*2.8.1. Effect of solvent*

The enzymatic synthesis of vanillyl-PUFA esters from fish oil and vanillyl alcohol in acetone solvent medium was studied by [10]. Lipase-catalyzed esterification of vanillyl alcohol with different fatty acids was carried out by [51] to the synthesis of capsiate analogs. Equimolar concentration of vanillyl alcohol and fatty acid was solubilized in *tert*-butanol and esterified

Enzymatic catalysis in solvent-free medium (SFM) has attracted considerable interest in the recent years [90]. It used as an efficient approach to the synthesis of natural products, pharmaceuticals and food ingredients. Under nonaqueous conditions, the industrial utility of enzymes can be improved, recovery of product and enzyme is eased, and the ability to catalyze reactions that are not favorable in aqueous solutions [91]. However, it would be technically beneficial if the enzymatic reactions were performed in mixtures of substrates in the absence of solvents. Lipase-catalyzed PL has been extensively studied in systems using organic solvents; however, if such a process is intended to be used in the food industry, it is preferred to develop solventfree systems. The downside of organic solvents is that they are expensive, toxic and flammable and their use involves higher investment costs to meet safety requirements [80]. On the other hand, solvent-free systems, which are a simple mixture of reactants and the biocatalyst, present the advantages of using nearly nonaqueous organic solvents while offering greater safety, reduction in solvent extraction costs, increased reactant concentrations and consequently higher volumetric productivity defined as kg product per unit of reactor volume [53, 80].

Phenolic lipids have been received increasing attention in the food area, since they are a good way for providing nutraceutical FA to consumers. Hong et al. [47] studied the esterification of vanillyl alcohol with conjugated linoleic acid under vacuum in solvent-free system. Further studies on the enzymatic synthesis of structured phenolic lipids in SFM have also been conducted by [34, 44, 92]. In these studies, phenolic acids were esterified with fatty acids resulted in the formation of more lipophilic constituents that can be used as a nutraceutical product. In addition, feruloylated mono- and diacylglycerols were synthesized in SFM using *C. antarctica*

Lipase-catalyzed synthesis in SFM has a number of advantages as compared to that in OSM, including the use of a smaller reaction volume, maximization of substrate concentration and with no additional solvent recovery. In addition, downstream processing is easier as fewer purification steps are required providing significant cost savings, as well as toxic organic solvents are completely avoided (clean conversions), and an increase in the volumetric productivity can be achieved [80]. However, there are some problems with the use of SFM, mainly, the high viscosity of the medium as well as the production of high amounts of glycerol, free FAs as by-products. These by-products affect the reaction equilibrium and limit the mass transfer rate [93]. Thus, the development of a bioprocess for the lipase-catalyzed synthesis in

**2.8. Parameters affecting the enzyme activity and conversion yield of phenolic lipids**

Grafting of phenolic compound substrates with lipids is the major difficulty to overcome in such lipase-catalyzed reactions. Several parameters must be considered in order to achieve

using *C. antartica* lipase (Novozym 435) at 55°C for 4 h.

100 Phenolic Compounds - Natural Sources, Importance and Applications

**2.7. Enzyme reactions in solvent-free medium (SFM)**

lipase, and the yield was 96% [92].

SFM is of major interest but with great challenge.

The interesting strategy is to carry out the synthesis reaction without using solvent. However, when it is not possible, the choice of an adequate solvent is important. The type of organic solvent employed can dramatically affect the reaction kinetics and catalytic efficiency of lipases [94]. Two factors must be considered when solvent is selected; solvent affects the enzyme activity and solvent effect on the equilibrium position of the desired reaction. Polarity of the solvents is an important characteristic which determine the effect of solvents on enzymatic catalysis reactions. Log *P* value, the partition coefficient between water and octanol, is used as the indicator of solvent polarity. Laane et al. [95] reported that solvents with log *P* < 2 are not suitable for enzyme-catalyzed systems, since they strip off the essential water from the enzyme and therefore inactivate them. However, solvents with log *P* values in the range of 2–4 were weak water distorters, in which enzymes display medium activity and solvents with log *P* > 4 are ideal media for enzyme-catalyzed systems since they do not distort the essential water from the enzyme. Therefore, intermediate polarity media are often chosen. Other factors that must be taken into account in determining the most appropriate solvent for given reaction include solubility of reactants, solvent inertness, density, viscosity, surface tension, toxicity, flammability, waste disposal, and cost [96]. A good contact between the substrates must be obtained, and the selected solvent must be solubilizing them at least partially.

Various authors have tried to find original strategies to improve enzyme activity in organic solvent [71, 93, 94]. The effect of solvent concentration on the conversion yield of phenolic lipids synthesized from flaxseed oil and phenolic acids was demonstrated by [41]. Solvent concentration of 7% was the best concentration with 61.1% of conversion yield.

### *2.8.2. Lipase conditioning*

Another important parameter in the synthesis reactions of phenolic lipids (PLs) is concerning with the enzyme itself and especially its conditioning. Various techniques for lipase conditioning have greatly improved during the last 10 years in the field of enzyme immobilization, chemical modification, or molecular engineering [97, 98]. Lipases are used after immobilization on a support. Different carrier materials are employed, and the resulting immobilized enzyme usually exhibits an improved thermostability compared to its free form. Moreover, the use of immobilized enzymes allows an easy removal and recovery of the biocatalyst once the reaction is over [99]. Lipase from *Candida rugosa* was immobilized onto montmorillonite via two techniques, i.e., adsorption and covalent-binding montmorillonite [100].

### *2.8.3. Influence of water activity*

Water content refers to the total amount of water present in the reaction system. Controlling of water activity is very important in lipid modification processes. Water content in the reaction system is a determining factor in whether the reaction equilibrium will progress toward hydrolysis or ester synthesis [101]. While ester synthesis depends on low water content, too low water activity prevents all reaction from occurring. The monolayer of water on the surface of enzyme is required to maintain the three-dimensional structure of the enzyme, which is essential to enzymatic activity [102]. This layer acts as a buffer between the enzyme surface and the bulk reaction medium. However, too much water can cause hydrolysis of the TAG [14]. The activity of lipases at different water activities is dependent on the source of the enzyme and the type of solvent and immobilization support used [103]. Lipases from molds have shown to be more tolerant to low water activity than bacterial lipases. The optimal water content for most interesterification reactions by different lipases has been reported to be in the range of 0.04 to 11% (w/v) [104].

However, the amount of water in the system should be minimized in order to decrease the byproducts. Lipases tend to retain the greatest degree of original activity, when immobilized on hydrophobic supports. Therefore, when the immobilized lipase contacts with oil in water emulsion, the oil phase tends to associate with and permeate the support, which can be assumed that an ordered hydrophobic network of lipid molecules will surround the support. Any water that reaches the enzyme for participation in the reaction must diffuse from the bulk emulsion. Thus, to avoid diffusional limitations, the oil phase must be well saturated with water [105].

Zhao et al. [106] investigated the effect of different reaction parameters on the enzymatic acidolysis of lard with capric acid catalyzed by Lipozyme TL-IM. They achieved the highest incorporation of capric acid (35.56 mol%) without added water. The amount of incorporation was almost constant up to 10% added water but decreased significantly above this amount. The current research work shows that Lipozyme TL-IM-catalyzed interesterification can easily be moved to the industrial sector for commercial exploitation. Both stirred tank reactors [107] and PBR [108, 109] can be used for the production of plastic fats, and the control of water activity in the system presents no particular difficulty, as is often the case in other lipase-application systems, in which the lipase activity was not affected by the reduction of water content in the system [107, 110].
