Determination of the critical aggregation concentration in water of Gum Arabic functionalized with curcumin oxidation products by micro-scale thermophoresis approach.

Gum Arabic underwent enzymatic modification with curcumin oxidation products, prompting self-assembly in water at lower concentrations than native gum Arabic, which was fully soluble. The resulting particles displayed a narrow size distribution, suggestive of a micellization mechanism akin to Critical Micellization Concentration (CMC) in surfactants or Critical Aggregation Concentration (CAC) in polymers. Accurately determining CAC is vital for utilizing polymers in molecule encapsulation, but precise measurement is challenging, requiring multiple techniques. Initially, CAC was probed via turbidity measurements, dynamic light scattering (DLS), and isothermal calorimetric titration (ITC), yielding a range of 0.0015 to 0.01 %. Micro-scale thermophoresis (MST) was then employed for the first time to define CAC more precisely, facilitated by the intrinsic fluorescence of modified gum Arabic. Using MST, CAC was pinpointed at 0.001 % (w/v), a novel approach. Furthermore, MST revealed a low EC50 value of 0.007 % (w/t) for self-assembly, signifying uniformity among GAC sub-units and assembly stability upon dilution.

Enzymatic Oxidation of Ferulic Acid as a Way of Preparing New Derivatives.

The ferulic acid (FA)-oxidation by Myceliophthora thermophila laccase was performed in phosphate buffer at 30 °C and pH 7.5 as an eco-friendly procedure. LC-MS analysis showed that oxidation products were four dehydrodimers (P1, P2, P3, P5) at MM = 386 g/mol, two dehydrotetramers (P6, P7) at MM = 770 g/mol and one decarboxylated dehydrodimer (P4) at MM = 340 g/mol. Structural characterization showed that FA-dehydrodimers were symmetric for P1 and P5 while asymmetric for P2, P3 and P4. Physicochemical characterization showed that oxidation products presented a higher lipophilicity than that of FA. Moreover, symmetric dimers and tetra dimers had a higher melting point compared to FA and its asymmetric dimers. Antioxidant and anti-proliferative assessments indicated that enzymatic oligomerization increased antioxidant and anti-proliferative properties of oxidation products for P2, P3 and P6 compared to FA. Finally, this enzymatic process in water could produce new molecules, having good antiradical and anti-proliferative activities.

Green synthesis of glyco-phenol by enzymatic coupling between ferulic acid and glucosamine: An ecofriendly procedure.

A new glyco-phenol was produced by the coupling between glucosamine (Glu) and ferulic acid (FA) using Myceliophthora thermophila laccase as biocatalyst in mild conditions (distilled water and 30°C) as an environmentally friendly process. Results indicated that the enzymatic reaction created a new derivative (FA-Glu), produced from coupling between Glu and FA by covalent bonds. By the high production of (FA-Glu) derivative and its stability, the optimal ratio of (FA:Glu) was of (1:1) at optimal time reaction of 6 h. Under these optimal conditions, almost 55% of -NH2 groups on Glu were bound with FA oxidation products. The new derivative showed higher hydrophobic character than Glu due to the presence of FA in its structure. Liquid chromatography-mass spectrometry analysis showed that (FA-Glu) derivative exhibited a molecular mass at MM 713 g/mol containing one Glu molecule and three FA molecules after decarboxylation. Furthermore, the new derivative presented good antioxidant and antiproliferative activities in comparison with Glu and FA. These results suggest that the enzymatic conjugation between Glu and FA is a promising process to produce a new glyco-phenol having good functional properties for potential applications.

Polymer functionalization through an enzymatic process: Intermediate products characterization and their grafting onto gum Arabic.

The modification of gum Arabic with ferulic acid oxidation products was performed in aqueous medium, at 30 °C and pH 7.5, in the presence of Myceliophthora thermophila laccase as biocatalyst. First, this study aimed to investigate the structures of the oxidation products of ferulic acid that could possibly be covalently grafted onto gum Arabic. HPLC analyses revealed that this reaction produced several oxidation products, whose structures were investigated using LC-MS/MS analyses (liquid chromatography-mass spectrometry with mass fragmentation analyses) and NMR experiments. The chemical structure of one intermediate reaction product was fully elucidated as the 2-(4-hydroxy-3-methoxyphenyl)-4-[(4-hydroxy-3-methoxyphenyl) methylidene] cyclobutane-1, 3-dione, called by the authors cyclobutadiferulone. Secondly, this study aimed to locate the grafting of the oxidation products onto gum Arabic by performing several NMR experiments. This study did not determine how much and specifically which oxidation products were grafted but some of them were undeniably present onto modified gum Arabic, close to the glucuronic acid C5 carbon or close to the galactose C6 carbon.

Enzymatic modification of polysaccharides: Mechanisms, properties, and potential applications: A review.

Polysaccharides are natural biopolymers found in almost all living organisms. They are used extensively in various industrial applications, such as food, adhesives, pharmaceuticals, and cosmetics. In many cases, their practical use is limited because of their weak solubility in neutral pH, their unsuitable hydrophilic/hydrophobic balance. In this context, chemical or enzymatic modification of their structure appears as a relevant way, to improve their properties, and thus to enlarge the field of their potential applications. Taking into account the reduction of the input energy and the environmental impact, and due to high specificity and selectivity properties, enzymatic bioprocesses have been investigated as attractive alternatives to toxic and non-specific chemical approaches. This review discusses the methods of enzymatic functionalization of four well-known polysaccharides, chitosan, cellulose, pectin and starch. Particular emphasis was placed on the methods, the reaction types and the enzymes implicated in the modification such as laccases, peroxidases lipases, tyrosinases, and transglutaminases. The impact of functionalization on the properties and the applications of polysaccharide derivatives were described.

Laccase mediated-synthesis of hydroxycinnamoyl-peptide from ferulic acid and carnosine.

Carnosine (CAR) dipeptide was functionalized with ferulic acid (FA) as substrate using laccase from Myceliophtora thermophila as biocatalyst. The enzymatic reaction was performed in aqueous medium under mild conditions (pH 7.5, 30°C) as an eco-friendly procedure. Results showed that this enzymatic process led to the synthesis of two new derivatives (P1, P2), from the coupling between CAR and FA derived products. Conditions allowing a high production of P1, P2 derivatives were determined with an optimal ratio of (FA: CAR) of (1:1.6) at optimal time reaction of 8h. Under these optimal conditions, the coupling between CAR and FA-products was demonstrated, resulting in the decrease of -NH2 groups (almost 50%) as quantified via derivatization. Due to the presence of FA in the structure of these new derivatives, they exhibited higher hydrophobic property than carnosine. Structural analyses by mass spectrometry showed that P1 and P2 (FA-CAR) derivatives exhibited the same molecular mass (MM 770g/mol) containing one CAR-molecule and three FA-molecules but with different chemical structures. Furthermore, these derivatives presented improved antioxidant (almost 10 times) and anti-proliferative (almost 18 times) properties in comparison with CAR. Moreover, P1 derivative exhibited higher antioxidant and anti-proliferative activities than P2 derivative, which confirmed the different structures of P1 and P2. These results suggested that the oxidized phenols coupling with carnosine is a promising process to enhance the CAR-properties.

Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan.

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco-friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA-chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EF-chitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications especially for tissue engineering. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:491-500, 2016.

Laccase-catalysed functionalisation of chitosan by ferulic acid and ethyl ferulate: evaluation of physicochemical and biofunctional properties.

Chitosan and its derivatives functionalized by laccase-catalyzed oxidation of ferulic acid (FA) and ethyl ferulate (EF) were characterised for their physico-chemical, antioxidant and antibacterial properties. The enzymatic grafting of oxidised phenols led to FA-coloured and EF-colourless chitosan derivatives with good stability of colour and grafted phenols towards the chemical treatment by organic solvents. The efficiency of FA-products grafting onto chitosan was higher than that of EF-products. Moreover, the enzymatic grafting of phenols onto chitosan changed its morphological surface, increased its molecular weight and its viscosity. Furthermore, the chitosan derivatives presented improved antioxidant properties especially for FA-chitosan derivative when compared with chitosan with good antioxidant stability towards thermal treatment (100°C/1h). Chitosan and its derivatives showed also similar antibacterial activities and more precisely bactericidal activities. This enzymatic procedure provided chitosan derivatives with improved properties such as antioxidant activity, thermal antioxidant stability as well as the preservation of initial antibacterial activity of chitosan.

Laccase-catalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: a green procedure for the synthesis of new compounds.

The enzymatic oxidation of ferulic acid (FA) and ethyl ferulate (EF) with Myceliophthora thermophila laccase, as biocatalyst, was performed in aqueous medium using an eco-friendly procedure to synthesize new active molecules. First, the commercial laccase was ultrafiltrated allowing for the elimination of phenolic contaminants and increasing the specific activity by a factor of 2. Then, kinetic parameters of this laccase were determined for both substrates (FA, EF), indicating a higher substrate affinity for ethyl ferulate. Additionally, enzymatic oxidation led to the synthesis of a FA-major product, exhibiting a molecular mass of 386 g/mol and a EF-major product with a molecular mass of 442 g/mol. Structural analyses by mass spectrometry allowed the identification of dimeric derivatives. The optical properties of the oxidation products showed the increase of red and yellow colours, with FA-products compared to EF-products. Additionally, enzymatic oxidation led to a decrease of antioxidant and cytotoxic activities compared to initial substrates. Consequently, this enzymatic procedure in aqueous medium could provide new compounds presenting optical, antioxidant and cytotoxic interest.

Combination of enzymatic hydrolysis and ethanol organosolv pretreatments: effect on lignin structures, delignification yields and cellulose-to-glucose conversion.

Enzymatic pre-hydrolysis using the industrial enzymatic cocktail Cellulyve® was assessed as a first step in a pretreatment process of Miscanthus biomass involving an aqueous-ethanol organosolv treatment. (13)C and (31)P Nuclear Magnetic Resonance and size exclusion chromatography were used to analyze the cellulose and lignin before and after treatment. It was demonstrated that despite a very low impact on the fibre structure (observed by Scanning Electron Microscopy) and composition (in terms of sugars and polyphenolics content), the enzymatic pre-treatment disrupted the lignocellulosic matrix to a considerable extend. This weakening permitted enhanced removal of lignin during organosolv pulping and increased hydrolysability of the residual cellulosic pulp for the production of monomeric glucose. Using this combined treatment, a delignification yield of 93% and an enzymatic cellulose-to-glucose conversion of 75% were obtained.

Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions.

Chitosan particles were functionalized with ferulic acid (FA) and ethyl ferulate (EF) as substrates using laccase from Myceliophtora thermophyla as biocatalyst. The reactions were performed with chitosan particles under an eco-friendly procedure, in a heterogeneous system at 30°C, in phosphate buffer (50mM, pH 7.5). The FA-chitosan derivative presented an intense yellow-orange color stable while the EF-chitosan derivative was colorless. The spectroscopic analyses indicated that the reaction products bound covalently to the free amino groups of chitosan exhibiting a novel absorbance band in the UV/Vis spectra between 300 and 350nm, at C-2 region by the duplication of C-2 signal in the 13C NMR spectrum, via Schiff base bond (NC) exhibiting novel bands in the FT-IR spectrum at 1640 and 1620cm-1. Additionally, antioxidant capacities of chitosan derivatives showed that the chitosan derivatives presented improved antioxidant properties, especially for FA-chitosan derivative (EC50 were 0.52±0.04, 0.20±0.02mg/ml for DPPH and ABTS+ scavenging, respectively).

Correlation between the release of sugars and uronic acid and free oil recovery following enzymatic digestion of oil seed cell walls.

Aqueous extraction of sunflower seeds and rapeseeds with mixtures of hydrolytic enzymes were carried out in 2-L reactor and uronic acid and reducing sugar concentrations were measured during digestion. The goal of the study was to determine if a correlation between those concentrations and free oil exists that would allow predicting free oil release from aqueous solution measurements. Similar concentrations of 4.5 and 4.7 g/L of reducing sugars and 43 and 55 g/L of uronic acid were found for sunflower and rapeseeds, respectively. The corresponding yields of free oil from the two seeds were 83% and 16% of total oil, and this difference was due to the formation of an emulsion in the rapeseed dispersion. Therefore, measurement of reducing sugars or uronic acid concentrations are not sufficient to predict a release of free oil from all oil seeds.

Production and biochemical characterization of a type B ferulic acid esterase from Streptomyces ambofaciens.

For the first time, the presence of a ferulic acid esterase (FAE) was demonstrated in Streptomyces ambofaciens. This extracellular enzyme was produced on a range of lignocellulosic substrates. The maximal level of activity was detected in the presence of either destarched wheat bran or oat spelt xylan as the sole carbon source. We found that 1% (m/v) of destarched wheat bran was the optimal concentration to induce its production. With this inducer, no ferulic acid dimers were released from the cell wall by the produced FAE. Interestingly, rape cattle cake (Brassica napus), which does not contain esterified ferulic acid, was also shown to induce the production of the FAE from S. ambofaciens. The FAE was partially purified from the culture supernatant. The purified enzyme was optimally active at pH 7 and 40 degrees C. The substrate specificity of the FAE from S. ambofaciens was investigated: the highest activity was determined with methyl p-coumarate, methyl ferulate, and methyl cinnamate. Furthermore, the FAE required a certain distance between the benzene ring and the ester bond to be active. According to these biochemical characteristics, the FAE from S. ambofaciens has been classified as a type B FAE.

Accumulation of a cocoa-butter-like lipid by Yarrowia lipolytica cultivated on agro-industrial residues.

Yarrowia lipolytica was cultivated on mixtures of saturated free fatty acids (an industrial derivative of animal fat called stearin), technical glycerol (the main by-product of bio-diesel production facilities), and glucose. The utilization of technical glycerol and stearin as co-substrates resulted in higher lipid synthesis and increased citric acid production than the combination of glucose and stearin. The lipids produced contained significant amounts of stearic acid (50-70%, wt/wt) and lower ones of palmitic (15-20%, wt/wt), oleic (7-20%, wt/wt), and linoleic (2-7%, wt/wt) acid. Single-cell oil having a composition similar to cocoa-butter up to 3.4 g/L was produced, whereas in some cases relatively increased citric acid quantities (up to 14 g/L) were excreted into the growth medium. The microorganism presented a high specificity for lauric, myristic, and palmitic acid, while a discrimination for the stearic acid was observed. As a conclusion, microbial metabolism could be directed by using mixtures of inexpensive saturated fats, glycerol, and glucose as co-substrates, in order to accumulate lipids with predetermined composition, e.g., cocoa-butter equivalents.