Processes APPLICATION OF SUPERCRITICAL FLUIDS FOR POLYPHENOLIC COMPOUNDS EXTRACTION FROM EXHAUSTED OLIVE POMACE Ashley Sthefanía Caballero 1, Juan Miguel Romero-García 2, Eulogio Castro 2, Carlos Ariel Cardona 1 1 Universidad Nacional de Colombia sede Manizales, Instituto de Biotecnología y Agroindustria. Laboratorio de Equilibrios Químicos y Cinética Enzimática. Departamento de Ingeniería Química. Manizales, Colombia 2 Center for Advanced Studies in Energy and Environment, University of Jaen, Spain, *Corresponding author: ccardonaal@unal.edu.co 1
CONTENT Introduction Methodology Results and discussion Conclusions Acknowledgments 2
Pruning Olive oil production process Biomass from pruning Olive leaves Olive pits Olive pomace Olive mill wastewater Bioethanol Oligosaccharides Lignin Antioxidants Nanocellulose Ceramic materials Xylitol Sugars Energy Techno-economic evaluation BIOREFINERY BASED ON OLIVE-DERIVED BIOMASS 3
OLIVE TREE BIOMASS from pruning Pruning OLIVE TREE FIELD Olive fruit Harvesting Olive washing wastewater (Washing)/Cleaning Leaves Crushing/Milling Malaxation Water DECANTER Three phase separation mode Two phase separation mode Waste water (3POMWW) Olive oil washing wastewater Olive pomace Olive oil WASHING Olive pomace Drying Olive stones Extra virgin OLIVE OIL Market Hexane extraction Distillation Exhausted Olive Pomace Crude Pomace Olive oil Oil refination Pomace Olive oil 4
Liquid Olive pomace Dry Olive pomace Rotary oven Hexane extraction Filtration Solid Miscela Distillation Exhausted Olive pomace Antioxidant valorisation PRODUCTION 300 kg exhausted olive pomace/1 hectare olive trees Hexane Olive Pomace Oil Total phenolic compouds DPPH (EC 50 ) Compound identification 5
INTRODUCTION: Polyphenolic compounds Anti-cancer Polyphenolic compounds are an important class of chemicals present in edible and inedible plants with interesting applications in the medical, food and cosmetic industry. Antiinflammatory Regulation of enzymatic inhibition Polyphenols Anti-toxic Anti-bacterial Oxidative stress Anti-viral Antioxidant Antiallergenic Antimutagenic Figure 1. Molecular structure of phenol. Taken from: https://goo.gl/1tunjk Figure 2. Properties contributed by polyphenolic compounds. 6
INTRODUCTION: Extraction technologies Obtaining polyphenolic compounds requires the use of extraction processes. The traditional (conventional) methods of extraction are characterized by the application of high temperatures, decrease in the size of the material, long operating time and low yield. However, the use of high temperatures can cause the degradation of the compounds due to their sensitivity. 1 2 3 4 6 Solvent extraction Percolation Mechanical extraction Soxhlet extraction Conventional Extractions Steam extraction 5 Supercritical fluid extraction 7 Ultrasound-assisted extraction 8 9 Molecular Distillation Non-Conventional Extractions Microwave assisted extraction Figure 3. Types of conventional and non-conventional extractions. Taken from 1. goo.gl/zdxc8q, 2. goo.gl/xk4m8u, 3. goo.gl/c9qupj, 4. goo.gl/24mqpu, 5. goo.gl/g3qlrl, 6. goo.gl/5dqdnr, 7. goo.gl/pztdmn, 8. goo.gl/jq7afz, 9. goo.gl/p9osnf. 7
INTRODUCTION: Extraction with supercritical fluids Table 1. Solvents most commonly used in supercritical fluid extraction. Solvent P c [MPa] T c [K] Solvent P c [MPa] T c [K] Carbon dioxide 7.38 304.15 Methane 4.60 190.4 Ethanol 6.14 513.9 Ammonia 11.35 405.55 Methanol 8.09 512.6 n-hexane 3.01 507.5 Propylene 4.60 364.95 Toluene 4.10 591.8 Propane 4.25 369.8 Sulfur dioxide 7.88 430.8 Acetone 4.70 508.1 Acetonitril 4.83 545.5 Ethyl acetate 3.83 523.25 Oxygen 5.04 154.6 Figure 4. Triple and critical point representation. Taken from goo.gl/2zm3fu Water 22.12 647.3 Carbon monoxide 3.50 132.9 Bencene 4.89 562.2 n-heptane 2.74 540.3 Isobutane 3.65 408.2 Hexane cycle 4.07 553.5 Di-ethyl amine 3.71 496.5 Propanediol 5.47 393.15 8
METHODOLOGY 9
Methodology Procedure HPLC Characterization of the raw material Extraction process -Solvent extraction Total phenolic content Antioxidant capacity Compound identification -Supercritical fluid extraction 10
Methodology Characterization of Olive Pomace (NREL procedure) OLIVE POMACE Ash LAP-005 (Calcination 575ºC) Biomass<1mm LAP-001 (Drying 105ºC) Total solids Elemental composition Proteins Fats Extracted biomass Hydrolysed biomass LAP-010 (Soxhlet extraction) LAP-002 (Acid hydrolysis in 2 stages) HPLC Extractives C5 and C6 sugars Total phenols (Aqueous extract) Starch Liquid Fraction Solid Fraction LAP-017 (HPLC) Acetic acid HPLC C5 and C6 sugars LAP-004 (Abs. 205 nm) LAP-003 (Drying 105ºC) LAP-005 (Calcination 575ºC) Insoluble acid lignin (IAL) Acetyl groups Cellulose Hemicellulose Soluble acid lignin (SAL) Insoluble acid ash (IAA) 11
Methodology Solvent Extraction Solvent: 60% ethanol (v/v) Solid-liquid ratio: 1:20 (w/v) Temperature: 25ºC Time: 8 hours Agitation: 300 rpm Vacuum filtration 12
Methodology Supercritical Fluid Extraction Exhausted Olive pomace in the thimble Solvent: Carbon dioxide Co-solvent: 60% ethanol (v/v) Solid-liquid ratio: 1:3 (w/v) Pressure: 200, 250 and 300 bars Temperature: 50ºC Time: 60 minutes Extracted Olive pomace 13
Methodology Determination of total phenolic content and antioxidantactivity Total phenolic content Folin-Ciocalteu % 1 100 Equation (1) Antioxidant activity 50 Equation (2) DPPH 1 Equation (3) 14
Methodology HPLC Table 1. Elution profiles of polyphenolic compounds. Figure 3. HPLC system (LC-2010A HT) with UVvisible detector. Time (min) (A) Acetic acid 0.5% v/v (B) Methanol Elution profile of chlorogenic acid 0 10 90 4 10 90 15 30 70 25 30 70 Elution profile of ferulic acid 0 20 80 4 45 55 9 45 55 12 80 20 25 80 20 Elution profile of vanillin 0 60 40 5 60 40 7 50 50 14 100 0 18 100 0 19 60 40 Elution profile of hydroxytyrosol 0 5 95 10 35 75 13 5 95 15 5 95 Elution profile of quercetin, caffeic acid and vanillinic acid 0 0 100 10 10 90 40 70 30 44 0 100 47 0 100 15
RESULTS AND DISCUSSION 16
RESULTS OLIVE POMACE TPC and Antioxidant activity Composition Total solid (%) 91.43 ± 0.15 Composition (% dry matter) Extractives 49.71 ± 0.61 Water-extract 45.78 ± 0.45 Glucose 7.63 ± 0.26 Xylose 0.45 ± 0.09 Galactose 1.37 ± 0.04 Arabinose 1.67 ± 0.06 Mannose 0.89 ± 0.01 Mannitol 5.03 ± 0.15 Total phenols* 6.14 ± 0.14 Ethanol-extract 3.93 ± 0.22 Cellulose 9.78 ± 0.34 Hemicellulose 10.71 ± 0.20 Xylose 9.90 ± 0.27 Galactose 0.98 ± 0.03 Arabinose 0.95 ± 0.01 Mannose 0.25 ± 0.04 Lignin 20.90 ± 0.08 Acid-soluble lignin 1.91 ± 0.01 Acid-insoluble lignin 18.99 ± 0.07 Acetyl groups 1.15 ± 0.06 Ash 8.70 ± 0.19 17
RESULTS OLIVE POMACE TPC and Antioxidant activity DPPH assay is a reliable method to determine the antioxidant capacity of biological substrates. EC50 is expressed as the amount of trolox that is quenched by 1 ml of extract Table 3. Total Phenolic Content and antioxidant activity of olive pomace Technology TPC DPPH (mg GAE/g) EC 50 (µg trolox/ml extract) SE 12.89 ± 0.22 69.19 ± 5.20 SFE-200 bar 9.18 ± 0.17 46.20 ± 3.58 SFE-250 bar 12.35 ± 0.25 64.72 ± 6.41 SFE-300 bar 14.01 ± 0.31 85.33 ± 7.04 18
RESULTAS OLIVE POMACE Identification of HPLC Table 4. Polyphenolic compounds present in exhausted olive pomace. Technology Hydroxytyrosol (mg/g) Chlorogenic acid (mg/g) Ferulic acid (mg/g) Quercetin (mg/g) Vanillic acid (mg/g) Caffeic acid (mg/g) SE 1.02 ± 0.008 0.31 ± 0.02 0.76 ± 0.008 0.06 ± 0.002 0.16 ± 0.01 0.09 ± 0.001 SFE-200 bar 0.91 ± 0.005 0.13 ± 0.005 0.99 ± 0.01 0.09 ± 0.005 0.07 ± 0.004 0.02 ± 0.001 SFE-250 bar 0.95 ± 0.007 0.11 ± 0.008 0.71 ± 0.003 0.05 ± 0.003 0.11 ± 0.01 0.04± 0.002 SFE-300 bar 1.25 ± 0.01 0.08 ± 0.003 0.52 ± 0.005 0.04 ± 0.02 0.13 ± 0.008 0.05 ± 0.002 19
CONCLUSIONS The use of olive pomace presents a high economic and environmental interest because of the the high potential for obtaining antioxidants. From the extraction of these residues, it was possible to identify the presence of a great variety of polyphenolic compounds: hydroxytyrosol, chlorogenic acid and ferulic acid, which have a high antioxidant, anticancer, antidiabetic capacity, among others. Additionally, the effect of the supercritical fluid extraction (SFE) was observed compared to conventional extraction (solvent extraction). In the SFE case there was a higher concentration of total polyphenolic compounds and higher antioxidant activity. Higher performance at high pressures (300 bar) was observed. In addition, the implementation of non conventional technologies such as the SFE is a promising alternative for future applications at the industrial level, requiring less time and quantity of solvent. 20
Acknowledgments The authors express their gratitude to the Universidad Nacional de Colombia sede Manizales and the Universidad de Jaén. THANK YOU!!! 21
Processes Biomass pretreatment and fractionation Biorefinery processes and products Economic evaluation, Sustainability and LCA Advances in Biotechnological Conversion Algal biomass and transformations Thermochemical processes Biopolymers and biomaterial Bioactive compounds from biomass Fermentation processes Green chemistry Bioeconomy 22