Techno-functional attribute and antioxidative capacity of edible insect protein preparations and hydrolysates thereof: Effect of multiple mode sonochemical action

Ultrasonics - Sonochemistry 58 (2019) 104676

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Techno-functional attribute and antioxidative capacity of edible insect protein preparations and hydrolysates thereof: Effect of multiple mode sonochemical action ⁎

T

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Benjamin Kumah Mintaha,b, , Ronghai Hea, , Mokhtar Dabboura,c, Jiahui Xianga, Akwasi Akomeah Agyekuma,d, Haile Maa a

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China ILSI-UG FSNTC, Department of Nutrition and Food Science, University of Ghana, P.O. Box LG 91, Legon, Accra, Ghana Department of Agricultural and Biosystems Engineering, Faculty of Agriculture, Benha University, Moshtohor, Qaluobia P.O. Box 13736, Egypt d Atomic Energy Commission, Applied Radiation Biology Centre, P.O. Box LG 61, Legon, Accra, Ghana b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Hermetia illucens Ultrasound Proteolysis Tecno-functional property Antioxidant

Hermetia illucens (edible insect) larvae protein, and hydrolysates were prepared using three pretreatment modes (conventional, fixed-frequency ultrasonic, and sweep-frequency). Protein subunit scores, microstructure, antioxidative activity, and techno-functional property of the respective isolates and hydrolysates were investigated. Alkaline protease hydrolysis significantly enhanced protein solubility, but impaired the emulsifying property and foaming stability. Isolates and hydrolysates treated by ultrasound exhibited highest antioxidative effect, and showed excellent solubility and foam expansion over wide (2–12) pH, likened the conventional. Ultrasonic, particularly sweep-frequency, treated hydrolysates overall showed superior solubility, foam, and antioxidative (ABTS, Superoxide scavenging, and Ferric-reducing) capacity than the remaining modes and isolates (p < 0.05). Treatment type influenced microstructure, functional attributes and antioxidative capacity of hydrolysates and isolates. Thus, functional/antioxidative property could be improved or modified for different food applications based on elected treatment. H. illucens isolate and hydrolysate preparations thereof could suitably be used in development of novel food formulations.

1. Introduction Insects are recently receiving attention in culinary [1,2] and research settings as sustainable protein/nutrient source to be exploited for food applications. They are, actually, projected to be the source of protein for humans and livestock in the next few decades [3]. Their rich nutritional profile [4] makes them important in human nutrition. Thus, the continuous consumption or inclusion of insects in the diet of humans may supplement the dietary requirement of many, ensuring food security, and consequently reducing development of malnutrition and other associated diseases [5]. That said, the consumption of/inclusion of insects in the diets of many, across the world, is limited [3]; for reasons such as the association of insects to dirt/waste and/or primitivism [6]. It follows that consumer acceptance is, and maybe, a prime challenge in the inclusion of such (insects) in the meals of many in the years to come. Opportunity to increase consumption (entomophagy) globally, however, is suggested. It is to isolate the constituents of insects

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for use in food products; thus cloaking their appearance [7–9]. Hermetia illucens (HI), an edible insect, presents greater benefits to humans in this regards, providing both manifest (i.e. nutrients) and latent benefits (e.g. waste degradation). The larvae of HI, besides other constituents (fibres, minerals, vitamins), are rich in fat as well as protein – making them good resource for food fortification. After the fat is extracted, the remaining meal (waste) is thus rich in protein – about 38–60% dry weight [4] with bioactive and techno-functional attributes. Thus, the protein can be extracted for use in food applications, with the aim of improving the nutritional, bioactive/antioxidative and techno-functional properties of targeted products. That notwithstanding, the protein extracts (obtained by conventional means) from HI, as it is for other plant and insect species, have poor solubility in water/aqueous medium, and this may impact it nutritional, bioactive and techno-functionality in food formulations/applications. Overcoming this challenge is therefore paramount for upscale utilization of HI protein isolate in industry. To improve functionality/bioactivity, methods, example, microwave-

Corresponding authors at: School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China. E-mail addresses: [email protected] (B.K. Mintah), [email protected] (R. He).

https://doi.org/10.1016/j.ultsonch.2019.104676 Received 7 May 2019; Received in revised form 7 June 2019; Accepted 4 July 2019 Available online 05 July 2019 1350-4177/ © 2019 Elsevier B.V. All rights reserved.

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2.2. methods

aided, solvent, and ultrasonication-assisted extraction of proteins are reported in literature [10,11]. Of these, ultrasonication, a green or physical processing technology, has been used recently in the field of food technology to improve/enhance the functional properties of food compositions [12]. Ultrasound-assisted (sonic-aided) extraction of proteins from seed flours, for example, has been reported; and the findings indicated that the extraction efficiency and functionality of extracts significantly improved [13]. Thus, this physical processing technology may impact the functional and/or bioactive functions of protein preparations from HI. Also, the functionality of HI proteins may be improved following hydrolysis. Hall et al. [14] reports that the techno-functionality (e.g. solubility, and foamability) of Gryllodes sigillatus protein extract improved following hydrolysis by enzymatic means. Further, protein hydrolysates (i.e. the product of hydrolysis) are endowed with bioactive/ antioxidative functions. This is because, they are rich in peptides (2–20 amino acids) which significantly impact their anti-oxidative potential, compared to unhydrolyzed protein [15,16]. To obtain protein hydrolysates (with bioactive function), various methods are applied. Of these (e.g. acid, alkaline), enzymolysis is deemed best [17]. This is linked to the milder/controllable reaction conditions, high quality reaction product, and the commercial availability of proteases [17]. The traditional approach of enzymolysis, however, is limited; due to protein conformational mismatch, making it difficult for protease to attack precise protein bonds that are enzyme-driven [18,19]. To overcome this limitation, (also) ultrasound treatment, again, of samples prior to hydrolysis has been found potentially useful in the generation of hydrolysates with enhanced bioactive/antioxidant capacity [20,21]. With their role in controlling the negative health effect of excess free radicals generated in humans, and preventing generation of undesirable organoleptic attributes in food, antioxidant has been the emphasis of several investigations in recent times. There are also more than few studies on functional attributes of insects derived protein [14,22], but there are no records explaining the impact of sonic-treatment on the functionality of insect (HI) protein preparations. Also, while several reports on protein hydrolysates derived by enzymolysis from sources such as fish [15], leaf [23], and whey [24] are available in extant literature, few reports on bioactivity of edible insects proteinhydrolysates are accessible [6,25]. Thus, as in the case of protein preparations, there are no data on the influence of sonic-aided enzymolysis on the techno-functionality and bioactivity of HI protein hydrolysates. Better understanding of the effect of sonication on the bioactivity and techno-functionality of edible insect proteins/hydrolysates may lead to operational utilization of such in different food systems. This study therefore, as maiden, sought to examine the antioxidative and technofunctional attributes of HI larvae protein preparations and hydrolysates thereof, treated by three (3) modes - conventional, fixed-frequency, and sweep ultrasound. Variations in the structural characteristic and subunits of the protein isolates and hydrolysates were also explored to explain the mechanism/influence of the treatment techniques on functionality of protein hydrolysates/isolates.

2.2.1. H. illucens larvae protein isolate (HILP) Extraction was done based on a slightly modified optimized protocol recently developed by our research group. HI meal suspension (1:25 w/v) was prepared with alkaline (0.25 M NaOH) and stirred (using impeller agitator, 100 rpm) at 50 °C (60 min). The resultant slurry was centrifuged at 4 °C, 4500×g for 15 min (Eppendorf 5810-R AG, 122339 Barkhausenweg, Hamburg, Germany), and supernatant pH adjusted to 4.4 (25 °C, 1 M HCl). The precipitate obtained after centrifugation was washed (distilled H2O) and centrifuged (under same conditions) twice. The pH was finally adjusted (7, using 0.25 M NaOH), and kept at −20 °C (24 h) and after, freeze-dried (Lyoquest – 85 Plus, Telstar, Parc Científic, Terrassa, Spain).

2.2.2. HILP treatments H. illucens larvae protein isolate (HILP) was used to make three (3) preparations (HILPs): control/traditional (HILP-T), fixed frequency ultrasonication pretreated (HILP-FF), and sweep frequency sonication pretreated (HILP-SF). A 20 g/L HILP suspension (distilled water as solvent) was made for the respective treatments/samples (HILP-T, HILP-FF, and HILP-SF). For HILP-T, the set portion was conditioned (pretreated) for 20 min by stirring (using impeller agitator, 100 rpm, 50 °C). The resultant (pH 7) was kept at −20 °C (24 h) and after, freeze-dried (Lyoquest – 85 Plus, Telstar, Parc Científic, Terrassa, Spain). The HILP-FF, and HILP-SF were prepared same as the HILP-T, except that the conditioning (pretreatment) was done with respective ultrasound modes (i.e. fixed, and sweep frequency) using a Multi Frequency Switch Ultrasound - MFSU (Fanbo Bio-Eng. Co., Wuxi, China). The MFSU equipment consisted of a sonication tank/bath (30.0 × 36.0 × 11.4 cm) to contain water, two operational frequency modes – sweep (SF) and fixed (FF), and two sonication/cavitation plates (lower and upper) with set acoustic operating power. In this study, the lower sonication plate was used. The sonication conditions for the HILP-SF were 500 ms sweep-cycle, 5.5 L water (in bath/tank), 15 s (on) and 5 s (off) pulse time, 600 W power, and frequency of 40 ± 2 kHz. The sonication conditions (sweep-cycle, volume of water in bath, pulse on-and-off time, and power) for the HILP-FF were same as that of the HILP-SF, but differed only in the frequency. Thus, the frequency for HILP-FF was 40 kHz (with no “ ± ” value). That is, the sweep frequency (pertaining to HILPSF) has unstable frequency (f) work model ([f − x] to [f + x] and viceversa) around a primary frequency with constant linear velocity [26]. Hence, the sweep frequency is arithmetically expressed as f ± x (where x = 2). The fixed frequency mode (pertaining to HILP-FF) however works under a steady frequency (f).

2.2.3. H. illucens larvae protein hydrolysates (HILPHs) As in the case of the HILPs, respective HILPHs: HILPH-T (control/ traditional hydrolysate), HILPH-FF, and HILPH-SF (hydrolysates processed from the fixed and sweep frequency ultrasonication treatment, correspondingly) were also prepared. The various portions (i.e. for control, and sonication) were conditioned (treated) at same time, temperature and sonication/impeller agitation limits as done for the HILPs (Section 2.2.2). After, each treated sample (proteolysis pH 9, achieved with 1 M NaOH, 50 °C) was reacted with alkaline protease (9000 U/g) and the pH maintained for the duration of proteolysis under impeller agitation (100 rpm). The proteolysis reaction was 90 min, after which the protease action was idled by heat (water at 90 °C, 10 min). Subsequently, centrifugation (4000g, 4 °C, 15 min) was done (Eppendorf 5810-R AG, 122339 Barkhausenweg, Hamburg, Germany), and the soluble fractions (pH 7) were stored (−20 °C), and then each (HILPH-T, HILPH-FF, and HILPH-SF) lyophilized for analytical investigations.

2. Materials and methods 2.1. Materials HI larvae meal (defatted with ethanol) were obtained from L-507B (FBE, Jiangsu University, China). The larvae were previously obtained from a Bio-Tech Company (Difei) in Jiangsu, China, and had been dried by microwave before grinding and defatting. Alkaline protease (1.17 g/ mL) with activity 150,000 U/mL was obtained from Novozymes, a Bioengineering company (China). Pyrogallic acid was acquired from Chem. Reagent Co., a Pharmaceutical Group (Shanghia, China). Other reagents, including FeCl3 used were of scientific investigative grade.

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reading. Increased absorbance of each sample mixture indicated increase in FRP [32].

2.3. Determination of techno-functional attributes 2.3.1. Solubility Solubility of HILP and HILPH suspension (3.0%) was determined at varying pH (2–12). The respective suspensions (solubilized) were centrifuged at 4000g for 15 min, and the protein content of supernatant was measured by the approach reported by [27].

2.4.3. Scavenging activity – superoxide radical (SRSC) The method outlined by [33] with slight modification was applied. The HILP and HILPH samples (1 mL, 4 mg/mL) was mixed with buffer (Tris-HCl, 3 mL, pH 8.2, 50 mM) containing 1.0 mM EDTA (ethylenediaminetetraacetic acid – C10H16N2O8) and 4.5 mM pyrogallic acid (0.5 mL). Mixture was vortexed (25 °C), absorbance read every 30 s at 320 nm for 4 min, for computation of slope (absorbance/min). Distilled H2O, in place the samples, was applied as blank. The SRSC was computed as:

2.3.2. Emulsion properties (EP) EP was analyzed by the approach of [28] altered by [29] with minor modification. 2 mL soybean oil and 6 mL of sample solution (0.2%) at varying pH (2, 4, 6, 8, 10, 12) were homogenized (2A-FSH; F. Ke Inst. Co., Ltd., Jiangsu., China) for 60 s and then the emulsion (50 μL) was picked from bottom of container and mixed with SDS solution (5 mL, 0.1%) at zero and 10 min subsequent to homogenization. Absorbance (500 nm) was read at zero (A0) and 10 min (A10) following emulsion formation, and was used to compute the EAI (emulsion activity index) and ESI (emulsifying stability index):

EAI(m2/g) =

Δspeed of sample Absorbance ⎞ SRSC (%) = ⎜⎛1 − ⎟ × 100 Δspeed of blank Absorbance ⎠ ⎝ 2.5. Amino acid evaluation

2 × 2.303 × 100 × Ab cp × 0.25 × 10000

Protein building block (%) of the HILPs and HILPHs was measured according to the report of [34], with slight modification. Protein subunit S-433D auto analyzer (GmbH Sykam Co., Germany) was used. The blast B1010-3 electric oven (Expt. Appliance Co. Ltd., Sh., China) for hydrolysis, and 0.22 μm film for sample filtration were applied.

where Ab is absorbance measured at 500 nm, and cp the concentratoin (g/ml) of protein.

ESI(min) =

A0 × 10 A0 − A10

2.6. Surface hydrophobicity (S0) The technique, Kato and Nakai [35] using ANS as fluorescence (visible/invisible radiation) probe was applied. Respective HILPs and HILPHs suspensions (0.01–0.05 mg/mL), phosphate buffer (0.01 mol/L, pH 8.0) as solvent, were used. The suspensions (vortexed, and allowed to stand (10 min, 25 °C) were centrifuged (4000g for 10 min). The supernatant (protein solution, 4 mL) was mixed with 8.0 mM ANS (20 µL) in 0.01 M PBS (8.0 pH), kept in dark (14 min) and subsequently measured the Fluorescence (relative intensity). The spectrophotometer – Cary Eclipse (Varian Inc., P. Alto, USA) was used (excitation at 270 nm; emission, 450–600 nm; 5 nm slit and scan speed, 120 nm/min). S0 was computed as initial slope (Is) of intensity (relative fluorescence) against mg/mL protein (by linear regression).

2.3.3. Foaming properties Foaming capacity (Cf) and foam stability (Sf) of HILPs and HILPHs were determined by following the protocol of [30]. Sample solutions (3 g/100 mL) were whipped (high speed, 2A-FSH; F. Ke Inst. Co., Ltd., Jiangsu., China) for 5 min, and transferred into graduated cylinder. Volume of foam was noted immediately. Change in foam volume at 20 min represented the stability in foam. Computations were completed as follows:

Cf (%) =

Volume after whipping − Volume prior to whipping × 100 Volume prior to whipping

Sf (%) =

Volume after set time × 100 Initial volume

2.7. Statistical analysis

2.4. Quantification of antioxidative action

The data described are mean ± standard errors of 3 (triplicate) observations. ANOVA with Tukey test was applied to determine variations between samples/mean scores at p < 0.05. The 16.0 version of Minitab, an arithmetic Software (PA, USA) was used. The OriginPro 9.0 was applied for graphs.

2.4.1. Scavenging capacity – ABTS radical (ABTSRSC) ABTSRSC of HILPs and hydrolysates (HILPHs) was determined by applying the method outlined by [31] with slight change. Seven millimolar ABTS mixed with Potassium persulphate (K₂O₈S₂, 2.450 mM) to generate the radical (ABTS) cation was stored in dark (25 °C, 16.5 h) in advance. The resulting solution (ABTS radical) was diluted (PBS phosphate-buffer, 7.4 pH, 0.2 M) to absorbance value 0.70 ± 0.02 (734 nm). Subsequently, 2 mL of the diluted was added to 200 µL of respective HILPs or HILPHs (2 mg/mL). Absorbance at 734 nm (10 min later) was read. Distilled H2O (200 µL) was the blank. The ABTSRSC was computed as:

3. Results and discussion 3.1. Techno-functional attributes of HILPs and HILPHs 3.1.1. Solubility The functionality of key ingredients is influenced by protein-water interactions. As a consequence, protein solubility is considered as a critical functional attribute in the development of novel protein ingredient [17], making it a desired attribute (in most instances) over other functionality in industry. Solubility of the HILPs and HILPHs at varying pH are displayed in Fig. 1. From the chart, the solubility of both HILPs and HILPHs were pH dependent. A decrease in solubility was observed as pH increased from 2 to 4 (linked to the isoelectric region), and then increased with increasing pH up to 12. In all respective treatments, the solubility of the ultrasonic treated (especially of the sweep frequency) samples were higher than control (p < 0.05). Solubility was enhanced in the order HILP-T < HILP-FF < HILP-SF (for the protein samples); whereas that

Sample absorbance ⎞ ABTSRSC (%) = ⎛1 − × 100 Blank absorbance ⎠ ⎝ 2.4.2. Ferric-reducing power (FRP) FRP was measured by mixing 1 mL HILP or HILPH (4 mg/mL) with phosphate buffer (1 mL, pH 6.6, 0.2 M) and 1 mL potassium ferricyanide (C6FeK3N6, 1% w/v). The resultant was incubated (50 °C, 20 min), cooled to 25 °C and 1 mL trichloroacetic acid (10%) was added, and then centrifuged (4000 g, 10 min). Distilled H2O (1 mL) was added to supernatant (1 mL), mixed with 1% FeCl3 for absorbance (700 nm) 3

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100

HILP-T HILP-FF HILP-SF HILPH-T HILPH-FF HILPH-SF

90 80

HILP-T HILP-FF HILP-SF HILPH-T HILPH-FF HILPH-SF

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Fig. 1. Solubility of HILPs (HILP-T, HILP-FF, HILP-SF) and HILPHs (HILPH-T, HILPH-FF, HILPH-SF) at varying pH.

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(b)

HILP-T HILP-FF HILP-SF HILPH-T HILPH-FF HILPH-SF

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for the protein hydrolysates was in the order HILPH-SF > HILPHFF > HILPH-T. However, the solubility of the HILPHs were enhanced more than the HILPs at all pH ranges (p < 0.05). Our findings are similar to what is reported for other protein hydrolysates treated with alkaline protease; showing enhanced solubility comparative to unhydrolyzed protein, along with increased solubility with increasing pH [14,36]. Similar observations are also reported for other insect species [14]. The enhanced solubility of the HILPHs over the HILPs could be linked to, first, a general unveiling of the enzymatic reaction, where the breakdown of protein units results in increased interactions (repulsive in nature) between peptides (proteins with lower molecular weight) and hydrogen bond (attractive – dipole-dipole) interactions with H2O molecules [37]. Secondly, sonication may have enhanced the partial unfolding of protein molecules, as well as reducing the particle size of samples, which enhanced enzyme action and eventually resulted in products (hydrolysates) that had increased interaction with H2O molecules. Our study findings depicts the potential for using the HILPs (HILP-T, HILP-FF, HILP-SF) and HILPHs (HILPH-T, HILPH-FF, HILPH-SF) in both acidic (e.g. acidified sauces) and non-acidic foods (i.e. over varying pH range) based on the order of their solubility, with the HILPHs (particularly, HILPH-SF) being superior. Also, the solubility of the HILPs and HILPHs could be modified by different treatments.

60

ESI (%)

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pH Fig. 2. EAI (a) and ESI (b) of HILPs (HILP-T, HILP-FF, HILP-SF) and HILPHs (HILPH-T, HILPH-FF, HILPH-SF) at different pH.

generating small peptides, which may buttress the reduction in the emulsion indexes of the HILPHs treated by ultrasonication (Fig. 2). The enhanced activity/stability of the HILPs, particularly the ultrasound treated, could be linked to denaturing of proteins induced by ultrasound. The sonic-cavitation effect of ultrasound may be the force behind the change in the tertiary/quaternary (3°/4°) structure resulting in formation of more disordered structure, and thus improving the adsorption potential at the interface [43]. Our findings are consistent with what is reported by other scholars [43] for other samples. The HILPHs values, as regards EAI, were found to be lower (particularly at alkaline pH) when compared to what is reported for hydrolysates obtained from migratory locust [44]. Although the HILPHs showed reduced emulsion properties, relative to the HILPs, our results (EAI) for both HILPHs and HILPs were higher than that reported for other target proteins/hydrolysates [43]. Increased emulsion activity values depicts the adsorption capacity of protein at oil-water interface, reflecting lesser circulated oil droplets [45]. Our results suggest the HILPs, especially the HILP-SF, relative to the HILPHs, may be better in ensuring less dispersed oil droplets in food systems, and thus may be beneficial in novel food application (where emulsion indexes may be required) in industry with the drive of increasing entomophagy.

3.1.2. Emulsifying activity and stability index (EAI, and ESI) EAI and ESI were compared for HILPs (HILP-T, HILP-FF, HILP-SF), and HILPHs (HILPH-T, HILPH-FF, HILPH-SF) over pH range 2–12 (Fig. 2a, and b). Emulsion activity and stability of the HILPs and HILPHs followed a similar trend as that observed for protein solubility. It is reported that the said indexes are related to protein solubility as the impact of pH on them may be similar (to that of protein solubility) [38]. However, relative to the HILPs, enzymolysis impaired the emulsifying activity/stability of HILPHs. The reduction in activity could be linked to generation of small peptides by the enzymolysis. Small peptides (as regards hydrolysates) migrate speedily and adsorb at the interface, but then demonstrate reduced efficacy in lessening the interfacial tension as they are unable to unfold (open) and re-arrange at the (oil-water) interface unlike the case of large peptides [39–41]; thus resulting in decreased emulsion activity. Further, and most importantly, the degree of amphiphilicity (which is low for peptides/hydrolyzed proteins) impacts emulsion properties [42], and this could support our current observation. Ultrasound is known to improve enzymolysis reaction in 4

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(a)

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HILP-T HILP-FF HILP-SF HILPH-T HILPH-FF HILPH-SF

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Foam capacity (%)

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stabilizing sub-units with increased likelihood of interaction at the airsolvent interface. As well, enzymolysis potentially improved the diffusion speed and flexibility of HILPHs, resulting in increased absorption rate and early stabilization of the foam interface and consequently improving foam expansion [47]. Further, the foam expansion of all samples treated by sonication were improved at the working pH (2–12). This could be linked to the rarefaction and compression of cavitation bubbles during ultrasonication, resulting in microjets/shear thrusts [48], and thus causing unfolding of protein molecules, and consequently making it possible for the said to migrate rapidly, open up and reposition at the gas-solvent interface to decrease tension at interface. Regarding stability of foam, the HILPs were more stable than the HILPHs (Fig. 3b). The stability of sonicated samples (particularly that treated by sweep frequency) were enhanced for the HILPs at all pH, but decreased for the HILPHs. This could be due to the assertion that some enzymolysis reactions of proteins, as plausibly observed with the HILPHs, are normally associated with decline in foam stability [49]. Since ultrasound treatment enhances enzymolysis reactions, sonication may have contributed to the observed decline in stability of foam of the HILPHs. It is noted that some hydrolysis reactions could improve interfacial activity, clarifying the enhanced initial foam of the HILPHs, they thus also reduces the molar mass of the hydrolyzed proteins (polypeptides). The size of hydrolyzed protein impacts the strength of interfacial film and formation of foam networks [14]. Thus, enzymolysis may negatively influence stability of foam with time. Further investigations to ascertain the impact of limited enzymolysis reaction on stability of HILPH foam is needed. Our study outcome suggest the HILPHs, in terms of foam expansion, may be better preference to the HILPs in food formulations requiring foam function; whereas the reverse is factual with stability of foam.

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3.2. Antioxidative activity of HILPs and HILPHs

10

3.2.1. aBTSRSC The ABTSRSC of the HILPs and HILPHs are shown in Table 1. As illustrated, the ABTSRSC of all hydrolysates (HILPH-T, HILPH-FF, and HILPH-SF) increased compared to the HILPs. However, the increase in terms of sonication treatment followed same trend for the HILPHs and HILPs. The increase was, thus, in the order: HILPH-T < HILPH-FF < HILPH-SF (p < 0.05), and HILP-T < HILP-FF < HILP-SF for the hydrolysate samples and isolates respectively. The high scavenging capacity presented by the HILPHs, likened the HILPs, could be due to the presence of high activity molecules as enzymolysis results in the generation of proteins with lower molecular mass in favor of antioxidative function. Further, the improvement in the sonicated samples could be linked to the sponge effect of sonication or its waves aiding the unfolding of both isolate and hydrolysate samples and consequently exposing hydrophobic ends, which in the case of the HILPH-FF and HILPH-SF created a better orientation for enhanced enzyme action, leading to the production of isolates/hydrolysates with enhanced antioxidantive

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pH Fig. 3. Foam capacity (a) and stability (b) of HILPs (HILP-T, HILP-FF, HILP-SF) and HILPHs (HILPH-T, HILPH-FF, HILPH-SF) at various pH.

3.1.3. Foaming property Unlike emulsions which are produced at an interface of oil and water (solvent), foams are formed by the trapping of minute gas (air) bubbles in liquid. A good foam need to migrate rapidly, unfold (open up) and reposition at the interface of air-water to decrease surface tension [46]. Fig. 3(a and b) shows the foam attributes of the HILPs and HILPHs. The HILPHs displayed increased foam expansion relative to the HILPs at all pH. Thus, the enzyme action may have unfolded surface-

Table 1 Antioxidative activity (%), and surface hydrophobicity of HILPs (HILP-T, HILP-FF, and HILP-SF) and corresponding HILPHs. Treatment

Antioxidative activity (%) ABTSRSC

HILP-T HILP-FF HILP-SF HILPH-T HILPH-FF HILPH-SF

60.95 63.47 68.57 71.05 76.19 85.00

± ± ± ± ± ±

S0 SRSC

1.20 d 1.09 d 2.11c 1.57c 2.00b 0.98 a

33.83 34.94 36.86 38.08 40.43 45.96

FRP ± ± ± ± ± ±

0.59 e 1.05 de 0.42 cd 0.78c 0.49b 0.97 a

1.02 1.20 1.27 1.29 1.32 1.49

± ± ± ± ± ±

0.02 d 0.03c 0.05 bc 0.03 bc 0.04b 0.04 a

Values are given as mean ± standard deviation; values in same column with different superscript are significantly different (p < 0.05). 5

642.69 689.54 755.06 783.99 820.07 864.67

± ± ± ± ± ±

4.32f 8.15 e 3.01 d 5.45c 3.67b 8.08 a

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the respective controls (HILP-T, and HILPH-T) reduced/masked a number of their antioxidant groups. The efficacy of reducing capacity of the HILPHs followed the order HILPH-SF > HILPH-FF > HILPH-T; and that of the HILPs followed similar trend, which agrees with previous report [54] for other samples. The (reasonably high) FRP values reported here, therefore, suggest that both HILPHs and HILPs, particularly the sonicated (sweep frequency) samples may be beneficial in development of novel foods (i.e. functional) with improved FRP. To precede a final point, the improvement of FRP in the sonicated samples may be credited to the wave/sponge effect as illustrated for the ABTSRSC; and the variations in reducing efficacy as regards the sonicated samples may be linked to the difference in the operational mode/ mechanism of the sweep (SF), and fixed frequency (FF) ultrasound – unstable frequency mode for SF, and steady frequency for the FF ultrasound. To end with, the observed variations in the reducing potential (of the investigated samples: HILPs and HILPHs) may be associated with the amino acid component of the respective isolates and/or hydrolysate peptides [55].

effect. This is in similitude with [50], when they stressed that ultrasonication pretreatment effects unfolding of proteins, an outcome which is desirable for enzyme action, and/or protein functionality/ bioactivity. This could be supported with protein sub-unit scores, and data on surface hydrophobicity (Sections 3.3 and 3.4). Our findings demonstrates an up effect of ultasonication on the scavenging capacity of protein isolates and/or hydrolysate. In particular, hydrolysis produced hydrolysates/peptide mixture possessing high activity. Both the HILPs and HILPHs may be useful in various food and/or pharmaceutic applications as the ABTSRSC were observably high (> 60%). 3.2.2. Scavenging activity - superoxide radical (SRSC) Superoxide radical (SR) anions, highly noxious species, are generated by several biological reactions. SR is a potential precursors of highly reactive species, like OH− (hydroxyl radical), and thus investigations on scavenging of such radical is imperatives [51]. The SRSC of HILPs and HILPHs were displayed in Table 1. The scavenging capacity of the HILPs, compared to the HILPHs, was low. That notwithstanding, the SRSC of the respective sonicated samples (HILP-FF/ HILP-SF, and HILPH-FF/HILPH-SF) increased relative to control (p < 0.05). The enhancement of the respective ultrasonicated samples, particularly the sweep frequency treated samples, may be attributed to, first, the unfolding of molecular structure (as a result of the sponge/ wave effect of ultrasound) which increased the hydrophobic units of the HILPs, and consequently (secondly) enhancing reactivity of the unfolded protein with enzyme (as regards the HILPHs), and thus generating hydrolysates (i.e. proteins with small molar mass) in the HILPHs. Finally, the difference of SRSC among the studied samples may be attributed to their amino acid scores [52].

3.3. Amino acid evaluation The functions of protein and/or hydrolysate peptide are generally dependent on composition of their sub-units. Antioxdant activities of protein extracts and hydrolysates are typically illustrated by their building blocks (sub-units). Proteins and/or hydrolysates containing Met, Tyr, His, and Lys are reported to possess improved antioxidative function. [56]. Chen and colleagues [57], indicated that the hydrophobic groups, precisely, Val, Ala, Leu and Phe have desireable radical scavenging capacity. His is also credited with same activity, ascribed to the breakdown of its imidazole (5-membered planer) ring [56,57]. Further, a close association between the hydrophobic attributes of proteins/hydrolysate peptides and antioxidant effects/activity has been documented [52]. Thus, hydrophobic residues (i.e. Ala, Leu, Val, Ile, Phe, Pro, Thr and Met) are mostly linked to antioxidant activity [58]. Nonetheless, presence of particular amino acids in order is not the only determinants of antioxidative function of proteins. The appropriate arrangement of subunits in sequence also impacts the antioxidative activity [52]. The size of the protein is also a contributing factor. For instance, proteins having < 20 or 16 down to 5 residues (e.g. hydrolysates) demonstrate significant antioxidantive effect [59]. The amino acid values of HILPs (HILP-T, HILP-FF, and HILP-SF) and the corresponding HILPHs are displayed in Table 2. Asp was the highest sub-unit in the HILPs, and Glu in the hydrolysate. The HILMPHs

3.2.3. Reducing power (FRP) FRP is one of the main index in the assessment of antioxidative potential of food samples. The test (antioxidant) sample is used as a reductant in a redox-link technique utilizing a reducible oxidant (Fe3+). When the +3 charge on iron in the Fe/ferricyanide complex reduces to +2 in a system with antioxidants (electron donating), a colourless to blue colour change occurs; and as a consequence, the absorbance (at 700 nm) is increased. A direct relationship exist between the absorbance values and reducing power [53]. That is, increased absorbance indicate stronger/increased FRP. The absorbance of the HILPs and HILPHs reflecting their FRP are shown in Table 1. The reducing power of the HILPs were lower than the HILPHs, but in each case the sonicated samples, especially the sweep frequency treated (p < 0.05), were more enhanced than control samples. Thus, it is obvious that the treatment of

Table 2 The amino acid (%) values of HILPs (HILP-T, HILP-FF, and HILP-SF) and corresponding HILPHs. Amino acid

HILP-T

HILP-SF

HILP-FF

HILPH-T

HILPH-SF

HILPH-FF

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Haa‡

15.40 ± 0.21 4.51 ± 0.05 3.81 ± 0.04 14.50 ± 0.13 4.50 ± 0.04 7.50 ± 0.16 6.40 ± 0.31 3.00 ± 0.06 2.40 ± 0.04 2.30 ± 0.07 6.17 ± 0.27 6.02 ± 0.31 4.01 ± 0.10 3.70 ± 0.21 8.01 ± 0.16 2.20 ± 0.06 33.29

13.02 ± 0.09 4.53 ± 0.02 3.78 ± 0.01 13.89 ± 0.11 4.66 ± 0.03 6.60 ± 0.07 6.82 ± 0.24 3.10 ± 0.07 2.60 ± 0.02 2.54 ± 0.04 6.38 ± 0.20 6.11 ± 0.12 4.4 ± 0.09 3.80 ± 0.19 4.2 ± 0.08 1.10 ± 0.04 35.03

12.00 ± 0.05 4.52 ± 0.04 3.74 ± 0.07 10.86 ± 0.18 4.54 ± 0.08 5.30 ± 0.20 6.67 ± 0.19 3.36 ± 0.04 2.37 ± 0.07 2.41 ± 0.09 6.23 ± 0.19 6.30 ± 0.16 4.05 ± 0.13 3.82 ± 0.30 3.6 ± 0.09 2.00 ± 0.08 34.15

9.73 ± 0.05 4.55 ± 0.02 2.83 ± 0.02 11.60 ± 0.16 5.10 ± 0.03 5.60 ± 0.17 7.70 ± 0.11 3.12 ± 0.05 2.50 ± 0.08 2.53 ± 0.05 5.4 ± 0.18 6.80 ± 0.21 4.39 ± 0.08 3.85 ± 0.16 3.15 ± 0.11 1.96 ± 0.07 35.29

10.40 ± 0.04 4.68 ± 0.03 2.85 ± 0.02 12.70 ± 0.11 5.80 ± 0.06 6.20 ± 0.19 8.80 ± 0.12 3.54 ± 0.04 2.74 ± 0.05 2.14 ± 0.03 5.36 ± 0.15 7.01 ± 0.19 4.57 ± 0.1 3.91 ± 0.12 2.10 ± 0.07 1.32 ± 0.04 37.63

9.80 ± 0.06 4.62 ± 0.04 2.83 ± 0.01 11.00 ± 0.19 5.46 ± 0.04 6.01 ± 0.21 8.16 ± 0.26 3.25 ± 0.05 2.57 ± 0.07 1.98 ± 0.09 5.46 ± 0.21 6.83 ± 0.30 4.52 ± 0.09 3.86 ± 0.31 3.15 ± 0.12 1.90 ± 0.09 36.02

‡ Hydrophobic amino acid (Ala, Leu, Val, Ile, Phe, Pro, Thr, and Met); HILP-T: control/traditional isolate, HILP-FF: fixed frequency ultrasonication pretreated isolate, and HILP-SF: sweep frequency sonication pretreated isolate; HILPH-T, HILPH-FF, HILPH-SF are the corresponding hydrolysates.

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treatment outcome depicts functional/antioxidative property could be improved or modified for different food applications based on elected treatment.

(treated by sonication) showed the highest sum of hydrophobic residues (Ala, Val, Leu, Ile, Phe, Pro, Thr and Met) compared to the HILPs. The increase was in the order HILPH-SF > HILPH-FF > HILPH-T. A similar trend was observed for the HILPs also: HILP-SF > HILP-FF > HILP-T. The said residues, as mentioned earlier, are linked to antioxidantive functions [58]. The observed increase in the HILPH-SF and HILPH-FF hydrolyzed with alkaline protease, suggest that they may contain protein with substantially 5–16 residues with C-terminus hydrophobic units than control. This is attributable to preferential cleavage of hydrophobic residues at the C-terminus of proteins by the protease [60]. The increases in hydrophobic sub-unit in the sonicated HILPHs (particularly HILPH-SF) likened to the HILPs (Table 2); illustrates why sonication treatment resulted in increased antioxidative activity of the hydrolysates. Also, complementing the function of hydrophobic subunits [61], His is credited with scavenging potential. Our results, aside hydrophobic groups, depict relative increase in His for the HILPHs, contrast to the HILPs; and was slightly higher in the respective sonicated samples (Table 2); and this could further support the observed increases in the ABTSRSC.

Acknowledgement The study was supported by the Primary Research and Development Plan of Jiangsu Province, China (BE2016352, BE2016355). And the Zhenjiang “1+1+N” New Agricultural Technology Extension Project (ZJNJ[2017]03). References [1] S. Belluco, C. Losasso, M. Maggioletti, C.C. Alonzi, M.G. Paoletti, A. Ricci, Edible insects in a food safety and nutritional perspective: a critical review, Compr. Rev. Food Sci. Food Saf. 12 (2013) 296–313. [2] M. Shelomi, The meat of affliction: Insects and the future of food as seen in Expo, Trends Food Sci. Technol. 56 (2016) (2015) 175–179. [3] D.A.T. Sosa, V. Fogliano, Insect Physiology and Ecology, IntechOpen, 2017. [4] K.B. Barragan-Fonseca, M. Dicke, J.J.A. van Loon, Nutritional value of the black soldier fly (Hermetia illucens L.) and its suitability as animal feed – a review, J. InsectsFood Feed 3 (2017) 105–120. [5] N. Mishra, N.C. Hazarika, K. Narain, J. 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3.4. Surface hydrophobicity (S0) S0 is imperative in protein conformational investigations, to indicate the extent to which hydrophobic units (normally at the core) are visible at the outer/periphery [62]. It measurement is aided by ANS which eliminate the limitation of low quantum/fluorescence yield by binding to the hydrophobic spots of proteins [63]. The influence of the sample treatments on S0 is shown in Table 1. S0 increased in sequence HILPHSF > HILPH-FF > HILPH-T for the hydrolysates (p < 0.05), and that for the protein isolates was also HILP-SF > HILP-FF > HILP-T (p < 0.05). It was obvious that the sonication treatment was key in the observed trend since S0 values for the respective controls (HILP-T, and HILPH-T) were found to be lower than the sonicated samples (p < 0.05). This outcome is consistent with other reports indicating sonication pretreatment enhanced S0 [20,64]; and may further illustrate why the sonicated samples, in the present study, generally exhibited improved functionality and antioxidative activity. Regarding the sonicated samples, the increase in S0 could be attributed to the cavitation action of ultrasound which stimulated molecular unfolding, disrupting hydrogen bonds and/or hydrophobic linkages/bonds of the protein molecules, and consequently causing exposure of hydrophobic units to the peripheral of the molecules. Further, the changes in S0 as regards the sonicated samples (HILP-FF/HILPH-FF, and HILP-SF/ HILPH-SF) may be related to the variation in the mechanism/operational mode of the sweep (SF), and fixed frequency (FF) ultrasound as mentioned earlier (Section 3.2.3). 4. Conclusion In this investigation, we demonstrated that various pretreatments (conventional, ultrasonic-fixed, and ultrasonic-sweep frequency) of HI larvae meal can successfully produce proteins and/or hydrolysates with enhanced techno-functionality and antioxidative capacity. Improved solubility of the hydrolysates and isolates, obtained following ultrasonication (particularly with the sweep frequency), makes them, respectively, suitable for food formulations over varied range pH. The increased emulsion property of HI protein, by way of sonication, highpoints the potential to resourcefully use such, especially HILP-SF, in food emulsions. The enhanced foamability of the hydrolysates, particularly HILPH-SF, joined with solubility makes HILPHs relatively useful in new and/or existing foods that support advancement of entomophagy amongst consumers, globally. The antioxidative capacity of the isolates and the hydrolysates, precisely that treated with ultrasound, displayed in this work imply sonicated HI isolates and hydrolysates respectively may be suitable choice (depending on desired activity) for therapeutic/functional food fortification. The various 7

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