Identification of rennet-whey solids in “traditional butter” by means of HPLC/ESI-MS of non-glycosylated caseinomacropeptide A

‘‘Traditional butter’’ (TB) is directly obtained from milk cream according to Reg. EC No. 2991/1994. Ascertainment of TB authenticity implies the development of an analytical method for detecting illegal addition of cream from rennet-whey cream (RWC) to milk cream. The reference HPLC method adopted for detecting the presence of rennet-whey solids in skim milk powder (EC Reg. No. 213/2001) is based on the determination of non-glycosylated caseinomacropeptide A (CMP A, i.e. k-CNA f 106–169). In this paper, the same method, coupled to ESI-MS, has been applied to the water phase of butter in order to detect CMP A deriving from usage of RWC for butter manufacturing. The reliability of this approach has been evaluated by studying the effect of both natural creaming and cream ripening in originating CMP A or peptides with CMP-like chromatographic behaviour. Results demonstrated that peptides other than CMP A, and interfering in the HPLC profile, can form in cream after prolonged ripening with commercial starters of lactic acid bacteria. Furthermore, proteolysis caused by psychrotrophic bacteria was studied by inoculating milk cream with Pseudomonas fragi ATCC 4973. In this case, different peptides, including CMP A, are cleaved from k-casein when bacterial count is >106 cfu/g. For these reasons, only LC/MS can unequivocally show the presence of non-glycosylated CMP A and hence the usage of RWC in manufacturing of TB. In this regard, monitoring of multicharged ions at m/z 1697.5 and 2263.2 was adopted for recognition of monophosphorylated CMP A. Control of commercial samples by means of HPLC/ESI-MS revealed that usage of RWC is sometimes performed in countries where butter consumption is not widespread and a large volume of rennet whey is available.