Isotopic comparison of gelatin extracted from bone powder with that from bone chunk and development of a framework for comparison of different extraction methods

Stable isotope analysis of collagen extracted from ancient bones has become a routine exercise. Collagen can be extracted from either bone powder or chunks, but little research has compared these two procedures in detail. In this study, yield, elemental concentration, and carbon and nitrogen stable isotope ratios of gelatin extracted from bone powder or chunks were compared, and a new framework to compare the quality of gelatin samples extracted from identical bone samples using different extraction methods was presented. Forty adult human skeletons from two sites in Japan dating from the Edo period (1603-1867 CE) were used. In the “chunk method,” bone chunks are demineralized, gelatinized, and filtered to obtain gelatin, while bone powder is used in the “powder method.” Using the chunk method, the gelatin yield (+5.29 ± 1.58%), %C (+0.62 ± 0.66%), and %N (+0.22 ± 0.22%) significantly increased, but the C/N ratio (−0.01 ± 0.03), δ13C (0.00 ± 0.09), and δ15N (−0.02 ± 0.21) did not significantly differ for 20 well-preserved bones. In 20 poorly preserved bones, the gelatin yield (+1.27 ± 1.33%), %N (+0.41 ± 0.49%), C/N ratio (−0.07 ± 0.07), and δ15N (−0.12 ± 0.37) significantly changed, but the %C (+0.37 ± 1.11%) and δ13C (−0.01 ± 0.09) were not significantly different using the chunk method. The observed significant differences have a sufficient statistical power (>0.8) with a significance level of 0.01 (except for the significant difference in δ15N of poorly preserved bones). The Fourier transform infrared spectra indicate that the extracted gelatin mostly consists of collagen without apparent contaminants. In general, in the context of palaeodietary studies, there are no actual differences in the δ13C and δ15N values obtained from the powder and chunk methods for gelatin from well preserved as well as poorly preserved bones.