Research Group in Chemical, Catalytic and Biotechnological 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 Research Group in Chemical, Catalytic and Biotechnological
Olive oil production process Pruning Biomass from pruning Olive leaves Olive pits Olive pomace Olive mill wastewater Ceramic materials Oligosaccharides BIOREFINERY BASED ON Lignin Energy Bioethanol OLIVE-DERIVED BIOMASS Antioxidants Xylitol Techno-economic Nanocellulose evaluation Sugars 3 Research Group in Chemical, Catalytic and Biotechnological
OLIVE TREE FIELD Pruning OLIVE TREE BIOMASS Harvesting from pruning Olive fruit Olive washing (Washing)/Cleaning Leaves wastewater Crushing/Milling Malaxation Water DECANTER Three ‐ phase separation mode Two ‐ phase separation mode Waste water Olive oil Olive pomace Olive pomace (3POMWW) Olive stones Olive oil washing WASHING Drying wastewater Extra virgin Hexane extraction OLIVE OIL Exhausted Olive Pomace Distillation Market Crude Pomace Olive oil Oil refination 4 Pomace Olive oil
Olive pomace Rotary oven Dry Olive pomace Hexane extraction Filtration Liquid Solid Exhausted Miscela Olive pomace Distillation Antioxidant valorisation Olive • Total phenolic compouds Hexane Pomace Oil • DPPH (EC 50 ) • Compound identification PRODUCTION 300 kg exhausted olive pomace/1 hectare olive trees 5 Research Group in Chemical, Catalytic and Biotechnological
INTRODUCTION: Polyphenolic compounds Anti-cancer Regulation of Polyphenolic compounds enzymatic Anti-toxic inhibition are an important class of chemicals present in edible and inedible plants with Anti- Anti-bacterial interesting applications in inflammatory the medical, food and Polyphenols cosmetic industry. Oxidative Anti-viral stress Anti- Antioxidant allergenic Anti- mutagenic Figure 1. Molecular structure of phenol. Taken from: Figure 2 . Properties contributed by polyphenolic compounds . https://goo.gl/1TuNjK 6 Research Group in Chemical, Catalytic and Biotechnological
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 3 2 4 6 Solvent extraction Percolation Mechanical extraction Soxhlet extraction Steam extraction Conventional Extractions 5 7 9 8 Supercritical fluid extraction Ultrasound-assisted Microwave assisted Molecular Distillation extraction extraction Non-Conventional Extractions 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 Research Group in Chemical, Catalytic and Biotechnological
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 Methane 7.38 304.15 4.60 190.4 Ethanol Ammonia 6.14 513.9 11.35 405.55 Methanol 8.09 512.6 n-Hexane 3.01 507.5 Propylene Toluene 4.60 364.95 4.10 591.8 Propane Sulfur dioxide 4.25 369.8 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 Carbon Water 22.12 647.3 3.50 132.9 monoxide Figure 4. Triple and critical point representation. Taken from goo.gl/2zM3Fu Bencene n-Heptane 4.89 562.2 2.74 540.3 Isobutane Hexane cycle 3.65 408.2 4.07 553.5 Di-ethyl amine 3.71 496.5 Propanediol 5.47 393.15 8 Research Group in Chemical, Catalytic and Biotechnological
METHODOLOGY 9 Research Group in Chemical, Catalytic and Biotechnological
Methodology Procedure HPLC Antioxidant capacity Compound Total phenolic identification content Extraction process Characterization of the raw -Solvent material extraction -Supercritical fluid extraction 10 Research Group in Chemical, Catalytic and Biotechnological
Methodology Characterization of Olive Pomace (NREL procedure) OLIVE POMACE LAP-001 LAP-005 Ash Biomass<1mm Total solids (Calcination 575ºC) (Drying 105ºC) LAP-010 Elemental Extractives (Soxhlet extraction) composition HPLC Extracted biomass Proteins C5 and C6 sugars Total phenols LAP-002 (Acid hydrolysis (Aqueous extract) in 2 stages) Fats Hydrolysed biomass Starch Liquid Fraction Solid Fraction LAP-003 LAP-017 (Drying 105ºC) LAP-004 (HPLC) HPLC (Abs. 205 nm) Insoluble acid lignin (IAL) C5 and C6 sugars Acetic acid LAP-005 (Calcination 575ºC) Cellulose Soluble acid lignin Insoluble acid ash Acetyl groups Hemicellulose (SAL) (IAA) 11 Research Group in Chemical, Catalytic and Biotechnological
Methodology Solvent Extraction Solvent: 60% ethanol (v/v) Solid-liquid ratio: 1:20 (w/v) Vacuum filtration Temperature: 25ºC Time: 8 hours Agitation: 300 rpm 12 Research Group in Chemical, Catalytic and Biotechnological
Methodology Supercritical Fluid Extraction Solvent: Carbon dioxide Exhausted Olive Extracted Olive pomace pomace in the thimble Co-solvent: 60% ethanol (v/v) Solid-liquid ratio: 1:3 (w/v) Pressure: 200, 250 and 300 bars Temperature: 50ºC Time: 60 minutes 13 Research Group in Chemical, Catalytic and Biotechnological
Methodology Determination of total phenolic content and antioxidant activity Total phenolic content Folin-Ciocalteu 1 � ��� ����� %���������� � � 100 Equation (1) ��� ����� Antioxidant �� �������� 50 � ��������� Equation (2) �� �� � activity �� ������� ����� DPPH ���� ���� �� 1 �� �� � Equation (3) �� �� ������������� ������� ���� ������ �������� � 14 Research Group in Chemical, Catalytic and Biotechnological
Methodology HPLC Table 1. Elution profiles of polyphenolic compounds. (A) Acetic acid 0.5% Time (min) (B) Methanol v/v 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 Figure 3. HPLC system (LC-2010A HT) with UV- 40 70 30 44 0 100 visible detector. 47 0 100 15 Research Group in Chemical, Catalytic and Biotechnological
RESULTS AND DISCUSSION 16 Research Group in Chemical, Catalytic and Biotechnological
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 Research Group in Chemical, Catalytic and Biotechnological
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 TPC DPPH Technology (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 Research Group in Chemical, Catalytic and Biotechnological
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