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Role of ns-LTP1 in the Development of Primary Gushing
Hecht, D., Hippeli, S.

Our investigations were focussed on ns-LTP1 (non specific lipid transfer protein 1) as the main inductor of the primary gushing phenomenon. We asserted that gushing beer contains more ns-LTP1 than non-gushing beer and these overbalance is responsible for over foaming. Surprisingly, less to non ns-LTP1 was detectable in a set of gushing beers. We showed that loss of ns-LTP1 depends on fungal infestation and heating procedure. ns-LTP1 degrading activity of heated culture filtrates of Fusarium culmorum and Fusarium graminearum support the assumption that heat stable extracellular proteinases secreted by the two Fusaria strains are responsible for protein degradation. Heating procedure also results in a destruction of naturally occurring proteinase inhibitors in wheat kernels. This is the condition precedent to an effective operation of the fungal proteases. Furthermore our results support the conception that not ns-LTP1 itself, but rather glycated peptides generated during proteolytic fragmentation of modified ns-LTP1 species initiate gushing activity. Gushing is a phenomenon of vigorous effervescence of beer. Upon the opening of a bottle a great number of fine bubbles are being formed spontaneously throughout the volume of beer, which ascend quickly under foam formation, and shoot out of the bottle. This infuriating gushing phenomenon has been and is still a problem of world-wide importance to the brewing industry. The research group of Carlsberg [1, 2] divide gushing into two types: "primary gushing" which occurs periodically and appears to be related to the quality of malt, and "secondary gushing" which is due to faults during beer production or to the incorrect treatments of packaged beer. In this paper primary gushing is discussed solely.Prentice and Sloey [3] and Sloey and Prentice [4] clearly showed that especially the Fusaria cause changes in the analytical values of malt samples, such as increase in α-amylase activity, soluble nitrogen, extract and gushing. Gjertsen et al. [5] confirmed in experimental malting tests that inoculation of several barley grains with Fusarium cultures during steeping could yield gushing malts. The addition to the mash of mycelial extracts or culture filtrate of Fusarium failed to show gushing in beer. The authors concluded that gushing is not caused by the Fusarium itself but as a result of an interaction between Fusarium and the germinating grain. Several research groups tried to isolate and characterise substances from culture filtrates of different moulds, but also from malt, mash, wort and gushing beer, which could induce malt-derived gushing [6-10]. But until now, the exact chemical nature and the physical and chemical mechanisms of these substances are unknown.In 2002 we published new hypotheses on the chemical nature of gushing inducing factors [11]. Beside the fungal hydrophobines the group of non specific lipid transfer proteins (ns-LTPs) synthesised in grains as a response to fungal infection and their modification during the brewing process, was postulated to be responsible for malt-derived gushing. ns-LTPs are ubiquitous lipid binding proteins in plants with a localization in extracellular layers, i. e. cell walls or cutin. Furthermore, ns-LTPs are abundant soluble proteins of the aleurone layers from grain endosperms. They have been comprehensively reviewed by Yamada [12] or Kader [13]. Two main groups of ns-LTPs, ns-LTP1 and ns-LTP2 have been identified with molecular masses of about 10 and 7 kDa, respectively. ns-LTP1 is water soluble, heat stable and surface-active [14]. The isoelectric point of ns-LTP ranges between 8.8 and 10 depending on the plant source. The alkaline nature of ns-LTP1 is in contrast to the acidic nature of the Japanese wort component [6, 9] and the gushing inducing substances from Weideneder [10]. S?rensen and co-workers [15] reported that barley ns-LTP1 is involved in beer foam formation. ns-LTP1 purified from beer created a good beer foam with high potential in a foam assay. In contrast ns-LTP1 purified from barley displayed only poor foaming properties. The authors concluded that ns-LTP1 is modified during the malting and brewing processes, whereas the modified ns-LTP1 species is the most important one for the beer foam potential. J?gou and co-workers [16] identified these modified ns-LTP1 species as sugar-adducts generated via Maillard-reaction. The Maillard-reaction is a well known non enzymatic reaction which can form covalent linkages between reducing sugars and free amino groups of proteins and is preferred by high-temperature treatments during the brewing processes. Available amino groups are the ε-NH2 of the side chain of lysine residues and the guanidino amino group of arginine residues. This is a very important point, because lysine as well as arginine are basic amino acids which are together with the histidine residues responsible for the basic nature of ns-LTP1. It was shown that under oxidative conditions histidine could convert into aspartic acid and proline into glutamic acid [17].

Descriptors: gushing, lipid tansfer protein 1, wheat, ELISA, Fusaria

BrewingScience Monatsschrift fr Brauwissenschaft, 60 (November/December 2007), pp. 1-9