Received: 25 January 2019  |  Revised: 27 April 2019  |  Accepted: 11 June 2019 DOI: 10.1111/jfpp.14093 O R I G I N A L A R T I C L E Effect of sonication pretreatment parameters and their optimization on the antioxidant activity of Hermitia illucens larvae meal protein hydrolysates Benjamin Kumah Mintah1,2  | Ronghai He1  | Mokhtar Dabbour1,3  | Moses Kwaku Golly1,4  | Akwasi Akomeah Agyekum1,5 | Haile Ma1 1School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Abstract China The study investigated the effect of sonication conditions on antioxidant activity 2Department of Nutrition and Food Science, of Hermetia illucens larvae meal protein hydrolysates. Three‐factor three‐level: pH ILSI‐UG FSNTC, University of Ghana, Legon, Ghana (7–9), time (10–30 min), and temperature (25–55°C) were optimized. Box–Behnken's 3Department of Agricultural and Biosystems design was applied to optimize sonication treatment. Ferrous ion chelating activity Engineering, Faculty of Agriculture, Benha University, Benha, Egypt (ICA), DPPH‐radical scavenging activity (DPPHRSA), Hydroxyl radical scavenging ac‐ 4Faculty of Applied Science and tivity (HRSA), and cupric ion chelating activity (CCA) were considered as responses. Technology, Sunyani Technical University, Findings demonstrated that sonication preceding enzymolysis significantly impacted Sunyani, Ghana 5Atomic Energy Commission, Applied on ICA, DPPHRSA, HRSA, and CCA. ANOVA showed the determination coefficient Radiation Biology Centre, Legon, Ghana (R2) were 0.98 (ICA), 0.99 (DPPHRSA), 0.98 (HRSA), and 0.88 (CCA); demonstrating Correspondence that the models were reasonably fit with experimental results. Optimum sonication Ronghai He, School of Food and Biological conditions were pH (9), time (29.84 min), and temperature (54.93°C). For these con‐ Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China. ditions, the experimental data obtained [ICA (37.84%), DPPHRSA (43.19%), HRSA Email: heronghai1971@126.com (71.01%), and CCA (68.93%)] were consistent with predicted values, higher than con‐ Funding information trol, and supported by protein subunits, fluorescence spectra and microstructure. Primary Research and Development Plan Practical applications of Jiangsu Province, Grant/Award Number: BE2016352, BE2016355; Zhenjiang With a rich nutrient profile, edible insects are potential ingredient for food applica‐ “1+1+N” New Agricultural Technology tions. Hermetia illucens is one of the most encouraging edible insect species for incor‐ Extension Project, Grant/Award Number: ZJNJ[2017]03 poration in food products as it has several benefits to the environment, coupled with the already available knowhow for their rearing. With the prediction that in the next few decades insects will be reliable source of protein for humans and livestock, it is logical that the antioxidants activity of insect larvae meal proteins are investigated for new product development. Ultrasound is reported to enhance enzyme action in the preparation of bioactive hydrolysates/peptides. The present study showed that the use of ultrasonication pretreatment in the enzymatic hydrolysis of HILMP to gener‐ ate hydrolysates with antioxidant constituents was efficient (likened to conventional approach). The study outcome could help the food and/or pharmaceutical industry to advance new bioactive products/ functional foods from HILMP hydrolysates. J Food Process Preserv. 2019;43:e14093. wileyonlinelibrary.com/journal/jfpp © 2019 Wiley Periodicals, Inc.  |  1 of 12 https://doi.org/10.1111/jfpp.14093 2 of 12  |     MINTAH eT Al. 1  | INTRODUC TION high, and the proteases are commercially available (McCarthy et al., 2013). Several factors such as hydrolysis time, temperature, pH, Over the last two decades, a growing demand among consumers enzyme, and substrate concentration influences the enzymolysis across the world for bioactive elements/antioxidants from natural process (in vitro), and consequently the bioactivity of protein hydro‐ sources as oppose synthetic ones has resulted in the search for same lysates. Conventional enzymolysis has also been noted to limit the by researchers for use as food ingredients or the development of enzymolysis of proteins owing to the incompatible conformation of functional foods. The search is driven by the continuous generation protein which makes it hard for the protease to attack the enzyme‐ of free radicals (reactive oxygen and/or nitrogen species) in humans, driven bonds of a protein (Halim & Sarbon, 2017; Jamil et al., 2016; as part of their usual metabolic process, which when in excess could Jian, Wenyi, & Wuyong, 2008; Qu et al., 2013). aid the development of certain diseases (e.g., stroke, cancer, inflam‐ The use of ultrasound (a modern physical processing technology) mation, or myocardial infraction). Free radicals may also contribute has been shown to enhance the process of enzymolysis of proteins to skin pathologies or the aging of skin cells in humans (Stadtman, in the generation of bioactive compounds (Jian et al., 2008; Pan, Qu, 2006; Umachigi et al., 2007). Antioxidants are documented as good Ma, Atungulu, & McHugh, 2012; Qu et al., 2013; Qu, Pan, & Ma, remedies for controlling, reducing, or eliminating these diseases in 2010; Wang, Sun, Cao, Tian, & Li, 2008). This is attributable to the humans (Lobo, Patil, Phatak, & Chandra, 2010; Ruiz‐Ruiz, Dávila‐ cavitation effect of ultrasound (acoustic in nature) which results in Ortíz, Chel‐Guerrero, & Betancur‐Ancona, 2013). As a consequence intense shear thrusts, turbulence, and shockwaves (Chandrapala, the search for antioxidants from diverse natural sources by food sci‐ Oliver, Kentish, & Ashokkumar, 2012). Accordingly, the use of ultra‐ entists for possible use as food ingredient or the development of sound to pretreat samples prior to enzymolysis has been shown by functional foods continuous. some scholars to improve the bioactivity of selected products (Jian Edible insects are potential ingredient in food applications, as et al., 2008). they are rich in nutrients such as protein, lipids/ fatty acids, fiber, Literature shows several studies on the bioactivity of several or micronutrients (Rumpold & Schlüter, 2013). Among the most food sources/products such as milk, cheese, yoghourt, bean, and rice encouraging species for the incorporation of edible insects in con‐ (Gobbetti et al., 2015; Karaś et al., 2015; Megías et al., 2007). Studies temporary food is the black soldier fly (Hermetia illucens), due to its on the techno‐functional properties of insect‐derived protein hydro‐ benefits to the human environment, coupled with its rich nutritional lysates are also documented (Hall, Jones, O’Haire, & Liceaga, 2017; profile: 37%–63% protein, 7%–39% fat, and 9%–28% ash (Barragan‐ Purschke, Meinlschmidt, Horn, Rieder, & Jäger, 2018). There are also Fonseca, Dicke, & van Loon, 2017). Also, there are already technol‐ few studies on the antioxidant activity of edible insect protein hy‐ ogies in place for the rearing of this insect on commercial basis for drolysates (Zhang et al., 2016; Zielińska, Baraniak, & Karaś, 2017), food and feed applications (Barroso et al., 2014). but no information has been documented explaining the effect of The incorporation of insects such as HI (H. illucens) in the diets ultrasonication treatment on the antioxidant properties of Hermicia of humans may well enrich their food in bioactive constituents/com‐ illucens larvae protein hydrolysate (HILPH). As a consequence, in this pounds—peptides (Zielińska, Karaś, & Jakubczyk, 2017). Bioactive study (for the first time), the influence of ultrasound aided enzy‐ compounds/peptides can be generated during gastrointestinal molysis of H. illucens larvae meal protein (HILMP) on the antioxidant digestion, or by in vitro proteolysis, resulting in the production of activity of the hydrolysate thereof, is considered. We optimized the small molecular weight compounds with many health benefits to hu‐ enzymolysis pretreatment conditions to obtain the protein hydroly‐ mans, such as antioxidant, antihypertensive, and anti‐inflammatory sate with the optimum antioxidative activity. ICA, DPPHRSA, HRSA, benefits (Gobbetti et al., 2015; Karaś, Jakubczyk, Szymanowska, and CCA were used to assess the antioxidative capacity of the hy‐ Złotek, & Zielińska, 2017; Zhang, Mu, Wang, & Sun, 2012). The pro‐ drolysate peptides. The metals, Fe2+ and Cu2+, were considered as tein hydrolysates derived from insects (e.g., H. illucens) may be ben‐ a result of their catalytic role in the generation of reactive oxygen eficial to humans in such regards. Protein hydrolysates are a better species in aerobic organisms (Perron & Brumaghim, 2009). alternative to intact proteins and/or elemental formulas in the de‐ velopment of functional foods to support the nutritional needs of 2  | MATERIAL AND METHODS individuals/patients (Clemente, 2001). With the increasing scientific proof associating bioactive compounds with reduced development 2.1 | Sample, and chemicals of certain diseases/illnesses in humans, analyzing the contribution of edible insects to the bioactive (antioxidant) capacity of human diet H. illucens larvae meal, defatted using ethanol (Zhao, Vázquez‐ is imperative. Gutiérrez, Johansson, Landberg, & Langton, 2016), was provided by The generation of bioactive hydrolysates/peptides (from pro‐ the research team of L‐507B (Jiangsu University's School of Food teins) by enzymatic means has been deemed most appropriate com‐ and Biological Engineering, China). The larvae had been previously pared to the use of acids or alkaline (Halim & Sarbon, 2017; Jamil, received from the Difei Biological Technology Company Limited Halim, & Sarbon, 2016; McCarthy, O’Callaghan, & O’Brien, 2013). (Jiangsu, China), and were microwave dried prior to milling and The reaction conditions are controllably milder, product quality is defatting. MINTAH eT Al.      |  3 of 12 Hydrogen peroxide (H2O2), paramagnetic solid (FeCl2), and ferro‐ optimum sonication pretreatment conditions in preparing hydro‐ zine were acquired from a chemical/reagent Company—Sinopharm lysates with best antioxidant capacity. Ltd (China). Enzyme (Alcalase—150,000 U/ml activity) was bought Thus, the sonication treatments were optimized using the fac‐ from a Biotech Company—Novozymes Ltd. (China). Remaining tors: pH, time, and temperature under the set ultrasound parame‐ chemicals as well as 1, 1‐DPPH (diphenyl‐2‐picryhydrazyl) were of ters. The results for the ICA, DPPHRSA, HRSA, and CCA were the analytical rating. dependent (response) variables. The three factor levels were coded as: −1 (low), 0 (average), and 2.2 | HILMP hydrolysates +1 (high), and the actual values for the factors were pH (a): 7, 8, and 9; sonication time (b): 10, 20, and 30; and sonication temperature (c): H. illucens larvae meal was used to prepare a suspension (distilled 25, 40, and 55, respectively. To expound the association between water as solvent) with an initial substrate concentration of 14.0 g/L. the dependent (response) and independent variables, the responses The mixture was continuously stirred using a JJ‐1 impeller agitator were fitted to a second order mathematical model: (J. Xichengxinrui Inst. Co., Jiangsu, China) set to 100 rpm, and the pH and temperature were set to the respective limits for the en‐ Y=0+1A+2B+3C+12AB+13AC+23BC+ A211 + 2 222B +33C zymolysis (Table 1). After, the respective suspensions/slurry were pretreated by sonication using multiple frequency ultrasound (MFU) where, Y represent the responses (ICA, DPPHRSA, HRSA, and CCA); with sweep cycle of 500 ms, 5.5 L tank volume, pulse time of 15 s A, B, and C are independent variables; β0 is the constant coefficient, (on) and 5 s (off), 600 W power, and 40 ± 2 kHz frequency. The hy‐ β1, β2, and β3 are the linear coefficient; β12, β13, and β23 are the cross drolysis reaction was carried out according to the set conditions product (interactive) coefficients; and β11, β22, and β33 are the qua‐ (Table 1), using the mentioned enzyme (9,000 U/g). The reaction was dratic coefficient. halted after 90 min, and the enzyme was inactivated by heat (i.e., A control experiment was also done to compare with the experi‐ placing sample in boiling water) for a duration of 10 min. The solu‐ mental values derived from the best predictive points for all the four ble fractions were collected by centrifuging at 4,000× g for 15 min responses. (16‐TGL, High‐Speed TTC, Nanjing, China), and were stored (−18°C) prior to analysis. 2.4 | Determination of ferrous ion (Fe2+) chelating activity (ICA) 2.3 | Experimental design The ICA of the HILMP hydrolysates was estimated with the method A three‐factor, three‐level Box and Behnken's design (BBD) with Box‐ reported by García‐Moreno et al. (2014), with minor modification. Wilson methodology (Response Surface) was used to investigate the The hydrolysate (3 ml) was transferred into test tube and then 0.1 ml TA B L E 1  Box and Behnken's design of three experimental factors with four responses Run A: pH B: Time (min) C: Temperature (°C) Fe2+ (%) DPPH (%) HRSA (%) Cu2+ (%) 1 9.00 20.00 25.00 32.22 43.21 63.39 51.83 2 7.00 30.00 40.00 35.15 38.38 67.10 39.48 3 8.00 20.00 40.00 29.12 30.01 62.99 52.46 4 7.00 10.00 40.00 30.79 28.06 65.98 59.15 5 7.00 20.00 55.00 28.72 36.71 66.59 57.61 6 8.00 30.00 25.00 26.00 53.15 65.55 44.02 7 8.00 10.00 25.00 31.37 37.75 68.20 59.81 8 8.00 30.00 55.00 34.37 40.47 72.68 64.15 9 9.00 30.00 40.00 34.39 35.97 68.10 62.26 10 8.00 20.00 40.00 28.34 30.94 63.07 52.54 11 9.00 10.00 40.00 39.51 26.78 69.12 60.58 12 8.00 10.00 55.00 28.87 32.96 70.48 59.94 13 8.00 20.00 40.00 29.48 32.08 63.94 60.55 14 8.00 20.00 40.00 28.20 30.80 62.99 52.46 15 9.00 20.00 55.00 35.52 38.12 69.49 62.89 16 7.00 20.00 25.00 28.42 48.42 60.04 49.51 17 8.00 20.00 40.00 28.20 30.80 63.88 53.35 4 of 12  |     MINTAH eT Al. of FeCl2 (ferrous chloride—2 mM) was added. After 5 min, 0.2 ml 2.7 | Cu2+ chelation assay of ferrozine (5 mM) was added, vortexed, and allowed to react for 10 min at 25°C. The absorbance was then read at 562 nm, and the The ability of HILM hydrolysates/peptides in solution to chelate 2+ inhibition of Iron (II)‐ferrozine complex formation was computed as Cu (prooxidative ion) was measured by the method reported by follows: Zhuang, Tang, and Yuan (2013) with minor modification. For the re‐ action, 1 ml of CuSO4 (2 mM) was mixed with 10% of pyridine (1 ml) ( ) ( ) Ahs−Ac and 0.1% of pyrocatechol violet (20 µl). Following the addition of ICA (%)= 1− ×100 A the hydrolysate (1 ml), change in color (i.e., loss of blue color) was wb observed for 7 min, and absorbance reading was done at 632 nm. where Ahs represent the absorbance of sample, A absorbance of The CCA was then computed as follows: c control (i.e., sample without ferrozine), and Awb the blank (distilled ( ( )) water used in place of sample). Ahs−AwpCCA (%)= 1− ×100 Awb 2.5 | Determination of 1,1‐DPPHRSA where Ahs represent the absorbance of sample, Aws absorbance of The DPPHRSA of the HILMPH hydrolysates was measured with sample without pyrocatechol violet, and Awb the blank sample (distil the technique reported by García‐Moreno et al. (2014) with slight water instead of sample). modification. The hydrolysates (0.25 ml) was added to 0.5 ml of Tris buffer (pH 2.8 | Amino acid evaluation 7.4, 50.0 mM). A 4.25 ml of fresh solution (0.1 mM) DPPH (metha‐ The protein subunits (%) of lyophilized sample (control and soni‐ nol as solvent) was added, vortexed, and the mixture kept in dark cated) at optimum condition was determined as described by (25°C, for 30 min) and the absorbance determined at 515 nm. Blank Marino et al. (2010), with minor modification, using protein subunit was prepared by replacing the hydrolysate with distilled water, and autoanalyzer (S‐433D, GmbH Sykam Co., Germany). The blast elec‐ control was likewise prepared for each hydrolysate sample using tric oven (B 1010‐3, Expt. Instrument Co. Ltd., Shanghai) for hy‐ CH3OH (methanol) instead of the DPPH solution. The DPPHRSA drolysis, and 0.22 µm membrane for sample filtration were applied. was computed with the formula as follows: ( ) ( ) A −A 2.9 | Intrinsic fluorescence examinationhs mc DPPHRSA (%)= 1− ×100 Awb The prepared HILMP hydrolysate samples (obtained at optimized condition—sonicated and control), freeze‐dried, were subjected to where A represent the sample absorbance, A control absorbance, intrinsic fluorescence (Fi) analysis at 24 ± 1°C using 0.05 mg/ml in hs mc and A the blank. 0.1 mol/L buffer (phosphate—PBS, pH 80). Cary Eclipse Fi spectro‐wb photometer (Varian‐Alto Palo Incorporated, USA), with 1 cm cell as regards path length was used. The excitation and emission wave‐ 2.6 | Determination of scavenging activity— length: 279 nm and 280–450 nm was applied; with slit width and Hydroxyl radical (HRSA) scan‐speed: 5 nm, and 10 nm/s in turn. PBS was utilized as spectrum The HRSA of the hydrolysates was estimated using the method re‐ blank, and scan spectra (10) expressed in mean scores were applied. ported by Wang, Wang, Dang, Zheng, and Zhang (2013) with some modifications. Briefly, 1,000 µl of FeSO4 solution (6 mM) was mixed 2.10 | Microstructure analysis with 1,000 µl of hydrolysate and 1,000 µl of H2O2 (6 mM) solu‐ The microstructure of lyophilized HILMPH samples pretreated with tion. The resultant mixture was vortex and left for 15 min (25°C). and without ultrasonication, obtained at optimized condition for all Subsequently, to the mixture, 1,000 µl of a lipophilic monohydroxy‐ the responses were analyzed using a smart light microscope (BX43‐ benzoic acid (salicylic acid, 6 mM) was added and the absorbance (at Olympus, Tokyo, Japan) installed with a V350D digital camera. The wavelength 510 nm) was read after 30 min. The blank preparation method outlined by Alenyorege and colleagues (Alenyorege et al., was done by substituting the sample with distilled water (H2O). The 2018) was used; and micrographs were captured at ambient tem‐ HRSA of the hydrolysate was calculated using equation as follows: perature (22 + 1°C) with 400× magnification. ( ) ( ) Ahs−Aws HRSA (%)= 1− ×100 A 2.11 | Statistical analysiswb All treatments were done thrice and the results were presented where Ahs represent the absorbance of sample, Aws absorbance of as mean values. Statistical analysis was worked out using Design sample devoid of of salicyclic acid, and Awb the blank. Expert software (v8061). MINTAH eT Al.      |  5 of 12 The model accuracy was evaluated using: the F‐test, the lack confirm the model adequacy, the lack of fit values were shown to of fit test, and R2 (coefficient of determination) at 0.5, 0.001, and be statistically insignificant (Table 2). 0.0001% significance levels. The optimal pretreatment conditions were achieved by plotting the response surface‐plots of each re‐ 3.1 | Effect of sonication parameters on ICA of sponse (ICA, DPPHRSA, HRSA, and CCA). HILMP hydrolysates Metal ions (e.g., iron—Fe) are required for physiological roles in hu‐ 3  | RESULTS AND DISCUSSION mans; but when in excess may cause serious cell/ tissue damage. Excess ferrous ions (Fe2+) in the body facilitates the formation of ox‐ Response surface methodology (RSM) is an arithmetic technique ygen containing radicals and consequently causing several diseases built on the appropriateness of quadratic (polynomial) equation to (Lobo et al., 2010). Antioxidants will, in this regards, be needed to an experimental data (Bezerra, Santelli, Oliveira, Villar, & Escaleira, create a balance with the free radical production. Thus, in this study, 2008). It well explains the nature of data set aiming to make arith‐ the ICA of HILMP hydrolysates was considered. metical prediction/forecasting. Compared to conventionally used The ICA of HILMP hydrolysates obtained through sonication single‐factor optimization, RSM is more favorable due to the fact pretreatments varied from 28.20–39.51% (Table 1). Figure 1 pres‐ that it saves time, sample usage, and space (Lee et al., 2012). ents the influence of sonication treatment conditions on ICA of the In this study, we have applied a RSM (Box–Behnken design) hydrolysate. From the results (based on p‐values), it is obvious that with three factors tested at three levels, with the aim of obtain‐ the principal factor influencing ICA, positively (p < 0.0001), was ing HILMP hydrolysates with optimum ICA, DPPHRSA, HRSA, and pH. Thus, ICA of the HILMP hydrolysates increased with increas‐ CCA. The values of the three factors considered in the present ing pH to a maximum PH of 9 (Figure 1a; Table 1). The influence study are shown in Table 1 together with the experimentally mea‐ of time was, however, not significant though positive. It was also sured values for the four (4) responses (DPPHRSA, ICA, HRSA, and evident in Figure 1b that the interaction between pH and tempera‐ CCA) for each experimental run as specified in the design. The ture positively increased the ICA of HILMP hydrolysates. That is, measured values of the response variables were fitted to polyno‐ increasing pH and temperature, together increased the ICA of the mial (quadratic) model, and ANOVA was utilized in assessing the hydrolysates. influence of the studied variables, factor interactions, and signif‐ Compared to pH, temperature showed lower positive, but yet icance of the model in arithmetic terms. The p‐ and F values of significant effect on the ICA. As illustrated in Figure 1b,c, ICA in‐ regression coefficients for the dependent (response) variables are creased with increasing temperature. Temperature is one factor presented in Table 2. From the significant p‐values of the models, that is known to influence, positively, most extraction processes it could be inferred that the quadratic polynomial model presented and/or activity of bioactive compounds; it exhibits remarkable ef‐ good estimations for the measured responses—demonstrating the fect on mass‐transfer processes. At increased temperatures, cellular fitness of the model. This was in agreement with the R2 (multiple structure degradation increases which makes cells more permeable determination coefficient) values which were 0.98, 0.99, 0.98, and (Jovanović et al., 2017). Thus, sonication treatments at increasing 0.88 for ICA, DPPHSA, HRSA, and CCA, respectively. To further temperature conditions favored the ICA of the hydrolysates. Hence, TA B L E 2  ANOVA for measured responses from Box and Behnken design Fe2+ DPPH HRSA Cu2+ F value p‐value F value p‐value F value p‐value F value p‐value Model 52.33 <0.0001 172.28 <0.0001 46.62 <0.0001 10.17 0.0155 A (pH) 100.57 <0.0001 13.27 0.0083 32.88 0.0007 11.95 0.0062 B (Time) 0.11 0.7469 426.09 <0.0001 0.035 0.8559 10.33 0.0093 C (Temp) 26.21 0.0014 277.87 <0.0001 148.05 <0.0001 18.34 0.0016 AB 52.53 0.0002 0.6 0.4632 2.81 0.1379 10.77 0.0083 AC 5.27 0.0553 20.67 0.0026 0.12 0.7375 0.21 0.6587 BC 69.15 <0.0001 29.48 0.0010 14.36 0.0068 9.44 0.0118 A2 133.27 <0.0001 7.25 0.031 0.058 0.8165 0.015 0.9073 B2 67.30 <0.0001 1.41 0.2742 187.21 <0.0001 0.56 0.4774 C2 12.50 0.0095 757.13 <0.0001 25.61 0.0015 0.64 0.4487 Lack of fit 1.55 0.3334 0.90 0.5143 2.66 0.1844 1.06 0.4575 R2 0.9854 0.9955 0.9836 0.8809 Note: p‐values < 0.05 are statistically significant. 6 of 12  |     MINTAH eT Al. F I G U R E 1  Response surface plot showing the effect of sonication (pretreatment) parameters on ICA of HILMP hydrolysate samples from the present study findings, we can infer that ICA of HILMP hy‐ highest DPPHRSA was achieved with pH 7, sonication time of 20 min, drlysates increases with increasing sonication temperature. and temperature of 25°C (Table 1). However, the lowest DPPHRSA Similar observation on the effect of increasing temperature has was obtained under the sonication conditions: pH 9, 10 min, and 40°C. been indicated by other scholars for somewhat different studies The investigated pretreatment conditions (pH and time) showed sig‐ but also on bioactive compounds (Arruda, Pereira, & Pastore, 2017; nificant positive impact on DPPHRSA (Table 2; Figure 2a–c). Chanioti & Tzia, 2017). Temperature effect was also significant, but with a decreasing The current study results also showed that the interactions be‐ activity (at some point) from 25°C to the mid‐ranged temperatures tween pH and time, as well as that between temperature and time (37–43°C), and then increase from this point to 55°C (Figure 2b,c). significantly influenced ICA. Furthermore, all the quadratic terms of Contrary to ICA, sonication treatment time showed the highest and the model showed significant effect on ICA of the hydrolysates. significant positive influence on the % DPPHRSA (Figure 2c). Other To study the association between the factor and dependent vari‐ studies using ultrasound aided extraction of bioactive compounds ables, a second‐degree quadratic equation was generated from the have indicated the positive effect of time in achieving best results regression analysis. The regression equation for describing the effi‐ (Rodrigues, Fernandes, de Brito, Sousa, & Narain, 2015). ciency of sonication pretreatment in achieving the optimum ICA of Our results also showed that, the interactions between pH and HILMP hydrolysates was: temperature, as well as time and temperature significantly impacted DPPHRSA of the hydrolysates, while remaining limits were insignif‐ ICA(%) =28.67+2.32A−0.078B+1.18C icant (Table 2). −2.37 +0.75 +2.72 +3.68 2AB AC BC A The quadratic equation for predicting and/or describing the effi‐ +2.62 2B −1.13 2C cacy of the sonication pretreatment in achieving optimum DPPHRSA of the HILMP hydrolysates was: On the optimum conditions for obtaining maximized ICA of HILMP hydrolysates, the study results showed that sonication pre‐ DPPHRSA (%) =30.93−0.94A+5.30B−4.28C−0.28AB treatment conditions could be set at pH 8.99, time 10.00, and tem‐ + 21.65AC−1.97BC+0.95A perature 35.34°C; with a maximum predicted value of 39.78%. + 2 20.42B +9.74C 3.2 | Effect of sonication parameters on The present study results showed that the optimum pretreat‐ DPPHRSA of HILMP hydrolysates ment conditions for obtaining maximal DPPHRSA of HILMP hydroly‐ sates were: pH 7.17, time 29.40 min, and temperature 25.09°C. The The usual cell function is characterized by the formation of free radi‐ maximum predictive value, however, was 55.03%. cals; but when in excess, may catalyze many diseases. To inhibit free radical catalyzed cell damage, antioxidants are thus needed (Young 3.3 | Effect of sonication parameters on HRSA of & Woodside, 2001). In this regards, 1,1‐DPPH technique was ap‐ HILMP hydrolysate plied in evaluating the antioxidant potential of HILMP hydrolysates. It (i.e., 1,1‐DPPH technique) is generally used in analyzing the scav‐ Hydroxyl radical is a significant oxygen containing free radicals in enging strength of protein‐derived hydrolysates due to its precision many disease forms as they can destroy important biological mol‐ (Laroque, Chabeaud, & Guérard, 2008). ecules (e.g., DNA, fats, starches, and proteins) (Young & Woodside, The DPPHRSA of the HILMP hydrolysates obtained using sonica‐ 2001). To control their action in humans, antioxidants from external tion pretreatment (in this study) ranged from 26.78 to 48.42%. And the sources may be required. MINTAH eT Al.      |  7 of 12 F I G U R E 2  Response surface plot showing the effect of sonication (pretreatment) parameters on DPPHRSA of HILMP hydrolysates F I G U R E 3  Response surface plot showing the effect of sonication (pretreatment) parameters on HRSA of HILMP hydrolysates Accordingly, the present study looked at the effect of three inde‐ HRSA (%) =63.38+1.30A−0.043B+2.76C pendent variables on the HRSA of HILMP hydrolysates. Our results − 20.54AB−0.11AC+1.21BC−0.075A showed that two of the variables, pH (A) and temperature (C), were 2 2 highly significant (Table 2). Temperature level was found to have the +4.27B +1.58C main effect on HRSA (Figure 3c), suggesting that temperature tend to increase the HRSA of HIMLP hydrolysates. Nonetheless, except The study results also showed that, the optimum conditions for A2 (p > 0.05), all the other quadratic terms B2 and C2, had signif‐ for sonication pretreatment the could yield predictively maximal icant effect on HRSA. Further, the results revealed that the inter‐ (73.57%) HRSA of the hydrolysates are: pH 8.96, time 29.91 min, and active term BC (p < 0.05) had positive effect on HRSA (Figure 3c); temperature 54.88°C. whereas the other interactions, though showed positive effect, were not statistically significant. 3.4 | Effect of sonication parameters on CCA of In the current work, the HRSA of the hydrolysates varied from HILMP hydrolysate 60.04 to 72.68%. The experimental conditions that showed high 2+ 2+ HRSA were: A = 8, B = 30 min, and C = 55°C; whereas the conditions: Just as in the case of Fe , excess Cu in the body facilitates the A = 7, B = 20 min, and C = 25°C recorded lowest HRSA. Deducing production of oxygen containing radicals and accordingly may cause from this is that, lower temperature and pH under pretreatment several tissue damages (Lobo et al., 2010). External sources of an‐ (sonication) conditions may not be desirable in generating HILMP tioxidants may therefore be required to balance the action of pro‐ hydrolysates with highest HRSA. duced free radical. This study, thus examined the CCA of HILMP The regression equation for explaining the efficacy of the ultra‐ hydrolysates. sound pretreatments in generating HILMP hydrolysates with opti‐ Analysis of the CCA model showed that all the linear terms (A, B, mal HRSA is as follows: and C) were significant variables. Temperature and pH had positive 8 of 12  |     MINTAH eT Al. F I G U R E 4  Response surface plot showing the effect of sonication (pretreatment) parameters on CCA of HILMP hydrolysate samples effect on the CCA of the hydrolysates, indicating that an increase in conditions for the generation of hydrolysates with antioxidant ac‐ the temperature and/or pH favored the chelating activity of the hy‐ tivity. The optimal pretreatment/sonication conditions (for all four drolysates. By contrast, time showed a negated effect (Figure 4a,c) responses) were: pH 9, time = 29.84 min, and temperature = 54.93. and this suggests that the CCA of the hydrolysates may be more The predicted responses (ICA = 38.26%, DPPHRSA = 41.39%, favorable at shorter sonication (pretreatment) time. HRSA = 73.55%, and CCA = 72.84%) under the optimal condi‐ The CCA of the HILMP hydrolysates obtained through ultra‐ tions were compared to experimental values (ICA = 37.84%, sound pretreatment varied from 39.48–64.15%; and the treatment DPPHRSA = 43.19%, HRSA = 71.01%, and CCA = 68.93%). The ex‐ with the highest CCA was achieved under the following condition: perimental data compared very well with the projected values; sug‐ A = 8, B = 30 min, and C = 55°C. The least CCA was, however, ob‐ gestive that the selected RSM (BBD) model was successfully applied tained under the following pretreatment limits: A = 7, B = 30 min, for ultrasound pretreatment of HILMP in order to generate hydro‐ and C = 40°C. This may be that sonication pretreatment under lower lysates with ICA, DPPHRSA, HRSA, and CCA. pH conditions may not be that suitable for the generation of HILMP Free radicals are largely unstable and reactive. In situations hydrolysates with high CCA thereafter. This could be supported by where free radicals engulf an organ system's ability to control them, the result that the second most linear term/factor influencing (posi‐ it results in an oxidative stress (OS). Consequently, they (free radi‐ tively) CCA of the hydrolysates was pH (Table 2). cals) undesirably alter the structure of proteins, fats, and DNA and Our results also indicated that the interactions among A and B initiate a couple of diseases in humans. To manage the OS, the uti‐ (pH and time), and B and C (time and temperature) significantly in‐ lization of antioxidants from external sources are recommended fluenced the CCA of the hydrolysates. Thus, as time decreases and (Lobo et al., 2010). Also, metal ion (e.g., Fe2+ and Cu2+) acts as pos‐ pH or temperature increases, the CCA of the hydrolysates also in‐ itive catalyst in the generation of reactive oxygen leading to same/ creases. The quadratic levels (A, B, and C) did not statistically influ‐ similar health conditions in humans. The present study findings ence the CCA of the hydrolysates. (DPPHRSA and HRSA, and/or ICA and CCA) suggest that the hy‐ From our study results, the predictive equation for explaining the drolysates derived from HILMP may be beneficial in controlling OS efficiency of the ultrasonication pretreatments in generating HILMP in humans. hydrolysates with optimum CCA was: CCA (%) =54.27+3.98A−3.70B+4.93C+5.34AB 3.6 | Comparison of sonication and conventional +0.74AC+ 25.00BC−0.21A pretreatments on ICA, DPPHRSA, HRSA, and CCA + 2 21.31B +1.40C Subsequent to validation of the model, the experimental val‐ The optimum parameters for the ultrasound pretreatment that ues (ICA = 37.84%, DPPHRSA = 43.19%, HRSA = 71.01%, and could yield 67.54% CCA of the hydrolysates, however, were: pH CCA = 68.93%) obtained using sonication pretreatment were com‐ 8.89, time 27.73 min, and temperature 51.76°C. pared to a conventional one (control) under the set optimized condi‐ tions: pH 9, time = 29.84 min, temperature = 54.93. The conventional 3.5 | Model verification and validation pretreatment (control) under the said parameters was done by re‐ placing ultrasound with a JJ‐1 impeller agitator (J. Xichengxinrui Inst. In order to verify the reliability of the model for all the investi‐ Co., Jiangsu, China). Regarding the outcome, the sonication pretreat‐ gated responses (ICA, DPPHRSA, HRSA, and CCA), an experiment ment/method resulted in hydrolysates with higher ICA, DPPHRSA, was done under the optimal (predictive) sonication pretreatment HRSA, and CCA than the conventional method. The ICA, DDHRSA, MINTAH eT Al.      |  9 of 12 HRSA, and CCA using the conventional method were 28.98, 31.53, likened to control (Table 3); and this support why sonication preced‐ 58.71, and 56.34%, respectively. It was noted that the pretreatment ing enzyme hydrolysis resulted in improved antioxidative activity. by sonication resulted in higher responses than the conventional treatment. 3.8 | F of HIL protein hydrolysates The higher ICA, DPPHRSA, and CCA of the hydrolysate, ob‐ i served with the ultrasound pretreatment, could be associated with Spectra from Fi are linked to protein subunits: Y (tyrosine), F (pheny‐ the sonication waves facilitating the unfolding of the HILMP and lalanine), and W (tryptophan) (Ma et al., 2011). Hence fluorescence consequently exposing hydrophobic ends and making it possible for spectra is used in illustrating the alterations of protein structure an enhanced enzyme action which eventually lead to the production of hydrolysates with improved antioxidant (bioactive) activity than TA B L E 3  Amino acid scores of control and sonication treated the traditional approach. This is in agreement with Gülseren, Güzey, HILMP hydrolysate (%) Bruce, and Weiss (2007) when they reported that sonication pre‐ treatment causes unfolding of proteins, an outcome which is good Amino acid Control Sonicated sample for enzyme action. Most importantly, to buttress the observed im‐ Asp 8.97 ± 0.17a 9.31 ± 0.14a provement in the antioxidant activity of the sonicated samples, is Thr 3.47 ± 0.05a 3.96 ± 0.12b how ultrasound woks (i.e., it mechanism in extraction of target com‐ Ser 3.08 ± 0.02a 3.17 ± 0.11a pounds). During ultrasonication treatment, cavitation bubbles (thin Glu 12.6 ± 0.38a 12.51 ± 0.34a liquid sphere enfolding air/gas) swing back and forth and collapse, Pro 5.10 ± 0.15a 5.51 ± 0.11b resulting in some physical effects such as shock waves, turbulence, Gly 5.64 ± 0.33a 5.98 ± 0.29a microjets, and shear thrusts (Kadam, Tiwari, Álvarez, & O’Donnell, Ala 7.76 ± 0.18a 8.05 ± 0.21a 2015; Tiwari, 2015). When the circulatory motion of the cavitation Val 2.98 ± 0.15a 3.17 ± 0.07a bubbles is intense as a function of the formation and collapse of bub‐ Met 2.20 ± 0.07a 2.38 ± 0.12a bles (rarefaction and compression), it results in what is termed as a a microstreaming (Margulis & Margulis, 2004). Thus, the cavitation in‐ Ile 2.36 ± 0.18 2.31 ± 0.12 a b fluence (shock waves/microjets) results in disruption (pore creation Leu 5.14 ± 0.22 5.68 ± 0.18 a a on surfaces) of cell wall of substrates/food samples, and decreased Tyr 4.80 ± 0.33 5.41 ± 0.35 particle size; which consequently improves permeability of the food Phe 3.84 ± 0.09a 4.13 ± 0.12b matrix, and mass transfer (Tiwari, 2015). Implicit from this is that, His 3.50 ± 0.18a 4.11 ± 0.24b the cellular matrix of the sonicated samples in the current study, be‐ Lys 3.10 ± 0.04a 3.61 ± 0.09b came more permeable, allowing solvent into its internal structure, Arg 3.04 ± 0.23a 1.90 ± 0.08b enhancing enzyme action, causing target components to dissolve in HAA‡  32.85 35.19 solution, and thus facilitating the production of hydrolysates with ‡Hydrophobic amino acid (Ala, Val, Leu, Ile, Phe, Pro, Thr, and Met); improved antioxidant activity. This could be supported with protein means with different superscript letters (in a row), are significantly dif‐ subunit scores, fluorescence intensity, and microstructural data. ferent (p < 0.05). 3.7 | Amino acid scores Proteins with less than 20 or 16 down to 5 subunits demonstrate potent antioxidative activity (Zhou et al., 2015). Also, the type, amount and order of protein subunit also have effect on the bioac‐ tivity of hydrolysates. The amino acid scores of control and sonica‐ tion treated samples are presented in Table 3. Glu is the main subunit in the control (conventional) hydrolysate. The HILMP hydrolysate (pretreated by sonication) showed the highest score of indispensa‐ ble subunits (His, Ile, Leu, Lys, Met, Phe, Thr, and Val), likened the control. Hydrophobic units (Phe, Pro, Val, and Ile), normally linked to bioactive functions such as antioxidant activity (Megías et al., 2004), were enhanced when hydrolyzed with Alcalase, subsequent to soni‐ cation. Thus, the sonicated HILMP hydrolysate may contain protein with significant 5–16 subunits with C‐terminus hydrophobic building blocks than control. This is due to the preferential cleavage of hy‐ drophobic units at the C‐terminus of proteins by Alcalase (Jia et al., F I G U R E 5   Fluorescence spectra of control and ultrasound 2010). The hydrophobic subunit in sonicated hydrolysate was higher treated HILMP hydrolysates 10 of 12  |     MINTAH eT Al. F I G U R E 6  Micrograph of the HILMP samples pretreated with (a) and without (b) sonication (Zhao, Dong, Li, Kong, & Liu, 2015) during product processing or stor‐ best ultrasound pretreatment conditions that could result in hydro‐ age (Keerati‐u‐rai, Miriani, Iametti, Bonomi, & Corredig, 2012). In this lysates with ICA of 38.26, DPPHRSA of 41.39, HRSA of 73.55, and study, samples treated with ultrasound showed a shift in the maximal CCA of 71.01%. Compared to conventional pretreatment, the hydroly‐ emission peak from 360 nm (control) to 365 nm, implying a rise in the sis preceded by ultrasonication resulted in hydrolysates with higher an‐ polarity of subunits like W and F as a consequence of molecular altera‐ tioxidant activity (ICA, DPPHRSA, HRSA, and CCA). The study showed tion (Figure 5). Further, the Fi peak of the ultrasonic treated HILMP hy‐ that the use of sonication pretreatment in the enzymolysis of HILMP to drolysates was intense than the conventional (control). It follows that generate hydrolysates with antioxidant components was efficient, but‐ sonication preceding ezymolysis modified the conformation of HILMP. tressed by amino acid scores, Fi spectra, and micrographs. This investi‐ That is, breaking hydrophobic interactions and consequently causing gation could help the food and/or pharmaceutical industry to develop release (unfolding) of hydrophobic groups from the interior of mole‐ new bioactive products/functional foods from HILMP hydrolysates. cule (Gülseren et al., 2007; Jambrak, Mason, Lelas, Herceg, & Herceg, Consequently, further studies on the in vivo functional properties of 2008), reflecting the intense fluorescence of the HILMP hydrolysate the HILMP hydrolysates is recommended. relative to control. This is consistent with the observed variations in the antioxidant properties of the hydrolysates, with ultrasonication treated samples demonstrating enhanced activity than control. ACKNOWLEDG MENT This research was supported by the Primary Research and 3.9 | Microstructure Development Plan of Jiangsu Province (BE2016352 and BE2016355). And the Zhenjiang “1+1+N” New Agricultural Technology Extension Microstructures (photomicrographs) of the HILMP samples pre‐ Project. treated with sonication, and without sonication (control) obtained at the optimized condition for the experimental responses are shown in Figure 6. The micrograph of the control showed distinc‐ CONFLIC T OF INTERE S T tive compact or intact morphology/particles. However, the spaces The authors have declared no conflicts of interest for this article. between particles of the sonication treated samples appear sepa‐ rated/loose—more distracted. Deducing from this is that, the cavi‐ tation effect produced at the set ultrasonication conditions was ORCID good enough to create a sponge effect (alternating compressions Benjamin Kumah Mintah https://orcid.org/0000‐0001‐5337‐5007 and expansions) resulting in the loose structure of the samples pretreated by sonication; and this could further explain why the Ronghai He https://orcid.org/0000‐0002‐0904‐0522 sonicated samples demonstrated superior antioxidant (bioactive) Mokhtar Dabbour https://orcid.org/0000‐0001‐6109‐9065 capacity in the current investigation. Moses Kwaku Golly http://orcid.org/0000‐0002‐5573‐4103 4  | CONCLUSION R E FE R E N C E S Alenyorege, E. A., Ma, H., Ayim, I., Aheto, J. H., Hong, C., & Zhou, C. In this study, response surface methodology was successfully used to (2018). Reduction of Listeria innocua in fresh‐cut Chinese cabbage set the optimized conditions for ultrasound pretreatment of HILMP for by a combined washing treatment of sweeping frequency ultrasound enzymolysis. The investigated pretreatment parameters (pH, time, and and sodium hypochlorite. LWT – Food Science and Technology, 101, 410–418. https ://doi.org/10.1016/j.lwt.2018.11.048 temperature) impacted on the antioxidant activity (ICA, DPPHRSA, Arruda, H. S., Pereira, G. A., & Pastore, G. M. (2017). Optimization HRSA, and CCA) of the hydrolysates. With the Box–Behnken design, of extraction parameters of total phenolics from Annona crassi‐ optimum pH, time, and sonication temperature were predicted for flora mart. (araticum) fruits using response surface methodology. MINTAH eT Al.      |  11 of 12 Food Analytical Methods, 10(1), 100–110. https ://doi.org/10.1007/ Jovanović, A. A., Đorđević, V. B., Zdunić, G. M., Pljevljakušić, D. S., s12161‐016‐0554‐y Šavikin, K. P., Gođevac, D. M., & Bugarski, B. M. (2017). Optimization Barragan‐Fonseca, K. B., Dicke, M., & van Loon, J. J. A. (2017). Nutritional of the extraction process of polyphenols from Thymus serpyllum L. value of the black soldier fly (Hermetia illucens L.) and its suitability herb using maceration, heat‐ and ultrasound‐assisted techniques. as animal feed—A review. Journal of Insects as Food and Feed, 3(2), Separation and Purification Technology, 179, 369–380. https ://doi. 105–120. https ://doi.org/10.3920/JIFF2 016.0055 org/10.1016/j.seppur.2017.01.055 Barroso, F. G., de Haro, C., Sánchez‐Muros, M. J., Venegas, E., Martínez‐ Kadam, S. U., Tiwari, B. K., Álvarez, C., & O’Donnell, C. P. (2015). Sánchez, A., & Pérez‐Bañón, C. (2014). The potential of various in‐ Ultrasound applications for the extraction, identification and deliv‐ sect species for use as food for fish. Aquaculture, 422–423, 193–201. ery of food proteins and bioactive peptides. Trends in Food Science and https ://doi.org/10.1016/j.aquac ulture.2013.12.024 Technology, 46(1), 60–67. https ://doi.org/10.1016/j.tifs.2015.07.012 Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. Karaś, M., Baraniak, B., Rybczyńska, K., Gmiński, J., Gaweł‐Bęben, A. (2008). Response surface methodology (RSM) as a tool for opti‐ K., & Jakubczyk, A. (2015). The influence of heat treatment of mization in analytical chemistry. Talanta, 76(5), 965–977. https: //doi. chickpea seeds on antioxidant and fibroblast growth‐stimulat‐ org/10.1016/j.talan ta.2008.05.019 ing activity of peptide fractions obtained from proteins digested Chandrapala, J., Oliver, C., Kentish, S., & Ashokkumar, M. (2012). under simulated gastrointestinal conditions. International Journal Ultrasonics in food processing. Ultrasonics Sonochemistry, 19(5), of Food Science and Technology, 50(9), 2097–2103. https ://doi. 975–983. https ://doi.org/10.1016/j.ultso nch.2012.01.010 org/10.1111/ijfs.12872 Chanioti, S., & Tzia, C. (2017). Optimization of ultrasound‐assisted ex‐ Karaś, M., Jakubczyk, A., Szymanowska, U., Złotek, U., & Zielińska, E. traction of oil from olive pomace using response surface technol‐ (2017). Digestion and bioavailability of bioactive phytochemicals. ogy: Oil recovery, unsaponifiable matter, total phenol content and International Journal of Food Science and Technology, 52(2), 291–305. antioxidant activity. LWT ‐ Food Science and Technology, 79, 178–189. https: //doi.org/10.1111/ijfs.13323 https: //doi.org/10.1016/j.lwt.2017.01.029 Keerati‐u‐rai, M., Miriani, M., Iametti, S., Bonomi, F., & Corredig, M. Clemente, A. (2001). Enzymatic protein hydrolysates in human nutrition. (2012, May). Structural changes of soy proteins at the oil‐water Trends in Food Science and Technology, 11(7), 254–262. https ://doi. interface studied by fluorescence spectroscopy colloids and sur‐ org/10.1016/S0924‐2244(01)00007‐3 faces B: Biointerfaces structural changes of soy proteins at the García‐Moreno, P. J., Batista, I., Pires, C., Bandarra, N. M., Espejo‐Carpio, oil—Water interface studied by fluorescence spectroscopy. Colloids F. J., Guadix, A., & Guadix, E. M. (2014). Antioxidant activity of and Surfaces B: Biointerfaces, 93, 41–48. https: //doi.org/10.1016/ protein hydrolysates obtained from discarded mediterranean fish j.colsu rfb.2011.12.002 species. Food Research International, 65(PC), 469–476. https: //doi. Laroque, D., Chabeaud, A., & Guérard, F. (2008). Antioxidant capacity org/10.1016/j.foodr es.2014.03.061 of marine protein hydrolysates. In I. J. P. Bergé (Ed.), Added value Gobbetti, M., Stepaniak, L., Angelis, M. D., Corsetti, A., Cagno, R. D., & to fisheries waste (pp. 147–161). Kerala, India: Transworld Research Stepaniak, L. … Di, R. (2015, December). Latent bioactive peptides Network. in milk proteins: Proteolytic activation and significance in dairy pro‐ Lee, A. Y., Kim, H. S., Jo, J. E., Kang, B. K., Moon, B. C., Chun, J. M., cessing. Critical Reviews in Food Science and Nutrition, 8398, 37–41. … Kim, H. K. (2012). Optimization of extraction condition for https: //doi.org/10.1080/104086 9029 0825538 major iridoid components in fruit of corni (Cornus officinalis) by Gülseren, I., Güzey, D., Bruce, B. D., & Weiss, J. (2007). Structural and UPLC‐PDA using response surface methodology. Food Science functional changes in ultrasonicated bovine serum albumin solutions. and Biotechnology, 21(4), 1023–1029. https ://doi.org/10.1007/ Ultrasonics Sonochemistry, 14(2), 173–183. https: //doi.org/10.1016/ s10068‐012‐0133‐y j.ultson ch.2005.07.006 Lobo, V., Patil, A., Phatak, A., & Chandra, N. (2010). Free radicals, antioxi‐ Halim, N. R. A., & Sarbon, N. M. (2017). A response surface approach dants and functional foods: Impact on human health. Pharmacognosy on hydrolysis condition of eel (Monopterus Sp.) protein hydrolysate Reviews, 4(8), 118. https ://doi.org/10.4103/0973‐7847.70902 with antioxidant activity. International Food Research Journal, 24(3), Ma, H., Huang, L., Jia, J., He, R., Luo, L., & Zhu, W. (2011). Effect of en‐ 1081–1093. ergy‐gathered ultrasound on Alcalase. Ultrasonics Sonochemistry, Hall, F. G., Jones, O. G., O’Haire, M. E., & Liceaga, A. M. (2017). Functional 18(1), 419–424. https ://doi.org/10.1016/j.ultso nch.2010.07.014 properties of tropical banded cricket (Gryllodes sigillatus) protein hy‐ Margulis, M. A., & Margulis, I. M. (2004). Mechanism of sonochemi‐ drolysates. Food Chemistry, 224, 414–422. https ://doi.org/10.1016/ cal reactions and sonoluminescence. High Energy Chemistry, 38(5), j.foodc hem.2016.11.138 285–294. https ://doi.org/10.1023/B:HIEC.000004 1338.11770.74 Jambrak, A. R., Mason, T. J., Lelas, V., Herceg, Z., & Herceg, I. L. (2008). Marino, R., Iammarino, M., Santillo, A., Muscarella, M., Caroprese, M., Effect of ultrasound treatment on solubility and foaming proper‐ & Albenzio, M. (2010). Technical note: Rapid method for determina‐ ties of whey protein suspensions. Journal of Food Engineering, 86(2), tion of amino acids in milk. Journal of Dairy Science, 93(6), 2367–2370. 281–287. https ://doi.org/10.1016/j.jfoode ng.2007.10.004 https ://doi.org/10.3168/jds.2009‐3017 Jamil, N. H., Halim, N. R. A., & Sarbon, N. M. (2016). Optimization of McCarthy, A., O’Callaghan, Y., & O’Brien, N. (2013). Protein hydrolysates enzymatic hydrolysis condition and functional properties of eel from agricultural crops—Bioactivity and potential for functional food (Monopterus sp.) protein using response surface methodology (RSM) development. Agriculture, 3(1), 112–130. https: //doi.org/10.3390/ Optimization of enzymatic hydrolysis condition and functional prop‐ agricu lture 3010112 erties of eel (Monopterus sp.) protei. International Food Research Megías, C., Yust, M. D. M., Pedroche, J., Lquari, H., Girón‐calle, J., Journal, 23(1), 1–9. Manuel, A., … Vioque, J. (2004). Purification of an ACE inhibitory Jia, J., Ma, H., Zhao, W., Wang, Z., Tian, W., Luo, L., & He, R. (2010). The peptide after hydrolysis of sunflower (Helianthus annuus L.) protein use of ultrasound for enzymatic preparation of ACE‐inhibitory pep‐ isolates. Journal of Agricultural and Food Chemistry, 52, 1928–1932. tides from wheat germ protein. Food Chemistry, 119(1), 336–342. https: //doi.org/10.1021/jf0347 07r https ://doi.org/10.1016/j.foodc hem.2009.06.036 Megías, C., Pedroche, J., Yust, M. M., Girón‐Calle, J., Alaiz, M., Millán, F., Jian, S., Wenyi, T., & Wuyong, C. (2008). Ultrasound‐accelerated enzy‐ & Vioque, J. (2007). Affinity purification of copper chelating peptides matic hydrolysis of solid leather waste. Journal of Cleaner Production, from chickpea protein hydrolysates. Journal of Agricultural and Food 16(5), 591–597. https ://doi.org/10.1016/j.jclep ro.2006.12.005 Chemistry, 55(10), 3949–3954. https ://doi.org/10.1021/jf063 401s 12 of 12  |     MINTAH eT Al. Pan, Z., Qu, W., Ma, H., Atungulu, G. G., & McHugh, T. H. (2012). activity, amino acid composition and functional properties. BMC Continuous and pulsed ultrasound‐assisted extractions of antiox‐ Research Notes, 6(1), 197. https: //doi.org/10.1186/1756‐0500‐6‐197 idants from pomegranate peel. Ultrasonics Sonochemistry, 19(2), Young, I., & Woodside, J. (2001). Antioxidants in health and disease. 365–372. https ://doi.org/10.1016/j.ultso nch.2011.05.015 Journal of Clinical Pathology, 54(3), 176–186. https ://doi.org/10.1136/ Perron, N. R., & Brumaghim, J. L. (2009). A review of the antioxidant jcp.54.3.176 mechanisms of polyphenol compounds related to iron binding. Cell Zhang, H., Wang, P., Zhang, A.‐J., Li, X., Zhang, J.‐H., Qin, Q.‐L., & Wu, Biochemistry and Biophysics, 53(2), 75–100. https: //doi.org/10.1007/ Y.‐J. (2016). Antioxidant activities of protein hydrolysates obtained s12013‐009‐9043‐x from the housefly larvae. Acta Biologica Hungarica, 67(3), 236–246. Purschke, B., Meinlschmidt, P., Horn, C., Rieder, O., & Jäger, H. (2018). https: //doi.org/10.1556/018.67.2016.3.2 Improvement of techno‐functional properties of edible insect pro‐ Zhang, M., Mu, T. H., Wang, Y. B., & Sun, M. J. (2012). Evaluation of free tein from migratory locust by enzymatic hydrolysis. European Food radical‐scavenging activities of sweet potato protein and its hydro‐ Research and Technology, 244(6), 999–1013. https ://doi.org/10.1007/ lysates as affected by single and combination of enzyme systems. s00217‐017‐3017‐9 International Journal of Food Science and Technology, 47(4), 696–702. Qu, W., Ma, H., Liu, B., He, R., Pan, Z., & Abano, E. E. (2013). Enzymolysis https ://doi.org/10.1111/j.1365‐2621.2011.02895.x reaction kinetics and thermodynamics of defatted wheat germ pro‐ Zhao, J., Dong, F., Li, Y., Kong, B., & Liu, Q. (2015). Effect of freeze‐thaw tein with ultrasonic pretreatment. Ultrasonics Sonochemistry, 20(6), cycles on the emulsion activity and structural characteristics of soy 1408–1413. https ://doi.org/10.1016/j.ultson ch.2013.04.012 protein isolate. Process Biochemistry, 50(10), 1607–1613. https ://doi. Qu, W., Pan, Z., & Ma, H. (2010). Extraction modeling and activities of an‐ org/10.1016/j.procbi o.2015.06.021 tioxidants from pomegranate marc. Journal of Food Engineering, 99(1), Zhao, X., Vázquez‐Gutiérrez, J. L., Johansson, D. P., Landberg, R., & 16–23. https ://doi.org/10.1016/j.jfoode ng.2010.01.020 Langton, M. (2016). Yellow mealworm protein for food purposes— Rodrigues, S., Fernandes, F. A. N., de Brito, E. S., Sousa, A. D., & Narain, Extraction and functional properties. PLoS ONE, 11(2), 1–17. https :// N. (2015). Ultrasound extraction of phenolics and anthocyanins from doi.org/10.1371/journ al.pone.0147791 jabuticaba peel. Industrial Crops and Products, 69, 400–407. https: // Zhou, C., Hu, J., Ma, H., Yagoub, A. E. A., Yu, X., Owusu, J., … Qin, X. doi.org/10.1016/j.indcr op.2015.02.059 (2015). Antioxidant peptides from corn gluten meal : Orthogonal Ruiz‐Ruiz, J., Dávila‐Ortíz, G., Chel‐Guerrero, L., & Betancur‐Ancona, design evaluation. Food Chemistry, 187, 270–278. https: //doi. D. (2013). Angiotensin i‐converting enzyme inhibitory and anti‐ org/10.1016/j.foodc hem.2015.04.092 oxidant peptide fractions from hard‐to‐cook bean enzymatic hy‐ Zhuang, H., Tang, N., & Yuan, Y. (2013). Purification and identification drolysates. Journal of Food Biochemistry, 37(1), 26–35. https ://doi. of antioxidant peptides from corn gluten meal. Journal of Functional org/10.1111/j.1745‐4514.2011.00594.x Foods, 5(4), 1810–1821. https ://doi.org/10.1016/j.jff.2013.08.013 Rumpold, B. A., & Schlüter, O. K. (2013). Potential and challenges of Zielińska, E., Baraniak, B., & Karaś, M. (2017). Antioxidant and anti‐ insects as an innovative source for food and feed production. inflammatory activities of hydrolysates and peptide fractions ob‐ Innovative Food Science and Emerging Technologies, 17, 1–11. https :// tained by enzymatic hydrolysis of selected heat‐treated edible doi.org/10.1016/j.ifset.2012.11.005 insects. Nutrients, 9(9), 1–14. https ://doi.org/10.3390/nu9090 970 Stadtman, E. R. (2006). Protein oxidation and aging. Free Radical Research, Zielińska, E., Karaś, M., & Jakubczyk, A. (2017). Antioxidant activity of 40(12), 1250–1258. https: //doi.org/10.1080/107157 6060 0918142 predigested protein obtained from a range of farmed edible insects. Tiwari, B. K. (2015). Trends in analytical chemistry ultrasound: A clean, International Journal of Food Science and Technology, 52(2), 306–312. green extraction technology. Trends in Analytical Chemistry, 71, https ://doi.org/10.1111/ijfs.13282 100–109. https ://doi.org/10.1016/j.trac.2015.04.013 Umachigi, S. P., Kumar, G. S., Jayaveera, K. N., Kishore, K. D. V., Ashok, K. C. K., & Dhanapal, R. (2007). Antimicrobial, wound healing and How to cite this article: Mintah BK, He R, Dabbour M, Golly antioxidant activities of Anthocephalus cadamba. African Journal of Traditional, Complementary and Alternative Medicines, 4(4), 481–487. MK, Agyekum AA, Ma H. Effect of sonication pretreatment Wang, J., Sun, B., Cao, Y., Tian, Y., & Li, X. (2008). Optimisation of ultrasound‐ parameters and their optimization on the antioxidant activity assisted extraction of phenolic compounds from wheat bran. Food of Hermitia illucens larvae meal protein hydrolysates. J Food Chemistry, 106(2), 804–810. https ://doi.org/10.1016/j.foodc hem. Process Preserv. 2019;43:e14093. https: //doi.org/10.1111/ 2007.06.062 Wang, J., Wang, Y., Dang, X., Zheng, X., & Zhang, W. (2013). Housefly lar‐ jfpp.14093 vae hydrolysate: Orthogonal optimization of hydrolysis, antioxidant