1. 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

2. PLan Objective of study Materials and methods Results and discussion
Conclusion

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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:

9. 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

10. 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

11. Results and discussion

12. LC-MS analysis of galacturonic acid (GA): main unit of pectin
FA-galactose-Laccase
FA-Laccase
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

13. 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

14. 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
/- 0.65
Pectin-POX
70.0 +/- 1.9
6.4 +/- 0.6
24.4 +/- 0,4
/- 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

15. 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

16. Antiradical activity Modified pectin has better
When EC the antioxidant activity
EC50 (mg/ml)
Pectin-N
Pectin-F
Pectin-POX
Scavenging ability on ABTS+•
/- 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

17. 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,

18. 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

19. 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