drying, enzymatic pretreatment, extraction method, operating - PowerPoint PPT Presentation

Optimization of polyphenols extraction from pomegranate peels – drying, enzymatic pretreatment, extraction method, operating conditions L. Papaoikonomou 1 , K. Kaderides 2 , A.M. Goula 2 , K.G. Adamopoulos 1 1 Department of Chemical Engineering, School of Engineering, Aristotle University, 541 24 Thessaloniki, Greece 2 Department of Food Science and Technology, School of Agriculture, Forestry and Natural Environment, Aristotle University, 541 24 Thessaloniki, Greece

Introduction Usually cultivated in Mediterranean climate (Spain, Turkey), many varieties (Wonderful, Berri etc.) Healing and medical properties, important Pomegranate source of antioxidants ( Punica Granatum L.) 24% Peel 14% Seeds 62% Juice

Composition of Pomegranate Peels Phenolic Content Reference fraction (mg/g dry matter) Punicalagin 16.67-64.98 Li et al., 2015; Gullon et al., 2016 Fisher et al., 2011; Ellagic acid 0.44-2.83 Rowashed et al., 2013; Li et al., 2015 Content Component Caffeic acid 0.60 Rowashed et al., 2013 (g/100 g dry peel) P-coumaric acid 0.18 Rowashed et al., 2013 Total solids 94.50 Catechin 8.68-12.65 Rowashed et al., 2013; Li et al 2015 Moisture 5.40 Cyanidin 0.22 Fisher et al., 2011 Total sugars 17.70 Pelargonidin 0.20 Fisher et al., 2011 Fisher et al., 2011; Protein 4.90 Gallic acid 12.58-25.90 Rowashed et al., 2013; Li et al., 2015 Fat 1.26 Al-Rawahi et al., 2013; Amani et al., Total phenolics 49.00-181.00 (Aguilar et al., 2008) 2014; Kaderides et al., 2015

Biological Activity Antioxidant activity (Cam et al., 2009) Anticancer properties (Hamad & Al-Momene, 2009) Phenolic compounds 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 Time Yield Reference (g GA Ε/100 g dry matter) extraction (min) Wang et al., 2011; 8.26 – 11.9 Normal Stirring 60 Pan et al., 2011 25.8 – 26.4 Pressurized liquid 15 Cam & Hisil, 2010 Ultrasound-assisted (Continuous) 6 14.8 Pan et al., 2011 (Pulsed) 8 14.5 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 propose an optimum method for isolation of  To study the effect of peels moisture content on priced compounds extraction yield from pomegranate  To study the enhancement of the optimum peels 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

Materials and Methods

Process for Extraction of Phenolics from Pomegranate Peels Pomegranate peels Folin Ciocalteu Filtration method Microwave assisted extraction Determination of Washing phenolic compounds Ultrasound assisted extraction Drying (40 ο C, 48h) Grinding-Sieving

Sieving of Peel Powder Sieving

Microwave-assisted Extraction Parameters Extraction time Microwave radiation power Solvent type Microwave system (Multiwave B30MC030A) Liquid/solids ratio (Anton Paar, Austria) Experiment Design - Response Surface Methodology Parameters Levels 50% aqueous 70% aqueous 50% aqueous 70% aqueous Solvent type (S) Water EtOH EtOH MeOH MeOH Power (W) 100 201 350 499 600 Liquid/solids ratio (LS, mL/g) 10 20 35 50 60

Ultrasound-assisted Extraction Parameters Extraction temperature Solvent type Liquid/solids ratio Amplitude level 130 W, 20 kHz VCX-130 Sonics and Materials Pulse duration/pulse interval ratio (Danbury, CT, USA), Ti – Al – V probe (13 mm) Extraction time Experiment Design - Response Surface Methodology Parameters Levels Solvent type (S) EtOH MeOH Water 50% aqueous Ethyl- MeOH acetate Extraction temperature (T, o C) 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

Effect of Peels Moisture Content on Extraction Yield 140 Yield (mg GAE/g dry 120 Peels 100 80 peel) 60 40 Drying (40 o C) 20 at different 0 0 10 20 30 40 50 60 70 moisture contents Peels moisture content (%) Microwave-assisted extraction at the optimum conditions Grinding Ultrasound-assisted extraction at the optimum conditions

Enzymatic Pre-treatment at the Optimum Conditions   Parameters  Time of pre-treatment (min)  Enzyme concentration (% of dry matter)  Pectinase/Cellulase ratio  Liquid/solids ratio (mL/g) Experiment Design - Response Surface Methodology 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 pH = 4.5-5.0 T = 50 ± 2 o C Liquid/solids ratio (mL/g) : 20/10 30/10 40/10 50/10 60/10

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 o C  At regular intervals, determination of:  The weight of the samples  The total phenolics content

Kinetic Modeling of Drying - Mechanistic model Diffusion approach MR : moisture ratio      2   2 X X 8 1 2 n 1 π Dt  X 0 : initial moisture content    e MR exp      2 2 2 X X π  4 L 2 n 1 X : moisture content at time t    n 1 0 e X e : equilibrium moisture content L : slab thickness t (min) 0,0 0 100 200 300 400 500 600 700 800 -0,5 2 π 8 Dt   ln MR ln 2 2 π -1,0 4 L lnMRexp. lnMR -1,5 Linear (lnMRexp.) -2,0 y = -0.0039x - 0.0827 R² = 0.991 Simplification for long drying periods -2,5 -3,0

First order kinetic model for thermal degradation of phenolic compounds during drying t E   a   2       k e R T A A X A X d t C 0 1 2 3  e 0 C o k o : frequency factor E a : activation energy X : peels moisture content T : drying temperature A 1 , A 2 , A 3 : constants

Results

Microwave Extraction – Effects of Studied Factors Optimum extraction time 9,5 4 min 9 8,5 Extraction yield (%) 8 7,5 7 6,5 6 5,5 5 0 4 8 12 16 20 Extraction time (min) Nmax Power : 499 W S : 70% EtOH L/S : 50/1 mL/g

Microwave Extraction - Optimization Determination of Empirical Regression analysis statistically modelling of significant factors extraction yield 257.6 mg GAE/g dry peel Y = 20.4395 – 0.0428 ∙ P + 0.0001 ∙ P 2 + 0.7630 ∙ S 2 – 0.0059 ∙ P ∙ S + 0.0003 ∙ P ∙ LP Y: g GA Ε/100 g R 2 = 0.78 Statistically significant parameters: Power (p = 0.002), Power 2 (p = 0.044)

Ultrasound Extraction – Effects of Studied Factors 7 methanol, LP =20/1, A =40%, PUL =7/6, T =25 o C 6 Extraction yield (g GAE/100 g) 5 water, LP =20/1, A =40%, PUL =7/6, T =25 o C 4 ethanol, LP =20/1, A =40%, PUL =7/6, T =25 o C N: g GA Ε/100 g 3 10 Ν max Extraction yield 8 (g GAE/100 g) : 35 ⁰ C 2 T 6 A : 40% 1 4 PUL : 1.2 2 0 LP : 35/1 0 10 20 30 40 50 60 70 0 methanol ethyl acetate water ethanol 50% aqueous Extraction time, t (min) methanol

Ultrasound Extraction - Optimization 111.9 mg GAE/g dry peel -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∙T 2 Y: g GA Ε/100 g – 0.018∙LP 2 – 2.129∙S 2 – 8.357∙PULSE 2 + 0.041∙LP∙S R 2 = 0.61 LP 2 (p = 0.039), S 2 (p = 0.020) Statistically significant parameters:

Effect of Peels Moisture Content on Extraction Yield   140 Extraction yield (mg /g dry peel) 120 100 Microwave Assisted 80 Extraction 60 Ultrasound 40 Assisted Extraction 20 0 0 10 20 30 40 50 60 70 80 14 45 Peels moisture content (% w.b.)

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 o C, extraction time = 10 min 87.1 mg GAE/g dry peel

Pomegranate Peels Drying Behavior 80 80 Drying temperature ( o C) Drying 70 temperature ( o C) 70 Moisture (% w.b.) 60 40 40 50 50 50 60 40 60 Moisture (% w.b.) 60 30 70 50 20 70 80 10 80 40 0 0 1000 2000 3000 30 Drying time (min) 20 10  The drying rate increased on 0 increasing the temperature 0 200 400 600  The drying occurred mostly in Drying time (min) the falling rate period  The controlling resistance to mass transfer was internal diffusion of moisture