Universidad Autónoma de Nayarit Secretaría

Universidad Autónoma de Nayarit Secretaría de Investigación y Posgrado

FORMATO DE REPORTE TÉCNICO DE AVANCE Y TÉRMINO DE PROYECTO

1

No. Registro: SIP18-084 .

TITULO DEL PROYECTO: Aplicación del ultrasonido de alta intensidad para el mejoramiento del secado de productos pesqueros y la funcionalidad de materiales proteínicos obtenidos a partir de subproductos agroindustriales

VIGENCIA: FECHA DE INICIO: 08/2018 FECHA DE TERMINO: 28/05/2019

RESPONSABLE TÉCNICO DEL PROYECTO: JOSÉ ARMANDO ULLOA CUERPO ACADÉMICO: TECNOLOGÍA DE ALIMENTOS INFORME 100% PERIODO AGO2018-MAYO2019 No. de Informe: 1er ( X ) 2do ( ) Final ( X ) I. RESUMEN DE LOS AVANCES DEL PERIODO: En el presente proyecto se han cubierto las actividades experimentales para evaluar el efecto del ultrasonido en propiedades químicas, fisicoquímicas, estructurales y funcionales de aislados proteínicos obtenidos a partir de las pastas de canola. II. LOGRO DE METAS, RESPECTO DE METAS COMPROMETIDAS: 100% de las correspondientes a las actividades experimentales para evaluar el efecto del ultrasonido en propiedades químicas, fisicoquímicas, estructurales y funcionales de aislados proteínicos obtenidos a partir de las pastas de canola y cártamo. III. LOGRO DE OBJETIVOS RESPECTO DEL COMPROMISO: Se ha logrado evaluar el efecto del ultrasonido en las propiedades químicas, fisicoquímicas, estructurales y funcionales de productos alimenticios y procesos alimenticios de interés regional y nacional, de acuerdo a lo comprometido. IV. PRODUCTOS OBTENIDOS: (Indicar con una X y entregar a la Secretaría copia del producto obtenido, en caso de tesis presentar la portada) Artículos (revista Indexada): ( x ) Artículos (revista arbitrada): ( ) Libros: ( ) Capítulo de libro: ( ) Memorias en extenso de congreso: ( ) Tesis: ( ) Material didáctico derivado de proyectos: ( ) Otros: _____________________________ ( ) V. GRUPOS DE TRABAJO: Nombre Profesor Actividades realizadas José Armando Ulloa Coordinación general del proyecto José Carmen Ramírez Ramírez proteicos Coordinación preparación aislados Pedro Ulises Bautista Rosales Coordinación de tratamiento ultrasonido, análisis

estructural Ranferi Gutiérrez Leyva Coordinación evaluación química Petra Rosas Ulloa Coordinación evaluación funcional Yessica Silva Carrillo Coordinación evaluación fisicoquímica, cinéticas

Universidad Autónoma de Nayarit Secretaría de Investigación y Posgrado

FORMATO DE REPORTE TÉCNICO DE AVANCE Y TÉRMINO DE PROYECTO

2

deshidratación Blanca Estela Ulloa Rangel Coordinación de análisis estadístico Nombre Estudiante Actividades realizadas Nitzia Thalía Flores Jiménez Ultrasonido a aislado de cártamo y su evaluación Juan Alberto Resendiz Vazquez Ultrasonido a aislado de semilla de jaca y su

evaluación Minerva Marisol Zúñiga-Salcedo Ultrasonido a aislado de cártamo y su evaluación Estefani Yaneth Hinojosa Delgado Preparación de muestras y evaluación

fisicoquímica Rodrigo Pintado Morales Preparación de muestras y evaluación química Hassel Humberto Soria Ramírez

Preparación de muestras y evaluación funcional

Julio Alberto Alegría Fuentes Preparación de muestras y evaluación funcional

NOTA: Hacer llegar a esta Secretaría en formato en archivo digital y en formato impreso.

ANEXO 1. Productos comprometidos

Se cubrió al 100% los productos compromisos correspondientes a 3 publicaciones (artículos científicos):

1) Effect of ultrasound-assisted enzymolysis on jackfruit (Artocarpus heterophyllus) seed proteins: structural characteristics, technofunctional properties and the correlation to enzymolysis (publicado, se adjunta artículo)

2) Effect of high-intensity ultrasound on the compositional, physicochemical, biochemical, functional and structural properties of canola (Brassica napus L.) protein isolate (publicado, se adjunta artículo)

3) Effect of ultrasound treatment on physicochemical, functional and nutritional properties

of a safflower (Carthamus tinctorius L) protein isolate (Aceptado para publicación en Italian Journal of Food Science; aparecerá en julio de 2019 (se adjunta e-mail de comunicación).

Volume 10 • Issue 5 • 1000796J Food Process Technol, an open access journalISSN: 2157-7110

Open AccessResearch Article

Journal of FoodProcessing & TechnologyJo

urna

l of F

ood Processing & Technology

ISSN: 2157-7110

Resendiz-Vazquez et al., J Food Process Technol 2019, 10:6DOI: 10.4172/2157-7110.1000796

*Corresponding authors: Judith Esmeralda Urías-Silvas, Food Technology, Center for Research and Assistance in Technology and Design of the State of Jalisco A.C., Normalistas Avenue 800, Colinas de la Normal, Guadalajara 44270, Jalisco, Mexico, Tel: (33) 33455200; E-mail: [email protected] Armando Ulloa, Doctoral Program in Agricultural Biological Sciences, Autonomous University of Nayarit, Carretera Tepic-Compostela Km 9, Xalisco 63780, Nayarit, Mexico, E-mail: [email protected] March 07, 2019; Accepted April 15, 2019; Published April 19, 2019

Citation: Resendiz-Vazquez JA, Urías-Silvas JE, Ulloa JA, Bautista-Rosales PU, Ramírez-Ramírez JC (2019) Effect of Ultrasound-assisted Enzymolysis on Jackfruit (Artocarpus heterophyllus) Seed Proteins: Structural Characteristics, Technofunctional Properties and the Correlation to Enzymolysis. J Food Process Technol 10: 796. doi: 10.4172/2157-7110.1000796

Copyright: © 2019 Resendiz-Vazquez JA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractThe aim of this study was to evaluate the effect of Ultrasound-Assisted Enzymolysis (UAE) on the techno-functional

properties and structure of Jackfruit Seed Protein (JSP). Before the hydrolysis by alcalase (60 min), protein solutions (10%, w/v) were exposed to ultrasound pretreatment (200 W, 400 W, 600 W for 15 min and 30 min). Compared with the control JSP, UAE improved the proteolysis process, as confirmed by an increase in the degree of hydrolysis (DH; p<0.05), as well as the Oil Holding Capacity (OHC) and emulsifying stability (ES). Moreover, the UAE treatment increased the protein solubility (PS), while the Least Gelation Concentration (LGC) did not exhibit significant changes. Scanning Electron Microscopy (SEM) demonstrated that UAE disrupted the microstructure of the JSP, exhibiting larger aggregates in comparison with control JSP. Fourier transform infrared (FT-IR) spectra indicated that the UAE treatments induced molecular unfolding by increasing the α-helix, β-turn and random coil content, as demonstrated by increased surface hydrophobicity (H0-ANS). The knowledge of this study could be selectively employed in the food industry for the development of conventional or novel foods based on jackfruit seed protein.

Effect of Ultrasound-assisted Enzymolysis on Jackfruit (Artocarpus heterophyllus) Seed Proteins: Structural Characteristics, Technofunctional Properties and the Correlation to EnzymolysisJuan Alberto Resendiz-Vazquez1, Judith Esmeralda Urías-Silvas2*, José Armando Ulloa1,3*, Pedro Ulises Bautista-Rosales1,3 and José Carmen Ramírez-Ramírez4

1Doctoral Program in Agricultural Biological Sciences, Autonomous University of Nayarit, Carretera Tepic-Compostela Km 9, Xalisco 63780, Nayarit, Mexico2Food Technology, Center for Research and Assistance in Technology and Design of the State of Jalisco A.C., Normalistas Avenue 800, Colinas de la Normal, Guadalajara 44270, Jalisco, Mexico3Food Technology Center, Autonomous University of Nayarit, City of Culture Amado Nervo, Tepic 63155, Nayarit, Mexico4Academic Unit of Veterinary Medicine and Zootechnics, Autonomous University of Nayarit, Compostela-Chapalilla Road Km 3.5, Compostela 63700, Nayarit, Mexico

Keywords: Jackfruit seed protein; Ultrasound-assisted enzymolysis; Technofunctional properties; Structural properties; Surface hydrophobicity

IntroductionProteins are the primary constituents of agricultural raw materials

with two main (complimentary) functions: bio-and techno function [1]. Bio functionality of proteins is related to their nutritional and physiological properties, while techno-functionality is related to their physicochemical properties affecting appearance, texture, and stability of food products (e.g., solubility, viscosity, foaming, emulsifying and gelling ability, fat absorption capacity) [2].

In the last years, there has been increasing demand of protein sources, mainly those that are of high nutritional value, adequate functionality, and low cost and that could be used as functional ingredients in the food industry [3]. A great opportunity to recovery proteins of nonconventional sources for human consumption could be the seeds proceedings of fruit processing. An example of these is the jackfruit seeds, a byproduct from the dehydration process of edible jackfruit bulbs in Nayarit, Mexico. Jackfruit seeds are a good source of starch (22%), dietary fiber (3.19%) [4] and protein with content that is 17.8-37% depending on the variety of jackfruit [5]. In addition, the proteins of jackfruit seed have a good balance of essential amino acids considering the amino acids requirements of FAO/WHO [4].

Another important reason for the search of nonconventional protein is the need for renewable and sustainable sources of proteins and the emerging dietary preferences (e.g., vegans, vegetarians) that demand novel food ingredients and plant-based products [6]. On the other hand, protein-rich fractions, protein isolates, and concentrates offer interesting functional properties, which are usually superior to those of the flour source [7-9]. In that sense, the food industry has a growing interest in producing plant protein isolates not only due to their increasing use as food functional additives but also because they

may also improve nutritive quality and functional properties of food products [9]. Hence, jackfruit seeds could be used as a nonconventional source of protein in the food industry.

To improve the functional properties of proteins, enzymatic hydrolysis is usually applied [10-12]. However, traditional enzymolysis has many disadvantages such as a low degree of hydrolysis and long enzymolysis time [13]. To overcome these drawbacks of conventional enzymatic hydrolysis method, many eco-innovative technologies such as microwave radiation assisted technology, ultrahigh pressure assisted technology and ultrasound-assisted technology has been applied [14]. According to the above, ultrasound technology, as a new nonthermal physical processing technology, has been widely applied in the food industry, especially in extraction [15] and enzymatic treatment [16]. Ultrasound pretreatment has been successfully used to improve bioactive peptides release and the enzymolysis efficiency of protein [16] such as alcalase based enzymolysis of wheat germ protein [12], corn protein [2] and wheat gluten [17]. In this context, because the jackfruit protein is mainly composed by glutelin (∑ 70%), which is alkali-soluble

mailto:[email protected]


Page 2 of 11


[4], ultrasound treatment prior to enzymolysis can improve mass transfer and increase the frequency of contact between the substrate and the enzyme [18]. Thus, we hypothesized that the UAE with alcalase can improve the techno-functional properties of the jackfruit seed proteins. Moreover, such treatment can change the secondary and microstructure of proteins [16,19]. In this regard, Fourier transforms infrared spectroscopy (FT-IR) is well suited to detect relative changes in protein secondary structure due to external factors, by analyzing the amide I band of proteins between 1700 cm-1 and 1600 cm-1 [20]. This band is influenced by hydrogen bonds, which are mainly affected during conformational changes in protein secondary structure (α-helix, β-sheet, β-turn, and random coil) [21]. FT-IR has been applied in the determination of structural changes of corn gluten meal hydrolysates [2], beef proteins [22] and rice protein [11]. Nevertheless, no research has been done on the effect of the UAE and its relationship between the techno-functional properties and the changes structural of jackfruit seed protein.

Therefore, the aims of this study were (i) to investigate the structural changes of jackfruit seed protein after UAE, including the comparison of degree of hydrolysis (DH), protein solubility (PS), microstructure (SEM), surface hydrophobicity (H0), techno-functional properties and secondary structure (FT-IR); (ii) to study the relationships among H0-ANS, PS, DH, techno-functional properties and secondary structural elements of the hydrolysates. It is also hoped that the results of this research will be of great value to support the use of jackfruit seed protein as a novel ingredient for the food industry.

Materials and MethodsMaterials

The 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 (México). Alcalase 2.4 L with an activity of 2.4 AU/g was purchased from Novozymes Co., LTD (Tianjing, China). The Bradford reagent, 2-4-6-trinitrobenzenesulfonic acid solution (TNBS; 5%) and 1-anilino-8-naphthalene-sulfonate (ANS) were purchased from Sigma-Aldrich Corp (St. Louis, Missouri, USA). Other chemicals and solvents used in the experiment were analytical grades. All solutions were prepared with distilled deionized water.

Preparation of the jackfruit protein

The jackfruit seeds were milled using a hammer mill to pass through a 1 cm sieve and then dried in a cabinet 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 µm) and then collected and stored in a polyethylene bag (20°C) prior to analysis. Subsequently, the slurry was prepared by mixing JSF in distilled water at a ratio of 1:20 and adjusting the pH to 12.0 using 1 M NaOH. The slurry was stirred for 30 min at 25°C and then centrifuged at 2875 g for 30 min. The pH of the supernatant was adjusted to a pH of 4.0 using 1.0 M HCl, and the slurry was stirred for 20 min at 25°C. The precipitate was separated by centrifugation at 2875 g for 20 min at 25°C. The protein precipitate was then subjected to an alcoholic extraction using 96% ethanol at a ratio of 1:2 (protein precipitate:ethanol). The protein precipitate was subsequently stirred for 10 min and separated by centrifugation at 2875 g for 10 min at 25°C. Finally, the precipitate was washed with ethyl ether (1:4; p/v) by vacuum filtration and dried (35°C; 1.5 m/s) to a constant weight, obtaining a powder of jackfruit seed protein isolate (JSPI) that was characterized according to AOAC

methods [23]. The protein, fat, and ash contents were 69.62% (N × 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. [24].

Ultrasound treatment of samples

Prior to enzymatic hydrolysis, the JSPI was pretreated by ultrasound. 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 using 1 M 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 the 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 with power output levels of 0 W, 200 W, 400 W, and 600 W for 15 min and 30 min (pulse duration: on-time, 5 s; off-time 1 s). The final temperature of the ultrasound process was 15 ± 2°C, 23 ± 1°C and 26 ± 1°C for the High-Intensity Ultrasound (HIU) application of 200 W, 400 W, and 600 W, respectively. After ultrasound treatment, all samples were centrifuged (2875 g for 30 min). Then, the JSPI suspensions with HIU pretreatment were lyophilized and stored at room temperature in airtight containers until its use for the treatment of enzymatic hydrolysis. The 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 (15 and 30 min), 400 W (15 and 30 min) and 600 W (15 and 30 min), the ultrasonic intensity was 40 ± 9 W cm-2, 62 ± 2 W cm-2 and 112 ± 1 W cm-2, respectively.

Enzymatic hydrolysis

The enzymatic hydrolysis was performed according to the methods by Zhang et al. [17] with some modifications. Batches (0.3 L) of JSPI suspensions (5%; w/v) with HIU pretreatment and of a control (JSPI without HIU pretreatment) were adjusted to pH 8.0 using 1 M NaOH, and 0.075 mL of alcalase were added to the protein suspensions at an enzyme-substrate ratio [E/S] of 1:200. The enzymatic hydrolysis was performed in a thermostatic bath with agitation (Boekel Scientific, 290400, Feasterville, PA, USA) at 50°C and 80 rpm. After 60 min of reaction, the hydrolysates were boiled for 10 min to terminate the reaction. Next, the hydrolysates were stored at -20°C for further analysis.

Degree of hydrolysis (DH)

The DH, expressed as the percentage of free amino groups, was determined in triplicate using the trinitrobenzene sulfonic (TNBS) method [25] as described by Connolly et al. [26] with modifications. Samples (5%; w/v) and leucine standard solutions were prepared in duplicate aliquots (0.064 mL) and 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. The 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:

t 0

tot 0

N - NDH (%) = 100h - N

α α×

α (1)

where αNt is the degree of dissociation of α-NH2 groups at a given


Page 3 of 11


time, αN0 is the degree of dissociation of α-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 6N HCl, sealing under vacuum, and heating to 110°C for 24 h. The hydrolysis was stopped by adding 4.5 mL of 6 N NaOH. The suspension was filtered, and the α-NH2 groups were determined as described previously.

Fourier transforms infrared spectra (FT-IR) measurement

The FT-IR spectra of the JSPI samples were scanned in the wavenumber range from 4000 cm-1 to 515 cm-1 using a Perkin-Elmer FT-IR spectrometer (LR-64912C, PerkinElmer, Inc. Norwalk, CT, USA) at room temperature (25°C). The spectra were an average of 28 scans. The data transformation, deconvolution and peak-separation analysis of the amide I band (1700 cm-1 to 1600 cm-1) were processed using the OriginPro8 software (OriginLab Corporation, Northampton, MA 01060, USA).

Surface hydrophobicity (H0-ANS) measurements

The H0-ANS was determined using 1-anilino-8-naphthalene-sulfonate (ANS) as a fluorescence probe according to the method given by Kato et al. [27] as described by Jiang et al. [28] with modifications. UAE-treated and control protein dispersions (1.5 mg/mL in 0.01 M phosphate buffer at pH 9.0) were centrifuged at 8,000 g at 17°C for 20 min. After determining the protein concentration in the supernatants according to the method given by Bradford [29], each supernatant was serially diluted with the same buffer to obtain protein concentrations ranging from 0.05 to 0.0001 mg/mL. Then, 25 µL of ANS (8.0 mM in 0.01 M phosphate buffer, pH 9.0) was added to 2 mL of sample. The fluorescence intensity (FI) was measured with a fluorescence spectrophotometer (Tecan Infinite 200 Pro, Grödig, Austria) at wavelengths of 364 nm (excitation) and 475 nm (emission). The initial slope of the 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.

Scanning electron microscopy (SEM)

The microstructure of the freeze-dried JSPI samples was observed with an SEM (SEC, Mini-SEM SNE-3200 M, South Korea) at an accelerating voltage of 30 kV. Before using the SEM, the samples were coated with gold using an ion sputter coater (MCM-100, SEC).

Protein solubility measurement (PS)

For PS measurement, 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 g for 20 min at 17°C. The protein content in the supernatant was measured using the Bradford method [29], and bovine serum albumin was used as the standard. PS-Bradford was expressed as mg/mL.

Techno-functional properties

Water holding capacity (WHC) and oil holding capacity (OHC): A previous procedure was used with slight modification to determine the WHC and OHC [30]. 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 g for 10 min. The supernatant was decanted, and the residue was weighed together with the centrifuge tube. The WHC was expressed as g of water held per

g of sample. An identical method was used to measure corn oil holding capacity, and the OHC was expressed as g of oil held per g of protein.

Emulsifying activity (EA) and emulsion stability (ES): A modified version of the method described by Ulloa et al. [31] was used to determine the EA and ES of the JSPI. Suspensions were prepared by dissolving 1 g of JSPI sample in 15 mL of 0.01 M phosphate buffer solution (pH 7.0). 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 of 20 for 1 min and centrifuged at 198 g for 5 min. The emulsion layer volume was recorded. The emulsifying activity (EA) was calculated as:

EA(%) = (height of emulsigfied 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. The emulsion stability was expressed as the percentage of emulsifying activity remaining after heating.

Least gelation concentration (LGC): LGC was determined using 2, 4, 6, 8, 10, and 12 g/100 mL JSPI dispersions for each JSPI sample in centrifuge tubes. The pH of the dispersions was adjusted to 4. 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 [30].

Statistical analysis

Statistical analysis was performed using Statgraphics Centurion Software version XV (Statpoint Technologies, Inc. Virginia, USA). All data are shown as the mean ± standard deviation (SD). Duncan’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. Pearson correlation analysis was conducted to evaluate relationships between structural characteristics, DH and techno-functional properties, and p<0.05 or p<0.01 were regarded as statistically significant.

Results and DiscussionDegree of hydrolysis (DH) and protein solubility (PS)

The DH gives an initial indication of a change in the molecular integrity, and thus, large complex structured protein molecules are broken down into smaller sized peptides and specific amino acids [10]. The results of the DH of JSPI after ultrasonic treatment at different power (200 W, 400 W, 600 W) and time (15 min and 30 min) combinations are shown in Figure 1. As shown in Figure 1, JSPI-UAE (200 W to 600 W, 15 or 30 min) increased the DH significantly (p<0.05) when compared to the enzymolysis sample. The DH increased from an initial DH of 2.4% enzymolysis treatment to 5.3% up to 6.0% after UAE (200 W to 600 W). However, the DH did not change significantly (p<0.05) at different times of UAE (15 min or 30 min). This indicates that the UAE was more efficient than traditional enzymolysis.

Li et al. [18,32] evaluated the kinetics of UAE (58 W L-1; 28 kHz) using rice proteins. These researchers reported that ultrasound treatment improved the enzymatic efficiency and, in turn, increased the DH (p<0.05) of the protein. Furthermore, Zhou et al. [33] studied the combined effect of ultrasound and/or heat on corn gluten enzymolysis. They reported that the ultrasound pretreatment (40 kHz) accelerated


Page 4 of 11


the reaction rate of the corn gluten hydrolysis and led to an increase in the DH of the protein. The reason might be that the ultrasound and sonochemistry effects helped disrupt strong solute-matrix interactions, which involved Van de Waals forces, hydrogen bonding and dipole attractions between the molecules [18]. The foregoing results were consistent with enzymolysis of corn gluten meal [34], wheat gluten [17], oats protein [35] and potato protein [36] after ultrasound pretreatment.

The solubility is the most practical measurement of protein denaturation and aggregation and is, therefore, a reliable index of protein functionality due to its considerable effect on other technological characteristics, particularly gelation, foaming, and emulsifying, which depend on an adequate initial solubility of proteins [10,37]. The PS of JSPI increased significantly (p<0.05) after UAE compared with the solubility of the enzymolysis treatment (Figure 1). This might be due to that in a natural state, proteins are present in the form of aggregates, and the physical factors of cavitation might disrupt the hydrogen bonds and hydrophobic interactions, which are responsible for intermolecular association of protein aggregates [8]. The PS increased from an initial value (enzymolysis) of 0.07 ± 0.00 mg/mL to 0.29 ± 0.06 mg/mL, 0.30 ± 0.01 mg/mL, and 0.28 ± 0.05 mg/mL after UAE at 200 W, 400 W and 600 W, respectively. Therefore, this increase in solubility may be due to conformational change during ultrasonic treatment and hydrolysis of the peptide bonds of the JSPI protein molecules. Positive correlations between PS and DH (r=0.9772; p<0.01) and between α-helix and DH (r=0.7699; p<0.05) were observed after UAE. According to the above, the ultrasound-enzymolysis synergistic effect caused an increase in PS, which is directly related to the conformational changes in the secondary structure and the hydrolysis of the peptide bonds of the JSPI protein molecules, in accordance with the studies of Li et al. [14] and Yang et al. [37] who reported that the UAE increased the PS of rice proteins and wheat germ, respectively.

Secondary structure (FT-IR)

FT-IR spectroscopy is a measurement of wavelength numbers and the intensity of the absorption of infrared (IR) radiation by the sample [33]. The polypeptide and protein repeat units give rise to nine characteristic IR absorption bands, namely, amide A, B, I, II, III, IV, V, VI, and VII [38]. The amide I (1600 cm-1 to 1700 cm-1) band represents the C=O and a small extent of the C=N stretching vibration, which provides information on the secondary structures of the proteins (α-helix, β-sheet, β-turn, and random coil) [2]. To obtain further information concerning protein structural changes, we analyzed the FT-IR spectra of the samples exposed to the UAE between wavelength numbers 1700 cm-1 and 1600 cm-1.

The FT-IR spectra of JSPI-UAE treatments in the region of 1750 ∑ 1450 cm-1 are shown in Figure 2. As presented in this Figure, there was an obvious difference between the enzymolysis treatment and JSPI subjected to UAE in terms of the absorption region and the intensity of the peaks. After deconvolution and overlapping component extraction procedures, the fitted peaks are shown in Figure 3. The deconvoluted amide I bands and the content assignment of the secondary structure of JSPI are shown in Table 1 according to some previous studies [2,21,22].

The UAE treatment had a significant effect on the secondary structure compared to the enzymolysis treatment (p<0.01). The relative content of α-helixes, β-turns, and random coils increased from the initial value (enzymolysis treatment) of 8.68%, 27.19% and 20.81% up to 10.40%, 30.06% and 23.18% after UAE treatments, respectively. In contrast, the relative content of β-sheets decreased from an initial value of 43.32% up to 36.88%.

When UAE was applied, the β-sheet content decreased (up to 15%, 200 W-30 min) while the α-helix (up to 16.54%, 400 W-15 min), β-turn (up to 9.55%, 200 W-30 min) and random coil (up to 10.22%, 400 W, 15)

Figure 1: Effect of enzymolysis (A) and ultrasound-assisted enzymolysis (B-G) on the degree of hydrolysis (DH) and protein solubility (PS) of jackfruit seed protein isolate. Different letters mean significant differences between values with different ultrasonic power and time (Duncan; p<0.05).


Page 5 of 11


content increased. In agreement with the above, a negative correlation was observed between α-helix and β-sheet (r=-0.9023; p<0.01) and random coil and β-sheet (r=-0.7820; p<0.05) content. Furthermore, a positive correlation was observed between α-helix and random coil (r=0.9141; p<0.01).

In general, these findings suggest that UAE results in unfolding of

the α-helical region followed by the formation of a β-turn and random coil as well as a decrease in the interaction of the amino acids that formed the β-sheet structure. The changes in the secondary structure content might be observed because the free radical, microjets, shear forces, shock waves, and turbulence, which were induced by ultrasound, have disrupted the interactions between the local sequences of amino acids and between different parts of the protein molecule [2].

Figure 2: Effect of enzymolysis (A) and ultrasound-assisted enzymolysis (B-G) on the FT-IR absorbance spectra (amide I band) of jackfruit seed protein isolate.

Figure 3: Deconvolution and peak-separation analysis of amide I band (1700-1600 cm-1) of jackfruit seed protein isolate treated by enzymolysis (A) and ultrasound-assisted enzymolysis (B-G).


Page 6 of 11


The results of the present study are different from those of Yang et al. [11] and Jin et al. [2]. These authors reported that dual-frequency power ultrasound treatment was responsible for decreased α-helix content and an increased β-sheet and random coil content, compared with the native rice protein and corn gluten meal, respectively. Different results have also been reported by researchers who have applied sonication to other types of proteins, such as walnut protein and soy protein [37,38]. Conversely, Zhang et al. [39] utilized ultrasound-assisted alkali to pretreat rice protein, and the results of FT-IR showed that the ultrasound increased the β-turn content and decreased the β-sheet and random coil content. These contradictory results, compared to our results, might arise from the differences in sonication conditions, such as the type of ultrasound used, the frequency, the ultrasonic intensity as well as the intrinsic characteristics of the sonicated material.

Microstructure

To understand the effect of different UAE conditions applied to JSPI, the microstructure of the lyophilized JSPI was observed by SEM. Figure 4 shows a set of SEM images of different JSPI at a 450-fold magnification factor. Compared with the enzymolysis treatment, samples B-G exhibited more disordered structures and irregular

fragments. In addition, the SEM images showed that on average, sample E (400 W-30 min) and G (600 W-30 min) was larger than D (400 W-15 min) and F (600 W-15 min), respectively, suggesting that a longer ultrasonic time could result in larger structures. The functional native structure of proteins is determined by the subtle balance between many non-covalent and covalent interactions; this balance can be easily disrupted by the mechanical/shear stresses from sonication leading to protein denaturation and change in the secondary structure [17]. With relatively low-power ultrasound treatment, the effect of turbulent forces and microstreaming might increase the speed of collision and aggregation; this typically results in the formation of unstable aggregates and an increase in the particle size [40,41]. When the power of the ultrasonic treatment is increased, the particles become smaller, and the particle size-distribution broadens [28]. Similar observations were reported by Malik et al. [3] for sunflower protein isolate. Kang et al. [22] showed that the exposure of the hydrophobic regions resulted in an increase in the β-sheet content, accompanied by a decrease in α-helix structures. In this context, these results might be due to the changes in the ultrasonic treatment leading to unfolding of the JSPI protein molecules and increased exposure of hydrophobic groups (see Section 3.4), and this result is confirmed by the increase in the β-sheet and random coil content (see Section 3.2), which could interact with each other and form larger aggregates during freeze drying.

Surface hydrophobicity (H0-ANS)

The surface hydrophobicity (H0-ANS) of proteins is one of the structural characteristics, is important for protein stability and conformation and has an impact on protein functionality [42]. Figure 5 shows that UAE significantly increased the H0-ANS of JSPI (p<0.05). This finding was consistent with previous studies that showed that UAE could cause an increase in the H0-ANS for wheat germ protein [12] and rice protein [12,32]. Comparing the H0-ANS of JSPI-UAE, it was observed that H0-ANS increased with ultrasonic intensity (from 200 W to 600 W) and time (15 min and 30 min). This indicates that ultrasonic treatment induces a certain degree of the 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

Figure 4: Effect of enzymolysis (A) and ultrasound-assisted enzymolysis B=200 W-15 min, C=200 W-30 min, D=400 W-15 min, E=400 W-30 min, F=600 W-15 min, G=600 W-30 min on microstructure of lyophilized jackfruit seed protein isolate by scanning electron microscopy. Scale bar is 200 µm in all cases.

Treatment

Secondary structure (%)

α-helix (1651-1652 cm-1)

β-sheet (1610-1634,1695

cm-1)

β-turn (1658-1689 cm-1)

Random coil (1640-1645

cm-1)Enzymolysis 8.68a 43.32g 27.19b 20.81a

UAE at:200 W 15 min 9.83c 41.40f 26.95a 21.83d

200 W 30 min 10.36d 36.88a 30.06g 22.70e

400 W 15 min 10.40d 37.67b 28.75e 23.18g

400 W 30 min 10.16d 39.16c 27.49c 23.17f

600 W 15 min 9.50b 39.72d 29.72f 21.06b

600 W 30 min 9.38b 40.99e 28.30d 21.33c

Within a column, means with different superscript letters indicated significant difference (Duncan; p<0.01)

Table 1: Effect of the enzymolysis and ultrasound-assisted enzymolysis (UAE) by alcalase on secondary structure of jackfruit seed protein isolate.


Page 7 of 11


exposed to the polar surrounding environment [4]. This aggregation protects the hydrophobic regions of the proteins [28]. However, the H0-ANS of the samples treated at an ultrasound power of 400 W and 600 W for prolonged times (30 min) decreased. High-intensity ultrasound treatment might also lead to partial denaturation of proteins, which might increase the extent of bonding and reduce the H0-ANS [43].

Similar results were obtained by Malik et al. [42], Wu et al. [44], Zhang et al. [39] and Yang et al. [37] in sunflower protein, whey protein, rice protein, and soy protein, respectively.

Functional properties

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

Figure 5: Effect of the enzymolysis (A) and ultrasound-assisted enzymolysis (B-G) on the surface hydrophobicity (H0-ANS) of jackfruit seed protein isolate (mean ± SD, n=2). Mean values with different letters were significantly different (Duncan; p<0.05).

Figure 6: Effect of the enzymolysis (A) and ultrasound-assisted enzymolysis (B-G) on water holding capacity (WHC; A) and oil holding capacity (OHC; B) of jackfruit seed protein isolate. Mean values with different letters were significantly different (Duncan; p<0.05). WHC and OHC: n=4 ± SD for control; n=3 ± SD for 0 W; n=2 ± SD for 200 W to 600 W for 15 min and 30 min.


Page 8 of 11


Figure 7: Effect of enzymolysis (A) and ultrasound-assisted enzymolysis (B-G) on emulsifying activity (EA) and emulsifying stability (ES) jackfruit seed protein isolate (mean ± SD, n=2). Different letters means significant differences between values with different ultrasonic power and time (Duncan; p<0.05).

Figure 8: Influence of enzymolysis (A) and ultrasound-assisted enzymolysis (B-G) on the least gelation concentration (LGC) of jackfruit seed protein isolate (mean ± SD, n=2).


Page 9 of 11


The WHC and OHC result from different JSPI samples are shown in Figure 6. The WHCs of the UAE samples were significantly (p<0.05) lower compared to the enzymolysis treatment (Figure 6a). The WHC decreased from an initial WHC of 3.60 g g-1 (enzymolysis treatment) up to 2.83 g g-1 after UAE at 200 W-15 min. However, at 600 W-30 min, the WHC decreased significantly (p<0.05) to 2.44 g g-1. One reasonable explanation is that the UAE after stronger ultrasound treatment might heavily denature the molecular structure of the protein and cause an increase in the free sulfhydryl groups to the surface of the JSPI, resulting in lower levels of WHC. These results are in agreement with those reported by Zhang et al. [45] who observed an increase in the WHC of myofibrillar protein at an ultrasound power of 200 W to 600 W, but at higher ultrasound power (≥ 800 ∑ ≤ 1000 W, 15 min) the WHC decreased. A negative correlation (r=-0.8006; p<0.05) between WHC and UAE was observed.

On the other hand, the OHC of JSPI subjected to UAE at 200 W–15 min was significantly (p<0.05) greater compared to the enzymolysis treatment. The OHC increased from an initial OHC of 1.82 g g-1 up to 2.18 g g-1 after UAE of 200 W-15 min (Figure 6b). The presence of OHC might be because of the exposure of hydrophobic groups after the UAE allowed the physical entrapment of oil [10]. Such exposure of hydrophobic groups can be observed by the formation of large aggregates of proteins in the dry state after freeze-drying JSPI samples treated with UAE in comparison with control JSPI (Figure 4).

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, milk and coffee whiteners [13,46]. The EA value of the JSPI subjected to enzymolysis was not significantly different (p<0.05) in comparison with JSPI treated with UAE in all conditions (Figure 7). It has been reported that the emulsifying properties of hydrolysates are closely related to the degree of hydrolysis [47], with a low DH (3 ∑ 5%) increasing and a high DH (≥ 8%) decreasing emulsifying properties [10]. However, in this study, the UAE increased the DH in the range from 2.9 to 3.5 with respect to enzymolysis but without effect on EA.

The ES of JSPI at 200 W-15 min and 400 W-30 min was increased ~ 6% after UAE with respect to the enzymolysis (Figure 7). The increase in the ES can be explained by the more favorable orientation of proteins resulting from the influence of turbulent behavior produced by the ultrasound-enzymolysis [43,48]. Xiong et al. [41], Hu et al. [46], Zhou et al. [13] and Zhang et al. [49] reported that high-intensity ultrasound increased the EA and ES of ovalbumin, soy β-conglycinin, soy glycinin, and peanut protein, respectively, which is in agreement with the results of ES obtained in this study for only two treatments (UAE at 200 W-15 min and 400 W-30 min).

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 the EA [47]. 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 greater adsorption rate [50] as well as increased activity and stability of the emulsion [46].

As in other properties, EA and ES depend upon the molecular flexibility and stability of the protein structure (such as the secondary and tertiary structure) [43]. In this sense, a negative correlation between EA and the β-turn content (r=-0.7815; p<0.05) was observed after UAE. According to these results, EA and ES improved mainly due

to the increase in the β-sheet content and random coil content of JSPI after UAE.

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 [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 [51]. In general, the LGC showed no significant change (p>0.01) after UAE compared to the enzymolysis (Figure 8). The LGC was 2.0 g/100 g in all JSPI treated with UAE. In a previous study [8], we reported that high-intensity ultrasound modified the LGC of jackfruit seed proteins and had values of 2.0 g/100 g to 8.0 g/100 g at pH 2 ∑ pH 10, respectively. In this investigation, all JSPI had the lowest LGC value (2.0 g/100 g), which is highly desirable in food gelling agents. Nevertheless, the evaluation of the microscopic structural organization and rheological properties could lead to a greater understanding of the type of network formed by the UAE.

ConclusionIn general, the ultrasound pretreatment of JSPI improved the

enzymolysis reaction by alcalase, as confirmed by an increase in DH (p<0.05). UAE improved the techno-functional properties in terms of the OHC and ES and modified the structure of JSPI. UAE decreased the β-sheet content and increased the β-turn content, the random coil content and the α-helix content. The effective promotion of techno-functional properties by UAE was achieved by increasing the degree of hydrolysis, the unfolding of the secondary protein structure and a reduction of intermolecular interactions, as demonstrated by an increase in H0-ANS, leading to improved PS of JSPI. The results presented here could be used in an industrial setting to develop conventional or novel foods based on jackfruit proteins.

AcknowledgmentThe authors would like to acknowledge the National Council for Science

and Technology of Mexico for the Ph.D. scholarship (418963) for Juan Alberto Resendiz Vazquez and the Patronage to Administer the Special Tax Destined to the Autonomous University of Nayarit for providing research funds (PUAN-CP-001/2018).

References1. Pojić M, Mišan A, Tiwari B (2018) Eco-innovative technologies for extraction of

proteins for human consumption from renewable protein sources of plant origin. Trends Food Sci Technol 75: 93-104.

2. Jin J, Ma H, Wang B, Yagoub AEA, Wang K, et al. (2016) Effects and mechanism of dual-frequency power ultrasound on the molecular weight distribution of corn gluten meal hydrolysates. Ultrason Sonochem 30: 44-51.

3. Malik MA, Saini CS (2018) Rheological and structural properties of protein isolates extracted from dephenolized sunflower meal: Effect of high intensity ultrasound. Food Hydrocoll 81: 229-241.

4. Ulloa JA, Villalobos-Barbosa MC, Resendiz-Vazquez JA, Rosas-Ulloa P, Ramírez-Ramírez JC, et al. (2017) Production, physico-chemical and functional characterization of a protein isolate from jackfruit (Artocarpus heterophyllus) seeds. J Food 15: 497-507.

5. Swami SB, Thakor NJ, Haldankar PM, Kalse SB (2012) Jackfruit and its many functional components as related to human health: A review. Compr Rev Food Sci Food F 11: 565-576.

6. De la Cruz-Torres LF, Pérez-Martínez J D, Sánchez-Becerril M, Toro-Vázquez JF, Mancilla-Margalli NA, et al. (2017) Physicochemical and functional properties of 11S globulin from chan (Hyptis suaveolens L. poit) seeds. J Cereal Sci 77: 66-72.

7. Bojórquez-Velázquez E, Lino-López GL, Huerta-Ocampo JA, Barrera-Pacheco A, Barba de la Rosa AP, et al. (2016) Purification and biochemical

https://www.sciencedirect.com/science/article/pii/S0924224417306854



https://doi.org/10.1016/j.ultsonch.2015.11.021



https://doi.org/10.1016/j.foodhyd.2018.02.052



https://doi.org/10.1080/19476337.2017.1301554

https://doi.org/10.1080/19476337.2017.1301554

https://doi.org/10.1080/19476337.2017.1301554

https://doi.org/10.1080/19476337.2017.1301554

https://doi.org/10.1111/j.1541-4337.2012.00210.x

https://doi.org/10.1111/j.1541-4337.2012.00210.x

https://doi.org/10.1111/j.1541-4337.2012.00210.x

https://doi.org/10.1016/j.jcs.2017.06.017





Page 10 of 11


characterization of 11S globulin from chan (Hyptis suaveolens L. Poit) seeds. Food Chem 192: 203-211.

8. Resendiz-Vazquez JA, Ulloa JA, Urías-Silvas JE, Bautista-Rosales PU, Ramírez-Ramírez JC, et al. (2017) Effect of high-intensity ultrasound on the technofunctional properties and structure of jackfruit (Artocarpus heterophyllus) seed protein isolate. Ultrason Sonochem 37: 436-444.

9. López DN, Ingrassia R, Busti P, Bonino J, Delgado JF, et al. (2018) Structural characterization of protein isolates obtained from chia (Salvia hispanica L.) seeds. LWT-Food Sci Technol 90: 396-402.

10. Meinlschmidt P, Sussmann D, Schweiggert-Weisz U, Eisner P (2015) Enzymatic treatment of soy protein isolates: Effects on the potential allergenicity, technofunctionality, and sensory properties. Food Sci Nutr 4: 11-23.

11. Yang X, Li Y, Li S, Oladejo AO, Ruan S, et al. (2017) Effects of ultrasound pretreatment with different frequencies and working modes on the enzymolysis and the structure characterization of rice protein. Ultrason Sonochem 38: 19-28.

12. Yang X, Li Y, Li S, Oladejo AO, Wang Y, et al. (2017) Effects of multi-frequency ultrasound pretreatment under low power density on the enzymolysis and the structure characterization of defatted wheat germ protein. Ultrason Sonochem 38: 410-420.

13. Zhou M, Liu J, Zhou Y, Huang X, Liu F, et al. (2017) Effect of high intensity ultrasound on physicochemical and functional properties of soybean glycinin at different ionic strengths. Innov Food Sci Emerg Technol 34: 205-213.

14. Li S, Ma H, Guo Y, Oladejo AO, Yang X, et al. (2018) A new kinetic model of ultrasound-assisted pretreatment on rice protein. Ultrason Sonochem 40-A: 644-650.

15. Pradal D, Vauchel P, Decossin S, Dhulster P, Dimitrov K (2016) Kinetics of ultrasound-assisted extraction of antioxidant polyphenols from food byproducts: extraction and energy consumption optimization. Ultrason Sonochem 32: 137-146.

16. Abadía-García L, Castaño-Tostado E, Ozimek L, Romero-Gómez S, Ozuna CS, et al. (2016) Impact of ultrasound pretreatment on whey protein hydrolysis by vegetable proteases. Innov Food Sci Emerg Technol 37-A: 84-90.

17. Zhang Y, Wang B, Zhou C, Atungulu GG, Xu K, et al. (2016) Surface topography, nano-mechanics and secondary structure of wheat gluten pretreated by alternate dual-frequency ultrasound and the correlation to enzymolysis. Ultrason. Sonochem 31: 267-275.

18. Li S, Yang X, Zhang Y, Ma H, Qu W, et al. (2016) Enzymolysis kinetics and structural characteristics of rice protein with energy-gathered ultrasound and ultrasound assisted alkali pretreatments. Ultrason Sonochem 31: 85-92.

19. Wang B, Meng T, Ma H, Zhang Y, Li Y, et al. (2016) Mechanism study of dual-frequency ultrasound assisted enzymolysis on rapeseed protein by immobilized Alcalase. Ultrason Sonochem 32: 307-313.

20. Baltacioglu H, Bayindirli A, Severcan F (2017) Secondary structure and conformational change of mushroom polyphenol oxidase during thermosonication treatment by using FTIR spectroscopy. Food Chem 214: 507-514.

21. Ulrichs T, Drotleff AM, Ternes W (2015) Determination of heat-induced changes in the protein secondary structure of reconstituted livetins (water-soluble proteins from hen’s egg yolk) by FTIR. Food Chem 172: 909-920.

22. Kang D, Zou Y, Cheng Y, Xing L, Zhou G, et al. (2016) Effects of power ultrasound on oxidation and structure of beef proteins during curing processing. Ultrason Sonochem 33: 47-53.

23. AOAC (1990) Official Methods of Analysis. Association of Official Analytical Chemists (15th edtn), Arlington, USA

24. Lima BNF, Lima FF, Tavares MIB, Costa AMM, Pierucci (2014) APTR Determination of the centesimal composition and characterization of flours from fruit seeds. Food Chem 151: 293-299.

25. Adler-Nissen J (1979) Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J Agric Food Chem 27: 1256-1262.

26. Connolly A, Piggott CO, FitzGerald RJ (2014) Technofunctional properties of a brewers' spent grain protein-enriched isolate and its associated enzymatic hydrolysates. LWT-Food Sci Technol 59: 1061-1067.

27. Kato A, Nakai S (1980) Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. BBA-Protein Struct 624: 13-20.

28. Jiang J, Wang J, Li Y, Wang Z, Liang J, et al. (2014) Effects of ultrasound on

the structure and physical properties of black bean protein isolates. Food Res Int 62: 595-601.

29. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.

30. Joshi AU, Liu C, Sathe SK (2015) Functional properties of select seed flours. LWT-Food Sci Technol 60: 325-331.

31. Ulloa JA, Rosas-Ulloa P, Ulloa-Rangel BE (2011) Physicochemical and functional properties of a protein isolate produced from safflower (Carthamus tinctorius L.) meal by ultrafiltration. J Sci Food Agric 91: 572-577.

32. Li S, Yang X, Zhang Y, Ma H, Liang Q, et al. (2016) Effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein. Ultrason Sonochem 31: 20-28.

33. Zhou C, Hu J, Yu X, Yagoub AEA, Zhang Y, et al. (2017) Heat and/or ultrasound pretreatments motivated enzymolysis of corn gluten meal: Hydrolysis kinetics and protein structure. LWT-Food Sci Technol 77: 488-496.

34. Ren X, Ma H, Mao S (2014) Effects of sweeping frequency ultrasound treatment on enzymatic preparations of ACE-inhibitory peptides from zein. Eur Food Res Technol 238: 435-442.

35. Wang B Atungulu GG, Khir R (2015) Ultrasonic treatment effect on enzymolysis kinetics and activities of ACE-inhibitory peptides from oat-isolated protein. Food Biophys 10: 244-252.

36. Cheng Y, Liu Y, Wu J, Donkor PO, Li T, et al. (2017) Improving the enzymolysis efficiency of potato protein by simultaneous dual-frequency energy-gathered ultrasound pretreatment: Thermodynamics and kinetics. Ultrason. Sonochem 37: 351-359.

37. Yang F, Liu X, Ren X, Huang Y, Huang C, et al. (2018) Swirling cavitation improves the emulsifying properties of commercial soy protein isolated. Ultrason Sonochem 42: 471-481.

38. Zhu Z, Zhu W, Yi J, Liu N, Cao Y, et al. (2018) Effects of sonication on the physicochemical and functional properties of walnut protein isolate. Food Res Int 106: 853-861.

39. Zhang L, Pan Z, Shen K, Cai X, Zheng B, et al. (2018) Influence of ultrasound-assisted alkali treatment on the structural properties and functionalities of rice protein. J Cereal Sci 79: 204-209.

40. Hu H, Wu J, Li-Chan ECY, Zhu L, Zhang F, et al. (2013) Effects of ultrasound on structural and physical properties of soy protein isolate (SPI) dispersions. Food Hydrocolloids 30: 647-655.

41. Xiong W, Wang Y, Zhang C, Wan J, Shah JBR, et al. (2016) High intensity ultrasound modified ovalbumin: Structure, interface and gelation properties. Ultrason Sonochem 31: 302-309.

42. Malik MA, Sharma HS, Saini CS (2017) High intensity ultrasound treatment of protein isolate extracted from dephenolized sunflower meal: Effect on physicochemical and functional properties. Ultrason Sonochem 39: 511-519.

43. Yanjun S, Jianhang C, Shuwen Z, Hongjuan L, Jing L, et al. (2014) Effect of power ultrasound pre-treatment on the physical and functional properties of reconstituted milk protein concentrate. J Food Eng 124: 11-18.

44. Wu Q, Zhang X, Jia J, Kuang C, Yang H (2018) Effect of ultrasonic pretreatment on whey protein hydrolysis by alcalase: Thermodynamic parameters, physicochemical properties and bioactivities. Process Biochem 67: 46-54.

45. Zhang Z, Regenstein JM, Zhou P, Yang Y (2017) Effects of high intensity ultrasound modification on physicochemical property and water in myofibrillar protein gel. Ultrason Sonochem 34: 960-967.

46. Hu H, Cheung IWY, Pan S, Li-Chan ECY (2015) Effect of high intensity ultrasound on physicochemical and functional properties of aggregated soybean β-conglycinin and glycinin. Food Hydrocolloids 45: 102-110.

47. Chen L, Chen J, Ren J, Zhao M (2011) Effects of ultrasound pretreatment on the enzymatic hydrolysis of soy protein isolates and on the emulsifying properties of hydrolysates. J Agric Food Chem 59: 2600-2609.

48. Jambrak AR, Lelas V, Mason TJ, Kresic G, Badanjak M (2009) Physical properties of ultrasound treated soy proteins. J Food Eng 93: 386-393.

49. Zhang Q, Tu Z, Xiao H, Wang H, Huang H, et al. (2014) Influence of ultrasonic treatment on the structure and emulsifying properties of peanut protein isolate. Food Bioprod Process 92: 30-37.

https://www.semanticscholar.org/paper/Effect-of-high-intensity-ultrasound-on-the-and-of-Resendiz-Vazquez-Ulloa/82f42fff6f59214abe5a6145f16cb11c63c82ab8




https://www.deepdyve.com/lp/elsevier/structural-characterization-of-protein-isolates-obtained-from-chia-6DAL26H3Y2



https://doi.org/10.1155/2017/7896037

https://doi.org/10.1155/2017/7896037

https://doi.org/10.1155/2017/7896037

https://scialert.net/fulltext/?doi=ajft.2016.240.252




https://doi.org/10.1016/j.ifset.2016.02.007



https://doi.org/10.1016/j.foodchem.2014.09.128






https://pubs.acs.org/doi/abs/10.1021/jf60226a042

https://pubs.acs.org/doi/abs/10.1021/jf60226a042

https://doi.org/10.1016/j.lwt.2014.06.054



https://doi.org/10.1016/0005-2795(80)90220-2

https://doi.org/10.1016/0005-2795(80)90220-2

https://doi.org/10.1016/0005-2795(80)90220-2

https://doi.org/10.1016/j.foodres.2014.04.022



https://doi.org/10.1016/0003-2697(76)90527-3

https://doi.org/10.1016/0003-2697(76)90527-3

https://doi.org/10.1016/0003-2697(76)90527-3

https://doi.org/10.1002/jsfa.4227









http://agris.fao.org/agris-search/search.do?recordID=US201400086707


















https://eurekamag.com/research/059/798/059798932.php



https://doi.org/10.1016/j.jfoodeng.2013.09.013



https://doi.org/10.1016/j.procbio.2018.02.007











https://doi.org/10.1016/j.fbp.2013.07.006




Page 11 of 11


50. Delahaije RJBM, Gruppen H, Giuseppin ML, Wierenga PA (2015) Towards predicting the stability of protein-stabilized emulsions. Adv Colloid Interfac 219: 1-9.

51. Maltais A, Remondetto GE, Subirade M (2008) Mechanisms involved in the formation and structure of soya protein cold-set gels: A molecular and supramolecular investigation. Food Hydrocolloids 22: 550-559.

https://www.semanticscholar.org/paper/Towards-predicting-the-stability-of-emulsions.-Delahaije-Gruppen/a79036aaef9c64ddf09bf578ab0a79659d484027

https://www.semanticscholar.org/paper/Towards-predicting-the-stability-of-emulsions.-Delahaije-Gruppen/a79036aaef9c64ddf09bf578ab0a79659d484027




Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Effect of high-intensity ultrasound on the compositional, physicochemical,biochemical, functional and structural properties of canola (Brassica napusL.) protein isolate

Nitzia Thalía Flores-Jiméneza, José Armando Ulloaa,c,⁎, Judith Esmeralda Urías Silvasb,José Carmen Ramírez Ramírezd, Petra Rosas Ulloac, Pedro Ulises Bautista Rosalesa,c,Yessica Silva Carrilloe, Ranferi Gutiérrez Leyvad

a Posgrado en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit, Carretera Tepic-Compostela Km 9, Xalisco 63780, Nayarit, Mexicob Tecnología Alimentaria, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A. C., Avenida Normalistas 800, Colinas de la Normal,Guadalajara 44270, Jalisco, Mexicoc Centro de Tecnología de Alimentos, Universidad Autónoma de Nayarit, Ciudad de la Cultura Amado Nervo, Tepic 63155, Nayarit, MexicodUnidad Académica de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Nayarit, Carretera Compostela-Chapalilla Km 3.5, Compostela 63700, Nayarit,MexicoeUnidad Académica de Agricultura, Universidad Autónoma de Nayarit, Carretera Tepic-Compostela Km 9, Xalisco 63780, Nayarit, Mexico

A R T I C L E I N F O

Keywords:Canola protein isolateHigh-intensity ultrasoundFunctional propertiesPhysicochemical propertiesStructural propertiesBiochemical propertiesCavitationUltrasound bath

A B S T R A C T

The objective of this study was to investigate the impacts of high-intensity ultrasound treatments on the com-positional, physicochemical, biochemical, functional and structural properties of canola protein isolates (CPI).Aqueous canola protein suspensions were sonicated at 40 kHz for 15min and 30min. The moisture content,water activity, bulk density and the L* and a* color parameters of the CPI decreased due to the ultrasound;however, the in vitro protein digestibility was not modified by the treatment. Glutelin (57.18%) was the mainprotein fraction in the canola protein isolate. SDS-PAGE demonstrated that there were no changes in the proteinelectrophoretic patterns, thus indicating that sonication did not break the covalent bonds. However, the ultra-sound treatment improved the protein solubility, oil absorption capacity and the emulsifying, gelation andfoaming properties, but these improvements depended on the pH and ultrasound exposure time. Scanningelectron microscopy revealed that the ultrasound treatment disrupted the microstructure of the CPI by exhibitinglarger aggregates as a lyophilized powder. In addition, there was an increase in the surface hydrophobicity and adecrease in the size of the particles of the canola protein due to the ultrasound effects, which indicates a de-struction of the particles or a dissociation of the protein aggregates in the canola protein dispersions. Theseresults suggest that ultrasound treatment is a valuable tool for improving the characteristics of canola proteinsfor use in foods.

1. Introduction

Canola (Brassica napus L.) is the second most important oilseedcultivated in the world after soybeans. According to the FAO(2018),> 68.8 million metric tons of canola was produced worldwidein 2016, with Canada as the largest producer, followed by China, India,France and Germany. Canola is an important oilseed crop grown pri-marily for its high oil content, which contains linoleic (omega-6) and α-linolenic acid (omega-3) essential fatty acids at a 2:1 ratio, thus makingit one of the healthiest cooking oils for food use. Furthermore, it has

many other applications including biofuels, cosmetics and other in-dustrial products (Wanasundara, McIntosh, Perera, Withana-Gamage, &Mitra, 2016). The national consumption of canola in México for theproduction of canola oil was 1703 thousand tons in 2016, with 92.5%of that being imported from Canada (SAGARPA, 2017). After oil ex-traction, canola meal contains 35–40 g/100 g protein, and it is usedmainly as an animal feed or as a fertilizer (Chang, Gupta, Timilsena, &Adhikari, 2016). However, this protein source could be used in humannutrition as protein isolates.

In addition, canola proteins contain a well-balanced amino acid

https://doi.org/10.1016/j.foodres.2019.01.025Received 24 September 2018; Received in revised form 23 December 2018; Accepted 10 January 2019

⁎ Corresponding author at: Centro de Tecnología de Alimentos, Universidad Autónoma de Nayarit, Ciudad de la Cultura Amado Nervo, Tepic 63155, Nayarit,Mexico.

E-mail addresses: [email protected], [email protected] (J.A. Ulloa).

Food Research International 121 (2019) 947–956

Available online 11 January 20190963-9969/ © 2019 Elsevier Ltd. All rights reserved.

T

http://www.sciencedirect.com/science/journal/09639969

https://www.elsevier.com/locate/foodres






http://crossmark.crossref.org/dialog/?doi=10.1016/j.foodres.2019.01.025&domain=pdf

composition, especially a high content of lysine (6.0 g/100 g) andsulfur-containing amino acids (3–4 g/ 100 g), and a high protein effi-ciency ratio (2.64) compared to that of soy protein (2.19), thus makingit favorable for human consumption (Jayaraman, MaIsaac, & Anderson,2016). Numerous studies conducted on the physicochemical, functionaland bioactive properties of canola proteins have indicated potential forit to be used in the food industry as protein concentrate and isolate(Gerzhova, Mondor, Benali, & Aider, 2015). However, canola proteinisolates are often prepared by direct alkaline extraction followed byacid precipitation, and the poor water solubility of such protein frac-tions leads to isolates with unsuitable technological and functional foodproperties (Pirestani, Nasirpour, Keramat, & Desobry, 2017).

On the other hand, numerous studies have shown that ultrasoundcan change the structural and/or the functional properties of foodproteins by altering their molecular characteristics (Ozuna, Paniagua-Martínez, Castaño-Tostado, Ozimek, & Amaya-Llano, 2015). Ultrasoundis an acoustic wave with a frequency>20 kHz (O'Sullivan, Murray,Flynn, & Norton, 2016). Ultrasound can be classified into two frequencyranges: low frequency (20–100 kHz; intensity in the range of10–1000W cm−2) and high frequency (100 kHz-1MHz; intensity< 1W cm2) (Jambrak, Mason, Lelas, Paniwnyk, & Herceg, 2014). A low-intensity ultrasound (high frequency) is most commonly applied as ananalytical technique to provide information on the physicochemicalproperties of food, such as its firmness, ripeness, sugar content, acidity,etc. (Hu et al., 2013). In contrast, a high-intensity ultrasound (lowfrequency) can be used to physically or chemically alter food properties(Hu, Li-Chan, Wan, Tian, & Pan, 2013).

The fundamental effects of high-intensity ultrasound on liquid sys-tems are primarily attributed to its ability to induce cavitation andmicrostreaming currents. During sonication, small gas bubbles areformed within the fluid that violently collapse, leading to extreme localtemperatures (up to 5000 K) and pressures (up to 1000 atm). The in-tense cavitation, turbulence, and shear stresses produced through thisprocess result in alterations of physicochemical properties and/orstructure and functional properties of proteins in solution without theuse of additives (chemical or biological) or excess heat (O'Sullivan,Park, Beevers, Greenwood, & Norton, 2017; Zhu et al., 2018). In ad-dition, high-intensity ultrasound is considered a technique of “greenfood processing”, as an alternative to conventional processing, pre-servation and extraction procedures (Chemat et al., 2017). Green foodprocessing may increase production efficiency and contribute to en-vironmental preservation by reducing the use of water and solvents,elimination of wastewater, fossil energy and generation of hazardoussubstances, as well as ensure a safe and high quality product (Chematet al., 2017).

Recent studies reporting the effects of ultrasound on the structuraland functional properties include those performed on walnuts (Zhuet al., 2018), faba beans (Martínez-Velasco et al., 2018), and sunflowerprotein isolates (Malik, Sharma, & Saini, 2017). However, the benefitsof implementing this technology depend on the inherent characteristicsof the protein source, the conditions of the ultrasound treatment, thepH, the temperature, the ionic strength, the time, and all of the vari-ables that have an effect on the physicochemical properties of proteins.Therefore, it is necessary to establish the particular conditions for eachtype of food (Higuera-Barraza, Del Toro-Sanchez, Ruiz-Cruz, &Márquez-Ríos, 2016).

Some physicochemical and functional properties of protein isolatesobtained from canola meals affected by different production conditionshave been recently reported. Kim, Varankovich, and Nickerson (2016)examined the gelation mechanism as a function of pH for a canolaprotein isolate in comparison with a commercial soy protein isolateproduct. Karaca, Low, and Nickerson (2011) studied the emulsifyingproperties of protein isolates produced by isoelectric precipitation andsalt extraction. Tan, Mailer, Blanchard, and Agboola (2011) comparedthe common alkaline extraction method to the Osborne method for theextraction of protein from meal derived from Australian canola species.

Tan, Mailer, Blanchard, Agboola, and Day (2014) determined the gel-ling properties of the protein fractions and protein isolate extractedfrom Australian canola meal. Kim, Varankovich, Stone, and Nickerson(2016) investigated the nature of the interactions involved during thegelation of a CPI using rheology and fractal imaging at a neutral pH as afunction of the protein concentration. Chang, Tu, Ghosh, and Nickerson(2015) evaluated the effect of the pH on the inter-relationships betweenthe physicochemical, interfacial and emulsifying properties for canolaprotein isolate. However, to the best of our knowledge, there are noreports on the effects of ultrasounds on the quality of CPI to be used as abetter ingredient in foods.

Therefore, the objectives of this study were to determine the che-mical, physicochemical, functional, digestibility and structural proper-ties of CPI as indicators of its potential use by the food industry.

2. Materials and methods

2.1. Materials

The canola meal (CM) used for the preparation of canola proteinisolate was provided by Forrajes y Alimentos San Cayetano, S. de R.L.de C.V. (Tepic, Nayarit, México). According to the AOAC (1990)methods, the crude protein (N × 6.25), moisture, ash, fat, and totalcarbohydrate contents of the CM were 39.72 ± 0.32%, 9.76 ± 0.14%,7.05 ± 0.04%, 2.96 ± 0.25%, and 31.74 ± 0.81%, respectively. Allchemicals that were used were of reagent grade and purchased fromSigma-Aldrich, Fermont and J.T. Baker (Ciudad de México, México).

2.2. Preparation of CPI and exposure to ultrasound

First, a study was conducted to determine the minimal and maximalpH (in the range of 2–12) of the protein extracted from the CM ac-cording to the method reported by Bernardino-Nicanor et al. (2013).Then, the protein isolate was obtained following the next procedure.Batches of CM suspensions were made by adding 1 part meal to 20 partsdistilled water, which was followed by mixing for 30min at roomtemperature (25 °C) by means of a propeller stirrer RW 28 basic (IKA,Staufen, Germany) at 800 rpm. The pH of the suspension was adjustedto the maximum value of the protein extraction with 1.0M NaOHduring mixing. The insoluble residue of the protein suspension wasseparated by filtration with a cotton cloth. Then, the pH of the proteinsuspension free of insoluble material was adjusted to the minimumvalue of the protein extraction with 1.0 M HCl, left 60min to allow theprotein to precipitate, and decanted. After that, centrifugation at8000×g for 10min at 4 °C followed. Finally, the protein precipitatewas re-suspended in distilled water in a ratio of 1:10 (precipitate:water) and adjusted to pH 7.0 with 1M NaOH. Each protein suspensionsample was placed in a 500mL beaker.

For the ultrasound treatment, a 40 kHz ultrasound bath (Branson,Model MTH-3510, 130W of power, a tank capacity of 5 L, and internaldimensions of 290× 150×150mm) was used. Two 500-mL glassbeakers containing the protein suspension samples were placed directlyinto the ultrasound bath, at the center of ultrasonic bath tank (indirectirradiation), to receive the ultrasound treatment at 25 °C for 15 or30min, and an additional control treatment without ultrasound wasprepared using a magnetic stirrer at 300 rpm. In this ultrasound ex-periment, the ultrasonic intensity was 1W/cm2, as measured by ca-lorimetry (Jambrak et al., 2014). The protein suspension samples sub-jected to the ultrasound and the control treatments were lyophilized ina FreeZone 12 L freeze dryer (Labconco, USA) to obtain the powdersamples of CPI, which were stored at room temperature in airtightcontainers until analysis.

N.T. Flores-Jiménez et al. Food Research International 121 (2019) 947–956

948

2.3. Composition and physicochemical properties

2.3.1. Analysis proximalThe moisture, crude protein (N×6.25), total carbohydrates and ash

contents were determined in triplicate according to the AOAC methods.The percentage of total carbohydrates was determined by the differ-ence.

2.3.2. Color analysis, water activity (aw) and bulk density (ρb)The color was determined using a Minolta CR-300 color meter

(Minolta Ltd., Co., Tokyo, Japan). The measured values were expressedaccording to the CIELAB color scale, where L*= lightness,+a*=redness, −a*=greenness, +b*=yellowness and–b*=blueness. The Ls∗, as∗, and bs∗ values of the white standard tile thatwas used as a reference were 94.43, 0.19 and 3.87, respectively. Totalcolor difference (ΔE) was calculated as follows:

= − + − + −∗ ∗ ∗ ∗ ∗ ∗Ε L L a a b bΔ ( ) ( ) ( )s s s

2 2 2 (1)

The aw was measured according to the powder samples of CPI usingan AquaLab 4TEV (Decagon Devices Inc., Pullman, WA, USA) wateractivity meter. ρb as determined using the method described byMonteiro and Prakash (1994) and was expressed as g/mL.

2.3.3. In vitro protein digestibility (IPD)The IPD was performed according to Hsu, Vavak, Satterlee, and

Miller (1977), with some modifications. The CPI suspensions (6.25 mg/mL) were prepared and adjusted at pH 8.0 using 0.1 M NaOH. A total of1.5 mL of a multienzyme solution was added to the CPI suspensions,which were mixed and incubated at 37 °C for 10min in a water bath,and the pH drop was measured. The multienzyme solution (pH 8.0) wasprepared with trypsin (1.6 mg/mL), chymotrypsin (3.1 mg/mL) andprotease (1.3 mg/mL). Casein was used as a control and the IPD wascalculated with the following equation:

= −IPD X(%) 210.46 18.10 f (2)

where Xf is the pH value of the solution after 10min of digestion withthe multienzyme solution.

2.4. Functional properties

2.4.1. Protein solubility profile of the CPIThe protein solubility profile of the CPI, as a function of pH, was

determined according to the method reported by Piornos et al. (2015).The protein content in the supernatant was determined by the Kjeldahlmethod. The solubilized protein was calculated and expressed as apercentage of the total protein.

2.4.2. Water absorption capacity (WAC) and oil absorption capacity(OAC)

The WAC was measured according to the method described byUlloa, Rosas-Ulloa, and Ulloa-Rangel (2011). The WAC was expressedas g water absorbed/g protein. The OAC was determined by the sameprocedure as for WAC by substituting water for canola oil (Aceites,Grasas y Derivados, S.A., Zapopan, Jalisco, México) and using 1 g ofCPI.

2.4.3. Emulsifying activity (EA) and emulsion stability (ES)A modified version of the method described by Ulloa et al. (2011)

was used to determine the EA and ES of CPI. Five suspensions wereprepared by dissolving 1 g of CPI in 15mL of water, and the pH valuesof the suspensions were adjusted to 2, 4, 6, 8 and 10 with 0.1 M HCl orNaOH. Subsequently, 15mL of canola oil was added to each suspension.Each mixture was stirred with an Ultra-Turrax T-25 homogenizer (IKAInstruments, Germany) at a speed of 12,000 rpm for 1min and cen-trifuged at 1200 × g for 5min. The volume of the emulsion layer was

recorded. The EA was calculated as follows:

= ×EAvolume of emulsified layer

volume of total volume in tube(%) 100

(3)

Additionally, to determine the emulsion stability, the samples wereheated at 80 °C for 30min in a water bath, cooled to 25 °C in runningwater and centrifuged as described above. The ES was expressed as thepercentage of the EA remaining after heating.

2.4.4. Least gelation concentration (LGC)The LGC was determined according to the method described by

Benelhadj, Gharsallaoui, Degraeve, Attia, and Ghorbel (2016). CPIsamples were mixed with distilled water in test tubes to obtain 2–20%protein concentrations, and the pH values of the suspensions were ad-justed to 2, 4, 6, 8 and 10 using 0.1 M HCl or NaOH. The test tubes wereheated for 1 h in a boiling water bath, cooled rapidly under running tapwater and further cooled for 2 h in a refrigerator at 4 °C. The LGC wasconsidered to be the lowest concentration at which the CPI sample inthe inverted test tube did not fall or slip.

2.4.5. Foaming propertiesTo determine the foaming properties, five suspensions were pre-

pared by dissolving 2 g of CPI in 100mL of distilled water, and the pHvalues of the suspensions were adjusted to 2, 4, 6, 8 and 10 using 0.1MHCl or NaOH. The suspensions were then whipped in an Ultra-Turrax T-25 homogenizer at a speed of 10,000 rpm for 1min at 25 °C. The re-sulting foam was poured into a 250-mL cylinder. The total foam volumewas recorded, and the foaming capacity was expressed as the percen-tage increase in the volume. The foam stability (FS) was determinedaccording to the method proposed by Kabirullah and Wills (1982). Thefoam volume was recorded 30min after whipping, and the FS wascalculated as follows:

= ×EAfoam volume after min

initial foam volume(%)

30100

(4)

2.5. Biochemical characterization

2.5.1. Protein fractionationProteins from the CPI were sequentially extracted at room tem-

perature in water, 0.5 M NaCl, 0.1M NaOH, and 70% ethanol based ontheir solubility according to the Osborne fractionation procedure asdescribed by Amza, Amadou, Balla, and Zhou (2015). The total proteincontent of each component (albumin, globulin, glutelin and prolamin)was measured using the Bradford (1976), and bovine serum albuminwas the standard.

2.5.2. Gel electrophoresisThe determination of the molecular weight distribution of the pro-

tein fractions from the CPI was conducted according to the methodreported by Laemmli (1970). The sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) test was carried out underreducing and nonreducing conditions using a buffer solution with andwithout 2-mercaptoethanol, respectively, on a gel slab comprised of12% separating gel and 4% stacking gel. Protein samples were in-cubated for 1 h at room temperature and heated at 95 °C for 5min.Aliquots of 25 μL containing 20 μg/mL of protein were loaded onto eachlane. Gels were run in a Miniprotean cell at 100 V. The gel was stainedwith coomassie brilliant blue G-250 (0.125% w/v), methanol (40% v/v), and acetic acid (7% v/v) and then destained with methanol (50% v/v) and acetic acid (10% v/v). A prestained standard (161–0373, Bio-Rad Laboratories Inc., Hercules, CA) was used as a protein molecularweight marker. The molecular weights of the samples were obtained bythe Quantity One 1-D analysis software (Bio-Rad Hercules, USA).


949

2.6. Structural characterization

2.6.1. Scanning electron microscopy (SEM)The microstructure of the lyophilized CPI samples was observed

with a SEM (SEC, Mini-SEM SNE-3200M, South Korea) at an accel-erating voltage of 30 kV. Before using the SEM, the samples were coatedwith gold using an ion sputter coater (MCM-100, SEC).

2.6.2. Surface hydrophobicity (H0)The H0 was determined using 1-anilino-8-naphthalene-sulfonate

(ANS) as a fluorescence probe as described by Jiang et al. (2014) withmodifications. The ultrasound-treated and control protein dispersions(1.5 mg/mL in 0.01M phosphate buffer at pH 9) were centrifuged at8000× g at 17 °C for 20min. After determining the protein con-centration in the supernatants, according to the method of Bradford(1976), each supernatant was serially diluted with the same buffer toobtain protein concentrations ranging from 0.05 to 0.001mg/mL. Then,25 μL of ANS (8.0mM in 0.01M phosphate buffer, pH 9.0) was added to2mL of sample. The fluorescence intensity (FI) was measured with afluorescence spectrophotometer (Tecan Infinite 200 Pro, Grödig, Aus-tria) at wavelengths of 364 nm (excitation) and 475 nm (emission). Theinitial slope of the FI versus the protein concentration (mg/mL) (cal-culated by linear regression analysis) was used as an index of the pro-tein H0.

2.6.3. Mean particle size (Dz) measurementThe particle sizes of CPI were determined using a laser particle size

analyzer (ZEN 3600, Malvern Instrument, UK). A total of 1mL of CPIsample (0.23 mg/mL) was transferred into the measuring cell. Theparticle size is reported as Dz, which is the intensity-weighted meanhydrodynamic size of the particles.

2.7. Statistical analysis

The results were reported as the mean ± standard deviation (SD) ofthe three determinations. The variance between the means was assessedby a One-Way ANOVA using the Statgraphics Centurion Software ver-sion XV (Statpoint Technologies, Inc. Virginia, USA). Significant dif-ferences (p < .05) between experiments were determined using theFisher's test.

3. Results and discussion

3.1. Protein extraction from CM

Fig. 1 shows the effects of the extracted pH on the protein solubilityfrom CM. According to the results, the maximal protein solubility was36.39% at pH 12, and the minimal protein solubility was 14.30% atpH 4, which was considered to be the isoelectric point. The results ofthe maximal of the protein extraction at pH 12 for the CM from thisstudy were lower than the results obtained by Akbari and Wu (2015),which had an extraction of 55% at pH 12, whereas the lowest proteinextraction was 25% at pH 4. Therefore, a pH of 12 was selected as thepH with the highest extraction yield and was used for the preparation ofthe CPI.

3.2. Physico-chemical properties

3.2.1. Proximate compositionThe protein, total carbohydrate, fat and ash contents of the CPI

exposed to the ultrasound for 15 and 30min were not significantly(p > .05) different with respect to the control treatment (Table 1).However, the moisture content of the CPI was reduced, decreasing from4.24% (control treatment) to 2.05% when it was exposed to the ultra-sound for 30min. The lower moisture content of such CPI is due to thehigher effects of the compressions and expansions induced by the sound

waves passing through the food medium, which increase the acousticdehydration (Awad, Moharram, Shaltout, Asker, & Youssef, 2012).

3.2.2. ColorColor is an important sensory parameter that is responsible for the

acceptability and marketability of food products (Nidhina &Muthukumar, 2015). The effects of the exposure time of the ultrasoundon the color of the CPI in comparison with the control treatment areshown in Table 2. The L* and ΔE* values of the CPI exposed to theultrasound for 15 and 30min increased significantly (p < .05), whilethe a* values decreased significantly (p < .05) in comparison with thecontrol treatment. In the case of the b* value, a significant (p < .05)decrease was observed in only the CPI exposed to ultrasound for30min. The effects of the ultrasound on the color in food depend on theintrinsic characteristics of the food and the ultrasound conditions (Bi,

Fig. 1. Protein solubility of canola meal as a function of pH. Error bars showstandard deviation.

Table 1Effect of ultrasound exposure time on the proximate composition of canolaprotein isolate.

Component (g/100 g) Ultrasound exposure time (min)

0 15 30

Protein (N x 6.25) 86.34 ± 0.49a 86.37 ± 0.36a 86.54 ± 0.12a

Moisture 4.24 ± 0.25a 3.76 ± 0.40a 2.05 ± 0.08b

Ash 6.78 ± 0.01ab 6.66 ± 0.04b 6.81 ± 0.07a

Fat 1.62 ± 0.01a 1.79 ± 0.41a 2.23 ± 0.34a

Total carbohydrates 1.02 ± 0.77a 1.42 ± 0.90a 2.36 ± 0.57a

The values are mean of triplicate± SD. Different superscripts in the same lineare significantly different by Fisher's test (p < .05).

Table 2Effect of ultrasound exposure time on the color parameters, water activity (aw)and bulk density (ρb) of the canola protein isolates.

Physical property Ultrasound exposure time (min)

0 15 30

ColorL* 51.27 ± 0.49a 49.55 ± 0.39b 49.03 ± 0.29b

a* 3.38 ± 0.06b 3.61 ± 0.09a 3.58 ± 0.07a

b* 15.17 ± 0.16a 14.64 ± 0.27ab 14.25 ± 0.35b

ΔE* 44.78 ± 0.43b 46.33 ± 0.35a 46.70 ± 0.20a

aw 0.24 ± 0.01a 0.21 ± 0.01b 0.12 ± 0.01c

ρb (g/cm3) 0.26 ± 0.01a 0.17 ± 0.02b 0.16 ± 0.01c

The values are mean of triplicate± SD. Different superscripts in the same lineare significantly different by Fisher's test (p < .05).


950

Hemar, Balaban, & Liao, 2015). Zhou, Zhang, Fang, and Liu (2015)reported that an ultrasound treatment on egg white protein decreasedthe L* value and increased the values of a* and b* because such atreatment induced an increase in the number of hydrophobic groups,thus resulting in an increase in the exposed riboflavin on the surface ofthe protein. Adekunte, Tiwari, Cullen, Scannell, and O'Donnell (2010)reported a decrease in the L*, a* and b* values of tomato juice aftersonication due to the degradation of lycopene, while Valero et al.(2007) found that ultrasound treatments had no significant effect on thecolor scoring in orange juice. On the other hand, Pingret, Fabiano-Tixier and Chemat (2013) reported the degradation or losses of otherscompounds (including pigments or antioxidants as anthocyanins, car-otenoids, ascorbic acid, and total phenolics content) by ultrasoundpretreatments, especially when a liquid medium during sonication wasapplied, suggesting as mechanisms of reactions the pyrolysis and oxi-dation by OH¯ radicals formed by cavitation. Recently Khadhraoui et al.(2018) proposed six types of mechanisms for the extraction of meta-bolites induced by ultrasound, which can be associated to color changesin plant tissues: erosion, shear forces, sonoporation, fragmentation,capillarity effect and detexturation; these mechanisms following eachother during ultrasound treatment, resulting in a gradual physical da-mage of tissues structure.

3.2.3. Bulk density (ρb)ρb depends on the interrelated factors such as the intensity of at-

tractive interparticle forces, the particle size and the number of contactpoints (Yuliana, Truong, Huynh, Ho, & Ju, 2014). The increase in theexposure time to the ultrasound significantly decreased (p < .05) theρb of the CPI (Table 2) with respect to the control treatment, whichcould be attributed to the formation of larger structures resulting fromthe influence of the ultrasound (Resendiz-Vazquez et al., 2017). Joshi,Adhikari, Aldred, Panozzo, and Kasapis (2011), Wani, Sogi, and Gill(2015), and Ogunbusola, Fagbemi, and Osundahunsi (2013) reportedprevious ρb values of 0.28 g/cm3, 0.17–0.21 g/cm3 and 0.21 g/cm3,respectively, in proteins from safflowers, beans, lentils and whitemelons.

3.2.4. Water activity (aw)aw describes the extent to which the water present in a food is

bound, and therefore available to participate in certain reactions, whichhas a major influence on microbial, chemical and physical stability offood (Amagliani, O'Regan, Kelly, & O'Mahony, 2016). The aw values forthe control and ultrasonicated CPI are presented in Table 2. The CPIexhibited a significant decrease (p < .05) in its aw due to the time ofexposure to the ultrasound, which went from an initial value of 0.24 forthe control treatment to 0.21 and 0.12 when the ultrasound treatmentswere 15min and 30min, respectively. As discussed previously, soundwaves passing through the food medium make the moisture removaleasier, which also influenced the reduction of the value of aw of theultrasound-treated CPI, as was observed in this study (Chemat, Zill-E-Huma, & Khan, 2011). However, the control treatment and the CPIexposed to the ultrasound had values of aw below the limit to ensuremicrobial stability because it is generally accepted that no microbialgrowth will occur at aw < 0.66 (Ulloa et al., 2015). Martins and Netto(2006) reported an aw value of 0.33 for whey protein isolate, whileAmagliani et al. (2016) reported 0.23 for rice protein concentrate.

3.2.5. Digestibility of proteinsThe digestibility of proteins is an important parameter to evaluate

their nutritional quality. No significant (p > .05) difference in IPD wasobserved between the unexposed and exposed CPI to ultrasound, aswell as to the casein used as a reference (Table 3). According toSullivan, Pangloli, and Dia (2018), the ultrasonication led to an im-provement in sorghum gluten-like flour digestibility by altering thesecondary structure of kafirin, which is the most abundant protein insorghum. On the other hand, the study by Martínez-Velasco et al.

(2018) revealed a decrease (∼3.6%) in the relative protein digestibilityof faba bean protein isolate due to the structural changes caused byultrasonication. The IPD values for other protein isolates have beenreported as follows: quinoa 78.37% (Elsohaimy, Refaay, & Zaytoun,2015), chia 49.4% (Olivos-Lugo, Valdivia-López, & Tecante, 2010), andL. clymenun 95.00% (Pastor-Cavada, Juan, Pastor, Alaiz, & Vioque,2010). Therefore, the results of the IPD of the CPI in this study werehigher than that of chia and quinoa protein isolates but lower than thatof L. clymenun protein isolate.

3.3. Functional properties

3.3.1. Protein solubility profile of CPIAll samples of CPI exhibited the typical bell-shaped curves, with the

minimum protein solubilities of 13.75%, 16.26% and 17.5% for thecontrol treatment, and the CPI exposed to ultrasound for 15min and30min, respectively, at approximately their isoelectric point (pH 4),and the maximum protein solubilities were 74.32%, 77.36, and77.96%, respectively, at pH 12 (Fig. 2). A significant increase (p < .05)in the protein solubility of the CPI exposed for 30min to the ultrasoundin the 6–8 pH range with respect to control treatment was observed,and it reached up to 10%. The increase in the protein solubility is due tothe protein molecules partially unfolding due to the ultrasound effect,which increases the interaction between the proteins and the watermolecules too (Holmberg et al., 2007; Resendiz-Vazquez et al., 2017).In other studies with black bean (Jiang et al., 2014) and soy (Karkiet al., 2009) protein isolates and milk protein concentrate (Yanjunet al., 2014), it was reported that the ultrasound treatment increasedthe protein solubility, which is in agreement with the results obtainedin this study.

3.3.2. Water absorption capacity (WAC) and oil absorption capacity(OAC)

Interactions of water and oil with proteins are very important in

Table 3Effect of ultrasound exposure time on in vitro protein digestibility (IPD)of canola protein isolate.

Ultrasound exposure time (min) IPD (%)

0 83.92 ± 2.05a

15 85.07 ± 1.35a

30 84.22 ± 1.37a

Caseín 88.04 ± 3.53ª

The values are mean of triplicate± SD. Means with different super-scripts are significantly different by Fisher's test (p < .05).

Fig. 2. Protein solubility of CPI as a function of pH at different ultrasoundexposure times. Error bars show standard deviation.


951

food systems because of their effects on the flavors and textures of foods(Elsohaimy et al., 2015). The WAC is an index of the ability of proteinsto absorb and retain water. The results of the WAC of the CPI exposed tothe ultrasound are shown in Table 4. The WAC decreased from 2.93 g/g(control treatment) to 2.72 g/g and 2.52 g/g for the CPI exposed to theultrasound for 15 and 30min, respectively. This decrease in the WAC isbecause the ultrasound treatment might denature the molecular struc-ture of the protein and causes an increase in the hydrophobic surface ofthe CPI, which can lead to low levels of WAC (Resendiz-Vazquez et al.,2017). In contrast, the CPI exposed to the ultrasound increased the OACin comparison with the control treatment from a value of 2.84 g/g to3.06 g/g and 3.26 g/g for the CPI treated with the ultrasound for 15minand 30min, respectively. Such an effect might be attributed to the ex-posure of nonpolar residue side chains after the ultrasound that interactwith hydrocarbon chains in fat molecules, thus resulting in better oiladsorption (Higuera-Barraza et al., 2016). The same behavior of theWAC and OAC from the ultrasound was observed in sunflower (Maliket al., 2017) protein isolate.

3.3.3. Emulsifying activity (EA) and emulsion stability (ES)The emulsifying properties express the interfacial area stabilized per

unit weight of a protein, thus characterizing the ability of a protein toabsorb to the oil-water interface. (Zhang et al., 2014). Table 5 showsthe effects of the ultrasound time on the EA and ES values of CPI. Theultrasound treatment for 30min at the pH values of 4, 8 and 10 sig-nificantly (p < .05) increased the EA of the CPI, as well as the ES at thepH values of 6 and 10, in comparison with the control treatment.However, the most notable effect of the ultrasound treatment on EA wasat pH 4, where it increased from 0 (control treatment) to 33% and41.6% for the ultrasound exposure times of 15min and 30min, re-spectively. The partial denaturation and formation of a more disorderedstructure could provide the protein with a better ability to adsorb at theoil-water interface to lead to an increase in EA (Jambrak, Lelas, Mason,

Krešić, & Badanjak, 2009). On the other hand, the highest increases ofES were observed at pH 10 for the CPI treated with the ultrasound for15 and 30min (51.9% and 55.7%, respectively) compared with the ESof the CPI without ultrasonication (46.7%) (Table 5). The increase in EScan be explained by the more favorable orientation of proteins resultingfrom the influence of the turbulent behavior produced by the ultra-sound and the integration of oil bubbles in the emulsion (Yanjun et al.,2014). Resendiz-Vazquez et al. (2017) and Zhang et al. (2014) reportedincreases in EA and ES due to the ultrasound on jackfruit seed andpeanut protein isolates, respectively. The results in Table 5 were inagreement with the observations of H0. Ultrasound-treated proteinsusually exhibit a high surface hydrophobicity, which enhances the EA,ES and OAC by exposing the hydrophobic groups (Yanjun et al., 2014).

3.3.4. Least gelation concentration (LGC)Gelation properties are attributed to the partial denaturation of

protein that allows for exposing functional groups with which proteinsinteract and generate the three-dimensional gel structure (Piornoset al., 2015). Table 5 shows the results of LGC for the CPI at different pHvalues (2−10). In comparison with the control treatment, only theultrasound exposure time of 30min at the pH values of 6 and 8 de-creased the LGC of the CPI from 12 g/100 g to 10 g/100 g and 14 g/100 g to 12 g/100 g, respectively. After the heating process, the partialdenaturation of protein allows for exposing functional groups withwhich proteins interact and generate the three-dimensional gel struc-ture. Thus, significant amounts of water are retained in the gel struc-ture, thus transforming the liquid sample to solid. According toResendiz-Vazquez et al. (2017), the partial denaturation of proteins byultrasound, which increased hydrophobic regions, improves the gela-tion properties of proteins. In contrast with other studies with chickpea(Kaur & Singh, 2007) and lupin protein (Piornos et al., 2015) isolates,where the LGC were 14–18% and 14–20%, respectively, the results ofLGC for the CPI exposed for 30min to the ultrasound at pH values of 6and 8 of this study were better (10–12 g/100 g) to those commentedabove.

3.3.5. Foaming propertiesThe effects of ultrasound exposure time at different pH values on the

foam capacity (FC) and foam stability (FS) of the CPI are shown inTable 5. The FC of the CPI exposed to the ultrasound for 30min at thepH values of 4, 6, and 8 significantly increased (p < .05) from173.13%, 190.33% and 234.68% to 177.46%, 198.36% and 239.20%,in comparison with the control treatment, respectively. On the otherhand, the FS of the CPI with ultrasound treatment for 30min at the pHvalues of 6 and 8 significantly (p < .05) increased from 64.25% and

Table 4Effect of ultrasound exposure time on water absorption capacity (WAC) and oilabsorption capacity (OAC) of canola protein isolate.

Property Ultrasound exposure time (min)

0 15 30

WAC, (g H2O/g protein) 2.93 ± 0.03a 2.72 ± 0.04b 2.52 ± 0.02c

OAC, (g oil/g protein) 2.84 ± 0.08c 3.06 ± 0.15b 3.26 ± 0.07a

The values are mean of triplicate± SD. Means with different superscripts in thesame line are significantly different by Fisher's test (p < .05).

Table 5Effect of ultrasound exposure time at different pH values on the emulsifying activity (EA), emulsifying stability (ES), foaming capacity (FC), foaming stability (FS) andleast gelation concentration (LGC) of canola protein isolates.

Ultrasound exposure time (min) pH EA (%) ES (%) FC (%) FS (%) LGC (g/100mL)

0 2 43.1 ± 1.5a 37.5 ± 0.4a 220.1 ± 1.2a 68.1 ± 0.5a 1215 2 41.8 ± 1.9a 36.2 ± 0.5b 220.7 ± 2.7a 68.7 ± 0.7a 1230 2 41.5 ± 0.8a 36.9 ± 0.9ab 219.7 ± 1.3a 67.9 ± 0.6a 120 4 ND ND 173.1 ± 1.1b 84.8 ± 0.5a 1615 4 32.9 ± 0.6b ND 175.4 ± 1.8ab 84.9 ± 0.1ª 1630 4 41.6 ± 1.9a ND 177.5 ± 1.2a 85.3 ± 0.1a 160 6 30.9 ± 2.4a 23.1 ± 0.9b 190.3 ± 2.7b 64.3 ± 0.7b 1215 6 30.3 ± 2.7a 23.4 ± 0.5b 192.6 ± 0.8b 65.2 ± 0.6b 1230 6 33.3 ± 1.0a 25.4 ± 0.6a 198.4 ± 1.4a 68.2 ± 0.6a 100 8 37.8 ± 1.0b 37.9 ± 0.8b 234.7 ± 2.4b 59.9 ± 0.5b 1415 8 39.6 ± 0.7b 39.5 ± 0.9a 235.8 ± 2.5b 60.2 ± 0.6a 1430 8 43.0 ± 0.9a 37.5 ± 0.7b 239.2 ± 1.1a 63.6 ± 0.4a 120 10 44.6 ± 0.7b 46.7 ± 0.1c 244.3 ± 0.9ª 74.0 ± 0.5a 1615 10 45.8 ± 1.0b 51.8 ± 0.8b 245.7 ± 1.1a 74.5 ± 0.5a 1630 10 55.5 ± 1.0a 55.7 ± 0.8a 246.4 ± 1.3a 74.4 ± 0.7a 16

The values are mean of triplicate± SD. Means with different superscripts for each functional property at same pH are significantly different by Fisher's test(p < .05). ND is not detected.


952

59.94% to 68.15% and 63.56%, in comparison with the control treat-ment, respectively (Table 5). The foaming capacity depends on thediffusion of the protein at the air-water interface due to the unfolding ofits structure, while the foaming stability is dependent on the formationof a thick cohesive layer around the air bubble (Khan et al., 2011). Theincreases in foaming properties might be explained by the additionalpartial denaturation by ultrasound, which generates a more flexibleprotein structure in aqueous solutions and interacts strongly at the air-water interface. Studies with pea protein (Xiong et al., 2018) and fababean (Martínez-Velasco et al., 2018) protein isolates showed the en-hancement of the foaming properties by ultrasound.

3.4. Biochemical characterization

3.4.1. CPI protein fractionsThe Osborne solubility-based protein fractionation data indicated

that the NaOH-soluble (glutelin) and water-soluble (albumin) proteinswere the predominant fractions in CPI, thus accounting for 57.18% and23.09%, respectively. Meanwhile, only 18.54% of the NaCl solublefraction (globulin) and 1.17% of the ethanol-soluble fraction (prolamin)were determined. The protein fraction distribution found in otherprotein isolates from fruit seeds as seinat (Cucumis melo var. tibish) wascomprised of 38.6% glutelins, followed by 34.5% albumins, 24.0%globulins and 2.7% prolamins (Siddeeg, Xu, Jiang, & Xia, 2015).Meanwhile, in the gingerbread plum, the glutelin, albumin, globulinand prolamin fractions were 40.6%, 27.6%, 25.8% and 64.8%, re-spectively (Amza et al., 2015). The protein fraction content in theprotein isolates depends of the protein fraction distribution of theprotein source and the procedure to obtain the protein isolate (Zhaoet al., 2012).

3.4.2. Molecular weight distributionThe electrophoretic profiles obtained by SDS-PAGE for the proteins

of the control treatment and the CPI exposed to ultrasound for 15 and30min under both the reducing and nonreducing conditions are shownin Fig. 3. Under the nonreducing conditions (−ME), the distribution ofthe molecular weights of the proteins of all CPIs consisted of five

fractions main with the following molecular weights: 50.90 kDa,30.15 kDa, 26.86 kDa, 19.20 kDa and 17.90 kDa.

However, under reducing conditions, the band at 50.90 kDa wasdissociated into its two polypeptides (~20 kDa and ~30 kDa), as re-ported by Akbari and Wu (2015). Therefore, the results of this studyconfirmed that the ultrasound did not cause the hydrolysis of the pro-teins of the CPI, which is in agreement with the results reported by Zhuet al. (2018) for walnut protein isolate and Higuera-Barraza et al.(2017) for squid mantle proteins. In contrast, Jambrak et al. (2014) andResendiz-Vazquez et al. (2017) observed that the ultrasound treatmentinduced a reduction in the molecular weight of the whey and jackfruitseed protein isolates, which suggests that the reduction in the molecularweight may depend on the protein type and sonication conditions.

3.5. Structural characteristics

3.5.1. Microstructure of the CPIFig. 4 show the microstructure of the CPI exposed to the ultrasound

for 15 and 30min and then freeze dried in comparison with the controltreatment, which exhibited more disordered structures and irregularfragments. According to Jiang et al. (2014), the application of ultra-sound induces an increase in the charges and the exposure of freesulfhydryl groups and hydrophobic groups, which could interact witheach other and form larger aggregates during freeze drying, as well asalso affect some functional properties such as the as foaming capacityand emulsifying properties, as happened in this study. Hu, Wu, et al.(2013) found that even though the aggregates of ultrasonic treatedsamples in dry states are larger, the aggregates in the dispersions aresmaller, which was the behavior that was observed in this study(Fig. 6).

3.5.2. Surface hydrophobicity (H0)Protein surface hydrophobicity (H0) is an index of the number of

hydrophobic groups on the surface of a protein molecule (Chen, Chen,Ren, & Zhao, 2011). Due to the macromolecular structure of proteins,the surface hydrophobicity has more influence on the functionalproperties than the total hydrophobicity (Chandrapala, Zisu, Palmer,Kentish, & Ashokkumar, 2011). As seen from Fig. 5, the CPI exposed tothe ultrasound for 30min significantly increased (p < .05) the H0 of17.6% with respect to the control treatment. Previous studies showedthat the ultrasonic treatment increased the surface hydrophobicity forsoy (Hu, Wu, et al., 2013), peanut (Zhang et al., 2014) and black bean(Jiang et al., 2014) protein isolates. The increase in H0 can be explainedby the effect of cavitation phenomenon produced by ultrasonic treat-ment, which induces a certain degree of molecular unfolding of theproteins, thereby causing an increase in the number of hydrophobicgroups and regions that are originally inside the molecules to becomeexposed to the polar surrounding environment (Hu, Li-Chan, et al.,2013).

3.5.3. Dz of proteinThe particle size of proteins is one of the factors that influences the

function of proteins (Hu, Cheung, Pan, & Li-Chan, 2015). The effect ofthe ultrasound exposure time on the Dz of the CPI is shown in Fig. 6.The ultrasound treatment for 30min reduced the Dz of CPI from 249.4to 219.4 nm. The size reduction is probably due to protein dissociationduring cavitation in which particles under sonication are violentlyagitated, thus resulting in broken aggregates particles (Jiang et al.,2014). Yanjun et al. (2014) and Zhang et al. (2014) found that ultra-sound reduces the particle size of proteins of milk protein concentrateand peanut protein isolate, respectively, which is in agreement with theresults of this study.

4. Conclusions

In this work, the effects of high-intensity ultrasound exposure time

Fig. 3. SDS-PAGE electrophoretic profiles of CPI: Lane M, molecular weightstandard (15–250 kDa); Lane 1, untreated CPI; Lane 2, ultrasound exposed CPIfor 15min; Lane 4, ultrasound exposed CPI for 30min in non-reducing (Lane1–3) and reducing (Lane 4–6) conditions.


953

on the compositional, physicochemical, biochemical, functional andstructural properties of canola protein isolate was investigated. Theultrasound was found to have a major influence on the structural, so-lubility, gelation and emulsifying properties. In particular, the ultra-sound treatment decreased the moisture content, aw, ρb, Dz, and LGC (atpH 6–8), but increased the solubility, OAC, H0, and the number of largeaggregates, and improved the emulsifying activity (at pH 4) of the ca-nola proteins. These effects are attributed to the ability of the high-intensity ultrasonic waves to alter the physical bonds between andwithin the globular protein molecules, thereby leading to some un-folding. These results may be important for increasing the potential

industrial application of canola proteins as natural plant-based func-tional ingredients in food and beverage proteins.

Acknowledgements

The authors would like to acknowledge the National Council forScience and Technology of México for the scholarship (443046) for IBQ.Nitzia Thalía Flores Jiménez and the Patronage to Administer theSpecial Tax Destined to the Autonomous University of Nayarit forproviding research funds (PUAN-CP-001/2018).

Fig. 4. Microstructure of untreated (A) and ultrasound exposed CPI for 15min (B) and 30min (C) determined by SEM.

Fig. 5. Changes in surface hydrophobicity (H0) of untreated and ultrasoundexposed CPI. Different letters indicate significant differences at p < .05. Errorbars show standard deviation.

Fig. 6. Effect of ultrasound exposure time on Dz of the CPI. Different lettersindicate significant differences at p < .05. Error bars show standard deviation.


954

References

Adekunte, A. O., Tiwari, B. K., Cullen, P. J., Scannell, A. G. M., & O'Donnell, C. P. (2010).Effect of sonication on colour, ascorbic acid and yeast inactivation in tomato juice.Food Chemistry, 122, 500–507.

Akbari, A., & Wu, J. (2015). An integrated method of isolating napin and cruciferin fromdefatted canola meal. Food Science and Technology, 64, 308–315.

Amagliani, L., O'Regan, J., Kelly, A. L., & O'Mahony, J. A. (2016). Physical and flowproperties of rice protein powders. Journal of Food Engineering, 190, 1–9.

Amza, T., Amadou, I., Balla, A., & Zhou, H. (2015). Antioxidant capacity of hydrolyzedprotein fractions obtained from an under-explored seed protein: Gingerbread plum(Neocarya macrophylla). Journal of Food Science and Technology, 52, 2770–2778.

AOAC, Association of Official Analytical Chemists (1990). Official methods of analysis(15th ed.). (Washington: Arlington).

Awad, T. S., Moharram, H. A., Shaltout, O. E., Asker, D., & Youssef, M. M. (2012).Applications of ultrasound in analysis, processing and quality control of food: A re-view. Food Research International, 48, 410–427.

Benelhadj, S., Gharsallaoui, A., Degraeve, P., Attia, H., & Ghorbel, D. (2016). Effect of pHon the functional properties of Arthrospira (Spirulina) platensis protein isolate. FoodChemistry, 194, 1056–1063.

Bernardino-Nicanor, A., Bravo-Delgado, C. H., Vivar-Vera, G., Martínez-Sánchez, C. E.,Pérez-Silva, A., Rodríguez-Miranda, J., & Vivar-Vera, M. A. (2013). Preparation,composition, and functional properties of a protein isolate from a defatted mameysapote (Pouteria sapota) seed meal. Cyta-Journal of Food, 12, 176–182.

Bi, X., Hemar, Y., Balaban, M. O., & Liao, X. (2015). The effect of ultrasound on particlesize, color, viscosity and polyphenol oxidase activity of diluted avocado puree.Ultrasonics Sonochemistry, 27, 567–575.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein–dye binding. AnalyticalBiochemistry, 72, 248–254.

Chandrapala, J., Zisu, B., Palmer, M., Kentish, S., & Ashokkumar, M. (2011). Effects ofultrasound on the thermal and structural characteristics of proteins in reconstitutedwhey protein concentrate. Ultrasonics Sonochemistry, 18, 951–957.

Chang, C., Tu, S., Ghosh, S., & Nickerson, M. T. (2015). Effect of pH on the inter-re-lationships between the physicochemical, interfacial and emulsifying properties forpea, soy, lentil and canola protein isolates. Food Research International, 77, 360–367.

Chang, P. G., Gupta, R., Timilsena, Y. P., & Adhikari, B. (2016). Optimisation of thecomplex coacervation between canola protein isolate and chitosan. Journal of FoodEngineering, 191, 58–66.

Chemat, F., Rombaut, N., Meullemiestre, A., Turk, M., Perino, S., Fabiano-Tixier, A., &Albert-Vian, M. (2017). Review of green food processing techniques. Preservation,transformation, and extraction. Innovative Food Science and Emerging Technologies, 41,357–377.

Chemat, F., Rombaut, N., Sicaire, A., Meullemiestre, A., Fabiano-Tixier, A., & Albert-Vian,M. (2017). Ultrasound assisted extraction of food and natural products. Mechanisms,techniques, combinations, protocols and applications. A review. UltrasonicsSonochemistry, 34, 540–560.

Chemat, F., Zill-e-Huma, & Khan, M. K. (2011). Applications of ultrasound in foodtechnology: Processing, preservation and extraction. Ultrasonics Sonochemistry, 18,813–835.

Chen, L., Chen, J., Ren, J., & Zhao, M. (2011). Effects of ultrasound pretreatment on theenzymatic hydrolysis of soy protein isolates and on the emulsifying properties ofhydrolysates. Journal of Agricultural and Food Chemistry, 59, 2600–2609.

Elsohaimy, S. A., Refaay, T. M., & Zaytoun, M. A. M. (2015). Physicochemical andfunctional properties of quinoa protein isolate. Annals of Agricultural Science, 60,297–305.

Food and Agriculture Organization of the United Nations (2018). FAO statistical databases.Rome: FAO/United Nations. Available online at http://faostat.fao.org, Accessed date:April 2018.

Gerzhova, A., Mondor, M., Benali, M., & Aider, M. (2015). A comparative study betweenthe electro-activation technique and conventional extraction method on the ex-tractability, composition and physicochemical properties of canola protein con-centrates and isolates. Food Bioscience, 11, 56–71.

Higuera-Barraza, O. A., Del Toro-Sanchez, C. L., Ruiz-Cruz, S., & Márquez-Ríos, E. (2016).Effects of high-energy ultrasound on the functional properties of proteins. UltrasonicsSonochemistry, 31, 558–562.

Higuera-Barraza, O. A., Torres-Arreola, W., Ezquerra-Brauer, J. M., Cinco-Moroyoqui, F.J., Rodríguez Figueroa, J. C., & Marquez-Ríos, E. (2017). Effect of pulsed ultrasoundon the physicochemical characteristics and emulsifying properties of squid (Dosidicusgigas) mantle proteins. Ultrasonics Sonochemistry, 38, 829–834.

Holmberg, M., Stibius, K. B., Ndoni, S., Larsen, N. B., Kingshott, P., & Hou, X. L. (2007).Protein aggregation and degradation during iodine labeling and its consequences forprotein adsorption to biomaterials. Analytical Biochemistry, 36, 120–125.

Hsu, H. W., Vavak, D. L., Satterlee, L. D., & Miller, G. A. (1977). A multienzyme techniquefor estimating protein. Journal of Food Science, 42, 1269–1273.

Hu, H., Cheung, I. W. Y., Pan, S., & Li-Chan, E. C. Y. (2015). Effect of high intensityultrasound on physicochemical and functional properties of aggregated soybeanβconglycinin and glycinin. Food Hydrocolloids, 45, 102–110.

Hu, H., Li-Chan, E. C. Y., Wan, L., Tian, M., & Pan, S. (2013). The effect of high intensityultrasonic pre-treatment on the properties of soybean protein isolate gel induced bycalcium sulfate. Food Hydrocolloids, 32, 303–311.

Hu, H., Wu, J., Li-Chan, E. C. Y., Zhu, L., Zhang, F., Xu, X., ... Pan, S. (2013). Effects ofultrasound on structural and physical properties of soy protein isolate (SPI) disper-sions. Food Hydrocolloids, 30, 647–655.

Jambrak, A. R., Lelas, V., Mason, T. J., Krešić, G., & Badanjak, M. (2009). Physical

properties of ultrasound treated soy proteins. Journal of Food Engineering, 93,386–393.

Jambrak, A. R., Mason, T. J., Lelas, V., Paniwnyk, L., & Herceg, Z. (2014). Effect of ul-trasound treatment on particle size and molecular weight of whey proteins. Journal ofFood Engineering, 121, 15–23.

Jayaraman, B., MaIsaac, J., & Anderson, D. (2016). Effects of derived meals from juncea(Brassica juncea), yellow and black seeded canola (Brassica napus) and multi-carbohydrase enzymes supplementation on apparent metabolizable energy in broilerchickens. Animal Nutrition, 2, 154–159.

Jiang, L., Wang, J., Li, Y., Wang, Z., Liang, J., Wang, R., ... Zhang, M. (2014). Effects ofultrasound on the structure and physical properties of black bean protein isolates.Food Research International, 62, 595–601.

Joshi, M., Adhikari, B., Aldred, P., Panozzo, J. F., & Kasapis, S. (2011). Physicochemicaland functional properties of lentil protein isolates prepared by different dryingmethods. Food Chemistry, 129, 1513–1522.

Kabirullah, M., & Wills, R. B. H. (1982). Functional properties of acetylated and succi-nylated sunflower protein isolate. International Journal of Food Science and Technology,17, 235–239.

Karaca, A. C., Low, N., & Nickerson, M. (2011). Emulsifying properties of canola andflaxseed protein isolates produced by isoelectric precipitation and salt extraction.Food Research International, 44, 2991–2998.

Karki, B., Lamsal, B. P., Grewell, D., Pometto, A. L., Van Leeuwen, J., Khanal, S. K., &Jung, S. (2009). Functional properties of soy protein isolates produced from ultra-sonicated defatted soy flakes. Journal of the American Oil Chemists' Society, 86,1021–1028.

Kaur, M., & Singh, N. (2007). Characterization of protein isolates from different Indianchickpea (Cicer arietinum L.) cultivars. Food Chemistry, 102, 366–374.

Khadhraoui, B., Turk, M., Fabiano-Tixier, A. S., Petitcolas, E., Robinet, P., Imber, R., ...Chemat, F. (2018). Histo-cytochemistry and scanning electron microscopy forstudying spatial and temporal extraction of metabolites induced by ultrasound.Towards chain detexturation mechanisms. Ultrasounds Sonochemistry, 42, 482–492.

Khan, S. H., Butt, M. S., Sharif, M. K., Sameen, A., Mumtaz, S., & Sultan, M. T. (2011).Functional properties of protein isolates extracted from stabilized rice bran by mi-crowave, dry heat, and parboiling. Journal of Agricultural and Food Chemistry, 59,2416–2420.

Kim, J. H., Varankovich, N. V., Stone, A. K., & Nickerson, M. T. (2016). Nature of protein-protein interactions during the gelation of canola protein isolate networks. FoodResearch International, 89, 408–414.

Kim, J. H. J., Varankovich, N. V., & Nickerson, M. T. (2016). The effect of pH on thegelling behaviour of canola and soy protein isolates. Food Research International, 81,31–38.

Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head of bac-teriophage-T4. Nature, 227, 680–685.

Malik, M. A., Sharma, H. K., & Saini, C. S. (2017). High intensity ultrasound treatment ofprotein isolate extracted from dephenolized sunflower meal: Effect on physico-chemical and functional properties. Ultrasonics Sonochemistry, 39, 511–519.

Martínez-Velasco, A., Lobato-Calleros, C., Hernández-Rodríguez, B. E., Román-Guerrero,A., Alvarez-Ramirez, J., & Vernon-Carter, E. J. (2018). High intensity ultrasoundtreatment of faba bean (Vicia faba L.) protein: Effect on surface properties, foamingability and structural changes. Ultrasonics Sonochemistry, 44, 97–105.

Martins, V. B., & Netto, F. M. (2006). Physicochemical and functional properties of soyprotein isolate as a function of water activity and storage. Food Research International,39, 145–153.

Monteiro, P. V., & Prakash, V. (1994). Functional properties of homogeneous proteinfractions from peanut (Arachis hypogaea L.). Journal of Agriculture and Food Chemistry,42, 274–278.

Nidhina, N., & Muthukumar, S. P. (2015). Antinutritional factors and functionality ofprotein-rich fractions of industrial guar meal as affected by heat processing. FoodChemistry, 173, 920–926.

Ogunbusola, E. M., Fagbemi, T. N., & Osundahunsi, O. F. (2013). In-vitro protein di-gestibility, amino acid profile, functional properties and utilization of white melon(Cucumeropsis mannii) protein isolates. African Journal of Food Science and Technology,4, 153–159.

Olivos-Lugo, B. L., Valdivia-López, M.Á., & Tecante, A. (2010). Thermal and physico-chemical properties and nutritional value of the protein fraction of Mexican chia seed(Salvia hispanica L.). Food Science and Technology International, 16, 89–96.

O'Sullivan, J., Murray, B., Flynn, C., & Norton, I. (2016). The effect of ultrasound treat-ment on the structural, physical and emulsifying properties of animal and vegetableproteins. Food Hydrocolloids, 53, 141–154.

O'Sullivan, J. J., Park, M., Beevers, J., Greenwood, R. W., & Norton, I. T. (2017).Applications of ultrasound for the functional modification of proteins and nanoe-mulsion formation: A review. Food Hydrocolloids, 71, 229–310.

Ozuna, C., Paniagua-Martínez, I., Castaño-Tostado, E., Ozimek, L., & Amaya-Llano, S. L.(2015). Innovative applications of high-intensity ultrasound in the development offunctional food ingredients: Production of protein hydrolysates and bioactive pep-tides. Food Research International, 77, 685–696.

Pastor-Cavada, E., Juan, R., Pastor, J. E., Alaiz, M., & Vioque, J. (2010). Protein isolatesfrom two Mediterranean legumes: Lathyrus clymenum and Lathyrus annuus. Chemicalcomposition, functional properties and protein characterisation. Food Chemistry, 122,533–538.

Pingret, D., Fabiano-Tixier, A., & Chemat, F. (2013). Degradation during application ofultrasound in food processing: A review. Food Control, 31, 593–606.

Piornos, J. A., Burgos-Díaz, C., Ogura, T., Morales, E., Rubilar, M., Maureira-Butler, I., &Salvo-Garrido, H. (2015). Functional and physicochemical properties of a proteinisolate from AluProt-CGNA: A novel protein-rich lupin variety (Lupinus luteus). FoodResearch International, 76, 719–724.


955

http://refhub.elsevier.com/S0963-9969(19)30025-0/rf0005






















































http://faostat.fao.org

















































































































Pirestani, S., Nasirpour, A., Keramat, J., & Desobry, S. (2017). Preparation of chemicallymodified canola protein isolate with gum Arabic by means of Maillard reaction underwet-heating conditions. Carbohydrate Polymers, 155, 201–207.

Resendiz-Vazquez, J. A., Ulloa, J. A., Urías-Silvas, J. E., Bautista-Rosales, P. U., Ramírez-Ramírez, J. C., Rosas-Ulloa, P., & González-Torres, L. (2017). Effect of high-intensityultrasound on the technofunctional properties and structure of jackfruit (Artocarpusheterophyllus) seed protein isolate. Ultrasonics Sonochemistry, 37, 436–444.

SAGARPA. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación(México) (2017). Oleaginosas canola, cártamo, girasol, soya mexicanas. PlaneaciónAgrícola Nacional, 2017–2030. https://www.gob.mx/cms/uploads/attachment/file/.../B_sico-Oleaginosas-parte_una.pdf, Accessed date: 17 March 2018.

Siddeeg, A., Xu, Y., Jiang, Q., & Xia, W. (2015). In vitro antioxidant activity of proteinfractions extracted from seinat (Cucumis melo var. tibish) seeds. Cyta-Journal of Food,13, 472–481.

Sullivan, A. C., Pangloli, P., & Dia, V. P. (2018). Impact of ultrasonication on the phy-sicochemical properties of sorghum kafirin and in vitro pepsin-pancreatin digestibilityof sorghum gluten-like flour. Food Chemistry, 240, 1121–1130.

Tan, S. H., Mailer, R. J., Blanchard, C. L., & Agboola, S. O. (2011). Extraction andcharacterization of protein fractions from Australian canola meals. Food ResearchInternational, 44, 1075–1082.

Tan, S. H., Mailer, R. J., Blanchard, C. L., Agboola, S. O., & Day, L. (2014). Gellingproperties of protein fractions and protein isolate extracted from Australian canolameal. Food Research International, 62, 819–828.

Ulloa, J. A., Ibarra-Zavala, S. J., Ramírez-Salas, S. P., Rosas-Ulloa, P., Ramírez-Ramírez, J.C., & Ulloa-Rangel, B. E. (2015). Chemical, physicochemical, nutritional, micro-biological, sensory and rehydration characteristics of instant whole beans (Phaseolusvulgaris). Food Technology and Biotechnology, 53, 48–56.

Ulloa, J. A., Rosas-Ulloa, P., & Ulloa-Rangel, B. E. (2011). Physicochemical and functionalproperties of a protein isolate produced from safflower (Carthamus tinctorius L.) mealby ultrafiltration. Journal of the Science of Food and Agriculture, 91, 572–577.

Valero, M., Recrosio, N., Saura, D., Muñoz, N., Martí, N., & Lizama, V. (2007). Effects of

ultrasonic treatments in orange juice processing. Journal of Food Engineering, 80,509–516.

Wanasundara, J. P. D., McIntosh, T. C., Perera, S. P., Withana-Gamage, T. S., & Mitra, P.(2016). Canola/rapeseed protein-functionality and Nutrition. Oilseeds & Fats Cropsand Lipids, 23, 1–15.

Wani, I. A., Sogi, D. S., & Gill, B. S. (2015). Physico-chemical and functional properties ofnative and hydrolysed protein isolates from Indian black gram (Phaseolus mungo L.)cultivars. LWT - Food Science and Technology, 60, 848–854.

Xiong, T., Xiong, W., Ge, M., Junhao Xia, J., Li, B., & Chen, Y. (2018). Effect of highintensity ultrasound on structure and foaming properties of pea protein isolate. FoodResearch International, 109, 260–267.

Yanjun, S., Jianhang, C., Shuwen, Z., Hongjuan, L., Jing, L., Lu, L., ... Jiaping, L. (2014).Effect of power ultrasound pre-treatment on the physical and functional properties ofreconstituted milk protein concentrate. Journal of Food Engineering, 124, 11–18.

Yuliana, M., Truong, C. T., Huynh, L. H., Ho, Q. P., & Ju, Y. H. (2014). Isolation andcharacterization of protein isolated from defatted cashew nut shell: Influence of pHand NaCl on solubility and functional properties. LWT - Food Science and Technology,55, 621–626.

Zhang, Q., Tu, Z., Xiao, H., Wang, H., Huang, X., Liu, G., ... Lin, D. (2014). Influence ofultrasonic treatment on the structure and emulsifying properties of peanut proteinisolate. Food and Bioproducts Processing, 92, 30–37.

Zhao, Q., Selomulya, C., Xiong, H., Chen, X. D., Ruan, X., Wang, S., ... Zhou, Q. (2012).Comparison of functional and structural properties of native and industrial process-modified proteins from long-grain indica rice. Journal of Cereal Science, 56, 568–575.

Zhou, B., Zhang, M., Fang, Z. X., & Liu, Y. (2015). Effects of ultrasound and microwavepretreatments on the ultrafiltration desalination of salted duck egg white protein.Food and Bioproducts Processing, 96, 306–313.

Zhu, Z., Zhu, W., Yi, J., Liu, N., Cao, Y., Lu, J., & Decker, E. A. (2018). Effects of sonicationon the physicochemical and functional properties of walnut protein isolate. FoodResearch International, 106, 853–861.


956








https://www.gob.mx/cms/uploads/attachment/file/.../B_sico-Oleaginosas-parte_una.pdf

https://www.gob.mx/cms/uploads/attachment/file/.../B_sico-Oleaginosas-parte_una.pdf



















































20/5/2019 Gmail - Publication ITALIAN JOURNAL OF FOOD SCIENCE

https://mail.google.com/mail/u/0?ik=f2ba02cd78&view=pt&search=all&permthid=thread-f%3A1633047544400339789&simpl=msg-f%3A16330475444… 1/1

José Armando Ulloa <[email protected]>

Publication ITALIAN JOURNAL OF FOOD SCIENCE 1 mensaje

chiara <[email protected]> 9 de mayo de 2019, 3:51Para: José Armando Ulloa <[email protected]>Cc: Costabello <[email protected]>

Dear Dr Jose’ Armando Ulloa we confirm you that your paper #1440 will be published in our Italian Journal of Food Science Issue 3/2019.This Issue will be online on July. Best CHIARA MANCUSI Web editor CHIRIOTTI EDITORI srl Viale Rimembranza, 60 - 10064 Pinerolo TO - Italia Tel. +39 0121 393127 www.chiriottieditori.it - www.foodexecutive.com www.pasticceriaextra.it - www.tuttogelato.it www.alimentifunzionali.it

Le informazioni contenute in questa e-mail sono riservate. I contenuti trasmessi non sono sicuri e possono essere visti e/o modificati da terzi non autorizzati. Qualora abbiate ricevuto questo messaggio per errore, vi preghiamo cortesemente di segnalarcelo immediatamente. The information in this e-mail is confidential. The contents may not be disclosed or used by anyone other than the addressee. QIf you are not the intended recipient, please notify us immediately at the above address.

https://maps.google.com/?q=Viale+Rimembranza,+60+-+10064+Pinerolo+TO+-+Italia&entry=gmail&source=g

http://www.chiriottieditori.it.it/

http://www.foodexecutive.com/

http://www.pasticceriaextra.it/

http://www.tuttogelato.it/

http://www.alimentifunzionali.it/

Universidad Autónoma de Nayarit Secretaría

Documents

Transcript of Universidad Autónoma de Nayarit Secretaría