APPLICATION OF SUPERCRITICAL FLUIDS FOR POLYPHENOLIC COMPOUNDS - - PowerPoint PPT Presentation

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APPLICATION OF SUPERCRITICAL FLUIDS FOR POLYPHENOLIC COMPOUNDS EXTRACTION FROM EXHAUSTED OLIVE POMACE

Ashley Sthefanía Caballero1, Juan Miguel Romero-García2, Eulogio Castro2, Carlos Ariel Cardona1

1Universidad Nacional de Colombia sede Manizales, Instituto de Biotecnología y

• Agroindustria. Laboratorio de Equilibrios Químicos y Cinética Enzimática. Departamento de

Ingeniería Química. Manizales, Colombia

2Center for Advanced Studies in Energy and Environment, University of Jaen, Spain,

*Corresponding author: ccardonaal@unal.edu.co

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CONTENT

Introduction Methodology Results and discussion Conclusions Acknowledgments

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Pruning Biomass from pruning Olive leaves Olive pits Olive pomace Olive mill wastewater Olive oil production process Ceramic materials Antioxidants Oligosaccharides Bioethanol Lignin Sugars Xylitol Techno-economic evaluation Energy

BIOREFINERY BASED ON OLIVE-DERIVED BIOMASS

Nanocellulose

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OLIVE TREE FIELD

Three‐phase separation mode

Waste water (3POMWW)

Olive oil

Olive pomace WASHING Olive oil washing wastewater Extra virgin OLIVE OIL Market Drying Hexane extraction Distillation Crude Pomace Olive oil

Exhausted Olive Pomace

Oil refination Pomace Olive oil Olive fruit Pruning OLIVE TREE BIOMASS from pruning Harvesting

Two‐phase separation mode

Olive pomace Olive stones Crushing/Milling Malaxation DECANTER Water (Washing)/Cleaning Olive washing wastewater Leaves

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Olive pomace

Rotary oven

Dry Olive pomace

Hexane extraction Filtration

Miscela

Distillation

Exhausted Olive pomace

Liquid Solid Hexane Olive Pomace Oil Antioxidant valorisation

PRODUCTION 300 kg exhausted olive pomace/1 hectare olive trees

• Total phenolic compouds
• DPPH (EC50)
• Compound identification

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INTRODUCTION: Polyphenolic compounds

Polyphenols

Anti-cancer Anti-toxic Anti-bacterial Anti-viral Anti- allergenic Anti- mutagenic Antioxidant Oxidative stress Anti- inflammatory Regulation of enzymatic inhibition

Figure 1. Molecular structure of phenol. Taken from: https://goo.gl/1TuNjK

Figure 2. Properties contributed by polyphenolic compounds.

Polyphenolic compounds are an important class of chemicals present in edible and inedible plants with interesting applications in the medical, food and cosmetic industry.

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INTRODUCTION: Extraction technologies

Solvent extraction Mechanical extraction Soxhlet extraction Percolation Steam extraction

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Conventional Extractions

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.

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.

Supercritical fluid extraction Microwave assisted extraction Molecular Distillation

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Non-Conventional Extractions

Ultrasound-assisted extraction

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INTRODUCTION: Extraction with supercritical fluids

Figure 4. Triple and critical point representation. Taken from goo.gl/2zM3Fu Solvent Pc [MPa] Tc [K] Solvent Pc [MPa] Tc [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

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

Table 1. Solvents most commonly used in supercritical fluid extraction.

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METHODOLOGY

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Procedure

Methodology

Extraction process

• Solvent

extraction

• Supercritical

fluid extraction

Antioxidant capacity Total phenolic content Characterization

• f the raw

material

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HPLC

Compound identification

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Characterization of Olive Pomace (NREL procedure)

Methodology

Biomass<1mm Extracted biomass Hydrolysed biomass Ash Total solids Extractives LAP-002 (Acid hydrolysis in 2 stages) Acetyl groups Solid Fraction Liquid Fraction HPLC C5 and C6 sugars Cellulose Hemicellulose Soluble acid lignin (SAL) Insoluble acid lignin (IAL) Insoluble acid ash (IAA) HPLC C5 and C6 sugars Total phenols (Aqueous extract) LAP-001 (Drying 105ºC) LAP-005 (Calcination 575ºC) LAP-010 (Soxhlet extraction)

OLIVE POMACE

Acetic acid LAP-003 (Drying 105ºC) LAP-005 (Calcination 575ºC) LAP-004 (Abs. 205 nm) LAP-017 (HPLC) Elemental composition Proteins Fats Starch Research Group in Chemical, Catalytic and Biotechnological

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Solvent Extraction

Methodology

Solvent: 60% ethanol (v/v) Solid-liquid ratio: 1:20 (w/v) Temperature: 25ºC Time: 8 hours Agitation: 300 rpm Vacuum filtration

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Supercritical Fluid Extraction

Methodology

Extracted Olive pomace 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 Exhausted Olive pomace in the thimble

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Determination of total phenolic content and antioxidant activity

Methodology

% 1

• 100
• 50
• 1
• Equation (1)

Equation (2)

Antioxidant activity DPPH Total phenolic content Folin-Ciocalteu

Equation (3)

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HPLC

Methodology

Time (min) (A) Acetic acid 0.5% v/v (B) Methanol Elution profile of chlorogenic acid 10 90 4 10 90 15 30 70 25 30 70 Elution profile of ferulic acid 20 80 4 45 55 9 45 55 12 80 20 25 80 20 Elution profile of vanillin 60 40 5 60 40 7 50 50 14 100 18 100 19 60 40 Elution profile of hydroxytyrosol 5 95 10 35 75 13 5 95 15 5 95 Elution profile of quercetin, caffeic acid and vanillinic acid 100 10 10 90 40 70 30 44 100 47 100

Table 1. Elution profiles of polyphenolic compounds. Figure 3. HPLC system (LC-2010A HT) with UV- visible detector.

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RESULTS AND DISCUSSION

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TPC and Antioxidant activity

RESULTS – OLIVE POMACE

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

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TPC and Antioxidant activity

RESULTS – OLIVE POMACE

Technology TPC (mg GAE/g) DPPH EC50 (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 Table 3. Total Phenolic Content and antioxidant activity of olive pomace

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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

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Identification of HPLC

RESULTAS – 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

Table 4. Polyphenolic compounds present in exhausted olive pomace.

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CONCLUSIONS

 The use of olive pomace presents a high economic and environmental interest because

• f 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.

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Acknowledgments

The authors express their gratitude to the Universidad Nacional de Colombia sede Manizales and the Universidad de Jaén.

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THANK YOU !!!

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Research Group in Chemical, Catalytic and Biotechnological 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