## 3 Results
### Raman Spectroscopy Analyses  
The investigation of the biochemical and structural changes present in the spores from bacilli after the thermal treatment was carried out using Raman spectroscopy. Based on the work of [PERSON] et al. (2020), it was possible to assign the peaks (Table 2).  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Raman Band/Peak (cm\({}^{-1}\))** & **Tentative Assignment of Functional Group** & **References** \\ \hline
622 & Proteins & [29] \\
661 & CaDPA & [30] \\
725 & DNA & [31] \\
780 & DNA & [31] \\
824 & CaDPA & [31] \\
864 & C-C stretch & [29] \\
1004 & Phenilanine & [32] \\
1017 & CaDPA & [29] \\ \hline \hline \end{tabular}
\end{table}
Table 2: Raman bands and the tentative assignment of their functional groups.  
Figure 4: Resistance to wet heat (98 \({}^{\circ}\)C) of spores from _B. horneckiae_ SBP3, _B. horneckiae_ DSM 23495 and _B. subtilis_: (**A**) survival curves of spores and (**B**) bar graph displaying the LD90 values (i.e., time to reduce spore survival to 10% of the initial spore culture).  
The Raman spectra of the untreated spores and their functional groups are reported in Figure 5. As demonstrated by the FTIR analysis, the major differences among the spectra of the untreated spores were detected in the regions assigned to saccharide band/carbohydrates (1060-1070 cm\({}^{-1}\)), to proteins and polypeptides (1645-1674 cm\({}^{-1}\)) and to CH stretching of lipids (1700-1736 cm\({}^{-1}\)).  
After treatment, changes in the Raman spectra were observed in the region of 800-900 cm\({}^{-1}\) attributed to DPA and nucleic acids, with the highest variation detected in BHO and the lowest in SBP3 (Figure 5). Compared to untreated spores, the spectrum of treated BHO spores showed evident peak shifts in the regions that referred to amide I (1660-1674 cm\({}^{-1}\)) and lipids (1700-1736 cm\({}^{-1}\)). Unlike untreated SBP3 spores, the spectrum of heated spores showed higher variations in the amide I region, while moderate peak variations were detected in the region attributed to lipids (Figure 5).  
\begin{table}
\begin{tabular}{c c c} \hline
**Raman Band/Peak (cm\({}^{-1}\))** & **Tentative Assignment of Functional Group** & **References** \\ \hline
1060–1070 & Glucose-saccharide band & [33] \\
1115 & Proteins & [29] \\
1200–1244 & Proteins, amide III & [29] \\
1336 & DNA & [31] \\
1395 & CaDPA & [30] \\
1448 & Proteins & [32] \\
1485 & DNA & [31] \\
1555 & CaDPA & [31] \\
1582 & CaDPA & [31] \\
1624 & Proteins & [34] \\
1645–1674 & Proteins, amide I, lipids & [32] \\
1700–1736 & Lipids & [35] \\ \hline \end{tabular}
\end{table}
Table 2: _Cont._  
Figure 5: Raman spectra of untreated and wet-heat-treated spores from _Bacillus horneckiae_ SBP3, _Bacillus horneckiae_ DSM 23495T (BHO) and _Bacillus subtilis_ 168 (BSU).  
To analyze the differences among the Raman spectra, a principal component analysis (PCA) was performed, and Figure 6 reports the data.  
Based on the major peaks in the Raman spectra of the spores, the PCA was performed to distinguish the spores. The three main axes explained 94.02% of the total variance. The PC1 axis, which explained 63.7% of the variance, was strongly associated with peaks related to amide I and to nucleic acids and DPA on the positive side and with nucleic acids on the negative side. The PC2 axis (16.2% of the variance) was mainly associated with lipids on the positive side and with carbohydrates on the negative side, whereas the PC3 axis (14.1% of the variance) was mainly associated with amide I and carbohydrates on the positive side and lipids on the negative side.