Innovative Food Science and Emerging Technologies 69 (2021) 102666

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Innovative Food Science and Emerging Technologies

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Fortification of wheat flour with black soldier fly prepupae. Evaluation of

technological and nutritional parameters of the intermediate doughs and
final baked products
Giuseppe Montevecchi a, *, Fabio Licciardello a, Francesca Masino a, Lucian T. Miron b, c,
Andrea Antonelli a
a
Department of Life Science (Agro-Food Science Area), BIOGEST - SITEIA Interdepartmental Centre, University of Modena and Reggio Emilia, Piazzale Europa 1, 42124
Reggio Emilia, Italy
b
Zetadec BV, Agro Business Park 44, 6708, PW, Wageningen, the Netherlands
c
Bioprocess Engineering, Department of Agrotechnology and Food Sciences, Wageningen University, PO Box 16, 6700, AA, Wageningen, the Netherlands

A R T I C L E I N F O A B S T R A C T

Keywords: Bread wheat flour presents specific nutrient deficiencies, such as some essential amino acids and this drawback
Bread could be tackled by adding ingredients that contain them in high amounts. Therefore, this study aims at
Hermetia illucens developing new types of flours as well as at analysing enriched flours and baked derived products, which
Essential amino acids
combine the compositional and sensory characteristics of wheat bread with some peculiar nutritional properties
Gluten
Insect food
of black soldier fly prepupae (Hermetia illucens). These composite flours were obtained by mixing “Italian type 1”
semi-whole wheat flour (W = 300) with flour obtained from prepupae of black soldier fly (20 g and 40 g/1 kg
composite flour). Chemical, physical, and rheological analyses were carried out for each of the flour mixtures.
Moisture, ash, gluten, total protein content, falling number, strength, tenacity, extensibility, amino acid profile of
doughs and breads were analysed as well. An increase in the content of essential amino acids in doughs and
baked products was obtained and also led to an improvement in bread texture.

1. Introduction wheat flour lacks some essential amino acids, notably lysine and thre­
onine (Shewry, 2007);(Cardoso, Fernandes, Gonzaléz-Paramás, Barros,
Nowadays, there is increasing consumer demand for value-added & Ferreira, 2019), hence their addition through an external source,
foods, with special regards for products with nutritional and health compatible with the production technology, could contribute to the
benefits (often referred to as nutraceuticals). Product innovation aimed increase of the nutritional value of flour and derived products (Siro,
at health-promoting properties consists in either the modification Kápolna, Kápolna, & Lugasi, 2008).
(usually through supplementation and fortification) of existing products In recent years, the use of insects has emerged as one of the most
having a consolidated market or in the creation of new products, which interesting alternative sources of proteins (Churchward-Venne, Pincka­
however need to gain the favour and acceptance of consumers. Bread ers, van Loon, & van Loon, 2017); (Atakan & Wansink, 2018). Although
and leavened bakery products are considered staple foods, whose basic insects are a common part of the diet for a large portion of the world
ingredient is soft wheat flour. Staple foods, thanks to their high con­ population (notably Asia, Africa, Australia, and South America), their
sumption and familiarity for consumers, are attracting items to be sup­ habitual consumption, especially in western countries, is still very
plemented in order to introduce valuable components and health- limited (Van Huis, 2013);(Shockley & Dossey, 2014): indeed, they
promoting elements in the diet (Patel, Watson, & Preedy, 2011). Soft perceive insects as a primitive food, with poor or unattractive sensory

Abbreviation: BD, bulk density; BSF, black soldier fly; BSFP, black soldier fly prepupae; BSFP20, black soldier fly prepupae flour (20 g) added to semi-whole wheat
flour to obtain a total of 1 kg of mixture; BSFP40, black soldier fly prepupae flour (40 g) added to semi-whole wheat flour to obtain a total of 1 kg of mixture; DEEMM,
diethyl 2-(ethoxymethylidene)propanedioate; FC, foam capacity; FN, falling number; L, dough extensibility; OAC, oil absorption capacity; P, dough tenacity; W,
dough strength; WAC, water absorption capacity.
* Corresponding author.
E-mail address: [email protected] (G. Montevecchi).

https://doi.org/10.1016/j.ifset.2021.102666
Received 16 December 2020; Received in revised form 6 March 2021; Accepted 13 March 2021
Available online 16 March 2021
1466-8564/© 2021 Elsevier Ltd. All rights reserved.
G. Montevecchi et al. Innovative Food Science and Emerging Technologies 69 (2021) 102666

qualities. Moreover, their scarce market availability, current price, in­ insects for the feed-food sector (Gasco, Biasato, Dabbou, Schiavone, &
sect phobia, feelings of disgust, and food neophobia (Palmieri, Perito, Gai, 2019). However, research into the use of edible insects and related
Macrì, & Lupi, 2019); (Barton, Richardson, & McSweeney, 2020); (Liu, technological applications are on the increase. Milled insects (Acheta
Li, & Gómez, 2020) contribute to the low consideration of insects as domesticus – house cricket; Gryllus assimilis – silent cricket; Schistocerca
food. Furthermore, many studies are focussing on the human health gregaria – grasshopper; Patanga succincta – Bombay locust; Alphitobius
hazards linked to eating insects, which include pathogens, mycotoxins, diaperinus – lesser mealworm or little beetle; Tenebrio molitor – meal­
pesticides, and heavy metals (viz. cadmium and mercury), as well as on worm; Bombyx mori – silkworm) or their derived products have already
the presence of anti-nutritional factors and the need to assess their been incorporated into novel foods, such as bread and bakery products
allergenicity (Birch & Bonwick, 2019); (Fasolin et al., 2019); (Fernan­ (Osimani et al., 2018); (Biró, Fodor, Szedljak, Pásztor-Huszár, & Gere,
dez-Cassi et al., 2019); (Murefu, Macheka, Musundire, & Manditsera, 2019); (da Rosa Machado & Thys, 2019); (González, Garzón, & Rosell,
2019); (Van der Spiegel, Noordam, & Van der Fels-Klerx, 2013); (Van 2019); (Haber, Mishyna, Martinez, & Benjamin, 2019); (Roncolini et al.,
Huis et al., 2013). One of the potential safety issues is linked to the 2019); (Akande, Jolayemi, Adelugba, & Akande, 2020); (Cappelli,
presence of spore-forming bacteria in bakery products (Osimani et al., Oliva, Bonaccorsi, Lorini, & Cini, 2020); (Indriani, Ab Karim, Bin Nali­
2018); another one is the mycotoxins problem, which has a multiplicity nanon, & Karnjanapratum, 2020); (Roncolini et al., 2020), while no
of implications, such as the tolerance that the specific insect has towards study has addressed the formulation of bread with BFS.
different toxic molecules, their accumulation, extraction, and biotrans­ This study is aimed at evaluating the enrichment of wheat flour with
formation with subsequent production of potentially toxic metabolites BSFP flour in order to produce a new kind of bread with an improved
(Van Huis, 2015). Although some studies have reported the ability that amino acids profile. Dough and bread properties were assessed in order
insects have of tolerating mycotoxins, the problem is far from being to evaluate possible technological effects of BSFP flour addition. The
solved (Bosch, Van Der Fels-Klerx, de Rijk, & Oonincx, 2017); amino acids chemical reduction caused by the baking process were also
(Camenzuli et al., 2018); (Purschke, Scheibelberger, Axmann, Adler, & investigated.
Jäger, 2017). As for the potential risk of allergies linked to insects’
consumption, a cross-reactivity between insects and other Arthropoda 2. Materials and methods
(crustaceans, mites) was ascertained in proteins found within insects’
muscle, in particular, tropomyosin was identified as the major pan- 2.1. Sampling of raw materials and preparation of the flour mixtures
allergen. The immunoreactivity can be observed in some protein frag­
ments even after protein hydrolysis has taken place, although some Semi-whole wheat flour (Italian type 1, legal specifications: moisture
suitable enzymatic processes can reduce the allergenic potential (Leni 14.5 g max/100 g; ash 0.80 g max/100 g D.W.; proteins 12 g min/100 g
et al., 2020); (Leni et al., 2020). D.W.) with W = 300 was provided from a local mill (Industria Molitoria
Despite all the drawbacks described above and the fact that risk Denti srl., Borzano di Albinea, Reggio Emilia, Italy). The black soldier fly
evaluation is ongoing, it is worth noting that there is also a part of prepupae grown on vegetable residues were collected from different
consumers (in particular young people) who are very attracted to novel suppliers located in Northern Italy. Other ingredients, such as water
foods since entomophagy is considered a thrilling experience (Roma, (minimally mineralised water – total dissolved solids 48 mg/L), table
Ottomano Palmisano, & De Boni, 2020). The number of potential con­ salt, and baker’s yeast were purchased from local markets.
sumers is expected to increase in the near future especially considering BSFP delivered frozen at − 20 ◦ C were dried in a ventilated oven at
the impact of the widespread awareness on this food as well as the power 60 ◦ C until constant weight: this temperature was selected to ensure a
mass media have in steering consumers’ decisions (Aquilanti et al., considerable reduction of the microbial charge and to avoid spoiling the
2020); (Liu et al., 2020); (Roma et al., 2020). Another interesting point biomass (Baker, 2002); (McCabe-Sellers & Beattie, 2004). Dried BSFP
is that insect rearing has numerous advantages over cattle breeding in were ground using an immersion blender (Pimmy metal, Ariete, Flor­
terms of sustainability in the current climate change scenario, ethical ence, Italy) and sifted using a 0.5 mm sieve (Certified test sieves, Giu­
opposition to meat consumption, and the attempt to limit the spread of liani Tecnologie, Turin, Italy). The flour mixtures were obtained with
diseases from animal to human beings (Van Huis, 2020); (Dicke, 2019); the geometric dilution method, using both kitchen utensils and an
(Ruschioni et al., 2020). electric mixer until thorough homogeneity was reached.
Hermetia illucens, commonly called black soldier fly (BSF), is a non- Two mixtures of flours were prepared using 20 g (BSFP20; 2% w/w)
infesting species as opposed to the common fly (Musca domestica), and 40 g (BSFP40; 4% w/w) of black soldier fly prepupae flour added to
which is attracted to human food and so it can become a potential carrier semi-whole wheat flour to obtain a total of 1 kg of mixture. The control
of pathogens (Furman, Young, & Catts, 1959); (Bahrndorff, De Jonge, sample consisted of semi-whole wheat flour.
Skovgård, & Nielsen, 2017); (Miranda, Cammack, & Tomberlin, 2020).
BSF larvae are voracious consumers of decaying organic matter (Bondari 2.2. Chemicals
& Sheppard, 1981), (Newton, Sheppard, Watson, Burtle, & Dove, 2005);
(Bortolini et al., 2020); (Jucker, Lupi, Moore, Leonardi, & Savoldelli, Pure reference compounds (arginine, cysteine, histidine, isoleucine,
2020), capable of efficiently bioconverting large quantities of waste or leucine, lysine, methionine, phenylalanine, threonine, tryptophan,
spoiled food into biomass, which is rich in proteins and lipids (Diener, tyrosine, and valine), acetic acid, acetonitrile, boric acid, diethyl 2-
Zurbrügg, & Tockner, 2009); (Hadj Saadoun et al., 2020); (Nuvoli et al., (ethoxymethylidene)propanedioate (DEEMM, CAS Number 87–13-8),
2021). As for its chemical components, the BSF prepupa (BSFP) is hydrochloric acid, methanol, sodium hydroxide were purchased from
composed on a dry weight (D.W.) basis of up to 37 g/100 g lipids, 32 g/ Sigma-Aldrich (Milan, Italy). Deionised water was obtained using an
100 g proteins, 19 g/100 g mineral salts (including micronutrients, such Elix® Essential3UV system (Merck-Millipore, Milan, Italy).
as iron and zinc), and 9 g/100 g chitin (Caligiani et al., 2018); (Smets
et al., 2020); (Montevecchi, Zanasi, Masino, Maistrello, & Antonelli, 2.3. Rheological and functional properties of flours
2020); (Hadj Saadoun et al., 2020); (Ravi et al., 2020).
Although in EU member countries the interpretation of the standard 2.3.1. Alveographic measurements
has never been univocal, from 1 January 2018 the Regulation (EU) The alveographic parameters, such as dough tenacity (P), dough
2015/2283 identifies all insects and their derivatives as novel foods and, extensibility (L), dough strength (W), and configuration of the curve (P/
as such, subject to authorization. The gaps and legislative constraints on L) were evaluated using a Chopin alveograph MA82 (Tripette et Renaud,
the use of edible insects in food production (Paganizza, 2017) have so far Villeneuve-la-Garenne, France), in accordance with the standard
hampered the development of industrial technologies to mass rear method (UNI EN ISO 27971:2015), employing only flours and water.

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G. Montevecchi et al. Innovative Food Science and Emerging Technologies 69 (2021) 102666

Based on the assumption that BSFP powder is not used in bread­ Table 1
making as it is, this ingredient has not been subjected to rheological Formulations of doughs obtained with the use of wheat flour (WF) and different
characterisation. blends of wheat flour fortified with 20 g and 40 g BSFP flour/1 kg composite
flour (namely, BSFP20 and BSFP40).
2.3.2. Determination of the falling number Ingredients Units Control BSFP20 BSFP40
The falling number (FN) was determined according to the standard (WF)
method (AACC, 2000) using a falling number instrument (1500 Perten, WF g 250 245 240
Esetek Instruments, Marino, RM, Italy). The analysis was performed in BSFP flour g not added 5 10
triplicate and the values were expressed in seconds. Water (minimally mineralised water – mL 100 100 100
total dissolved solids = 48 mg/L)
NaCl (aqueous solution 10 g/100 g) mL 50 50 50
2.3.3. Water absorption capacity (WAC) and oil absorption capacity Baker’s yeast g 0.5 0.5 0.5
(OAC)
The ability of flours to bind to water (WAC) and oil (OAC) in specific
conditions was determined using the method described by Kaur, the dough was measured using a Vernier calliper. The dry residue was
Sandhu, Arora, and Sharma (2015). determined in an oven at 105 ◦ C until constant weight and expressed as
WAC and OAC were calculated in triplicate as gwater and goil per gdry matter/100 gfresh dough.
gsample on a dry basis, respectively, by applying the following equations:
( / ) 2.6. Dynamometric tests on the doughs
WHC gwater gsample powders = [(fresh residue–dry residue)/dry residue ]
( / ) Dynamometer tensile tests were performed on risen dough samples
OHC goil gsample powders = [(fresh residue–dry residue)/dry residue ] (Z1.0 Zwick Roell). The doughs (20 g) were placed in a 25-mL beaker
and a 2-cm diameter spherical tool, which was initially completely
2.3.4. Bulk density immersed in the sample, was used for the measurements. The test was
The bulk density (BD) of the various flour mixtures was assessed carried out at 10 mm/min speed and it was stopped once 80% of the
using the method described by Kaur et al., 2015. The measurements maximum force (Fmax) was reached. The breaking ε values were evalu­
were made in three replicates and the density was calculated as gflour/10 ated to obtain information about the extent of deformation, and there­
mL. fore about elasticity, while the maximum strength gave indications
regarding the tenacity of the dough. For each sample, the analysis was
2.3.5. Determination of foam capacity conducted in duplicate.
The foam capacity (FC) was determined using the method described
by Chandra, Singh, and Kumari (2015). Deionised water (50 mL, 30 ◦ C) 2.7. Preparation of baked products
was added to each sample (1 g) in a 100 mL graduated cylinder. Sub­
sequently, the suspension was mixed for 5 min to obtain the foam. The For the preparation of products, 250 g of risen dough was baked for
volume was measured both before (BW) and after (AW) mixing. The 35 min at 220 ◦ C in a ceramic ramekin inside an oven. The height of the
measurements were made in triplicate and the FC was calculated ac­ baked products was measured using a Vernier calliper, while the weight
cording to the following formula: was quantified using a weighing scale. The dry residue was determined
FC = (volumeAW − volumeBW )/volumeBW in an oven at 105 ◦ C until a constant weight was reached and was
expressed as gdry matter/100 gfresh bread.

2.4. Chemical analysis (moisture, protein, gluten, and ash)

2.8. Determination of total and free amino acids

The moisture was determined using a thermobalance (HB43-S

Samples were analysed in both hydrolysed and native form in order
Halogen Mettler Toledo, Bühler S.p.A., Segrate, MI, Italy) at 105 ◦ C. The
to determine the concentration of total and free amino acids. After
analysis was performed in triplicate and the moisture was expressed in
extraction of the hydrolysed and native samples, a derivatization step
gwater/100 g of flour. Total protein content was determined through a
was carried out before HPLC-UV/Vis determination.
NIR spectrophotometer (1241 Infratec, FOSS Italia S.p.a., Padova, Italy)
As for the hydrolysis of protein amino acids, 0.5 g of each sample was
and expressed in gproteins/100 g D.W. of flour. In the same determination,
introduced into a glass test tube and 3 mL of 6 M HCl was added (Hirs,
NIR was also used to measure the moisture.
Moore, & Stein, 1956). Sealed test tubes were subsequently vortexed for
Dry gluten was determined on 10 g of flour according to the standard
1 min and the reaction was allowed in a thermoblock at 110 ◦ C for 24 h.
method (AACC, 2000) using a Glucomatic device (2200 Perten, Esetek
Subsequently, the samples were centrifuged at 2065.7 g for 6 min at
Instruments, Marino, RM, Italy). To calculate the gluten content in 100 g
room temperature. The supernatant was collected and the pH was
of flour the following formula was used:
adjusted to 9 using NaOH solution and borate buffer.
/
gdry gluten 100 g D.W. flour = m × 10 × 100 U The extraction of free amino acids was performed according to the
method described by Benedito De Barber, Prieto, and Collar (1989).
where m is the mass of dry gluten, and U is the percentage moisture Each sample (0.5 g) was introduced into a test tube, and 1.5 mL of 0.01
of the sample. M acetic acid was added. The mixture was vortexed for 5 min and then
Ash content was determined using a standard method (AOAC, 2000) brought to 3 ◦ C. Subsequently, the test tube was centrifuged for 10 min
in a muffle furnace (550 ◦ C for 6 h) and the result was expressed as gash/ at 1464.8 g and the supernatant was collected in a 5-mL volumetric flask.
100 g D.W. of flour. The sediment was extracted again with 0.01 M acetic acid. The whole
process was repeated a third time and the pooled extract was made up to
2.5. Preparation of dough and leavening 5 mL with a borate buffer solution at pH 9.
Amino acids’ derivatisation was carried out using 200 μL of the
With regards to the dough, 250 g of total flour were prepared as sample, 750 μL of borate buffer (pH 9), 300 μL of methanol, and finally
described in Table 1. The dough was obtained using a kneading machine 30 μL of pure DEEMM. The reaction took place in a sealed test tube at
(Prospero KM28, Kenwood, Bologna, Italy) and left to rise for 24 h at 70 ◦ C and lasted 2 h.
25 ◦ C inside plastic 3-L containers closed with their lids. The height of The derivatised sample for free amino acids determination (non-

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G. Montevecchi et al. Innovative Food Science and Emerging Technologies 69 (2021) 102666

hydrolysed) was diluted 1:100, while the derivatised sample for total Table 2
amino acids determination (hydrolysed) was diluted 1:500. The samples Results of chemical analyses carried out of semi-whole wheat flour (Control)
were injected in triplicate. alone and in combination with 20 g and 40 g BSFP flour/1 kg composite flour
The determination of amino acids was carried out using an HPLC (BSFP20 and BSFP40). Control: wheat flour (type 1 W300).
system (PU4180, Jasco Europe Srl, Cremella, LC, Italy) equipped with a Units Control BSFP20 BSFP40 ANOVA
UV/Vis detector (UV4070, Jasco) set at 280 nm. Through an autosam­ (WF) (Fvalue)
pler, 20 μL of each sample was injected into the RP-C18 column Moisture g/100 12.48 ± 12.33 ± 12.11 ± 121***
(Poroshell, 120 SB-C18, Agilent Technologies, D.T.O. Servizi Srl, Spinea, (thermobalance) gFW 0.04 c 0.04 b 0.01 a
VE, Italy). The solvent system was prepared according to the method Moisture (NIR) g/100 12.75 ± 12.25 ± 11.32 ± 336***
gFW 0.07 c 0.07 b 0.02 a
described by Gómez-Alonso, Hermosín-Gutiérrez, and García-Romero Gluten g/100 11.00 ± 11.15 ± 11.35 ± 19**
(2007) with some minor adjustments and was composed of eluent A: gDW 0.14 a 0.07 a 0.07 b
11.6 mL of 2 M NaOH and 1.5 mL of glacial acetic acid brought to 1 L Ash g/100 0.75 ± 2.56 ± 3.60 ± 187***
with deionised water (subsequently, the pH meter was used to adjust the gDW 0.07 a 0.44 b 0.01 c
Protein (NIR) g/100 13.10 ± 14.35 ± 14.85 ± 20**
pH to 5.34); and eluent B: methanol-acetonitrile 20:80. Elution (70 min
gDW 0.14 a 0.78 b 0.35 b
run) was carried out according to the following gradient (mL/100 mL B): Falling number sec 447 ± 1 b 412 ± 12 407 ± 6 a 16*
0 at 0 min, 4 at 7 min, 6 at 18 min, 8 at 23 min, 8 at 25 min, 15 at 28 min, a
23 at 45 min, 45 at 50 min, 60 at 55 min, 80 at 57 min, 80 at 64 min, 0 at
Results of the one-way ANOVA and Tukey’s test are reported as Fvalues and
66 min, and 0 at 70 min with a flow rate of 0.6 mL/min at ambient lowercase letters (“c” > “b” > “a”), respectively. Different letters identify
temperature (pressure of about 180 bar at run start). significantly different samples (p ≤ 0.05).* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤
Identification and quantification steps were carried out by injecting a 0.001; ns: non-significant. FW = fresh weight; DW = dry weight.
set of pure-standard solutions of known concentrations prepared in the
same conditions used for the samples. Quantification took place through has already been observed at a higher extent for higher replacement
external calibration. levels with cricket flour (Osimani et al., 2018).
As for gluten, ANOVA shows an increase in the sample BSFP40. This
2.9. Statistical analysis surprising finding could be linked to the properties of a particular
fraction of BSFP proteins. Indeed, Caligiani et al. (2018) described the
Univariate analyses were carried out on the data set. Differences presence of water-insoluble proteins, such as glutelins and prolamins, in
among cases were assessed through the analysis of variance (one-way BSFP. However, this hypothesis deserves further investigation. As a
ANOVA) based on three replicates for each treatment. When a signifi­ matter of fact, it cannot be excluded that insoluble BSF biopolymers of
cant effect (at least p ≤ 0.05) was observed, comparative analyses were different nature could play a role in the increase of this value.
carried out via the post hoc Tukey’s multiple comparison test. All tests
were performed using Statistica v8.0 software (Stat Soft Inc., Tulsa,
USA). 3.2. Results of the technological analyses carried out on flour enriched
with BSFP
3. Results and discussion
Looking at Table 3, the ANOVA shows a significant increase of
Bread was chosen as the preferred food product on which to test the tenacity (P) and P/L and a decrease of dough extensibility (L) with
performance of BSFP flour, on the one hand, because it is a staple food increasing BSFP flour replacement levels. The addition of protein com­
consumed worldwide as part of the human diet, on the other, because it ponents could act on various gluten proteins by promoting the formation
is subject to low hygienic-microbiological risks. Indeed, food- of additional interprotein bonds, directly or through some reaction by-
manufacturing methods such as baking, as well as boiling, frying, and products (Bonomi, Ferranti, & Mamone, 2014). Furthermore, an effect
roasting, are known to increase the safety, and, in turn, the acceptability on P due to the higher presence of ash (cations) should be taken into
of insect-based foods (Murefu et al., 2019). However, lower acceptance consideration (Chieh, 2008). Similarly, Osimani et al. (2018) observed
levels from the panellists as well as negative technical characteristics an increase of P/L in composite dough made of wheat flour with an
were observed in a study where cricket powder was incorporated in high addition of 10% and 30% cricket flour. These authors also observed a
amounts (30%) (Osimani et al., 2018). significant reduction of W value. In the present study, the dough
During preliminary sample preparation tests, it was observed that strength showed a decreasing trend when BSFP was added, even if dif­
adding BSF flour in percentages higher than 4% led to a drastic drop of ferences were not statistically significant due to the wide variability of
dough strength value (W). For this reason, it was decided not to exceed the values around the average. However, taking into account the low
4% and also to use flour with a high W (i.e. 300) to avoid an excessive replacement level (2–4%) used in the present study as compared to the
drop of this parameter caused by the presence of BSFL flour. cited paper, these effects of insect flour on the rheological properties of
dough are confirmed.
3.1. Chemical composition of flours It should be stressed that the Chopin alveograph was designed and
optimised for the rheological evaluation of doughs made exclusively
The chemical composition of flour samples is shown in Table 2. The with wheat flour (Bouazizi, Montevecchi, Antonelli, & Hamdi, 2020).
ANOVA highlights significant differences for all the parameters taken The centroid of the sample set (BSFP20) showed an intermediate value
into consideration. In particular, in all the samples where BSFP flour had between the control and BSFP40 for all the parameters observed. The
been added, the content of ash and total proteins significantly increased, increasing and decreasing trends highlighted by the present study
as to testify the high contribution of BSFP flour at even small levels of coincided with the technological variables’ trends, described in the
substitution (2–4%). On the other hand, the addition of BSFP flour led to literature, that concern the addition of other kinds of insects to wheat
a decrease in moisture and falling number. A residual enzymatic pool of flour (Osimani et al., 2018); (Cappelli et al., 2020).
BSFP could have contributed to the partial hydrolysis of starch and, The ANOVA also shows a significant increase of the Fmax in the
therefore, to the reduction of the falling number. This advantageous samples containing BSFP when compared to the control, while the ε
feature could also reduce the demand for bakery additives, such as malt, break does not show any significant difference among the samples due to
malted cereals, and amylases (Edwards, 2007). The decrease in the the large variation observed around the average (high standard de­
falling number using insect-based flour in replacement of wheat flour viations) (Osimani et al., 2018).

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Table 3 Table 4
Alveographic measurements, rheological, and functional properties of semi- Measurements of dough and baked products obtained using semi-whole wheat
whole wheat flour (Control) alone and in combination with 20 g and 40 g flour (Control) alone and in combination with 20 g and 40 g BSFP flour/1 kg
BSFP flour/1 kg composite flour (BSFP20 and BSFP40). Control: wheat flour composite flour (BSFP20 and BSFP40). Control: wheat flour (type 1 W300).
(type 1 W300). Units Control BSFP20 BSFP40 ANOVA
Units Control BSFP20 BSFP40 ANOVA (WF) (Fvalue)
(WF) (Fvalue)
Dough Pre mm 9.7 ± 11.5 ± 12.3 ± n.s.
Alveographic and leavening 1.5 3.3 1.2
properties bread Post mm 39.0 ± 40.3 ± 37.7 ± n.s.
W 10− 4
J 322.0 ± 302.6 ± 292.2 ± n.s. height leavening 1.8 2.1 4.7
17.4 45.9 25.9 Post mm 57.2 ± 57.8 ± 66.2 ± n.s.
P mm 102.0 ± 110.6 ± 121.8 ± 16*** cooking 5.8 4.2 8.0
4.4 a 6.5 a 5.5 b Dough Pre g 250.0 250.1 250.0 n.s.
L mm 94.2 ± 5.8 79.2 ± 67.2 ± 7.1 11** and cooking
b 13.1 ab a bread Post g 216.9 217.4 217.0 n.s.
P/L Ratio 1.09 ± 1.42 ± 1.83 ± 22*** weight cooking
0.09 a 0.19 b 0.22 c Water loss g 33.1 32.6 33.0
Rheological (13%) (13%) (13%)
properties Dry Dough g/ 53.6 ± 54.8 ± 55.5 ± 22*
ε break % 582.29 ± 735.76 ± 749.34 ± n.s. weight 100 0.1 a 0.3 ab 0.3 b
77.77 84.39 18.04 gFW
Fmax N 2.58 ± 3.10 ± 3.45 ± 23** Bread g/ 64.3 ± 62.7 ± 63.1 ± n.s.
0.14 a 0.17 b 0.16 b 100 2.9 1.7 0.4
Functional gFW
properties
Results of the one-way ANOVA and Tukey’s test are reported as Fvalues and
BD g/mL 0.87 ± 0.79 ± 0.80 ± 81***
0.01 b 0.01 a 0.01 a lowercase letters (“b” > “a”), respectively. Different letters identify significantly
WAC gwater/ 1.87 ± 1.84 ± 1.86 ± n.s. different samples (p ≤ 0.05).* = p ≤ 0.05; ns: non-significant. FW = fresh weight.
gsample 0.01 0.02 0.01
OAC goil/ 1.86 ± 1.82 ± 1.80 ± n.s.
Bread height is related to bread volume development. González et al.
gsample 0.13 0.06 0.08
FC g/100 0.18 ± 0.17 ± 0.11 ± 19*
(2019) observed a significant reduction of bread specific volume
mL 0.00 b 0.02 b 0.01 a following a 5% BSF replacement, whether whole or defatted, to wheat
flour. Volume reduction is usually related to a weakening of the gluten
Results of the one-way ANOVA and Tukey’s test are reported as Fvalues and
network, which was not observed in our study. Here, no significant
lowercase letters (“c” > “b” > “a”), respectively. Different letters identify
significantly different samples (p ≤ 0.05).* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ difference was observed for dough and bread height following replace­
0.001; ns: non-significant. ment with 2% and 4% BSFP flour. However, P/L values significantly
P = dough tenacity; L = dough extensibility; W = dough strength; P/L = increased, with an increase in P and a reduction in L values at increasing
configuration of the curve; BD = bulk density; WAC = water absorption ca­ BSFP flour replacement levels (Table 3): this change may explain the
pacity; OAC = oil absorption capacity; FC = foaming capacity. trend observed in the dough leavening degree. This value, deriving from
the ratio between the height of leavened dough and the height of pre-
The results of the technological parameters carried out on the sample leavened dough, was about 400%, 350%, and 300% in the control, the
set are shown in Table 3. ANOVA showed significant differences for BD BSFP20, and the BSFP40 dough samples, respectively, thus showing a
and FC, indeed both decreased with the progressive addition of BSFP decreasing trend when BSFP was added. In other words, BSF flour seems
flour, while WAC and OAC were not affected. The water and oil ab­ to induce higher stiffness and lower extensibility in the dough, thus
sorption capacity are mainly to be attributed to the physical entrapment limiting the volume expansion during leavening.
of water or oil. For this reason, the addition of low percentages of BSFP
(2–4%) did not influence WAC and OAC. Generally, the addition of
3.4. Free and total amino acids’ determination
flours with a high amount of hydrophilic proteins increases the hydra­
tion and water absorption of dough. Results showed that water ab­
The free amino acids were determined for three main reasons: to
sorption capacity was not influenced by the BSFP replacement: this
verify their presence in the ingredients, the effect of baker’s yeast on
finding agrees with the results from Osimani et al. (2018) for wheat
them during fermentation, and their chemical modifications linked to
dough obtained with cricket flour replacement and supports the above-
the thermal treatment. However, they were also quantified based on the
mentioned hypothesis of water-insoluble proteins contributed by BSF
assumption that, when the BSFP are killed, a more or less extensive
(Caligiani et al., 2018). On the other hand, González et al. (2019)
enzymatic hydrolysis may take place, which certainly involves the li­
observed a reduction of water absorption capacity of dough following a
pases (Montevecchi et al., 2020), but could also potentially affect the
5% BSF replacement, whether whole or defatted, to wheat flour, and
proteases and therefore the release of amino acids. A significant increase
they attributed this result to the specific nature of proteins and amino
of all free amino acids in the flours was observed with increasing levels
acids contained in the insect flour (Rumpold & Schlüter, 2013).
of BSFP flour, the only exceptions being methionine and cysteine
(Table 5).
3.3. Results of the technological analyses carried out on dough and baked The total concentrations of histidine, threonine, arginine, valine,
products leucine, and lysine gradually increased in the samples added with BSFP
flour, while, in other cases, the large variation around the average did
Table 4 shows the measurements of the dough and baked products. not allow the same trend to be observed (Table 5).
Overall, the ANOVA does not show significantly different values, except All free amino acid concentrations in BSFP-enriched products were
for the dry weight of the doughs, which increased in parallel with the significantly higher, except methionine and cysteine, thus showing a
BSFP flour content, while no differences in height, weight, and dry consistent behaviour with the one observed in the flours (Table 6). The
residue of the bread samples were observed. The slight difference due to results of the ANOVA carried out on the free amino acid concentrations
the lower moisture of the samples added with BSFP flour was completely in the flours and in the baked products to evaluate the thermal degra­
lost during baking. dation of the amino acids is shown in Table 6. The concentrations of free

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G. Montevecchi et al. Innovative Food Science and Emerging Technologies 69 (2021) 102666

Table 5
Free amino acid concentrations (expressed as mgaa/kgDW) and total amino acid concentrations (expressed as mgaa/kgDW) in semi-whole wheat flour (Control) alone
and in combination with 20 g and 40 g BSFP flour/1 kg composite flour (BSFP20 and BSFP40). Control: wheat flour (type 1 W300).
Free amino acids Total amino acids

Control BSFP20 BSFP40 ANOVA Control BSFP20 BSFP40 ANOVA

Hys 53.0 ± 9.3 a 98.8 ± 0.9 b 151.7 ± 2.6 c 156*** 2879 ± 70 a 3209 ± 139 ab 3768 ± 182 b 21*
Thr 41.3 ± 4.0 a 160.7 ± 1.7 b 297.1 ± 30.5 c 103** 2839 ± 151 a 3265 ± 4b 3884 ± 64 c 62**
Arg 120.9 ± 4.2 a 320.0 ± 1.1 b 510.6 ± 26.9 c 307*** 4203 ± 6 a 4755 ± 184 a 5652 ± 174 b 50**
Tyr 10.3 ± 0.9 a 44.8 ± 1.7 b 83.5 ± 0.1 c 2182*** 2076 ± 84 2093 ± 38 2180 ± 11 n.s.
Val 55.7 ± 3.8 a 205.3 ± 0.3 b 369.0 ± 6.4 c 2664*** 6032 ± 149 a 6661 ± 36 a 8055 ± 352 b 44**
Met 33.7 ± 0.5 28.1 ± 2.1 31.5 ± 5.7 n.s. 1931 ± 171 1989 ± 172 2204 ± 39 n.s.
Cys < LOD < LOD < LOD n.s. 2931 ± 209 2637 ± 34 3108 ± 325 n.s.
Ile + Trp 126.0 ± 1.6 a 155.6 ± 2.7 b 198.5 ± 5.5 c 196*** 4204 ± 249 4268 ± 131 5221 ± 448 n.s.
Leu 108.1 ± 5.4 a 289.0 ± 4.1 b 398.2 ± 7.9 c 1196*** 9607 ± 9 a 9122 ± 240 a 12,275 ± 234 b 154***
Phe 188.1 ± 1.7 a 190.1 ± 5.7 a 401.5 ± 23.4 b 155*** 6239 ± 333 6488 ± 251 6850 ± 302 n.s.
Lys 55.7 ± 2.0 a 151.0 ± 0.3 b 242.1 ± 15.4 c 215*** 2328 ± 23 a 2611 ± 101 a 3278 ± 153 b 42**

Results of the one-way ANOVA and Tukey’s test are reported as Fvalues and lowercase letters (“c” > “b” > “a”), respectively. Different letters identify significantly
different samples (p ≤ 0.05).* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; ns: non-significant.

Table 6
Concentrations of free amino acids (expressed as mgaa/kgDW) in baked products obtained with semi-whole wheat flour (Control) alone and in combination with 20 g
and 40 g BSFP flour/1 kg composite flour (BSFP20 and BSFP40) and evaluation of the thermal degradation of free amino acids by comparing flours and baked products
(ANOVA). Control: semi-whole wheat flour (type 1 W300).
Free amino acids in baked products Thermal degradation evaluation of the free amino acids

Control BSFP20 BSFP40 ANOVA Control BSFP20 BSFP40 Control BSFP20 BSFP40 ANOVA
bread bread bread flour flour flour bread bread bread

Hys 27.8 ± 0.2 a 81.4 ± 2.3 b 103.1 ± 6.9 c 172*** b cd e a c d 152***

Thr 91.7 ± 5.5 a 98.3 ± 14.2 a 226.1 ± 18.7 59** a b d a ab c 71***
b
Arg 151.4 ± 1.6 478.1 ± 22.2 746.5 ± 13.6 785*** a b c a c d 479***
a b c
Tyr 15.1 ± 2.8 a 42.8 ± 1.3 b 118.9 ± 9.5 c 173*** a b c a b d 202***
Val 18.2 ± 0.4 a 218.0 ± 9.4 b 482.6 ± 16.6 895*** b c d a c e 211***
c
Met < LOD 2.0 ± 0.8 1.6 ± 0.7 n.s. b b b a a a 84***
Cys < LOD < LOD < LOD n.s. n.s.
Ile + 52.7 ± 2.3 a 83.3 ± 0.3 b 158.8 ± 8.6 c 225*** c d e a b d 287***
Trp
Leu 93.4 ± 3.5 a 176.3 ± 0.2 b 368.4 ± 10.5 983*** a c e a b d 910***
c
Phe 140.3 ± 5.6 282.6 ± 15.1 449.4 ± 16.0 280*** a a c a b c 174***
a b c
Lys 72.2 ± 4.5 a 149.6 ± 2.0 b 297.8 ± 8.8 c 777*** a b c a b d 311***

Results of the one-way ANOVA and Tukey’s test are reported as Fvalues and lowercase letters (“e” > “d” > “c” > “b” > “a”), respectively. Different letters identify
significantly different samples (p ≤ 0.05). ** = p ≤ 0.01; *** = p ≤ 0.001; ns: non-significant.

amino acids were, generally speaking, significantly higher in the flours 4. Conclusions
compared to the baked products, specifically histidine, threonine,
methionine, isoleucine and leucine. In some cases, the concentrations The use of semi-wholemeal flours enriched with BSFP to make bread
were similar in the raw and baked products, while, in some other cases, has given satisfactory results in terms of rheological and physicochem­
the baked products showed higher concentrations than flours. ical characteristics. The free and total essential amino acids contribu­
The concentration values of total amino acids in the baked products tions to the flours and baked products were also assessed and the
are shown in Table 7. Amino acid concentrations in BSFP enriched thermal damage they suffered was evaluated.
products were significantly higher for all amino acids, except for tyro­ BSFP flour, on which many studies are currently focussing when
sine, methionine, cysteine and phenylalanine, which did not show any incorporated at concentrations of 2% and 4%, did not give rise to any
relevant difference also due to high standard deviations. The results of significant differences in terms of strength (W). The processing of the
the ANOVA carried out on the total amino acid concentrations in the doughs was not difficult in technological terms. The samples containing
flours and in the baked products in order to evaluate the thermal BSFP highlighted two important results: on one hand, the falling number
degradation of the amino acids are shown in Table 7. In many cases, the decreased, and this was likely due to the presence of an additional
concentrations of total amino acids were similar in the flours and baked enzymatic activity, on the other hand, the gluten content increased, for
products while in other cases, the baked products showed slightly lower reasons which are not yet clear. An increase of P/L was observed with
concentrations than flours. The concentrations of total amino acids in BSFP addition, which led to a slight decrease of leavening degree,
many cases showed only narrow differences between the flour samples however differences among bread samples were hardly noticeable at the
and the baked products, thus proving the degree of protection the low replacement levels selected in this study.
polyamide structure provides against the baking process carried out in With regards to the amino acids’ profile, an overall increase in
the conditions of the present research study. essential amino acids was observed, ascribable to the presence of the BSF
flour. The concentrations of total amino acids did not show any sub­
stantial difference between the baked products and the original flours,

6
G. Montevecchi et al. Innovative Food Science and Emerging Technologies 69 (2021) 102666

Table 7
Concentrations of total amino acids (expressed as mgaa/kgDW) in baked products obtained with semi-whole wheat flour (Control) alone and in combination with 20 g
and 40 g BSFP flour/1 kg composite flour (BSFP20 and BSFP40) and evaluation of the thermal degradation of free amino acids by comparing flours and baked products
(ANOVA). Control: semi-whole wheat flour (type 1 W300).
Total amino acids in baked products Thermal degradation evaluation of the total amino acids

Control BSFP20 BSFP40 ANOVA Control BSFP20 BSFP40 Control BSFP20 BSFP40 ANOVA
bread bread bread flour flour flour bread bread bread

Hys 2700 ± 26 a 3192 ± 171 3426 ± 46 b 26* ab bc cd a bc d 19**

b
Thr 2760 ± 9 a 3479 ± 64 b 3909 ± 109 c 126** a b c a b c 69***
Arg 4369 ± 40 a 5019 ± 55 b 5348 ± 192 b 36** a bc e ab cd de 36***
Tyr 2069 ± 15 2138 ± 81 2203 ± 14 n.s. n.s.
Val 5103 ± 120 6002 ± 185 5954 ± 256 b 13* b b c a b b 45***
a b
Met 1829 ± 110 2042 ± 92 2058 ± 292 n.s. n.s.
Cys 3027 ± 40 2778 ± 92 3030 ± 294 n.s. n.s.
Ile + 3916 ± 6 a 4728 ± 88 b 4640 ± 134 b 46** a a b a ab ab 9**
Trp
Leu 9916 ± 60 a 10,246 ± 84 12,162 ± 711 17* a a b a a b 34***
a b
Phe 6642 ± 95 6821 ± 66 7286 ± 562 n.s. n.s.
Lys 2246 ± 12 a 2426 ± 61 a 2888 ± 127 b 33** a ab c a a b 34***

Results of the one-way ANOVA and Tukey’s test are reported as Fvalues and lowercase letters (“e” > “d” > “c” > “b” > “a”), respectively. Different letters identify
significantly different samples (p ≤ 0.05).* = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; ns: non-significant.

while the degradative phenomenon was more evident for the free amino Benedito De Barber, C., Prieto, J. A., & Collar, C. (1989). Reversed-phase high-
performance liquid chromatography analysis of changes in free amino acids during
acids. It can, therefore, be concluded that the essential amino acids
wheat bread dough fermentation. Cereal Chemistry, 66(4), 283–288.
supplied to wheat flour by the BSFP flour are maintained throughout the Birch, C. S., & Bonwick, G. A. (2019). Ensuring the future of functional foods.
production steps, thus resulting in an effective fortification of the International Journal of Food Science & Technology, 54(5), 1467–1485. https://doi.
finished products. org/10.1111/ijfs.14060
Biró, B., Fodor, R., Szedljak, I., Pásztor-Huszár, K., & Gere, A. (2019). Buckwheat-pasta
enriched with silkworm powder: Technological analysis and sensory evaluation.
Declaration of Competing Interest LWT, 116, 108542. https://doi.org/10.1016/j.lwt.2019.108542
Bondari, K., & Sheppard, D. C. (1981). Soldier fly larvae as feed in commercial fish
production. Aquaculture, 24, 103–109. https://doi.org/10.1016/0044-8486(81)
The authors declare that they have no known competing financial 90047-8
interests or personal relationships that could have appeared to influence Bonomi, F., Ferranti, P., & Mamone, G. (2014). Wheat flour: Chemistry and biochemistry.
the work reported in this paper. In Bakery Products Science and Technology (pp. 55–74). Wiley Online Library. https://
doi.org/10.1002/9781118792001.
Bortolini, S., Macavei, L. I., Saadoun, J. H., Foca, G., Ulrici, A., Bernini, F., …
Acknowledgements Maistrello, L. (2020). Hermetia illucens (L.) larvae as chicken manure management
tool for circular economy. Journal of Cleaner Production, 121289. https://doi.org/
10.1016/j.jclepro.2020.121289
The authors wish to thank Dr. Lisa Salani, Dr. Valeria Paganizza Bosch, G., Van Der Fels-Klerx, H. J., de Rijk, T. C., & Oonincx, D. G. A. B. (2017).
(consultant on legal matters), and Nicola Montevecchi for their valuable Aflatoxin B1 tolerance and accumulation in black soldier fly larvae (Hermetia
contribution to the present study. illucens) and yellow mealworms (Tenebrio molitor). Toxins, 9(6), 185. https://doi.
org/10.3390/toxins9060185
In addition, we would like to acknowledge the English-language
Bouazizi, S., Montevecchi, G., Antonelli, A., & Hamdi, M. (2020). Effects of prickly pear
reviewing work done by Dr. Sara Ronconi. (Opuntia ficus-indica L.) peel flour as an innovative ingredient in biscuits formulation.
LWT, 124, 109155. https://doi.org/10.1016/j.lwt.2020.109155
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