(175m) Development of Artificial Garlic Cell with Potent in Vitro Bactericidal Effect

Over time, humans learned to use numerous plants growing in their vicinity for their healing properties in different forms (e.g. powders, extracts, wound dressings). The World Health Organisation estimates that herbal extracts or their active constituents are used as folk medicine in traditional therapies among 80 % of the world´s population. Therefore, such plants deserve scientific attention for their various healing properties and possible applications. The well-known spice and remedy plant garlic (Allium sativum) belongs to those group of plants. The pharmacological effects (such as antibacterial, antiviral and antifungal) are mainly linked to sulphur-containing components (especially allicin). Allicin is produced by the action of a single enzyme (S-alk(en)yl-L-cysteine sulphoxide lyase - also known as alliinase) with natural substrate (+)-S-allyl-L-cysteine sulphoxide (alliin). The enzyme and the substrate are localised separately in garlic cells and come into contact only when a cell is damaged. Such compartmentalised architecture of alliinase/alliin system is an example of a capable defence system.

This study aims to extract, stabilise alliinase, fabricate antimicrobial microparticles containing alliinase and alliin separately to mimic the unique mechanism of allicin biosynthesis in the garlic cell and to determinate the antimicrobial activities of in situ generated allicin. The extraction of the enzyme alliinase from garlic cloves was performed by fractional precipitation, and its purity was determined by the SDS-PAGE (higher than 95%). The specific activity of the alliinase was assayed via coupled NADH (β‑Nicotinamide adenine dinucleotide) - dependent reduction of the pyruvate (released as a co-product of enzymatic reaction) in the presence of enzyme lactate dehydrogenase. The UV-VIS spectrometry measured the decrease of the NADH. The optimal temperature and pH for the reaction were experimentally determined to be 37°C and 7, respectively. Additionally, the influence of additives (i.e. salts, antioxidants) on enzymatic activity was tested. The preparation of particles was performed by encapsulation of the bioactive components using various types of encapsulation techniques. The encapsulation of both alliin and alliinase was optimized in terms of process parameters to ensure the highest enzymatic activity. For testing of antimicrobial activity against bacteria E. coli, disc diffusion method was used. The stabilisation of enzyme in the polymer matrix with additives supporting enzymatic activity is essential for the development of artificial garlic cell.