# Development of Indirect Sandwich ELLA for Detection of Insets in Food
## 4 Discussion  
Despite the fact that insect consumption in the European Union does not have a tradition compared to other countries such as Africa, Latin America, and Asia, its use is gradually increasing, especially with regard to the environmental benefits of insect protein production [31]. The fear of insects that is often associated with insect consumption in Western countries is also gradually decreasing [32]. As such, insects are used for direct consumption after basic technological operations, but in Europe, their highest potential is associated with their use as insect-derived materials in other products. In view of the above, the use of insects in Europe is regulated by the Novel Food Regulation (EU) 2015/2283 [1]. According to [PERSON], the production of insects and insect-based foods in Europe is expected to develop significantly. The author expects production of up to 260,000 tonnes by 2030 [33]. Significant applications are particularly expected in the field of feed production. Despite the undeniable benefits, the EU takes a moderate approach and does not underestimate the risks associated with the consumption of insects and insect products. According to the EFSA's opinion [34], insects pose risks mainly in terms of allergy potential and chemical and biological risks, but there are also concerns about the environmental impact associated with the introduction of new invasive species into the EU. Ensuring the detection of insects in food products is crucial for consumer protection, food quality, and authenticity verification. According to [PERSON] [35], the inclusion of insects as food ingredients affects consumer acceptance, as these products offer high-quality proteins, minerals, and essential fatty acids. With insect-based foods recognized \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Chitosan** & **Mean \(\pm\) SD** & **CV (\%)** & **Relative Error (\%)** \\ \hline \multicolumn{4}{c}{Intra-day precision} \\
0.1 mg/mL & 0.15 \(\pm\) 0.02 & 15.41 & 48.79 \\
1.0 mg/mL & 0.99 \(\pm\) 0.06 & 5.66 & 0.34 \\
10 mg/mL & 11.51 \(\pm\) 0.03 & 2.45 & 15.10 \\ Insect product (AD 93\%) 25 mg/mL & 7.58 \(\pm\) 1.38 & 18.19 & - \\ Insect product (AD 93\%) 50 mg/mL & 15.05 \(\pm\) 1.55 & 10.29 & - \\ _Tenebro molitor_ 25 mg/mL & 7.17 \(\pm\) 2.17 & 30.29 & - \\ _Tenebro molitor_ 50 mg/mL & 11.45 \(\pm\) 2.7 & 23.59 & - \\ \multicolumn{4}{c}{Inter-day precision} \\
0.1 mg/mL & 0.15 \(\pm\) 0.02 & 12.87 & 53.75 \\
1.0 mg/mL & 0.96 \(\pm\) 0.09 & 8.92 & 3.61 \\
10 mg/mL & 11.4 \(\pm\) 0.04 & 0.36 & 14.37 \\ \hline \hline \end{tabular}  
Chitosan concentrations were detected using indirect sandwich ELLA (\(n\) = 5). SD: standard deviation, CV: coefficient of variation, AD: _Acheta domesticus_, - not relevant.  
\end{table} 
Table 4: Precision and relative error of the indirect sandwich ELLA developed in this study. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Sample** & \(n\) & **Chitosan (mg/g, Mean \(\pm\) SD)** \\ \hline Insect processed products & 11 & 9.69 \(\pm\) 2.15\({}^{\text{a}}\) \\ Insect products & 17 & 12.32 \(\pm\) 3.45\({}^{\text{b}}\) \\ Non-insect product & 11 & 4.26 \(\pm\) 1.5\({}^{\text{c}}\) \\ \hline \hline \end{tabular}  
Different letters indicate statistical differences (\(p\) < 0.05). \(n\): number of samples analyzed.  
\end{table} 
Table 5: Summarized chitosan content in commercial products. as a potential future trend to address global food needs, effective detection methods enable regulatory authorities to verify the presence or absence of insects, aligning with manufacturers' declarations. Given the allerggenic potential of insect proteins, which may provoke allergic reactions in some consumers [36], reliable detection is also essential for managing allergen risks and maintaining consumer confidence. Our developed ELLA method is focused on detecting insects in food without differentiating between permitted and non-permitted species, for which PCR methods are applicable [11; 12].