The identification of a barley haze active protein that influences beer haze stability: Cloning and characterisation of the barley SE protein as a barley trypsin inhibitor of the chloroform/methanol type

Previous pilot brewing trials have demonstrated that in the absence of a molecular weight (MW) ∼12,000 (barley silica eluate (SE) protein), the beer brewed from the malt of these SE −ve varieties formed less haze after accelerated ageing than beers brewed using SE +ve malt varieties. The previously described SE protein was characterised using comparative two-dimensional (2-D) gel electrophoresis immunoblots of barley seed extracts from both SE +ve and SE −ve varieties. The SE protein spot identified was excised and its partial sequence determined, after in-gel cleavage using trypsin and separation of the resulting fragments by reversed-phase high performance liquid chromatography (HPLC). N-terminal sequence analysis of the tryptic peptides from SE +ve and SE −ve varieties identified the SE protein as the barley trypsin inhibitor-CMe precursor (BTI-CMe). The mature BTI-CMe protein is 13.3 kDa and its functional gene is located on chromosome 3H. Cloning of the BTI-CMe protein demonstrated that both SE −ve and SE +ve barley varieties contain a BTI-CMe protein family member that is similar but has a consistently different sequence, primarily in the last 30 amino acid residues of their C-termini.