Accepted Manuscript

Effects of thermal treatments on the colloidal properties, antioxidant capacity and in-
vitro proteolytic degradation of cricket flour

Tatyana David-Birman, Gayle Raften, Uri Lesmes

PII: S0268-005X(17)31211-0
DOI: 10.1016/j.foodhyd.2017.11.044
Reference: FOOHYD 4169

To appear in: Food Hydrocolloids

Received Date: 13 July 2017

Revised Date: 21 November 2017
Accepted Date: 25 November 2017

Please cite this article as: David-Birman, T., Raften, G., Lesmes, U., Effects of thermal treatments on
the colloidal properties, antioxidant capacity and in-vitro proteolytic degradation of cricket flour, Food
Hydrocolloids (2017), doi: 10.1016/j.foodhyd.2017.11.044.

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 ACCEPTED MANUSCRIPT
Effects of thermal treatments on the colloidal properties, antioxidant

capacity and In-vitro proteolytic degradation of cricket flour

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1 Effects of thermal treatments on the colloidal

2 properties, antioxidant capacity and In-vitro
3 proteolytic degradation of cricket flour

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4 Tatyana David-Birmana, Gayle Raftenb and Uri Lesmesa

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5

a
6 Laboratory of Chemistry of Foods and Bioactives, Department of Biotechnology and Food

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7 Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel

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8 Dept. of Food Science, University of Massachusetts – Amherst, MA, USA
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9 * Corresponding Author: Associate Prof. Uri Lesmes

10 Laboratory of Chemistry of Foods and Bioactives

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11 Department of Biotechnology and Food Engineering,

12 Technion – Israel Institute of Technology, Haifa 32000,

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13 Israel
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14 Tel. 972-77-8871869; Fax.972-4-8293399

15 Email: lesmesu@bfe.technion.ac.il
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17 Running title: Impact of thermal processing on cricket flour digestibility

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18 Journal: Food Hydrocolloids

19 Submitted: July 2017

20 Revised: November 2017

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21 Abstract
22 Insects are gaining increased interest as rich, viable and sustainable sources of

23 edible proteins. However, low consumer acceptance of insects as human food

24 stimulates a need to mask the insects' appearance through various processing

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25 operations, such as grinding, cooking and baking. This research studied the

26 implications of thermal processing of cricket (Acheta domesticus) flour on its

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27 physiochemical properties, antioxidant capacity and bioaccessibility in the

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28 gastrointestinal tract. Thermal processing led to marginal changes in colloid sizes

29 and zeta-potentials at 3.0<pH<7.0. Concomitantly, sample color changes upon

30

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processing (delta E) were found to at least double when processed in the presence
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31 of fructose compared to processing without fructose (evaluated by L*a*b*color

32 values). Overall, processed cricket flour changes were all ascribed to thermally-
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33 induced phenomena, such as protein denaturation, cross-linking, Maillard glycation

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34 and/or aggregation. Next, antioxidant activity examined by FRAP and ORAC

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35 assays revealed thermally-treated cricket flour had a significantly (p<0.05)

36 diminished electron transfer ability with a simultaneous rise in its proton abstraction
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37 capability exceeding 400 uM Trolox equivalents. This improved antioxidant activity

38 may be attributed to conformational changes in cricket proteins exposing proton-

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39 donating residues, namely cysteine. Finally, dynamic in vitro digestion of untreated

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40 and treated cricket flours showed that baking increased the gastric proteolysis of

41 cricket proteins and their subsequent bioaccessibility. Thus, justifying further

42 investigation of industrial processes that could be harnessed to fabricate palatable

43 and nourishing insect-based products.

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44 Key words: Insect protein, In vitro digestion, Antioxidant capacity, thermal

45 processing, Maillard reaction

46 1. Introduction

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47 Dietary proteins are the main source of amino acids that are imperative for

48 human health and numerous physiological functions. Nowadays, one of the

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49 most prevalent nourishment challenges is the adequate provision of protein

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50 and/or energy (Alexandratos & Bruinsma, 2003; Huis et al., 2013; Klunder,

51 Wolkers-Rooijackers, Korpela, & Nout, 2012). From the industrial perspective,

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52 proteins are also important for their techno-functionalities, as stabilizers,
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53 emulsifiers, foaming and gelling agents, as well as antioxidants. To this end,

54 insect proteins are vigorously explored as a viable, cost-effective and more

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55 sustainable alternative to animal-based proteins, e.g. meat and poultry (Alston,

56 Beddow, & Pardey, 2009; Delgado, 2003; Henning Steinfeld, Pierre Gerber, T.
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57 D. Wassenaar, 2006; Huis et al., 2013; Msangi & Rosegrant, 2012). Such
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58 proteins have been found to have great nutritional potential, as they are rich in

essential amino acids (Belluco et al., 2013; Blásquez, Moreno, & Camacho,
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60 2012; Finke, 2002; B A Rumpold & Schluter, 2013; Birgit A. Rumpold &
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61 Schlüter, 2013; Verkerk, Tramper, Van Trijp, & Martens, 2007). However,
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62 introducing insects into Western diet consumption is inhibited by low consumer

63 acceptance, and stimulates a need to eliminate the insect appearance

64 (Caparros Megido et al., 2014; Vanhonacker, Van Loo, Gellynck, & Verbeke,

65 2013). Additionally, processing of insects may modulate their bioaccessibility,

66 bioavailability and overall nutritional value.

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67 Currently, the quality of proteins in food is determined by their digestible-

68 indispensable amino acid score (DIAAS) measured in humans (Consultation,

69 2011; Leser, 2013). This value is strongly affected by protein bioaccessibility to

70 proteolysis in the gastro-intestinal tract. In this respect, researchers

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71 increasingly utilize in vitro human digestion models to study the underlying

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72 principles of bioaccessibility and the delivery of bioactive compounds through

73 food (Benshitrit, Levi, Tal, Shimoni, & Lesmes, 2012; Hur, Lim, Decker, &

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74 McClements, 2011; Palafox-Carlos, Ayala-Zavala, & González-Aguilar, 2011;

75 Rodríguez-Roque et al., 2015; Wickham, Faulks, & Mills, 2009). Overall,

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numerous studies have shown that processing alters protein physicochemical
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77 properties and consequently their bioaccessibility, bioavailability and overall

bioactivity (Mackie & Macierzanka, 2010; van Boekel et al., 2010). For example,
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79 one recent study has shown milk processing affects dairy protein digestibility
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80 and the generation of bioactive peptides during digestion (Kopf-Bolanz et al.,

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81 2014). Other studies have shown that thermally-induced Maillard glycation of

82 whey proteins may alter their physicochemical properties, e.g. antioxidant

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83 (AOX) capacity, and in turn modulate their susceptibility to digestive proteolysis

84 (Joubran, Moscovici, Portmann, & Lesmes, 2017; Moscovici et al., 2014).

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85 Regarding insects, a recent work focused on the digestibility of Tenebrio molitor

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86 using a static digestion system to highlight differences in soluble and insoluble

87 protein fractions(Yi, Van Boekel, Boeren, & Lakemond, 2016). However, scant

88 scientific literature exists on the impact of thermal processing on insect

89 proteins. Most recently some studies have implied that the protein content of

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90 insects may not be as high as previously believed due to various analytical and

91 biological considerations (Janssen, Vincken, Van Den Broek, Fogliano, &

92 Lakemond, 2017; Jonas-Levi & Martinez, 2017; Payne, Scarborough, Rayner,

93 & Nonaka, 2016).

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94 Various on-going endeavors seek to mask the appearance of insects in

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95 commonly accepted products, e.g. snack bars, chips, shakes and pastry

96 products. In many cases, the production of such products involves various

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97 processing operations, including thermal processes (for example cooking or

98 baking), that may have beneficial or deleterious effects on protein digestive

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fate. Therefore, this research aimed to elucidate the impact of thermal
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100 processing on the physiochemical properties, bioaccessibility and AOX

potential of cricket (Acheta domesticus) flour, as a model system. The research

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102 tested the hypothesis that controlled thermal processing of cricket flour in the
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103 presence or absence of a reducing sugar, i.e. fructose, which leads to chemical
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104 or physical modifications in the flour, thus, in turn, altering the flour's

105 physiochemical properties and digestive fate. Obtaining this scientific evidence
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106 will offer food and health care professionals with valuable information needed

107 for the rational design of nutritious insect-containing products.

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108
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109 2. Materials and methods

110 2.1. Materials

111 Finely Milled Whole Cricket Powder was purchased from Cricket flours LLC,

112 USA. Pepsin from porcine gastric mucosa (P7000, enzymatic activity 537

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113 U/mg); Trypsin from porcine pancreas (T0303 enzymatic activity 15008 U/mg);

114 α-Chymotrypsin from bovine pancreas (C4129, enzymatic activity 65.622

115 U/mg); Mucin from porcine stomach; Sodium glycodeoxycholate and

116 Taurocholic acid sodium salt hydrate were purchased from Sigma Aldrich. All

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117 chemicals used were of analytical grade and did not undergo additional

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118 purification.

119

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120 2.2. Methods

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121 2.2.1. Sample preparation and protein content determination
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122 Cricket flour (CF) was initially analyzed by CHNS Elemental analysis using Flash

123 2000 Organic elemental analyzer (Thermo Scientific) to determine its nitrogen
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124 content. Characterized Cricket flour samples were treated thermally in the

presence and absence of fructose under conditions simulating cooking and baking.
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125
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126 Cooked CF with and without fructose (CCF+ and CCF-, respectively). Cooked

127 CF samples (CCF+) were prepared by augmenting CF solutions of 0.5% (w/w) with
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128 1.5 % (w/w) fructose using double distilled water (DDW) equilibrated to pH=7.0.

129 Control samples without fructose addition (CCF-) and CCF+ suspensions were
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130 held in a shaking water bath at 70˚C for 4 hours. Then, samples were frozen and
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131 lyophilized for 48h, pulverized using a mortar and pestle (as possible) and were

132 kept in a desiccator for further use.

133 Baked CF with and without fructose (BCF+ and BCF-, respectively): CF-

134 solutions 5%(w/w) were prepared using DDW, pH=7, for CCF+ samples 1.5 %

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135 fructose was added and stirred overnight at room temperature(RT). Later, sample

136 was lyophilized for 48h and baked for 30 minutes at 180˚C in a controlled relative

137 humidity environment (in a closed chamber over saturated KBr). Samples were ,

138 pulverized similarly to the cooked smaples and stored in a desiccator until further

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139 use.

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140 2.2.2. Color and UV-vis analyses of Maillard reaction products

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141 Evaluation of change in color due to the various treatments was performed using

142 a Minolta CR-399 chroma meter (Konica Minolta Sensing Americas, Ramsey, NJ)

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143 according to the CIELAB scale. Difference in color between original CF and the
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144 treated flours was determined using Equation (1) (Joubran, Moscovici, & Lesmes,

145 2015):
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146 ∆E=[(Ltreated-Loriginal)2+(atreated-aoriginal)2+(btreated-boriginal)2]1/2 (Eq.1)

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147 Where L* - black-white axis, a* - green-red axis and b* - yellow-blue axis. Each
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148 sample was measured 5 times.

Samples were diluted in citrate-phosphate buffers (pH ranging 3-7) to the

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150 concentration of 1.5%(w/v). Measurements were performed using a UV/visible

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151 spectrophotometer (OPTIZEN POP, MECASYS). UV spectra was scanned at the

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152 range of 200-680nm. Citrate-phosphate buffers at corresponding pH values were

153 used as blank references.

154 2.2.3. Colloidal properties of dispersions based on thermally processed

155 Cricket flour samples

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156 Colloidal stability for pH: CCF-, CCF+, BCF- or BCF+ solutions (0.5% w/v) in a

157 citrate- phosphate buffer, pH=7 were stirred overnight at RT pH adjusted to values

158 of 3.0, 4.0, 5.0, 6.0 and 7.0 using 1M HCl and 1M NaOH solutions and analyzed for

159 particle size distribution, ζ- Potential, turbidity and UV spectra in a citrate-

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160 phosphate buffer of a corresponding pH value.

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161 Particle size distribution was measured using static light scattering on a

Mastersizer 3000 instrument (Malvern Mastersizer 3000, Malvern Instruments, UK)

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163 in citrate-phosphate buffer formulated to attain 3.0<pH<7.0 values, and analyzed

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164 using the general Fraunhofer model.
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165 ζ- Potential of the samples was determined using Dynamic Light Scattering (Nano-

166 ZS, Malvern Instruments, Worcestershire, UK). Samples were diluted in citrate-
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167 phosphate buffers (pH ranging 3-7) to a concentration of 0.17% (w/v) and electrical

168 charge (ζ- Potential) of the particles was calculated using the Smoluchowski
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169 model.
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170 Turbidity: Turbidity data was acquired by absorbance measurements at 600nm.

Citrate-phosphate buffers at corresponding pH values were used as blank

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172 references. All samples were prepared in a triplicate.

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173 2.2.4. Antioxidant capacity of thermally processed Cricket flour samples

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174 Solutions of CCF-, CCF+, BCF-or BCF+ (0.5%(w/v) in DDW based on cricket flour

175 weight) were rotated overnight at RT and analyzed for antioxidant capacity via two

176 major possible mechanisms of electron or hydrogen transfer (Huang, Boxin, &

177 Prior, 2005). Specifically, this study applied the Ferric Reducing Antioxidant Power

178 (FRAP) and Oxygen Radical Antioxidant Capacity (ORAC) assays.

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179 FRAP assay was used to measure reducing ability of the samples as described

180 previously (Benzie & Strain, 1996). Briefly, FRAP reagent was prepared by mixing

181 10mM TPTZ, 20mM FeCl3·6H2O and 300mM acetate buffer, pH=3.6 in the ratio of

182 1:1:10 respectively. Samples were rotated by a Fisher Scientific spin-down device

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183 for 10sec, 7µl of sample’s supernatant phase was aliquoted into a 96-well plate,

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184 and 210µl of FRAP reagent was added into each well simultaneously using a

185 multichannel pipette followed by an absorbance measurement at 593nm using

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186 Eon™ Microplate Spectrophotometer, BioTek Instruments, Inc., USA. Antioxidant

187 power of samples was determined as ascorbic acid equivalents compared to a

188
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calibration curve of ascorbic acid at 0-1000µM.
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189 ORAC assay was performed according to a previously introduced protocol (Huang,
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190 Ou, Hampsch-Woodill, Flanagan, & Prior, 2002) with some modifications. Briefly,

191 Fluorescein stock [0.1mM] was prepared fresh daily by dissolving fluorescein in a
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192 phosphate buffer [75mM], pH 7.4, and diluted further adjacent to the measurement
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193 to a concentration of 4×10-6mM. 25µl of samples were introduced into wells. Later,

194 150µl of fluorescein working solution were added to the wells using multichannel
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195 pipette. Afterwards the plate was inserted into a Varioskan Flash, Thermo
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196 Scientific, USA for 15 min incubation at 37˚C. After the incubation, 25µl AAPH
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197 [153mM] (diluted in a phosphate buffer [75mM], pH 7.4) were added rapidly using a

198 multichannel pipette and the reading was initiated as follows: readings were

199 performed every 25 seconds for 150 minutes, preceded by a 20 sec shaking

200 operation before each reading. Excitation wavelength was set to 485nm and

201 emission was set to 528nm.

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202 2.2.5. Digestive fate of thermally processed Cricket flour samples

203 Thermally processed cricket flour samples and an unprocessed control were

204 dispersed overnight at RT in DDW to obtain 5% (w/v) suspensions. These

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205 suspensions were subjected to a bi-compartmental dynamic digestion model

206 operated under adult conditions (Levi & Lesmes, 2014). Briefly, each CF sample

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207 was vigorously mixed (1:1 v/v) for 30 sec with an adult simulated saliva solution

208 (SSF) (Shani Levi, Goldstein, Portmann, & Lesmes, 2017) to a final volume of

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209 40ml. This oral bolus was poured into a gastric bioreactor filled with 57ml of

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210 simulated gastric fluid (SGF, pH=1.3, preheated to 37˚C). Bioreactor pH was
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211 adjusted to 4.5, followed by pepsin and CaCl2 addition prior to the control software

212 initiation. This software generated a gastric pH gradient, controlled gastric

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213 emptying rate into the intestinal bioreactor as well as the rate of bile secretion into

214 the intestinal bioreactor. Trypsin and α-chymotrypsin were added in two pulses to
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215 the intestinal compartment, the first after 10min and the second after 50min from
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216 the beginning of the experiment. Samples were collected both from the gastric

217 bioreactor (at 0, 10, 30, 60 and 120 minutes) and the intestinal bioreactor (15, 30,
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218 60, 120 minutes). Gastric digesta samples were inactivated using 1M ammonium
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219 bicarbonate, whereas intestinal digesta samples were supplemented with PMSF
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220 (phenylmethylsulfonyl fluoride) to inhibit the activity of pancreatic proteases.

221 Digesta aliquots were frozen at -20 ˚C until further analysis by SDS-polyacrylamide

222 gel electrophoresis (PAGE). This analysis was carried out on 10% acrylamide gels

223 and resolved at 180V for 50min in a Tris/Glycine/SDS running buffer. Gels were

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224 stained using Comassie Brilliant blue stain and subsequently imaged using

225 Microtek 9800XL Plus scanner (Microtek, Carson, CA).

226 2.2.6. Statistical analyses

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227 Experiments were carried out in triplicates, with each repetition was measured

228 three times; results are presented as the calculated mean and standard deviation

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229 of the nine measurements. Digestion experiments were performed in duplicates.

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230 Statistical analyses were performed using Excel 2013 data analysis toolpack, and

231 were based on one way ANOVA and t-test assuming equal variances.

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233 3. Results and discussion

234 The constant need to ensure food security and safety maintains a need to

235 investigate the true nutritional potential of novel food sources, such as insects.

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236 This work sought to elucidate the effects of common thermal processing

237 conditions, equivalent to cooking and baking, on the characteristics, functionality

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238 and digestive proteolysis of cricket flour. CHNS elemental analysis of the cricket

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239 flour revealed it contains 10.3% (w/w) nitrogen, which using the common nitrogen-

240 to-protein conversion factor of 6.25 would translate into 64.4% (w/w) protein

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241 content. However, a recent study has established that the nitrogen-to-protein factor
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242 of three insect species is 4.76 or 5.60 for extracted and purified insect proteins

243 (Janssen et al., 2017). In accordance, the protein content of the cricket flour is
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244 better estimated to contain 49.0% (w/w) protein. This allows the classification of

245 cricket flour as a protein-rich product.

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246 When thermally processed, protein-rich products may show non-enzymatic

247 browning, also known as the Maillard reaction, which may alter the functionality
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248 and nutritional value of the product (Oliver, Melton, & Stanley, 2006; Somoza,

249 2005; van Boekel et al., 2010). Thus, this study explored the possible cross
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250 reactivity between cricket proteins and fructose under the experimental thermal
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251 processing conditions. Sample's color and UV absorbance were measured using a

252 tristimulous color analyzer and spectrophotometer. Results given in Figure 1 affirm

253 that each thermal treatment caused a significant (p<0.05) change in the color of

254 the samples (Figure 1A). Further, the addition of fructose led to more pronounced

255 changes in color compared to similarly treated samples without the addition of

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256 fructose. This was also accompanied by a significant change in the UV absorbance

257 of the samples in the range of 290-305nm (Figure 1B). This range of wavelengths

258 has been previously shown to be indicative of Maillard reaction products'

259 (MRPs)formation (Ajandouz, Tchiakpe, Ore, Benajiba, & Puigserver, 2001; Zhu,

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260 Damodaran, & Lucey, 2008).

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261 Studies have shown that MRPs have modulated protein functionality that may be of

262 industrial interest (Augustin, Sanguansri, & Bode, 2006; Oliver et al., 2006). One of

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263 the key techno-functional properties of edible proteins is their colloidal behavior.

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264 Hence, the change of suspension particle size, ζ- potential and turbidity at 600nm
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265 were analyzed at a range of pH values relevant to food systems. The results of

266 these measurements appear in Figure 2. Figure 2A demonstrates the mean

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267 particle size of the CF, CCF-, CCF+, BCF- and BCF+ at pH values between 3 and

268 7 does not vary significantly (ANOVA, p<0.05). ζ- Potential measurements (Figure
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269 2B) also showed no significant difference between any of the samples. However,
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270 the repeatability of the size measurements (Figure 2A) for the baked samples

271 (BCF+) was low compared to the other samples and throughout the pH range
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272 examined. This may arise from extensive Maillard reaction accelerated by the
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273 elevated temperatures of baking.

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274 The formation of MRPs may involve protein cross-linking and/or formation of

275 aggregates (Oliver et al., 2006). The kinetics for the formation of cross-linked or

276 aggregated structures is highly affected by temperature. Therefore, one may

277 expect the formation of larger entities in the baked samples compared to the

278 cooked samples. Since all samples were of a fixed concentration, the mass

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279 partitioning of the cricket flour would differ and correlate with the aggregate size. In

280 this study, evaluation of the "by number" mean particle sizes [D1,0] (Figure 2A

281 insert) shows that cooked samples (CCF+) yielded smaller sized particles than the

282 baked counterparts (BCF+). This supports the notion that the elevated temperature

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283 of baking leads to the formation of larger MRP-based hydrocolloids compared to

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284 the milder temperature of the cooking process. Consequently, the partitioning of

285 the substances in the baked sample (BCF+) would produce a less homogenous

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286 sample and challenge the experimental repeatability.

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287 The solubility and appearance of a solution or suspension are important techno-
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288 functional properties that may be modulated by the occurrence of the Maillard

289 reaction (Oliver et al., 2006; van Boekel et al., 2010). The turbidity of samples
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290 measured at 600nm in the pH range of 3-7 (Figure 2C) demonstrated a similar

291 trend in which thermally processed samples in the presence of fructose (CCF+ and
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292 BCF+) showed increased variability in the findings. Again, this can be attributed to
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293 physicochemical changes induced by the heat, moisture and reducing sugar

294 leading to Maillard-type protein cross-linking, glycosylation and aggregation. One

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295 intriguing functionality of food proteins is their ability to interfere oxidative reactions,
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296 i.e. act as antioxidants (Elias, Kellerby, & Decker, 2008). Yet, the action of proteins
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297 as antioxidants can stem from their ability to donate protons, terminate radicals or

298 neutralize pro-oxidative agents, namely chelation of metal ions (Huang et al., 2005;

299 Moon & Shibamoto, 2009). Therefore, this study determined the antioxidant

300 capacity (AOX) of processed and unprocessed cricket flour samples using FRAP

301 and ORAC AOX assays (Figure 3). The findings suggest a mixed trend in which

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302 processing both deteriorated and elevated the antioxidant capacity of the cricket

303 flour.

304 Specifically, the electron transfer capacity of the samples measured via FRAP

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305 assay (Figure 3A) showed to be significantly (p<0.05) diminished by thermal

306 processing of CF in terms of ascorbic acid equivalents. This can be attributed to

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307 the physical or chemical blocking of amino acids that may undergo one-electron

308 oxidation, namely Methionine, Tryptophan, Tyrosine, Phenylalanine or Histidine

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309 (Elias et al., 2008; Liang & Kitts, 2014). Yet, no significant difference was found

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310 between any of the thermally-treated samples (ANOVA, alpha<0.05). This may
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311 imply that the observed reduction in electron transfer capacity is a consequence of

312 physical seclusion of relevant amino acids due to structural changes in protein
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313 structure, protein cross-linking or protein aggregation. Moreover, the FRAP method

314 has been suggested to show poor sensitivity to antioxidant compounds containing
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315 thiol groups (Liang & Kitts, 2014; Svilaas et al., 2004); Thus, rendering the method
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316 incompatible to monitor the AOX capacity of proteins that originates from thiol

317 groups of cysteine residues.

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318 Concomitantly, the ORAC assay was applied to the samples (Figure 3B) to gain
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319 insight into samples' hydrogen abstraction activity. The results suggest that thermal
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320 processing operations (CCF-, BCF-) slightly increased the AOX activity of CF.

321 Moreover; thermal treatments in the presence of fructose appreciably increased

322 the antioxidant activity to a bigger extent. This supports the notion that the AOX

323 activity of treated CF involves hydrogen abstraction that may originate from

324 cysteine residues (Elias et al., 2006). Further, the Maillard reaction that occurred in

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325 CCF+ and BCF+ samples improved the AOX activity of the samples. This may

326 arise from conformational changes in protein structure exposing cysteine residues,

327 previously buried within the original tertiary structure of the proteins. Yet, one

328 cannot exclude the possibility that the AOX activity of CF samples may arise from

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329 other elements.

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330 Overall, the obtained results imply that thermal processing of CF impaired its ability

331 to serve as an electron donor to quench radicals, possibly due to seclusion of

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332 some amino acids (e.g. Tryptophan or Histidine). At the same time, ORAC findings

333 demonstrated an increased AOX activity of as a proton donor, attributed to the

334
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exposure of cysteine's thiol groups. In addition, this effect was more pronounced in
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335 samples that underwent the Maillard reaction. This concurs with recent studies

showing a similar mixed trend in the AOX capacity of whey proteins following
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337 thermal processing and/or Maillard glycation (Drusch et al., 2009; Joubran et al.,
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338 2015, 2017; Moscovici et al., 2014). Alternatively, this study can't exclude the
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339 possibility that the AOX of CF samples may arise from non-protein compounds or

340 side reactions, e.g. thermally induced release of antioxidant lipids from flour
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341 particles. This observed phenomena should be studied in more detail to fully

342 understand the underlying causes

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343 The bio-relevance of the ORAC assay (Huang et al., 2005; Moon & Shibamoto,

344 2009) stimulated a need to understand the bioaccessibility and digestive fate of the

345 processed CF samples. Therefore, samples were fed into a dynamic in-vitro GI

346 model mimicking the gastro-intestinal digestion of a healthy adult to evaluate the

347 proteolytic breakdown of the CF proteins (Figure 4). These analyses revealed that

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348 thermal treatments altered the gastric breakdown of treated CF samples (CCF-,

349 CCF+, BCF-, BCF+) compared to the breakdown of unprocessed CF (Figure 4A).

350 Additionally, cooking and baking in the absence of fructose (Figure 4B and Figure

351 4C, respectively) and in its presence (Figure 4D and Figure 4E, respectively)

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352 presented a differential impact on the proteolytic fate of CF proteins in the gastric

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353 phase. No differences were detected in the digesta samples collected from the

354 intestinal phase.

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355 As can be seen in Figure 4B and Figure 4C, cooking but not baking of CF altered

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356 the persistence of various protein bands during the gastric phase compared to the
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357 untreated CF sample (Figure 5A). The most evident differences are highlighted in

358 the closed boxes (corresponding to bands at MW of ~95 kDa, 72-55 kDa and ~17
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359 kDa). Figure 4B gave an indication that cooking of CF increased the susceptibility

360 of certain proteins to gastric proteolysis. For example, the band at ~17 kDa
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361 persisted for only 30min compared to 60 min in the untreated control (Figure 4A).
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362 Interestingly, Figures 4D and 4E revealed the formation of higher MW bands

363 compared to the control (Figure 4A). This noted increase in MW can be attributed
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364 to some protein glycation, cross-linking or aggregation. Yet, both of the samples,
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365 CCF+ and BCF+, exhibited rapid proteolysis with no discrete bands observed after
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366 30min of gastric digestion, as opposed to the untreated control (Figure 4A) or the

367 samples treated without the addition of fructose (Figure 4B and Figure 4C). This

368 concurs with prior evidence showing that early stage Maillard reaction products

369 may exhibit increased proneness to proteolysis (Corzo-Martínez, Soria, Belloque,

370 Villamiel, & Moreno, 2010; Hernandez-Hernandez, Moreno, Kolida, Rastall, &

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371 Sanz, 2011; Joubran et al., 2017; Moscovici et al., 2014; Oliver et al., 2006). Such

372 modulated liability to proteolytic degradation mandate further investigation of the

373 potential ramifications to the nutritional quality of CF.

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374 In conclusion, this study made an effort to apply realistic cooking and baking

375 conditions on cricket flour that are expected to alter the flour's physicochemical and

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376 colloidal properties (e.g. color and zeta potential) and subsequently the digestive

377 fate of proteins in the flour. Based on turbidity and particle size analyses, the

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378 changes in cricket flour techno-functionality have been suggested to stem from

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379 various events, such as protein denaturation, cross-linking and/or aggregation.
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380 Antioxidant activity analyses revealed that the processing of cricket flour reduced

381 its electron transfer ability but improved its aptitude as a proton donor. This mixed
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382 trend has been attributed to the seclusion of some amino acid residues that react

383 via a one-electron oxidation mechanism, while exposing proton-donating residues,

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384 namely cysteine. Last, in vitro digestion of untreated and treated cricket flours
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385 showed that cooking had a marginal effect on the digestive fate of the cricket

386 proteins while baking increased the proteolytic breakdown of cricket proteins.
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387 Overall, this work demonstrates that thermal processing of cricket flour can
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388 modulate its functionality, improve its function as a proton donating antioxidant and
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389 enhance its protein bioaccessibility. Thus, justifying further investigation of

390 industrial processing technologies and conditions that will not only yield palatable

391 insect-based products, but also help fully exploit their potential to beneficially affect

392 consumer health.

393

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394 Acknowledgements

395 The research was supported by the Israel Ministry of Health Research Projects and

396 Fellowships Fund on Food and Nutrition with implications on Public health (grant

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397 number 12834). Additional support was granted by the Laura Gurwin Flug Family

398 Fund. The authors would also like to thank Dr. Carmit Shani-Levi for her technical

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399 assistance and support.

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400

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Effects of thermal treatments on the colloidal properties, antioxidant

capacity and In-vitro proteolytic degradation of cricket flour

List of captions:

Figure 1. Optical properties of untreated cricket flour (CF), cooked cricket

flour (CCF), baked cricket flour (BCF-), cooked cricket flours with added

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fructose (CCF+) and baked cricket flour with added fructose (BCF+) . [A] ∆E
of color differences between CF and CCF-, CCF+, BCF- and BCF+

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(statistically different samples denoted by different letters, p<0.05) and [B] UV-
VIS spectra of CF, CCF-, CCF+, BCF- and BCF+ measured at pH=7.

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Figure 2. Colloidal properties of untreated cricket flour (CF), cooked cricket
flour (CCF), baked cricket flour (BCF-), cooked cricket flours with added

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fructose (CCF+) and baked cricket flour with added fructose (BCF+) in [A]
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mean particle size d(4,3) (Insert: ean particle size by number d(1,0) of CCF+ and
BCF+ as a function of pH value), (B) ζ-potentials and (C) absorbance at 600 nm.
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Figure 3. Antioxidant capacity of untreated cricket flour (CF), cooked cricket

flour (CCF-), baked cricket flour (BCF-), cooked cricket flours with added
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fructose (CCF+) and baked cricket flour with added fructose (BCF+). [A]
Results of FRAP analysis and [B] ORAC analysis. Letters indicate statistically
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different values (p<0.05).

Figure 4. SDS PAGE analyses of in-vitro GI digestion under adult conditions

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of [A] untreated cricket flour (CF) [B] Cooked cricket flour (CCF-) [C] Baked
cricket flour (BCF-) [D] Cooked cricket flours with added fructose (CCF+) and
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[E] Baked cricket flour with added fructose (BCF+). Boxes highlight protein
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bands that differ between samples. Proteolytic enzymes are indicated by arrows
(Pepsin, Trypsin and chymotrypsin indicated as pep., trp. and ctrp.).
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Effects of thermal treatments on the colloidal properties, antioxidant

capacity and In-vitro proteolytic degradation of cricket flour

List of figures:
Figure 1:

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Figure 2:

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Effects of thermal treatments on the colloidal properties, antioxidant

capacity and In-vitro proteolytic degradation of cricket flour

Highlights

• Crickets are explored a potential innovative sources of proteins.

•

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Cooking and baking of cricket flour alter the colloidal properties of the
flour.
• Processing of cricket flour improves its hydrogen abstraction capacity.

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• Digestive proteolysis of the flour is accelerated by processing and by

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the Maillard reaction.

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