LWT - Food Science and Technology 184 (2023) 115012

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LWT
journal homepage: www.elsevier.com/locate/lwt

Nutritional characteristics, microbial loads and consumer acceptability of

cookies enriched with insect (Ruspolia differens) meal
Brian O. Ochieng a, b, **, Joseph O. Anyango b, Fathiya M. Khamis a, Sunday Ekesi a,
James Peter Egonyu a, Sevgan Subramanian a, John M. Nduko b, Dorothy Nakimbugwe c,
Xavier Cheseto a, Chrysantus M. Tanga a, *
a
International Centre of Insect Physiology and Ecology (icipe), P.O. BOX 30772, 00100, Nairobi, Kenya
b
Department of Dairy and Food Science and Technology, Egerton University, P.O. Box 536, 20115, Njoro, Kenya
c
Department of Food Technology and Nutrition, School of Food Technology, Nutrition and Bioengineering, Makerere University, P.O. Box 7062, Kampala, Uganda

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

Keywords: Utilization of Ruspolia differens Serville as functional food ingredient is rapidly gaining popularity. This study
Edible grasshoppers evaluated the nutrient quality, microbial safety and consumers’ acceptability of cereal-based cookies fortified
Ruspolia differens processing with various processed products of R. differens meals. Cookies fortified with blanched, boiled, and toasted
Microbial quality
R. differens meals had higher protein, fat and energy levels, respectively, than the control cookies. Enrichment of
Nutritional composition
Safe cookie products
cookies with differentially processed R. differens meals had elevated levels of isoleucine and leucine. Omega-3
fatty acid, methyl (9Z,12Z,15Z)-octadecatrienoate, was detected only in cookies prepared from wheat-insect
meals blends. Blanched and boiled R. differens meal significantly (p < 0.05) boosted iron (1.70-folds) and zinc
(1.12–1.16-folds) contents of the cookies. The cookie products had reduced Enterobacteriaceae, S. aureus, yeast
and mould with permissible exposure limits for human consumption. The overall acceptability of insect-enriched
cookie product by male and female respondents ranged between 57 and 80%. The survey revealed that the
flavour, colour, mouthfeels and texture of the cookie products were important motivation for consumers to
accept grasshoppers as a food source. Further research on the flavour of cookie products enriched with grass­
hopper meal would be required to increase acceptability to market-driven consumer appealing food products.

1. Introduction safety of these insects before integration into products since, the
collection of edible insects by entomophagists in developing countries is
The exponential rise in global population has consistently escalated unregulated from the wild and the value chain is characterized by
the demand for quality proteins, prompting researchers to evaluate ineffective implementation of hygienic practices. (Imathiu, 2020).
other sustainable sources like edible insects, which offer comparable or Ruspolia differens Serville (Orthoptera:Tettigonidae) is a seasonally
superior nutritional profiles to conventional animal sources (Acos­ occurring edible insect in 21 African countries, constituting an impor­
ta-estrada, Reyes, Rosell, Rodrigo, & Ibarra-herrera, 2021; Rumpold & tant customary delicacy among various communities (Kinyuru, Kenji,
Schlüter, 2013; van Huis et al., 2013). Edible insects are hence ideal for Njoroge, & Ayieko, 2010; Ng’ang’a et al., 2019). Several researchers
enriching widely recognized cereal-based products like wheat products have investigated and reported its rich nutritional value, laying
with demonstrated nutritional protein inferiority (Pencharz, Elango, & emphasis on their high protein content and unsaturated fats (Kinyuru,
Wolfe, 2016; Sozer, Nordlund, & Poutanen, 2017). The development of Kenji, Muhoho, & Ayieko, 2010; Rutaro et al., 2018; Ssepuuya, Smets,
insect-based snacks presents a worthy vehicle for delivering essential Nakimbugwe, Van Der Borght, & Claes, 2019). It is therefore recom­
insect nutrients to target consumers (Awobusuyi, Pillay, & Siwela, 2020) mended for supplementation and fortification of foods that are nutrient
especially those with historic reluctance to consume whole insect deficient like cereal porridges (Mmari, Kinyuru, Laswai, & Okoth, 2017)
(Olamide, Samuel, Adeniyi, & Taiwo, 2020). Of great concern is the and bakery goods. However, because they are wild-sourced, they may be

* Corresponding author.
** Corresponding author. International Centre of Insect Physiology and Ecology (icipe), P.O. BOX 30772, 00100, Nairobi, Kenya.
E-mail addresses: [email protected] (B.O. Ochieng), [email protected] (C.M. Tanga).

https://doi.org/10.1016/j.lwt.2023.115012
Received 1 January 2023; Received in revised form 16 June 2023; Accepted 23 June 2023
Available online 23 June 2023
0023-6438/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
B.O. Ochieng et al. LWT 184 (2023) 115012

associated with microbial hazards (Belluco et al., 2013; Garofalo et al., subsequently oven-dried at 60 ◦ C for 24 h following procedures outlined
2019; Imathiu, 2020). Such hazards reportedly exacerbate edible by Ochieng et al. (2022). The samples were milled in a three-speed
insect-aversion amongst individuals unaccustomed to their consumption Waring laboratory blender (Camlab, Over, UK) before being screened
(Lange & Nakamura, 2021). through a 0.1 mm stainless steel laboratory sieve. They were then vac­
Ng’ang’a, Fombong, Kiiru, Kipkoech, and Kinyuru (2021) evaluated uum packaged in sterile Ziploc bags, labeled and stored at 4 ◦ C for
and reported microbial hazards and heavy metals as the major health further analysis. A representation of the basic nutritional characteristics
risks in edible grasshopper. High total viable counts, lactic acid bacteria, of the processed R. differens and the wheat flour is shown in Table 1.
Enterobacteriaceae, Salmonella, Staphylococcus aureus, bacterial endo­
spores, yeasts and moulds are the main pathogenic and spoilage mi­ 2.2. Blending of flours and cookies preparation
croorganisms that have been reported in raw harvested R. differens,
posing health risk to potential consumers (Labu et al., 2021; Ng’ang’a Processed R. differens-based cookies and the control cookies were
et al., 2019, 2021, Nyangena et al., 2020). Interestingly, most thermal prepared according to a method described by Noor Aziah et al. (2012),
processes applicable to R. differens have been discovered to markedly with a few modifications. Based on a continuously proven marginal
subdue a majority of these microbes to acceptable limits (Labu et al., acceptance of sensory features of bakery products previously manufac­
2021; Ng’ang’a et al., 2019, 2021). These processing methods also tured with insect flours at a 10% (w/w) inclusion level, self-raising
destroy degradative enzymes, increase or decrease allergenicity and wheat was substituted with processed R. differens at 10% (w/w) (Ade­
improve the sensory quality by introducing new aroma compounds boye, Bolaji, & Fatola, 2016; Awobusuyi, Siwela, & Pillay, 2020;
(FAO, 2021; Melgar-Lalanne, Hernández-Álvarez, & Salinas-Castro, González, Garzón, & Rosell, 2019; Ogunlakin, Oni, & Olaniyan, 2018;
2019). Ojinnaka, Ofoelo, & Ezenwa, 2015; Osimani et al., 2018). Also, this was
Cookies have become one of the most popular snacks among both based on Kenya Bureau of Standards’ (KEBS) proposed minimum
children and adults across nations due to its inexpensive manufacturing allowable level of edible insects in products of 10% (w/w) (KEBS,
costs, increased convenience, long shelf life and ready-to-eat nature 2020a). The control cookies, contained chicken eggs as main protein
(Ayensu, Lutterodt, Annan, Edusei, & Loh, 2019; Noor Aziah, Mohamad source, specifically resembling conventional cookie formulations while
Noor, & Ho, 2012). Cookies are the best choice as excellent carriers of a the insect-based cookies contained only processed insect ingredients as
blend of different and varied functional ingredients, due to their protein sources. Approximately 172.2 g sugar and 3.4 g salt were sieved
formulation simplicity and ease of production, without compromising and mixed with 408.2 g of self-raising wheat flour (pre-mixed with
the sensory quality or shelf stability of the resulting products. As a result, food-grade improvers) for 5 min. Approximately 172.2 g of shortening
their formulations are amenable to inclusion of other ingredients (hydrogenated palm oil) was added and mixed in a bakery mixer
intended for nutritional enhancement (Ndife, Kida, & Fagbemi, 2017). (BJY-BM10, Berjaya, Malaysia) for 15 min to produce a creamy mixture.
In this respect, several researchers have capitalized on the high value About 84 g of whole eggs (for control) or processed R. differens flours
nutrient-dense biomass of edible insects to develop nutritious insect- was added, and the mixture mixed for another 10 min. This was followed
based food products. For instance, edible insect meals such as that of by hand-kneading of the dough for 5 min to yield a firm and uniform
mealworm (Tenebrio molitor), giant mealworms (Zophobas atratus Fab­ dough weighing about 180 g a piece. The dough was rolled out to a
ricius), silkworm (Bombyx mori Linnaeus), house cricket (Acheta thickness of 5 mm using a rolling pin and cut into 5 cm diameter circles
domesticus Linnaeus), desert locust (Schistocerca gregaria Forskål) and on a wooden board. The cut-out cookie doughs were placed 50 mm apart
termites (Macrotermes spp.), have been used to develop healthier on greased baking trays and baked in a preheated oven for 15 min at
nutrient-dense biscuit and cookie products (Awobusuyi, Siwela, & Pil­ 180 ◦ C, 30% relative humidity of air (BISTROT 665; BestFor®, Ferrara,
lay, 2020; Bawa, Songsermpong, Kaewtapee, & Chanput, 2020; Olamide Italy). The formulation and baking process was repeated thrice for the
et al., 2020; Sriprablom, Kitthawee, & Suphantharika, 2022; Tedjaku­ doughs with eggs (control), blanched, boiled, toasted and deep-fried
suma, Linggadiputra, Cahya, & Surya, 2022; Torres et al., 2022). The R. differens ingredient culminating into 15 experimental materials.
developed insect-based products were characterized by higher protein Cookies from each replicate dough were placed in sterile plastic Ziploc
levels, essential amino acid and mineral profiles than their conventional bags and marked correspondingly for microbial analysis. The remaining
counterparts, hence, offering valuable nutritional supply to consumers. cookie samples were stored in a cold room at − 10 ◦ C awaiting nutri­
Therefore, incorporation of insect meals into popular value-added baked tional analyses.The schematic representation of R. differens processing,
products, such as cookies, could promote their fortification and utili­ cookies preparation and experiments conducted is shown in Fig. 1.
zation as functional food source. However, the use of R. differens in
cookie products have received limited research attention, globally. The 2.3. Proximate determinations
current study aimed at evaluating the nutritional content, microbial
quality and consumer responses to sensory characteristics of wheat Proximate parameters (moisture content, carbohydrates, protein
cookies enriched with differentially processed R. differens flours as an content, ash content, dry matter, fat content and fibre content) of the
alternative to chicken eggs, which is conventionally used as a protein cookies were assayed following the Association of Official Analytical
source in baking. Chemists methods (AOAC, 2000) methods. Briefly, dry matter and
moisture were measured by sample drying in a conventional oven (WTB
2. Methodology binder, Tuttlingen, Germany) at 130 ◦ C for 3 h. Protein content was
estimated using a Kjeldahl analyzer (Velp UDK 159, Velp Scientifica,
2.1. Materials Europe) and subsequently converted to protein using a factor of 6.25 for
quantification as reported by Boulos, Tännler, and Nyström (2020). Fat
Wild harvested and plucked (wings, appendages and ovipositors content was solvent-extracted in petroleum ether in a Soxhlet extraction
removed) R. differens weighing 20 kg were purchased from Masaka (0 system (Velp SER 148, Velp Scientifica, Europe). Ash content was
20′ 28.0′′ S 31 44′10.0′′ E) and Kampala (0.3476 N, 32.5825 E) in assessed by sample incineration in a muffle furnace (Her­
Uganda. The samples were put into sterile sample collecting plastic aeus-Kundendienst, Düsseldorf, Germany) at 550 ◦ C for 12 h. Fibre
containers, stored in cold boxes with flaked ice (4–7 ◦ C) and transported content was acid and base-digested in a fibre analyzer (FIWE, Velp
to the International Centre of Insect Physiology and Ecology (icipe) Scientifica, Europe) and evaluated based on loss upon ignition. Carbo­
laboratory. Ruspolia differens samples of 700 g each were blanched hydrate content was estimated by subtracting the fat, protein, ash and
(100 ◦ C/5 min), boiled (100 ◦ C/15 min), toasted (for 10 min) and deep- moisture content from 100. The total energy [kcal/100 g] was calcu­
fried in palm cooking oil (175 ◦ C until crunchy/dark brownish) and lated with the following Atwater formula: Total Energy = 4 ×

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B.O. Ochieng et al. LWT 184 (2023) 115012

Table 1
Basic characteristics (proximate components) of processed R. differens and wheat flours used in cookies preparation.
Flour type Dry matter (g/ Moisture content (g/ Protein content (g/ Fat content (g/ Ash content (g/ Fibre content (g/ Carbohydrate (g/
100 g db) 100 g db) 100 g db) 100 g db) 100 g db) 100 g db) 100 g db)
§
Blanched 98.2 ± 0.05c 1.8 ± 0.05c 40.1 ± 1.33c 43.8 ± 0.41c 2.2 ± 0.00b 11.2 ± 0.01c N.D.
R. differens flour
a e d b bc c
§
Boiled R. differens 85.6 ± 0.10 14.4 ± 0.10 43.1 ± 1.60 36.3 ± 1.06 2.3 ± 0.09 10.9 ± 0.19 N.D.
flour
§
Toasted R. differens 98.4 ± 0.06d 1.6 ± 0.06b 44.7 ± 1.03d 46.0 ± 0.82c 2.4 ± 0.17bc 9.0 ± 0.74b N.D.
flour
§
Deep-fried 99.2 ± 0.03e 0.8 ± 0.03a 7.8 ± 0.59a 83.0 ± 1.54d 1.2 ± 0.16a 8.7 ± 0.39b N.D.
R. differens flour
b d b a c a
Wheat flour 87.9 ± 0.00 12.1 ± 0.00 12.6 ± 0.10 1.7 ± 0.10 2.5 ± 0.04 0.6 ± 0.04 70.4 ± 0.18

Values are expressed as means ± standard deviations. Means in the same column followed by different small superscript letters are significantly different at p < 0.05
§
Source: (Ochieng et al., 2022).

Fig. 1. A schematic summary of R. differens processing, cookies preparation and experiments conducted. CTRC=Control cookies with eggs; BCRC=Blanched
R. differens-based cookies; BLRC=Boiled R. differens-based cookies; TSRC = Toasted R. differens-based cookies; DFRC = Deep fried R. differens-based cookies.

protein (%) + 4 × carbohydrate(%) + 9 × fat(%) (FAO, 2003). All the Waters Technologies, USA) in scan mode. Chromatographic separation
determinations were performed in triplicates. was performed on a ACQUITY UPLC I-class system (Waters Corp., Mil­
ford, MA) fitted with an ACQUITY UPLC BEH C18 column (2.1 mm ×
2.4. Amino acid profile 150 mm, 1.7 μm particle size; Waters Corp., Wexford, Ireland, oven
temp 45 ◦ C). The autosampler tray was cooled to a temperature of 5 ◦ C.
The amino acid composition was determined as previously described The mobile phase comprised of (A) water and (B) methanol (solvent B)
by Musundire, Osuga, Cheseto, Irungu, and Torto (2016) with some both acidified with 0.002 mol/L formic acid. The gradient system used
modification. Briefly, on an analytical scale, cookie samples (10 mg) was 0–2 min, 5% B, 2–4 min, 40% B, 4–7 min, 40% B, 7–8.5 min 60% B,
were carefully weighed and transferred into 5 mL Supelco® 8.5–10 min 60% B, 10–15 min, 80% B, 15–19 80% B, 19–20.5 min,
micro-reaction vials. Afterwards, 1.5 mL of 6 mol/L HCl was added, 100% B, 20.5–23 min, 100% B, 23–24 min 95% B, 24–26 min, 95% B.
followed by nitrogen and finally capped. To achieve full hydrolysis, the The flow rate was maintained at 0.2 mL/min throughout the experi­
samples were conditioned at 110 ◦ C for 24 h. The hydrolysates were then ment. The UPLC was connected to a Waters Xevo TQ-S electrospray
vacuum evaporated to complete dryness, reconstituted in 1 mL of a 95:5 ionization (ESI) full scan MS in positive ionization mode. The m/z range
mixture of 0.002 mol/L formic acid and acetonitrile, vortex-shaken for 40–1000 with a capillary voltage of 0.5 kV, sampling cone voltage of 30
30 s, sonicated at 50 kHz for 30 min and centrifuged (Eppendorf AG, V, source temperature 150 ◦ C desolvation temperature of 120 ◦ C. The
22331 Hamburg, Germany) at 14 000 rpm. The supernatants were nitrogen desolvation flow rate was 800 L/h. Data was acquired using
filtered into 2 mL clear glass vial (Supelco, Bellefonte, PA, USA) and MassLynx version 4.1 SCN 712 (Waters). Mass spectrometric data,
immediately analysed (0.2 μL) by UPLC-MS/MS (Waters XEVO TQ-S, retention time, and co-injection of the hydrolysate with an authentic

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B.O. Ochieng et al. LWT 184 (2023) 115012

standard amino acid mixture were used to identify the amino acids. under study in the samples and standard solution. The ICP-OES had the
Amino acid standard solution (AAS 18) obtained from Sigma-Aldrich following operational parameters: Radio frequency power-1450 W;
(Chemie GmbH, Munich, Germany) was used for external quantifica­ Plasma gas flow rate-15 L/min; Auxiliary gas flow rate-0.2 L/min;
tion of the amounts of each amino acid present. This was repeated three Nebulizer gas flow rate-0.8 L/min; Sample flow rate1.5 L/min; View
times using different batches of replicated cookie samples. mode-axial mode; The read-peak area; Source equilibration time-10 s;
Read delay time-10 s; Replicates-1; Background correction-2-point
2.5. Fatty acids profile (manual point correction); Spray chamber-Scott type; Nebulizer
cross-Flow GemTip Nebulizer (HF resistant); The detector-CCD; Purge
The fatty acids were assessed as fatty acid methyl esters (FAMEs) of gas-nitrogen; Shear gas-air; Plasma gas-nitrogen. The distinctive
the cookies (100 mg each) following modified protocols previously used elemental spectra of each mineral were measured at the following
(Cheseto, Baleba, Tanga, Kelemu, & Torto, 2020). Sodium methoxide wavelengths: Magnesium – 285.213 nm, Iron – 259.939 nm, Manganese
solution (15 mg/mL) of 1 mL was introduced into each sample then the – 257.61 nm, Calcium – 317.933 nm, Phosphorous – 213.617 nm, Po­
mixture vortexed for 1 min, ultrasonicated for 10 min, and incubated for tassium – 766.49 nm, Aluminium – 396.153 nm, Copper – 224.7 nm and
1 h in a water bath at 70 ◦ C. To quench the reaction, 100 μL of distilled Cobalt – 228.616 nm. ICP-OES mix standard CatNo.43843 (Sigma-Al­
deionized water was added, followed by another 1 min of vortexing. To drich, USA) prepared by serially diluting with 0.4 mol/L nitric acid to
extract the resultant FAMEs, 1 mL of gas chromatography (GC)-grade generate calibration standards of 400, 800, 2000, and 4000 g/L were
hexane (Sigma–Aldrich, St. Louis, MO, USA) was added and centrifuged also assessed by the ICP-OES, yielding linear calibration curves with
at 14,000 rpm for 5 min. The supernatant was filtered, dried over elemental correlation coefficients of R2 = 0.999 for all the minerals
anhydrous sodium sulphate and analysed (1.0 μL) by GC-MS on a 7890A investigated. On a PerkinElmer Winlab 32 software (PerkinElmer, USA),
gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) external standard calibration and data gathering were accomplished. All
linked to a 5975 C mass selective detector (Agilent Technologies Inc., of the mineral elements were quantified using the data gathered. The
Santa Clara, CA, USA). The GC was fitted with a (5%-phenyl)-­ determinations were replicated three times with different batches of
methylpolysiloxane (HP5 MS) low bleed capillary column (30 m _ 0.25 cookies.
mm i.d., 0.25_μm; J&W, Folsom, CA, USA). At a flow rate of 1.25
mL/min, helium was used as the carrier gas. The oven temperature was 2.7. Microbial assays
programmed to rise from 35 to 285 ◦ C at a rate of 10 ◦ C min− 1 with the
initial and final temperatures set to hold for 5 min and 20.4 min, The sanitary qualities of the cookies and their respective processed
respectively. The temperatures of the ion source and quadrupole mass R. differens flours were established. In a sterile filter stomacher bag
selective detector were kept at 230 and 180 ◦ C, respectively. The elec­ (Bagmixer 400W, Interscience, St. Nom, France), 5 g of samples were
tron impact (EI) spectrum masses were measured at a 70 eV acceleration blended with 45 mL of sterile peptone physiological salt solution (0.14
energy. In full scan mode, fragment ions were studied throughout a mass mol/L sodium chloride, 1 g/L peptone (OXOID LP0034), and pH of 7.0
range of 40–550 m/z. The duration of the filament delay was set at 3.3 ± 0.2 at 25 ◦ C and homogenized at normal speed for 1 min (Seward, 400
min. Authentic standard, methyl octadecanoate (0.2–125 ng/L), pre­ circulator, West Sussex, UK). A 10-fold serial dilution of the homogenate
pared from octadecanoic acid (95% purity) (Sigma-Aldrich, St. Louis, with 1 mL was then performed for TVC, Enterobacteriaceae, Staphylo­
MO) and serially diluted, was also analysed by GC-MS in full scan mode coccus aureus and yeast and moulds except for Salmonellae. These mi­
to generate a linear calibration curve (peak area vs. concentration) with crobial analyses were specifically selected because they serve as
the equation below; indicators of food safety and contamination. The homogenates (0.1 mL)
[ ] [ ] were aliquoted and inoculated onto approximately 20 mL of freshly
Y = 5 × 107 x + 2 × 107 with R2 = 0.9997 Equation 1 prepared, sterile, solidified media. TVC was determined using Plate
Count Agar (Oxoid CM0463) and incubated at 30 ◦ C for 48 h (Klunder,
Where; the gradient = 5 × 107 , y-intercept = 2 × 107 and R2 = coeffi­ Wolkers-Rooijackers, Korpela, & Nout, 2012). Lactose Positive Entero­
cient of determination bacteriaceae was assessed on MacConkey agar (Oxoid CM0007) and
External quantification of the various fatty acids from the samples incubated at 37 ◦ C for 24 h (Nyangena et al., 2020). Staphylococcus
was done using this regression equation. The following integration pa­ aureus were evaluated on a Baird Parker agar (Oxoid CM1127) enriched
rameters were used to generate peak spectral masses using ChemStation with 50 g/L of Egg Yolk Tellurite Emulsion (Oxoid CM0276) and later
B.02.02. acquisition software: 3 for initial threshold, 0.010 for initial incubated at 35 ◦ C for 48 h. Colonies of characteristic circular (2–3 mm
peak width, 1 for initial area reject, and ’on’ for shoulder detection. NIST diameter), smooth, convex, moist appearance were enumerated (Ram­
05, 08, and 11 mass spectra and retention durations were compared to ashia, Tangulani, Mashau, & Nethathe, 2020). For Salmonellae, the
authentic standards and reference spectra published in library–MS da­ samples (25 g) were first enriched in 225 mL of nutrient broth
tabases. The FAMES in all of the samples were determined in triplicates. comprising 5 g peptone, 5 g NaCl, 1 g Lab-Lemco beef extract, and 2 g
yeast extract per 1L of water, pH 7.4 (Oxoid CM0067), and then incu­
2.6. Mineral profile bated at 35 ◦ C for 24 h. Homogenates (25 mL) were further selectively
enriched in 225 mL of Rappaport-Vassiliadis broth (Oxoid CM0669) and
The mineral profiles of the cookies were analysed in accordance with incubated at 37 ◦ C for 24 h. Rappaport-Vasiliadis broth culture was
AOAC (2000). Ground samples weighing 0.5 g were weighed into collected using a sterile wire loop, streaked over Salmonella- Shigella
digestion tubes containing, mixed with 8.0 mL concentrated (16.2 Agar (Oxoid CM0099), and plates incubated at 37 ◦ C for 24 h. Salmonella
mol/L) nitric acid and 2 mL of 9.8 mol/L hydrogen peroxide, and spp. were detected as colourless colonies with black centres (Nyangena
permitted to stand overnight in a fume hood. The samples were then et al., 2020). Yeast and moulds were cultured on Potato Dextrose Agar
transferred to a microwave digestion machine (Multiwave Go Plus, (PDA) (Oxoid Ltd., United Kingdom) then incubated at 25 ◦ C for 5 days
Anton Paar, VA, US) and exposed to a temperature-time digestion after which colonies were examined and enumerated using a magnifying
schedule of 100 ◦ C for 10 min and then adjusted to 180 ◦ C for 10 min. lens (Ramashia et al., 2020).
Cooled clear solutions, signifying completely digested materials, were
placed into 25 mL falcon tubes and adjusted to the mark with 0.4 mol/L 2.8. Consumer acceptability
nitric acid. Inductively coupled plasma optical emission spectrometry
(ICP-OES) measurements (Optima 2100TMDV ICP-OES, PerkinElmer The sensory perception of the fortified cookies was assessed ac­
Massachusetts, USA) were used to examine the contents of the minerals cording to Dubost, Shewfelt, and Eitenmiller (2003). Briefly, 143

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B.O. Ochieng et al. LWT 184 (2023) 115012

untrained panellists (72 males and 71 females) of age ranging 18-50 3.2. Amino acid profiles of the cookies
years, were randomly chosen for the exercise. The evaluation of the
cookies was based on the following parameters: colour, flavour, texture, Eleven amino acids (five essential amino acids and six non-essential
mouthfeel and overall acceptability. A 3-point acceptability scale (1 = amino acids) were detected from the prepared cookies (Table 3).
dislike, 2 = neither like nor dislike and 3 = like) was adopted for the Isoleucine and leucine of the essential amino acids and tyrosine and
rating of the experimental cookies. The panellists were informed of the cystine of the non-essential amino acids were significantly (p < 0.05)
aim of the study and instructed to consent to the study on the sensory higher in BCRC, BLRC and TSRC than in the CTRC and DFRC. DFRC were
questionnaire before commencement. This sensory study was approved characterized with significantly (p < 0.05) lower amino acid levels.
by the Institutional Animal Care and Use Committee (IACUC) of Kenya Valine, glutamic acid, and alanine were the most abundant amino acids
Agricultural and Livestock Research Organization (KALRO)-Veterinary in all the cookie types.
Science Research Institute (VSRI); Muguga North upon compliance with
all provisions vetted under and coded: 3.3. Fatty acid profile of the cookies
KALRO-VSRI/IACUC028/16032022. This study was reviewed and
approved by Egerton University and the National Council for Science The fatty acid spectra and total fatty acid categories of the cookie
Technology and Innovation in Kenya (NACOSTI/P/21/8303). Further, types are shown in Table 4 and Fig. 2, respectively. A total of 32 fatty
we also obtained the informed consent of the participants via the acids were detected from the four cookies types. Saturated fatty acids
statement "I am aware that my responses are confidential, and I agree to (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty
participate in this survey as well as affirming that I can withdraw from acids (PUFAs) constituted 52.8%, 45.8%, and 1.4%, respectively in the
the survey at any time without giving a reason. The products tested were control cookies and 24.6–49.4%, 36.3–49.8%, and 0.8–30.0%, respec­
safe for consumption. tively in the cookies enriched with insect meals. The most abundant fatty
acids compositionally were discovered to be methyl hexadecanoate of
2.9. Statistical analysis the SFAs, methyl 9Z-octadecenoate of the MUFAs, and methyl (9Z,12Z)-
octadecadienoate of the PUFAs. The PUFA, methyl (9Z,12Z,15Z)-octa­
Statistical analyses were conducted using R Studio software version decatrienoate, was only detected in insect meal enriched cookies with
1.3.1093–1 (R Core Team, 2020) for windows. One-way ANOVA was BCRC and BLRC exhibiting higher concentrations. However, methyl
performed to determine significant differences in proximate compo­ (9Z,12Z)-octadecadienoate and methyl 9Z-octadecenoate displayed
nents, amino acids profiles, fatty acids profiles, mineral profiles and incredible quantities in the cookies enriched with insect meals than in
individual microbial counts for the cookies enriched with R. differens the control cookies.
flours prepared by blanching, boiling, toasting and deep-frying. Tukey
HSD multiple comparison test was used to identify statistically hetero­ 3.4. Mineral profiles of the cookies
geneous subsets at α = 0.05. All microbial counts were expressed as log
cfu/g. Means and standard deviation of three replicates were reported The mean concentration of minerals in the cookies, with the excep­
for each analysis. tion of P, showed significant (p < 0.05) variations (Table 5). Calcium
level was significantly (p < 0.05) higher in control cookies (CTRC) than
3. Results in the cookies enriched with R. differens meals. On the other hand, BCRC
exhibited zinc (4.33 mg/100g) and iron (7.11 mg/100g) whereas BLRC
3.1. Proximate composition of the cookies exhibited zinc (4.17 mg/100g) and iron (7.12 mg/100g) which were
significantly (p < 0.05) higher than those detected from control cookies
The proximate parameters; moisture content, dry matter, protein (CTRC).
content, fat content, ash content, carbohydrate and energy varied
significantly (p < 0.05) across the cookie types except for fibre (Table 2). 3.5. Microbial quality of the cookies and their respective insect ingredients
The moisture content of the control cookies was significantly (p < 0.05)
higher than the insect-based cookies. Protein content of the insect-based The total viable counts (TVC), Enterobacteriaceae and Staphylococcus
cookies (BCRC, BLRC, and TSRC) were significantly (p < 0.05) greater aureus significantly (p < 0.05) differed among the four processed insect
than the CTRC and DFRC. The insect-based cookies (BCRC, BLRC, TSRC meals whereas Salmonella spp., and yeast and moulds colonies were both
and DFRC) manifested significantly (p < 0.05) higher levels of fat and not identified (Table 6). Blanched R. differens meals recorded signifi­
energy than the control cookies (CTRC). cantly higher counts of TVC, Enterobacteriaceae and S. aureus than the
other processed insect meals. The cookies expressed low microbial levels
characterized by non-detection of both S. aureus or Salmonella spp., low

Table 2
Proximate composition and gross energy of processed R. differens based cookies.
Enriched Moisture (g/ DM (g/ Protein content (g/ Fat content (g/ Ash content (g/ Fibre content (g/ CHO (g/100 Energy (kcal/
cookies 100g wb) 100g) 100 g db) 100 g db) 100 g db) 100 g db) g db) 100 g db)

CTRC 6.17 ± 1.26b 93.83 ± 7.74 ± 0.40a 17.76 ± 0.41a 2.64 ± 0.02b 0.06 ± 0.02a 65.63 ± 453.33 ± 6.70a
1.26a 1.12b
BCRC 2.83 ± 0.29a 97.17 ± 11.09 ± 0.65b 19.61 ± 1.08ab 2.06 ± 0.01a 0.17 ± 0.14a 64.23 ± 477.81 ± 4.10b
0.29b 1.45ab
BLRC 3.00 ± 0.87a 97.00 ± 10.90 ± 0.08b 20.68 ± 0.20b 2.23 ± 0.29ab 0.22 ± 0.33a 62.97 ± 481.59 ± 1.28b
0.87b 0.86a
TSRC 2.33 ± 0.29a 97.67 ± 10.99 ± 0.24b 19.84 ± 0.53b 2.56 ± 0.01b 0.07 ± 0.03a 64.21 ± 479.37 ± 3.70b
0.29b 0.20ab
DFRC 1.33 ± 0.58a 98.67 ± 6.78 ± 0.14a 25.82 ± 0.89c 2.20 ± 0.29ab 0.08 ± 0.02a 63.79 ± 514.69 ± 6.52c
0.58b 0.43ab

Values are presented as mean ± standard deviation of triplicate determinations. Means in the same column followed by different small superscript letters are
significantly different at p < 0.05. kcal = Kilocalories; CHO= Carbohydrate; DM = Dry matter; CTRC=Control cookies with eggs; BCRC=Blanched R. differens-based
cookies; BLRC=Boiled R. differens-based cookies; TSRC = Toasted R. differens-based cookies; DFRC = Deep fried R. differens-based cookies.

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B.O. Ochieng et al. LWT 184 (2023) 115012

Table 3
Amino acid profiles detected in cookies enriched with different processed R. differens.
Amino acid CTRC (mg/g) BCRC (mg/g) BLRC (mg/g) TSRC (mg/g) DFRC (mg/g) F(4,10) p-value

Essential Amino Acids

Phenylalanine 4.41 ± 0.28a 4.91 ± 0.42a 4.08 ± 0.40a 4.53 ± 0.50a 3.93 ± 0.54a 2.4 ns
Isoleucine 4.00 ± 0.84a 5.62 ± 0.82b 6.07 ± 0.33b 5.56 ± 0.34b 3.39 ± 0.16a 12.47 p < 0.001
Leucine 7.93 ± 0.68ab 8.97 ± 0.42b 8.60 ± 0.22b 8.57 ± 0.22b 7.32 ± 0.28a 7.93 p < 0.01
Methionine 2.49 ± 0.16a 3.08 ± 0.33a 2.82 ± 0.07a 2.65 ± 0.42a 2.72 ± 0.18a 2.11 ns
Valine 20.37 ± 0.72a 22.41 ± 2.46a 19.40 ± 1.82a 19.77 ± 4.93a 16.38 ± 1.45a 1.95 ns
Non-Essential Amino acids
Tyrosine 3.41 ± 0.06a 4.18 ± 0.12b 4.17 ± 0.12b 4.08 ± 0.17b 3.57 ± 0.11a 28.08 p < 0.001
Arginine 11.88 ± 0.48b 13.73 ± 0.25c 11.27 ± 0.91b 12.68 ± 0.80bc 9.51 ± 0.50a 18.61 p < 0.001
Cystine 4.54 ± 0.21a 8.75 ± 1.44bc 9.96 ± 0.32c 6.72 ± 1.32ab 5.83 ± 0.49a 17.21 p < 0.001
Proline 4.14 ± 1.09a 7.53 ± 0.55b 5.25 ± 0.24a 6.17 ± 0.85ab 5.48 ± 0.82ab 7.98 p < 0.01
Glutamic acid 18.00 ± ’1.88a 21.54 ± 0.68a 19.58 ± 0.48a 20.69 ± 2.66a 18.67 ± 0.20a 2.74 ns
Alanine 21.63 ± 0.37b 24.13 ± 0.76b 23.21 ± 1.37b 23.14 ± 2.47b 14.75 ± 0.75a 23.55 p > 0.001

Values are presented as mean ± standard deviation of triplicate determinations. Means in the same row followed by same small superscript letters are not significantly
different at p < 0.05. ns = not significant; CTRC=Control cookies with eggs; BCRC=Blanched R. differens-based cookies; BLRC=Boiled R. differens-based cookies; TSRC
= Toasted R. differens-based cookies; DFRC = Deep fried R. differens-based cookies.

counts of TVC (less than 30), and no observation of Enterobacteriaceae, Standards (KEBS) requirement of 15 g/100 g (KEBS, 2020b). To enhance
and yeast and moulds. the protein levels in such insect-based products, utilization of defatted
R. differens as an enrichment ingredient is imperative as earlier
3.6. Consumer preferences demonstrated in another insect (Lee et al., 2020). This strategy could be
effective due to the fact that fats are compositionally the highest prox­
Fig. 3 illustrates a comparison of the consumer preference of the imate components in R. differens (Ssepuuya, et al., 2019) and therefore
various cookie products based on different attributes between male and their removal would proportionally enhance protein levels.
female respondents. A larger proportion of male (90.3% and 79.2%) and The fat and energy content of the insect-based cookies was higher
female (85.9% and 81.7%) respondents liked the colour of CTRC and than the control cookies. This is unsurprising because, R. differens has a
DFRC. Only 52.8% of the male respondents and 40.8% of the female fat content of 42.2–54.3 g/100 g (Ssepuuya, et al., 2019) which may
respondents liked the flavour of BCRC. However, 75% of the male re­ have been reflected in the cookies. Similar results were replicated in
spondents liked the flavour of BLRC and DFRC whereas for the females, cookies enriched with sorghum-termite blends (Awobusuyi, Siwela, &
77.5% liked the CTRC with 71.8% liking the DFRC flavour. Both the Pillay, 2020). These fats, on the other hand, are high in polyunsaturated
male (73.6%) and female (73.2%) respondents preferred the texture of fatty acids (PUFAs) and monounsaturated fatty acids (MUFAs), which
CTRC. Higher number of both male (83.3% and 73.6%) and female may reduce the shelf life of the insect-based cookies even further if the
(77.5% and 78.9%) respondents generally accepted CTRC and DFRC insect ingredients are not defatted. However, this study did not consider
(Fig. 4). defatting the insect ingredients as one of the aims was to compare the
fatty acids profiles. These fat levels play a critical role in influencing the
cookies’ energy levels (Omoba & Omogbemile, 2013) since the carbo­
4. Discussion
hydrate levels were comparable (Table 2). Due to the carry-over effect
from deep fat frying of R. differens, fat was conspicuously higher in the
The higher moisture and lower dry matter exhibited in control
DFRC (Table 1) compared to the other cookie types. Apparently, the
cookies (CTRC) compared to the insect-based cookies was as a conse­
control cookies (CTRC) had the lowest energy compared to the
quence of direct inclusion of raw eggs in the control cookies formulation
insect-based cookies which corroborates the report by Awobusuyi, Pil­
mix. Eggs reportedly contain higher moisture content of 78 g/100g wb
lay, and Siwela (2020).
(Ogunwole, Yinka, & Ojelade, 2015) compared with the processed
Amino acids profiles of the CTRC compared favourably with the
R. differens flours (Ochieng et al., 2022) explaining the high moisture in
insect-based cookies. Alanine, aspartic acid, glutamic acid, and essential
the CTRC. The moisture content of the insect-based cookies reflected the
amino acids leucine, lysine, and valine have been reported in raw
moisture content of palm weevil flour-fortified biscuits reported by
R. differens in appreciable levels (Ssepuuya, et al., 2019). This is clearly
Ayensu et al. (2019). This suggest that insect enriched cookies have low
evident in Table 3 where alanine, glutamic acid and valine were the
moisture contents as the insect ingredients used are often subjected to
most quantitatively dominant amino acids, particularly in cookies sup­
dehydrative pre-processes. The low moisture content of processed insect
plemented with blanched, boiled, and toasted R. differens, which had
flours and insect-based cookie products was ideal to ensure improved
higher mean protein levels (Ochieng et al., 2022). On the other hand,
and extended shelf life as well as reduced the susceptibility to microbial
eggs are regarded as standard and valuable source of all essential amino
and chemical related deteriorations.
acids including lysine (Attia, Al-harthi, & Korish, 2020). Despite the fact
The improved protein contents observed in the cookie products could
that lysine is abundant in both eggs and R. differens, it was not detected
be attributed to the source of protein used and their inclusion levels.
in the cookies, which could be due to processing-related losses (Cheng,
Whole eggs used in CTRC formulation has been reported to contain
Jin, & Zhang, 2014). Specifically, the R. differens underwent a two-stage
protein levels of 11.54–11.56 g/100 g db (Ogunwole et al., 2015)
cooking (initial blanching, boiling, toasting and deep-frying then
compared to 40.1–44.7 g/100 g db in blanched, boiled and toasted
baking) which may have rendered the amino acids unavailable in the
R. differens (Ochieng et al., 2022) and may account for the differences
baked foods. Typically, this manifested in DFCR, which had relatively
observed in the respective cookies. Deep-fried R. differens, on the other
low amounts of the majority of amino acids due to the fact that it was
hand, had a low protein content associated to a sequence of biochemical
made from deep-fried R. differens exposed to the harshest processing in
processes that markedly disintegrates proteins (Bordin, Tomihe Kuni­
oil at 175 ◦ C (Ochieng et al., 2022).
take, Kazue Aracava, & Silvia Favaro Trindade, 2013). The low protein
Methyl hexadecanoate, methyl 9Z-octadecenoate and methyl
levels of the deep-fried R. differens were mirrored in the baked cookies
(9Z,12Z)-octadecadienoate were the most abundant fatty acids (FAs) in
since they were used directly in dough formulation. The protein contents
SFAs, MUFAs and PUFAs, respectively. The identified dominant FAs and
in all of the insect-enriched cookies fell short of the Kenya Bureau of

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B.O. Ochieng et al. LWT 184 (2023) 115012

Table 4
Fatty acid composition (μg/mg) of cookies made from different processed R. differens analysed by Gas Chromatography coupled to Mass Spectrometry (GC-MS).
tR FAME (μg/mg) Corresponding Fatty ω-n(Δn) CTRC BCRC BLRC TSRC DFRC
(min) acid

14.81 Methyl octanoate C8:0 N.D. N.D. N.D. N.D. N.D.

16.85 Methyl decanoate Capric acid C10:0 0.01 ± 0.00a 0.01 ± 0.00a N.D. 0.01 ± 0.00a 0.01 ±
0.01a
14.88 Methyl nonanoate C9:0 N.D. N.D. N.D. N.D. N.D.
17.36 Methyl butanoate C4:0 N.D. N.D. N.D. N.D. N.D.
18.95 Methyl dodecanoate Lauric acid C12:0 0.11 ± 0.01a 0.22 ± 0.06a 0.18 ± 0.17 ± 0.11a 0.36 ±
0.05a 0.21a
20.83 Methyl 12-methyltridecanoate Tridecyclic acid iso-methyl-C13:0 N.D. N.D. N.D. N.D. N.D.
21.24 Methyl tetradecanoate Myristic acid C14:0 0.62 ± 0.10a 0.69 ± 0.55a 0.59 ± 0.64 ± 0.20a 0.98 ±
0.48a 0.22a
21.8 Methyl 4,8-dimethylnonanoate iso-dimethyl-C9:0 N.D. 0.01 ± 0.00 N.D. N.D. N.D.
23.52 Methyl hexadecanoate Palmitic acid C16:0 10.58 ± 5.02 ± 8.32a 9.99 ± 10.37 ± 12.73 ±
1.33ab 2.85ab 2.53ab 0.59b
25.49 Methyl octadecanoate Stearic acid C18:0 2.43 ± 0.18a 3.07 ± 0.20a 2.84 ± 2.60 ± 0.47a 2.84 ±
0.35a 0.13a
26.99 Methyl eicosanoate Arachidic acid C20:0 N.D. N.D. 0.59 ± 0.02 N.D. N.D.
26.99 Methyl 18-methylnonadecanoate iso-methyl-C19:0 N.D. 0.78 ± 0.02 N.D. N.D. N.D.
28.61 Methyl docosanoate Behenic acid C22:0 0.11 ± 0.01 N.D. N.D. N.D. N.D.
29.37 Methyl tricosanoate Trycosylic acid C23:0 0.06 ± 0.02a N.D. 0.04 ± N.D. 0.08 ±
0.01a 0.03a
30.13 Methyl tetracosanoate Lignoceric acid C24:0 0.15 ± 0.16 ± 0.01b 0.13 ± 0.08 ± 0.01a 0.14 ±
0.00b 0.02ab 0.04ab
32.08 Methyl hexacosanoate Cerotic acid C26:0 0.07 ± 0.06 ± 0.05 ± 0.01 ± 0.00a 0.06 ±
0.01b 0.00ab 0.02ab 0.02ab
∑
SFA 14.14 10.02 14.42 13.9 17.21
20.97 Methyl 11Z-tetradecenoate Vaccenic acid C14:1 (n-3) 0.01 ± 0.00 N.D. N.D. N.D. N.D.
20.97 Methyl 4Z-octenoate C8:1(n-4) N.D. N.D. 0.01 ± 0.00 N.D. N.D.
22.09 Methyl 5Z-dodecenoate C12:1(n-7) N.D. N.D. N.D. 0.01 ± 0.00a 0.01 ±
0.01a
22.15 Methyl 13-Methyl 9Z-tetradecenoate Myristoleic iso-methyl-C14:1 N.D. N.D. N.D. N.D. 0.01 ± 0.00
(n-5)
23.12 Methyl 9Z-hexadecenoate Palmitoleic acid C16:1(n-7) 0.26 ± 0.03a 0.77 ± 0.15b 0.73 ± 0.62 ± 0.56 ±
0.17b 0.23b 0.03ab
24.1 Methyl 10Z-heptadecenoate C17:1 (n-7) 0.10 ± 0.12 ± 0.02b 0.09 ± 0.04 ± 0.00a 0.11 ±
0.01ab 0.03ab 0.03ab
25.22 Methyl 9Z-octadecenoate Oleic acid C18:1(n-9) 11.55 ± 17.20 ± 13.16 ± 12.08 ± 16.19 ±
0.37a 2.24a 3.05a 2.17a 4.01a
26.79 Methyl 11Z-eicosenoate C20:1(n-9) 0.32 ± 0.38 ± 0.04b 0.32 ± 0.14 ± 0.01a 0.37 ±
0.05b 0.09b 0.08b
28.43 Methyl 11Z-docosenoate C22:1(n-11) N.D. 0.04 ± 0.00a N.D. 0.05 ± 0.05a N.D.
28.43 Methyl 13Z-docosenoate Erucic acid C22:1(n-9) N.D. N.D. 0.05 ± N.D. 0.04 ±
0.00a 0.005a
29.95 Methyl 15Z-tetracosenoate C24:1(n-9) 0.03 ± N.D. 0.02 ± N.D. 0.05 ±
0.00b 0.00a 0.00c
49.35 Methyl 8E-octadecenoate C18:1(n-10) N.D. 0.02 ± 0.00 N.D. N.D. N.D.
∑
MUFA 12.27 18.53 14.38 12.94 17.34
24.69 Methyl (12,15)-octadecadienoate C18:2(n-3) N.D. 0.04 ± 0.00a N.D. 0.01 ± N.D.
0.00b
25.18 Methyl (9Z,12Z)-octadecadienoate Linoleic acid C18:2(n-6) 0.19 ± 0.05a 12.10 ± 10.71 ± 5.46 ± 0.24 ±
10.31c 9.16c 9.25b 0.05a
26.3 Methyl (9Z,12Z,15Z)- α Linolenic acid C18:3(n-3) N.D. 2.12 ± 0.97b 1.55 ± 0.49 ± 0.01a 0.34 ±
octadecatrienoate 0.21ab 0.00a
26.45 Methyl (5Z,8Z,11Z,14Z)- Arachidonic acid C20:4(n-6) 0.14 ± 0.09 N.D. N.D. N.D. N.D.
eicosatetraenoate
∑
PUFA 0.33 14.26 12.26 5.96 0.58
∑
n-6 PUFA 0.33 12.10 10.71 5.46 0.24
∑
n-3 PUFA N.D. 2.16 1.44 0.50 0.34
∑
n-6/n-3 N.D. 5.60 7.44 10.92 0.71
∑
PUFA/SFA 0.02 1.42 0.85 0.43 0.03

Values are presented as Mean ± SD (standard deviation) of triplicate determinations. N.D. = Not determined; tR= Retention time, SFA = saturated fatty acids, MUFA
= monounsaturated fatty acids, PUFA = polyunsaturated fatty acids, CTRC=Control cookies with eggs; BCRC=Blanched R. differens-based cookies; BLRC=Boiled
R. differens-based cookies; TSRC = Toasted R. differens-based cookies; DFRC = Deep fried R. differens-based cookies; FAME=Fatty acids methyl ester.

the proportions of SFAs, MUFAs and PUFAs in the insect-based cookies The appearance and disappearance of particular FAs in cookies in
reflected the pattern demonstrated in their respective processed insects comparison to processed R. differens could be due to dough formulation
(Ochieng et al., 2022). Likewise, the trend characterized by higher inconsistencies and the influence of baking circumstances. In the CTRC,
PUFAs in BDRC and BLRC and higher SFAs in TSRC and DFRC was the proportions of FAs followed the increasing order: SFA (14.1%)
witnessed by Ochieng et al. (2022) in the respective processed >MUFA (12.3%) >PUFA (0.3%). This implies that eggs, which were
R. differens used for the cookies development. A similar trend was re­ incorporated in the CTRC, contained appreciably higher levels of SFAs
ported by Cheseto et al. (2020), which revealed that baking likely had no than MUFAs and PUFAs. The detection of appreciable levels, methyl
effect on the fatty acid quality and patterns. (9Z,12Z,15Z)-octadecatrienoate (α linolenic acid) (omega 3), methyl

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B.O. Ochieng et al. LWT 184 (2023) 115012

Fig. 2. Categories of the total fatty acids (%) detected in the processed R. differens-based cookies. CTRC=Control cookies with eggs; BCRC=Blanched R. differens-
based cookies; BLRC=Boiled R. differens-based cookies; TSRC = Toasted R. differens-based cookies; DFRC = Deep fried R. differens-based cookies.

Table 5
Mineral profiles of processed R. differens-based cookies.
Enriched Cookies CTRC BCRC BLRC TSRC DFRC F(4,10) p-value

Macro minerals
Mg (mg/100g) 24.55 ± 0.59b 25.15 ± 0.01b 24.12 ± 0.20ab 23.53 ± 0.47ab 22.34 ± 0.40a 6.73 p < 0.01
Ca (mg/100g) 20.17 ± 0.52d 17.78 ± 0.11c 16.64 ± 0.85bc 15.09 ± 0.76a 15.30 ± 0.02ab 40.97 p < 0.001
P (mg/100g) 290.80 ± 3.52a 298.12 ± 19.80a 289.33 ± 21.64a 281.50 ± 4.14a 275.80 ± 5.49a 1.22 ns
Na (mg/100g) 905.63 ± 8.92b 922.32 ± 28.50b 852.89 ± 14.03a 886.05 ± 3.89ab 917.27 ± 8.57b 10.17 p < 0.01
K (mg/100g) 145.22 ± 3.87a 145.87 ± 6.66a 151.88 ± 4.42ab 160.59 ± 2.69b 147.30 ± 7.30ab 4.38 p < 0.05
Trace Elements
Fe (mg/100g) 4.19 ± 0.01a 7.11 ± 0.10c 7.12 ± 0.37c 5.09 ± 0.02b 5.67 ± 0.43b 72.69 p < 0.001
Cu (μg/100g) 128.83 ± 5.38a 301.30 ± 7.65bc 346.79 ± 21.74d 331.08 ± 21.74cd 273.30 ± 8.50b 115.37 p < 0.001
Mn (mg/100g) 681.28 ± 20.80a 808.71 ± 45.14c 760.99 ± 16.25c 748.75 ± 17.33bc 692.95 ± 0.92ab 13.45 p < 0.001
Zn (mg/100g) 3.73 ± 0.12a 4.33 ± 0.08b 4.17 ± 0.03b 3.86 ± 0.16a 3.79 ± 0.00a 21.82 p < 0.001
Al (mg/100g) 0.75 ± 0.03a 2.52 ± 0.01d 1.94 ± 0.03c 1.92 ± 0.11c 1.45 ± 0.04b 413.91 p < 0.001

Results are presented as mean ± standard deviation. Same small superscript letters within rows indicate no significant differences of the minerals at p < 0.05.
CTRC=Control cookies with eggs; BCRC=Blanched R. differens-based cookies; BLRC=Boiled R. differens-based cookies; TSRC = Toasted R. differens-based cookies;
DFRC = Deep fried R. differens-based cookies.

Table 6
Microbial levels (Log cfu/g) and detection of Salmonellae in the processed R. differens flours and respective cookie types developed.
Processing TVC (Log cfu/ Lac + Enterobacteriaceae (Log cfu/g) Staphylococcus aureus (Log cfu/g) Salmonellae (Log cfu/g) Yeast and Moulds (Log cfu/g)
method g)

Blanching 4.54 ± 0.11c 3.04 ± 0.22b 4.83 ± 0.02b N.D. N.D.

Boiling 2.57 ± 0.05a N.D. 2.61 ± 0.17a N.D. N.D.
Toasting 3.38 ± 0.10b 1.01 ± 0.03a 3.70 ± 0.33a N.D. N.D.
Deep-frying 2.42 ± 0.33a N.D. N.D. N.D. N.D.

Cookie types TVC (Log cfu/ Lac þ Enterobacteriaceae (Log cfu/ Staphylococcus aureus (Log cfu/ Salmonellae (Log cfu/ Yeast and Moulds (Log cfu/
g) g) g) g) g)

CTRC <30 N.D. N.D. N.D. N.D.

BCRC <30 N.D. N.D. N.D. N.D.
BLRC <30 N.D. N.D. N.D. N.D.
TSRC <30 N.D. N.D. N.D. N.D.
DFRC <30 N.D. N.D. N.D. N.D.

Means on the same column followed by the same small letters are not significantly different (p < 0.05; n = 3). N.D. = Not determined; TVC = Total viable counts;
YMC=Yeast and moulds; Lac + ve = Lactose positive; CTRC=Control cookies; BCRC=Blanched R. differens-based cookies; BLRC=Boiled R. differens-based cookies;
TSRC = Toasted R. differens-based cookies; DFRC = Deep fried R. differens-based cookies.

(9Z,12Z)-octadecadienoate (linoleic acid) (omega 6) and methyl 9Z- cardioprotective (Blondeau et al., 2015). Oleic acid and linoleic acid are
octadecenoate (oleic acid) in the insect-based cookies than in the control known to be anti-inflammatory, anti-dermatitis, anticancer agents,
cookies, implies that insect-neophobic consumers can still access the suppress coronary disorders, play a role in glycolysis, hypertension
health benefits of oils endowed in insects through developed products. α control, insulin sensitivity and resistance (Atowa et al., 2021; Glick &
Linolenic acid (ALA) plays a key role as precursors for the synthesis of Fischer, 2013)
prostaglandin, thromboxane and leukotriene and are both neuro and Ruspolia differens has been demonstrated to furnish considerable

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B.O. Ochieng et al. LWT 184 (2023) 115012

Fig. 3. Distribution of respondents (%) based on their sensory perception of the cookies. A: Colour; B: Flavour; C: Mouthfeel and D: Texture and E. CTRC = “Control”
(i.e., cookies without processed R. differens meal); BCRC=Blanched R. differens-based cookies; BLRC=Boiled R. differens-based cookies; TSRC = Toasted R. differens-
based cookies; DFRC = Deep fried R. differens-based cookies. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web
version of this article.)

Fig. 4. Overall consumer acceptability of various cookie products. CTRC = “Control” (i.e., cookies without processed R. differens meal); BCRC=Blanched R. differens-
based cookies; BLRC=Boiled R. differens-based cookies; TSRC = Toasted R. differens-based cookies; DFRC = Deep fried R. differens-based cookies.

quantities of macro minerals K, P, and Mg, which, with the exception of entomophagy.
Ca, favourably equate with or surpass animal products including beef, Similar to other insects, R. differens has a lot of moisture, a good pH,
pork, and eggs (Ssepuuya, et al., 2019). Except for DFRC, this assump­ and is nutrient dense, thus it’s conducive for colonization by a variety of
tion is clearly supported by Table 5, which shows that the levels of microbiological organisms (Klunder et al., 2012; Ssepuuya, et al., 2019).
minerals in all cookies showed negligible variation. The calcium levels Furthermore, elevated microbial counts in harvested insects could be
in CTRC were significantly higher than those in the insect-based cookies. due to wild collection in an unregulated environment, unsanitary
This is because R. differens has lower levels of Ca attributable to the lack handling environments, and poorly cleansed hands, or handling equip­
of mineralized skeleton (Kinyuru, Kenji, Njoroge, & Ayieko, 2010) as ment (Gatheru et al., 2019; Ng’ang’a et al., 2019). Therefore, eating
opposed to chicken eggs. R. differens also had significant quantities of edible insects may endanger consumers’ health by offering serious
the trace elements Fe, Zn, Cu and Mn, exceeding those found in pork and health risks (Belluco et al., 2013). To reduce these health risks, proper
chicken (Ssepuuya, et al., 2019). This study reinforces the earlier report processing, handling, and storage practices have been advocated
by indicating significant differences in relation to the levels of these (Rumpold & Schlüter, 2013). The processing strategies used in this
trace minerals between the CTRC and insect-based cookies (mainly investigation had a significant impact on the counts of microorganisms.
BCRC, BLRC and TSRC) (Table 5). These findings replicate reports by Higher levels of TVC, Staphylococcus aureus, and Enterobacteriaceae
Ayensu et al. (2019) where the levels of trace minerals proportionally observed in blanched R. differens demonstrate the inefficiency of
increased with increasing levels of palm weevil incorporation. This blanching in suppressing a variety of microorganisms. TVC values in
suggests that the average mineral in the final baked products may have blanched R. differens are comparable to those found in blanched meal­
mirrored the patterns of minerals in the respective ingredients. There­ worm larvae after 10 s (Vandeweyer, Lenaerts, Callens, & Van Cam­
fore, fortification of cookies with R. differens can be used to combat penhout, 2017), 4 min blanched house cricket, 1 min blanched termites,
micronutrient related deficiency among populations unaccustomed to 5 min blanched Bingula caterpillar and 1 min blanched mealworm

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B.O. Ochieng et al. LWT 184 (2023) 115012

larvae (Caparros-Megido et al., 2017). Despite their greater levels in BRCR, BLRC, TSRC and DFRC. Most respondents generally accepted the
blanched R. differens, the TVC were within acceptable limits of less than CTRC and DFRC, which could be indicative of their congruity that these
Log10 7 cfu/g, a measure of hygienic condition of food (Nyangena et al., novel consumer familiar products could be a good alternative
2020; Ramashia et al., 2020). This hygiene criterion was adapted from market-driven products.
the Superior Health Council (SHC) and the Federal Agency for the Safety
of the Food Chain (FASFC) guideline, which suggested using process 5. Conclusion
hygiene standards for minced meat described in EU Regulation (EC) No.
1441/2007 for edible insects (Ssepuuya, et al., 2019). In comparison to This study demonstrates that cookies supplemented with blanched,
toasted samples, lower TVC levels were discerned in the boiled and boiled and toasted R. differens flour are richer in proteins, amino acids
deep-fried samples. This is because boiling and deep-frying cause higher (alanine, glutamic acid, and valine), minerals (Fe, Zn, Cu, and Mn) than
heat dispersion into the food matrices (Nyangena et al., 2020) than in the control and those with deep-fried R. differens meal. This uncovers the
toasting. In this investigation, deep-frying resulted in low levels of detrimental effects of deep-frying on key nutrients retention in food
Staphylococcus aureus, Enterobacteriaceae, yeast and moulds, and products development. Ruspolia differens meal fortified cookie products
completely subdued Salmonellae. The effectiveness of deep-frying in showed higher energy levels than cookies without processed R. differens.
decontaminating edible insects has been reported by other authors Microbial loads were substantially suppressed in boiled and deep-fried
(Gatheru et al., 2019; Labu et al., 2021). Because of their great heat R. differens meals. Remarkably, all the baked cookie products with in­
sensitivity, Enterobacteriaceae numbers were entirely eliminated after clusion of R. differens revealed that there was complete microbial
boiling and deep-frying (Ng’ang’a et al., 2019). Their existence in elimination. The high level of acceptability of the insect meal enriched
toasted samples, on the other hand, indicates that heat transfer to the cookie products reflects the consumer preference for novel insect-based
core (guts) was ineffective throughout this process (Klunder et al., food products, thus providing more insights for advanced production of
2012). The non-detection of Salmonellae spp. in all the samples justifies market-driven consumer insect-based food products that entices the
their sensitivities to heat, culminating into products compliant with growing entomophagists populace. However, future studies on full scale
microbiological guidelines on food safety (KEBS, 2020b). Low yeast and sensory evaluation including the nine-point hedonic scale would be
mould counts in all processed samples indicates that they were severely crucial to assess the degree of consumers’ liking of various insect-based
suppressed during processing, resulting in counts below the suggested food products based on their sensory appeals.
maximum limits of 5 log10 CFU/g (Ramashia et al., 2020; Vandeweyer
et al., 2017). CRediT authorship contribution statement
Despite Ssepuuya, et al. (2019) reporting pathogenic microorgan­
isms of public health concern from raw R. differens, the insect Brian O. Ochieng: Conceptualization, Methodology, Formal Anal­
based-cookies in the current study expressed permissible microbial ysis, Investigation, Data Curation, Writing – Review & Editing, and
levels and can be regarded safe for consumption. Similar results were Visualization. Joseph O. Anyango: Conceptualization, Methodology,
reported on biscuits fortified with palm weevil larvae (Ayensu et al., Visualization, Validation, Investigation, Writing – Review & Editing and
2019) and energy dense-biscuits enriched with silkworm pupae and Supervision. Fathiya M. Khamis: Conceptualization, Methodology,
locusts (Olamide et al., 2020). It is possible that use of pre-treated insect Investigation, Writing – Review & Editing. Sunday Ekesi: Project
ingredients and maintenance of a high degree of human and environ­ Administration, Funding Acquisition, Writing – Review & Editing.
mental hygiene throughout ingredient formulations contributed to the Sevgan Subramanian: Project Administration, Resources, Writing –
low microbial counts. Furthermore, the baking temperatures Review & Editing. James P. Egonyu: Project Administration, Writing –
(180 ◦ C/15 min) may have markedly subdued majority of the micro­ Review & Editing. John M. Nduko: Conceptualization, Validation,
organisms while the stringent adherence to post-baking hygiene stan­ Writing – Review & Editing and Supervision. Xavier Cheseto: Meth­
dards, with prompt packaging into sterile zip lock bags may have curbed odology, Software, Formal Analysis, Investigation, Writing – Review &
post-baking contamination from the surroundings. Finally the low water Editing and Visualization. Dorothy Nakimbugwe: Funding Acquisition,
activities and pH in cookies have also been reported to restrain bacterial Conceptualization, Writing – Review & Editing, Visualization and
growth and colonization (Khan, Hashmi, & Saleem, 2017). Validation. Chrysantus M. Tanga: Conceptualization, Methodology,
Consumer acceptability of insect-based products are partly a subject Investigation, Validation, Resources, Writing – Review & Editing,
to organoleptic characteristics such as flavour, aroma, colour, texture, Visualization, Supervision, Project Administration and Funding Acqui­
taste and appearance (Wilkinson et al., 2018; Yazici & Ozer, 2021). The sition. All authors read, reviewed, and approved the final manuscript.
larger proportion of both male and female respondents liking CTRC and
DFRC colours may be due to the familiar golden-brown colour they Funding sources
exhibited, as in common bakery products. Relatively few respondents
liked the dark-brown colours of BCRC, BLRC and TSRC which emerged Financial support for this research was provided by the BioInnovate
from intense Maillard reaction (Ogunlakin et al., 2018), stimulated by Africa Programme (INSBIZ—Contribution ID No. 51050076), the Curt
the high levels of available proteins in the respective processed in­ Bergfors Foundation Food Planet Prize Award, Bill & Melinda Gates
gredients (Ochieng et al., 2022). The increased dislike of the flavour of Foundation (INV-032416), Norwegian Agency for Development Coop­
BCRC in both male and female respondents may be linked to high con­ eration, the Section for research, innovation, and higher education
centration of residual displeasing aroma compounds since, they were (RAF–3058 KEN–18/0005); Australian Centre for International Agri­
formulated with mildly processed R. differens, characterized by limited cultural Research (ACIAR) (ProteinAfrica – Grant No: LS/2020/154),
aroma transformation. The greater disparity in the liking of the flavour the Swedish International Development Cooperation Agency (Sida); the
of BCRC between male (52.8%) and female (40.8%) respondents is ar­ Swiss Agency for Development and Cooperation (SDC); Australian
ticulative to the reports elucidated by Kröger, Dupont, Büsing, and Centre for International Agricultural Research (ACIAR), the Federal
Fiebelkorn (2022) citing insect acceptance to positively correlate with Democratic Republic of Ethiopia and the Government of the Republic of
masculinity but to negatively associate with femininity. The liking of Kenya. The first author, Brian O. Ochieng, was financially supported by
flavour of DFRC and BLRC may be accredited to processing-induced the Center of Excellence in Sustainable Agriculture and Agribusiness
formation of pleasant aroma during R. differens processing. A majority Management (CESAAM) of Egerton University, Kenya. The funders had
of the respondents preferred the texture of the CTRC as compared to the no role in the study design, data collection, and analysis, decision to publish,
insect-based cookies. This may be explained by the gritty effect of the or preparation of the manuscript. Therefore, the views expressed herein do
insect exoskeleton (Ojinnaka et al., 2015) used in the formulation of not necessarily reflect the official opinion of the donors.

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B.O. Ochieng et al. LWT 184 (2023) 115012

Declaration of competing interest consumption : A state-of-the-art review. Food Research International, 125, Article
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The authors declare that they have no known competing financial (2019). Impact of processing methods on microbial load of reared and wild-caught
interests or personal relationships that could have appeared to influence edible crickets (Scapsipedus icipe and Gryllus bimaculatus) in Kenya. Journal of Insects
the work reported in this paper. as Food and Feed, 5, 171–183. https://doi.org/10.3920/jiff2018.0042
Glick, N. R., & Fischer, M. H. (2013). The role of essential fatty acids in human health.
Evidence-based Complementary and Alternative Medicine, 18, 268–289. https://doi.
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