Milica Carević

Structural Elucidation of Enzymatically Synthesized Galacto-oligosaccharides Using Ion-Mobility Spectrometry–Tandem Mass Spectrometry

Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with β-galactosidases. Enzymatic transgalactosylation results in a mixture of compounds of various degrees of polymerization and types of linkages. Because structure plays an important role in terms of prebiotic activity, it is of crucial importance to provide an insight into the mechanism of transgalactosylation reaction and occurrence of different types of β-linkages during GOS synthesis. Our study proved that a novel one-step method, based on ion-mobility spectrometry-tandem mass spectrometry (IMS-MS/MS), enables complete elucidation of GOS structure. It has been shown that β-galactosidase from Aspergillus oryzae has the highest affinity toward formation of β-(1→3) or β-(1→6) linkages. Additionally, it was observed that the occurrence of different linkages varies during the reaction course, indicating that tailoring favorable GOS structures with improved prebiotic activity can be achieved by adequate control of enzymatic synthesis.

The influence of reaction media on CdIn2S4 and ZnIn2S4 nanocrystallite formation and growth of mesocrystal structures

A hot-injection method for the synthesis of CdIn2S4 in three different compositions of organic media/solvents was studied. Nanosized CdIn2S4 is successfully synthesized in an oleic acid/oleylamine mixture of complexing/capping agents. The obtained mesocrystals of 20–30 nm in diameter are self-organized in marigold-like structures. The estimated band-gap of synthesized semiconductor is in the visible spectral region and has a value of about 2.1 eV. The potential of the band edges is calculated using an empirical equation. The as-prepared material was successfully transferred from organic to aqueous media by using 2-mercaptoethanol in a surface ligand exchange process. Using a similar synthetic procedure, ZnIn2S4 synthesis was performed. The obtained materials were characterized using UV/vis spectroscopy, XRD and TEM. Formation and growth mechanisms of the synthesized materials are proposed.

Influence of fatty acid on lipase-catalyzed synthesis of ascorbyl esters and their free radical scavenging capacity

Fatty acid (FA) ascorbyl esters are recently emerging food, cosmetic, and pharmaceutical additives, which can be prepared in an eco‐friendly way by using lipases as catalysts. Because they are amphiphilic molecules, which possess high free radical scavenging capacity, they can be applied as liposoluble antioxidants as well as emulsifiers and biosurfactants. In this study, the influence of a wide range of acyl donors on ester yield in lipase‐catalyzed synthesis and ester antioxidant activity was examined. Among saturated acyl donors, higher yields and antioxidant activities of esters were achieved when short‐chain FAs were used. Oleic acid gave the highest yield overall and its ester exhibited a high antioxidant activity. Optimization of experimental factors showed that the highest conversion (60.5%) in acetone was achieved with 5 g L−1 of lipase, 50 mM of vitamin C, 10‐fold molar excess of oleic acid, and 0.7 mL L−1 of initial water. Obtained results showed that even short‐ and medium‐chain ascorbyl esters could be synthesized with high yields and retained (or even exceeded) free radical scavenging capacity of l‐ascorbic acid, indicating prospects of broadening their application in emulsions and liposomes.

Novel β-galactosidase nanobiocatalyst systems for application in the synthesis of bioactive galactosides

In this study, unmodified, amino modified and cyanuric chloride functionalized amino modified nonporous fumed nano-silica particles (FNS, AFNS and CCAFNS, respectively) were used for the development of efficient nanobiocatalysts for application in the biosynthesis of bioactive galactosides, galacto-oligosaccharides (GOS). Hence, in an attempt to elucidate the mechanism of immobilization, based on available enzyme conformation, the effects of immobilization parameters (initial enzyme concentration, immobilization time and pH) were analyzed. Among all three used nano-sized supports, the one with amino groups (AFNS) exhibited the best β-galactosidase binding capacity of 220 mg g−1 support with the efficiency of ∼90% at pH 4.5 and immobilization time 3 h. The highest hydrolytic activity of ∼2200 IU g−1 was achieved, which is far higher than previously reported. Meanwhile, β-galactosidases covalently immobilized on CCAFNS and adsorbed on FNS nanoparticles were found to have similar trends with respect to immobilization efficiency (58–71%) and hydrolytic activity (∼600 IU g−1). Furthermore, thermal stability at 60 °C was increased by β-galactosidases immobilized on AFNS and CCAFNS (4 and 1.4-fold, respectively) due to the protective effect of interactions formed between enzyme molecules and the surfaces of nanoparticles. Since β-galactosidase immobilized on AFNS was the nanobiocatalyst with the highest activity and stability, it was applied in GOS production. With AFNS–β-galactosidase GOS production of 90 g L−1 h−1 was achieved as compared to 30 g L−1 h−1 by free β-galactosidase, meaning that AFNS enhanced the selectivity of β-galactosidase for transgalactosylation, which is a crucial advantage for its application in GOS production.

Optimization of galacto-oligosacharides synthesis using response surface methodology

Galacto-oligosaccharides (GOS) are important lactose-derived compounds, considered to be a prebiotics, based on abundant scientific evidence about their unique physical properties and physiological effects. This consequently allows their widespread application as supplement in food and feed industry. They are preferably produced by the enzymatic transgalactosylation action of βgalactosidase. However, this enzyme simultaneously performs its primary biological function of lactose hydrolysis, and it is of crucial importance to gain an insight into the influence of different reaction conditions, and provide favorization of transgalactosylation, particularly GOS synthesis reaction. In this study, the response surface methodology (RSM) was applied in terms of individual experimental factors effect estimation, their mutual interaction identification and finally, the determination of optimum conditions for highest GOS yield achievement. Having said that, it can be observed that the temperature and pH have no significant impact on the GOS yield, while on the other hand, the lactose concentration of 400 g/l, enzyme concentration of 13.5 g/l and reaction time of 13 min represent the optimum conditions for achieving the highest GOS yields.