Combination Of Enzymatic Treatments Research Articles

Identification of Bioactive Peptides from Nannochloropsis oculata Using a Combination of Enzymatic Treatment, in Silico Analysis and Chemical Synthesis.

In vitro ACE-1 inhibitory peptides were characterised previously from a number of microalgal species including Spirulina platensis (peptide IAPG), Chlorella vulgaris (peptides FDL, AFL, VVPPA), Isochrysis galbana (peptide YMGLDLK), Chlorella sorokiniana (peptides IW and LW) and indeed Nannochloropsis oculata (peptides GMNNLTP and LEQ). The isolation of protein from Nannochloropsis oculata using a combination of ammonium salt precipitation and xylanase treatment of resulting biomass combined with molecular weight cut off filtration to produce a permeate and characterisation of bioactive peptides is described. The Angiotensin-1-converting enzyme (ACE-1) IC50 value for the generated permeate fraction was 370 µg/mL. Ninety-five peptide sequences within the permeate fraction were determined using mass spectrometry and eight peptides were selected for chemical synthesis based on in silico analysis. Synthesized peptides were novel based on a search of the literature and relevant databases. In silico, simulated gastrointestinal digestion identified further peptides with bioactivities including ACE-1 inhibitory peptides and peptides with antithrombotic and calcium/calmodulin-dependent kinase II (CAMKII) inhibition. This work highlights the potential of Nannochloropsis oculata biomass as both a protein and bioactive peptide resource, which could be harnessed for use in the development of functional foods and feeds.

Enzymatic treatments of raw, germinated and fermented spelt (Triticum spelta L.) seeds improve the accessibility and antioxidant activity of their phenolics

Our study aimed to investigate the effect of cellulase, xylanase, esterase, α-amylase and protease on the enhanced release of phenolics from spelt seeds, and to demonstrate the co-effect of enzymatic treatment, germination or fermentation on the spelt phenolics content and their in vitro and cellular antioxidant activities. Bound phenolics of spelt seeds were successfully released by different combinations of cellulase, xylanase, feruloyl esterase, α-amylase and protease. Samples treated with all five enzymes together showed significantly improved extractable p-coumaric, ferulic and p-hydroxybenzoic acid contents, particularly efficient antioxidant activity in ABTS•+ scavenging activity and the highest extractable TPCs. By combining enzymatic treatment with germination and fermentation, extractable TPCs were increased by 28% and 458%, respectively. A combination of enzymatic treatment with germination and fermentation led to enormously increased extractable trans-ferulic acid content (5899% and 8263%, respectively). The bound fraction of enzymatic treated raw seeds showed lower decrease in intracellular oxidation than the corresponding extractable fraction, but a 24-fold higher uptake of hydroxycinnamic acids. Principal component analysis showed some similarities between extractable and bound fractions of enzymatic treated raw, germinated and fermented samples, mainly due to the increase in extractable acids content (biochemical parameters), especially cis-ferulic, caffeic and p-hydroxybenzoic acids.

Glycosaminoglycans and glycoproteins influence the elastic response of synovial fluid nanofilms on model oxide surfaces

Synovial fluid (SF) is the natural lubricant found in articulated joints, providing unique cartilage surface protecting films under confinement and relative motion. While it is known that the synergistic interactions of the macromolecular constituents provide its unique load-bearing and tribological performance, it is not fully understood how two of the main constituents, glycosaminoglycans (GAGs) and glycoproteins, regulate the formation and mechanics of robust load-bearing films. Here, we present evidence that the load-bearing capabilities, rather than the tribological performance, of the formed SF films depend strongly on its components' integrity. For this purpose, we used a combination of enzymatic treatments, quartz crystal microbalance with dissipation (QCM-D), and the surface forces apparatus (SFA) to characterize the formation and load-bearing capabilities of SF films on model oxide (i.e., silicates) surfaces. We find that, upon cleavage of proteins, the elasticity of the films is reduced and that cleaving GAGs results in irreversible (plastic) molecular re-arrangements of the film constituents when subjected to confinement. Understanding thin film mechanics of SF can provide insight into the progression of diseases, such as arthritis, but may also be applicable to the development of new implant surface treatments or new biomimetic lubricants.

Characterization of dietary fiber from residual cellulose sausage casings using a combination of enzymatic treatment and high-speed homogenization

The objective of this study was to characterize dietary fiber from residual cellulose sausage casings using a combination of enzymatic hydrolysis and high-speed homogenization. To modify structure and function of the cellulose, residual cellulose casings (40 or 50 mg mL−1) were subjected to enzymatic hydrolysis followed by high-speed homogenization to obtain dietary fiber at a targeted yield of 70%. Total dietary fiber of the sausage casing ingredient was 50% (w:w) on a dry basis, of which 45% was insoluble fiber and 5% soluble fiber when the treatment of 50 mg mL−1 against total dietary fiber was used. The obtained fiber using 50 mg mL−1 solids had the following characteristics: swelling capacity, 3.9 mL water·g sample−1; water binding capacity, 2.5 g water·g sample−1; and oil binding capacity, 2.7 g oil·g sample−1. The particle size exhibited a bimodal distribution with the surface mean value of 65 μm and volume mean of 377 μm. The rheological behavior of the fiber dispersion was characterized as shear thinning, which could be described accurately with the Power Law model with equation of σ=0.82γ˙0.3 , with at an apparent viscosity at 100 s−1 of 27 mPa s. These results indicated that residual cellulose casings can be used as an inexpensive raw material to obtain an ingredient with adequate content and the inherent characteristics of dietary fiber.

A Novel Methodology for the Quantification of B-Vitamers in Breast Milk

With this report we present development, validation and application of an analytical methodology for the quantification of 18 water soluble vitamers and secreted or biological forms in breast milk. On a relatively low amount of breast milk (200 µL), we applied isotope dilution-based sample preparation based on a combination of enzymatic treatment and protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Compounds separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. To perform the quantification of 18 water soluble vitamers, procured pooled breast milk was used to build matrixmatched calibration curves, as labelled internal standards were not available for each vitamer. The analytical approach has been validated according to the EMA guidelines. The overall performance of the method was considered adequate, with 0.3- 28.3% and 0.9-32.6% intra and inter-day precision respectively and averaged accuracy reaching 92.2-107.5%. In addition, performed freeze/thaw stability studies showed the potential degradation of some vitamers. We therefore recommend particular attention in sample collection with rather having dedicated aliquots with small volumes. The feasibility of this analytical approach has been evaluated by quantifying various breast milk samples that were procured from an external supplier. The main forms found in breast milk were thiamine monophosphate for B1, flavin adenine nucleotide for B2, nicotinamide for B3, pyridoxal for B6 and 5-methyl tetrahydrofolic acid for B9. In addition, we newly reported nudifloramide as B3 form present in breast milk. With this analytical approach, it will give more confidence to provide a comprehensive assessment of the presence of water soluble vitamins in breast milk. This will enable the accurate evaluation of the nutritional requirements of infants.

Color remediation of chemimechanical pulping effluent using combination of enzymatic treatment and Fenton reaction

This research investigated the efficiency of Advanced Oxidation Processes, Enzymatic treatment, and combined enzymatic/AOPs sequences on color remediation of CMP pulp and paper mills effluent. Regarding enzymatic treatment two kinds of fungal enzymes; Laccase (EC: 1.10.3.2) from Terametes Versicolor and Versatile Peroxidase (EC: 1.11.1.7) from Bjerkandera adusta were chosen and applied. Also, the effect of external mediator on the enzyme based degradations was studied. It was found that both VP from Bjerkandera adusta and Laccase from Terametes versicolor decolorized the deep brown effluent to a clear light yellow solution. It has been found that, concomitant use of enzymes and photo-Fenton process produces a considerable effect on color remediation. The data analysis of sequence treatment indicated that, chemical treatment after the enzymatic stage (photo-Fenton as a post treatment unit) yield a better performance for the CMP effluent.

Culture of embryonic C. elegans cells for electrophysiological and pharmacological analyses

Despite the considerable advantages that C. elegans offers for studying gene function in vivo, this system is quite challenging for in vivo electrophysiological analysis of channel function, particularly in neurons. A major problem is that C. elegans neurons are confined in a pressurized and hard-to-penetrate cuticle. Recently, a method for culturing C. elegans embryonic cells has been developed and numerous researchers have already applied this option to study a variety of native ion channels and transporters using various configurations of the patch-clamp technique. C. elegans embryonic cells are obtained from eggs harvested from synchronized gravid adults and then are dissociated using a combination of enzymatic treatment and manual pipetting. Once plated on a surface covered with peanut lectin, cells adhere and differentiate into neurons, muscle and epithelial cells. Cultured embryonic cells recapitulate the expression of differentiation markers and are found in the culture in proportion to their cell type in the mature embryo. Differentiated cells survive well for at least 2 weeks. It should be noted that postembryonic cells do not appear to be generated in these cultures. Cultures can be used for electrophysiological study, testing of pharmacological sensitivities, and for RNAi. C. elegans cell culture thus constitutes the basis for the application of experimental procedures that are not easily applicable to the intact nematode.

Modification of the nitrogen solubility properties of soy protein isolate following proteolysis and transglutaminase cross-linking

The effect of (a) limited hydrolysis [0.5–2.0% degree of hydrolysis (DH)] with Alcalase™, (b) cross-linking with transglutaminase (TGase) and (c) combinations of these modifications on the nitrogen solubility (pH 3–8) of soy protein isolate (SPI) was investigated. Between pH 3.0 and 5.0, SPI hydrolysates, hydrolysates of cross-linked SPI and the cross-linked products of SPI hydrolysates displayed significant ( P<0.05) increases in solubility compared to unmodified SPI. Cross-linking pre- or post hydrolysis did not alter the overall trend of increased ( P<0.05) solubility relative to the unmodified control at low pH. At 2% DH, cross-linking pre- or post-hydrolysis resulted in greater solubility ( P<0.05) than that observed in hydrolysates per se at low pH. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the 22 kDa 11S basic polypeptide was relatively resistant to Alcalase hydrolysis and that the 18 and 22 kDa 11S basic polypeptides were not susceptible to TGase cross-linking. The results demonstrate that a combination of enzymatic treatments and the order in which they are applied may have potential for creating novel food ingredients with improved functional properties, especially those properties that are dependant on high solubility at low pH.

Characterization of the Contents of Oil Bodies from the Liverwort Radula complanata1

Abstract: With a combination of enzymatic treatment and micromanipulation, the contents of oil bodies of Radula complanata were obtained. A GC‐MS analysis revealed that 3‐methoxybibenzyl is the main constituent of these compartments. Its structure was elucidated by NMR spectroscopy.

Long-term culture of microvascular endothelial cells derived from Mongolian gerbil brain.

A method for long-term culture of microvascular endothelial cells from Mongolian gerbil brain and their biologic properties in vitro are described. Microvessels were isolated from Mongolian gerbil brain by a combination of enzymatic treatment, filtration, and centrifugation and were seeded onto a gelatin-coated dish. A morphologically homogeneous cell plaque showing a cobblestone appearance was removed 2 to 3 weeks after the seeding, and the cells were subcultured. The cultured cells grew as monolayers of flat polygonal cells and were carried for more than 20 passages without morphologic change. These cells synthesized prostacyclin and retained an endothelial specific marker, factor VIII-related antigen. When the cells were cultured in a collagen gel, they rapidly formed capillarylike tubular structures without endothelial cell growth factor or special substrata. Long-term culture of purified microvascular endothelial cells derived from Mongolian gerbil brain will facilitate the study of the function of microvascular endothelial cells in human brain under normal and pathologic conditions.