Optimization of polyphenols extraction from pomegranate peels –

drying, enzymatic pretreatment, extraction method, operating conditions

1Department of Chemical Engineering, School of Engineering, Aristotle University, 541 24 Thessaloniki,

Greece

2Department of Food Science and Technology, School of Agriculture, Forestry and Natural Environment,

Aristotle University, 541 24 Thessaloniki, Greece

L. Papaoikonomou1, K. Kaderides2, A.M. Goula2, K.G. Adamopoulos1

Introduction

Pomegranate

(Punica Granatum L.)

Usually cultivated in Mediterranean

climate (Spain, Turkey), many varieties

(Wonderful, Berri etc.)

Healing and medical properties, important

source of antioxidants

24% Peel 14% Seeds 62% Juice

ComponentContent

(g/100 g dry peel)

Total solids 94.50

Moisture 5.40

Total sugars 17.70

Protein 4.90

Fat 1.26

(Aguilar et al., 2008)

Composition of Pomegranate Peels

Phenolic

fraction

Content

(mg/g dry matter)Reference

Punicalagin 16.67-64.98 Li et al., 2015; Gullon et al., 2016

Ellagic acid 0.44-2.83Fisher et al., 2011;

Rowashed et al., 2013; Li et al., 2015

Caffeic acid 0.60 Rowashed et al., 2013

P-coumaric acid 0.18 Rowashed et al., 2013

Catechin 8.68-12.65 Rowashed et al., 2013; Li et al 2015

Cyanidin 0.22 Fisher et al., 2011

Pelargonidin 0.20 Fisher et al., 2011

Gallic acid 12.58-25.90Fisher et al., 2011;

Rowashed et al., 2013; Li et al., 2015

Total phenolics 49.00-181.00Al-Rawahi et al., 2013; Amani et al.,

2014; Kaderides et al., 2015

Biological Activity

Phenolic

compounds

Antioxidant activity (Cam et al., 2009)

Anticancer properties (Hamad & Al-Momene, 2009)

Reduce risk of coronary heart disease (Aviram et al., 2000)

Antimicrobial activity (Duman et al., 2009)

Methods of Phenolics Extraction from Pomegranate Peels

Technique of

extraction

Time

(min)

Yield

(g GAΕ/100 g dry matter) Reference

Normal Stirring 60 8.26 – 11.9Wang et al., 2011;

Pan et al., 2011

Pressurized liquid 15 25.8 – 26.4 Cam & Hisil, 2010

Ultrasound-assisted

(Continuous)

(Pulsed)

6

8

14.8

14.5

Pan et al., 2011

Microwave-assisted 1 21.5 Zheng et al., 2011

New ‘‘green’’ techniques – use less solvent and energy

Ultrasound-assisted Microwave-assisted

Objectives

To compare new methods for extraction of

phenolic compounds from pomegranate peels

• Microwave-assisted extraction

• Ultrasound-assisted extraction

To study the effect of peels moisture content on

extraction yield

To study the enhancement of the optimum

extraction treatment by enzymatic pre-treatment

To study the drying behavior of pomegranate peels

and the kinetics of total phenolics degradation

during the drying process

To propose an

optimum method

for isolation of

priced compounds

from pomegranate

peels

Materials and Methods

Washing

Microwave assisted extraction

Ultrasound assisted extraction

FiltrationFolin Ciocalteu

method

Determination of phenolic compounds

Pomegranate peels

Drying (40 οC, 48h) Grinding-Sieving

Process for Extraction of Phenolics from Pomegranate Peels

Sieving

Sieving of Peel Powder

Microwave-assisted Extraction

Microwave system (Multiwave B30MC030A)

(Anton Paar, Austria)

Parameters Levels

Solvent type (S) Water50% aqueous

EtOH

70% aqueous

EtOH

50% aqueous

MeOH

70% aqueous

MeOH

Power (W) 100 201 350 499 600

Liquid/solids ratio (LS, mL/g) 10 20 35 50 60

Experiment Design - Response Surface Methodology

Parameters

Extraction time

Microwave radiation power

Solvent type

Liquid/solids ratio

130 W, 20 kHz VCX-130 Sonics and Materials

(Danbury, CT, USA), Ti–Al–V probe (13 mm)

Extraction temperature

Solvent type

Liquid/solids ratio

Amplitude level

Pulse duration/pulse interval ratio

Extraction time

Parameters Levels

Solvent type (S) EtOH MeOH Water 50% aqueous

MeOH

Ethyl-

acetate

Extraction temperature (T, oC) 25 30 35 40 45

Liquid/solids ratio (LP, mL/g) 10/1 20/1 30/1 40/1 50/1

Amplitude level (A, %) 20 30 40 50 60

Pulse duration/pulse interval ratio (PULSE, -) 5/15 3/4 7/6 19/12 2/1

Experiment Design - Response Surface Methodology

Parameters

Ultrasound-assisted Extraction

Effect of Peels Moisture Content on Extraction Yield

Peels

Drying (40 oC)

at different

moisture contents

Ultrasound-assisted extraction

at the optimum conditions

Microwave-assisted extraction

at the optimum conditionsGrinding

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70Yie

ld (

mg G

AE

/g d

ry

pee

l)

Peels moisture content (%)

Enzymatic Pre-treatment at the Optimum Conditions

Time of pre-treatment (min)

Enzyme concentration (% of dry matter)

Pectinase/Cellulase ratio

Liquid/solids ratio (mL/g)

Time (min) : 60 105 150 195 240

Enzyme concentration (% of dry matter) : 2 2.5 3 3.5 4

Pectinase/Cellulase ratio (% pectinase) : 0 25 50 75 100

Liquid/solids ratio (mL/g) : 20/10 30/10 40/10 50/10 60/10

Experiment Design - Response Surface Methodology

pH = 4.5-5.0

T = 50±2 oC

Parameters

Pomegranate Peels Drying

Pomegranate peels, with 75.28 ± 1.08% moisture

content, were used for all drying experiments

Pomegranate peels slabs of 6 mm thickness

30 x 19 cm aluminum dishes

In a tray dryer with an air velocity of 1.2 m/s

Five drying temperatures: 40, 50, 60, 70, 80 oC

At regular intervals, determination of:

The weight of the samples

The total phenolics content

2

2

2 4

8lnln

L

Dtπ

πMR

MR : moisture ratio

X0 : initial moisture content

X : moisture content at time t

Xe : equilibrium moisture content

L : slab thickness

Simplification for long drying periods

Kinetic Modeling of Drying

12

22

22

0 4

12exp

12

18

ne

e

L

Dtπn

nπXX

XXMR

y = -0.0039x - 0.0827

R² = 0.991

-3,0

-2,5

-2,0

-1,5

-1,0

-0,5

0,0

0 100 200 300 400 500 600 700 800

lnM

R

t (min)

lnMRexp.

Linear (lnMRexp.)

- Mechanistic model

Diffusion approach

ko : frequency factor

Ea : activation energy

X : peels moisture content

T : drying temperature

A1, A2, A3 : constants

t

TR

aE

tXAXAek

o

eC

C0

23210 dA

First order kinetic model for thermal degradation

of phenolic compounds during drying

Results

Microwave Extraction – Effects of Studied Factors

Nmax

Power : 499 W

S : 70% EtOH

L/S : 50/1 mL/g

5

5,5

6

6,5

7

7,5

8

8,5

9

9,5

0 4 8 12 16 20

Ex

trac

tion

yie

ld (

%)

Extraction time (min)

Optimum extraction time

4 min

Regression analysisDetermination of

statistically significant factors

Empirical modelling of

extraction yield

Y = 20.4395 – 0.0428 ∙ P

+ 0.0001 ∙ P2 + 0.7630 ∙ S2

– 0.0059 ∙ P∙S + 0.0003 ∙ P∙LP

R2 = 0.78

Microwave Extraction - Optimization

Y: g GAΕ/100 g

Statistically significant parameters:

Power (p = 0.002), Power2 (p = 0.044)

257.6

mg GAE/g dry peel

Ultrasound Extraction – Effects of Studied Factors

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70

Extr

acti

on y

ield

(g G

AE

/100 g

)

Extraction time, t (min)

water, LP=20/1, A=40%, PUL=7/6, T=25oC

ethanol, LP=20/1, A =40%, PUL=7/6, T=25oC

methanol, LP=20/1, A=40%, PUL=7/6, T=25oC

Νmax

T : 35 ⁰C

A : 40%

PUL : 1.2

LP : 35/10

2

4

6

8

10

methanol ethyl acetate water ethanol 50% aqueous

methanol

Extr

acti

on

yie

ld

(g G

AE

/100 g

)

N: g GAΕ/100 g

Ultrasound Extraction - Optimization

R2 = 0.61

Statistically significant parameters: LP2 (p = 0.039), S2 (p = 0.020)

-2 : MeOH

-1 : Ethylacetate

0 : Water

1 : EtOH

2 : 50% aqueous MeOH

Y = - 88.056 + 3.417∙T + 1.197∙LP + 21.161∙PULSE - 0.048∙T2

– 0.018∙LP2 – 2.129∙S2 – 8.357∙PULSE2 + 0.041∙LP∙SY: g GAΕ/100 g

111.9

mg GAE/g dry peel

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70 80

Ex

trac

tio

n y

ield

(m

g /

g d

ry p

eel)

Peels moisture content (% w.b.)

Microwave

Assisted

Extraction

Ultrasound

Assisted

Extraction

14 45

Effect of Peels Moisture Content on Extraction Yield

Enzymatic Pre-treatment - Microwave Assisted Extraction

Power = 600 W, Solvent/peels ratio = 60/1 mL/g, Extraction time = 4 min

Solvent = 50% aqueous EtOH, Peels moisture content = 45% w.b.

Optimum yield: 172.8 mg GAE/g dry peel

Pre-treatment time = 240 min

Enzyme concentration = 2% of dry matter

Enzyme type = 100% pectinase

Liquid/solids ratio = 20/10 mL/g

Enzymatic Pre-treatment-Ultrasound Assisted Extraction

Solvent = water, Solvent/peels ratio = 32.2/1 mL/g, amplitude level = 39.8%,

Pulse duration/pulse interval = 1.2, temperature = 34.7 oC, extraction time = 10 min

87.1 mg GAE/g dry peel

Pomegranate Peels Drying Behavior

0

10

20

30

40

50

60

70

80

0 200 400 600

Mois

ture

(%

w.b

.)

Drying time (min)

40

50

60

70

80

Drying temperature (oC)

The drying rate increased on

increasing the temperature

The drying occurred mostly in

the falling rate period

The controlling resistance to

mass transfer was internal

diffusion of moisture

0

10

20

30

40

50

60

70

80

0 1000 2000 3000

Mo

istu

re (

% w

.b.)

Drying time (min)

40

50

60

70

80

Drying

temperature (oC)

y = -3401.6x – 12.07

R² = 0.929

-23,2

-23,0

-22,8

-22,6

-22,4

-22,2

-22,0

-21,8

-21,6

-21,4

0,0028 0,0029 0,0030 0,0031 0,0032 0,0033

lnD

eff

(Def

fin

m2/s

)

1/T (K-1)

𝐃 = 𝐃𝟎𝐞𝐱𝐩(−𝐄𝐚𝐑𝐓

)

Pomegranate Peels Drying Behavior

Kinetics of Total Phenolics Degradation

• Higher temperatures caused more loss of

phenolics

• Drying of pomegranate peel can result in

significant phenolics degradation

Parameter Value

ko (min-1) 21.6∙10-5

Ea (kJ/mol) 15.8

A1 4084.5

A2 1932.3

A3 -58.4

R2 SSE

0.846 0.16

t

TR

aE

tXAXAek

o

eC

C0

23210 dA

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

0 200 400 600 800 1000 1200 1400

C/C

o

Drying time (min)

4050607080

Temperature (oC)

0,0

0,2

0,4

0,6

0,8

1,0

0,0 0,2 0,4 0,6 0,8 1,0

C/C

o -

exp

erim

enta

l

C/Co - predicted

40 oC50 oC60 oC70 oC80 oC

Conclusions

The optimum extraction yield by microwave extraction was 257.6 g

GAE/g dry peel at 600 W, 50% EtOH as solvent, liquid/solid ratio of

60/1 mL/g and extraction time of 4 min.

The optimum extraction yield by ultrasound extraction was 111.8 g

GAE/g dry peel at 34.7 οC, water as solvent, liquid/solid ratio of 32.2/1

mL/g, amplitude level up to 39.8%, pulse duration/pulse interval ratio of

7/6 and extraction time of 10 min.

The optimum moisture content in order to maximize the extraction

yield was 45% w.b. for the microwave extraction and 14% w.b. for the

ultrasound extraction.

The enzymatic pre-treatment using cellulase and pectinase did not

enhance the extraction.

Conclusions

The drying rate increased on increasing the temperature, whereas the

drying occurred mostly in the falling rate period.

The effective diffusivity values varied between 7.12∙10-9 and 2.68∙10-8

m2/s and the activation energy was found to be 28.28 kJ/mol.

Phenolics content of pomegranate peel decreased by 60-75% after

drying at 40-80 oC and higher temperatures caused more loss of

phenolic substance.

The degradation of phenolics during drying can be described by a 1st

order kinetic model.

Conclusions

Thank you

for your attention!