The aim of this study was to define the simplest and least expensive protocol for total protein extraction for three different macroalgae of the genus Caulerpa (the invasive C. taxifolia and C. cylindracea and the autochthonous C. prolifera). Five multi-step protein extraction procedures, set up for other macroalgal species, were tested. For each of them, different pre-treatment and extraction conditions were simultaneously examined, according to a factorial design, considering the starting material, the solvent-to-biomass ratio, and the incubation temperature. Protein yield in the obtained extracts was estimated with the Bradford method. Further, polyacrylamide gel electrophoresis (SDS-PAGE) was used to resolve proteins, assessing their quality and integrity. Significant differences in protein yield were observed among the extraction protocols and the conditions tested, also in relation to the considered species. Profiles having an acceptable quality were obtained for C. prolifera and C. cylindracea, and from the obtained results, the best method to obtain high yield and quality protein extracts for the two above-mentioned species appears to require the use of a primary TCA/acetone extraction buffer followed by a lysis buffer with NaCl, KCl, urea, Triton, SDS and a protease inhibitor. The best results, in particular, were obtained starting from fresh pulped material with a buffer-to-biomass ratio of 10:1 and an incubation temperature of 4°C. For C. taxifolia, instead, none of the tested protocols produced satisfactory results and further studies will be required.

This study aims to determine the untargeted lipidome of cow milk during non-staphylococcal sub-clinical mastitis. Dairy cow mastitis severely impacts the dairy industry by reducing milk yield and quality and increasing culling rate. Among the pathogens that cause bovine sub-clinical mastitis, non-aureus staphylococci (NAS) have become the most frequently isolated bacteria from milk samples. The molecular mechanisms regulating the mammary gland inflammatory responses to NAS are unclear. Although the application of system biology approaches to mastitis has provided pivotal information by investigating the transcriptome, proteome, and metabolome, the milk lipidome remains undisclosed. We determined the lipidome of 17 dairy cows with NAS sub-clinical mastitis (SM) and we compared the results with the lipidome of the milk of 13 healthy cows. The study was carried out following a liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) approach. Sixteen subclasses of lipids were identified in both groups of animals. From 2,506 measured lipids, 631 were significantly changed more than 10 folds (FDR < 0.01 in milk from cows with mastitis as compared to healthy cows. Our results point out the significant influence of NAS on the milk lipidome, contribute to the understanding of inflammatory processes in the bovine udder, and highlight potential novel biomarkers for improving mastitis diagnosis. Part of this work was carried out in “OMICs”, an advanced mass spectrometry platform established by the Università degli Studi di Milano.