Food Control 115 (2020) 107276

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Antioxidant, quenching, electrophoretic, antifungal and structural T

properties of proteins and their abilities to control the quality of Amaranthus
industrial products
María A. Lozano-Grandea, Alma Leticia Martínez-Ayalaa, Rajamohamed Beema Shafreenb,∗∗,
Arkadiusz Szterkc, Zenon Jastrzębskic, Hanna Leontowiczd, Jerzy Drzewieckie, Pawel Paskof,
Aviva Ezrag, Shela Gorinsteing,∗
a
Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, San Isidro, 62731, Yautepec, MOR, Mexico
b
Department of Biotechnology, Alagappa University, Science Campus, Karaikudi, 630003, Tamil nadu, India
c
National Medicines Institute, 30/34 Chełmska, 00-725, Warsaw, Poland
d
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Warsaw, Poland
e
Plant Breeding and Acclimatization Institute, National Research Institute, Radzików, Poland
f
Department of Food Chemistry and Nutrition, Faculty of Pharmacy, Medical College, Jagiellonian University, 30-688, Kraków, Poland
g
Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

ARTICLE INFO ABSTRACT

Keywords: The present study showed the bioactive properties such as antioxidant, quenching, structural and antifungal in
Amaranth six Amaranthus (A.) hypochondriacus laboratory and industrial products. The values of CUPRAC radical
Protein scavenging, quenching and antifungal capacities in industrial samples were higher than in laboratory. Such
Multiproperties properties can be explained by the presence of polyphenols in the samples. Higher molecular weights and
Biofilm inhibition
corresponding protein bands separated sodium dodecyl sulphate - polyacrylamide gel electrophoresis
Control
(SDS–PAGE) were observed in laboratory protein isolates rather than in industrial samples. Interaction with
Industrial products
Stability human serum albumin (HSA) was used to determine the functional properties of the products. As character-
ization of antifungal properties, all amaranth products at 30 μg/mL concentration significantly reduced (< 50%
inhibition) the metabolic activity of Candida (C.) albicans cells within the biofilm. The structural studies of
proteins by FTIR, fluorescence and UV spectroscopy with SDS denaturation showed that after the industrial
treatment the proteins were relatively stable. The investigated proteins could be considered as an indicator of
food control in commercial industrial products and a source of food additives with health benefits.

1. Introduction after simulated gastrointestinal digestion and showed a potential anti-

Pseudocereals such as amaranth, quinoa, and buckwheat, have at-
tracted the attention of consumers and scientists all over the world due
to their nutritional properties (Banerji, Ananthanarayan, & Lele, 2018).
Amaranth is a rich source of proteins, fats, fibers and minerals. As a
pseudocereal amaranth contains a balanced amino acid profile and is
rich in lysine. The consumption of plant foods is increasing, introducing
new cultivars with their high quality (Aguilar et al., 2015). The use of
amaranth proteins, as a potential food alternative ingredient for func-
tional foods, was investigated in a number of recent reports based on
nutritional, physical, thermal, mechanical, structural, pharmacological
and health-promoting properties. Amaranth peptides were released
proliferative activity over HT-29 tumor cells. This effect was connected
to the induction of cell apoptosis, supporting that amaranth proteins
can serve in functional foods (Sabbione et al., 2019). The hydro-
ethanolic extract of Amaranthus caudatus, containing sugars, poly-
phenols and amino acids which is consumed in food preparations in
Bolivia, showed a positive effect in type 2 diabetic Goto-Kakizaki rats
and healthy Wistar rats (Zambrana et al., 2018). The amaranth products
can be used as alternative in malnutrition, cow milk lactose and casein
intolerance, diabetes, overweight and obesity (Soriano Garcia, 2015).
Staple foods like wheat chapatti in India can be supplemented with
amaranth flour for nutritional and rheological improvement of the
product (Banerji et al., 2018). Characterization of biological active

∗
Corresponding author. Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, 9112001, Israel.
∗∗
Corresponding author.
E-mail addresses: beema.shafreen@gmail.com (R.B. Shafreen), gorin@cc.huji.ac.il (S. Gorinstein).

https://doi.org/10.1016/j.foodcont.2020.107276
Received 11 February 2020; Received in revised form 24 March 2020; Accepted 25 March 2020
Available online 30 March 2020
0956-7135/ © 2020 Elsevier Ltd. All rights reserved.
M.A. Lozano-Grande, et al.
peptides extracted from amaranth was reviewed. The properties of
amaranth proteins were studied in different stages, evaluating protein
hydrolysates that reduced in spontaneously hypertensive rats systolic
blood pressure after the supplementation to the diets. Then such hy-
drolysates as a supplementation can be used also for functional foods
(Ramirez-Torres et al., 2017; Tovar-Perez, Lugo-Radillo, & Aguilera-
Aguirre, 2019). Protein isolates from gluten-free plants are important
food products for everyday human diet, because of replacement of
animal proteins (Drzewiecki et al., 2018; Lopez, Galante, Raimundo,
Spelzini, & Boeris, 2019). Physico-chemical and structural changes by
amaranth protein as a result of different treatments have been eval-
uated in the characteristics of the interfacial film as in the foam stability
and were connected to the increased unfolding, greater flexibility and
net charge of amaranth proteins at acidic conditions (Bolontrade,
Scilingo, & Anon, 2016). There are many possibilities for antibiofilm
activities in vitro experiment not only by the use of proteins, but also by
essential oils and plant extracts (Bazargani & Rohloff, 2016). The nu-
tritional properties of Amaranthus species, Chenopodium quinoa and
Fagopyrum esculentum and industrial products based on these pseudo-
cereals were studied by their polyphenol content, antioxidant capacities
and binding properties by interaction with serum protein (Paśko et al.,
2019; Shafreen et al., 2019). As a summary of the cited recent literature
it was shown that seed storage proteins from maize, common bean,
amaranth, quinoa and chia are associated with a wide range of bene-
ficial effects on the human health such as antihypertensive, anti-cho-
lesterolemic, antioxidant, anti-inflammatory, anticancer, antimicrobial
and immunomodulatory properties. Nowadays the molecular properties
of proteins in pseudocereals have been investigated in order to add into
food formulations or to replace conventional cereals (Orona-Tamayo,
Valverde, & Paredes-López, 2019). Based on all information mentioned
in the recent literature the subject of this study was to estimate the
effects of total phenolic, protein, antioxidant, binding, antifungal and
structural properties of six Amaranthus (A.) hypochondriacus products
after laboratory and industrial treatments. The multi-functional prop-
erties of proteins were evaluated by antioxidant assay, electrophoresis,
fluorescence, UV and FTIR measurements and denaturation with so-
dium dodecyl sulphate.
2. Materials and methods
2.1. Chemicals
Trolox (6-hydroxy-2,5,7,8,-tetramethyl-chroman-2-carboxylic acid);
Folin–Ciocalteu reagent (FCR); Tris, tris (hydroxymethy1) amino-
methane; 2,2′-azobis-2-methyl-propanimidamide; lanthanum (III)
chloride heptahydrate; CuCl2 x2H2O; and 2,9-dimethyl-1,10-phenan-
throline (neocuproine), sodium dodecyl sulphate, and human serum
albumin were obtained from Sigma Chemical Co., St. Louis, MO, USA.
All reagents were of analytical grade. Deionised and distilled water was
used throughout the experiments.
2.2. Preparation of the samples
Six A. hypochondriacus products were investigated in this research:
laboratory samples P1, P2, P3, protein isolate, defatted flour and flour;
industrial samples P4, P5, P6, protein dietary antidepressant, natural
amaranth and protein drink. The industrial samples were bought at
Gastronomia Molecular, Puebla, Mexico, 2018. Protein isolate was
prepared using defatted flour in water with pH 10, and then the su-
pernatant was adjusted to pH 5, and then centrifuged according to
Avanza and Añón (2007). The liquid samples were freeze-dried.
2.3. Elemental analysis, polyphenols, antioxidant and binding capacities
2.3.1. Chemical composition
Dry matter, ash protein content was determined according to AOAC
2
Food Control 115 (2020) 107276
(1996). Protein content was carried out by the Kjeldahl technique,
using conversion factor of 5.85 (AOAC, 1996).
2.3.2. Polyphenols
Polyphenols were extracted with methanol (concentration 20 mg/
mL) for 1 h in an ultrasonic bath at room temperature. Total poly-
phenols (mg gallic acid equivalents (GAE)/g DW) were determined by
Folin-Ciocalteu method (Singleton, Orthofer, & Lamuela-Raventos,
1999).
2.3.3. Antioxidant capacities
Cupric reducing antioxidant capacity (CUPRAC) was determined
with the mixture of 1 mL of copper (II)-neocuproine and NH4Ac buffer
solution, acidified and non acidified methanol extracts of protein (or
standard) solution (x, in mL) and H2O [(1.1−x) mL] were added ribbed
to make the final volume of 4.1 mL. The absorbance was measured at
450 nm (Apak, Güçlü; Özyürek, & Karademir, 2004).
2.3.4. Binding capacities by fluorescence measurements
Three dimensional fluorescence measurements (3D-FL) for protein
extracts at a concentration of 0.01 mg/mL were recorded on a model
FP-6500, Jasco spectrofluorometer, serial N261332, Japan. The 3D-FL
spectra were collected with subsequent scanning emission spectra from
200 to 780 nm at 1.0 nm increments by varying the excitation wave-
length from 200 to 450 nm at 10 nm increments. All solutions for
protein interaction were prepared in 0.05 mol/L Tris–HCl buffer (pH
7.4), containing 0.1 mol/L NaCl (Hamid et al., 2017).
2.4. Protein extraction, 1-D sodium dodecyl sulphate–polyacrylamide gel
electrophoresis (SDS–PAGE), densitometry measurements
40 mg of samples 2–6 or 15 mg (sample 1) were extracted with 1 mL
of Sample Buffer, Laemmli 2X (1:1). Samples were boiled 5 min and
centrifuged. Tris-Glycine 4–12% gradient gels (Jule, Inc., Milford, CT)
were used for protein separation with loading of 10 μL of each sample
from 2 to 6, in the case of sample 1 loading was 5 μL. The gel size was
140 × 160 × 1.5 mm. Tris-Glycine running Buffer was used during 4 h
of electrophoretic run separation, in current 50/30 mA per gel
(Laemmli, 1970). Serva molecular weight marker (6.5–116 kDa) in li-
quid mix was used. Gels were stained in Coomassie Brilliant Blue G-250
stain to visualize proteins. The electrophoregrams were measured for
polymorphism using software BIO GENE (Vilber Lourmat, France, ver.
99.03). Similarity coefficients were calculated. The dendrograms were
generated from homology values using UPGMA algorithm (Nei & Li,
1979). The band separation was estimated according to the peak top
detection. The densitometry profiles of proteins were found as curves of
peaks height of maximum intensity (Drzewiecki et al., 2018).
2.5. Antibiofilm activities of investigated samples against C. albicans
2.5.1. Preparation of culture inoculums and metabolic activity of C.
albicans biofilm
C. albicans were maintained in Sabourand Dextrose Agar (SDA). The
overnight culture (ATCC: 90028) was grown at 37 °C in 2 mL Sabourard
Dextrose broth (SDB) kept in orbital shaker at 75 rpm. Prior to all the
assays, 100 μL of the suspension containing 107 cells mL−1 were used
for further assays. The effect of the six Amaranthus products (P1–P6) to
inhibit the biofilms of C. albicans was tested in 24-well microtiter plates.
Amaranthus products (P1–P6) at three different concentrations
(10–30 μg/mL) were added in SDB containing the fungal suspension at
107 cells/mL and incubated at 37 °C for 24 h. Fresh SDB (900 mL),
90 mL of XTT salt solution (0.5 mg/mL) and 10 mL menadione solution
(1 mM) were added to the preformed C. albicans, and the plates were
incubated at 37 °C for 5 h under dark condition. The conversion of XTT
tetrazolium salt to XTT formazan by the metabolically active cells of C.
albicans biofilm resulted in a colorimetric change, which was read
M.A. Lozano-Grande, et al.
Table 1a
Composition of laboratory and industrial A. hypochondriacus samples.
Índices P1 P2
a b
Protein, N x 5.85,% 80.8 ± 1.9 16.5 ± 1.3
Dry matter, % 89.6 ± 3.4 b 90.4 ± 3.6 ab
Ash, % 2.4 ± 0.4 b 3.2 ± 0.3a
CUPRAC, μMTE/g 5.67 ± 0.4ab 2.86 ± 0.2d
Polyphen, mgGAE/g 0.94 ± 0.07 b 0.62 ± 0.02 c
Quenching,% 37.82 ± 2.4 ab 21.14 ± 1.1c
Mean ± SD (standard deviation) of 3 measurements. Average in rows marked with different letters differ significantly (P < 0.05).
spectrophotometrically at 490 nm (SpectraMax M3, USA) (Cocuaud,
Rodier, Daniault, & Imbert, 2005). The concentration, at which 50% of
C. albicans biofilm is reduced, is determined as the biofilm inhibitory
concentration (BIC).
2.5.2. Cytotoxicity assay
HeLa cells were purchased from National centre for cell science,
pune, India. The cells were grown in DMEM medium, incubated at 37 °C
in 5% of CO2. The cells were passaged for 3–4 days before use. To ex-
amine the cytological effect of Amaranthus products (P1–P6), the cells
(5 × 104/mL) were plated onto 96 well microtitre plates and incubated
at 37 °C for 24 h. The media from the plates were removed, and the cells
were washed twice with PBS (0.01 M, pH 7.4). To this, 20 μL of MTT
solution (5 mg/mL in PBS) was added to each well and incubated for
3 h. After complete solubilisation of formazan the media was replaced
with 150 μL of DMSO. Absorbance was then determined at 540 nm
(PerkinElmer, EnSpire, USA), using a multimode reader (Das &
Jayaprakash, 2018). The cytotoxic effect was expressed as the % cell
viability.
2.5.3. Zebrafish embryonic toxicity studies
Zebrafish were maintained in the re-circulating system
(Aquaneering, standalone system USA) at 28 °C for 10 h dark and 14 h
light conditions. Embryos were collected from breeding tank and wa-
shed with deionised water. For each batch of experiment, ten embryos
per well was used. Embryos were incubated in 1 mL of egg water in 12
well microtitre plates as a positive control (Falcao, de Souza, Dolabella,
Guimaraes, & Walker, 2018). Zebrafish embryos were exposed to three
different concentrations (10–30 μg/mL) of samples (P1–P6) and their
survival and effects on embryos were studied under stereo microscope
(Leica M165FC).
2.6. Structural stability of investigated samples
2.6.1. FTIR spectra
The FTIR spectra of the samples were collected in a Shimadzu
IRAffinity-1 spectrophotometer in ATR sampling mode (attenuated
total reflectance), equipped with LabSolutions IR software to obtain
data. The scan was performed in the mid-infrared region in the 4000-
550 cm−1 spectrum at room temperature (25 °C) with a resolution of
4 cm−1 and 64 scan time. The samples (1 mM) were deposited in the
ATR plate performing three repetitions by sample. The spectra were
processed in the Shimadzu software to obtain the data in absorbance
mode. To evaluate the changes in the secondary structure of amaranth
protein, the spectra were analyzed with the Software Origin 7.0 Pro®
(OriginLab Co., USA) in the Amide I region (1700-1600 cm−1). The
spectra were deconvoluted and fitted with a Lorentzian function to
subsequently obtain the spectral values and calculation of secondary
proteins contains (Kong and Yu, 2007).
2.6.2. Evaluation of the stability of protein samples by fluorescence and UV
measurements after denaturation with SDS
For the fluorescence analysis, Multi-mode Varioskan-LUX device of
ThermoFisher Scientific® was used, equipped with a fluorescence
Food Control 115 (2020) 107276
P3 P4 P5 P6
b bc b
15.5 ± 0.8 13.9 ± 1.1 15.5 ± 0.7 5.4 ± 0.3 c
90.4 ± 4.6 ab 90.4 ± 3.7 ab 90.6 ± 4.4 ab 92.4 ± 3.8 a
3.2 ± 0.3 a 2.3 ± 0.2 b 3.2 ± 0.4 a 1.4 ± 0.1 c
5.53 ± 1.1b 6.04 ± 0.8b 9.18 ± 1.3a 7.16 ± 0.9ab
0.89 ± 0.03 b 0.97 ± 0.03 b 1.45 ± 0.14 a 1.15 ± 0.11 b
36.17 ± 1.9 b 40.51 ± 3.2a 43.30 ± 2.3a 41.26 ± 2.8a
microplate reader and Skanlt software® to draw and export data. All
fluorescence measurements were performed at room temperature in a
scanned spectrum of 300–400 nm, with monochrome filter and in
fluorescence emission mode at 200 nm. For the scanning of the UV
spectra, the UV micro-drop (μ-Drop) accessory was used in the emission
range of 250–450 nm with 5 μL of sample and absorbance mode be-
tween 0 and 6 nm. Sample preparation was carried out by dissolving
protein samples (1:10 v/v) in different aqueous media: phosphate buffer
(5 mM, pH = 7.0) and SDS solution (20%).
2.7. Statistical analysis
All the experiments were performed in triplicates and the results
shown in bar diagrams is the representation of standard deviation. The
data was statistically compared and significant difference was studied
using student t-test with P < 0.05 was considered as statistically sig-
nificant.
3. Results and discussion
3.1. Chemical and biological composition of investigated samples (proteins,
polyphenols, antioxidant and binding properties)
The highest protein content (%) was in Amaranthus protein isolate
(80.8 ± 1.9 for P1, Table 1a), following by defatted flour (laboratory
sample). Natural flour (laboratory sample) and natural amaranth (in-
dustrial) were similar in all indices, except bioactivity (Table 1a). The
amount of protein in amaranth protein dietary antidepressant (P4) was
slightly lower than in natural flour (15.5 ± 0.7). Amaranth protein
drink showed the lowest elemental composition. The obtained results,
showing the amount of proteins in the concentrate, were in line with
other reports, where electrospinning technique was used, and the pro-
tein content of amaranth concentrate obtained commercially was
30.9 ± 0.4% and after purification protein content increased and was
about 85.5 ± 0.2% (Abugoch, Martínez, & Añón, 2010; Aceituno-
Medina, Lopez-Rubio, Mendoza, & Lagaron, 2013).
Similar results were obtained in the present report (Table 1a).
Polyphenols (mg GAE/g) were in range of 0.62 ± 0.02 and
1.45 ± 0.14 and the antioxidant capacities (μMTE/g) determined by
CUPRAC were 2.86 ± 0.2 and 9.18 ± 1.3 as the lowest for P2 and the
highest for P5, respectively. The concentrations of polyphenols (mg
GAE/g) in conventionally processed protein isolates, extracted with
methanol and water were 2.51 ± 0.06 and 3.85 ± 0.07, respectively,
in comparison with the present results of polyphenols in methanol ex-
tract of 1.45 ± 0.14, showing their highest value. Antioxidant capa-
cities (mg TE/g) determined by DPPH assay were 1.69 ± 0.05 and
1.44 ± 0.02 in methanol and water extracts in comparison with higher
values determined by CUPRAC (Table 1A). The obtained results were in
line with other reports (Castel, Andrich, Netto, Santiago, & Carrara,
2014). Polyphenols and proteins found in complexes and present se-
parately play an important role in the quality of the final product. So,
pomace extract from wild blueberry with soy protein isolate produced
polyphenol-protein particles. Complexed polyphenols retained their
structural integrity and bioactive potency for both lyophilized and
3
M.A. Lozano-Grande, et al. Food Control 115 (2020) 107276

Fig. 1. Three-dimensional fluorescence spectra (3D-FL) of A and Aa: HSA, count image of HSA and its 3D-FL spectra in methanol (peak a with measurements of λex/
λem = 228/347 nm and fluorescence intensity of 745.3 arbitral units; peak b with measurements of λex/λem = 280/350 nm and fluorescence intensity of 854.25
arbitral units); B and Bb: count image of HSA in interaction with P5 and its 3D-FL spectra (peak a with measurements of λex/λem = 280/357 nm and fluorescence
intensity of 428.88 arbitral units; peak b with measurements of λex/λem = 280/357 nm and fluorescence intensity of 749.18 arbitral units). Abbreviations: P5,
industrial natural Amaranthus hypochondriacus; human serum albumin (HSA), measurements were done with methanol as a solvent with concentration of the sample
of 30 mg/mL; HSA at 2M x 10−5.

spray dried treatments with biological activities (Hoskin, Xiong,
Esposito, & Lila, 2019). There are reports showing the interaction be-
tween polyphenols and proteins, because such interactions have sig-
nificant effects on the activity of phenolic compounds (Jakobek, 2015).
Proteins from amaranth, chia and quinoa seeds were characterized and
evaluated by their digestibility. The cited data of the biological activ-
ities and properties of the proteins from Amaranthus (Lopez et al., 2019)
agreed with the present results. The present results of relatively high
antioxidant activity (Table 1a) are in line with other reports (Castel
et al., 2014; Ramirez-Torres et al., 2017), showing that amaranth pure
peptides with potential anti-atherosclerotic effect possess high anti-
oxidant activity.
The fluorescence measurements are presented on Fig. 1 and
Table 1a. The calculated results of quenching capacities of the six in-
vestigated samples were in the following order: P5 > P6 > P4 >
P1 > P3 > P2. The maximum of fluorescence intensity of two peaks a
and b differ in the investigated samples. The quenching of HSA during
interaction was higher in peak a than in peak b (Fig. 1B, Bb). The
quenching capacities of P5 were higher than other samples, and this can
be explained by the amount of the polyphenols in the sample. Peak a
showed in all samples bigger changes than peak b, therefore the cal-
culations were done according to changes in the fluorescence intensity
of peak a. This conclusion is in line with the binding properties of
phenolic acids with human serum. Caffeic acid showed high affinity to
HSA, which is important for the drug metabolism in humans (Li et al.,
2016). Most of the interactions with HSA are shown with phenolic
compounds, but HSA has a variety of external and internal ligands
participating in the regulation of pH in blood such as alpha-1-acid
glycoprotein. This factor is important in drugs pharmacokinetics
(Huang & Ung, 2013). Traces of polyphenols in amaranth proteins make
them bioactive. Interactions between resveratrol, as many food-con-
taining polyphenols, and albumin, showed the changes in the fluores-
cence intensity. These results support a role played by polyphenols-
albumin interactions in the plasma for the bio-activities of these food
microcomponents in the body (Latruffe, Menzel, Delmas, Buchet, &
Lancon, 2014).
3.2. Electrophoretic characterization of the products
The SDS-PAGE spectrum of protein electrophoregrams consists of
17–34 bands of different intensities with molecular weights of 6–92 kDa
(high molecular weight bands, more than 67 kDa are present only in
samples 4 and 5, Fig. 2A). The major protein bands were in zone of
14–56 kDa (Fig. 2A). All industrial samples have fewer bands than the
laboratory ones, and the bands are weaker, less distinguishable, espe-
cially in two zones of spectrum: 29–45 kDa and 14 kDa (Fig. 2 A). Bands
of protein isolate (sample 1) were more intensive by the amount of
protein stained on the gel than other samples. The highest number of
bands (more than 30) was detected in electrophoregrams of laboratory
samples 4 and 5. Nevertheless number of bands in defatted flour
(sample 4) was less than in sample 5 (natural amaranth). A level of
biochemical homology (by coefficient of similarity) between protein
samples varied from 0.68 to 0.95 (Fig. 2B). The highest homology of
proteins was shown for samples 3 and 6 (Fig. 2B). Dendrogram analysis
of protein spectrum was able to group samples 3 and 6 to one distinct
cluster of 95% of homology. Sample 1 is clearly distinguishable from
other samples as a separate line, not generating common clusters with
other samples (Fig. 2C). The densitometry analysis of protein profiles
has generated lines of curves (Fig. 2D and E). Partial covering of the
lines was found, and in laboratory and industrial groups the highest
curve showed sample 1 (Fig. 2D and E). It means that in the course of
densitometry lanes-curves composed of peaks, quantitative differences
by their height were strong, except curves 3 and 6, representing sam-
ples manufactured by industry. From the results of SDS-electrophoresis
it can be concluded that the main protein bands which present in
treated by different factors by laboratory or industry technology

4
M.A. Lozano-Grande, et al. Food Control 115 (2020) 107276

Fig. 2. Protein composition of Amaranthus hypochondriacus products: A, Electrophoretical patterns of proteins extracted with Sample Buffer 2x (1:1) from 40 mg
weighted samples (samples 2–6, protein loading 10 μL) and 15 mg (sample 1, protein loading 5 μL) and separated by SDS-PAGE. B, The matrix of biochemical
similarity of proteins: similarity values are shown as decimal fractions. C, UPGMA dendrogram: biochemical similarity values are shown in percentages, as whole
numbers. D, The value of densitometry profiles of protein bands are shown as curves of peak bands height (0–250). The comparison is made on laboratory samples: 1,
4 and 5. E, The value of densitometry profiles of protein bands are shown as curves of peak bands height (0–250). Industrial samples (2, 3, 6) were compared with
protein isolate (sample 1). Numbers of amaranth protein samples in A–E: 1, protein isolate; 2, protein dietary antidepressant; 3, protein drink; 4, defatted flour; 5,
flour; 6, natural amaranth. Molecular weight markers in A: β- Galactosidase 116 kDa, Albumin (bovine) 67 kDa, Ovalalbumin (egg), 45 kDa, Carbonic anhydrase
29 kDa, Trypsin inhibitor (soybean) 21 kDa, Lysozyme 14.3 kDa and Aprotinine 6.5 kDa. The results in B, C are based on the protein electrophoregrams.

significantly differ from the protein isolate. Protein isolate had the
highest amount of protein in comparison with industrial samples, which
was confirmed by protein patterns and densitometry curves of all ex-
amined samples. According to the results obtained by electrophoresis
(Fig. 2A), sample 1 consisted of a mixture of different proteins with
molecular weights, ranging from 10 kDa, as was reported by other
authors (Abugoch et al., 2010; Martínez & Añon, 1996). The results of
protein separation show the differences occurred in laboratory and
industry technology. Amaranth isolate had more proteins than in-
dustrial samples, and this phenomenon occurs in electrophoretic se-
paration due to the following features as protein patterns and densi-
tometry curves of all examined samples.
3.3. Antibiofilm activities of investigated samples against Candida albicans
3.3.1. Metabolic activity of C. albicans biofilm
Amaranthus products (P1–P6) at their BIC reduced the metabolic
activity of C. albicans biofilms cells. The development of drug resistance
is primarily associated with development of metabolically active cells in
a biofilm. From in vitro XTT assay, it elucidated that cells undergo
mitrochondrial respiration during biofilm development which is sig-
nificantly reduced by Amaranthus products (P1–P6). Among them P5
showed 67% inhibition at a concentration of 10 μg/mL, and for P6 at
20 μg/mL there was 69% inhibition. Thus from the results, all the
amaranth products at 30 μg/mL concentration significantly reduced
(< 50% inhibition) the metabolic activity of C. albicans cells within the
biofilm (Table 1b).
3.3.2. Cytotoxicity assay
In vitro cytotoxicity of Amaranthus products at a concentration
ranging from 10 μg −30 μg/mL was investigated on HeLa cells using
MTT. Amaranthus products (P1–P6) showed dose dependent reduction
of cell proliferation (Fig. 3A). At high concentration 30 μg/mL, P5 and
P6 showed 24% and 30% cell viability, respectively.
3.3.3. Zebrafish embryonic toxicity studies
In vivo cytotoxic effect of Amaranthus products (P1–P6) was in-
vestigated on developing embryos. This stage was determined as the

5
M.A. Lozano-Grande, et al. Food Control 115 (2020) 107276

Table 1b also been investigated against food borne pathogens and has shown
Investigated samples of Amaranthus products against Candida albicans biofilm antifungal properties against these pathogens that are harmful for
formation. human when consumed. The aim of the present study was to investigate
Samples % of Biofilm Inhibition six amaranth samples for their antibiofilm activity and in vitro and in
vivo evaluvation to determine the degree of selective toxicity (toxicity
10 μg 20 μg 30 μg against HeLa cell lines and zebrafish embryos infection model). For
biofilm assay C. albicans was used for investigation since it is an op-
P1 42.9* 44.9 57.5*
P2 24.6 35.8 57.7* portunistic fungal pathogen which can cause infection when the im-
P3 42.1 52.9* 57.4 mune system of the human is feeble. Formation of biofilm by C. albicans
P4 50.8* 54.4 56.1 protects the yeast cells from immune cells and conventional antifungal
P5 67.8* 68.8* 69.9*
agents leading to the survival of the organisms and establishes the in-
P6 48.5* 69.3* 74.2*
fection. Hence from XTT reduction assay, it was evident that amaranth
Bold indicates the Biofilm inhibitory concentration (BIC) of the Amaranthus samples significantly inhibited the metabolic activity of the yeast cells
products; * Indicates the statistically significant with a P < 0.05. within the biofilm. Owing to its promise as antibiofilm agent, in vitro
cytotoxicity assay showed the intrinsic ability of amaranth to inhibit
most crucial stage of embryonic development where organogenesis HeLa cell proliferation. Similarly, in vivo toxicity with zebrafish embryo
takes place. There was no malformation and lethal effect even at higher revealed that embryo are highly sensitive to toxic compounds and leads
concentration of 30 μg/mL (Fig. 3B). to structural malformations, because organogenesis is the critical stage
Amaranthus as one of gluten-free pseudocereals became useful in where there is high rate of proliferation of the cells (Cebrian et al.,
everyday consumption, based on its nutrition, especially proteins and 2019). Therefore the study will be first of its kind to discuss that
antioxidants, having several health benefits with high nutritional value amaranth samples are not toxic to the zebra embryos and thereby
containing the essential amino acids, dietary fibre and antioxidant promotes post-fertilization. Among the six amaranth samples used for
property (Drzewiecki et al., 2018; Mosovska et al., 2012). Amaranth has investigation P5 and P6 have shown promising antibiofilm and

Fig. 3. A, Viability of HeLa cells were examined on exposure to Amaranthus products after 24 h incubation. The cytotoxicity was determined by MTT assay. Data are
expressed as the percentage of inhibition compared with negative control in which cell survival was assumed to be 100%. Statistically significant was indicated as *
with P < 0.05. B, Effect of Amaranthus products at 30 μg/mL on embryos of Zebrafish for 48 hpf showed that there was no toxic effect on the embryo which entered
the larval stage.

6
M.A. Lozano-Grande, et al.
antiproliferative effect. They are also reported as non toxic to zebrafish
embryos. Therefore from the above study P5 and P6 were rendered
most suitable for therapeutic purposes with their defining concentration
ranges for safe and rational administration. The present results are in
accordance with other reports showing that many plants were screened
for C. albicans antibiofilm activity. The active extract was noncytotoxic
as in this report. Chemical characterization showed the presence of
phenols which always play the main role and showed activity on C.
albicans. Maximum killing reached 90% of C. albicans cells in suspen-
sion and 65% of cells in biofilms (Brighenti et al., 2017). Analyzing the
present results it can be concluded that even low concentrations of
polyphenols in complexes with proteins increased the inhibition of C.
albicans, which is in line with Bazargani and Rohloff (2016) that all
plant extracts with high antioxidant activities inhibited bacteria cell.
Rutin with its antimicrobial property, showed anti-biofilm potential
against Pseudomonas aeruginosa, which is a model biofilm forming pa-
thogenic bacterium, in combination with conventional antibiotic gen-
tamicin (Deepika et al., 2018). The comparison of the results of la-
boratory samples and industrial products showed that antioxidant and
binding capacities, antifungal and inhibition abilities were higher in the
industrial samples. This can be explained by the presence of higher
amount of polyphenols in the industrial samples compare to laboratory
ones. This is in line with others, who suggested that antioxidant, anti-
microbial and antihaemolytic properties were significantly improved
upon hydrolysis of quinoa and amaranth proteins (Mudgil, Omar,
Kamal, Kilari, & Maqsood, 2019).
3.3.4. Evaluation of the stability of protein samples by FTIR, fluorescence
and UV measurements before and after denaturation with SDS
The peptide groups were observed in the characteristic bands of the
amides in the FTIR spectrum of all amaranth samples (Fig. 4, I). The
band at 3500 cm−1 corresponds to amide A. Amide B, amide II and the
stretching vibration of the N–H groups correlate with the region of
3100–3000 cm−1. The intensity of this band was observed in greater
proportion in amaranth isolate and flours (P1–P3), while the industrial
samples (P4–P6) show the lowest intensity of this band, due to a lower
content of amides and N–H groups. The two bands of the spectrum in
the fingerprint region between 2000 and 1500 cm−1 correspond to
amides I and II. In protein spectra amide I has the most intense ab-
sorption band, which is mainly governed by the stretching vibrations of
groups C=O and C–N with the frequency in the range of
1700–1600 cm−1. The exact position is determined by the conforma-
tion of the spine and the hydrogen-bonding pattern. Amide II, on the
other hand, results from the bending vibration of the N–H group and
the stretching vibration of C–N, so this band is generally sensitive to
conformation (Kong and Yu, 2007).
The contribution of the different vibrations, which were adjusted to
a sum of components, was determined in order to evaluate the sec-
ondary structure of the investigated samples. The structures of the
plates β-turns, α-helix, and β-sheets were evaluated and presented in
the deconvoluted spectra in the range 1700–1600 cm−1, corresponding
to amide I bands (Fig. 4, II). The frequencies of the beta chain structures
as the main bands, were found in all samples. Beta chain structures
form the backbone of amino acids and can deduce the absorption of
amide I, regardless of the sequence of amino acids and their hydrophilic
and hydrophobic properties. Sample P1 (Fig. 4, II) had a maximum at
1640 cm−1 and a minimum at 1610 cm−1. Similar frequency showed
sample P2 with maximum at 1640-1630 cm−1 and a minimum at
1610 cm−1. Samples P3 and P4 demonstrated the highest frequency in
the β-sheet with the maximum at 1640-1630 cm−1. P5 and P6 revealed
the highest frequency in the β-sheet with the maximum at 1640 cm−1,
and with a minimum at 1630-1620 cm−1. These results indicate that
the structure leads mainly to absorption of amide I at that wavelength,
and the samples showed similar values. For the α-helix structures, it
was found that the mean frequency was 1660–1640 cm−1 for the P3
and P4, while the protein isolate has a very low frequency at
7
Food Control 115 (2020) 107276
1660 cm−1. These absorption bands correlate mainly with amide II and
the width of the band indicates the stability by the union of the residues
between α-helix and hydrogen. This can indicate that the industrial
protein isolates have higher H+ content and their structures are in a
compact coiled similar of tertiary globular proteins rather than linear or
helical chains of secondary structures. The industrial samples contained
β-sheet structure which was similar to amaranth defatted flour (Fig. 4,
II; Table 1c). The β-turn structure involves 4 amino acid residues that
form a loop due to chain segments that adopt an antiparallel orientation
and form a hydrogen bond. The amaranth isolate P1 demonstrated
higher frequency at 1690-1670 cm−1 than amaranth flour and in-
dustrial products (Fig. 4, II). In addition Table 1c confirms that the
structure β-turn is in slightly greater proportion then for the rest of the
analyzed samples. According to Barth (2007), the rotation structures
can be type I (not helical), type II and type III (rotation of up to 310
propellers). The protein isolate has type II and III β-turn structures,
while amaranth flour and industrial samples contain type I β-turn
structures.
As it was mentioned above, in addition to FTIR measurements
fluorescence studies were carried out in order to verify the stability of
the investigated samples. The fluorescence spectrum of the sample
depends on the amino acid composition and conformational state of the
protein. As the protein undergoes from a native (folded) to a denatured
(unfolded) state, the local environment surrounding of the aromatic
amino acids changes, which affects the fluorescence properties. These
changes in intrinsic protein fluorescence are used to monitor protein
unfolding. Fluorescence analysis of the protein isolate was used to de-
termine the bioavailability and biocompatibility associated with in-
adequate protein deployment. In addition, the native state of proteins
can be altered during the extraction process, either using high tem-
perature, chemical agents or pH adjustments. As the protein unfolds,
the amino acids in the innermost hydrophobic nucleus of the protein
are exposed to the solvent or surrounding medium. This exposure
promotes susceptibility to quenching agents by reducing the fluores-
cence of compounds such as tryptophan, tyrosine and phenylalanine.
The fluorescence spectrum of investigated samples in buffer with
adjustment of pH 7.0 and after denaturation with SDS evaluated the
change in fluorescence intensity of proteins (Fig. 5A and B). All spectra
show changes in the fluorescence intensity due to the unfolding process
of protein in a linear way to the concentration of the denaturant. This is
observed stronger for samples P1 and P2, but the samples P4, P5, and
P6 showed less deployment due to interactions between protein chains
that contribute significantly to the conformational stability of the whole
protein. It was found that the emission spectra for P4, P5, and P6
samples were the least displaced at wavelengths below 300 nm, where
sample P5 showed the best results, because is maintained the stability
at 288 nm, even at concentration of a strong denaturant such as SDS
(buffer, SDS + buffer). Studies carried out on different proteins have
shown that the unfolding process varies in a linear way to the con-
centration of the denaturant (Sánchez-Miguel et al., 2010). The change
of the fluorescence emission spectra occurs as well, depending on the
degree of exposure of phenylalanine, tyrosine, and tryptophan. When
these emission spectra are inside the protein and hidden and their
fluorescence spectrum is between 280–320 nm, then the residues are
exposed to aqueous solvents and their fluorescence emission moves to
other fluorescence lengths, what was evaluated in the present results.
Such phenomena can be also explained due to protein aggregate con-
formation which affects bioactivity and the immune response (Poole,
Hawe, Jiskoot, & Braeckmans, 2012, ch. 9). The maximum fluorescence
intensity at 350 nm for all treatments with pH changes corresponds to
the maximum fluorescence emission reported for tryptophan and
305 nm for tyrosine fluorescences (Kong and Yu, 2007; Poole et al.,
2012). The fluorescence measurements of proteins in denatured state
showed that the industrial samples remained their stability and can be
placed in descending order as: P5, P6, P4, P3, P2 and P1. This is re-
inforced by the results of the FTIR analysis and the quantification of the
M.A. Lozano-Grande, et al. Food Control 115 (2020) 107276

Fig. 4. I, ATR-FTIR analysis of amaranth samples in the region from 4000 to 400 cm−1; II, A, B, C, D, Deconvoluted FTIR spectra corresponding to amides (1600-
1700 cm−1). P1, P2, P3, P4, P5, P6, protein isolate, defatted flour, flour, protein dietary antidepressant, natural amaranth, protein drink.

8
M.A. Lozano-Grande, et al. Food Control 115 (2020) 107276

Table 1c secondary structures of the proteins. In the case of P1, P2 and P3

Quantification of secondary structures of laboratory and industrial A. hy- samples, the α-helix and β-turns structures dominate, which modifies
pochondriacus samples. the conformational structure and the active sites of the proteins due to
Samples β-turns (%) α-helix (%) Random (%) β-sheets (%) the destabilization of the electrostatic interactions of the secondary
structure (Table 1c).
P1 24.55 10.71 10.61 54.12 To complement the FTIR and fluorescence results, absorption
P2 21.51 20.96 – 57.52
spectra (Uv–vis) were performed in the visible light range
P3 14.06 53.66 – 32.28
P4 14.92 32.07 – 53.01 (300–400 nm) with the same treatment as it was done above, using SDS
P5 19.33 22.04 – 58.64 as a denaturant. The spectra (Fig. 6) present an absorbance intensity
P6 15.96 17.39 – 66.66 behavior similar to the fluorescence test. However, the correlation of
these results with the conformation of aggregates that trigger an ac-
Abbreviations: P1–P6, investigated Amaranthus samples: protein isolate, de-
cumulation of toxic conglomerates requires analysis with an extrinsic
fatted flour, flour protein dietary antidepressant, natural amaranth, and
dye or using green fluorescent protein (GFP) as a marker (Poole et al.,
amaranth protein drink.
2012).
As it was shown above, the most valuable parts of the pseudocereal

Fig. 5. Fluorescence analysis of amaranth protein samples before (A) and after (B) denaturation with SDS. P1, P2, P3, P4, P5, P6, protein isolate, defatted flour, flour,
protein dietary antidepressant, natural amaranth, protein drink. Sample preparation was carried out by dissolving protein samples (1:10 v/v) in different aqueous
media: A, phosphate buffer (5 mM, pH = 7.0) and B, buffer + 20% SDS.

9
M.A. Lozano-Grande, et al. Food Control 115 (2020) 107276

Fig. 6. Visible ultraviolet spectral (300–400 nm) of amaranth protein samples in: A, buffer solution (5 mM, pH = 7.0) and B, buffer + 20% SDS. P1, P2, P3, P4, P5,
P6, protein isolate, defatted flour, flour, protein dietary antidepressant, natural amaranth, protein drink.

10
M.A. Lozano-Grande, et al.
Amaranthus are proteins and polyphenols and can be an additional
complementary nutritional source (Aguilar et al., 2015; Drzewiecki
et al., 2018). The present results are in line with the recently published
reports (Blanco-Padilla, López-Rubio, Loarca-Piña, Gómez-Mascaraque,
& Mendoza, 2015; Tovar-Perez et al., 2019) where was suggested to
consider amaranth as a potential source of nutrition and wide appli-
cation in foods and films. As an example of such application can be
protein drink, produced from the whole amaranth seed, having a long
lasting storing and shelf life. The protein drink contained three times
higher content of protein compared to cow milk (Soriano Garcia, 2015).
The stability of proteins after the industrial invention is in line with the
cited reports and our recent studies, that by products of pseudocereals
showed relatively high binding properties and their cytotoxic activity
against human cervical carcinoma cell lines (Paśko et al., 2019).
4. Conclusions
For this study, Amaranthus laboratory and industrial samples were
characterized by antioxidant, protein and polyphenol contents using
radical scavenging assay, electrophoresis, UV–vis, fluorimetry and FT-
IR spectral analyses and denaturation.
The electrophoretic evaluation showed that the laboratory samples
had higher number of protein subunits that the industrial. Results
showed that industrial products at their minimum inhibitory con-
centration assay (MIC) reduced the metabolic activity of C. albicans
biofilms cells. Two industrial products showed 67% inhibition at a
concentration of 10 μg/mL and at 20 μg/mL there was 69% inhibition.
Evaluation and comparison of antioxidant, binding, and antifungal
properties showed that industrial products of Amaranthus higher than
laboratory ones, what can be explained by incorporation of polyphenols
in the proteins. The structural stability of proteins maintained during
the industrial treatments. Protein properties can be used for the control
of industrial products particularly in this study and for food industry of
cereals and pseudocereals in general.
CRediT authorship contribution statement
María A. Lozano-Grande: Data curation, Investigation, Writing -
original draft. Alma Leticia Martínez-Ayala: Methodology,
Visualization. Rajamohamed Beema Shafreen: Conceptualization,
Investigation, Writing - original draft. Arkadiusz Szterk: Visualization,
Investigation. Zenon Jastrzębski: Methodology, Formal analysis.
Hanna Leontowicz: Formal analysis, Resources. Jerzy Drzewiecki:
Data curation, Resources. Pawel Pasko: Software, Validation. Aviva
Ezra: Software, Validation. Shela Gorinstein: Conceptualization,
Supervision, Writing - review & editing.
Declaration of competing interest
All authors of the present submitted article “Antioxidant,
quenching, electrophoretic, antifungal and structural properties of
proteins and their abilities to control the quality of Amaranthus in-
dustrial products” don't have any conflict of interest.
Acknowledgements
The author, R. Beema Shafreen acknowledge RUSA 2.0 [F. 24–51/
2014-U, Policy (TN268 Multi-Gen), Dept of Edn, GOI]. The authors are
thankful to Dr. Elena Katrich (School of Pharmacy, Hebrew University
of Jerusalem) for her technical assistance in determination of anti-
oxidant status in pseudocereal products.
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