Brewing with barley: Comparing protease activities with the resulting proteins and peptides in beer using activity-based protein profiling and LC-MS/MS

Technical Session 02: Analytical I Session
Lone Baekgaard, R&D, Novozymes A/S, Bagsvaerd, Denmark
Co-author(s): Renier van der Hoorn and Joji Villamor, Plant Chemetics Lab, Max Planck Institute for Plant Breeding Research, Cologne, Germany; Christian Jørgensen, Carsten Sönksen, and Niels Elvig, R&D, Novozymes A/S, Bagsvaerd, Denmark; Stefan Kreisz, Carlsberg Research Laboratory, Copenhagen V., Denmark; Hans-Peter Heldt Hansen, R&D, Novozymes A/S, Bagsvaerd, Denmark

ABSTRACT: Today it is possible to brew beer with 100% unmalted barley using the enzyme product Ondea Pro. Although the barley beer is very similar to a malt beer in many aspects, differences could be observed when we analyzed the beers at the proteomic level. The overall content of proteins and peptides was higher in barley beer compared to malt beer. Using LC-MS/MS we have shown that the peptides mainly originated from the proline rich hordeins. This was in line with previous results, where we have seen a lower concentration of the less fermentable free amino acids, especially proline, in the barley wort. These results show that the proteolytic activities are different when you brew a barley beer with Ondea Pro compared with a pure malt beer. Ondea Pro contains a protease that works in synergy with endogenous proteases in barley (S. Aastrup [2010], Scand. Brew. Rev. 67:28-33.). Thus, to improve our understanding of protease activities in barley compared to malt, we analyzed barley extracts (with and without the Ondea Pro protease) and malt extract using technique activity-based protein profiling (ABPP). This is a new and powerful technique to be applied in brewing related research. The technique employs specific probes for different classes of proteases that bind irreversibly to the active site of the proteases, but only when the proteases are active and, thus, it is not dependent on substrates as many other protease assays are (I. Kolodziejek et al. [2010], Curr. Opin. Biotechnol. 21:225-233). Four different probes were tested under different brewing relevant pH and temperature conditions for papain-like cysteine proteases (PLCPs), serine hydrolases (including serine proteases such as carboxy peptidases), proteasome (threonine proteases), and vacuolar processing enzymes (cysteine proteases). Clear differences in protease activities were observed between malt and barley under the different conditions, which likely play a role during the brewing process. For example, PLCPs were very dominant in malt, whereas no activity was seen in barley. These results support previous findings, where it has been shown that PLCPs such as EP-A and EP-B are produced during germination (S. M. Koehler et al. [1990], Plant Cell 2:769-783). However, when the Ondea Pro protease was added to the barley extract, some PLCP activity could be seen. This shows that the Ondea Pro protease surprisingly is able to activate endogenous proteases in barley and in this way work in synergy with the barley proteases.

Lone Baekgaard has a Ph.D. degree in plant physiology from Copenhagen University (2005). From 2005 to 2009, she had a post-doc position at Copenhagen University, where she worked with biochemical characterization of plant enzymes. Since April 2009, she has been working as a research scientist in the Department of Brewing and Alcoholic Beverages, R&D, Novozymes A/S, focusing on protein chemistry within brewing.