Gabriela Bahrim

Analysis of the Thermally Induced Structural Changes of Bovine Lactoferrin

Bovine lactoferrin (LF) is subjected to thermal processing during isolation for commercial use and while preparing milk products intended for infant nutrition. The present study is focused on the heat-induced structural changes of LF in buffer solution. Fluorescence spectroscopy, molecular modeling, and enzymatic hydrolysis studies were combined to extensively characterize LF thermal behavior. The temperature-induced changes induced on LF conformation were analyzed through intrinsic and ANS fluorescence parameters (intensity, maximum position, and parameter A value), the phase diagram method, and quenching experiments using acrylamide and iodide. A higher exposure of hydrophobic residues was highlighted through the molecular modeling approach, with a decrease in α-helix content from 23.5% to 21.2% when increasing the temperature from 25 °C to 80 °C. The experimental results demonstrate a more flexible conformation of the protein at higher temperature, thus facilitating the enzymatic hydrolysis by thermolysin.

Production of medium chain saturated fatty acids with enhanced antimicrobial activity from crude coconut fat by solid state cultivation of Yarrowia lipolytica.

Fatty acids profiles and antimicrobial activity of crude coconut fat hydrolysates obtained in solid-state cultivation system with a selected yeast strain Yarrowia lipolytica RO13 were performed. A preliminary step regarding extracellular lipase production and solid state enzymatic hydrolysis of crude fat at different water activity and time intervals up to 7 days was also applied. Gas chromatography-mass spectrometry analysis was used for quantification of medium chain saturated fatty acids (MCSFAs) and the results revealed a higher concentration of about 70% lauric acid from total fatty acids. Further, antimicrobial activity of fatty acids against some food-borne pathogens (Salmonella enteritidis, Escherichia coli, Listeria monocytogenes and Bacillus cereus) was evaluated. The minimum inhibitory concentration of the obtained hydrolysates varied from 12.5 to 1.56 ppm, significantly lower than values reported in literature. The results provide substantial evidence for obtaining biopreservative effects by coconut fat enzymatic hydrolysis.

Effect of buckwheat flour and oat bran on growth and cell viability of the probiotic strains Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and their combination SYNBIO®, in synbiotic fermented milk.

Fermented foods have a great significance since they provide and preserve large quantities of nutritious foods in a wide diversity of flavors, aromas and texture, which enrich the human diet. Originally fermented milks were developed as a means of preserving nutrients and are the most representatives of the category. The first aim of this study was to screen the effect of buckwheat flour and oat bran as prebiotics on the production of probiotic fiber-enriched fermented milks, by investigating the kinetics of acidification of buckwheat flour- and oat bran-supplemented milk fermented by Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and their 1:1 combination named SYNBIO®. The probiotic strains viability, pH and sensory characteristics of the fermented fiber-enriched milk products, stored at 4 °C for 28 days were also monitored. The results showed that supplementation of whole milk with the tested probiotic strains and the two vegetable substrates results in a significant faster lowering of the pH. Also, the stability of L. rhamnosus IMC 501®, L. paracasei IMC 502® and SYNBIO® during storage at 4 °C for 28 days in buckwheat flour- and oat bran-supplemented samples was remarkably enhanced. The second aim of the study was to develop a new synbiotic product using the best combination of probiotics and prebiotics by promoting better growth and survival and be acceptable to the consumers with high concentration of probiotic strain. This new product was used to conduct a human feeding trial to validate the fermented milk as a carrier for transporting bacterial cells into the human gastrointestinal tract. The probiotic strains were recovered from fecal samples in 40 out of 40 volunteers fed for 4 weeks one portion per day of synbiotic fermented milk carrying about 10(9) viable cells.

Extraction and characterization of volatile compounds and fatty acids from red and green macroalgae from the Romanian Black Sea in order to obtain valuable bioadditives and biopreservatives

Three species of macroalgae, Ceramium virgatum (Rhodophyta), Ulva intestinalis, and Cladophora vagabunda (Chlorophyta), harvested from the Romanian Black Sea coast, were studied as sources of valuable compounds that could be used as additives and biopreservatives. Volatile compounds including hexanal (11.2xa0%), octane (9.8xa0%), nonanal (7.0xa0%), octanal (6.7xa0%), 2,5,5-trimethyl-2-hexene (4.7xa0%), 3-hexen-2-one (4xa0%), and o-cymene (3.6xa0%) were identified as the major components in the biomass extract of C. vagabunda. In C. virgatum, the major volatile components were 3-hexen-2-one (27.9xa0%), acetone (12.4xa0%), hexanal (3.4xa0%), and o-cymene (2.7xa0%). The major volatile compounds of U. intestinalis were hexanal (14.6xa0%), trichloromethane (7.3xa0%), nonanal (5.6xa0%), 3-hexen-2-one (5.3xa0%), and octanal (3.1xa0%). Some of these compounds have industrial applications as additives in the food, pharmaceutical, or cosmetics industries. The U. intestinalis extract had a greater content of mono- and polyunsaturated fatty acids around 46.0xa0% as compared with 42.0xa0% for C. vagabunda and 31.9xa0% for C. virgatum. The most abundant fatty acids were palmitic acid (C16:0), arachidonic acid (C20:4n-6), and oleic acid (C18:1ω-9cis). The antimicrobial effect of fatty acid extracts was tested against four pathogenic bacteria. The minimum inhibitory concentrations of C. vagabunda, C. virgatum, and U. intestinalis fatty acids extracts were 1.8, 3.8, and 3.8xa0mgxa0mL−1, respectively, for all bacterial strains. This study can help the efforts of finding new, value-added uses for natural marine resources.

Advances in structure-function relationships of tyrosinase from Agaricus bisporus - investigation on heat-induced conformational changes.

A combination of fluorescence spectroscopic measurements, inactivation kinetics and in silico prediction was used in the present study to investigate the heat induced behaviour of tyrosinase from Agaricus bisporus. The phase diagram indicated the existence of at least two distinct species induced by the temperature increase up to 75°C. Regardless of calcium ion presence, the fluorescence intensity results suggest that tyrosinase tends to form aggregates after 10min at 75°C. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of tyrosinase at higher temperature. Detailed insights into tyrosinase structure after performing molecular dynamics simulations, suggest important structural rearrangements of the protein with the temperature increase. The copper coordinating His(94) residue was predicted to be involved in salt bridge formation with Glu(98), therefore causing significant alteration of the substrate binding site with increasing temperature. These significant changes in tyrosinase structure at temperatures over 60°C might lead to enzyme inactivation.

Probing thermal behaviour of microbial transglutaminase with fluorescence and in silico methods

BACKGROUNDnKnowledge of transglutaminase behaviour at thermal treatment allows efficient applications in food processing. The heat-induced conformational changes of microbial transglutaminase were studied by fluorescence spectroscopy and a molecular modelling approach.nnnRESULTSnThe experimental results indicate the unfolding of transglutaminase in a single-phase reaction, at temperatures over 60 °C. The incidence of conformational changes is also supported by the increase of both intrinsic and 1-anilino-8-naphthalene sulfonate fluorescence intensity with temperature. Changes in the secondary and tertiary structure of transglutaminase were outlined after running molecular dynamics simulations at temperatures ranging from 25 °C to 80 °C.nnnCONCLUSIONnThe motifs particularities varied with the temperature, suggesting structural rearrangements of the protein, mainly in helices. The largest deviation from the structure equilibrated at 25 °C was observed at 80 °C.

Response surface optimization of experimental conditions for carbamazepine biodegradation by Streptomyces MIUG 4.89.

Carbamazepine an iminostilbene derivative compound with a tricyclic structure is one of the most widely prescribed drugs for the treatment of epilepsy. It is hardly or not degraded during the conventional technology used in wastewater treatment plants (WWTPs) (up to 7%) and many studies have found it ubiquitous in various environmental matrices in concentrations typically ranging from μg L(-1) to ng L(-1). Streptomyces MIUG 4.89 was previously studied for its ability in carbamazepine biodegradation (up to 14%) during cultivation in submerged system under aerobic conditions at an initial CBZ concentration of 0.2 mg L(-1). The influence of some factors (independent variables) upon biodegradation potential was examined by Plackett-Burman analysis. Central composite design of experiments (CCD) and response surface methodology (RSM) were used to get more information about the significant effects and their interactions of the five parameters selected upon their biodegradation potential in order to increase the elimination yield of this drug from a liquid medium. The investigated ranges of the independent variables were: 1.0-3.0 g L(-1) yeast extract, 3.0-10.0 g L(-1) glucose, 4.0-10.0% (v/v) inoculation level, pH 5.0-7.0 and 50-250 mL of medium at a constant initial concentration of carbamazepine (CBZ) of 0.2 mg L(-1). The response surface analysis results showed that the capacity of the selected strain Streptomyces MIUG 4.89 to degrade carbamazepine was high in submerged cultivation system by cultivation in a liquid medium containing 6.5 g L(-1) glucose and 2 g L(-1) yeast extract, inoculated at 7% (v/v) and cultivated at pH 6.0, during 7 days of incubation at 25 °C and 150 rpm. Under these culture conditions the achieved experimental CBZ biotransformation yield was 30%.

pH‐induced structural changes of tyrosinase from Agaricus bisporus using fluorescence and in silico methods

BACKGROUNDnTyrosinases are involved in enzymatic browning reactions in damaged fruits during post-harvest handling and processing. The overall structure of tyrosinase from Agaricus bisporus mushrooms at different pH values was monitored using fluorescence spectroscopy and molecular dynamics simulations.nnnRESULTSnWhen the pH value was increased from 6.0 to 9.0, the protein passed through several structural intermediates, including the tetramer, trimer and dimer stages. Changes in the secondary and tertiary structure of tyrosinase at neutral pH were outlined after running molecular dynamics simulations. A detailed check at the single-molecule level by means of molecular modeling tools suggested that the most important contribution to the fluorescence intensity is given by the H subunits with seven Trp and nine Tyr residues exposed to the solvent, whereas the lectin-like folded L subunits have only six Trp and three Tyr residues, of which only Trp(15) , Trp(59) and Trp(93) are partially exposed to the solvent.nnnCONCLUSIONSnThe results indicated that the enzyme was sensitive to pH. The experimental results revealed the unfolding of the native tetrameric enzyme in acidic pH range, causing exposure of the hydrophobic residues.

Potential of newly isolated wild Streptomyces strains as agents for the biodegradation of a recalcitrant pharmaceutical, carbamazepine

Carbamazepine (CBZ) is a recalcitrant xenobiotic pharmaceutical pollutant highly stable in soil and wastewater during treatment. The biodegradation of CBZ using streptomycetes has been few studied up to now. Sixteen newly filamentous bacteria belong to genus Streptomyces spp. isolated from different Romanian soil samples and three strains from a collection of microorganisms (MIUG) were morphologically characterized, tested based on their resistance against CBZ toxicity and then selected as agents for bioremediation. Five Streptomyces spp. strains coded MIUG 4.88, MIUG 4.89, SNA, LP1 and LP2 showed CBZ tolerance at all of the tested concentrations, i.e. 0.05, 0.2, 1, 5 and 8 mg L−1. Two of these (MIUG 4.89 and SNA strains) were selected based on their resistance to target compound and were then assessed for CBZ biodegradation. The strain Streptomyces MIUG 4.89 showed an interesting efficiency for CBZ removal, with a yield of 35% when it was cultivated in submerged conditions on a minimal medium supplemented with 5 g L−1 glucose. This ability was linked to extracellular laccase production. These results are promising for the use of these filamentous bacteria as bioremediation agents.

pH- and heat-induced structural changes of bovine α-lactalbumin in response to oleic acid binding

Bovine α-lactalbumin (α-LA) is able to interact with fatty acids, resulting in structural changes that are potentially responsible for the HAMLET/BAMLET role. Different states of α-LA induced by pH, temperature and fatty acid binding have been examined. Evidences of the structural changes of α-LA in molten globule and native states in correlation with oleic acid (OA) binding are shown using fluorescence spectroscopy and in silico approach. In addition, the α-LA was subjected to automated docking analysis, to better understand the interaction with oleic acid, using the PatchDock algorithm. The experimental results demonstrate a more flexible conformation of the protein at pH 2.5 when compared to neutral pH, thus facilitating the oleic acid binding to α-LA. The quenching experiments indicate the remarkable increase in the content of molten globule state at pH 2.5 and a more compact and rigid structure for α-LA–OA complexes at pH 7.0. The docking results are consistent with the experimental data concerning the thermal stability of the α-LA–OA complex. α-LA in different conformations/complexes was sensitive to pH and temperature. Several different molecular species induced by pH, heat treatment and oleic acid binding were suggested. The structure of the protein was more flexible at acidic pH, therefore favoring the hydrophobic exposure.