Novel phenolic derivatives of pectin: enzymatic synthesis and properties

Nadine KARAKI

International Conference and Exhibition on Biopolymers and Bioplastics

August 10-12,2015 San Francisco, USA

Supervisors: Lionel Muniglia, Abdulhadi Aljawish, Catherine Humeau, Jordane Jasniewski

• Objective of study

• Materials and methods

• Results and discussion

• Conclusion

PLAN

IMPORTANCE OF THE STUDY

OXYDATION

Novel architecture (supra) molecular –

antioxidant

ANTIOXIDANTS

Direct and indirect enzymatic graftingIngredient with double functionality

and novel color

SYSTEM DEFINITION

1. Biopolymer: Citrus pectin

2. Phenolic compound: Ferulic acid

3. Enzyme: Laccase from Myceliophtora thermophila

STUDIED BIOPOLYMER: PECTIN

Poly-Galacturonic acid

(polyanionic)

• Texturant• Gelling Agent• Stabilizing agent

• Cosmetics• Pharmaceutics• Food

Citrus Pectin

Rajpurohit et al., 2010

LimitationFeruloylated pectin is taxonomically widespread but of low abundance

HG-I , RG-I and RG-II(Hairy region)

(Smooth region),

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

C.V.D.Cardiovascular disease

AsthmaHypertension

3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid

Kumar et al., 2014

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

OH

OH

R

O

O

R

OH

OH

R

O

O

R

OH

R

O

R

.

Laccase

O2

Laccase

O2

Laccase

O2

+ H2O

+ H2O

+ H2O

o-diphenol o-quinone

p-diphenol p-quinone

Mono-phenol Semi-quinone

(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

PLANNED STRATEGY

Direct functionalization Indirect functionalization

Ferulic acid+ enzyme POXPOX+ pectin Pectin-POX

Ferulic acid + pectin + enzyme pectin-F

Enzymatic functionalization

Solutions are freeze-driedPowder washed with organic solvent

Storage in dessicator

Pectin-FPectin-POX

UV-Vis

Physico-chemical properties

Phenolic content Hygroscopy

Radical scavengingactivity

Structuralcharacterization

Native pectin(Pectin-N)

CHARACTERIZATION OF PECTIN DERIVATIVES

SEM DPPH

ABTS

Color intensity

LC-MS

FTIR

NMR

RESULTS AND DISCUSSION

LC-MS ANALYSIS OF GALACTURONIC ACID (GA): MAIN UNIT OF PECTIN

FA-Laccase

FA-GA

GA-Laccase

FA-GA-Laccase

FA-galactose-Laccase

A novel peak corresponding to the product formed having m/z=629 The carboxyl group is implicated in the enzymatic modification

P.S: FA=Ferulic acid

Positive modewww.wikepedia.com

SURFACE ANALYSIS (SEM)

Pectin-POX Pectin-F

Pectin-N

• 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

SamplesConcentration 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

Direct enzymatic oxidation leading to pectin-F showed the highest phenolic content

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

The color is due to the oxidation reaction

Pectin-F has the most intense color the highest content of phenols

Color parameters (Datacolor)Phenolic content (Folin & Ciocalteu)

(Color intensity)

(C) = (a*2 + b*2)1/2

HYGROSCOPY (DYNAMIC VAPOR SORPTION)

0 10 20 30 40 50 60 70 800

5

10

15

20

25

30

35

40

45

50pectin-N pectin-F pectin-POX

Relative humidity (%)

Mas

s cha

nge

(%)

• 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

ANTIRADICAL ACTIVITY

EC50 (mg/ml) Pectin-N Pectin-F Pectin-POXScavenging 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

Modified pectin has better

antiradical activity due

to it highest content of phenol

When EC50 the antioxidant activity

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:

1. Increased the pectin phenolic content, its antiradical activity and its hydrophobic

nature

2. Led to colored pectin derivatives

3. Decreased the roughness of surface

Nancy, France

Lebanon

JEITA-LEBANON

Thank you for your attention