Evaluation and Implementation of Biocompatible Methods for the Cross-linking of Plasma Proteins

Autologous plasma proteins can be used to fabricate patient specific cardiovascular implants but need to be cross-linked to increase their mechanical strength and reduce water solubility. Glutaraldehyde is the state-of-the-art solution but its reaction products have been shown to be cytotoxic and pro-inflammatory. In this work, it has been shown, that cross-linking of plasma proteins with biocompatible alternatives to glutaraldehyde is possible. This was achieved by identifying four candidate substances (thrombin, transglutaminase, genipin, EDC) from current literature and investigating their ability to cross-link porcine plasma proteins in vitro. The degree of crosslinking was examined using calorimetric (DSC) and spectroscopic (FTIR, Raman) methods, mapping the influence of cross-linking on the denaturation temperature and primary amino-group content of the proteins. It could be shown that thrombin, genipin and EDC are able to cross-link plasma proteins to a satisfactory degree and thus represent useful alternatives to glutaraldehyde. Transglutaminase, on the other hand, could not sufficiently cross-link the plasma proteins and was therefore ruled out as an alternative.

Effects of Ethanolic Extract of Kiam Wood/Cashew Bark and Commercial Phenolic Compounds Oxidized Under Alkaline Condition on Gel Property of Gelatin from Cuttlefish Skin

The effect of ethanolic extracts of kiam wood (EKW) or cashew bark (ECB) and commercial phenolic compounds oxidized under alkaline condition (pH 9) on gel properties of gelatin extracted from cuttlefish skin was investigated. All the oxidized compounds increased gel strength (GS) of gelatin, in which the highest value was noticed for gels containing oxidized catechin (CH-G) and gallic acid (GA-G) (P<0.05). Among the ethanolic extracts, the gel added with EKW (EKW-G) had higher GS than that containing ECB gel (ECB-G) (P<0.05). Both extracts yielded gels with similar GS to those added with oxidized ferulic and tannic acids (P>0.05). Lightness and free amino group content of gels were decreased with the addition of oxidized compounds, regardless of their types. Gels added with oxidized compounds showed lower solubility and amino group content as compared to the control, indicating the formation of nondisulphide covalent bonds in the gel matrix. The treated samples showed a gel network with thicker strands and larger voids, compared with the control gel. Overall, oxidized EKW extract had a similar impact on the gel properties of gelatin to the oxidized phenolic compounds, especially catechin and gallic acids.

Thermal Conductivity Analysis of Chitin and Deacetylated-Chitin Nanofiber Films under Dry Conditions

Chitin, a natural polysaccharide polymer, forms highly crystalline nanofibers and is expected to have sophisticated engineering applications. In particular, for development of next-generation heat-transfer and heat-insulating materials, analysis of the thermal conductivity is important, but the thermal conductivity properties of chitin nanofiber materials have not been reported. The thermal conductivity properties of chitin nanofiber materials are difficult to elucidate without excluding the effect of adsorbed water and analyzing the influence of surface amino groups. In this study, we aimed to accurately evaluate the thermal conductivity properties of chitin nanofiber films by changing the content of surface amino groups and measuring the thermal diffusivity under dry conditions. Chitin and deacetylated-chitin nanofiber films with surface deacetylation of 5.8% and 25.1% showed in-plane thermal conductivity of 0.82 and 0.73 W/mK, respectively. Taking into account that the films had similar crystalline structures and almost the same moisture contents, the difference in the thermal conductivity was concluded to only depend on the amino group content on the fiber surfaces. Our methodology for measuring the thermal diffusivity under conditioned humidity will pave the way for more accurate analysis of the thermal conductivity performance of hydrophilic materials.

Study on TEMPO-Mediated Oxidation of N-Succinyl Chitosan and the Water Retention Property

C-6 oxidized chitosan is of great interest in obtaining a new moisture retention polymer like hyaluronic acid. The direct C-6 specific oxidation of chitosan mediated by the TEMPO/NaClO/NaBr system has proven to be difficult because of the high crystalline and high C-2 amino group content. In this work, the pre-modification of chitosan by N-succinylation was investigated and followed by the TEMPO-mediated C-6 specific oxidation under homogeneous conditions. The desired 6-oxidized N-succinyl chitosan product was obtained within 15 min with a yield of about 92%. The structure of these chitosan derivatives was confirmed by FTIR and NMR spectroscopy. Moreover, it was observed that the selective oxidation led to a great improvement in water solubility and moisture retention ability. These results present a wide range of possibilities for expanding the utilization of chitosan resources.

Anti-Oxidative and Anti-Aging Activities of Porcine By-Product Collagen Hydrolysates Produced by Commercial Proteases: Effect of Hydrolysis and Ultrafiltration

To investigate methods for improving the processing of porcine waste, porcine skin was hydrolyzed using different commercially available proteases (Alcalase, Flavorzyme, Neutrase, Bromeline, Protamex, and Papain) under several optimal conditions. Following enzymatic hydrolysis, the collagen hydrolysates (CHs) were fractionated by molecular weight (3 kDa) via membrane ultrafiltration. The CHs were analyzed for physical properties (pH, protein recovery, free amino group content, molecular weight distribution, and amino composition) as well as for functional properties (antioxidant activities and anti-aging activities). Among the CHs, CHs hydrolyzed by Alcalase (CH-Alcalase) exhibited the highest degree of hydrolysis compared to other CHs. Both “CH-Alcalase” and “CH-Alcalase < 3 kDa” fractions showed a considerably high antioxidant activity and collagenase inhibition activity. Therefore, resulting bioactives have potential for development as antioxidants and anti-aging ingredients in the food, cosmetics, and pharmaceuticals, from animal by-products.

Enhancement of Hydrophobicity of Fish Skin Gelatin via Molecular Modification with Oxidized Linoleic Acid

Fish gelatin possesses hydrophilicity in nature since it contains a few hydrophobic amino acids and a large portion of hydrophilic amino acids. Low hydrophobicity of fish gelatin results in poor water vapor barrier and water resistance of gelatin films. This can limit its usage as packaging material. To overcome this drawback, the molecular attachment of hydrophobic domains can effectively improve the hydrophobicity of fish gelatin. In this study, fish skin gelatin modified with oxidized linoleic acid (OLA) prepared under various conditions using different molar ratios of OLA/free amino group content was characterized. When OLA was prepared at 60 and 80°C for various oxidation times (0–24 h), the peroxide value (PV) increased continuously up to 9 and 12 h when reaction was performed at 80 and 60°C, respectively. Consequently, the PV decreased until the end of the reaction (24 h). Thiobarbituric acid reactive substances (TBARS) of OLA were increased sharply up to 12 h, regardless of reaction temperatures. Thus, primary and secondary lipid oxidation products mainly occurred within the first 12 h. As gelatin was modified with different OLA at various OLA/free amino group molar ratios, the one modified with OLA prepared at 60°C for 24 h at a molar ratio of 10 : 1 had the highest increases in surface hydrophobicity and carbonyl content with the coincidentally lowest free amino group content, compared with control gelatin (without OLA modification). Fourier transforms infrared (FTIR) spectra also reconfirmed the presence of fatty acid covalently attached to resulting gelatin. Therefore, OLA could be used to modify gelatin and increase its hydrophobicity.

Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.