International Journal of Biological Macromolecules 165 (2020) 3206–3214

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Characteristics of chitin extracted from black soldier fly in different life stages

Lise Soetemans a,b, Maarten Uyttebroek a, Leen Bastiaens a,⁎
a
Flemish Institute for Technological Research (VITO), Mol, Belgium
b
University of Parma, Parma, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Chitin was collected and extracted along different lifecycle stages of the Black Soldier Fly (BSF) (larvae, prepupae,
Received 7 May 2020 pupae, flies, shedding & cocoons). The chitin content in the collected biomass ranged between 8% and 24%, with
Received in revised form 27 October 2020 sheddings and cocoons being most rich in chitin. Purified chitin was subjected to a physicochemical evaluation
Accepted 6 November 2020
based on FTIR, XRD, and TGA as well as a deacetylation step. The data indicated that BSF chitin was α-chitin
Available online 9 November 2020
with FTIR profiles matching closely to shrimp chitin and showing some differences compared to squid pen chitin
Keywords:
(β-chitin). Small physicochemical differences were observed among the different BSF samples. Prepupae and co-
Black soldier fly coon chitin was more crystalline while chitin from larvae and sheddings had a lower thermal degradation tem-
Chitin perature. In addition, sheddings were more difficult to purify. Further processing to chitosan showed that a
Deacetylation deacetylation degree of 89% could be obtained for all samples after 3 h, although sheddings were found to be
less reactive in the deacetylation process. Overall, the small differences in physicochemical properties that
were detected between the BSF chitin samples did not prevent further processing of chitin to chitosan with
the same degree of deacetylation via the same treatment.
© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction addition, indications of chitin being a non-digestible fiber that nega-

Chitin, a naturally occurring biopolymer, is an under-utilized re-
source partially due to its crystalline nature but has many unique prop-
erties and broad range in applications. The polymer, consisting of a
mixture of mainly N-acetyl-D-glucosamine and a small amount of D-
glucosamine, can easily be converted to chitosan by deacetylation (>
60% of the monosaccharides are D-glucosamine) [1,2]. This derivative
has an increased solubility in an acid environment which facilitates fur-
ther processing into marketable products. Currently, the main sources
for commercialized chitin and chitosan are crustaceans [3], and to a
lesser extent mushrooms [4,5] and both chitin and chitosan find appli-
cations in healthcare, (waste)water treatment, agrochemicals, food &
beverages and many more [6,7]. Predictions of increasing chitin/chito-
san application and the growth expectancy of the two main application
sectors (healthcare and wastewater treatment) have led to an expected
growth of the chitin market of 3.3 times by 2027 [8]. To keep up with
this demand and the exploration of new applications, there is a search
for more and new chitin sources [9]. Insect chitin, a byproduct for the
feed industry, is investigated as a potential source [10]. The number of
industrial insect farms is growing worldwide to provide a new protein
source for feed & food [11] or as a waste management tool [12]. With
this new livestock chain, new byproducts are formed such as cocoons,
sheddings from molting and dead insects, all containing chitin. In
⁎ Corresponding author at: VITO, Boeretang 200, 2400 Mol, Belgium.
E-mail address: Leen.bastiaens@vito.be (L. Bastiaens).
tively influences the digestibility of proteins and lipids, have led to a
tendency of removing chitin for food and feed applications. The larvae
of the Hermetia illucens, also known as the black soldier fly (BSF) are
the most studied insect type and are reared on large scale worldwide
[12]. Starting from egg hatching, the BSF larvae grow from instar to
prepupae in 14 days to 2 months, depending on the growing conditions
[13]. In this period, they go through five larval stadia where the first four
stages are difficult to differentiate, except for body size and molding be-
tween every stage [14]. The sheddings derived from the moldings steps
are the first chitin-rich byproduct. The fifth instar is a fully-grown larvae
and this stage is used for harvesting for feed/food applications. Further
rearing leads to the full-grown larvae to mold one last time into
prepupae. The prepupae change color from light brown to dark brown
and eventually black [15–17]. In this phase, the digestion track is emp-
tied and removed and the prepupae migrate to a more dry location to
pupate [13,18]. Prepupae are also often harvested and both (prepupae
and larvae) are a second source for chitin-rich byproduct if, for example,
chitin is separated from the proteins and lipids to increase digestibility
or when technical applications such as biofuel are envisioned [19].
Without molding, the prepupae become pupae by altering the soft tis-
sue from larvae to fly. This phase is indicated by a completely rigid
state with no elasticity [16]. After 10 days, the fly crawls out and leaves
an empty casing behind, called a cocoon [14,20] which is a third chitin-
rich byproduct. The fly lives for only a few days to lay eggs and then dies.
Since rearing companies rear year-round and keep insect species sepa-
rately, disadvantages regarding crustacea chitin isolation such as a

https://doi.org/10.1016/j.ijbiomac.2020.11.041
0141-8130/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
L. Soetemans, M. Uyttebroek and L. Bastiaens
discontinuous supply due to seasonal variations and high heterogeneity
of the biomass (combination of different species) would be tackled. A
best-case scenario would be to combine all chitin-rich byproducts to
employ one purification process to obtain a robust, homogeneous prod-
uct with well-defined characteristics. In this case, the characteristics of
chitin in all life stages should be similar. To our knowledge, no research
has been reported on the chitin extracted from all life cycle stages of the
black soldier fly except for the study of Wάsko et al. [21] who examined
the flies and the pupae of the BSF. In respect to crystalline formation of
chitin, three types (α, β and γ) have been reported in literature with dif-
ferent characteristics. α-Chitin is considered the most crystalline forma-
tion [22] whereas β-chitin is a softer material with a higher affinity for
solvents and a higher reactivity [23]. The third formation, γ-chitin, is
considered to be a mixture or an intermediate form of α- and β-chitin
[22]. Most literature data state that insect chitin has an α-crystallinity.
However, Merzendorfer and Zimoch [24] reported that all three crystal-
line forms can be found in insects. The α-form is more common but β-
and γ-chitin can be found in cocoons and peritrophic matrices of
some species. For example, the Australian spider beetle Ptinus tectus
has γ-chitin in its cocoon, the larvae of the silkworm Antheraea perny
and the sawfly larvae Phymatocera aterrima contain γ-chitin and β-
chitin was found in the cocoons of the figwort weevils Cionus and
Cleopus [24,25]. Further, the degree of crystallinity within a crystalline
formation can differ and affects its characteristics and further process-
ing. For example, gray shrimp, pink shrimp, and red crab all contained
α-chitin but the time to reach the some degree of deacetylation
(DDA) under the same conditions varied from 2 h for the gray shrimp,
to 3 to 4 h for the pink shrimp and 6 to 7 h for the red crab [22]. The dif-
ference could be related to different physical structures like particle size,
the isolation process or the nature of the chitin [6,26,27]. In the current
study, chitin from different life stages of BSF biomass (larvae, prepupae,
pupae, sheddings of the larvae, cocoons, and flies) was extracted for ex-
amining their physicochemical properties. More specifically, purified
chitin samples of BSF biomass, as well as shrimp and squid chitin,
were subjected to FTIR, XRD, TGA and a deacetylation step to evaluate
among other their chitin formation and crystallinity. In addition, the
chitin content of the raw samples was measured and reported. To our
knowledge, this is the first study that reports on the physicochemical
properties of the prepupae, pupae, sheddings, and cocoons of the BSF.
2. Material and method
2.1. Starting material
The BSF larvae were purchased from a local breeder (Millibeter/Cir-
cular Organics, Turnhout, Belgium). The larvae were harvested in the
Fig. 1. Different life stages of BSF and their characteristics for identification.
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International Journal of Biological Macromolecules 165 (2020) 3206–3214
last larval stage, removed from the substrate and delivered alive. After
a few hours, the bins contained sheddings that were collected by gently
blowing air on top. Some larvae were further reared in small containers
on moisturized broiler feed to collect all life stages. The prepupae were
collected after they crawled out of the substrate and were distinguished
by the dark color, a flexible body and they did not move unless exposed
to bright light. A considerable amount of prepupae was allowed to
search a pupation spot in cardboard pieces. The pupal stage was indi-
cated after an examination where the pupae had to meet four condi-
tions, (1) being stiff, (2) exhibiting an s-shape (see Fig. 1), (3) white
eyes (see Fig. 1) and (4) absence of a dark digestion track. Some
pupae were left to pupate and after flies emerged, the cocoons were
identified (small hole in the top) and collected. Flies were collected
after death. Commercial chitin was purchased from Sigma-Aldrich
(from shrimp shells, C9213), while squid pen was kindly provided by
a local restaurant (1.3 kg of fresh squid provided 1.05 g wet squid pen).
2.2. Extraction of chitin
The collected insect-based samples were dried (105 °C for 48 h) and
grounded in a mortar to break the exoskeleton. The particle size of the
obtained fractions was gravimetrically determined by shaking the sam-
ples (Fritsch Pulverisette – mortar grinder mill, amplitude 7) during
2 min over Retsch sieves of different mesh sizes and a particle distribu-
tion of 25% > 2 mm, 49% between 2 and 1 mm, 17% between 1 and
0.5 mm and 9% < 0.5 mm was obtained. Next, chitin was extracted in
triplicate for every life stage by a multi-step process. First, demineraliza-
tion was performed based on the method of Marei et al. [28] at a solid:
liquid ratio of 1:10 (m/v) with HCl 1 M at room temperature for 1 h.
Deproteinization included a 1 M NaOH treatment (solid:liquid ratio of
1:25 (m/v), 1 h at 80 °C) that was repeated until the absence of color
(12 times) similar to the method of Sagheer et al. [29]. Solids comprising
the chitin were separated from the liquid by filtration (pore size 25 μm,
49 PA (25/14), Solana). Finally, the chitin was washed with
demineralized water until neutral pH and dried at 105 °C for 48 h.
2.3. Characterization of extracted chitin
Fourier-transform infrared spectroscopy (FT-IR) spectra of the
purified chitin samples were measured in an ALPHA spectrometer
under Attenuated Total Reflectance (ATR) mode (Bruker, Alpha,
Ettlingen Germany) with a resolution of 4 cm−1 and 32 accumulations.
The data was processed in OPUS software. All samples were measured
in triplicate.
X-ray diffraction (XRD) measurements were performed in tripli-
cate on the purified chitin samples. Prior to the measurement, each
L. Soetemans, M. Uyttebroek and L. Bastiaens International Journal of Biological Macromolecules 165 (2020) 3206–3214

sample was further grounded in a mortar. XRD patterns were recorded
with an Empyrean system equipped with a cobalt tube (PANalytical,
Empyrean, Almeloo the Netherlands). Data were collected at 40 mA,
45 kV and a scan speed of 0.067335°s−1 with a PIXcel detector and a
scan angle between 0 and 45°. Highscore Plus (PANalytical) software
was used to convert the cobalt measurement to copper. The crystallinity
index (Icr) was calculated from the intensity (counts) at 15.0° (Iam,
amorphous phase) and 19.3° (I110) as indicated in formula (1) [30].
I110−Iam
Icr ¼ ∗100
I110
Thermogravimetric analysis (TGA) was obtained by Netzsch-
Jupiter-STA-449C, Eke Belgium. The conditions were the following:
about 15 mg of sample, temperature ramp from 25 °C to 625 °C at
10 °C min−1, nitrogen atmosphere with a flow of 70 ml min−1. The chi-
tin samples after purification were measured in triplicate. All samples
were measured in triplicate.
Scanning electron microscopy (SEM) was performed using a Nova
Nanosem 450 FEI (Eindhoven the Netherlands) after the samples were
coated with +1.5 nm Pt/PD 80/20 wt% by a Sputter Coater HP208
Cressington (Watford England).
2.4. Deacetylation
Deacetylation was performed by applying a heat treatment at alka-
line pH (1:30 m:v sample in 50 m% NaOH, 90 °C, 1 or 3 h, under stirring)
on dried, purified chitin samples in pressure-resistant glass vials. Next,
samples were filtered over a Whatman 589/2 (4–12 μm pore) filter
with water until a neutral pH was reached, washed three times with
ethanol and dried for 48 h at 105 °C. Deacetylation and analyses were
performed in triplicate unless stated otherwise.
2.5. Chemical analysis
The chitin-containing biomass samples were dried at 105 °C for 48 h
and dry matter (DM) was determined gravimetrically. The chitin con-
tent (on dry matter basis) in the biomass was determined by two
methods. The first method involves a gravimetric analysis described
by Kaya et al. [31]. The biomass obtained after the purification (demin-
eralization and deproteinization) was presumed pure chitin and the
proximate chitin content was calculated based on dry matter as detailed
in formula (2).
Crude or approximate chitin%
m after demineralization and deproteinisation ðdmÞ
¼  100
m biomass ðdmÞ
The second method was based on the quantification method de-
scribed by D'Hondt et al. [32], where the (non-purified, dried) biomass
was first hydrolyzed to release glucosamine and acetate followed by
UPLC-MS/MS (Waters, Quattro Premier, Milford USA) analysis to deter-
mine the glucosamine concentration and HPLC-RID (Agilent Technolo-
gies, T200 serie, Strasse Germany) for acetate measurements. The
degree of acetylation (DA) and degree of deacetylation (DDA) was cal-
culated based on the molar concentration of acetate and glucosamine
determined in the sample as was also described by D'Hondt et al. [32].
2.6. Statistical analysis
ð1Þ
All measurements were performed in triplicate unless stated other-
wise. The data were expressed as the average with the standard devia-
tion. Analysis with three replicas were evaluated on significant
differences by ANOVA (p < 0.05) and a Tukey post hoc test was per-
formed by using IBM SPSS software. The Shapiro–Wilk test was used
to control the data on a normally distribution (p < 0.05) and Levene's
test was used to judge the variance of the population (p < 0.05).
When these terms were not met, a Kruskal–Wallis analysis was per-
formed with a pairwise comparison, and significant values were ad-
justed by Bonferroni correction. All measurements of FTIR, XRD, and
TGA, were done in triplicate and a singular measurement of every sam-
ple is shown after evaluating the similarity of the triplicate.
3. Results and discussion
3.1. Chitin content of non-purified samples
The dry matter and chitin content for all BSF samples as well as the
squid pen are summarized in Table 1. The dry matter of the BSF samples
varied between 30% and 94% (Table 1), with the lowest value for the BSF
larvae and the highest for the BSF cocoons and the ash content varied
between 11 and 25%. In general, during the first phases of the insect
life cycle, the chitin content stays similar (not significantly different ac-
cording to a Kruskal-Wallis analysis) to decrease in flies (<9%). Signifi-
cantly higher values were measured in the sheddings, cocoons (>22%)
and the squid pen (36%). When comparing the two chitin quantification
approaches, although the general trends were similar, differences were
observed. The first method may have underestimated the chitin content
due to 1) partial deacetylation during deproteinization leading to losses
of acetate and 2) losses of chitinous material during the extensive wash-
ing process. The observation that method 1 resulted in a higher chitin
content compared to method 2, could point towards an incomplete
purification process for some samples. Especially for the sheddings,
the chitin content differed significantly (31.1% versus 23.7%). An addi-
tional test was performed for the sheddings with a more extensive
ð2Þ
deproteinization process (15 repeats instead of 12, method 1) and a
decreased chitin content of 26.6% was measured which is more in line
with the result of the second method. These findings suggest that the
sheddings of the BSF were more challenging to clean compared to the

Table 1
Dry matter, chitin content and degree of deacetylation of non-purified and purified chitin samples, n = 3.

DM (%) Whole biomass % DA (method 2) Purified chitin samples

b
Ash content (%) % chitin (method 1) % chitin (method 2) % chitin (method 2)

BSF larvae 29.5 ± 0.3 12.0 ± 0.3 9.5 ± 0.6 7.8 ± 0.3 92.0 ± 6.1 96.3 ± 3.7b
BSF prepupae 51.5 ± 1.1 12.2 ± 0.5 9.1 ± 0.02 10.9 ± 0.7 77.9 ± 4.2 94.5 ± 1.5b
BSF pupae 60.9 ± 0.9 10.7 ± 0.3 10.3 ± 0.7 10.7 ± 0.1 96.7 ± 3.2 93.9 ± 2.0b
BSF shedding 92.3 ± 0.5 24.5 ± 0.4 31.1 ± 0.3 23.7 ± 1.9 93.4 ± 3.3 75.7 ± 4.0b
BSF cocoon 94.2 ± 0.9 19.4 ± 0.2 23.8 ± 1.5 22.4 ± 0.9 89.8 ± 1.6 96.8 ± 1.8b
BSF flies 60.5 ± 3.1 nd 5.6 ± 0.4 8.4 ± 1.9 nd 95.7a
Squid pen 90.5a nd 35.5a nd nd 107.5a
Shrimp chitin 93,5 ± 0,1 nd nd nd nd 97.6 ± 2.0

Method 1: gravimetric based; Method 2: glucosamine-measurement based; nd: not determined because of limited sample amount or the sample was already purified when purchased.
a
Single measurement.
b
Duplicate measurement.

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L. Soetemans, M. Uyttebroek and L. Bastiaens
other samples. As method 2 (glucosamine determination) was identi-
fied as a more accurate chitin quantification approach, further discus-
sion will be based on these values. The high chitin content in
sheddings and cocoons (±23%) was expected since these samples con-
sist only of exoskeletons without flesh. Ash content was also much
higher for these samples. Prepupae and pupae were richer in chitin
(11%) compared to the larvae (8%), pointing towards increased produc-
tion of chitin during these stages (the weight in dry matter of the larvae
and pupae was similar), potentially for increased protection during the
delicate pupate phase. No literature data were found to support this hy-
pothesis. The chitin content of the larvae was in line with the range re-
ported in literature which was between 2 and 11% [10,33,34]. This large
variation can be explained by different quantification methods but also
by the rearing conditions which impact larval weight and length and the
larval stage at harvest. The chitin content was comparable to that of
other commercially reared insects such as grasshoppers species (be-
tween 5.3 and 7.4% chitin on DM of the adult [35]) and the lesser meal-
worm (between 4.4 and 9.1% chitin on DM of the mealworm [34]). A
slightly higher chitin content for prepupae (11% versus 6–9% [36,37]),
and for sheddings and cocoons (23% versus 20% [10]) was measured
compared to literature. The chitin content of the insect shells (cocoons
and sheddings) was very similar to the chitin content of the crustacean
shells (around 25%, before purification) [38,39] but less than the chitin
content found in the cuticle of the silkworm Bombyx mori (36–62%
[40]). The degree of acetylation of the chitin samples was around 90%
whit the exception of the prepupae that had a significantly lower DA
of 78%.
3.2. Characterization of purified chitin
All samples underwent the same extraction procedure, except for
commercial shrimp shells that were extracted by the supplier with an
unknown method. The chitin content of all samples was above 95%
(Table 1) confirming clean samples, except for the sheddings where a
lower value of 86% confirmed the hypothesis that chitin from this source
was more difficult to purify. All samples were characterized using differ-
ent methods to study the chitin in more detail.
3.2.1. FTIR
Every organic bound has a characterized resonation frequency in the
infrared spectra and thus FTIR can be used to study the molecular struc-
ture by producing a spectrum showing at which wavelengths infrared
was absorbed by the sample. For chitin, characteristic wavelength at
Fig. 2. FTIR spectrum of controls and BSF chitins: A) squid, B) commercial shrimp, C) larvae, D) prepupae, E) pupae, F) cocoon, G) fly, H) sheddings.
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International Journal of Biological Macromolecules 165 (2020) 3206–3214
3480–3450 cm−1 (O\\H stretch); 1660, 1627 cm−1 or 1656 cm−1
(C_O stretch); 1560 cm−1 (N\\H bend, C\\N stretch); 1420 cm−1
(CH2 ending and CH3 deformation); 1380 cm−1 (CH bend, CH3 sym-
metrical deformation); 1020 cm−1 (C\\O\\C asymmetric stretch in
phase ring) and 890 cm−1 (CH rind stretch) have been reported repeat-
edly and are independent of the source (shrimp, prawn, crab, lobsters,
silkworm or grasshopper) [35,41–43]. Fig. 2 shows the FTIR spectra for
the shrimp, squid and BSF samples. Two regions from 3500 to
2800 cm−1 and especially in the fingerprint region from 1700 till
500 cm−1 showed high absorbance. Some samples also exhibited
some negative absorbance from 2300 till 1900 cm−1 which can be-
explained by the variance in concentration of atmospheric water and
carbon dioxide. The similarity of the spectra suggests the same chitin
quality except for two additional absorbance bands at 2920 cm−1 and
2850 cm−1 for the BSF samples. This may indicate an impurity in the
sample, perhaps a compound that is also responsible for the slight color-
ation of the sample. The characteristic absorbance bands for chitin were
all present in the spectrum of the BSF samples.
FTIR spectroscopy is also commonly used to identify the crystalline
form (α, β or γ) of chitin as differences in hydrogen bonding can be de-
tected [30]. The carbonyl group of α-chitin is involved in two hydrogen
bonds, one intramolecular (between the carbonyl group and\\CH2OH)
that has an absorbance at 1620 cm−1, and one intermolecular (between
\\NH\\ and the carbonyl group) at 1660 cm−1. β-Chitin only exhibits
one signal at 1650 cm−1 as a result of the weaker intramolecular hydro-
gen bond [29,44]. Kaya et al. [30] stated that γ chitin also shows a di-
vided amide I band but with a less pronounced band at 1660 cm−1
and a clear and sharp at 1620 cm−1. In the current case, squid pen is
known to contain β-chitin and exhibited a band at 1636 cm−1. Com-
mercial shrimp chitin showed two absorbance bands which were
expected since shrimp is a well- known α-chitin source. The samples
of the BSF all showed two sharp bands at 1654–1652 cm−1 and
1619–1618 cm−1 and thus were identified as α-chitin. Most insects
that are studied in published work were identified as α-chitin, for
example the Melolontha melolontha [45], different grasshoppers
[35,46], the potato beetle [47] and crickets [48]. Waśko et al. [21] also
found that the larvae and flies of the BSF contained α-chitin.
3.2.2. XRD
XRD involves the diffraction of X-rays by a crystal structure in the
sample and thus allows the study of the atomic and molecular structure
of the crystal. Fig. 3a visualizes the XRD spectra of all chitin samples. The
insect samples all have similar peaks between 4 and 25° (around 9.4°;
L. Soetemans, M. Uyttebroek and L. Bastiaens
Fig. 3. (a) XRD spectrum of controls and BSF chitins: A) squid, B) commercial shrimp, C) larvae, D) prepupae, E) pupae, F) sheddings, G) cocoon, H) fly; (b) Crystallinity index of the
samples, Kruskal-Wallis analysis, n = 3.
13.0°; 19.3°; 20.8°, 23.2°, and 29.5°) and the same peaks were found in
the commercial shrimp sample (9.4°; 12.9°; 19.4°; 20.9° and 23.6°). The
spectra of squid pen, however, showed only two, more broad peaks at
8.4° and 19.7° indicating that squid pen is less crystalline than the
others. The results of the current study were in line with published
data regarding the shrimp chitin and squid pen. Considering the BSF
samples, Waśko et al. [21] found similar peaks for the BSF larvae and
flies (at 9°, 19°, 22°, 24°, and 30°). Fig. 3b illustrates the crystallinity
index (Icr) for all samples. The BSF larvae, sheddings and fly had a sim-
ilar Icr as commercial chitin. However, extraction was different and
could have an impact. For instance, Gbenebor et al. [49] reported a
shift between 79% and 87% on the same sample but with a different pu-
rification method. The BSF samples and the squid pen were extracted
the same way and a significant increased Icr of the BSF samples was ob-
served. This was predicted since squid pen has β-chitin with a lower
crystallinity. Significant differences between the BSF samples were
also detected (p = 0.004, Kruskal-Wallis analysis). The chitin in
prepupae and cocoons was significantly more crystalline (94%) than
the chitin originating from the sheddings and the larvae (89%). Kurita
[22] and Kaya et al. [35] already reported different Icr for different α-
chitin species of crustacea and grasshoppers (Icr from 63% to 73%)
[35,22] and the current results suggest that Icr-values can also vary
throughout the life stages.
XRD is also suitable for the identification of the chitin formation. Jang
et al. [44] stated that α-chitin exhibits four sharp crystalline peaks at
9.6; 19.6; 21.1 and 23.7° whereas β-chitin only has two broader peaks
at 9.1 and 20.3°. Kaya et al. [30] reported that the first relatively sharp
peak appeared at 8.59° for β chitin, at 9.35° for γ chitin and at 9.46°
for α chitin. The second sharp peak is at 12.7° for α and γ chitin while
for β chitin, this peak is more broad and located at 12.3°. Based on the
results, the insect samples were identified as α-chitin. Nevertheless,
some small differences were detected between the BSF samples, even
though they were all characterized as α-chitin. For example, the shoul-
der of peak 19.6° (at 21.1°) became less pronounced with the sample of
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International Journal of Biological Macromolecules 165 (2020) 3206–3214
the fly. This peak indicates a small decrease in crystallinity for fly chitin.
Kaya et al. [51] also found small differences in location of peaks between
α-chitin of different insect species. The same is notable in the study with
seven grasshopper species where the extraction method was kept con-
stant. The authors suggested that not only the isolation method but also
the species has an impact on the crystallinity [35,51].
3.2.3. Thermogravimetry analysis (TGA)
A thermogravimetric analysis reports the thermal characteristics of
the sample and the degradation temperature of a more crystalline sam-
ple will be higher. The first derivative of the TGA curve (DTG) for the
samples is given in Fig. 4a. Previous studies reported two mass losses
for chitin samples, one representing water loss and one for the thermal
decomposition of chitin [29]. A similar profile was measured in the cur-
rent study for all samples where a very small peak was detected at 60 °C
(water loss) and a high mass loss around 370 °C. The samples were
dried in the oven at 105 °C, explaining the small peak at 60 °C. Com-
pared to the other chitins, squid chitin decomposed at a much lower
temperature, which is in accordance with a lower crystallinity for β-
chitin [30]. Small deviations in the profile involve a shoulder at 336 °C
for commercial shrimp chitin, as well as a second mass loss at 467 °C
for BSF sheddings. Both indicate the presence of impurities. In respect
to the shedding, a 56% weight loss was recorded at 365 °C (chitinous
material) and 16% was further burned at 467 °C. This confirms the pres-
ence of impurities as was observed by the chitin quantification (86%).
The degradation temperature (Tm) of the samples, defined as the tem-
perature at the greatest rate of change in the TGA curve, is reported in
Fig. 4b. Squid pen chitin was degraded around 320 °C and the BSF sam-
ples and shrimp between 360 °C and 390 °C. No significant differences
in Tm were measured between the insect samples of the different life
stages (p = 0.73, ANOVA analysis). However, BSF larvae (p = 0.03)
and sheddings (p = 0.02) did have a decreased thermal stability com-
pared to commercial chitin. A comparison of data generated in different
studies is challenging as Tm varies with the extraction method and
L. Soetemans, M. Uyttebroek and L. Bastiaens International Journal of Biological Macromolecules 165 (2020) 3206–3214

Fig. 4. (a) DTG curve of spectra for controls and BSF chitins: A) squid, B) shrimp, C) larvae, D) prepupae, E) pupae, F) sheddings, G) cocoon, H) fly; (b) The degradation temperatures for the
samples, n = 3, ANOVA analysis * duplicates.

(a) Larvae (b) Prepupae (c) Pupae

(d) Shedding (e) Cocoon (f) Fly

(g) Shrimp
Fig. 5. SEM images of chitin samples from different life stages of the BSF and commercial shrimp chitin (magnitude 250×).

3211
L. Soetemans, M. Uyttebroek and L. Bastiaens
species. For example, Jang et al. [44] reported a Tm around 367 °C for α-
chitin (shrimp) and 362 °C for β-chitin (squid) whereas Sagheer et al.
[29] measured a Tm of 326 °C for α-chitin (crab) and 303 °C for β-
chitin (cuttlefish). Nevertheless, α-chitin has a more stable structure
than β-chitin, resulting in a higher degradation temperature [29]
which is also measured in the current study. Regarding insect chitin,
other insects also contained α-chitin and exhibited a Tm between
360 °C and 390 °C [35,45–47,51]. The current study did not show
significant differences between the life stages of the BSF regarding the
Tm, which is in line with the findings of Kaya et al. [31] who also
found similar Tm for the larvae, pupae, and adults of the Vaspa
Crabro wasp.
3.2.4. Scanning electron microscopy (SEM)
The surface morphologies of the extracted chitin samples of the BSF
were studied and compared to commercial shrimp chitin. Firstly, a
honeycomb-like structure was noticed for the larvae, prepupae,
pupae, sheddings, and cocoon chitin samples at a 250 x magnitude,
see Figure 5. This was not observed in the chitin samples of the fly nor
the commercial shrimp chitin. These findings are in line with Waśko
et al. [21] who studied the chitin of the flies and larvae of the BSF. The
surface morphology at this magnitude of chitin in flies and of commer-
cial shrimp showed a non-specific pattern with scattered nanofibers.
(a) Larvae
(d) Shedding
(g) Shrimp
Fig. 6. SEM images of chitin samples from different life stages of the BSF and commercial shrimp chitin (magnitude 25,000×).
International Journal of Biological Macromolecules 165 (2020) 3206–3214
At a 25,000× magnitude, only non-adherent nanofibers were de-
tected in chitin derived from larvae whereas chitin from prepupae
showed a small number of pores (see Figure 6). However, the surface
was, like the larvae, largely non-adherent, dense nanofibers. Chitin ex-
tracted from the pupae composed more pores but had still an overall
nanofiber dominating surface with hardly any porosity. The fibers
were more adherent to each other and a uniform lamellar structure
was observed. Chitin from cocoons and sheddings exhibited more and
larger pores (the pores were visible at a 5000× magnitude) and the
nanofibers were disorganized and non- adherent, resulting in an overall
spongy surface appearance. Chitin obtained from the fly showed differ-
ent types of morphologies. Some areas had large and many pores in a
dense, adherent nanofiber matrix while other areas were without or
with very few pores where the nanofibers were firmly adherent to
each other. The different morphologies of the fly can be explained by
the sample characteristics. The sample contained different body parts
of the fly as well as a mixture of different sexes. This would result in dif-
ferent cuticle structures that can explain the different morphologies.
Erdogan and Kaya [52] also found different morphologies in the same
individual grasshopper and Kaya et al. [53] showed different morphol-
ogies (especially in porosity) between females and males grasshoppers.
In general, the BSF insect samples had a different morphology compared
to that of shrimp chitin. Chitin of shrimp simply consisted of different
(b) Prepupae (c) Pupae
(e) Cocoon (f) Fly
3212
L. Soetemans, M. Uyttebroek and L. Bastiaens
Fig. 7. Degree of deacetylation of BSF chitins after deacetylation, n = 3 with standard
deviation,*duplicate, **single analysis, regular letter represent the results of the Tukey
post hoc analysis whereas capital letters represent the result of the Kruskal-Wallis analysis.
nanofibers with variable thickness without any pores. Between the dif-
ferent BSF samples, some differences were observed. This has been pre-
viously reported, for example, Kaya et al. [47] found that the larvae and
the adults of the Colorado potato beetle both had a nanofiber structure
with pores but the number of pores was much higher in chitin obtained
from the adults. However, this is not always the case as Erdogan and
Kaya [52] found similar surface morphologies between adult and
nymph grasshoppers.
3.3. Impact on processing chitin to chitosan
All samples were subjected to a chemical deacetylation process
(50 m% NaOH at 90 °C) to evaluate whether the potential difference in
chitin structure impacts further processing to chitosan. After 1 h of
deacetylation, a significant difference was observed between the BSF
samples (p = 0.0009, ANOVA analysis, Figure 7). For BSF sheddings a
DDA of 50% was recorded whereas for all others, significantly higher
values were reached. Sheddings performed poorly but TGA illustrated
that the sample contained impurities that may have influenced the
deacetylation. The other BSF samples reached a similar DDA compared
to commercial chitin after 1 h. By prolonging the deacetylation time to
3 h, the samples all reached a similar DDA of 89% (p = 0.255, Kruskal-
Wallis analysis). This suggests that 1) a longer reaction time made it
possible to overcome the differences that influence the deacetylation,
or 2) that BSF samples have a maximum reachable DDA. The latter
would imply that prepupae and pupae reached this maximum DDA
(89%) earlier than the other samples. Literature data confirm that the
deacetylation of insect-chitin generates chitosan with a DDA below
90%. Even under more harsh conditions (100 °C), the chitosan obtained
from the Potato beetle larvae or adults only had a DDA of maximum 82%
(3 h, 50 m% NaOH) [47]. Chae et al. [54] performed deacetylation for 9 h
at 95 °C with 50 m% NaOH on crickets and found a DDA of 67%. The DDA
reached in the current study was much higher which suggests that the
BSF samples were more accessible for deacetylation. In addition, a sim-
ilar DDA was measured when commercial chitin was deacetylated.
4. Conclusion
Chitin from different BSF biomass samples collected along the
lifecycle was purified and physicochemically characterized to identify
potential differences. Based on FTIR analysis, XRD spectra and TGA, the
chitin extracted from every BSF life stage was appointed as α-chitin.
The FTIR spectra of the BSF samples and commercial shrimp chitin
3213
International Journal of Biological Macromolecules 165 (2020) 3206–3214
match closely. Both FTIR and TGA indicated the presence of some impu-
rities, especially for the sheddings. Prepupae and cocoon chitin were
more crystalline than chitin from sheddings, larvae, and flies with a
crystallinity index of 94% compared to 89%. Sheddings showed a lower
deacetylation reactivity. In conclusion, chitin containing byproducts
from BSF rearing was found to be a suitable source for chitin with a chi-
tin content of 8% to 24%. Small differences in physicochemical character-
istics between the chitin samples were observed but they did not
strongly influence the further processing of chitosan leading to the ad-
vantage that all chitin-containing fraction of the BSF can be collected
and processed to chitosan with the same DDA.
CRediT authorship contribution statement
Soetemans L.: Conceptualization, Methodology, Validation, formal
analysis, Investigation, Writing original draft, Visualization.
Bastiaens L.: Conceptualization, Writing review & editing, visualiza-
tion, supervision, project administration.
Uyttebroek M.: Conceptualization, Writing review & editing,
supervision.
Acknowledgments
The authors would like to thank Myrjam Mertens for her support in
XRD analysis, and Stefano Sforza for his critical view of the manuscript.
This research was funded by VITO-PhD-grant.
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