A Study on the Regioselective Acetylation of Flavonoid Aglycons Catalyzed by Immobilized Lipases.

This study aimed to explore the capacity of immobilized lipases on the acetylation of six aglycon flavonoids, namely myricetin, quercetin, luteolin, naringenin, fisetin and morin. For this purpose, lipase B from Candida antarctica (CaLB) and lipase from Thermomyces lanuginosus (TLL) were immobilized onto the surface of ZnOFe nanoparticles derived from an aqueous olive leaf extract. Various factors affecting the conversion of substrates and the formation of monoesterified and diesterified products, such as the amount of biocatalyst and the molar ratio of the substrates and reaction solvents were investigated. Both CaLB and TLL-ZnOFe achieved 100% conversion yield of naringenin to naringenin acetate after 72 h of reaction time, while TLL-ZnOFe achieved higher conversion yields of quercetin, morin and fisetin (73, 85 and 72% respectively). Notably, CaLB-ZnOFe displayed significantly lower conversion yields for morin compared with TLL-ZnOFe. Molecular docking analysis was used to elucidate this discrepancy, and it was revealed that the position of the hydroxyl groups of the B ring on morin introduced hindrances on the active site of CaLB. Finally, selected flavonoid esters showed significantly higher antimicrobial activity compared with the original compound. This work indicated that these lipase-based nanobiocatalysts can be successfully applied to produce lipophilic derivatives of aglycon flavonoids with improved antimicrobial activity.

Enzymatic Modification of Pomace Olive Oil with Natural Antioxidants: Effect on Oxidative Stability.

Enzymatic lipophilization has been proposed as a cost-effective strategy to produce new liposoluble antioxidant compounds. In this study, modified oils rich in structured phenolipids were prepared via one-pot enzymatic acylation of hydroxytyrosol (HTYR), vanillyl alcohol (VA) and homovanillyl alcohol (HVA) with pomace olive oil (POO) in solvent-free conditions using immobilized lipase on biogenic nanoparticles. The effect of temperature (30-70 °C) and enzyme concentration (0.1-1%, w/w) on the efficiency of the bioprocess as well as the reusability of the nanobiocatalyst were thoroughly investigated. The modified oils exhibited increased antioxidant activity compared to the control oil according to DPPH and CUPRAC assays (p < 0.05). The oxidative stability of pomace olive oil was also significantly enhanced after modification, as depicted by the K232 values and TBARS contents under accelerated oxidation at 60 °C (p < 0.05). Moreover, a fortified mayonnaise containing modified oil with HTYR was prepared that was noticeably stable compared to the control mayonnaise at 28 °C for 5 months (p < 0.05). Enzymatically modified oils have great potential for application in the nutraceutical and food industry, encouraging the exploitation of immobilized lipases as effective and green catalytic tools.

Detection and Quantification of Acrylamide in Second Trimester Amniotic Fluid Using a Novel LC-MS/MS Technique to Determine Whether High Acrylamide Content during Pregnancy Is Associated with Fetal Growth.

INTRODUCTION: Acrylamide, an organic compound, is, chemically speaking, a vinyl-substituted primary amide. It is produced industrially, principally as a precursor to polyacrylamides, for use in such products as plastics and cosmetics. This same compound, however, forms naturally in certain foods, both home-cooked and packaged, especially when prepared at high temperatures. We developed and validated a novel reliable technique for the determination of acrylamide in amniotic fluid. Multiple reaction monitoring (MRM) is a targeted mass spectrometry (MS) technique which enables the detection and quantification of particular molecules in a complex mixture. Thanks to its throughput, selectivity, and sensitivity, MRM-MS has been identified as offering an alternative to antibody-based studies for the purpose of biomarker verification. Our aim was to investigate the presence of acrylamide in amniotic fluid and, via the MRM-MS technique, to determine whether there is any correlation between maternal exposure to acrylamide, through a woman's diet, and fetal growth. METHODS: Our amniotic fluid bank included 40 samples from various fetal growth rates, as objectively denoted by the neonatal weight centile at delivery, while our analytical detection method was based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). Acrylamide was determined with reversed phase chromatography and monitoring of two multiple reaction monitoring (MRM) transitions. Quantification was performed using the matrix-matched calibration curve. RESULTS: Acrylamide was detected at concentrations between 7.1 and 1468 ng/mL in six out of the total of 40 amniotic fluid samples that were used. Our method limit of detection and quantification was 1.4 ng/mL and 4.6 ng/mL, respectively. The repeatability of our method ranged between 11 and 14%, expressed as relative standard deviation levels between 5 and 100 ng/mL. CONCLUSIONS: Detection of acrylamide in early second trimester amniotic fluid, for the first time in the literature to our knowledge, raises concerns about fetal health, given that published data on animal studies have attributed a number of birth defects to acrylamide. Our novel LC-MS/MS method for the determination of acrylamide in amniotic fluid proved to be effective and its performance in practice was very accurate, simple, and fast. Validation of the method revealed that the use of a matrix-matched curve is necessary for the quantification.

A Bi-enzymatic Immobilized Nanobiocatalyst for the Biotransformation of Oleuropein to Hydroxytyrosol.

Multi-enzymatic assemblies offer the opportunity of bringing in proximity several enzymes that are enabled to work together for the catalysis of multi-step reactions. Especially, the development of robust nanobiocatalytic systems comprising of several enzymes has gained considerable attention over the last few years for the catalysis of complex reactions and the production of high added-value products. In the present chapter, we describe the methodology for the development of a bi-enzymatic nanobiocatalyst consisting of the enzymes ß-glucosidase from Thermotoga maritima and lipase A from Candida antarctica (CalA) co-immobilized on chitosan-coated magnetic nanoparticles. This nanobiocatalyst can be efficiently applied for the biotransformation of oleuropein to hydroxytyrosol, a reaction of increased biotechnological interest. Several techniques, as well as methodologies that are required for the characterization of the structure and the activity of such systems are also comprehensively described.

3D printed PLA enzyme microreactors: Characterization and application for the modification of bioactive compounds.

Process sustainability of biocatalytic processes is significantly empowered with the use of continuous-flow technologies that offer high productivity, minimal wastes and low volumetric consumption. Combining microreactor design with 3D printing technology can broaden the engineering potentials. This work proposes a protocol to modify the surface of 3D-printed PLA scaffolds, based on chitosan deposition. Mimicking the concept of microplates, multi-well plates were designed to facilitate parameter testing. Immobilization of laccase from Trametes versicolor was successfully performed, while chitosan and cross-linker concentration and incubation time were optimized. Τhe developed protocol was applied for the continuous flow bioconversion of hydroxyyrosol, yielding a TTN of 438.6 × 103 for a total of 10 h continuous use. Also, a peristaltic flow pattern seemed to favor the system performance, reaching 95% bioconversion efficiency in a total of 1 h reaction time. The potential of the developed system was further evaluated for the biotransformation of different biophenols from dietary sources, proving the efficiency of the system as a versatile biotechnological tool.