Study of

processing
technologies
in edible
insects
João Pedro Correia de Matos Marques
Mestrado em Ciências do Consumo e Nutrição
Departamento de Geociências, Ambiente e Ordenamento do Território
2022

Orientador
Doutor Luís Miguel Cunha, Professor Associado
(GreenUPorto, DGAOT-FCUP)

Coorientadora
Doutora Lilia Ahrné, Professora Catedrática
(UCPH FOOD)
Declaração de Honra

Eu, João Pedro Correia de Matos Marques, inscrito no Mestrado em Ciências do

Consumo e Nutrição da Faculdade de Ciências da Universidade do Porto declaro, nos
termos do disposto na alínea a) do artigo 14.º do Código Ético de Conduta Académica
da U.Porto, que o conteúdo da presente dissertação reflete as perspetivas, o trabalho
de investigação e as minhas interpretações no momento da sua entrega.

Ao entregar esta dissertação, declaro, ainda, que a mesma é resultado do meu próprio
trabalho de investigação e contém contributos que não foram utilizados previamente
noutros trabalhos apresentados a esta ou outra instituição.

Mais declaro que todas as referências a outros autores respeitam escrupulosamente as

regras da atribuição, encontrando-se devidamente citadas no corpo do texto e
identificadas na secção de referências bibliográficas. Não são divulgados na presente
dissertação quaisquer conteúdos cuja reprodução esteja vedada por direitos de autor.

Tenho consciência de que a prática de plágio e auto-plágio constitui um ilícito

académico.

João Pedro Correia de Matos Marques

Lamego, 30 de setembro de 2022

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Acknowledgements

First of all, I would like to thank the Faculty of Sciences of the University of Porto
(FCUP) and the Faculty of Nutrition and Food Science of the University of Porto
(FCNAUP) for providing me an exceptional scientific knowledge and experience during
these last two years.
Words cannot express my gratitude to Professor Luís Miguel Cunha for all the
support and guidance throughout this project. Thank you for all the kindness and for
always finding time to help me. Thank you for being such a remarkable person, mentor,
and teacher.
To Professor Lilia Ahrné, that provided me the wonderful opportunity to intern in
her research team and for always expressing her passion and knowledge regarding food
science. Thank you for the warm welcome and for making me feel at home at the
University of Copenhagen. Thank you for all the guidance and for being there for me in
the toughest times.
A big thank you to José Carlos Ribeiro for all the caring and patience, for always
believing in me, and for teaching me, not only about hard work but also about life. There
will never be enough words to describe how grateful I am for everything you have taught
me and for all the strength you have given me to always move forward. You are a true
role model.
I also would like to thank all the Dairy and Processing research group at the
University of Copenhagen, especially Professor Markus Walkling-Ribeiro, for teaching
me valuable laboratory skills, for being supportive, and for always having a friendly word
in the toughest moments.
All of you have taught me something valuable and made me grow in some way. It
was a pleasure to work with such great professionals.
This dissertation would not be possible without the amazing support system of my
family members, girlfriend and friends. A special thanks to my mom, Ana Isabel Matos,
for always believing in me, even when I did not, and for supporting all my decisions
throughout my academic journey. Words cannot describe how grateful I am for having
you as my mom.
Lastly, I would like to acknowledge the European Commission for the financial
support during my six months in Denmark as part of my Erasmus + Traineeship.
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ii Study of processing technologies in edible insects

Agradecimentos

Em primeiro lugar, gostaria de agradecer à Faculdade de Ciências da

Universidade do Porto (FCUP) e à Faculdade de Ciências da Nutrição e Alimentação da
Universidade do Porto (FCNAUP) por me proporcionarem um conhecimento e
experiência científica excecionais durante estes dois últimos anos.
Não há palavras para expressar a minha gratidão ao Professor Doutor Luís Miguel
Cunha por todo o apoio e orientação ao longo deste projeto. Obrigado por toda a
gentileza e por encontrar sempre tempo para me ajudar. Obrigado por ser uma pessoa,
um orientador, e um professor tão marcante.
À professora Lilia Ahrné que me proporcionou a excelente oportunidade de
estagiar na sua equipa de investigação e por me demonstrar a sua paixão e
conhecimento sobre ciência alimentar. Obrigado pela incrível receção e por me ter feito
sentir em casa na Universidade de Copenhaga. Obrigada por toda a orientação e por
ter estado ao meu lado nos momentos mais difíceis.
Um enorme obrigado ao José Carlos Ribeiro por toda a amizade e paciência, por
sempre acreditar em mim e me ensinar, não só a trabalhar arduamente, mas também
sobre a vida. Nunca haverá palavras suficientes para descrever o quanto estou grato
por tudo que me ensinaste e por toda a força que me deste para sempre seguir em
frente. És um verdadeiro exemplo de investigador e de pessoa.
Também gostaria de agradecer a todo o grupo de investigação em Laticínios e
Processamento da Universidade de Copenhaga, especialmente ao professor Markus
Walkling-Ribeiro, por me ensinar importantes competências de laboratório, por me ter
apoiado e por ter sempre uma palavra amiga nos momentos mais difíceis.
Todos vocês me ensinaram algo valioso e me fizeram crescer de alguma forma.
Foi, sem dúvida alguma, um prazer trabalhar com profissionais que tanto admiro.
Esta dissertação não seria possível sem o apoio dos meus familiares, namorada
e amigos. Um agradecimento especial à minha mãe, Ana Isabel Matos, por sempre
acreditar em mim, mesmo quando eu não acreditava, e por apoiar todas as minhas
decisões ao longo do meu percurso académico. Não existem palavras para descrever o
quão grato estou por ter como minha mãe.
Por último, gostaria de agradecer à Comissão Europeia pelo apoio financeiro
durante os meus seis meses na Dinamarca como parte do meu Erasmus + Estágio.
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Abstract

Over the last few decades, a major increase in the world’s population led to an
intensified demand for food production in the most diverse sectors of the global food
system. As global dietary patterns are rich in meat, which has been associated with a
considerable environmental impact and greenhouse gases emissions, it is crucial to find
alternative and sustainable food sources to solve this environmental problematic, without
compromising nutritional values and dietary requirements. In the last few years, trends
regarding sustainability have emerged and revealed edible insects and insect-based
food products as promising solutions to an ever-growing demand in meat production.
Edible insects were shown to have lower greenhouse-gases emissions and NH3
production, a high feed-conversion efficiency and percentage of digestible biomass, a
high protein and polyunsaturated fatty acids content, and can also be rich in several
vitamins and minerals. Thus, since scientific research is limited in this subject, it is
important to investigate how different processing technologies affect crucial parameters
on edible insects in order to overcome barriers related to food safety, food quality and
consumer acceptance.

Therefore, the main purpose of this project was to deepen the knowledge about
how different processing technologies in edible insects affect different parameters, and
it is divided in three distinct parts.

On the first one, a systematic review on lipid extraction methodologies on edible

insects was conducted, and it was evaluated the effectiveness of the extractions and
subsequent characterizations and applications. The use of solvents is the most common
procedure for lipid extraction in edible insects with conventional and Soxhlet extractions
being the most applied methodologies. In general, it was noticed that solvent extractions
resulted in higher efficiencies that can be affected by the polarity of the solvents that
were used. Aqueous extractions lead to a better lipid quality comparatively to solvent
extractions despite showing a lower efficiency of extraction. Additionally, few were the
studies that did further application of the extracted lipids even though scientific literature
can be found on the use of edible insects’ lipids for animal feed and sensory studies.

In the second part, a study on pre-treatment conditions of yellow mealworm was

performed, different blanching, drying and conservation techniques were compared and
their impacts on dry matter, water activity and colour were assessed. In general,
immersion blanching was superior to steam blanching by displaying a higher L* value, a
higher dry mater percentage and a lower water activity value on oven dried and freeze-
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dried samples. Concerning the different drying methods, oven drying had the highest dry
matter percentage and lowest water activity value. Nonetheless, freeze-drying with
immersion blanched samples could also be a good alternative to oven drying specially
when aiming for a higher L* value and a lower water activity value.

Lastly, as part of an Erasmus + Traineeship at the Department of Food Science of

the University of Copenhagen, soluble proteins from defatted yellow mealworm and
house cricket powders were extracted using different concentrations of defatted powder,
pH and extraction technologies. Additionally, protein solubility, molecular weight, gelling
ability and rheological properties of previously extracted soluble proteins were studied
and analysed. In general, increases in pH of extraction and concentration led to higher
protein solubulity percentages with no differences between ultrasound and non-
ultrasound treated samples despite slightly higher results in ultrasound treated samples.
In addition, pellets and full samples exhibited better gelling ability in comparison to
supernatants. The rheological tests confirmed the very weak gelling ability found in
soluble protein dispersions in both insects.

Keywords: Entomophagy; Tenebrio molitor; Acheta domesticus; food quality;

conservation; blanching; drying; lipid extraction; protein extraction; ultrasound treatment;
gelling; rheological properties.
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Resumo

Durante as últimas décadas, um grande aumento da população mundial levou a

uma procura intensificada pela produção de alimentos nos mais diversos setores do
sistema alimentar global. Como os padrões alimentares globais são ricos em carne, que
tem sido associada a um considerável impacto ambiental e emissões de gases de efeito
estufa, é necessário encontrar fontes alimentares alternativas e sustentáveis para
resolver esta problemática, sem comprometer os valores nutricionais e as necessidades
alimentares. Nos últimos anos, surgiram tendências em relação à sustentabilidade e
revelaram os insetos comestíveis e os produtos alimentícios à base de insetos como
soluções promissoras para uma procura cada vez maior na produção de carne. Insetos
comestíveis apresentaram menores emissões de gases de efeito estufa e produção de
NH3, uma alta eficiência de conversão alimentar e percentagem de biomassa digestível,
alto teor de proteínas e ácidos gordos polinsaturados, bem como podem ser ricos em
diversas vitaminas e minerais. Deste modo, como a pesquisa científica é limitada nesta
área, é importante investigar como diferentes tecnologias de processamento afetam
parâmetros cruciais em insetos edíveis, a fim de superar barreiras relacionadas à
segurança e qualidade alimentar bem como à aceitação por parte do consumidor.

Desta forma, o objetivo principal deste projeto foi aprofundar o conhecimento

sobre como diferentes tecnologias de processamento em insetos edíveis afetam
diferentes parâmetros, e encontra-se dividido em três partes distintas.

Na primeira, foi realizada uma revisão sistemática sobre metodologias de extração

de lípidos em insetos edíveis, avaliando-se a eficácia das extrações e subsequentes
caracterizações e aplicações. O uso de solventes é o procedimento mais comum para
extração de lípidos em insetos edíveis, sendo as extrações convencionais e pelo método
de Soxhlet as metodologias mais aplicadas. De maneira geral, percebeu-se que as
extrações com solventes resultaram em maiores eficiências que podem ser afetadas
pela polaridade dos solventes utilizados. As extrações aquosas levam a uma melhor
qualidade lipídica comparativamente às extrações com solvente, apesar de
apresentarem uma menor eficiência de extração. Além disso, poucos foram os estudos
que fizeram aplicação dos lípidos extraídos, embora exista literatura científica sobre o
uso de lípidos de insetos edíveis para alimentação animal e estudos sensoriais.

Na segunda parte, foi realizado um estudo sobre as condições de pré-tratamento

na larva da farinha. Diferentes técnicas de branqueamento, secagem e conservação
foram comparadas e seus impactos na matéria seca, atividade de água e cor foram
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avaliados. Em geral, o branqueamento por imersão foi superior ao branqueamento a

vapor ao apresentar maior valor de L*, maior percentagem de matéria seca e menor
valor de atividade de água em amostras secas no forno e liofilizadas. Em relação aos
diferentes métodos de secagem, a secagem no forno apresentou a maior percentagem
de matéria seca e o menor valor de atividade de água. No entanto, a liofilização com
amostras branqueadas por imersão pode ser uma boa alternativa à secagem no forno,
especialmente quando se procura um valor de L* maior e um menor valor de atividade
de água.

Por último, no âmbito de um estágio de Erasmus + no Departamento de Ciência

Alimentar da Universidade de Copenhaga, foram extraídas proteínas solúveis de
farinhas desengorduradas de larva da farinha e de grilo doméstico usando diferentes
concentrações de farinha desengordurada, valores de pH e tecnologias de extração.
Além disso, a solubilidade proteica, o peso molecular, a capacidade de gelificação e as
propriedades reológicas das proteínas solúveis previamente extraídas foram estudadas
e analisadas. Em geral, os aumentos no pH e na concentração levaram a maiores
percentagens de solubilidade proteica sem diferenças entre amostras tratadas com
ultrassons e não tratadas com ultrassons, apesar de se verificar resultados ligeiramente
mais altos nas amostras tratadas com ultrassons. Complementarmente, os pellets e as
amostras completas exibiram melhor capacidade de gelificação em comparação com os
sobrenadantes. Os testes reológicos confirmaram a gelificação muito fraca encontrada
em dispersões de proteínas solúveis em ambos os insetos.

Palavras-chave: Entomofagia; Tenebrio molitor; Acheta domesticus; qualidade

alimentar; conservação; branqueamento; secagem; extração lipídica; extração proteica;
ultrassons; gelificação; propriedades reológicas.
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Table of Contents

Acknowledgements ........................................................................................................ i

Agradecimentos .............................................................................................................ii

Abstract ........................................................................................................................ iii

Resumo ........................................................................................................................ v

Table of Contents ......................................................................................................... 1

List of Figures ............................................................................................................... 6

List of Tables .............................................................................................................. 10

List of Abbreviations ................................................................................................... 12

1. Introduction .......................................................................................................... 14

1.1 The global food system: An ever-growing demand for food production ......... 14

1.2 Entomophagy: Insects as an alternative protein source................................. 15

1.3 Benefits of edible insects............................................................................... 17

1.3.1 Environment and sustainability ............................................................... 17

1.3.2 Nutritional value...................................................................................... 18

1.3.2.1 Protein quality.................................................................................. 20
1.3.2.2 Fat quality ........................................................................................ 23
1.3.2.3 Vitamins and minerals ..................................................................... 26

1.4 Major challenges to the consumption of edible insects .................................. 26

1.4.1 Food safety and allergen risk.................................................................. 26

1.4.2 Legislation .............................................................................................. 27

1.4.3 Consumer perception and acceptability .................................................. 29

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1.5 Novel insect-based food products and processing technologies.................... 30

2. Aims..................................................................................................................... 34

Part A – Systematic review on lipid extraction methodologies from edible insects ...... 35

3. Materials and methods ......................................................................................... 35

3.1 Inclusion and exclusion criteria...................................................................... 35

3.2 Study selection and data analysis ................................................................. 36

4. Results and Discussion ........................................................................................ 37

4.1 Lipid extraction methodologies ...................................................................... 38

4.2 Efficiency of the extractions ........................................................................... 39

4.3 Characterization ............................................................................................ 43

4.3.1 Chemical composition ............................................................................ 43

4.3.2 Oxidative stability ................................................................................... 46

4.3.3 Physical properties ................................................................................. 47

4.3.4 Bioactive properties ................................................................................ 48

4.3.5 Microscopic changes .............................................................................. 49

4.4 Applications ................................................................................................... 49

5. Conclusion ........................................................................................................... 50

Part B – Study of the effect of blanching, drying and conservation conditions on quality
parameters of Tenebrio molitor larvae ........................................................................ 51

6. Materials and methods ......................................................................................... 51

6.1 Insects .......................................................................................................... 51

6.2 Study design ................................................................................................. 51

6.2.1 Pre-conservation .................................................................................... 52

6.2.2 Blanching ............................................................................................... 52

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6.2.3 Post-conservation................................................................................... 53

6.2.4 Drying ..................................................................................................... 53

6.2.4.1 Electrical oven drying ...................................................................... 54
6.2.4.2 Microwave-assisted drying .............................................................. 54
6.2.4.3 Freeze-drying .................................................................................. 54

6.3 Characterization ............................................................................................ 55

6.3.1 Dry matter .............................................................................................. 55

6.3.2 Water activity .......................................................................................... 55

6.3.3 Colour .................................................................................................... 56

6.4 Statistical analysis ......................................................................................... 57

7. Results and Discussion ........................................................................................ 58

7.1 Effect of blanching and conservation on colour ............................................. 58

7.2 Colour variation over time (effect of post-conservation) ................................. 60

7.3 Post-drying effects of blanching and conservation on dry matter and water
activity .................................................................................................................... 61

7.4 Post-drying effects of blanching and conservation on colour ......................... 63

7.5 Comparison between drying methodologies on dry matter, water activity and
colour ..................................................................................................................... 67

8. Conclusion ........................................................................................................... 70

Part C – Study of different protein extraction conditions, gelling ability and rheological
properties of proteins from Tenebrio molitor larvae and Acheta domesticus ............... 71

9. Materials and methods ......................................................................................... 71

9.1 Insects and chemicals ................................................................................... 71

9.2 Defatting........................................................................................................ 72

9.3 Study design ................................................................................................. 73

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9.4 Protein extraction .......................................................................................... 73

9.5 Characterization ............................................................................................ 75

9.5.1 Protein solubility ..................................................................................... 75

9.5.2 Gelling ability of the full samples, supernatants and pellets .................... 75

9.5.3 Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-

PAGE)… ............................................................................................................ 76

9.6 Freeze-drying ................................................................................................ 77

9.7 Gelling ability of the proteins ......................................................................... 77

9.8 Rheological properties of the proteins ........................................................... 77

9.9 Statistical analysis ......................................................................................... 78

10. Results and Discussion ........................................................................................ 79

10.1 Defatting........................................................................................................ 79

10.2 Protein solubility ............................................................................................ 79

10.2.1 Protein solubility of Tenebrio molitor larvae defatted powder .................. 79

10.2.1.1 Effect of sample and pH value of extraction on protein solubility ...... 79
10.2.1.2 Comparison between samples on protein solubility ......................... 80
10.2.1.3 Comparison between pH values of extraction on protein solubility ... 81

10.2.2 Protein solubility of Acheta domesticus defatted powder ........................ 82

10.2.2.1 Effect of sample and pH value of extraction on protein solubility ...... 82
10.2.2.2 Comparison between samples on protein solubility ......................... 84
10.2.2.3 Comparison between pH values of extraction on protein solubility ... 85

10.3 Gelling ability of the full samples, supernatants and pellets ........................... 86

10.4 Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE).. 89

10.5 Gelling ability of the proteins ......................................................................... 92

10.6 Rheological properties of the proteins ........................................................... 94

10.6.1 Rheological properties of Tenebrio molitor larvae proteins ..................... 94

10.6.2 Rheological properties of Acheta domesticus proteins............................ 97

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11. Conclusion ......................................................................................................... 101

12. Dissemination of results ..................................................................................... 102

13. References ........................................................................................................ 103

14. Appendices ........................................................................................................ 129

Appendix 1. Supplementary table of the included studies in the systematic review

about lipid extraction in edible insects .................................................................. 129

Appendix 2. Supplementary table with freeze-drying conditions overview ............ 142

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List of Figures

Figure 1 - Quantity of edible insect species consumed per country worldwide (From:
Jongema (2012); van Huis et al. (2013)). .................................................................... 16

Figure 2 – Diagram of the article’s inclusion and exclusion. ....................................... 37

Figure 3 - Sankey diagram representation of the studies division into order, species and
life stages. .................................................................................................................. 38

Figure 4 – Example of euthanized Tenebrio molitor larvae used in this study. ........... 51

Figure 5 – Study design from part B. .......................................................................... 52

Figure 6 – Effect of different blanching treatments on colour of non-dried samples (a, b,

c – significantly different results between samples subjected to different blanching
treatments, where: p < 0.05; ns – not significantly different results between samples
subjected to different blanching treatments, where: p ≥ 0.05). .................................... 58

Figure 7 - Effect of different pre-conservation methods on colour of non-dried samples

(a, b – significantly different results between samples subjected to different pre-
conservation methods, where: p < 0.05; ns – not significantly different results between
samples subjected to different pre-conservation methods, where: p ≥ 0.05). .............. 59

Figure 8 – Colour variation (ΔE*) over 120 hours in refrigeration for samples: pre-frozen
with immersion blanching treatment, pre-frozen with steam blanching treatment, pre-
refrigerated with immersion blanching treatment and pre-refrigerated with steam
blanching treatment. ................................................................................................... 60

Figure 9 – Post-drying effect of different pre-conservation methods on colour (a, b –

significantly different results between samples subjected to different pre-conservation
methods in each drying method, where p < 0.05; ns – not significantly different results
between samples subjected to different pre-conservation methods in each drying
method, where: p ≥ 0.05). ........................................................................................... 64

Figure 10 - Post-drying effect of different blanching treatments on colour (a, b –

significantly different results between samples subjected to different blanching
treatments in each drying method, where p < 0.05; ns – not significantly different results
between samples subjected to different blanching treatments in each drying method,
where: p ≥ 0.05). ......................................................................................................... 65
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Figure 11 - Post-drying effect of different post-conservation methods on colour (a, b –

significantly different results between samples subjected to different post-conservation
methods in each drying method, where p < 0.05; ns – not significantly different results
between samples subjected to different post-conservation methods in each drying
method, where: p ≥ 0.05). ........................................................................................... 66

Figure 12 - Effect of different drying methods on colour (a, b, c – significantly different

results between samples subjected to different drying methods, where: p < 0.05). ..... 68

Figure 13 - Effect of different blanching treatments on colour (a, b – significantly different

results between samples subjected to different blanching treatments, where: p < 0.05;
ns – not significantly different results between samples subjected to different blanching
treatments, where: p ≥ 0.05). ...................................................................................... 69

Figure 14 – Example of euthanized Tenebrio molitor larvae and Acheta domesticus used
in this study................................................................................................................. 71

Figure 15 – Study design from part C. ........................................................................ 73

Figure 16 - Study design for the protein extraction and subsequent characterization. 74

Figure 17 - Effect of different samples on protein solubility of Tenebrio molitor larvae (a,
b – significantly different results between samples subjected to different concentrations
and/or treatments in each pH value of extraction, where: p < 0.05). ............................ 81

Figure 18 - Effect of different pH values of extraction on protein solubility of Tenebrio

molitor larvae (a, b, c – significantly different results between samples subjected to
different pH values of extraction in each sample, where: p < 0.05). ............................ 82

Figure 19 - Effect of different samples on protein solubility of Acheta domesticus (a, b, c

– significantly different results between samples subjected to different concentrations
and/or treatments in each pH value of extraction, where: p < 0.05). ............................ 84

Figure 20 - Effect of different pH values of extraction on protein solubility of Acheta

domesticus (a, b – significantly different results between samples subjected to different
pH values of extraction in each sample, where: p < 0.05). .......................................... 85

Figure 21 - Non-reduced (A) and reduced (B) SDS-PAGE gels with supernatant proteins
from Tenebrio molitor larvae and Acheta domesticus extracted with different conditions
(TM – Tenebrio molitor larvae; AD – Acheta domesticus; 1 – Marker; 2 – TM 25%
concentration (w/v) pH4 non-ultrasound; 3 – TM 25% concentration (w/v) pH4
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ultrasound; 4 – TM 25% concentration (w/v) pH7 non-ultrasound; 5 – TM 25%

concentration (w/v) pH7 ultrasound; 6 – TM 25% concentration (w/v) pH10 non-
ultrasound; 7 – TM 25% concentration (w/v) pH10 ultrasound; 8 – Marker; 9 – AD 25%
concentration (w/v) pH4 non-ultrasound; 10 – AD 25% concentration (w/v) pH4
ultrasound; 11 – AD 25% concentration (w/v) pH7 non-ultrasound; 12 – AD 25%
concentration (w/v) pH7 ultrasound; 13 – AD 25% concentration (w/v) pH10 non-
ultrasound; 14 – AD 25% concentration (w/v) pH10 ultrasound; 15 – Marker). ........... 90

Figure 22 – Gelling samples from Tenebrio molitor larvae (1 to 6) and Acheta domesticus
(7 to 12) soluble proteins at different concentrations (w/v) and with or without 0.1M Ca2+
addition (TM – Tenebrio molitor larvae; AD – Acheta domesticus; 1 – TM 5%
concentration (w/v) without calcium; 2 – TM 10% concentration (w/v) without calcium; 3
– TM 15% concentration (w/v) without calcium; 4 – TM 5% concentration (w/v) 0.1M
Ca2+; 5 – TM 10% concentration (w/v) 0.1M Ca2+; 6 – TM 15% concentration (w/v) 0.1M
Ca2+; 7 – AD 5% concentration (w/v) without calcium; 8 – AD 10% concentration (w/v)
without calcium; 9 – AD 15% concentration (w/v) without calcium; 10 – AD 5%
concentration (w/v) 0.1M Ca2+; 11 – AD 10% concentration (w/v) 0.1M Ca2+; 12 – AD
15% concentration (w/v) 0.1M Ca2+). .......................................................................... 93

Figure 23 – Temperature ramp of 10% (w/v) protein dispersions of Tenebrio molitor

larvae (TM) – Elastic modulus (G’) (Pa), viscous modulus (G’’) (Pa) and temperature (ºC)
as a function of time (s) for samples with and without calcium addition. ...................... 94

Figure 24 - Frequency sweep of 10% (w/v) protein dispersions of Tenebrio molitor larvae
(TM) – Elastic modulus (G’) (Pa) and viscous modulus (G’’) (Pa) as a function of
frequency (Hz) for samples with and without calcium addition. ................................... 96

Figure 25 - Amplitude sweep of 10% (w/v) protein dispersions of Tenebrio molitor larvae
(TM) – Elastic modulus (G’) (Pa) and viscous modulus (G’’) (Pa) as a function of shear
strain (%) for samples with and without calcium addition. ........................................... 97

Figure 26 - Temperature ramp of 10% (w/v) protein dispersions of Acheta domesticus

(AD) – Elastic modulus (G’) (Pa), viscous modulus (G’’) (Pa) and temperature (ºC) as a
function of time (s) for samples with and without calcium addition............................... 98

Figure 27 - Frequency sweep of 10% (w/v) protein dispersions of Acheta domesticus

(AD) – Elastic modulus (G’) (Pa) and viscous modulus (G’’) (Pa) as a function of
frequency (Hz) for samples with and without calcium addition. ................................... 99
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Figure 28 - Amplitude sweep of 10% (w/v) protein dispersions of Acheta domesticus

(AD) – Elastic modulus (G’) (Pa) and viscous modulus (G’’) (Pa) as a function of shear
strain (%) for samples with and without calcium addition. ......................................... 100
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List of Tables

Table 1 – Most commonly consumed insect groups around the world (Data from: van
Huis et al. (2013)). ...................................................................................................... 17

Table 2 - Approximate ranges of macronutrient composition (protein and fat) compiled

across various sources for common edible insect species (From: Halloran et al. (2018)).
................................................................................................................................... 19

Table 3 – Digestibility of several edible insect species (From: Noyens et al. (2020)). . 21

Table 4 – Omega-6/3 ratio in different edible insects (Data from: Bednářová et al. (2013);
Finke (2002); Ghosh et al. (2017); Paul et al. (2017); Tzompa-Sosa et al. (2014)). ..... 24

Table 5 – Overview of processing technologies of insects (From: Noyens et al., 2021).31

Table 6 – Main fractions from insects, potential applications and extracting technologies
(From: Noyens et al., 2021) ........................................................................................ 31

Table 7 – Colour values at 0 and 120 hours in refrigeration for samples: pre-frozen with
immersion blanching treatment, pre-frozen with steam blanching treatment, pre-
refrigerated with immersion blanching treatment and pre-refrigerated with steam
blanching treatment. ................................................................................................... 60

Table 8 – Effect of different blanching treatments on dry matter and water activity of dried
samples. ..................................................................................................................... 61

Table 9 - Effect of different pre-conservation methods on dry matter and water activity of
dried samples. ............................................................................................................ 62

Table 10 - Effect of different drying methodologies and blanching treatments on dry

matter and water activity. ............................................................................................ 67

Table 11 - Effect of different pH values of extraction and samples on protein solubility of

Tenebrio molitor larvae. .............................................................................................. 79

Table 12 - Effect of different pH values of extraction and samples on protein solubility of

Acheta domesticus. .................................................................................................... 83
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Table 13 - Gelling ability of the full samples, supernatants and pellets from Tenebrio
molitor larvae extracted at different concentrations of powder (w/v), pH values and
methodologies. ........................................................................................................... 86

Table 14 - Gelling ability of the full samples, supernatants and pellets from Acheta
domesticus extracted at different concentrations of powder (w/v), pH values and
methodologies. ........................................................................................................... 87

Table 15 - Gelling ability of freeze-dried soluble proteins from Tenebrio molitor larvae
and Acheta domesticus at different concentrations of soluble proteins (w/v) and with or
without addition of 0.1M Ca2+. ..................................................................................... 92
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List of Abbreviations

AAs – Amino acids

ANOVA – Analysis of variance

AV – Acid value

aw – Water activity

ca. – Circa

CaCl2 – Calcium chloride

CH4 – Methane

CO2 – Carbon dioxide

CP – Crystallization point

CTAB – Hexadecyl trimethyl ammonium bromide

CVD – Cardiovascular disease

DAGs – Diacylglycerols

DGAV – Direção Geral da Alimentação e Veterinária

DIAAS – Digestible indispensable amino acid score

DM – Dry matter

EFSA – European Food Safety Authority

ERGOs – Ergosterols

EU – European Union

FA – Fatty acid

FAO – Food and Agriculture Organization of the United Nations

FCE – Feed-conversion efficiency

G’ – Elastic modulus

G” – Viscous modulus

GHGs – Greenhouse gases

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HHP – High hydrostatic pressure

IAA – Indispensable amino acid

IV – Iodine value

MAGs – Monoacylglycerols

MC – Moisture content

MP – Melting point

MUFAs – Monounsaturated fatty acids

N – Nitrogen

N2O – Nitrous oxide

NaCl – Sodium chloride

NH3 – Ammonia

PCE – Protein-conversion efficiency

PDCAAS – Protein digestibility-corrected amino acid score

PUFAs – Polyunsaturated fatty acids

PV – Peroxide value

SB – Sulfobetaine

SC-CO2 - Supercritical carbon dioxide

SDBS – Sodium dodecylbenzene sulfonate

SDS-PAGE – Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis

SFAs – Saturated fatty acids

T – Time

T2D – Type-2 diabetes

TAGs – Triacylglycerols

TPC – Total phenolic content

TPP – Three phase partitioning

Tween-20 – Polyoxyethylene (20) sorbitan monolaurate

UFAs – Unsaturated fatty acids

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1. Introduction
1.1 The global food system: An ever-growing demand for food
production

Over the last few decades, a major increase in the world population is noteworthy,
resulting in an intensified demand for food production in the most diverse sectors of the
global food system (FAO, 2017, 2018a). According to the Food and Agriculture
Organization of the United Nations (FAO), it is expected that the world population will
reach 9.7 billion by the year 2050, ensuing in a 32% population growth when compared
to statistics from 2015 (FAO, 2018a). Subsequently to this massive increase, an upsurge
in livestock production and meat consumption is also prominent, specifically in poultry,
beef, pork and sheep meat, estimating total meat consumption values of 410 billion kg
per year until mid-century, corresponding to a 102% increase since the beginning of the
millennium (Boland et al., 2013).

This intensification in meat consumption is not only the result of a large increase
in population but also a consequence of globalization, westernization, economic
(increase in incomes and market liberalization) and social drivers (migration, sedentary
lifestyles and employment of women) that led to dietary convergence and dietary
adaptation even in the most rural environments, therefore contributing to significant
changes in dietary patterns over the last decades (FAO, 2004; Sans & Combris, 2015).

Although the increase in meat consumption and large-scale production of animal-

based proteins has been associated with economic growth and employment for the meat
industry, it has been globally criticized and associated with environmental and
sustainability problems (FAO, 2018a; Garnett, 2011; Godfray et al., 2010). Livestock
production is the leading anthropogenic greenhouse gases (GHGs) source of methane
(CH4) and nitrous oxide (N2O) accounting for 18% of total emissions, mostly due to
ruminants’ enteric fermentation through digestive processes and feed production
respectively (FAO, 2016; Steinfeld et al., 2006; Wang et al., 2018). An increase in carbon
dioxide (CO2) production is also a considerable challenge that results from the clearing
of forests for pasture (FAO, 2016). Despite industrialized stock systems contributing to
fewer GHGs emissions per unit of product compared to livestock, it is worth mentioning
that they also have other major environmental impacts, namely higher levels of pollution
and water withdrawals, intense use of antimicrobials with a higher possible risk of
antimicrobial resistance and a higher risk of zoonotic diseases outbreaks (FAO, 2017,
2018b; Tomley & Shirley, 2009).
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Regarding health and longevity, epidemiological studies on meat consumption,

specifically concerning red meat and processed meat, show that there is a potential
correlation between all-cause mortality and the consumption of this type of meat,
especially when consumed in larger quantities (Abete et al., 2014; Larsson & Orsini,
2014; O'Sullivan et al., 2013). Furthermore, a higher intake of red meat and processed
meat were also associated with a higher risk of cardiovascular disease (CVD) mortality
corresponding to a 16% and 18% increase respectively (Abete et al., 2014). Concerning
type-2 diabetes (T2D) specifically, several meta-analyses verify that a high intake of
processed meat could increase the risk of this pathology (Aune et al., 2009; Feskens et
al., 2013; Micha et al., 2010; Pan et al., 2011). Moreover, accordingly to World Cancer
Research and American Institute for Cancer Research, the consumption of red meat
might increase the likelihood of cancer development, while processed meat consumption
is considered to be carcinogenic to humans (IARC, 2018; Vieira et al., 2017).

From an economic standpoint, meat production has a low feed conversion

efficiency especially in cattle, as well as a low percentage of edible mass, since only 40%
to 55% of the produced biomass of cattle, pigs and poultry are utilized directly as food
(Pimentel & Pimentel, 2003; van Huis et al., 2013; Wilkinson, 2011). Additionally,
substantial land and water usage combined with an ever-growing cost for energy and
feed lead to different scenarios anticipating that meat prices tend to go even higher until
2050 (Sulser et al., 2021; Taghizadeh-Hesary et al., 2019).

Decreasing conventional meat consumption values has so become extremely

relevant in the past few years (Godfray et al., 2018; Rust et al., 2020). This challenging
task has driven many initiatives that consider alternative protein sources that can be
produced more sustainably without compromising nutritional values and attending to
consumer trends and dietary needs (Maurya & Kushwaha, 2019). Grains (wheat and
zein), seeds (rapeseed and chia), pulses (peas, beans and lentils), aquatic plants
(duckweed), algae (microalgae and seaweed), fungus (mycoproteins), milk (whey
proteins), and insects are examples of emerging and sustainable sources of protein that
can be explored to meet world’s growing food demand (Maurya & Kushwaha, 2019; Van
der Spiegel et al., 2013; van der Weele et al., 2019).

1.2 Entomophagy: Insects as an alternative protein source

In 2013, in a FAO forestry paper published by van Huis et al. (2013), the practice
of eating insects, also known as entomophagy, was pointed out as a possible and
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promising alternative to this intensive meat production by the global food system while
simultaneously assuring food and feed security.

The practice of entomophagy is not a new concept since it is a habit and tradition
in many different parts of the world, estimating a total of at least 2 billion people consume
a wide variety of edible insects’ species regularly worldwide (Mitsuhashi, 2016; van Huis
et al., 2013). As exhibited in figure 1, it is noteworthy that Mexico is the leading country
in the consumption of the most edible species of insects, with a total of 549 species
consumed, followed by the Amazon region with a value of 428, Africa with 250 and some
Asian countries also with significant values (Chen et al., 2009; Paoletti et al., 2000;
Ramos-Elorduy, 2008; van Huis, 2005; van Huis et al., 2013; Yhoung-aree &
Viwatpanich, 2005). On the other hand, Europe and North America have very few
species being consumed since consumers’ psychological factors (mainly food neophobia
and disgust towards insects), culture, tradition and availability are crucial components in
countries’ dietary choices (Tan & House, 2018). Nevertheless, and as a consequence of
sustainability trends, the interest in entomophagy is growing in some European countries
in recent years (Jensen & Lieberoth, 2019; Lombardi et al., 2019).

Figure 1 - Quantity of edible insect species consumed per country worldwide (From: Jongema (2012); van
Huis et al. (2013)).

Whole insects can be consumed at different life cycle stages, including eggs,
larvae, pupae, and adults, although the majority of the documented species are
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consumed as larvae or pupae (Lucas et al., 2020). Some species are also consumed in
different life stages (Mitsuhashi, 2016; van Huis et al., 2013). According to van Huis et
al. (2013), and as represented in table 1, the most consumed insects’ orders are
Coleoptera (31%) followed by Lepidoptera (18%), Hymenoptera (14%), Orthoptera
(13%), Hemiptera (10%), Isoptera (3%), Odonata (3%) and Diptera (2%).

Table 1 – Most commonly consumed insect groups around the world (Data from: van Huis et al. (2013)).

Most common Consumption around

Insect order Common name in English
consumption stage the world (percentage)

Coleoptera Beetles Larvae 31%

Lepidoptera Butterflies and moths Larvae 18%

Hymenoptera Ants, bees and wasps Adults/larvae 14%

Cockroaches, crickets,
Orthoptera Adults 13%
grasshoppers and locusts

Cicadas, leafhoppers, planthoppers,

Hemiptera Adults/larvae 10%
scale insects and true bugs

Isoptera Termites Adults 3%

Odonata Dragonflies Adults/larvae 3%

Diptera Flies Larvae 2%

1.3 Benefits of edible insects

1.3.1 Environment and sustainability

Regarding the environmental challenges of the XXI century and the sustainability
of the world, insect farming and entomophagy seem to be advantageous options in
comparison to traditional meat productions (Gahukar, 2016; van Huis et al., 2013). It
requires lower water usage, a smaller land-use (reducing the impact on soil fertility and
deforestation) and has a lower risk of transmitting zoonotic infections to humans in
comparison to birds and mammals (Halloran et al., 2018; van Huis & Oonincx, 2017; van
Huis et al., 2013).

Insects also present a higher digestible biomass percentage when compared to

conventional meat (van Huis et al., 2013). Acheta domesticus (house cricket) and
Tenebrio molitor larvae (yellow mealworm) display digestible biomass percentages of
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80% and 100% respectively, considerable higher values when compared to beef (40%),
pork (55%) or even chicken (55%) (Heckmann et al., 2018; van Huis et al., 2013).

Another major benefit of insect farming is a high feed-conversion efficiency (FCE):

since insects are poikilotherms (“cold-blooded” animals), an extra amount of substrate
to preserve body temperature is not necessary, resulting in a lower ratio of kilogram of
feed intake per kilogram of gained body mass (Collavo et al., 2005; Premalatha et al.,
2011; van Huis et al., 2013). As an example, house crickets’ FCE ranges between 1.3-
1.8 kg of dry feed in order to produce 1 kg of live weight, while in chicken, pig and beef
cattle these values are around 1.7-2.3, 5.9 and 12.7 kg respectively (Lundy & Parrella,
2015). Likewise, a higher protein-conversion efficiency (PCE) ranging from 23–35% is
also noticed in house crickets, in comparison to beef cattle, pigs, and chickens that reveal
PCE values of 5%, 13%, and 25% respectively (Lundy & Parrella, 2015).

Concerning GHGs and ammonia (NH3) production, insects are by far superior
sustainable choices when compared to traditional meat (Oonincx et al., 2010; Steinfeld
et al., 2006). Mealworm larvae, crickets, and locusts release 100 times fewer GHGs and
10 times less NH3 than pigs and beef cattle (Oonincx et al., 2010). Moreover, energy
used in agriculture to produce feed crops for livestock is also accountable for higher
GHGs emissions (FAO, 2016; Gahukar, 2016). In contrast, insects that can be farmed
on organic side streams and “agricultural or food by-products” (such as expired products
from grocery stores, yeasts from beer and wine production or portions of crops that are
not used for human consumption), thus reducing food waste from the food supply chain
as well as displaying economic advantages (Gahukar, 2016; Lundy & Parrella, 2015;
Oonincx & de Boer, 2012; Oonincx et al., 2010).

1.3.2 Nutritional value

Since there are more than 2000 species of edible insects in the world, it is expected
that the nutritional composition would vary substantially between them (van Huis et al.,
2013). Furthermore, insects are eaten at different metamorphological life stages, have
different origins, diets and can be eaten as males or females, all of these factors
contributing to even larger variations between nutritional compositions (Kouřimská &
Adámková, 2016; van Huis et al., 2021; van Huis et al., 2013). Some species, such as
the weaver ant (Oecophylla sp), are usually consumed as eggs, others such as
mealworms (Tenebrio molitor, Alphitobius diaperinus) in a larvae stage, while others are
only consumed in adult stages such as crickets (Gryllus bimaculatus, Acheta
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domesticus) and locusts (Locusta migratoria) (Halloran et al., 2018; van Huis et al.,
2013).
The macronutrient profile of insects can be comparable, to a certain extent, to the
ones found in meat and fish (Halloran et al., 2018). In general, the protein content of
insects ranges between 40-70% of dry matter (DM) with fat levels usually fluctuating
between 5-40% of DM, however fat content as high as 70% of DM is also reported in the
literature in certain species such as the palm weevil larvae (Rhynchophorus phoenicis)
(Rumpold & Schlüter, 2013). Moisture content (MC) in insects is usually around 65%
(Rumpold & Schlüter, 2013). In table 2, displayed in the book Edible Insects in
Sustainable Food by Halloran et al. (2018), it is represented the diverse ranges of protein
and fat content in common edible insect species.

Table 2 - Approximate ranges of macronutrient composition (protein and fat) compiled across various
sources for common edible insect species (From: Halloran et al. (2018)).

Insect order and

Common name in English Life-stage used Protein (% of DM) Fat (% of DM)
species

Crickets Order: Orthoptera

House cricket Acheta domesticus Adults 60-75 7-20

Tropical house cricket Gryllodes sigillatus Adults 60-75 7-20

Grasshoppers/locusts Order: Orthoptera

Migratory locust Locusta migratoria Adults 40-60 10-25

Schistocerca
American grasshopper Adults 40-60 10-25
americana

Flies Order: Diptera

Common house fly Musca domestica Larvae 55-70 10-25

Black soldier fly Hermetia illucens Larvae/prepupae 40-60 20-40

Beetles Order: Coleoptera

Yellow mealworm Tenebrio molitor Larvae 45-55 25-35

Zophobas atratus/
Giant mealworm Larvae 40-50 40-45
morio

Lesser mealworm Alphitobius diaperinus Larvae 45-60 25-30

Moths Order: Lepidoptera

Greater wax moth Galleria mellonella Larvae 35-45 40-60

Lesser wax moth Achroia grisella Larvae 35-45 40-60

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Silkworm Bombyx mori Larvae/pupae 50-70 8-10

1.3.2.1 Protein quality

Protein consumption is crucial when addressing the world’s current public health
challenges such as obesity and age-related muscle loss problems such as sarcopenia
(Schoufour et al., 2021; Walston, 2012). Meat, eggs, and milk are traditional animal-
based, protein-dense foods that are considered high-quality protein sources since they
fulfil all of the current indispensable amino acid (IAA) needs and are effectively digested
and absorbed (defined in terms of the balance of amino acids (AAs) across the small
intestine) (van Vliet et al., 2015; Wolfe, 2015).
Concerning insects, the orders Coleoptera, Hymenoptera, Lepidoptera, and
Orthoptera often have AA contents that meet or even surpass IAA requirements for
adults (Churchward-Venne et al., 2017). However, the protein and AA content of edible
insects is quite variable since different feeds, development stages, gender, place and
time of collection and processing methods play a huge role in the total assessments
(Churchward-Venne et al., 2017; van Huis et al., 2013).
Notwithstanding meeting the IAA requirements for adults, the proteins’ digestibility
needs to be known in order to provide a complete picture regarding protein quality
(Churchward-Venne et al., 2017; Halloran et al., 2018). This protein quality has been
assessed using a variety of methods, such as the protein efficiency ratio, biological value,
chemical score, net protein utilization, and protein digestibility-corrected amino acid
score (PDCAAS) (Churchward-Venne et al., 2017; FAO, 2013). The PDCAAS method
evaluates protein digestibility over the total gut (referred to as ‘faecal’ protein or
digestibility) and was the standard index for assessing protein quality for many years
(FAO, 2013). However, the digestible indispensable amino acid score (DIAAS) is
currently the accepted method for assessing protein quality for use in human nutrition
(FAO, 2013). DIAAS assessment of protein quality is determined on the AA requirement
pattern and the AA digestibility at the end of the small intestine (from the mouth to the
terminal ileum; also referred to as ‘ileal’ digestibility) and is usually only performed in rats
or pigs since an ileostomy is needed to collect digesta of the individuals in repeated trials
(Churchward-Venne et al., 2017; FAO, 2013). Thus far, no DIAAS evaluations on insect
protein have been published.
Nonetheless, there are several studies in the current literature about insects’
apparent digestibility (ratio of nitrogen ingested to the one that is excreted) and true
digestibility (difference between the AA given to the individual and the ones found in the
digesta) (Tomé, 2021). Table 3, retrieved from Noyens et al. (2020), shows a compilation
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of studies performed in-vitro and in-vivo (in pigs, rats and chickens), on protein
digestibility of several edible insect species. As can be observed, the majority of the
species have digestibility values ranging from 66-95%. Nevertheless, methodologies for
assessing digestibility vary throughout the different studies and should be standardised
and additional species of edible insects should be explored on this specific subject
(Churchward-Venne et al., 2017; Noyens et al., 2020). Furthermore, none of the
experiments were conducted on humans, thus the results could change significantly
(Noyens et al., 2020).

Table 3 – Digestibility of several edible insect species (From: Noyens et al. (2020)).

Edible insects Digestibility (%) Comments Reference

Coleoptera (beetles, grubs)

Holotrichia paralela larvae 78.4 Assessed in-vitro Yang et al. (2014)

Yellow mealworm (Tenebrio

Assessed in pigs;
molitor larvae) (mixed meal 90.3 Jin et al. (2016)
apparent digestibility
containing 1.5% larvae)

T. molitor larvae (mixed meal Assessed in pigs;

91.3 Jin et al. (2016)
containing 3.0% larvae) apparent digestibility

T. molitor larvae (mixed meal Assessed in pigs;

92.2 Jin et al. (2016)
containing 4.5% larvae) apparent digestibility

T. molitor larvae (mixed meal Assessed in pigs;

93.0 Jin et al. (2016)
containing 6.0% larvae) apparent digestibility

Assessed in chickens;
T. molitor larvae 86.0 Marco et al. (2015)
apparent digestibility

T. molitor larvae 91.3 Assessed in-vitro Bosch et al. (2014)

T. molitor larvae 66.3 Assessed in-vitro Marono et al. (2015)

T. molitor larvae 66.7 Assessed in-vitro Marono et al. (2015)

T. molitor larvae 65.5 Assessed in-vitro Marono et al. (2015)

T. molitor larvae 66.2 Assessed in-vitro Marono et al. (2015)

T. molitor larvae 65.8 Assessed in-vitro Marono et al. (2015)

T. molitor larvae 66.2 Assessed in-vitro Marono et al. (2015)

Giant mealworm (Zophobas morio

92.0 Assessed in-vitro Bosch et al. (2014)
larvae)
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Edible insects Digestibility (%) Comments Reference

Diptera (flies)

Black soldier fly (Hermetia illucens)

67.1 Assessed in-vitro Marono et al. (2015)
larvae

Assessed in chickens;
H. illucens larvae 68.0 Marco et al. (2015)
apparent digestibility

H. illucens larvae 67.3 Assessed in-vitro Marono et al. (2015)

H. illucens larvae 67.6 Assessed in-vitro Marono et al. (2015)

H. illucens larvae 68.7 Assessed in-vitro Marono et al. (2015)

H. illucens larvae 66.8 Assessed in-vitro Marono et al. (2015)

H. illucens larvae 66.0 Assessed in-vitro Marono et al. (2015)

H. illucens larvae 89.7 Assessed in-vitro Bosch et al. (2014)

H. illucens pupae 77.7 Assessed in-vitro Bosch et al. (2014)

Common house fly (Musca

84.3 Assessed in-vitro Bosch et al. (2014)
domestica) pupae

Hemiptera (true bugs)

Ramos-Elorduy et al.
Ahuahutle 89.3 Assessed in-vitro
(1997)

Ramos-Elorduy et al.
Axayacatl 98.0 Assessed in-vitro
(1997)

Hymenoptera (ants, bees)

Assessed in rats;
European honey bee (Apis
apparent digestibility,
mellifera) (18.8% of mixed meal; 62.0 Ozimek et al. (1985)
not corrected for N in
whole dried)
chitin

Assessed in rats;
A. mellifera (18.8% of mixed meal;
68.5 apparent digestibility, Ozimek et al. (1985)
whole dried)
corrected for N in chitin

Assessed in rats; true

A. mellifera (18.8% of mixed meal;
71.5 digestibility, not Ozimek et al. (1985)
whole dried)
corrected for N in chitin

Assessed in rats; true

A. mellifera (18.8% of mixed meal;
79.8 digestibility, corrected Ozimek et al. (1985)
whole dried)
for N in chitin
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Edible insects Digestibility (%) Comments Reference

A. mellifera (16.6% of mixed meal; Assessed in rats;

87.6 Ozimek et al. (1985)
protein concentrate) apparent digestibility

A. mellifera (16.6% of mixed meal; Assessed in rats; true

94.3 Ozimek et al. (1985)
protein concentrate) digestibility

Ramos-Elorduy et al.
Leaf cutter ant (Atta Mexicana) 87.6 Assessed in-vitro
(1997)

Mexican honey wasp Ramos-Elorduy et al.

85.2 Assessed in-vitro
(Brachygastra mellifica) (1997)

Ramos-Elorduy et al.
Polybia parvulina 86.4 Assessed in-vitro
(1997)

Southern yellowjacket wasp Ramos-Elorduy et al.

76.6 Assessed in-vitro
(Vespula squamosa) (1997)

Orthoptera (crickets, hoppers,

termites)

Tree locust (Anacridium

49.9 Assessed in-vitro Hassan et al. (2008)
melanorhodon)

Tropical house cricket (Gryllodes

76.2 Assessed in-vitro Stone et al. (2019)
sigillatus)

Winged termite (Macrotermes

90.1 Assessed in-vitro Kinyuru et al. (2009)
subhylanus)

Longhorn grasshopper (Ruspolia

79.6 Assessed in-vitro Kinyuru et al. (2009)
differens)

1.3.2.2 Fat quality

The fatty acid (FA) composition of foods establishes their nutritional fat quality
(Halloran et al., 2018). Thus, this fat quality is characterized by the amount of saturated
fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty
acids (PUFAs) in a specific food (Halloran et al., 2018; Rumpold & Schlüter, 2013).
Replacing SFAs with PUFAs in human eating habits has been shown to decrease the
incidence of CVD and T2D, alongside with the consumption of long-chained omega-3
PUFAs being advantageous for infants and young children’s brain development (FAO,
2010; Lauritzen et al., 2001; Lenighan et al., 2019).
Even though fat quality on edible insects is very variable and feed dependant,
edible insects can be considered good sources of essential omega-3 (α-linolenic acid)
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and omega-6 (linoleic acid) FAs in general (Barroso et al., 2017; Kouřimská &
Adámková, 2016; Rumpold & Schlüter, 2013).
As humans, essential omega-3 and omega-6 FAs must come from our diet, and
while omega-6 FAs may be obtained from a variety of dietary sources, omega-3 FAs
may be insufficient in some eating patterns, particularly the ones low on animal products
(Halloran et al., 2018; Panchal & Brown, 2021). Therefore, the omega-6/3 ratio is
frequently used to characterize FA quality, since a lower ratio indicates a good source of
omega-3 FAs (Halloran et al., 2018; Panchal & Brown, 2021).
In edible insects, the omega-6/3 ratio is extremely variable between different
species (Bednářová et al., 2013; Finke, 2002; Ghosh et al., 2017; Paul et al., 2017;
Tzompa-Sosa et al., 2014). As represented in table 4, the majority of edible insects have
an inadequate FA quality, since they have a high omega-6/3 ratio (Paul et al., 2017;
Tzompa-Sosa et al., 2014). However, in some specific species, such as in the meadow
grasshopper (Chorthippus parallelus) and the silkworm (Bombyx mori) pupae, this ratio
was found to be 1:3, thus being lower and more beneficial to the ratio found in meat
sources and comparable to the ones observed in marine fishes and seafood (Bednářová
et al., 2013; DiNicolantonio & O'Keefe, 2021; Finke, 2002; Michaelsen et al., 2011; Paul
et al., 2017).

Table 4 – Omega-6/3 ratio in different edible insects (Data from: Bednářová et al. (2013); Finke (2002);
Ghosh et al. (2017); Paul et al. (2017); Tzompa-Sosa et al. (2014)).

Edible insects Omega-6/3 ratio Extraction method Reference

Coleoptera (beetles, grubs)

Allomyrina dichotoma larvae 36.00 Soxhlet extraction Ghosh et al. (2017)

Lesser mealworm (Alphitobius

20.42 Soxhlet extraction Tzompa-Sosa et al. (2014)
diaperinus larvae)

A. diaperinus larvae 21.96 Folch extraction Tzompa-Sosa et al. (2014)

A. diaperinus larvae 19.04 Aqueous extraction Tzompa-Sosa et al. (2014)

Protaetia brevitarsis
25.00 Soxhlet extraction Ghosh et al. (2017)
larvae

Yellow mealworm (Tenebrio

25.98 Soxhlet extraction Tzompa-Sosa et al. (2014)
molitor larvae)

T. molitor larvae 27.15 Folch extraction Tzompa-Sosa et al. (2014)

T. molitor larvae 24.35 Aqueous extraction Tzompa-Sosa et al. (2014)

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Edible insects Omega-6/3 ratio Extraction method Reference

Conventional solvent
T. molitor larvae 204.15 Paul et al. (2017)
extraction

T. molitor larvae 69.73 Soxhlet extraction Ghosh et al. (2017)

T. molitor larvae 17.76 Not determined (ND) Finke (2002)

Giant mealworm (Zophobas morio

29.18 ND Finke (2002)
larvae)

Hymenoptera (ants, bees)

European honey bee (Apis

1.89 Soxhlet extraction Bednářová et al. (2013)
mollifera) brood

Lipidoptera (moths, butterflies)

Silkworm (Bombyx mori larvae) 2.5 ND Finke (2002)

B. mori pupae 0.35 Soxhlet extraction Bednářová et al. (2013)

Greater waxworm (Galleria

21.16 Soxhlet extraction Bednářová et al. (2013)
mellonella)

Orthoptera (crickets, hoppers,

termites)

House cricket (Acheta domesticus) 13.26 Soxhlet extraction Tzompa-Sosa et al. (2014)

A. domesticus 16.85 Folch extraction Tzompa-Sosa et al. (2014)

A. domesticus 12.82 Aqueous extraction Tzompa-Sosa et al. (2014)

Conventional solvent
A. domesticus 37.04 Paul et al. (2017)
extraction

A. domesticus 38.17 ND Finke (2002)

Meadow grasshopper Conventional solvent

0.33 Paul et al. (2017)
(Chorthippus parallelus) extraction

Long-winged conehead Conventional solvent

25.08 Paul et al. (2017)
(Conocephalus discolor) extraction

Two-spotted cricket (Gryllus

53.13 Soxhlet extraction Ghosh et al. (2017)
bimaculatus)

Migratory locust (Locusta

5.55 Soxhlet extraction Bednářová et al. (2013)
migratoria) nymph

Black field cricket (Teleogryllus

43.13 Soxhlet extraction Ghosh et al. (2017)
emma)
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1.3.2.3 Vitamins and minerals

Insects are often consumed entirely, with the edible portion comprising all tissue
types, including the head and internal organs, thus providing a higher content of vitamins
and minerals when compared to animal-source foods where some parts are not
considered edible for humans (Halloran et al., 2018; van Huis et al., 2013).
Although mineral values in edible insects vary widely between species and orders,
insects are generally rich sources of minerals like iron and zinc, which are also minerals
which are frequently insufficient in the eating patterns of low- and middle-income nations
(Finke, 2015; IFPRI, 2016; Kinyuru et al., 2013; Rumpold & Schlüter, 2013).
Despite generally showing low amounts of calcium and potassium per 100 g of
DM, edible insects are not only an excellent source of iron and zinc but also potentially
good sources of magnesium, phosphorous and selenium (Finke, 2002; Rumpold &
Schlüter, 2013). Additionally, most edible insects have adequate amounts of copper and
manganese with only termites and beetles displaying low manganese levels (Finke,
2002; Rumpold & Schlüter, 2013).
In terms of vitamin requirements in human adults, a 100 g serving of insects based
on DM is often high in riboflavin, pantothenic acid, and biotin (Kouřimská & Adámková,
2016; Rumpold & Schlüter, 2013). Moreover, insects of the order Coleoptera and
Orthoptera are abundant in folic acid (Rumpold & Schlüter, 2013). However, 100 g of
insects based on DM do not provide a sufficient amount of vitamin A, vitamin C, niacin,
or, in most cases, thiamine (Kouřimská & Adámková, 2016; Rumpold & Schlüter, 2013).

1.4 Major challenges to the consumption of edible insects

As previously acknowledged, there are several challenges to the introduction of

edible insects into Western countries, with three of them being the major ones:
uncertainty about safety and allergen risk; legislation in terms of consumption and
production; and Western consumers’ perception and acceptability towards entomophagy
(FAO, 2021; La Barbera et al., 2018; Ribeiro et al., 2018; Wendin & Nyberg, 2021).

1.4.1 Food safety and allergen risk

Last year, FAO published a report that provides an outline of numerous food safety
concerns that may be linked to edible insects (FAO, 2021). In this report, biological
(bacteria, viruses, fungus, and parasites), chemical (mycotoxins, heavy metals,
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pesticides, and antimicrobials), and physical hazards are seen as food safety issues
(FAO, 2021).
The likelihood of allergenicity is also explored in this report and concludes that
more research is necessary (FAO, 2021). It is noteworthy that individuals who are allergic
to crustaceans and house dust mites are more prone to allergic responses to edible
insects due to allergen cross-reactivity (FAO, 2021; Ribeiro et al., 2018). There is also a
risk of developing sensitization to unidentified allergens present in insects (FAO, 2021;
Ribeiro et al., 2018).
Entomophagy has safety concerns depending on the species, the feed, the habitat
where they are produced or harvested, and also on production and processing
techniques applied (Belluco et al., 2013; FAO, 2021). Nonetheless, it is important for
well-established organizations such as FAO, to recognize and assess these potential
food safety threats, in order to establish good hygiene and manufacturing practices
around the world (FAO, 2021).
Insects are usually eaten whole, and contamination from feed or housing materials
might accumulate (Belluco et al., 2013; FAO, 2021). In addition, because of their small
size, collecting insects to decontaminate can be a difficult task, and any contamination
may be carried throughout the manufacturing and processing chain (FAO, 2021;
Rumpold & Schlüter, 2013).
Insect farming under regulated hygienic conditions and the application of sanitary
processing procedures should decrease several risks, such as microbial contamination
(FAO, 2021; Imathiu, 2020). Moreover, precautions should be made to prevent insects
from escaping from manufacturing facilities since certain food and feed species, such as
cockroaches and houseflies, are also pests and vectors of foodborne diseases (Belluco
et al., 2013; FAO, 2021). If the insect species being farmed escape to the wild and are
not native to the area, it is likely to have an influence on the local environment and
ecosystem (FAO, 2021).
However, to determine the safety and shelf-stability of insect-based products used
in human food and animal feed, more scientific research is required (FAO, 2021; Imathiu,
2020). New reports on human consumption of edible insects will promote insights into
potential exposure to microbiological and chemical contaminants (Belluco et al., 2013).

1.4.2 Legislation

In most countries, there is no insect-specific legislation, guidelines, labelling, or

other regulation in action to regulate the production and commercialization of insects in
feed and food supply chains (FAO, 2021; Imathiu, 2020). This lack of such a framework
is a serious obstacle to the establishment of economies for insects and insect-based
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products (FAO, 2021). Governments in both developing and developed economies must
focus the utmost priority on safety policies and regulations to assure a risk-free supply
of edible insects or edible insect food products to the final customer from farm to fork
(FAO, 2021; Imathiu, 2020). Due to consumers' growing awareness of their legal rights
to high-quality and food safety, many consumers' eating habits have dramatically altered
in recent years, thus being undoubtedly relevant for Western consumers, who tend to be
particularly hesitant to adopt entomophagy (Imathiu, 2020).
In regions where entomophagy is prevalent, which is usually the case in developing
countries, it is often seen as a local tradition which is not highly regulated (Dobermann
et al., 2017). However, the scenario is extremely different in Western nations, where the
majority of the countries are in the process of creating, evaluating, and/or implementing
policies and guidelines on this specific issue (FAO, 2021).
In the European Union (EU), and under the regulation (EU) No 2015/2283, all
insect-based foods intended for human consumption are seen as novel foods (EU, 2015;
FAO, 2021). According to this legislation, consumers in the EU have access to a broad
variety of safe, distinct, and innovative food choices, including those from developing
nations (EU, 2015). Since last year and until now, the European Food Safety Authority
(EFSA) ruled that the yellow mealworm (Tenebrio molitor larvae), the house cricket
(Acheta domesticus), the migratory locust (Locusta migratoria), and the lesser mealworm
(Alphitobius diaperinus larvae) were safe for human consumption under the
recommended dosages and applications (Turck et al., 2021a, 2021b, 2021c, 2021d,
2022a, 2022b). In addition to this law, several EU member countries, such as Holland,
Denmark, Austria and Belgium have their own legal policies regarding edible insect
commerce for consumption as human food according to the respective food safety
authorities (Imathiu, 2020).
For instance, in Portugal, the Direção Geral da Alimentação e Veterinária (DGAV)
authorized the inclusion of seven species of edible insects to be used and
commercialized in Portuguese territory, under transitory measurements, as long as they
follow under two conditions, accordingly to the 2015/2283 EU Commission legislation
regarding novel foods (DGAV, 2021; EU, 2015). The first condition is that these novel
foods need to be legally on the market, in an EU country, before the 1st of January 2018,
while the second condition is that the product’s commercialization must have been
requested prior to the 1st of January 2019 (DGAV, 2021). Taking that into consideration,
the following species can be produced, commercialized and incorporated into the
Portuguese market: Acheta domesticus, Alphitobius diaperinus, Apis mellifera (male
pupae), Gryllodes sigillatus, Schistocerca gregaria, Locusta migratoria and Tenebrio
molitor (DGAV, 2021). These species must be commercialized as an entire insect (dead),
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or ground, for example, as flour, although separated fractions (lipid and protein) cannot
be integrated into food products at the moment (DGAV, 2021).

1.4.3 Consumer perception and acceptability

Despite an emerging focus on entomophagy, only a few Western consumers are

inclined to incorporate insects into their current diets (Caparros Megido et al., 2014;
Cunha & Ribeiro, 2019). This Western aversion toward edible insects is the total opposite
of what happens in numerous countries in Africa, Asia and Latin America where a wide
range of species are valued and marketed for their delicious taste (Halloran et al., 2018;
Menozzi et al., 2017; van Huis et al., 2021).
Although there are several psychological factors to the acceptance of edible
insects, disgust and neophobia are frequently mentioned as the two primary ones for the
rejection of this food (Cunha et al., 2014; Mandolesi et al., 2021). These factors are quite
expected since our relationship with food is defined by insecurity or even fear of new and
unknown products, particularly insects, which are supposed to be dangerous, as well as
interest in exploring different types of foods (Wendin & Nyberg, 2021). This interest factor
is particularly important when overcoming disgust, as acceptance is also influenced by
emotions such as excitement, wildness, and sensation seeking (Lammers et al., 2019;
Nyberg et al., 2021; Tuccillo et al., 2020).
An increase in familiarity, by trying to increase exposure to a certain food, is also
a key component of consumer acceptance and a decrease in feelings of fear and disgust
(Barton et al., 2020; Batat & Peter, 2020; Castro Delgado et al., 2020). Various authors
have demonstrated the impact of incorporating insects into familiar products (such as
cookies, bars, burgers, pasta and bread) or adding them to favourite recipes to enhance
familiarity (Batat & Peter, 2020; Delicato et al., 2020; Sidali et al., 2019; van Huis et al.,
2021). Nonetheless, the wide variety of edible insect species and the different serving
techniques should be considered regarding the level of familiarity (Lombardi et al., 2019;
Wendin & Nyberg, 2021). A familiar physical and social context, such as a food festival
or a friends’ gathering, can also be important for an increase in willingness to try insect-
based foods (Motoki et al., 2020).
The sensory attributes of insects and insect-based foods are critical for them to be
assessed as appealing and pleasurable by consumers (Cunha & Ribeiro, 2019;
Lammers et al., 2019; Ribeiro et al., 2019; Sogari et al., 2019; van Huis et al., 2021;
Wendin & Nyberg, 2021). Insect visibility in food products is an important sensory
attribute since visible insects or insect parts (head, body, wings or legs) may induce
negative associations (Castro Delgado et al., 2020; Cicatiello et al., 2020; Gedrovica,
2019; Thielen et al., 2018). Nonetheless, additional sensory attributes such as taste,
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texture and odour are also major concerns (Cunha & Ribeiro, 2019; Gedrovica, 2019;
Rumpold & Langen, 2019). It is noteworthy that odour and flavour attributes can be
negatively impacted by the use of whole-fat insect powders in comparison to the defatted
ones when included in fruit and cereal bars (Ribeiro et al., 2019).
Social and cultural interactions with friends, co-workers, family and even social
media can also change the consumer perception of certain food products (Batat & Peter,
2020). In the acceptance of edible insects, this factor is quite noticeable since it is still
considered a food taboo for the majority of Western consumers (Iannuzzi et al., 2019;
Schäufele et al., 2019). Moreover, gender seems to have an impact on moral and ethical
concerns regarding entomophagy, with men usually showing a higher level of
acceptance compared to women (Lammers et al., 2019; Tuccillo et al., 2020).
The lack of knowledge available about the importance of changing protein sources
sustainability-wise, and about the different cooking methods and dishes involving edible
insects is also a significant obstacle to entomophagy (Gedrovica, 2019). Thereby,
education allied with tasting sessions at food-related events or canteens could be a smart
approach to battle this issue (Jones, 2020; Wendin & Nyberg, 2021).

1.5 Novel insect-based food products and processing technologies

Over the last decade, numerous insect-based food products were created in other
to improve their overall acceptance (Mandolesi et al., 2021; van Huis et al., 2021). These
products include insects as a whole or as a powder, a wide variety of insect-based
snacks (bars, crisps, cookies), insects as a meat substitution (sausages, burgers), insect
flours mixed in staple foods (bread, pasta) and even in products such as butter or ice
cream (Caparros Megido et al., 2016; Carcea, 2020; Delicato et al., 2020; Hartmann &
Siegrist, 2016; Hernández Toxqui et al., 2021; Kim et al., 2016; Ribeiro et al., 2019;
Roncolini et al., 2019; Sriprablom et al., 2022).
Nonetheless, procedures such as processing, cooking, packaging, conservation
and transportation are able to impact sensory attributes (aroma, appearance, taste and
texture), as well as changes in quality, microbial load and shelf life time to the final
product, thus affecting the acceptance by the market and potential consumers (Elhassan
et al., 2019; Marone, 2016). Since there are relatively few studies focusing on specific
insects in terms of quality, conservation and microbial load, more research is necessary
in these fields (Elhassan et al., 2019).
Regarding processing specifically, several technologies are applied to various
insect species in order to create insect-based food products, and can be categorized into
four subcategories: heat, cold, shred treatments and others (table 5) (Noyens et al.,
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2021). These technologies are usually linked together to provide a complete process
flow that includes multiple stages, transforming the whole insect into an ingredient in a
specific product (Noyens et al., 2021).

Table 5 – Overview of processing technologies of insects (From: Noyens et al., 2021).

Heat treatments Cold treatments Shred treatments Others

Ultrasound-assisted
Steaming Freeze-drying Crushing
extraction

Roasting Freezing Pulverizing Fluidized bed drying

Cold atmospheric pressure

Smoking Grinding Marinate
plasma

Frying Milling Fermentation

Stewing Mashing Microwave-drying

Drying (Sun-, Oven-) Mincing Spray drying

Toasting Dry fractionation

Curing

Blanching

Apart from these technologies previously mentioned, it is also possible to obtain

different fractions from edible insects, mostly protein, lipids and chitin, thus expanding
their range of applications and economic interest even further (Baiano, 2020; Noyens et
al., 2021). The main methods and applications for protein, lipid and chitin extractions are
exhibited in table 6.

Table 6 – Main fractions from insects, potential applications and extracting technologies (From: Noyens et
al., 2021)

Fraction Application/use Extraction method

Chemical de-proteinization
Chitin Biomaterial (weak acid and alkaline
extraction)

Antimicrobial

Oil-binder

Protein Protein supplement Aqueous extraction:

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Fraction Application/use Extraction method

Functional ingredient - Ionic strength

- pH shift

Organic solvents

Enzymatic hydrolysis

Dry fractionation (sieve)

Defatting

Sonication

Microwave

Lipid Edible oils Aqueous extraction

Source of essential fatty acids Defatting (organic solvent)

Supercritical CO2

Ultrasounds

Chitin extraction from insects is usually performed due to its ability to bond with
numerous links, forming materials such as fibers, films, hydrogels, sponges and
membranes (Zainol Abidin et al., 2020). Chitosan production derived from chitin
extraction is also receiving plenty of attraction since it can be used in agriculture,
biomedicine and cosmetical and pharmaceutical industries (Anitha et al., 2014; Danti et
al., 2019; Mezzana, 2008; Puvvada et al., 2012; Xing et al., 2015).
Protein extraction can be performed subsequently to lipid extraction or at the same
time as oils are being fractionated and the most common methods use pH shift or
isoelectrical solubilization precipitation, enzymatic extractions and ionic strength by
adding sodium chloride (NaCl) to an aqueous extraction buffer solution (Jiang et al.,
2021; Purschke et al., 2018b; Queiroz et al., 2021; Smets et al., 2020). Thermal
treatments prior to the extraction are also popular, although they can lead to protein
modification when executed at higher temperatures (Noyens et al., 2021).
Lipid extractions are most commonly used as a step prior to protein extraction but
they can also be utilized to extract edible oils or for conversion into energy through
biodiesel production (Alves et al., 2019; Lee et al., 2021; Son et al., 2020; Tzompa-Sosa
et al., 2019; Wang et al., 2017). Supercritical CO2 (SC-CO2), aqueous and organic
solvent extractions are the most frequent types of lipid extractions in edible insects (Kim
et al., 2019; Ribeiro et al., 2019; Sipponen et al., 2018; Tzompa-Sosa et al., 2019;
Tzompa-Sosa et al., 2014).
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Nonetheless, in order to understand the suitability of insect fractions as ingredients

in food products, techno-functional properties need to be evaluated (Mishyna et al.,
2021). Since different processing technologies have an influence on solubility, water and
oil-holding capacity, foaming, emulsifying, and gelling properties, it is crucial to fully
understand these effects to identify appropriate food applications for certain insect
ingredients (Mishyna et al., 2021; Villaseñor et al., 2021). For instance, very few research
was conducted regarding gelling ability and rheological properties of insect proteins thus
being a noteworthy area of investigation (Kumar et al., 2022; Villaseñor et al., 2021).
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2. Aims

Upon scientific research, it is noteworthy that there are noticeable differences in

entomophagy practices and acceptance around the globe, as insects are often perceived
in Western countries with feelings of disgust and neophobia. However, in the last few
years, trends regarding sustainability have emerged and revealed edible insects and
insect-based food products as promising solutions to an ever-growing demand in meat
production. Yellow mealworms (Tenebrio molitor larvae) and house crickets (Acheta
domesticus), are two of the main edible insects associated with this upsurge of food
products, since they have lower GHGs and NH3 production, a high FCE and percentage
of digestible biomass, a high protein and PUFAs content, and rich in several vitamins:
such as riboflavin, pantothenic acid, and biotin; and minerals: such as iron and zinc.
Therefore, since research is limited in this field, it is important to investigate how different
processing technologies affect crucial parameters on edible insects.

Firstly, in the initial stage of the project, it was conducted a systematic review on
lipid extraction methodologies from edible insects, evaluating the effectiveness of the
extractions and subsequent characterizations and applications.

Furthermore, a study on pre-treatment conditions of Tenebrio molitor larvae was

performed at GreenUPorto - Sustainable Agrifood Production Research Centre, in
Vairão. Different blanching, drying and conservation techniques were compared and
their impacts on dry matter, water activity and colour were assessed.

Lastly, as part of an Erasmus + Traineeship at the Department of Food Science of

the University of Copenhagen, soluble proteins from defatted Tenebrio molitor larvae
and Acheta domesticus powders were extracted using different concentrations of
defatted powder, pH and extraction technologies. Additionally, protein solubility,
molecular weight, gelling ability and rheological properties of previously extracted soluble
proteins were studied and analysed.
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Part A – Systematic review on lipid extraction

methodologies from edible insects

3. Materials and methods

In order to perform this systematic review, scientific research was conducted on

three online databases (PubMed/Medline, Scopus and Web of Science) in November of
2021. The same query was used in all three databases: ("edible insect" OR
entomophagy OR mealworm OR cricket OR grasshopper OR locust OR silkworm OR
termite OR fly OR moth OR beetle OR cockroach OR cicada OR bee OR wasp) AND
(fat OR oil OR lipid) AND (defat* OR extract* OR fraction*). There were no restrictions
implemented neither on the date of publication nor the language of the articles.

In addition, studies previously known that were suitable and acceptable based on
the inclusion criteria described in section 3.1, were also included in the review.
References of included studies and scientific reviews regarding entomophagy and edible
insects were assessed to identify possible studies that could be appropriate to be
included and to confirm the query applied on the online databases.

3.1 Inclusion and exclusion criteria

In this systematic review, only original publications that were written in English
were taken into consideration. Therefore, this assessment did not include studies written
in other languages, book chapters, review articles or conference abstracts.

Concerning the inclusion criteria, original articles that applied lipid extraction
methodologies and quantified the efficiency of the techniques were included in this study.
Articles working with commercial powders were included when subsequent defatting was
performed and evaluated.

In regards to exclusion criteria, articles only assessing the nutritional composition

of the extracted oil or that exclusively characterized the defatted fraction were not
included.
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3.2 Study selection and data analysis

The screening of the articles obtained from the database research was conducted
independently by two authors. After duplicates removal, authors selected articles for full-
text reading by analysing their title and abstract. Following full-text evaluation, authors
decided on the inclusion of the articles for in-depth analysis. Moreover, references that
were cited in the included articles were assessed by the authors for relevance and
matching of the inclusion criteria, since they might not have been included through the
online database research. For the included studies, data were collected concerning
authors, year of publication, insect species, life stage, pre-treatment, extraction methods
and conditions, efficiency of extraction (determined by lipid content, yield and/or oil
recovery), characterization and application of the extracted lipids and main conclusions
about the study. Any disagreement in the inclusion of a particular article was solved by
consensus between the authors.
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4. Results and Discussion

A total of 7557 articles were obtained from the scientific research and, following
the removal of 2408 duplicates that were in the three online databases, 5149 articles
were selected for reading of the title and abstract. Following this preliminary assessment,
a total of 77 articles were collected for full-text reading, of which 46 of them were included
in this systematic review. Furthermore, three additional publications, that were not
present in the online database research, were added to this review as they were relevant
and met the inclusion criteria stated in section 3.1 (figure 2).

Figure 2 – Diagram of the article’s inclusion and exclusion.

These 49 included articles were equivalent to 68 studies, since each insect species
and/or life stage were counted as a single study. The most studied species were H.
illucens (n = 26, 38.2%), T. molitor (n = 17, 25%), A. domesticus (n = 7, 10.3%) and B.
mori (n = 5, 7.4%). Concerning insects’ order, the majority of the studies were on Diptera
(n = 26, 38.2%), Coleoptera (n = 22, 32.4%) and Orthoptera (n = 11, 16.2%). As for life
stages, larvae (n = 46, 67.6%) was the most researched followed by adult (n = 13,
19.1%), pupae (n = 7, 10.3%) and prepupae (n = 2, 2.9%) (figure 3).
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Figure 3 - Sankey diagram representation of the studies division into order, species and life stages.

In appendix 1, a supplementary table developed for a better understanding of the

included studies on this systematic review is available. This table, comprises data
regarding the reference of the article, the species and life stage of the studied insect, the
pre-treatments used, the extraction methods, the efficiency of the extraction (determined
by lipid content, yield and/or oil recovery) and also the characterization and application
of the extracted oils. For a clearer interpretation, this table is alphabetically organized,
firstly by the insects’ order, secondly by the scientific name and life stage of the studied,
thirdly by the first author’s last name and lastly by the year of the publication.

4.1 Lipid extraction methodologies

It is noteworthy that, of the 68 analysed studies, a total of 103 methodologies were

used to perform the lipid extractions. The vast majority (n = 88, 85.4%) worked with dried
whole insects or powders, 14 (13.6%) performed the extractions with non-dried products
and 1 (1.0%) worked with aqueous extracts.

Out of these 103 methodologies, the most commonly applied ones were
conventional extraction (n = 29, 28.2%), Soxhlet extraction (n = 24, 23.3%), aqueous
extraction (n = 10, 9.7%), Supercritical carbon dioxide (SC-CO2) extraction (n = 8, 7.8%)
and microwave-assisted extraction (n = 5, 4.9%). Folch extraction (n = 4, 3.9%),
ultrasound-assisted extraction (n = 4, 3.9%), screw press extraction (n = 3, 2.9%) and
several other methods were also applied however with less frequency.
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In regards to conventional extraction, that was defined by the authors as an

extraction with homogenization with a solvent followed by filtration and/or centrifugation
with posterior removal of the solvent, the most frequently used solvents were hexane (n
= 14, 28.8%), n-hexane (n = 8, 17.0%) and ethanol (n = 5, 10.6%). Additionally, several
conventional extractions were also performed with mixed solvents (n = 10, 21.3%).
Concerning Soxhlet extraction, the most commonly used solvents were petroleum ether
(n = 16, 38.1%), hexane (n = 9, 21.4%) and ethanol (n = 5, 11.9%). As for microwave-
assisted extraction, petroleum ether (n = 4, 23.5%), n-hexane (n = 3, 17.6%) and
chloroform (n = 2, 11.8%) were the most preferred solvents.

4.2 Efficiency of the extractions

Tzompa-Sosa et al. (2014) explored the lipids of four different insect species
extracted with three different methods: aqueous extraction, Soxhlet extraction with
petroleum ether and Folch extraction with a mixture of dichloromethane and methanol.
In all four different species, the lowest yields were observed in aqueous extractions
(Blaptica dubia adult – 3.1%; Alphitobius diaperinus larvae – 5.5%; Tenebrio molitor
larvae – 7.8%; Acheta domesticus adult – 1.6%). Soxhlet extractions resulted the highest
yields for B.dubia adult (7.6%) and A.diaperinus larvae (10.7%) and Folch extractions
led to the highest yields for T.molitor larvae (12.9%) and A.domesticus adult (8.0%).

Similarly, T. K. Kim et al. (2021) reported that aqueous extraction led to the lowest
efficiency on Protaetia brevitarsis larvae. The lipid content was the highest on the
defatted powder extracted with aqueous extraction (14.82%) in comparison to
conventional extractions with ethanol (0.13%), n-hexane (0.32%) and methanol (1.17%).

In a study by Saviane et al. (2021) on Hermetia illucens larvae, aqueous extraction

also resulted in a lower efficiency (yield – 4% to 6.55%) when compared to conventional
extraction with ether as a solvent (yield – 25.55%) and screw press extraction (yield –
26.11%) despite showing a higher overall efficiency comparatively to Naviglio extraction
(yield – 0.28% to 5.31%).

Sun et al. (2018) assessed the yields of Clanis bilineata larvae lipids extracted with
two different methodologies and discovered that aqueous extraction with an ultrasound
assistance (yield – 5.39% to 19.49%) also led to lower efficiencies comparatively to
Soxhlet extraction with petroleum ether as a solvent (yield – 24.56%).

Laroche et al. (2019) explored the lipids from T.molitor larvae and A.domesticus
adult extracted with three different methods: Soxhlet extraction (with four different
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solvents), three phase partitioning (TPP) and SC-CO2. Both species had the lowest
efficiencies with SC-CO2. Regarding T.molitor larvae, Soxhlet extraction led to higher
yields with all four different solvents (ethanol – 28.8%; ethyl acetate – 25.7%; hexane –
25.5%; petroleum ether – 24.3%), followed by TPP (23.7%) and lastly SC-CO2 (22.1%).
As for A.domesticus adult, Soxhlet extraction with ethanol led to highest yield (22.7%)
followed by TPP (19.3%), Soxhlet extraction with the three other solvents (ethyl acetate
– 15.1%; petroleum ether – 14.7%; hexane – 14.6%) and lastly SC-CO2.

Otero et al. (2020) also explored the lipids from T.molitor larvae and A.domesticus
adult but with two different methodologies: ultrasound-assisted extraction and
pressurized-liquid extraction; and using two different solvents: ethanol and a mixture of
ethanol and water in a ratio of 1:1 (v/v). Concerning T.molitor larvae, pressurized-liquid
extractions resulted in higher yields (ethanol:water – 33.87%; ethanol – 32.37%) in
comparison to ultrasound-assisted extractions (ethanol – 28.85%; ethanol:water –
17.14%). As far as A.domesticus adult, the different solvents resulted in major
differences in yields of pressurized-liquid extractions (ethanol – 24.85%; ethanol:water –
5.02%) while having relatively similar results in both ultrasound-assisted extractions
(ethanol – 15.48%, ethanol:water – 15.05%).

The lipids from the two previous species were also studied by Ugur et al. (2021)
but with conventional extraction and high hydrostatic pressure (HHP)-assisted
extraction, both using hexane as a solvent. T.molitor larvae and A.domesticus adult
showed higher overall yields with conventional extractions (T.molitor larvae – 24.07% to
24.22%; A.domesticus adult – 18.05% to 18.09%) comparatively to HHP-assisted
extractions (T.molitor larvae – 22.75% to 22.90%; A.domesticus adult – 16.17% to
17.41%).

Purschke et al. (2017) compared the oil recovery of SC-CO2 and conventional
extraction methodologies on T.molitor larvae. In this study, conventional extraction with
n-hexane led to a higher efficiency of extraction (oil recovery – 96.56%) relatively to SC-
CO2 even at optimal conditions of extraction of 250 bar at 45ºC for 105 minutes (oil
recovery – 95.30%).

Son et al. (2019) also studied T.molitor larvae but compared conventional
extraction with hexane as a solvent with different screw press extractions. As a result,
the defatted powder from conventional extraction had a lower lipid content (1.98%)
compared to the defatted powder from screw press extractions (13.22% to 14.45%).

Zhao et al. (2016) also performed conventional extractions on T.molitor larvae but
using ethanol and a mixture of hexane and isopropanol in a ratio of 3:2 (v/v) with the
addition of Na2SO4. Conventional extractions with ethanol resulted in higher yields
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(33.1% to 34.8%) in comparison to the extraction with the mixture of hexane and
isopropanol (31.6%).

Regarding H.illucens larvae, C. Wang et al. (2017) investigated the effectiveness

of microwave-assisted extractions with several different solvents. Thus, the highest yield
was achieved with methanol (43.26%), followed by a mixture of chloroform and methanol
in a ratio of 2:1 (v/v) (34.06%), chloroform (33.75%), a mixture of hexane and methanol
in a ratio of 3:2 (v/v) (32.51%), hexane (31.78%), petroleum ether (31.32%) and lastly
acetone (25.58%).

Feng et al. (2018) also investigated the effectiveness of microwave-assisted

extractions with three different solvents in H.illucens larvae. Extractions with chloroform
led to a higher oil recovery (82% to 89%), followed by n-hexane (78% to 83%) and
petroleum ether (76% to 83%). In a follow-up study in the same species, Feng et al.
(2020) tested microwave-assisted extractions with eight different solvents. Oil recovery
was the highest in the mixture of petroleum ether and isopropanol in a ratio of 1:1 (v/v)
(75.92%) and the lowest when ethanol was used as a solvent (29.34%). All solvents that
were a mixture of different solvents resulted in higher oil recoveries in contrast to solvents
used individually. In an additional follow-up, also in H.illucens larvae, Feng et al. (2021)
compared conventional extraction with ethyl acetate as a solvent with surfactant-assisted
extractions with ethyl acetate as a solvent and using different types of surfactants
solutions. All surfactant-assisted extractions improved the extraction to some degree by
having a higher oil recovery (CTAB – 78.99%; SB – 72.95%; SDBS – ca. 70%; Tween-
20 – ca. 65%) in comparison to conventional extraction (64.29%).

Hao et al. (2021) also studied the yield of extraction of H.illucens larvae lipids by
using either microwave-assisted extractions or Soxhlet extraction with petroleum ether
and noticed that this last one led to higher yields (34.24%) in comparison to microwave-
assisted extractions with different microwave powers, sample to solvent ratios and
extraction times (8.9% to 31.0%).

Similarly, Ishak et al. (2018) also performed Soxhlet extractions on H.illucens

larvae and found that ethanol led to a higher lipid yield (58%) relatively to petroleum ether
(56%) and acetone (50%).

Also concerning H.illucens larvae, Lee et al. (2021) verified a higher yield with oil
expeller press extraction (37.23%) comparatively to conventional extraction with hexane
as a solvent (26.97%).

Ravi et al. (2019) accessed the efficiency of extraction of H.illucens larvae lipids
with Soxhlet extraction or multistage cross-current lipid extraction, both methodologies
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using either n-hexane or 2-methyloxolane as solvents. As verified, both solvents led to

higher yields in Soxhlet extraction (2-methyloxolane – 35.83%; n-hexane – 32.51%) in
comparison to multistage cross-current lipid extraction (2-methyloxolane – ca. 20%; n-
hexane – ca. 15%). Nonetheless, 2-methyloxolane revealed higher yields in both
methodologies relatively to n-hexane.

S. W. Kim et al. (2019) found that SC-CO2 led to a lower lipid content on the
defatted powder of H.illucens larvae relatively to hot water extraction (15.97%).

In a study by Smets et al. (2021) on H.illucens larvae lipids, it was evaluated the
yields of Soxhlet extractions and wet lipid extractions with either 2-methyltetrahydrofuran
or hexane as solvents. In both methodologies, 2-methlytetrahydrofuran (Soxhlet
extraction – 43.23%; wet lipid extraction – 41.02%) led higher yields in comparison to
hexane (Soxhlet extraction – 40.34%; wet lipid extraction – 28.36%).

Also on H.illucens larvae, Su et al. (2019) investigated the effects of enzymatic pre-
treatments on a conventional extraction with n-hexane as a solvent. All five different
enzymes led to higher yields of extraction (protamex – 18.17% to 36.10%; flavourzyme
– 13.52%; papain – 12.14%; champzyme – 11.68%; bromelain – 11.62%) relatively to
the conventional extraction without previous enzymatic treatment (8.10%).

Similarly, Caligiani et al. (2018) tested four different enzymes and their
effectiveness on extracting lipids from H.illucens prepupae relatively to conventional
extraction with petroleum ether. As a result, conventional extraction led to a substantially
higher oil recovery (87%) in comparison to all four enzymatic assisted extractions
(B.licheniformis protease – 10%; papain – 10%; pepsin – 10%; pancreatin – 10%).

T. Wang et al. (2020) compared different solvents used for conventional extraction
of H.illucens larvae lipids. A mixture of petroleum ether and isopropanol in a ratio of 1:1
(v/v) resulted in the highest yield (38.01%) relatively to four other solvents (ethyl
acetate:water – 27.12% to 29.04%; ethyl acetate – 28.71%; n-hexane – 15.60%;
petroleum ether – 13.92%).

In a study by Pan et al. (2012) on Antheraea pernyi pupae, Soxhlet extraction

resulted in a higher efficiency of extraction (yield – 28.08%) when compared to SC-CO2
(yield – 26.18%).

Regarding Bombyx mori pupae, Hu et al. (2017) assessed the yields of extraction
with Soxhlet extraction with n-hexane as a solvent and with microwave-assisted
extractions with five different solvents. As a result, Soxhlet extraction led to a relatively
similar yield (30.42%) comparatively to microwave-assisted extraction at optimal
conditions with a mixture of ethanol and n-hexane in a ratio of 1:1 (30.16%).
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Likewise, Wei et al. (2009) compared the yield of B.mori pupae oil extraction with
Soxhlet extraction with hexane (30.34%) to SC-CO2 at optimal conditions (324.5 bar,
39.6ºC, 131.2 minutes and 19.3 L/h) (29.48%).

Amarender et al. (2020) explored the effectiveness of extraction of cricket (species

not specified) lipids with conventional extraction with either ethanol or hexane as
solvents. Ethanol led to a lower lipid content on the defatted powder (9.27%) relatively
to hexane (11.98%).

In a study by Ribeiro et al. (2019), lipids from two species of crickets were extracted
via Soxhlet with five different solvents. Regarding A.domesticus adult, the higher yield
was found in extractions performed with ethanol (28.2%), followed by acetone (24.6%),
petroleum ether (21.3%), diethyl ether (20.8%) and lastly hexane (18.9%). As for
Gryllodes sigillatus adult, ethanol also led to the highest yield (28.4%), followed by
acetone (23.5%), diethyl ether (20.8%), hexane (20.8%) and lasty petroleum ether
(20.2%).

Also on crickets, Jeong et al. (2021) performed conventional extractions with three
different solvents on Gryllus bimaculatus adult. As a result, the lipid content of the
defatted powders was lower when ethanol was used as a solvent (0.73%), followed by
n-hexane (1.83%) and acetone (2.56%).

4.3 Characterization

Out of the 68 studies, the most common characterizations of the extracted lipids
were on fatty acid (FA) composition (n = 41, 60.3%), lipid classes (n = 12, 17.6%),
peroxide value (PV) (n = 8, 11.8%) and acid value (AV) (n = 7, 10.3%).

The different characterizations were arranged, accordingly to their assessments,

in five categories: chemical composition, oxidative stability, physical properties, bioactive
properties and microscopic changes.

4.3.1 Chemical composition

Tzompa-Sosa et al. (2014) assessed FA compositions, lipid classes and free

cholesterol of B.dubia adult, A.diaperinus larvae, T.molitor larvae and A.domesticus adult
lipids extracted with aqueous extraction, Soxhlet extraction or Folch extraction. As main
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findings, the authors concluded that for all four insect species, aqueous extractions had
the highest contents of omega-3 FAs and Folch extractions led to the lowest contents of
omega-6 FAs with the exception of B.dubia adult. Free FAs and partial glycerides were
found in lipids from Folch and Soxhlet extractions while absent in lipids from aqueous
extractions. In addition, Folch extractions led to the highest contents of free cholesterol
while the lowest contents were found in aqueous extractions for all four insect species.
The authors address that the extractions where organic solvents were used (Soxhlet and
Folch) extract wide ranges of lipid classes in comparison to aqueous extraction that
predominantly extract non-polar lipids.

Laroche et al. (2019) evaluated FA compositions of T.molitor larvae and

A.domesticus adult lipids extracted with Soxhlet extraction (with hexane, petroleum
ether, ethyl acetate or ethanol as solvents), TPP or SC-CO2. As primary results for both
insects, Soxhlet extractions with ethanol as a solvent and TPP led to the lowest C16:0
contents while the highest contents were found in lipids from SC-CO2 extractions. For
Acheta domesticus adult, the highest C18:0 contents were found in Soxhlet extractions
with hexane as a solvent and TPP. For both insects, the highest contents of
transvaccenic acid were found in SC-CO2 extractions. Additionally, and regarding both
species, the highest C18:2 contents were observed in Soxhlet extractions with ethanol
as a solvent and TPP. According to the authors, the polarity of the solvents is a crucial
factor regarding the extraction of different FAs, since polar solvents such as ethanol and
t-butanol (used in Soxhlet extraction and TPP respectively) are more effective at
extracting C18:2, as non-polar solvents exhibited higher C18:1 and C16:0 contents.

In a study by Otero et al. (2020), FA compositions, lipid classes and cholesterol

contents of T.molitor larvae and A.domesticus adult lipids extracted with ultrasound-
assisted extraction or pressurized-liquid extraction, both using either ethanol or a mixture
of ethanol and water in a ratio of 1:1 (v/v) as solvents, were assessed. Ultrasound-
assisted extractions with the mixture of ethanol and water resulted in the most favourable
fatty acid profiles by exhibiting the highest PUFAs contents and the lowest MUFAs, SFAs
and cholesterol contents as well as the lowest atherogenicity and thrombogenicity
indices for lipids of both insects.

Also concerning T.molitor larvae and A.domesticus adult, Ugur et al. (2021)
characterized FA compositions from lipids extracted with HHP-assisted or conventional
extraction, both with hexane as a solvent, and at temperature of either 30ºC or 40ºC. As
main findings, the authors concluded that overall, for T.molitor larvae, extractions at 40ºC
for both conventional and HHP-assisted extractions result in the highest UFAs contents,
as for A.domesticus adult, the highest UFAs contents were observed at 40ºC for HPP-
assisted extractions. Furthermore, total FA contents are significantly lower at 30ºC in
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HHP-assisted extractions in comparison to conventional extractions at the same

temperature for both species.

Purschke et al. (2017) compared T.molitor larvae lipids extracted with conventional
extraction with n-hexane as a solvent and the ones extracted with SC-CO2 extraction in
terms of FA compositions. Lipids extracted with conventional extraction exhibited a
significantly higher UFAs content in regards to C16:1, C18:1 and C18:2 contents, in
comparison to lipids extracted with SC-CO2, despite showing no significant differences
between C14:0, C16:0, C:18:0 and C18:3 contents.

Regarding H.illucens larvae, Feng et al. (2021) determined FA compositions of

lipids extracted with surfactant-assisted extractions with ethyl acetate as a solvent and
using different types of surfactants solutions. As a result, CTAB-assisted extraction led
to the highest C10:0 content, SB-assisted extraction to the highest C12:0 content, SDBS-
assisted extraction to the highest C14:0 and C18:0 contents and Tween-20-asisted
extraction to the highest C16:1 content in the larvae lipids. The conventional extraction
with ethyl acetate led to the highest C16:0, C18:1 and C18:2 contents comparatively to
the surfactant-assisted extractions.

Also on H.illucens larvae, Ravi et al. (2019) explored FA compositions and lipid
classes of lipids extracted with Soxhlet extraction with either n-hexane or 2-
methyloxolane as solvents. SFAs, MUFAs and PUFAs contents were slightly higher in
lipids extracted with n-hexane. N-hexane extracted lipids led to higher monoacylglycerols
(MAGs), ergosterols (ERGOs) and triacylglycerols (TAGs) relative contents while 2-
methyloxolane led to higher diacylglycerols (DAGs) and free FAs relative contents.
Phospholipids were only found in lipids extracted with 2-methyloxolane.

Smets et al. (2021) also compared FA compositions and lipid classes of H.illucens
larvae lipids extracted with Soxhlet extractions or wet lipid extractions with either 2-
methyltetrahydrofuran or hexane as solvents. Overall, FA compositions did not differ
significantly between both methodologies. Lipids extracted with Soxhlet extraction with
both solvents, led to higher TAGs, free FAs, cholesterols, phosphatidylcholines and
phosphatidylethanolamines contents in comparison to wet lipid extractions with both
solvents. Soxhlet extracted lipids also led to lower DAGs (only in hexane samples) and
lower MAGs contents in comparison to wet lipid extraction (when settings were optimized
for achieving highest recovery).

In a study by Hu et al. (2017), B.mori pupae was explored in terms of FA

compositions of lipids extracted with Soxhlet extraction with n-hexane as a solvent or
microwave-assisted extraction with a mixture of ethanol and n-hexane in a ratio of 1:1
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(v/v) as a solvent. As a result, no significant differences in FA compositions were found

between lipids extracted with the two methodologies.

Sun et al. (2018) assessed FA compositions of C.bilineata larvae lipids extracted

with ultrasound-assisted aqueous extraction or extracted with Soxhlet extraction with
petroleum ether as a solvent and discovered that lipids extracted with ultrasound-
assisted aqueous extraction led to significantly higher PUFAs and lower MUFAs. No
significant differences between methodologies were found in SFAs and omega-3/6 ratio
of extracted lipids.

4.3.2 Oxidative stability

Ugur et al. (2021) studied PVs of T.molitor larvae and A.domesticus adult lipids
extracted with HHP-assisted or conventional extraction, both with hexane as a solvent,
and at temperature of either 30ºC or 40ºC. For T.molitor larvae, the increase in
temperature caused an increase in the PV regardless of the methodology. As for
A.domesticus adult, significant differences were found in all extraction conditions with
the lowest PV observed in lipids extracted with conventional extraction at 30ºC.

Purschke et al. (2017) examined T.molitor larvae lipids extracted with conventional
extraction with n-hexane as a solvent and the ones extracted with SC-CO2 extraction in
terms of AVs and free FAs but no significant differences were found between the two
methodologies on both measurements.

Feng et al. (2020) determined saponifiable lipids purities of H.illucens larvae lipids
extracted with microwave-assisted extractions with eight different solvents. Petroleum
ether and a mixture of petroleum ether and isopropanol in a ratio of 1:1 (v/v) were the
solvents that led to lipids with significantly higher saponifiable lipids purities in
comparison to the remaining solvents. The lipids extracted with ethanol displayed the
lowest percentage. In a follow-up study in the same species, Feng et al. (2021) compared
saponifiable lipids purities of lipids extracted with surfactant-assisted extractions with
ethyl acetate as a solvent and using different types of surfactants solutions even though
no significant differences were found between the different surfactants solutions on this
measurement.

Hu et al. (2017) studied PVs, Avs and iodine values (IVs) of B.mori pupae lipids
extracted with Soxhlet extraction with n-hexane as a solvent or microwave-assisted
extraction with a mixture of ethanol and n-hexane in a ratio of 1:1 (v/v) as a solvent.
Although IVs between lipids from both methodologies showed no significant differences,
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lipids extracted with microwave-extraction had a significantly lower AV and PV

comparatively to lipids extracted with Soxhlet extraction. According to the authors, lower
PV and AV could be good quality indicators for microwave-assisted extracted lipids. In
addition, the authors assert that a lower AV could also indicate less rancidity.

In regards to C.bilineata larvae, Sun et al. (2018) examined IVs, AVs, PVs,
saponification values and p-Anisidine values of lipids extracted with ultrasound-assisted
aqueous extraction or extracted with Soxhlet extraction with petroleum ether as a
solvent. Although lipids extracted with ultrasound-assisted aqueous extraction led to a
significantly higher IV and saponification value, they exhibited a significantly lower AV,
PV and p-Anisidine value relatively to lipids extracted with Soxhlet extraction.

4.3.3 Physical properties

Ugur et al. (2021) analysed crystallization (CP) and melting points (MP) of T.molitor
larvae and A.domesticus adult lipids extracted with HHP-assisted or conventional
extraction, both with hexane as a solvent, and at temperature of either 30ºC or 40ºC.
Regarding T.molitor larvae, HHP-assisted extraction led to lower CPs with no significant
differences observed in MPs. The authors say that a possible explanation for these
results could be that a higher UFAs content in HHP-assisted extracted lipids of T.molitor
larvae may have caused lower CPs. As for A.domesticus, HHP-assisted extracted lipids
led to lower CPs. In regards to MP, it was the highest in lipids extracted with conventional
extraction at 30ºC and the lowest in lipids extracted with HHP-assisted extraction at
40ºC. According to the authors, since hydrophobic interactions between lipids are fairly
sensitive to pressure and since liquid lipids at room temperature form crystals under
pressure, this could increase the MP of triglycerides in A.domesticus adult.

Concerning B.mori pupae, Hu et al. (2017) studied refractive indices and specific
gravity of lipids extracted with Soxhlet extraction with n-hexane as a solvent or
microwave-assisted extraction with a mixture of ethanol and n-hexane in a ratio of 1:1
(v/v) as a solvent. Both physical properties exhibited no significant differences between
methodologies.

Sun et al. (2018) examined density and thermal stability of C.bilineata larvae lipids
extracted with ultrasound-assisted aqueous extraction or extracted with Soxhlet
extraction with petroleum ether as a solvent. Despite no significant differences in density
between lipids extracted with both methodologies, lipids extracted with ultrasound-
assisted aqueous extraction had a higher thermal stability.
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4.3.4 Bioactive properties

Ugur et al. (2021) assessed total phenolic contents (TPC) and antioxidant activities
of T.molitor larvae and A.domesticus adult lipids extracted with HHP-assisted or
conventional extraction, both with hexane as a solvent, and at temperature of either 30ºC
or 40ºC. Regarding TPCs of T.molitor larvae, it was noticed that lipids extracted with
HHP-assisted extraction increased TPC at 30ºC but reduced it at 40ºC in comparison to
lipids extracted with conventional extraction. As for TPCs of A.domesticus adult, lipids
extracted with HHP-assisted extraction had an increased TPC at 30ºC in comparison to
lipids extracted with conventional extraction, with no significant changes between both
methodologies upon an increase in temperature. Overall, antioxidant activities increased
in lipids extracted with HHP-assisted extraction and with an increase in temperature in
both species. The authors presume that lipids extracted with HHP-assisted extraction
could disrupt the cells walls due to pressure, releasing antioxidant compounds into the
extracellular environment that can have result in different antioxidant activities.
Furthermore, the authors mention that an increase in temperature might increase the
extraction rate and the subsequent recovery of antioxidant compounds.

Regarding H.illucens larvae, Ravi et al. (2019) analysed TPCs and radical
scavenging capacities of lipids extracted with Soxhlet extraction with either n-hexane or
2-methyloxolane as solvents. As a results, both TPC and radical scavenging capacity
were higher in lipids extracted with 2-methyloxolane. Authors indicated that bioactive
properties of lipids extracted with 2-methyloxolane could result in a lower lipid
peroxidation at different temperatures and consequently improving the functionality of
these lipids for potential applications.

As for B.mori pupae, Hu et al. (2017) determined TPCs and antioxidant activities
of lipids extracted with Soxhlet extraction with n-hexane as a solvent or microwave-
assisted extraction with a mixture of ethanol and n-hexane in a ratio of 1:1 (v/v) as a
solvent. Lipids extracted with microwave-assisted led to a higher TPC and antioxidant
activity comparatively to lipids extracted with Soxhlet extraction. According to the
authors, the increase in TPC in lipids extracted with microwave-assisted extraction could
be due a rupture of the cells by the microwave treatment that lead to a higher release of
phenolics into the oils in short time. In addition, the presence of ethanol, a polar solvent,
in microwave-assisted extraction, could also lead to a higher extraction of phenolics. In
regards to antioxidant activity, authors presume that a higher activity in lipids extracted
with microwave-assisted extraction could in part be due to a higher TPC.
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Sun et al. (2018) compared C.bilineata larvae lipids extracted with ultrasound-
assisted aqueous extraction and with Soxhlet extraction with petroleum ether as a
solvent and discovered that lipids extracted with ultrasound-assisted aqueous extraction
had a significantly higher antioxidant activity when submitted to DPPH radical
scavenging and β-carotene bleaching tests relatively to lipids extracted with Soxhlet
extraction.

4.3.5 Microscopic changes

Hu et al. (2017) evaluated the microscopic changes, via scanning electron

microscopy, of B.mori pupae lipids extracted with Soxhlet extraction with n-hexane as a
solvent or microwave-assisted extraction with a mixture of ethanol and n-hexane in a
ratio of 1:1 (v/v) as a solvent. As a result, the scanning electron microscopy micrographs
exhibited that the microwave-assisted extraction promoted the cell disruption and the
release of lipids from the pupae, thus being a promising methodology for lipid extraction.

4.4 Applications

The vast majority of the studies did not use the extracted lipids for further
applications (n = 58, 85.3%). Even so, eight articles (11.8%) used the extracted lipids for
biodiesel related purposes, one article (1.5%) incorporated the lipids in fish diets and one
article (1.5%) used them for nano-emulsions.

Despite not addressing the efficiency of the lipid extractions, several articles can
be found in the scientific literature that used insects from several edible insects’ species
for animal feed as aim for optimal nutrition and health (Belghit et al., 2018; Benzertiha et
al., 2019; Gasco et al., 2019; Kierończyk et al., 2018; Li et al., 2016; Sypniewski et al.,
2020). Moreover, sensory studies can also be found in the scientific literature where
edible insects’ lipids were used (Tzompa-Sosa et al., 2021, 2022). Nonetheless,
additional studies regarding deodorization and optimization of sensory attributes on
edible insects’ lipid fractions are needed.
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5. Conclusion

The current systematic review provided a deepening in knowledge on lipid

extraction methodologies from edible insects, evaluating the effectiveness of the
extractions and subsequent characterizations and applications.

The most studied insects’ order was Diptera, the species was H.illucens and the
life stage was larvae. As for methodologies, the use of solvents is the most common
procedure for lipid extraction in edible insects with conventional and Soxhlet extractions
being the most applied ones. Overall, it was noticed that solvent extractions resulted in
higher efficiencies that can be affected by the polarity of the solvents that were used.
Despite aqueous extractions generally resulting in lower efficiencies they can lead to a
better lipid quality comparatively to solvent extractions. Furthermore, few were the
studies that did further application of the extracted lipids even though scientific literature
can be found on the use of edible insects’ lipids for animal feed and even for sensory
studies.

Future studies on edible insects should aim to use greener solvents or more
sustainable methodologies for lipid extractions such as aqueous extractions, ultrasound-
assisted extractions, and press extractions. Moreover, further studies on deodorization
and optimization of sensory attributes on edible insects’ lipid fractions are also needed.

In brief, the aims of the current systematic review were accomplished. The review
summarized the scientific literature on edible insects’ lipids for more sustainable, mindful
and conscious choices by the food industry and research colleagues.
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Part B – Study of the effect of blanching, drying

and conservation conditions on quality
parameters of Tenebrio molitor larvae

6. Materials and methods

6.1 Insects

Frozen T.molitor larvae (figure 4) were kindly supplied by TecmaFoods

(Matosinhos, Portugal) to GreenUPorto - Sustainable Agrifood Production Research
Centre, located in Vairão, Portugal. All the insects were transferred from the Styrofoam
freezer box to press seal bags before conservation. For the purpose of the study, some
larvae were kept at refrigeration temperatures (4ºC) while others at freezing
temperatures (-24ºC).

Figure 4 – Example of euthanized Tenebrio molitor larvae used in this study.

6.2 Study design

As displayed in figure 5, the focus of the study was on pre-treatment conditions of

Tenebrio molitor larvae. Different blanching, drying and conservation techniques were
compared and their impacts on dry matter (DM), water activity (aw) and colour were
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assessed. These three characterization methodologies (DM, aw and colour) were

selected to be evaluated since they can be good indicators of quality in certain foods.

Figure 5 – Study design from part B.

6.2.1 Pre-conservation

Following the reception of the frozen larvae, some were kept in press seal plastic
bags in the refrigerator at 4ºC while others in the freezer at -24ºC, in order to evaluate
the conservation impact before the blanching treatments that were performed 24 hours
afterwards.

6.2.2 Blanching

Both larvae at 4ºC and at -24ºC were subjected for blanching treatments by
immersion or steam.

Immersion blanching was performed according to Santos et al. (2021). Briefly, the
larvae were immersed in water at 100ºC for 5 minutes in a ratio of 1:10 (w/v). After
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blanching, the water with the larvae was poured onto a granulometric sieve, leaving the
blanched larvae that were transferred to identified press seal plastic bags.

Steam blanching was carried out in a steam cooker machine (Tefal® model
VS400333 VitaCuisine Compact). Before the placement of the larvae in the basket, the
steam cooker was filled with water, until the recommended maximum level, and it was
used for 1 minute, in order to warm the water before the blanching. The larvae were
placed in the perforated basket until the full covering of the bottom part. The basket with
the larvae was inserted in the steam cooker machine and the blanching was performed
for 5 minutes. Following blanching, the excess water was removed from the holes in the
perforated basket and the blanched larvae were transferred to identified press seal
plastic bags.

6.2.3 Post-conservation

After the blanching treatment, the larvae from the four different conditions (pre-
refrigeration and immersion blanching; pre-refrigeration and steam blanching; pre-
freezing and immersion blanching; pre-freezing and steam blanching) were divided
according to post-conservation. Therefore, and similarly to the pre-conservation
conditions, half of the larvae from each condition were kept in press seal plastic bags in
the refrigerator at 4ºC while the other half was kept in the freezer at -24ºC.

6.2.4 Drying

Three different drying methodologies were investigated to see how they might
affect the quality parameters of T.molitor larvae: electrical oven drying, microwave-
assisted drying and freeze-drying. These methodologies were applied to all the eight
conditions, with the exception of the freeze-drying that was not performed in samples
with a refrigerated post-conservation, since sample must be frozen for the freeze-drying
procedure to work properly. For all drying conditions, samples were conserved for 24
hours before drying.
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6.2.4.1 Electrical oven drying

Electrical oven drying was performed in a conventional oven (Unox® model XF016-
TG) and accordingly to Purschke et al. (2018a). Briefly, larvae were placed in a tray, until
the bottom was completely filled, and then the tray was placed into the conventional oven
at 80ºC for 7 hours. Soon after, the larvae were ground in a kitchen robot (Kenwood®
Major Titanium with the multi mill attachment model AT320A) for further characterization.

6.2.4.2 Microwave-assisted drying

Microwave-assisted drying was also executed, as previously studied by

Vandeweyer et al. (2017) and Lenaerts et al. (2018), however with different conditions
and in a conventional microwave (Teka® MW 21 IVS INOX). In summary, the larvae were
distributed in a ceramic plate and dried at 800 W for 5 minutes. Then, the larvae were
ground in the kitchen robot with the multi mill attachment for further characterization.

6.2.4.3 Freeze-drying

For freeze-drying, the frozen larvae were placed into the drying racks that were
then transferred to the shelves of the freeze-dryer (Telstar® LyoQuest) chamber. The
freeze-dryer was used with a vacuum pump (Ulvac® model GLD136C) for 3 hours of
freezing, 3 hours of cool + vacuum (0.250 mbar) and 24 hours of heat shelves (35°C.
0.250 mbar). Heat shelves times of 48 and 72 hours were also tested. After freeze-drying,
the larvae were ground in the kitchen robot with the multi mill attachment for further
characterization.

As mentioned previously in section 6.2.4, freeze-drying was not performed in

samples with a refrigerated post-conservation, since sample must be frozen for the
freeze-drying procedure to work properly.
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6.3 Characterization

To examine the quality of the T.molitor larvae when submitted to different

methodologies of conservation, blanching and drying, three characterizations were
assessed: dry matter (DM), water activity (aw) and colour.

6.3.1 Dry matter

To determine the DM content of the samples, petri dishes were identified and
weighed (W1), and samples were then weighed (W2) in each one of the petri dish.
Subsequently, the petri dishes with the samples were placed in an air-flow lab incubator
(Binder® APT. line series model ED115) at 95ºC for 24 hours. After this time, the petri
dishes with the dried samples were weighed (W3). The calculation of the DM content
was accomplished by the use of the following formula:

𝑾𝟑 − 𝑾𝟏
𝑫𝒓𝒚 𝑴𝒂𝒕𝒕𝒆𝒓 (%) = + × 𝟏𝟎𝟎
𝑾𝟐

where,

W1 – weight of the petri dish (g);

W2 – weight of the sample (g);

W3 – weight of the petri dish with the sample after 24 hours in the air-flow lab
incubator at 95 ºC (g).

DM content was determined on both non-dried and dried samples. For non-dried
samples, DM was assessed before blanching (either frozen or refrigerated samples) and
in post-blanched conserved samples (either frozen or refrigerated samples). For the non-
dried samples, the analysis was performed in the whole larvae, while the dried samples
were ground prior to analysis. All measurements were performed in triplicates and with
the assistance of an analytical balance (Ohaus Explorer® model E01140).

6.3.2 Water activity

The aw value was measured in triplicates on all the samples that were dried and
ground with the assistance of a water activity meter (Rotronic® HygroLab 3).
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6.3.3 Colour

For colour determination it was used a colourimeter (Minolta® Chroma Meter model
CR-400) in a CIELAB colour space (L*a*b*), where the L* is the lightness value (that
defines black as 0 and white as 100), the a* axis corresponds to the green–red opponent
colours (where negative values correspond towards green and positive ones towards
red) and the b* axis corresponds to blue-yellow opponent colours (where negative values
correspond towards blue and positive ones towards yellow).

The measurements were performed in four different study stages as earlier

displayed in the study design in section 6.2.

In the first stage colour measurements were used as a control, since it was
measured in pre-conserved whole larvae samples, both refrigerated and frozen, before
any blanching treatment.

In the following stages, colour measurements were performed in blanched and

post-conversed whole larvae samples to investigate the total colour variation over five
different time (T) periods (T0, T3, T24, T48, T120) for a total of 120 hours. The colour
from the 0 hours’ time stand (T0) was determined right after the blanching treatments of
the samples, without any post-conservation. The colour at T3, T24, T48 and T120 were
determined for the post-refrigerated samples while the colour was only measured at the
end of the time study (T120) for the post-frozen samples.

Lastly, colour assessments were made following the different drying processes in
the dried and ground larvae powder.

The colour measurements performed were repeated six times for each sample and
it was calculated the mean value for each axis. Additionally, it was possible to determine
the total colour variation to access the differences in colour in the time study, according
to the following formula:

𝟏/𝟐
𝜟𝑬∗ = [(𝜟𝑳∗ )𝟐 + (𝜟𝒂∗ )𝟐 + (𝜟𝒃∗ )𝟐 ]

where,

ΔL* = L0 – L, being L0 the initial L* colour value and L the final L* colour value;

Δa* = a0 – a, being a0 the initial a* colour value and a the final a* colour value;

Δb* = b0 – b, being b0 the initial b* colour value and b the final b* colour value.
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6.4 Statistical analysis

i

For statistical analysis of the collected data, it was used the IBM® SPSS® Statistics
27 program for Windows®. The results are displayed as mean values of the
measurements with the corresponding standard deviation value.

To assess the influence of the pre-conservation methods and blanching

treatments, prior to drying, on the DM and colour of the samples it was applied a Two-
Way ANOVA (Analysis of variance) followed by Tukey’s range test, using the pre-
conservation methods and blanching treatments as factors.

For the colour variation over time in the post-refrigerated samples, the impact of
the different pre-conservations and blanching treatments on the total colour variation
(ΔE*) was evaluated. Thus, for each of the measurements it was applied a Two-Way
ANOVA followed by Tukey's range test, using the pre-conservation methods and
blanching treatments as factors. To assess the post-conservation method (refrigerated
or frozen) impact at 0 hours and 120 hours, it was applied a Three-Way ANOVA, using
the pre-conservation methods, the blanching treatments and the post-conservation
methods as factors.

The evaluation of the effect of pre-conservation methods, blanching treatments

and post-conservation methods after drying on the colour of the samples was done
individually in function of the drying methods applied. Therefore, it was applied a Two-
Way ANOVA followed by Tukey’s range test, using the drying treatment and the other
condition studied as factors.

To compare the effect of drying or blanching treatments on the DM, aw and colour
of the frozen samples (pre- and post-frozen) it was applied a One-Way ANOVA followed
by Tukey’s range test.

All the previously described tests were applied with a confidence level of 95%.
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7. Results and Discussion

7.1 Effect of blanching and conservation on colour

The effects of different blanching treatments and pre-conservation methods on

colour of T.molitor larvae prior to drying are represented in figure 6 and figure 7,
respectively.

70,00
a
60,00
b
c
50,00

40,00 ns ns
ns

30,00

20,00 a c b

10,00

0,00 L* a* b*

-10,00 L* a* b*
Non-Blanched Immersion Steam

Figure 6 – Effect of different blanching treatments on colour of non-dried samples (a, b, c – significantly
different results between samples subjected to different blanching treatments, where: p < 0.05; ns – not
significantly different results between samples subjected to different blanching treatments, where: p ≥ 0.05).

The blanching treatments had a significant effect on colour, specifically on the L*

and a* values. Blanched (by immersion or steam) samples had significantly higher L*
values and lower a* values comparatively to non-blanched samples, without significant
differences in b* values. Therefore, blanched samples resulted in lighter samples with
less redness. One possible explanation for the results could be that as blanching purpose
is to inactivate several enzymes, such as polyphenol oxidase that cause losses in colour,
these enzymes if not inactivated can cause contribute to darker colour values in non-
blanched samples. These darkness in non-blanched samples could also be justified by
a higher microbiological load comparatively to blanched samples that could have
reduced these amounts of microorganisms (such as Enterobacteriaceae, endospores,
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yeasts and moulds) as they were submitted to a thermal treatment at high temperatures
for 5 minutes.

Additionally, it is noteworthy that immersion blanched samples had significantly

higher L* values and lower a* values in comparison to steam blanched samples, without
no significant differences in b* values. Thus, immersion blanched samples not only had
higher lightness values but also less redness. Immersion blanching seems to be more
effective than steam blanching for preservation of colour of T.molitor larvae.

70,00
a
60,00
b
50,00
a
40,00 b
30,00
ns
20,00 ns

10,00

0,00
L* a* b*
-10,00

Freezer Refrigeration

Figure 7 - Effect of different pre-conservation methods on colour of non-dried samples (a, b – significantly
different results between samples subjected to different pre-conservation methods, where: p < 0.05; ns –
not significantly different results between samples subjected to different pre-conservation methods, where:
p ≥ 0.05).

Regarding pre-conservation methods, is it noticeable that samples in the freezer

had significantly higher L* and b* values compared to refrigerated samples, with not
significant differences in a* values (despite samples in the freezer showing slightly higher
a* values). These results mean that refrigerated samples showed not only a higher
darkness but also a lower yellowness than samples in the freezer. Since samples in the
freezer are conserved at -24ºC and since this low temperature prevents the growth of
microorganisms, it can justify that refrigerated samples at 4ºC led to a higher microbial
load and microbiological activity that consequently resulted in a less preserved colour
with a higher darkness and a lower yellowness.
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7.2 Colour variation over time (effect of post-conservation)

The results of L*a*b* values at 0 and 120 hours are displayed in table 7. Colour
values were only represented at this time stands for a clearer interpretation. The results
of colour variation (ΔE*) over time are displayed in figure 8.

Table 7 – Colour values at 0 and 120 hours in refrigeration for samples: pre-frozen with immersion blanching
treatment, pre-frozen with steam blanching treatment, pre-refrigerated with immersion blanching treatment
and pre-refrigerated with steam blanching treatment.

Samples
Colour values Hours
Pre-Frozen Pre-Frozen Pre-Refrigerated Pre-Refrigerated
Immersion Steam Immersion Steam

0 50.21 ± 1.25 48.48 ± 1.82 44.75 ± 1.22 40.19 ± 1.25

L*
120 42.83 ± 1.56 37.79 ± 1.60 31.18 ± 1.40 36.88± 1.64

0 5.35 ± 0.15 6.79 ± 0.27 5.93 ± 0.15 6.76 ± 0.15

a*
120 8.78 ± 0.25 6.29 ± 0.69 7.54 ± 0.34 6.68 ± 0.30

0 26.25 ± 0.71 27.54 ± 1.72 27.40 ± 0.23 25.45 ± 0.71

b*
120 30.60 ± 0.56 23.98 ± 2.18 23.50 ± 0.87 23.65 ± 1.26

16,00

14,00

12,00

10,00
ΔE*

8,00

6,00

4,00

2,00

0,00
6 24 48 120
Time (h)

Pre_Frozen_Immersion Pre_Refrigerated_Immersion
Pre_Frozen_Steam Pre_Refrigerated_Steam

Figure 8 – Colour variation (ΔE*) over 120 hours in refrigeration for samples: pre-frozen with immersion
blanching treatment, pre-frozen with steam blanching treatment, pre-refrigerated with immersion blanching
treatment and pre-refrigerated with steam blanching treatment.
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Despite ΔE* being relatively similar at 120 hours between three samples (pre-
frozen and immersion blanching: 9.32; pre-frozen and steam blanching: 11.46; and pre-
refrigerated and immersion blanching: 14.22), it is noteworthy that the sample with pre-
refrigeration and steam blanching revealed lower ΔE* values (3.92) ate the end of this
period. However, and considering table 7, this sample was also the one that was most
affected prior to the actual ΔE* test. This can be seen by looking at L* value for this
sample that was fairly low compared to the other samples. Since the L* value was already
relatively low at 0 hours (40.19 ± 1.25) it did not change as drastically at 120 hours
(36.88± 1.64) and the total ΔE* of this specific sample was lower than the other samples.

The total ΔE* correlates L*, a* and b* values according to the initial ones.
Therefore, the data evaluation could be difficult to perform since different ΔE* does not
consider individual axis of colour and the different colour values at 0 hours.

7.3 Post-drying effects of blanching and conservation on dry matter

and water activity

The post-drying effects of different blanching treatments and pre-conservation

methods on dry matter (DM) and water activity (aw) of T.molitor larvae are represented
in table 8 and table 9 respectively. The results are displayed as mean values with
corresponding standard deviations with significant results compared separately in each
drying method. Only effects of pre-conservation and blanching are displayed since post-
conservation had no significant effects on the DM and aw values of dried samples.

Table 8 – Effect of different blanching treatments on dry matter and water activity of dried samples.

Oven Microwave Freeze-drying

Characterization
Immersion Steam Immersion Steam Immersion Steam

99.4 98.6 98.1 96.2 97.1 87.2

Dry matter (%) a b a b a b
± 0.9 ± 0.8 ± 1.2 ± 1.6 ± 1.1 ± 5.6
0.088 0.191 0.361 0.239 0.071 0.592
aw b a a b b a
± 0.016 ± 0.056 ± 0.053 ± 0.053 ± 0.038 ± 0.038

– significantly different results between samples subjected to different blanching treatments in each drying
a, b

method (p < 0.05).

ns – not significantly different results between samples subjected to different blanching treatments methods
in each drying method (p ≥ 0.05).
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In regards to the different blanching treatments in oven dried samples, it is

noticeable that immersion blanching had a significantly higher DM result in comparison
to steam blanching (immersion – 99.4 ± 0.9%; steam – 98.6 ± 0.8%) as well as a lower
aw value (immersion – 0.088 ± 0.016; steam – 0.191 ± 0.056). For microwave dried
samples, immersion blanching also had a significantly higher DM result in comparison to
steam blanching (immersion – 98.1 ± 1.2%; steam – 96.2 ± 1.6%) however it had a higher
aw value (immersion – 0.361 ± 0.053; steam – 0.239 ± 0.053). The freeze-dried samples
had the biggest differences between the different blanching treatments, since immersion
blanching had a significantly higher DM result in comparison to steam blanching
(immersion – 97.1 ± 1.1%; steam – 87.2 ± 5.6%) as well as a substantially lower aw value
(immersion – 0.071 ± 0.038; steam – 0.592 ± 0.038).

In summary, immersion blanching led to significantly higher DM results between

all the different drying methods in comparison to steam blanching. Immersion blanching
also led to better results for aw , since it had lower values, for oven dried and freeze-dried
samples but it had worse results for microwave dried samples.

It is clear that immersion is the most beneficial approach for blanching, especially
for oven dried and freeze-dried samples since it causes high DM results and lower aw
values. Nonetheless, it is noteworthy to mention that for microwave dried samples steam
blanching causes lower aw values in comparison to immersion blanching.

Table 9 - Effect of different pre-conservation methods on dry matter and water activity of dried samples.

Oven Microwave Freeze-drying

Characterization
Freezer Refrigerated Freezer Refrigerated Freezer Refrigerated

99.4 98.6 97.6 96.7 95.6 89.7

Dry matter (%) a b a b a b
± 0.7 ± 0.9 ± 1.2 ± 1.9 ± 2.7 ± 7.2
0.106 0.172 0.303 0.298 0.221 0.441
aw b a ns ns b a
± 0.031 ± 0.079 ± 0.097 ± 0.059 ± 0.203 ± 0.368

a, b – significantly different results between samples subjected to different pre-conservation methods in each
drying method (p < 0.05).

ns – not significantly different results between samples subjected to different pre-conservation methods in
each drying method (p ≥ 0.05).

Concerning different pre-conservation methods in oven dried samples, it is

noticeable that samples in the freezer showed a significantly higher DM result compared
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to refrigerated samples (freezer – 99.4 ± 0.7%; refrigerated – 98.6 ± 0.9%) as well as a

lower aw value (freezer – 0.106 ± 0.031; refrigerated – 0.172 ± 0.079). For microwave
dried samples, the samples in the freezer had a significantly higher DM result in
comparison to refrigerated samples (freezer – 97.6 ± 1.2%; refrigerated – 96.7 ± 1.9%)
with no significant differences for aw values between both pre-conservation methods
(freezer – 0.303 ± 0.097; refrigerated – 0.298 ± 0.059). The freeze-dried samples were
where the biggest differences were noticed with samples in the freezer showing a
significantly higher DM result when compared to refrigerated samples (freezer – 95.6 ±
2.7%; refrigerated – 89.7 ± 7.2%) and a substantially lower aw value (freezer – 0.221 ±
0.203; refrigerated – 0.441 ± 0.368).

In brief, pre-conservation in the freezer led to significantly higher DM results across

all the drying methods in comparison to pre-conservation in the refrigerator. Pre-
conservation in the freezer also led to better aw values, since it had lower values, for oven
dried and freeze-dried with not statistically different aw values for microwave dried
samples between freezer and refrigerated samples.

Pre-conservation (prior to any blanching treatment) in the freezer at -24ºC seems

to better than in the refrigerator at 4ºC since it has better DM results between all the
drying methods as well as most optimal aw values in oven dried and freeze-dried
samples.

7.4 Post-drying effects of blanching and conservation on colour

The post-drying effects of different pre-conservation methods, blanching

treatments and post-conservation methods on colour of T.molitor larvae are displayed in
figure 9, figure 10 and figure 11 respectively. It is noteworthy to mention that post-
conservation was not studied on freeze-dried samples since it is a required that samples
are previously frozen for this method to work properly.
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60,00
a
50,00
a a b

40,00
b a a
a b
b
30,00 b
b
a a
20,00 b
a b b

10,00

0,00
L* a* b*
-10,00

Oven_Pre_Frozen Oven_Pre_Refrigerated
Microwave_Pre_Frozen Microwave_Pre_Refrigerated
Freeze-drying_Pre_Frozen Freeze-drying_Pre_Refrigerated

Figure 9 – Post-drying effect of different pre-conservation methods on colour (a, b – significantly different
results between samples subjected to different pre-conservation methods in each drying method, where p <
0.05; ns – not significantly different results between samples subjected to different pre-conservation methods
in each drying method, where: p ≥ 0.05).

In regards to different pre-conservation methods in oven dried samples, it is

noticeable that samples in the freezer had significantly higher L* and b* values and
significantly lower a* values in comparison to refrigerated samples (despite a lower
significance for a* values). For both microwave dried and freeze-dried samples, samples
in the freezer had significantly higher L*, a* and b* values. These results mean that
refrigerated samples exhibited a higher darkness and a lower yellowness in all the
different drying methods as well as a lower redness in both microwave dried and freeze-
dried samples. As mentioned earlier in section 7.1, since samples in the freezer are
conserved at -24ºC and since this low temperature prevents the growth of
microorganisms, it can justify that refrigerated samples at 4ºC had a higher microbial
load and microbiological activity that consequently resulted in a less preserved colour
with a higher darkness and a lower yellowness in all the different drying methods.
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60,00
a
50,00 b
a
40,00 a
b b
b a
a a
30,00 b b

ns ns b a
20,00 b a

10,00

0,00
L* a* b*

-10,00
Oven_Immersion Oven_Steam
Microwave_Immersion Microwave_Steam
Freeze-drying_Immersion Freeze-drying_Steam

Figure 10 - Post-drying effect of different blanching treatments on colour (a, b – significantly different results
between samples subjected to different blanching treatments in each drying method, where p < 0.05; ns –
not significantly different results between samples subjected to different blanching treatments in each drying
method, where: p ≥ 0.05).

Concerning different blanching treatments in oven dried samples, it is noticeable

that immersion blanching had significantly higher L* and b* values and significantly lower
a* values in comparison to steam blanching. For microwave dried samples, L* and b*
values were also significantly higher in immersion blanching samples, however no
significant differences were found between a* values of both blanching treatments. As
for freeze-dried samples, immersion blanching had significantly higher L* values and
significantly lower a* and b* values comparatively to refrigerated samples. Therefore,
refrigerated samples resulted in a higher darkness in all drying methods, higher redness
in both oven dried and freeze-dried samples (with no differences between microwave
dried samples) and lower yellowness in oven dried and microwave dried samples (with
higher yellowness in freeze-dried samples). In summary, immersion blanching seems
that it is more effective in preservation of a lighter colour of T.molitor larvae in all the
different drying methods.
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45,00
a
40,00 a
b
b
35,00
a
a
30,00 b b

25,00

20,00 ns b a
ns
15,00

10,00

5,00

0,00
L* a* b*

Oven_Post_Frozen Oven_Post_Refrigerated
Microwave_Post_Frozen Microwave_Post_Refrigerated

Figure 11 - Post-drying effect of different post-conservation methods on colour (a, b – significantly different
results between samples subjected to different post-conservation methods in each drying method, where p
< 0.05; ns – not significantly different results between samples subjected to different post-conservation
methods in each drying method, where: p ≥ 0.05).

In regards to different post-conservation methods in oven dried samples, it is

noticeable that frozen samples had significantly higher L* and b* values in comparison
to refrigerated samples with no significant differences in a* values between both
methods. As for microwave dried samples, frozen samples had significantly higher L*
and b* values and lower a* values comparatively to refrigerated samples. These results
mean that refrigerated samples showed a higher darkness and a lower yellowness in
both drying methods, yet a higher redness was found in microwave dried samples. As
mentioned earlier, these results could be caused by a higher microbial load and
microbiological activity in the refrigerated samples, that was triggered by a higher
temperature (4ºC) in comparison to frozen samples (-24ºC), that consequently resulted
in a less preserved colour with a higher darkness and a lower yellowness in both drying
methods.
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7.5 Comparison between drying methodologies on dry matter, water

activity and colour

The effects of different drying methodologies and blanching treatments on DM and

aw of T.molitor larvae is exhibited in table 10. The results are displayed as mean values
with corresponding standard deviations with significant results compared separately to
either different drying methods or different blanching treatments. The effects of the
different drying methods and blanching treatments on colour of T.molitor larvae are
displayed in figure 12 and figure 13. For the comparison between drying methods only
results from frozen samples (for both pre-conservation and post-conservation) were
considered.

Table 10 - Effect of different drying methodologies and blanching treatments on dry matter and water activity.

Drying Blanching
Characterization
Oven Microwave Freeze-drying Immersion Steam

a b c a b
Dry matter (%) 99.2 ± 0.4 97.0 ± 1.1 95.6 ± 2.7 98.3 ± 1.0 96.2 ± 2.6

c a b b a
aw 0.107 ± 0.038 0.312 ± 0.135 0.221 ± 0.203 0.179 ± 0.190 0.247 ± 0.122

a, b, c – significantly different results between samples subjected to either different drying methods or different
blanching treatments blanching (p < 0.05).

Regarding DM, all drying methods had significantly different results as the oven
had the best one (99.2 ± 0.4%), followed by the microwave (97.0 ± 1.1%) and lastly the
freeze-drying (95.6 ± 2.7%). As for aw, the results were also significantly different
between drying methods as oven had the most optimal aw value (0.107 ± 0.038), followed
by the freeze-drying (0.221 ± 0.203) and lastly the microwave (0.312 ± 0.135). Oven
drying at 80ºC for 7 hours seems to be the most optimal approach in comparison both
microwave drying at 800 W for 5 minutes and freeze-drying for 24 hours. It is noteworthy
to mention that despite freeze-drying showing the lowest results on DM and a fairly high
aw value, these results were mainly because of steam blanched samples, as seen in
section 7.3, that resulted on substantially low DM results (87.2 ± 5.6%) and high aw
values (0.592 ± 0.038). Therefore, freeze-drying for 24 hours with immersion blanched
samples could also be a good alternative to oven drying at 80ºC for 7 hours specially
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68 Study of processing technologies in edible insects

when aiming for low aw values. For microwave drying, despite showing high DM results
(97.0 ± 1.1%), it resulted in high aw values (0.312 ± 0.135) that can potentially cause a
lower shelf life and eventually a lower quality of the larvae.

As for the different blanching treatments, both DM and aw were the most optimal in
immersion blanching (DM – 98.3 ± 1.0%; aw – 0.179 ± 0.190) in comparison to steam
blanching (DM – 98.3 ± 1.0%; aw – 0.179 ± 0.190). Nonetheless, it is important to mention
that total steam blanching results were extremely affected by the freeze-drying method,
as previously discussed in section 7.3, that had the lowest DM result (87.2 ± 5.6%) and
the highest aw value (0.592 ± 0.038) from all the conditions.

60,00
a
50,00 b b

40,00 a c
b

30,00

b a c
20,00

10,00

0,00
L* a* b*
-10,00
Oven Microwave Freeze-drying

Figure 12 - Effect of different drying methods on colour (a, b, c – significantly different results between
samples subjected to different drying methods, where: p < 0.05).

In regards to colour of the different drying methods, the L* value was significantly
the highest in the freeze-drying samples, followed by oven drying and microwave drying
with no statistical differences between both methods. The a* value was significantly the
highest in microwave dried samples, followed by oven dried samples and lastly by
freeze-dried samples. As for the b* value, this was the highest in microwave dried
samples, followed by oven dried samples and lastly freeze-dried samples. In summary,
freeze-dried samples were the ones with the highest lightness and the lowest yellowness
and redness (being the last two the highest in microwave dried samples).
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60,00
a
50,00 b

40,00 ns ns

30,00

b a
20,00

10,00

0,00
L* a* b*

Immersion Steam

Figure 13 - Effect of different blanching treatments on colour (a, b – significantly different results between
samples subjected to different blanching treatments, where: p < 0.05; ns – not significantly different results
between samples subjected to different blanching treatments, where: p ≥ 0.05).

Concerning to colour of the different blanching treatments, immersion blanching

had a significantly higher L* value and significantly lower a* value comparatively to steam
blanching, with no significant differences between b* values of both treatments.
Therefore, immersion blanching led higher lightness and lower redness in comparison to
steam blanching.
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8. Conclusion

The present research allowed a deepening of knowledge on how different

blanching, drying and conservation techniques affect quality parameters, such as dry
matter, water activity and colour of T.molitor larvae.

In general, pre-conservation in the freezer led to better results comparatively to

refrigeration as it revealed a higher L* value, a higher dry matter percentage and a lower
water activity value on oven dried and freeze-dried samples. Similarly, immersion
blanching was superior to steam blanching by displaying a higher L* value, a higher dry
mater result and a lower water activity value on oven dried and freeze-dried samples.
Concerning the different drying methods, oven drying had the highest dry matter
percentage and the lowest water activity value despite the highest L* value being on
freeze-dried samples. Notwithstanding oven drying having the most optimal dry matter
and water activity values, freeze-drying with immersion blanched samples could also be
a good alternative specially when aiming for a higher L* value and a lower water activity
value.

To better understand the implications of these results, future studies could focus
on different drying technologies, access safety parameters such as microbiological load
or use different edible insects’ species.

In summary, the aims of the current research were accomplished. The effects of
different processing methodologies on quality parameters of T.molitor larvae reveal new
valuable findings for the food industry and could develop similar studies on different
edible insects so that entomophagy could be better accepted by Western consumers.
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Part C – Study of different protein extraction

conditions, gelling ability and rheological
properties of proteins from Tenebrio molitor
larvae and Acheta domesticus

9. Materials and methods

9.1 Insects and chemicals

Frozen T.molitor larvae and A.domesticus (Figure 14) were kindly supplied by
Tecmafoods (Matosinhos, Portugal) and GotanBug (Santarém, Portugal) respectively,
to GreenUPorto - Sustainable Agrifood Production Research Centre, located in Vairão,
Portugal. All insects were submitted to a blanching treatment by immersion in water at
100ºC for 5 minutes in a ratio of 1/10 (w/v). After blanching, the insects were dried in a
conventional oven (Unox® model XF016-TG) at 80ºC for 7 hours and ground with the
assistance of a kitchen robot (Kenwood® Major Titanium with the multi mill attachment
model AT320A). Both powders were stored in closed glass bottles surrounded by
aluminium foil and kept in dark conditions. Later, the powders were vacuum sealed in
secure plastic bags and shipped to the Department of Food Science at University of
Copenhagen where the study was conducted.

All the chemicals used in the study were from Honeywell International Inc.
(Muskegon, Michigan, United States).

Figure 14 – Example of euthanized Tenebrio molitor larvae and Acheta domesticus used in this study.
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9.2 Defatting

A defatting process of the insect powders was performed prior to the actual study
to remove excess lipids that could interfere with the efficacy of the protein extractions.

The defatting was carried out in a Soxhlet extractor (Gemini B.V.® Tecator Soxtec
extraction unit model HT 1043) and ethanol 96% was the selected solvent. Firstly, 10g
of sample (W1) was added into each extraction thimble (W2) and then they were lifted
into the capacitors. Cotton was added on top to prevent sample loss. Soon after, 50mL
of ethanol 96% was added to each extraction cup and then they were lifted to fit the
opening in the capacitors. After engaging the safety catch, the machine was started at a
temperature of 160ºC with cooling water running during the entire process. When the
solvent started flowing, the taps were open and the thimbles were left into boiling position
for 2 hours, in rising position for 1 hour and in evaporation for 10 minutes. The extraction
thimbles with the wet defatted samples were placed in an air-flow lab incubator
(Memmert® Incubator I model MEM_6004) overnight at 80ºC to remove the remaining
solvent.

Following evaporation of the solvent, the weight of the thimbles with the dried
defatted samples was registered (W3) to determine the lipid content that was extracted
from the samples, accordingly to the formula:

𝑾𝟑 − 𝑾𝟐
𝑳𝒊𝒑𝒊𝒅 𝒄𝒐𝒏𝒕𝒆𝒏𝒕 𝒆𝒙𝒕𝒓𝒂𝒄𝒕𝒆𝒅 (%𝑫𝑴) = × 𝟏𝟎𝟎
𝑫𝑴(%)
𝑾𝟏 × 𝟏𝟎𝟎

where,

W1 – weight of the sample (g);

W2 – weight of the extraction thimble (g);

W3 – weight of the dried extraction thimble with the dried defatted sample (g);

DM – Dry matter, accessed as ≈97% for both species accordingly to the formula in
section 6.3.1.

The measurements for the determination of the lipid content extracted were taken
six times for both insect powders.
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9.3 Study design

As displayed in figure 15, the emphasis of this study was on the effect of different
protein extraction conditions (such as concentration, pH and treatment technologies) on
the protein solubility, the gelling ability of full samples, supernatants and pellets and on
the molecular weight of the extracted proteins. Moreover, the study also focused on
subsequent gelling ability of freeze-dried soluble proteins at different concentrations and
in the assessment of the rheological properties of both insects’ proteins.

Figure 15 – Study design from part C.

9.4 Protein extraction

As exhibited in Figure 16, protein extraction was performed at different

concentrations of powder (w/v): 5%, 15% and 25%; and at different pH values: pH4, pH7
and pH10. Samples at 25% concentration of powder were duplicated since half was
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submitted to an ultrasound treatment at 300W for 5 minutes, in order to assess if it would

influence the subsequent characterization.

Firstly, and depending on the concentration, 2.5g, 7.5g, or 12.5g of defatted

powder were weighed for each concentration that was tested and added to a glass bottle
followed by the addition of 50mL of Milli-Q® ultrapure water. Then, the pH of the samples
was adjusted with 1M HCl or 1M NaOH, depending on the chosen pH, with the
assistance of a pH meter (Hach® model HQ440) and a magnetic stirrer (IKA® Magnetic
stirrer model 0003671000). Right after, all samples were submitted to treatment with an
Ultra-Turrax homogenizer (IKA® digital Ultra-Turrax® model T25 with the stem model
S25KV-25F) at 12000 rpm for 5 minutes and then all samples were stirred (IKA®
Magnetic stirrer model RT 15) overnight at 600 rpm at 25ºC. In the following day, half of
the 25% concentration samples were submitted to an ultrasound treatment (Hielscher®
Ultrasonic processor model UP400St) at a power of 300W, amplitude of 72%, pulse of
90%, at 25ºC for 5 minutes. All samples were transferred into 10mL test tubes,
centrifuged (Thermo Scientific® model SL 16R) at 3200 rpm, at 25ºC for 20 minutes and
all the supernatants were separated from the pellets. The protein extraction was
performed twice and in duplicates for each sample.

Figure 16 - Study design for the protein extraction and subsequent characterization.
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9.5 Characterization

9.5.1 Protein solubility

The nitrogen (N) percentage of the supernatants and the defatted powders was
determined using the Dumas (Elementar® Rapid MAX N exceed) high-temperature
combustion method. All measurements were performed twice and in duplicates using
200 mg crucibles. Aspartic acid was used to calibrate the instrument. The protein
percentage of the samples was calculated using a protein-to-nitrogen conversion factor
of 6.25 since it is the value that has been the most accepted for insects (Finke, 2013;
Rumpold & Schlüter, 2013; Yi et al., 2013; Zhao et al., 2016). The calculation was
achieved by using the formula:

𝑷𝒓𝒐𝒕𝒆𝒊𝒏 (%) = 𝑵𝟏 × 𝟔. 𝟐𝟓

where,

N1 – nitrogen percentage in the sample, determined by the Dumas (%).

Likewise, the protein solubility of the samples it was determined by the following
formula:

𝑷𝟏
𝑷𝒓𝒐𝒕𝒆𝒊𝒏 𝑺𝒐𝒍𝒖𝒃𝒊𝒍𝒊𝒕𝒚 (%) = × 𝟏𝟎𝟎
𝑪𝑷
𝑷𝟐 × 𝟏𝟎𝟎

where,

P1 – protein percentage in the sample (%);

P2 – protein percentage in the defatted powder (%);

CP – concentration of defatted powder used for the protein extraction (%).

9.5.2 Gelling ability of the full samples, supernatants and pellets

The gelling ability of the full samples, supernatants and pellets were tested for each
sample twice and in duplicates. The full samples were transferred to 10mL test tubes
after either the magnetic stirring overnight or, if it was applied, after the ultrasound
treatment. For gelling ability of the supernatants and the pellets, the samples were
centrifuged in 10mL test tubes, and the supernatant was transferred to a new test tube
while the pellet test tube was filled up with Milli-Q® ultrapure water to 10mL.
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Gelling of the samples was performed according to Kumar et al. (2022) with slight
modifications. Briefly, the samples were vortexed (IKA® MS 3 basic) until they had a
homogeneous appearance and heated at 85ºC for 30 minutes in a water bath (Julabo®
13 water bath with circulator). Then, the samples were cooled in an ice bath for 30
minutes and left in refrigeration overnight at 4ºC. The gelling ability results were
determined in the following morning by inverting the test tubes and visualizing if the gel
would fall down or slip (Sathe et al., 1982). The strength (+) or weakness (-) of the gels
was quantified accordingly to how much solidification on the top of the tube was when
they were inverted using the scale:

0-25% solidification: Very weak gel ( - - )

26-50% solidification: Weak gel (-)

51-75% solidification: Strong gel (+)

76-100% solidification: Very strong gel ( + + )

9.5.3 Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Following protein extraction, supernatants of each sample at 25% concentration of

defatted powder (w/v) were submitted to an SDS-PAGE protocol to examine if the
extraction conditions had a significant effect on the molecular weight of the extracted
proteins. The SDS-PAGE was performed according to the UCPH FOOD department’s
protocol.

Firstly, samples were diluted with Milli-Q® ultrapure water in a 1:1 ratio (v/v). 15μL
of sample were mixed with 5μL of 4xNuPAGE™ LDS loading buffer and 2μL of deionized
water (for the non-reduced gel) or 100mM of DTT (for the reduced gel). Then, samples
were vortexed (IKA® MS 3 basic), heated (Eppendorf® Thermomixer Compact model
5350) at 70ºC for 10 minutes at 600 rpm and centrifuged (Eppendorf® MiniSpin Plus
microcentrifuge model Z618543) at 13800 x g for 15 seconds.

The gels (Invitrogen™ NuPAGE™ Novex™ 12% Bis-Tris Gel, 1.0 mm, 15 Well)
were placed in the electrophoresis trays with MES buffer. Then, the marker (Invitrogen™
Novex™ Sharp Pre-stained Protein Standard) and the samples were placed into the
wells and ran at 150V.

The gels were then stained overnight with Coomassie™ Blue dye-based stain. The
next day, the gels were dyed for 15 minutes thrice and, between each time, rinsed with
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deionised water until subsequent scanning (Bioimager® gel imager /scanner model Bio-
6000T).

9.6 Freeze-drying

The protein extraction sample with the conditions that resulted in a higher protein
solubility for each species was chosen for the freeze-drying of the soluble proteins that
were used in the following experiences. In appendix 2, there is available a supplementary
table developed for a better overview of the freeze-drying process. Briefly, after protein
extraction, the supernatants were transferred into plastic cups with parafilm with small
holes on top to allow air to circulate during the process. Samples were left 48 hours in
the freeze-dryer (Buch & Holm A/S model not specified) and then stored in dark
conditions until further use.

9.7 Gelling ability of the proteins

The gelling ability of the soluble proteins was tested in 5%, 10% and 15%
concentrations (w/v) and with or without the addiction of calcium ions since Ca2+ has
been proven to increase gel strength in small amounts (Xiao et al., 2021).

After weighing the proteins depending on each concentration,10mL of water was

added to each 10mL test tube. In the conditions with the Ca2+ addition, 0.1M of calcium
chloride (CaCl2) was weighed and added to the tubes. Gelling and gelling ability
determination of the dispersions was conducted accordingly to the method described at
section 9.5.2. Additional notes regarding the gels’ appearance were took in the following
day. The procedure was performed in duplicates for each sample.

9.8 Rheological properties of the proteins

The rheological properties of the proteins’ dispersions were studied at 10%

concentration (w/v) of soluble proteins for both insects since it was the protein
concentration also used by Kumar et al. (2022) for the rheological properties of black
soldier fly (Hermetia illucens) larvae proteins. All the rheological measurements were
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performed in duplicates with freshly prepared dispersions for both insects and for the
conditions with and without 0.1M Ca2+ addition.

The study was conducted using Malvern® Kinexus Pro rheometer with a serrated
concentric cylinder bob (Malvern® C25G A0009SS: PC25 SPLINED) and a serrated cup
(Malvern® PC25G A0008AL) geometries. The protein dispersions were consecutively
subjected to a temperature ramp, frequency sweep, and amplitude sweep. Elasticity
modulus (G’) and viscous modulus (G’’) were determined in each measurement.
Accordingly to Yi et al. (2013), the elastic modulus is a measure that reveals the stiffness
of a gel by measuring the elastic energy stored reversibly during its deformation; while
the viscous modulus is measured by the energy dissipated during deformation as a
consequence of viscous friction.

The conditions for the rheological measurements were based on Kumar et al.
(2022). Briefly, 15mL of the dispersion was loaded into the serrated cup. A ring of paraffin
oil was placed on top of the sample to avoid evaporation during the measurements.
Immediately after placing the paraffin oil and closing the security cap the temperature
ramp was initiated. The heating was done from 25ºC to 85ºC at 5ºC per minute, 85ºC for
10 minutes and cooling from 85ºC to 25ºC at 3ºC per minute. A constant strain of 0.05%
and frequency of 0.1 Hz was maintained during the temperature ramp. Then, a frequency
sweep was initiated with frequency varying from 0.5 to 20 Hz at a constant temperature
of 25ºC and a constant strain of 0.01%. Following the frequency sweep, an amplitude
sweep was performed with a strain increase from 0.1% to 5000% at a constant
temperature of 25ºC and a constant frequency of 1 Hz.

9.9 Statistical analysis

For statistical analysis of the protein solubility collected data, it was used the IBM®
SPSS® Statistics 27 program for Windows®.

Initially, in order to analyse the overall effects of sample (5%, 15%, 25% non-
ultrasound treatment and 25% with ultrasound treatment) and pH (pH4, pH7 and pH10)
on protein solubility, it was applied a Two-way ANOVA followed by Tukey's range test.
Then, conditions were grouped by either pH or sample and it was applied a One-Way
ANOVA followed by Tukey’s range test. The results are displayed as mean values of the
measurements with the corresponding standard deviation value.

All the previously described tests were applied with a confidence level of 95%.
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10. Results and Discussion

10.1 Defatting

The defatting process via Soxhlet extraction methodology using 96% ethanol as a
solvent resulted in lipid extraction values of 39.92 ± 1.23% for T.molitor larvae and 23.57
± 0.74% for A.domesticus. Protein content of T.molitor larvae and A.domesticus defatted
powders were 72.11 ± 0.22% and 81.03 ± 0.20% respectively. Defatted powders of both
insects resulted in a noticeable lighter colour when compared to the whole fat dried
powders.

10.2 Protein solubility

10.2.1 Protein solubility of Tenebrio molitor larvae defatted powder

10.2.1.1 Effect of sample and pH value of extraction on protein solubility

The effects of different samples and pH values of extraction on protein solubility of

T.molitor larvae are represented in table 11. The results are displayed as mean values
with corresponding standard deviations with significant results compared separately to
either different pH values of extraction or different samples.

Table 11 - Effect of different pH values of extraction and samples on protein solubility of Tenebrio molitor larvae.

pH Sample
Characterization
4 7 10 5% Non-US 15% Non-US 25% Non-US 25% US

10.23 11.17 15.27 8.77 12.21 13.68 14.24

Protein solubility (%) c b a c b a a
± 2.04 ± 1.86 ± 3.33 ± 1.10 ± 3.56 ± 2.43 ± 2.63

a, b, c – significantly different results between samples subjected to either different pH values of extraction or
different samples (p < 0.05).

Note. US - Ultrasound treatment.

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When analysing different pH values of extraction on protein solubility of T.molitor

larvae, it is noticeable that a higher pH resulted in a significantly higher protein solubility
as these values were higher at pH10 (15.27 ± 3.33%), then at pH7 (11.17 ± 1.86%) and
lastly at pH4 (10.23 ± 2.04%). Bußler et al. (2016) also reported that increases in pH
resulted in gradual increases in protein solubility from pH4 to pH10 in T. molitor defatted
powders. The authors also found that from pH2 to pH4 there was a drastically decrease
in protein solubility as well as a slight decrease from pH10 to pH12. According to the
authors, the isoelectric point of T.molitor larvae proteins was around the pH4 region thus
leading to a lower protein solubility at this pH of extraction. At extreme acidic or basic pH
values it is noticed a higher protein solubility that could possibly be caused by a higher
level of unfolding of the proteins that lead to a higher exposure of hydrophobic groups in
the solution.

Regarding the different samples on protein solubility of T.molitor larvae, it can be

observed that a higher concentration of defatted powder led to a higher protein solubility,
regardless of the application of an ultrasound treatment. Therefore, the highest protein
solubility was found in samples at 25% concentration with ultrasound treatment (14.24 ±
2.63%), followed by samples at 25% concentration with non-ultrasound treatment (13.68
± 2.43%), samples at 15% concentration with non-ultrasound treatment (12.21 ± 3.56%)
and lastly samples at 5% concentration with non-ultrasound treatment (8.77 ± 1.10%). It
is noteworthy to mention that there were no significant differences between the protein
solubility of samples at 25% concentration with ultrasound and without ultrasound (p =
0.143) despite a slight increase in ultrasound-treated samples. Possible explanations for
this slight increase in protein solubility on ultrasound-treated samples could be linked to
either: the use of the ultrasounds that generate rapid movement of solvents, resulting in
a higher mass transfer speed; or not from the use of the ultrasound itself but from an
increase in temperature that occurs from the application of this treatment for five minutes
at 300 W.

10.2.1.2 Comparison between samples on protein solubility

The effects of different samples across different pH values of extraction on protein

solubility of T.molitor larvae are displayed in figure 17.
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22,00%
20,00%
a
18,00% a a
16,00%
Protein solubility (%)

14,00% a a a
a a
12,00% b
10,00% b b b
8,00%
6,00%
4,00%
2,00%
0,00%
pH4 pH7 pH10

5% Concentration; Non-Ultrasound Treatment 15% Concentration; Non-Ultrasound Treatment

25% Concentration; Non-Ultrasound Treatment 25% Concentration; Ultrasound Treatment

Figure 17 - Effect of different samples on protein solubility of Tenebrio molitor larvae (a, b – significantly
different results between samples subjected to different concentrations and/or treatments in each pH value
of extraction, where: p < 0.05).

Concerning different samples at pH4, it is noticeable that samples at 25%

concentrations of both ultrasound and non-ultrasound treatment, resulted in significantly
higher protein solubility values in comparison to samples at 15% and 5% concentration
with non-ultrasound treatment. For samples at both pH7 and pH10, samples at 25%
concentrations of both ultrasound and non-ultrasound treatment, and the sample at 15%
concentration with non-ultrasound treatment, had significantly higher protein solubility
values compared to the sample at 5% concentration with non-ultrasound treatment.

10.2.1.3 Comparison between pH values of extraction on protein solubility

The effects of different pH values of extraction across different samples on protein

solubility of T.molitor larvae are represented in figure 18.
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22,00%
20,00%
a
18,00% a a

16,00%
Protein solubility (%)

14,00% b b
b b b
12,00% a
10,00% a a c

8,00%
6,00%
4,00%
2,00%
0,00%
5% Concentration; 15% Concentration; 25% Concentration; 25% Concentration;
Non-Ultrasound Non-Ultrasound Non-Ultrasound Ultrasound Treatment
Treatment Treatment Treatment

pH4 pH7 pH10

Figure 18 - Effect of different pH values of extraction on protein solubility of Tenebrio molitor larvae (a, b, c
– significantly different results between samples subjected to different pH values of extraction in each
sample, where: p < 0.05).

When analysing different pH values of extraction at 5% concentration with non-

ultrasound treatment, it is evident that there were no significant differences between the
different pH values of extraction. As for pH values at 15% concentration with non-
ultrasound treatment, it is observed that pH10 was significantly the highest pH in terms
of protein solubility, followed by pH7 and lastly pH4. For pH values of extraction at both
25% concentrations, with and without ultrasound treatment, it is clear that pH10 was
significantly the highest pH in terms of protein solubility, with no significant differences
between pH7 and pH4.

10.2.2 Protein solubility of Acheta domesticus defatted powder

10.2.2.1 Effect of sample and pH value of extraction on protein solubility

The effects of different samples and pH values of extraction on protein solubility of

A.domesticus are represented in table 12. The results are displayed as mean values with
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corresponding standard deviations with significant results compared separately to either

different pH values of extraction or different samples.

Table 12 - Effect of different pH values of extraction and samples on protein solubility of Acheta domesticus.

pH Sample
Characterization
4 7 10 5% Non-US 15% Non-US 25% Non-US 25% US

9.24 17.65 17.89 11.36 15.63 16.11 16.61

Protein solubility (%) b a a c b a,b a
± 1.35 ± 2.41 ± 4.64 ± 3.19 ± 4.46 ± 4.55 ± 4.71

a, b, c – significantly different results between samples subjected to either different pH values of extraction or
different samples (p < 0.05).

Note. US - Ultrasound treatment.

In regards to different pH values of extraction on protein solubility of A. domesticus,

it can be observed that protein solubility was significantly higher at pH10 (17.89 ± 4.64%)
and at pH7 (17.65 ± 2.41%) comparatively to pH4 (9.24 ± 1.35%). Brogan et al. (2021)
described that increases in pH resulted in gradual increases in protein solubility from
pH4 to pH12 in A.domesticus powders. In this present study, an increase in protein
solubility also occurred with a higher pH value of extraction. However, from pH7 to pH10,
the differences were not significant (p = 0.343). Similarly to what happened to T.molitor
larvae protein solubility in section 10.2.1.1, pH4 for also led to the lowest protein solubility
value, indicating that the isoelectric point of A.domesticus could also be around the pH4
region, explaining the lower solubility found at this value.

When examining the different samples on protein solubility of A.domesticus, it can

be noticed that the highest protein solubility was found in samples at 25% concentration
with ultrasound treatment (16.61 ± 4.71%), followed by samples at 25% concentration
with non-ultrasound treatment (16.11 ± 4.55%), samples at 15% concentration with non-
ultrasound treatment (15.63 ± 4.46%) and lastly samples at 5% concentration with non-
ultrasound treatment (11.36 ± 3.19%). Samples at 5% concentration had a significantly
lower protein solubility than all the other samples. The ultrasound treatment did not
reveal significant differences between both samples at 25% concentrations (p = 0.070).
However, samples at 25% with ultrasound treatment led to a significantly higher protein
solubility in comparison to samples at 15% concentration with non-ultrasound, while
samples at 25% with non-ultrasound treatment did not exhibit significant differences (p
= 0.093). As mentioned previously in section 10.2.1.1 regarding protein solubility of
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T.molitor larvae, these slight improvement in protein solubility on ultrasound-treated

samples could be linked to either: the application of the ultrasound treatment itself; or an
increase in temperature that occurs from this treatment, since higher temperatures of
extraction are usually linked to a higher protein solubility.

10.2.2.2 Comparison between samples on protein solubility

The effects of different samples across different pH values of extraction on protein

solubility of A.domesticus are displayed in figure 19.

22,00%
a a
a,b a a
20,00% b
18,00%
16,00%
b
Protein solubility (%)

14,00% c

12,00%
a a
a
10,00%
b
8,00%
6,00%
4,00%
2,00%
0,00%
pH4 pH7 pH10

5% Concentration; Non-Ultrasound Treatment 15% Concentration; Non-Ultrasound Treatment

25% Concentration; Non-Ultrasound Treatment 25% Concentration; Ultrasound Treatment

Figure 19 - Effect of different samples on protein solubility of Acheta domesticus (a, b, c – significantly
different results between samples subjected to different concentrations and/or treatments in each pH value
of extraction, where: p < 0.05).

Regarding different samples at pH4 and pH10, samples at 25% concentrations of

both ultrasound and non-ultrasound treatment, and the sample at 15% concentration
with non-ultrasound treatment, resulted in significantly higher protein solubility values
compared to the sample at 5% concentration with non-ultrasound treatment. As for pH7,
the sample at 5% concentration had a significantly lower protein solubility than all the
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other samples. Furthermore, the sample at 25% concentration with non-ultrasound

treatment led to no significant differences between the sample at 25% concentration with
ultrasound treatment and the sample at 15% concentration with non-ultrasound
treatment, despite these last two having significant differences between each other.

10.2.2.3 Comparison between pH values of extraction on protein solubility

The effects of different pH values of extraction across different samples on protein

solubility of A.domesticus are represented in figure 20.

22,00%
a a
a a
20,00% a a
18,00%
16,00%
a
Protein solubility (%)

14,00% a

12,00% b
b b
10,00%
b
8,00%
6,00%
4,00%
2,00%
0,00%
5% Concentration; 15% Concentration; 25% Concentration; 25% Concentration;
Non-Ultrasound Non-Ultrasound Non-Ultrasound Ultrasound Treatment
Treatment Treatment Treatment

pH4 pH7 pH10

Figure 20 - Effect of different pH values of extraction on protein solubility of Acheta domesticus (a, b –
significantly different results between samples subjected to different pH values of extraction in each sample,
where: p < 0.05).

Concerning different pH values of extraction of the four different samples, it is

noticeable that all the pH values of extraction exhibit similar statistical results between
them throughout the different samples. Both pH10 and pH7 led to statistically higher
protein results relatively to pH4, however with no statistical differences between both.
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10.3 Gelling ability of the full samples, supernatants and pellets

Gelling ability results of the full samples, supernatants and pellets at different
extraction conditions are presented in table 13 and table 14, for both T.molitor larvae and
A.domesticus respectively. The gelling ability designation is displayed beneath each
table.

Table 13 - Gelling ability of the full samples, supernatants and pellets from Tenebrio molitor larvae extracted
at different concentrations of powder (w/v), pH values and methodologies.

Conditions
Sample Gelling Ability
Concentration (w/v) pH Methodology

TM Full sample 5% 4 Ultra-Turrax + Magnetic stirring --

TM Pellet 5% 4 Ultra-Turrax + Magnetic stirring --
TM Supernatant 5% 4 Ultra-Turrax + Magnetic stirring --
TM Full sample 15% 4 Ultra-Turrax + Magnetic stirring -
TM Pellet 15% 4 Ultra-Turrax + Magnetic stirring -
TM Supernatant 15% 4 Ultra-Turrax + Magnetic stirring --
TM Full sample 25% 4 Ultra-Turrax + Magnetic stirring ++
TM Pellet 25% 4 Ultra-Turrax + Magnetic stirring ++
TM Supernatant 25% 4 Ultra-Turrax + Magnetic stirring --
TM Full sample 5% 7 Ultra-Turrax + Magnetic stirring --
TM Pellet 5% 7 Ultra-Turrax + Magnetic stirring --
TM Supernatant 5% 7 Ultra-Turrax + Magnetic stirring --
TM Full sample 15% 7 Ultra-Turrax + Magnetic stirring +
TM Pellet 15% 7 Ultra-Turrax + Magnetic stirring +
TM Supernatant 15% 7 Ultra-Turrax + Magnetic stirring --
TM Full sample 25% 7 Ultra-Turrax + Magnetic stirring ++
TM Pellet 25% 7 Ultra-Turrax + Magnetic stirring ++
TM Supernatant 25% 7 Ultra-Turrax + Magnetic stirring --
TM Full sample 5% 10 Ultra-Turrax + Magnetic stirring --
TM Pellet 5% 10 Ultra-Turrax + Magnetic stirring --
TM Supernatant 5% 10 Ultra-Turrax + Magnetic stirring --
TM Full sample 15% 10 Ultra-Turrax + Magnetic stirring ++
TM Pellet 15% 10 Ultra-Turrax + Magnetic stirring ++
TM Supernatant 15% 10 Ultra-Turrax + Magnetic stirring --
TM Full sample 25% 10 Ultra-Turrax + Magnetic stirring ++
TM Pellet 25% 10 Ultra-Turrax + Magnetic stirring ++
TM Supernatant 25% 10 Ultra-Turrax + Magnetic stirring --
TM Full sample 25% 4 Ultra-Turrax + Magnetic stirring + Ultrasound ++
TM Pellet 25% 4 Ultra-Turrax + Magnetic stirring + Ultrasound ++
TM Supernatant 25% 4 Ultra-Turrax + Magnetic stirring + Ultrasound --
TM Full sample 25% 7 Ultra-Turrax + Magnetic stirring + Ultrasound ++
TM Pellet 25% 7 Ultra-Turrax + Magnetic stirring + Ultrasound ++
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TM Supernatant 25% 7 Ultra-Turrax + Magnetic stirring + Ultrasound --

TM Full sample 25% 10 Ultra-Turrax + Magnetic stirring + Ultrasound ++
TM Pellet 25% 10 Ultra-Turrax + Magnetic stirring + Ultrasound ++
TM Supernatant 25% 10 Ultra-Turrax + Magnetic stirring + Ultrasound --

Note. TM - Tenebrio molitor larvae; 0-25% solidification: Very weak gel ( - - ); 26-50% solidification: Weak
gel (-); 51-75% solidification: Strong gel (+); 76-100% solidification: Very strong gel ( + + ).

T.molitor larvae full samples and pellets had the same gelling ability results for all
tested conditions. Generally, concentration of defatted powder had an impact in gelling
ability of full samples and pellets. Higher concentrations of defatted powder resulted in
stronger gelling abilities at pH4 and at pH7. At pH10, the gelling ability increased from
5% concentration of defatted powder to 15%, reaching the strongest ability at this
concentration, thus remaining the same at 25% concentration. When the pH value of the
extractions was compared at the same concentration of defatted powder it was visible
that it had no effect on 5% and 25% concentrations. However, at 15% concentration of
defatted powder an increase in pH resulted in a stronger gel. Regarding the application
of the ultrasound treatment, it was revealed that it did not affect the gelling ability of full
samples and pellets.

As for T.molitor larvae supernatants, these were categorized as very weak gels
across all samples since the different concentrations of defatted powder, pH of
extractions and methodologies had no improvements in the gelling ability of this fraction.

Table 14 - Gelling ability of the full samples, supernatants and pellets from Acheta domesticus extracted at
different concentrations of powder (w/v), pH values and methodologies.

Conditions
Sample Gelling Ability
Concentration (w/v) pH Methodology

AD Full sample 5% 4 Ultra-Turrax + Magnetic stirring -

AD Pellet 5% 4 Ultra-Turrax + Magnetic stirring -
AD Supernatant 5% 4 Ultra-Turrax + Magnetic stirring --
AD Full sample 15% 4 Ultra-Turrax + Magnetic stirring +
AD Pellet 15% 4 Ultra-Turrax + Magnetic stirring +
AD Supernatant 15% 4 Ultra-Turrax + Magnetic stirring --
AD Full sample 25% 4 Ultra-Turrax + Magnetic stirring ++
AD Pellet 25% 4 Ultra-Turrax + Magnetic stirring ++
AD Supernatant 25% 4 Ultra-Turrax + Magnetic stirring --
AD Full sample 5% 7 Ultra-Turrax + Magnetic stirring -
AD Pellet 5% 7 Ultra-Turrax + Magnetic stirring -
AD Supernatant 5% 7 Ultra-Turrax + Magnetic stirring --
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AD Full sample 15% 7 Ultra-Turrax + Magnetic stirring +

AD Pellet 15% 7 Ultra-Turrax + Magnetic stirring +
AD Supernatant 15% 7 Ultra-Turrax + Magnetic stirring --
AD Full sample 25% 7 Ultra-Turrax + Magnetic stirring ++
AD Pellet 25% 7 Ultra-Turrax + Magnetic stirring ++
AD Supernatant 25% 7 Ultra-Turrax + Magnetic stirring --
AD Full sample 5% 10 Ultra-Turrax + Magnetic stirring -
AD Pellet 5% 10 Ultra-Turrax + Magnetic stirring -
AD Supernatant 5% 10 Ultra-Turrax + Magnetic stirring --
AD Full sample 15% 10 Ultra-Turrax + Magnetic stirring +
AD Pellet 15% 10 Ultra-Turrax + Magnetic stirring +
AD Supernatant 15% 10 Ultra-Turrax + Magnetic stirring --
AD Full sample 25% 10 Ultra-Turrax + Magnetic stirring ++
AD Pellet 25% 10 Ultra-Turrax + Magnetic stirring ++
AD Supernatant 25% 10 Ultra-Turrax + Magnetic stirring --
AD Full sample 25% 4 Ultra-Turrax + Magnetic stirring + Ultrasound ++
AD Pellet 25% 4 Ultra-Turrax + Magnetic stirring + Ultrasound ++
AD Supernatant 25% 4 Ultra-Turrax + Magnetic stirring + Ultrasound --
AD Full sample 25% 7 Ultra-Turrax + Magnetic stirring + Ultrasound ++
AD Pellet 25% 7 Ultra-Turrax + Magnetic stirring + Ultrasound ++
AD Supernatant 25% 7 Ultra-Turrax + Magnetic stirring + Ultrasound --
AD Full sample 25% 10 Ultra-Turrax + Magnetic stirring + Ultrasound ++
AD Pellet 25% 10 Ultra-Turrax + Magnetic stirring + Ultrasound ++
AD Supernatant 25% 10 Ultra-Turrax + Magnetic stirring + Ultrasound --

Note. AD - Acheta domesticus; 0-25% solidification: Very weak gel ( - - ); 26-50% solidification: Weak gel (-
); 51-75% solidification: Strong gel (+); 76-100% solidification: Very strong gel ( + + ).

A.domesticus full samples and pellets also had the same gelling ability results for
all tested conditions. In both of these fractions, concentration of defatted powder had an
impact in gelling since a higher concentration results in a stronger gelling ability at pH4,
pH7 and pH10. When the pH value of the extractions was compared at the same
concentration of defatted powder it was observed that there were no effects on gelling
ability in any of the tested concentrations. The application of an ultrasound treatment did
not improve the gelling ability of full samples and pellets since non-ultrasound treatment
samples had the same results as the ultrasound treated ones.

Supernatants from A.domesticus had the same gelling ability as the ones from
T.molitor as these were also categorized as very weak gels across all samples. The
different concentrations of defatted powder, pH of extractions and methodologies had no
improvements in the gelling ability of this fraction.
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10.4 Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis

(SDS-PAGE)

SDS-PAGE results are displayed in figure 21A and 21B for both non-reduced and
reduced gels respectively. The wells are numbered from 1 to 15 and the correspondent
labelling is beneath the figures. Wells 1, 8 and 15 are the molecular weight markers and
the weight of their bands is identified, in kDa, at the beginning and at the end of each of
the gels. Areas with visible bands were surrounded by a black rectangle for a clearer
interpretation.
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Figure 21 - Non-reduced (A) and reduced (B) SDS-PAGE gels with supernatant proteins from Tenebrio
molitor larvae and Acheta domesticus extracted with different conditions (TM – Tenebrio molitor larvae; AD
– Acheta domesticus; 1 – Marker; 2 – TM 25% concentration (w/v) pH4 non-ultrasound; 3 – TM 25%
concentration (w/v) pH4 ultrasound; 4 – TM 25% concentration (w/v) pH7 non-ultrasound; 5 – TM 25%
concentration (w/v) pH7 ultrasound; 6 – TM 25% concentration (w/v) pH10 non-ultrasound; 7 – TM 25%
concentration (w/v) pH10 ultrasound; 8 – Marker; 9 – AD 25% concentration (w/v) pH4 non-ultrasound; 10
– AD 25% concentration (w/v) pH4 ultrasound; 11 – AD 25% concentration (w/v) pH7 non-ultrasound; 12 –
AD 25% concentration (w/v) pH7 ultrasound; 13 – AD 25% concentration (w/v) pH10 non-ultrasound; 14 –
AD 25% concentration (w/v) pH10 ultrasound; 15 – Marker).
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Both non-reduced and reduced SDS-PAGE gels exhibit bands in the same wells
with the same molecular weights. However, the non-reduced gel displays a stronger
colour intensity throughout the 15 wells as also more intense bands, in comparison to
the reduced gel. Therefore, the dithiothreitol (DTT) used in the reduced gel in order to
reduce disulphide bridges in proteins so that they would adopt a random coil
conformation, did not improved the separation of the soluble proteins from T.molitor
larvae and A.domesticus.

When analysing the molecular weight of T.molitor larvae protein bands at pH4 it is
detectable that there are several visible protein bands with weights from 8-9kDa to 55-
58kDa without any visible bands at pH7 and pH10.

As for A. domesticus protein bands at pH4, there are visible bands from 9-10kDa
to 24-26kDa with a small single band between 80 and 110 kDa that is more visible in the
ultrasound treated sample. At pH7, the bands from 9-10kDa to 24-26kDa are no longer
visible, only being visible bands from 43-46 kDa to 80-110 kDa in the non-ultrasound
treated sample. At pH10 there were no visible bands.

After the examination of both gels’ results it is clearly noticeable that pH had an
impact on bands’ formation for both insects since higher pH values of extraction had less
formation of bands. At pH4 it is observed the highest formation of bands while at pH10
there were no visible bands on both insects’ proteins. At pH7, a few number of bands
are visible but only on non-ultrasound treatment samples. Regarding the intensity of
colour of the migrated proteins, it is evident that a stronger intensity of blue was exhibited
in higher pH values of extraction despite being associated with no visible bands. This
could be linked to having higher protein concentrations at higher pH values of extraction
that was also evident in the protein solubility results mentioned earlier in section 10.2,
since a higher protein solubility was found at pH10, then at pH7 and lastly at pH4. These
results could indicate that: either wells from pH7 and pH10 samples had an excessive
amount of proteins and that a higher dilution is needed for proteins to properly run the
gel; or that the proteins’ conformation was severely affected with higher pH values of
extraction.

Regarding the effect of the ultrasound treatment on bands’ formation, bands from
proteins extracted this treatment appear to be more visible at pH4 values of extraction
on both insects compared to the non-ultrasound samples at the same pH. However,
A.domesticus protein bands that were visible at pH7 without the ultrasound treatment,
faded away in the ultrasound treated samples at the same pH. The intensity of colour of
the migrated proteins appears to be stronger in the ultrasound treated samples, specially
at pH7 and pH10, despite not showing visible bands. This stronger intensity of blue could
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also be related to a higher concentration of proteins that is supported by a higher protein

solubility found in section 10.2.1 and 10.2.2 in samples treated with ultrasounds
compared to the ones without this treatment.

10.5 Gelling ability of the proteins

Gelling ability results of the freeze-dried soluble proteins from T.molitor larvae and
A.domesticus are displayed in table 15. The gelling ability designation is displayed
beneath the table.

Table 15 - Gelling ability of freeze-dried soluble proteins from Tenebrio molitor larvae and Acheta
domesticus at different concentrations of soluble proteins (w/v) and with or without addition of 0.1M Ca 2+.

Conditions
Sample Gelling ability
Concentration (w/v) Addition
TM soluble proteins 5% No addition --
TM soluble proteins 10% No addition --
TM soluble proteins 15% No addition --
2+
TM soluble proteins 5% 0.1M Ca --
TM soluble proteins 10% 0.1M Ca2+ --
2+
TM soluble proteins 15% 0.1M Ca --
AD soluble proteins 5% No addition --
AD soluble proteins 10% No addition --
AD soluble proteins 15% No addition --
2+
AD soluble proteins 5% 0.1M Ca --
AD soluble proteins 10% 0.1M Ca2+ --
2+
AD soluble proteins 15% 0.1M Ca --

Note. TM - Tenebrio molitor larvae; AD – Acheta domesticus; 0-25% solidification: Very weak gel ( - - ); 26-
50% solidification: Weak gel (-); 51-75% solidification: Strong gel (+); 76-100% solidification: Very strong gel
( + + ).

At concentrations of soluble proteins (w/v) of 5%, 10% and 15%, and with or
without the addition of 0.1M of Ca2+, it was noticed that all samples would fall when the
test tubes were inverted in the day after gelation. As a result, all samples were classified
as very weak gels ( - - ). Despite this classification in all the samples from both insects,
it was clearly noticeable visible differences between the samples (Figure 22).
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Figure 22 – Gelling samples from Tenebrio molitor larvae (1 to 6) and Acheta domesticus (7 to 12) soluble
proteins at different concentrations (w/v) and with or without 0.1M Ca2+ addition (TM – Tenebrio molitor
larvae; AD – Acheta domesticus; 1 – TM 5% concentration (w/v) without calcium; 2 – TM 10% concentration
(w/v) without calcium; 3 – TM 15% concentration (w/v) without calcium; 4 – TM 5% concentration (w/v) 0.1M
Ca2+; 5 – TM 10% concentration (w/v) 0.1M Ca2+; 6 – TM 15% concentration (w/v) 0.1M Ca2+; 7 – AD 5%
concentration (w/v) without calcium; 8 – AD 10% concentration (w/v) without calcium; 9 – AD 15%
concentration (w/v) without calcium; 10 – AD 5% concentration (w/v) 0.1M Ca2+; 11 – AD 10% concentration
(w/v) 0.1M Ca2+; 12 – AD 15% concentration (w/v) 0.1M Ca2+).

Concentration had a significant impact on the colour of the samples from both
insects, with lower concentrations of soluble proteins showing lighter colours when
compared to higher concentrations. However, this darkness with higher concentrations
was more noticeable from 5% concentration to 10% then from 10% to 15%. Furthermore,
the addition of 0.1M Ca2+ also had an effect on the colour of the samples since samples
from the same insect at the same concentration of soluble proteins revealed a lighter
colour when calcium ions were added in comparison to samples without calcium addition.

It was also noticeable that concentration had no significant impact in foam

formation since this formation was quite similar between samples with the same
conditions apart from concentration. Nevertheless, foam formation was clearly superior
in samples where 0.1M Ca2+ was added when compared to samples without calcium
ions addition.

It is worth mentioning that all samples appeared to have a liquid yogurt-like

viscosity that was visible when the test tubes were inverted. Viscosity also appeared to
be increased with an increase in concentration.

Since all gels were labelled as very weak gels, the middle concentration of soluble
proteins, 10% (w/v), was chosen for the rheological tests of T.molitor larvae and
A.domesticus. Likewise, dispersions at 10% (w/v) concentrations were previously used
by Kumar et al. (2022) to study rheological properties of dispersions from black soldier
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fly (Hermetia illucens) larvae thus being an additional validation of the chosen
concentration.

10.6 Rheological properties of the proteins

10.6.1 Rheological properties of Tenebrio molitor larvae proteins

The temperature ramp, the frequency sweep and the amplitude sweep tests of
T.molitor larvae protein dispersions with and without the addition of 0.1M Ca2+, are
displayed in figures 23, 24 and 25 respectively.

10 90

9 80

8
70
7
60

Temperature (ºC)
6
G'/G'' (Pa)

50
5
40
4
30
3
20
2

1 10

0 0
70
0

771

1051
1121
1191
1261
1336
1420
1504
1588
1672
1756
1840
1924
2008
2092
2176
2260
2344
2428
2512
140
210
280
350
420
490
560
630
701

841
911
981

Time (s)
G' TM (No calcium) G'' TM (No calcium)
G' TM (Calcium) G'' TM (Calcium)

Figure 23 – Temperature ramp of 10% (w/v) protein dispersions of Tenebrio molitor larvae (TM) – Elastic
modulus (G’) (Pa), viscous modulus (G’’) (Pa) and temperature (ºC) as a function of time (s) for samples
with and without calcium addition.

During the heating period from 25ºC to 85ºC, there were no increases in both
elastic and viscous modulus for both samples. In the start of the constant heating period
at 85ºC, it was noticeable a small increase in both modulus for both samples that was
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rapidly reduced. Across the remaining constant heating period, there were small
increases in both elastic and viscous modulus but only in the sample with calcium
addition, while the sample without calcium exhibited no changes. Accordingly to Yi et al.
(2013) and Kumar et al. (2022), these small increases could be due to the formation of
aggregates that lead to the sol-gel transition. In the cooling period, both the elastic and
viscous modulus had a clear increase but only on the sample with calcium addition with
no significant increases on the sample without calcium. Yi et al. (2013) and Kumar et al.
(2022) state that these increases could be a result of the formation of hydrogen bonds
for the development of the gel structure. Therefore, in the sample with calcium addition,
this last step could be crucial for the formation of the gel network, specially due to the
increase in the elastic modulus (Hu et al., 2013; Kumar et al., 2022). Additionally, it is
noteworthy that the sample with calcium exhibited a dominant elastic behaviour
throughout the heating and cooling periods when compared to the viscous behaviour.

The temperature ramp on T.molitor larvae samples proved the results found in
section 10.5, since samples have a very weak formation of gels, not surpassing values
of 8 Pa for the elastic modulus and 2 Pa for the viscous modulus throughout the entire
temperature ramp. Nonetheless, an increase in gelling formation is visible when 0.1M
Ca2+ was added to the protein dispersions. Similar finding were also found by Xiao et al.
(2021) when studying cellulose nanocrystals-whey protein isolate composite gels.
According to the authors, Ca2+ in low concentrations (0.05M to 0.15M) can improve
gelation by inducing conformational modifications (Xiao et al., 2021).
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40

35

30

25
G'/G'' (Pa)

20

15

10

Frequency (Hz)

G' TM (No calcium) G'' TM (No calcium)

G' TM (Calcium) G'' TM (Calcium)

Figure 24 - Frequency sweep of 10% (w/v) protein dispersions of Tenebrio molitor larvae (TM) – Elastic
modulus (G’) (Pa) and viscous modulus (G’’) (Pa) as a function of frequency (Hz) for samples with and
without calcium addition.

When analysing the frequency sweep, it is visible that there was a general increase
in both modulus in the end of the sweep at 20 Hz except for the viscous modulus for the
sample with calcium that a decrease from 17.15 Hz to 20 Hz, exhibiting lower values at
the end comparatively to the start. Furthermore, it is noticeable that despite the sample
with calcium starting with higher elastic and viscous modulus, upon an increase in
frequency (at 7 Hz for the elastic modulus and 15 Hz for the viscous modulus), the
sample without calcium had higher elastic and viscous modulus until the end of the
sweep. Thus, it is noteworthy to mention that the calcium addition might interfere to a
certain degree in gel behaviour when gels are submitted to higher frequencies, since
both modulus exhibit lower values when calcium is added to the dispersions.
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10

6
G'/G'' (Pa)

4
3

158,48

292,86
116,60

215,45

398,09
541,18
735,59
10,00
13,60
18,48
25,12
34,14
46,42
63,09
85,76
0,07
0,09
0,12
0,25
0,34
0,46
0,63
0,86
1,17
1,58
2,16
2,93
3,98
5,40
7,36

1 000,15
1 359,19
Shear strain (%)

G' TM (No calcium) G'' TM (No calcium)

G' TM (Calcium) G'' TM (Calcium)

Figure 25 - Amplitude sweep of 10% (w/v) protein dispersions of Tenebrio molitor larvae (TM) – Elastic
modulus (G’) (Pa) and viscous modulus (G’’) (Pa) as a function of shear strain (%) for samples with and
without calcium addition.

As for the amplitude sweep, the sample with calcium addition started with higher
elastic and viscous modulus that remained relatively stable at a lower shear strain. When
the shear strain was increased both elastic and viscous modulus were reduced in the
sample with calcium addition, with a crossover of the viscous modulus over the elastic
modulus at a shear rate around 40%. The sample without calcium addition exhibited both
modulus values close to 0 Pa throughout the whole amplitude sweep.

In brief, T.molitor larvae showed extremely low gelation throughout the three
rheological tests. Moreover, calcium addition could be beneficial for gel formation in
T.molitor larvae protein dispersions however it could negatively interfere with gel
behaviour when frequency is increased.

10.6.2 Rheological properties of Acheta domesticus proteins

The temperature ramp, the frequency sweep and the amplitude sweep tests of
A.domesticus protein dispersions with and without the addition of 0.1M Ca2+, are
displayed in figures 26, 27 and 28 respectively.
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10 90

9 80
8 70
7
60

Temperature (ºC)
6
G'/G'' (Pa)

50
5
40
4
30
3

2 20

1 10

0 0
70
0

630

1051
1121
1191
1261
1336
1420
1504
1588
1672
1756
1840
1924
2008
2092
2176
2260
2344
2428
2512
140
210
280
350
420
490
560

701
771
841
911
981

Time (s)

G' AD (No calcium) G'' AD (No calcium)

G' AD (Calcium) G'' AD (Calcium)

Figure 26 - Temperature ramp of 10% (w/v) protein dispersions of Acheta domesticus (AD) – Elastic
modulus (G’) (Pa), viscous modulus (G’’) (Pa) and temperature (ºC) as a function of time (s) for samples
with and without calcium addition.

Similar to the findings in section 10.6.1 on T.molitor larvae, A.domesticus

temperature ramp also proved the results found in section 10.5, since samples exhibited
no formation or very weak formation of gels, not surpassing values of 2 Pa in both elastic
and viscous modulus in both samples throughout the entire temperature ramp. Minimal
increases in both modulus were found during the constant heating period and cooling
period in the sample with calcium addition comparatively to the one without calcium.
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40

35

30

25
G'/G'' (Pa)

20

15

10

Frequency (Hz)

G' AD (No calcium) G'' AD (No calcium)

G' AD (Calcium) G'' AD (Calcium)

Figure 27 - Frequency sweep of 10% (w/v) protein dispersions of Acheta domesticus (AD) – Elastic modulus
(G’) (Pa) and viscous modulus (G’’) (Pa) as a function of frequency (Hz) for samples with and without calcium
addition.

In regards to the frequency sweep, it is noticeable that there was an increase in

elastic and viscous modulus in both samples with an increase in frequency. Moreover,
at the end of the sweep, it is visible that the sample without calcium had higher elastic
and viscous modulus comparatively to the sample with calcium addition. These finding
were similar to the ones found in section 10.6.1 on T.molitor larvae, where the calcium
addition also appeared to interfere to a certain degree in gel behaviour when higher
frequencies were tested.
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10

6
G'/G'' (Pa)

85,76

398,09
116,60
158,48
215,45
292,86

541,18
735,59
10,00
13,60
18,48
25,12
34,14
46,42
63,09
0,07
0,09
0,12
0,25
0,34
0,46
0,63
0,86
1,17
1,58
2,16
2,93
3,98
5,40
7,36

1 000,15
1 359,19
Shear strain (%)

G' AD (No calcium) G'' AD (No calcium)

G' AD (Calcium) G'' AD (Calcium)

Figure 28 - Amplitude sweep of 10% (w/v) protein dispersions of Acheta domesticus (AD) – Elastic modulus
(G’) (Pa) and viscous modulus (G’’) (Pa) as a function of shear strain (%) for samples with and without
calcium addition.

Concerning the amplitude sweep, both samples started the sweep with extremely
low elastic and viscous modulus (< 1 Pa). Even so, the sample with calcium addition
started with higher elastic and viscous modulus that remained relatively stable at a lower
shear strain. With an increase in shear strain, both elastic and viscous modulus were
reduced in the sample with calcium addition, with a crossover of the viscous modulus
over the elastic modulus at a shear rate around 50%. Despite the values being close to
0 Pa, the sample without calcium addition had a crossover of the viscous modulus over
the elastic modulus at a shear rate around 0.46%.

In sum, and similarly to T.molitor larvae results, A.domesticus revealed extremely

low gelation throughout the three rheological tests. Contrarily to T.molitor larvae, calcium
addition led minimal differences in gel formation of A.domesticus protein dispersions and
negatively interfered with gel behaviour at higher frequencies.
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11. Conclusion

The current experiments provided a better understanding on how different protein

extraction conditions, such as pH of extraction, concentration of powder and application
of ultrasounds, influence the protein solubility, the gelling ability of full samples,
supernatants and pellets and the molecular weight of T.molitor larvae and A.domesticus
extracted proteins. In addition, the study also explored the gelling ability of soluble
proteins at different concentrations and assessed rheological properties.

In general, increases in pH of extraction and concentration of defatted powder led

to higher protein solubility percentages in T.molitor larvae. As for A.domesticus overall
results, only the pH4 led to lower protein solubility percentages in comparison to the
other pH values, and only the 5% concentration of powder led to lower protein solubility
percentages in comparison to the other concentrations of powder. Both insects had no
significant differences between ultrasound and non-ultrasound treated samples in terms
of protein solubility despite being slightly higher in ultrasound treated samples. In regards
to SDS-PAGE, bands were only noticeable when proteins were extracted with lower pH
values. When gelling ability was assessed, it was visible that pellets and full samples
exhibited better gelling ability in comparison to supernatants in both insects. Also
regarding gelling ability, the freeze-dried soluble proteins revealed a very weak gelling
ability at 5%, 10% and 15% concentrations of soluble proteins regardless calcium
addition. As for rheology, the three rheological tests proved that the protein dispersions
from both species had extremely low gelation. The addition of calcium in the dispersions
might be beneficial for gel formation, especially on T.molitor larvae, despite negatively
affecting gel behaviour at 20 Hz in both insects.

To have a deeper understanding of the impact of these experiments, future studies

should aim to analyse different methodologies for protein extraction that could lead to
relevant techno-functional properties and sustainability. Furthermore, the rheological
properties of the protein dispersions from both species could be assessed at higher
concentrations of soluble proteins and at higher concentrations of calcium ions to see if
gelling formation can be improved.

In brief, the aims of the current research were accomplished. These findings reveal
additional information for the scientific community that could lead to further progress in
research regarding protein extraction, techno-functional properties of edible insects and
future food applications.
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12. Dissemination of results

-Oral presentation:

Ribeiro, J. C., Marques, J. P., Fernandes, T. R., Pintado, M. E., Carvalho, S. M. P., Cunha,
L. M., (2022, September) “Effect of conservation, blanching and drying conditions on the
safety and quality of Tenebrio molitor” INSECTA, Gießen, Germany.

-Publication of the systematic review article in an international scientific journal:

It is currently being elaborated a manuscript, to be submitted at the Critical Reviews in

Food Science and Nutrition journal, named “Systematic review of edible insects’
fractionation: extraction methods and characterization of chemical, techno-functional and
biological properties” that will comprise results from the Part A of this dissertation.
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14. Appendices

Appendix 1. Supplementary table of the included studies in the systematic review about lipid extraction in
edible insects

Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Blattodea
Aqueous extraction

OR

Soxhlet extraction (petroleum

Aqueous extraction – 3.1% (Yield) FA composition
Tzompa-Sosa et al. Argentinian wood roach Ground and dried ether)
Soxhlet (petroleum ether) – 7.6% (Yield) Lipid classes ND
(2014) (Blaptica dubia) adult (Soxhlet and Folch)
Folch (dichloromethane:methanol, 2:1) – 7.5% (Yield) TAG profile
OR

Folch extraction
(dichloromethane:methanol,
2:1)
Polarized light
microscopy
Tzompa-Sosa et al. FA composition
B. dubia adult NA Aqueous extraction ND ND
(2019) DSC
Colour
Volatile compounds
Coleoptera
Lesser mealworm Aqueous extraction Aqueous extraction – 5.5% (Yield) FA composition
Tzompa-Sosa et al. Ground and dried
(Alphitobius diaperinus) Soxhlet (petroleum ether) – 10.7% (Yield) Lipid classes ND
(2014) (Soxhlet and Folch)
larvae OR Folch (dichloromethane:methanol, 2:1 ratio) – 9.4% (Yield) TAG profile
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Soxhlet extraction (petroleum

ether)

OR

Folch extraction
(dichloromethane:methanol,
2:1)
Polarized light
microscopy
Tzompa-Sosa et al. FA composition
A. diaperinus larvae NA Aqueous extraction ND ND
(2019) DSC
Colour
Volatile compounds
Coconut borer (Pachymerus Conventional extractiona Toxicological
Alves et al. (2019) Dried ND ND
nucleorum) larvae (hydro-ethanolic solvent) profile
Conventional extractiona
(methanol, ethanol and n- Dried larvae – 21.2% (LC)
hexane) Defatted powder (aqueous extraction) – 14.82% (LC)
T. K. Kim et al. White-spotted flower chaffer
Dried and ground Defatted powder (ethanol) – 0.13% (LC) ND ND
(2021) (Protaetia brevitarsis) larvae
OR Defatted powder (n-hexane) – 0.32% (LC)
Defatted powder (methanol) – 1.17% (LC)
Aqueous extraction
African palm weevil Lipid classes
Fogang Mba et al. Conventional extractiona Conventional extractiona (chloroform) - 0.29 mg/mL to
(Rhynchophorus phoenicis) Aqueous extracts Total lipids ND
(2021) (chloroform) 1.42 mg/mL (LC)
larvae FA composition
Yellow mealworm (Tenebrio Conventional extractiona Toxicological
Alves et al. (2019) Dried ND ND
molitor) larvae (hydro-ethanolic solvent) profile
Bußler et al. Conventional extractiona Whole powder – 19.1% (LC)
T. molitor larvae Dried and ground ND ND
(2016) (hexane) Defatted powder after extraction – 2.8% (LC)
Conventional extractiona
Choi et al. (2017) T. molitor larvae Dried and ground Defatted powder – 0.69% (LC) ND ND
(hexane)
del Hierro et al. Conventional extractiona
T. molitor larvae Dried and ground ND ND ND
(2021) (hexane)
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

AND/OR

Ultrasound-assisted extraction
(methanol, ethanol or ethyl
acetate)
Gravel et al. Dried, ground and Whole powder – 28.5% (LC)
T. molitor larvae Soxhlet extraction (hexane) ND ND
(2021) sieved Defatted powder (Soxhlet - hexane) – 0.4% (LC)
Incorporation in Red
Conventional extractiona Seabream (Pargus
Ido et al. (2019) T. molitor larvae Commercial powder Defatted powder (hexane:ethanol, 9:1) - 5.6% (LC) ND
(hexane:ethanol, 9:1) major) diet
formulation
Soxhlet extraction (hexane,
petroleum ether, ethyl acetate
or ethanol)
Soxhlet (hexane) – 25.5% (Yield)
Soxhlet (petroleum ether) – 24.3% (Yield)
OR
Laroche et al. Soxhlet (ethyl acetate) – 25.7% (Yield)
T. molitor larvae Commercial powder FA composition ND
(2019) Soxhlet (ethanol) – 28.8% (Yield)
Three-phase partitioning
Three-phase partitioning – 23.7% (Yield)
SC-CO2 – 22.1% (Yield)
OR

SC-CO2 extraction
Ultrasound-assisted extraction
Ultrasound-assisted extraction (ethanol) – 28.85% (Yield)
(ethanol or ethanol:water, 1:1)
Ultrasound-assisted extraction (ethanol:water; 1:1) –
FA composition
17.14% (Yield)
Otero et al. (2020) T. molitor larvae Dried and ground OR Lipid classes ND
Pressurized-liquid extraction (ethanol) – 32.37% (Yield)
Cholesterol content
Pressurized-liquid extraction (ethanol:water; 1:1) – 33.87%
Pressurized-liquid extraction
(Yield)
(ethanol or ethanol:water, 1:1)
SC-CO2 extraction
Extraction kinetics
Purschke et al. OR SC-CO2 – 20.83%-95.30% (OR) Acid value
T. molitor larvae Dried and ground ND
(2017) Conventional extractiona (n-hexane) – 96.56% (OR) Free FA analysis
Conventional extractiona (n- FA composition
hexane)
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Saponification value
Acid value
Iodine value
Peroxide value
Volatile matter
Biodiesel production
Sen Siow et al. Soxhlet extraction (petroleum Insoluble impurities
T. molitor larvae Ground and dried Soxhlet (petroleum ether) – 37.54% (Yield) via
(2021) ether) Moisture content
transesterification
Cloud point
Phosphorus, iron
and copper content
TGA
FA composition
Sipponen et al. FA composition
T. molitor larvae Dried and ground SC-CO2 extraction SC-CO2 – 3.5% (LC of defatted fraction)/74% (OR) ND
(2018) Lipid classes
Conventional extractiona
(hexane)
Whole powder – 37.55% (LC)
Son et al. (2019) T. molitor larvae Dried Defatted powder (screw press) – 13.22%-14.45% (LC) ND ND
OR
Defatted powder (hexane) – 1.98% (LC)
Screw press extraction
FA composition
Specific gravity
Viscosity
Colour
Peroxide value
Acid value
TBARS value
Conventional extractiona (n-
Son et al. (2020) T. molitor larvae Dried and ground 29.5% (Yield) Induction time ND
hexane)
Tocopherols content
Squalene content
Sterols content
Polyphenol content
Predicted shelf-life
Anti-inflammatory
activity
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Aqueous extraction

OR

Soxhlet extraction (petroleum

Aqueous extraction – 7.8% (Yield) FA composition
Tzompa-Sosa et al. Ground and dried ether)
T. molitor larvae Soxhlet (petroleum ether) – 12.7% (Yield) Lipid classes ND
(2014) (Soxhlet and Folch)
Folch (dichloromethane:methanol, 2:1) – 12.9% (Yield) TAG profile
OR

Folch extraction
(dichloromethane:methanol,
2:1)
Polarized light
microscopy
Tzompa-Sosa et al. FA composition
T. molitor larvae NA Aqueous extraction ND ND
(2019) DSC
Colour
Volatile compounds

HHP-assisted extraction FA composition

(hexane) HHP-assisted extraction (hexane) – 22.75%-22.90% DSC
(Yield) Peroxide value
Ugur et al. (2021) T. molitor larvae Commercial powder OR ND
Conventional extractiona (hexane) – 24.07%-24.22% Total phenolic
(Yield) content
Conventional extractiona
Antioxidant activity
(hexane)
Conventional extractiona Conventional extractiona (ethanol) – 33.1%-34.8% (Yield)
Zhao et al. (2016) T. molitor larvae Dried and ground (hexane:isopropanol or Conventional extractiona (hexane:isopropanol, 3:2) – FA composition ND
ethanol) 31.6% (Yield)
Diptera
pH adjustment (7) followed NB:AE – 80% (OR – Cream)
Blanched (B) OR non-
by centrifugation (AE) NB:S-AE – 70% (OR – Cream)
blanched (NB) followed
Azzollini et al. Black soldier fly (Hermetia B:AE – 50% (OR – 20% Cream + 30% Lipid) Microstructural
by mechanical ND
(2020) illucens) larvae OR B:S-AE – 50% (OR – 40% Cream + 10% Lipid) characterization
processing in a slow
B:S-H-AE – 60% (OR – 15% Cream + 45% Lipid)
juicer
Sonication + pH adjustment B:H-AE – 60% (OR – 15% Cream + 45% Lipid)
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

(7) followed by centrifugation

(S-AE)

OR

Sonication + Hydrolysis + pH
adjustment (7) followed by
centrifugation (S-H-AE)

OR

Hydrolysis + pH adjustment
(7) followed by centrifugation
(H-AE)
Bußler et al. Conventional extractiona Whole powder – 20.0% (LC)
H. illucens larvae Dried and ground ND ND
(2016) (hexane) Defatted powder after extraction – 2.8% (LC)
Microwave-assisted extraction (methanol) – 43.26%
(Yield)
Microwave-assisted extraction (chloroform:methanol, 2:1)
Microwave-assisted – 34.06% (Yield)
extraction (methanol, Microwave-assisted extraction (chloroform) – 33.75% FA composition Biodiesel production
C. Wang et al. chloroform:methanol 2:1, (Yield) FTIR via
H. illucens larvae Homogenized and dried
(2017) chloroform, hexane:methanol Microwave-assisted extraction (hexane:methanol, 3:2) – DSC transesterification
3:2, hexane, petroleum ether 32.51% (Yield) TGA process
or acetone) Microwave-assisted extraction (hexane) – 31.78% (Yield)
Microwave-assisted extraction (petroleum ether) – 31.32%
(Yield)
Microwave-assisted extraction (acetone) – 25.58% (Yield)
Nano-emulsions:
- TEM
Conventional extractiona
- Droplet Size, PDI,
(hexane) followed by
Chou et al. (2020) H. illucens larvae Dried and ground ND and Zeta Potential Nano-emulsions
purification (degumming,
- Rheology
dewaxing, and bleaching)
- DSC
- Fluorescence
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Polarization
- Stability
Conventional extractiona
(hexane)
Ethanol 100% - 1.89% (LC)
del Hierro et al.
H. illucens larvae Dried and ground AND/OR Ethanol 70% - 1.29% (LC) ND ND
(2021)
Ethanol 50% - 0.76% (LC)
Ultrasound-assisted extraction
(ethanol 100%, 70% or 50%)
Microwave-assisted extraction (petroleum ether) – 28.5%-
31.5% (Yield)/76%-83% (OR)
Microwave-assisted Kinetics and
Microwave-assisted extraction (n-hexane) – 29.5%-31.5%
Feng et al. (2018) H. illucens larvae Ground and dried extraction (petroleum ether, thermodynamics of ND
(Yield) /78%-83% (OR)
n-hexane, and chloroform) extraction
Microwave-assisted extraction (chloroform) – 31.0%-
33.5% (Yield)/ /82%-89% (OR)
FA composition
Microwave-assisted Energy FAMEs biodiesel
Feng et al. (2019) H. illucens larvae Ground and dried 47-85% (OR)
extraction (n-hexane) consumption conversion
evaluation
n-Hexane – 46.38% (OR)
Conventional extractiona (n-
Petroleum ether – 51.35% (OR)
hexane, petroleum ether, Saponifiable lipids
Ethanol – 29.34% (OR)
ethanol, isopropanol, n- purity
Isopropanol – 34.69% (OR) FAMEs biodiesel
Feng et al. (2020) H. illucens larvae NA hexane:ethanol, n- FA composition
n-Hexane:ethanol, 1:1 – 65.79% (OR) conversion
hexane:isopropanol, Kinetics of
n-Hexane:isopropanol, 1:1 – 70.34% (OR)
petroleum ether:ethanol, extraction
Petroleum ether:ethanol, 1:1 – 67.57% (OR)
petroleum ether:isopropanol)
Petroleum ether:isopropanol, 1:1 – 53.42%-75.92% (OR)
Conventional extractiona Conventional extractiona – 64.29% (OR)
(ethyl acetate) CTAB (Hexadecyl trimethyl ammonium bromide) assisted Saponifiable lipids
extraction – 78.99% (OR) purity
OR SDBS (Sodium dodecylbenzene sulfonate) assisted FA composition FAMEs biodiesel
Feng et al. (2021) H. illucens larvae NA
extraction – ca. 70% (OR) Kinetics and conversion
Surfactant-assisted extraction SB (Sulfobetaine) assisted extraction – 72.95% (OR) thermodynamics of
(ethyl acetate + different Tween-20 (Polyoxyethylene (20) sorbitan monolaurate) extraction
types of surfactants solution) assisted extraction – ca. 65% (OR)
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Firmansyah and Dried larvae – 35.5% (LC)

H. illucens larvae Dried Soxhlet extraction (n-hexane) ND ND
Abduh (2019) Defatted larvae – 3.5% (LC)
Microwave-assisted
extraction (petroleum ether)
Microwave-assisted extraction (petroleum ether) – 8.9%-
FTIR
Hao et al. (2021) H. illucens larvae Dried and ground OR 31.0% (Yield) ND
DSC
Soxhlet ( petroleum ether) – 34.24% (Yield)
Soxhlet extraction (petroleum
ether)
Partially defatted Conventional extractiona
Ido et al. (2021) H. illucens larvae Defatted powder (hexane) - 8.3% (LC) ND ND
commercial powder (hexane)
Production of
biodiesel via acid-
Soxhlet extraction (petroleum Soxhlet extraction (ethanol) – 58% (Yield)
catalyzed
Ishak et al. (2018) H. illucens larvae Dried ether, ethanol or acetone) Soxhlet extraction (petroleum ether) – 56% (Yield) ND
esterification and
followed by purification Soxhlet extraction (acetone) – 50% (Yield)
alkaline-catalyzed
transesterification
Conventional extractiona
(hexane)
Conventional extractiona (hexane) – 26.97% (Yield) FA composition
Lee et al. (2021) H. illucens larvae Dried ND
OR Expeller press – 37.23% (Yield)/86.66% (LC) Acid value

Oil expeller press

FA composition
Sterol composition
Tocopherols and
tocotrienols content
Oil press assisted extraction
TAG profile
followed by refining process
Matthaus et al. Dried larvae – 33.7% (LC) DSC
H. illucens larvae Dried and ground (degumming, chemical ND
(2019) Defatted powder – 8-10% (LC) DSC
neutralization, bleaching and
Iodine value
deodorization)
Saponification value
Peroxide value
Free FA analysis
Colour
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Phosphorus content
Oxidative stability
Soxhlet extraction (n-hexane Kinetics of
or 2-methyloxolane) extraction
Soxhlet (n-hexane) – 32.51% (Yield)
FA composition
Soxhlet extraction (2-methyloxolane) – 35.83% (Yield)
OR Lipid classes
Ravi et al. (2019) H. illucens larvae Dried and ground Multistage cross-current (n-hexane) – ca. 15% (Yield) ND
Total polyphenol
Multistage cross-current (2-methyloxolane) – ca. 20%
Multistage cross-current lipid content
(Yield)
extraction (n-hexane or 2- Radical scavenging
methyloxolane) capacity
Dried and ground
FA composition
Ravi et al. (2020) H. illucens larvae (different devitalization Soxhlet extraction (hexane) ND ND
Lipid classes
techniques)
Hot water extraction
Dried larvae – 32.5% (LC)
S. W. Kim et al. Defatted powder after hot water extraction – 15.97% (LC)
H. illucens larvae Dried and ground OR FA composition ND
(2019) Defatted powder after optimal SC-CO2 extraction – 4.58%
(LC)
SC-CO2 extraction
Conventional extractiona
(ether)

OR
Conventional extractiona (ether) – 29.55% (Yield) FA composition
Aqueous extraction
Aqueous extraction (whole larvae) – 4%-6.55% (Yield) Free FA analysis
Saviane et al.
H. illucens larvae Dried Naviglio extraction – 0.28%-5.31% (Yield) Peroxide value ND
(2021) OR
Screw press extraction – 26.11% (Yield)/63.8% Antimicrobial
(OR)/87.0% (LC) assays
Naviglio extraction

OR

Screw press extraction

Soxhlet extraction (petroleum
Smets et al. (2020) H. illucens larvae Dried and ground ND FA composition ND
ether)
Ground and dried Soxhlet extraction (hexane or Soxhlet (2-methyltetrahydrofuran) - 43.23% (Yield) FA composition
Smets et al. (2021) H. illucens larvae ND
(Soxhlet extraction) 2-methyltetrahydrofuran) Soxhlet (hexane) – 40.34% (Yield) Lipid classes
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Wet lipid extraction (2-methytetrahydrofuran) – 41.02%

OR OR (Yield)/94.9% (OR)
Wet lipid extraction (hexane) – 28.36% (Yield)/70.3%
Ground (wet lipid Wet lipid extraction (hexane (OR)
extraction) or 2-methyltetrahydrofuran)
Conventional extractiona (n- Protamex – 18.17%-36.10% (Yield) Acid value
Biodiesel production
hexane) with optional Flavourzyme – 13.52% (Yield) Iodine value
via two step
enzymatic pretreatment Papain – 12.14% (Yield) Saponification value
Su et al. (2019) H. illucens larvae Dried and ground esterification-
(Protamex, flavourzyme, Champzyme – 11.68% (Yield) FA composition
transesterification
papain, champzyme or Bromelain – 11.62% (Yield) Biodiesel quality
process
bromelain) Conventional extractiona (n-hexane) – 8.10% (Yield) parameters
Conventional extractiona Petroleum ether:isopropanol, 1:1 – 38.01% (Yield)
(petroleum ether, n-hexane, Ethyl acetate:water – 27.12%-29.04% (Yield)
T. Wang et al.
H. illucens larvae NA petroleum ether:isopropanol, Ethyl acetate – 28.71% (Yield) ND ND
(2020)
ethyl acetate or ethyl n-Hexane – 15.60% (Yield)
acetate:water) Petroleum ether – 13.92% (Yield)
Conventional extractiona Conventional extractiona (petroleum ether) - 32.5% (Yield)/
(petroleum ether) 87% (OR)
Caligiani et al. B. licheniformis protease - 10% (OR)
H. illucens prepupae Ground ND ND
(2018) OR Papain - 10% (OR)
Pepsin – 10% (OR)
Enzymatic assisted extraction Pancreatin - 10% (OR)
Soxhlet extraction (petroleum
Smets et al. (2020) H. illucens prepupae Dried and ground ND FA composition ND
ether)
Soxhlet extraction (petroleum
Smets et al. (2020) H. illucens pupae Dried and ground ND FA composition ND
ether)
Lepidoptera
SC-CO2 extraction

Chinese tasar moth OR SC-CO2 – 26.18% (Yield)

Pan et al. (2012) Dried and ground FA composition ND
(Antheraea pernyi) pupae Soxhlet (petroleum ether) – 28.08% (Yield)
Soxhlet extraction (petroleum
ether)
Silkworm (Bombyx mori) Conventional extractiona
Choi et al. (2017) Dried and ground Defatted powder – 0.34% (LC) ND ND
pupae (hexane)
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

FA composition
Microwave-assisted Refractive index
extraction (ethanol, ethyl Specific gravity
acetate, petroleum ether, Acid value
Soxhlet (n-hexane) – 30.42% (Yield)
Dried, ground and diethyl ether or n-hexane) Peroxide value
Hu et al. (2017) B. mori pupae Microwave-assisted extraction (ethanol:n-hexane; 1:1) – ND
sieved Iodine value
30.16% (Yield)
OR Total phenolic
content
Soxhlet extraction (n-hexane) Antioxidant activity
SEM
Production of
Ravikumar et al.
B. mori pupae Dried and ground Soxhlet extraction (hexane) ND ND biodiesel via
(2019)
transesterification
FA composition
Free FA analysis
Saviane et al.
B. mori pupae Dried Screw press extraction 28.06% (Yield)/78.4% (OR)/83.9% (LC) Peroxide value ND
(2021)
Antimicrobial
assays
SC-CO2 extraction
SC-CO2 – 29.48% (Yield)
Wei et al. (2009) B. mori pupae Dried and ground OR FA composition ND
Soxhlet (hexane) – 30.34% (Yield)
Soxhlet extraction (hexane)
Density
Ultrasound-assisted aqueous
Acid value
extraction
Saponification value
Ultrasound-assisted aqueous extraction – 5.39%-19.47%
Two-line velvet hawkmoth Iodine value
Sun et al. (2018) Dried and ground OR (Yield) ND
(Clanis bilineata) larvae FA composition
Soxhlet (petroleum ether) – 24.56% (Yield)
Antioxidant
Soxhlet extraction (petroleum
activities
ether)
TGA
Orthoptera
Whole powder – 20.86% (LC)
Amarender et al. Crickets (species not Conventional extractiona
Commercial powder Defatted powder (ethanol) – 9.27% (LC) ND ND
(2020) specified) (ethanol or hexane)
Defatted powder (hexane) – 11.98% (LC)
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Soxhlet extraction (hexane,

petroleum ether, ethyl acetate
or ethanol)
Soxhlet (hexane) – 14.6% (Yield)
Soxhlet (petroleum ether) – 14.7% (Yield)
OR
Laroche et al. House cricket (Acheta Soxhlet (ethyl acetate) – 15.1% (Yield)
Commercial powder FA composition ND
(2019) domesticus) adult Soxhlet (ethanol) – 22.7% (Yield)
Three-phase partitioning
Three-phase partitioning – 19.3% (Yield)
SC-CO2 – 11.9% (Yield)
OR

SC-CO2 extraction
Ultrasound-assisted extraction
Ultrasound-assisted extraction (ethanol) – 15.48% (Yield)
(ethanol; ethanol:water)
Ultrasound-assisted extraction (ethanol:water; 1:1) –
FA composition
15.05% (Yield)
Otero et al. (2020) A. domesticus adult Dried and ground OR Lipid classes ND
Pressurized-liquid extraction (ethanol) – 24.85% (Yield)
Cholesterol content
Pressurized-liquid extraction (ethanol:water; 1:1) – 5.02%
Pressurized-liquid extraction
(Yield)
(ethanol or ethanol:water, 1:1)
Soxhlet (acetone) – 24.6% (Yield)
Soxhlet extraction (petroleum Soxhlet (diethyl ether) – 20.8% (Yield)
Ribeiro et al.
A. domesticus adult Dried and ground ether, hexane, acetone, diethyl Soxhlet (ethanol) – 28.2% (Yield) ND ND
(2019)
ether and ethanol) Soxhlet (petroleum ether) – 21.3% (Yield)
Soxhlet (hexane) – 18.9% (Yield)
Sipponen et al. FA composition
A. domesticus adult Dried and ground SC-CO2 extraction SC-CO2 – 4.8% (LC of defatted fraction)/79% (OR) ND
(2018) Lipid classes
Aqueous extraction

OR
Aqueous extraction – 1.6% (Yield) FA composition
Tzompa-Sosa et al. Ground and dried
A. domesticus adult Soxhlet extraction (petroleum Soxhlet (petroleum ether) – 6.0% (Yield) Lipid classes ND
(2014) (Soxhlet or Folch)
ether) Folch (dichloromethane:methanol, 2:1) – 8.0% (Yield) TAG profile

OR
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Reference Species Pre-treatment Extraction method Lipid content (LC)/Yield/ Oil recovery (OR) Characterization Application

Folch extraction
(dichloromethane/methanol,
2:1)
Polarized light
microscopy
Tzompa-Sosa et al. FA composition
A. domesticus adult NA Aqueous extraction ND ND
(2019) DSC
Colour
Volatile compounds
HHP-assisted extraction
FA composition
(hexane)
HHP-assisted extraction (hexane) – 16.17%-17.41% DSC
(Yield) Peroxide value
Ugur et al. (2021) A. domesticus adult Commercial powder OR ND
Conventional extractiona (hexane) – 18.05%-18.09% Total phenolic
(Yield) content
Conventional extractiona
Antioxidant activity
(hexane)
Two-spotted cricket (Gryllus Conventional extractiona
Choi et al. (2017) Dried and ground Defatted powder – 1.07% (LC) ND ND
bimaculatus) adult (hexane)
Whole powder – 17.30% (LC)
Conventional extractiona Defatted powder (ethanol) – 0.73% (LC)
Jeong et al. (2021) G. bimaculatus adult Dried and ground ND ND
(ethanol, n-hexane or acetone) Defatted powder (n-hexane) – 1.83% (LC)
Defatted powder (acetone) – 2.56% (LC)
Soxhlet (acetone) – 23.5% (Yield)
Soxhlet extraction (petroleum Soxhlet (diethyl ether) – 20.8% (Yield)
Ribeiro et al. Tropical house cricket
Dried and ground ether, hexane, acetone, diethyl Soxhlet (ethanol) – 28.4% (Yield) ND ND
(2019) (Gryllodes sigillatus) adult
ether and ethanol) Soxhlet (petroleum ether) – 20.2% (Yield)
Soxhlet (hexane) – 20.8% (Yield)

Note. NA – Not applicable, ND – Not determined, ca. – Circa, SC-CO2 – Supercritical carbon dioxide, HHP – High hydrostatic pressure, FA – Fatty acid, TAG –
Triacylglycerol, FTIR - Fourier-transform infrared spectroscopy, DSC – Differential scanning calorimeter, TGA – Thermogravimetric analysis, TBARS – Thiobarbituric acid
reactive substances, SEM – Scanning electron microscopy, TEM – Transmission electron microscopy, PDI – Polydispersity index.

a – Defined by the authors as an extraction with homogenization with a solvent followed by filtration and/or centrifugation with posterior removal of the solvent.
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Appendix 2. Supplementary table with freeze-drying conditions overview