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 , 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. , 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. , 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.
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