Ultrasonics - Sonochemistry 58 (2019) 104700

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Ultrasonics - Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Structural modification of quinoa seed protein isolates (QPIs) by variable T

time sonification for improving its physicochemical and functional
characteristics
⁎
Nisar A. Mir, Charanjit S. Riar , Sukhcharn Singh
Department of Food Engineering & Technology, Sant Longowal Institute of Engineering & Technology, Longowal, Punjab 148106, India

A R T I C LE I N FO A B S T R A C T

Keywords: High intensity ultrasound treatment (HIUS) by probe method is a novel technique to impart desirable physical,
Quinoa protein isolates structural and functional characteristics to the native proteins structures. In this concern, effect of HIUS treat-
Dynamic rheology ment at variable intervals from 5 to 35 min on quinoa seed protein isolates (QPIs) characteristics was analyzed. A
Structural properties typical dynamic rheological characteristic curve of QPIs had been obtained as a result of HIUS treatments at
Functional properties
variable time intervals. Higher sonication resulted in the formation of large protein aggregates with higher
SEM
Acoustic energy
particle size which increased QPIs turbidity. Temperature and frequency sweep tests had shown dominance of
storage modulus over loss modulus, thus described strong gelling behavior of treated QPIs. HIUS treatment
reduced particle size of QPIs with improved its flow properties. No splitting of bands had occurred due to
sonication, whereas, more intensity of bands of treated QPIs depicted its greater water solubility. HIUS treatment
decreased fluorescence intensity of QPIs whereas, no significant changes in Amide-II & III regions of QPIs oc-
curred except decrease in wave number. The effects of HIUS on QIPs isolates had shown completely different
response than those of results of quinoa protein extracts. Moreover, the studies conducted on quinoa protein
extracts provided detailed information about the effect of HIUS on structural changes and its impact on phy-
sicochemical, functional and rheological characteristics.

1. Introduction oxidative stress related diseases like cancer [2]. In India, little in-
formation available about the nutritional and health benefits of proteins
Quinoa (Chenopodium quinoa) belongs to the family Chenopodiaceae extracted from quinoa seed crop grown. Since majority of Indian po-
and is a highly valuable pseudo-cereal seed crop because of its excellent pulation rely upon traditional cereal crops like wheat, maize, barley etc.
nutritional profile [1]. Quinoa comprises approximately 250 species therefore, protein isolates extracted from quinoa seeds can be used to
and has been domesticated in the Andean regions of Peru and Bolivia. enrich different kinds of foods like beverages, bakery products and
In Peru, Argentina, Ecuador and Bolivia it is commonly known as breakfast cereals in order to increase their nutritional value.
quinoa. In the recent years, production of quinoa has increased re- HIUS treatment, particularly by probe method is a novel approach
markably because of its gluten free nature, high quality protein (par- to impart desirable structural characteristics to the native structure of
ticularly good content of essential amino acids), excellent fatty acid proteins which are otherwise usually lacking in these protein sources.
profile, dietary fiber and presence of vitamins and minerals [2]. HIUS is defined as a clean and green technology which has been used
Moreover, the quality of protein in terms of nutritional characteristics from laboratory to industrial scale in a wide range of food products to
like amino acid score, protein efficiency ratio, essential amino acid improve their physicochemical properties from the technological point
index, biological value, and nutritional index is also far better than the of view [4]. HIUS is characterized either by low frequency
traditional cereal crops like wheat, maize, rice and barley [3]. Studies (16–100 kHz, power 10–1000 Wcm−2) to high frequency
conducted on peptides released by quinoa protein under simulated (100 kHz–1 MHz, power < 1 Wcm−2) [5]. Low intensity or high fre-
gastrointestinal digestion have shown promising results against quency ultrasound is also a non-destructive type of technique which is

Abbreviations: WBC, Water binding capacity; OBC, Oil binding capacity; HIUS, High intensity ultrasound treatment; QPIs, Quinoa protein isolates; SDS-PAGE,
Sodium dodecyl sulphate polyacrylamide gel electrophoresis; FTIR, Fourier transform infra-red spectroscopy; SEM, Scanning electron microscope
⁎
Corresponding author.
E-mail address: charanjitriar@yahoo.com (C.S. Riar).

https://doi.org/10.1016/j.ultsonch.2019.104700
Received 4 April 2019; Received in revised form 5 July 2019; Accepted 16 July 2019
Available online 17 July 2019
1350-4177/ © 2019 Elsevier B.V. All rights reserved.
N.A. Mir, et al. Ultrasonics - Sonochemistry 58 (2019) 104700

used to evaluate certain characteristics of foods like determination of 2.2.3. Acoustic energy determination
quality characteristics of food products and inspecting packaging sys- The amount of acoustical energy information is valuable for ob-
tems, whereas, high intensity or low frequency ultrasonication is used taining the energetic yield of sonochemical reactions which corre-
to alter the physicochemical and functional characteristics of different sponds to chemical acoustical energy and gives the information related
types of food constituents. Overall, the success of protein isolates before to formation of radical and molecular products as per the proposition
employing them in food systems mainly depends upon its functional given by Grotthus-Draper law for photochemical reactions. The calcu-
properties. Gelation is considered as one of the most important func- lation for dissipated acoustic power absorbed in a volume of liquid was
tional characteristics of food systems which can be altered or improved calculated according to the calorimetric method of Jiang et al. [11].
by different types of approaches. Gelation properties of sunflower Ultrasound treatment with the frequency of 20-kHz and power output
protein isolates obtained from dephenolized sunflower meal has shown of 500 W generated ultrasonic intensities of 43 W/cm2 respectively
a significant improvement after the application of HIUS [6]. In addi- [12].
tion, functional properties like emulsification and water binding capa-
cities have also shown significant improvement after treating with HIUS 2.2.4. Physicochemical characteristics
[7,8]. This improvement in gelation and functional characteristics may 2.2.4.1. Emulsification properties. Emulsifying properties such as
possibly be due to the cavitation mechanism, shear stress turbulences or emulsion activity and emulsion stability of native and HIUS treated
may be due to some structural modifications caused by HIUS treatment quinoa protein isolates were determined by the method of Lawal et al.
[9]. Moreover, the literature focusing on the potential impact of HIUS [13].
treatment on QPIs is also scarce. Till this date, there has been only one
study conducted by Vera et al. [10] regarding the effect of HIUS 2.2.4.2. Turbidity. Turbidity of native and HIUS treated QPIs have been
treatment on quinoa proteins extracts containing around 47% protein. determined by the method of Jiang et al. [14].
However, the present study aims at to analyze the effects of HIUS with
varying time intervals on quinoa protein isolate containing about 94%, 2.2.4.3. WBC and OBC. WBC and OBC were determined by the method
db, protein. Moreover, Vera et al. [10] have studied the effect of HIUS of Timilsena et al. [15] as modified by Mir et al. [3].
on conformational and physicochemical characteristics of quinoa pro-
tein extracts. In the present study, the authors have focused on detailed 2.2.5. Dynamic rheology
information about the structural modifications caused by HIUS in QPIs Dynamic rheology of HIUS treated QPIs was determined according
and its impact on functional, rheological, and physicochemical char- to the method of Sun and Arntfield [16].
acteristics, which are very important from the processing and techno-
logical point of view. The results obtained from the present investiga- 2.2.6. SDS-PAGE electrophoresis
tion can provide valuable information about the utilization of quinoa SDS-PAGE electrophoresis of native and HIUS treated quinoa pro-
proteins in a diverse range of food products particularly gluten free and tein isolates was performed according to the method of Laemmli [17]
essential amino acid rich foods in the near future, which could be an using Electrophoresis equipment (GE Healthcare Laboratories, Rich-
alternative to the products prepared from traditional cereal crops. mond, CA, USA) and Mni VE electrophoresis tank (Amersham Bios-
ciences Company, USA).
2. Materials and methods
2.2.7. Fourier transform infra-red (FTIR) spectroscopy
2.1. Materials FTIR analysis of native and HIUS treated QPIs was determined by
the method of Kumar et al. [18].
Quinoa (Chenopodium quinoa) seeds were obtained from a certified
seed center in Rajasthan (India). The grains were cleaned, sieved to 2.2.8. Intrinsic fluorescence
remove foreign matters and then ground in a lab scale grinder (Agrosaw Intrinsic fluorescence spectrum of native and HIUS treated QPIs was
Private Limited, India). The mixture thus obtained was passed through obtained by the method as described by Xiong et al. [19].
a 60-mesh sieve for obtaining flour of uniform particle size. The flour
was packed and sealed in an air tight container and stored at 4 °C till 2.2.9. SEM
further analysis. Millipore water obtained from water purification sys- The morphological characteristics of native and HIUS treated QPIs
tems (Elix 3 Serial No. F7CA82546B, Merk, India) was used in all the have been determined as per the method of Zhu et al. [20]
experiments. All other chemicals used in this study were of analytical
grade. 2.3. Statistical analysis

2.2. Methods The measurements presented in figures represent the average of

three readings and error bars represent standard deviation ( ± SD).
2.2.1. Preparation of quinoa protein isolates (QPIs) Data was analyzed by one way analysis of variance (ANOVA). Duncan’s
QPIs were prepared according to the method of Mir et al. [3]. The multiple range tests (p ≤ 0.05) was done to determine significant dif-
resultant protein isolate contained moisture (10.08 ± 0.08), protein ferences among the mean values by using the software SPSS 16.0 ver-
(84.54 ± 0.53), ash (4.13 ± 0.08), crude fiber (1.23 ± 0.03), and sion.
carbohydrates (0.02 ± 0.01).
3. Results and discussion
2.2.2. High intensity ultrasound treatment (HIUS) of QPIs
Quinoa protein isolate dispersions (8%, w/v) were prepared by 3.1. Effect of HIUS treatment time on physicochemical characteristics of
stirring protein isolate powder in deionized water. Mixing of the protein QPI
dispersion was carried out with the help of Swirlex-Vortex Shaker
(ABDOS Labtech Pvt. Ltd.) at 25 °C for 4 h. Quinoa protein isolates were 3.1.1. Emulsion activity and emulsion stability
sonicated at a frequency of 20 kHz (500 W and 25% amplitude) for 5, The effect of HIUS treatment on emulsification properties of quinoa
15, 25 and 35 min intervals. After sonication treatments, the protein protein isolates is shown in Fig. 1a. The significant (p ≤ 0.05) and
dispersions were freeze dried and kept under refrigerated conditions for desirable changes were observed in emulsification properties of QPIs
further analysis. when the HIUS treatment time was increased from 5 to 25 min wherein

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N.A. Mir, et al. Ultrasonics - Sonochemistry 58 (2019) 104700

a 120 Similarly, emulsion stability of QPIs was also improved by the ap-
plication of HIUS as compared to native protein isolates. Highest values
Emulsion activity and Emulsion stability (%)

a
100 for emulsion stability were observed for quinoa protein isolates treated
a b
b with HIUS for 25 min followed by 35 min, 15 min and 5 min intervals.
c This significant (p ≤ 0.05) improvement in the emulsion stability of
80 c quinoa protein isolates by HIUS treatments may be due to the favorable
d
e d conditions created by HIUS which resulted in the better orientation of
60 e protein molecules and favored adsorption of oil droplets in emulsion.
EA
ES
However, when treatment time for sonication was increased up to
40 35 min, the emulsion stability of QPIs was reduced. This may be due to
the over processing which was promoted by larger duration of sonica-
tion time that resulted in extensive protein unfolding and aggregation
20
and thus decreased emulsion stability. Zhu et al. [20] also observed
similar type of results for emulsion stability values of walnut protein
0 isolates.
0 5 15 25 35

Time (min)

b 3 3.1.2. Turbidity
a Effect of high intensity ultrasound time treatment of quinoa protein
2.5 isolates on turbidity is shown in Fig. 1b. It was observed that sonication
b
b c has induced significant (p ≤ 0.05) changes in turbidity of quinoa pro-
2 d tein isolates. The HIUS treatment up to 25 min resulted in a sharp de-
cline in the turbidity of quinoa protein isolates whereas, a further in-
Turbidity

1.5 crement in the treatment time of 35 min, the turbidity of quinoa protein
isolates had shown an increasing trend. A similar trend was observed
1 for sunflower protein isolates by Malik et al. [24]. Shen et al. [25], have
observed a decline in the turbidity values of whey protein treated with
0.5 high intensity ultrasound. They proposed that the decrease in turbidity
is closely related to the particle size reduction due to which surface area
0 of proteins gets reduced and results in the maximum scattering of light.
0 5 15 25 35 Turbidity represents the size and quantity of protein particles sus-
Time (min) pended in a solution thus it is an important criterion to determine the
formation of aggregates at macroscopic level [26]. However, higher
c 180 a
treatment time of sonication resulted in the formation of large protein
b
c
160 c aggregates with higher particle size which could be responsible for the
d
140 a b increase in turbidity of QPIs.
c
WBC/OBC (%)

120 WBC
d
e 0BC
100 3.1.3. WBC and OBC
80
The effect of HIUS at varying time intervals on water and oil binding
capacities of quinoa protein isolates has been shown in Fig. 1c. It can be
60
observed that there was a significant improvement in the properties of
40 QPIs subjected to HIUs treatments as compared to native quinoa protein
20
isolates. The highest values of WBC were found for QPIs treated with
HIUS for 25 min followed by 15 min and 5 min treatments. The results
0
0 5 15 25 35
of the present investigation are consistent with those observed by Hu
et al. [7] for soybean protein isolates treated with HIUS treatment.
Time (min)
Riener et al. [27], also observed similar type of results for water holding
Fig. 1. Effect of high intensity ultrasound treatments on a) emulsion activity capacity of yoghurt gels treated with HIUS treatment. The reduction in
and stability, b) turbidity, c) water and oil binding capacities of quinoa protein particle size and the improvement in the solubility due to HIUS treat-
isolates. ments were the possible reasons for improvement in the water holding
capacity of quinoa protein isolates. Interestingly, it was also observed
the emulsification properties were improved as compared to native that quinoa protein isolates sonicated for longer duration of time led to
QIPs. Similar results were obtained by Yang et al. [8] for commercial a decline in the values of water holding capacity which might be due to
soy protein isolate. Various researchers have observed an improvement the denaturation of molecular structure of quinoa protein isolate.
in emulsion capacity due to sonication process for different types of Significant (p ≤ 0.05) difference was also observed for OBC of na-
protein isolates such as egg protein [21], soy protein, [22] and peanut tive QPIs and HIUS treated QPIs. The trend shown by oil binding ca-
protein, [23]. The improvement in emulsifying capacity by HIUS pacity was almost similar to that of water binding results. Highest oil
treatment may be due to the availability of greater number of soluble binding capacity was observed in quinoa protein isolates treated with
protein fractions which might have absorbed water at the oil water HIUS for 25 min followed by 35 min, 15 min and 5 min. This increase
interface and also due to the changes in the surface chemistry of the might be due to the exposure of hydrophobic groups of proteins mo-
proteins induced by HIUS treatments. However, further increment in lecules to the external environment after the application of HIUS which
treatment time up to 35 min decreased the emulsion activity of quinoa allows entrapping oil molecules thus increases the oil absorbing capa-
protein isolates. This might be due to disruption of protein structure city [28]. Similar result for OBC was also observed by Malik et al. [24]
and diminishing of oil water interfaces. for sunflower protein isolates.

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N.A. Mir, et al. Ultrasonics - Sonochemistry 58 (2019) 104700

60000
a 700000 b
600000 50000

Native
500000
Storage Modulus G' (Pa)

40000

Loss Modulus G" (Pa)

5 Min Native

400000 15 Min 5 Min

30000 15 Min
25 Min
300000 35 Min 25 Min
20000 35 Min
200000
10000
100000

0
0 0 20 40 60 80 100 120
0 20 40 60 80 100 120
Tempertaure (ºC) Temperature (ºC)

c 10,00,000 d 1,00,000
9,00,000 90,000
8,00,000 80,000
Storage Modulus G' (Pa)

Loss Modulus G" (Pa)

7,00,000 70,000
6,00,000 Native 60,000 Native
5 Min 50,000 5 Min
5,00,000
15 Min 15 Min
4,00,000 40,000
25 Min 25 Min
3,00,000 30,000
35 Min
35 Min
2,00,000 20,000
10,000
1,00,000
0
0
0 20 40 60 80 100 120
0 20 40 60 80 100 120
Frequency (Hz) Frequency (Hz)

Fig. 2. Rheological characteristics of native and HIUS treated quinoa protein isolate such as: (a) Storage modulus as a function of temperature sweep (b) Loss
modulus as a function of temperature sweep (c) Storage modulus as function of frequency sweep (d) Loss modulus as a function of frequency sweep.

3.2. Effect of HIUS treatment time on dynamic rheology of QPIs isolates treated with HIUS. However, the loss moduli values were less as
compared to storage moduli values suggesting the dominance of elastic
The typical dynamic rheological characteristic curves of quinoa components over viscous components. The improvement in the storage
protein isolates which were subjected to HIUS treatments at variable modulus and loss modulus characteristics of quinoa protein isolates by
time are shown in the Fig. 2a. It can be depicted that storage moduli of the application of HIUS can be utilized for improving the mouth feel
all the HIUS treated quinoa protein isolates were higher than native and regulating the texture of different food products [32].
quinoa protein isolates. Among all the HIUS treated quinoa protein Frequency sweep tests values shown in Fig. 2c and d are particularly
isolates, highest storage moduli values were shown by quinoa protein useful tests to enable the viscoelastic properties of samples to be de-
isolates for treatment time of 35 min followed by 25 min, 15 min and termined as a function of time scale. These had been carried out to
5 min intervals. The gelling characteristics of HIUS treated quinoa determine the effect of high intensity ultrasound treatment on quinoa
protein isolates was studied in three phases. The first phase occurred protein isolates parameters such as storage (elastic) modulus (G′), the
from temperature of 25–95 °C, this phase has been known as the heating viscous (loss) modulus (G″), and the complex viscosity (η*). From the
step which was followed by a holding period of 15 min at 95 °C, and results it can be observed that storage and loss moduli of high intensity
finally the cooling phase which started from 95 °C down to 25 °C. It can ultrasound quinoa protein isolates were higher than native quinoa
be observed from the Fig. 2b that in all the three phases, loss moduli protein isolates. Among all the HIUS treated quinoa protein isolates, the
values showed a continuous increase. Similar type of observation has highest values of storage and loss moduli were shown by quinoa protein
been reported by Shen et al. [25] for whey protein gels treated with isolates treated for 35 min followed by 25 min, 15 min and 5 min. Like
high intensity ultrasound. This increase has been associated with the temperature sweep tests, frequency sweep tests had also shown the
strengthening of protein gel network attributed to the stronger ag- dominance of storage modulus over loss modulus values, thus de-
gregation promoted by the hydrophobicity due to high intensity ultra- scribing the strong gelling behavior of quinoa protein isolates as a result
sound treatment [29]. Highest increase was observed in quinoa protein of HIUS treatments. HIUS can reduce the particle size of protein isolates
isolates sonicated for 35 min followed by 25 min, 15 min, and 5 min, which results in an improvement in the flow properties of protein dis-
intervals. This means that stronger gels were produced in quinoa pro- persions [33,34]. Shen et al. [35], obtained similar results for whey
tein isolates treated for 35 min. This abrupt increase in the storage protein gels as a result of HIUS treatments.
moduli of HIUS treated quinoa protein isolates resulted in the change in
the dispersion phase into solid phase thus produced more elastic and 3.3. Effect of HIUS treatment time on SDS-PAGE electrophoresis of QPI
stronger gels. Khatkar et al. [30], also observed the similar type of re-
sults for whey proteins after application of HIUS. Sun and Arntfield The electrophoretic pattern of native and HIUS treated quinoa
[16], proposed that ultrasound shear forces results in the disruption of protein isolates is shown in Fig. 3. The results had shown two intense
disulfide bonds and enhances protein-protein aggregation. Zisu et al. bands in the molecular weight range of 30–40 kDa and 50–65 kDa for
[31], have found that HIUS has a positive impact on the solubility of native and HIUS treated quinoa protein isolates, respectively. It in-
proteins by manipulating the disulfide bonds which results in the for- dicated that no splitting of bands had occurred due to sonication pro-
mation of the relatively rigid structures like protein gels. cess. The more intensity of bands in the sonicated samples as compared
For the loss modulus a similar trend was observed for quinoa protein to native quinoa protein isolates depicted the greater water solubility of

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N.A. Mir, et al. Ultrasonics - Sonochemistry 58 (2019) 104700

200

116.3

97.4

66.2

45

Native 5 min 15 Min 25 Min 35 Min Marker kDa

Bio-rad
Fig. 3. Effect of high intensity ultrasound treatments on SDS-PAGE pattern of quinoa protein isolates (QPI).

sonicated samples. Similar results were observed by Higuera-Barraza with high intensity ultrasound have resulted in decrease in the wave
et al. [36], for squid mantle protein isolates, Hu et al. [7], for soy number as compared to native quinoa protein isolates. In native quinoa
protein isolates, Zhang et al. [23], for peanut protein isolates, and Jiang protein isolates the corresponding peaks for amide-I region were ob-
et al. [11], for black bean protein isolates. The bands present in the served in the wave number at 1664 cm−1 and in case of HIUS treated
molecular weight ranging from 50 to 65 kDa corresponds to 11S glo- quinoa protein isolates this peak was observed in the wave number at
bulin known as chenopodin which are in accordance with the results of 1660 cm−1. A decline in the wave number was also observed in the
Vera et al. [10], for quinoa protein fractions treated with HIUS. They region of 3000–3500 cm−1 in HIUS treated quinoa protein isolates
also observed some bands with molecular weight greater than 190 kDa which determine the consumption of eOH groups of amino acids
but in our case no such kind of bands with this molecular weight were during HIUS process. This may be attributed to the OeH and NeH
obtained. The results are also in contradiction with the results of Re- bonding which results in the formation of hydrogen bonds through
sendiz-Vazquez et al. [28] who observed a reduction in molecular carbonyl groups of protein molecules [39]. The results obtained thus
weight of jack fruit seed protein isolates due to HIUS treatments. suggest that HIUS has resulted in an alteration in the secondary struc-
Likewise, a reduction in molecular weight might have been possible due ture of quinoa protein isolates.
to variation in ultrasound intensity which might have caused higher
shear stress turbulence effects and resulted in the splitting of higher 3.5. Effect of HIUS treatment time on the intrinsic fluorescence pattern of
molecular bands into lower molecular bands. In our case the varying QPI
time treatment might not have caused that much of shear stress tur-
bulences which are responsible for splitting of higher molecular bands Fluorescence spectrum is based on the amino acids residues in a
into lower molecular bands. protein like tryptophan, tyrosine, phenylalanine and particularly tryp-
tophan which are the most important determinants of conformational
3.4. Effect of HIUS treatment time on FTIR analysis of QPIs changes in the tertiary structure of protein molecule. If λmax is less than
330 nm, it is believed that tryptophan residues are buried and are
Impact of high intensity ultrasound treatment with varying time on present in the non-polar environment, however, if the λmax is greater
quinoa protein isolates is shown in Fig. 4. The infrared spectrum ob- than 330 nm the tryptophan residues gets located in the polar en-
tained for HIUS treated quinoa protein isolates can be explained on the vironment due to the conformational changes occurring in the tertiary
basis of three bands which are expressed as amide-I: C]O stretching structure of proteins as a result of sonication process [40]. The fluor-
zone occurring in the wave number from 1700 to 1600 cm−1 and re- escence spectra of native and HIUS treated quinoa protein isolate is
garded as a vulnerable zone for changes in secondary structure as shown in the Fig. 5. It can be observed that the wavelength where the
compared to amide-II band. The amide-II band which is associated with maximum (λmax) fluorescence intensity had occurred was found at
NeH stretching and ranges form 1575 to 1480 cm−1 whereas, the 348 nm. The highest fluorescence intensity (λmax) was found in case of
amide-III band which occurs from 1400 to 1200 cm−1 has been asso- native quinoa protein isolate followed by quinoa protein isolates
ciated with C-N stretching and NeH bending, [37,38]. Our results treated with HIUS for 5 min, 15 min 25 min and 35 min intervals. A
shows that there was no significant change in the amide-II and amide-III similar trend for fluorescence spectra was found by Zhu et al. [20] for
band regions of FTIR spectra of native and HIUS treated quinoa protein walnut protein isolates, Xiong et al. [19] for pea protein isolates, and
isolates, however, it was observed that quinoa protein isolates treated Jiang et al. [11] for black bean protein isolates. However, the results are

5
N.A. Mir, et al. Ultrasonics - Sonochemistry 58 (2019) 104700

90

80

70

Transmittance (%)
60

50 Native
5 Min
40
15 Min
30 25 Min

20 35 Min

10

0
0 500 1000 1500 2000 2500 3000 3500 4000
Wavelength (cm-1)

Fig. 4. Effect of high intensity ultrasound treatments on Fourier transform infrared spectroscopy pattern of quinoa protein isolates.

in contradiction with the results of Vera et al. [10] who observed a isolates (94%) and the variation on the basis of region [10].
reverse kind of trend for quinoa protein fractions treated with high
intensity ultrasound with varying time and pulse durations. They ob- 3.6. Effect of HIUS treatment time on the morphological characteristics of
served the occurrence of peak emission spectra for tryptophan residues QPIs
in the wavelength of ∼340 nm for high intensity ultrasound treated
quinoa protein extracts and a wavelength of ∼346 nm for the native Impact of HIUS treatment on quinoa protein isolates observed under
samples. However, in our results the occurrence of peak emission scanning electron microscopy has been shown in Fig. 6. As compared to
spectra for native as well as HIUS treated quinoa protein isolates the native quinoa protein isolate, HIUS treated protein isolate had
samples have been found in the wave length of ∼348 nm. Moreover, shown the more rough and irregular surfaces. It was also observed that
the fluorescence intensity of control sample was higher than HIUS sonication time also influenced the roughness and geometry of quinoa
treated quinoa protein isolates in our study, than those reported by protein isolates. The results indicated that the quinoa protein isolates
Vera et al. [10] for the quinoa protein extracts. The possible reasons for sonicated for 35 min represented more irregular and rough surfaces as
this may be due to the variation in the pulse duration during sonication compared to quinoa protein isolates treated for lower time intervals.
process, the lower concentration of protein in quinoa protein extracts Our results are in agreement with that of Jiang et al. [11] for black bean
with an average protein content of about 47% as compared to protein protein isolates. This might be due to the cavitation force exerted by the

450

400 Native
5 minutes
350
15 minutes
25 minutes
Fluorescence Intensity

300
35 minutes
250

200

150

100

50

0
300 320 340 360 380 400 420 440 460
Wavelength (nm)
Fig. 5. Fluorescence spectroscopy analysis of native and high intensity ultrasound treated quinoa protein isolates.

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N.A. Mir, et al. Ultrasonics - Sonochemistry 58 (2019) 104700

a b

c d

Fig. 6. Scanning electronic micrographs of (a) native, and (b–e) high intensity ultrasound treated quinoa protein isolates for 5 min, 15 min, 25 min, and 35 min.

probe and the generation of micro streaming and turbulence during corresponds to 11S globulin known as chenopodin in native and HIUS
high intensity ultrasound treatment. It is also noteworthy that sonica- treated quinoa protein isolates. Other functional properties like emul-
tion had resulted in the reduction of fragment size of quinoa protein sifying capacity, emulsifying stability, water and oil binding capacities
isolates. Smaller fragments of irregular shapes and sizes were produced and turbidity have also been improved by the application of HIUS
when quinoa protein isolates were sonicated for 25 min. These results treatment as compared to native quinoa protein isolates. Thus, overall
can be attributed to the changes in particle size of quinoa protein iso- improvement in the rheological, functional and structural character-
lates due to sonication. Some researchers have observed that sonication istics is desirable in different types of foods. Considering, the quinoa
for shorter time results in the production of smaller particles. However, protein isolates having excellent nutritional profile and desirable
when sonication was carried out for longer periods of time, a significant functional, gelling and structural characteristics obtained after HIUS
increase in the particle size of chicken myofibrillar proteins isolate was treatment could be used in a number of food formulations and can also
observed Resendiz-Vazquez et al. [28]. serve as protein diets in the form of protein supplements.

4. Conclusion Declaration of Competing Interest

In this study HIUS treatment with varying time treatment has been The authors declare no conflict of interest in the present study.
proved to be a very important tool for improving the gelling, functional
and structural characteristics of quinoa protein isolates. Favorable re-
sults were obtained for many important parameters of quinoa protein Acknowledgements
isolates after the application of HIUS as compared to native quinoa
protein isolates. Among all the parameters studied like gelling char- First author would like to extend his appreciation to the Ministry of
acteristics were most influenced by HIUS. HIUS led to an improvement Human Resource Development, Govt. of India for providing the fi-
in the gelling characteristics and moreover, the improvement was nancial support in the form of Institutional Fellowship. The authors
proportional with the treatment time of HIUS. HIUS did not produce would also like to thank Deptt. of Zoology (Delhi University), Deptt. of
any changes in the molecular weight of QPI, however, two bands were Chemistry (SLIET, Longowal), and SAIF Labs (Panjab University) for
produced in the molecular weight ranging from 50 to 65 kDa which helping in sample analysis.

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N.A. Mir, et al. Ultrasonics - Sonochemistry 58 (2019) 104700

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