## 5 Conclusions  
Fourier-transformed infrared spectroscopy has been used for over a decade to monitor the secondary structure of silk fibroin-based materials [43]. It was essential to examine this technique and improve the significance of its results.  
The oxidation estimator \(\mathrm{E_{AmidI/II}}\) appears to be the most stable and least affected by the degradation conditions, thus being the most age-correlated estimator. Its consistent increase indicates that peptide bond oxidation occurs from the initial hours of artificial aging and continues throughout long-term degradation under natural and artificial conditions. The oxidation rate is slightly accelerated with the introduction of water, but it is not affected by changes in crystallinity. Fibroin oxidases lead to the formation of ketones (including o-quinone), aldehydes, \(\alpha\)-keto-acids with carboxylic groups, and dicarboxylic groups along the amino acid chain.  
On the other hand, the hydrolysis estimator \(\mathrm{E_{COOH}}\) is more sensitive to the environment, and its trends change with the introduction of water, as expected. The presence of water accelerates the breaking of amino acid chains and leads to the formation of COOH groups. This hydrolysis strongly impacts the crystalline structure. Water entering in between pleats (as calculated by [PERSON], [PERSON] et al. [44]) plays a role in L to D amino acids transition in both parallel and antiparallel \(\beta\)-sheet domains [41; 42]. Still, it cannot be excluded that hydrolysis reaction in dry conditions may not be followed by crosslinking or further oxidation of -COOH groups (as the vibration of free dicarboxylic amino acids (COOH-NH-\(\ldots\)-COOH) weakens considerably with prolonged ex situ artificial aging).  
Crystallinity for samples with a dichroic ratio \(R\)-value close to zero exhibits high hydrolysis and oxidation product levels. Polarization of the infrared laser beam (\(\mathrm{E_{CPOL}}\)) enables the determination of lost antiparallel sheet crystalline structures, thereby defining the point at which the FTIR-derived information about crystallinity no longer reflects the sample's overall crystallinity level (\(R\) below zero). The loss of order in the amino acid structure produces similar products in both antiparallel and parallel pleated domains. This can be explained by the natural conversion of L-amino acids to their D form, altering the hydrogen bonding symmetry in the silk's crystalline regions [41; 44].  
Figure 10: Hydrolysis (\(\mathrm{E_{COOH}}\)), oxidation (\(\mathrm{E_{AmidI/II}}\)), crystallinity (\(\mathrm{E_{CFIR}}\)), and crystallinity corrected by polarizer (\(\mathrm{E_{CFIRpol}}\)) estimators calculated from ATR-FTIR for model silk sample (MS), in situ thermo-aged MS in the air (\(\mathrm{isO_{2}}\)), ex situ thermo-aged MS in the air (\(\mathrm{esO_{2}}\)), in the humid closed reactor (\(\mathrm{esO_{2}}\)/\(\mathrm{VOC/H_{2}O}\)) and historic: Inscription Banner (IB); Court Banner (CB); [PERSON]’s Funeral Banner (FB).  
**Author Contributions:** Conceptualization, J.P.-P. and M.A.K.; methodology, M.A.K.; software, M.A.K.; validation, M.A.K. and J.B.; formal analysis, M.A.K., K.G., M.M.Z.-O., D.P. and M.S.; investigation, M.A.K., J.B. and J.P.-P.; resources, M.A.K. and E.B.; data curation, M.A.K.; writing--original draft preparation, M.A.K.; writing--review and editing, J.B. and M.A.K.; visualisation, M.A.K.; supervision, J.P.-P. and E.B.; project administration, E.B.; funding acquisition, M.A.K. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the National Centre of Science (Narodowe Centrum Nauki), grant number NCN FUGA 2016/20/S/ST4/00149.  
Not applicable.  
Data Availability Statement: Data is available online: [[https://koperska.info/fuga/](https://koperska.info/fuga/)]([https://koperska.info/fuga/](https://koperska.info/fuga/)) (accessed on 29 June 2023).  
The authors thank the Wavel Castle Museum for their help and support.  
The authors declare no conflict of interest.