Donkey-hide gelatin, also called Ejiao (colla corii asini), is commonly used as a food health supplement and valuable Chinese medicine. Its growing popular demand and short supply make it a target for fraud, and many other animal gelatins can be found as adulterants. Authentication remains a quality concern. Peptide markers were developed by searching the protein database. However, donkeys and horses share the same database, and there is no specific marker for donkeys. Here, solutions are sought following a database-independent strategy. The peptide profiles of authentic samples of different animal gelatins were compared using LC-QTOF-MS/MS. Fourteen specific markers, including four donkey-specific, one horse-specific, three cattle-specific, and six pig-specific peptides, were successfully found. As these donkey-specific peptides are not included in the current proteomics database, their sequences were determined by de novo sequencing. A quantitative LC-QQQ multiple reaction monitoring (MRM) method was further developed to achieve highly sensitive and selective analysis. The specificity and applicability of these markers were confirmed by testing multiple authentic samples and 110 batches of commercial Ejiao products, 57 of which were found to be unqualified. These results suggest that these markers are specific and accurate for authentication purposes.

Type I collagen hydrolysate with haematopoietic activity has shown tremendous potential for application in the functional food fields. The identification of peptide with haematopoietic activity to ensure the efficiency of functional foods remains challenging because of the vast combinatorial potential of short peptides from collagen hydrolysates. In the present study, we identified a novel marker peptide (the I-1 peptide, GAAGLpGPK, p: hydroxyproline) for type I collagen with haematopoietic capabilities via theoretical sequence analysis and computational prediction of activity. The I-1 peptide was shown to increase the survival rate of haematopoietic stem cells following injury induced by 5-fluorouracil (5-FU), increasing the survival rate from 65.40 to 105.28%, thereby exhibiting significant haematopoietic activity. More importantly, the mechanism underlying the haematopoietic activity of the I-1 peptide was analogous to activate the JAK/STAT signalling pathway mediated by granulocyte‒macrophage colony‒stimulating factor. On this basis, a high-precision method for the detection of type I collagen hydrolysates was developed using the I-1 peptide (relative standard deviation = 1.57%). This study not only provides novel perspectives for the exploration of haematopoietic peptides but also offers a new methodology for controlling the bioactivity of collagen-based food products. Graphical Abstract