Alkyl Ferulates Research Articles

Antioxidant properties of ferulic acid-based lipophenols in oil-in-water (O/W) emulsions

Butyl ferulate (BF) and octyl ferulate (OF) were investigated to enhance oxidative stability of 1.0% vegetable oil-in-water (O/W) emulsions at pH 3.5 and 7.0 at different concentrations (5, 10, 15, 20, and 25 mg/L of emulsion). Particle size distribution, droplet surface charge, hydroperoxides (PV), hexanal and nonanal were monitored for 14 days at 35 °C. Higher concentrations of BF and OF generated smaller particles at pH 7.0 but not at pH 3.5. Control showed the highest negative surface charge (−58.38 ± 0.31 mV, pH 7.0) compared to BF and OF (−46.71 ± 0.06 mV at levels >0.10 mg/L). Both alkyl ferulates counteracted lipid oxidation, especially at pH 3.5, where PV decreased up to 96% compared to the control and delayed hexanal and nonanal formation. A low content of lipophenols allowed to achieve a tremendous shelf-life extension, demonstrating that BF and OF could be of great interest for food application.

Synthesized alkyl ferulates with different chain lengths inhibited the formation of lipid oxidation products in soybean oil during deep frying

The hydrophilic nature of ferulic acid limits its applications under lipophilic conditions. This study set out to evaluate the antioxidant efficacy of alkyl ferulates with different chain lengths in soybean oil under frying conditions. Ferulic acid was esterified with four unbranched fatty alcohols (C4:0-C16:0), and tert-butylhydroquinone (TBHQ) served as a standard for comparison. The antioxidant effect of alkyl ferulates increased with the alkyl chain length. The addition of antioxidants could inhibit increases in the levels of p-anisidine, total polar compounds, conjugated dienes, conjugated trienes, oxidized triglyceride monomers, triglyceride dimers, triglyceride oligomers, and glycerol core aldehydes efficiently, and the inhibitory effects of hexadecyl ferulate was the strongest. Moreover, hexadecyl ferulate and TBHQ exhibited better inhibitory effects on the generation of n-alkanals, (E)-2-alkenals, and 4-oxo-alkanals determined by 1H nuclear magnetic resonance than others. Hence, the long-chain alkyl ferulates meet the industrial demands for ideal antioxidants with strong antioxidant capacity at high temperatures.

Chemical composition of the lipophilic compounds from the rind and pith of papyrus (Cyperus papyrus L.) stems.

Papyrus (Cyperus papyrus L.) is a sedge plant with a high rate of biomass productivity that represents an interesting raw material to produce chemicals, materials and fuels, which are currently still obtained from fossil resources, in the context of a lignocellulosic biorefinery. In this work, the content and chemical composition of the lipids present in papyrus stems were thoroughly studied. For this, the papyrus stems were separated into the rind and the pith. The lipid content accounted for 4.1% in the rind and 4.9% in the pith (based on dry matter). The main compounds identified in both parts of the papyrus stem were hydrocarbons, n-fatty acids, 2-hydroxyfatty acids, alcohols, alkylamides, mono- and diglycerides, steroids (sterols, ketones, hydrocarbons, esters and glycosides), tocopherols, tocopherol esters, phytol, phytol esters, alkyl ferulates, ω-carboxyalkyl ferulates (and their monoglycerides), and acylglycerol glycosides. The rind presented a predominance of n-fatty acids (6790 mg/kg; that represented 28.6% of all identified compounds), steroid compounds (6255 mg/kg; 26.3%), phytol and phytol esters (4985 mg/kg; 21.0%), and isoprenoid hydrocarbons, namely phytadiene and squalene (2660 mg/kg; 11.2%), while the most abundant lipids in the pith were steroids (8600 mg/kg; 44.4% of all identified compounds) and fatty acids (6245 mg/kg; 32.2%). Due to the great diversity and significant abundance of the compounds identified in papyrus, it can be considered as a potential raw material for biorefineries to obtain valuable phytochemicals of interest to various industrial sectors.

Chitosan-based CLEAs from Aspergillus niger type A feruloyl esterase: high-productivity biocatalyst for alkyl ferulate synthesis.

The enzymatic synthesis of alkyl ferulates is an important reaction in cosmetic and pharmaceutical chemistries, since it may allow to expand the biorefinery concept valorizing biomass wastes enriched in ferulic acid. However, robust biocatalysts for that purpose are scarce. Herein, we have immobilized the type A feruloyl esterase from Aspergillus niger (AnFaeA) as cross-linked enzyme aggregates, employing chitosan as co-feeder (ChCLEAs). High immobilization yields and relative activity recovery were attained in all assessed conditions (> 93%). Furthermore, we enhanced the thermal stability of the soluble enzyme 32-fold. AnFaeA-ChCLEAs were capable to quantitatively perform the solvent-free direct esterification of short- to medium-chain alkyl ferulates (C4-C12) in less than 24 h. By raising the operational temperature to 50 °C, AnFaeA-ChCLEAs transformed 350 mM ferulic acid into isopentyl ferulate with a space-time yield of 46.1 g of product × L-1 × day-1, 73-fold higher than previously reported. The overall sustainability of this alkyl ferulate production bioprocess is supported by the high total turnover number (TTN 7 × 105) and the calculated green metrics (E factor = 30). Therefore, we herein present a robust, efficient, and versatile heterogeneous biocatalyst useful for the synthesis of a wide diversity of alkyl ferulates. KEY POINTS: • CLEAs of feruloyl esterase A from A. niger using chitosan as co-feeder were obtained. • Microenvironment of the biocatalysts allowed to obtain C1 to C18 alkyl ferulates. • Biocatalyst at boundary conditions showed a high productivity of 46 g/L day. Graphical Abstract.

Characterizing the interaction between methyl ferulate and human serum albumin by saturation transfer difference NMR.

Methyl ferulate (MF) is an alkyl ferulate ester that widely exists in edible plants and has application value in the food and medicine industries. Thus, its effect on biological macromolecules should be considered. In this study, we exploit saturation transfer difference NMR (STD-NMR) to characterize the interaction of all protons of MF with human serum albumin (HSA) at the molecular level. STD-NMR and Ka (1.298 × 103 M−1) revealed that protons H1–6 and H8 bound to HSA with a medium affinity. Binding epitope mapping further showed that the aromatic ring played a key role in the HSA–MF interaction. STD-NMR site-marker-displacement experiments and circular dichroism spectroscopy revealed that MF prefered to bind to site II of HSA without changing the basic skeleton of HSA. Computer simulations confirmed these experimental results. Overall, this work elucidates the molecular level interaction of MF with HSA and provides new insights into the possibility of the potential applications of MF in the food and medicine industries.

Alkyl Ferulate Esters as Multifunctional Food Additives: Antibacterial Activity and Mode of Action against Escherichia coli in Vitro.

This work aims to prepare ferulic acid alkyl esters (FAEs) through the lipase-catalyzed reaction between methyl ferulate and various fatty alcohols in deep eutectic solvents and ascertain their antibacterial activities and mechanisms. Screens of antibacterial effects of FAEs against Escherichia coli ATCC 25922 ( E. coli) and Listeria monocytogenes ATCC 19115 ( L. monocytogenes) revealed that hexyl ferulate (FAC6) exerted excellent bacteriostatic and bactericidal effects on E. coli and L. monocytogenes (minimum inhibitory concentration (MIC): 1.6 and 0.1 mM, minimum bactericidal concentration (MBC): 25.6 and 0.2 mM, respectively). The antibacterial mechanism of FAC6 against E. coli was systematically studied to facilitate its practical use as a food additive with multifunctionalities. The growth and time-kill curves implied the partial cell lysis and inhibition of the growth of E. coli caused by FAC6. The result related to propidium iodide uptake and cell constituents' leakage (K+, proteins, nucleotides, and β-galactosidase) implied that bacterial cytomembranes were substantially compromised by FAC6. Variations on morphology and cardiolipin microdomains and membrane hyperpolarization of cells visually verified that FAC6 induced cell elongation and destructed the cell membrane with cell wall perforation. SDS-PAGE analysis and alterations of fluorescence spectra of bacterial membrane proteins manifested that FAC6 caused significant changes in constitutions and conformation of membrane proteins. Furthermore, it also could bind to minor grooves of E. coli DNA to form complexes. Meanwhile, FAC6 exhibited antibiofilm formation activity. These findings indicated that that FAC6 has promising potential to be developed as a multifunctional food additive.

Multifunctional alkyl ferulate esters as potential food additives: Antibacterial activity and mode of action against Listeria monocytogenes and its application on American sturgeon caviar preservation

The alkyl ferulate esters were prepared through lipase-catalyzed reactions by using green deep eutectic solvent-water binary mixtures. Antibacterial effects screening of alkyl ferulate esters against Listeria monocytogenes demonstrated that hexyl ferulate (FAC6) exerted both bacteriostatic and bactericidal effects on Listeria monocytogenes (minimum inhibitory concentration: 0.1 mM, minimum bactericidal concentration: 0.2 mM). The antibacterial mechanism of FAC6 were further investigated to provide more information on practical applications as a multi-functional food additive. The growth curves and time-kill assay also showed the occurrence of cell lysis and significant inhibition of the growth of L. monocytogenes caused by FAC6. PI uptake analysis and massive leakage of cell constituents (K+, proteins and nucleotide) demonstrated that the membrane integrity and permeability were undermined by FAC6. Alterations in morphology and membrane hyperpolarization of L. monocytogenes cells treated with FAC6 further clearly confirmed that it disrupted the cell membrane. Meanwhile, FAC6 interacted with membrane proteins and affected the protein expression system, causing a significant change in contents, constitution and conformation of membrane proteins. Moreover, FAC6 could bind to L. monocytogenes DNA grooves to form complexes. Overall, this research highlights the effectiveness of FAC6 against L. monocytogenes, suggesting that FAC6 with both antioxidant and antibacterial activities can be used as an effective and safe multifunctional food additive for American (Amer) sturgeon caviar preservation.

Hydrolysis of Nonpolar n-Alkyl Ferulates by Feruloyl Esterases.

Ferulic acid is one of the major phenolic acids in plants and can be found esterified to plant cell wall components, but also as long-chain n-alkyl and steryl esters. Microbial feruloyl esterases may play a role in the bioavailability of phenolic acids during human and animal digestion. It is therefore of interest if feruloyl esterases are capable of hydrolyzing nonpolar ferulic acid esters. A series of n-alkyl ferulates with increasing lipophilicity were enzymatically synthesized, and the kinetic constants of their hydrolysis by four feruloyl esterases and a lipase as control were determined. A decrease in Km and kcat could be observed with decreased substrate polarity for all of the feruloyl esterases. Only one feruloyl esterase and the control lipase showed hydrolytic activity toward octadecyl ferulate. These results led to the conclusion that lipophilic ferulates are poor substrates for known feruloyl esterases and more specific esterases/lipases need to be identified.

Formation of ethyl ferulate from feruloylated oligosaccharide by transesterification of rice koji enzyme under sake mash conditions

Formation of ethyl ferulate (EF) and ferulic acid (FA) under sake mash conditions was studied using feruloylated oligosaccharide (FO), prepared from rice grains, as the substrate for rice koji enzyme. EF and FA were produced from FO over six times faster than from alkyl ferulates however, under the same ethanol concentration, only small differences were observed between the EF/FA ratios when either FO or methyl ferulate were used as substrates. Esterification and hydrolysis of FO or methyl ferulate showed similar pH dependencies and similar EF/FA ratios for each substrate in all of the pH ranges tested. Ethanol concentration clearly affected the EF/FA ratio; the ratio increased as ethanol concentration increased. Formation of EF and FA in the sake mash simulated rice digest was accelerated by addition of exogenous FO. These results indicated that supply of FO to sake mash is a crucial step for EF and FA formation, and ethanol is an influencing factor in the EF/FA ratio. The rice koji enzyme reaction suggested that EF and FA are formed through a common feruloylated enzyme intermediate complex by transesterification or hydrolysis, and these reactions occur competitively.

Alkyl chain length impacts the antioxidative effect of lipophilized ferulic acid in fish oil enriched milk

Lipophilization of phenolics by esterification with fatty alcohols may alter their localization in an emulsion and thereby their antioxidant efficacy. In this study, synthesized unbranched alkyl ferulates were evaluated as antioxidants in fish oil enriched milk. Lipid oxidation was determined by peroxide values and concentration of volatile oxidation products. A cut-off effect in the antioxidant efficacy in relation to the alkyl chain length was observed. The most efficient alkyl ferulate was methyl ferulate followed by ferulic acid and butyl ferulate, whereas octyl ferulate was prooxidative and the prooxidative effect increased further with an increment in the alkyl chain length from C8 to C12. Further elongation of the alkyl chain length to C16 and C20 resulted in weak prooxidative effects to weak antioxidative effects depending on the different volatile oxidation compounds developed.

Potent and selective butyrylcholinesterase inhibitors from Ficus foveolata

Four stilbenes (1–4), one inseparable mixture of two alkyl diferulates (5a, 5b), one alkyl ferulate (6) and four flavonoids (7–10) were isolated from Ficus foveolata Wall. Except for quercetin (10), the other ten constituents were isolated from F. foveolata for the first time. In addition, one of the two components in mixture 5 is a new compound identified as (1E,22E)-1,22-docosanediol diferulate (5a). All nine isolated compounds, plus the mixture of 5a and 5b known as 5, exhibited a low or no activity against acetylcholinesterase. However, and interestingly, the stilbenes 1–4 showed a high inhibition towards butyrylcholinesterase. Gnetol (4) had the lowest IC50 value of 1.3μM towards butyrylcholinesterase and showed a reversible and competitive inhibition in the kinetic study.

Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila

A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.

Hydroxycinnamates in suberin formation

Hydroxycinnamates are found associated with suberin in several forms: covalently linked to the aliphatic suberin; in the residue after suberin-removal; and in the non-polar extractives of suberized tissues in the form of alkyl ferulates. Suberin-associated hydroxycinnamates have been found mainly as ferulic acid-derivatives, sometimes as feruloylamides and in a lesser extent as caffeates. Ferulic acid esters of long-chain ω-hydroxyacids are prevalent in the partial depolymerisation products of suberin. Also, enzymes able to catalyze the feruloylation of ω-hydroxyacids were found timely-associated with the suberization process. It is proposed that ferulic acid, and its dimers, through esterification to ω-hydroxyacids, covalently link the suberin aliphatic polyester to suberin-associated polyaromatics. In this case, the known role of ferulates, and related hydroxycinnamates, as cross-linkers of structurally different polymers would be enlarged to suberized cell-walls.

Highly Efficient Esterification of Ferulic Acid Under Microwave Irradiation

A highly efficient synthesis of alkyl ferulates under microwave irradiation is described. The time of these reactions ranged from 3 to 5 minutes, which was much shorter than the traditional synthetic methods, and the alkyl ferulates were obtained in higher yields.

Cloning, characterization and functional expression of an alkalitolerant type C feruloyl esterase from Fusarium oxysporum

A hypothetical protein FoFaeC-12213 of Fusarium oxysporum was found to have high amino acid sequence identity with known type C feruloyl esterases (FAEs) containing a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase. The putative FAE from the genomic DNA was successfully cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in Pichia pastoris X-33 to confirm that the enzyme exhibits FAE activity. The molecular weight (62 kDa) and pI (6.8) were in agreement with the theoretical calculated values indicating the correct processing of the secretion signal in P. pastoris. The recombinant FAE was purified to its homogeneity and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme is a type C FAE showing broad hydrolytic activity against the four methyl esters of hydroxycinnamic acids and strong preference for the hydrolysis of n-propyl ferulate. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from Trichoderma longibrachiatum (a maximum of 67% total FA released after 1-h incubation). The esterase showed broad pH stability making it an important candidate for alkaline applications such as pulp treatment in the paper industry.

Preparation of multipurpose cross-linked enzyme aggregates and their application to production of alkyl ferulates

Commercial multicomponent enzyme preparations, Ultraflo L, Depol 740L and Depol 670L, with feruloyl esterase activity, were tested for the transesterification of methyl ferulate to 1-butyl ferulate in their free and immobilized form using as a reaction system a ternary water–organic mixture consisting of n-hexane, 1-butanol and water. A number of factors affecting enzymes precipitation and cross-linking into cross-linked enzyme aggregates (CLEAs) have been investigated. Consecutive optimization of the precipitant type and cross-linker concentration resulted in CLEAs showing higher operational stability and synthetic activity compared to the free enzymes’ forms. Under certain optimization conditions, conversion yields of 97%, 87% and 5%, were obtained by CLEAs prepared from Ultraflo L, Depol 740L and Depol 670L, respectively. The activities initially present in the three commercial preparations were completely retained after cross-linking resulting in multipurpose biocatalysts which have the potential to carry out different and independent reactions. This work is consistent to the novel CLEA concept called combi-CLEA.

Constituintes químicos das cascas do caule de Cenostigma macrophyllum: ocorrência de colesterol

Phytochemical investigation of the bark of Cenostigma macrophyllum (Leguminosae-Caesapinioideae) resulted in the isolation and identification of valoneic acid dilactone, ellagic acid, lupeol, alkyl ferulate, four free sterols (cholesterol, campesterol, stigmasterol and sitosterol), a mixture of sitosteryl ester derivatives of fatty acids, sitosterol-3-O-b-D-glucopyranoside, stigmasterol-3-O-b-D-glucopyranoside and saturated and unsaturated fatty acids. The structures of the isolated compounds were identified by 1H and 13C NMR spectral analysis and comparison with literature data. The mixtures of 3-b-hydroxysterols and fatty acids were analysed by GC/MS.

Highly Efficient Preparation of Lipophilic Hydroxycinnamates by Solvent-free Lipase-Catalyzed Transesterification

Various medium- or long-chain alkyl cinnamates and hydroxycinnamates, including oleyl p-coumarate as well as palmityl and oleyl ferulates, were prepared in high yield by lipase-catalyzed transesterification of an equimolar mixture of a short-chain alkyl cinnamate and a fatty alcohol such as lauryl, palmityl, and oleyl alcohol under partial vacuum at moderate temperature in the absence of solvents and drying agents in direct contact with the reaction mixture. Immobilized lipase B from Candida antarctica was the most effective biocatalyst for the various transesterification reactions. Transesterification activity of this enzyme was up to 56-fold higher than esterification activity for the preparation of medium- and long-chain alkyl ferulates. The relative transesterification activities found for C. antarctica lipase were of the following order: hydrocinnamate > cinnamate > 4-hydroxyhydrocinnamate > 3-methoxycinnamate > 2-methoxycinnamate approximately 4-methoxycinnamate approximately 3-hydroxycinnamate > hydrocaffeate approximately 4-hydroxycinnamate > ferulate > 2-hydroxycinnamate > caffeate approximately sinapate. With respect to the position of the hydroxy substituents at the phenyl moiety, the transesterification activity of C. antarctica lipase B increased in the order meta > para > ortho. The immobilized lipases from Rhizomucor miehei and Thermomyces lanuginosus demonstrated moderate and low transesterification activity, respectively. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate and 3-(3,4-dimethoxyphenyl)propyl oleate, were obtained by C. antarctica lipase-catalyzed transesterification of fatty acid methyl esters with the corresponding 3-phenylpropan-1-ols in high yield, as well.

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60-90 degrees C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90 degrees C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90 degrees C for at least two weeks.

The feruloyl esterase system of Talaromyces stipitatus: Determining the hydrolytic and synthetic specificity of TsFaeC

The active site of the recombinant Talaromyces stipitatus type-C feruloyl esterase (TsFaeC) was probed using a series of C1-C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of k(cat) and k(cat)/K(m) values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of feruloyl esterase to catalyze feruloyl group transfer to l-arabinose, using as a reaction system a ternary water-organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.