# Unveiling the singular pattern of chitin in Black Soldier Fly pupal shells during extraction: a structural study for a better understanding of chitin's association in biological matrix

Alexis Falgayrac, Susana C.M. Fernandes, Cécile Terrol

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Alexis Falgayrac, Susana C.M. Fernandes, Cécile Terrol. Unveiling the singular pattern of chitin in Black Soldier Fly pupal shells during extraction: a structural study for a better understanding of chitin's association in biological matrix. The 14th International Conference of the European Chitin Society (EUCHIS 2023), Sep 2023, Siglufjörður, France. hal-04718441

HAL Id: hal-04718441

https://univ-pau.hal.science/hal-04718441v1

Submitted on 2 Oct 2024

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<figure><img src="image_1.png" /></figure>

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# Unveiling the singular pattern of chitin in Black Soldier Fly pupal shells during extraction: a structural study for a better understanding of chitin's association in biological matrix

Alexis Falgayrac <fnref n="1" />,<fnref n="2" />,<fnref n="3" />, Cécile Terrol <fnref n="3" />, Susana C. M. Fernandes <fnref n="1" />,<fnref n="2" />

## INTRODUCTION

The emergence of a new industry based on the production of alternative proteins from insects has brought to light the potential of their rearing by-products as an alternative source for biomolecules. In this context, companies involved in this activity are seeking solutions to valorise them by creating a circular model of economy.

On an industrial scale, the rearing of Black Soldier Fly (Hermetia illucens) provides several types of by-products displaying a chitin-rich exoskeleton associated with proteins, fats, minerals and pigments. From a structural point of view, a deep understanding of this matrix could represent a milestone in the optimal exploitation of those products as alternative chitin sources.

## METHODS

<figure><img src="image_2.png" /><figcaption>Black Soldier Fly by-products</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Chitin extraction process</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Soxhlet
Diethyl ether</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>100 °C
1 M HCl</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>90 °C
2 M NaOH</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>90 °C
5% H₂O₂</figcaption></figure>

<figure><img src="image_8.png" /></figure>

### Characterisation methodology

### Techniques and properties

### Materials

<figure><img src="image_9.png" /></figure>

* Raw pupal shells (puparium)

* Whole intact puparium at each extraction step

* FTIR - Chemical structure of internal and external membrane

* TGA (Air) - Thermal properties & inorganic content (residue after combustion > 800 °C)

* WAXS - Crystalline structure

* SEM - Surface morphology of the external part

Evaluate the impact of each treatment step on the physico-chemical properties of the puparium through the combination of characterisation techniques

<figure><img src="image_10.png" /></figure>

## RAW SAMPLE CHARACTERIZATION

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>ATR-FTIR</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>TGA UNDER AIR ATM</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>SR-WAXS</figcaption></figure>

<figure><img src="image_15.png" /></figure>

## STRUCTURE EVOLUTION THROUGH EXTRACTION

<figure><img src="image_16.png" /><figcaption>Deproteinized</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>Demineralized</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>Bleached</figcaption></figure>

<figure><img src="image_19.png" /></figure>

### Several morphology changes during the process

¹st – Similar to raw sample with structured matter on top of a hexagonal pattern

²nd – Reveal an alveolus hexagonal pattern filled with holes

³rd – Loss of topology and binding borders between hexagons' appearance

<figure><img src="image_20.png" /></figure>

Combined with WAXS and TGA analysis – Inorganic matters is hypothesized to fill the hexagons holes inner and border

<figure><img src="image_21.png" /><figcaption>Thermal behavior (TGA)</figcaption></figure>

<figure><img src="image_22.png" /></figure>

<table><thead><tr><th>Sample</th><th>Degradation stage (Air)</th><th>Related T (°C)</th></tr></thead><tbody><tr><td rowspan="3">Defatted</td><td rowspan="3">3</td><td>353.5</td></tr><tr><td>569.6</td></tr><tr><td>716.4</td></tr><tr><td rowspan="2">Demineralized</td><td rowspan="2">2</td><td>353</td></tr><tr><td>(529.4)</td></tr><tr><td rowspan="2">Deproteinized</td><td rowspan="2">2</td><td>360.9</td></tr><tr><td>(568.9)</td></tr><tr><td rowspan="2">Bleached</td><td rowspan="2">2</td><td>344.3</td></tr><tr><td>(520.7)</td></tr></tbody></table>

<table><caption>Chemical structure (FTIR)</caption><thead><tr><th>Sample</th><th>Inorganic content (%)</th></tr></thead><tbody><tr><td>Defatted</td><td>20.4</td></tr><tr><td>Demineralized</td><td>-0.09</td></tr><tr><td>Deproteinized</td><td>0.61</td></tr><tr><td>Bleached</td><td>0.27</td></tr></tbody></table>

<figure><img src="image_23.png" /><figcaption>EXTERNAL LAYER</figcaption></figure>

<figure><img src="image_24.png" /></figure>

## CONCLUSION & OUTLOOK

A combination of several characterisation techniques showed an interesting morphology change in the pupal shells during the chitin extraction process

Considering this hypothesis, the conventional extraction process could be optimised by adapting the extraction steps' order

More surface-specific characterisation techniques should be performed and the whole structural study extended to all the resources

According to these results, we believe that the upper layer covering the outer part of the puparium is reinforced with a mineral layer (CaCO₃)

## References :

Hahn, T. et al. 2021. « Purification of Chitin from Pupal Exuviae of the Black Soldier Fly ». *Waste and Biomass Valorization*. Waśko, Adam et al. 2016. « The First Report of the Physicochemical Structure of Chitin Isolated from Hermetia Illucens ». *International Journal of Biological Macromolecules* 92: 316-20.
McReynolds, Colin et al. 2022. « Double Valorization for a Discard—α-Chitin and Calcium Lactate Production from the Crab Polybius Henslowii Using a Deep Eutectic Solvent Approach ». *Marine Drugs* 20(11): 717.

## Acknowledgments:

This research was carried out in the framework of an industrial collaboration supported by the ANRT between the company Agronutris and the CNRS under the CIFRE program no. 2022/0421.
The authors thank the European Chitin Society for allowing them to present this research work.

<footnote n="1">¹ Université de Pau et des Pays de l'Adour, CNRS, IPREM, E2S UPPA, 64000 Pau, France</footnote>

<footnote n="2">² MANTA - Marine Materials Research Group, Université de Pau et des Pays de l'Adour, E2S UPPA, 64600 Anglet, France</footnote>

<footnote n="3">³ Agronutris, 31650 Saint-Orens de Gameville, France</footnote>