To observe the effects of sodium hypochlorite on spore properties, LTRS was used to compare the Raman spectra of spores treated with sodium hypochlorite for 0, 5, 10, 15, and 20 min (Figure 2 (b) ). We found substantial changes in the Raman spectra of treated spores, with the most obvious change in the characteristic peak of CaDPA. Taking 1017 cm^-1 as an example, its relative intensity decreased dramatically as the treatment timeincreased (Figure 2(c) ). The percentages of spores releasing CaDPA increased rapidly with an increase in treatment time (Figure 3 ). These two results indicate that sodium hypochlorite treatment caused the spores to release CaDPA and had greater effects on spores over time. It is worth noting that CaDPA is located in the core of spores, and the inner membrane controls the entry and exit of materials from the core. Therefore, the release of CaDPA from spores after sodium hypochlorite treatment is sufficient evidence that sodium hypochlorite disrupts the permeability barrier of the spores, including the inner membrane [14, 23]. The mechanism by which sodium hypochlorite promotes the release of CaDPA from the spores is not fully understood; however, Raman spectroscopy and phase contrast micrography results suggest that CaDPA is released from spores after treatment and that the number of individuals releasing CaDPA increases with the duration of the treatment are in agreement with the results of a previous report [14]. By comparing the Raman peaks of DNA at 782 cm^-1, we detected a slight shift after sodium hypochlorite treatment, although the relative Raman intensity was largely unchanged (Figure 2(d) ). These results indicated that sodium hypochlorite may damage the DNA of spores, without affecting the DNA content. Among the 30 spectra for single spores measured in each group at 0, 5, 10, 15, and 20min, the spectra of 0, 0, 2, 6, and 15 spores had shifted peaks, respectively, indicating that DNA denaturation occurred and intensified as the treatment time increase [14]. It can be speculated that sodium hypochlorite entered the spores after destroying the permeability barrier, resulting in damage to the DNA but not the loss of DNA. It is

Figure 2 (a) Survival of spores after sodium hypochlorite treatment obtained using plate culture and live cell imaging; (b) average Raman spectra of 30 Bacillus subtilis spores after different times of sodium hypochlorite treatment; (c) average Raman spectra in the CaDPA bands and the number of spores releasing CaDPA at different time; and (d) average Raman spectra in the DNA band and the offset at 782 cm^-1. a.u., arbitrary units.

worth noting that the 1004 cm^-1 attributed to phenylalanine decreased after treatment, because sodium hypochlorite reacts with amino acids in a neutralizing manner, damaging spore proteins [24].