Simplified determination of the content and average degree of acetylation of chitin in crude black soldier fly larvae samples

Article abstract of DOI:10.1016/j.carres.2019.107899

Insects are considered a promising alternative protein source for food and feed, but contain significant amounts of chitin, often undesirable due to indigestibility, disagreeable texture and negative effect on nutrients intake. Fractionation strategies are thus increasingly being applied to isolate and valorize chitin separately. The analysis of chitin generally requires an intensive pretreatment to remove impurities, and derivatization to generate sufficient detector response. In this work, a liquid chromatography method, without pretreatment nor derivatization, was developed for the simultaneous determination of chitin content and degree of acetylation in non-purified samples of black soldier fly (BSF) larvae. The method is found to be more suitable, compared to traditional methods, for assessing high degrees of acetylation. For the first time, the degree of acetylation of BSF chitin (81 ± 2%) is reported. Additionally, the chitin content of BSF soft tissues is estimated at approximately 20% of the total chitin content (8.5 ± 0.1%).

Full text of DOI:10.1016/j.carres.2019.107899

Journal Pre-proof
Simplified determination of the content and average degree of acetylation of chitin in
crude black soldier fly larvae samples
Els D'Hondt, Lise Soetemans, Leen Bastiaens, Miranda Maesen, Vincent Jespers,
Bert Van den Bosch, Stefan Voorspoels, Kathy Elst
PII:
S0008-6215(19)30319-2
DOI:
https://doi.org/10.1016/j.carres.2019.107899
CAR 107899
Reference:
To appear in: Carbohydrate Research
Received Date: 24 May 2019
Revised Date: 28 November 2019
Accepted Date: 20 December 2019
Please cite this article as: E. D'Hondt, L. Soetemans, L. Bastiaens, M. Maesen, V. Jespers, B. Van
den Bosch, S. Voorspoels, K. Elst, Simplified determination of the content and average degree of
acetylation of chitin in crude black soldier fly larvae samples, Carbohydrate Research (2020), doi: https://
doi.org/10.1016/j.carres.2019.107899.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.

1
2
Simplified determination of the content and average degree of acetylation of chitin in crude
black soldier fly larvae samples
3
4
5
Els D’Hondt,a, Lise Soetemansa, Leen Bastiaensa, Miranda Maesena, Vincent Jespersa, Bert Van
den Boscha, Stefan Voorspoelsa, Kathy Elst*,a
6
7
8
a: Flemish Institute for Technological Research (VITO), Boeretang 200, 2400 Mol (Belgium)
els.dhondt@icats.be
9
10
11
12
13
14
15
16
17
18
19
lise.soetemans@vito.be
leen.bastiaens@vito.be
miranda.maesen@vito.be
vincent.jespers@vito.be
bert.vandenbosch@vito.be
stefan.voorspoels@vito.be
kathy.elst@vito.be (* Corresponding author)
Highlights
20
21
22
23
Determination of chitin content and acetylation degree on black soldier fly larvae.
Simultaneous determination of chitin content and acetylation degree.
Applicable on crude samples without extensive pretreatment or derivatization.
Assessment of total chitin content, including soft tissues.
24
25
Determination of acetylation degree of highly acetylated chitin samples.
1

26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Abstract
Insects are considered a promising alternative protein source for food and feed, but contain
significant amounts of chitin, often undesirable due to indigestibility, disagreeable texture and
negative effect on nutrients intake. Fractionation strategies are thus increasingly being applied
to isolate and valorize chitin separately. The analysis of chitin generally requires an intensive
pretreatment to remove impurities, and derivatization to generate sufficient detector
response. In this work, a liquid chromatography method, without pretreatment nor
derivatization, was developed for the simultaneous determination of chitin content and
degree of acetylation in non-purified samples of black soldier fly (BSF) larvae. The method is
found to be more suitable, compared to traditional methods, for assessing high degrees of
acetylation. For the first time, the degree of acetylation of BSF chitin (81±2%) is reported.
Additionally, the chitin content of BSF soft tissues is estimated at approximately 20% of the
total chitin content (8.5±0.1 %).
Keywords
Chitin content, degree of acetylation, UPLC-MS, analysis, crude samples, black soldier fly
larvae, fast method
2

51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
1. Introduction
Chitin is the most abundant natural polymer next to cellulose and is found in crustaceans,
insects and some microorganisms. Because of their biodegradability and low toxicity, chitin
and chitosan (produced via deacetylation of chitin), and derivatives thereof have a variety of
applications in multiple sectors such as medicine, food, textile, cosmetics and wastewater
treatment. Since Western countries nowadays show an increased interest in insects as a
sustainable and local protein source for food and feed, increasing amounts of insect chitin are
expected to become available on the market. This is especially expected for black soldier fly
(Hermetia Illucens; BSF), as it is already reared commercially and is one of the only seven insect
species recently allowed in aqua feed applications. [1]
Chitin is present in insects in the cuticle, gut lining, peritrophic matrix, salivary gland,
trachea, eggshells and muscle attachment points. [2] Chitin in the cuticle can be relatively easy
isolated and used as resource for bio-based products. Determination of the chitin content and
degree of acetylation (DA) of biological samples, however, requires time intensive and high
chemical consuming sample preparation to remove minerals, catechols, proteins and lipids. [3]
To quantify the chitin content in biological samples several methods are available in
literature, including methods for sample clean-up prior to characterization via either NMR,
spectroscopic and/or chromatographic procedures. Even though 1H-NMR and especially 13C-
NMR are well suited to determine the chitin content and its acetylation degree [4], the
technique is not universably available. Hence, a performant chromatographic approach is
expected to provide a more accessible alternative for implementation within chitin processing
facilities and laboratories. An overview of techniques to determine the chitin content by
chromatographic methods is well described by Zhu et al..[3] To summarize, most methods to
quantify the chitin content of biological samples include 3 steps, i.e. (i) sample clean-up, (ii)
acidic hydrolysis and (iii) derivatization of the monomer glucosamine with a fluorenyl group via
3

76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
the amine function to generate an spectroscopic response. [3,5–9] Derivatization (iii) can be
omitted when using mass spectrometric or refractive index detection. [10,11] The sample
clean-up step (i) requires an acidic thermal treatment to remove minerals and catechols. This is
followed by neutralization to subsequently subject the sample to a thermal alkaline treatment
to remove proteins and ends with a neutralization step.[5,7,12,13] This lengthy procedure is
not only time consuming, also generates a large amount of chemical waste even for small
samples sizes. Additionally, the chitin structure is often altered by these procedures, i.e.
partially depolymerized and deacetylated.[14,15] Crespo et al. already partially avoided the
lengthy pretreatment in the case of crab shells, by setting up a chromatographic method
suitable to determine the chitin content after removing the protein-rich soft tissue.[10]
Nevertheless, to the best of our knowledge, no chromatographic methodology is currently
available to achieve a fast and efficient quantification of chitin in crude biological samples such
as whole BSF larvae.
Another challenge in chitin characterization concerns the accurate quantification of its DA,
especially for highly acetylated chitin. Knowing and controlling the acetylation degree of chitin
is very important for its properties. [16] Various methods were described in literature and
discussed in a review by Kasaai. [17] Fourier-Transform Infrared (FT-IR) is the most frequently
used method, but as commonly known, its accuracy is insufficient at high acetylation degrees.
[18] Additionally, interpretation of FT-IR data and subsequent calculation of the acetylation
degree varies largely between research groups and is affected by the presence of impurities.
[19] Moreover, the extensive pretreatment procedure needed for chitin quantification affects
the acetylation degree of the chitin sample, making the simultaneous determination of both
parameters challenging.
The main aim of this article is to provide a simplified chromatographic method, avoiding
lengthy pretreatment steps, for the characterization of chitin in biological samples, with a
4

101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
prime focus on its application on the increasingly important BSF larvae. To this, a method is
developed that allows the determination of the chitin content on crude, unprocessed samples,
thus with many impurities such as proteins and lipids, without clean-up or derivatization steps.
Moreover, the method is designed to provide simultaneous information on the content and DA
of the chitin. The proposed methodology is based on acidic hydrolysis, followed by analysis of
the chitin via LC-MS/MS and LC-RID. Another goal of the article is to characterize for the first
time the DA of unprocessed BSF larvae chitin and to assess the proportion of chitin contained
in the soft tissues of BSF larvae.
2. Materials and methods
2.1. Materials
Materials were purchased from different chemical providers, i.e., HCl, NaOH, sulphuric
acid 95-97 %, glacial acetic acid 100%, acetonitrile analytical grade from VWR [Radnor,
Pennsylvania, US]; N-acetylglucosamine (> 99 %) from Alfa-Aesar [Ward Hill, Massachusetts,
US]; glucosamine sulphate (98 %) from Sigma-Aldrich [St Louis, Missouri, US]. Chitin (from
shrimp shells), and chitosan were bought on the market. The acetylation degree was
determined by the supplier with an in-house method based on the measurement of picric acid
sorption and desorption [20], and was reported to be 98 % for the commercial chitin and 22 %
for the commercial chitosan. Fresh and living BSF larvae were kindly provided by Millibeter
[Turnhout, Belgium]. The larvae used in this work were grown on crumb for 18 to 20 days.
2.2. Raw materials and their pretreatments
In this study, 3 different types of raw chitin materials were used with increasing impurity
and thus also complexity. First, commercially available pure chitin and chitosan were applied
as reference materials for process optimization. Subsequently, clean and crude exoskeletons of
5

125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
BSF larvae were analyzed. The study was ended with the assessment of dried whole BSF larvae.
The preparation and pretreatment of the different materials was done as follows.
The purchased chitin was used without any further purification, and only dried in an oven
at 105 °C for 48 h and grinded in a ball mill to reduce its particle size.
To produce the clean and crude exoskeletons, fresh and living larvae of the BSF were
mechanically treated to separate the exoskeletons from the liquid internal organs with a mesh
size of 0.3 mm to 0.5 mm and thus removing the chitin contained in the soft tissue. The dry
matter content of both fractions was determined after drying in an oven at 105 °C for 48 h.
The slaying mechanical treatment of the BSF larvae was found to result in 40.6 % (w/w) of
exoskeletons and 59.4 % (w/w) of dry intestines (not used as sample) on dry matter base. A
fraction of the dried crude exoskeletons was used without further pre-treatment and is further
addressed in the manuscript as “crude pellet”. The other fraction was cleaned in several
successive steps based on literature methods.[21,22] The washing procedure was performed
in triplicate experiments. First, the dried exoskeletons were washed twice with demineralized
water (solid to water ratio 1:75; w/w) with intermediate filtration to remove most of non-
bound proteins and lipids. To remove the remaining proteins, the water-washed exoskeletons
were treated in several subsequent washing cycles at 80 °C for 1 h with 1 M NaOH until the
liquid was clear (13 cycles with solid to solvent ratio of 1:50; w/w). After the alkaline
treatment, the solids were washed with demineralized water until neutral pH, resulting in the
clean exoskeletons. The 3 batches of clean exoskeletons were merged to obtain 1
homogeneous batch for the hydrolysis experiments and are further on noted as “clean pellet”.
During the washing procedure, in total 77.0 ± 1.2 % (w/w) of impurities were removed from
the crude pellets on dry weight base. Per gram of dry clean pellet produced, this lengthy
cleaning procedure consumed roughly 650 mL of demineralized water in 2 steps and 2.8 L of 1
M NaOH in 13 deproteination steps of each 1 h at 80 °C.
6

150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
All exoskeleton fractions, i.e. all replicates from the “crude and clean pellets”, were dried in an
oven at 105 °C and grinded in a ball mill to reduce the particle size.
Finally, also samples of whole larvae were prepared. To this, a batch of fresh larvae were oven
dried at 105 °C for 48 h and grinded with ball mill to reduce their particle size.
The particle size of all samples was determined by laser diffraction (Microtrac, S3500 with Flex
software). The commercial chitine and clean BSF pellet had an average particle size of 200 µm,
the crude BSF pellet and grinded whole larvae was lower at 80-100 µm.
2.3. Acidic hydrolysis
The optimization of the hydrolysis was performed with either pretreated commercially
available chitin or monomeric N-acetylglucosamine. In each experiment, 50 mg of the chosen
substrate was transferred to heat and pressure resistant glass vials and 2.5 mL of 3 N or 6 N
HCl were added. The hydrolysis was executed in closed vials under N2 atmosphere in an
aluminum block on a magnetically stirred heating plate. The reaction was performed in
function of time between 0.5 h and 16 h at 90 °C and 110 °C.
For the 3 types of biological samples, i.e. the clean pellet, crude pellet and whole ground
dry larvae, 50 mg of the dried powdered sample was combined with 5 mL 6N HCl in heat and
pressure resistant glass vials. The stirred reaction at 110 °C was monitored between 2 to 16 h.
After reaction, the closed vials were immediately cooled in a laminar air flow. The
reaction mixture was centrifuged to remove the residual non-dissolved material and
subsequently filtered over 0.45 µm and used for chromatographic analysis as detailed in
section 2.4. All experiments were executed in triplicate.
2.4. Chromatographic analysis and method validation
To quantify the chitin content of the starting raw material, the filtered hydrolysate was
analyzed for its free acetate (as acetic acid), glucosamine and N-acetylglucosamine content.
7

174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
The acetate concentration (as acetic acid) was determined with the method of De Sitter et
al.[23], equally validated for other organic acids.[24] In brief, 250 µL of the filtered supernatant
hydrolysate samples were partially neutralized with 750 µL of 400mM NaOH. Twenty-five µL of
diluted samples were injected on an Agilent 1200 series HPLC containing an Agilent Hi-Plex H
column (300 × 7.7 mm, 8µm particles) at 55 °C and an Agilent 1260 refractive index detector at
55 °C. The mobile phase was 0.01 M H2SO4 at a flow rate of 0.6 mL/min. The retention time of
acetate (measured as acetic acid) was 15.49 minutes. Calibration was performed by injecting
11 different stock dilutions of acetic acid reference standards with a concentration ranging
from 50 to 25000 mg/L and a linear order calibration curve was fitted (R² > 0.999). The limit of
detection was found to be 9.6 mg/L.
Two quality control samples, with a known concentration of 12500 and 1000 mg/L
respectively and independently prepared from the calibration standards, were added at the
start of the series, in the middle and at the end. For extended measurement series, the
measurement of both quality controls was repeated at least every 20 samples. No drift was
observed during any of the analytical batches. The recovery was found to be on average 100.8
± 3.1 % (n=34) for the quality control of 12500 mg/L and 101.4 ± 3.4 % (n=32) for 1000 ml/L.
For the determination of glucosamine and N-acetylglucosamine content, an aliquot of 0.5
mL of filtered supernatant was diluted to 10 mL with milliQ water. Then 40 µL of this diluted
sample was added to 960 µL of a mixture prepared from 50 mL acetonitrile, 45 mL water and 1
mL 1 N NaOH for Ultra High-Performance Liquid Chromatography-Mass Spectrometry (UPLC-
MS/MS) analysis. Five µL of diluted and neutralized samples were injected on a Waters
Quattro Premier UPLC-MS/MS system equipped with a Waters UPLC BEH HILIC 2.1 × 100 mm,
1.7 µm column at 40 °C. Chromatographic resolution of the analytes was obtained by 40 °C
isothermal gradient elution using water with (A) 20 mM ammoniumformiate and 0.1% formic
acid (pH = 3) and acetonitrile (B) with 0.1 % formic acid. A constant flow of 0.4 mL/min was
8

199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
used with following gradient settings: 5-25 % A (0-3 min), 25 % A (3-4 min), 25-5 % A (4-4.1
min), 5 % A (4.1-7 min). The retention times of N-acetylglucosamine and glucosamine were
1.75 and 3.25 min respectively.
Peak detection and quantification was done using a triple quadrupole MS operated in
Multiple Reaction Monitoring mode. After electron spray ionization, the following m/z pairs
were recorded: 222 → 204 and 222 -> 168 for N-acetylglucosamine and 180 → 162 and 180 ->
84 for glucosamine. After confirmation of linear detector response, calibration was performed
by injecting stock dilutions of N-acetylglucosamine and glucosamine sulphate reference
standards with a concentration ranging from 0.4 to 400 mg/L and a linear (glucosamine) and
second order (N-acetylglucosamine) calibration curve was fitted (R² > 0.999). The limit of
quantification was found to be 4 times lower than the lowest standard, so 0.1 mg/L. The fitted
calibration equation was used to calculate the monomer content. To ensure reliability of the
analytical method, a quality assurance scheme was in place. Apart from the fit and working
range as mentioned above, method precision was assessed and monitored by means of
random replicate samples analysis. During the course of the experiments, 33 samples were
measured in duplicate. Variance of the results was used to assess method precision. Results of
n paired measurements were used to estimate the precision according to EUROCHEM/CITAC
Guide CG 4 [25], resulting in a precision uncertainty of 5.2 % for glucosamine (n = 108) and 5.4
% for acetylglucosamine (n = 11). This precision was considered fit-for-purpose to evaluate the
experiments. Trueness was investigated by means of repetitive analysis of spiked samples. To
this extent, random samples were fortified with a known amount of analyte and measured.
Recovery was used as a measure for trueness and was 91 ± 7 % for glucosamine and 77 ± 8 %
for N-acetylglucosamine. N-acetylglucosamine was shown not to be present in hydrolysis
samples (see 2.6 and 3.1.1), thus the low recovery did not affect the interpretation of the
results of this article. All glucosamine measurements were corrected with the recovery. To
9

224
225
226
227
228
229
230
ensure system stability, reference standards were inserted in the analytical sequence every 10
samples. No drift was observed during any of the analytical batches.
2.5. Fourier-Transform Infrared and elemental analysis
FT-IR spectra of commercial chitin, commercial chitosan, and clean pellet were run in
Attenuated Total Reflectance mode on an ALPHA spectrometer [Bruker, Evere, Belgium] with a
resolution of 4 cm-1 and 32 accumulations. The average DA was calculated via Eq. (1) described
by Czechowska-Biskup et al.. [26]
ꢇ
ꢇ
ꢐ
ꢐ
ꢄꢅꢅ
ꢈꢉꢊꢈ ꢄꢋꢅꢅꢌꢄꢍꢅꢅ ꢎꢏ
ꢂ ꢃ
ꢀꢁ % = ꢄ.ꢆꢆ
231
∗
Eq. (1)
ꢈꢉꢊꢈ ꢑꢍꢅꢅꢌꢆꢒꢅꢅ ꢎꢏ
232
233
234
235
236
The carbon and nitrogen contents (mole%) were determined for the clean
exoskeletons (clean pellet) and commercial chitin through elemental analysis (C/N) with a
Vario micro cube [Elementar, Langenselbold, Germany]. 2.5 mg of dry sample was weighted
and treated with a combustion temperature of 1150 °C and a reduction temperature of 850 °C.
To convert the C/N ratio to the average DA, the formula of Xu et al. was used (Eq. (2)). [27]
ꢓ/ꢔꢌꢋ.ꢄꢕ
ꢂ ꢃ
ꢀꢁ % = 100 ∗
237
Eq. (2)
ꢄ.ꢍꢑ
238
239
240
241
242
243
244
245
246
2.6. Calculations
The stability of the monomers N-acetylglucosamine and glucosamine under hydrolysis
conditions was evaluated over time by calculating the monomers reduction, X(tx), via Eq. (3).
N-acetylglucosamine, as being the prime chitin monomer, was added at an initial molar
ꢇ
ꢐ
concentration N − acetylglucosamine t0. As N-acetylglucosamine easily converts to
glucosamine, both monomers were measured to evaluate the monomer stability. So, the time
dependent (tx) monomer molar concentration was determined by the sum of both
glucosamine and N-acetylglucosamine. In practice, however, mostly glucosamine was
observed due to the immediate deacetylation at the hydrolysis conditions.
ꢇ
ꢐ
ꢠꢡꢏ ꢏꢢꢎꢢꢏꢊꢉꢠ ꢣꢤ
ꢂ
ꢃꢂ ꢃ
247
ꢖ ꢗꢘꢙꢘꢗꢚꢛꢜ ꢝꢞ ꢞꢟ % = 100 − 100 ∗ ꢇ
Eq. (3)
ꢐ
ꢔꢌꢈꢥꢊꢣꢦꢧꢨꢧꢡꢥꢢꢠꢈꢏꢩꢎꢊ ꢣꢅ
10

248
249
250
251
To account for monomer degradation during hydrolysis, the molar concentration of
glucosamine measured in the hydrolyzed samples was corrected with the slope of trendline
equation of monomer conversion, i.e. X = 1.846*t(h) (see 3.1). By applying Eq. (4), ‘corrected’
glucosamine values were obtained ([glucosamine]c) that were used in all further calculations.
ꢄꢅꢅ
ꢇ
ꢐ
ꢇ
ꢐ
252
ꢪꢫꢬꢭꢘꢜꢝꢗꢮꢙꢚ ꢭ = glucosamine measured ∗ ꢄꢅꢅꢌꢄ.ꢯꢕꢒ∗ꢣꢂꢰꢃ
Eq. (4)
253
254
255
256
The amount of hydrolyzed chitin (mg/ml) was derived from the analytically determined
molar concentrations of glucosamine, acetate and N-acetylglucosamine in the hydrolysate. The
conversion to mass was performed by considering their respective molecular masses of the
monomer unit as present in the chitin chain; i.e. 161.15, 42.04 and 203.19.
257
ꢱꢲꢳꢛꢘꢫꢲꢴꢚꢳ ꢭℎꢮꢞꢮꢙ ꢵꢶꢶꢷꢸ ꢹ =
ꢇ
ꢐ
ꢇ
ꢐ
ꢇ
ꢐ
258
259
260
261
glucosamine c ∗ 161.15 + acetate ∗ 42.04 + N − acetylglucosamine ∗ 203.19 Eq. (5)
The fraction of sample liberated as hydrolyzed chitin as function of hydrolysis time,
X(tx), was determined via Eq. (6) with the calculated amount of hydrolyzed chitin (mg/ml) of
Eq. (5) as the numerator and the amount of loaded sample (mg/ml) as denominator.
ꢽꢾ
ꢽꢿ
ꢺꢦꢻꢉꢢꢧꢦꢼꢊꢻ ꢥꢰꢩꢣꢩꢎ ꢂ
ꢃ
ꢂ
ꢃꢂ ꢃ
262
ꢖ ꢭℎꢮꢞꢮꢙ ꢝꢞ ꢞꢟ % = 100 ∗
Eq. (6)
ꢽꢾ
ꢽꢿ
ꣀꢢꢈꢻꢊꢻ ꢠꢈꢏꣁꢧꢊ ꢵ
ꢹ
263
264
265
266
To convert the chitin content of the pellet samples (calculated via Eq. (6)) into the
chitin content of the insect, the dry weight of fractions and the loss of insect mass during pre-
treatment were taken into account (see section 2.2). Eq. (7) and Eq. (8) were used for the clean
and crude pellet data respectively.
ꢂ ꢃ
ꣂꢙꢜꢚꢭꢞ ꢭℎꢮꢞꢮꢙ ꢭꢘꢙꢞꢚꢙꢞ ꣃꢮꢝ ꢭꢫꢚꢝꢙ ꣄ꢚꢫꢫꢚꢞ % =
ꢂ
ꢇ
ꢐꢃ
ꣅ꣆꣆꣇ ꣈꣉ ꣊ꢿ꣋꣌꣍꣎꣍ꢾ ꢿ꣏꣐꣐
ꢇ
ꢐ
ꢓꢰꢩꢣꢩꢎ ꢥꢢꢎꢣꢊꢎꢣ ꢥꢧꢊꢈꢎ ꣁꢊꢧꢧꢊꢣ ∗
∗ꢇ% ꢊꢤꢢꢠ꣑ꢊꢧꢊꢣꢢꢎ ꣒꣓ ꣔ꢉꢈꢥꢣꢩꢢꢎꢐ
ꣅ꣆꣆
267
268
Eq. (7)
Eq. (8)
ꢄꢅꢅ
ꢂ ꢃ
ꣂꢙꢜꢚꢭꢞ ꢭℎꢮꢞꢮꢙ ꢭꢘꢙꢞꢚꢙꢞ ꣃꢮꢝ ꢭꢛꢬꢳꢚ ꣄ꢚꢫꢫꢚꢞ % =
ꢇ
ꢐ
ꢓꢰꢩꢣꢩꢎ ꢥꢢꢎꢣꢊꢎꢣ ꢥꢉꢡꢻꢊ ꣁꢊꢧꢧꢊꢣ ∗ ꢇ% ꢊꢤꢢꢠ꣑ꢊꢧꢊꢣꢢꢎ ꣒꣓ ꣔ꢉꢈꢥꢣꢩꢢꢎꢐ
ꢄꢅꢅ
11

269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
The average DA of the chitin, was determined by dividing the molar concentration of acetate
by the corrected molar concentration of glucosamine (cfr. Eq. (4)).
3. Results and discussion
N-acetylglucosamine (chitin monomer) and commercially available crustacean chitin were
first used as reference substrates in section 3.1 to develop a procedure to determine both the
chitin content and average DA via hydrolysis and LC analysis. Monomer stability was evaluated
under the applied conditions, and the overall method was verified for commercially available
chitin by calculating the mass balance. Thereafter in section 3.2, biological samples of black
soldier fly larvae with increasing amounts of impurities were subjected to hydrolysis to validate
the method for insect chitin and to evaluate the effect of impurities on the accuracy of the
method. All triplicate hydrolysis results generated as function of time were evaluated to
develop a protocol to calculate the chitin content and average DA of crude biological chitinous
samples. The results were compared to each other and literature to establish a standard
procedure. Finally, the applicability of the procedure and generated results were evaluated
(see section 3.3).
3.1. Hydrolysis of commercial chitin and chitin monomers
Previous experiments with commercial chitin and chitin monomers as substrate (not
shown) indicated that 110 °C and 6 N HCl were the best conditions to fully hydrolyze chitin
with minimal monomer degradation. Milder conditions resulted in incomplete chitin
conversion while stronger conditions resulted in excessive monomer degradation. To quantify
monomer degradation and allow the determination of the chitin content and average DA, N-
acetylglucosamine, the chitin monomer, was subjected to hydrolysis. Subsequently,
commercial chitin of known purity was hydrolyzed to construct a mass balance to verify the
calculation method and determine the optimal hydrolysis time range.
12

294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
3.1.1. Monomer stability
In first instance, N-acetylglucosamine was subjected to the hydrolysis procedure to
evaluate its stability. It must be noted that during hydrolysis N-acetylglucosamine was
immediately deacetylated to glucosamine since only glucosamine was detected in LC analysis.
References to monomers in all discussions below are therefore related to the detection of
glucosamine solely. Figure 1A shows the evolution of the monomer concentration as function
of time (see Eq. (3)). The linear decrease indicates a zeroth kinetic order for monomer
decomposition implying that the degradation rate is independent of its concentration. Figure
1B shows that the monomer percental conversion (secondary y-axis) is significant and
increases linearly at a rate of X(t) (%) = 1.846*t(h), wherein X(t) is the percentage of monomer
conversion/degradation as function of time. Because of significant glucosamine degradation
during hydrolysis, in this work it was opted to always correct the measured glucosamine
concentration in hydrolysis experiments with this time dependent correction factor (see Eq.
(4)) to calculate the chitin content.
3.1.2. Commercial chitin hydrolysis
When commercial chitin is used as reference substrate (Figure 1B), chitin hydrolysis
increases from 0 h to 8 h, remains stable for two hours and declines subsequently. When
monomer decomposition as function of time is considered via Eq. (4), the curve results into a
plateau after 8 h which indicates maximal chitin conversion whereof the average value of the
plateau is 95.9 ± 1.4 %. This is in line with the expected chitin content of this commercial
sample suggesting a complete hydrolysis. Moreover, the stable plateau indicates that
monomer decomposition was compensated sufficiently without overestimation of the
glucosamine content in the time range studied. In the discussions below and in all figures, the
data corrected for monomer decomposition are referred to as ‘corrected’ and are always
13

318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
displayed with grey symbols. ‘Uncorrected’ data on the other hand are displayed as black
symbols.
3.2. Hydrolysis of insect samples
The different types of samples of BSF larvae were subjected to hydrolysis to evaluate
the performance of the analytical procedure and the quantification of the chitin content and
average DA of BSF larvae chitin. As indicated above, samples with increasing amount of
impurities, i.e. purified exoskeletons (clean pellet), crude exoskeletons (crude pellet) and
whole larvae, were prepared as detailed in section 2.2. Subsequently, hydrolysis experiments
were performed and the chitin content and DA of these samples were calculated via the
method of this article, i.e. by hydrolysis and LC analysis (see section 3.2.1 and 3.2.1). At the
end, based on the various observations, a simplified standard protocol is derived to
characterize chitinous samples (see section 3.3).
3.2.1. Evaluation of hydrolysis results
As a first step, the release of acetate and (corrected) glucosamine was investigated in
function of hydrolysis time for the various sample types. Figure 2 shows the molar release of
acetate and (corrected) glucosamine, normalized per gram of dry matter. At shorter hydrolysis
times all samples show a similar pattern, with first a systematic increase in glucosamine and
acetate concentration indicative of the proceeding hydrolysis, followed by a stabilization
related to maximal hydrolysis. The similar behavior of glucosamine and acetate as function of
time is coherent with the hypothesis that released N-acetylglucosamine, the monomer unit of
chitin, is instantaneously deacetylated with the formation of glucosamine and acetate, as was
also observed in the monomer stability experiments (see section 3.1.1). At hydrolysis times of
more than 10 h, a different behavior is observed between commercial crustacean chitin and
the two insect derived pellet fractions. The corrected glucosamine remains stable for
commercial chitin (see Figure 1B) but increases for the crude and clean pellet (Figure 2A). The
14

343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
different behavior at long hydrolysis times is attributed to the simplified correction applied for
monomer degradation. The correction model assumes that all N-acetylglucosamine is liberated
at the start and then degrades during hydrolysis in a similar way as was observed for the
monomer. In practice, however, the hydrolysis kinetics and the details of the N-
acetylglucosamine release depend on the sample characteristics, such as for instance
crystallinity and/or chitin content. The biological samples of this work are known to have
crystalline regions, of about 35 % according to Wasko et al.. [8] Additionally, the pellet cleaning
with a hot aqueous medium (see section 2.2) is known to be able to induce re-crystallization.
The crystalline regions are expected to slow down hydrolysis, resulting in a delayed release of
N-acetylglucosamine and hence overestimation of the loss of glucosamine due to degradation.
Very similarly, as indicated above, the more “pure” samples (commercial chitin > clean pellet >
crude pellet > whole insect) have an extended hydrolysis period (see Figures 1B and 2), likely
related to their higher chitin content. The more pure BSF samples have a delayed release of N-
acetylglucosamine which is expected to lead to inaccuracies in the correction applied at long
hydrolysis times. As a result, a hydrolysis time of at most 10 h is recommended for the
determination of the chitin content. Under these circumstances, the monomer degradation is
estimated to be at most 20 % for the worse case in which all monomers are immediately
released at the start of hydrolysis (see Figure 1A), limiting the possible errors induced by the
simplified model used for correction.
3.2.2. Insect chitin content and degree of acetylation
If impurities do not interfere with the analysis, the calculated insect chitin content
determined via the crude and clean pellet samples should result in a similar value. Subjecting
the whole larvae to hydrolysis should lead to a higher insect chitin content considering the
presence of chitin in the inner parts which were removed from the pellet samples in the first
mechanical clean up step (see 2.2).
15

368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
Figure 3A shows the calculated insect chitin content as a function of time for the different
samples. As explained above, the hydrolysis time is restricted to 10 h to limit the inaccuracy
that might be induced by the correction applied for monomer degradation. The plateau values,
averaged between time intervals as well as at relevant discrete points are summarized in Table
1. In case of the whole insect, all data points result in an insect chitin content in accordance
with the result of Caligiani et al., i.e. 9 ± 1 %, obtained after hydrolysis, derivatization and gas
chromatography/mass spectrometry analysis. [28] The insect chitin content calculated via the
average value of the plateau is 8.0 ± 0.4 % (plateau 2 h – 8 h in Table 1). For the crude and
clean pellet, the average values of the plateaus between 4 h and 10 h for are respectively 6.9 ±
0.4 % and 6.5 ± 0.6 % (Table 1) which are in accordance with each other and with literature.
Spranghers et al. determined an insect chitin content of 5.6 % to 6.7 % [29] after applying the
time consuming clean-up method of Liu et al. [12] which is similar to the clean-up method
used in this work to produce the clean pellet. This agreement with literature for the three
biological samples, supports the validity of the followed approach for the determination of the
chitin content of BSF larvae. Moreover, the difference measured between the chitin content of
the whole larvae and the one of the pellets, suggests a chitin content of about 1 – 2 % in the
soft tissues. It is well known that, besides in the cuticle, insects also contain chitin in their gut
lining, peritrophic matrix, salivary gland, trachea, eggshells and muscle attachment points. [2]
An important characteristic of the chitin concerns its DA. The acetyl group typically
contributes to the formation of hydrogen bonds that can stabilize the crystalline structure of
the chitin, influencing important material properties such as solubility and mechanical
strength. [30] Hence, this work aimed to determine the average DA simultaneously with the
chitin content. As an approach, the released acetate was measured during hydrolysis (see
Figure 2), and the DA was determined as the ratio between the molar concentrations of
acetate and the corrected glucosamine. Figure 3B gives an overview of the average DA for the
16

393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
various samples in function of hydrolysis time. After an initial transition period, presumably
related to differences in the release of glucosamine and acetate during the proceeding
hydrolysis, the DA remains relatively constant between 4 h and 10 h. Averaging the plateau
values within the same time ranges as used for the chitin calculation, i.e. 4 to 10 h for the
pellets and 2 to 8 h for the whole insect, the average DA values obtained for the chitin are 78.1
± 3.4 % for the whole insect, 78.6 ± 2.4 % for the crude pellet and 72.0 ± 3.7 % for the clean
pellet (Table 1). As would be expected, the DA of the crude pellet and whole larvae are similar
(78.6 ± 2.4 % versus 78.1 ± 3.4 %), but the one of the clean pellet is slightly lower (72.0 ± 3.7)
probably related to the harsh cleaning procedure which is known to result in minor
deacetylation. [14,15]
Since to the best of our knowledge, no public data are available on the average DA of BSF
larvae chitin, these results could not be positioned against literature. Therefore, the method to
determine DA was additionally validated for selected samples by comparison with
conventional techniques, i.e., FT-IR and C/N analysis. Since it is well-known that both FT-IR or
C/N have limitations in the presence of impurities [18], the assessment was concentrated on
the pure samples only, i.e. commercial chitosan for low DA, commercial chitin for high DA and
the clean pellet (see Table 1). For commercial chitosan and chitin, the results were also
analyzed against the supplier’s specifications. To ensure an equal comparative base, for these
samples a slightly different calculation method was applied. As these methodologies assume
that the sample is uniquely composed of N-acetylglucosamine and glucosamine, the DA was
based on the acetate analysis only and assuming a pure chitin/chitosan.
FT-IR analysis of commercial chitosan resulted in 18 %, a value in accordance with the DA
result obtained via LC after 8 h of hydrolysis (20.5 % for oven dried and 20.2 % for untreated,
see Table 1). These values are also in agreement with the specification of the supplier (22%)
based on picric acid adsorption, indicating the validity of the LC-approach for low DA samples.
17

418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
FT-IR analysis of commercial chitin and the clean pellet resulted in low DA values, i.e. 31 %
and 23 % respectively, which is a known drawback of the technique, i.e. samples with high DA
cannot be analyzed accurately with FT-IR. The second benchmark technique, C/N analysis,
resulted in very high DA values, i.e. 96 % and 147 %, for respectively commercial chitin and the
clean pellet (C/N resp. 6.79 and 7.66). Elemental analysis of commercial chitin resulted the
expected DA as communicated by the supplier (DA 98 %) while the clean pellet showed a value
that is typical for impure chitin samples (DA >> 100 %, cfr. Wasko et al. [8]). The DA values
attained in this work for the untreated commercial chitin after 6 and 8 h of hydrolysis (92.0 -
91.4 %, see Table 1), are in good agreement with the C/N result and the specifications
provided by the supplier, which supports the validity of the approach for high DA samples. In
case of the oven-treated commercial chitin, the LC method provides a slightly lower DA of 88%
o
as compared to the untreated sample, suggesting that the drying at 105 C overnight might
influence the chitin. The slightly lower value obtained with the LC-method as compared to the
specifications is within the uncertainty that can be expected from the different methodologies
applied. For instance, it has been reported that the reference method based on picric acid
might be affected by the low solubility of the chitin sample, which might hamper complete
picric acid adsorption and result in an overestimation of the DA. [31] The full destruction of the
samples followed by acetic acid determination applied in this work probably ensures a more
fair measurement of the real DA in case of limited solubility.
3.3. The standardized protocol and its applicability
Based on the results obtained in section 3.2, an assessment was done to standardize
the procedure to determine both the content and average DA of chitin in biological samples.
Moreover, recommendations were derived on its overall applicability which are discussed in
more detail below.
18

442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
When hydrolysis followed by LC analysis is used to determine the chitin content and
corresponding DA, the hydrolysis times should be limited to at most 10 h to avoid the
previously mentioned uncertainties related to the precise correction of monomer degradation.
To determine the best hydrolysis time, LC plateau data and literature data are compared to
data obtained at discrete hydrolysis times for both the chitin content and DA (see Table 1).
For the whole larvae, both 4 h and 6 h of hydrolysis gave the same chitin content and
average DA as the plateau data (within the error margin of the plateau). For both crude and
clean pellet, the values obtained after a hydrolysis time of 6 h and 8 h are in line with the
plateau values reported in section 3.2.2. Moreover, at these times the results for the crude
pellet have similar results as those from whole larvae. This indicated that for the crude and
clean pellet, hydrolysis times between 6 h and 8 h seem the most suitable. Based on Figure 1B,
8 h will also be most efficient for analyzing commercially available chitin since both purified
samples have a higher chitin content compared to the crude samples. Summarizing all above
information, it is concluded that crude biological samples are preferably hydrolyzed for 4 h to 6
h to determine both the chitin content and DA while purified biological samples, thus with a
higher chitin content, are preferably hydrolyzed for 6 h to 8 h. Based on all results, the
following procedure is suggested: 50 mg of crude grinded sample is stirred during 6 h at 110 °C
in 5 mL of 6 N HCl as sample preparation method prior to analysis by LC-MS and LC-RID to
determine the chitin content and average DA of crude biological samples. For purified
chitinous samples, the same procedure can be used, but with a hydrolysis time of 8 h.
For the first time the average DA of black soldier fly larvae chitin is reported carefully as
81.0 ± 2.1 % (6 h, crude pellet result). Since literature data are not available, further
confirmation is needed in future work. Additionally, it must be noted that fresh and living
larvae were used to generate the three biological samples. For the accurate determination of
acetylation degree, it is important that chitin is the only source leading to the formation of
19

467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
acetate. Therefore, careful storage of the samples is needed to avoid decay of the biological
material when crude pellets are used. Moreover, it is recommended that for every new
biological resource a comparison between crude and clean pellet is performed to exclude the
contribution of non-chitin sources to acetate formation. In view of applications, the insect
chitin content of 8.5 ± 0.1 % (6 h) determined by analyzing whole larvae is interesting from a
biological point of view. However, when targeting chitin valorization, it is best to use the chitin
content determined by measuring the crude pellet, i.e. 6.7 ± 0.4 % (6 h), to prevent
overestimation of the revenues since chitin present in the soft tissues cannot be isolated
easily. Both results imply that chitin in the soft tissues represents about 1.8 wt.% of the total
dry insect which is which about 20 % of the total chitin content.
In summary, an LC method, without pretreatment nor derivatization, was developed for
the simultaneous determination of the chitin content and degree of acetylation in crude
samples of BSF larvae with up to 90% impurities on dry matter base. As compared to the
traditional methodologies, the method avoids the labor intensive and harsh clean up
procedure and allows the assessment at high acetylation degrees of acetylation. For the first
time, the DA of BSF larval chitin (81±2%) is reported. Additionally, the chitin content of BSF
larval soft tissues is estimated at 20% of the total chitin content (8.5±0.1 %). In further
research, the generic nature of this method should be validated with other samples and
resources since the effect of chitin crystallinity or other types of impurities was not evaluated.
List of Abbreviations
BSF
Black Soldier Fly
C/N
DA
Elemental analysis
Degree of acetylation
Fourier Transform Infrared Radiation
FT- IR
20

492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
HPLC
High Performance Liquid Chromatography
(UP)LC-MS
(Ultra High Performance) Liquid Chromatography-Mass Spectrometry
Acknowledgements
Insect samples were kindly provided by Millibeter. The pre-trajectory of this work was
supported by FISCH-IWT funding of the CHITINSECT project [grand number: 130787], currently
known as Catalisti-VLAIO funding.
Declarations of interest
None
References
[1]
European Commision, COMMISSION REGULATION (EU) 2017/893, Off. J. Eur. Union. L
138 (2017) 92–116.
[2]
S. Muthukrishnan, H. Merzendorfer, Y. Arakane, Kramer, Chitin Metabolism in Insects,
in: Insect Mol. Biol. Biochem., 2012: pp. 193–235. doi:10.1016/B978-0-12-384747-
8.10007-8.
[3]
[4]
X. Zhu, J. Cai, J. Yang, Q. Su, Determination of glucosamine in impure chitin samples by
high-performance liquid chromatography, Carbohydr. Res. 340 (2005) 1732–1738.
doi:10.1016/j.carres.2005.01.045.
M.R. Kasaai, Determination of the degree of N-acetylation for chitin and chitosan by
various NMR spectroscopy techniques: A review, Carbohydr. Polym. 79 (2010) 801–810.
doi:10.1016/j.carbpol.2009.10.051.
[5]
[6]
A. Ekblad, T. Nasholm, Determination of chitin in fungi and mycorrhizal roots by an
improved HPLC analysis of glucosamine, Plant Soil. 178 (1996) 29–30.
B. Li, J. Zhang, F. Bu, W. Xia, Determination of chitosan with a modified acid hydrolysis
21

517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
and
HPLC
method,
Carbohydr.
Res.
366
(2013)
50–54.
doi:10.1016/j.carres.2012.11.005.
[7]
[8]
J. Lopez-Cervantes, D. Sanchez-Machado, K. Delgado-Rosas, Quantitation of
Glucosamine From Shrimp Waste Using HPLC, J. Chromatogr. Sci. 45 (2007) 195–199.
A. Wasko, P. Bulak, M. Polak-Berecka, K. Nowak, C. Polakowski, A. Bieganowski, The
first report of the physicochemical structure of chitin isolated from Hermetia illucens,
Int. J. Biol. Macromol. 92 (2016) 316–320. doi:10.1016/j.ijbiomac.2016.07.038.
X. Yan, H.M. Evenocheck, Chitosan analysis using acid hydrolysis and HPLC / UV,
Carbohydr. Polym. 87 (2012) 1774–1778. doi:10.1016/j.carbpol.2011.09.091.
[9]
[10] M. Punin Crespo, M. Vilasoa Martinez, J. Lopez Hernandez, M. Lage Yusty, High-
performance liquid chromatographic determination of chitin in the snow crab ,
Chionoecetes
opilio,
J.
Chromatogr.
A.
1116
(2006)
189–192.
doi:10.1016/j.chroma.2006.03.068.
[11] J. Li, L. Chen, Z. Meng, G. Dou, Development of a mass spectrometry method for the
characterization of a series of chitosan, Int. J. Biol. Macromol. 121 (2019) 89–96.
doi:10.1016/j.ijbiomac.2018.09.194.
[12] S. Liu, J. Sun, L. Yu, C. Zhang, J. Bi, F. Zhu, M. Qu, C. Jiang, Q. Yang, Extraction and
characterization of chitin from the beetle Holotrichia parallela motschulsky, Molecules.
17 (2012) 4604–4611. doi:10.3390/molecules17044604.
[13] S. Zaku, S. Emmanuel, O. Aguzue, S. Thomas, Extraction and characterization of chitin; a
functional biopolymer obtained from scales of common carp fish (Cyprinus carpio l.): A
lesser known source., African J. Food Sci. 5 (2011) 478–483.
[14] S. Kaur, G.S. Dhillon, Recent trends in biological extraction of chitin from marine shell
wastes:
A
review,
Crit.
Rev.
Biotechnol.
35
(2015)
44–61.
doi:10.3109/07388551.2013.798256.
22

542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
[15] A. Percot, C. Viton, A. Domard, Optimization of chitin extraction from shrimp shells,
Biomacromolecules. 4 (2003) 12–18. doi:10.1021/bm025602k.
[16] I. Younes, M. Rinaudo, Chitin and chitosan preparation from marine sources. Structure,
properties
and
applications,
Mar.
Drugs.
13
(2015)
1133–1174.
doi:10.3390/md13031133.
[17] Kasaai R, Various Methods for Determination of the Degree of N-Acetylation of Chitin
and Chitosanꢀ: A Review, J. Agric. Food Chem. 57 (2009) 1667–1676.
[18] M. Lavertu, Z. Xia, A.N. Serreqi, M. Berrada, A. Rodrigues, D. Wang, M.D. Buschmann, A.
Gupta, A validated 1 H NMR method for the determination of the degree of
deacetylation of chitosan, J. Pharm. Biomed. Anal. 32 (2003) 1149–1158.
doi:10.1016/S0731-7085(03)00155-9.
[19] M.L. Duarte, M.C. Ferreira, M.R. Marvao, J. Rocha, An optimised method to determine
the degree of acetylation of chitin and chitosan by FTIR spectroscopy, Int. J. Biol.
Macromol. 31 (2002) 1–8.
[20] W.A. Neugebauer, E. Neugebauer, R. Brzezinski, Determination of the degree of N-
acetylation of chitin-chitosan with picric acid, Carbohydr. Res. 189 (1989) 363–367.
[21] W. Arbia, L. Arbia, L. Adour, A. Amrane, Chitin extraction from crustacean shells using
biological methods -A review, Food Technol. Biotechnol. 51 (2013) 12–25.
[22] S. Liu, J. Sun, L. Yu, C. Zhang, J. Bi, F. Zhu, M. Qu, C. Jiang, Q. Yang, Extraction and
Characterization of Chitin from the Beetle Holotrichia parallela Motschulsky, Molecules.
17 (2012) 4604–4611. doi:10.3390/molecules17044604.
[23] K. De Sitter, L. Garcia-Gonzalez, C. Matassa, L. Bertin, H. De Wever, The use of
membrane based reactive extraction for the recovery of carboxylic acids from thin
stillage, Sep. Purif. Technol. 206 (2018) 177–185. doi:10.1016/j.seppur.2018.06.001.
[24] G. Kaur, K. Elst, Development of reactive extraction systems for itaconic acid: A step
23

567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
towards in situ product recovery for itaconic acid fermentation, RSC Adv. 4 (2014).
doi:10.1039/c4ra06612j.
[25] S.R.L. Ellison, A. Williams, eds., Eurachem/CITAC guide: Quantifying Uncertainty in
Analytical Measurement, Third edit, Available from www.eurachem.org, 2012.
www.eurachem.org.
[26] R. Czechowska-biskup, D. Jarosińska, B. Rokita, P. Ulański, J.M. Rosiak, Determination of
Degree of Deacetylation of Chitosan - Comparison of Methods, Prog. Chem. Appl. Chitin
Its Deriv. XVII (2012) 5–20.
[27] J. Xu, S.P. McCarthy, R.A. Gross, D.L. Kaplan, Chitosan Film Acylation and Effects on
Biodegradability, Macromolecules. 29 (1996) 3436–3440.
[28] A. Caligiani, A. Marseglia, G. Leni, S. Baldassarre, L. Maistrello, A. Dossena, S. Sforza,
Composition of black soldier fly prepupae and systematic approaches for extraction and
fractionation of proteins, lipids and chitin, Food Res. Int. 105 (2018) 812–820.
doi:10.1016/j.foodres.2017.12.012.
[29] T. Spranghers, M. Ottoboni, C. Klootwijk, A. Ovyn, S. Deboosere, B. De Meulenaer, J.
Michiels, M. Eeckhout, P. De Clercq, S. De Smet, Nutritional composition of black soldier
fly (Hermetia illucens) prepupae reared on different organic waste substrates, J. Sci.
Food Agric. (2016) n/a--n/a. doi:10.1002/jsfa.8081.
[30] J. Cui, Z. Yu, D. Lau, Effect of Acetyl Group on Mechanical Properties of Chitin / Chitosan
Nanocrystalꢀ: A Molecular Dynamics Study, Int. J. Mol. Sci. 17 (2016) 1–13.
doi:10.3390/ijms17010061.
[31] H. Sato, S. Mizutani, S. Tsuge, H. Ohtani, K. Aoi, A. Takasu, M. Okada, S. Kobayashi, T.
Kiyosada, S.-I. Shoda, Determination of the Degree of Acetylation of Chitin / Chitosan by
Pyrolysis-Gas Chromatography in the Presence of Oxalic Acid, Anal. Chem. 70 (1998) 7–
12. doi:10.1021/ac9706685.
24

592
593
594
595
596
Graphical abstract
597
598
599
600
601
602
603
604
605
606
607
608
609
610
25

611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
Captions
Figure 1: (A) Kinetic plot of monomer degradation and (B) hydrolysis of commercial chitin and
chitin monomers with standard deviations as error bars.
Figure 2: Molar release of (corrected) glucosamine and acetate normalized per gram of dry
sample as function of time during acidic hydrolysis of (A) whole larvae, (B) crude pellet and (C)
clean pellet with standard deviations as error bars.
Figure 3: (A) LC determined chitin content in biological samples and (B) average degree of
acetylation as function of time with standard deviations as error bars.
Table 1: Characterization of chitinous samples based on LC data and standard techniques for
DA determination. The data were derived from the corrected glucosamine contents. * The DA
the commercial chitin/chitosan was determined based assuming a 100 % pure sample to allow
comparison with specifications. ^ Result for undried sample.
26

626
627
Table 1: Characterization of chitinous samples with the LC method using the corrected glucosamine. Comparison of DA against standard techniques.
DA the commercial chitin/chitosan was determined assuming a 100 % pure sample to allow comparison with specifications. ^ For undried sample.
*
Method →
Sample ↓
FT-IR C/N
LC: Average of plateau
Average DA Chitin content Chitin content Discrete
LC: Discrete hydrolysis time
Chitin content Chitin content
Average DA
(%)
Time
range
Average DA (%)
(%)
of sample (%)
of insect (%)
time
of sample (%)
of insect (%)
chitosan
(DA<25%)
18
n.d.
8h
20.5
20.2
±
±
0.01*
0.01*^ 103.7
101.9
84.2
n.d.
n.d.
n.d.
/
/
±
1.8
4.2
10 h
6h
/
/
chitin
(DA>95%)
31
96
6h-8h
n.d.
n.d.
89.2
92.0
±
±
7.0
6.9
±
92.0
±
0.03*
91.4
88.0
±
±
0.01*
0.02*^
6h-10h
8h
94.1 ± 2.2
/
8h-10h
4h-8h
4h-10h
6h-8h
6h-10h
8h-10h
4h-8h
4h-10h
6h-8h
6h-10h
8h-10h
2h-6h
2h-8h
4h-6h
4h-8h
6h-8h
n.d.
95.8 ± 2.4
4.1 64.1 5.1 6.3
72.0 ± 3.7 66.3 ± 6.1 6.5
10h
4h
n.d.
±
97.6 ± 2.0
/
23
147
n.d.
n.d.
72.8
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.5
0.6
0.5
0.5
0.2
0.3
0.4
0.2
0.3
0.2
0.3
0.4
0.4
0.5
0.7
69.0
77.1
72.2
69.6
1.1
5.2
1.7
0.3
60.1
±
0.7 5.9
±
±
±
±
0.1
0.3
0.2
0.1
clean BSF
pellet
6h
±
62.2 ± 3.2
69.8 ± 1.3
6.1
6.9
7.2
74.7
73.0
70.9
79.4
±
±
±
±
3.5 66.0
3.8 68.3
1.8 71.4
2.2 16.6
±
±
±
±
5.4 6.5
5.5 6.7
2.2 7.0
0.7 6.8
8h
±
10h
±
73.0
±
0.7
crude BSF
pellet
4h
6h
76.9
81.0
80.4
76.3
±
±
±
±
1.2
2.1
1.8
2.6
16.0
±
0.3 6.5
±
±
±
±
0.3
0.4
0.5
0.5
n.d.
n.d.
78.6 ± 2.4 17.0 ± 0.9 6.9
16.6 ± 0.4 6.7
17.3 ± 0.5 7.0
80.7
79.2
78.4
77.8
±
±
±
±
0.4 17.0
2.6 17.3
2.9 17.7
±
±
±
±
0.5 6.9
0.7 7.0
0.5 7.2
0.3 8.2
8h
10h
18.0
±
0.5 7.3
whole BSF
larvae
4.1
8.2
2h
4h
6h
8h
73.0
80.0
80.3
79.0
±
±
±
±
4.5
2.1
8.8
4.6
8.0
±
0.3 8.0
±
±
±
±
0.3
0.2
0.1
0.2
78.1 ± 3.4
8.0 ± 0.4 8.0
8.0 ± 0.2 8.0
8.5 ± 0.1 8.5
80.2
79.8
79.6
±
±
±
0.2
0.7
0.9
8.3
8.0
8.0
±
±
±
0.4 8.3
0.5 8.0
0.7 8.0
7.5
± 0.2 7.5
27

628
Figures
629
630
28

631
632
Figure 1: (A) Kinetic plot of monomer degradation and (B) hydrolysis of commercial chitin and chitin monomers with standard deviations as error bars.
633
634
29