1. Introduction

2. Methodology

3. Results

3.2. Standarization of Demineralization Process

A Tukey test was performed, revealing that experimental conditions 3, 4, 7, and 8 exhibited statistical equality and included the highest %DM values, marked in red (Table 1). Among these conditions, number seven was selected (HCl concentration 2M at 30ºC for 3 h) since it represents the shortest reaction time and the lowest reaction temperature. This choice renders the process more environmentally friendly and less energetically costly without statistically impacting the %DM.

Table 2 displays the results of DM experiments conducted to standardize this process. The percentage of demineralization (%DM) was calculated by comparing the ash content with the initial value in the proximal analysis (4.3%). A significant decrease in mineral content can be observed under all experimental conditions, with values ranging 86.79% to 93.86% for %DM.

Table 2. DM process experimental conditions and results for Acheta domesticus cricket flour*.

Table 2. DM process experimental conditions and results for Acheta domesticus cricket flour*.

Experimental condition	HCl concentration [M]	Time (h)	Temperature (ºC)	Ash content (%)	%DM
1	1	6	65	0.37 ± 0.03	92.1 ± 0.69 A, B, C, D
2	1	3	30	0.47 ± 0.05	90.09 ± 0.98 B, C, D, E
3	2	3	65	0.29 ± 0.11	93.86 ± 2.34 A
4	1	3	65	0.53 ± 0.05	88.84 ± 1.08 D, E
5	1	6	30	0.62 ± 0.06	86.79 ± 1.27 E
6	2	6	30	0.49 ± 0.03	89.56 ± 0.59 C, D, E
7	2	3	30	0.36 ± 0.03	92.25 ± 0.67 A, B, C
8	2	6	65	0.33 ± 0.04	93.02 ± 0.92 A, B

*Results are the mean value of a triplicate, ± standard deviation. Mean values with different superscripts indicate statistical differences (Tukey test, 95% confidence).

Considering the obtained results, an analysis of variance (ANOVA) was conducted, revealing that the temperature and acid concentration factors were statistically significant, whereas the reaction time was not (Table 3). It was determined that a concentration of HCl at 2M and a temperature of 65ºC represent the levels that maximize the %DM, corresponding to experimental conditions 3 and 8. Condition number 8 was excluded due to observation of an unfavorable reaction in the sample (manifested as a black and viscous appearance), suggesting potential protein denaturation or alteration to the chitin structure.

Finally, the condition that maximizes the %DM was implemented to demineralize the previously degreased cricket flour. Samples were taken to perform ash determination to verify the maximization of the %DM, and the result was 95.85 ± 0.012%.

3.3. Optimization of Deproteinization Process

Table 4 displays the conditions and results of the Box-Behnken design experiments to optimize the DP process. The percentage of protein (dry weight) was used to calculate the %DP, with values ranging from 27.13 to 52.61.

Table 4. Experimental conditions and results for deproteinization (DP) process optimization for Acheta domesticus cricket flour according to Box-Behnken design*

Table 4. Experimental conditions and results for deproteinization (DP) process optimization for Acheta domesticus cricket flour according to Box-Behnken design*

Experimental condition	NaOH concentration [M]	Time (min)	Temperature (ºC)	Protein (%)	DP (%)
1	3	45	70	30.67 ± 3.63	44.08 ± 6.44
2	2	30	70	25.99 ± 1.84	52.61 ± 3.27
3	2	45	80	30.3 ± 0.08	44.76 ± 0.14
4	2	15	80	34.15 ± 0.3	37.73 ± 0.53
5	1	30	60	38.57 ± 1.69	29.69 ± 3.00
6	2	30	70	36.69 ± 2.11	33.11 ± 3.75
7	1	30	80	34.64 ± 0.31	36.84 ± 0.54
8	1	15	70	39.97 ± 0.88	27.13 ± 1.56
9	3	15	70	32.27 ± 1.97	41.16 ± 3.50
10	3	30	60	34.39 ± 0.97	37.31 ± 1.73
11	1	45	70	35.32 ± 0.72	35.6 ± 1.27
12	3	30	80	32.13 ± 2.88	41.42 ± 5.11
13	2	15	60	34.22 ± 0.38	37.62 ± 0.68
14	2	30	70	33.54 ± 4.07	38.84 ± 7.22
15	2	45	60	31.43 ± 2.98	42.7 ± 5.30

* Results for protein (%) and %DP are the mean of value of a triplicate, ± standard deviation.

After conducting the 15 experiments in triplicate, a statistical analysis of the data was performed using a Box-Behnken surface response model in MINITAB® Version 19.2020.1.0 software. The resulting ANOVA used the %DP as the response variable, as depicted in Table 5. The analysis revealed that reaction time, NaOH concentration, and the concentration*concentration interaction were significant factors affecting the response variable. The regression equation for the model was determined (equation 11), where C represents the concentration of NaOH (M), T is temperature of the reaction (ºC), and t is the reaction time (minutes). Next, a prediction of the optimal conditions to maximize the %DP value was performed, resulting in the following parameters: 80ºC for temperature, 45 minutes for reaction time, and 2.56M for NaOH concentration. The %DP was predicted to reach 46.37 ± 3.86% with a 95% confidence interval, using the optimal established conditions.

$$\%\mathrm{DP}==-3.84{\mathrm{C}}^2+19.70\mathrm{C}+0.17\mathrm{T}+0.197\mathrm{t}-1.33$$

(11)

The experimental value of the optimal condition was determined in triplicate to validate the %DP predicted by the model, yielding a value of 43.23 ± 1.25%. This value falls within the predicted confidence interval.

4. Discussion

4.4. Characterization: Identification of Chitin and Chitosan’s Functional Groups Using Fourier Transform Infrared Spectroscopy (FTIR)

FTIR analyzes how matter interacts with infrared radiation, enabling the identification of chemical species by determining the frequencies at which different functional groups absorbs in the IR spectra [39]. This technique effectively confirms the presence of both chitin and chitosan as it identifies their characteristic functional groups in the spectra. These include alcohols, amides (found in chitin), amines (in chitosan), carbonyl groups and ethers, appearing at wavelengths 3650 cm^-1, 1640 cm^-1, 3500 cm^-1, 1700 cm^-1 and ~1100 cm^-1, respectively [3,40,41].

Figure 2 shows the FTIR spectrum of whitened chitin obtained under the standardized and optimized conditions demineralization and deproteinization, as outlined in previous sections. The spectrum clearly reveals the presence of all functional groups characteristic of chitin’s structure. A broad band at 3434 cm^-1 indicates the stretching of the hydroxyl group (-OH), while a more intense band at 3256 cm^-1, indicates the stretching of the -NH group, associated with a secondary amide, typical of chitin. Additionally, lower intensity bands at 3097 and 2876 cm^-1, correspond to the stretching of the -CH and -CH3 groups, respectively. The medium intensity bands near 1645 cm^-1, attributed to the carbonyl group C=O stretching, confirm the α (alpha) crystalline structure of the chitin obtained. This identification is because chitin exists in three crystalline structures in nature: α-chitin, with two bands around 1650 cm^-1, β-chitin showing a single band at 1650 cm^-1, and γ-chitin of which not much information can be found in the scientific literature [42].

A medium-intensity band at approximately 1551 cm^-1 in the FTIR spectrum corresponds to the bending of the -NH group and the stretching of the C-N bond. Notably, the absence of a band around 1540 cm^-1 in this region indicates successful deproteinization. A signal in this wave number typically indicates the presence of peptide bonds. Its absence therefore implies a minimal presence of proteins in the whitened chitin (Figure 2) [43]. The next band, at around 1375 cm^-1 shows bending of the hydroxyl group bond (-OH), verifying its presence in the obtained compound as it complements the band at 3434 cm^-1 (Figure 2). Finally, intense bands can be seen at 1067, 1010 and 720 cm^-1 in the fingerprint region, corresponding to the stretching of the C-OH bond of the primary alcohol, the stretching of the C-O bond corresponding to the cyclic 5-carbon ether, and the bending of the N-H bond of the amide group, respectively, further delineating the molecular structure of chitin (Figure 2).

Figure 2. IR spectrum of chitin (left) and chitosan (right) extracted from Acheta domesticus crickets showing its characteristic and dominant functional groups.

Figure 2. IR spectrum of chitin (left) and chitosan (right) extracted from Acheta domesticus crickets showing its characteristic and dominant functional groups.

The chitosan obtained from the whitened chitin showed notable differences with the chitin IR spectrum, as shown in Figure 2 on the right side. A difference is the band at 3437 cm^-1 which corresponds to the stretching of the -NH bond in the primary amine, a feature of the molecular structure of chitosan following deacetylation process of chitin. Additionally, the band at 3257 cm^-1 is indicative of the hydroxyl group bond (-OH) stretching similar to that in chitin. Lower intensity bands at 3095 and 2876 cm^-1, represent the stretching of the -CH and -CH3 groups, respectively. The carbonyl group stretching is observed at 1645 cm^-1 and the bending of the -NH bond, characteristic of an amine, appears around 1555 cm^-1 (Figure 2). These bands are typical in the IR spectra for chitosan as it is not completely deacetylated. Therefore, the presence of a carbonyl group in the IR spectrum depends on the degree of deacetylation of the chitosan the sample [8].

Since the degree of deacetylation (%DA) obtained was 99.47%, it can be considered that the time and conditions under which the deacetylation process was performed were extremely efficient. This result is comparable to those reported for chitin deacetylation from other insect sources such as silkworm Bombyx mori, in which a 94% DA was obtained using NaOH 40% for 4 h at 110ºC [35]. The results agree with reports where it is stated that the deacetylation of insects’ chitin is easier than chitin from crustacean. There are reports of only 75% DA for samples extracted from P. monodon shrimps using HCl 1M at 25ºC for demineralization, NaOH 1M at 100ºC for 8 h for deproteinization and NaOH 50% at 100ºC for 8 h for deacetylation [35].

Finally, Figure 3 shows the differences in the bands present in the chitin and chitosan that were obtained in this study (Chitin E and Chitosan E) in comparison to commercially available chitin and chitosan extracted form crab (Chitin C and Chitosan C) reported by Sáenz et al. [44].

The intensity of the stretching bands of the hydroxyl group -OH and the -NH bond around 3500 cm^-1 and 3450 cm^-1, respectively, are slightly more intense in the experimental chitin. However, both samples show the characteristic bands of the functional groups, with similar intensities between them, including the C=O bond stretch around 1645 cm^-1, the bending of the N-H bond and the stretching of the C-N bond of the secondary amide around 1551 cm^-1, the stretching of the -CH and -CH3 bonds which are visible around 3097 and 2876 cm^-1, respectively, the stretching of the C-OH bond of the primary alcohol at 1067 cm^-1, the stretching of the C-O group corresponding to the 5-carbon cyclic ether at 1010 cm^-1 and the bending of the -NH bond in the amide group around 720 cm^-1.

On the other hand, the IR spectrum for the chitosan from this work and commercial chitosan differ from each other mainly in the intensity and clarity of the characteristic bands. Nevertheless, the main functional groups can be distinguished, such as the -NH and -OH bond stretching bands at around 3400 and 3300 cm^-1, respectively. The stretching of the C=O bond (1645 cm^-1), the bending of the primary amine N-H bond (1555 cm^-1), and the stretching of the C-O bond corresponding to the 5-carbon cyclic ether at around 1009 cm^-1.

Because of the great similarity between IR spectra from chitin and chitosan from this work and commercial reference, it can be stated that we successfully obtained these polysaccharides from Acheta domesticus in accordance with the conditions established in this study [44].

Figure 3. IR spectrum of chitin (E) and chitosan (E) extracted from Acheta domesticus cricket flour in comparison to commercially available chitin (C) and chitosan (C) reported by Sáenz et al. [44].

Figure 3. IR spectrum of chitin (E) and chitosan (E) extracted from Acheta domesticus cricket flour in comparison to commercially available chitin (C) and chitosan (C) reported by Sáenz et al. [44].

4.5. Characterization: Identification of Chitin and Chitosan’s Crystalline Structure Using X-Ray Diffraction (XRD)

XRD is an analytical technique that measures the diffraction that an X-ray shows when it interacts with the atoms of a sample. It is especially useful to identify the crystalline structure and the purity of a sample without an extensive sample preparation [45]. In previous studies, XRD showed distinctive peaks for chitin at 9.1º and 19.3º and for chitosan at 9.1º and 19.1º in 2θ, where it is possible to identify 3 crystalline forms of chitin (α, β and γ) due to the presence of hydrogen bonds in its structure [4].

The result is a diffractogram that shows the diffraction angle of the X-ray in 2θ and the intensity of the received signal when diffracting occurs when the ray encounters the chitin and chitosan. As well, it allows the identification of the crystallographic planes and the crystalline network of both compounds [45,46].

Figure 4 shows the XRD analysis results for chitin and chitosan obtained in this study from Acheta domesticus cricket flour, compared to commercially available chitin and chitosan extracted from crab reported by Escobar et al. [14]. These diffractograms show the most representative peaks for commercial chitin and chitosan, which match, in shape and position, those found in samples obtained in this report, confirming the identity of the compounds. Additionally, the crystalline peaks around 9.6, 19.6, 21.1, and 23.7 degrees in 2θ confirm that the obtained chitin is α-chitin, as previously reported in other publications. This observation demonstrates that the crystalline form obtained aligns with the most commonly obtained form of chitin from crustaceans available in the industry [47,48].

The crystallinity index was also calculated for the samples, resulting in 93.86% and 90.60% for chitin and chitosan, respectively. In comparison, 81.84% and 58.14% were reported for commercially available chitin and chitosan. It has been reported that crystallinity indexes for chitin vary from 83.4% to 85.21% and 50.1% to 49.1% for chitosan extracted from grasshopper and shrimp husk sources, respectively [7,48]. A higher crystallinity index represents higher thermal stability for the compounds, as it indicates the presence of a minimum quantity of amorphous regions in the samples and therefore the premature degradation of its structure at high temperatures can be avoided (Section 4.7).

Figure 4. Obtained XRD difractograms for chitin and chitosan obtained in this study (chitin E and chitosan E) from Acheta domesticus cricket flour in comparison to results reported by Escobar et al. [14] using XRD on commercially available chitin and chitosan obtained from crab (chitin C and chitosan C).

Figure 4. Obtained XRD difractograms for chitin and chitosan obtained in this study (chitin E and chitosan E) from Acheta domesticus cricket flour in comparison to results reported by Escobar et al. [14] using XRD on commercially available chitin and chitosan obtained from crab (chitin C and chitosan C).

4.7. Characterization: Analysis of Chitin and Chitosan Thermal Behaviour Using Differential Scanning Calorimetry (DSC)

DSC was used to know the thermic stability and transition temperatures of the extracted chitin and chitosan. There were no DSC studies for these molecules from sources similar to Acheta domesticus [7,48].

Figure 6 shows the thermogram obtained by DSC of the chitin and chitosan obtained from cricket flour. In the case of chitin, no peak or slope can be perceived which may indicate the crystallization temperature or the glass transition temperature of the material. The crystallinity index of the chitin is very high and without the presence or scarce presence of amorphous regions, making the thermal behavior of the polymer very stable under high temperatures. It is estimated that changes in chitin structure may occur when surpassing 300ºC, due to decomposition of residual monomers of N-acetyl glucosamine and N-glucosamine [50]. An exothermic peak would be expected around 110 to 160ºC corresponding to water on the polymer chain [50,51]; however, since the sample was previously dried for its analysis, this could not be appreciated in the thermogram.

The chitosan thermogram shows that an exothermic peak at 185ºC corresponding to the crystallization temperature (T_c). Similar to chitin, it was expected that a significant exothermic peak may appear at 110 to 160ºC corresponding to water present in the polymer chains, this peak was visible at low intensity at around 140ºC [50,51]. The thermal behavior of chitosan is consistent with its crystalline structure. It has been reported in the scientific literature that this molecule presents a lower crystallinity index than chitin, as observed in the present study, which means that it contains more amorphous regions that turn crystalline when reaching the T_c. Therefore, chitosan is a more reactive chemical compound than chitin and consequently has a wider variety of applications (biomedicine, cosmetics, residual water treatment, etc.), being a highly sought-after compound in different industries [50,51].

Figure 6. DSC thermogram for chitin and chitosan extracted from Acheta domesticus cricket flour.

Figure 6. DSC thermogram for chitin and chitosan extracted from Acheta domesticus cricket flour.

5. Conclusions

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