Development of artificial garlic cell with potent in vitro bactericidal effect

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22340%2F19%3A43919596" target="_blank" >RIV/60461373:22340/19:43919596 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Development of artificial garlic cell with potent in vitro bactericidal effect

Popis výsledku v původním jazyce

Garlic (Allium sativum) has been used for centuries for its flavour and health promoting properties such as antibacterial, antithrombotic, antioxidant and antidiabetic properties. Garlic is rich in sulphur – containing compounds, including the nonproteinogenic amino acid alliin (S-allyl-L-cysteine sulfoxide). Alliin is a precursor of allicin, which is a product of the enzymatic reaction of the substrate (alliin) and enzyme alliinase. Once the garlic plant is mechanically or microbial damaged, alliin and alliinase, contained in storage and vacuole cells respectively, come in contact with each other; alliin is converted directly into volatile alk(en)yl sulfinyl compounds, the most known is allicin, which exhibits antimicrobial properties. The enzyme stability is essential from a commercialization viewpoint; unfortunately, it also constitutes a long-standing biotechnological challenge. Pure alliinase is known to be thermolabile, irreversibly inactivated by denaturants, and rather unstable upon storage. One of the most attractive methods for improving the stability of pure enzymes is by incorporating of additives. 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.

Název v anglickém jazyce

Development of artificial garlic cell with potent in vitro bactericidal effect

Popis výsledku anglicky

Garlic (Allium sativum) has been used for centuries for its flavour and health promoting properties such as antibacterial, antithrombotic, antioxidant and antidiabetic properties. Garlic is rich in sulphur – containing compounds, including the nonproteinogenic amino acid alliin (S-allyl-L-cysteine sulfoxide). Alliin is a precursor of allicin, which is a product of the enzymatic reaction of the substrate (alliin) and enzyme alliinase. Once the garlic plant is mechanically or microbial damaged, alliin and alliinase, contained in storage and vacuole cells respectively, come in contact with each other; alliin is converted directly into volatile alk(en)yl sulfinyl compounds, the most known is allicin, which exhibits antimicrobial properties. The enzyme stability is essential from a commercialization viewpoint; unfortunately, it also constitutes a long-standing biotechnological challenge. Pure alliinase is known to be thermolabile, irreversibly inactivated by denaturants, and rather unstable upon storage. One of the most attractive methods for improving the stability of pure enzymes is by incorporating of additives. 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.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

20401 - Chemical engineering (plants, products)

Projekt

<a href="/cs/project/TJ01000313" target="_blank" >TJ01000313: Vývoj biomimetických částic pro antibakteriální aplikace</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2019

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů