Investigation On New Process For Gelatin Extraction And Method Development For Tracing The Species Origin Of Gelatin

Gelatin is a mixture of peptides obtained by partial hydrolysis of collagens, and has been widely used in food, cosmetics, medicine and other industry for its unique physical-chemical properties. The traditional method of gelatins extraction, such as alkaline or acid based process, shows many disadvantages such as the low controllability of extraction process and wide molecular weight range of gelatin. Gelatins from different animal sources generally show large similarity in chemical properties, which makes it difficult to differentiate their animal source. The purpose of this research is to establish a new process for gelatin extraction and investigate the possibility for gelatin differentiation based on marker peptides detection using mass spectrometric method.First, the release process of collagen from porcine skin is investigated in batch operation. The results indicate that the extraction temperature and extraction time are key factors affecting the collagen degradation. And the influence of time and temperature is less in continues operation process. Then, the possibility to extract gelatin using multistage counter-current solid-liquid extraction is investigated. The results indicate that the gelatin molecular weight range is controllable by set the retention time, the ratio of water and skin during multistage counter-current solid-liquid extraction. The optimal operational parameters are obtained. Compared to commercial products, the prepared gelatin shows narrower molecular weight range, and higher mean molecular weight.Alignment-based sequence analysis indicates that the amino acid sequences of collagens from different animals are not identical. The possibility to differentiate gelatins based on detection of amino acid variants containing peptide was investigated. The porcine and bovine gelatins were used as model gelatin, were digested by trypsin.The resulting peptides were analyzed by high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The marker peptides specific for bovine and porcine were successfully detected in the digested bovine and porcine gelatin, respectively. Thus gelatin differentiation by marker peptides detection is possible, and it was found the enzymatic degradation of gelatin, marker peptide identification and analytical condition of HPLC/MS were the key factors affecting marker peptides detection. Then, the method was successfully used to identify fish-skin derived gelatin, differentiate the porcine bone gelatin from porcine skin gelatin. Further investigation indicated that the method could be used for quality control of gelatin-containing medicine, such as Lujiaojiao and Guijiaojiao. This research demonstrated that detection of marker peptide using tandem mass spectrometry coupled with tryptic digesting is an effective method for gelatin differentiation.