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S-Alk(en)yl cysteine sulfoxides have been observed in several plants, mainly belonging to the onion family (Alliaceae), which are of high commercial interest (e.g. garlic, Allium sativum). The quality of most garlic containing herbal remedies produced from garlic powder is determined by their content of the cysteine sulfoxide alliin. Therefore, a comprehensive method for the documentation of alliin amounts present in the fresh plant material through to the final remedy is desirable. The newly developed biosensoric method described in this paper was designed in order to fulfil these demands. In contrast to conventional HPLC-methods, neither a pre-column derivatization nor a chromatographic separation are required allowing a high throughput of samples. This technique is based on immobilized alliinase (EC 4.4.1.4), which was combined with an ammonia-gas electrode. The enzyme was either placed in a small cartridge or was immobilized in direct contact of the electrode surface giving detection limits of 3.7 x 10(-7) and 5.9 x 10(-6) M. Founded on these experiments, a pH-sensitive electrolyte/insulator/semiconductor (EIS) layer structure made of Al/p-Si/SiO(2)/Si(3)N(4) was also combined with immobilized alliinase. Measurements could be performed in a range between 1 x 10(-5) and 1 x 10(-3) M alliin. All sensors were operated in the flow-through modus. A high specificity for alliin could be demonstrated for the electrode and a number of garlic samples were analyzed. Results gained with the new method showed a good correlation with those obtained with conventional HPLC-methods. In addition, onion and a variety of wild Allium species were analyzed in order to determine the amount of isoalliin or total cysteine sulfoxides present, respectively.

Various Allium hybrids, obtained by the crossbreeding of Allium cepa (onion) as the mother plant and six taxonomically distant wild species obtained by embryo rescue, were investigated with special respect to their individual profiles of cysteine sulfoxides as well as enzymically and nonenzymically formed aroma substances. Alliinase (EC 4.4.1.4) catalyzes the conversion of odorless (+)-S-alk(en)yl-L-cysteine sulfoxides into volatile thiosulfinates. These thiosulfinates were converted to a variety of sulfides by steam distillation. SPME-gas chromatography (GC) and high-performance liquid chromatography (HPLC) used for the analysis of aroma components and their precursors permitted a high sample throughput, so that numerous gene bank accessions and Allium breeding materials were analyzed within a comparatively short time. Cysteine sulfoxides as well as alliinase activity were found in all investigated samples at different levels, but (+)-S-methyl-L-cysteine sulfoxide (methiin) was the most abundant sulfoxide present. (+)-S-(trans-1-Propenyl)-L-cysteine sulfoxide (isoalliin) is typical for onion and was found in all investigated hybrids. The pattern of the other cysteine sulfoxides depended strongly on the parent plants used. The profile of aroma components corresponded with the related pattern of aroma precursors (cysteine sulfoxides). Successful hybridization was proven by randomly amplified polymorphic DNA analysis. Together with these established marker techniques, HPLC and SPME-GC analysis provide support to breeding projects designed to improve the health and aroma properties of Allium hybrids.

Membranes and powders prepared from PTFE (polytetrafluorethylene) were investigated for their potential use as multifunctional supports for enzymes. The obtained bioactive materials are valuable for the construction of biosensors and enzyme reactors. To allow covalent coupling of enzymes to PTFE, the surface of the material was treated with elementary sodium followed by oxidation with ozone or hydrogen peroxide.%Derivatization steps were optimized in order to achieve highest enzyme loading and short reaction times. Alliinase (EC 4.4.1.4) and L-lactic dehydrogenase (EC 1.1.1.27) were chosen as model enzymes and were either immobilized by covalent coupling or fixed indirectly by a sugar-lectin binding. For the latter method, the sugar mannan was bound to the membrane surface as an anchor for layers of the lectin concanavalin A and the alliinase. Highest alliinase loading was achieved at 0.2 microg x cm(-2). Immobilization of alliinase via the lectin concanavalin A and a bifunctional epoxide gave the best long-term stability.%L-Lactic dehydrogenase was most sufficiently immobilized by using benzoquinone as spacer. These procedures show several advantages: 1) enzymes can be immobilized under physiological conditions, 2) an enzyme-multilayer can be achieved, and 3) protein layers are renewable., (Copyright 2001 John Wiley & Sons, Inc.)

Membrane filters prepared from porous aluminum oxide (Anopore) were investigated for their potential use as a durable support for enzymes. Alliinase (EC 4.4.1.4) was chosen as a model enzyme for immobilization experiments. To allow for smooth fixation, the enzyme was immobilized indirectly by sugar-lectin binding. Monomolecular layers of the lectin concanavalin A and alliinase were applied by self-assembling processes. As an anchor for these layers, the sugar, mannan, was covalently coupled to the membrane surface. This procedure exhibits several advantages: (i) enzyme immobilization can be carried out under smooth conditions; (ii) immobilization needs little time; and (iii) protein layers may be renewed., (Copyright 2000 John Wiley & Sons, Inc.)

In recent years, numerous clinical trials were undertaken in order to elucidate the active principle of garlic (Allium sativum L., Alliaceae). The most prominent effect of garlic preparations is a contribution to the prevention of stroke and arteriosclerosis. Allicin[(2-propenyl)-2-propenethiosulfinate] and other sulfur containing compounds were suggested as active compounds. The extremely unstable allicin itself is liberated from the more stable alliin [S-(+)-2-propenyl-L-cysteine sulfoxide] by the enzyme alliinase (EC 4.4.1.4) if fresh garlic is crunched or garlic powder is moistened. Therefore, an active enzyme is required in alliin containing remedies like those prepared from garlic powder. In order to investigate enzyme stability, alliinase was isolated from garlic powder. The partially purified enzyme could be stabilized over several months by addition of sodium chloride, sucrose, and pyridoxal-5'-phosphate. Alliinase may also be freeze-dried. This allows combinations of synthetic alliin and purified alliinase as components of an acid resistant tablet or capsule. In the intestine, the pro-drug alliin would be enzymatically converted to allicin. In clinical trials, highly dosed preparations of this kind should yield a precise information about the physiological effects of allicin. In addition, alliin-homologues substances which bear a modified alkyl side chain and do not occur in nature may be tested.

Alliinase (EC 4.4.1.4) has been isolated from commercially available garlic (Allium sativum L., Alliaceae) powder and was investigated with respect to its use as ingredient of herbal remedies. The enzyme was purified to apparent homogeneity and results were compared with those obtained from a sample of fresh A. sativum var. pekinense. The purification of the enzyme involved a gel filtration step as well as affinity chromatography on concanavalin-A agarose. Vmax using L-(+)-alliin as substrate (252 mumol min-1 mg-1) was at the lower range of data given in the literature (214-390 mumol min-1 mg-1). L-(-)-Alliin was also accepted as substrate (54 mumol min-1 mg-1). Vmax for alliinase from A. sativum var. pekinense was at 332 mumol min-1 mg-1 and 90 mumol min-1 mg-1 for L-(+)- and L-(-)-alliin, respectively. The Km values for alliinase from garlic powder were estimated to be 1.6 mM for L-(+)-alliin and 2.8 mM for L-(-)-alliin. In contrast to literature values, both temperature and pH optima were somewhat higher (36 degrees C and pH 7.0 versus 33 degrees C and pH 6.5, respectively). The enzyme was found to be active in a range from pH 5 to pH 10. Gel electrophoresis gave evidence that the alliinase obtained from garlic powder consisted of two slightly different subunits with molecular weights of 53 and 54 kDa whereas alliinase obtained from fresh garlic consists of two identical subunits. It is assumed that the alliinase gets significantly altered during the drying process of garlic powder but is still capable to convert alliin to allicin.