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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

Component Content (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 0.44-2.83 Fisher 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 12.58-25.90 Fisher et al., 2011; Rowashed et al., 2013; Li et al., 2015 Total phenolics 49.00-181.00 Al-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 – 11.9 Wang 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

• f

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

• f

the

• ptimum

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

• ptimum method

for isolation of priced compounds from pomegranate peels

Materials and Methods

Washing Microwave assisted extraction Ultrasound assisted extraction Filtration Folin 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) Water 50% 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 conditions Grinding

20 40 60 80 100 120 140 10 20 30 40 50 60 70

Yield (mg GAE/g dry peel) 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) : 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 8 ln ln L Dt π π MR  

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

   



 

            

1 2 2 2 2 2

4 1 2 exp 1 2 1 8

n e e

L Dt π n n π X X X X MR

y = -0.0039x - 0.0827 R² = 0.991

• 3,0
• 2,5
• 2,0
• 1,5
• 1,0
• 0,5

0,0 100 200 300 400 500 600 700 800 lnMR 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 T R a E

t X A X A e k

• e

C C

2 3 2 1

d A

First order kinetic model for thermal degradation

• f 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 4 8 12 16 20

Extraction yield (%) Extraction time (min) Optimum extraction time 4 min

Regression analysis Determination 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

1 2 3 4 5 6 7 10 20 30 40 50 60 70 Extraction yield (g GAE/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 : 35/1

2 4 6 8 10 methanol ethyl acetate water ethanol 50% aqueous methanol Extraction yield (g GAE/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∙S

Y: g GAΕ/100 g

111.9 mg GAE/g dry peel

 

20 40 60 80 100 120 140 10 20 30 40 50 60 70 80

Extraction yield (mg /g dry peel) 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

10 20 30 40 50 60 70 80 200 400 600 Moisture (% 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

10 20 30 40 50 60 70 80

1000 2000 3000 Moisture (% 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 lnDeff (Deff in m2/s) 1/T (K-1) 𝐄 = 𝐄𝟏𝐟𝐲𝐪(− 𝐅𝐛 𝐒𝐔)

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 T R a E

t X A X A e k

• e

C C

2 3 2 1

d A

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 200 400 600 800 1000 1200 1400

C/Co Drying time (min)

40 50 60 70 80

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/Co - experimental C/Co - predicted 40 oC 50 oC 60 oC 70 oC 80 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

• C and higher temperatures caused more loss of

phenolic substance.

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

• rder kinetic model.

Conclusions

Thank you for your attention!