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EXTRACTION OF POLYPHENOLS FROM OLIVE LEAVES AND HYDROLYSIS OF - - PowerPoint PPT Presentation
EXTRACTION OF POLYPHENOLS FROM OLIVE LEAVES AND HYDROLYSIS OF - - PowerPoint PPT Presentation
EXTRACTION OF POLYPHENOLS FROM OLIVE LEAVES AND HYDROLYSIS OF OLEUROPEIN FOR THE PRODUCTION OF 3-HYDROXYTYROSOL G.K. LAMPROU , A. VLYSIDIS, A.G. VLYSSIDES LABORATORY OF ORGANIC CHEMICAL TECHNOLOGY SCHOOL OF CHEMICAL ENGINEERING NATIONAL
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STATE OF THE ART
Phenolic compounds, known as “polyphenols”, have attracted the attention of research community due to
their beneficial action in human health.
Their physical role is to provide protection in plants against pathogens, predators or/and stress caused by
abiotic factors such as such as hydric deficiency, salinity, low fertilization and climatic conditions.
These molecules have been recognized for their anti-carcinogenic, anti-inflammatory, and antimicrobial
- actions. In 2012, EU Committee established a list of permitted health claims made on foods, based on
scientific research made by EFSA. This was a basic factor, in order to become extra virgin oil for its health benefits.
In 2017, EU published Regulation 2017/2373, through which authorized the placing on the market of
hydroxytyrosol as a novel food ingredient intended for the general population, excluding children under the age of three years, pregnant women, and lactating women. (hydroxytyrosol (HT) as a food additive to fish and vegetable oils up to 215 mg/kg and to margarines up to 175 mg/kg).
HT, may be found in many plants. Olea europeaea has a high concentration of this molecule. Olive oil, olive
mill waste and leaves are the main sources for the extraction this compound.
Although HT is expensive, there is no industrial activity in this field.
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ACID HYDROLYSIS OF OLEUROPEIN
Recovery of polyphenols from olive leaves is an important challenge for food and pharmaceutical industrial applications. Oleuropein is the major phenolic compound in olive leaves, and large amounts of HT can be obtained using hydrolysis of
- leuropein. HT production requires enzymatic or chemical hydrolysis. The natural mechanism that occurs when the olive
tree forms free HT is enzymatic hydrolysis, and specific native β-glycosidase and esterase are implicated. On the contrary, acid hydrolysis is the more used mechanism in the laboratory and industrial processes.
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METHODOLOGY
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EXPERIMENTAL METHODOLOGY FOR EXTRACTING THE PHENOLIC COMPOUNDS
Acid hydrolysis process (extraction time 300 mins) Lab scale experiments with initial amount of olive leafs 10 g (14.2% moisture) Design of a 33 factorial experiment in order to measure the effect of three important process
parameters
T
emperature (X1)
Quantity of the strong acid (X2) Dilution of Olive Leafs (X3)
At the end of each run the aqueous phase was separated and analyzed:
- TPC (Folin method)
- Concentration of 3-Hydroxytyrosol,
Tyrosol, Oleuropein (HPLC)
Level T emperature oC (X1) H2SO4 % (v/w) (X2) L/S (w/v) (X3)
- 1.682
20.0 2.0% 10.0
- 1
24.1 3.6% 14.1 30.0 6.0% 20,0 1 36.0 8.4% 26.0 1.682 40.0 10.0% 30.0
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EXPERIMENTAL RESULTS OF THE FACTORIAL EXPERIMENT
RUNS Temperature (oC) H2SO4 % (v/w) L/S (v/w) TPC (mg/g db) Oleuropein (mg/g db) Hydroxytyrosol (mg/g db) Tyrosol (mg/g db) 1 36.0 (1) 8.4 (1) 26.0 (1) 80,20 22,96 7,84 1,58 2 36.0 (1) 8.4 (1) 14.1(-1) 63,42 9,84 8,33 1,52 3 36.0 (1) 3.6 (-1) 26.0 (1) 75,92 42,15 3,92 2,30 4 36.0 (1) 3.6 (-1) 14.1(-1) 45,76 23,17 2,33 1,22 5 24.1 (-1) 8.4 (1) 26.0 (1) 80,04 30,63 4,83 2,32 6 24.1 (-1) 8.4 (1) 14.1(-1) 58,99 15,75 6,08 1,51 7 24.1 (-1) 3.6 (-1) 26.0 (1) 62,78 43,20 2,06 2,17 8 24.1 (-1) 3.6 (-1) 14.1(-1) 72,85 38,14 2,57 0,79 9 40.0 (1,682) 6.0 (0) 20.0(0) 72,38 30,03 0,71 1,09 10 20.0 (-1,682) 6.0 (0) 20.0(0) 59,37 24,81 0,33 1,09 11 30.0 (0) 10.0 (1,682) 20.0(0) 69,13 11,05 6,51 1,13 12 30.0 (0) 2.0 (-1,682) 20.0(0) 84,99 35,10 0,00 1,31 13 30.0 (0) 6.0 (0) 30.0 (1,682) 80,51 31,78 0,00 1,87 14 30.0 (0) 6.0 (0) 10.0 (-1,682) 65,88 11,46 5,92 0,59 15 30.0 (0) 6.0 (0) 20.0(0) 82,55 24,62 0,00 1,37 16 30.0 (0) 6.0 (0) 20.0(0) 74,82 25,51 0,00 1,37 17 30.0 (0) 6.0 (0) 20.0(0) 83,36 30,94 0,00 1,42 18 30.0 (0) 6.0 (0) 20.0(0) 84,17 30,05 0,00 1,10
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Experimental and modelling values for the TPC (mg/g)
Parameter Estimate t Ratio β0 83.556 β1 0.9167 0.47 β2
- 0.09762
- 0.05
β3 6.0427 3.09 β4 2.3175 0.91 β5 4.495 1.76 β6 2.2175 0.87 β7
- 7.021455
- 3.45
β8
- 3.066961
- 1.51
β9
- 4.433445
- 2.18
β10
- 5.5625
- 2.17
MODELLING RESULTS OF THE FACTORIAL DESIGN
- Regarding the TPC, the significance of the model was adequate
- R2 between the experimental and model predictions is 0.83
- Important parameters are the square of the T
emperature and the L/S ratio
ΥTPC = β0 + β1 Χ1 + β2 Χ2 + β3 Χ3 + β4 Χ1 Χ2 + β5 Χ1 Χ3 + β6 Χ2 Χ3 + β7 Χ1 X1 + β8 Χ2 X2 + β9 Χ3 X3 + β10 Χ1 X2 Χ3
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Experimental and modelling values for the OLEUROPEIN (mg/g)
Parameter Estimate t Ratio β0 27.5662 β1 ‐1.524585 ‐1.08 β2 ‐7.90278 ‐5.6 β3 6.3128762 4.48 β4 0.305 0.17 β5 1.52 0.82 β6 0.495 0.27 β7 0.8294436 0.57 β8 ‐0.706746 ‐0.48 β9 ‐1.221166 ‐0.83 β10 ‐1.96 ‐1.06
MODELLING RESULTS OF THE FACTORIAL DESIGN (CONT’)
- Regarding the OLEUROPEIN, the significance of the model was also adequate
- R2 between the experimental and model predictions is 0.89
- Important parameters are the amount of the sulphuric acid and the L/S ratio
ΥOleuropein = β0 + β1 Χ1 + β2 Χ2 + β3 Χ3 + β4 Χ1 Χ2 + β5 Χ1 Χ3 + β6 Χ2 Χ3 + β7 Χ1 X1 + β8 Χ2 X2 + β9 Χ3 X3 + β10 Χ1 X2 Χ3
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OPTIMUM EXTRACTION CONDITIONS AND VALIDATION ON THE OPTIMUM VALUE
Optimum conditions For X1 was 0.305 (31.8 oC) For X2 was 0.168 (6.40 v/w) For X3 was 0.840 (25.0 v/w) YTPC = 86.43 mg/g
HYDROXYTYROSOL mg/g db TYROSOL mg/g db OLEUROPEIN mg/g db TPC mg/g db 5.69 1.65 7.53 86.4
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Factor Description Level Value X1 T0C 0,30456 31,81 X2 H2SO4 % (v/w) 0,16772 6,40 X3 L/S 0,84015 24,99 20 40 60 80 100 50 100 150 200 250 300 350 Total Phenolic Content
Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
0,00 5,00 10,00 50 100 150 200 250 300 350 Oleuropein
Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
0,00 0,50 1,00 1,50 2,00 50 100 150 200 250 300 350
Tyrosol
Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
0,00 1,00 2,00 3,00 4,00 5,00 6,00 50 100 150 200 250 300 350
Hydroxytyrosol
Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
Optimization per T
- tal Phenolic Content
1. Theoretical optimum TPC 86.43 mg/g db. Experimental result 86.39 mg/g db 2. Water and Leaves pH 4.27 pH 1.12 After the acidification 3. Total Phenolic Content is enhanced by the presence of sulfuric acid. 4. Concentration of Tyrosol is not affected by pH 5. Concentration of Oleuropein in positively affected by H2SO4 6. Hydroxytyrosol is strongly dependent on the pH
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0,00 2,00 4,00 6,00 8,00 10,00
50 100 150 200 250 300 350 Oleuropein
Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
Factor Description Level Value X1 T0C 1,6818 40 X2 H2SO4 % (v/w)
- 1,6818
2.0 X3 L/S 0,84015 30
1,45 1,50 1,55 1,60 1,65 1,70 1,75 1,80 1,85 50 100 150 200 250 300 350
Tyrosol
Blank (mg/g d.b.) H2SO4 (mg/g d.b.)
Optimization per Oleuropein
1. Concentration and time of extraction for Oleuropein in positively affected by H2SO4 2. Tyrosol highest concentration raised at 95min. 3. Water and Leaves pH 4,01 pH 1,90 After the acidification 4. There was no 3-hydroxytyrosol produced in this pH
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CONCLUSIONS
We illustrated an extraction process of the phenolic compounds from olive leaves
using acid hydrolysis
The Factorial Experiment gave us an overall extraction of Hydroxytyrosol 8.33 mg/g
- f dry OMW and a TPC of 85.33 mg/g of dry base
Water can be used as a solvent with satisfying results. The extraction of the polyphenols is increased in low pH. Concentration of Oleuropein, Hydroxytyrosol & Tyrosol is increased under the
presence of sulfuric acid, under certain circumstances.
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