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Brewing is a relatively simple process which many people can claim to understand, bearing in mind the huge numbers who have tried the process at home. But home brewing is different to commercial brewing in that you are given a malt extract to begin working with. In the industry, brewers generally malt their own cereal grains so that they can control the malting process. Malting is one of the key operations in brewing. During this process, the cereal grains are encouraged to germinate, which releases enzymes used by the seeds to convert the starch into sugars and the proteins into amino acids. These enzymes are required for brewing, so once the grains begin to sprout, they are heat-dried to suspend the enzyme activity. When brewing is due to begin, the brewer will reactivate the enzymes by soaking the malted barley in hot water, then the starch will be converted into sugar ready for conversion into alcohol by yeast. The proteins in the cereal grains are also affected by malting, many of them being glycated by the addition of sugar groups. Some of the modified proteins that are rich in proline residues can cause haze in beer while others are important for foam formation and stabilisation. Lipid transfer proteins (LTP) and protein Z, which originate from barley, are the principal proteins in beer and they constitute about 10% of the high-molecular-mass fraction in foam, the rest being largely composed of carbohydrates. The modified forms of barley LTP1, produced by malting, are said to contribute to foam formation whereas the unmodified forms are inactive. Glycated protein Z improves the stability of beer foams. Since these post-translationally modified proteins are central to brewing, scientists from that fine brewing country, the Czech Republic, have been looking at ways to identify them in malted barley. Josef Chmelik and Janette Bobalova from the Research Centre for the Study of Extract Compounds of Barley and Hop at the IAC had been using one-dimensional polyacrylamide gel electrophoresis (PAGE) with mass spectrometry but could not assign the protein modifications unambiguously. Apart from those problems, PAGE suffers from a lack of speed. In an industrial setting, it would be preferable if faster techniques could be substituted. The duo decided to investigate the suitability of a recently developed chromatographic approach based on the use of convective interaction media (CIM). This employs porous monolithic polymers as the stationary phase and is available in various types for ion exchange, reversed-phase, affinity, and hydrophobic interaction chromatography. A combination of the separation power and capacity of regular columns with the convective mass transport associated with membrane technology imparts much faster separations with high throughput. For the modified barley proteins, the researchers chose an anion exchange disk-shaped CIM column with diode array detection to separate the proteins in an aqueous extract of barley malt. With a salt gradient, the eluate was divided into three fractions, the first being a sharp peak at 0-1.0 min, the second and third being sections of a broader peak, at 1.0-1.6 and 1.6-3.4 min. All proteins were eluted within this time, confirming the speed of the process. The proteins in each fraction were identified by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS), which reveals the protein molecular masses. In fraction 1, a protein at 9991 Da corresponded to LTP1 with a 14-hydroxy-10,13-dioxo-7-heptadecenoic acid aspartate ester (CHDH) modification. Three further peaks belonged to LTP1 with CHDH plus one, two or three hexose modifications formed by the Maillard reactions of the sugars with the lysine residues in the protein. Fraction 2 contained a hexose-modified form of LTP2 and fraction 3 contained a C-terminal fragment of protein Z in unmodified and hexose-added forms, as well as LTP2 with 1-3 hexose residues. Further proteins were identified in all fractions from their molecular masses. The proteins were confirmed by in-solution digestion and, following SDS-PAGE, by in-gel digestion. This procedure also identified unmodified LTP1 in fraction 1 and intact unmodified protein in fractions 2 and 3. Of the proteins found, Chmelik and Bobalova propose that LTP1 would be a good marker for post-translational modifications during barley malting and could be used as a malting quality parameter. It can be tracked using CIM chromatography and is eluted on the first, sharp fraction with 1 minute. The CIM process is ideal for an industrial setting where results are required quickly in order to keep production going. The total analysis taking just 15 minutes compared with 2 days for 2D electrophoresis. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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