Food Hydrocolloids 79 (2018) 48e54

Contents lists available at ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Effects of thermal treatments on the colloidal properties, antioxidant

capacity and in-vitro proteolytic degradation of cricket flour
Tatyana David-Birman a, Gayle Raften b, Uri Lesmes a, *
a
Laboratory of Chemistry of Foods and Bioactives, Department of Biotechnology and Food Engineering, Technion e Israel Institute of Technology, Haifa
32000, Israel
b
Dept. of Food Science, University of Massachusetts, Amherst, MA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Insects are gaining increased interest as rich, viable and sustainable sources of edible proteins. However,
Received 13 July 2017 low consumer acceptance of insects as human food stimulates a need to mask the insects' appearance
Received in revised form through various processing operations, such as grinding, cooking and baking. This research studied the
21 November 2017
implications of thermal processing of cricket (Acheta domesticus) flour on its physiochemical properties,
Accepted 25 November 2017
Available online 30 November 2017
antioxidant capacity and bioaccessibility in the gastrointestinal tract. Thermal processing led to marginal
changes in colloid sizes and zeta-potentials at 3.0 < pH < 7.0. Concomitantly, sample color changes upon
processing (delta E) were found to at least double when processed in the presence of fructose compared
Keywords:
Insect protein
to processing without fructose (evaluated by L*a*b*color values). Overall, processed cricket flour changes
In vitro digestion were all ascribed to thermally-induced phenomena, such as protein denaturation, cross-linking, Maillard
Antioxidant capacity glycation and/or aggregation. Next, antioxidant activity examined by FRAP and ORAC assays revealed
Thermal processing thermally-treated cricket flour had a significantly (p < 0.05) diminished electron transfer ability with a
Maillard reaction simultaneous rise in its proton abstraction capability exceeding 400 mM Trolox equivalents. This
improved antioxidant activity may be attributed to conformational changes in cricket proteins exposing
proton-donating residues, namely cysteine. Finally, dynamic in vitro digestion of untreated and treated
cricket flours showed that baking increased the gastric proteolysis of cricket proteins and their subse-
quent bioaccessibility. Thus, justifying further investigation of industrial processes that could be har-
nessed to fabricate palatable and nourishing insect-based products.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Dietary proteins are the main source of amino acids that are
imperative for human health and numerous physiological func-
tions. Nowadays, one of the most prevalent nourishment chal-
lenges is the adequate provision of protein and/or energy
(Alexandratos & Bruinsma, 2003; Klunder, Wolkers-Rooijackers,
Korpela, & Nout, 2012; van Huis et al., 2013). From the industrial
perspective, proteins are also important for their techno-
functionalities, as stabilizers, emulsifiers, foaming and gelling
agents, as well as antioxidants. To this end, insect proteins are
vigorously explored as a viable, cost-effective and more sustainable
alternative to animal-based proteins, e.g. meat and poultry (Alston,
Beddow, & Pardey, 2009; Delgado, 2003; Steinfeld, Gerber,
* Corresponding author.
E-mail address: lesmesu@bfe.technion.ac.il (U. Lesmes).
Wassenaar, V.C, 2006; van Huis et al., 2013; Msangi & Rosegrant,
2012). Such proteins have been found to have great nutritional
potential, as they are rich in essential amino acids (Belluco et al.,
2013; Bl
asquez, Moreno, & Camacho, 2012; Finke, 2002; Rumpold
& Schlüter, 2013; Rumpold & Schluter, 2013; Verkerk, Tramper,
Van Trijp, & Martens, 2007). However, introducing insects into
Western diet consumption is inhibited by low consumer accep-
tance, and stimulates a need to eliminate the insect appearance
(Caparros Megido et al., 2014; Vanhonacker, Van Loo, Gellynck, &
Verbeke, 2013). Additionally, processing of insects may modulate
their bioaccessibility, bioavailability and overall nutritional value.
Currently, the quality of proteins in food is determined by their
digestible-indispensable amino acid score (DIAAS) measured in
humans (Consultation, 2011; Leser, 2013). This value is strongly
affected by protein bioaccessibility to proteolysis in the gastro-
intestinal tract. In this respect, researchers increasingly utilize
in vitro human digestion models to study the underlying principles
of bioaccessibility and the delivery of bioactive compounds through

https://doi.org/10.1016/j.foodhyd.2017.11.044
0268-005X/© 2017 Elsevier Ltd. All rights reserved.
 T. David-Birman et al. / Food Hydrocolloids 79 (2018) 48e54
food (Benshitrit, Levi, Tal, Shimoni, & Lesmes, 2012; Hur, Lim,
Decker, & McClements, 2011; Palafox-Carlos, Ayala-Zavala, &
Gonz
alez-Aguilar, 2011; Rodríguez-Roque et al., 2015; Wickham,
Faulks, & Mills, 2009). Overall, numerous studies have shown that
processing alters protein physicochemical properties and conse-
quently their bioaccessibility, bioavailability and overall bioactivity
(Mackie & Macierzanka, 2010; van Boekel et al., 2010). For example,
one recent study has shown milk processing affects dairy protein
digestibility and the generation of bioactive peptides during
digestion (Kopf-Bolanz et al., 2014). Other studies have shown that
thermally-induced Maillard glycation of whey proteins may alter
their physicochemical properties, e.g. antioxidant (AOX) capacity,
and in turn modulate their susceptibility to digestive proteolysis
(Joubran, Moscovici, Portmann, & Lesmes, 2017; Moscovici et al.,
2014). Regarding insects, a recent work focused on the di-
gestibility of Tenebrio molitor using a static digestion system to
highlight differences in soluble and insoluble protein fractions (Yi,
Van Boekel, Boeren, & Lakemond, 2016). However, scant scientific
literature exists on the impact of thermal processing on insect
proteins. Most recently some studies have implied that the protein
content of insects may not be as high as previously believed due to
various analytical and biological considerations (Janssen, Vincken,
Van Den Broek, Fogliano, & Lakemond, 2017; Jonas-Levi & Marti-
nez, 2017; Payne, Scarborough, Rayner, & Nonaka, 2016).
Various on-going endeavors seek to mask the appearance of
insects in commonly accepted products, e.g. snack bars, chips,
shakes and pastry products. In many cases, the production of such
products involves various processing operations, including thermal
processes (for example cooking or baking), that may have beneficial
or deleterious effects on protein digestive fate. Therefore, this
research aimed to elucidate the impact of thermal processing on
the physiochemical properties, bioaccessibility and AOX potential
of cricket (Acheta domesticus) flour, as a model system. The research
tested the hypothesis that controlled thermal processing of cricket
flour in the presence or absence of a reducing sugar, i.e. fructose,
which leads to chemical or physical modifications in the flour, thus,
in turn, altering the flour's physiochemical properties and digestive
fate. Obtaining this scientific evidence will offer food and health
care professionals with valuable information needed for the
rational design of nutritious insect-containing products.
2. Materials and methods
2.1. Materials
Finely Milled Whole Cricket Powder was purchased from Cricket
flours LLC, USA. Pepsin from porcine gastric mucosa (P7000,
enzymatic activity 537 U/mg); Trypsin from porcine pancreas
(T0303 enzymatic activity 15 008 U/mg); a-Chymotrypsin from
bovine pancreas (C4129, enzymatic activity 65.622 U/mg); Mucin
from porcine stomach; Sodium glycodeoxycholate and Taurocholic
acid sodium salt hydrate were purchased from Sigma Aldrich. All
chemicals used were of analytical grade and did not undergo
additional purification.
2.2. Methods
2.2.1. Sample preparation and protein content determination
Cricket flour (CF) was initially analyzed by CHNS Elemental
analysis using Flash 2000 Organic elemental analyzer (Thermo
Scientific) to determine its nitrogen content. Characterized Cricket
flour samples were treated thermally in the presence and absence
of fructose under conditions simulating cooking and baking.
Cooked CF with and without fructose (CCFþand CCF-,
respectively). Cooked CF samples (CCFþ) were prepared by
49
augmenting CF solutions of 0.5% (w/w) with 1.5% (w/w) fructose
using double distilled water (DDW) equilibrated to pH ¼ 7.0. Con-
trol samples without fructose addition (CCF-) and
CCF þ suspensions were held in a shaking water bath at 70  C for
4 h. Then, samples were frozen and lyophilized for 48 h, pulverized
using a mortar and pestle (as possible) and were kept in a desic-
cator for further use.
Baked CF with and without fructose (BCFþand BCF-, respec-
tively): CF- solutions 5% (w/w) were prepared using DDW, pH ¼ 7,
for CCF þ samples 1.5% fructose was added and stirred overnight at
room temperature (RT). Later, sample was lyophilized for 48 h and
baked for 30 min at 180  C in a controlled relative humidity envi-
ronment (in a closed chamber over saturated KBr). Samples were,
pulverized similarly to the cooked samples and stored in a desic-
cator until further use.
2.2.2. Color and UV-vis analyses of Maillard reaction products
Evaluation of change in color due to the various treatments
was performed using a Minolta CR-399 chroma meter (Konica
Minolta Sensing Americas, Ramsey, NJ) according to the CIELAB
scale. Difference in color between original CF and the treated flours
was determined using Equation (1) (Joubran, Moscovici, & Lesmes,
2015):
DE ¼ [(Ltreated-Loriginal)2þ(atreated-aoriginal)2þ(btreated-boriginal)2]1/2(1)
Where L* - black-white axis, a* - green-red axis and b* - yellow-blue
axis. Each sample was measured 5 times.
Samples were diluted in citrate-phosphate buffers (pH ranging
3e7) to the concentration of 1.5% (w/v). Measurements were per-
formed using a UV/visible spectrophotometer (OPTIZEN POP,
MECASYS). UV spectra was scanned at the range of 200e680 nm.
Citrate-phosphate buffers at corresponding pH values were used as
blank references.
2.2.3. Colloidal properties of dispersions based on thermally
processed cricket flour samples
Colloidal stability for pH: CCF-, CCFþ, BCF- or BCF þ solutions
(0.5% w/v) in a citrate-phosphate buffer, pH ¼ 7 were stirred
overnight at RT pH adjusted to values of 3.0, 4.0, 5.0, 6.0 and 7.0
using 1M HCl and 1M NaOH solutions and analyzed for particle size
distribution, z- Potential, turbidity and UV spectra in a citrate-
phosphate buffer of a corresponding pH value.
Particle size distribution was measured using static light
scattering on a Mastersizer 3000 instrument (Malvern Mastersizer
3000, Malvern Instruments, UK) in citrate-phosphate buffer
formulated to attain 3.0 < pH < 7.0 values, and analyzed using the
general Fraunhofer model.
z- Potential of the samples was determined using Dynamic
Light Scattering (Nano-ZS, Malvern Instruments, Worcestershire,
UK). Samples were diluted in citrate-phosphate buffers (pH ranging
3e7) to a concentration of 0.17% (w/v) and electrical charge (z-
Potential) of the particles was calculated using the Smoluchowski
model.
Turbidity: Turbidity data was acquired by absorbance mea-
surements at 600 nm. Citrate-phosphate buffers at corresponding
pH values were used as blank references. All samples were pre-
pared in a triplicate.
2.2.4. Antioxidant capacity of thermally processed cricket flour
samples
Solutions of CCF-, CCFþ, BCF-or BCFþ (0.5% (w/v) in DDW based
on cricket flour weight) were rotated overnight at RT and analyzed
for antioxidant capacity via two major possible mechanisms of
electron or hydrogen transfer (Huang, Boxin, & Prior, 2005).
50 T. David-Birman et al. / Food Hydrocolloids 79 (2018) 48e54

Specifically, this study applied the Ferric Reducing Antioxidant
Power (FRAP) and Oxygen Radical Antioxidant Capacity (ORAC)
assays.
FRAP assay was used to measure reducing ability of the samples
as described previously (Benzie & Strain, 1996). Briefly, FRAP re-
agent was prepared by mixing 10 mM TPTZ, 20 mM FeCl3$6H2O and
300 mM acetate buffer, pH ¼ 3.6 in the ratio of 1:1:10 respectively.
Samples were rotated by a Fisher Scientific spin-down device for
10sec, 7 ml of sample's supernatant phase was aliquoted into a 96-
well plate, and 210 ml of FRAP reagent was added into each well
simultaneously using a multichannel pipette followed by an
absorbance measurement at 593 nm using Eon™ Microplate
Spectrophotometer, BioTek Instruments, Inc., USA. Antioxidant
power of samples was determined as ascorbic acid equivalents
compared to a calibration curve of ascorbic acid at 0e1000 mM.
ORAC assay was performed according to a previously intro-
duced protocol (Huang, Ou, Hampsch-Woodill, Flanagan, & Prior,
2002) with some modifications. Briefly, Fluorescein stock [0.1 mM]
was prepared fresh daily by dissolving fluorescein in a phosphate
buffer [75 mM], pH 7.4, and diluted further adjacent to the mea-
surement to a concentration of 4  106mM. 25 ml of samples were
introduced into wells. Later, 150 ml of fluorescein working solution
were added to the wells using multichannel pipette. Afterwards the
plate was inserted into a Varioskan Flash, Thermo Scientific, USA for
15 min incubation at 37  C. After the incubation, 25 ml AAPH
[153 mM] (diluted in a phosphate buffer [75 mM], pH 7.4) were
added rapidly using a multichannel pipette and the reading was
initiated as follows: readings were performed every 25 s for
150 min, preceded by a 20 s shaking operation before each reading.
Excitation wavelength was set to 485 nm and emission was set to
528 nm.
mean and standard deviation of the nine measurements. Digestion
experiments were performed in duplicates. Statistical analyses
were performed using Excel 2013 data analysis toolpack, and were
based on one way ANOVA and t-test assuming equal variances.
3. Results and discussion
The constant need to ensure food security and safety maintains
a need to investigate the true nutritional potential of novel food
sources, such as insects. This work sought to elucidate the effects of
common thermal processing conditions, equivalent to cooking and
baking, on the characteristics, functionality and digestive proteol-
ysis of cricket flour. CHNS elemental analysis of the cricket flour
revealed it contains 10.3% (w/w) nitrogen, which using the com-
mon nitrogen-to-protein conversion factor of 6.25 would translate
into 64.4% (w/w) protein content. However, a recent study has
established that the nitrogen-to-protein factor of three insect
species is 4.76 or 5.60 for extracted and purified insect proteins
(Janssen et al., 2017). In accordance, the protein content of the
cricket flour is better estimated to contain 49.0% (w/w) protein. This
allows the classification of cricket flour as a protein-rich product.
When thermally processed, protein-rich products may show

2.2.5. Digestive fate of thermally processed cricket flour samples

Thermally processed cricket flour samples and an unprocessed
control were dispersed overnight at RT in DDW to obtain 5% (w/v)
suspensions. These suspensions were subjected to a bi-
compartmental dynamic digestion model operated under adult
conditions (Levi & Lesmes, 2014). Briefly, each CF sample was
vigorously mixed (1:1 v/v) for 30 s with an adult simulated saliva
solution (SSF) (Shani Levi, Goldstein, Portmann, & Lesmes, 2017) to
a final volume of 40 ml. This oral bolus was poured into a gastric
bioreactor filled with 57 ml of simulated gastric fluid (SGF, pH ¼ 1.3,
preheated to 37  C). Bioreactor pH was adjusted to 4.5, followed by
pepsin and CaCl2 addition prior to the control software initiation.
This software generated a gastric pH gradient, controlled gastric
emptying rate into the intestinal bioreactor as well as the rate of
bile secretion into the intestinal bioreactor. Trypsin and a-chymo-
trypsin were added in two pulses to the intestinal compartment,
the first after 10min and the second after 50min from the beginning
of the experiment. Samples were collected both from the gastric
bioreactor (at 0, 10, 30, 60 and 120 min) and the intestinal biore-
actor (15, 30, 60, 120 min). Gastric digesta samples were inactivated
using 1M ammonium bicarbonate, whereas intestinal digesta
samples were supplemented with PMSF (phenylmethylsulfonyl
fluoride) to inhibit the activity of pancreatic proteases. Digesta al-
iquots were frozen at 20  C until further analysis by SDS-
polyacrylamide gel electrophoresis (PAGE). This analysis was car-
ried out on 10% acrylamide gels and resolved at 180 V for 50min in a
Tris/Glycine/SDS running buffer. Gels were stained using Comassie
Brilliant blue stain and subsequently imaged using Microtek
9800XL Plus scanner (Microtek, Carson, CA).
Fig. 1. Optical properties of untreated cricket flour (CF), cooked cricket flour (CCF),
baked cricket flour (BCF-), cooked cricket flours with added fructose (CCFþ) and baked
2.2.6. Statistical analyses cricket flour with added fructose (BCFþ). [A] DE of color differences between CF and
Experiments were carried out in triplicates, with each repetition CCF-, CCFþ, BCF- and BCFþ (statistically different samples denoted by different letters,
was measured three times; results are presented as the calculated p < 0.05) and [B] UV-VIS spectra of CF, CCF-, CCFþ, BCF- and BCF þ measured at pH ¼ 7.
 T. David-Birman et al. / Food Hydrocolloids 79 (2018) 48e54 51

non-enzymatic browning, also known as the Maillard reaction,
which may alter the functionality and nutritional value of the
product (Oliver, Melton, & Stanley, 2006; Somoza, 2005; van Boekel
et al., 2010). Thus, this study explored the possible cross reactivity
between cricket proteins and fructose under the experimental
thermal processing conditions. Sample's color and UV absorbance
were measured using a tristimulous color analyzer and spectro-
photometer. Results given in Fig. 1 affirm that each thermal treat-
ment caused a significant (p < 0.05) change in the color of the
samples (Fig. 1A). Further, the addition of fructose led to more
pronounced changes in color compared to similarly treated sam-
ples without the addition of fructose. This was also accompanied by
a significant change in the UV absorbance of the samples in the
range of 290e305 nm (Fig. 1B). This range of wavelengths has been
previously shown to be indicative of Maillard reaction products'
(MRPs)formation (Ajandouz, Tchiakpe, Ore, Benajiba, & Puigserver,
2001; Zhu, Damodaran, & Lucey, 2008).
Studies have shown that MRPs have modulated protein func-
tionality that may be of industrial interest (Augustin, Sanguansri, &
Bode, 2006; Oliver et al., 2006). One of the key techno-functional
properties of edible proteins is their colloidal behavior. Hence, the
change of suspension particle size, z-potential and turbidity at
600 nm were analyzed at a range of pH values relevant to food
systems. The results of these measurements appear in Fig. 2. Fig. 2A
demonstrates the mean particle size of the CF, CCF-, CCFþ, BCF- and
BCF þ at pH values between 3 and 7 does not vary significantly
(ANOVA, p < 0.05). z- Potential measurements (Fig. 2B) also showed
no significant difference between any of the samples. However, the
repeatability of the size measurements (Fig. 2A) for the baked
samples (BCFþ) was low compared to the other samples and
throughout the pH range examined. This may arise from extensive
Maillard reaction accelerated by the elevated temperatures of
baking.
The formation of MRPs may involve protein cross-linking and/or
formation of aggregates (Oliver et al., 2006). The kinetics for the
formation of cross-linked or aggregated structures is highly
affected by temperature. Therefore, one may expect the formation
of larger entities in the baked samples compared to the cooked
samples. Since all samples were of a fixed concentration, the mass
partitioning of the cricket flour would differ and correlate with the
aggregate size. In this study, evaluation of the “by number” mean
particle sizes [D1,0] (Fig. 2A insert) shows that cooked samples
(CCFþ) yielded smaller sized particles than the baked counterparts
(BCFþ). This supports the notion that the elevated temperature of
baking leads to the formation of larger MRP-based hydrocolloids
compared to the milder temperature of the cooking process.
Consequently, the partitioning of the substances in the baked
sample (BCFþ) would produce a less homogenous sample and

Fig. 2. Colloidal properties of untreated cricket flour (CF), cooked cricket flour (CCF), baked cricket flour (BCF-), cooked cricket flours with added fructose (CCFþ) and baked cricket
flour with added fructose (BCFþ) in [A] mean particle size d (4,3) (Insert: ean particle size by number d (1,0) of CCFþ and BCF þ as a function of pH value), (B) z-potentials and (C)
absorbance at 600 nm.
52 T. David-Birman et al. / Food Hydrocolloids 79 (2018) 48e54
challenge the experimental repeatability.
The solubility and appearance of a solution or suspension are
important techno-functional properties that may be modulated by
the occurrence of the Maillard reaction (Oliver et al., 2006; van
Boekel et al., 2010). The turbidity of samples measured at 600 nm
in the pH range of 3e7 (Fig. 2C) demonstrated a similar trend in
which thermally processed samples in the presence of fructose
(CCFþ and BCFþ) showed increased variability in the findings.
Again, this can be attributed to physicochemical changes induced
by the heat, moisture and reducing sugar leading to Maillard-type
protein cross-linking, glycosylation and aggregation. One
intriguing functionality of food proteins is their ability to interfere
oxidative reactions, i.e. act as antioxidants (Elias, Kellerby, &
Decker, 2008). Yet, the action of proteins as antioxidants can stem
from their ability to donate protons, terminate radicals or
neutralize pro-oxidative agents, namely chelation of metal ions
(Huang et al., 2005; Moon & Shibamoto, 2009). Therefore, this
study determined the antioxidant capacity (AOX) of processed and
unprocessed cricket flour samples using FRAP and ORAC AOX as-
says (Fig. 3). The findings suggest a mixed trend in which pro-
cessing both deteriorated and elevated the antioxidant capacity of
the cricket flour.
Specifically, the electron transfer capacity of the samples
measured via FRAP assay (Fig. 3A) showed to be significantly
(p < 0.05) diminished by thermal processing of CF in terms of
ascorbic acid equivalents. This can be attributed to the physical or
chemical blocking of amino acids that may undergo one-electron
Fig. 3. Antioxidant capacity of untreated cricket flour (CF), cooked cricket flour (CCF-),
baked cricket flour (BCF-), cooked cricket flours with added fructose (CCFþ) and baked
cricket flour with added fructose (BCFþ). [A] Results of FRAP analysis and [B] ORAC
analysis. Letters indicate statistically different values (p < 0.05).
oxidation, namely Methionine, Tryptophan, Tyrosine, Phenylala-
nine or Histidine (Elias et al., 2008; Liang & Kitts, 2014). Yet, no
significant difference was found between any of the thermally-
treated samples (ANOVA, alpha<0.05). This may imply that the
observed reduction in electron transfer capacity is a consequence of
physical seclusion of relevant amino acids due to structural changes
in protein structure, protein cross-linking or protein aggregation.
Moreover, the FRAP method has been suggested to show poor
sensitivity to antioxidant compounds containing thiol groups
(Liang & Kitts, 2014; Svilaas et al., 2004); Thus, rendering the
method incompatible to monitor the AOX capacity of proteins that
originates from thiol groups of cysteine residues.
Concomitantly, the ORAC assay was applied to the samples
(Fig. 3B) to gain insight into samples' hydrogen abstraction activity.
The results suggest that thermal processing operations (CCF-, BCF-)
slightly increased the AOX activity of CF. Moreover; thermal treat-
ments in the presence of fructose appreciably increased the anti-
oxidant activity to a bigger extent. This supports the notion that the
AOX activity of treated CF involves hydrogen abstraction that may
originate from cysteine residues (Elias et al., 2006). Further, the
Maillard reaction that occurred in CCFþ and BCF þ samples
improved the AOX activity of the samples. This may arise from
conformational changes in protein structure exposing cysteine
residues, previously buried within the original tertiary structure of
the proteins. Yet, one cannot exclude the possibility that the AOX
activity of CF samples may arise from other elements.
Overall, the obtained results imply that thermal processing of CF
impaired its ability to serve as an electron donor to quench radicals,
possibly due to seclusion of some amino acids (e.g. Tryptophan or
Histidine). At the same time, ORAC findings demonstrated an
increased AOX activity of as a proton donor, attributed to the
exposure of cysteine's thiol groups. In addition, this effect was more
pronounced in samples that underwent the Maillard reaction. This
concurs with recent studies showing a similar mixed trend in the
AOX capacity of whey proteins following thermal processing and/or
Maillard glycation (Drusch et al., 2009; Joubran et al., 2017, 2015;
Moscovici et al., 2014). Alternatively, this study can't exclude the
possibility that the AOX of CF samples may arise from non-protein
compounds or side reactions, e.g. thermally induced release of
antioxidant lipids from flour particles. This observed phenomena
should be studied in more detail to fully understand the underlying
causes.
The bio-relevance of the ORAC assay (Huang et al., 2005; Moon
& Shibamoto, 2009) stimulated a need to understand the bio-
accessibility and digestive fate of the processed CF samples.
Therefore, samples were fed into a dynamic in-vitro GI model
mimicking the gastro-intestinal digestion of a healthy adult to
evaluate the proteolytic breakdown of the CF proteins (Fig. 4).
These analyses revealed that thermal treatments altered the gastric
breakdown of treated CF samples (CCF-, CCFþ, BCF-, BCFþ)
compared to the breakdown of unprocessed CF (Fig. 4A). Addi-
tionally, cooking and baking in the absence of fructose (Fig. 4B and
C, respectively) and in its presence (Fig. 4D and E, respectively)
presented a differential impact on the proteolytic fate of CF proteins
in the gastric phase. No differences were detected in the digesta
samples collected from the intestinal phase.
As can be seen in Fig. 4B and C, cooking but not baking of CF
altered the persistence of various protein bands during the gastric
phase compared to the untreated CF sample (Fig. 4A). The most
evident differences are highlighted in the closed boxes (corre-
sponding to bands at MW of ~95 kDa, 72-55 kDa and ~17 kDa).
Fig. 4B gave an indication that cooking of CF increased the sus-
ceptibility of certain proteins to gastric proteolysis. For example,
the band at ~17 kDa persisted for only 30min compared to 60 min in
the untreated control (Fig. 4A). Interestingly, Fig. 4D and E revealed
 T. David-Birman et al. / Food Hydrocolloids 79 (2018) 48e54 53

Fig. 4. SDS PAGE analyses of in-vitro GI digestion under adult conditions of [A] untreated cricket flour (CF) [B] Cooked cricket flour (CCF-) [C] Baked cricket flour (BCF-) [D] Cooked
cricket flours with added fructose (CCFþ) and [E] Baked cricket flour with added fructose (BCFþ). Boxes highlight protein bands that differ between samples. Proteolytic enzymes
are indicated by arrows (Pepsin, Trypsin and chymotrypsin indicated as pep., trp. and ctrp.).

the formation of higher MW bands compared to the control
(Fig. 4A). This noted increase in MW can be attributed to some
protein glycation, cross-linking or aggregation. Yet, both of the
samples, CCFþ and BCFþ, exhibited rapid proteolysis with no
discrete bands observed after 30min of gastric digestion, as
opposed to the untreated control (Fig. 4A) or the samples treated
without the addition of fructose (Fig. 4B and C). This concurs with
prior evidence showing that early stage Maillard reaction products
may exhibit increased proneness to proteolysis (Corzo-Martínez,
Soria, Belloque, Villamiel, & Moreno, 2010; Hernandez-
Hernandez, Moreno, Kolida, Rastall, & Sanz, 2011; Joubran et al.,
2017; Moscovici et al., 2014; Oliver et al., 2006). Such modulated
liability to proteolytic degradation mandate further investigation of
the potential ramifications to the nutritional quality of CF.
In conclusion, this study made an effort to apply realistic
cooking and baking conditions on cricket flour that are expected to
alter the flour's physicochemical and colloidal properties (e.g. color
and zeta potential) and subsequently the digestive fate of proteins
in the flour. Based on turbidity and particle size analyses, the
changes in cricket flour techno-functionality have been suggested
to stem from various events, such as protein denaturation, cross-
linking and/or aggregation. Antioxidant activity analyses revealed
that the processing of cricket flour reduced its electron transfer
ability but improved its aptitude as a proton donor. This mixed
trend has been attributed to the seclusion of some amino acid
residues that react via a one-electron oxidation mechanism, while
exposing proton-donating residues, namely cysteine. Last, in vitro
digestion of untreated and treated cricket flours showed that
cooking had a marginal effect on the digestive fate of the cricket
proteins while baking increased the proteolytic breakdown of
cricket proteins. Overall, this work demonstrates that thermal
processing of cricket flour can modulate its functionality, improve
its function as a proton donating antioxidant and enhance its pro-
tein bioaccessibility. Thus, justifying further investigation of in-
dustrial processing technologies and conditions that will not only
yield palatable insect-based products, but also help fully exploit
their potential to beneficially affect consumer health.
Acknowledgements
The research was supported by the Israel Ministry of Health
Research Projects and Fellowships Fund on Food and Nutrition with
implications on Public health (grant number 12834). Additional
support was granted by the Laura Gurwin Flug Family Fund (grant
number 3-43831). The authors would also like to thank Dr. Carmit
Shani-Levi for her technical assistance and support.
References
Ajandouz, E. H., Tchiakpe, L. S., Ore, F. D., Benajiba, A., & Puigserver, A. (2001). Effects
of pH on caramelization and Maillard reaction kinetics in fructose-lysine model
systems. Journal of Food Science, 66(7), 926e931. http://doi.org/10.1111/j.1365-
2621.2001.tb08213.x.
Alexandratos, N., & Bruinsma, J. (2003). World agriculture: Towards 2015/2030: An
FAO perspective. Land Use Policy, 20(4), 1e147. http://doi.org/10.1016/S0264-
8377(03)00047-4.
Alston, J. M., Beddow, J. M., & Pardey, P. G. (2009). Agricultural research, produc-
tivity, and food prices in the long run. Science, 325(5945), 1209e1210.
Augustin, M. A., Sanguansri, L., & Bode, O. (2006). Maillard reaction products as
encapsulants for fish oil powders. Journal of Food Science, 71(2), E25eE32.
http://doi.org/10.1111/j.1365-2621.2006.tb08893.x.
Belluco, S., Losasso, C., Maggioletti, M., Alonzi, C. C., Paoletti, M. G., & Ricci, A. (2013).
Edible insects in a food safety and nutritional perspective: A critical review.
54 T. David-Birman et al. / Food Hydrocolloids 79 (2018) 48e54
Comprehensive Reviews in Food Science and Food Safety, 12(3), 296e313.
Benshitrit, R. C., Levi, C. S., Tal, S. L., Shimoni, E., & Lesmes, U. (2012). Development of
oral food-grade delivery systems: Current knowledge and future challenges.
Food & Function, 3(1), 10e21. http://doi.org/10.1039/C1fo10068h.
Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a
measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry,
239(1), 70e76. http://doi.org/10.1006/abio.1996.0292.

squez, J. R.-E., Moreno, J. M. P., & Camacho, V. H. M. (2012). Could grasshoppers
Bla
Be a nutritive meal? Food and Nutrition Sciences, 3(2), 164e175.
van Boekel, M., Fogliano, V., Pellegrini, N., Stanton, C., Scholz, G.,
Lalljie, S., … Eisenbrand, G. (2010). A review on the beneficial aspects of food
processing. Molecular Nutrition and Food Research, 54(9), 1215e1247. http://doi.
org/10.1002/mnfr.200900608.
Caparros Megido, R., Sablon, L., Geuens, M., Brostaux, Y., Alabi, T.,
Blecker, C., … Francis, F. (2014). Edible insects acceptance by belgian Con-
sumers: Promising attitude for entomophagy development. Journal of Sensory
Studies, 29(1), 14e20. http://doi.org/10.1111/joss.12077.
Consultation, R. F. E. (2011). Dietary protein quality evaluation in human nutrition.
Food and Agriculrure Organization of the United Nations, 92, 1e79.
Corzo-Martínez, M., Soria, A. C., Belloque, J., Villamiel, M., & Moreno, F. J. (2010).
Effect of glycation on the gastrointestinal digestibility and immunoreactivity of
bovine b-lactoglobulin. International Dairy Journal, 20(11), 742e752. http://doi.
org/10.1016/j.idairyj.2010.04.002.
Delgado, C. L. (2003). Rising consumption of meat and milk in developing countries
has created a new food revolution. The Journal of Nutrition, 133(11),
3907Se3910S.
Drusch, S., Berg, S., Scampicchio, M., Serfert, Y., Somoza, V., Mannino, S., et al.
(2009). Role of glycated caseinate in stabilisation of microencapsulated lipo-
philic functional ingredients. Food Hydrocolloids, 23(3), 942e948. http://doi.org/
10.1016/j.foodhyd.2008.07.004.
Elias, R. J., Bridgewater, J. D., Vachet, R. W., Waraho, T., McClements, D. J., &
Decker, E. A. (2006). Antioxidant mechanisms of enzymatic hydrolysates of b-
lactoglobulin in food lipid dispersions. Journal of Agricultural and Food Chem-
istry, 54(25), 9565e9572. http://doi.org/10.1021/jf062178w.
Elias, R. J., Kellerby, S. S., & Decker, E. A. (2008). Antioxidant activity of proteins and
peptides. Critical Reviews in Food Science and Nutrition, 48(5), 430e441.
Finke, M. D. (2002). Complete nutrient composition of commercially raised in-
vertebrates used as food for insectivores. Zoo Biology, 21(3), 269e285.
Hernandez-Hernandez, O., Moreno, F. J., Kolida, S., Rastall, R. A., & Sanz, M. L. (2011).
Effect of glycation of bovine b-lactoglobulin with galactooligosaccharides on the
growth of human faecal bacteria. International Dairy Journal, 21(12), 949e952.
http://doi.org/10.1016/j.idairyj.2011.06.002.
Huang, D., Boxin, O. U., & Prior, R. L. (2005). The chemistry behind antioxidant
capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841e1856.
http://doi.org/10.1021/jf030723c.
Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J. A., & Prior, R. L. (2002). High-
throughput assay of oxygen radical absorbance capacity (ORAC) using a
multichannel liquid handling system coupled with a microplate fluorescence
reader in 96-well format. Journal of Agricultural and Food Chemistry, 50(16),
4437e4444. http://doi.org/10.1021/jf0201529.
Hur, S. J., Lim, B. O., Decker, E. A., & McClements, D. J. (2011). In vitro human
digestion models for food applications. Food Chemistry, 125(1), 1e12. http://doi.
org/10.1016/j.foodchem.2010.08.036.
van Huis, A., Van Itterbeeck, J., Klunder, H., Mertens, E., Halloran, A., Muir, G., &
Vantomme, P. (2013). Edible insects. Future prospects for food and feed security.
Food and Agriculture Organization of the United Nations (Vol. 171). Rome: Food
and agriculture organization of the United nations (FAO). http://doi.org/10.1017/
CBO9781107415324.004.
Janssen, R. H., Vincken, J. P., Van Den Broek, L. A. M., Fogliano, V., &
Lakemond, C. M. M. (2017). Nitrogen-to-Protein conversion factors for three
edible insects: Tenebrio molitor, Alphitobius diaperinus, and hermetia illucens.
Journal of Agricultural and Food Chemistry, 65(11), 2275e2278. http://doi.org/10.
1021/acs.jafc.7b00471.
Jonas-Levi, A., & Martinez, J.-J. I. (2017). The high level of protein content reported in
insects for food and feed is overestimated. Journal of Food Composition and
Analysis. http://doi.org/10.1016/j.jfca.2017.06.004.
Joubran, Y., Moscovici, A., & Lesmes, U. (2015). Antioxidant activity of bovine alpha
lactalbumin Maillard products and evaluation of their in vitro gastro-duodenal
digestive proteolysis. Food & Function, 6(4), 1229e1240.
Joubran, Y., Moscovici, A., Portmann, R., & Lesmes, U. (2017). Implications of the
Maillard reaction on bovine alpha-lactalbumin and its proteolysis during
in vitro infant digestion. Food & Function, 8, 2295e2308. http://doi.org/10.1039/
C7FO00588A.
Klunder, H. C., Wolkers-Rooijackers, J., Korpela, J. M., & Nout, M. J. R. (2012).
Microbiological aspects of processing and storage of edible insects. Food Control,
26(2), 628e631. https://doi.org/10.1016/j.foodcont.2012.02.013.
Kopf-Bolanz, K. A., Schwander, F., Gijs, M., Verge res, G., Portmann, R., & Egger, L.
(2014). Impact of milk processing on the generation of peptides during diges-
tion. International Dairy Journal, 35(2), 130e138. http://doi.org/10.1016/j.idairyj.
2013.10.012.
Leser, S. (2013). The 2013 FAO report on dietary protein quality evaluation in human
nutrition: Recommendations and implications. Nutrition Bulletin, 38(4),
421e428. http://doi.org/10.1111/nbu.12063.
Levi, C. S., & Lesmes, U. (2014). Bi-compartmental elderly or adult dynamic digestion
models applied to interrogate protein digestibility. Food & Function, 5(10),
2402e2409.
Liang, N., & Kitts, D. (2014). Antioxidant property of coffee Components: Assess-
ment of methods that define mechanisms of action. Molecules, 19(11),
19180e19208. http://doi.org/10.3390/molecules191119180.
Mackie, A., & Macierzanka, A. (2010). Colloidal aspects of protein digestion. Current
Opinion in Colloid & Interface Science, 15(1), 102e108.
Moon, J. K., & Shibamoto, T. (2009). Antioxidant assays for plant and food compo-
nents. Journal of Agricultural and Food Chemistry, 57(5), 1655e1666. http://doi.
org/10.1021/jf803537k.
Moscovici, A. M., Joubran, Y., Briard-Bion, V., Mackie, A., Dupont, D., & Lesmes, U.
(2014). The impact of the Maillard reaction on the in vitro proteolytic break-
down of bovine lactoferrin in adults and infants. Food & Function, 5(8),
1898e1908. http://doi.org/10.1039/c4fo00248b.
Msangi, S., & Rosegrant, M. W. (2012). Feeding the Future's changing Diets: Impli-
cations for agriculture markets, nutrition, and policy. In S. Fan, & R. Pandya-
Lorch (Eds.), Reshaping agriculture for nutrition and health (pp. 65e71).
Oliver, C. M., Melton, L. D., & Stanley, R. A. (2006). Creating proteins with novel
functionality via the Maillard reaction: A review. Critical Reviews in Food Science
and Nutrition, 46(4), 337e350. http://doi.org/10.1080/10408690590957250.
Palafox-Carlos, H., Ayala-Zavala, J. F., & Gonza
lez-Aguilar, G. A. (2011). The role of
dietary fiber in the bioaccessibility and bioavailability of fruit and vegetable
antioxidants. Journal of Food Science, 76(1), R6eR15. http://doi.org/10.1111/j.
1750-3841.2010.01957.x.
Payne, C. L. R., Scarborough, P., Rayner, M., & Nonaka, K. (2016). Are edible insects
more or less “healthy” than commonly consumed meats? A comparison using
two nutrient profiling models developed to combat over- and undernutrition.
European Journal of Clinical Nutrition, 70(3), 285e291. http://doi.org/10.1038/
ejcn.2015.149.
Rodríguez-Roque, M. J., de Ancos, B., Sa
nchez-Moreno, C., Cano, M. P., Elez-
Martínez, P., & Martín-Belloso, O. (2015). Impact of food matrix and processing
on the in vitro bioaccessibility of vitamin C, phenolic compounds, and hydro-
philic antioxidant activity from fruit juice-based beverages. Journal of Functional
Foods, 14, 33e43. http://doi.org/10.1016/j.jff.2015.01.020.
Rumpold, B. A., & Schluter, O. K. (2013). Potential and challenges of insects as an
innovative source for food and feed production. Innovative Food Science &
Emerging Technologies, 17, 1e11. http://doi.org/10.1016/j.ifset.2012.11.005.
Rumpold, B. A., & Schlüter, O. K. (2013). Nutritional composition and safety aspects
of edible insects. Molecular Nutrition & Food Research, 57(5), 802e823. http://
doi.org/10.1002/mnfr.201200735.
Shani Levi, C., Goldstein, N., Portmann, R., & Lesmes, U. (2017). Emulsion and protein
degradation in the elderly: Qualitative insights from a study coupling a dynamic
in vitro digestion model with proteomic analyses. Food Hydrocolloids, 69,
393e401. http://doi.org/10.1016/j.foodhyd.2017.02.017.
Somoza, V. (2005). Five years of research on health risks and benefits of Maillard
reaction products: An update. Molecular Nutrition and Food Research, 49(7),
663e672. http://doi.org/10.1002/mnfr.200500034.
Steinfeld, H., Gerber, P., Wassenaar, T., Castel, V., Rosales, M., & de Haan, C. (2006).
Livestock's long shadow - environmental issues and options. Food and Agri-
culture Organization of the United Nations, 3(1), 1e377. http://doi.org/10.1007/
s10666-008-9149-3.
Svilaas, A., Sakhi, A. K., Andersen, L. F., Svilaas, T., Stro € m, E. C.,
Jacobs, D. R., … Blomhoff, R. (2004). Intakes of antioxidants in coffee, wine, and
vegetables are correlated with plasma carotenoids in humans. The Journal of
Nutrition, 134(3), 562e567.
Vanhonacker, F., Van Loo, E. J., Gellynck, X., & Verbeke, W. (2013). Flemish consumer
attitudes towards more sustainable food choices. Appetite, 62, 7e16.
Verkerk, M. C., Tramper, J., Van Trijp, J. C. M., & Martens, D. E. (2007). Insect cells for
human food. Biotechnology Advances, 25(2), 198e202.
Wickham, M., Faulks, R., & Mills, C. (2009). In vitro digestion methods for assessing
the effect of food structure on allergen breakdown. Molecular Nutrition and Food
Research, 53(8), 952e958. http://doi.org/10.1002/mnfr.200800193.
Yi, L., Van Boekel, M. A. J. S., Boeren, S., & Lakemond, C. M. M. (2016). Protein
identification and in vitro digestion of fractions from Tenebrio molitor. Euro-
pean Food Research and Technology, 1e13.
Zhu, D., Damodaran, S., & Lucey, J. A. (2008). Formation of whey protein isolate
(WPI)-dextran conjugates in aqueous solutions. Journal of Agricultural and Food
Chemistry, 56(16), 7113e7118. http://doi.org/10.1021/jf800909w.