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Processing methods for the black soldier fly (Hermetia

illucens) larvae : From feed withdrawal periods to killing


methods

Mémoire

Jennifer Larouche

Maîtrise en sciences animales - avec mémoire


Maître ès sciences (M. Sc.)

Québec, Canada

© Jennifer Larouche, 2019


Processing methods for the black soldier fly (Hermetia
illucens) larvae
From feed withdrawal periods to killing methods

Mémoire

Sciences animales

Jennifer Larouche

Sous la direction de :

Grant W. Vandenberg, directeur de recherche


Résumé

Les larves de mouches soldats noires représentent un ingrédient alternatif prometteur pour le bétail, mais les
étapes de transformation peuvent affecter leur qualité. Les périodes de vidange gastrique utilisées pour évacuer
les excréments du tractus digestif afin de réduire sa charge microbienne, et les méthodes d’abattage sont
variables et peu documentées. Ce projet vise à optimiser la vidange gastrique et l’abattage des larves pour en
maximiser la qualité. En effet, un jeûne prolongé et une méthode d’abattage inadéquate pourraient altérer la
composition et la microbiologie du produit. Le temps d’évacuation du tractus digestif des larves alimentées de
Gainesville a été déterminé en suivant l’excrétion des fèces aux douze heures. Puis, l’impact du temps de jeûne
sur la composition et la contamination ont été mesurés quotidiennement pendant quatre jours. Également, les
effets de dix méthodes d’abattage sur la composition, la microbiologie et la coloration ont été comparés :
ébouillantage (40 s), dessiccation (60 °C, 30 min), congélation (-20 °C et -40 °C, 1 h; azote liquide, 40 s),
hautes pressions hydrostatiques (3 min, 600 MPa), broyage (2 min) et asphyxie (CO2 et conditionnement sous
vide, 120 h; N2, 144 h). Bien que le temps d’évacuation du tractus digestif médian fût de 72 h, un jeûne de 96 h
n’a pas permis de réduire la contamination. Certaines méthodes d’abattage ont affecté le pH, la stabilité de la
couleur ainsi que la charge microbienne. De plus, ébouillantage, asphyxie et dessiccation ont affecté la
composition proximale et l’oxydation des lipides. Malgré l’incapacité de la vidange gastrique à réduire la
contamination des larves, l’ébouillantage apparait comme la méthode la plus appropriée en réduisant la charge
microbienne et l’humidité tout en minimisant l’oxydation des lipides. Nous proposons donc un protocole pour
abattre les larves répondant aux exigences réglementaires canadiennes en matière de transformation des
insectes.

ii
Abstract

Black soldier fly (BSF) larvae represents a promising alternative ingredient for animal feed, but post-production
processing can affect their quality. Feed withdrawal periods (FWP) used to evacuate fecal matter from the
gastrointestinal tract, reducing the microbial load (ML), and killing methods are variable and poorly documented.
This project aims to optimize the FWP and killing methods of BSF larvae to maximize product quality. Indeed, a
prolonged FWP and an inappropriate killing method could alter larvae composition and ML. The gastrointestinal
evacuation time (GET) of BSF larvae fed on coloured Gainesville diet was determined by following frass
excretion every 12 h for 108 h. Then, FWP impact on the proximate composition and ML was measured daily
over four days. Finally, the effects on the chemical composition, ML and colour of 10 killing methods were
compared, i.e., blanching (B = 40 s), desiccation (D = 60 °C, 30 min), freezing (F20 = − 20 °C, 1 h;
F40 = − 40 °C, 1 h; N = liquid nitrogen, 40 s), high hydrostatic pressure (HHP = 3 min, 600 MPa), grinding
(G = 2 min) and asphyxiation (CO2 = 120 h; N2 = 144 h ; vacuum conditioning, V = 120 h). Although, the median
GET was 72 h, a 96 h FWP did not reduce larvae ML. Certain killing methods affected the pH (B, asphyxiation),
total moisture (B, asphyxiation and D), ash (B), lipid content (asphyxiation) and lipid oxidation (B, asphyxiation
and D), as well as the colour stability during freeze-drying. FWP were ineffective in reducing the ML. Blanching
appeared as the most appropriate method since it minimizes lipid oxidation, reduces ML and total moisture
(78.1 ± 1.0%). Our studies propose a standardize protocol to kill BSF that meet the Canadian regulatory
requirements of the insect production and processing industry.

iii
Table of contents

Résumé ................................................................................................................................................ ii
Abstract.................................................................................................................................................iii
Table of contents ................................................................................................................................. iv
Table of figures .................................................................................................................................... vi
Table of tables .....................................................................................................................................vii
List of abbreviations and acronyms .................................................................................................... viii
Remerciements.................................................................................................................................... xi
Avant-propos .......................................................................................................................................xii
Introduction ........................................................................................................................................... 1
Chapter 1. Literature review ................................................................................................................. 2
1.1 Edible insects ............................................................................................................................ 3
1.2 Black soldier fly (Hermetia illucens) ........................................................................................... 4
1.2.1 Life cycle of the black soldier fly ........................................................................................ 4
1.2.2 Proximate composition of the black soldier fly ................................................................... 5
1.2.3 Fatty acid profile ................................................................................................................ 5
1.2.4 Microbiota of the black soldier fly ...................................................................................... 5
1.3 Insect processing....................................................................................................................... 9
1.3.1 Harvesting ......................................................................................................................... 9
1.3.2 Post-harvest processing steps .......................................................................................... 9
1.3.3 Killing ............................................................................................................................... 10
1.3.4 Decontamination.............................................................................................................. 13
1.3.5 Drying .............................................................................................................................. 16
1.3.6 Other processing steps.................................................................................................... 17
1.4 Insect quality............................................................................................................................ 18
1.4.1 Proximal composition (macronutrient metabolism) .......................................................... 19
1.4.2 Lipid quality: peroxidation ................................................................................................ 20
1.4.3 Protein quality: digestibility .............................................................................................. 21
1.4.4 Microbial safety................................................................................................................ 22
1.4.5 Colour stability ................................................................................................................. 23
1.5 Aim and outline ........................................................................................................................ 27
References .................................................................................................................................... 29
Chapter 2. Effect of feed withdrawal periods on the proximate composition and microbial load of black
soldier fly (Hermetia illucens) larvae ................................................................................................... 36
2.1 Résumé ................................................................................................................................... 37
2.2 Abstract ................................................................................................................................... 37
2.3 Introduction .............................................................................................................................. 38
2.4 Materials and methods ............................................................................................................ 39
2.4.1 Gastrointestinal evacuation time ..................................................................................... 39
2.4.2 Impact of feed withdrawal period ..................................................................................... 40
2.4.3 Proximate composition and pH ........................................................................................ 40
2.4.4 Microbial analysis ............................................................................................................ 41
2.4.5 Statistical analysis ........................................................................................................... 41
2.5 Results..................................................................................................................................... 41
2.5.1 Gastrointestinal evacuation time ..................................................................................... 41
2.5.2 Effects of feed withdrawal period on proximate composition ........................................... 42
2.5.3 Effects of feed withdrawal period on the microbial load ................................................... 44

iv
2.6 Discussion ............................................................................................................................... 45
2.6.1 Gastrointestinal evacuation time ..................................................................................... 45
2.6.2 Effect of starvation on the initiation of pupariation. .......................................................... 46
2.6.3 Effects of a prolonged feeding period on the larvae ........................................................ 46
2.6.4 Effects of a feed withdrawal period on the larvae ............................................................ 46
2.7 Conclusion ............................................................................................................................... 47
References .................................................................................................................................... 49
Chapter 3. Effects of killing methods on lipid oxidation, colour and microbial load of black soldier fly
(Hermetia illucens) larvae ................................................................................................................... 51
3.1 Résumé ................................................................................................................................... 52
3.2 Abstract ................................................................................................................................... 52
3.3 Introduction .............................................................................................................................. 53
3.4 Materials and Methods ............................................................................................................ 54
3.4.1 Biological material ........................................................................................................... 54
3.4.2 Killing methods ................................................................................................................ 55
3.4.3 Analysis ........................................................................................................................... 56
3.4.4 Statistical analysis ........................................................................................................... 58
3.5 Results..................................................................................................................................... 58
3.5.1 Chemical composition and pH ......................................................................................... 58
3.5.2 Lipid oxidation.................................................................................................................. 59
3.5.3 Larval colour .................................................................................................................... 59
3.5.4 Microbial analysis ............................................................................................................ 61
3.6 Discussion ............................................................................................................................... 64
3.6.1 Chemical composition ..................................................................................................... 64
3.6.2 Colour .............................................................................................................................. 65
3.6.3 Microbiology .................................................................................................................... 65
3.6.4 Heating ............................................................................................................................ 66
3.6.5 Freezing .......................................................................................................................... 67
3.6.6 Asphyxiation .................................................................................................................... 68
3.6.7 Mechanical disruption ...................................................................................................... 68
3.7 Conclusions ............................................................................................................................. 69
Appendix A .................................................................................................................................... 71
References .................................................................................................................................... 72
General conclusions ........................................................................................................................... 76
Bibliography ........................................................................................................................................ 78

v
Table of figures

Figure 1.1 Life cycle of the black soldier fly ......................................................................................... 4


Figure 1.2 High hydrostatic pressure batch system ........................................................................... 15
Figure 1.3 Atmospheric direct and indirect cold plasma process ....................................................... 16
Figure 1.4 Tyrosine related substrates, reactions and enzymes involved in enzymatic browning,
together with subsequent non-enzymatic reactions in the black soldier fly larvae. ............................. 24
Figure 1.5 Representations of reactions involved in BSF browning at different pH. The colour behind
the structures represents the colour shade induce by the reaction..................................................... 25
Figure 2.1 Black soldier fly larvae excreting coloured Gainesville diet on a moist Whatman filter ..... 40
Figure 2.2 Gastrointestinal evacuation time and pupariation time of starving and isolated 12-d-old
black soldier fly larvae ........................................................................................................................ 42
Figure 2.3 Effects of feeding and starving on individual black soldier fly larvae composition: moisture;
lipids; proteins; carbohydrates ............................................................................................................ 44
Figure 3.1 Colour analysis of thawed and freeze-dried and granulated BSF larvae after different
killing methods: lightness; colour change between thawed and freeze-dried and granulated larvae;
chroma; hue angle .............................................................................................................................. 60
Figure 3.2 Freeze-dried and granulated BSF larvae killed by different methods: desiccation;
blanching; freezing; grinding; asphyxiation; high hydrostatic pressures ............................................. 61

vi
Table of tables

Table 1.1 Proximal composition, polyunsaturated fatty acids, minerals content and production
characteristics of the black soldier fly larvae and prepupae, mealworms larvae and domestic cricket. 3
Table 1.2 Fatty acid profile of the black soldier fly larvae and prepupae .............................................. 5
Table 1.3 Microbial load of black soldier fly larvae under various rearing conditions ........................... 8
Table 1.4 Killing methods used in industry according to Erens et al., 2012 ....................................... 11
Table 1.5 Microbial load reduction of decontamination methods applied to insects ........................... 14
Table 1.6 Impact of processing methods on protein quality of insects and shrimps ........................... 22
Table 1.7 Microbial safety criteria for ready-to-eat food according to Health Canada ........................ 23
Table 1.8 Impacts of processing technologies on polyphenol oxidase activity of crustaceans........... 26
Table 2.1 Proximal composition of fed and starved black soldier fly larvae at 27 °C ......................... 43
Table 2.2 Microbial load of fed and starved 10 d-old black soldier fly larvae at 27 °C ....................... 44
Table 3.1 Chemical composition, primary and secondary lipid oxidation levels, and pH of black
soldier fly larvae killed by different methods ....................................................................................... 59
Table 3.2 Microbial load of thawed black soldier fly larvae killed by different methods ...................... 62
Table A1. Colour measures, colour intensity, hue angle and colour change while drying of thawed
and freeze-dried and granulated BSF larvae killed by different methods. .......................................... 71
Table 4.1 Summary of the impact of ten killing methods on the black soldier fly larvae ..................... 77

vii
List of abbreviations and acronyms

AKH Adipokinetic hormone


ANOVA Analysis of Variance
AOAC Association of Official Analytical Chemists
AOCS American Oil Chemists’ Society
Aw Water activity
B Blanching
BSF Black soldier fly
CFU Colony forming unit
CHP Cumene hydroperoxide
CO2 Carbon dioxide
CPS Coagulase positive Staphylococci
C-F-C Cetrimide, fucidin and cephalosporin
C* Chroma
D Desiccation
DM Dry matter
E Enterobacteriaceae
EE Ether extract
F20 Freezing at -20 ºC
F40 Freezing at -40 ºC
FAO Food and Agriculture Organization of the United Nations
FDG Freeze-dried and granulated
Fe2+ Ferrous iron
Fe3+ Ferric iron
FOX Ferrous oxidation-xylenol orange assay
FWP Feed withdrawal period
G Grinding
GET Gastrointestinal evacuation time
GRIPHA Groupe de Recherche Intégré en Physiologie et Sciences Animales, Université Laval
h Hue angle
HHP High hydrostatic pressure
LAB Lactic acid bacteria
LH Lipid hemolytic
Log logarithms to the base 10
LOOH Lipid hydroperoxides
LOO· Peroxide radical
LO· Alkoxy radical
LTA Laboratoire de Transformation des Aliments, Université Laval
L· Lipid radical
L* Lightness
MDA Malondialdehyde
ML Microbial load
MRS deMan, Rogosa and Sharp agar
n. d. Not detected
N Liquid nitrogen
N2 Nitrogen
O2 Oxygen
PCA Plate count agar
pH Potential hydrogen
PPO Polyphenol oxidase

viii
ROS Reactive oxygen species
spp. Species
TBA Thiobarbituric acid
TBARS Thiobarbituric acid reactive substance
TI Total inactivation
TVC Total aerobic viable count
V Vacuum packaging
VRBA Violet Red Bile Agar
VRBG Violet Red Bile Glucose Agar
∆E Colour change

ix
À Philippe, my Sun and Stars.
À mon grand-père Raynald, dont la curiosité
et l’amour pour la nature ont su m’inspirer.

x
Remerciements

Au terme de ma maîtrise en sciences animales, j’aimerais souligner les personnes qui m’ont soutenu et appuyé,
et qui m’ont encouragé à me dépasser pour pouvoir vous offrir ce mémoire.

Je tiens d’abord à remercier Prof. Grant W. Vandenberg, mon directeur de recherche, qui m’a toujours
encouragé à laisser libre place à ma créativité dans la mise en place de mon projet et l’élaboration de mes
hypothèses et théories, me permettant même de valider toutes celles qui me semblaient les plus passionnantes.
Son écoute, sa disponibilité, ses conseils et surtout sa confiance m’ont permis de progresser à une vitesse que
je ne me croyais pas capable. La seconde personne à l’honneur est Dr Marie-Hélène Deschamps, la
professionnelle de recherche au laboratoire, mais surtout mon modèle qui a su m’aider à garder le cap sur mes
objectifs. Sa franchise, son jugement et son empathie font d’elle une personne qui nous motive à nous améliorer
et à attendre la critique avec confiance.

Je me dois également de remercier le programme Innov’Action agroalimentaire, "un programme issu de l’accord
cultivons l’avenir 2 conclu entre le ministre de l’Agriculture, des Pêcheries et de l’Alimentation du Québec, et
Agriculture et Agroalimentaire Canada", pour le soutien financier du projet. J’aimerais également remercier les
organismes subventionnaires m’ayant accordé des bourses d’études me permettant de me concentrer sur mon
projet, le Conseil de recherches en sciences naturelles et en génie du Canada et le Fonds de recherche du
Québec – Nature et technologie.

Je tiens également à remercier toutes les personnes qui ont contribué de près ou de loin à ce projet, que ce soit
par une aide technique ou un simple conseil ou encouragement : Prof. Linda Saucier, Prof. Alain Doyen, Yolaine
Lebeuf, Dominic Gagné, l’ensemble des étudiants du laboratoire Vandenberg, le personnel technique du Groupe
de recherche en physiologie animale (GRIPHA) et l’équipe du Laboratoire de recherche en sciences aquatique
(LARSA).

Merci aux membres de ma famille qui ont su m’encourager à persévérer en célébrant toutes les petites victoires
que j’ai connues lors de mon parcours. Un merci particulier à mes parents, Nathalie Verreault et Guy Larouche,
mon frère et ma sœur, Eddy et Véronick Larouche, à mes grands-parents, à ma belle-famille, les Clavet, et à
mes amis qui ont toujours été présents pour moi.

Finalement, mais non le moindre, un merci particulier à mon amoureux, Philippe Clavet, qui m’a supporté dans
ma décision de poursuivre mes études et lors des défis qui ont suivi. Ta présence réconfortante, ta joie de vivre
et ton énergie contagieuse m’ont encouragé à me lever chaque matin, les yeux rivés vers mon objectif et prête
à affronter tous les obstacles se trouvant sur ma route. Tu es le meilleur.

xi
Avant-propos

Ce mémoire présente les résultats de deux études visant à optimiser le conditionnement préabattage et
l’abattage des larves de mouches soldats noires, une source de protéines alternatives pour l’alimentation
humaine et animale. Ces travaux ont permis de proposer un protocole optimisé pour la transformation des
insectes, de la vidange gastrique jusqu’à l’abattage.

Les travaux réalisés dans le cadre de ce mémoire et portant sur l’optimisation de la vidange gastrique feront
prochainement l’objet d’une courte communication scientifique dont je serai l’auteur principal accompagné de
M.-H. Deschamps, Y. Lebeuf, L. Saucier, M. Cissé et G.W. Vandenberg.

De plus, les résultats obtenus lors de l’optimisation des méthodes d’abattage ont conduit à la publication d’un
article scientifique, avec comité de lecture, dans le journal Animals en avril 2019 intitulé « Effects of Killing
Methods on Lipid Oxidation, Colour and Microbial Load of Black Soldier Fly (Hermetia illucens) Larvae »1.

1 Larouche, J.; Deschamps, M.-H.; Saucier, L.; Lebeuf, Y.; Doyen, A.; Vandenberg, G.W. Effects of killing methods on lipid
oxidation, colour and microbial load of black soldier fly (Hermetia illucens) larvae. Animals 2019, 9 (4), 182;
https://doi.org/10.3390/ani9040182.

xii
Introduction
The Food and Agriculture Organization of the United Nations (FAO) estimates that food production will need to
increase by 50% to sustain projected 2050 global population growth [1]. Furthermore, nearly a third of the
comestible food currently produced is wasted (~ 1.3 GT/year) and diverted to landfills and other waste
management options [2]. In North America, this represents nearly 300 kg of high-value organic matter per capita
per year [2]. Since farming conventional livestock has important impacts on the environment [3], several studies
have focused on the development of alternative protein sources to support future food and feed demand [4].
Insects are particularly interesting due to their high nutritional quality and bioconversion rate when fed on waste
organic residues, their short life cycle as well as low water and space requirements [5-6]. The black soldier fly
(BSF) larvae, Hermetia illucens (Diptera: Stratiomyidae), a saprophagous insect, stands out from other edible
species by its higher ability to convert residual organic matter into a high-quality protein and lipid source, and as
a soil amendment [7]. Since the production of insects on an industrial scale is quite recent, it is necessary to
establish standardized production and processing methods to ensure the quality and safety of insect products.

The next chapter will focus on the edible insect species, insects processing methods and their impact on the
quality of the product. Therefore, it will summarize earlier research on the studied field while emphasing on the
quality of the product and recommended threshold. The second chapter describes the effects of a feed
withdrawal period on the product quality while the third one compares ten killing methods and their effects on
the product quality.

1
Chapter 1. Literature review

2
1.1 Edible insects
Among the edible insect species, the domestic cricket (Acheta domesticus), the mealworm (Tenebrio molitor)
and the BSF are the most often farmed [8]. The BSF appears very promising for large scale rearing systems
because it possesses the shortest development time, the lowest food conversion ratio (i.e., kg of feed needed
to produce 1 kg of biomass) and offers a great nutritional quality as shown in Table 1. In human nutrition,
polyunsaturated fatty acids contribute in the prevention of inflammatory-related disease including, among others,
arthritis, asthma and cardiovascular heart disease and thus should be part of the diet [9]. Even if the BSF
contains less omega-6 than mealworms and crickets, its n-6/n-3 ratio is closer to the recommended value for
human consumption [10]. Furthermore, feeding BSF larvae for only 3 h with 40% fish meal inclusion, allows to
reduce this ratio to 2.8 while increasing the total unsaturated fatty acid content by 5% [11]. Furthermore, the BSF
possesses two to four times more minerals than mealworms and crickets. Since they are required in all animals
feed, it could reduce the need for supplementation [12]. Indeed, on a dry basis, BSF larvae contains 26 times
more calcium than crickets and mealworms, at least twice less sodium and fourfold more iron [13]. Finally, the
BSF has high-quality protein since their essential amino acid profile corresponds to fish meal requirements [14].

Table 1.1 Proximal composition, polyunsaturated fatty acids, minerals content and production
characteristics of the black soldier fly (Hermetia illucens) larvae and prepupae, mealworms (Tenebrio
molitor) larvae and domestic cricket (Acheta domesticus) [13-17].
Species H. illucens T. molitor A. domesticus
Stage Larvae Prepupae Larvae Adult
Proximal composition (%, dry basis)
Crude protein (N x 6.25) 36.2 40.7 58.4 73.1
Crude lipid 18.0 15.6 30.1 15.9
Ash 9.3 19.7 3.5 5.6
Nitrogen-free extract 36.5 24.0 8.0 5.4
Polyunsaturated fatty acid
Omega-3 (% lipid) 0.6 – 1.5 0.1 – 0.9 0.4 – 1.5
Omega-6 (% lipid) 4.2 – 17.3 13.6 – 25.6 23.6 – 34.9
Omega-6/omega-3 ratio 6 – 11 24 – 102 15 – 29
Minerals content (mg/100 g; dry basis)
Calcium 2,900 3,000 100 166
Phosphorus 350 620 760 860
Sodium 100 50 174 363
Iron 200 8 6 6
Zinc 61 3 11 14
Production characteristics
Food conversion ratio 1.4 – 2.3 3.8 – 6.1 2.3 – 10.0
Development time (d) 21 – 37 116 – 227 48 – 167

3
1.2 Black soldier fly (Hermetia illucens)
1.2.1 Life cycle of the black soldier fly
The BSF, a tropical and temperate dipteran insect [18], is adapted to large-scale production considering its short
life cycle, the great size of its immature stage, the elevated number of eggs and its bioconversion efficiency.
Females lay between 320 and 620 eggs [19]. Four days later, the larvae hatch and go through six larval instars,
including the prepupae, then pupate [20-21]. The larvae are beige and possess photoreceptors which allow them
to flee light [22]. They can eat a wide variety of organic matter such as manure, fruits and vegetables, food waste
and fish offal [23-24], and can reduce, up to 50%, their feeding substrate on a dry basis [18]. They also have a
high conversion efficiency since they can produce 1 kg of larvae biomass with 1.4 kg of ingested feed compared
to cricket and mealworms which require 2.3 and 3.8 kg, respectively [15]. The BSF larvae reach the prepupal
stage in 10 to 52 days and weigh 300 mg depending on the feed offered and the rearing temperature [23, 25]. It
then stops eating and initiates melanisation, resulting in a darker colouration of the cuticle a few hours before
moulting to become a prepupa [26-27]. During this stage of 7 to 10 d, the prepupa migrates to a dry place to
metamorphose into a pupa [18, 28]. The pupal stage, during which larvae do not move nor eat for at least 8
days, ends with the adult emergence [29]. Because the imago does not eat, it uses its accumulated reserves to
meet its metabolic needs [19]. The fly mates and lays its eggs inside its 8 to 9 living days [30]. The most nutritious
stages are the larval ones (larva and prepupa) and are, therefore, the most often harvested by BSF producers
[17].

Figure 1.1 Life cycle of the black soldier fly (adapted from: http://uniquebiotechnology.com/).

4
1.2.2 Proximate composition of the black soldier fly
The nutritional quality of the BSF larvae mainly refers as its lipid and protein content [31], oxidation and
digestibility as well as its minerals and vitamins content [8]. The composition of the BSF is highly variable and
depends on the feeding substrate and the stage of harvest as shown in Table 1.1. There are two stages of
interest for the use of BSF as food or feed, the larvae and the prepupae. The pupae are less suitable for use as
a feed since they have lost about 20% of their lipid content compared to the larval stage [17]. When reared in
the same conditions, the larvae contain more lipids and nitrogen-free extract, but lower ash and protein contents
than the prepupae and the pupae [14, 17]. The prepupae also possess 20% more water than larvae [15, 32] and
therefore require a longer drying time. From a nutritional point of view, the choice of the harvested stage will
depend on the later use of the BSF meal.

1.2.3 Fatty acid profile


The BSF fatty acid profile is also variable depending on the development stage and contains mostly saturated
fatty acid allowing it to tolerate a wide temperature range, as shown in Table 1.2 [33]. Indeed, because the BSF
cannot regulate its body temperature and must survive at temperatures up to 40 °C, high concentration of lauric
acid allows it to reduce the fluidity of the membrane and its oxidation [33]. Moreover, the composition of the BSF
lipid also depends on the feeding substrate [17, 24]. Indeed, it is possible to increase the proportion of valuable
unsaturated fatty acid up to 37% of the lipid content by feeding the larvae a diet containing fish meal inclusions
a few hours prior killing it [24, 34]. However, unsaturated fatty acids are vulnerable to oxidation which could
reduce its nutritional value if not inhibited [35]. It is therefore essential to maintain the quality of the fatty acids
by preventing their oxidation.

Table 1.2 Fatty acid profile of the black soldier fly larvae and prepupae.
Fatty acid (% lipid) Larvae Prepupae
References [15, 17] [17, 32]
Lauric (C12 :0) 29 – 61 44 – 73
Myristic (C14 :0) 7 – 10 7 – 10
Palmitic (C16 :0) 8 – 17 6 – 10
Palmitoleic (C16 :1n-7) 3–7 1–8
Stearic (C18 :0) 1–3 0–2
Oleic (C18 :1n-9) 8 – 18 3–8
Linoleic (C18 :2n-6) 4 – 17 5 – 12
Alpha-Linolenic (C18 :3n-3) 0–2 0–1
Unsaturated fatty acid 18 – 37 10 – 24
Saturated fatty acid 53 – 80 63 – 91

1.2.4 Microbiota of the black soldier fly


The BSF is associated with an elevated microbiological risk related to the diversity of the feeding conditions. To
our knowledge, only three studies investigated the microbiota of the BSF and only one its mycobiota. The
mycobiota diversity is influenced by the diet and can be reduced by a prolonged feeding time [36]. According to

5
the authors, the majority of the yeasts detected on the BSF fed on several substrates are producers of
antimicrobial compounds [36]. The impact of the feed on the microbiota has been evaluated on the larvae entire
digestive tract [37] and on specific parts of the midgut as well [38]. The microbiota of all BSF stages has also
been tracked when fed the Gainesville diet [39], a reference diet for Diptera (50% wheat bran, 30% alfalfa meal
and 20% corn meal). The microbiota of the whole larva contained 54% Bacteroidetes, 20% Firmicutes, 28%
Proteobacteria and 9% Actinobacteria [39] which is similar to its midgut microbiota when fed on Gainesville diet
[38]. It seems that the bacteria taxa of the BSF provided with a balance diet are dominated by Bacteroidetes
(i.e., peptidoglycan degraders) and contains 9–20% Firmicutes and 16–28% Proteobacteria [38-39]. Hence,
when fed an unbalanced diet such as 100% fish meal and 100% cocked rice, the microbial diversity completely
changes. Indeed, the larva digestive tract do not contain Bacteroidetes anymore, but is now colonized by
Proteobacteria (54–56%) and Firmicutes (43–47%), which may be problematic since most food-poisoning
microorganisms are part of these taxa [37-38]. However, it is not necessarily problematic since lactic acid
bacteria also belong to the Firmicutes.

Proteobacteria is a group of gram-negative bacteria, meaning that they possess an external membrane of
lipopolysaccharides and a thin layer of peptidoglycan, and which includes some important pathogen bacteria
[40]. Indeed, Escherichia coli, which some are known to induce gastroenteritis, Salmonella spp., responsible for
salmonellosis, and Shigella spp., which provoke shigellosis in humans, are members of the Enterobacteriaceae
family (Gammaproteobacteria) which are Proteobacteria [41]. Another well-known Gammaproteobacteria is
Pseudomonas aeruginosa, which is an opportunist pathogen [42]. Campylobacter spp. are also Proteobacteria
(Epsilonbacteria) which is one of the most often reported etiologic agent of gastroenteritis (i.e.,
campylobacteriosis) [41].

Firmicutes is a group of gram-positive bacteria which possess a thick layer of peptidoglycan allowing them to
increase their resistance to physical disruption, heat and desiccation, but are more vulnerable to antibiotics and
other chemical antimicrobial compounds than gram-negative bacteria [43]. Firmicutes also includes major
pathogens which are mainly in two classes, Clostridia and Bacilli [42]. Clostridia, including Clostridium
perfringens, a sporulated food-spoilage bacteria, are often found in vacuum-packed food or food poorly
refrigerated [41]. Other important food-poisoning microorganisms are Listeria monocytogenes, Bacillus cereus
and coagulase positive Staphylococci (Bacilli) [41].

In addition to the feeding substrates that greatly influence the microbial load of the BSF larvae, the stage at
harvest is also important to consider. Indeed, when fed the Gainesville diet, it has been shown that microbial
community of prepupae contains 20% less Bacteroidetes, 10% less Firmicutes and 25% more Proteobacteria
than larvae [39]. As a result, the prepupae may include more pathogens and should be further investigated. For
some microorganisms, even the presence of few bacteria is enough to induce toxicity while for others, it requires

6
higher counts [41]. It is therefore critical to quantify the presence of these pathogens per gram to establish the
risk they represent. The Table 1.3 reports the only published microbial load data on the BSF larvae or prepupae
until now. As far as we know, Listeria spp. and Shigella spp. has not been detected in BSF larvae, but B. cereus,
E. coli, Salmonella spp., Staphylococcus spp., Pseudomonas spp. and Clostridia have been. Further processing
steps must focus on the reduction of the microbial load to ensure product safety.

7
Table 1.3 Microbial load (log CFU/g) of black soldier fly larvae under various rearing conditions.
Yeast and Aerobic Listeria Salmonella
Processing TVC LAB Enterobacteria Others
molds endospore spp. spp.
Total anaerobic: 5.6
Killed at -20 °C, 1h;
5.3 5.2 4.3 n. d. 4.0 Clostridia: n. d. [44]
Dried 50 °C
E. coli: 3.8
Killed at -20 °C, 1 h;
Dried at 80 °C, 2 h; 7.2 6.8 8.1 n. d. 6.1 E. coli: 5.9 [45]
Ground
Washed 1 min; B. cereus : n. d.
9.8 6.4 6.1 9.7 6.5 n. d.1 n. d.2 [46]
Stored at 4 °C, 24 h CPS : n. d.
Washed 1 min; B. cereus : n. d.
9.1 7.5 6.2 8.8 6.1 n. d.1 n. d.2 [46]
Stored at 4 °C, 24 h CPS : n. d.
Washed 1 min; B. cereus : n. d.
8.9 8.5 6.8 8.2 5.7 n. d.1 n. d.2 [46]
Stored at 4 °C, 24 h CPS : n. d.
Washed 1 min; B. cereus : n. d.
8.9 < 4.8 4.0 8.1 7.5 n. d.1 n. d.2 [46]
Stored at 4 °C, 24 h CPS : n. d.
Washed 1 min; B. cereus : n. d.
8.0 5.0 4.9 7.5 5.8 n. d.1 n. d.2 [46]
Stored at 4 °C, 24h CPS : n. d.
Washed 1 min; B. cereus : n. d.
8.1 5.4 3.5 7.3 4.5 n. d.1 n. d.2 [46]
Stored at 4 °C, 24h CPS : n. d.
Washed 1 min; B. cereus : 2.3
8.0 < 4.1 < 3.1 7.4 3.7 n. d.1 n. d.2 [46]
Stored at 4 °C, 24h CPS : n. d.
Coliforms: n. d.
Dried/cooked at 350 °C,
3.2 n. d. n. d. n. d. Clostridia: 2.1 [47]
15 min
E. coli: n. d.
Blanched 5 min;
5.5 n. d. 3.0 detected E. coli: 1.4 [48]
Sun-dried
n. d. = not detected; CPS = coagulase positive Staphylococci; 1 Listeria monocytogenes; 2 Salmonella enterica

8
1.3 Insect processing
Insect processing starts with harvesting and should end in obtaining a safe and stable product from a
microbiological and physico-chemical point of view. Processing has a great influence on the final product quality
and can reduce the microbial load while enhancing the colour and nutritional stability of the product. It can also
increase or decrease the nutritional quality and change protein functional properties. Depending on the final use
of the insect product, processing steps may include post-harvest processing, killing, macronutrient extraction
(i.e., proteins, lipids and chitin), decontamination, drying and grinding. In industry, every processing step should
aim to reduce microbial load while increasing or maintaining the nutritional quality.

1.3.1 Harvesting
Harvesting represents the separation of the larvae from their feeding substrate and is carried on by sieving or
by using their behaviours to let them collect themselves, also called auto-collecting. The choice of the method
used will depend on the BSF stage (i.e., larvae and prepupae) at harvest and the feeding substrate humidity and
particle size. The first method, and the most used in industry, consists to sieve the substrate while keeping the
larvae or prepupae into the sieve. Sieving, using a rotary drum system or a vibrating sieve, can be applied to
both stages and allows collecting the substrate which represents a potential soil amendment [49]. Therefore, the
substrate must have been completely digested, the particle size must be small enough to be easily sieved and
it must contain a maximum of 50% humidity at time of harvest to prevent sieve obstruction [49]. The second
harvesting method is that via autocollection, which can only harvest prepupae since it takes advantage of their
migration behaviour to a drier place prior to pupate, but tolerates higher humidity substrate [18, 50]. Because
they try to flee the substrate, it is possible to orient their movement on a ramp to let them fall into a collecting
bucket [30]. It has the advantage to ensure that only the living ones are harvested, but may induce energetic
reserve mobilization, reducing the nutritional value of the product, though this still need to be investigated further.
The harvesting efficiency is also lower since not all prepupae will migrate according to plan.

1.3.2 Post-harvest processing steps


To obtain a stable insect product and to enhance its quality, large-scale production uses post-harvest processing
steps. Although not every producer uses them, they could contribute in enhancing the microbial quality of the
product. They include a feed withdrawal period and a washing step.

1.3.2.1 Feed withdrawal period


Because it is difficult to remove the insect gastrointestinal tract, undigested feed, frass and microbial load
associated with them usually become a part of the resulting insect meal. To prevent it, insects are sometimes
degutted, i.e., applying pressure on it to force its content evacuation, or starved. The FAO recommends including
a feed withdrawal period (FWP), also called post-harvest starvation or gut purging, to promote the reduction of

9
the insect microbial load [6]. The egestion time, which means the time required for a feed bolus to be excreted
when feed is continuously ingested, is of 100 min for Musca domestica and between 60 min and 90 min for
Lucilia sericata, two Diptera larvae, respectively [51-52]. The egestion time increase when no more feed is
available and corresponds to the gastrointestinal evacuation time (GET). For instance, the GET of adult Blatella
requires 3 days while the egestion time is of 120 min [53]. Because there is only little information on insects
GET, it is generally assumed that a FWP of 12 to 48 hours is enough to empty most of the digestive tract [54-
56]. To date, only mealworms were investigated for the suitability of applying a FWP to reduce microbial load
[57]. The authors concluded that a FWP of up to 48 h with or without faecal contact does not affect the
mealworms microbial load [57]. However, since the GET of mealworms is unknown yet, frass may have remained
in the digestive tract resulting in a remaining high microbial load. Besides, starvation may induce energetic
reserve metabolism and its evaluation appears necessary when estimating the optimal FWP [58]. Since the BSF
larvae eat contaminated residual organic matter, research should focus on the effectiveness of a FWP since it
could reduce the microbial load at low cost.

1.3.2.2 Washing
As for many crustaceans, some insect species have been reported to be washed with water prior or after killing
to remove any feed or frass remaining on their cuticle, including Coleoptera (T. molitor) and Diptera (M.
domestica, Piophila casei, H. illucens) larvae [59-60]. Washing carcasses is an effective process frequently used
in animal production since it allows to physically remove organic matter and the microbes it may contain at low
cost and enhance product self-life [61]. Washing can be done by water dipping or by spraying. Washing can be
optimized by increasing pressure, duration, temperature, dipping repetition and by adding sanitizers into the
water, such as chlorine and organic acids (acetic, lactic and propionic acids) [61]. The effect of dipping in agitated
water during 1 min on the microbial load of T. molitor has been investigated and showed no significant microbial
reduction [57]. However, as far as we know, this is the only study reporting the efficiency of rinsing edible insects
and further research should focus on the optimization of the washing process. Although pre-consumption diet is
currently used as feed for BSF larvae, washing the larvae should be investigated since it could allow them to be
used to convert highly contaminated residual organic matter.

1.3.3 Killing
Killing is a key step in insect processing since it can impact on the nutritional quality, microbial safety, colour
stability and taste of the product [62]. Being necessary in any animal production, killing should be fast and
effective and contribute to reducing the microbial load while maintaining the nutritional quality of the product.
Invertebrates are killed by several methods such as cold, heat, asphyxiation and mechanical disruption.
Therefore, the killing method should be adapted to the specie requirement. Freezing and blanching are the most
frequently used methods, but many other methods have potential to be just as effective [5, 8]. The Table 1.4
summarises killing methods applied at industrial scales.

10
1.3.3.1 Killing by cold
Freezing is frequently used since it allows product preservation while slowly killing the insect. Because insects
are poikilotherms, killing by cold allows reducing metabolism of the insects preventing any potential pain or
suffering. It also has the advantages of reducing microbial growth and spoilage-enzyme activity. It can be carried
out by freezing, or freeze-drying, and immersion in ice water [63]. A few studies investigated the impact of killing
BSF larvae by freezing on the lipid and protein quality and on the colour stability [64-65]. Since enzymes remain
active after freezing, spoilage enzyme has been reported to induce lipid degradation and polyphenol oxidation
[64-65]. In the BSF larvae, enzyme responsible for lipolysis has been shown to be highly active, even at freezing
temperature, inducing lipid deterioration during storage at -20 °C [64]. Consequently, it appears very important
to inhibit those enzymes rapidly in order to maintain the nutritional quality of the insect.

Table 1.4 Killing methods used in industry according to Erens et al., 2012 [66].
Individual or company Species Killing method
Tarique Arsiwalla, H. illucens Mechanically crushed by centrifugation
Protix Biosystems
Jagran B. V. Insect Rearing M. domestica Grinding and freezing;
Concepts experimenting asphyxia with high N2/low 02.
Various insects Small insects: stunned with CO2 and sprayed
Leon Westerd,
with hot water
Wageningen University
Large insects: freeze-drying
Orthoptera, Coleoptera Freeze or freeze-drying
Kreca
and Diptera
Van der Ven T. molitor Boiling or freeze-drying

1.3.3.2 Killing by heat


Killing by heat has been used for many invertebrates including lobster, shrimps and insects. Blanching is one of
the most frequent killing methods used in insects production since it allows microbial reduction while being very
fast [67]. Some insects start dying at 50 °C but higher temperature is generally used to inactivate
microorganisms at the same time [68]. It can be carried on by dry heat, in an oven, or by moist heat such as
blanching and steaming. In addition to reducing the microbial load of the insects, heat treatment can induce
endogenous spoilage-enzyme denaturation thus limiting product deterioration during storage [69]. Dry heat can
allow a high level of non-enzymatic browning by Maillard reaction compared to blanching which minimize it
because of the high-water content [70]. Immersive method such as blanching has also been reported to reduce
the protein and water content in shrimp, which could reduce the drying time [71]. In insects, blanching has been
investigated as a killing method for BSF and mealworms. It was reported to increase the pH by 0.5 unit for at
least 48 h, indicating a higher product stability compared to raw product [72]. Blanching also reduces the water-
holding capacity of insects, reducing the drying time and inactivating spoilage enzymes while maintaining colour
and nutritional quality [64-65, 72].

11
1.3.3.3 Asphyxia
The use of carbon dioxide (CO2) is one of the most popular methods to anesthetize invertebrates and requires
exposition time between 3 and 60 minutes depending on the insect species [66, 73]. A concentration of CO2
above 40% induces neuron depolarization without affecting its conductance inducing insects immobilization [74].
However, when applied for a longer period of time, it can also be lethal by reducing the hemolymph pH and
inducing insect dehydration by promoting spiracle opening [75-76]. In addition, to increase their survival time
under anoxic condition, oxygen is spared by reducing their metabolic activity [77]. However, in the presence of
CO2 the metabolism activity is maintained promoting oxygen depletion and thus, reducing their survival [77]. The
time required to effectively kill insects with carbon dioxide is highly variable and depends on the species, the
insect stage, the temperature, the CO2 and O2 concentration, as well as the humidity [78]. Pure carbon dioxide
is lethal for Drosophila melanogaster (Diptera) larvae in only 30 min [79] while it requires 72 h in Cadra cautella
(Lepidoptera) and Tribolium castaneum (Coleoptera) larvae [78]. Even if carbon dioxide asphyxiation requires a
relatively long time, it can anesthetize the insect prior to killing it, and reduce drying time by initiating dehydration
resulting from spiracle opening.

Asphyxiation can also be applied by air saturation with N2 and requires that the oxygen level is below 3% [80].
Because air saturation can be hard to achieve on a large scale, vacuum packaging and drowning can also be
used [81]. Insects have different resistance to asphyxia amongst species and stages. Indeed, M. sexta pupae
can survive 5 d when immersed in water while the larvae only survive 4 h [82]. Adult grasshopper species can
survive between 7.5 to 22 h when immersed in water, while the nymph of the same species can only survive
between 3 and 13 hours [83]. The mortality of larval stages of the Ephestia cautella (Lepidoptera) to 98% N2 is
also highly variable and requires between 96 to 144h which is 1 to 2 days longer than aerobic atmosphere with
60% CO2 [80]. The BSF larvae have not been investigated for its resistance to anoxic condition and could be as
efficient as for D. melanogaster exposed to 100% CO2. However, prolonged anaerobic conditions could allow
anaerobic microbial growth and thus could require a drastic decontamination step.

1.3.3.4 Mechanical disruption


Mechanical disruption of the neuronal system appears to be a promising killing method because of its low cost
and rapidity. It can be applied by grinding or centrifugation according to Protix [66]. Even if mechanical disruption
appears to be applied in industry (Table 1.4), no study has evaluated the impact of this method on the quality of
the resulting product [66]. It was reported that grinding induces browning for several insect species [13, 84]. It
could also allow lipid oxidation by exposing insect constituents to oxygen [85]. Besides, grinding could enhance
drying capacity by increasing surface evaporation [85]. In addition, Jagran, a housefly rearing company, stated
that among others, grinding appeared to be most animal-friendly killing method [66].

12
1.3.3.5 Other killing methods
Other killing methods have been reported for invertebrates such has electrocution for crustaceans [86].
Electrocution at 120 volts and 20 amps for 10 s has been reported to effectively stun crustaceans if applied for
1 s and to kill lobsters and crabs if applied for 5 and 10 s respectively [87]. However, electrocution has not been
investigated yet as a stunning or killing method for insects. In summary, a high diversity of methods is used to
kill insects, but only blanching and freezing have been under focus. Therefore, it appears primordial to optimize
the killing process for BSF larvae in order to maximize its quality while minimizing potential pain and suffering.

1.3.4 Decontamination
After killing, the resulting product is the whole wet larva, whose microbial load has potentially been reduced,
depending on the killing method used, and whose pH is close to neutrality [46] indicating that it is a product of
perishable nature and that it is treated as a low-acid food [88]. It is therefore necessary to process the larvae to
extend their shelf life. Several decontamination techniques have been developed for the food industry, but only
a few have been applied to insects. Amongst them, blanching, desiccation, high hydrostatic pressures, direct
and indirect cold plasma and microwave have been investigated for their capacity to reduce the microbial load
of insects as shown in Table 1.5.

13
Table 1.5 Microbial load reduction of decontamination methods applied to insects.
Microbial load reduction (log CFU/g)
Treatment Species
TVC Other microorganisms
HHP: 350 MPa, 7 min H. illucens 3.2 Salmonella typhimurium: 5.0 [44]
Salmonella typhimurium: TI (6.5)
HHP: 350 MPa, 15 min H. illucens TI (8.0) [44]
Yeast and molds: 1.0
HHP: 400 MPa, 7 min H. illucens < 1.0 Yeast and molds: TI (4.5) [45]
HHP: 600 MPa, 10 min T. molitor 3.0 [89]
Blanching, 90 °C, 10 min T. molitor 4.0 [89]
Enterobacteriaceae: TI (6.1)
Yeast and molds: TI (1.5)
Blanching, 100 °C, 40 s T. molitor 5.6 [90]
Psychrotophic: TI (5.5)
Aerobic endospore: < 1.0
Enterobacteriaceae: TI (4.2)
Blanching, 100 °C, 1 min A. domesticus 4.1 [91]
Aerobic endospore: < 1.0
Blanching, 100 °C, 1 min, in
A. domesticus 4.2 Aerobic endospore: 1.0 [91]
vinegar + 5% acetic acid
Enterobacteriaceae: TI (6.8)
Blanching, 100 °C, 10 min T. molitor TI (7.7) [91]
Aerobic endospore: TI (2.1)
Total anaerobic count: 5.6
Psychrotophic: 5.4
Steamed, 5 min T. molitor 5.2 [92]
Enterobacteriaceae: 5.6
Anaerobic endospore: 1.2
Enterobacteriaceae: TI (4.6)
Roasting, 10 min T. molitor TI (7.7) [91]
Aerobic endospore: < 1.0
Desiccated 90 °C, 15 min T. molitor 1.2 [89]
Desiccated 100 °C, 15 min T. molitor 2.6 [93]
Desiccated 120 °C, 15 min T. molitor TI (6.8) [93]
Indirect cold plasma, 10 min T. molitor < 1.0 [89]
Direct cold plasma, 5 min T. molitor < 1.0 [89]
Semi-direct old plasma,15 min T. molitor 3.0 [93]
Enterobacteriaceae: 5.3
Microwave 10 min T. molitor 5.2 Yeast and molds: TI (2.8) [90]
Aerobic endospore: < 1.0
Enterobacteriaceae: TI (6.4)
Blanched 40 s + microwave
T. molitor 6.0 Yeast and molds: TI (2.8) [90]
drying 13 min
Aerobic endospore: < 1.0
TI = Total inactivation; HHP = High hydrostatic pressure

1.3.4.1 High hydrostatic pressure


High hydrostatic pressure (HHP) technology is a non-heating decontamination technique able to maintain most
product quality indices while reducing its microbial load. In a batch system, the product will enter a room that will
be filled with a compressible fluid followed by the air removal (Figure 1.2). The fluid will then be compressed
directly or indirectly resulting in an instantaneous and uniform pressure on the product allowing it to maintain its
structure [94]. Although HHP is considered as a non-thermal treatment, a high temperature might be applied
during pressurization to increase effectivity [28]. Three parameters must be optimized to increase the efficiency
of the treatment, the pressure applied (MPa), the pressure holding time, and the temperature [28].

14
Figure 1.2 High hydrostatic pressure batch system (from https://www.hiperbaric.com/en/high-pressure).

Although HHP is still very expensive, it is increasingly used in food processing. This technology offers many
advantages over traditional methods depending on the conditions used such as the possibility of pressurizing
foods at low temperatures, reducing the load of some bacteria, yeast and moulds, inhibiting enzyme activity and
allowing to create new functional foods [94-98]. High pressure is very effective in reducing gram-negative
bacteria, yeast and moulds, but less in reducing gram-positive and spore-forming bacteria. When applied to
insects, HHP has highly various effects on total viable counts (from < 1.0 to 8.0 log reduction) depending on the
research resulting from the presence of spore-forming bacteria able to resist the treatment [44-45]. Therefore,
HHP should be optimized according to the microbial community of the product which in turn, varies from insect
species, stages and facilities.

Very few studies have attempted to demonstrate the ability of HHP to inhibit enzymatic activity in insects.
However, HHP treatment at 400 and 500 MPa for 3 min were able to prevent browning in mealworms suggesting
that enzymatic inhibition did occur [72]. It was also reported that HHP reduces water-holding capacity in
mealworms which this effect increased with higher pressure [72]. Lower water-holding capacity could also
reduce drying time which would be a great advantage for the industry. However, HHP can also have negative
effects on the quality of the product since it can promote lipid peroxidation [99] as it was reported for giant tiger
shrimp where lipid oxidation levels increased from 300 MPa [100]. Considering the wide variety of effects of
HHP, it is necessary to further evaluate the potential utility of this technology for insects, especially BSF larvae.

1.3.4.2 Cold atmospheric pressure plasma


Cold atmospheric pressure plasma is a non-thermal technology that allows reducing a wide range of
microorganisms including spore-forming bacteria and viruses. Although this technology is gaining interest in food
processing, it is an expensive one [93]. Cold plasma is composed of neutral particles and a lower quantity of
charged ones; their bombardment inactivates microorganisms by erosion of their cell membrane [101].

15
Therefore, gram-positive bacteria and spores are more resistant to this treatment [101]. As shown in Figure 1.3,
the plasma discharge can be applied directly, semi-directly or indirectly on a product resulting in different
antibacterial effects [101]. Applied on fresh mealworms, direct and indirect cold plasma has little effect [89],
however, it could reduce by 3 log total viable counts when applied semi-directly on the powder [93]. Cold
atmospheric pressure plasma could be useful in controlling microbial loads of insect powders and should be
further investigated.

Figure 1.3 Atmospheric direct and indirect cold plasma process (adapted from Almeida et al. 2015)
[102].

1.3.4.3 Other decontamination methods


Several other decontamination methods can be used to reduce the microbial load of insects such as pulse
electric field, low-energy electron beam, ultrasound, microwave and heat treatment [101]. Among them, only
microwave and heat treatment have been used on insects to inactivate microorganisms. Microwave has been
highly effective in reducing the microbial load of T. molitor, but was not able to reduce spore-forming bacteria
[90]. Heat treatment can be separate in two kinds, moist and dry (desiccation) heat. Blanching during only 40 s
in water is very effective in reducing most microorganisms, but spore-forming bacteria remain unaffected [90].
However, blanching in an acidic solution appears to be more effective on aerobic endospores in A. domesticus
[91]. Moreover, increasing the blanching time to 10 min allows to totally inactivate aerobic endospores (2.2 log)
in T. molitor [91]. At 100 °C, dry heat appears to be less effective than blanching, but higher temperature can be
achieved. Desiccation at 120 °C for 15 min was able to totally inactivate total viable counts (6.8 log) [93].
However, no information is available on its effect on insect spore-forming bacteria. Since spore-forming bacteria
are the most challenging type of microorganism, decontamination methods should focus on their inactivation.

1.3.5 Drying
Drying insect products has many advantages since it improves preservation by reducing microbial growth and
spoilage-enzyme activity. Several drying methods can be applied to insects which have different effects on the
final dry product. Indeed, the method can affect the colour, water activity, proximate composition, lipid oxidation
and protein solubility. Sun-drying, oven-drying, freeze-drying and microwave-drying are the most frequent
methods used in insect processing and its optimization is currently the aim of several research [8].

16
1.3.5.1 Oven drying
Oven drying has been shown to have a great potential as a drying method for insects [103-104]. Many kind of
system can be used such as vacuum and rotating oven which may have different effects on the product [105].
Temperatures between 50 °C and 120 °C are generally applied from an hour to a few days, but lower
temperature is favourable in order to maintain protein solubility and to reduce Maillard reaction, shrinkage and
tissue collapsing [105-107]. Drying at 60 °C appears as the optimal drying temperature to prevent those effects
while reducing the drying time [108].

1.3.5.2 Freeze-drying
Freeze-drying is a non-thermal treatment frequently used in insect processing, but is relatively expensive and
requires a minimum of 24-53 h to dry mealworms [105, 109]. It is one of the best drying methods to maintain
insect colour since it does not induce Maillard reaction resulting in the whitest powder [109]. However, it also
induces a slight protein solubility reduction of 10% [105]. Moreover, freeze-drying induces the greatest lipid
oxidation resulting in important quality loss [105], but blanching prior freeze-drying was able to reduce by half
the oxidation [109]. In addition, it can enhance BSF larvae customer acceptability since the whole freeze-dried
larvae appears inflated instead of shrank.

1.3.5.3 Microwave drying


Microwave is also expensive but is the fastest drying method for insects requiring only 10 to 15 min depending
on the microwave parameters [109]. It also produces inflated whole dried larvae but allows browning to occur
[103]. Microwave drying of mealworms has been reported to reduce protein solubility of 40% [105]. On BSF
larvae, microwave drying induces protein polymerization resulting in a lower digestible amino acid score and
digestibility, and in a powder of bigger particle sizes compared to oven-drying at 60 °C [103].

1.3.5.4 Other drying methods


Other drying methods may be used on insects such as sun drying and fluidized bed drying. Sun drying is a
traditional low-cost drying method, considered as a low temperature and extended-time treatment, which has
been applied to many insect species including grasshopper, caterpillar, termite and maggot [60, 107]. Fluidized
bed drying is a fast, high-temperature, short-time technology that can also be used since it induces only a small
lipid oxidation increase when applied at 130 °C during 110 min, but could induce browning [105]. Most drying
studies on edible insects focused on T. molitor and few have investigated the effects of drying on the BSF larvae
or prepupae quality. Therefore, a better understanding of the effects of drying on the BSF could contribute in
improving the final product quality.

1.3.6 Other processing steps


Once the product has been decontaminated and dried, it is now stable and should be able to be preserved for
at least a few months without much nutritional quality change. Further processing steps are optional since the

17
product is already stable enough to be shipped and store but could give a plus value or enhance the quality of
the product.

1.3.6.1 Grinding
Grinding can appear as particularly attractive, since edible insects are frequently used as a powder in a diet or
in a meal. For food, insects are more easily accepted by Western customers if they cannot be perceived [110]
and thus incorporated as a powder into a recipe. However, grinding fresh insects has been reported to induce
browning in many insects resulting from enzymatic and non-enzymatic reaction involved with polyphenols, which
may reduce product quality [13, 55, 84]. Therefore, grinding should be applied after product stability is achieved.

1.3.6.2 Extraction of insect components


Extraction of lipids, proteins and chitin may enhance the value of the product by increasing the control of the
proportion of their inclusion during diet formulation. However, extraction needs to be optimized for every species
in order to maximize extraction yield and purity and to obtain a product with desirable functional protein
properties. Indeed, extraction can alter the flavour, the colour and protein functionality, induce protein
denaturation and reduce protein extraction yield of the residual defatted meal [54, 111]. Lipid extraction can be
done by mechanical pressing, aqueous or solvent extraction and by differential density. Protein extraction is
generally applied to defatted insects and can be done by alkali or sonication [112-113]. Finally, chitin extraction
consists of product demineralization followed by deproteination and can be done using chemicals or lactic acid
bacteria [8]. New extraction technologies should also be further investigated such as supercritical CO2 ,
microwave and ultrasound [106, 114]. Some processing steps can enhance component extractability, such as
HHP and blanching [65, 115]. Therefore, processing methods should be considered as a whole from the harvest
until obtaining the desire final product. Indeed, several processing methods discussed above can contribute in
many ways to the final product characteristics allowing the reduction of the number of processing steps or
enhancing the final quality. As an example, blanching can be used to kill, decontaminate and to enhance
extraction. Further study should focus on the interactions of processing steps in order to provide effective
pathways in obtaining different quality products.

1.4 Insect quality


Insect processing can greatly affect the insect product including its nutritional and microbiological quality as well
as colour stability. The nutritional quality refers mainly to the proximate composition, the lipid oxidation and the
protein digestibility, while the microbial quality refers to the abundance of spoilage and pathogenic
microorganisms. Understanding mechanism involved in food spoilage and how processing impacts them is
primordial to obtain a product of great quality. Therefore, even if criteria have not been established to evaluate
insect product quality, some references in other food systems may be applied to insects.

18
1.4.1 Proximal composition (macronutrient metabolism)
Processing steps from harvesting to killing can induce a stress on the insects resulting in energy mobilization as
for starvation. In addition, most processing techniques can also affect the insect composition depending on the
method applied. Understanding the impact of processing on the chemical composition of the insect is primordial
in order to increase the control of the resulting product.

1.4.1.1 Stress response


From harvesting to killing, every processing step can induce significant stress on the insect which could strongly
affect the composition of the final product. The stress response in insects varies with the development stage,
the duration, the nature and the intensity of the stress [116-117]. The response begins with the secretion of
octopamine, a stress hormone equivalent to mammalian adrenaline, which promotes gluconeogenesis and
glycogenolysis by inducing adipokinetic hormone (AKH) secretion, a neurohormone synthesized and stored in
cardiac bodies [118]. In insect larvae, AKHs induce glycogen mobilization, glycogenolysis, whereas in the adult
stage they induce the mobilization of triglycerides by lipolysis [117].

The amount of energy stored during the larval stages is even more important in insects in which the imago stage
is not feeding such as in BSF. Therefore, the larva must have accumulated enough energy in the form of
glycogen and triglycerides to allow reproduction and oviposition [117]. Though the amount of glycogen stored in
the fat body is lower than for triglycerides, glycogen is the primary source of energy being mobilized in response
to starvation or environmental changes [117]. Glycogen phosphorylase, an enzyme used to mobilize glycogen
in the form of trehalose, is active since the larval stage and its activity increases particularly in the prepupal stage
[117] in anticipation of a very energy-intensive stage, the pupa. It should be recommended to minimize the stress
impose on insects prior and during killing in order to prevent any glycogen or triglycerides loss.

1.4.1.2 Starvation
During the fasting period, triglycerides are normally mobilized after depletion of glycogen [117]. Triglycerides are
stored as droplets in association with cholesterol and surrounded by phospholipids in the fat body [117]. Lipase
access to triglycerides is regulated by the presence of droplet-associated proteins similar to perilipins in
mammals [117]. One of these proteins, Lsd1, is present at the prepupal stage and is activated by AKH which
activates lipolysis [117]. The other regulatory protein, Lsd2, is present at all stages of development [117]. It is
associated with lipid accumulation and promote droplet formation. The absence of trehalose in the haemolymph
under fasting conditions, caused by the depletion of glycogen, induces the mobilization of triglycerides resulting
in production of glycerol and fatty acids [117]. Depending on the fasting length, this mechanism could highly
reduce the lipids content of the insect. In addition, protein can also be mobilized as a source of energy.

The effect of starvation on lipid and protein metabolism is highly variable amongst insect species. Starved adult
crickets and grasshoppers start to metabolize their lipid after 1 d, but oxidize differently their protein [119].

19
Crickets start to oxidize their proteins after 2.5 d, while grasshoppers do not really use them [119]. Their survival
under starvation is also quite different since 50% of crickets died after 5 d while it takes 16 d for grasshoppers
[119]. Cockroaches initiate lipolysis after 3 d and metabolize their proteins after 36 d. Furthermore, 50% of them
die after 52 d which makes them extremely resistant to starvation [119]. Mealworm larvae initiate lipolysis after
2 d but do not rely on their proteins and 50% die in 5.5 d, while M. sexta larvae do not rely on their lipids nor
their proteins and 50% die in 3 d [119]. To our knowledge, the effect of starvation on the BSF has not been
investigated yet and should be evaluated considering the great diversity of strategy used in insects to survive
starvation.

1.4.1.3 Effects of endogenous degradation enzyme post-killing


For BSF prepupae, slow killing methods such as freezing promotes glycogenolysis, lactic acid production from
anaerobic metabolism, consumption of citric acid and lipolysis [64-65]. It seems that lipolytic enzymes remain
active during cold storage, promoting triglyceride degradation and resulting in lower lipid content [64]. Therefore,
it would be useful to inactivate these enzymes as fast as possible to minimize lipid degradation.

1.4.2 Lipid quality: peroxidation


Lipid quality generally refers to their level of oxidation as it reduces fatty acid availability. Because valuable
unsaturated fatty acids are highly vulnerable to oxidation, processing methods must focus on minimizing their
oxidation in order to increase product quality [35]. Indeed, oxidized polyunsaturated fatty acids are no longer
available when ingested and can be potentially harmful via the production of secondary products such as
alkenals and malondialdehyde [35]. In addition, the oxidation and hydrolysis of the unsaturated fatty acids lead
to the formation of volatile compounds, a sign of lipid degradation, resulting in alteration of sensory properties,
notably smell and taste [35]. It is therefore important to minimize oxidation in order to optimize the quality of the
product. Considering that in certain circumstances, BSF larvae is enriched in polyunsaturated fatty acid [11], it
is important to prevent lipid oxidation during processing.

Lipid oxidation takes place in three stages, namely initiation, propagation and termination. Initiation is generally
induced by the presence of reactive oxygen species (ROS) and corresponds to the lipid hemolytic (LH) cleavage
liberating water and a radical (L·) [120]. The autocatalytic propagation immediately follows the initiation and
corresponds to a reaction between molecular oxygen and L· to form a peroxide radical (LOO·) [120]. The
propagation represents the attack of a peroxide radical on an oxygen atom from another lipid resulting in the
formation of hydroperoxides (LOOH) and a lipid radical (L·) [120]. The termination phase corresponds either to
the combination of two hydroperoxides, to form various products such as volatile compounds, or to the
decomposition of the monohydroperoxide by an alkoxy radical (LO·) resulting in the formation of 4-
hydroxyalkenals, 2-alkenals and malondialdehyde (MDA) [35, 120]. Lipid oxidation is measured from the
propagation and the termination steps, also called primary and secondary lipid oxidation, respectively [121]. The

20
primary lipid oxidation can be established by measuring the level of hydroperoxides while the secondary one is
measured by the MDA content [122]

It is already known that light, heat, the presence of oxygen and iron as well as the presence of reaction producing
ROS, such as mitochondrial electron transport and metal-catalyzed oxidation reaction, contribute to the initiation
of the peroxidation [120]; ferrous iron can promote initiation by activating oxygen [123]. Therefore, inhibition of
lipid peroxidation initiation may be done by reducing the concentration of ROS and ferrous iron (Fe2+) and by
promoting antioxidant activity [124]. Antioxidant enzymes such as superoxide dismutase, catalase and
peroxidase catalyze the conversion of ROS into water while ferrous iron can be inactivated by oxidation into
ferric iron by ferroxidase or physically by chelation [85, 125-126].

Several processing methods put the larvae under such conditions which can initiate lipid peroxidation. Low
temperature can promote peroxidation since enzymes’ antioxidant activity is reduced resulting in an increase in
ROS [127]. Heating and HHP can promote lipid oxidation by protein denaturation, which reduces antioxidant
activity and increase iron by releasing them from haem proteins [99]. In fact, heat treatment can reduce the
activation energy required to initiate peroxidation while high pressures from 300 MPa promote the reduction of
ferric iron into active ferrous iron [99]. However, processing methods can also prevent lipid peroxidation by
oxygen removal using modified atmosphere or vacuum packaging and by Maillard reaction products, resulting
from heating, which can exhibit antioxidant properties [128]. Moreover, blanching and microwave drying did not
promote insect lipid oxidation while freeze-drying did [109]. Because no acceptable threshold for insect lipid
oxidation has been established yet, reference quality value for fish oil is being used. Therefore, secondary lipid
oxidation values below 1.5 mg MDA/kg on a wet basis are considered as not rancid (excellent quality), between
1.6 and 3.6 as slightly rancid (good quality) and above 3.7 as rancid (unacceptable quality) [129].

1.4.3 Protein quality: digestibility


The protein quality refers to the amino acid profile, crude protein content and protein digestibility. Since the
amino acid profile and the crude protein content of the BSF was established to be of good quality [14], the main
concern is to maintain them as much as possible during processing. The high protein digestibility of the BSF
makes it an interesting ingredient for food and feed. Because BSF prepupae has higher chitin content than BSF
larvae reaching 82-90% and 78%, respectively, larvae are more digestible and appears as a better choice for
use as an ingredient [12]. Processing greatly affects protein digestibility and therefore should be optimized in
order to maximize it. The degutting of mopane caterpillar increases crude protein content by 35% and increases
protein digestibility by 6%, while roasting decreases protein content and digestibility by 10% and 4%, respectively
[12]. As shown in Table 1.6, heat can have different effects on insect protein digestibility depending on the
species. While heat treatment affects protein digestibility of grasshoppers, it does not affect that of termites [130].
In response to heat, proteins can be denatured, i.e. unfolding of the polypeptide, thus increasing its availability

21
to digestive enzymes [131]. However, depending on the amino acid composition of the polypeptide, denaturation
can also reduce protein digestibility [131]. Indeed, denatured proteins are also subject to form aggregates;
exposed sulfhydryl groups being available to form intermolecular disulfide bonds [132], reducing their solubility
hence reducing their digestibility [131]. It does not appear to be the case for tropomyosin, a protein in shrimp,
for which denaturation by HHP enhance its digestibility while moist heat does not affect it [131]. Therefore,
processing has different effects depending on the protein amino acid composition. Insect protein digestibility has
been reported to be especially high when the cuticle could be removed and reach 90% of the protein content for
some insects [133]. Thus, further studies should focus on the effects of different processing methods on the BSF
protein digestibility.

Table 1.6 Impact of processing methods on protein quality from insects and shrimps.
Processing Species Protein Digestibility Allergenicity Reference
Degutting Mopane caterpillar + 6% [12]
Roasting Mopane caterpillar - 4% [12]
Toasting M. subhylanus No effects [130]
R. differens - 2%
Sun-drying (30 °C) M. subhylanus No effects [130]
R. differens - 4%
Moist heat Shrimp Unaltered Increased [131]
Tropomyosin
High pressure Shrimp Increase Decreased [131]
Tropomyosin
Ultrasound Shrimp Increase Unaltered [131]
Tropomyosin

1.4.4 Microbial safety


In Canada, to ensure product safety and quality, counts of pathogenic and spoilage microorganisms must not
exceed specific limits, which are defined by Health Canada and the Canadian Food Inspection Agency (CFIA).
As discussed in the section 1.2.4, minimally processed BSF larvae are highly contaminated hence must be
subject to a thermal treatment or otherwise decontaminated [134]. Moreover, as shown in Table 1.3, several
pathogens have been detected in BSF larvae such as E. coli, B. cereus, Salmonella spp. and Clostridium spp.
which could represent a health risk if eaten. Although CFIA investigated the presence of Salmonella spp. and E.
coli in edible insects [134], there is no specific recommendation on microbial load limits applied to insect products
to be used as food and feed in Canada. Therefore, the strict criteria of Health Canada for ready-to-eat food
(Table 1.7) can be used as a guideline for the insect industry [135].

Similarly, no guideline on the methodology to be used to establish microbial counts in insects is available.
However, while no microorganisms were detected on whole blanched mealworms, the analysis of the crushed
insect showed 2.5 log CFU/g of TVC and 2.5 log CFU/g of bacterial endospore [91]. Moreover, preliminary work
in our lab comparing counts from larvae either mixed using a stomacher at 230 rpm for 3 min or homogenized

22
during 20 s (VDI 12, VWR) has shown that homogenization gives more accurate microbial load (1.2 log fold
increase in total viable count compared to stomacher). Therefore, insects should be shredded in order to
estimate accurately the microbial load and assess the safety of the product.

Table 1.7 Microbial safety criteria for ready-to-eat food according to Health Canada (log CFU/g) [135].
Microorganisms Satisfactory Marginal Unsatisfactory
Total viable aerobic count
Totally cooked or processed < 4.0 < 5.0 ≥ 5.0
Partially cooked or processed < 6.0 < 7.0 ≥ 7.0
Indicator organisms
Coliforms < 2.0 < 3.0 ≥ 3.0
E. coli < 1.0 < 2.0 ≥ 2.0
Pathogens
Salmonella spp. n. d. in 25 g Detected
Campylobacter spp. n. d. in 25 g Detected
Shigella spp. n. d. in 25 g Detected
E. coli 0 157: H7 & VTEC n. d. in 25 g Detected
L. monocytogenes n. d. in 25 g 1.0 ≤ 2.0 Detected
V. cholerae n. d. in 25 g Detected
V. parahaemolyticus n. d. in 25 g < 2.0 2.0 – 3.0
Clostridium perfringens < 1.0 1.3 – 2.0 2.0 – < 4.0
Coagulase positive staphylococci < 1.4 < 2.0 2.0 – < 4.0
B. cereus and other Bacillus < 1.7 < 3.0 < 4.0

1.4.5 Colour stability


The insect product must not only have an acceptable microbiological quality while complying with the health and
safety standards, but the product should not be perceptible in food to promote consumer acceptability. The
appearant quality, in crustaceans, includes the maintenance of the body colour over time [136]. This represents
a challenge because as soon as the animal dies, cuticle staining can be observed if it is not kept in cold or
treated accordingly [137]. This phenomenon, where the cuticle of arthropods takes a darker colour after death
because of enzymatic reaction, is called melanosis, or “black spot”. It is also present in insects and result from
enzymatic oxidation [136]. Although melanosis is not responsible for any safety problem for human or animal
health per se, this phenomenon could reduce protein digestibility and create consumer aversion for the product
which could turn out as economic challenges for the industry [136, 138]. Moreover, insect browning can also
result from non-enzymatic reactions, such as complex formation between iron and polyphenol [13] and Maillard
reactions [139]. The BSF is known to develop a particular darker colour when processed, compared to other
insect species and should be prevented during processing [140]. As a reference, in many food system, a
perceptible colour alteration is represented by a colour change (∆E) higher than 2 [141].

1.4.5.1 Melanosis
Melanosis has been well described in crustaceans considering its important impact on the product commercial
value. Several enzymatic and non-enzymatic reactions occurs during melanosis resulting in melanin formation,

23
a brown pigment [136]. The endogenous spoilage enzymes inducing melanosis in crustaceans include
polyphenol oxidase (PPO), or tyrosinase, peroxidase, laccase, tyrosine hydroxylase and DOPA decarboxylase
[136]. According to a recent study in which the activity of these different enzymes toward L-tyrosine has been
quantified, it seems that only PPO activity was detected in three insect species namely T. molitor, A. diaperinus
and H. illucens [138]. Therefore, as shown in Figure 1.4, PPO appears to be the principal enzyme responsible
for melanosis in those insects which catalyze two reactions in the presence of oxygen: the conversion of a
monophenol, L-tyrosine, into a diphenol, L-DOPA, and the conversion of a diphenol, L-dopamine or L-DOPA,
into an o-quinone, L-DOPA quinone or L-Dopamine quinone [84].

Figure 1.4 Tyrosine related substrates, reactions and enzymes involved in enzymatic browning, together
with subsequent non-enzymatic reactions in the black soldier fly larvae (adapted from Janssen et al.
2017) [84].

This PPO, which is found in the insect cuticle, is also found in zymogenic form in their digestive tract and in their
hemolymph [136, 142-143]. It is associated with sclerotization of the cuticle, that is hardening of the exoskeleton
after moulting, and immunity [144]. Insects PPO can be activated by ethanol, lipids and cetylpyridiunium chloride
and, depending on the source of activation, PPO will show different activity levels and produce different
intermediates [145]. The reaction leading to the formation of melanin by the PPO, as described in Figure 1.4,
requires the presence of an amino acid i.e., tyrosine, oxygen and its coenzyme, copper. Several factors can
therefore influence the speed of the reaction, namely the concentration of PPO and tyrosine, the presence of
copper and oxygen, the animal species, the water content, the pH and the larval stage (proximity of a moult).
[136, 146]. In BSF, the PPO has its higher activity at pH 7 and is completely inhibited at pH 4 [138]. Moreover,
the activity of the PPO in BSF larvae appears to be at least two times lower than for T. molitor and A. diaperinus,
which is surprising since the BSF shows the greatest colour change upon grinding [138]. Janssen and
collaborators found that another reaction involving polyphenol might be responsible, the complex formation
between iron and polyphenol [13]. Even if the PPO activity is relatively low in BSF larvae, processing should aim
at inhibiting it since it still contributes to some product browning.

24
1.4.5.2 Complex formation between iron and polyphenols
Because the BSF larvae contain elevated levels of minerals (Table 1.1), Janssen et coll. also investigated their
possible involvement in browning [13]. According to their research, iron appears to be the only mineral involved
in the reaction. Since the BSF larvae contains four times more iron than T. molitor and A. diaperinus, the reaction
between iron and polyphenol could explain why the BSF larvae appear darker than the other species [13]. In
addition, the iron content of the BSF larvae is 26 times higher than that of the prepupae which developed a dark
colouration, thus concurring with the iron involvement in darkening [17]. In fact, Janssen concluded that ferric
iron (Fe3+) can interact with at least two L-DOPA resulting in a green colour development which develops faster
than polyphenol oxidation [13]. This reaction, requiring oxygen, appears to be reversible and occurring at a
neutral and basic pH [13]. Auto-oxidation of polyphenols can also occur at basic pH resulting in a reddish colour
[13]. Therefore, at acidic pH, ferric iron can be reduced in ferrous iron (Fe2+) allowing a covalent cross link
between two L-DOPA resulting in a whitish colour [13]. This reaction seems to be the best-case scenario to
produce a lighter insect powder with a high colour stability. Figure 1.5 represents a summary of the polyphenol
reactions occurring in BSF larvae including their optimal pH and resulting colour change. More information is
needed on the molecules at play during the colour formation in order to develop effective methods to prevent it.

Figure 1.5 Representations of reactions involved in BSF browning at different pH. The colour behind the
structures represents the colour shade induced by the reaction (from Janssen et al. 2019) [13]

1.4.5.3 Maillard reaction


Numerous efforts have focused on Maillard reactions in many food products since they are responsible for non-
enzymatic browning. Because some products of this reaction would have antiprotease properties in some animal

25
species, heat treatments inducting Maillard reactions should be avoided in order to promote nutritional quality of
the product [147]. Maillard reactions represent a series of non-enzymatic reactions which begins with a
condensation between a reducing sugar with a free amino group. This series of reactions also require water and
involve water production, but too much water in a food product decreases the reaction speed [70]. Therefore, a
water activity (Aw) lower than 0.5 or higher than 0.8 will reduce the reaction speed, thus the browning [70]. When
drying a product, this Aw range should be passed over as fast as possible and at the lowest temperature possible
to minimize browning [70]. The pH also has an important effect on Maillard reactions, thus on product colour and
taste. A basic pH promotes the production of dicarbonyls from Amadori products by fission or dehydration while
an acidic pH promotes aldehyde production by Strecker degradation, which has less browning potential [70].
Maillard reactions are poorly documented in insects. However, it seems that fructose, a reducing sugar, may be
involved in Maillard reactions in cricket, while glucose seems involved in mealworms [107, 148].

1.4.5.4 Polyphenol oxidase inhibition


Considering the magnitude of financial losses associated with melanosis in crustaceans, several strategies for
inhibiting PPO have been established and can be separated into two categories, chemical and physical.
Chemical methods include the use of reducing agents (such as sulphites), chelating agents, complexing agents,
acidulants and enzyme inhibitors [136, 149-150]. Although these methods are effective in inhibiting melanosis,
they cause an alteration of product composition and can be perceived pejoratively by the consumer who tends
to prefer natural products [136]. In contrast, physical methods do not necessarily change the composition as it
is the case for HHP. Even if some methods only delay the onset of melanosis (freezing, dehydration and modified
atmosphere packaging), some allow a definitive inhibition by denaturing the endogenous spoilage enzymes
involved (heat and HHP) [69]. Heat treatment has been effective in reducing PPO activity in crustaceans as
shown in Table 1.8. However, PPO activity appears to have different thermostability depending on the species.
High pressure treatment from 500 MPa seems particularly interesting since it would prevent the use of heat
favouring the maintenance of product quality [151]. The application of a lower pressure, such as 300 MPa, seems
to induce the activation of the zymogen form of the PPO resulting in an increase in its activity [151]. Melanosis
is still relatively misunderstood since the phenomenon varies widely among species [152]. Considering the great
potential of larval products for food and feed, it is necessary to better understand this phenomenon in BSF larvae
and to define methods to inhibit it in order to promote customer acceptability.

Table 1.8 Impacts of processing technologies on polyphenol oxidase (PPO) activity of crustaceans.
Treatment Species Activity of PPO (%) Reference
High hydrostatic pressure
300 MPa, 1.5 min Litopenaeus vannamei 158 [151]
500 MPa, 2 min Litopenaeus vannamei 3 [151]
600 MPa, 1 min Litopenaeus vannamei 3 [151]
Heat

26
60 °C, 30 min Litopenaeus vannamei 39 [151]
65 °C, 30 min Nephrops norvegicus 40 [142]
70 °C, 30 min Parapenaeus longirostris 0 [137]
90 °C, 20 min Panulirus cygnus 0 [153]
90 °C, 2 min Litopenaeus vannamei 13 [69]
100 °C, 1.5 min Litopenaeus vannamei Almost totally inactivated [151]

1.5 Aim and outline


Most research on insects as food and feed have focused on the production factors influencing their composition
and only few investigated the effect of processing on their quality. The BSF is highly perishable considering it
neutral pH, its high-water content, its high microbial load and low colour stability at harvest. It is therefore
essential to optimize BSF processing methods in order to maintain its quality.

Although, feed withdrawal period could reduce the microbial load of larvae, a prolonged period could also induce
energetic mobilisation, thus reducing their quality. Moreover, killing method could contribute in reducing larvae
contamination. In addition, some methods could enhance larvae preservation by promoting endogenous
spoilage enzyme inactivation. Therefore, this thesis focused on the ability of feed withdrawal periods and of
killing methods to reduce the microbial load of BSF larvae while maintaining their nutritional quality and colour.

The specific aims of this project were to:

(i) Estimate the gastrointestinal evacuation time of black soldier fly larvae;

(ii) Evaluate the impact of a prolonged feed withdrawal period of up to the gastrointestinal evacuation time
previously established on the proximate composition and the microbial load of the larvae and,

(iii) Compare the effects of ten killing methods on the chemical composition, lipid oxidation, colour stability and
microbial load.

In Chapter 2, the specific gastrointestinal evacuation time of BSF larvae fed on a Gainesville diet has been
established and the effects on the microbial load and proximate composition of a feed withdrawal period until
complete gastrointestinal evacuation was evaluated.

In Chapter 3, the effects of ten killing methods such as by heat (blanching and desiccation), cold (freezing at -
20 °C, -40 °C and in liquid nitrogen), asphyxiation (CO2, N2 and under vacuum) and mechanical disruption
(grinding and HHP) on the chemical composition, lipid oxidation and colour stability and microbial load was
compared.

27
We concluded with recommendations for BSF larvae mass-rearing in order to maximize product quality and
insect processing.

28
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fitness in monarch butterflies. Physiol Biochem Zool 2016, 89 (5), 389-401.
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of l-DOPA. Arch Insect Biochem Physiol 2018, 98 (4), e21457.
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tobacco hornworm, Manduca sexta (L.) and other lepidoptera : Identification of ß-D-glucopyranosyl-O-L-tyrosine and
other metabolites. Arch Biochem Biophys 1980, 205 (1), 146-155.
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2016, 463, 89-96.
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Chapter 2. Effect of feed withdrawal periods on the
proximate composition and microbial load of black
soldier fly (Hermetia illucens) larvae

Jennifer Larouche, Marie-Hélène Deschamps, Linda Saucier, M’ballou Cisse and


Grant W. Vandenberg

36
2.1 Résumé
L’utilisation de larves de mouches soldats noires comme aliment alternatif pour le bétail permettrait de réduire
les pêcheries et le gaspillage alimentaire, mais leur qualité devra être maximisée par l’utilisation de méthodes
de transformation appropriées. Les périodes de jeûne préabattage utilisées pour évacuer leurs fèces afin de
réduire leur charge microbienne, sont variables et peu documentées. Pourtant, un jeûne prolongé pourrait
réduire leur qualité par mobilisation des réserves énergétiques. Le temps d’évacuation du tractus digestif (GET)
des larves a été déterminé en suivant la production de fèces aux 12 h, puis l’impact du temps de jeûne sur la
charge microbienne et la composition ont été déterminés quotidiennement pendant 96 h. Bien que le GET
médian était de 72 h, 96 h de jeûne n’a pas réduit la contamination, mais a affecté la composition. Finalement,
un délai de 48 h entre la récolte et l’abattage pourrait être tolérée par les larves.

2.2 Abstract
The use of black soldier fly (BSF) larvae (Hermetia illucens) as alternative feed for livestock could help to
decrease organic wastes while providing novel animal feed ingredients. Rearing, killing and processing are
crucial to maximizing their nutritional quality. Feed withdrawal periods, used to evacuate fecal matter from the
gastrointestinal tract prior killing to reduce the microbial load, are variable and poorly documented. Indeed, a
prolonged starving period can reduce the quality of larvae by inducing energetic mobilization. The
gastrointestinal evacuation time (GET), at 27 °C of 12-d-old larvae weighing on average 184 ± 11 mg fed on
Gainesville diet coloured with carmine red, was determined by following frass excretion every 12 h over a 108 h
period. The impact of starvation time on the proximate composition (% dry basis) and microbial load was
measured over time (0, 24, 48, 72 & 96 h). The median GET was 72 h and 99% of the larvae had an empty gut
after 84 h. Following a 96 h feed withdrawal period, the microbial load was not affected by starvation while protein
and ash increased by about 2.3% and 1.0%, respectively on an absolute basis. Therefore, the industry should
not apply FWP on BSF larvae to reduce its microbial load. Finally, a delay of 48 h between harvest and killing
could be tolerated by BSF larvae without affecting proximate composition.

37
2.3 Introduction

The growing population requires an increase of high-quality food production [1]. However, feed production
already imposes a great pressure on the environment [3]. Insects appear to be a promising feed ingredient since
they need less time, space, water than conventional ingredients, thus reducing their environmental impact [154].
Specifically, the black soldier fly (BSF), Hermetia illucens, is interesting considering its high bioconversion rate
and great feeding substrate diversity [155]. Besides being rich in essential amino acids and lipids, BSF larvae
can transform residual organic matter into insect biomass and soil amendments [156]. However, BSF larvae are
perishable considering their neutral pH, high moisture and protein content, and their high microbial load at
harvest [46]. Therefore, they must be processed accordingly to ensure microbial safety and preservation of the
product. Since insect production is a developing industry, processing methods have not yet been optimized to
secure economic sustainability.

To maximize product quality, processing methods must be adapted to every insect species. Although some
research has focused on production and processing aspects, limited work has investigated the effect of post-
harvest conditioning on BSF larvae. However, processing can greatly stress the larvae and thus affect their
quality. In industry, post-harvest insect conditioning often includes a feed withdrawal period (FWP) and a
washing step prior to killing [57]. The FWP is carried out by starving the larvae until the gastrointestinal tract is
completely emptied reducing the microbial load associated to its feeding substrate and frass [6]. Considering
that the BSF is consumed in its entirety, emptying its gastrointestinal tract prior killing could allow reducing its
microbial load at low cost [56]. Because the gastrointestinal evacuation time (GET) of most insect species are
unknown, they are generally starved for a few hours to a few days depending on the species before eating them
as recommended by the FAO [6]. Larvae of BSF effectively convert their diets into biomass but their
effectiveness depends on the feeding substrate, just as is the retention time in the digestive tract [15]. Indeed,
the more the feed is nutritious for the insect, the longer the gastrointestinal evacuation time is [157].

The nutritional quality of the larvae mainly refers to their lipid and protein composition [31], as well as fatty acid
and amino acid profiles. The larva is composed of up to 46% protein and 35% lipids on a dry basis, but this
varies according to the feed provided and larval stage [15, 24]. Therefore, the challenge during post-harvest
conditioning is to prevent lipid and protein metabolism by the insect. Furthermore, glycogen and triglycerides
stored in the fat body can be mobilized through starvation [117]. In response to starvation, glycogen is first
mobilized and can be followed by lipids and/or proteins [119]. Because strategies used to tolerate starvation
varies amongst species [119], this dynamic must be better understood prior applying FWP. Depending on the
FWP, this mechanism could significantly reduce the lipid and protein content of the product.

38
The longer the FWP, the greater the energetic metabolism can be, resulting in a decrease of lipids and proteins
over time. This project aims at optimizing BSF larvae FWP, which constitute a compromise between the microbial
load reduction and maintaining quality. Indeed, optimized FWP could greatly contribute in reducing the microbial
load of the larvae. Therefore, it was necessary to estimate the GET of the BSF larvae to determine the minimum
starving time required to evacuate residual frass. Subsequently, we assessed the effects of a prolonged FWP
over 96 h on the proximate composition and microbial load.

2.4 Materials and methods


2.4.1 Gastrointestinal evacuation time
The GET was determined by a method adapted from Day and Powning1949) in adult Blattella (Blattodea:
Blattellidae), by observing the progression of coloured Gainesville diet until frass egestion [53]. The BSF eggs
used in this experiment were provided by Imnature insects (Québec, Canada) and their hatching occurred in
climate-controlled incubators (Growth Cabinet, MLR-350, Sanyo, Osaka, Japan) in the dark at 27 °C with 80%
relative humidity. Five clutches of eggs were suspended above 50 g of Gainesville diet (50% bran wheat, 20%
corn, 30% alfalfa, 70% moisture [158], milled at 1 mm using a vertical cyclone mill (CT 193, Cyclotec, FOSS,
Nanterre) to uniformize particle size [159]), inside a Masson jar (Bernardin Ltd., Toronto, Canada) covered with
a doubled net (Agryl P-12, Agryl, Courbevoie, France). Every day, the eggs were transferred to another jar to
ensure that it contained only the larvae hatched within a 24-hour window. Gainesville (50 g) was provided daily
until day 4. Then, the 4-day-old larvae were mixed with dry Gainesville, sieved, counted manually, and split into
five-litre round containers covered with a net (600 larvae/container, 1.2 larvae/cm 2) [160]. From days 4 to 11,
the larvae were maintained at 27 ± 1 °C and 17 ± 2% humidity and fed daily with 175 g of Gainesville at 70%
humidity, corresponding to 163 mg/larva/day on a dry basis [160].

On the 12th day, larvae were sieved (3 mm), washed with distilled water and fed 200 g of coloured Gainesville
diet with carmine red at a concentration of 0.1% (m:m) for five hours in the dark. They were then harvested and
washed with distilled water to remove any coloured feed from their silk. To ensure that the larvae had consumed
the diet, only larvae which the coloured diet was clearly visible in their digestive tract, as shown in Figure 2.1,
were selected and 24 amongst them were randomly picked. They were then transferred individually into culture
wells (4 multi-well culture plates, n = 6) with a perforated lid containing white microfibre filters (Whatman
No. 1827 047) humidified with 250 µL of distilled water. Observation and replacement of the moist filters were
done every 12 h. Observation consisted of red frass presence/absence and melanization initiation. The
experiment ended when greenish frass was deposited on the filter or no new red frass has been observed for
more than 24 h. The GET corresponded to the time where no new red frass was deposited on the filter while the
pupariation time represented the melanization initiation time. The larval initial weight was 184 ± 11 mg and the

39
larvae were also weighted at the end of the experiment. The experiment was performed in triplicates (24
larvae/replicate).

Figure 2.1 Black soldier fly larvae excreting coloured Gainesville diet on a moist Whatman filter.

2.4.2 Impact of feed withdrawal period


To determine the impact of the FWP on the composition and microbial load of BSF larvae, starved and fed larvae
were compared over time. Commercially produced small larvae (8,000 larvae; 3–5mm) of unknown ages were
shipped to our laboratory. At arrival, the larvae were sieved, counted manually, and split inside round containers
of 5 L covered with a net (600 larvae/tray, 1.2 larvae/cm 2) [160] and maintained in the same rearing conditions
then the previous experiment. After 10 feeding days, the larvae (120 ± 1 mg) were sieved, washed with distilled
water, partially dried on absorbent papers and pooled. A 20 g sample was then collected for the initial
composition and microbial load analysis while the rest was divided at random inside two 5-L plastic containers
covered with a net (1 container/treatment; 600 larvae/treatment; n = 3) and then 175 g of Gainesville at 70%
humidity were added to the control treatment. To evaluate the effects of starvation as opposed to feeding on
BSF larvae, 140 larvae/treatment were randomly collected every 24 hours until 96 h, weighted and the prepupae
were counts. While 2 g of larvae were kept on ice for microbial analysis the rest was frozen at -20 °C, freeze-
dried (-40 °C/40 °C) and milled with a coffee grinder (Black & Decker, Baltimore, USA) before being stored at -
20 °C until proximate analysis.

2.4.3 Proximate composition and pH


The samples collected during the FWP experiment were used to perform proximate analysis in duplicate. Dry
matter (DM), ash, ether extract (EE), protein and carbohydrates were analyzed by standard methods
(AOAC 934.01, AOAC 942.05, AOCS AM 5-04, AOAC 955.04 and AOAC 986.25, respectively) [161-162]. The
DM (%) was determined by drying the samples in a vacuum oven at 98 °C until constant weight (Isotemp
Standard Lab Oven, Model 230F, Thermo Fisher Scientific, Waltham, USA). The ash content (%; dry basis) was
determined by incineration (Lindberg/Blue M LGO Furnace Box, Thermo Fisher Scientific, Waltham, USA) of the

40
dry matter at 600 °C for 2 h. Protein content was measured using the Kjeldahl method and calculated by
multiplying the nitrogen content by 4.76 [140]. The EE (%; dry basis) was determined by extraction with
petroleum ether for 120 minutes at 90 °C in TX4 filters (ANKOM XT15 Extractor, ANKOM Technology, New
York, USA). Total carbohydrates, which includes chitin, were calculated by difference of total solids minus
proteins, EE and ash. Results were also expressed as mg/larva. The pH was measured on thawed larvae
according to Larouche et al. 2019 [163].

2.4.4 Microbial analysis


Microbiological analysis was carried out within the next 12 h on 2.0 g of thawed larvae mixed in 198 mL of
peptone water (1% m:v; Difco™, Becton Dickinson [BD], Franklin Lakes, USA) using a stomacher at 230 rpm
for 3 min (Stomacher® 400C, Seward Laboratory Systems Inc., London, UK) [91]. The total viable aerobic counts
(TVC), presumptive lactic acid bacteria (LAB), presumptive Pseudomonas spp., Enterobacteriaceae and
presumptive Listeria spp. were determined according to Larouche et al. 2019 [163]. Counts were transformed in
logarithms in base 10 of colony forming units per gram on a dry basis (log CFU/g).

2.4.5 Statistical analysis


The experiments were carried out in a completely randomized design. The GET and the pupariation time
corresponded to the average of median times of each replicate. A Pearson correlation was used to determine
the presence of a correlation between GET and pupariation time. One-way ANOVA was used to evaluate the
impact of starvation and continuous feeding on every parameter. Tukey tests were then used to indicate
differences between treatments. A confidence interval of 95% (p < 0.05) was used to confirm a significant
difference between means. Dixon tests were applied to exclude any aberrant data. The Kruskal-Wallis test was
performed as a nonparametric test when required, followed by Nemenyi’s test as multiple comparisons test.

2.5 Results
2.5.1 Gastrointestinal evacuation time
The GET is important to consider when applying a feed withdrawal period in order to be able to empty the gut.
As shown in Figure 2.2, the median gastrointestinal evacuation time of 12-d-old larvae was of 72 h, but varied
from 48 h to 96 h, while the median pupariation time was of 84 h and varied from 60 h to 108 h. After starving
for 84 h, 99% of the larvae had an empty gut. Moreover, a strong correlation between the GET and the
pupariation time was observed (r = 0.90), which occurred 12 h later.

41
Figure 2.2 Gastrointestinal evacuation time and pupariation time of starving and isolated 12-d-old black
soldier fly larvae (n = 3; 24 larvae/replicate; r = 0.90).

2.5.2 Effects of feed withdrawal period on proximate composition


As shown in Table 2.1, a prolonged FWP had several effects on the proximate composition and on the individual
larval weight. However, it had no effect on the proportion of prepupae, the total moisture and lipid content. The
proportion of prepupae was similar for both treatments over time and reached 28% for starved larvae and 30%
for fed larvae after 96 h. The total moisture of starved and fed larvae decreased over time and was significantly
different of the initial one from 48 h and 72 h, respectively. At 96 h, total moisture had decreased by 4% for both
treatments. The ash content increased by 1% for starved larvae after 96 h while it decreased by 1% for the fed
ones. Feeding had little effect on the protein content while starvation significantly increased it by 2.7% after 72 h
and remained almost the same until 96 h (40.0% and 39.6%, respectively). Starvation had no effect on the lipid
content while feeding increased it by 4% until 72 h and 7% after 96 h. Starvation induced weight loss after 72 h
and 96 h, 18 mg and 30 mg respectively, compared to the initial weight (105 ± 1 mg), while fed larvae were
23 mg and 28 mg heavier. The pH ranged from 6.7 to 7.3 and was not affected by starvation (p = 0.532).

42
Table 2.1 Proximal composition of fed (control) and starved (starvation) black soldier fly larvae at 27 °C.

Parameters Treatment Initial 24 h 48 h 72 h 96 h

Prepupae Control 0.0 ± 0.1 a 3.1 ± 0.8 ab 9.2 ± 1.4 bc 17.7 ± 2.2 d 30.4 ± 3.1 f
(%) Starvation 2.9 ± 0.7 ab 10.5 ± 0.4 c 23.7± 3.0 de 27.5 ± 4.5 ef
Larval weight Control 105 ± 1 c 123 ± 5 d 126 ± 3 de 128 ± 3 de 133 ± 3 e
(mg) Starvation 104 ± 3 c 98 ± 4 c 87 ± 2 b 75 ± 1 a
Total moisture Control 76.3 ± 0.1 cd 76.4 ± 0.3 cd 75.5 ± 0.2 bc 74.5 ± 0.4 b 72.4 ± 0.8 a
(%) Starvation 76.8 ± 0.2 d 74.6 ± 0.4 b 74.4 ± 0.5 b 72.3 ± 0.5 a
Ash Control 10.5 ± 0.2 bcd 10.1 ± 0.2 ab 10.2 ± 0.1 abc 9.8 ± 0.4 ab 9.4 ± 0.1 a
(%; dry basis) Starvation 10.8 ± 0.3 cde 11.0 ± 0.3 de 11.2 ± 0.3 de 11.5 ± 0.4 e
Ether extract Control 20.3 ± 1.3 ab 19.9 ± 1.1 a 22.8 ± 0.3 bc 24.6 ± 0.4 c 27.6 ± 1.4 d
(%; dry basis) Starvation 19.5 ± 1.0 a 19.2 ± 0.4 a 20.6 ± 0.7 ab 18.5 ± 0.7 a
Crude protein Control 37.3 ± 0.2 bc 35.0 ± 0.2 a 36.6 ± 0.1 bc 36.7 ± 0.2 bc 35.9 ± 0.3 ab
(%; dry basis) Starvation 38.0 ± 0.5 c 37.3 ± 0.4 bc 40.0 ± 0.9 d 39.6 ± 0.9 d
Different letter for the same parameters indicates a significant difference (p < 0.05); mean ± standard deviation; n = 3;
ANOVAs p < 0.001 for all parameters.

To better understand the impact of starvation on the larvae, the proximate composition was also expressed as
mg/larva. As shown in Figure 2.3, important changes occurred for both treatments. The proportional decreased
of the moisture and dry matter content with starved larvae was not perceptible when compared on a dry basis
because they followed the same proportion then the fed ones. However, when starved for 72 h, the larvae lost
15.9 mg of water and 2.8 mg of dry matter (p < 0.001); the differences are more important when starved for 96 h
(25.8 and 4.0 mg, respectively), while the ash content was not affected. Starvation did impact the composition
of the larvae for which protein and lipid decreased over time, but was only significant from 96 h with a loss of
1.0 mg and 1.2 mg per larvae, respectively. On the other hand, feeding the larvae over 96 h did increase the
quantity of protein, lipid and ash of the larvae of 3.9 mg, 5.0 mg and 0.9 mg, respectively (p < 0.001). Moreover,
the moisture increased by 14.0 mg in the first 24 h of feeding, but remained the same afterward.

43
Figure 2.3 Effects of feeding (control) and starving (starvation) on individual black soldier fly larvae
composition (mg/larva): (a) moisture; (b) lipids; (c) proteins; (d) carbohydrates (p < 0.001 for all
parameters). Different letter for the same parameters indicates a significant difference (p < 0.05).

2.5.3 Effects of feed withdrawal period on the microbial load


As shown in Table 2.2, starvation had no effect on TVC, presumptive LAB, presumptive Pseudomonas spp. and
Enterobacteriaceae since counts were no different on a dry basis than the initial ones and the fed ones of the
same day. Listeria spp. is the only microorganism that was affected over time. Since there is no significance
difference between controls and starved larvae, the higher Listeria counts observed from 24 h are more likely to
be associated with the larval development.

Table 2.2 Microbial load of fed (control) and starved (starvation) 10 d-old black soldier fly larvae at 27 °C.

44
Microbial counts
Initial 1d 2d 3d 4d
(log CFU/g; dry basis)
Total viable counts Control 8.4 ± 0.3 abc 8.6 ± 0.1 abc 8.6 ± 0.1 c 8.6 ± 0.1 bc 8.5 ± 0.1 abc
p = 0.003 Starvation 8.2 ± 0.1 a 8.6 ± 0.1 abc 8.3 ± 0.1 ab 8.3 ± 0.1 ab
Lactic acid bacteria Control 7.8 ± 0.1 ab 7.8 ± 0.1 ab 7.6 ± 0.1 ab 8.4 ± 0.2 b 8.1 ± 0.1 ab
p = 0.002 1 Starvation 7.1 ± 0.1 ab 7.6 ± 0.1 a 8.3 ± 0.1 b 8.0 ± 0.2 ab
Pseudomonas spp. Control 8.1 ± 0.1 bcd 8.2 ± 0.1 bcd 8.0 ± 0.1 ab 8.3 ± 0.1 d 8.2 ± 0.1 cd
p < 0.001 Starvation 8.2 ± 0.1 bcd 7.9 ± 0.1 a 8.0 ± 0.2 abc 8.1 ± 0.1 bcd
Enterobacteriaceae Control 6.5 ± 0.1 bcd 6.4 ± 0.1 ab 6.1 ± 0.1 ab 6.5 ± 0.1 bc 6.6 ± 0.1 d
p < 0.001 Starvation 6.5 ± 0.1 bc 6.3 ± 0.1 a 6.4 ± 0.1 ab 6.5 ± 0.1 cd
Listeria spp. Control 6.0 ± 0.3 a 7.0 ± 0.1 cd 7.5 ± 0.1 e 6.9 ± 0.1 bc 7.0 ± 0.1 cd
p < 0.001 Starvation 6.9 ± 0.1 bc 7.3 ± 0.1 de 6.7 ± 0.1 b 6.8 ± 0.1 bc
Different letter for the same microorganism indicates a significant difference (p < 0.05); mean ± standard deviation; n = 3.
1Kruskal-Wallis test, followed by Nemenyi’s test of multiple comparisons.

2.6 Discussion
Insect post-harvest conditioning, which includes the processing steps until killing, is still poorly documented, but
can greatly affect the quality of the larvae and should be optimized in order to maximize product quality and
reduce processing costs. Among those steps, a post-harvest feed withdrawal period (FWP) from 12 h to 48 h
has been recommended and applied in industry for many insect species [54-56, 60]. However, its effectiveness
in reducing the microbial load has not been investigated for BSF larvae. Moreover, without knowing its
gastrointestinal evacuation time, it is not possible to ensure that 48 h is long enough to empty BSF larvae gut.
This study focused on estimating the gastrointestinal evacuation time of BSF larvae and evaluating its impact
on the quality of the resulting product.

2.6.1 Gastrointestinal evacuation time


It is important to evaluate the gastrointestinal evacuation time of an insect prior applying a FWP since remaining
fecal content could decrease the potential microbial load reduction. The GET corresponded to the starving time
when no frass was deposited on the filter for 24 h; when the excretion was greenish instead of red; which meant
that all food was first excreted, or when pupariation began. In this study, the median GET of individualized 12-
d-old BSF larvae (184 ± 11 mg) was of 72 h which corresponded to the time requires to empty the gut of adult
Blatella [53]. In scientific studies, FWP usually applied on BSF larvae are between 12 and 24 hours [54-56].
However, according to our study, no larvae had emptied their gut in this length of time. In fact, after 72 h, the gut
of 78% of the larvae was empty while 99% was empty after starving for 84 h. Since the egestion time varies
depending on the feed and the temperature, the GET should be established under the conditions applied [164].
In addition, according to the surface law, the basal metabolism of animals is proportional to the surface of the
body [165]. Therefore, in this study, the estimated GET corresponds to larvae whose initial weight was 184 mg
which may vary depending on the larval weight at harvest. In addition, since larvae are usually produced and
processed as a pool, their isolation could have an impact on the GET and the contamination, and should be
investigated [166].

45
2.6.2 Effect of starvation on the initiation of pupariation.
It was also suggested that the FWP should not exceed 48 h since it could induce pupariation, metamorphosis
into prepupae [57]. Therefore, we also observed the time at which pupariation began in those conditions and
found that the median pupariation time when larvae were starved was of 84 h, 12 h after the GET. A strong
correlation was detected between the GET and the pupariation time suggesting that the emptiness of the gut
could trigger pupariation (r = 0.90). To further investigated this, the proportion of prepupae when starved and
fed was also followed over time (initial weight = 105 mg). Pupariation started after only 24 h for both treatment
and the proportion of prepupae increased over time. Since there was no difference between the percentage of
prepupae between fed and starved larvae, we cannot conclude to an effect between the emptiness of the gut
and the pupariation initiation, but this should be further investigated further at the molecular level.

2.6.3 Effects of a prolonged feeding period on the larvae


The weight of the larvae during the experiment was higher to that of prepupae fed Gainesville diet [19] but lower
than those fed chicken feed [17], both consider as equivalent diets. The initial proximate composition of the BSF
larvae corresponded to that of larvae between 9 and 12 days fed chicken feed [17]. Moreover, the lipids/protein
ratio (w/w) were similar to those of 9 d-old larvae (0.51) and increased in four days to 0.71 which correspond to
the one of 14-d-old larvae [17]. In this study, the quantity of proteins and lipids showed a smaller increase than
other studies when fed during 96 h [17] which could be related to the shipping stress imposed on the larvae prior
to reach the rearing facility. The initial microbial load of harvested and rinsed BSF were elevated (8.4 log UFC/g
for TVC) but within the range of unprocessed BSF [46]. Listeria spp. were present in the larvae as for a previous
study resulting from the initial contamination of the Gainesville diet used as feed in the experiment [163, 167].

2.6.4 Effects of a feed withdrawal period on the larvae


In this experiment, the impact of a prolonged FWP on BSF larvae proximate composition and microbial quality
was investigated. In general, a FWP < 96 h did not impact the microbial load, but affected the proximate
composition of the product. Indeed, as for a FWP of 48 h on mealworm larvae, microbial counts observed were
similar for the fed and starved larvae suggesting that starvation do not reduce the microbial load [57]. Moreover,
the proximate composition of larvae subjected to a FWP was affected by the treatment from 72 h until the end
of the experiment. Indeed, the protein content increased by 2.7% on a dry basis from 72 h, while the ash content
increased by 1.0% from 96 h suggesting that starvation did induce energetic metabolism by reducing lipids and
carbohydrates content [58].

During starvation, many insects mobilise their glycogen and triglyceride reserves to support their metabolic
needs in order to spare protein [119]. However, the metabolism of insects under such condition is highly variable
and depends mostly on the specie and the development stage [119]. Tenebrio molitor (Coleoptera) larvae have
been reported to initiate lipolysis after two days of feed withdrawal and not to rely on their proteins while Manduca

46
sexta (Lepidoptera) larvae do not rely on their lipids nor proteins, but both are poorly resistant to starvation and
starts dying in 3 to 6 days [119]. In addition, metabolism under starvation of adult Drosophila melanogaster
(Diptera) was also investigated showing a glycogen depletion in 30 h corresponding to a peak of carbohydrates,
a continuous mobilization of lipids and, in smaller amount, of soluble proteins [168]. In this study, BSF larvae
under starvation seemed to rely on carbohydrates at first, reached a peak of carbohydrates at 48 h, and later
start metabolizing their lipids and proteins which were significantly lower per larva than the initial content after
96 h of starvation. In M. sexta, the trehalose content in the haemolymph has been shown to increase during the
first 24 hours of starvation and then return to the normal level after 48 hours [169] which could explain the
carbohydrate peak observed at the same time. In addition, a high concentration of trehalose in insect
haemolymph is known to inhibit lipolytic activity which could explain why lipids were not affected until 72 h [170].
To confirm this, the trehalose content in the haemolymph should be measured during starvation as well as the
lipid content. Moreover, in our conditions, the moisture content of BSF larvae under food and water restriction
quickly started to decrease from 24 h reaching its two thirds (54 mg) after 96 h. Schimpf and coll. (2011), which
investigated the impact of starvation on metabolism and water loss on cockroach, found that during starvation,
water deprivation reduced their survival time and suggested that those insects may have died from dehydration
rather than starvation [171]. Therefore, considering the high water loss from larvae in this study, it is possible
that the impact of dehydration represented a greater risk to their survival than starvation hence impacting on
their metabolism. Future research should focus on the impact of a prolonged feed withdrawal period on the black
soldier fly larvae without water restriction.

2.7 Conclusion
The estimation of the effectiveness of a feed withdrawal period in reducing the microbial load is the first step in
a long process in optimizing the post-harvest processing steps of BSF larvae. This study demonstrated that a
feed withdrawal period long enough to empty the digestive tract of the larvae, corresponding to the
gastrointestinal evacuation time of 72 h and beyond, was unable to reduce its microbial load, but affected its
proximate composition. In addition, since a FWP of 48 h did not impact the proximate composition, the industry
could have a delay of 48 h between the moment of harvesting and killing. However, this could impact on the
microbial load, notably with Listeria spp. which increased during this study, and should be further investigated.
Finally, since an empty gut does not provide any benefits in terms of safety, gut loading should be considered
as a quality-enhancing step since it could enhance the flavour and taste of the larvae while increasing its
nutritional quality.

Author Contributions: conceptualization, J.L., M.-H.D., L.S., and G.W.V.; methodology, J.L. and M.C.;
software, J.L.; validation, J.L., M.-H.D and G.W.V.; formal analysis, J.L.; investigation, J.L.; resources, J.L. and

47
M.-H.D.; data curation, J.L.; writing—original draft preparation, J.L.; writing—review and editing, M.-H.D., G.W.V.
and L.S.; visualization, J.L.; supervision, G.W.V.; funding acquisition, G.W.V.

Funding: This work was supported by funding from the Programme Innov’Action Agroalimentaire, a program
under Growing Forward 2 between the Ministère de l’agriculture, des pêcheries et de l’alimentation du Québec
and Agriculture and Agri-Food Canada.

Acknowledgments: We thank the Vandenberg laboratory team for their assistance in counting the larvae and
the other members of the research project, Alain Doyen, Cristina Ratti, Lucie Beaulieu from Université Laval,
Charles Lavigne, Celine Georlette from the CDBQ and the technical professionals of the LARSA.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

48
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163. Larouche, J.; Deschamps, M.-H.; Saucier, L.; Lebeuf, Y.; Doyen, A.; Vandenberg, G.W. Effects of killing methods on
lipid oxidation, colour and microbial load of black soldier fly (Hermetia illucens) larvae. Animals 2019, 9 (4), 182;
https://doi.org/10.3390/ani9040182.
164. Carvalho, D.A.; Collins, P.A.; De Bonis, C.J. Gut evacuation time of Macrobrachium borellii (Caridea: Palaemonidae)
feeding on three types of prey from the littoral-benthic community. J Crustac Biol 2011, 31 (4), 630-634.
165. Kleiber, M. Body size and metabolism. J Agr Sci 1932, 6 (11), 315-353.
166. Connat, J.L.; Delbecque, J.P.; Glitho, I.; Delachambre, J. The onset of metamorphosis in Tenebrio molitor larvae
(Insecta, Coleoptera) under grouped, isolated and starved conditions. J Insect Physiol 1991, 37 (9), 653-662.
167. Larouche, J.; Vandenberg, G.W. (Département des sciences animales, Université Laval, Québec, CAN), Microbial
load analysis of the Gainesville diet use as feed for black soldier fly (Hermetia illucens) larvae in the Laboratoire des
sciences aquatiques of Université Laval, 2019.
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Comp Biochem Physiol B Biochem Mol Biol 2018, 226, 26-35.
169. Thompson, S.N. Trehalose – The insect ‘blood’ sugar. Adv In Insect Phys 2003, 31, 205-285.
170. Arrese, E.L.; Rojas-Rivas, B.I.; Wells, M.A. The use of decapitated insects to study lipid mobilization in adult Manduca
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171. Schimpf, N.G.; Matthews, P.G.; White, C.R. Cockroaches that exchange respiratory gases discontinuously survive
food and water restriction. Evolution 2012, 66 (2), 597-604.

50
Chapter 3. Effects of killing methods on lipid
oxidation, colour and microbial load of black
soldier fly (Hermetia illucens) larvae

Jennifer Larouche, Marie-Hélène Deschamps, Linda Saucier, Yolaine Lebeuf,


Alain Doyen and Grant W. Vandenberg

Larouche, J.; Deschamps, M.-H.; Saucier, L.; Lebeuf, Y.; Doyen, A.; Vandenberg, G. W.
Effects of killing methods on lipid oxidation, colour and microbial load of black soldier fly
(Hermetia illucens) larvae. Animals 2019, 9 (4) 182; https://doi.org/10.3390/ani9040182

51
3.1 Résumé
Les larves de mouches soldats noires représentent un ingrédient alternatif prometteur pour l’alimentation
animale, mais la préservation de leur qualité par une transformation appropriée est primordiale. Un abattage
adapté pourrait contribuer à maintenir leur qualité nutritionnelle et microbiologique. Ainsi, les effets de dix
méthodes d’abattage ont été comparés : ébouillantage (40 s), dessiccation (60 °C, 30 min), congélation (-20 °C
et -40 °C, 1 h; azote liquide, 40 s), hautes pressions hydrostatiques (3 min, 600 MPa), broyage (2 min) et
asphyxie (CO2 et ; conditionnement sous vide, 120 h; N2, 144 h). Ébouillantage, asphyxie et dessiccation ont
affecté le pH, la composition et l’oxydation des lipides. Certaines méthodes ont également affecté la stabilité de
la couleur et leur charge microbienne. L’ébouillantage s’est démarqué en minimisant l’oxydation lipidique et en
réduisant la charge microbienne. Nous proposons un protocole d’abattage des larves répondant aux exigences
canadiennes en matière de transformation des insectes.

3.2 Abstract
Black soldier fly (BSF) larvae represent a promising alternative ingredient for animal feed. Post-production
processing can, however, affect their quality. This project aimed to optimize larval killing by comparing the effects
on the nutritional and microbiological quality of 10 methods, i.e., blanching (B = 40 s), desiccation (D = 60 °C,
30 min), freezing (F20 = −20 °C, 1 h; F40 = −40 °C, 1 h; N = liquid nitrogen, 40 s), high hydrostatic pressure
(HHP = 3 min, 600 MPa), grinding (G = 2 min) and asphyxiation (CO2 = 120 h; N2 = 144 h; vacuum conditioning,
V = 120 h). Some methods affected the pH (B, asphyxiation), total moisture (B, asphyxiation and D) and ash
contents (B, p < 0.001). The lipid content (asphyxiation) and their oxidation levels (B, asphyxiation and D) were
also affected (p < 0.001). Killing methods altered the larvae colour during freeze-drying and in the final product.
Blanching appears to be the most appropriate strategy since it minimizes lipid oxidation (primary = 4.6 ± 0.7 mg
cumen hydroperoxide (CHP) equivalents/kg; secondary = 1.0 ± 0.1 mg malondialdehyde/kg), reduces microbial
contamination and initiates dehydration (water content = 78.1 ± 1.0%). We propose herein, an optimized
protocol to kill BSF that meet the Canadian regulatory requirements of the insect production and processing
industry.

52
3.3 Introduction
The larval stage of the black soldier fly (BSF) represents a promising animal feed ingredient considering its high
protein and lipid content (46% and 35% on a dry basis, respectively) [15], and has been suggested as a
sustainable ingredient for animal feed, especially for fish, poultry and swine [172]. Furthermore, BSF larvae can
accumulate up to 38% unsaturated fatty acids depending on the larval stage and the feed offered [17, 24].
However, BSF larvae are highly perishable considering their neutral pH and their high water and protein content
[46]. The microbial load associated with the larvae is also variable considering the great diversity of the feeding
substrates (colony-forming unit = CFU; total viable aerobic counts = 7.1 to 9.8 log CFU/g; presumptive lactic
acid bacteria = 4.1 to 8.5 log CFU/g; enterobacteria = 7.3 to 9.7 log CFU/g; endospores = 3.7 to 7.5 log CFU/g;
yeast and moulds = 3.1 to 5.8 log CFU/g) [7, 45-46]. Some studies have reported the presence of pathogenic
bacteria such as Salmonella, Escherichia coli and Bacillus cereus [44-46]. Compared to other insect species,
the BSF larvae develop a particularly dark colouration during processing [138]. In short, BSF larvae represent a
promising ingredient but it is required to optimize processing techniques to maintain the nutritional quality and
colour while ensuring feed safety.

Insect processing steps usually applied in industry include killing, microbial decontamination by heat,
dehydration and grinding [8]. Invertebrates are killed by several methods, but freezing and blanching appear as
the most frequent methods employed [5, 8]. Other methods have proved effective such as carbon dioxide
asphyxiation for crickets [74], electrocution for lobsters [86], desiccation for maggots and cockroaches [60] and
immersion in tepid water for maggots [60]. Several processes have been investigated to reduce the microbial
load of insects such as high hydrostatic pressures, blanching, desiccation, direct and indirect cold plasma and
microwaves [44-45, 89-90]. However, those treatments are mostly effective in vegetative cells since some
bacteria can sporulate into highly resistant endospore [44-45]. Finally, the product is dehydrated using
convective, contact or radiation drying methods and ground [8]. Several studies reported a colour change of the
insect during processing [13, 65] which may potentially reduce the market value of the product. Processing the
BSF remains a challenge because of the presence of highly oxidable unsaturated fatty acid, the high initial
microbial load and the colour alteration during processing.

Processing methods can significantly influence lipid oxidation, colouration and microbial load of the final product.
Indeed, various reactions can be responsible for the colour change in insects such as enzymatic polyphenol
oxidation [138] and complex formation between iron and polyphenols [13], as well as Maillard reaction products
[148]. Reactions involving polyphenol oxidation can be temporarily reduced (low temperature and water content,
and anaerobic condition) [136] or inhibited permanently via enzyme denaturation (blanching and sulfites) [138].
Methods such as high hydrostatic pressure, heat treatment, sulfite addition and acidification have reported being
effective in reducing enzymatic browning in crustaceans [137-138, 151]. In addition, because the BSF larvae

53
might contain valuable unsaturated fatty acids, oxidation prevention is important. Initiation of lipid peroxidation
is influenced by many factors such as exposure to light, heat and oxygen [120]. The peroxidation is carried out
in three stages, initiation, propagation and termination, generating by-products such as hydroperoxides (primary
oxidation) and malondialdehyde (secondary oxidation) [120]. The analysis of the primary and secondary
oxidation level thus makes it possible to obtain an accurate determination of lipid stability [109]. Methods such
as freeze-drying and high hydrostatic pressures (HHP) may increase lipid oxidation, by denaturation of
antioxidants, while microwave processing reduces it, resulting from the antioxidant activity of Maillard reaction
products [99, 109]. Although larvae can inactivate several pathogens present in their feeding substrate [173],
their microbial load still remains high at harvest and requires a decontamination step to ensure the safety of the
product. For instance, the Canadian Food Inspection Agency recommends including thermal treatment to reduce
microbial counts to an acceptable level [134]. It is therefore important to optimize processing methods to the
specific challenges of the BSF larvae product.

Killing is a key stage in insect processing since it can affect proximate composition [64-65, 174], colour [65],
microbial load [45] and taste of the final product [62]. Indeed, a longer killing time is related to increased
metabolism of energy reserves (metabolism of triglycerides into acylglycerol and free fatty acids) and promotes
their oxidation [64]. Furthermore, other killing methods should be investigated such as grinding, microwave
cooking [109] and HHP [45]. Considering the great diversity of insects, killing methods used in the industry
should be adapted for each species. In addition to being able to maximize the nutritional and microbiological
quality of the final product, insect killing methods should apply the precautionary principle to minimize the
discomfort or pain as well as being fast [8, 89].

A number of studies have demonstrated the degree by which killing BSF larvae by freezing and blanching
impacts protein and lipid quality and colour stability [64-65]; however, the effect of other killing methods on insect
product quality has not been investigated. Hence, the objective of this study was to compare the impact of 10
killing methods on the chemical composition (water content, ash, ether extract and lipid oxidation), microbial
load, pH and colour of the resulting BSF larvae meal in order to propose a killing method adapted to BSF larvae
while being able to fulfill quality criteria for the industry.

3.4 Materials and Methods


3.4.1 Biological material
The BSF eggs used in this study were provided by three facilities in order to support the required biomass for
the experiments. The Centre de développement bioalimentaire du Québec (CDBQ), the hatchery of the
Laboratoire des sciences aquatiques (LARSA) in Université Laval and the company Imnature Insects (Québec,
QC, Canada) provided the BSF eggs used in this experiment. Egg hatching occurred in climate-controlled

54
incubators (Growth Cabinet, MLR-350, Sanyo, Osaka, Japan) in the dark at 27 °C with 80% relative humidity.
10 clutches of eggs were suspended above 50 g of Gainesville diet (50% bran wheat, 20% corn, 30% alfalfa,
70% moisture) [158] inside a Masson jar (Bernardin Ltd. Brampton, Canada), covered with a doubled net
(Agryl P-12, Agryl, Courbevoie, France). Egg clutches were transferred to new jar daily to ensure that jars
contained larvae hatched within a 24-hour window. Gainesville diet (50 g) was provided every day until day 4.
Four day-old larvae batches were pooled, mixed with the dry diet, sieved, counted manually and split into 18 five
liters containers covered with a net (600 larvae/tray, 1.2 larvae/cm 2) [160]. From days 4 to 9, the larvae were
fed daily with 175 g of Gainesville diet at 70% humidity, corresponding to 163 mg/larvae/day on a dry basis [160].
On day 10, larvae were sieved (3 mm mesh), washed with distilled water, partially dried on absorbent papers,
combined, and divided at random into 10 containers (one container per killing method). Initial larvae weight was
measured prior to killing of three samples of 50 larvae picked randomly (0.07 ± 0.02 g). The 10 killing methods
were conducted on 50 g of larvae (~ 750 larvae) and the experiment was repeated four times (n = 4).

3.4.2 Killing methods


Killing methods were carried out at the Groupe de Recherche Intégré en Physiologie et Sciences Animales
(GRIPHA) and at the Laboratoire de Transformation des Aliments (LTA) at Université Laval depending on the
killing method. 10 larvae/treatment were kept under observation for 24 hours at 21 ± 1 °C in an aerated Petri
dish after each killing experiment to validate the capacity of the method to kill the larvae. After the application of
every killing method, larvae were brought back to room temperature and then 25 g of larvae/treatment were
vacuum packaged and frozen at −20 °C to perform microbial, lipid oxidation and pH analysis on thawed larvae;
the remaining larvae were dried. The remaining sample was frozen at −20 °C, freeze-dried (−40 °C/40 °C) until
constant weight and granulated with a coffee grinder (Black and Decker, Baltimore, MD, USA) before being
stored at –20 °C in the dark until chemical analysis. Colour analyses were performed prior freeze-drying
(thawed) and after, on the granulated product (freeze-dried and granulated).

3.4.2.1 Mechanical disruption


Grinding (G) consisted of homogenizing 50 g of larvae for 2 min at 15,000 rpm (Ultra Turrax T18 digital, IKA,
Wilmington, MD, USA). The high hydrostatic pressure (HHP) method was carried out on 50 g of larvae packed
under a 95% vacuum followed by a pressure treatment at 600 MPa during 3 min at room temperature (21 °C) in
a laboratory-scale system (ISO-LAB, model S-IL-085-09-AO, Stansted Fluid Power, Harlow, United Kingdom).

3.4.2.2 Heating
Blanching (B) was carried out by immersing 50 g of larvae in a sterile Stomacher filter into boiling water for 40 s
[16]. Desiccation (D) consisted of putting 50 g of larvae on a metal screen into an air oven set at 60 °C for 30 min
(Shel Lab SMO28-2 Laboratory Forced Air Oven, 27.5 CU FT, 230 V, Stellar Scientific, Baltimore, MD, USA)
[68].

55
3.4.2.3 Freezing
Freezing was performed by placing 50 g of larvae on a metal screen at −20 °C (F20) or at −40 °C (F40) for 1 h.
The liquid nitrogen method (N) was carried out by immersing 50 g of larvae vacuum packaged in liquid nitrogen
for 40 s.

3.4.2.4 Asphyxiation
Vacuum packaging (V) consisted of bagging 50 g of larvae under vacuum and keeping them in the dark at the
rearing temperature (27 °C) for 120 h. Asphyxiation methods consisted of vacuum packaging the larvae with
introduction of a modified atmosphere, either 100% CO2 or N2 using a syringe through a silicone stopper; bags
were stored at 27 °C in the dark for 120 h and 144 h, respectively [80].

3.4.3 Analysis
3.4.3.1 Chemical composition
The dry matter (DM), ash and ether extract (EE) content of the larvae were evaluated in triplicate on the freeze-
dried larvae by standard methods (AOAC 934.01, AOAC 942.05 and AOCS AM 5-04, respectively) on freeze-
dried and granulated larvae [161-162]. Dry matter (DM) content was determined by drying the samples into a
vacuum oven at 98 °C (Isotemp Standard Lab Oven, Model 230F, Thermo Fisher Scientific, Waltham, UK) until
constant. Ash content was determined by incineration (Lindberg/Blue M LGO Furnace Box, Thermo Fisher
Scientific, Waltham, UK) of the dry matter at 600 °C for 2 h. The EE was determined on hydrolyzed samples
(4 N hydrochloric acid, 60 min, 90 °C) using extraction with petroleum ether for 120 min at 90 °C in TX4 filters
(XT15 Extractor, ANKOM Technology, New York, NY, USA). Thawed larvae were analyzed in duplicate for pH
measurement after homogenizing 1 g of larvae (VDI 25, VWR, Radnor, PA, USA) for 30 s in 9 mL of distilled
water [175]. The pH of the homogenate was then measured using a digital pH meter (AB15 pH meter, BASIC
accumulator, Thermo Fisher Scientific, Waltham, MA, USA).

3.4.3.2 Primary lipid oxidation


Primary lipid oxidation, which refers to lipid hydroperoxides, was measured using a spectrophotometric method,
ferrous oxidation-xylenol orange (FOX) assay, adapted from Hermes-Lima et al. [176] and Grau et al. [177]. The
cumene hydroperoxide (CHP) equivalent concentration for FOX analysis was determined on the supernatant
after homogenizing (VDI 25, VWR, Radnor, PA,USA) 3.5 g of thawed larvae in 100% HPLC grade cold methanol
(1:5, m:v in dry basis) and centrifuging at 3000 g for 10 min at 4 °C. In a new tube, the following solutions were
added in this order and mixed: 250 μL of 1 mM aqueous solution of (NH4)2 Fe (SO4)2, 100 μL of a methanolic
solution of 25 mM H2SO4, 100 μL of a methanolic solution of 0.1 mM xylenol orange, 450 μL MeOH and 100 µL
of the supernatant. The standards consisted of 100 µL of a solution of known concentration of CHP. The
hydroperoxide level (FOX; mg of CHP equivalents/kg; wet basis) corresponded to the absorbance at 580 nm
(Varioskan, Thermo Fisher Scientific, Waltham, MA, USA) after 60 min of incubation in the dark.

56
3.4.3.3 Secondary lipid oxidation
A spectrophotometric method adapted from Uchiyama and Mihara [178] and Joanisse and Storey [122] was
used to determine the concentration of malondialdehyde (MDA) for the analysis of thiobarbituric acid reactive
substances (TBARS). Thawed larvae (3.5 g) homogenized in cold 1.15% phosphoric acid (1:10, m:v on a dry
basis) was centrifuged at 3000 g for 10 min at 4 °C. The supernatant (400 µL) was mixed with 400 μL of 1%
thiobarbituric acid (TBA) solution and 0.1 mM of butylated hydroxytoluene in 0.05 M NaOH while only 400 μL of
3 mM HCl was used for the blank. Standards solution corresponded to 400 μL of known concentration of MDA
with 400 μL of TBA solution. All tubes were then treated with 200 μL of 7% phosphoric acid and incubated for
15 min in a water bath at 100 °C. After cooling to room temperature in the dark during 10 min, every tube
received 1.5 mL of n-butanol and was centrifuged at 2000 g for 5 min at 4 °C. The MDA concentration (mg
MDA/kg; wet basis) represented the difference of the absorbance reading between 532 nm and 600 nm.

3.4.3.5 Larval colouration and colour change occurring while drying


Colour measurement was performed on killed larvae prior freeze-drying (thawed) and after, on the dry product,
(freeze-dried and granulated). Larvae colour was defined by the CIE L*a*b* colour space [179] and measured
in triplicate using a chromameter (chromameter CR400/410, Konica Minolta, Tokyo, Japan). Parameters used
to compare larvae colours were the lightness (L*), the colour intensity (chroma, C*) and the hue angle (h). The
larvae colour change (∆E) while drying was also calculated using equation (1) [106]. The colour change was
determined using thawed and ground larvae colour from the same killing method. Equation (2) represents the
chroma, while equation (3) represents the hue angle [180]:

∆E = √(∆L∗ )2 + (∆a∗ )2 + (∆b ∗ )2 (1)

C* = √(a∗ 2 + b ∗ 2 ) (2)

h = Tan−1 (b ⁄a∗ ) (3)

3.4.3.6 Microbiological analysis


Microbiological analyses were carried out on 2.0 g of thawed larvae homogenized (VDI 12, VWR) in 18 mL of
peptone water (1% m:v; Difco™, Becton Dickinson [BD], Franklin Lakes, NJ, USA) for 20 s [91]. The
homogenizing tip was disinfected under laminar flow hood prior to every use by operating it in a hydrogen
peroxide solution (2.5% v:v; Oxivir Plus, Diversey, Charlotte, NC, USA). It was then rinsed three times in sterile
water to remove the residual hydrogen peroxide.

Total viable aerobic counts (TVC) were enumerated on Plate Count Agar (PCA; Difco™, BD) incubated for 48 h
at 35 °C (MFHPB-18) [181]. Analyses of presumptive lactic acid bacteria (LAB) were performed on deMan,
Rogosa and Sharp agar (MRS; Difco™, BD) incubated anaerobically for 24 h at 35 °C using anaerobic jars with

57
CO2 generators (BBL™, BD) [181]. Presumptive Pseudomonas spp. were enumerated on Pseudomonas Agar
Base (Oxoid™, Thermo Scientific, Nepean, Canada) supplemented with a vial of cetrimide, fucidin and
cephalosporin supplement (C-F-C supplements; Oxoid™, Thermo Scientific) rehydrated in 2 mL of 1:1
ethanol: sterile distilled water and incubated for 48 h at 25 °C [182]. Yeast and mould counts were performed
using Rose Bengal Agar Base (Difco™, BD) supplemented with 0.05 g of chloramphenicol rehydrated in 2 mL
of ethanol 95% and incubated for 72 h at 25 °C (MFHPB-22) [181]. Enterobacteriaceae were enumerated on
Violet Red Bile Glucose Agar (VRBG; Difco™, BD) incubated for 48 h at 35 °C (MFLP-43) [181]. Coliform counts
were determined on Violet Red Bile Agar (VRBA Difco™, BD) and incubated for 24 h at 35 °C (MFHPB-34)
[181]. Presumptive Listeria spp. counts were determined using PALCAM Listeria Selective Agar (Milipore Sigma,
St. Louis, MO, USA) incubated for 48 h at 35 °C (MFHPB-07) [181]. Clostridia and other anaerobes were
enumerated on Reinforced Clostridial Agar (HiMedia Laboratories, Mumbai, India) and incubated anaerobically
for 48 h at 35 °C (MFHPB-01) [181]. Counts were expressed as logarithmic (base 10) of colony forming units
per gram on a dry basis (log CFU/g).

3.4.4 Statistical analysis


The experiment was carried out in a completely randomised block design, where each block represented the
analyses of a different day. Two-way ANOVA was used to compare means of chemical composition (DM, ash,
and EE), lipids oxidation (FOX and TBARS), colour change (ΔE), pH, and microbial load. Two-way ANOVA, for
paired data, was used to compare means of the colouration (L*, a*, b*, C* and h) where factors are the killing
methods and the larval conditions (thawed and freeze-dried and ground). Tukey tests were used to indicate
differences between methods. A confidence interval of 95% (p < 0.05) was used to confirm a significant
difference between means. Dixon tests were applied to exclude any aberrant data.

3.5 Results
3.5.1 Chemical composition and pH
The total moisture content of BSF was influenced by the killing methods (p < 0.001; Table 3.1). Those employing
heat resulted in the lowest moisture (78.4 ± 0.4%; p = 0.048) while asphyxiation obtained the highest
(83.4 ± 0.2%; p = 0.045) compared to other methods (81.0 ± 0.5%). The killing methods also had different
effects on the chemical composition of freeze-dried and granulated larvae. Asphyxiated larvae demonstrated
lower DM content (CO2, N2 and V = 75.8 ± 0.4%; p = 0.002) than desiccated and ground larvae (D and
G = 87.5 ± 1.5%), but were not significantly different than the frozen counterparts (F20, F40 and
N = 79.8 ± 1.3%; p = 0.944). Blanching induced the highest DM content (B = 96.0 ± 0.8; p = 0.042). Only the
ash content of the blanched larvae (B = 8.5 ± 0.3%; p = 0.005) was higher versus asphyxia, freezing and HHP-
treated (7.1 ± 0.1%). Finally, EE was significantly higher for asphyxia (CO2, N2 and V = 16.1 ± 0.4%; p = 0.004)
than for methods based on cooling, mechanical disruption and desiccation (12.5 ± 0.6%). Moreover, the killing

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methods had different effects on the pH of thawed larvae. Asphyxiated larvae showed a slightly acidic pH (CO2,
N2 and V = 6.2 ± 0.1; p < 0.001) compared to the neutral pH of most methods (D, F20, F40, N, G and
HHP = 7.5 ± 0.2) while blanching obtained a basic pH value (B = 8.7 ± 0.1; p < 0.001).

Table 3.1 Chemical composition, primary (ferrous oxidation-xylenol orange assay) and secondary
(thiobarbituric acid reactive substances) lipid oxidation levels, and pH of black soldier fly (BSF) larvae
killed by different methods.
FOX
Total Moisture Ash Ether Extract TBARS
Killing (mg CHP
Content (%; Dry (%; Dry (mg MDA/kg; pH
Methods equivalents/kg;
(%; Wet Basis) Basis) Basis) Wet Basis)
Wet Basis)
Heating
D 78.1 ± 1.0 a 7.9 ± 0.6 ab 13.4 ± 1.6 ab 5.8 ± 2.6 a 2.5 ± 0.2 c 7.8 ± 0.5 c
B 78.6 ± 0.7 a 8.5 ± 0.3 b 14.5 ± 0.5 bc 6.4 ± 2.3 a 1.0 ± 0.1 a 8.7 ± 0.1 d
Freezing
F20 81.6 ± 0.3 b 7.1 ± 0.4 a 12.8 ± 0.5 ab 7.2 ± 0.9 a 1.7 ± 0.2 b 7.4 ± 0.3 bc
F40 81.3 ± 0.4 b 7.0 ± 0.5 a 12.4 ± 0.7 a 7.4 ± 1.0 a 2.0 ± 0.3 bc 7.4 ± 0.3 bc
N 80.8 ± 1.5 b 7.1 ± 0.3 a 12.6 ± 1.2 a 7.9 ± 2.2 a 1.8 ± 0.3 b 7.3 ± 0.3 bc
Asphyxiation
CO2 83.5 ± 0.3 c 7.1 ± 0.4 a 15.9 ± 1.6 cd 19.4 ± 4.8 b 2.0 ± 0.2 b 6.1 ± 0.3 a
N2 83.2 ± 1.3 c 7.3 ± 0.7 a 16.6 ± 1.6 d 18.6 ± 7.1 b 1.6 ± 0.1 b 6.3 ± 0.1 a
V 83.6 ± 0.5 c 7.3 ± 0.4 a 15.9 ± 1.9 cd 18.7 ± 7.2 b 1.9 ± 0.3 b 6.2 ± 0.4 a
Mechanical disruption
G 80.3 ± 0.2 b 7.9 ± 0.3 ab 11.9 ± 0.3 a 6.7 ± 1.2 a 2.0 ± 0.3 b 7.5 ± 0.2 bc
HHP 80.8 ± 1.0 b 7.0 ± 0.6 a 12.0 ± 0.7 a 7.3 ± 3.4 a 1.6 ± 0.3 b 7.2 ± 0.1 b
Different letter in the same column indicates a significant difference (p < 0.05); mean ± standard deviation; D = Desiccation,
B = Blanching, F20 = Freezing at −20 °C, F40 = Freezing at −40 °C, N = Freezing in liquid nitrogen, CO2 = Asphyxia with
CO2, N2 = Asphyxia with N2, V = Asphyxia under vacuum, G = Grinding, HHP = High hydrostatic pressures.

3.5.2 Lipid oxidation


The primary lipid oxidation level was two times higher for asphyxiated larvae (FOX; CO2, N2 and
V = 14.5 ± 0.7 mg CHP equivalents/kg; p = 0.001) than for those killed by other methods (5.8 ± 1.5 mg CHP
equivalents/kg). The secondary oxidation level was lower for blanched larvae (TBARS, B = 1.0 ± 0.1 mg
MDA/kg; p = 0.005) compared to the others (F20, F40, N, CO2, N2, V, G and HHP = 1.8 ± 0.1 mg MDA/kg) while,
desiccation showed higher values (D = 2.8 ± 0.7 mg MDA/kg; p = 0.019).

3.5.3 Larval colour

3.5.3.1 Impact of Freeze-Drying on the Colour Change


Depending on the killing method applied, freeze-drying of larvae induced different patterns of colour changes
(Figure 3.1; Table A1). The drying process allows to increase the hue angle (p = 0.012) of larvae for all killing
methods, except for grinding. Compared to the colour of thawed larvae, freeze-drying of larvae killed by heating
decreased the lightness (p = 0.001) and by desiccation also increased the intensity of the colour (p = 0.026).
Freeze-dried larvae killed by grinding induced a specific colour change pattern resulting in decreased colour
lightness and intensity (p < 0.001). The magnitude of the colour change, represented by the ∆E, was variable

59
among the killing methods. Asphyxiation obtained a significantly lower ∆E (CO2, N2 and V = 2.2 ± 0.2; p = 0.025)
than methods using heat (D and B = 5.9 ± 0.4; p < 0.001) while grinding induced the most significant colour
change (G, ΔE = 17.5 ± 3.6; p < 0.001). Indeed, the colour change of ground larvae was three times higher than
for the other methods.

Figure 3.1 Colour analysis of thawed and freeze-dried and granulated BSF larvae after different killing
methods (D = Desiccation; B = Blanching; F20 = Freezing at –20 °C; F40 = Freezing at –40 °C;
N = Freezing in liquid nitrogen; CO2 = Asphyxia with CO2; N2 = Asphyxia with N2; V = Asphyxia under
vacuum; G = Grinding; HHP = High hydrostatic pressures): (a) lightness; (b) colour change between
thawed and freeze-dried and granulated larvae; (c) chroma; (d) hue angle.

3.5.3.2. Colour of the Freeze-Dried and Granulated Product


As shown in Figure 3.1, the killing methods had different effects on the colouration of the resulting freeze-dried
and granulated (FDG) BSF larvae, resulting in beige-coloured meals. Killing by asphyxiation (CO2, N2 and V;
L* = 44.8 ± 2.4; C* = 10.4 ± 0.1; h = 1.52 ± 0.01) and by cold (F20, F40 and N; L* = 42.2 ± 1.5, C* = 10.2 ± 0.5;

60
h = 1.46 ± 0.02) resulted in similar colours unlike mechanical disruption (B and H) and heating methods (D and
E) which demonstrated significantly different colours. Indeed, the colour intensity of FDG larvae killed by
desiccation (D = 11.4 ± 0.4) was significantly higher than that of blanched larvae whose colour matched those
of the frozen ones (B = 9.8 ± 0.1; p < 0.001). Additionally, HHP obtained a FDG product with a significantly
higher lightness and colour intensity (HHP; L* = 45.9 ± 2.0; C* = 10.7 ± 0.7) than with grinding (G;
L* = 37.2 ± 2.6; C* = 6.6 ± 0.8; p < 0.001), which resulted in the lowest colour intensity among all killing methods
(p < 0.001). Finally, asphyxiation produced FDG larvae with a higher lightness and hue angle than methods by
heat (D and B; L* = 39.3 ± 1.9; h = 1.43 ± 0.01; p = 0.035; Figure 3.2 (a) and (b), respectively) resulting in
colouration closer to yellow for asphyxiated larvae as shown on Figure 3.2 (e).

Figure 3.2 Freeze-dried and granulated BSF larvae killed by different methods: (a) desiccation; (b)
blanching; (c) freezing (liquid nitrogen); (d) grinding; (e) asphyxiation (vacuum); (f) high hydrostatic
pressures.

3.5.4 Microbial analysis


Under our rearing and feeding conditions, contamination of larvae frozen at −20 °C and thawed contain 9.0 log
for TVC, 8.2 log for LAB, 8.4 log for Pseudomonas spp., 6.9 log for yeast and moulds, 7.2 log for enterobacteria,
7.0 log for coliforms, 7.5 log for Listeria spp., 8.7 log for Clostridia and other anaerobes, 7.2 log for anaerobic
endospores, 7.3 log for aerobic endospores and no detection of Salmonella spp. and Listeria monocytogenes in
25 g [183]. The results from the current study demonstrate that killing methods affect the microbial load of BSF
larvae (Table 3.2). In general, blanching resulted in the lowest counts followed by HHP while asphyxiated larvae
demonstrated the highest counts. Asphyxiation methods (9.7 ± 0.1 log CFU/g) demonstrated TVC at least 1 log
higher than other methods, while blanching and HHP gave the lowest (B and HHP = 6.0 log CFU/g). Killing
methods had the same effect on LAB as TVC, but LAB were more vulnerable to blanching than TVC. Blanching

61
resulted in the lowest LAB counts (3.1 ± 0.8 log CFU/g) followed by HHP (6.7 ± 0.9 log CFU/g) which were
3 logs higher. The killing methods which demonstrated the highest Pseudomonas spp. were freezing at −40 °C
and in liquid nitrogen, asphyxia and grinding (F40, N, CO2, N2, V and G = 7.1 log CFU/g). Desiccation and
freezing at −20 °C obtained lower Pseudomonas spp. counts (D and F20 = 5.2 log CFU/g) while blanching and
HHP reduced it below the detection limit (< 2.1 ± 0.1 log CFU/g). Yeast and moulds had higher counts in larvae
subjected to freezing and grinding (F20, F40, N and G = 6.2 log CFU/g) followed by asphyxiated and desiccated
larvae (CO2, N2, V and D = 5.1 log CFU/g) while they were not detected after blanching and HHP
(< 2.1 ± 0.1 log CFU/g).

The killing methods had the same effects on enteric microbial indicators such as enterobacteria and coliforms
(Table 3.2). Asphyxiation resulted in the highest counts of Enterobacteriaceae and coliforms (7.2 log CFU/g),
followed by freezing, desiccation and grinding (F20, F40, N, D and G; Enterobacteriaceae = 4.6 log CFU/g;
coliforms = 4.4 log CFU/g). Finally, no indicator of enteric contamination was detected in blanched larvae
(< 1.1 log) while only few coliforms were detected in HHP treated larvae (1.3 ± 0.4 log CFU/g). The killing
methods had little effect on Listeria spp., but asphyxiation methods (6.7 log CFU/g) resulted in higher counts
than the other methods (5.3 log CFU/g). Finally, asphyxia and grinding (CO2, N2, V and G = 9.5 log CFU/g)
obtained the higher counts of Clostridia and other anaerobes, followed by freezing and desiccation (F20, F40,
N and D = 8.4 log CFU/g), HHP (6.1 ± 0.6 log CFU/g) and finally blanching (4.8 ± 0.3 log CFU/g).

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Table 3.2 Microbial load (log CFU/g; dry basis) of thawed black soldier fly larvae killed by different methods.
Total Viable Clostridia and
Killing Lactic Acid Pseudomonas Yeast and
Aerobic Enterobacteria Coliforms Listeria spp. other
Methods Bacteria spp. Moulds
Count anaerobic
Heating
D 7.7 ± 0.7 b 7.8 ± 0.7 bc 4.8 ± 0.4 b 5.1 ± 0.5 bc 4.1 ± 0.8 b 3.5 ± 1.3 b 5.2 ± 0.2 a 7.9 ± 0.5 c
B 5.6 ± 0.3 a 3.1 ± 0.8 a < 2.1 ± 0.1 a < 2.1 ± 0.1 a < 1.1 ± 0.1 a < 1.1 ± 0.1 a 5.2 ± 0.1 a 4.8 a ± 0.3
Freezing
F20 8.3 ± 0.5 bc 8.4 ± 0.6 cd 5.6 ± 0.3 b 6.3 ± 0.8 c 4.5 ± 0.9 b 4.5 ± 0.9 b 5.5 ± 0.2 ab 8.6 ± 0.2 cd
F40 8.5 ± 0.1 c 8.6 ± 0.1 cde 6.7 ± 0.7 c 6.4 ± 0.7 c 5.3 ± 0.9 b 5.1 ± 0.8 bc 5.1 ± 0.1 a 8.4 ± 0.1 cd
N 8.3 ± 0.2 bc 8.7 ± 0.1 cde 7.0 ± 0.4 cd 6.1 ± 0.7 c 4.6 ± 0.7 b 4.5 ± 0.7 b 5.2 ± 0.1 a 8.4 ± 0.3 cd
Asphyxiation
CO2 9.8 ± 0.2 d 9.9 ± 0.2 f 7.8 ± 0.4 d 5.8 ± 0.6 bc 7.5 ± 0.6 c 7.6 ± 0.6 d 7.0 ± 0.6 c 9.9 ± 0.1 f
N2 9.6 ± 0.2 d 9.7 ± 0.1 ef 7.3 ± 0.3 cd 5.3 ± 0.9 bc 7.3 ± 0.6 c 7.2 ± 0.4 d 6.7 ± 0.7 c 9.6 ± 0.1 ef
V 9.6 ± 0.2 d 9.7 ± 0.3 ef 7.3 ± 0.2 cd 4.2 ± 1.2 b 6.9 ± 0.7 c 6.7 ± 0.5 cd 6.3 ± 0.8 bc 9.6 ± 0.2 ef
Mechanical disruption
G 8.5 ± 0.5 c 9.1 ± 0.3 def 6.5 ± 0.3 c 6.1 ± 0.4 c 4.7 ± 0.7 b 4.6 ± 0.6 b 5.2 ± 0.3 a 8.9 ± 0.3 de
HHP 6.3 ± 0.5 a 6.7 ± 0.9 b < 2.1 ± 0.1 a < 2.1 ± 0.1 a < 1.1 ± 0.1 a 1.3 ± 0.4 a 5.5 ± 0.5 ab 6.1 ± 0.6 b
Different letter in the same column indicates a significant difference (p < 0.05); mean ± standard deviation; D = Desiccation, B = Blanching, F20 = Freezing at −20 °C, F40 = Freezing
at −40 °C, N = Freezing in liquid nitrogen, CO2 = Asphyxia with CO2, N2 = Asphyxia with N2, V = Asphyxia under vacuum, G = Grinding, HHP = High hydrostatic pressures.

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3.6 Discussion
Killed BSF larvae are highly perishable due to their high water content (78%–84%), their neutral pH (6–9) and
their high microbial load [15, 46]. It is therefore critical to maximize BSF larvae preservation by including
decontamination and processing steps that reduce contamination and subsequent microbial growth. Therefore,
the product must respect some criteria of lipid and colour stability and microbiological safety.

In this study, killing methods used on BSF larvae have been shown to affect the chemical composition, lipid
oxidation, pH, colour and microbial load of the larvae. In the following, industry criteria for the product quality are
briefly discussed with emphasis on advantages and disadvantages of every method considered.

3.6.1 Chemical composition


The nutritional quality of a product refers to its nutrient contents (proteins, lipids, minerals and vitamins),
digestibility and oxidation of its constituents [8]. In this study, we focused on lipid content (EE) and oxidation
(FOX and TBARS), and on total moisture and ash content of the product. The larval ether extracts obtained, in
this study, were lower than those reported for BSF larvae [15, 45, 184], which may result from the samples
hydrolysis prior the extraction [185]. Therefore, we recommend that no hydrolysis should be applied on BSF
prior lipid extraction in future research.

The killing methods affected the total moisture, since heating methods allow water evaporation [186] and reduce
the water-holding capacity of proteins by denaturing them [187]. A lower water content can reduce drying time,
thus resulting in energy saving. Ash content of feed ingredients is generally associated with mineral levels.
Furthermore, because minerals are important components in feed for all animals and that the BSF larvae have
a high ash content, BSF larvae inclusion could reduce the need for mineral supplementation in diet formulations
[12]. In this study, most killing methods used had little effects on the larvae ash content except for blanching
which increased it.

The pH is an important parameter to consider when predicting product shelf life. To ensure growth inhibition of
pathogenic microorganisms, the Canadian Food Inspection Agency regulated processing and preservation of
low-acid food, i.e., possesses a pH higher than 4.6 and an aw higher than 0.85 [88]. To promote preservation of
a low-acid food product, such as insects, the product must be sterilized by heat if it is packed hermetically or it
must remain refrigerated or frozen [88]. However, dehydrating the insect product under 5% moisture [8, 88], thus
being considered a low-moisture food, appears to be the most economical and effective approach to preserve
its quality [90].

Lipid oxidation is associated with a loss of nutritional value, the development of a rancid odour and taste, and
can potentially lead to toxicity [121]. Many factors influence the rate of oxidation, such as the levels of

64
unsaturated fatty acids, the concentration of oxygen, temperature, pH and water activity [85]. The unsaturated
fatty acid content of BSF larvae can reach up to 38% of the lipids, and thus be vulnerable to oxidation [24].
However, to our knowledge, no acceptable oxidation limit has been proposed for BSF products. Nevertheless,
oxidation limits in products with a high oxidative potential, such as fishmeal, could be used as a reference in
insects. According to Connell, lipids can be characterized as not rancid (TBARS value < 1.5 mg MDA/kg), slightly
rancid (1.6 < TBARS value < 3.6) and rancid (3.7 < TBARS value) [129]. During this study, lipids were at most
slightly rancid since all TBARS value were below 3.6 MDA/kg, suggesting that it is not a critical factor for BSF
products in the tested conditions. However, the BSF larvae feeding substrate can influence its lipid content and
fatty acid profile [24, 34], and thus, should be further considered in terms of product quality appreciation.

3.6.2 Colour
The BSF larvae, whose colour is usually beige, has the potential to appear darkish [65, 138], which may reduce
protein solubility and alter their functional properties [152]. Most BSF larvae colour alteration results, in the
presence of oxygen, from polyphenol oxidation and complex formation between iron and polyphenols [13, 138].
Since some polyphenol oxidation reactions are catalyzed by enzymes, those can be inhibited by denaturation
using heating treatments or HHP [138, 188]. However, heating treatments can also induce browning resulting of
Maillard reactions, non-enzymatic reactions between amino acids and reducing sugars such as fructose or
glucose [107, 148]. In addition, drying can alter a product colour perception resulting from the difference in
refractive index between water and air [186] and product structural shrinkage [180]. A product containing less
water may reflect less light and thus have a lower lightness. Because the perception of discolouration is variable
among food products, the minimal colour change required to be perceptible should be evaluated for insects.
Moreover, since a ΔE ≤ 2 represents an undetectable colour change for many food products [141], this value
was applied for BSF larvae. During this study, we have seen that the killing methods used greatly affected the
colouration of the final BSF product. Several processes (irradiation and controlled atmosphere) or additives
(sulfites, chelating agent and antioxidants) able to minimize browning could be useful to reduce colour alterations
during the transformation of BSF in the future [136, 138].

3.6.3 Microbiology
The microbial load of BSF larvae is highly variable and depends on, the feeding substrate and the rearing
conditions [46]. In fact, Listeria spp. are not usually detected in BSF larvae [44-45], but were detected in this
study ranging from 5.2 to 7.0 log CFU/g. The Listeria might be derived from the Gainesville diet, which
demonstrated almost 4 log CFU/g [167]. Health Canada is the federal institution responsible for emitting
microbial limits of food in Canada [135]; thus far, absence of Salmonella in animal feed is the main concern.
Since no recommendations on the microbial load of insects have been yet established, contamination of BSF
larvae was compared to ready-to-eat processed food requirements: TVC < 4 log CFU/g, coliforms

65
< 2 log CFU/g, E. coli < 10 CFU/g, Staphylococcus aureus coagulase positive < 25 CFU/g, Bacillus spp.
< 50 CFU/g, Clostridium perfringens < 10 CFU/g and no detection of pathogens microorganisms in 25 g
(Salmonella spp., Campylobacter spp., Shigella spp. and L. monocytogenes) [135]. Many preservation
technologies (acidification, use of preservatives, heat and physical technologies) may be used to inactivate or
inhibit growth (low temperature, reduction in water activity, vacuum and modified-atmosphere packaging) of
food-poisoning and food-spoilage microorganisms in insects [189]. Many killing methods used in this study also
include one of these preservation mechanisms and thus could reduce the microbial load. However, some
methods do not inhibit microbial growth (grinding) or are selective in the bacteria inhibited (asphyxiation).
Therefore, because harvested BSF larvae are highly contaminated, it is expected that these methods may only
obtain a limited microbial growth according to the Jameson Effect [190]. The Jameson effect indicates that
microbial growth may be reduced when the maximum microbial population density is achieved [190]. The killing
is not in itself a decontamination process, but according to our results, the choice of the methods being used,
such as blanching or HHP, can contribute to improving product safety.

3.6.4 Heating
3.6.4.1 Desiccation
Desiccation at 60 °C is a slow killing method as it takes almost 30 min to kill larvae. According to the
precautionary principle, this method should be optimized to reduce the application time, since the insect activity
increases with the increasing temperature. However, desiccation has the advantage to reduce manipulation and
equipment requirement to produce the final insect powder. In this study, desiccation as had little effect on the
microbial load, resulted in slightly rancid lipids and a larval meal with a higher colour intensity. Microbial
inactivation was too small for the product to be used as such, since TVC was 3.7 log higher than the
recommended limit [135]. In fact, Pseudomonas spp. was the only microorganism affected by desiccation; its
survival rate is known to decrease from 48 °C [191]. Furthermore, even if the secondary compound of lipid
oxidation increased (TBARS = 2.5) [129], the lipid quality was acceptable since the TBARS value was under 3.6
[192]. The colour alteration of the product while drying was perceptible (∆E = 5.6) and associated with a
decrease in lightness [180, 186] and an increase of the hue angle as well as colour intensity for which Maillard
reactions are likely to be involved [148]. In addition, desiccation for only 30 min at 60 °C, including the larval
rising temperature time, appears to initiate drying resulting in a lower total moisture [108].

3.6.4.2 Blanching
Blanching is used to improve shelf life of several food products [193] and to kill some invertebrates [60, 65] at
low cost. It is being considered as an acceptable way to kill insects because of its rapidity. Blanching have many
advantages such as reducing the microbial load [90], inhibiting spoilage enzymes [64], thus mitigating lipid
oxidation [109] and increasing colour stability [65], and enhancing flavour and taste [194]. Indeed, blanching of
mealworms during 40 s highly decreases the TVC, LAB, Enterobacteriaceae and yeast and moulds, but had

66
little effect on bacterial endospore [90]. On the other hand, immersion of a product in boiling water may induce
vitamin and mineral losses [138, 193-194], and protein denaturation, thus changing their functionality such as
water-holding capacity which may reduce drying time [187]. In this study, blanching appears as a promising
procedure since it maintained the chemical composition, with the lowest microbial counts, while reducing the
total moisture, which may result from the reduction of the protein water-holding capacity [187]. Indeed, blanching
did not lead to reduced ash since it was higher in larvae compared to other methods. Furthermore, blanching is
the only method that resulted in excellent lipid quality since they were not rancid (TBARS = 1.0) according to the
limit for fish oil (< 1.5) [109, 192]. Furthermore, blanching resulted in the highest pH (8.7), which may be caused
by ascorbic acid leachate [193]. Even if the pH was far from the recommended value of 4.6 [88], a basic pH can
inhibit microbial growth as it is the case notably in egg yolk [195]. The freeze-drying of blanched larvae produced
a meal of similar colour than with most of the methods tested by reducing lightness and increasing yellowness
resulting from a higher hue angle compared to thawed larvae [180, 186]. However, blanching did not prevent
colour alteration during drying (∆E = 6.2), as observed for mealworms treated the same way [106].
Pseudomonas spp., yeast and moulds, enterobacteria and coliforms were not detected in blanched larvae [89-
90]. Even if most microorganisms were reduced [89], TVC, Listeria spp., Clostridia and other anaerobes were
still too high to be considered as a ready-to-eat ingredient [135]. Indeed, TVC was 1.6 log higher than Health
Canada recommendations. Therefore, the presence of specific food-poisoning microorganisms should be further
investigated. In the future, blanching should be optimized to increase microbial inactivation and/or be combined
with other decontamination treatments to ensure product safety.

3.6.5 Freezing
Because insects are ectotherms, freezing appears to be an acceptable way to kill them since it will slow their
activity until death is achieved [8, 174]. Although freezing may be expensive in terms of time, energy and
equipment, it is one of the most frequent methods used [65]. Freezing allows food preservation by reducing the
available amount of water [196], thus inhibiting microbial growth, enzyme activity and biochemical reactions.
Freezing will also impact the colour, texture and flavour of the product [197]. The speed of the freezing process
depends mostly on the temperature that is applied. At low freezing speed, large ice crystals will be formed
inducing cells damage and increasing osmotic pressure that will result in a high water loss [189] and bacteria
inactivation [196]. Freezing at -20 °C as a killing method for the BSF larvae has been recently investigated [64-
65]. The authors demonstrated that the slow killing induced by freezing does not prevent lipolysis by endogenous
enzymes and browning while influencing chemical and physical properties of protein [64-65]. In the current study,
every method by freezing had a similar impact on the product. For all freezing methods, lipids were of good
quality (TBARS = 1.7–2.0) [192], powder colour was similar to blanched larvae, but perceptible colour alteration
while drying did occur (∆E = 3.7–5.0). Even if the action of freezing and thawing can induce a reduction of
contamination of several microorganisms [196], it is not considered an effective method to reduce microbial

67
growth. Freezing treatments applied in this study did not reduce contamination compared to other methods
except for freezing at −20 °C whose Pseudomonas spp. counts were 1 log lower than other freezing methods
[196]. The TVC was 4.8 log too high to reach Health Canada standards for ready-to-eat food, and therefore
requires an additional decontamination step [135]. Thus, killing methods using cold temperatures gives very few
industrial benefits while requiring space, time and equipment.

3.6.6 Asphyxiation
Carbon dioxide has been used on invertebrates as anaesthesia [198] and as a killing method [75]. Quite efficient
on crickets (CO2 = 15 min and N2 = 5 min) [74], this approach allows to quickly anesthetise an important quantity
of insects at low cost. In this study, asphyxiation methods have shown to be highly ineffective to kill BSF larvae.
Indeed, BSF larvae killed by asphyxiation required a very long time (120 h–144 h) inducing starvation and,
possibly, microbial growth. Even if larvae killed by asphyxiation showed a higher-lipid content that may result
from microbial metabolism [199-200], these lipids appear to be highly prone to oxidation, resulting in a higher
primary lipid oxidation level. In addition, asphyxiated larvae showed a lower pH which might be due to the
production of lactic acid from anaerobic metabolism [76] and the dissolution of CO2 in water and lipids [201].
Furthermore, TVC, LAB, Clostridia and other anaerobes, proliferated during asphyxia methods [202]; the small
increase in Listeria spp. and indicators of enteric contamination could be attributable to the Jameson Effect [190].
The development of Pseudomonas spp. was inhibited by anoxic conditions [202] and the slight decrease in yeast
and moulds may be attributable to CO2 toxicity [203]. Even if asphyxia was the only method that did not induce
a perceptible colour change, it induced lipid oxidation and obtained the highest total moisture and microbial load.
The TVC was 5.7 log higher than the recommended limit, requiring further efficient decontamination technique
to allow the product to reach acceptable microbial standards [135].

3.6.7 Mechanical disruption

3.6.7.1 Grinding
Grinding is a fast and effective killing method [174]. It can reduce drying time by increasing the surface area for
drying [187], resulting in a saving for the industry due to relatively low equipment cost. However, grinding is
known to enhance browning in insects [13] and to promote lipid oxidation by exposing the constituents to oxygen
[85]. In this study, killing BSF larvae by grinding did not affect the pH and the chemical composition whose lipids
were considered of good quality. The high DM content obtained after drying was expected because of the
increased drying efficiency [187]. Additionally, the high microbial load obtained for ground larvae limits its use
as food since TVC was 4.5 log too high compared to the Health Canada recommendation [135]. Moreover,
ground larvae obtained the highest colour change during drying, which may result from the polyphenol oxidation
and the complex formation between iron and polyphenol [13]. As a result, freeze-drying of ground larvae highly
reduce the lightness and the chroma of the product while having no effect on the hue angle. The powder obtained

68
from ground larvae appeared grey and would probably be less appealing to customers. Grinding could be further
optimized by including additives while grinding and by a subsequent decontamination step.

3.6.7.2 High hydrostatic pressures


High hydrostatic pressures are not usually employed as a killing method but as a decontamination method. This
process has been used in many food products to inactivate microorganisms at low temperatures while
maintaining food flavour and texture [204]. In our preliminary trials, we observed that BSF larvae are highly
resistant to pressures and were able to fully recover from a 100 MPa treatment for 3 min, requiring a higher
pressure to kill them. High pressures can denature most spoilage enzymes [188], thus inhibiting discolouration,
but can also denature antioxidants that may result in higher lipid oxidation [99]. Even if HHP may reduce most
microorganisms from many foods, the inactivation efficiency varies depending on the food composition, the
pressure applied, the holding time, the temperature and the initial contamination of the food product [204].
Microbial inactivation by HHP is achieved by inducing internal fluid leakage of cells and protein denaturation
which is more efficient on gram-negative bacteria, yeast and moulds [205]. With insects, HHP such as 350–
600 MPa can highly reduce Salmonella spp., E. coli and yeast and moulds [44-45], but have little effect on TVC,
which was 2.3 log higher than the recommended limit, because of the presence of bacterial endospores on
which the effect of HHP is very limited [89, 204]. In this study, BSF larvae killed by HHP showed slightly rancid
lipids, a powder of colouration close to others, and the second-lowest microbial load [129]. However, the DM
content was not high enough to ensure inhibition of microbial growth [8] and the colour change (∆E = 3.3) upon
drying was high enough to be perceptible [141] resulting from the increase of the hue angle. Furthermore,
although the HHP obtained a low microbial load, counts were still too high to be acceptable [135]. Indeed, HHP
obtained similar result to blanching except for LAB and Clostridia and other anaerobic whose counts were 3.6
and 1.3 log higher, respectively. It is possible that the structural component of BSF larvae, such as chitin and
lipid, may reduce the effect of pressure on bacteria resulting in a better survival rate [206]. Even if HHP requires
very expensive equipment, it can enhance subsequent extractability of proteins, chitin and lipids [115].

3.7 Conclusions
The killing method has a significant influence on the quality of the BSF larvae meals. In fact, killing impacts total
moisture, ash, lipids (EE) content, lipid oxidation, pH, microbial population count and on the colour of the BSF
end product, as well as on its preservation (pH). In this study, the blanching appears to be the preferred method
considering low lipid oxidation, the reduction of the total moisture which may reduce drying time, the increase of
the pH above 8, the colour stability, the significant reduction of the microorganisms and its execution speed.
However, blanching should be optimized or other decontamination methods should be applied to reduce Listeria
spp., Clostridia and other anaerobes below an acceptable threshold for feed.

69
Author Contributions: conceptualization, J.L., M.-H.D., L.S., A.D. and G.W.V.; methodology, J.L. and Y.L.;
software, J.L.; validation, J.L., M.-H.D. and G.W.V.; formal analysis, J.L.; investigation, J.L.; resources, J.L. and
M.-H.D.; data curation, J.L.; writing—original draft preparation, J.L.; writing—review and editing, M.-H.D.,
G.W.V., L.S., A.D. and Y.L.; visualization, J.L.; supervision, G.W.V.; funding acquisition, G.W.V.

Funding: This work was supported by funding from the Programme Innov’Action Agroalimentaire, a program
under Growing Forward 2 between the Ministère de l’agriculture, des pêcheries et de l’alimentation du Québec
and Agriculture and Agri-Food Canada. J.L. is the recipient of graduate scholarships from the Fonds de
recherche Québec and the Natural Sciences and Engineering Sciences Canada.

Acknowledgments: We thank the Vandenberg laboratory team for their assistance in counting the larvae and
the other members of the research project, Cristina Ratti, Lucie Beaulieu from Université Laval, Charles Lavigne,
Celine Georlette from the CDBQ and the technical professionals of the LARSA.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

70
Appendix A
Table A1. Colour measures (L*, a* and b*), colour intensity (chroma), hue angle and colour change while
drying (∆E) of thawed and freeze-dried and granulated BSF larvae killed by different methods.
Chroma Hue angle
Killing methods L* a* b* ∆E
(C*) (h)
Thawed
Heating
D 45.8 ± 1.0 de 2.9 ± 0.3 gh 9.5 ± 0.7 d 9.9 ± 0.7 cd 1.28 ± 0.03 ab 5.6 ± 0.9 b
B 46.0 ± 0.7 de 1.9 ± 0.4 def 8.7 ± 0.3 bc 8.9 ± 0.4 bc 1.36 ± 0.04 bce 6.2 ± 0.9 b
Freezing
F20 45.0 ± 0.7 cde 3.3 ± 0.2 h 8.9 ± 0.3 bc 9.5 ± 0.4 bcd 1.22 ± 0.02 a 3.7 ± 0.9 ab
F40 45.0 ± 0.4 cde 3.2 ± 0.2 h 8.9 ± 0.6 bc 9.5 ± 0.6 bcd 1.22 ± 0.02 a 5.0 ± 1.9 ab
N 41.9 ± 1.5 bc 2.7 ± 0.4 fgh 7.9 ± 0.9 ab 8.4 ± 0.9 b 1.24 ± 0.05 a 3.7 ± 1.4 ab
Asphyxiation
CO2 45.5 ± 0.2 cde 2.2 ± 0.5 efg 10.5 ± 0.2 de 10.7 ± 0.2 de 1.37 ± 0.05 cdefg 2.6 ± 0.9 a
N2 45.6 ± 0.6 cde 2.2 ± 0.2 efg 9.6 ± 0.4 cd 9.8 ± 0.4 cd 1.34 ± 0.02 bcd 2.2 ± 0.2 a
V 46.5 ± 0.9 e 2.2 ± 0.4 efg 10.1 ± 0.7 cde 10.3 ± 0.7 cde 1.36 ± 0.04 bcdef 2.5 ± 1.1 a
Mechanical disruption
G 54.1 ± 2.5 f 1.6 ± 0.4 cde 10.6 ± 0.4 de 10.8 ± 0.4 de 1.42 ± 0.04 cdefgh 17.5 ± 3.6 c
HHP 47.5 ± 0.7 e 3.0 ± 0.2 gh 10.0 ± 0.3 cde 10.4 ± 0.3 de 1.28 ± 0.02 ab 3.3 ± 1.0 ab
Freeze-dried and granulated
Heating
D 40.7 ± 1.5 ab 1.6 ± 0.2 bcde 11.3 ± 0.4 e 11.4 ± 0.4 e 1.43 ± 0.02 efgh
B 39.9 ± 1.1 ab 1.5 ± 0.2 bcde 9.7 ± 0.1 cd 9.8 ± 0.1 cd 1.42 ± 0.02 dfgh
Freezing
F20 42.4 ± 1.2 bcd 1.3 ± 0.3 abcd 10.1 ± 0.6 cde 10.1 ± 0.6 cde 1.45 ± 0.02 ghi
F40 40.7 ± 1.6 bc 1.1 ± 0.3 abcd 10.5 ± 0.3 de 10.6 ± 0.3 de 1.47 ± 0.03 hij
N 41.9 ± 1.7 bc 1.0 ± 0.2 abc 9.7 ± 0.5 cd 9.7 ± 0.6 bcd 1.47 ± 0.02 hij
Asphyxiation
CO2 44.8 ± 1.1 bcde 0.6 ± 0.4 a 10.5 ± 0.5 de 10.5 ± 0.5 de 1.52 ± 0.04 ij
N2 44.8 ± 0.8cde 0.4 ± 0.3 a 10.3 ± 0.3 de 10.3 ± 0.3 cde 1.53 ± 0.02 j
V 44.8 ± 0.7 cde 0.6 ± 0.3 a 10.5 ± 0.5 de 10.5 ± 0.5 de 1.51 ± 0.03 ij
Mechanical disruption
G 37.2 ± 2.6 a 0.8 ± 0.1 ab 6.6 ± 0.8 a 6.6 ± 0.8 a 1.45 ± 0.01 hij
HHP 45.9 ± 2.0 de 1.1 ± 0.2 abcd 10.6 ± 0.7 de 10.7 ± 0.7 de 1.46 ± 0.01 hij
Different letter in the same column indicates a significant difference (p < 0.05); mean ± standard deviation; the ∆E
represents the colour difference between thawed and freeze-dried and granulated larvae.

71
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General conclusions
Although insects appear as promising feed ingredients, several processing methods still remain to be optimized
for the various species of interest as well as how they impact them. This project aimed at contributing to the
enhancement of the current knowledge on processing methods of black soldier fly larvae.

This project demonstrated that, contrary to other farm animals such as pig, feed withdrawal period in BSF larvae
do not provide any advantage with respect to safety since it did not induce significant microbial reductions.
Moreover, it impacted on the composition of the product which could reduce its quality. Furthermore, since it
required three days to evacuate the gastrointestinal tract of most larvae, a prolonged feed withdrawal period
reaching this point could be criticized from an ethical point of view should the welfare of insects become an
issue. It would thus be interesting to investigate the ability of gut loading substrates to improve the product
nutritional quality.

This project demonstrates that killing methods should be chosen carefully since several methods appeared to
reduce the quality of the product while others were able to enhance it, such as blanching and high pressure
processing. Indeed, asphyxiation methods appeared to be less appropriate as they allowed microbial growth
and lipid oxidation to occur and required a long period of time to kill the insects.

From the comparison of the ten killing methods studied (resume in Table 4.1), blanching appears as the most
promising one since it reduced the microbial load, maintained the product colour and minimized lipid oxidation
while being one of the fastest methods. However, after killing, the microbial reduction did not meet load limits for
ready-to-eat food of the Canadian regulatory autorities. Future research should thus focus on decontamination
methods in order to inactivate the residual Listeria spp. and Clostridium and other anaerobes. Moreover, in this
study, blanching was applied using submersion, but several other treatments using moist heat should be
investigated such as rinsing with boiling and steaming water. Finally, other killing methods should also be
evaluated for their impact on the product quality such as electrocution and microwave.

76
Table 4.1 Summary of the impact of ten killing methods on the black soldier fly larvae.
Mechanical
Heat
Killing methods Cold Asphyxia disruption
Desiccation Blanching Grinding HHP
Time 30 min < 40 s 40 s – 1 h 6–7d < 40 s 3 min
Total moisture – – +/- + +/- +/-
Lipids (%) +
Lipid oxidation +/- – +/- + +/- +/-
pH Neutral Basic Neutral Acid Neutral Neutral
Colour change + +/- +/- – +++ +/-
Microbial load reduction
Total viable counts +/- –– +/- + +/- –
Enteric indicators +/- –– +/- + +/- ––
Pathogens +/- –– +/- + +/- –

77
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