Received: 13 February 2019  |  Revised: 22 June 2019  |  Accepted: 26 June 2019 DOI: 10.1111/jfbc.12982 F U L L A R T I C L E Sonochemical action and reaction of edible insect protein: Influence on enzymolysis reaction‐kinetics, free‐Gibbs, structure, and antioxidant capacity Benjamin Kumah Mintah1,2  | Ronghai He1 | Mokhtar Dabbour1,3  | Akwasi Akomeah Agyekum1,4 | Zheng Xing1 | Moses Kwaku Golly1,5 | Haile Ma1 1School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Abstract China We investigated the impact of sonochemical action and the reaction of Hermetia 2ILSI‐UG FSNTC, Department of Nutrition illucens larvae meal protein (HILMP) as regards enzymolysis under varied enzyme and Food Science, University of Ghana, Accra, Ghana concentration and temperature to explain the mechanism and effect of sonication on 3Department of Agricultural and Biosystems molecular conformation, limits of kinetics, free‐Gibbs energy, and antioxidative capac‐ Engineering, Faculty of Agriculture, Benha University, Moshtohor, Egypt ity. Control treatment was used for comparison. The results showed sonochemical 4Atomic Energy Commission, Applied treatment enhanced HILMP‐enzymolysis efficiency at various enzyme volume, and Radiation Biology Centre, Accra, Ghana temperature. Enzymolysis‐kinetics revealed sonochemical treatment increased the 5Faculty of Applied Science and Technology, Sunyani Technical University, rate constant (p < .05) by 17.21%, 25.06%, 26.91%, and 41.38% at 323, 313, 303, and Sunyani, Ghana 293 K, respectively. On free‐Gibbs, sonochemical treatment reduced the reactants‐ Correspondence reactivity energy, enthalpy, and entropy by 30.53%, 35.05%, and 10.71%, respec‐ Ronghai He and Benjamin Kumah Mintah, tively (p < .05). Changes in spectra of UV and fluorescence, and micrographic imaging School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, indicated alterations of HILMP by sonochemical treatment. Antioxidative activity of Zhenjiang 212013, China. sonochemically‐treated HILMP increased, compared to control. Thus, sonochemical Email: heronghai1971@126.com (R. H.) and b.minta20@gmail.com (B. K. M.) treatment may be beneficial in the production of edible insect proteins with smaller molecular weights for different food and/or pharmaceutical applications. Funding information Primary Research and Development Plan Practical applications of Jiangsu Province, Grant/Award Number: Sonochemical pretreatment of HILMP positively impacted it enzymolysis rate‐reac‐ BE2016352 and BE2016355; Zhenjiang “1+1+N” New Agricultural Technology tion, stability of reaction products, structure, and bioactivity. Thus, the technique Extension Project, Grant/Award Number: may be beneficial to industry in the processing/development of new (bioactive/phar‐ ZJNJ[2017]03 maceutical) products involving enzymolysis of edible insects (e.g., Hermetia illucens) protein; particularly at such a time where edible insects are projected to be a source of protein for human nutrition and livestock in the next few years. K E Y W O R D S antioxidant activity, edible insect protein, enzymolysis reactivity energy, molecular conformation, sonochemical treatment J Food Biochem. 2019;43:e12982. wileyonlinelibrary.com/journal/jfbc © 2019 Wiley Periodicals, Inc.  |  1 of 11 https://doi.org/10.1111/jfbc.12982 2 of 11  |     MINTAH eT Al. 1  | INTRODUC TION et al., 2015; Mintah et al., 2019; Zhang et al., 2015), as well as the rate of reaction, and conversion of substrates (Dabbour, He, The global demand for protein in human and livestock nutrition is Mintah, Tang, & Ma, 2018; Jin et al., 2015). This improvement, has projected to increase in the next two/three decades (Aiking, 2011). been associated with the role of ultrasonication pretreatments in Already, existing appraisal point to it that millions of tons of proteins altering the protein structure (Dabbour et al., 2018) and conse‐ are on demand each year (Jin et al., 2015). At the same time, a vast quently reducing the free‐Gibbs (△G) and Activation (Ea) energy number of protein resources are underutilized. One such resource is of reaction (Jin et al., 2015). It is also reported that sonication Hermetia illucens, an edible insect. The larvae of H. illucens is rich in has significantly positive impact on enzymolysis kinetics as it protein—about 38%–60% dry weight (Barragan‐Fonseca, Dicke, & improves the preliminary rate of reaction through reduction of van Loon, 2017). This can be extracted for food applications, with Michaelis constant (Jin et al., 2015). the aim of improving the nutritional and techno‐functional proper‐ Literature shows several studies on the preparation of bioactive ties of food products. This technique is deemed very appropriate hydrolysates from several products/produce (milk, cheese, yoghourt, since it masks the appearance of insects and thus creates an atmo‐ bean, and rice) using conventional enzymolysis technique (Gobbetti, sphere for increased consumption across the world (Sosa & Fogliano, Stepaniak, Angelis, Corsetti, & Cagno, 2002; Megías et al., 2007). 2017). However, just as in the case of proteins from plant sources Studies on the techno‐functional attributes of insect derived pro‐ (Jin et al., 2015) as well as for most insect species (Nongonierma & tein‐hydrolysates obtained, again, by conventional enzymolysis are FitzGerald, 2017), protein extract from the larvae of H. illucens has also documented (Hall et al., 2017; Purschke, Meinlschmidt, Horn, limited utilization in food applications due to its poor solubility in Rieder, & Jäger, 2018). There are also some studies on edible insect water. There are reports suggesting that the solubility of H. illucens protein hydrolysates obtained through traditional hydrolysis for bio‐ larvae meal protein (HILMP) could be augmented by hydrolysis (Hall, active functions (Zhang et al., 2016; Zielińska, Baraniak, & Karaś, Jones, O'Haire, & Liceaga, 2017). 2017), but no data has been documented to explain the effect of Hydrolysis of proteins by enzymatic means results in the gener‐ ultra‐sonication (multi frequency control ultrasound [MFCU]) action ation of bioactive peptides (Lin, Deng, & Huang, 2014; O'Sullivan, on the enzymatic hydrolysis of HILMP. Data on proteolysis reaction Lafarga, Hayes, & O'Brien, 2017; Sae‐Leaw et al., 2017; Saisavoey, kinetics is imperative for the hydrolysis of proteins and enhancing Sangtanoo, Reamtong, & Karnchanatat, 2016), and has been judged the utilization of edible insect protein in food applications thereof. most adequate compared to alkaline or acid hydrolysis (Clemente, As a consequence, in this study (as the first), the influence of ultra‐ 2001; McCarthy, O'Callaghan, & O'Brien, 2013; Panyam & Kilara, sound (sonochemical) pretreatment (action) on the enzymolysis of 1996). This is attributed to the fact that the reaction conditions are HILMP (reaction) under varied enzyme concentration, and tem‐ controllably milder, product quality is high, and the enzymes/pro‐ perature was considered to explain the mechanism and effect of teases are commercially available (Clemente, 2001; McCarthy et sonication on the enzymolysis efficacy, molecular conformation, al., 2013; Panyam & Kilara, 1996). Nonetheless, conventional enzy‐ and the limits of kinetics and free‐Gibbs energy (thermodynam‐ molysis has been noted to limit the enzymolysis of proteins owing ics). The antioxidative capacity of hydrolysates were also, explored. to the incompatible conformation of protein which makes it hard for The findings of the present study, therefore, could be beneficial in the protease to attack the enzyme‐driven bonds of a protein (Jian, setting the scientific basis with technological backing for extended Wenyi, & Wuyong, 2008; Qu et al., 2013). As a consequence, the research/application in the production of edible insect proteins with duration of enzymolysis is extended and the volume of energy con‐ smaller molecular weights for different food and/or pharmaceutical sumed is increased (Qu et al., 2013). It follows that, improving on applications. the conventional enzymolysis approach will benefit industry in many applications such as in the production of functional bioactive prod‐ 2  | MATERIAL S AND METHODS ucts with reduced energy cost. In that regards, ultrasound applica‐ tion may be a preferred option. 2.1 | Materials The use of ultrasound, a contemporary green‐processing technology, has been shown to augment the process of enzy‐ Defatted H. illucens larvae meal (<60 mesh‐size, stored at 4°C, pro‐ molysis of proteins in preparing bioactive compounds (Jian et al., tein content by Kjeldahl technique 51.62%) was provided by the RH‐ 2008; Pan, Qu, Ma, Atungulu, & McHugh, 2012; Qu et al., 2013; research group (Jiangsu University—School of Food and Biological Qu, Pan, & Ma, 2010; Wang, Sun, Cao, Tian, & Li, 2008). This is Engineering, China). The larvae were previously obtained from linked to the cavitation effect of ultrasound (acoustic in nature) the Difei Bio‐Technology Company, Jiangsu (China), and had been which results in intense shear thrusts, vibration, and shockwaves freeze‐dried prior to milling and defatting. Endopeptidase (Alcalase) (Chandrapala, Oliver, Kentish, & Ashokkumar, 2012). Accordingly, with density 1.17 g/ml and activity 150,000 U/ml was acquired from the use of ultrasound for/to treat samples prior to enzymolysis a Biotechnology company in China (Novozymes). Other reagents, has been shown by some researchers to improve the bioactivity including sodium hydroxide (NaOH), used in this work were of ana‐ of a number of products (Jia et al., 2010; Jian et al., 2008; Jin lytical rating. MINTAH eT Al.      |  3 of 11 2.2 | MFCU pretreatment of HILMP and htp, total content of peptide bonds in the protein. The htp (7.6) and  (1.0) indicated by Adler‐Nissen (1986), and Nielsen, Petersen, and MFCU pretreatment of HILMP preceded the enzymolysis reaction. Dambmann (2001) were used. The MFCU equipment (Fanbo Bio‐Engineering Co., Ltd., Wuxi, China) consisted of a sonication tank (30.0 × 36.0 × 11.4 cm), two operational frequency modes—sweeping (SFU) and fixed (FFU), 2.5 | Estimation of hydrolyzed protein and two sonication plates (lower and upper) with acoustic oper‐ concentration (Hpc) ating power of 600 W. Each frequency mode has five frequency In order to evaluate the enzymolysis reaction (kinetic) limits of (f ) options (22, 28, 33, 40, and 68 kHz). Compared to the FFU, HILMP, the hydrolyzed protein (HILMP) concentration was meas‐ the SFU has unstable frequency work model (i.e., from [f − x] to ured using the method described by Galvão, Pinto, Jesus, Giordano, [f + x] and vice‐versa) around a central frequency with constant and Giordano (2009), computed as: linear speed (Ren, Ma, Mao, & Zhou, 2014). Accordingly, the SFU frequency is expressed as f ± x (where x = 2). It follows that the FFU operates under a stable frequency (f ). In studying the effect Hpc=Cs×DH×0.01 of sonication on HILMP, the SFU with lower sonication plate was used. The sweep‐cycle, pulse‐on time, pulse‐off time, frequency, where, Hpc is the hydrolyzed HILMP concentration (g/L), and Cs the sonication‐time, and temperature were 500 s, 15 s, 5 s, 40 ± 2 kHz, starting HILMP concentration (g/L). 30 min, and 50°C, respectively; and HILMP suspension (14.0 g/L) was transferred into 0.50 L sample/sealed bags and placed in the ultrasonic tank containing water (5.5 L). The temperature of the 2.6 | Enzymolysis reaction‐rate (k) water tank is controlled by a digital water heating probe. A con‐ The first‐degree (1°) reaction‐rate model was used to measure the trol (without sonication treatment) was studied with the aid of an rate constant k of HILMP (Kadkhodaee & Povey, 2008). The reac‐ impeller‐JJ1 agitator ((J. Xichengxinrui Instrument Co., Jiangsu, tion‐rate (kinetic) equation was given as: China) at 100 rpm. dCt 2.3 | Enzyme hydrolysis of HILMP =−kCdt t The hydrolysis set‐up consisted of a digital acidity/basicity meter (10‐PB, Sartorius Science instruments, China), a JJ1‐impeller agi‐ Following integration, the kinetic model was expressed as: tator (J. Xichengxinrui Inst. Co., China) at a 100 rpm, and a ther‐ mostatic bath (6A‐HH, Zhongzheng apparatus manufacturing InCt=−kt+ In Cs Co., Jintan, China), and centrifuge (16‐TGL, HS‐Tabletop, Pioway, Nanjing, China). Subsequent to the sonication (SFU) and control where, k denotes the rate constant (min−1), whereas Ct is the concen‐ pretreatments, the HILMP suspensions were adjusted to pH 9.0 tration (g/L) of HILMP at a given time t (min). using 1 M NaOH after preconditioning (17 min heating) at speci‐ Since it is difficult to measure the reduction of HILMP, the rate‐ fied temperature (20, 30, 40, and 50°C), the respective enzyme reaction was estimated from the increase in amount of peptides volumes (0.56, 0.70, 0.84, and 0.98 ml/L) were added to initiate released in solution. the hydrolysis reaction. The pH of the respective suspensions ( )At a set temperature and pressure, Cs=Vmax and Ct= Vmax − Vt . were maintained, over an enzymolysis time of 100 min, with the Hence, the equation InCt=−kt+ In Cs could be rearranged as: NaOH. ( ) In V − V =−kt+ In V 2.4 | Measure of hydrolysis max t max The extent of hydrolysis, also hydrolysis degree (DH), of HILMP was where Vmax is the maximal peptide concentration (µg/ml) obtained determined using the pH‐stat technique (Nissen, 1987): under optimal SFU pretreatment conditions and the optimum enzyme conditions (pH 9.0, and 323 K for 5 hr), Vt the peptide con‐ centration at time t. The k value was computed from the gradient of BcNsh D ( )H (%)= ×100 M h the plot In Vmax − Vt against t (i.e., enzymolysis time). hp tp As sonication aided enzymolysis reaction could be influenced by cavitation and thermal effect, likened to control (traditional enzy‐ where, Bc is the amount of alkaline (NaOH) consumed (ml); Nsh, alka‐ molysis), the k in the sonication aided enzymolysis was expressed as: line concentration (mol/L); , mean extent of dissociation of alpha‐ amino group (α‐NH2) in HILMP, Mhp, mass (g) of hydrolyzed protein; k=ko+kse 4 of 11  |     MINTAH eT Al. where ko and kse denotes the rate‐reaction constant attributable to (2:1 volume ratio) and kept at 25°C (30 min); and then centrifuged thermal effect in the control‐enzymolysis, and sonication‐aided enzy‐ (4,000 ×g, 15 min).The absorbance was estimated at 680 nm with molysis, respectively. TU‐1810 spectrophotometer (Pui‐General Insts. Co. Ltd., Beijing, China). Sample blank was deionized water. 2.7 | Thermodynamics of HILMP enzymolysis 2.9 | Preparation of HILMP isolates The Arrhenius Svante equation was used to describe the association between the rate‐reaction constant, k, and hydrolysis temperature, A 500 ml HILMP suspension (14.0 g/L) was pretreated under the set T (Parkin, 2007): sonication (SFU) conditions (Section 2.2). The pretreated (sonicated) suspension was adjusted to pH 11.5 (achieved with 1 M NaOH), and then stirred continuously (100 rpm) at 50°C (for 60 min), and the slurry −Ea k=Ae RT was centrifuged at (4,500 ×g, 15 min, 4°C). The resultant (supernatant) was precipitated at isoelectric pH 4.4, allowed to stand (30 min) and where A is the pre exponential factor per minute; T, the absolute again centrifuged (idem). The precipitate (protein isolate) was lyophi‐ (thermodynamic) temperature (K); Ea, the minimal (activation) energy lized (1.2‐ALPHA, Martin Christ Inc., Osterode, Germany) and kept at for reaction (J/mol); R, molar gas constant (8.3144 J/K mol). −20°C prior to analysis. The control isolate was obtained with a JJ1‐ The Arrhenius Svante equation was transformed to: impeller agitator (100 rpm) instead (i.e., without sonication treatment). Ea k=− + A 2.10 | Ultraviolet‐visible spectra analysisIn In RT The prepared HILMP samples were dissolved in 0.01 mol/L (pH 8.0) And the plot of In(k) to 1/T was used to estimate E ; whereas the buffer solution (phosphate—PBS), to 1.5 mg/ml. The ultraviolet‐vis‐a Eyring transition state (ETS) theory was used to determine changes ible spectra (UV‐VS) of the resultants were examined in the range in the thermodynamic parameters (ΔG, ΔH, and ΔS) of the hydrolysis 200–400 nm using a Varian‐100 Cary UV‐Spectrophotometer (Varian process (Parkin, 2007): Incorporated, Palo‐Alto, USA), 10.0 mm quartz cell path‐length, at 25°C. The scan‐rate and band‐width settings were 60 nm/min and 2.0 nm, respectively. The blank spectrum was realized with the PBS solution. ( ) kBT ΔG k= exp − h RT 2.11 | Intrinsic fluorescence analysis where, k is the Boltzmann value (1.38 × 10−23B J/K); h, the Planck Intrinsic fluorescence analysis (IFA) of the prepared HILMP samples constant (6.6256 × 10−34 J s); ΔG, Gibbs energy (J/mol). (control, and sonication treated) was done at 24 ± 1°C using 0.05 mg/ The free (Gibbs) energy (ΔG) was quantified as: ml in 0.1 mol/L PBS (pH 80). Fluorescence Cary‐Eclipse spectropho‐ tometer (Varian Incorporated, Palo‐Alto, USA), path cell length 1 cm was used. Sample excitation wavelength, 279 nm; and emission wave‐ ΔG=ΔH−TΔS length, 280–450 nm was used; while the slit width and scan speed were 5 nm, and 10 nm/s, respectively. PBS was used as spectrum where, ΔH, activation enthalpy (J/mol); and ΔS, the activation blank, and 10 scan spectra expressed in mean values were applied. entropy (J/mol K). By combining the ETS and Gibbs equation, a transformation of the ETS equation was given as: 2.12 | Micrographic imaging analysis (MIA) The microstructure of the HILMP samples (sonication treated, and [ ] k ΔH 1 kB ΔS control), were analyzed using a digital light‐microscope (BX43‐ In =− × + In + T R T h R Olympus, Tokyo, Japan) installed with a V35‐0D camera. The method described by Alenyorege and colleagues (Alenyorege et al., A linear plot of kIn and 1 was used to determine the values of ΔS 2018) was used; and micrographs were taken at ambient tempera‐ T T (from intercept) and ΔH (slope). ture (24 + 1°C) with 400× magnification. 2.8 | Hydrolysate peptide concentration 2.13 | Scavenging activity—hydroxyl radical analyses The peptide concentration (μg/ml) of HILMP hydrolysate was deter‐ mined with method of Ledoux and colleague (Ledoux & Lamy, 1986). The scavenging activity—hydroxyl radical (SAHR) of the HILMP HILMP hydrolysate was reacted with 15% trichloroethanoic acid hydrolysates (SFU treated, and control) were estimated at various MINTAH eT Al.      |  5 of 11 enzymolysis time with the protocol outlined by Juan Wang, Wang, 3.0 0.56 (mL/L) Dang, Zheng, and Zhang (2013) with little modifications. Briefly, 0.70 (mL/L) (a) control 0.84 (mL/L) 1,000 µl of FeSO4 solution (6 mM) was mixed with 1,000 µl of 2.5 0.98 (mL/L) hydrolysates and 1,000 µl of H2O2 (6 mM) solution. The resultant mix was vortexed and left for 15 min (25°C). Subsequently, to the 2.0 mixture, 1,000 µl of a lipophilic monohydroxybenzoic acid (salicylic acid, 6 mM) was added and the absorbance (at wavelength 510 nm) 1.5 was read after 30 min. The blank preparation was done by substitut‐ ing the sample with distilled H2O (water). The SAHR of the hydro‐ 1.0 lysate was calculated using: 0.5 ( ) ( ) Ahs−Ads SAHR (%)= 1− ×100 A 0.0wb 0 20 40 60 80 100 Time (min) where Ahs represent the absorbance of sample, Ads absorbance of sample devoid of salicyclic acid, and Awb the blank. 3.0 0.56 (mL/L) (b) Sonication pretreatment 0.70 (mL/L) 0.84 (mL/L) 2.14 | Statistical analysis 2.5 0.98 (mL/L) All experiments were in triplicate, and the outcomes were presented 2.0 as average (±) standard deviations. ANOVA (analysis of variance) was done (with Minitab 17.0 Software) to measure the effect of sonica‐ 1.5 tion treatment at p < .05. One‐way ANOVA (using Tukey's pairwise test) was utilized to liken the differences between individual groups. 1.0 The computations and graphs were done with Microsoft Excel (MSE 2016) and Origin Pro (v. 8.0) Software, respectively. 0.5 3  | RESULTS AND DISCUSSION 0.0 0 20 40 60 80 100 3.1 | Influence of sonication and control Time (min) pretreatments on HILMP enzymolysis F I G U R E 1  Hydrolyzed HILMP concentration in (a) traditional (control) and (b) sonication assisted enzymolysis at various enzyme The influence of each treatments (sonication and control) on hydro‐ concentrations (pH = 9.0, Cs = 14 g/L, and T = 323 K) lyzed protein (g/L) at various enzyme concentration, and tempera‐ ture are displayed in Figures 1 and 2, respectively. Under varying enzyme concentrations, Figure 1 showed that the In the present study, the influence of temperature (293, 303, hydrolysed HILMP concentration increased correspondingly with 313, and 323 K) was shown in Figure 2a,b. From the curves, a direct increases in enzyme concentration for the duration of enzymoly‐ relation between temperature and concentration of hydrolyzed pro‐ sis. Compared to the control, however, the sonication treated sam‐ tein was observed (up to about 80 min). That is, increasing tempera‐ ples showed a relatively sharp increase from 0 to 16 min when the ture caused an increase in the concentration of hydrolyzed protein enzyme concentration was 0.84 and/or 0.98 ml/L. This observation, for both samples (control and sonication‐aided enzymolysis). The according to Gülseren, Güzey, Bruce, and Weiss (2007), is as a result sonication pretreatment resulted in noticeable increase in hydro‐ of the sonication treatment causing the protein molecules to unfold; lyzed protein than the control at the set temperatures, particularly at an outcome which was conducive for improved enzyme action. It 313 and/or 323 K. At these temperatures, the curves revealed that also indicate that as more of the enzyme got into the system, colli‐ the hydrolyzed protein concentration obtained through sonication sion among the molecules increased, which resulted in commensu‐ pretreatment increased by 12.49% and 14.01% (p < .05) when com‐ rate levels of hydrolysed HILMP. pared to the control treatment. This observation, for hydrolysis time When the intensity of heat in a system is raised, the reacting mol‐ of 100 min, suggest that the sonication pretreatment resulted in an ecules gain more energy and bounces around. As a consequence, the enhanced enzymolysis than the control, through collision and combi‐ molecules are most likely to collide and as well combine. The reverse nation effect of the reacting molecules. Our findings agree with that is, however, true when the temperature in a system is decreased. reported by Jin et al. (2015). Hydrolyzed HILMP concentration Hydrolyzed HILMP concentration 6 of 11  |     MINTAH eT Al. 3.0 293 (K) (a) Control 303 (K) 313 (K) 2.5 323 (K) 2.0 1.5 1.0 0.5 0.0 0 20 40 60 80 100 Time (min) 3.0 293 (K) (b) Sonication pretreatment 303 (K) 313 (K) 2.5 323 (K) 2.0 1.5 1.0 0.5 0.0 0 20 40 60 80 100 Time (min) ( ) F I G U R E 2  Hydrolyzed HILMP concentration in (a) traditional F I G U R E 3  Values of In Vmax − Vt against enzymolysis time (t) (control) and (b) sonication assisted enzymolysis at different for sonication treatment and control (conventional enzymolysis) at temperature (pH = 9.0, Enzyme = 0.42 ml/L, and Cs = 14 g/L) various temperatures with substrate and enzyme concentrations 14 g/L and 0.42 ml/L, respectively 2 3.2 | Effects of sonication pretreatment and control the determination coefficient (R ) values which were greater than enzymolysis on rate‐reaction constant k 0.90 for both sonication pretreatment and Control samples. The results support our use of the primary kinetic model in determin‐ As indicated earlier, the ETS and Arrhenius equations were employed ing the rate‐reaction constant fit. Table 1 shows the rate‐reaction in calculating the thermodynamic parameters. The rate‐reaction constant kse (sonication aided enzymolysis) and ko (control) at varied constant is dependent on the enzymolysis temperature, making it an temperature. The kse and ko were estimated from the gradient of the ( ) important variable in the said equations. Accordingly, the changes linear plots of In Vmax − Vt against hydrolysis time. in the rate‐reaction (k) values due to the sonication pretreatment For both control and ultrasonic‐treated samples (Table 1), the k and control (conventional) enzymolysis was studied under hydrolysis values increased (but gradually) with increases in enzymolysis reac‐ reaction using: HILMP (14 g/L), enzyme concentration (0.84 ml/L), tion temperature (from 293 to 323 K). Implicit from this is that, the at temperatures (293–323 K), and pH 9.0; taken into consideration thermal effect on the hydrolysis reaction of HILMP was positive. ( ) the linear plots between 2 and 10 min. The plots of In Vmax − Vt According to Ma et al. (2011), such increases in k could be due to against enzymolysis reaction time in control and sonication pretreat‐ increased enzyme activity resulting from increased collisions among ment samples at the set temperatures are presented in Figure 3. The enzyme and substrate at increased temperature. That is, as more curves for both sonication‐aided enzymolysis and control (conven‐ collisions occurred in the enzymolysis system, more combinations tional) of the HILMP demonstrated good linear association with of the reacting molecules occurred as they bounced into each other, reaction time at set temperatures. This relationship is supported by allowing the molecules to complete the reaction. Compared to the Hydrolyzed HILMP concentration Hydrolyzed HILMP concentration MINTAH eT Al.      |  7 of 11 TA B L E 1   Rate‐reaction constants and 2R in sonochemical treatment and conventional (control) enzymolysis at varied absolute Adj temperatures Enzymolysis technique T (K) k (min−1) k k R2o se Adj Conventional 293 0.0119 ± 0.0007a 0.0119 ± 0.0007 0 0.985 303 0.0190 ± 0.0014cd 0.0190 ± 0.0014 0 0.978 313 0.0287 ± 0.0041b 0.0287 ± 0.0041 0 0.924 323 0.0332 ± 0.0038b 0.0332 ± 0.0038 0 0.951 Sonication‐aided 293 0.0203 ± 0.0021bcd 0.0119 ± 0.0007 0.0084 0.958 303 0.0260 ± 0.0038bc 0.0190 ± 0.0014 0.0070 0.920 313 0.0383 ± 0.0016a 0.0287 ± 0.0041 0.0096 0.993 323 0.0401 ± 0.0056a 0.0332 ± 0.0038 0.0069 0.927 Note: Means ± SD (n = 3), average values with different character set within a column implies significant difference at p < .05; ko is the rate‐reaction constant in control‐enzymolysis, kse is the constant for sonication‐aided enzymolysis; k, enzymolysis reaction‐rate constant. traditional (control), the sonication‐aided enzymolysis increased by 41.38%, 26.90%, 25.07%, and 17.21% at 293, 303, 313, and 323 K, respectively (p ≤ .05). Meaning, the reaction was faster with sonica‐ tion than the conventional (control enzymolysis). Similar outcomes have been reported in literature for Alcalase suspension (Ma et al., 2011) and cellulase (Subhedar & Gogate, 2014). Drawing from our study findings, it could be concluded that the sonication‐treated enzymolysis, compared to control, was improved since the k values increased significantly (indicating an enhanced enzyme action). 3.3 | Effects of sonication pretreatment and control enzymolysis on thermodynamic limits The minimum energy required to start a reaction (Ea) impacts strongly on enzymolysis reaction kinetics. That is, Ea causes sta‐ ble molecules to react, and consequently impact on reaction rate (Qu et al., 2010). In most cases, Ea ranged from 40 to 400 kJ/mol. At a particular instance where Ea falls below 40 kJ/mol, the reac‐ tion goes to completion faster (Qu et al., 2013). The Ea for the sonication treated and control enzymolysis was estimated as slope from Arrhenius curves—In(k) against 1/T (Figure 4). The values for Ea, however, were shown in Table 2. The Ea for the sonication‐aided enzymolysis was 19.14, whereas that for the control was 27.55 kJ/ mol. It was obvious that the sonication‐aided enzymolysis required less Ea, leading to a rapid reaction among the reactants (HILMP and the enzyme). Accordingly, the sonication‐aided enzymolysis caused a reduction in the Ea (30.53%, p < .05). It follows that the sonication‐aided enzymolysis reaction was faster than the control, inference to the experimental data. In agreement, other scholars also reported decreases in Ea following ultrasonication pretreat‐ ment (Ma et al., 2011; Qu et al., 2013). Thus, sonochemical pre‐ F I G U R E 4  Linear‐fit curves of (a) lnk against 1/T, and (b) lnk/T treatment may well advance the enzymolysis process of HILMP. against 1/T for sonication treatment and conventional (control) Regarding changes in the entropy (ΔS), free/Gibbs energy (ΔG), enzymolysis and enthalpy (ΔH), the ETS equation was utilized. The line‐plot of ln(k/T) against 1/T ( Figure 4b) was used to estimate the values of the ΔG values at varying temperature. From the results, the notice‐ ΔS (i.e., intercept) and ΔH (i.e., slope). Table 2 shows the thermody‐ able decrease in ΔH is indicative that sonication changed the pro‐ namic results. The equation ΔG=ΔH−TΔS was used in computing tein structure or precisely, disrupted the hydrophobic interactions, 8 of 11  |     MINTAH eT Al. TA B L E 2  Thermodynamic limits for conventional (control) enzymolysis and sonochemical pretreatment ∆G (kJ/mol) Enzymolysis technique Ea (kJ/mol) ∆H (kJ/mol) ∆S (J/mol K) 293 K 303 K 313 K 323 K Conventional 27.55 ± 0.41a 24.99 ± 0.35a −229.83 ± 1.25a 92.33 94.65 96.95178 99.25008 Sonication‐aided 19.14 ± 0.23b 16.23 ± 0.21b −254.44 ± 1.02b 90.78 93.34 95.886 98.43035 Decrease (%) −30.53 −35.05 −10.71 −1.68 −1.38 −1.10 −0.83 Note: Means ± SD (n = 3), average values with different character set within a column implies significant difference at p < .05; Ea, activation energy; H, enthalpy; S, entropy; G, Gibbs. leading to enhanced enzyme action through the conversion of (a) 3.5 the enzyme‐substrate interaction from a lower energy level to Control an excited one (Cheng et al., 2017). The low value of ΔH (which is 3.0 Sonication treatment endothermic) implied lower energy cost in driving the enzymoly‐ 2.5 sis reaction to completion (products). The ΔS value also decreased (approximately 10.00%, p < .05) when compared to the control 2.0 (conventional enzymolysis), supporting the assertion that sonica‐ tion‐aided enzymolysis enhances enzyme‐substrate interaction. 1.5 Entropy (S), typically explains the local disordering of reactants in a 1.0 reaction vessel. Thus, the reduction in ΔS (shown by negative val‐ ues) could be as a result of more orderly arrangement of the reac‐ 0.5 tants (HILMP and enzyme) following the sonication pretreatment, which improved the interaction among the reactants. Regarding 0.0 200 250 300 350 400 changes in Gibbs (ΔG), a relational increase with temperature Wavelength (nm) (from 293 to 323 K) was observed (Table 2). Compared to the con‐ ventional hydrolysis (control), the sonication‐aided values (ΔG) (b) decreased by 1.68%, 1.38%, 1.10%, and 0.83% when the degree Control 250 Sonication treatment of heat increased from 293 to 323 K, respectively. Further, the observed positive ΔG values shows that the enzymolysis reaction 200 was not spontaneous, resulting in formation of stable products, rel‐ ative to the starting molecules (reactants). From the current find‐ ings, it could be inferred that sonication pretreatment enhanced 150 the enzymolysis process of HILMP, when compared to the control (conventional enzymolysis). 100 50 3.4 | UV‐VS analysis The UV‐VS of the HILMP subjected to sonication and control (with‐ 0 out sonication) are displayed in Figure 5a. The typical absorption 300 320 340 360 380 400 at 278 nm may be ascribed to the combined effect of tryptophan Wavelength (nm) (~279 nm), tyrosine (~275 nm) and phenylalanine (~257 or 262 nm) F I G U R E 5   (a) UV and (b) fluorescence spectra of conventional (Grimsley & Pace, 2004; Zhou et al., 2013). (control) and sonication‐treated HILMP From the spectra (Figure 5a), the absorbance intensity of HILMP pretreated by sonication was strengthened, implying that the hydro‐ Hence, sonication treatment preceding enzymatic hydrolysis may be phobic groups which were characteristically buried (at the interior) a better option to conventional techniques. got exposed. Thus, the sonication treatment might have brought about the unfolding of the HILMP molecule as a result of breaking 3.5 | Intrinsic fluorescence analysis the bonds/interactions between them, which eventually exposed more protein to increase the absorption (UV) intensity. This may IFA provides spectra that aids the observation of protein attributes and support why the hydrolyzed protein concentration, rate‐reaction/ conformational changes during processing and/or storage (Keerati‐u‐ thermodynamic (free energy) property, and antioxidant capacity of rai, Miriani, Iametti, Bonomi, & Corredig, 2012). Spectra from IFA are the sonication‐treated samples improved, compared to the control. attributable to the residues: Tyr, Phe, and Trp (Ma et al., 2011). In most Fluorescence intensity Abs MINTAH eT Al.      |  9 of 11 (a) (b) F I G U R E 6  Microstructure of (a) conventional (control) and (b) sonochemically treated HILMP instance, the Trp residues are more sensitive to the polarity of sol‐ bio‐molecules (e.g., DNA, starches, fats, and proteins) (Young & vent. Thus, the quantum yield of Trp fluorescence could be employed Woodside, 2001), particularly when it accumulate or it is unchecked in characterizing the tertiary structural alterations of proteins (Zhao, over a long period (Aluko, 2012). To control their action in humans Dong, Li, Kong, & Liu, 2015). As shown in Figure 5b, the fluores‐ (and/or food products), antioxidants from external sources may cence spectra of the control HILMP and that subjected to sonication be applied. In the current study, the SAHR of HILMP hydrolysates (excited at 279 nm) were indicative at 360 nm. It was observed that (sonochemically treated and control) were thus investigated at fluorescence peak (intensity) of the sonochemically treated HILMP various enzymolysis time (Figure 7). The results indicated that the was more intense than the untreated (control). Deducing from this is inhibition capacity of the hydrolysates varied with time, and was that, sonication pretreatment altered the protein (HILMP) conforma‐ highest at 80 min with a slight decrease at 100 min for both sonica‐ tion; and this alteration caused molecules (hydrophobic groups) in the tion‐aided and control (conventional) enzymolysis. Deducing from interior to unfold/exposed, thus resulting in the intense fluorescence this is that, the scavenging activity of HILMP hydrolysates may of HILMP. This could explain why the hydrolysis, kinetics/thermody‐ not have direct relationship with different/increasing enzymoly‐ namic, and antioxidative attributes of the sonication‐treated samples sis time. Our findings shows that HILMP hydrolysate obtained were enhanced than the conventional (control). On another observa‐ at enzymolysis time 60, 80, and/or 100 min may be beneficial in tion (Figure 5b), there seems to have been a second fluorescence peak preventing cell damages due to oxidative stress, particularly the appearing at 395 nm for the sonication‐treated sample. This probable sonochemically treated hydrolysates. Protein hydrolysates, due shift of emission fluorescence to extended wavelengths (red‐shift), to their generally low molecular weight peptide content, exhibit compared to the control, could be indicative of fluorophores being potent antioxidative effect (Nwachukwu & Aluko, 2019). Thus, exposed to hydrophilic environment (Keerati‐u‐rai et al., 2012). the increase in inhibition activity of the sonication‐treated sam‐ ples over the conventional (control) hydrolysates could be due 3.6 | Micrographic imaging analysis Microstructures (photomicrographs) of the HILMP pretreated with 80 Control sonication, and without sonication (control) are displayed in Figure 6. Sonication treatment 70 The micrograph of the control showed distinctive compact or intact morphology/particles (Figure 6a). On thecontrary, spaces between 60 particles of the sonication treated samples appear loose. Inferring 50 from this is that, the cavitation effect produced by the sonochemical pretreatment was effective to create a sponge impact (alternating 40 compression and expansion) resulting in the loose structure of the 30 sonochemically treated samples; and this could further clarify why the sonicated samples demonstrated superior attributes, as regards 20 the study variables, in the current investigation. 10 0 3.7 | Scavenging activity (SAHR) of HILMP 20 40 60 80 100 hydrolysate samples Enzymolysis time (min) Hydroxyl radical (OH•) is a major oxygen‐containing free radical F I G U R E 7  Scavenging activity (SAHR) of HILMP hydrolysates in various disease forms, since it cable of destroying important pretreated with and without sonication SAHR (%) 10 of 11  |     MINTAH eT Al. to the cavitation effect of ultrasound which enhanced the reac‐ Barragan‐Fonseca, K. B., Dicke, M., & van Loon, J. J. A. (2017). 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