Application of biosensors to L-lactate assay in commercial juices and concentrates

Celem projektu badawczego QUALI-JUICE finansowanego przez UE w ramach 6 Ramowego Programu Badań i Rozwoju Technologii (COLL-CT-2005-012461) jest rozwój systemu wczesnego ostrzegania o skażeniu mikrobiologicznym w europejskim przemyśle soków owocowych. Na potrzeby projektu wybrano cztery komercyjnie dostępne biosensory, tj. Senzytec 1 (Tectronic srl, Włochy), Biosen_C Line sport (EKF−diagnostic GmbH, Niemcy), LactatProfi 3000 (ABT GmbH, Niemcy) i Olga (Sensolytics, Niemcy). Celem badań własnych było oznaczanie stężenia L−mleczanów w komercyjnych sokach jabłkowych i koncentratach za pomocą dwóch biosensorów, tj.: Biosen_C Line sport (EKF, Niemcy) i Lactat-Profi 3000 (ABT, Niemcy). Badaniom poddano dwanaście komercyjnych soków jabłkowych i dwanaście koncentratów dostarczonych przez firmę VINKON (Konin, Polska). Stężenia mleczanów mierzono przy użyciu biosensorów oraz za pomocą testów enzymatycznych Megazyme (Irlandia). Stwierdzono, że testowane biosensory mogą być wykorzystywane do pomiaru stężenia L–mleczanów w sokach jabłkowych i koncentratach, przy czym należy wprowadzić określone poprawki w instrukcji obsługi dołączonej przez ich producentów. Biosen_C Line sport charakteryzuje się wysoką odtwarzalnością i powtarzalnością wykonywanych pomiarów, ale jest typowym urządzeniem laboratoryjnym. Przy użyciu biosensora LactatProfi 3000 uzyskuje się mniejszą dokładność wykonywanych pomiarów, ale jest to urządzenie podręczne, które może być wykorzystywane na każdym etapie produkcji soków.The objective of the QUALI-JUICE Collective Research Project financed by the European Union under the 6th Framework Programme for Research and Technological Development (FP6) (COLL-CT-2005-012461) is to develop an early warning system alerting to microbiological contamination for use in the European fruit juice industry. For the purpose of this Project, four commercially available biosensors were selected, i.e. Senzytec 1 (Tectronic srl., Italy), Biosen_C Line sport (EKF-Diagnostic GmbH, Germany), LactatProfi 3000 (ABT GmbH, Germany), and Olga (Sensolytics GmbH, Germany). The objective of the research conducted by the authors of this paper was to determine the concentration rate of L – lactates in the commercial apple juices and in concentrates using two biosensors: Biosen_C Line sport (EKF, Germany) and LactatProfi 3000 (ABT, Germany). Twelve commercial apple juices and twelve concentrations supplied by the VINKON company (Konin, Poland) were analysed. The concentration rates of L-lactates were measured using both the biosensors and Megazyme enzyme kits (Ireland). It was found that the biosensors tested could be applied to measure the concentration rates of L-lactates in apple juices and concentrates provided that specific corrections be incorporated into the instruction manuals attached by their manufacturers. The Biosen_C Line sport biosensor is characterized by a high reproducibility and repeatability of measurements taken; however, it is a typical laboratory device. With the LactatProfi 3000 biosensor applied, the accuracy of measurements taken is lower, but this instrument is very practical and can be used at any stage of the fruit juice production

Optical properties of (3-(acetamidomethyl)phenyl)boronic acid and its interactions with selected sugars

(3-(Acetamidomethyl)phenyl)boronic acid (3AAPBA)has at pH 7 absorbance maximum at 270 nm with molar absorbance coefficient 516 M-1cm-1. 3AAPBA exhibits weak fluorescence with maximum at 297 nm and quantum yield 0.062 ± 0.001. Fluorescence decay is monoexponential and the lifetime is 2.05 ± 0.01 ns. Interactions of 3AAPBA with selected sugars were studied by absorbance, steady-state and time-resolved fluorescence measurements. At pH 7 fluorescence of 3AAPBA is quenched only by fructose (with quenching constant 67.9 M-1) and to some extend by galactose. Addition of these two monosaccharides causes also changes of absorbance spectra of 3AAPBA. Acid-base dissociation of free 3AAPBA and its esters with sugars was studied by absorbance and steady-state fluorescence measurements in pH range from 4.5 to 11.00. Esterification of phenylboronic acid derivatives by sugars leads to increased acidity of them. In case of 3AAPBA the obtained values of pK indicate that affinity of studied sugars towards it can be ordered as follows: fructose > galactose > glucose > maltose > lactose > sucrose. At pHs higher than pK the fluorescence decays turn to biexponential with additional shorter component in lifetime which we propose to attribute to anionic form of 3AAPBA or its esters

Characterization of secondary structure and FAD conformational state in free and sol–gel immobilized glucose oxidase

Immobilization procedures are a fundamental step in the technological use of enzymes. Among the different immobilization procedures sol–gel technique is widely recognised as a valuable approach to obtain very high quality catalytic supports. In this paper different optical techniques have been used and compared to investigate structural and dynamic properties of glucose oxidase (GOD) prior and after sol–gel immobilization process. In particular, using Fourier Transform infrared micro-spectroscopy and time-resolved fluorescence the secondary structure of GOD and the flavin adenine dinucleotide (FAD) conformational changes have been respectively investigated. Infrared spectroscopy measurements have confirmed that enzymatic activity is preserved and a predominant b-sheet subcomponent is retained by immobilized GOD. By time-resolved FAD fluorescence a three-exponential decaying behaviour has been observed for both free and immobilized enzymes with three different lifetimes, each being characteristic of a peculiar conformational state of the FAD structure. The comparison between lifetime values for free and immobilized GOD has not shown significant differences, while the fractional steady-state intensities of the single exponential components have been changed by immobilization procedure. All results reported and discussed in this paper have confirmed once again the efficacy of the adopted sol–gel immobilization procedure for enzymes and proteins. In addition, the joint use of different optical spectroscopic techniques has shown to be a very valuable tool for getting a better insight into structural and dynamic properties of immobilized enzymes

Decellularized adipose tissue scaffolds for soft tissue regeneration and adipose-derived stem/stromal cell delivery

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Surgically discarded adipose tissue is not only an abundant source of multipotent adipose-derived stem/stromal cells (ASCs) but can also be decellularized to obtain a biomimetic microenvironment for tissue engineering applications. The decellularization methods involve processing excised fat through a series of chemical, mechanical, and enzymatic treatment stages designed to extract cells, cellular components, and lipid from the tissues. This process yields a complex 3D bioscaffold enriched in collagens that mimics the biochemical and biomechanical properties of the native extracellular matrix (ECM). For ASC culture and delivery, decellularized adipose tissue (DAT) provides a cell-supportive platform that is conducive to adipogenesis. While DAT can be applied in its intact form as an off-the-shelf adipogenic matrix, it can also be used as an ECM source for the fabrication of an array of other scaffold formats including adipose ECM-derived microcarriers and porous foams. In this chapter, we describe the methods developed in our lab to decellularize human adipose tissue and to further process it into a variety of scaffolding materials for a range of applications in soft tissue regeneration, wound healing, and cell culture