*2.8.8. Agitation speed*

the BY decreased to 45 and 11%, respectively, when *p*-coumaric and caffeic acids were used as substrates. The inhibitory effect of p-OH substituent was most likely due to their electronic donating effect rather than to their steric hindrance in the enzyme-active site as the inhibition was much less significant (56%) in the presence of a double bond on the side chain conjugated

Temperature changes effect on different parameters including enzyme stability, affinity, and preponderance of the competing reactions [71]. Temperature normally affects lipase activity, and high temperatures usually increase the initial transesterification rate. However, high reaction temperatures deactivate the enzyme due to its protein nature [35]. The optimal temperature used in transesterification reactions is mainly based on considering properties of feedstock, such as melting behavior at different temperatures as well as the reaction system that is with or without solvent. In a solvent-free system, the temperature is maintained high

The optimal temperature for the most immobilized lipases ranges from 30–60°C, while it tends to be lower for free lipases. Heat stability of lipase also depends on whether a substrate is present. This is because substrates remove excess water from the immediate vicinity of the enzyme, hence limiting its overall conformational mobility. Ishihara et al. [116] studied the effect of temperature on vanillyl alcohol acylation with nonanoic acid to give vanillyl nonanoate in *n*-hexane solvent medium. The authors found that the optimum temperature for enzymatic acylation was 70°C. Higher temperatures than 70°C lead to decrease the conversion yield due to the deactivation of enzyme at high temperature. The effect of temperature on the synthesis of capsiate analog by lipase-catalyzed esterification of vanillyl alcohol and conjugated linoleic acid (CLA) was presented [47]. The range of temperature tested was from 30 to 60°C. The results demonstrated that the yield increased when the temperature increased from 30 to 50°C. However, when temperature increased to 60°C, there is no increase effect on

Normally, as the enzyme concentration increases, the reaction equilibrium will be shifted quickly toward the synthesis [117]. However, for economic reasons, it is important to reduce the enzyme loading and the reaction time. In addition, the presence of high enzyme concentration in the reaction medium may increase the probability of its collision with the substrate subsequently enhancing the reaction rate [118]; however, after reaching certain enzyme concentration, the conversion yield was constant. Carrin et al. [117] reported that during the Lipozyme TL-IM-catalyzed acidolysis of sunflower oil with palmitic acid and stearic acid mixture, the extent of palmitic and stearic acids incorporation was enhanced by increasing the amount of enzyme in the reaction; however, when the enzyme concentration was greater than 8% by weight of substrates, there was no significant increase in the esterification yield. The effects of lipase concentration on the synthesis of capsiate analog were depicted in the

with the aromatic ring of DHPA.

enough to keep the substrates in liquid state [40].

104 Phenolic Compounds - Natural Sources, Importance and Applications

*2.8.6. Reaction temperature*

the yield.

work of [47].

*2.8.7. Enzyme concentration*

In a heterogeneous enzymatic system, it is important to ensure that the rate of substrate diffusion will not limit the rate of the synthesis reaction. The increase in agitation speed may decrease the boundary liquid layer surrounding the porous support, leading to lower diffusion limitations. Lue et al. [102] reported an increase of the enzymatic activity from 108.6 to 156.5 nmol/g/min, when the agitation speed of the system was increased from 0 to 200 rpm. The increase in the enzymatic activity indicated that external diffusion limitations of substrates did occur within the range of agitation applied. Kumari et al. [118] reported that carrying the reaction at the optimum agitation speed can limit the external mass transfer limitations, in the case of immobilized enzymes, where the reactants need to diffuse from the bulk oil to the external surface of the enzyme particles and from there, subsequently to the interior pores of the catalyst. In addition [44] investigated the effect of agitation speed on the conversion yield of phenolic lipids synthesized from flaxseed oil and DHCA; the results have shown that the conversion yield increased significantly from 39 to 62.5% when the agitation speed was increased from 50 to 150 rpm, before it was decreased to 44.8% at agitation speed of 250 rpm. The low conversion yield could be attributed to insufficient agitation rate, a condition in which a hydrophilic layer of glycerol may be formed around the enzyme, limiting hence the mass transfer rate of the oil to the surface of the lipase.

### *2.8.9. Carbon chain length*

The effect of carbon chain length of fatty alcohols on the reaction rate was examined by [119]; the esterification of C4–C18 straight-chain fatty alcohol with dihydrocaffeic acid (DHCA), as a model of phenolic acid, was systematically evaluated. The results indicated that the conversion of DHCA was significantly affected by the number of carbon chain of fatty alcohols. Conversion yield of 95% was achieved within 3 days when hexanol was used as an acyl acceptor, while only 56 and 44% conversions were achieved when 1-butanol and octadecanol were employed, respectively. The conversions of ferulic and caffeic acids under the same conditions were much lower than was that of DHCA. In another by [120], various alkyl cinnamates were formed in high to moderate yield by lipase-catalyzed esterification of cinnamic acid and its analogs with fatty alcohols in vacuo at moderate temperatures in the absence of drying agents and solvents.

Several carboxylic acids of different chain lengths from acetic, propionic, butyric, caproic, and caprylic acids were tested via an enzymatic esterification reaction to produce hexyl ester in *n*-hexane and supercritical carbon dioxide (SCCO2 ). The reactions were carried out at 40°C, and the amount of enzyme used was 13.8 g/mol alcohol. Substrates were added at equimolar concentrations, with sufficient stirring to avoid external diffusion control. The results in both solvents show that the reaction rate increases with the chain length of the acid, but the final yields were similar.

### **2.9. Analysis and characterization of phenolic lipids**

The structural analyses of phenolic lipids have been carried out using a wide range of various techniques. These mainly include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), and liquid chromatographymass spectrometry (LC-MS). Thin-layer chromatography has been used for initial qualitative analyses of substrates by employing a wide range of organic solvent mixtures. Products from the esterification reactions are characterized and analyzed by TLC using silica gel G-25 plates [10, 47]. The elution solvents used depend on the nature of synthesized compounds. In the study of [10], the elution solvent used was chloroform/methanol mixture (80:20, v/v) and pure chloroform; the plates were visualized under UV light (254 nm), meanwhile, in the work of Hong et al. [47], the elution solvent was n-hexane/diethyl ether/formic acid (160:40:5.5, v/v/v), and the plate was visualized with 0.2% (w/v) 2,7 dichlorofluorescein in methanol solution under UV light.

High-performance liquid chromatography (HPLC) has often been used over other instrumentations and has shown scientifically to be the overall preferred method of choice for quantification and separation of phenolic lipids following synthesis reactions. Phenolic lipids were separated on C18 reverse-phase column using a gradient elution system with UV detection at 280 nm [10]. Gas-liquid chromatography (GLC) analysis has been conducted for determining the fatty acid composition of the synthesized phenolic lipids. REF has reported on the GC analysis of phenolic lipid esters through the use of a CP-Sil CB-MS column linked to an FID detector.

Recent research on phenolic lipids has also made using liquid chromatography-mass spectrometry (LC-MS) that is considered being one of the most powerful techniques used for the characterization of biomolecules due to its high sensitivity and specificity. Generally, its application is oriented toward the specific detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture). LC-MS has been used for the structural characterization of lipids and phenolic lipids [10, 116].

Many lipid systems have been studied by Fourier transform infrared spectroscopy (FTIR) in order to determine several aspects including the degree and the form of unsaturation of the acyl groups as well as their length [121]. The infrared region of the electromagnetic spectrum extends from 14,000 to 50 cm−1 and is divided into three areas: the far infrared from 400 to 50 cm−1, the mid infrared region from 4,000 to 400 cm−1, and the near infrared from 14,000 to 4,000 cm−1 [122].
