Accepted Manuscript

Effect of high-intensity ultrasound on the technofunctional properties and struc-

ture of jackfruit (Artocarpus heterophyllus) seed protein isolate

J.A. Resendiz-Vazquez, J.A. Ulloa, J.E. Uras-Silvas, P.U. Bautista-Rosales,

J.C. Ramrez-Ramrez, P. Rosas-Ulloa, L. Gonzales-Torres

PII: S1350-4177(17)30049-4
DOI: http://dx.doi.org/10.1016/j.ultsonch.2017.01.042
Reference: ULTSON 3536

To appear in: Ultrasonics Sonochemistry

Received Date: 1 June 2016

Revised Date: 13 September 2016
Accepted Date: 27 January 2017

Please cite this article as: J.A. Resendiz-Vazquez, J.A. Ulloa, J.E. Uras-Silvas, P.U. Bautista-Rosales, J.C.
Ramrez-Ramrez, P. Rosas-Ulloa, L. Gonzales-Torres, Effect of high-intensity ultrasound on the technofunctional
properties and structure of jackfruit (Artocarpus heterophyllus) seed protein isolate, Ultrasonics Sonochemistry
(2017), doi: http://dx.doi.org/10.1016/j.ultsonch.2017.01.042

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Effect of high-intensity ultrasound on the technofunctional properties and
structure of jackfruit (Artocarpus heterophyllus) seed protein isolate

J. A. Resendiz-Vazqueza, J. A. Ulloaac*, J. E. Uras-Silvasb*, P. U. Bautista-Rosalesc,

J.C. Ramrez-Ramrezd, P. Rosas-Ulloa, L. Gonzales-Torres.

a
Posgrado en Ciencias Biolgico Agropecuarias, Universidad Autnoma de Nayarit,
Carretera Tepic-Compostela, Xalisco 63780, Nayarit, Mxico.

b
Tecnologa Alimentaria, Centro de Investigacin y Asistencia en Tecnologa y Diseo
del Estado de Jalisco A. C., Avenida Normalistas 800, Colinas de la Normal,
Guadalajara 44270, Jalisco, Mxico.

c
Centro de Tecnologa de Alimentos, Universidad Autnoma de Nayarit, Ciudad de la
Cultura Amado Nervo, Tepic 63155, Nayarit, Mxico.

d
Unidad Acadmica de Medicina Veterinaria y Zootecnia, Universidad Autnoma de
Nayarit, Carretera Compostela-Chapalilla Km 3.5, Compostela 63700, Nayarit, Mxico.

e
Unidad Acadmica de Ciencias Bsicas e Ingenieras, Ciudad de la Cultura Amado
Nervo, Tepic 63190, Nayarit, Mxico.

*Corresponding authors: E-mail address: arulloa5@gmail.com (J.A.Ulloa) and

jurias@ciatej.mx (J. E. Uras-Silvas)

1
ABSTRACT

The influence of high-intensity ultrasound (HIU) on the technofunctional properties and

structure of jackfruit seed protein isolate (JSPI) was investigated. Protein solutions
(10%, w/v) were sonicated for 15 min at 20 kHz to the following levels of power
output: 200, 400, and 600 W (pulse duration: on-time, 5 s; off-time 1 s). Compared with
untreated JSPI, HIU at 200 W and 400 W improved the oil holding capacity (OHC) and
emulsifying capacity (EC), but the emulsifying activity (EA) and emulsion stability
(ES) increased at 400 W and 600 W. The foaming capacity (FC) increased after all HIU
treatments, as opposed to the water holding capacity (WHC), least gelation
concentration (LGC), and foaming stability (FS), which all decreased except at pH 4 for
FS. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-
PAGE) showed changes in the molecular weight of protein fractions after HIU
treatment. Scanning electron microscopy (SEM) demonstrated that HIU disrupted the
microstructure of JSPI, exhibiting larger aggregates. Surface hydrophobicity and protein
solubility of the JSPI dispersions were enhanced after ultrasonication, which increased
the destruction of internal hydrophobic interactions of protein molecules and accelerated
the molecular motion of proteins to cause protein aggregation. These changes in the
technofunctional and structural properties of JSPI could meet the complex needs of
manufactured food products.

Key words: Jackfruit protein isolate, High-intensity ultrasound, Technofunctional

properties, Surface hydrophobicity, Scanning electron microscopy, Tricine-SDS-PAGE.

2
1. Introduction

Jackfruit (Artocarpus heterophyllus Lam.) is widely cultivated in tropical countries

including Mexico. The fruit contains edible bulbs with yellow flesh and seeds [1],
which are enclosed in a white aril encircling a thin brown spermoderm that covers
fleshy white cotyledons [2]. Jackfruit seeds, which represent 8-15% of the total fruit
weight, are normally discarded, steamed and eaten as a snack, or used in local dishes
[3]. These seeds have fairly high carbohydrate (67%) and protein (20%) contents [1,4].
Jackfruit seeds also contain two lectins, namely jacalin and artocarpin, which have
antibacterial, antifungal and anticarcinogenic properties [3]. Jacalin has proven to be
useful for the evaluation of the immune status of patients infected with human
immunodeficiency virus 1 and the detection of tumors [5].

Recent years have seen an increase in the use of protein vegetables as functional
ingredients in food manufacturing because of their role in human nutrition and health
[3,6]. Proteins are of particular interest in food systems such as gelation, emulsification,
foaming and water/oil binding [7, 8, 9].

On the other hand, high-energy ultrasound has been implemented and used as a new
alternative to carry out studies on the technofunctional properties of proteins [10,11].
Ultrasound is an acoustic wave with a frequency greater than 20 kHz [12]. Ultrasound
can be classified into two frequency ranges: low frequency (20-100 kHz; intensity in the
range of 10 to 1000 W cm-2) and high frequency (100 kHz-1 MHz; intensity <1 W cm-2)
[13,14]. Low-intensity ultrasound (LIU; high frequency) is most commonly applied as
an analytical technique to provide information on the physicochemical properties of
food such as its firmness, ripeness, sugar content, acidity, etc. [15]. In contrast, high-
intensity ultrasound (HIU; low frequency) can be used to physically or chemically alter
food properties [13].

The effects of ultrasound on liquid systems are mainly attributed to the cavitation
phenomenon [16]. Cavitation bubbles, which are formed during sonication, are rapidly
formed and violently collapsed, leading to extreme temperatures (5000 C) and
pressures (1000 atm) that can produce very high shear energy waves and turbulence in
the cavitation zone [12,17]. Ultrasonic treatment not only represents a rapid, efficient
and reliable alternative to improve the quality of food but also has the potential to
develop new products with a unique functionality [8,15].

3
The application of ultrasound to proteins has been related to effects on the structural and
functional properties of soybean proteins [11,15], rice proteins [18], peanut protein
isolate [8], black bean proteins [19], and whey protein isolate [14]. Several studies have
demonstrated that ultrasound treatments could facilitate heat-induced gelation properties
of soy protein isolate [11,20]. Moreover, foaming [6] and emulsifying properties [8]
were found to be improved by HIU. However, there are contradictory reports on the
effect of ultrasound on the molecular weight of proteins [9]. For example, ultrasound
treatment of 20 and 40 kHz for 30 min resulted in a significant decrease in molecular
weight for whey protein concentrate, whey protein isolate [4] and -lactalbumin [21]. In
other studies, sonication at 20 kHz for 30 min with varying power intensities was
reported to have no significant effect on the molecular weight of soy protein isolate
[9,22]. In addition, no significant changes in molecular weight were reported for egg
white protein treated with ultrasound at 55 kHz for 12 min [9,23]. Furthermore, by
treating the peanut protein [8], soy protein [15] and black bean protein [19] with HIU, it
was found that ultrasound-induction increases the surface hydrophobicity.

Jackfruit seeds (JS) are a by-product of the dehydration process of edible jackfruit bulbs
in Nayarit, Mexico. Hence, JS could be used as a non-conventional source of protein in
the food industry. However, it is necessary to further investigate the effects of
ultrasound on the structural and technofunctional properties of proteins when a new
source is proposed to produce protein as a food ingredient. Currently, there is not
literature that focuses on the effect of HIU on the structure and technofunctional
properties of JSPI.

Therefore, the objective of this work was to determine which ultrasound intensities most
significantly affect the technofunctional properties, molecular weight, and
microstructure of jackfruit seed protein isolate (JSPI).

2. Materials and methods

2.1 Materials

Fresh jackfruit seeds were provided by Mexican Tropical Organics S. de R.L. de C.V.
located at Carretera Los Cocos-Aticama s/n San Blas, Nayarit (Mexico). The seeds were
milled using a hammer mill to pass through a 1-cm sieve and then dried in a cabinet

4
dryer (35 C; 3.0 m/s) until they reached a constant weight. Dry seed pieces were
pulverized in a mill (Cyclotec Mod. 1093, Foss Tecator, Slangerupgade, Denmark)
equipped with 0.8-mm mesh. The jackfruit seed flour (JSF) was sieved (150 m) and
then collected and stored in a polyethylene bag (20 C) prior to analysis. The centesimal
composition of JSF was determined according to AOAC methods [24]. The protein, fat,
and ash contents were 13.67% (N x 6.25), 1.89%, and 3.49%, respectively. The
percentage of carbohydrates (80.95%) was determined by the difference following the
method of Lima et al. [4].

2.2. Preparation of the protein isolate

First, a slurry was prepared by mixing JSF in distilled water at a ratio of 1:20 and
adjusting the pH value to 12.0 with 1 mol/L NaOH. The slurry was stirred for 30 min at
25C and then centrifuged at 2875 x g for 30 min. The pH of the supernatant was
adjusted to a pH of 4.0 with 1.0 M HCl, and the slurry was stirred for 20 min at 25C.
The precipitate was separated by centrifugation at 2875 x g for 20 min at 25C. The
protein precipitate was then subjected to an alcoholic extraction with 96% ethanol using
a ratio of 1:2 (protein precipitate:ethanol). The protein precipitate was subsequently
stirred for 10 min and separated by centrifugation at 2875 x g for 10 min at 25C.
Finally, it was washed with ethyl ether (1:4; p/v) by vacuum filtration and dried (35C;
1.5 m/s) to constant weight. The final JSPI product was characterized according to
AOAC methods [24]. The protein, fat, and ash contents were 69.62% (N x 6.25),
0.72%, and 2.34%, respectively. The percentage of carbohydrates (27.32%) was
determined by the difference following the method of Lima et al. [4].

2.3 Ultrasound treatment of samples

JSPI dispersions (10%, w/v) were prepared by adding JSPI into distilled water, gently
stirring for 30 min, and adjusting to a pH of 12 with 1 mol/L NaOH. An ultrasound
processor (Model CPX750, Cole-Parmer Instruments, Vernon Hills, Illinois, U.S.A.)
equipped with a 2.54-cm-diameter titanium probe was used to sonicate 500 mL of JSPI
dispersions in a 1000-mL glass beaker. The solution was placed in an ice-water bath for
15 min, maintained at a temperature below 15 C, and treated at 20 kHz at levels of
power output of 0 W (control), 200 W, 400 W, and 600 W for 15 min (pulse duration:
on-time, 5 s; off-time 1 s). The final temperature of ultrasound process was 15 2 C,
23 0 C and 26 1 C to HIU of 200 W, 400 W and 600 W, respectively. After

5
ultrasound treatment, all samples were centrifuged (2875 x g for 30 min). The
supernatant was lyophilized and stored at room temperature in airtight containers until
analysis.

2.4 Determination of ultrasound power and intensity

Ultrasonic energy, which is considered a type of mechanical energy, is partly lost in the
form of heat when ultrasound passes through a medium [11]. The ultrasonic treatment
of a liquid produces heat; therefore, after recording the change in the liquids
temperature as a function of time, the acoustic power can be estimated using the
equation: P = M x Cp x (dT/dt), where P is power (W); M is the mass of the sonicated
liquid (g); Cp is the specific heat of the medium at a constant pressure, which is
dependent upon the composition and volume of the medium (J K-1); and dT/dt is the
slope at the origin of the curve of a plot of temperature against time [19]. Ultrasonic
intensity was measured by calorimetry using a thermocouple (Cole-Parmer Instruments,
04711-50, Vernon Hills, Illinois, U.S.A.) and expressed in W cm-2. Using ultrasonic
treatment with the 20 kHz probe at a power output of 200 W, 400 W and 600 W, the
ultrasonic intensity was 41 8 W cm-2, 61 0 W cm-2 and 113 0 W cm-2,
respectively.

2.5 Water holding capacity (WHC) and oil holding capacity (OHC)

A previous procedure was used [25] with slight modification for the determination of
WHC and OHC. Duplicate samples (1 g) were rehydrated with 10 mL of distilled water
in centrifuge tubes (15 mL) and dispersed with a vortex mixer for 30 s. The dispersion
was allowed to stand at room temperature for 15 min and was then centrifuged at 1238
x g for 10 min. The supernatant was decanted, and the residue was weighed together
with the centrifuge tube. WHC was expressed as grams of water held per gram of JSPI
sample. An identical method was used to measure corn oil holding capacity, and OHC
was expressed as grams of oil held per gram of protein.

2.6 Emulsifying capacity (EC)

A 2g sample of JSPI was blended with 100 mL of distilled water in an Osterizer blender
(Tlalnepantla, Edo. de Mexico) at high speed for 30 s. Corn oil was gradually added
from a graduated burette while the mixture was being homogenized. Emulsion capacity
was determined at the inversion point where an oil-in-water emulsion turns into a water-

6
in-oil emulsion [26]. A drop in consistency (from a maximum), judged by a decrease in
resistance to blending, was considered to be the point of discontinuation of oil addition
[27]. The amount of oil added at this point was interpreted as the emulsifying capacity
of the sample. The result was expressed as milliliters of oil required to reach the
breakpoint of the emulsion per gram of protein.

2.7 Emulsifying activity (EA) and emulsion stability (ES)

A modified version of the method described by Ulloa et al. [27] was used to determine
the EA and ES of the JSPI. Five suspensions were prepared by dissolving 1 g of JSPI in
15 mL of water, and the pH of the suspensions was adjusted to 2, 4, 6, 8 or 10 with 0.1
mol/L HCl or NaOH. Subsequently, 15 mL of corn oil was added to each suspension.
Each mixture was stirred in a Tissue-Tearor Homogenizer (Model 985370-07, Biospec
Products, Inc.) at speed setting 20 for 1 min and centrifuged at 198 x g for 5 min. The
emulsion layer volume was recorded. Emulsifying activity (EA) was calculated as:

EA (%) = (height of emulsified layer/ height of total content in tube) 100

Finally, to determine emulsion stability, the samples were heated at 80 C for 30 min in
a water bath, cooled to 25 C in running water and centrifuged as described above.
Emulsion stability was expressed as the percentage of emulsifying activity remaining
after heating.

2.8 Least Gelation Concentration (LGC)

LGC was determined using 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 g/100 mL JSPI
dispersions for each JSPI in centrifuge tubes. The samples were heated for 1 h in a
boiling water bath, cooled rapidly under running tap water and further cooled for 2 h at
4 C. The LGC is the minimum concentration at which the cooked and subsequently
cooled sample from the inverted centrifuge tube did not fall or slip from the wall of the
tube [25].

2.9 Foaming properties

Foaming capacity (FC) and foam stability (FS) were measured using a modified version
of the method described by Klompong et al. [28]. Five suspensions were prepared (50
mL; 20 mg/mL); the pH of the suspensions was adjusted to 2, 4, 6, 8, and 10. The
suspensions were then whipped in an Osterizer blender at medium speed for 1 min to

7
incorporate the air at room temperature. The resultant foam was poured into a 250-mL
cylinder. The total foam volume was recorded, and FC was expressed as the percentage
increase in volume. The FS was calculated according to the following equation:

FS (%) = (foam volume after 30 min / initial foam volume) x 100

2.10 Degree of hydrolysis (DH)

The DH, expressed as the percentage of free amino groups, was determined in triplicate
using the trinitrobenzenosulfonic (TNBS) method [29] as described by Connolly et al.
[30] with modifications. Samples (5%; w/v) and leucine standard solutions were
prepared in duplicate aliquots (0.064 mL) of test or standard solutions, which were
added to test tubes containing 1.0 mL of sodium phosphate buffer (0.2125 M; pH 8.2).
TNBS reagent (0.250 mL) was then added to each tube, followed by mixing and
incubation at 50 C for 30 min in a covered water bath. After incubation, the reaction
was stopped by the addition of 0.1 M sodium sulfite (1 mL) to each tube. Samples were
then allowed to cool at room temperature for 15 min, and the absorbance values were
measured at 420 nm.

The DH was calculated according to the following equation:

were Nt is the degree of dissociation of -NH2 groups at a given time, 0 is the

degree of dissociation of -NH2 groups at time 0, and htot is the total number of peptide
bonds in the protein substrate. Total hydrolysis was performed by adding 0.5 mL of
protein suspension (5%; w/v) to a 50-mL tube, adding 4.5 mL of 6 N HCl, sealing under
vacuum, and heating to 110 C for 24 hours. The hydrolysis was stopped by adding 4.5
mL of 6 N NaOH. The suspension was filtered, and the -NH2 groups were determined
as described previously.

2.11 Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-

PAGE)

The polypeptide composition of all samples was conducted according to Condes et al.
[31] using Tricine-SDS-PAGE under reducing conditions with slight modification.
Samples were analyzed in gels formed by 4% w/v and 16% w/v acrylamide gels

8
(stacking and separating, respectively). The following continuous buffer system was
used: 1 mol L1 TrisHCl, 1 g L1 SDS, pH 8.45 for the gel buffer; 200 mM Tris-HCl,
pH 6.8, 40% glycerol, 2% SDS, 5% -mercaptoethanol, 0.04% Coomassie blue G-250
for the sample buffer; and TrisTricine (p.a., Sigma Chemical Co.) buffer system as
running buffer [0.1 mol L1 TrisHCl, pH 8.9 as an anode buffer and 0.1 mol L1 Tris,
0.1 mol L1 Tricine,1 g L1 SDS, pH 8.25 as a cathode buffer (p.a., Sigma Chemical
Co.)]. The following protein MW markers (161-0326, Bio-Rad Hercules, USA) were
used: triosephospate isomerase (26.6 k a); myoglobin (16.9 k a); -lactalbumin (14.4
k a); aprotinin (6.5 k a); insulin chain, oxidized (3.5 k a); and bacitracin (1.4 kDa).
Silver staining was realized according to protocol silver stain plus TM kit (161-0449,
Bio-Rad Hercules, USA). The molecular weights of the samples were obtained by
Quantity One 1-D analysis software (Bio-Rad Hercules, USA).

2.12 Scanning electron microscopy (SEM)

The microstructure of the freeze-dried JSPI samples was observed with a SEM (SEC,
Mini-SEM SNE-3200M, South Korea) at an accelerating voltage of 20 kV. Before
using the SEM, the samples were coated with gold using an ion sputter coater (MCM-
100, SEC).

2.13 Surface hydrophobicity (H0-ANS) measurements

H0-ANS was determined using 1-anilino-8-naphthalene-sulfonate (ANS) as a

fluorescence probe according to the method of Kato and Nakai [48] as described by
Jiang et al. [19] with modifications. HIU-treated and control protein dispersions (1.5
mg/ mL in 0.01 M phosphate buffer at pH 9.0) were centrifuged at 8,000 xg at 17 C for
20 min. After determining the protein concentration in the supernatants according to the
method of Bradford [49], each supernatant was serially diluted with the same buffer to
obtain protein concentrations ranging from 0.05 to 0.0001 mg/mL. Then 25 L of ANS
(8.0 mM in 0.01M phosphate buffer, pH 9.0) was added to 2 mL of sample.
Fluorescence intensity (FI) was measured with a fluorescence spectrophotometer (Tecan
infinite 200 pro, Grdig, Austria), at wavelengths of 364 nm (excitation) and 475 nm
(emission). The initial slope of FI versus protein concentration (mg/mL) (calculated by
linear regression analysis) was used as an index of the protein H0-ANS. All
determinations were performed in triplicate.

9
2.14 Solubility measurement
For measurement solubility, 60 mg of the protein sample was mixed with 40 mL 0.01 M
phosphate buffer solution (pH 9.0). The solution was stirred for 60 min and then
centrifuged at 8,000 x g for 20 min at 17 C. The protein content in the supernatant was
measured using the Bradford method [49] and bovine serum albumin was used as
standard. Solubility was expressed as mg/mL.

2.15 Statistical analysis

Statistical analysis was performed using Statgraphics Centurion Software version XV

(Statpoint Technologies, Inc. Virginia, USA). All data are shown as mean standard
deviation (SD). Tukey`s test was used to test for significant differences between the
groups analyzed, and the differences were considered to be significant at p < 0.05 or p
< 0.01. A simple Pearson correlation analysis was used to determine correlations
between surface characteristics and solubility, and functional properties. p < 0.05 was
regarded as statistically significant.

3. Results and discussion

3.1. Water holding capacity (WHC) and oil holding capacity (OHC)

The results of WHC and OHC of different JSPI samples are shown in Fig. 1a. The
WHC decreased from an initial WHC of 3.35 g g-1 to 3.20 g g-1, 1.99 g g-1, and 1.88 g g-
1
after HIU to 200 W, 400 W and 600 W, respectively. One reasonable explanation was
that the HIU might denature the molecular structure of protein [11] and cause an
increase of the hydrophobic surface of the JSPI (see, section 3.8), which can lead to low
levels of WHC. In contrast, the OHC of all JSPI-HIU was significantly (p < 0.05)
higher compared to the control (0 W) JSPI. The OHC increased from an initial OHC of
1.92 g g-1 to 3.60 g g-1, 4.01 g g-1, and 3.22 g g-1 after HIU to 200 W, 400 W and 600 W,
respectively. A negative correlation (r = -0.55; p > 0.05) between WHC and OHC was
observed after HIU. The presence of OHC might be attributed to the exposure of
hydrophobic groups after HIU allowed the physical entrapment of oil [32]. Such
exposure of hydrophobic groups can be observed by the formation of large aggregates
of proteins in the dry state after freeze-drying samples of JSPI treated with HIU in
comparison with untreated JSPI (Fig. 7). Hu et al. [11, 13] reported that samples of soy
protein treated with HIU at 400 W for 5, 20, and 40 min increased WHC, while Reiner

10
et al. [33] noted that thermosonication for 10 min at an ultrasound amplitude of 24 kHz
improved the WHC of yogurts, in contrast with the results obtained in this investigation.
According to Higuera-Barraza et al. [10], the technofunctional properties depend on the
inherent characteristics of the protein, source, intensity and frequency of the ultrasound,
as well as the pH, temperature, ionic strength, time, and all other variables that have an
effect on the physicochemical properties of proteins.

3.2. Emulsifying capacity (EC)

Emulsification levels can be modified by varying the cavitation intensity [34]. As

shown in Fig. 1b, the EC of JSPI increased after the application of HIU. The EC
increased from an initial EC of 76.6 mL g-1 to 223.0 mL g-1, 292.0 mL g-1, and 296.0
mL g-1 after HIU to 200 W, 400 W and 600 W, respectively. A positive correlation (r =
0.88; p > 0.05) between OHC (Fig. 1a) and EC (Fig. 1b) was observed after HIU. When
the emulsifying properties of a protein are evaluated, it is very important to specify the
initial protein concentration to determine the maximum concentration of oil that can
emulsify [35]. Ulloa et al. [27], Gao et al. [36] and Khalil et al. [37] reported previous
EC values of 200.5 mL g-1, 69-76 mL g-1 and 130 mL g-1 in proteins from safflower, pea
and sesame, respectively. Thus, the present results indicate that JSPI after HIU
exhibited greater EC than these other plant proteins. In addition, the EC was
proportional to the increase in ultrasonic intensity. Therefore, ultrasound is considered
to be an effective tool for improving the EC of jackfruit seed protein.

3.3. Emulsifying activity (EA) and emulsion stability (ES)

The ability of proteins to assist in the formation and stabilization of emulsions is of

critical importance for many applications such as frozen desserts, salad dressings,
comminuted meats, mayonnaise, cake batters, milks and coffee whiteners [38,39].
These emulsifying properties depend on the molecular flexibility and stability of the
protein structure [39]. The EA of JSPI increased significantly (p < 0.01) in the range of
pH 2 to 8 for the HIU treatments of 400 W (Fig. 2c) and 600 W (Fig. 2d) in comparison
with the control treatment (0 W) (Fig. 2a). The EA increased from an initial value of
65.83% at pH 4 to a maximum value of 100% for the HIU treatments of 400 W and 600
W. Similarly, the EA increased from initial values of 76.02% and 78.36% at pH 6 and
pH 8, respectively, to 100% for the HIU treatments of 400 W and 600 W. In contrast,
the EA value of JSPI was significantly lower (p < 0.01) when the HIU was 200 W (Fig.

11
2b) compared to the control (0 W) JSPI. Moreover, the EA decreased from an initial
EA of 84.63% at pH 10 to 41.67%, 70.27% and 72.97% after HIU to 200 W, 400 W and
600 W, respectively.

A higher ES was observed in all samples of JSPI treated with HIU compared with the
ES of JSPI without ultrasonication (Fig. 2). The ES of JSPI at pH 6 and pH 8 showed
values of 100% after HIU to 400 W (Fig. 2c) and 600 W (Fig. 2d), respectively, as well
as a value of 100% for conditions of pH 4 and HIU to 600 W. The increase in the ES
can be explained by the more favorable orientation of proteins resulting from the
influence of turbulent behavior produced by ultrasound [34,40].

Xiong et al. [41], Hu et al. [39], Zhou et al. [38] and Zhang et al. [8] reported that HIU
increased the EA and ES of ovalbumin, soy -conglycinin, soy glycinin and peanut
protein, which is in agreement with the results obtained in this study for the samples of
JSPI treated with HIU at 400 W and 600 W. Partial denaturation of the tertiary and
quaternary structure and formation of a more disordered structure that could provide the
protein a better potential to adsorb at the oil-water interface were speculated to lead to
an increase in EA [40]. For a protein with good solubility, an increase in exposed
hydrophobicity has been described to decrease the barrier for adsorption to the oil-
water, resulting in a higher adsorption rate [42] as well as increased activity and
stability of the emulsion [39]. Thus, a significant relationship between EA and ES with
exposing hydrophobic groups (Section 3.8, Fig. 8), OHC (Fig. 1a) and EC (Fig. 1b)
could be the reason for the improved emulsifying properties of JSPI after HIU (Fig. 2).

3.4. Least gelation concentration (LGC)

Gelation is often the aggregation of denatured molecules; this aggregation may be

primarily driven by physical interactions in which the aggregation is random [27,41].
Heating a protein solution causes molecular unfolding, which leads first to aggregation
and then to gelation when the amount of aggregated protein exceeds a critical
concentration [43]. As shown in Fig. 3, the LGC increased significantly (p < 0.01) from
an initial LGC of 2.0 g/100 g at pH 4 (point isoelectric) to 6.0 g/100 g after HIU to 200
W, 400 W and 600 W. This effect could be attributed to partial denaturation of proteins
by HIU, which affected the solubility due to the increased hydrophobic regions. In
contrast, the LGC decreased significantly (p < 0.01) to pH 2, pH 6, pH 8 and pH 10
after HIU to 200 W, 400 W and 600 W, respectively, compared to the control (0 W)

12
JSPI. The LGC showed values of 4.0 g/100 g at pH 2 and pH 6 after HIU in all of the
JSPI. Moreover, the LGC showed values of 6.0 g/100 g and 8.0 g/100 g at pH 8 and pH
10, respectively, after HIU to 200 W, 400 W and 600 W. According to these results,
there appeared to be no correlation (r = 0; p < 0.05) between HIU and LGC for the JSPI
treated with HIU.

Kaur & Singh [44] and Horax et al. [45] reported an LGC of 14-18 g/100 g, 12 g/100 g
and 10 g/100 g in chickpea protein, cowpea protein and soy protein, respectively. These
results indicate that the JSPI, even without ultrasound (0 W), can be used as a strong
gelling agent.

3.5. Foaming properties

Protein adsorption is important for the stabilization of interfaces and thereby the
formation of foams [41]. In general, the FC increased significantly (p < 0.05) after HIU
to 200 W, 400 W and 600 W (Fig. 4). The JSPI exhibited a higher increase in FC
between pH 2.0 and pH 6.0 after HIU to 200 W (Fig. 4b), 400 W (Fig. 4c) and 600 W
(Fig. 4d) compared to the control (0 W) JSPI (Fig. 4a). The FC increased from an initial
FC of 251.3% at pH 8 to 289.0%, 301.2% and 296.3% after HIU to 200 W, 400 W and
600 W, respectively. These results indicate that HIU to 200 W, 400 W and 600 W
positively affected the FC of the JSPI. The higher increases in FC observed could be a
result of the denaturation [46] of jackfruit seed protein. Morales et al. [6] evaluated the
effect of ultrasound on the foaming properties of soy proteins subject to HIU (20 kHz,
150 W) for 20 min; a higher FC in such proteins was obtained as a result. Similar results
were obtained by Xiong et al. [41] on ovalbumin protein from egg whites.

On the other hand, the FS of JSPI was significantly greater (p < 0.05) at pH 4 after HIU
to 200 W (Fig. 4b), 400 W (Fig. 4c) and 600 W (Fig. 4d) with values of 118.5%,
106.9% and 103.3%, respectively, compared to the initial FS (0 W) of 38.3% (Fig. 4a).
In contrast, the FS decreased significantly (p < 0.05) at pH 2, pH 6, pH 8 and pH 10
after HIU to 200 W, 400 W and 600 W.

In a study carried out by Zhang et al. [46], the effect of HIU (20 kHz, 10 min) at three
different intensities of ultrasound (500 W, 720 W, and 900 W) on the foaming property
of gluten proteins from wheat was evaluated. They reported a higher air-water diffusion
interface due to an increase in the cohesiveness and flexibility of the foams, which was

13
attributed to the partial denaturation during the ultrasound application. An increase in
temperature (60 C) was observed that exposed the hydrophobic regions and, in turn,
allowed better interaction with the interface leading to greater stability. In this study, the
FC and FS showed a negative correlation (r = -0.98; p < 0.05) after HIU, to a greater
value for the FC, while the FS was lower in all JSPI treated with HIU.

3.6. Degree of hydrolysis (DH) and Tricine-SDS-PAGE

The electrophoretic profiles obtained by Tricine-SDS-PAGE for untreated and

ultrasonicated JSPI are shown in Fig. 5. The electrophoretic profile of JSPI (0 W)
subunits contains seven main classes (line 1). The distribution of molecular weights of
JSPI (0 W) consists of the following: molecular weight of 26.82 kDa (A), 22.94 kDa
(B), 21.52 kDa (C), 18.05 kDa (D), 15.85 kDa (E), 10.96 kDa (F) and 6.08 kDa (G).
Compared to the control (0 W) JSPI, HIU induced changes in the electrophoretic
profiles of JSPI to 200 W, 400 W and 600 W (lines 2-4), indicating that HIU changes
the primary structure of the JSPI. The distribution of molecular weights of JSPI
fractions after ultrasound treatments to 200 W, 400 W and 600 W consists of the
following: molecular weight of 26.82 kDa (a), 21.84 kDa (b), 18.84 kDa (c), 17.90 kDa
(d), 16.48 kDa (e), 13.64 kDa (f), 10.81 kDa (g) and 6.08 kDa (h). It is clear that the
changes in molecular weight of JSPI samples occurred after ultrasonication. The
ultrasonication of JSPI samples at these intensities showed no evidence for the presence
of aggregates.
These results are in agreement with those reported by Jambrak et al. [14, 21], who
observed a reduction in the molecular weight of whey protein isolate (2 k z, 48 W
cm-2 and 15 min) and -lactalbumin (20 kHz and 40 kHz, 15 min) treated by ultrasound.
In contrast, O`Sullivan et al. [47], Xiong et al. [41] and O`Sullivan et al. [12] observed
that ultrasound treatment induced no changes in the molecular weight of whey protein
isolate and soy protein isolate (2 k z, 34 W cm-2 and 40 min), ovalbumin (2 k z,
35-48 W cm-2 and 40 min) and egetable proteins (2 k z, 34 W cm-2 and 2 min). The
difference between our results and those of O`Sullivan et al. [12, 47] and Xiong et al.
[41] could be resulted from the different ultrasonic intensity applied to the JSPI. In our
investigation, the ultrasonic intensity was 41 W cm-2, 61 W cm-2 and 113 W cm-2 to 200
W, 400 W and 600 W, respectively. This ultrasound intensity might have caused higher
shear stress and turbulence effects in the JSPI solutions and resulted in the split of the
molecular structure of the protein.

14
Fig. 6 shows the changes in DH of JSPI after HIU to 200 W, 400 W and 600 W. As
depicted, HIU increased the DH significantly (p < 0.05) when compared to control (0
W) JSPI. In addition, it can be observed that DH increased significantly (p < 0.05) to
200 W and 600 W compared to treatment of 400 W. These results might have been
generated because of the cavitational forces exerted by the probe during ultrasonic
treatment as well as microstreaming and turbulent forces [15,19].

3.7. Scanning electron microscopy (SEM)

Fig. 7 presents a set of microstructures of JSPI exposed to distinct ultrasonication

treatments; the SEM images were obtained at 1K (1000x) magnification. Compared
with sample A (0 W), samples B-D, which had been ultrasonicated to various degrees
and freeze dried, exhibited more disordered structures and irregular fragments. In
addition, the SEM images showed that on average, sample D was smaller than other
samples (B-C) after HIU treatment. With relatively low-power ultrasound treatment, the
effect of turbulent forces and micro-streaming might increase the speed of collision and
aggregation; this typically results in the formation of unstable aggregates and an
increase in particle size [15,41]. When the power of the ultrasonic treatment is
increased, the particles become smaller and particle size-distribution broadens [19].
These results might be due to the changes in ultrasonic treatment leading to unfolding of
the JSPI molecules, and increased exposure of hydrophobic groups (see Section 3.8)
and free SH groups [15] at the surface of the molecules, which could interact with each
other and form larger aggregates during freeze drying. However, as shown by Tricine-
SDS-PAGE (Fig. 5), no aggregation was observed. This result suggests that the
formation of aggregates is a non-covalent bond [41].

3.8 Surface hydrophobicity (H0-ANS) and solubility

Protein surface hydrophobicity (H0-ANS) is an index of the number of hydrophobic

groups present on the surface of protein molecules and is closely associated with protein
functional properties [50]. Fig. 8 showed that HIU treatment significantly increased the
H0-ANS of JSPI (p < 0.05). This finding was consistent with previous studies, which
showed that ultrasound treatment could cause the increase of H0-ANS for peanut protein
[8], soy protein (15), ovalbumin [41], beef protein [50] and myofibrillar protein [51].
Comparing the H0-ANS of JSPI sonicated under different conditions, it was observed
that H0-ANS was increased with ultrasonic intensity (from 200 to 400 W) at the same

15
ultrasonic time (15 min). This indicates that ultrasonic treatment induces a certain
degree of molecular unfolding of the proteins, and thereby causes an increase in the
number of hydrophobic groups and regions that are originally inside the molecules to
become exposed to the polar surrounding environment [15]. This aggregation protects
the hydrophobic regions of the proteins [19]. However, the H0-ANS of the samples
treated with HIU (600 W 15 min) decreased. HIU treatment might also lead to partial
denaturation of proteins, which might increase the extent of bonding and reduce the H0-
ANS [34]. Similar results were obtained by Jiang et al. [19] on black bean protein
isolate.
Solubility is the most practical measurement of protein denaturation and aggregation an
it is, therefore, a reliable index of protein functionality [19]. The solubility of JSPI
increased significantly (p < 0.05) after ultrasonic treatment, as compared with the
solubility of the untreated sample (Fig. 8). This might be due to that: in natural state,
proteins were present in the form of aggregates, the physical factors of cavitation
phenomenon might disrup the hydrogen bonds and hydrophobic interactions which
were responsible for intermolecular association of protein aggregates [51]. Furthermore,
a correlation analysis showed that protein solubility was positively correlated with H0-
ANS (r = 0.56; p > 0.05). This result agrees with the results of previous studies, which
showed that ultrasound treatment caused an increase in protein solubility and surface
hydrophobicity of black bean protein [19], soy protein [15] and myofibrillar protein
[51].
Protein solubility increased from an initial value (0 W) of 0.06 mg/mL to 0.66 mg/mL,
0.38 mg/mL, and 0.57 mg/mL after HIU to 200 W, 400 W and 600 W, respectively. The
highest solubility was observed in sample 200 W, and solubility decreased with further
increased in HIU. Therefore, this increased in solubility may be due to conformational
change during ultrasonic treatment and hydrolysis of peptide bonds of the JSPI protein
molecules, as shown by Tricine-SDS-PAGE profile of the JSPI after HIU (see Section
3.6). A positive correlation (r = 0.97; p < 0.05) between protein solubility and GH was
observed after HIU.

4. Conclusions

The HIU treatment changed the technofunctional properties of JSPI. Compared with
control (0 W) JSPI, ultrasound processing increased oil holding capacity and decreased

16
water holding capacity, which subsequently improved the emulsifying capacity,
emulsifying activity and foaming capacity. In general, the emulsifying stability did not
change among all of the samples. However, the foaming stability decreased due to
increased ultrasound intensity. Additionally, the LGC of the HIU-treated samples was
lower than that of the control (0 W) JSPI, except at pH 4. The molecular weight of the
protein was modified after HIU, and in turn, the DH increased significantly (p <0.05)
after HIU in all samples. SEM revealed that the microstructure of HIU-treated JSPI was
altered, with larger aggregates for HIU-treated JSPI than non-treated (0 W) JSPI.
Surface hydrophobicity and protein solubility of JSPI dispersions were both increased
after ultrasonic treatment in all samples. These changes in JSPI properties could meet
the complex needs of manufactured food products, but further study is required to
understand the detailed mechanism.

Acknowledgements

The authors would like to acknowledge the National Council for Science and
Technology of Mexico for the scholarship for M.Sc. Juan Alberto Resendiz Vazquez
and for providing research funds.

References

[1] P. L. Tran, D. H. D. Nguyen, V. H. Do, Y. Kim, S. Park, S. Yoo, S. Lee, Y. Kim,

Physicochemical properties of native and partially gelatinized high-amylose
jackfruit (Artocarpus heterophyllus Lam.) seed starch, Lebensm. Wiss. Technol.
62 (2015) 1091-1098.
[2] K. Rengsutthi, S. Charoenrein, Physico-chemical properties of jackfruit seed
starch (Artocarpus heterophyllus) and its application as a thickener and stabilizer
in chilli sauce, Lebensm. Wiss. Technol. 44 (2011) 1309-1313.
[3] S. B. Swami, N. J. Thakor, P. M. Haldankar, S. B. Kalse, Jackfruit and its many
functional components as related to human health: A review, Compr. Rev. Food
Sci. Food Saf. 11 (2012) 565-576.
[4] B. N. B. Lima, F. F. Lima, M. I. B. Tavares, A.M. M. Costa, A.P. T. R. Pierucci,
Determination of the centesimal composition and characterization of flours from
fruit seeds, Food Chem. 151 (2014) 293-299.

17
[5] S. Kabir, Jacalin: a jackfruit (Artocarpus heterophyllus) seed-derived lectin of
versatile applications in immunobiological research, J. Immunol. Methods 212
(1998) 193-211.
[6] R. Morales, K. D. Martnez, V. M. P. Ruiz-Henestrosa, A. M. R. Pilosof,
Modification of foaming properties of soy protein isolate by high ultrasound
intensity: Particle size effect, Ultrason. Sonochem. 26 (2015) 48-55.
[7] M. C. Tan, N. L. Chin, Y. A. Yusof, F. S. Taip, J. Abdullah, Gel strength and
stability characterization of ultrasound treated whey protein foams, Agric. Agric.
Sci. Procedia 2 (2014) 144-149.
[8] Q. Zhang, Z. Tu, H. Xiao, H. Wang, X. Huang, G. Liu, C. Liu, Y. Shia, L. Fan, D.
Lin. Influence of ultrasonic treatment on the structure and emulsifying properties
of peanut protein isolate, Food Bioprod. Process. 92 (2014) 30-37.
[9] J. OSulli an, M. Arellano, R. Pichot, I. Norton, The effect of ultrasound
treatment on the structural, physical and emulsifying properties of dairy proteins,
Food Hydrocolloids 42 (2014) 386-396.
[10] O.A. Higuera-Barraza, C.L. Del Toro-Sanchez, S. Ruiz-Cruz, E. Mrquez-Ros,
Effects of high-energy ultrasound on the functional properties of proteins,
Ultrason. Sonochem. 31 (2016) 558-562.
[11] H. Hu, X. Fan, Z. Zhou, X. Xu, G. Fan, L. Wang, X. Huang, S. Pan, L. Zhu, Acid-
induced gelation behavior of soybean protein isolate with high intensity ultrasonic
pre-treatments. Ultrason. Sonochem. 20 (2013) 187-195.
[12] J. O'Sullivan, B. Murray, C. Flynn, I. Norton, The effect of ultrasound treatment
on the structural, physical and emulsifying properties of animal and vegetable
proteins, Food Hydrocolloids, 53 (2016) 141-154.
[13] H. Hu, E. C. Y. Li-Chan, W. Li, M. Tian, S. Pan, The effect of high intensity
ultrasonic pre-treatment on the properties of soybean protein isolate gel induced
by calcium sulfate, Food Hydrocolloids 32 (2013) 303-311.
[14] A. R. Jambrak, T.J. Mason, V. Lelas, L. Paniwnyk, Z. Herceg, Effect of
ultrasound treatment on particle size and molecular weight of whey proteins, J.
Food Eng. 121 (2014) 15-23.
[15] H. Hu, J. Wu, E. C.Y. Li-Chan, L. Zhu, F. Zhang, X. Xu, G. Fan, L. Wang, X.
Huang, S. Pan, Effects of ultrasound on structural and physical properties of soy
protein isolate (SPI) dispersions. Food Hydrocolloids 30 (2013) 647-655.

18
[16] A. C. Soria, M. Villamiel, Effect of ultrasound on the technological properties and
bioactivity of food: a review, Trends Food Sci. Technol. 21 (2010) 323-331.
[17] Arzeni, K. Martnez, P. Zema, A. Arias, O. E. Prez, A. M. R. Pilosof,
Comparative study of high intensity ultrasound effects on food proteins
functionality, J. Food Eng. 108 (2012) 463472.
[18] S. Li, X. Yang, Y. Zhang, H. Ma, Q. Liang, W. Qu, R. He, C. Zhou, G. K.
Mahunu, Effects of ultrasound and ultrasound assisted alkaline pretreatments on
the enzymolysis and structural characteristics of rice protein, Ultrason. Sonochem.
31 (2016) 20-28.
[19] L. Jiang, J. Wang, Y. Li, Z. Wang, J. Liang, R. Wang, Y. Chen, W. Ma, B. Qi, M.
Zhang, Effects of ultrasound on the structure and physical properties of black
bean protein isolates, Food Res. Int. 62 (2014) 595-601.
[20] C. Tang, X. Wang, X. Y, L. Li, Formation of soluble aggregates from insoluble
commercial soy protein isolate by means of ultrasonic treatment and their gelling
properties, J. Food Eng. 92 (2009) 432-437.
[21] A. R. Jambrak, T. J. Mason, V. Lelas, G. Krei, Ultrasonic effect on
physicochemical and functional properties of -lactalbumin. Lebensm. Wiss.
Technol. 43 (2010) 254-262.
[22] Karki, B. P. Lamsal, S. Jung, J. (Hans) van Leeuwen, A. L. Pometto III, Grewell,
S. K. Khanal, Enhancing protein and sugar release from defatted soy flakes using
ultrasound technology, J. Food Eng. 96 (2010) 270-278.
[23] K.M. Krise, The effects of microviscosity, bound water and protein mobility on
the radiolysis and sonolysis of hen egg white, in: PhD Thesis, Penn State
University, 2011.
[24] AOAC, Association of Official Analytical Chemists, Official Methods of
Analysis (15th Ed.) Arlington, VI (1990).
[25] A. U. Joshi, C. Liu, S. K. Sathe, Functional properties of select seed flours,
Lebensm. Wiss. Technol. 60 (2015) 325-331.
[26] A. C. Karaca, N. Low, M. Nickerson, Emulsifying properties of canola and
flaxseed protein isolates produced by isoelectric precipitation and salt extraction,
Food Res. Int. (2011) 2991-2998.
[27] J. A. Ulloa, P. Rosas-Ulloa, B. E. Ulloa-Rangel, Physicochemical and functional
properties of a protein isolate produced from safflower (Carthamus tinctorius L.)
meal by ultrafiltration, J. Sci. Food Agric. 91 (2011) 572-577.

19
[28] V. Klompong, S. Benjakul, D. Kantachote, F. Shahidi, Antioxidative activity and
functional properties of protein hydrolysate of yellow stripe trevally (Selaroides
leptolepis) as influenced by the degree of hydrolysis and enzyme type, Food
Chem. 102 (2007) 1317-1327.
[29] J. Adler-Nissen, Determination of the degree of hydrolysis of food protein
hydrolysates by trinitrobenzenesulfonic acid, J. Agric. Food Chem. 27 (1979)
1256-1262.
[30] A. Connolly, C. O, Piggott. R. J. FitzGerald, Technofunctional properties of a
brewers' spent grain protein-enriched isolate and its associated enzymatic
hydrolysates, Lebensm. Wiss. Technol. 59 (2014) 1061-1067.
[31] M. C. Conds, F. Speroni, A. Mauri, M. C. An, Physicochemical and structural
properties of amaranth isolates treated with high pressure, Innovative Food Sci.
Emerg. Technol. 14 (2012) 11-17.
[32] P. Meinlschmidt, D. Sussmann, U. Schweiggert-Weisz, P. Eisner, Enzymatic
treatment of soy protein isolates: effects on the potential allergenicity,
technofunctionality, and sensory properties, Food Sci. Nutr. (2015) 1-13.
[33] J. Riener, F. Noci, D.A. Cronin, D.J. Morgan, J.G. Lyng, The effect of
thermosonication of milk on selected physicochemical and microstructural
properties of yoghurt gels during fermentation, Food Chem. 114 (2009) 905911.
[34] S. Yanjun, C. Jianhang, Z. Shuwen, L. Hongjuan, L. Jing, L. Lu, H. Uluko, S.
Yanling, C. Wenming, G. Wupeng, L. Jiaping, Effect of power ultrasound pre-
treatment on the physical and functional properties of reconstituted milk protein
concentrate, J. Food Eng. 124 (2014) 11-18.
[35] L. Shen, C. Tang, Emulsifying properties of vicilins: Dependence on the protein
type and concentration, Food Hydrocolloids, 36 (2014) 278-286.
[36] L. Gao, K. D. Nguyen, A. C. Utioh, Pilot scale recovery of proteins from a pea
whey discharge by ultrafiltration, Lebensm. Wiss. Technol. 34 (2001) 149-158.
[37] M. M. Khalil, Factors affecting production of melon seed kernel protein: Yield,
composition, and protein isolates quality, Nahrung 42 (1998) 295-297.
[38] M. Zhou, J. Liu, Y. Zhou, X. Huang, F. Liu, S. Pan, H. Hu, Effect of high
intensity ultrasound on physicochemical and functional properties of soybean
glycinin at different ionic strengths, Innovative Food Sci. Emerg. Technol. 34
(2016) 205-213.

20
[39] H. Hu, I. W.Y. Cheung, S. Pan, E. C.Y. Li-Chan, Effect of high intensity
ultrasound on physicochemical and functional properties of aggregated soybean -
conglycinin and glycinin, Food Hydrocolloids, 45 (2015) 102-110.
[40] A. R. Jambrak, V. Lelas, T. J. Mason, G. Kresic, M. Badanjak, Physical properties
of ultrasound treated soy proteins, J. Food Eng. 93 (4) (2009) 386-393.
[41] W. Xiong, Y. Wang, C. Zhang, J. Wan, B. R. Shah, Y. Pei, B. Zhou, J. Li, B. Li,
High intensity ultrasound modified ovalbumin: Structure, interface and gelation
properties, Ultrason. Sonochem. 31 (2016) 302-309.
[42] R.J.B.M. Delahaije, H. Gruppen, M.L. Giuseppin, P.A. Wierenga, Towards
predicting the stability of protein-stabilized emulsions, Adv. Colloid Interface Sci.
219 (2015) 1-9.
[43] A. Maltais, G. E. Remondetto, M. Subirade. Mechanisms involved in the
formation and structure of soya protein cold-set gels: A molecular and
supramolecular investigation, Food Hydrocolloids, 22 (2008) 550-559.
[44] M. Kaur, N. Singh, Characterization of protein isolates from different Indian
chickpea (Cicer arietinum L.) cultivars, Food Chem. 102 (2007) 366-374.
[45] R. Horax, N. S. Hettiarachchy, P. Chen, M. Jalaluddin, Functional properties of
protein isolate from cowpea (Vigna unguiculata L. Walp.), J. Food Sci. 69 (2004)
119-121.
[46] H. Zhang, I. P. Claver, K. Zhu, H. Zhou, The effect of ultrasound on the
functional properties of wheat gluten, Molecules 16 (2011) 4231-4240.
[47] J. O'Sullivan, M. Park, J. Beevers, The effect of ultrasound upon the
physicochemical and emulsifying properties of wheat and soy protein isolates, J.
Cereal Sci. 69 (2016) 77-84.
[48] A. Kato, S. Nakai, Hydrophobicity determined by a fluorescence probe method
and its correlation with surface properties of proteins. Biochim. Biophys. Acta,
Protein Struct. 624 (1) (1980) 13-20.
[49] M. M. Bradford, A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72 (1976) 248-254.
[50] D. Kang, Y. Zou, Y. Cheng, L. Xing, G. Zhou, W. Zhang, Effects of power
ultrasound on oxidation and structure of beef proteins during curing processing,
Ultrason. Sonochem. 33 (2016) 47-53.

21
[51] Z. Zhang, J. M. Regenstein, P. Zhou, Y. Yang, Effects of high intensity
ultrasound modification on physicochemical property and water in myofibrillar
protein gel. Ultrason. Sonochem. 34 (2017) 960-967.

22
Figure captions

Fig. 1. Effect of HIU on water holding capacity (WHC), oil holding capacity (OHC) and
emulsifying capacity (EC) of JSPI. (a)-- WHC, (a) --OHC, (b) --EC. Mean values
with different letters were significantly different (p < 0.05). WHC and OHC: n = 5 SD
for control (0 W); n = 2 SD for 200 W to 600 W. EC n = 3 SD for control (0 W); n =
2 SD for 200 W to 600 W.

Fig. 2. Effect of HIU on emulsifying activity (EA) and emulsifying stability (ES) of
JSPI at ultrasonic power of 0 W (a), 200 W (b), 400 W (c), 600 W (d) and different pH
values (mean SD, n = 2). Different letters means significant differences between
values with different ultrasonic power and pH (p < 0.01; except to 0 W (p < 0.05).

Fig. 3. Influence of HIU on the least gelation concentration (LGC) of JSPI at different
pH values (mean SD, n = 2). Different letters means significant differences between
values with different ultrasonic power and pH (p < 0.01).

Fig. 4. Foaming capacity (FC) and foaming stability (FS) of HIU-treated JSPI at
ultrasonic power of 0 W (a), 200 W (b), 400 W (c), 600 W (d) and different pH values.
Different letters means significant differences between values with different ultrasonic
power and pH (p < 0.05). FC and FS: n = 3 SD for control (0 W); n = 2 SD for 200
W to 600 W.

Fig. 5. Tricine-SDS-PAGE electrophoretic profiles of molecular weight marker (M),

untreated (1) and HIU-treated JSPI (2-4).

Fig. 6. Degree of hydrolysis (DH) of untreated and HIU-treated JSPI (mean SD, n =
2). Mean values with different letters were significantly different (p < 0.05).

Fig. 7. SEM micrographs at 1K (1000x) of JSPI: (A) untreated 0 W, HIU-treated to (B)

200 W, (C) 400 W and (D) 600 W. Scale bar is 100 m in all cases.

23
Fig. 8 Changes in surface hydrophobicity (H0-ANS) and protein solubility of untreated
and HIU-treated JSPI (mean SD, n = 3). Different letters means significant differences
between values with different ultrasonic power (p < 0.05).

24
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Highlights

Ultrasonic effect on technofunctional properties of JSPI was assessed

HIU treatment significantly improved the emulsifying activity and stability of JSPI.

HIU processing remarkably increased the foaming ability of JSPI.

The surface hydrophobicity and protein solubility of HIU-treated JSPI were increased.

Tricine-SDS-PAGE confirmed HIU have effect on the molecular weight of JSPI

33