Optimization of polyphenols extraction from pomegranate peels drying, enzymatic pretreatment, extraction method, operating conditions

Transcription

1 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, Thessaloniki, Greece 2 Department of Food Science and Technology, School of Agriculture, Forestry and Natural Environment, Aristotle University, Thessaloniki, Greece

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

3 Composition of Pomegranate Peels Phenolic fraction Content (mg/g dry matter) Reference Punicalagin Li et al., 2015; Gullon et al., 2016 Component Content (g/100 g dry peel) Total solids Moisture 5.40 Total sugars Ellagic acid 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 Rowashed et al., 2013; Li et al 2015 Cyanidin 0.22 Fisher et al., 2011 Pelargonidin 0.20 Fisher et al., 2011 Protein 4.90 Fat 1.26 (Aguilar et al., 2008) Gallic acid Total phenolics Fisher et al., 2011; Rowashed et al., 2013; Li et al., 2015 Al-Rawahi et al., 2013; Amani et al., 2014; Kaderides et al., 2015

4 Biological Activity Antioxidant activity (Cam et al., 2009) Anticancer properties (Hamad & Al-Momene, 2009) Reduce risk of coronary heart disease (Aviram et al., 2000) Phenolic compounds Antimicrobial activity (Duman et al., 2009)

5 Methods of Phenolics Extraction from Pomegranate Peels Technique of extraction Time (min) Yield (g GAΕ/100 g dry matter) Reference Normal Stirring Wang et al., 2011; Pan et al., 2011 Pressurized liquid Cam & Hisil, 2010 Ultrasound-assisted (Continuous) (Pulsed) Pan et al., 2011 Microwave-assisted Zheng et al., 2011 New green techniques use less solvent and energy Ultrasound-assisted Microwave-assisted

6 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 extraction yield To study the enhancement of the optimum extraction treatment by enzymatic pre-treatment priced compounds from pomegranate peels To study the drying behavior of pomegranate peels and the kinetics of total phenolics degradation during the drying process

7 Materials and Methods

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

9 Sieving of Peel Powder Sieving

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

11 Ultrasound-assisted Extraction Parameters Extraction temperature Solvent type Liquid/solids ratio Amplitude level Pulse duration/pulse interval ratio Extraction time 130 W, 20 khz VCX-130 Sonics and Materials (Danbury, CT, USA), Ti Al V probe (13 mm) Experiment Design - Response Surface Methodology Parameters Levels Solvent type (S) EtOH MeOH Water 50% aqueous MeOH Ethylacetate Extraction temperature (T, o C) Liquid/solids ratio (LP, ml/g) 10/1 20/1 30/1 40/1 50/1 Amplitude level (A, %) Pulse duration/pulse interval ratio (PULSE, -) 5/15 3/4 7/6 19/12 2/1

12 Yield (mg GAE/g dry peel) Effect of Peels Moisture Content on Extraction Yield Peels Drying (40 o C) at different moisture contents Grinding Peels moisture content (%) Microwave-assisted extraction at the optimum conditions Ultrasound-assisted extraction at the optimum conditions

13 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) : Enzyme concentration (% of dry matter) : Pectinase/Cellulase ratio (% pectinase) : Liquid/solids ratio (ml/g) : 20/10 30/10 40/10 50/10 60/10 ph = T = 50±2 o C

14 Pomegranate Peels Drying Pomegranate peels, with ± 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

15 lnmr Kinetic Modeling of Drying - Mechanistic model Diffusion approach MR X X X 8 e exp X e π n1 2n n 1 2 L 2 π 2 Dt MR : moisture ratio X 0 : initial moisture content X : moisture content at time t X e : equilibrium moisture content L : slab thickness 0,0 t (min) ln MR ln π 8 2 π 2 4L Dt 2-0,5-1,0 lnmrexp. -1,5 Linear (lnmrexp.) Simplification for long drying periods -2,0-2,5 y = x R² = ,0

16 First order kinetic model for thermal degradation of phenolic compounds during drying C 0 C o e t Ea k RT 0 e A1 A2 X A3 X dt 2 k o E a : frequency factor : activation energy X : peels moisture content T : drying temperature A 1, A 2, A 3 : constants

17 Results

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

19 Microwave Extraction - Optimization Regression analysis Determination of statistically significant factors Empirical modelling of extraction yield mg GAE/g dry peel Y = P P S P S P LP Y: g GAΕ/100 g R 2 = 0.78 Statistically significant parameters: Power (p = 0.002), Power 2 (p = 0.044)

20 Extraction yield (g GAE/100 g) Extraction yield (g GAE/100 g) Ultrasound Extraction Effects of Studied Factors 7 methanol, LP=20/1, A=40%, PUL=7/6, T=25 o C water, LP=20/1, A=40%, PUL=7/6, T=25 o C ethanol, LP=20/1, A =40%, PUL=7/6, T=25 o C 10 3 N: g GAΕ/100 g methanol ethyl acetate Extraction water time, ethanol t (min) 50% aqueous methanol Νmax T : 35 ⁰C A : 40% PUL : 1.2 LP : 35/1

21 Ultrasound Extraction - Optimization mg GAE/g dry peel Y = T LP PULSE T 2 R 2 = LP S PULSE LP S Statistically significant parameters: LP 2 (p = 0.039), S 2 (p = 0.020) -2 : MeOH -1 : Ethylacetate 0 : Water 1 : EtOH 2 : 50% aqueous MeOH Y: g GAΕ/100 g

22 Extraction yield (mg /g dry peel) Effect of Peels Moisture Content on Extraction Yield Microwave Assisted Extraction Ultrasound Assisted Extraction Peels moisture content (% w.b.)

23 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: mg GAE/g dry peel Pre-treatment time Enzyme concentration Enzyme type Liquid/solids ratio = 240 min = 2% of dry matter = 100% pectinase = 20/10 ml/g

24 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

25 Moisture (% w.b.) Moisture (% w.b.) Pomegranate Peels Drying Behavior Drying temperature ( o C) Drying temperature ( o C) Drying time (min) Drying time (min) 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

26 lnd eff (Deff in m 2 /s) Pomegranate Peels Drying Behavior 1/T (K -1 ) 0,0028 0,0029 0,0030 0,0031 0,0032 0, ,4-21,6-21,8-22,0-22,2-22,4 y = x R² = ,6-22,8 D = D 0 exp( E a RT ) -23,0-23,2

27 C/Co - experimental C/Co Kinetics of Total Phenolics Degradation 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0, ,0 Temperature ( o C) Drying time (min) Higher temperatures caused more loss of phenolics Drying of pomegranate peel can result in significant phenolics degradation C 0 C o e t Ea k RT 0 e A1 A2 X A3 X dt 2 0,8 Parameter Value 0,6 40 oc 0,4 50 oc 60 oc 0,2 70 oc 80 oc 0,0 0,0 0,2 0,4 0,6 0,8 1,0 C/Co - predicted k o (min -1 ) E a (kj/mol) 15.8 A A A R 2 SSE

28 Conclusions

29 Conclusions The optimum extraction yield by microwave extraction was 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 g GAE/g dry peel at 34.7 ο C, 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.

30 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 and m 2 /s and the activation energy was found to be kj/mol. Phenolics content of pomegranate peel decreased by 60-75% after drying at o C and higher temperatures caused more loss of phenolic substance. The degradation of phenolics during drying can be described by a 1 st order kinetic model.

31 Thank you for your attention!