Synthesis of phenolic lipids (PL) compounds
Phenolic lipids (PL) are types of fats and oils modified to improved nutritional or physical properties by incorporate phenol compound on the glycerol backbone. Phenolic lipids play an important role as antioxidant and biological active compounds, but their contents in the nature are minor and the procedures for separation and purification are not easy, very expensive and take a long time, which makes their applications in the food or cosmetic industry very inconvenient. Consequently, the synthesis of PL has attracted more attention in recent years due to it is a good way to improve the hydrophobic nature of phenolic compounds, which could be achieved by chemical or enzymatic synthesis.
Chemical synthesis of phenolic lipids
Chemical synthesis is a traditional method that used for PL preparation. Synthesis of PL through chemical synthesis could be done by using Friedel–Crafts acylation reaction or Fisher acid catalysis esterification. These processes are generally carried out at relatively high temperatures and pressures under anhydrous conditions, using rather unspecific alkali metal or alkali catalysts. Some related works have been provided in this topic, one of them is the work of (Qianchun et al., 2011) about the chemical synthesis of phytosterols esters of polyunsaturated fatty acids (PUFA), that could be used in different formulations of functional foods. Direct esterification of phytosterols with PUFA was catalyzed by sodium bisulfate to produce sterol esters of PUFA without organic solvent. The modeling of sodium bisulfate with superfluous fatty acids as solvents to synthesize phytosterols esters of PUFA was successfully performed with degree of esterification up to 96% and less oxidative products in the reaction process (Qianchun et al., 2011).
The chemical esterification of flavonoids with some fatty acids was provided by (Mainini et al., 2013) and its product exhibited lipophilic, antiradical and antioxidant properties. Works reported by (Zhong andShahidi, 2011;2012) on epigallocatechin gallate (EGCG) the predominant catechin in tea was structurally modified by esterification with fatty acids, including stearic acid (SA), docosapentaenoic acid (DPA), eicosapentaenoic (EPA) and docosahexaenoic (DHA). The esterification of EGCG with these fatty acid using acylating agents, namely, the acyl chlorides, resulted in yields of 65.9, 42.7 and 30.7 for SA, EPA and DHA respectively (Zhong andShahidi, 2011). This esterification leads to produce various compounds that have anti-inflammatory effect and also showing higher inhibition effect against hydroxyl and peroxyl radical-induced DNA scission (Zhong andShahidi, 2012). Chemical synthesis of PL meets some partial needs to a certain extent, this pattern possesses a low degree of regioselectivity and is generally accompanied by drastic reaction conditions, many intermediary stages and purification steps to remove byproducts and catalyst residues and generating extra wastes to dispose of. The main drawbacks to chemical transesterification are: (1) non-selectivity leading to random distribution of FAs, (2) isomerization of sensitive PUFAs by the alkali catalyst, (3) production of fatty acid soaps and unwanted by products and (4) requiring substantial post treatment and downstream processes, especially when food applications are concerned.
Enzymatic synthesis of phenolic lipids (PL)
Enzymes are used more and more in various applications of different fields such as pharmaceutical, cosmetic or food industry. In the past years, a better understanding of enzymes functionalities and catalytic behaviors, together with the progress of molecular engineering has led to new applications for various types of enzymes. Enzymatic synthesis of PL from fats and oils is receiving a lot of attention as a method for their modification because of the enzymes offers the advantages of milder reaction conditions, minimization of side reactions and by product formation, a selective specificity, a wider variety of pure synthetic substrates, fewer intermediaries and purification steps, and a more environmentally friendly process (Torres de Pinedo A. T. et al., 2005b). Even if most reactions of enzymes may be more expensive than chemical reagents, the enzyme catalyzed acylation is a well-mastered technique for synthesis of selectively modification of PL at present. A high degree of conversion to the desired products could be achieved under the optimal reaction conditions. The enzymatic processes can be used in the production of fats and oils containing beneficial fatty acids and phenolic compounds. Some reviews have given a comprehensive understanding and shown a whole outline on the enzymatic synthesis of PL (Aziz andKermasha, 2014b ; Karboune et al., 2008 ; Sabally Kebba et al., 2006a ; Sorour N. et al., 2012b ; Tan et al., 2012 ; Zheng Y. et al., 2010). In particular, enzymes appear to be very effective for the synthesis of molecules involving the grafting of a lipophilic moiety or a hydrophilic one. This review will be described and discussed some of the recent works in the field of enzymes assisted acylation of fatty acids with phenolic compounds in order to modify the hydrophilic/lipophilic properties of the initial molecules to obtain new products with multi-functional properties combining for example, antimicrobial, antioxidant and emulsifying properties. The enzymatic synthesis of phenolic lipids have been reported previously (Hong et al., 2012 ; Mbatia B. et al., 2011 ; Speranza andMacedo, 2012 ; Tan et al., 2012 ; Tan andShahidi, 2013). A lot of enzymes can be used in the synthesis of PL and selectivity is the most important characteristics of enzymes used in phenolic lipid synthesis. Lipase is the most enzymes used in this type of process because of high selectivity, lower overall reaction time and fewer side reactions when compared with chemical methods (Li C. et al., 2014). An example of a synthesis reaction catalyzed by the lipase is shown in Figure 1.6. This overwhelming interest is based largely on consumers’ desire to maintain overall well-being with minimal effort and an industries’ ability to respond to this need. Furthermore, with the consumption of manufactured foods continually on the rise, there is a distinct advantage to providing more healthful choices for consumers. The concept of a natural phenolic lipid composed of a long-chain aliphatic and phenolic moiety readily fits this mould, particularly since the inclusion of unsaturated lipids into these compounds could result in additional nutritional benefits. Lipases constitute the most important group of biocatalysts for biotechnological applications.
Enzyme reactions in organic solvent media (OSM)
Enzymes in organic solvents have manifested good selectivity and stability; however, catalytic activities in this environment are generally lower than in aqueous solutions. This could be partly explained by the fact that in low water environments, enzymes are less flexible. On the other hand, the activities of enzymes also depend on the type of organic solvent, since some are known to inactivate or denature biocatalysts. Meanwhile the advantages of using organic solvent media are increased solubility of hydrophobic compounds that permits for greater interactions between substrates and enzymes as well as advantageous, partitioning of substrates and products; specifically, this is because partitioning of products away from the enzyme can decrease the possibility of inhibition due to excess product around the biocatalyst (Reddy et al., 2011 ; Sanchez et al., 2014).
When enzymes are placed in OSM they exhibit novel characteristics such as altered chemo- and stereo-selectivity, enhanced stability, increased rigidity, insolubility and high thermal stability (Dossat et al., 2002). It has also been reported that the thermal stability of lipases can be improved in organic solvent systems since the lack of water prevents the unfolding of the lipase at high temperatures (Rahman et al., 2005). The activity of lipase in OSM depends on the nature and concentration of the substrate and source of the enzyme. Moreover, the organic solvent used can dramatically affect the activity of the lipase. Lipases are more active in n-hexane, n-heptane and isooctane as compared to other solvents, such as toluene, ethyl acetate and acetonitrile (Lima et al., 2004 ; Namal Senanayake andShahidi, 2002). It has been reported that the hydrophobicity of the solvent can affect the degree of acyl migration during transesterification using a 1,3-specific lipase (Kim et al., 2002). Since the choice of organic solvents based on minimization of acyl migration may conflict with maximization of transesterification, acyl migration is usually minimized by reducing reaction times (Jennings andAkoh, 1999). With increasing concern for the environment, synthesis of PL in solvent-free systems (Chaibakhsh et al., 2009 ; Sun et al., 2007 ; Zheng Yan et al., 2009b) and ionic liquids systems (Guo andXu, 2006) has been extensively studied.
(Mbatia B. et al., 2011) study the enzymatic synthesis of vanillyl esters from fish oil and vanillyl alcohol in acetone solvent medium. Lipase catalyzed esterification of vanillyl alcohol with different fatty acids was carried out by (Reddy et al., 2011) to synthesis of capsiate analogues. Equimolar concentration of vanillyl alcohol and fatty acid were solubilized in tert-butanol and esterified using Candida antartica lipase (Novozyme 435) at 55°C for 4h.
Enzyme reactions in solvent free medium (SFM)
Enzymatic catalysis in solvent-free medium (SFM) has attracted considerable interest in the recent years (Feltes et al., 2012). It used as an efficient approach to synthesis of natural products, pharmaceuticals, and food ingredients. Under non-aqueous conditions, the industrial utility of enzymes can be improved; recovery of product and enzyme are ease, and the ability to catalyze reactions that are not favorable in aqueous solutions (Jin et al., 2003). However, it would be technically beneficial if the enzymatic reactions were performed in mixtures of substrates in the absence of solvents. Lipase catalyzed PL have 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 solvent-free 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 (Dossat et al., 2002). On the other hand, solvent-free systems, which are a simple mixture of reactants and the biocatalyst, present the advantages of using nearly non-aqueous 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 (Dossat et al., 2002 ; Sandoval et al., 2012b).
Lipase-catalyzed transesterification in SFM is important in industrial applications, and several studies reported that the immobilized Candida antarctica lipase (Novozym 435) could effectively catalyze the transesterification of oils in SFM (Aziz et al., 2012 ; Feltes et al., 2012). A study of (Dossat et al., 2002) on transesterification of sunflower oil with butanol-1 by Lipozyme® was carried out in a SFM, and the reactor was maintained without any loss in activity for 3 months. This result was very different to that obtained using hexane, which leads to a total loss of the enzyme activity within a few hours. The mixture has interesting lubricant and surfactant properties.
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., 2012) 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 (Sorour Noha et al., 2012a ; Sorour N. et al., 2012b ; Sun et al., 2012). 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 Candida antarctica lipase, and the yield was 96% (Sun et al., 2012).
Enzymatic synthesis of DHA-VE in organic medium
Enzymatic acylation reactions were achieved in organic solvent, under atmospheric pressure. Reaction media were prepared by solubilizing VA (100 mM, 15.4 g/L) and DHA-EE (200 mM) in 2 mL of acetonitrile. The solvent was pre-dried on 4-Å molecular sieves before use, aiming to low water activity below 0.1. Reactions were performed in 10-mL amber tubes submitted to orbital shaking (300 rpm) and initiated by adding 20 g/L of Novozym 435®. This protocol is further referred as solvent system.
Synthesis of DHA-VE in molten media
Reactions were performed under either atmospheric or reduced pressure. For syntheses achieved under atmospheric pressure, reaction media were prepared by solubilizing VA (200 mM, 30.8 g/L that corresponds to the maximal solubility of the substrate at 50°C) in a large excess of DHA-EE as acyl donor (2 mL), at 50°C. Reactions were performed in 10-mL amber tubes and initiated by adding 20 g/L of Novozym 435®. After 72 h of reaction, shaking was stopped allowing the decantation of enzyme particles. The supernatant was removed thus ending the reaction. This protocol led to reaction system A.
Syntheses achieved under reduced pressure were performed in the sample flask of a rotary evaporator Figure 9. Temperature and pressure conditions were set to 37°C and 500 mbar, so that the by-product of the reaction, i.e. ethanol, could be eliminated during the syntheses while avoiding VA evaporation. A rotation speed of 250 rpm was applied. Reaction media were prepared by solubilizing VA (162 mM, 25 g/L that corresponds to the solubility of the substrate at 37 °C) in 10 mL of DHA-EE. Reactions were started by adding 20 g/L of Novozym 435®. After 72 h, the enzyme was eliminated by filtering the reaction media. This protocol led to reaction system B.
Table of contents :
Chapter 1: Literature review
1.1. Fatty acids
1.2. Health Benefits of omega-3 fatty acids
1.3. Phenolic compounds
1.3.1. Nutritional and antioxidant properties
4. Synthesis of phenolic lipids (PL) compounds
1.4.1. Chemical synthesis of phenolic lipids
1.4.2. Enzymatic synthesis of phenolic lipids (PL)
1.5.1. Definition, sources and applications
1.5.2. Mechanism of action
5. 2. 3. Transesterification
1.5.3. Selectivity and Specificity of lipase
1.6. Enzyme reactions in organic solvent media (OSM)
1.7. Enzyme reactions in solvent free medium (SFM)
1.8. Parameters affecting the enzyme activity and conversion yield ofM phenolic lipids
1.8.1. Influence of solvent
1.8. 2. Lipase conditioning
1.8. 3. Influence of water activity
1.8.4. Molecular sieve
1.8.5. Substrate composition and concentration (molar ratio)
1.8.6. Reaction Temperature
1.8.7. Enzyme concentration
1.8.8. Agitation Speed
1.8.9. Carbon chain length
1.9. Analysis and characterization of phenolic lipids
1.10. Application of phenolic lipids
Chapter 2: Materials and Methods
2.1.1. Chemicals and enzyme
2.2.1. Enzymatic synthesis of DHA vanillyl ester (DHA-VE)
22.214.171.124. Enzymatic synthesis of DHA-VE in organic medium
126.96.36.199. Synthesis of DHA-VE in molten media
188.8.131.52. Process intensification
2.2.2. Kinetic following of the syntheses
2.2.3. Purification of DHA-VE by flash chromatography
2.2.4. Structural analyses
184.108.40.206. Liquid chromatography–mass spectrometry (LC–MS)
220.127.116.11. Nuclear magnetic resonance (NMR)
2.2.5. Evaluation of antioxidant activity
18.104.22.168. Radical scavenging activity
22.214.171.124.1. DPPH• Radical Scavenging Activity method
126.96.36.199.2. ABTS+• method
188.8.131.52. Inhibition of DNA scission
2.2.6. Study the oxidative stability of DHA phenolic esters (DHA-VE)
184.108.40.206. Accelerated oxidation test
220.127.116.11 Determination of conjugated dienes
18.104.22.168 FTIR Instrumentation
22.214.171.124 Spectral Acquisition
2.2.7. Biological activities and bioavailability
126.96.36.199. Primary cell cultures and treatments
188.8.131.52. Animals and diets
184.108.40.206. Fatty-acid analysis
2.2.8. Applied the esterification method with salmon oil
220.127.116.11. Enzymatic extraction of oil from salmon heads
18.104.22.168. Preparation and analysis of fatty acid methyl esters by GC
22.214.171.124. Lipid class analysis by thin-layer chromatography
126.96.36.199. Synthesis enzymatic reaction
188.8.131.52. Analysis and monitoring of reaction mixtures by HPLC and LC-MS
2.2.9. Oxidative stability of esterified oil
2.2.10. Application of synthesis phenolic lipids in food emulsion
184.108.40.206. Oil in water (O/W) emulsions preparation
220.127.116.11. Oxidative stability experiments
18.104.22.168.1. Peroxide value (PV)
22.214.171.124.2. Conjugated diene value (CD)
126.96.36.199.3. Anisidine value (p-An.v)
188.8.131.52.4. Thoibarbituric acid reactive substances (TBARS) assay
2.2.11. Statistical analysis
Chapter 3: Results and Discussion
Partie 1: Enzymatic production of bioactive docosahexaenoic acid phenolic ester
1.2. Enzymatic production of bioactive docosahexaenoic acid phenolic ester
1.3. Contribution de l’article
Partie 2: Oxidative stability of DHA phenolic ester
2.2. Oxidative stability of DHA phenolic ester
3.3. Contribution de l’article
Partie 3: Enzymatic synthesis of vanillyl fatty acid esters from salmon oil in solvent-free medium.
3.2. Enzymatic synthesis of vanillyl fatty acid esters from salmon oil in solventfree medium.
3.3. Contribution de l’article
Partie.4: Applications of phenolic lipids synthesized to food systems
4.2. Assessment of antioxidant capacity of DHA phenol ester in food emulsions
4.3. Contribution de l’article
Conclusions and perspectives