Natural products from olive tree(olea europeaea) byproducts

Transcription

1 Natural products from olive tree(olea europeaea) byproducts Supervisor:Nicolas Kalogerakis Name : Manuela Moreira dos Santos InSolEx project, Bi annual meeting, 4 th December London, UK

2 Olives in the Mediterranean basin Olive cultivation Today

3 Olive oil Byproducts Why de Use of OMWW for extraction of natural compounds The olive phenols are amphiphilic in nature and are more soluble in the water than in the oil phase.. Phenols Contents Olive fruit contens 1 3% Processing of olive Release or formed Olive cake/extracted pomace (45%) OMWW (53%)

4 Olive tree History Member of the family Oleaceae, which comprises 30 species such as jasmine, ash, lilac, and privet. The only edible species is Olea europeaea L, which is cultivated for its plump, fleshy, and oil-containing fruits Grew wild in the Middle East and its fruits have been used since prehistoric times First cultivation of the olive tree worldwide took place in Greece, and more specific in Crete. There are at least fifty different varieties of olive

5 Average world production of olive oil Olive Oil Producer Fig. 1- Main producer countries in 2003[ANONYMOUS, International Olive Oil Council,

6 Olive tree culture and olive processing industry produce large amounts of byproducts Quantities During the season 2007/2008, tons were produced in several thousand of olive oil mills [ANONYMOUS, International Olive Oil Council, Fig. 2: Produced olive oil in tonnes. [ANONYMOUS, Food and agriculture organization of the United Nations, Estimations of the total amount of OMWW produced annually range from 7 to over 30 million m3. The manufacturing process of the olive oil olive oil (20%) semi-solid waste (30%) aqueous liquor (50%).

7 Olive Fruit Composition The average composition of an olive: water (50.0%),oil (22.0%) sugars (19.1%) cellulose (5.8%)proteins (1.6%), and ash (1.5%), phenolic compounds (1-3%) of the fresh plup) The olive fruit weighs from 2 12 g. Harvesting season (from November to February) Hundreds of cultivars for olives: for oil, for table, and for both uses Extraction of olive oil is not an easy process.

8 Olive oil Byproducts OMWW Average characteristics: Strong offensive smell. COD values up to 220 g/l and a COD=BOD5 ratio between 2.5 and 5 ph between 3 and % of the Olive Fruit OMWW (53%) Olive Cake (45%) High content of polyphenols (up to 80 g/l) High content of solid matter (total solids up to 20 g/l).

9 Olives processing olive oil Fig. 3: Flow schemes of the 3 different olive oil production processes: a) Traditional process, b) 3 phase decanter process, c) 2 phase decanter process.

10 Findings-2008 in Natural Compounds 22-April 2008 Hydroxytyrosol, a natural polyphenol from olives, may extend the shelf-life of fish products to the same extent as synthetic preservatives, suggests new research. 27-Aug-2008 Extracts from olive leaves may help reduce cholesterol and blood pressure levels, according to a small human study from Israel-based supplier Frutarom 12-Sep-2008 Virgin olive oil extracts can benefit heart and circulation health by reducing platelet aggregation in a study conducted on rats.

11 Olive Mill waste phenols More than 30 different phenolic compounds have been detected in OMWW and reported by several authors. Main compounds: Bioactivity and Analysis of Biophenols Recovered from Olive Mill Wastewater J. Agric. Food Chem. 2005, 53,

12 Objectives Main Goal: Development of extraction method for obtaining high purity antioxidant compounds such as Hydroxytyrosol, Tyrosol and Oleuropein, among others, from Olive Oil Mill Wastewater Isolation and purification of Natural Compounds HVNCs, Antioxidant and antimicrobial activity Study of the relationship between the phenolic compound and activity

13 Problem Definition OMWW Complex Matrix It varies according to: The OMWW composition is not constant both qualitatively and quantitatively 1) Composition of the vegetation water; 2) Olive oil extraction process; Olive variety, Maturity of the olives, Olive s water content, Cultivation soil, Harvesting time, Presence of pesticides and fertilizers, Climatic conditions 1 Phenolic monomers formation 3) Storage time. 2 Medium and high MW polyphenolsfrom the polymerization and autoxidation of phenolic compounds of the first group

14 Problem Definition Recovery problems of polyphenols from OMWW 1) Poor Reproducibility 2) Scaling up problems 3 )Purity and isolation problems Show great variability depending on a number of unpredictable and diverse factors

15 Why so important? Properties: Anti viral, Anti fungal, Anti bacterial Anti Inflammatory Anti oxidants evidence indicating the involvement of oxygen derived free radicals in pathologic processes, such as cancer and atherosclerosis, heart diseases Availability and Prices : Biopurify Hydroxytyrosol 99% pure 5 euros/gr Olive Mill Waste Water PROFIT Oleuropein 98% pure 2,4 euros/gr OMW is potential rich source of a diverse range of biophenols with a wide array of biological activities.

16 Working Plan

17 What has been done by other researchers Lately, several techniques have been developed for the efficient and economic extraction of antioxidants: 2007 A New Membrane Process for the Selective Fractionation and Total Recovery of Polyphenols, Water and Organic Substances From Vegetation Waters 1) Investigation of Microfiltration (MF) and ultrafiltration(uf) ), operating without preliminary centrifugation step, operating with pilot plants with fixed process parameters. Productivity, fouling and cleaning of membranes with different cut off (statistical pore size distribution), material and configuration are compared 2) MF/UF permeates are concentrated in reverse osmosis (RO) 2007 A New Process for the Management of Olive Oil Mill Waste Water and Recovery of Natural Antioxidants 1) Filtration stages to reduce suspended solids. 2) Recovery of phenols by adsorbent resins/deodoring and decolorization of the wastewater 3) Thermal evaporation and recovery of the organic solvents mixture

18 Among different extraction methods, the liquid liquid extraction was preferred for its simplicity and convenience for recovery of antioxidant compounds 2007 Characterization and fractionation of phenolic compounds extracted from olive oil mill wastewaters 1)Verify the efficiency of the liquid liquid extraction (LLE) for the recovery; 2)Fractionate the phenolic extracts by Solid Phase Extraction (SPE), in order to obtain purified compounds As solvent for the extraction, ethyl acetate was chosen which is frequently used to extract biophenols from aqueous matrices such OMWW. It was demonstrated that ethyl acetate exhibits a higher extraction power respect to other solvents (such as methyl isobutyl ketone, methyl ethyl ketone, diethyl ether).. Ethyl acetate is the most efficient solvent for the recovery of phenolic monomers from OMW.

19 2004 Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters 1) different parameters were optimized: solvent nature, ph of OMWW, volumetric ratio between solvent and OMWW, number of extraction stages. 2) recovery of higher yield of hydroxytyrosol was achieved from OMW using a 3 staged continuous counter current liquid liquid extraction The task of recovery is complex as OMWW has high enzyme and hence extreme care must be taken to ensure correct extraction, devoid of chemical modification, which will invariably result in artifacts. Artifactual changes for ex, hydrolysis, oxidation, polymerization Sample handling strategies for the determination of biophenols in food and plants. Methods of protecting the compounds from those deterioratives processes have included etanol, HCl, additives,etc,enzyme during use of inert atmosphere and absence or presence of light. The analyses of phenols is influenced by several factors

20 Several, researchers reported that OMW acidified with HCL from ph=5,5 to ph=3,5 prevents phenols oxidation 2008 Effect of Processing Conditions, Prestorage Treatment, and Storage Conditions on the Phenol Content and Antioxidant Activity of Olive Mill Waste The effects of different storage were studied: Air drying, Oven-drying, Freeze-drying, room temperature storage, storage at 4 C addition of etanol and acetic acid in dark It was claimed that none of these process could stop the loss of total phenols under the current experimental conditions. stability data are seemingly contradictory Lessage Meessen et al. used 30% v/v ethanol to stabilize OMW samples and claimed long term stability of phenols at 4 C in dark storage More recently, hydroxytyrosol concentration increased during storage when 0% or 5% ethanol was added to the waste at 25 C in dark storage. A stability study of phenols in their sample matrix is a prerequisite for any decision taken toward adding value to OMW.

21 Our approach Sample handling Optimal method for HT conservation in OMWW Improve reproducibilty Selection of the most suitable method to prevent or minimize the degradation or oxidation of biophenols during spontaneous OMW fermentation Study the effect of storage treatment and storage condition on the phenol content and antioxidant activity to maximize recovery of biophenol Experimental variable: Time Temperature Light/Dark Acid Absence of air/air Additives Preparation of the sample uniformly enriched in the components of interest and free from interfering matrix components.

22 Our approach Pilot plant: Membrane assisted solvent Extraction of biophenols Selection or study of the membrane Study of distribution coefficient Study of mass transfer coefficient Study the type of flow pattern Possibility of Scalling up Optimize the production of HT

23 Our approach Technical Goal: 1) Obtain a HIGH PURITY product (i.e., concentration of polyphenols >99%) 2) Allow a LOW PRODUCT RECOVERY Since there is a very large amount of OMWW available, there is no need to recover all the polyphenols present in it. The recovery should be such that the remaining OMWW is not phytotoxic (i.e., polyphenols are less than 1.5 g/l in the treated OMWW).

24 Antioxidant Measurements Antioxidant Activity Great variety of methods/lack of validated assay Routine and as standards method Hydrogen atom transfer -HAT- quantify the hydrogen atom capacity ORAC oxygen radical absorbance Adapted for high productivities assay Measure both lipophilic and hydrophilic Utilize peroxyl radical which its predominant in biological systems Chemiluminescence TRAP modified total radical trapping antioxidant parameter Low detection limit Rapid, simple and sensitive approach for estimating phenolic antioxidant in OMWW Single electron transfer-et-measure antioxidants reducing capacity TEAC Trolox equivalent Antioxidant Capacity Assay Utilize ABST+ radical Simple, fast Measure both lipophilic and hydrophilic Antioxidant activity is a function of many parameters

25 Taking advantage of similarity of the compounds Oleuropein. Increase Purity and isolation An enzyme only binds to specific molecules as it is very selective, and the particular molecule or reactant that an enzyme works on is called its substrate. Retling cells Genetic modified microorganism

26 Obied, H. K.; Allen, M. S.; Bedgood, D. R.; Prenzler, P. D.;Robards, K. Investigation of Australian olive mill waste for recovery of biophenols. J. Agric. Food Chem. 2005, 53, Allouche, N.; Fki, I.; & Sayadi, S. (2004). Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters. Journal of Agricultural and Food Chemistry, 52, Russo C.,A new membrane process for the selective fractionation and total recovery of polyphenols, water and organic substances from vegetation waters (VW). Journal of Membrane Science 288 (2007) Agalias,A.; Magiatis, P.; Skaltsouni, A.; Mikros,E.; Tsarbopoulos,A.;GikasE.; Spanos, I; Manios, T. A New Process for the Management of Olive Oil Mill Waste Water and Recovery of Natural Antioxidants. J. Agric. Food Chem. 2007, 55, De Marco,E; Savarese,M; Paduano, A.; Sacchi,R. Characterization and fractionation of phenolic compounds extracted from olive oil mill wastewaters Food Chemistry 104 (2007)

27 THE END Thank You Obrigado ευχαριστώ Děkuju