Določevanje antioksidativnih učinkov izbranih živil v mikropretočnem sistemu z imobiliziranimi sporami Bacillus subtilis

Belej, Jure (author), Žnidaršič Plazl, Polona (mentor)

Mikropretočni sistemi omogočajo hitre in ponovljive reakcije, učinkovitost, ekonomičnost in nadzor nad kemijskimi procesi. Cilj magistrske naloge je bil razviti oz. sestaviti mikropretočni sistem, ki deluje kot pretočni biosenzor za analizo antioksidativne kapacitete izbranih živil. Sistem je zasnovan na mikroreaktorju z imobiliziranimi sporami Bacillus subtilis z magnetnimi mikrodelci, ki so v reaktorju zadržani na osnovi magnetnega polja. Test je osnovan na metodi ekvivalentov Troloxa za antioksidativno kapaciteto (ang. Trolox equivalent antioxidant capacity method, TEAC). Metoda temelji na redukciji radikala ABTS*+ ob interakcijah z antioksidanti, prisotnih v suspenzijah živil. Pridobivanje obarvanega radikala ABTS*+ temelji na oksidaciji brezbarvnega ABTS s CotA proteinom v plašču Bacillus subtilis, ki izkazuje lakazno aktivnost. V preliminarnih študijah smo opredelili vpliv temperature gojenja in načina odmrzovanja spor na njihovo lakazno aktivnost, ter vpliv pH in razmerja med magnetnimi mikrodelci in sporami na pretvorbo ABTS v radikal ABTS*+. Nastajanje radikala na izstopu iz mikropretočnega sistema smo merili s pretočnim UV/VIS miniaturiziranim sprektrofotometrom (ODcube). Za določanje antioksidativne kapacitete smo pred ODcube skozi injektor v sistem mikrokanalov injicirali vzorec živila, ki je reagiral z radikalom ABTS*+ pridobljenem v mikroreaktorju. Prisotnost antioksidantov v hrani je povzročila znižanje koncentracije radikala, kar smo spremljali z ODcube. Vrednost antioksidativne kapacitete smo izračunali glede na padec koncentracije v primerjavi z začetno vrednostjo koncentracije radikala. Z razvitim mikropretočnim sistemom smo tako analizirali vzorce različnih sokov in zelenih čajev in dobljene vrednosti primerjali s standardom za določanje antioksidativne kapacitete Trolox. Mikropretočni sistem je bil v primerjavi s šaržnim veliko bolj učinkovit in ekonomičen, saj je omogočal stabilno večdnevno delovanje z možnostjo analize velikega števila vzorcev v kratkem časovnem intervalu in brez potrebe po pripravi novega biokatalizatorja.

• Language: Slovenian
• Keywords: miniaturiziran biosenzor, Bacillus subtilis, antioksidativna kapacitete živil, magnetni mikrodelci
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2024
• PID: 20.500.12556/RUL-164887
• Publication date in RUL: 15.11.2024

Secondary language

• Language: English
• Title: Evaluation of antioxidant capacity of selected food using microflow system with immobilized Bacillus subtilis spores

Abstract

Microfluidic systems enable fast and reproducible reactions, efficiency, cost-effectiveness, and control over chemical processes. The aim of the master’s thesis was to develop/assemble a microfluidic system that functions as a flow biosensor for analyzing the antioxidant capacity of selected foods. The system is based on a microreactor with immobilized Bacillus subtilis spores on magnetic microparticles, which are retained in the reactor by a magnetic field. The test is based on the Trolox Equivalent Antioxidant Capacity (TEAC) method. The method relies on the reduction of the ABTS*+ radical during interactions with antioxidants present in food suspensions. The formation of the colored ABTS*+ radical is based on the oxidation of the colorless ABTS by the CotA protein in the Bacillus subtilis spore coat, which exhibits laccase activity. In preliminary studies, we determined the effect of growth temperature and thawing method of the spores on their laccase activity, as well as the effect of pH and the ratio between magnetic microparticles and spores on the conversion of ABTS into the ABTS*+ radical. The formation of the radical at the outlet of the microfluidic system was measured using a flow UV/VIS miniaturized spectrophotometer (ODcube). To determine the antioxidant capacity, a food sample was injected into the system’s microchannels through an injector prior to the ODcube, where it reacted with the ABTS*+ radical formed in the microreactor. The presence of antioxidants in the food caused a decrease in the radical concentration, which we monitored with the ODcube. The antioxidant capacity was calculated based on the decrease in concentration compared to the initial radical concentration. With the developed microfluidic system, we analyzed samples of various juices and green teas, and compared the obtained values with the antioxidant capacity standard Trolox. Compared to batch processes, the microfluidic system was much more efficient and cost-effective, as it allowed stable operation for several days with the ability to analyze a large number of samples in a short time without the need for new biocatalyst preparation.

• Keywords: miniaturized biosensor, Bacillus subtilis, Antioxidant capacity, magnetic microparticles