IJFS#1440_bozza Ital. J. Food Sci., vol. 31, 2019 - 591 PAPER EFFECT OF ULTRASOUND TREATMENT ON PHYSICOCHEMICAL, FUNCTIONAL AND NUTRITIONAL PROPERTIES OF A SAFFLOWER (CARTHAMUS TINCTORIUS L.) PROTEIN ISOLATE M.R. ZUÑIGA-SALCEDOa, J.A. ULLOA*a,b, P.U. BAUTISTA-ROSALESa,b, P. ROSAS-ULLOAb, J.C. RAMÍREZ-RAMÍREZc, Y. SILVA-CARRILLOd, R. GUTIÉRREZ-LEYVAc and C. HERNÁNDEZe aPrograma de Maestría en Ciencias Biológico Agropecuarias. Unidad Académica de Agricultura, Universidad Autónoma de Nayarit, Carretera Tepic-Compostela Km 9, 63780 Xalisco, Nayarit, México bCentro de Tecnología de Alimentos, Universidad Autónoma de Nayarit. Ciudad de la Cultura Amado Nervo, 63155 Tepic, Nayarit. México cUnidad Académica de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Nayarit, Carretera Compostela-Chapalilla Km 3.5, 63700 Compostela, Nayarit, México dUnidad Académica de Agricultura, Universidad Autónoma de Nayarit, Carretera Tepic-Compostela Km 9, 63780 Xalisco, Nayarit, México eCentro de Investigación en Alimentación y Desarrollo Unidad Mazatlán, Av. Sábalo Cerritos S/N, 82100 Mazatlán, Sinaloa, México *Corresponding author: Tel.: +523112118851; Fax +523112118861 E-mail address: arulloa5@gmail.com ABSTRACT The effect of ultrasound exposure time (15 and 30 min) at 130 W and 40 kHz on the physicochemical, functional, and nutritional properties of a safflower protein isolate (SPI) was evaluated. The moisture content, bulk density, and aw of the SPI were significantly (P < 0.05) decreased by ultrasound compared to the untreated control. In contrast to the control, the SPI exposed to ultrasound for 30 min had increased protein solubility (by 9.7% and 3.7% at pH 6 and 7, respectively), least gelation concentration (by 2.0% at pH 6), and oil absorption capacity (by 3.0%). No significant differences (P < 0.05) were observed in amino acid composition, chemical score, or predicted protein efficiency ratio of the control and the SPI exposed to ultrasound. Ultrasound treatment would benefit the application of SPI in the food industry for ground meat formulations, meat substitutes and extenders, doughnuts, baked goods, and soups. Keywords: ultrasound treatment, safflower protein isolate, physicochemical properties, functional properties, nutritional properties Ital. J. Food Sci., vol. 31, 2019 - 592 1. INTRODUCTION Safflower (Carthamus tinctorius L.) is one of the oldest cultivated crops and usually is grown for flowers that are used for coloring, flavoring foods, dyes, medicinal properties, and livestock feed (PEIRETTI, 2017). Safflower is gaining attention due to its oil yield potential and the ability to grow under high temperatures, drought, and high salinity (HUSSAIN et al., 2016). Safflower is produced in over 20 countries, but in 2016, the Russian Federation, Kazakhstan, Mexico, and the USA were the main producers, producing about 71% of the 948,516 t of safflower produced worldwide (FAO, 2018). Oil and meal are the two main products that come from safflower production. Oil is the primary product and has both food and industrial uses. The seed oil content of safflower ranges from 30 to 45% (LIU et al., 2016). Safflower meal, a by-product of the safflower-oil industry, contains approximately 20-25% protein, and is currently marketed as animal feed (TIRIL and KERIM, 2015). However, according to some studies, a protein isolate from safflower meal could represent an opportunity to recover proteins for human consumption (ULLOA et al., 2011; PAREDES-LÓPEZ and ORDORICA-FALOMIR, 1986). On the other hand, the application of ultrasonic technology in the food industry is currently attracting much attention (HU et al., 2013). Ultrasound is an acoustic wave with a frequency >20 kHz (O’SULLIVAN et al., 2016a). High intensity ultrasound (HIUS, 20- 100kHz) might have a wide variety of applications in the food industry (ZHANG et al., 2014) because it can alter the physicochemical properties and/or structure and functional properties due to cavitation effects on vegetable proteins such as soy protein isolate (JAMBRAK et al., 2009), black bean protein isolate (JIANG et al., 2014), and jackfruit seed protein isolate (RESENDIZ-VAZQUEZ et al., 2017). A recent study by XIONG et al. (2018) showed that ultrasonic treatment caused partial unfolding of the proteins of pea protein isolate, which improved foam ability and stability. According to MALIK et al. (2017), solubility, emulsifying capacity, emulsion stability, foaming capacity, foam stability, and oil binding capacity of a sunflower protein isolate were improved significantly after application of HIUS. ZHANG et al. (2018) found that the HIUS treatment modified the protein structure and significantly enhanced the solubility of rice proteins, as well as emulsifying properties and foaming properties. However, the results of the application of ultrasound treatment on protein properties depend on some conditions such as frequency, amplitude, time, temperature, ionic strength, pH, and concentration, as well as intrinsic characteristics of the protein source (HIGUERA- BARRAZA et al., 2016). To our knowledge, no studies have focused on the potential effects of HIUS application on safflower protein isolate (SPI). Therefore, the objective of this study was to evaluate the effect of HIUS on the physicochemical, functional, and nutritive properties of a protein isolate obtained from safflower meal. 2. MATERIAL AND METHODS 2.1. Materials and chemicals The safflower meal (23% protein, moisture 8%, 5.4% ash, 1.4% fat, and total carbohydrates 62.2%) used in this study was purchased from Aceitera La Junta, S.A. de C.V. (Limited Company of Variable Capital) (Guadalajara, Jalisco, Mexico). All chemical reagents were J.T. Baker analytical grade and purchased from Diseño Tecnológico en Laboratorios, S. A. de C.V. (Guadalajara, Jalisco, México). Ital. J. Food Sci., vol. 31, 2019 - 593 2.2. Preparation of protein isolate The SPI was prepared according to the method reported by ULLOA et al. (2011). Briefly, the protein was recovered in batches of safflower meal suspension (120 L) by adding one part safflower meal to 30 parts filtered tap water, which was then mixed for 45 min at room temperature (25°C). The pH of the suspension was adjusted to 9.0 with diluted NaOH during mixing. The insoluble residue of the protein suspension was separated by continuous centrifugation. The protein extract was concentrated using a pilot-scale ultrafiltration unit (Osmonic Inc., Minnetonka, USA) equipped with a polysulfonate membrane cartridge with a molecular weight cut-off of 100 kDa. Extracts were concentrated to one-fifth of their original volume, then diluted with filtered tap water and concentrated by diafiltration for further purification of protein retentate. Dilution consisted of adding an amount of water equal to four times the retentate volume after the first concentration. Three cycles of diafiltration were required to produce a protein isolate with a protein content ≥90.0% dry weight. Finally, the diafiltrated protein extract was spray dried in a Model Tower No. 1 drier (Niro Atomizer, SA, Monterrey, Nuevo León, Mexico) to obtain the powder of protein isolate. 2.3. Ultrasound treatment Aqueous suspensions of SPI containing 10% of protein (w/v) were prepared in 100-mL beakers by magnetic stirring for 15 min. Ultrasound treatment was performed in an ultrasound bath Branson Model MTH-3510 (130 W and 40 kHz; a tank capacity of 5 L; internal dimensions of 290 × 150 × 150 mm; an acoustic energy density of 0.026 W cm-3). Beakers containing SPI suspension were placed directly into the ultrasound bath to receive the treatment for 15 or 30 min, while the control SPI suspension was placed into a water bath at 25°C; the temperature during ultrasound treatment increased by <2°C. The ultrasonic intensity introduced in the system measured by calorimetry according to JAMBRAK et al. (2014) was 1 Wcm-2. Afterward the suspensions of SPI exposed to ultrasound and the control were lyophilized in a FreeZone Freeze-Dry System (Labconco, Kansas City, MO, USA) and stored at room temperature in sealed containers for further analysis. 2.4. Physicochemical and microstructure characteristics Moisture, protein (N × 6.25), and ash contents were determined according to AOAC methods (1995). Water activity (aw) was measured at 25°C using an AquaLab 4TEV (Decagon Devices Inc., Pullman, Washington, USA), on coarse powder samples (3 g). Prior to testing samples, the water activity meter was turned on and allowed to warm up for 30 min and calibrated by filling a plastic disposable cup half filled with a saturated sodium chloride solution. The accuracy of water activity values was ±0.003. The color was determined with a Minolta CR-400 color meter (Konica Minolta Sensing Ltd, Inc., Tokyo, Japan). The measured values were expressed according to the CIELAB color scale L* (lightness), a* (redness-greenness), and b* (yellowness–blueness). The 𝐿!∗ , 𝑎!∗, and 𝑏!∗ values of the white standard tile used as reference were 94.44, -0.23 and 3.89, respectively. Total color difference (∆E) was calculated as: ∆𝐸∗ = 𝐿!∗ − 𝐿∗ ! + 𝑎!∗ − 𝑎∗ ! + 𝑏! ∗− 𝑏∗ ! !/! (1) The bulk density was determined in triplicate using the method described by PIORNOS et al. (2015) and expressed as g mL-1. Ital. J. Food Sci., vol. 31, 2019 - 594 The microstructure of the freeze-dried SPI samples was observed with a scanning electron microscope (SEM; SEC, Mini-SEM SNE-3200M, South Korea) at an accelerating voltage of 20 kV. Before using the SEM, the samples were coated with gold using an ion sputter coater (MCM-100, SEC). 2.5. Functional properties Protein solubility profile of the SPI as a function of pH was determined according to the method reported by PIORNOS et al. (2015). The water absorption capacity (WAC) and oil absorption capacity (OAC) were measured according to the method described by ULLOA et al. (2011) and expressed as g water or oil absorbed per g protein. Soy oil (Fábrica de Aceites la Central, S. A. de C.V., Guadalajara, Jalisco, México) was used in the determination of OAC. The emulsifying activity (EA) and emulsion stability (ES) were determined according to the method reported by DENG et al. (2011) using soy oil. The least gelation concentration (LGC) at pH 2, 4, 6, 8, and 10 was determined according to the method reported by BENELHADJ et al. (2016). Foaming capacity (FC) and foam stability (FS) were measured using the method described by STONE et al. (2015). 2.6. Amino acid composition and protein nutritive quality Hydrolysis and quantification of amino acids were performed according to the methods described by VÁZQUEZ-ORTÍZ et al. (1995) using a Pro Star-210 Varian high-performance liquid chromatographic system (Varian Associates, Inc. USA). Amino acids were expressed on a protein basis, equivalent to g per 16 g of protein. The tryptophan content was not determined. The nutritive quality of proteins was estimated by determination of chemical score (CS) and predicted protein efficiency ratio (PER) according to the procedure described previously by ULLOA et al. (2015). 2.7. Statistical analysis Analyses of samples were done in triplicate and data were analyzed using one-way ANOVA. Significant differences (P < 0.05) between samples were determined from a Tukey’s test using SPSS Statistics Version 20 (IBM Corporation, New York, USA). 3. RESULTS AND DISCUSSION 3.1. Physicochemical and microstructure characteristics The effect of ultrasound exposure time on the physicochemical characteristics of SPI is shown in Table 1. The protein and ash contents of the SPI exposed to ultrasound for 15 and 30 min were not significantly (P > 0.05) different from the control (Table 1). However, the moisture content of SPI was significantly (P < 0.05) reduced from 5.08% (control) to 4.01% and 4.53% when samples were exposed to ultrasound for 15 and 30 min, respectively. The lower moisture content of SPI treated with ultrasound was due to a higher effect of compressions and expansions induced by the sound waves passing through the food medium, which make moisture removal easier (AWAD et al., 2012). Bulk density is a property used to characterize powder products. It is of great importance for economical and functional reasons, for example, for reducing packaging costs, which depends on the combined effects of interrelated factors, like particle size, number of Ital. J. Food Sci., vol. 31, 2019 - 595 contact points, and intensity of attractive inter-particle forces (PIORNOS et al., 2015). As shown in Table 1, the exposure to ultrasound of SPI significantly decreased the bulk density 4.9–6.5% as compared to the control. Such a decrease in the bulk density was due to the samples being treated with ultrasound and freeze drying, as these samples had larger and more heterogeneous structures in protein isolates in comparison with the control (HU et al., 2013; RESENDIZ-VAZQUEZ et al., 2017). The aw of a food system is a thermodynamic property, which is defined as the ratio of water vapor pressure of food to the saturated water vapor pressure at a given temperature. It is considered to be a good quality indicator for the safety and stability of foods with respect to microbial growth and biochemical reactions (TADAPANENI et al., 2017). The aw of SPI exposed for 15 and 30 min to ultrasound was lower than that of the control (Table 1). As discussed previously, sound waves passing through the food medium make moisture removal easier, which also influenced the reduction in aw of SPI as was observed in this study. However, both the control and the SPI exposed to ultrasound had values of aw below a limiting level to ensure microbial stability, because it is generally accepted that no microbial growth will occur at aw < 0.66 (ULLOA et al., 2015). Table 1. Effect of ultrasound exposure time on the physicochemical properties of safflower protein isolate. Property Ultrasound exposure time (min) 0 (control) 15 30 Protein content (%) 87.54±0.52 88.17±0.13 87.03±1.01 Ash content (%) 6.92±0.11 6.88±0.12 6.81±0.13 Moisture content (%) 5.08a±0.13 4.01c±0.12 4.53b±0.11 Bulk density (g mL-1) 0.61a±0.1 0.57b±0.1 0.58b±0.1 Water activity 0.341a±0.002 0.286c±0.007 0.300b±0.004 Color L* (lightness) 60.62±0.11 60.35±0.19 60.19±0.21 a* (redness-greenness) 4.21±0.06 4.23±0.03 4.18±0.08 b* (yellowness-blueness) 17.45±0.29 17.41±0.17 17.77±0.25 ΔE (color difference) 36.70±0.17 36.94±0.21 37.22±0.27 Values are mean±standard deviation of three determinations. Values followed by different superscript letters in the same line are significantly different (P < 0.05). The color characteristics of SPI exposed to ultrasound for 15 and 30 min in comparison with the control treatment are shown in Table 1. There were not significant (P > 0.05) differences in L*, a*, b*, and ∆E values among samples of control SPI and those exposed to ultrasound. The effect of ultrasound on color in food depends on intrinsic characteristics of food and ultrasound conditions (BI et al., 2015). ADEKUNTE et al. (2010) reported a decrease in L*, a* and b* values of tomato juice after sonication due to the degradation of lycopene. CHENG et al. (2007) found that guava juice treated by ultrasound for 30 min showed a significant change in the ∆E value due to a decrease in L* value and an increase in a* and b* values. VALERO et al. (2007) found that ultrasound treatments had no significant effect on color scoring in orange juice. The degradation of color might be due to the effect of cavitation of ultrasound that could induce both chemical (e.g., by generation of radicals) and mechanical degradation of biomolecules (ADEKUNTE et al., 2010); however, such a phenomenon was not observed in the SPI exposed to ultrasound in this study. In general, a similar behavior on the effect of ultrasound exposure time on the Ital. J. Food Sci., vol. 31, 2019 - 596 physicochemical properties of safflower protein isolate of this study was reported by FLORES-JIMÉNEZ et al. (2019) for canola protein isolate. Fig. 1 shows a set of SEM images of control SPI (A) and SPI exposed to ultrasound for 15 (B) and 30 min (C). It was observed that samples B and C obtained after ultrasonic treatments and freeze-drying had larger and more heterogeneous structures than sample A (untreated SPI). These results might have been caused by ultrasound treatment inducing the unfolding of proteins and increasing the surface hydrophobic groups of the SPI molecules (HU et al., 2013; JIANG et al., 2014). Figure 1. Effect of ultrasound exposure time (control = A, 15 min = B and 30 min = C) on the microstructure of safflower protein isolate. 3.2. Functional properties Solubility is one of the most important functional attributes of proteins because it affects the texture, color, and the sensory properties of products containing them. It is closely associated with water retention capacity and other physicochemical and functional properties. Solubility of proteins depends on the composition, molecular weight, and surface characteristics of constituent amino acids and environmental factors such as pH, temperature, and ionic strength, and it can be affected by some chemical and physical treatments (TIMILSENA et al., 2016). Table 2 shows the effect ultrasound exposure time on the protein solubility of SPI at different pH values. Only at pH 6 and pH 7, did the SPI exposed to ultrasound for 30 min have a beneficial effect on protein solubility, increasing 9.7% and 3.7%, respectively, in comparison to the control. This increase in protein solubility may be due to the conformational change during ultrasonic treatment and formation of soluble protein aggregates from insoluble protein (HU et al., 2013). However, such an increase in protein solubility depends on ultrasonic conditions (JIANG et al., 2014), as well as the pH at which the proteins are solubilized, as was observed in this study. Besides, for safflower proteins, the solubility values were minimum at the pH value of 5.0, which is the isoelectric point of safflower proteins, for ultrasound treatments and control treatment. The results of WAC and OAC by effect of ultrasound exposure time of SPI are shown in Table 3. The WAC decreased from an initial value of 2.14 g H2O g-1 protein to 1.94 g H2O g-1 protein after ultrasound treatment for 30 min. According to RESENDIZ-VAZQUEZ et al. (2017), the ultrasound treatment might denature the molecular structure of proteins and cause an increase in the hydrophobic surface, which can lead to low values of WAC, as was observed in proteins from jackfruit seeds. In contrast, the OAC of samples of SPI exposed to ultrasound for 15 and 30 min were significantly (P < 0.05) higher compared to the control SPI. The OAC increased from an initial value of 0.99 g oil g-1 protein to 1.19 g oil g-1 protein and 1.30 g oil g-1 after exposure to ultrasound for 15 and 30 min, respectively. The Ital. J. Food Sci., vol. 31, 2019 - 597 increase of OAC in proteins might be attributed to the exposure of hydrophobic groups after ultrasound treatment (HIGUERA-BARRAZA et al., 2016; ZHOU et al., 2016), which allowed the physical entrapment of oil (MEINLSCHMIDT et al., 2016). Table 2. Effect of ultrasound exposure time at different pH on protein solubility (%) of safflower protein isolate. pH Ultrasound exposure time (min) 0 (control) 15 30 2 90.95±0.68 87.22±1.43 88.10±0.06 3 81.06a±0.68 65.32b±0.68 67.16b±0.69 4 17.98a±0.68 12.59b±0.01 13.73b±0.01 5 14.57a±0.01 12.11b±0.66 13.20ab±0.69 6 49.61b±1.37 50.32b±0.01 54.41a±0.61 7 61.16b±0.01 57.94c±1.28 63.44a±0.04 8 89.75a±0.74 71.88b±0.68 73.49b±1.43 9 92.32a±0.12 86.34b±1.31 88.32ab±1.38 10 95.72a±0.68 90.26b±1.37 93.00ab±0.06 Values are mean±standard deviation of three determinations. Values followed by different superscript letters in the same line are significantly different (P < 0.05). Table 3. Effect of ultrasound exposure time on water and oil absorption capacity of safflower protein isolate. Property Ultrasound exposure time (min) 0 (control) 15 30 Water absorption capacity (g H2O g -1 protein) 2.14a±0.03 2.15a±0.03 1.94b±0.02 Oil absorption capacity (g oil g-1 protein) 0.99c±0.01 1.19b±0.02 1.30a±0.01 Values are mean±standard deviation of three determinations. Values followed by different superscript letters in the same line are significantly different (P < 0.05). Proteins are of particular interest as emulsifying agents in food formulations such as frozen desserts, salad dressings, comminuted meats, mayonnaise, cake batters, milks, and coffee whiteners, due to their ability to adsorb and form viscoelastic films at oil-water interfaces (O’SULLIVAN et al., 2016b). According to the results presented in Fig. 2, ultrasound treatment had no effect on EA and ES of SPI. YANJUN et al. (2014) reported that ultrasound pretreatment increased the emulsifying activity index (EAI) and ES index of milk proteins. ZHOU et al. (2016) found that the ultrasound treatment increased or decreased EAI of soybean glycinin depending on the ionic strength. In another study, the EA of defatted rice bran protein concentrate was higher (P < 0.05) than that of the ultrasound treatment, but the ES was not significantly different (P > 0.05) from the control (CHITTAPALO and NOOMHORM, 2009). The emulsifying properties of food proteins depend upon the solubility, molecular flexibility, surface hydrophobicity, and stability of the protein structure (ZHOU et al., 2016), which can be modified or not when are exposed to different ultrasound conditions as was observed in this study. Ital. J. Food Sci., vol. 31, 2019 - 598 Figure 2. Effect of ultrasound exposure time on the emulsifying activity (EA) and stability (ES) of safflower protein isolate. Gel properties are important functional characteristics of protein isolates, which are widely used as gelling agents to improve the texture and water holding capacity of meat products. Gelation is often an aggregation of denatured proteins, which involves the formation of a network, retaining significant amounts of water and transforming liquid sample to solid, which exhibits a certain degree of order where both covalent and non- covalent interactions are involved (CHEN et al., 2016). LGC indicates the gelling capacity of protein. Fig. 3 shows that the SPI exposed to ultrasound for 15 and 30 min at pH 2-10 had no effect on LGC, except for the ultrasound treatment of 30 min at pH 6, where the LGC increased from 6.0% (w/w) to 8.0% (w/w), which implies a reduction of the gelling capacity. The behavior of the LGC of SPI by effect of pH in this study was similar to that showed for lupin (PIORNOS et al., 2015), jackfruit (RESENDIZ-VAZQUEZ et al., 2017), and cashew nut shell (YULIANA et al., 2014) protein isolates. Figure 3. Effect of ultrasound exposure time on the last gelation concentration (LGC) of safflower protein isolate. Ital. J. Food Sci., vol. 31, 2019 - 599 The FC and FS of protein isolates are functional properties that determine their application in food systems, where aeration and overrun is required (e.g. baked foods, whipped toppings and ice cream mixes) (SHEVKANI et al., 2015). Because of surface-active properties, the proteins form foam when they are whipped (MALIK et al., 2017). According to the results obtained in this study, the exposure to ultrasound for 15 and 30 min did not modify the FC and FS of SPI with respect to the control (Fig. 4). Figure 4. Effect of ultrasound exposure time on the foaming capacity (FC) and foam stability (FS) of safflower protein isolate. Some reports showed that FC and FE of protein isolates from whey (JAMBRAK et al., 2008) and soy (ZHANG et al., 2014) were improved after ultrasound treatment for both 20 kHz and 40 kHz treatments, but no effect in foaming at 500 kHz treatment was observed, as occurred for SPI in this study. The improvement of the foaming properties in protein suspensions by ultrasound may be due to protein partial denaturation, which causes a higher air-water diffusion interface due to an increase in cohesiveness and flexibility of the foams (HIGUERA-BARRAZA et al., 2016). 3.3. Amino acid composition and protein nutritive quality Amino acid composition and nutritive quality of proteins in terms of CS and PER of SPI exposed to the ultrasound treatments in comparison with the control (Table 4) were not significantly different (P < 0.05). Of all the amino acids present in SPI, half corresponded to essential amino acids. On the other hand, glutamic acid was the amino acid with higher concentration in the SPI, as well as in the hempseed, soy (WANG et al., 2008), and pennycress protein isolates (HOJILLA-EVANGELISTA et al., 2014). According to amino acid requirements for adults (FAO/WHO, 1991), the first limiting amino acid of SPI was lysine. Therefore lysine was considered for calculating CS values for the SPI exposed to ultrasound for 15 min, 30 min, and the control, which were 48.4, 48.2, and 48.9 respectively, and were not significantly different (P > 0.05) from one another (Table 4). Ital. J. Food Sci., vol. 31, 2019 - 600 Table 4. Effect of ultrasound exposure time on the composition of amino acid and protein quality of safflower protein isolate. Parameter Amino acid composition (g/16 g N) Reference for adults (FAO/WHO, 1991) Ultrasound exposure time (min) 0 (Control) 15 30 Essential amino acid Lysine 2.69±0.08 2.66±0.15 2.65±0.05 5.5 Threonine 5.08±0.25 4.78±0.21 4.31±0.57 4.0 Valine 2.93±0.09 3.01±0.44 2.70±0.14 5.0 Methionine+cysteine 2.13±0.01 1.93±0.37 2.21±0.14 3.5 Cysteine Isoleucine 2.25±0.46 2.36±0.10 2.20±0.20 4.0 Leucine 6.39±0.94 7.06±0.36 6.57±0.14 7.0 Phenylalanine+tyrosine 4.10±0.94 4.16±1.12 3.56±0.60 6.0 Tyrosine 2.58±0.10 2.97±0.73 2.40±0.20 Tryptophan ND ND ND Total essential amino acids 25.58±0.70 25.96±1.10 24.19±0.80 No essential amino acid Histidine 3.84±0.05 3.42±0.32 4.09±0.46 Arginine 18.65±1.08 18.57±0.74 19.08±0.56 Aspartic acid 6.78±0.57 6.73±0.41 6.60±0.48 Serine 8.25±0.58 7.20±0.51 7.99±0.60 Glutamic acid 20.96±0.86 22.28±0.73 21.17±0.83 Glycine 8.83±0.52 9.09±0.58 9.61±0.26 Alanine 7.11±0.37 6.75±0.55 7.27±0.86 Total non-essential amino acid 74.42±0.81 74.04±0.75 75.81±0.90 Nutritive quality Chemical score 48.9±1.53 48.4±2.7 48.2±0.83 PER 2.16±0.22 2.42±0.20 2.26±0.04 Values are mean±standard deviation of three determinations. Values followed by different superscript letters in the same line are significantly different (P < 0.05). ND = Not determined. PER = Predicted protein efficiency ratio. With respect to the PER, the values obtained for the SPI exposed to ultrasound for 15 min, 30 min, and the control were not significantly different (P < 0.05) from one another and had values of 2.42, 2.26, and 2.16, respectively, which were higher than the values 2.14 and 2.04 for desi chickpea and soy protein isolates, respectively (WANG et al., 2010). 4. CONCLUSIONS Application of ultrasound at 130 W and 40 kHz for 30 min to SPI increased the protein solubility at pH range of 6-7 and the OAC. It was demonstrated that the ultrasound did not affect the color parameters of L*, a*, b* and ∆E, or the amino acid composition and nutritional quality of proteins of SPI. The improvement of the OAC of SPI could benefit its Ital. J. Food Sci., vol. 31, 2019 - 601 application in the food industry for ground meat formulations, meat substitutes and extenders, doughnuts, baked goods, and soups. ACKNOWLEDGMENTS The authors wish to thank CONACYT-Mexico for the scholarship granted to QFB Minerva Marisol Zúñiga- Salcedo during her master’s studies. REFERENCES Adekunte A.O., Tiwari B.K., Cullen P.J., Scannell A.G.M. and O’Donnell C. 2010. Effect of sonication on colour, ascorbic acid and yeast inactivation in tomato juice. Food Chem. 122:500-507. AOAC.1995. Official Methods of Analysis. Association of Official Analytical Chemists, Arlington, USA. Awad T.S., Moharram H.A., Shaltout O.E., Asker D. and Youssef M.M. 2012. Applications of ultrasound in analysis, processing and quality control of food: A review. Food Res. Int. 48:410-427. Benelhadj S., Gharsallaoui A., Degraeve P., Attia H. and Ghorbel D. 2016. Effect of pH on the functional properties of Arthrospira (Spirulina) platensis protein isolate. Food Chem. 194:1056-1063. Bi X., Hemar Y., Balaban M.O. and Liao X. 2015. The effect of ultrasound on particle size, color, viscosity and polyphenol oxidase activity of diluted avocado puree. Ultrason. Sonochem. 27:567-575. Chen Z., Shi X., Xu J., Du Y., Yao M. and Guo S. 2016. Gel properties of SPI modified by enzymatic cross-linking during frozen storage. Food Hydrocoll. 56:445-452. Cheng L.H., Soh C.Y., Liew S.C. and Teh F.F. 2007. Effects of sonication and carbonation on guava juice quality. Food Chem. 104:1396-1401. Chittapalo T. and Noomhorm A. 2009. Ultrasonic assisted alkali extraction of protein from defatted rice bran and properties of the protein concentrates. Int. J. Food Sci. Technol. 44, 1843-1849. Deng Q., Wang L., Wei F., Xie B., Huang F., Huang W., Shi J., Huang Q., Tian B. and Xue S. 2011. Functional properties of protein isolates, globulin and albumin extracted from Ginkgo biloba seeds. Food Chem. 124:1458-1465. FAO. 2018. FAO Statistical Database. Food and Agriculture Organization. www.fao.org/faostat/es/#data/QC (accessed 13.06.18). FAO/WHO. 1991. Protein Quality Evaluation. Rome, Food and Agriculture Organization. Flores-Jiménez, N. T., Ulloa, J.A., Urías Silvas, J. E., Ramírez Ramírez, J. C., Rosas Ulloa, P., Bautista Rosales, P. U., Silva Carrillo, Y. and Gutiérrez Leyva, R. 2019. Effect of high-intensity ultrasound on the compositional, physicochemical, biochemical, functional and structural properties of canola (Brassica napus L.) protein isolate. Food Res. Int. In press. DOI: doi.org/10.1016/j.foodres.2019.01.025 Higuera-Barraza O.A., Del Toro-Sanchez C.L., Ruiz-Cruz S. and Márquez-Ríos, E. 2016. Effects of high-energy ultrasound on the functional properties of proteins. Ultrason. Sonochem. 31:558-562. Hojilla-Evangelista M.P., Selling G.W., Berhow M.A. and Evangelista R.L. 2014. Preparation, composition and functional properties of pennycress (Thlaspi arvense L.) seed protein isolates. Ind. Crops Prod. 55:173-179. Hu H., Wu J., Li-Chan E.C., Zhu, L. Zhang, F., Xu X., Fan G., Wang L., Huang X. and Pan, S. 2013. Effects of ultrasound on structural and physical properties of soy protein isolate (SPI) dispersions. Food Hydrocoll. 30:647-655. Hussain M.I., Dionyssia-Angeliki L., Farooq M., Nikoloudakis N. and Khalid N. 2016. Salt and drought stresses in safflower:a review. Agron. Sustain. Dev. 36:1-31. Jambrak A.R., Lelas V., Mason T.J., Kresic G. and Badanjak M. 2009. Physical properties of ultrasound treated soy proteins. J. Food Eng. 93:386-393. Jambrak A.R., Mason T.J., Lelas V., Herceg Z. and Herceg L. 2008. Effect of ultrasound treatment on solubility and foaming properties of whey protein suspensions. J. Food Eng. 86:281-287. Ital. J. Food Sci., vol. 31, 2019 - 602 Jambrak A. R., Mason T. J., Lelas V., Paniwnyk L. and Herceg, Z. 2014. Effect of ultrasound treatment on particle size and molecular weight of whey proteins. J. Food Eng. 121:15-23. Jiang L., Wang J., Jiang Y.L., Wang J., Li Y., Wang Z., Chen Y., Ma W., Qi B., and Zhang, M. 2014. Effects of ultrasound on the structure and physical properties of black bean protein isolates. Food Res. Int. 62:595-601. Liu L., Guan LL., Wu W., and Wang L. 2016. A review of fatty acids and genetic characterization of safflower (Carthamus tinctorius L.) seed oil. Organic Chem. Curr. Res. 5:160. Malik M. A., Sharma H. K., and Saini C. S. 2017. High intensity ultrasound treatment of protein isolate extracted from dephenolized sunflower meal:Effect on physicochemical and functional properties. Ultrason. Sonochem. 39:511-519. Meinlschmidt P., Sussmann D., Schweiggert-Weisz U. and Eisner P. 2016. Enzymatic treatment of soy protein isolates:effects on the potential allergenicity, technofunctionality, and sensory properties. Food Sci. Nutr. 4:11-23. O'Sullivan J., Murray B., Flynn C. and Norton I. 2016a. The effect of ultrasound treatment on the structural, physical and emulsifying properties of animal and vegetable proteins. Food Hydrocoll. 53:141-154. O'Sullivan J., Park M. and Beevers J. 2016b. The effect of ultrasound upon the physicochemical and emulsifying properties of wheat and soy protein isolates. J. Cereal Sci. 69:77-84. Paredes-López O. and Ordorica-Falomir C. 1986. Production of safflower protein isolates:composition, yield and protein quality. J. Sci. Food Agric. 37:1097-1103. Peiretti P.G. 2017. Nutritional aspects and potential uses of safflower (Carthamus tinctorius L.) in livestock. Ch. 1. In: “Agricultural Research Updates”. Vol. 19. P. Gorawala and S. Mandhatri S. (Ed.), pp. 3-22. Nova Science Publishers, Inc., NY. Piornos J.A., Burgos-Díaz C., Ogura T., Morales E., Rubilar M., Maureira-Butler I. and Salvo-Garrido, H. 2015. Functional and physicochemical properties of a protein isolate from Aluprot-CGNA:A novel protein-rich lupin variety (Lupinus luteus). Food Res. Int. 76:719-724. Resendiz-Vazquez J.A., Ulloa J.A., Urías-Silvas J.E., Bautista-Rosales P.U., Ramírez-Ramírez J.C., Rosas-Ulloa P. and González-Torres L. 2017. Effect of high-intensity ultrasound on the technofunctional properties and structure of jackfruit (Artocarpus heterophyllus) seed protein isolate. Ultrason. Sonochem. 37:436-444. Shevkani K., Singh N., Kaur A. and Rana, J.C. (2015). Structural and functional characterization of kidney bean and field pea protein isolates:A comparative study. Food Hydrocoll. 43:679-689. Stone A.K., Karalash A., Tyler R.T., Warkentin T.D. and Nickerson M.T. 2015. Functional attributes of pea protein isolates prepared using different extraction methods and cultivars. Food Res. Int. 76:31-38. Tadapaneni R.K., Syamaladevi R.M., Villa-Rojas R. and Tang J. 2017. Design of a novel test cell to study the influence of water activity on the thermal resistance of Salmonella in low-moisture foods. J. Food Eng. 208:48-56. Timilsena Y.P., Adhikaria R., Barrow C.J. and Adhikaria B. 2016. Physicochemical and functional properties of protein isolate produced from Australian chia seeds. Food Chem. 212:648-656. Tiril S.U. and Kerim M. 2015. Evaluation of safflower meal as a protein source in diets of rainbow trout [Oncorhynchus mykiss, Walbaum, 1792)]. J. Appl. Ichthyol. 31:895-899. Ulloa J.A., Ibarra-Zavala S.J., Ramírez-Salas S.P., Rosas-Ulloa P., Ramírez-Ramírez J.C. and Ulloa-Rangel B.E. 2015. Chemical, physicochemical, nutritional, microbiological, sensory and rehydration characteristics of instant whole beans (Phaseolus vulgaris). Food Technol. Biotech. 53:48-56. Ulloa J.A., Rosas-Ulloa P. and Ulloa-Rangel B.E. 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. Valero M., Recrosio N., Saura D., Muñoz N., Martí N. and Lizama, V. 2007. Effects of ultrasonic treatments in orange juice processing. J. Food Eng. 80:509-516. Vázquez-Ortiz F.A., Caire G., Higuera-Ciapara I. and Hernández G. 1995. High performance liquid chromatographic determination of free amino acids in shrimp. J. Liq. Chromatogr. R T 18:2059-2068. Wang X., Gao W., Zhang J., Zhang H., Li J., He X. and Ma H. 2010. Subunit, amino acid composition and in vitro digestibility of protein isolates from Chinese kabuli and desi chickpea (Cicer arietinum L.) cultivars. Food Res. Int. 43:567- 572. Ital. J. Food Sci., vol. 31, 2019 - 603 Wang X.S., Tang C.H., Yang X.Q. and Gao W.R. 2008. Characterization, amino acid composition and in vitro digestibility of hemp (Cannabis sativa L.) proteins. Food Chem. 107:11-18. Xiong T., Xiong W., Ge M., Xia, J., Li, B. and Chen, Y. 2018. Effect of high intensity ultrasound on structure and foaming properties of pea protein isolate. Food Res. Int. 109:260-267. Yanjun S., Jianhang C., Shuwen Z., Hongjuan L., Jing L., Lu L., Uluko H., Yanling S., Wenming C., Wupeng G. and Jiaping L. 2014. Effect of power ultrasound pre-treatment on the physical and functional properties of reconstituted milk protein concentrate. J. Food Eng. 124:11-18. Yuliana M., Truong C.T., Huynh L.H., Ho Q.P. and Ju, Y. 2014. Isolation and characterization of protein isolated from defatted cashew nut shell: Influence of pH and NaCl on solubility and functional properties. LWT-Food Sci. Technol. 55:621-626. Zhang L., Jin Y., Xie Y., Wu X. and Wu T. 2014. Releasing polysaccharide and protein from yeast cells by ultrasound: Selectivity and effects of processing parameters. Ultrason. Sonochem. 21:576-581. Zhang L., Zheng P., Shen, K., Cai X., Zheng B. and Miao, S. 2018. Influence of ultrasound-assisted alkali treatment on the structural properties and functionalities of rice protein. J. Cereal Sci. 79:204-209. Zhou M., Liu J., ZhouY., Huang J., Liu F., Pan S. and Hu H. 2016. 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. Paper Received November 13, 2018 Accepted March 12, 2019