Novel phenolic derivatives of pectin: enzymatic synthesis and properties Nadine KARAKI Supervisors: Lionel Muniglia, Abdulhadi Aljawish, Catherine Humeau, Jordane Jasniewski International Conference and Exhibition on Biopolymers and Bioplastics August 10-12,2015 San Francisco, USA

PLan Objective of study Materials and methods Results and discussion Conclusion

Importance of the study OXYDATION ANTIOXIDANTS First, we are going talk about where did the idea of this work come from. In fact, the food oxidation is one of the problem encountered in food industry. It is caused by the pollution, the air, the radiation and many other factors. In order to face this problem, the antioxidants are one solution to reduce free radicals content and to avoid to damage cellular components. That’s why, we had the idea of synthetizing a novel supramolecular structure composed of polymers used in food industry with antioxidant properties. To do so, a direct and an indirect enzymatic grafting of phenolic compounds having antioxidant properties were carried on to prepare a double function food ingredient (antioxidant texturing agent) with novel color. Novel architecture (supra) molecular –antioxidant Direct and indirect enzymatic grafting Ingredient with double functionality and novel color

System definition Biopolymer: Citrus pectin Phenolic compound: Ferulic acid Enzyme: Laccase from Myceliophtora thermophila First of all, we are going to define our system composed of the biopolymer used for the study which is the pectin, the phenolic compound and the enzyme used

Studied biopolymer: pectin (Smooth region), Poly-Galacturonic acid HG-I , RG-I and RG-II (Hairy region) Limitation Feruloylated pectin is taxonomically widespread but of low abundance Citrus Pectin Biosynthesis of Pectin; Harholt et al, 2010 (polyanionic) It is an anionic biopolymer, composed of b-1.4 galacturonic acid forming the homogalacturonan chain called also the smooth region, and rhamnogalacturonan I and II ramified chains forming the hairy region. The pectin is used in food industry as a texturant, Gelifiant, Stabilising agent… It is also used in cosmetics and pharmaceutical fields But why it we need to modify the pectin: 2 Reasons 1-feruloylated pectin 2-hard dissolution, swelling behavior Although it is widely used, pectin has some limitation such as its bad dissolution in water forming lumbs and agglomeration, its swelling behavior and its hydrophilic nature (insoluble hydrophilic coating for sustained release delivery by interfacial complexation process) So, modifying the pectin with a hydrophobic phenolic compound having an antioxidant activity (such as the ferulic acid) can enhance certain properties of pectin. The second point is related to the possibility of Mimicking the nature because feruloylated pectin is taxonomically widespread but of low abundance so adding the ferlic acid to th epectin by an enzymatic approach can be a way to mimic the nature Rajpurohit et al., 2010 Texturant Gelling Agent Stabilizing agent Cosmetics Pharmaceutics Food Forte exprtise sur le chitosane, thèse d’Abdul, un des rares poly cationiques Pectine: gélifiante en fonction de la force ionique -naturellement féruloylées Notre but est donc: assembler des composés naturelles -integrer des composés phénoliques qui se trouvent dans la nature pour aug l’activité antioxy -remimer la nature car on essaie de faire pec ferulo

phenolic compound: Ferulic acid Secondary metabolite in plants, red fruits Hydroxycinnamic acid derivative Natural antioxidant reactive against the Radical Oxygen Species (ROS) Structure: Diabetes Aging Cancer Ferulic acid is a secondary metabolite in plants, exist in a huge quantity in red fruits. It is a hydroxycinnamic acid derivative used as a natural antioxidant reactive against the Reactive oxygen species. It is effective against aging and many diseases such as diabetes cancer… Hypertension Asthma Kumar et al., 2014 C.V.D. Cardiovascular disease 3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid

Polyphenol oxidase: laccase (EC.1.10.3.2) Copper-containing oxidase enzymes Found in many plants, fungi, and microorganisms. Oxidize phenolic compounds  oligomers and thus coupling phenoxy radicals (Sun et al., 2013) or reacting with nucleophilic functions in polymers and thus allowing the grafting. Delignification of lignocellulosics, crosslinking of polysaccharides, bioremediation of the toxic chemicals As for the last member of the system: the biocatalysor responsible of the synthesis of the novel phenolic derivative of pectin is named laccase The laccases are members of the family of Polyphenol oxidase; it is a Copper-containing oxidase enzyme, found in many plants, fungi, and microorganisms. (Aljawish et al.,2015)

Objectives Verification of the feasibility of an enzymatic modification of pectin Evaluation of the impact of enzymatic modification on the structure and the properties of the pectin : Optical propriety Antioxidant activity Physico-chemical proprieties After clarifying the importance of the subject and defining the system, we can cite the main objectives of the study, which are:

Planned strategy Enzymatic functionalization Direct functionalization Indirect functionalization Ferulic acid + pectin + enzyme  pectin-F Ferulic acid+ enzyme POX POX+ pectin Pectin-POX Now, we will explain the functionalization methods used to modify the pectin: the direct and the indirect one Solutions are freeze-dried Powder washed with organic solvent Storage in dessicator

CHARACTERIZATION OF PECTIN DERIVATIVES Native pectin (Pectin-N) UV-Vis FTIR ABTS Pectin-F Pectin-POX Structural characterization LC-MS Radical scavenging activity DPPH SEM NMR After collecting the modified powders: pectin-F and pectin-POX, several experiments were carried out to evaluate the influence of these modifications on the structure and properties of the pectin Physico-chemical properties Phenolic content Hygroscopy Color intensity

Results and discussion

LC-MS analysis of galacturonic acid (GA): main unit of pectin FA-galactose-Laccase FA-Laccase www.wikepedia.com Positive mode P.S: FA=Ferulic acid FA-GA GA-Laccase Because analysing the hole structure gave so much peaks which make the analysis so difficult, In order to determine the mass of the new product formed when adding FA-GA-Laccase A novel peak corresponding to the product formed having m/z=629 The carboxyl group is implicated in the enzymatic modification

Surface analysis (sem) Pectin-N Pectin-POX Pectin-F Pectin-N rough surface As for the surface analysis done by scanning electron microscopy, as you can see in the pictures Pectin-POX (scaly surface) physical adsorption of POX onto pectin Pectin-F (smooth surface) the aromatics were intercalated inside the structure The morphology changes were attributed to effects of the functionalization reaction

Phenolic content and samples color Phenolic content (Folin & Ciocalteu) Color parameters (Datacolor) (C) = (a*2 + b*2)1/2 Samples Concentration of gallic acid Equivalent mg /g of dry weight Native pectin 9.9 +/- 0.8 Pectin-F 54.7 +/- 0.3 Pectin-POX 32.6 +/- 1.2 Samples L* (Light) a* (Red) b* (Yellow) C* Pectin-N 78.8 +/- 0.6 3.2 +/- 0.1 12.7 +/- 0.6 13.1 +/- 0.65 Pectin-F 57.7 +/- 0.3 10.7 +/- 0.1 26.4 +/- 0.1 28.47 +/- 0.65 Pectin-POX 70.0 +/- 1.9 6.4 +/- 0.6 24.4 +/- 0,4 25.23 +/- 0.69 (Color intensity) Starting with the phenolic content of pectin, if we are grafting a phenolic compound to the pectin, the dosage of phenols should show this. The second property tested is the color, this property is tested because the oxidation of ferulic acid give a certain color to the mixture so if the pectin is modified its color must change also. So, as you can see in the right table Direct enzymatic oxidation leading to pectin-F showed the highest phenolic content The color is due to the oxidation reaction Pectin-F has the most intense color  the highest content of phenols

Hygroscopy (Dynamic vapor sorption) RH < 45 %  pectin-F is the less hygroscopic RH > 45 % pectin-N is the less hygroscopic The storage of pectin-F and pectin-POX is more stable inside an environment of less than 50 % of humidity The addition of phenolic group to pectin makes it more hydrophobic The addition of a hydrophobic phenol compound must affect the hydrophilic nature of pectin. To ckeck this a DVS measurement is done

Antiradical activity Modified pectin has better When EC50 the antioxidant activity   EC50 (mg/ml) Pectin-N Pectin-F Pectin-POX Scavenging ability on ABTS+• 116.2 +/- 3.9 11.2 +/- 0.8 11.5 +/-1.2 Scavenging ability on DPPH• 29.5 +/- 0.3 1.4 +/- 0.2 9.0 +/- 0.1 As for the last property studied and the most important is to test the antiradicalar activity of the modified pectin in comparison to the native one. Modified pectin has better antiradical activity due to it highest content of phenol

Enzymatic oxidation of FA and its grafting on pectin: Conclusion Two enzymatic approaches were described to modify the biopolymer pectin: the direct and the indirect one. Structural and physico-chemical properties were investigated : structure, surface, hygroscopy and antioxidant Enzymatic oxidation of FA and its grafting on pectin: Increased the pectin phenolic content, its antiradical activity and its hydrophobic nature Led to colored pectin derivatives Decreased the roughness of surface To summarize the work done,

Lebanon Finally i would like to thank all my colleagues and supervisors in LIBIO family in Nancy France for their friendship and help. And my lovely family in my beloved country Lebanon for their support and blessing And for Erasmus mundus program for their financial support Nancy, France

Thank you for your attention Jeita-lebanon AND A BIG THANKS FOR YOU LADIES AND GENTELMAN FOR YOUR ATTENTION. I will be pleased to answer any question you have. Thank you for your attention