Application of design-of-experiments procedures to optimize efficiently pretreatment of lipase for use in a nonaqueous reaction

A variety of different pretreatments can improve the performance of enzymes in nonpolar reaction media. These pretreatments have primarily been studied in isolation; however, interactions between some pairs of pretreatments are known to exist. The presence of these interactions complicates the design of an optimum multifactor pretreatment. Modern design-of-experiments techniques allow the simultaneous optimization of two or more variables. To improve the performance of lipase in a model reaction, we used a technique called the method of steepest ascent to optimize three variables simultaneously: pretreatment pH and sodium phosphate concentration, and the concentration of acetic acid (one of the reactants) in the reaction mixture. In only 26 experimental runs, this optimization process determined a combination of variable settings that yielded a reaction product approx 180 times faster than achieved with untreated enzyme. Evidence is presented to demonstrate that locating this optimum with single-factor experiments would be inefficient. This article demonstrates the efficiency of the method of steepest ascent particularly for evaluation of enzymatic reaction conditions exhibiting significant interactions.     

聚乙二醇-聚乳酸共聚物胶束溶液的冷冻干燥及胶束体外释药动力学研究

合成了一系列亲水、疏水链段质量比例不同的聚乙二醇-聚乳酸(PEG-PLA)嵌段共聚物胶束, 并以两性霉素B为模型药物制备了载药胶束. 为获得稳定性良好的、可长期储存的载药胶束剂型, 对胶束进行了冷冻干燥. 使用不同浓度的糖类(包括甘露糖、海藻糖、葡萄糖)、泊洛沙姆188 (Pluronic F68)、聚乙二醇作为冻干保护剂, 以冻干产品的重分散性、冻干前后胶束的粒径及多分散性为指标评价各种保护剂的保护效果. 结果发现, 当嵌段聚合物中聚乳酸链段的质量百分比小于或等于聚乙二醇时, 糖类、Pluronic F68和PEG均可以起到有效的冻干保护作用; 而对于聚乳酸链段质量比例较大的共聚物胶束, 只有PEG和Pluronic F68能够起到较好的冻干保护作用. 对载药胶束体外释放研究表明, 聚合物胶束的体外释放缓慢, 符合一级动力学特征.     

Impact of Sample Pretreatment and Extraction Methods on the Bioactive Compounds of Sugar Beet (Beta vulgaris L.) Leaves

To find the most optimal green valorization process of food by-products, sugar beet (Beta vulgaris L.) leaves (SBLs) were freeze-dried and ground with/without liquid nitrogen (LN), as a simple sample pretreatment method, before ultrasound-assisted extraction (UAE) of polyphenols. First, the water activity, proximate composition, amino acid (AA) and fatty acid (FA) profiles, and polyphenol oxidase (PPO) activity of dried and fresh SBLs were evaluated. Then, conventional extraction (CE) and UAE of polyphenols from SBLs using water/EtOH:water 14:6 (v/v) as extracting solvents were performed to determine the individual and combined effects of the sample preparation method and UAE. In all the freeze-dried samples, the specific activity of PPO decreased significantly (p ≤ 0.05). Freeze-drying significantly increased (p ≤ 0.05) the fiber and essential FA contents of SBLs. The FA profile of SBLs revealed that they are rich sources of oleic, linoleic, and α-linolenic acids. Although freeze-drying changed the contents of most AAs insignificantly, lysine increased significantly from 7.06 ± 0.46% to 8.32 ± 0.38%. The aqueous UAE of the freeze-dried samples without LN pretreatment yielded the most optimal total phenolic content (TPC) (69.44 ± 0.15 mg gallic acid equivalent/g dry matter (mg GAE/g DM)) and excellent antioxidant activities. Thus, combining freeze-drying with the aqueous UAE method could be proposed as a sustainable strategy for extracting bioactive compounds from food by-products.     

Chitosan Based Scaffolds by Lyophilization and sc.CO2 Assisted Methods for Tissue Engineering Applications

Porous chitosan scaffolds with possible tissue engineering applications were synthesized by using lyophilization and supercritical carbon dioxide (sc.CO₂) drying technique. 1% Chitosan (CS) solution in aq. acetic acid was treated with 37% formaldehyde solution; the resulting hydrogels were subjected to solvent-exchange prior to the final treatment procedures. Their morphology, pore structure, and physical properties were characterized by Fourier transform infrared spectroscopy (FTIR), thermal analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and the specific surface areas and porosities of scaffolds were determined by using N₂ adsorption. The sc.CO₂ treated scaffolds showed a much greater surface area in comparison to the lyophilized one. Hence, sc.CO₂ treated scaffolds is better for cell proliferation. We further investigated the bioactivity of sc.CO₂ treated scaffolds using simulated body fluid (SBF). The sc.CO₂ assisted chitosan scaffold prepared by using green chemistry approach is highly pure and from a hygienic point of view, it is an ideal material for tissue engineering applications.     

Characterization of Betabel Extract (Beta vulgaris) Encapsulated with Maltodextrin and Inulin

Betalains are powerful antioxidants contained in beets. These are divided into betacyanins (red-violet) and betaxanthins (yellow-orange), and they can be used as natural colorants in the food industry. The effects of freeze-drying pure beet juice (B) and the encapsulation of beet juice with a dextrose equivalent (DE) 10 maltodextrin (M) and agave inulin (I) as carrier agents were evaluated. The powders showed significant differences (p < 0.05) in all the variables analyzed: water absorption index (WAI), water solubility index (WSI), glass transition temperature (T_g), total betalains (TB), betacyanins (BC), betaxanthins (BX), total polyphenols (TP), antioxidant activity (AA, via 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and total protein concentration (TPC). The highest values of antioxidant activity were found in the non-encapsulated beet powder, followed by the powder encapsulated with maltodextrin and, to a lesser extent, the powder encapsulated with inulin. The glass transition temperature was 61.63 °C for M and 27.59 °C for I. However, for B it was less than 18.34 °C, which makes handling difficult. Encapsulation of beet extract with maltodextrin and inulin by lyophilization turned out to be an efficient method to increase solubility and diminish hygroscopicity.     

The management and exploitation of naturally light-emitting bacteria as a flexible analytical tool: A tutorial

Conventional detection of toxic contaminants on surfaces, in food, and in the environment takes time. Current analytical approaches to chemical detection can be of limited utility due to long detection times, high costs, and the need for a laboratory and trained personnel. A non-specific but easy, rapid, and inexpensive screening test can be useful to quickly classify a specimen as toxic or non toxic, so prompt appropriate measures can be taken, exactly where required. The bioluminescent bacteria-based tests meet all these characteristics. Bioluminescence methods are extremely attractive because of their high sensitivity, speed, ease of implementation, and statistical significance. They are usually sensitive enough to detect the majority of pollutants toxic to humans and mammals.     

Improving Activity of Salt-Lyophilized Enzymes in Organic Media

Lyophilization with salts has been identified as an important method of activating enzymes in organic media. Using salt-activated enzymes to transform molecules tethered to solid surfaces in organic phase requires solubilization of enzymes in the solvents. Methods of improving performance of salt-lyophilized enzymes, further, via chemical modification, and use of surfactants and surfactants to create fine emulsions prior to lyophilization are investigated. The reaction system used is transesterification of N-acetyl phenylalanine ethyl ester with methanol or propanol. Initial rate of formation of amino acid esters by subtilisin Carlsberg (SC) was studied and found to increase two to sevenfold by either chemical modification or addition of surfactants in certain solvents, relative to the salt (only)-lyophilized enzyme. The method to prepare highly dispersed enzymes in a salt-surfactant milieu also improved activity by two to threefold. To test the effect of chemical modification on derivatization of drug molecules, acylation of bergenin was investigated using chemically modified SC.     

Nanostructured Lipid Carriers Can Enhance Oral Absorption of Khellin,a Natural Pleiotropic Molecule

A novel formulation based on nanostructured lipid carriers (NLCs) was developed to increase solubility and intestinal absorption of khellin. K-NLCs were prepared with stearic acid, hempseed oil, Brij S20, and Labrafil M 1944 CS, using the emulsification-ultrasonication method. Developed nanoparticles were chemically and physically characterized by liquid chromatography, light scattering techniques, and electron microscopy. The size, about 200 nm, was optimal for oral delivery, and the polydispersity index (around 0.26), indicated high sample homogeneity. Additionally, K-NLCs showed a spherical morphology without aggregation by microscopic analysis. The encapsulation efficiency of khellin was about 55%. In vitro release studies were carried out in media with different pH to mimic physiological conditions. K-NLCs were found to be physically stable in the simulated gastric and intestinal fluids, and they preserved about 70% of khellin after 6 h incubation. K-NLCs were also successfully lyophilized testing different lyoprotectants, and obtained freeze-dried K-NLCs demonstrated good shelf life over a month. Lastly, permeability studies on Caco-2 cells were performed to predict khellin passive diffusion across the intestinal epithelium, demonstrating that nanoparticles increased khellin permeability by more than two orders of magnitude. Accordingly, developed NLCs loaded with khellin represent a versatile formulation with good biopharmaceutical properties for oral administration, possibly enhancing khellin’s bioavailability and therapeutic effects.     

Carbamide Peroxide Determination in Tooth Whitening Using a Reagentless HRP-Biosensor

Abstract

A rapid and effective method for carbamide peroxide (CP) quantification in pharmaceuticals is proposed. The reagentless biosensor was prepared by using lyophilized turnip extract as horseradish peroxidase (HRP) enzyme source. The biosensor presented the best performance. The measurements were carried out in 0.1 mol L^?1 Pipes buffer (pH 6.0). At ?100 mV vs. Ag|AgCl, a linear response range from 3.0 to 25.0 mmol L^?1 was obtained. The quantification and detection limits were 1.2 and 0.4 mmol L^?1, respectively, and the response time was 0.5 s. The biosensor repeatability, storage, and lifetime were excellent, allowing a satisfactory CP quantification in real pharmaceutical samples, when compared with those obtained by the official method.