FTIR spectroscopy was used to analyse the amount of sericin on
the cocoons. Attenuated total reflectance allowed the selective
probing of the inner and outer surfaces of the cocoon. To assign
the sericin and fibroin bands, spectra of pure sericin powder and
converted fibroin were acquired.
The fibroin and sericin spectra have very distinctive infrared
signatures (Fig. 3). The main differences are due to the presence
of b-sheets peaks present in the converted fibroin at 1700, 1617,
997, and 975 cm1 [10–15]. In contrast to sericin, fibroin also
shows the asymmetric bending mode at 441 cm1 and the symmetric
bending mode of CH3 at 1334 cm1 [10,16]. The
1160 cm1 band assigned to the N–Ca stretching is absent in the fibroin
mode [10]. On the other hand, sericin has distinctive bands at
1394 cm1 and 1041 cm1 due to C–H and C–C bending vibrations
respectively [17]. Since not all the frequencies vary independently,
a principal component analysis has been performed on the dataset
(n = 30) to extract the principal components explaining the main
differences between the samples and remove the co-linearity
[18]. As shown in Fig. 3, the first principal component has negative
peaks at frequencies representative of sericine and positive bands
at the wavenumber of fibroin modes. The relative amount of both
proteins can be linked to the score of the first principal component
representing 50% of the spectra variance.
In Fig. 4, the first principal component scores from the inner and
outer surfaces of cocoons are completely resolved. The unidimensional
test of equality of the means of the classes proves that the
two sides of the cocoon have significantly different infrared spectra
and chemical composition (p < 0.0001). Since the peaks of the first
principal component match those observed for the pure proteins,
we conclude that the inner surface has significantly less sericin
than the outermost surface.