3.2. Acidic depolymerisation of chitosan
Figure 2 shows the dynamics of acidic hydrolysis of chitosans obtained from H. illucens larvae.
Low molecular weight chitosans were prepared by using nitric acid of various concentrations – 3.3, 6.6, 9.9, 13.1 and 16.4%. As the acid concentration increased, degree of deacetylation of obtained chitosan also increased: 92, 95, 97, 97, and 98%, respectively. The drop of molecular weight values was initially observed with increasing nitric acid concentration, however, reached plateau and did not fall below 33 kDa.
Using acidic hydrolysis low molecular weight chitosans (33–88 kDa) can be obtained and further used for studying their biological activity (Table 1). The purity of initial chitosan samples was confirmed by FTIR (Fig. 2) analyses.
Table 1
The physicochemical characteristics of crab and insect chitosan samples.
| Sample | Abbreviation | Mw, kDa | Mn, kDa | Mp, kDa | PDI | DDA, % |
| 1 | 88h | 88 | 39 | 63 | 2.24 | 91.8 |
| 2 | 53h | 53 | 27 | 36 | 1.99 | 94.8 |
| 3 | 39h | 39 | 17 | 24 | 2.31 | 96.7 |
| 5 | 33h | 33 | 14 | 23 | 2.32 | 97.9 |
| 6 | 45cr | 45 | 16 | 22 | 2.99 | 85.4 |
| 7 | 25cr | 25 | 14 | 15 | 1.81 | 88.6 |
3.3 FTIR analyses of insect chitosan and crab chitosan
To clarify the structural characteristics of chitosan, FTIR spectral analysis of H. illucens chitosan and P. camtschaticus chitosan was performed in the frequency region of 4000 − 400 cm− 1 (Fig. 3).
Key IR bands at 3435, 2918, and 2881 cm− 1 were identified in both products, corresponding to stretching vibrations of OH, NH, and CH bonds, respectively. Additional bands near 1656 and 1576 cm− 1, denoting amide I and II vibrations in chitosan, were observed, with the former exhibiting a contribution from the scissoring vibration of water δ(H2O). The FTIR spectra revealed weak amide bands, confirming the high deacetylation of these products. The absence of the band at ~ 1540 cm− 1, typical for proteins, further affirmed effective deproteinisation. Bands at 1418 and 1380 cm− 1 originated from bending vibrations of CH2 and CH3 groups δ(CH2), δas(CH3), and δs(CH3), respectively, consistent with previous studies (Lee et al. 2022; Peng et al. 2022). The band at 1151 cm− 1, indicative of polysaccharides, was assigned to COC stretching vibrations at glycosidic bonds. Intense bands at 1083 and 1034 cm− 1 were associated with CO stretching in pyranoid rings.
Despite minor differences in absorption spectra, the characteristic peaks of H. illucens chitosan closely resembled those of P. camtschaticus chitosan, with enhanced intensities in most absorption peaks. This affirmed their structural similarity through spectral analysis.
To assess the quantitative content of certain functional groups in chitosan, a comparison was made in relative units. The absorption intensity of the studied absorption band was compared with the intensity of band 3435 cm-1 (NH, OH) according to the following principle – the intensity of the studied band was divided by the intensity of the band with maximum absorption (OH, NH) and multiplied by 100 to show the percentage. This method was used for relative comparison of the intensity of chitosan bands from different raw materials (Table 2).
Table 2
Comparison of characteristic peaks from the FTIR spectra of H. illucens chitosan and P. camtschaticus chitosan
| Absorption band | Chitosan from H. illucens | Chitosan from P. camtschaticus |
| Absolute absorption value, % | Comparative value | Absolute absorption value, % | Comparative value |
| OH, NH (3435 cm-1) | 55.15 | - | 62.85 | - |
| C-H (2918 cm-1) | 24.58 | 44.5 | 24.36 | 38.7 |
| C-H (2881 cm-1) | 23.29 | 42.2 | 25.22 | 40.1 |
| Amide I (1656 cm-1) | 27.36 | 49.6 | 26.07 | 41.4 |
| Amide II (1576 cm-1) | 35.48* | 64.3 | 20.09 | 31.9 |
| CH2 (1418 cm-1) | 29.43 | 53.3 | 16.88 | 26.8 |
| CH3 (1380 cm-1) | 22.80 | 41.3 | 19.87 | 31.6 |
| C-O-C (1151 cm-1) | 27.00 | 48.9 | 33.34 | 53 |
| C-O (1083 cm-1) | 43.61 | 79 | 56.22 | 89.4 |
| C-O (1034 cm-1) | 38.41 | 69.6 | 48.31 | 76.8 |
| * The values of peaks that have significant differences for chitosans obtained from H. illucens and P. camtschaticus are highlighted in bold. |
While common functional groups were identified in both chitosan varieties, the spectrum of H. illucens chitosan exhibited notably more intense bands in the N-H bond (1570 cm− 1, amides, proteins), CH2 (1415 cm− 1), and CH3 (1380 cm− 1) functional groups compared to chitosan from P. camtschaticus. This suggests that H. illucens chitosan may have slightly longer aliphatic hydrocarbon chains, indicating potential variations in the chemical extraction method or the distinct species involved. Differences may arise from impurities like melanin or other contaminants, potentially forming complexes with chitosan and influencing its chemical environment, leading to alterations in vibrational frequencies and peak shifts.
3.4 Antifungal activity
The study investigated the fungicidal activity of chitosans obtained from H. illucens and P. camtschaticus on CDA medium with two standard antiseptics and a control (no additives) against 12 filamentous fungi previously isolated in STG (Zhgun et al. 2020b). The fungi belonged to the following genera: Aspergillus, Cladosporium, Simplicillium, Microascus, Ulocladium, Penicillium and Mucor. These fungi have been intensively studied recently, both from the point of view of the possibility of using their metabolic potential for the needs of biotechnology (Zhgun et al. 2022b), and with the aim of creating targeted antiseptics against them with a broad spectrum of action (Alexandrova et al. 2021, 2022; Makarov et al. 2023). However, the influence of cultivation conditions on the characteristic micromorphology of these fungi has not been previously studied. And this is important, since antiseptics can have different effects on different morphological forms of fungi. In this regard, in the current study at the first stage, the micromorphology of fungi from STG, cultivated on Czapek-Dox agar medium, was studied by scanning electron microscopy (Fig. 4). It turned out that the micromorphology of fungi does not change compared to their characteristic structure when growing on painting materials. Thus, we have shown for the first time that these cultivation conditions are suitable for studying their sensitivity to antiseptics.
We then determined the antifungal activity of the studied compounds. It was quantified as the percentage inhibition of growth of filamentous fungi colonies on CDA medium with added chitosans relative to growth on control medium. Inhibition dynamics were tested using 1 mg/ml chitosans, 0.1 mM benzalkonium chloride (BAC), and 0.2 mM sodium pentachlorophenolate (NaPCP). Results were recorded every 3 days after inoculation. Figure 5 demonstrates the characteristic growth of these STG-strains after 14 days of cultivation. The cultures were incubated at 26°C for 41 days.
In our experiment there was an opposite tendency – in the series 33-39-53-88 kDa, chitosans with the lowest Mw showed the highest inhibition efficiency, after 39 kDa the inhibitory activity was lower (Figs. 6, 7). All variants of the tested chitosans and standard antiseptics completely inhibited members of the genera Simplicillium and Microascus (Fig. 6). Chitosans from H. illucens and P. camtschaticus demonstrated high antifungal activity (100%) against Cladosporium species compared to BAC, whose activity decreased from 100 to 30% after 41 days of incubation. Chitosans with molecular weight 33 and 39 kDa from H. illucens completely inhibited almost all representatives of the Aspergillus genus during the whole incubation period, but the growth inhibition of A. versicolor (STG-86) was present during the first 10 days of incubation, then gradually decreased and reached 20% at the end of incubation (Fig. 7). Chitosans with Mw 53 and 88 kDa also inhibited representatives of this genus, in particular strains Aspergillus creber (STG-93W) and A. protuberus (STG-106); the inhibitory effect of other representatives decreased with time (up to 25–70%). Chitosans from H. illucens with Mw 33–53 kDa completely inhibited Ulocladium sp. AAZ-2020a (STG-36), while the toxic effect of chitosan with Mw 88 kDa decreased from 98 to 65% over time.
It is important to note that the growth inhibition of P. chrysogenum (STG-117) and M. circinelloides (STG-30) was less than 70% at the beginning of the incubation period and almost no inhibition was observed after 20 days of cultivation (Figs. 6, 7). Only when NaPCP was added to the medium throughout cultivation, their complete inhibition was recorded.
Chitosans from P. camtschaticus showed similarly high results as in (Zhgun et al. 2020c) work – complete growth inhibition of most strains except for A. versicolor (STG-86), P. chrysogenum (STG-117) and Mucor circinelloides (STG-30). However, it is noteworthy that crab chitosan with MM 25 kDa showed the highest antifungal activity against A. versicolor STG-86, completely inhibiting the strain during the first 14 days of incubation.
A characteristic change in micromorphology of P. chrysogenum STG-117, expressed as a change in pigment colouration, was induced by the addition of black soldier fly chitosan, not by chitosan from crab (Fig. 8). The main pigments in P. chrysogenum are sorbicillin and chrysogine, which can be synthesised in response to several stimulus and give the strain growing on agar medium a characteristic greenish-yellow hue (Zhgun and Eldarov 2021). The phenotypic difference of P. chrysogenum STG-117 growing on agar medium supplemented with chitosans from various sources clearly demonstrates their differences at the molecular level, leading to different effects on the secondary metabolism of the mold.
The level of inhibition at 9, 21 and 41 days for all strains tested is shown in Fig. 9. The chitosans with Mw 33 and 39 kDa from black soldier fly and 25 and 45 kDa from crab were the most active compounds, outperforming the standard antiseptic NaPCP in terms of inhibition activity. BAC and chitosans from black soldier fly with Mw 53 and 88 kDa showed the lowest activity. The graph illustrates that chitosans from H. illucens with MW 33 and 39 kDa and from P. camtschaticus with Mw 25 and 45 kDa can act as alternative antiseptics against filamentous fungi.
Chitosans' antifungal activity is related to their interaction with fungal cell walls and membranes (El Ghaouth et al. 1992). However, the inhibition efficiency depends on the systematic group of the fungus. The lipid composition of the membranes is the main factor known to influence the efficacy of chitosan against filamentous fungi (Palma-Guerrero et al. 2010). Filamentous fungi with a high content of polyunsaturated fatty acids, such as linoleic acid, are more sensitive to chitosan. Conversely, fungi with a high content of saturated acids in the membrane are resistant to the action of chitosan. Previous studies have shown that chitosan's positive amino groups interact with the negative charges of plasma membrane phospholipids, which may lead to changes in plasma membrane permeability (Wang et al. 2014; Hua et al. 2019). The study (Xing et al. 2018) demonstrated that chitosan induces necrotic cell death and can penetrate the cell membrane during the early stages of fungal development without causing visible membrane disruption. This leads to a significant reduction in intracellular substance content and severe damage to the membrane structure.
It was earlier reported (Kulikov et al. 2014) that the biological activity of chitosan increases with a higher DDA. However, contradictory data exists regarding the relationship between antifungal activity and chitosan Mw. Some studies (Garcia et al. 2020; Sheng et al. 2022) have suggested a decrease in biocidal activity with an increase in chitosan Mw, while others have found higher activity in high molecular weight chitosans compared to low molecular weight ones (Guo et al. 2008; Garcia et al. 2018; Li et al. 2022).
The conflicting results in the activity-Mw relationship are attributed to variations in characteristics of molecular weight, DDA, and preparation methods among chitosan samples used by different investigators (Qin et al. 2006; Hernández-Lauzardo et al. 2008). The reported differences in biological effects could also stem from the presence of various types and quantities of lowest and highest chitooligosaccharides in the samples (Kulikov et al. 2014). Moreover, discrepancies may arise from variations in the chemical structure of terminal groups and acetyl-group distribution along oligochitosan chains due to differences in hydrolysis methods (Kulikov et al. 2014). The effectiveness of chitosan with diverse molecular weights in inhibiting growth is also influenced by the fungal species involved (Younes et al. 2014).
The ambiguity in experimental data on the correlation between biocidal activities and physicochemical characteristics of chitosan types is primarily due to molecular heterogeneity. Therefore, it is essential to characterise each chitosan sample by its molecular weight, degree of acetylation, and polydispersity before conducting investigations (Kulikov et al. 2012).
Existing literature highlights the antimicrobial properties of chitosan, yet limited information is available from the exploration of chitosan derived from insects. It was shown that the activity can be influenced by the developmental stage of H. illucens (e.g., larvae, pupal exuviae, or dead adults), as well as the extraction method (bleached versus unbleached chitosan samples) (Guarnieri et al. 2022). This pattern was similarly observed in other insect species like Periplaneta americana, Blattella germanica (Basseri et al. 2019), Antheraea mylitta (Jena et al. 2023). The studies’ findings (Guarnieri et al. 2022; Khayrova et al. 2022) were also consistent with those obtained from chitosan derived from crustaceans, affirming that chitosan sourced from insects, particularly H. illucens, stands as a viable alternative to commercial chitosan for antimicrobial activity.
