crop. Crops Soil 32:1315
4. Abdel-Aal ESM, Hucl P, Sosulski FW (1997) Structural and com-
positional characteristics of canary seed (Phalaris canariensis L.). J
Agric Food Chem 45:30493055
5. Abdel-Aal ESM, Hucl P, Miller SS, Patterson CA, Gray D (2011a)
Microstructure and nutrient composition of hairless canary seed
and its potential as a blending flour for food use. Food Chem
6. Abdel-Aal ESM, Hucl P, Patterson CA, Gray D
(2011b)Phytochemicals and heavy metals content of hairless canary
seed: a variety developed for food use. LWT-Food Sci Technol 44:
7. Putnam DH, Oelke EA, Oplinger ES, Doll JD, Peters JB (1990)
Annual canary grass. University of Wisconsin, Retrieved
November 15, 2014 from: " http://www.hort.purdue.
edu/newcrop/afcm/cangras.html "
8. Putnam DH, Miller PR, Hucl P (1996) Potential for production and
utilization of annual canary grass. Cereal Foods World 41:7583
Plant Foods Hum Nutr
9. Estrada-Salas PA, Montero-Morán GM, Martínez-Cuevas PP,
González C, Barba de la Rosa AP (2014) Characterization of anti-
diabetic and antihypertensive properties of canary seed (Phalaris
canariensis L.) peptides. J Agric Food Chem 62:427433
10. Boye JI, Achouri A, Raymond N, Cleroux C, Weber D, Koerner
TB, Hucl P, Patterson CA (2013) Analysis of glabrous canary seeds
by ELISA, mass spectrometry, and western blotting for the absence
of cross-reactivity with major plant food allergens. J Agric Food
Chem 61:61026112
11. Magnuson BA, Patterson CA, Hucl P, Newkirk RW, Ram JI,
Classen HL (2014) Safety assessment of consumption of glabrous
canary seed (Phalaris canariensis L.) in rats. Food Chem Toxicol
12. Novas MJ, Jiménez AM, Asuero AG (2004) Determination of an-
tioxidant activity of canary seed infusions by chemiluminescence. J
Anal Chem 59:8486
13. Pérez-Gutiérrez RM, Madrigales AD, Horcacitas MDC, García-
Baez E, Cruz-Victoria T, Mota-Flores JM (2014)
Ameliorativeeffect of hexane extract of Phalaris canariensis on
high fat diet-induced obese and streptozotocin-induced diabetic
mice. Evid Based Complement Alternat Med 2014:145901. doi:
10.1155/2014/145901 .
14. Passos CS, Carvalho LN, Pontes RB, Campos RR, Ikuta O, Boim
AM (2012) Blood pressure reducing effects of P. canariensis in
normotensive and spontaneously hypertensive rats. Can J Physiol
Pharmacol 90:201208
15. Inkanatural (2014) Canary seed: properties and contraindications.
Retrieved November, 2014 from: "http://www.inkanatural.
16. Sandoval-Oliveros R, Paredes-López O (2013) Isolation and char-
acterization of proteins from chia seeds (Salvia hispanica L.). J
Agric Food Chem 61:193201
17. Wang W, Bringe NA, Berhow MA, González de Mejía E (2008) B-
conglycinins among sources of bioactive in hydrolysates of differ-
ent soybean varieties that inhibit leukemia cells in vitro. J Agric
Food Chem 56:40124020
18. Luna-Suárez S, Medina-Godoy S, Cruz-Hernández A, Paredes-
López O (2010) Modification of the amaranth 11S globulin storage
protein to produce an inhibitory peptide of the angiotensin
converting enzyme, and its expression in Escherichia coli.J
Biotechnol 148:240247
19. Orona-Tamayo D, Valverde ME, Nieto-Rendón B, Paredes-López
O (2015) Inhibitory activity of chia (Salvia hispanica L.) protein
fractions against angiotensin I-converting enzyme and antioxidant
capacity. LWT-Food Sci Technol 64:236242
20. Zhou H, Chen X, Wang C, Ye J, Chen H (2012) Purification and
characterization of a novel ~18 kDa antioxidant protein from
Ginkgo biloba seeds. Molecules 17:1477814794
21. Martínez-Cruz O, Paredes-López O (2014) Phytochemical profile
and nutraceutical potential of chia seeds (Salvia hispanica L.) by
ultra high performance liquid chromatography. J Chrom A 1346:
22. Orsini-Delgado MC, Galleano M, Añón C (2015) Amaranth pep-
tides from simulated gastrointestinal digestion: antioxidant activity
against reactive species. Plant Foods Hum Nutr 70:2734
23. Meneses EP, Villa-Hernández O, Hernández-Orihuela L, Castro-
Franco R, Pando V, Aguilar MB, Batista CVF (2011) Peptidomic
analysis of the skin secretions of the frog Pachymedusa dacnicolor.
Amino Acids 40:113122
24. Minkiewicz P, Dziuba J, Iwaniak A, Dziuba M, Darewicz M
(2008)BIOPEP database and other programs for processing bio-
active peptide sequences. J AOAC Int 91:965980
25. Holding R (2014) Recent advances in the study of prolamin storage
protein organization and function. Front Plant Sci 5:19
26. Rajamohamed SH, Aryee ANA, Hucl P, Patterson CA, Boye JI
(2013) In vitro gastrointestinal digestion of canary seed proteins
as affected by variety and thermal treatment. Plant Foods Hum
Nutr 68:306312
27. Huang WY, Davidge ST, Wu J (2013) Bioactive natural constitu-
ents from food sources-potential use in hypertension prevention and
treatment. Crit Rev Food Sci Nutr 53:615630
28. Velarde-Salcedo AJ, Barrera-Pacheco A, Lara-González S,
Montero-Morán GM, Díaz-Gois A, González de Mejia E,
Barba de la Rosa AP (2013) In vitro inhibition of dipeptidyl
peptidase IV by peptides derived from the hydrolysis of ama-
ranth (Amaranthus hypochondriacus L.) proteins. Food Chem
29. Montoya-Rodríguez A, Gómez-Favela MA, Reyes-Moreno C,
Milán-Carrillo J, González de Mejía E (2015) Identification of
bioactive peptide sequences from amaranth (Amaranthus
hypochondriacus) seed proteins and their potential role in the
prevention of chronic diseases. Compr Rev Food Sci Food Saf
30. CapriottiAL,CarusoG,CavaliereC,SamperiR,VenturaS,
Chiozzi RZ, Laganà A (2015) Identification of potential bioactive
peptides generated by simulated gastrointestinal digestion of
soybean seeds and soy milk proteins. J Food Compos Anal
Plant Foods Hum Nutr
... [10]. Due to its high content of carotenoids, phenolic acids [11], and bioactive peptides [12,13], HCS represents a promising functional food ingredient. The potential use of HCS as a proper ingredient for the gluten-free foods [14] and wholegrain healthy products [11,[15][16][17] can widen commercial applications and consumption of these compounds through diet. ...
... Although the number of carotenoids and free ferulic acid in wheat/HCS bread was smaller than the suggested daily requirements, wheat/HCS bread can still boost the daily consumption of these healthy components [1,25]. In addition to carotenoids and ferulic acid, HCS is a recognized source of bioactive peptides [12,13], while wheat is a good source of dietary fiber. These compositional differences could make both grains complement each other in terms of health-enhancing attributes. ...
Full-text available
Using a simulated gastrointestinal digestion model combined with a Caco-2 cell model, this study aims to assess the bioaccessibility and cellular uptake of dietary lutein, zeaxanthin, and ferulic acid from muffins and bread prepared from blends of hairless canary seed (HCS), wheat, and corn. Residual digestive enzymes damaged the Caco-2 monolayer and necessitated the requirements for the additional clean-up of the digesta. Several digesta cleanup treatments were examined, and the C18 column, along with AEBSF inhibitor, was selected as the most effective treatment. However, the cleanup treatment reduced lutein, zeaxanthin, and ferulic acid concentrations. The bioaccessibility of lutein from muffins was high at 92–94% (without clean-up) and 81–86% (with cleanup); however, the cellular uptake was low (7–9%). The bioaccessibility and cellular uptake (4–11%) of zeaxanthin were lower than lutein. Ferulic acid from muffins exhibited a wide range of bioaccessibility for non-cleanup (105–229%) and clean-up (53–133%) digesta samples; however, cellular uptake was very low (0.5–1.8%). Bread made from wheat/HCS had higher lutein bioaccessibility (47–80%) than the control bread (42%), with an apical cellular uptake ranging from 4.3 to 9.2%. Similar to muffins, the bioaccessibility of zeaxanthin from bread was lower than lutein, while ferulic acid had a fairly high bioaccessibility at 98–103% (without clean-up) and 81–102% (with cleanup); however, zeaxanthin cellular uptake was low (0.2%). These results suggest that muffins and bread could boost the daily consumption of lutein, zeaxanthin, and ferulic acid, allowing for a small portion to be absorbed in the small intestine.
... Recently, hairless canary seed (Phalaris canariensis L.), an annual cereal grain, emerged as a novel and safe food ingredient with various nutritional and techno-functional attributes due to its small starch granules, phytochemical profile, and high protein content (19-24%) [10]. Interestingly, over decades, hairless canary seeds have been processed and employed as a traditional medicinal drink, known as "leche de alpiste" (alpiste milk) in Latin American countries to alleviate chronic conditions like cardiovascular diseases, diabetes, hypertension, oxidative damage, and obesity [11][12][13]. In vitro studies have associated the biological activities of canary seed with biopeptides produced during gastrointestinal digestion of the hydrophobic proteins present in the seed [13][14][15]. ...
... Interestingly, over decades, hairless canary seeds have been processed and employed as a traditional medicinal drink, known as "leche de alpiste" (alpiste milk) in Latin American countries to alleviate chronic conditions like cardiovascular diseases, diabetes, hypertension, oxidative damage, and obesity [11][12][13]. In vitro studies have associated the biological activities of canary seed with biopeptides produced during gastrointestinal digestion of the hydrophobic proteins present in the seed [13][14][15]. In this respect, research from our group has unveiled that canary seed peptides produced from commercial proteolysis with Alcalase ® have high in vitro bioavailability and are remarkably stable as inhibitors of metabolism-regulating enzymes like pancreatic lipase, angiotensin-converting enzyme (ACE), and dipeptidyl peptidase-IV (DPP-IV) even after simulated gastrointestinal digestion [16]. ...
Full-text available
Previous research showed that canary seed (Phalaris canariensis L.) peptides (CSP) possess robust in vitro antiobesity properties via inhibition of pancreatic lipase (PL). Nevertheless, no studies have yet explored their antiobesity properties in vivo. Consequently, we investigated the effects of CSP in C57BL/6J mice under a Western diet (WD). Mice were assigned into groups and fed a normal diet (ND) or a WD accompanied by an oral dose of CSP (250 or 500 mg/kg/day), orlistat (40 mg/kg/day), or distilled water. The results showed that consuming CSP can provide metabolic benefits, including preventing weight gain by up to 20%, increasing glucose tolerance, and reducing insulin, leptin, and LDL/VLDL levels in plasma. Conversely, total ghrelin was unaffected by CSP-500, but decreased by CSP-250, and amplified by orlistat. Surprisingly, CSP-250 was more effective in preventing weight gain and promoting satiety than CSP-500. Parallel to this, protein absorption in CSP-500 was decreased, supported by a rise in fecal crude protein (+3.5%). Similarly, fecal fat was increased by orlistat (38%) and was unaffected by CSP-250 (3.0%) and CSP (3.0%), comparatively to WD (2.5%). Despite this, both CSP treatments were equally effective in decreasing hepatic steatosis and avoiding hyperlipidemia. Furthermore, the enzymatic analysis showed that CSP-PL complexes dissociated faster (15 min) than orlistat-PL complexes (41 min). Lastly, CSP did not affect expression of hepatic lipid oxidation genes ACO and PPAR-α, but reduced the expression of the hydrolase gene LPL, and lipogenesis related genes FAS and ACC. Taken together, these results suggest that CSP antiobesity mechanism relies on lipid metabolism retardation to increase fat transit time and subsequently suppress hunger.
... The antioxidant activities of these proteins were likely to be due to the release of antioxidant peptides IY and VY upon pepsin treatment [23]. In addition, prolamin was found to exert bioactive activities which could activate anti-leukemia immunity in human [24,25], with additional hypoglycemic as well as antioxidant and antihypertensive effects [26,27]. ...
Full-text available
Rice protein is considered an important dietary protein source. Information regarding rice nutrient-related protein expression is still scarce, hence further study investigating this aspect is highly needed. Herein, we applied sequential window acquisition of all theoretical mass spectra (SWATH-MS) for a comparative proteomic analysis across six different Malaysian rice varieties. These consisted of black rice (BR: PH9 and BALI), red rice (RR: MRQ100 and MRM16), and white rice (WR: MRQ76 and MR297). This study aimed to unravel rice nutrient-related proteins and if their expressions were significantly different across varieties. A total of 4022 quantified proteins were found to be significantly expressed across all varieties with a false discovery rate (FDR) < 1% and p < 0.05. While among 1792 differentially expressed proteins (DEPs) that were identified, 74 DEPs had functions related to nutrient biosynthesis. There were significantly higher expressions of key enzymes for the carotenoid and amylopectin biosynthesis pathways and seed storage proteins, i.e., prolamins and glutelins in RR. Glycoproteins such as cupin and germin-like protein, as well as enzymes that are involved in the biosynthesis of thiamine and anthocyanin were abundantly found in BR. WR was particularly enriched with biosynthesis enzymes for essential amino acids (methionine and arginine), vitamin B, and unsaturated fatty acid. This study provides us insights into the differential expressions of storage and functional proteins with nutrient-related properties in shaping rice grain pigmentations and plant immunity, as well as in contributing diverse health benefits as daily functional food for human consumption.
... Among these compounds, those from proteinaceous nature are the most promising. Peptides from different food sources such as sesame and hemp seeds, corn, spinach, egg white proteins, rapeseed, meat, flaxseed, insects, birdseed, and amaranth seed have been characterised by their antihypertensive properties [11][12][13]. ...
Full-text available
This study aimed at determining the effect of cocoa proteins (CP) on the blood pressure, using in silico, in vitro and in vivo approaches. The in silico assay showed 26 Criollo cocoa peptides with alignment in the Blast® analysis. Peptide sequences ranged from 6 to 16 amino acids, with molecular weight ranging from 560.31 to 1548.76 Da. The peptide sequences LSPGGAAV, TSVSGAGGPGAGR, and TLGNPAAAGPF showed the highest theoretical affinity with −8.6, −5.0, and −10.2 kcal/mol, for the angiotensin-converting enzyme (ACE), renin, and angiotensin II type 1 receptor (AT1-R), respectively. The Criollo CP hydrolysates (CPH) presented in vitro ACE inhibitory activity with an IC50 value of 0.49 mg/mL. Furthermore, the orogastric administration of 150 mg CP/kg/day in rats fed a high-fat (HF) diet (HF + CP group) showed a significant decrease in systolic blood pressure (SBP) by 5% (p < 0.001) and diastolic blood pressure (DBP) by 7% (p < 0.001) compared with the HF group. The human equivalent dose (HED) of CP for an adult (60 kg) is 1.45 g per day. These results suggest that the consumption of CP could reduce blood pressure by blocking ACE, and could be used as an ingredient in the elaboration of antihypertensive functional foods.
Full-text available
Objetivo: Conhecer as práticas e saberes, em fitoterapia, dos profissionais de saúde de Unidades Básicas de Saúde (UBS) no município de Campina Grande, Paraíba. Metodologia: Estudo transversal do tipo exploratório e descritivo, realizado mediante um questionário apropriado. Após a análise estatística descritiva, algumas correlações entre variáveis foram realizadas mediante o teste qui-quadrado e o teste exato de Fischer, com nível de significância de 5%. Resultados: Entre os profissionais (n=93) pouco mais da metade (50,6%) relataram ter adquirido conhecimento sobre fitoterapia por iniciativa própria. Apenas 25,8% desses profissionais prescreviam ou aconselhavam o uso de plantas medicinais para os pacientes. Em todos esses casos, a relação entre espécie vegetal citada e uso indicado apresentou respaldo na literatura científica; apesar disso, apenas 30 (32,3%) responderam que se sentem capacitados para prescrever/orientar o uso das plantas medicinais e fitoterápicos. Os entrevistados (98,9%) se manifestaram favoráveis à implantação de um programa de fitoterapia na rede municipal de saúde. Como correlações estatisticamente significativas, tivemos que entre os profissionais que possuem um conceito adequado sobre fitoterapia, predominam as mulheres (p=0,027), e que os profissionais de nível superior prescrevem ou orientam o uso de plantas medicinais e fitoterápicos, mais que os profissionais de nível médio (p=0,047). Conclusão: A prescrição e/ou orientação para o uso de plantas medicinais e/ou fitoterápicos não é uma realidade entre a maioria dos entrevistados, sendo necessário investimentos em capacitação desses profissionais de saúde, para a promoção da fitoterapia racional na Atenção Primária em Saúde.
Full-text available
During oxidative stress, degenerative diseases such as atherosclerosis, Alzheimer’s, and certain cancers are likely to develop. Recent research on canary seed (Phalaris canariensis) peptides has demonstrated the high in vitro antioxidant potential. Thus, this study aimed to assess the cellular and in vivo antioxidant capacity of a low-molecular-weight (<3 kDa) canary seed peptide fraction (CSPF) using Caco-2 cells and the Caenorhabditis elegans model. The results show that the CSPF had no cytotoxicity effect on Caco-2 cells at any tested concentration (0.3–2.5 mg/mL). Additionally, the cellular antioxidant activity (CAA) of the CSPF was concentration-dependent, and the highest activity achieved was 80% by the CSPF at 2.5 mg/mL. Similarly, incubation with the CSPF significantly mitigated the acute and chronic oxidative damage, extending the lifespan of the nematodes by 88 and 61%, respectively. Furthermore, it was demonstrated that the CSPF reduced the accumulation of reactive oxygen species (ROS) to safe levels after sub-lethal doses of pro-oxidant paraquat. Quantitative real-time PCR revealed that the CSPF increased the expression of oxidative-stress-response-related gene GST-4. Overall, these results show that the CSPFs relied on GST-4 upregulation and scavenging of free radicals to confer oxidative stress protection and suggest that a CSPF can be used as a natural antioxidant in foods for health applications.
The bioactivity of canary seed peptides (CSP) towards metabolism-regulating enzymes was evaluated. Peptides with angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPP-IV), and pancreatic lipase activity remained stable (p < 0.05) to simulated gastrointestinal digestion (SGD). CSP-SGD were transported efficiently (>10%) through the Caco-2 monolayer, indicating absorption through the intestinal epithelium. Lineweaver-Burk plots demonstrated that CSP-SGD act as mixed-type inhibitors for DPP-IV and α-glucosidase. Furthermore, CSP-SGD were potent as antihypertensive and antiobesity agents. Molecular docking and in silico analyses were targeted to understand CSP-SGD interactions with ACE and pancreatic lipase. ACE-inhibitory peptides (LHPQ, QTPHQ, KPVPR, and ELHPQ) acted as non-competitive inhibitors by destabilization of the transition state and Zn(II) coordination in ACE. The uncompetitive inhibition of pancreatic lipase by peptides (VPPR, LADR, LSPR, and TVGPR) destabilized the open-lid conformation of pancreatic lipase. The results of this study showed that canary seed proteins could serve as a source of biologically active peptides.
The present chapter shows an overview of the production of bioactive peptides (BAPs) obtained from food matrices, using fermentation processes. It shows that it is possible to obtain BAPs from milk, meat, and vegetable proteins and emphasizes scientific production and the proven benefits that milk protein-derived BAPs provide to health. It also emphasizes a promising outlook in BAP production by fully using meat and vegetable proteins using food industry by-products, which also helps to mitigate waste environmental issue. For viable and safe BAPs industrial production, advances about in vivo research and adaptations of biotechnological processes for this scale of production are required.
Fermented food and beverages constitute a significant part of the human diet (5%–40%) worldwide. Fermentation has been used for preservation and to augment the flavor, texture, and nutritional qualities of the food, since antiquity. During fermentation, the bioavailability of vitamins, minerals, and other constituents increases due to the microorganisms’ metabolic activities. Besides enhancing nutritional quality, fermented foods contain live organisms reported to prevent/treat many health disorders. Types of the fermentation process are also classified based on these microorganisms. In developing countries, fermented foods were usually prepared using traditional methods without any standardized techniques. Considering the beneficial effects of fermented foods, industrial-level production requires consistent specific microorganisms, fermentation methods, evaluation of nutritional compositions, and food safety testing. This chapter discusses the fermented foods and associated organisms, different sources available for the consumption of fermented foods, and food component’s effect on microorganism’s efficacy.
Full-text available
In this work, the bioactive peptides produced during in vitro gastrointestinal digestion of soybean seeds and soy milk were investigated. The analysis was performed on extracted protein samples from soybean seeds and milk or directly on untreated soy milk. Proteins samples were subjected to simulated gastrointestinal digestion and then analyzed by nano-liquid chromatography coupled to tandem mass spectrometry for peptide sequencing. The identified peptides were 1173 in soybean seed samples, 1364 in untreated soy milk samples and 1422 in soy milk samples in which proteins were extracted by precipitation. The peptide identifications were then employed to search specific databases and look for the presence of bioactive peptides in the investigated samples, either with known biological activity or with potential antimicrobial activity. Results pointed out that soybean proteins underwent an extensive degradation process during gastrointestinal digestion and generated a large number of bioactive peptides, some with established activity, some with predicted antimicrobial activity. Finally, the supernatants collected after protein precipitation with acetone from both soybean seeds and soy milk were also analyzed to evaluate the presence of peptides produced by the action of endogenous proteases. Likely, peptides found in soy milk samples could be formed during food processing.
Full-text available
Amaranth (Amaranthus hypochondriacus) is a pseudocereal with higher protein concentration than most cereal grains. Enzymatic hydrolysis and food processing could produce biopeptides from amaranth proteins; however, there is limited information about the bioactivity of peptides from amaranth proteins. The objective of this comprehensive review was to determine bioactive peptide sequences in amaranth proteins that may prevent cardiovascular disease, cancer, and diabetes. Amaranth proteins, reported in UniProt database, were evaluated for potential bioactive peptide using BIOPEP database. The 15 main proteins present in amaranth seed are 11S globulin, 7S globulin, α-amylase inhibitor, trypsin inhibitor, antimicrobial proteins, nonspecific lipid-transfer-protein-1, superoxide dismutase, ring-zinc finger protein, prosystemin, amaranth albumin 1, glucose-1-phosphate adenyltransferase, glucosyltransferase, polyamine oxidase, granule-bound starch synthase 1, and acetolactate synthase. All proteins showed high occurrence frequencies of angiotensin-converting enzyme-inhibitor peptides (A = 0.161 to 0.362), as well as of dipeptidyl peptidase IV inhibitor (A = 0.003 to 0.087). Other proteins showed antioxidative (A = 0.012 to 0.063) and glucose uptake-stimulating activity (A = 0.023 to 0.042), and also antithrombotic (A = 0.002 to 0.031) and anticancer sequences (A = 0.001 to 0.042). The results of this study support the concept that amaranth grain could be part of a “healthy” diet and thereby prevent chronic human diseases.
Full-text available
We evaluated the capacity of simulated gastrointestinal digests or alcalase hydrolysates of protein isolates from amaranth to scavenge diverse physiologically relevant reactive species. The more active hydrolysate was obtained with the former method. Moreover, a prior alcalase treatment of the isolate followed by the same simulated gastrointestinal digestion did not improve the antioxidant capacity in any of the assays performed and even produced a negative effect under some conditions. Gastrointestinal digestion produced a strong increment in the scavenging capacity against peroxyl radicals (ORAC assay), hydroxyl radicals (ESR-OH assay), and peroxynitrites; thus decreasing the IC50 values to approximately 20, 25, and 20 %, respectively, of the levels attained with the nonhydrolyzed proteins. Metal chelation (HORAC assay) also enhanced respect to isolate levels, but to a lesser extent (decreasing IC50 values to only 50 %). The nitric-oxide- and superoxide-scavenging capacities of the digests were not relevant with respect to the methodologies used. The gastrointestinal digests from amaranth proteins acted against reactive species by different mechanisms, thus indicating the protein isolate to be a potential polyfunctional antioxidant ingredient.
Full-text available
Prolamin storage proteins are the main repository for nitrogen in the endosperm of cereal seeds. These stable proteins accumulate at massive levels due to the high level expression from extensively duplicated genes in endoreduplicated cells. Such abundant accumulation is achieved through efficient packaging in endoplasmic reticulum localized protein bodies in a process that is not completely understood. Prolamins are also a key determinant of hard kernel texture in the mature seed; an essential characteristic of cereal grains like maize. However, deficiencies of key essential amino acids in prolamins result in relatively poor grain protein quality. The inverse relationship between prolamin accumulation and protein quality has fueled an interest in understanding the role of prolamins and other proteins in endosperm maturation. This article reviews recent technological advances that have enabled dissection of overlapping and non-redundant roles of prolamins, particularly the maize zeins. This has come through molecular characterization of mutants first identified many decades ago, selective down-regulation of specific zein genes or entire zein gene families, and most recently through combining deletion mutagenesis with current methods in genome and transcriptome profiling. Works aimed at understanding prolamin deposition and function as well as creating novel variants with improved nutritional and digestibility characteristics, are reported.
Full-text available
Obesity is one of the major factors to increase various disorders like diabetes. The present paper emphasizes study related to the antiobesity effect of Phalaris canariensis seeds hexane extract (Al-H) in high-fat diet- (HFD-) induced obese CD1 mice and in streptozotocin-induced mild diabetic (MD) and severely diabetic (SD) mice.AL-H was orally administered to MD and SD mice at a dose of 400 mg/kg once a day for 30 days, and a set of biochemical parameters were studied: glucose, cholesterol, triglycerides, lipid peroxidation, liver and muscle glycogen, ALP, SGOT, SGPT, glucose-6-phosphatase, glucokinase, hexokinase, SOD, CAT, GSH, GPX activities, and the effect on insulin level. HS-H significantly reduced the intake of food and water and body weight loss as well as levels of blood glucose, serum cholesterol, triglyceride, lipoprotein, oxidative stress, showed a protective hepatic effect, and increased HDL-cholesterol, serum insulin in diabetic mice. The mice fed on the high-fat diet and treated with AL-H showed inhibitory activity on the lipid metabolism decreasing body weight and weight of the liver and visceral adipose tissues and cholesterol and triglycerides in the liver. We conclude that AL-H can efficiently reduce serum glucose and inhibit insulin resistance, lipid abnormalities, and oxidative stress in MD and SD mice. Our results demonstrate an antiobesity effect reducing lipid droplet accumulation in the liver, indicating that its therapeutic properties may be due to the interaction plant components soluble in the hexane extract, with any of the multiple targets involved in obesity and diabetes pathogenesis.
Canary grass is used as traditional food for diabetes and hypertension treatment. The aim of this work is to characterize the biological activity of encrypted peptides released after gastrointestinal digestion of canary seed proteins. Canary peptides showed 43.5% inhibition of dipeptidyl peptidase IV (DPPIV) and 73.5% inhibition of angiotensin-converting enzyme (ACE) activity. An isolated perfused rat heart system was used to evaluate the canary seed vasoactive effect. The nitric oxide (NO), a major vasodilator agent, was evaluated in the venous effluent from isolated perfused rat heart. Canary seed peptides (1 μg/mL) were able to induce the production of NO (12.24 μM) in similar amounts to those induced by captopril (CPT) and bradikynin (BK). These results show that encrypted peptides in canary seed have inhibitory activity against DPPIV and ACE; enzymes that are targets for diabetes and hypertension treatments.