HANDBOOK on BIOACTIVE COMPOUNDS from OLIVE PROCESSING RESIDUES

Transcription

1 HANDBOOK on BIOACTIVE COMPOUNDS from OLIVE PROCESSING RESIDUES Assessment and dissemination of strategies for the extraction of bioactive compounds from tomato, olive and grape processing residues (BIOACTIVE-NET) BIOACTIVE-NET MANUAL HANDBOOK on BIOACTIVE COMPOUNDS from OLIVE PROCESSING RESIDUES Visit our Website: BIOACTIVE-NET

2 HANDBOOK on BIOACTIVE COMPOUNDS from OLIVE PROCESSING RESIDUES

3 HANDBOOK on BIOACTIVE COMPOUNDS from OLIVE PROCESSING RESIDUES The aim of this handbook is to provide an overview about the bioactive compounds in olive processing residues, the extraction techniques and the application possibilities in the food and cosmetic industry. The Bioactive-net manual is a collection of three publications as part of the project BIOACTIVE-NET. The aim of this project is to collect most relevant knowledge and technologies related to bioactive compounds in tomato, olive and grape processing residues, from techniques of extraction, to application fields and economic feasibility of the extraction, and make it accessible to the public. This publication has been carried out with the support of the European Commission, priority 5 (food quality and safety): contract number FOOD-CT , Specific Support Action (SSA) Assessment and dissemination of strategies for the extraction of bioactive compounds from tomato, olive and grape processing residues. It does not necessarily reflect the views of the Commission and in no way anticipates the Commission s future policy on this area. Additionally, BIOACTIVE-NET foresees dissemination workshops addressed to tomato processors, olive oil mills and wine producers in the South European countries. The Bioactive-net manual constitutes a key part of that dissemination action and will be available on the project website and upon request. 3

4 BIOACTIVE-NET Membres de BIOACTIVE-NET: The BIOACTIVE-NET project is a Specific Support Action (SSA) funded by the European Commission under the 6th Framework Program. The primary objective of bioactive-net is to assess and disseminate to the SME processors, strategies for the extraction of bioactive compounds from tomato, olive, and grape processing residues and therefore allow the: Creation of a broad information platform regarding the extraction of bioactive compounds from tomato, olive, and grape processing residues as well as their application facilities in the food and cosmetic industry. Implementation of dissemination workshops in the south European countries (Spain, Italy, Greece and France) aimed at transferring know-how and evaluating economic feasibilities of the extraction of bioactive compounds to the residue generating companies (SMEs), to the technology providers, to the industrial residue extractors and to the end-users of the respective natural ingredients. Strengthening of the European market on natural ingredients, which has an enormous economic potential due to high availability of the raw materials. Increase of the competitiveness of the European food industry by pre-empting the competition in the use of bio-active compounds derived from natural, renewable and economic source processing residue. Increase of the use of bio-active compounds in the European diet. Project details: Type of instrument: Specific Support Action (SSA) Priority 5: Food Quality and Safety Project number: Project duration: 2 years ( ) Project coordinator: ttz Bremerhaven (Germany) ainia centro tecnológico (Spain) CCAE - Confederación de Cooperativas Agrarias de España (Spain) AMITOM - Mediterranean International Association of the Processing Tomato (France) VIGNAIOLI PIEMONTESI S.C.A (Italy) Union of Agricultural Cooperatives in Peza (Greece) ANFOVI - L organisme de formation des Vignerons Indépendants (France) Tecnoalimenti S.C.p.A. (Italy) This handbook has been developed by Elvira Casas (ainia), Marianna Faraldi (Tecnoalimenti) and Marie Bildstein (ttz Bremerhaven) for inclusion in the Bioactive-net manual. BIOACTIVE-NET mbildstein@ttz-bremerhaven.de 4 5

5 TABLE of content 1 Introduction Bioactive compounds in olive processing residues Olive Polyphenols Hydroxytyrosol Oleuropein Best Available Techniques for the extraction and purification of bioactive compounds from olive processing residues Pre-treatment of the olive processing residues Tray dryer Drum dryer Fluid bed dryer Milling and Homogenisation the olive processing residues Extraction of the dried and homogeneous olive processing residues Solvent extraction Supercritical Fluid Extraction (SFE) SC-CO 2 extraction Purification of the extracts Chromatographic techniques Membranes filtration techniques Crystallization Drying of the purified bioactive extracts Freeze Drying Spray Drying Rotary Vacuum Drying Application fields for bioactive compounds in food and cosmetic products Legislation Olive polyphenols Hydroxytyrosol Oleuropein Existing and potential markets for natural ingredients from olive processing residues Assessment of the economic feasibility for the extraction of olive bioactive compounds from processing residues Extraction of bioactive compounds from olive processing residues Economic analysis of the extraction of olive bioactive compounds from processing residues Extraction of Polyphenol containing oil and olive fibres Extraction of Polyphenol powder and olive fibres Yearly profit and break-even point Acknowledgements References Other related projects and links

6 1. INTRODUCTION At European scale, 3 million tons olives are processed in olive oil per year (with a yield in oil of about tons). Olive oil production is divided into three activity fields: Oil mills, which process the olives into oil, waste water and solid waste. Refineries, where the non-consumable oil is refined. Plants where the oil cake is processed and residual oil is extracted from the waste resulting from olive oil extraction. The wastes obtained after the oil milling process, as showed in the following graphic, are mainly Olive Mill Waste Water (OMWW), alpeorujo, pomace and margine. The European olive oil milling leads to 4,5 millions m3 of waste water per year. The oil mills, mostly of small dimensions, some isolated, others inserted in urban contexts have evident difficulties to unify to recover the wastes. Following the General EU Legislation on wastes (Directive 2006/12/EC), Member States shall take the necessary measures to ensure that waste is recovered or disposed of without endangering human health and without using processes or methods which could harm the environment. Currently the olive processing residues are disposed against payment from the olive oil mills or sold to oil extractors at low price. Is it possible to gain more added value from the olive processing residues for the companies? Can the health benefits from olive be obtained from olive processing residues? Various research studies demonstrated the high level of organic substances contained in olive pomace and Olive Mill Waste Water. Milling OLIVE Washing Crushing & grinding Crushing & grinding Malaxation Malaxation Malaxation Pressing Paste centrifugation Paste centrifugation Oil & Water Pomace Oil & aqueous phase Olive oil Decantation Vegetable water TRADITIONAL PROCESS Aqueous phase + oil Centrifugation Olive oil Vegetable water THREE PHASE PROCESS Water Pomace Olive oil Pomace & vegetable water (alperujo) TWO PHASE PROCESS 2. BIOACTIVE COMPOUNDS in OLIVE processing residues The main bioactive compounds extractable from olive processing residues are Polyphenols and more specifically Hydroxytyrosol and Oleuropein Olive Polyphenols Ingredient description Polyphenols are a group of chemical substances found in plants, characterized by the presence of more than one phenol group per molecule. Polyphenols are generally further subdivided into tannins, and phenylpropanoids such as lignins and flavonoids. Tannin chemistry originated in the importance of the eponymously named tannic acid to the tanning industry; lignins to the chemistry of soil and plant structure; and flavonoids to the chemistry of plant secondary metabolites for plant defense, and flower color (e.g. from anthocyanins). Notable sources of polyphenols include berries, tea, beer, wine, olive oil, chocolate/cocoa, walnuts, peanuts, yerba mate, and other fruits and vegetables. High levels of polyphenols can generally be found in the fruit skins. Metabolic known effects Research indicates that polyphenols may have antioxidant characteristics with potential health benefits. They may reduce the risk of cardiovascular disease and cancer. 8 9

7 Polyphenols have also been investigated as a source of additional health benefit in organic products, but no conclusion was made. It is believed that polyphenols can bind with nonheme iron (from plant sources) and decrease its absorption by the body. The suggested daily dosage of polyphenols is 100mg. Therefore it would be necessary to consume 200ml olive oil a day (~40 teaspoons or one glass). Amount of extractable molecule The Polyphenols concentrations in oil oscillate from 100 to 1000 mg total polyphenols per kg olive oil. In alpeorujo the polyphenols concentration is around 8500mg polyphenols /kg alpeorujo Hydroxytyrosol Ingredient description Hydroxytyrosol is a phytochemical. It is responsible for the bitter taste of extra virgin olive oil. The wastewaters generated during olive processing contain high levels of hydroxytyrosol, most of which can be recovered to produce hydroxytyrosol extracts. Metabolic known effects Hydroxytyrosol has antioxidant properties. It is believed to be the antioxidant with the highest free radical scavenging capacity: double than that of quercetin (wine antioxidant) and more than 3 times that of epicatechin (tea antioxidant). Studies by Visioli et al (2000) showed that a low dose of hydroxytyrosol reduces the consequences of sidestream smoke-induced oxidative stress in rats. Amount of extractable molecule The hydroxytyrosol concentration is around 400 µ g/kg in OMWW (Olive Mill Waste Water) Oleuropein Ingredient description Oleuropein is a polyphenolic fraction derived from the fruit, leaves, bark and roots of the olive tree, which help make it strongly resistant to damage from insects and other factors. Oleuropein is known as an iridoid, a type of plant chemical found throughout the olive tree and in olive oil. Metabolic known effects Within Oleuropein is a chemical agent called elenolic acid, which has been shown to assist the body's immune defense. Research studies have found that elenolic acid helps the body to balance levels of friendly bacteria and support the immune system. Like resveratrol, the polyphenolic component of red wine, oleuropein imparts some important antioxidant benefits that may help prevent the oxidation of LDL cholesterol and support healthy heart function. The oxidation of LDL cholesterol the socalled "bad" cholesterol can severely damage the walls of arteries. Along with resveratrol, oleuropein is high on the list of beneficial components of the acclaimed Mediterranean Diet (which includes olive oil), believed to be responsible for the reduced incidence of heart disease in those who habitually partake in the Diet. 3. BEST AVAILABLE TECHNIQUES for the extraction and purification of BIOACTIVE COMPOUNDS from olive processing residues To extract bioactive compounds out of olive processing residues different steps have to be followed: Pre-treatment of the olive processing residues. Extraction of the dried and homogeneous olive processing residues. Purification of the extracts. Drying of the purified extracts Pre-treatment of the olive processing residues Olive processing residues contain a lot of water. Depending on the extraction process it is necessary or not to dewater the samples before the extraction process. For supercritical fluid extraction, for example dewatering of the olive residues is necessary. Moreover the drying of the olive residues facilitates the transport and storage of the bioactive rich residues. To enable an efficient extraction, it is also necessary to mill the dried residues in order to guaranty the homogeneity of the extractor feed. Several techniques and equipments may be applied in order to prepare olive processing residues. Some of the most used drying and milling techniques are presented here

8 Tray dryer Olive residues are spread out thinly on trays, inside a cabin which is connected to a source of air heated by gas diesel or biomass to remove the moist vapours. Depending on the cabinet design, there are batch tray dryers, semi-continuous tray dryers and cross flow chambers. Door No. 1 Door No. 2 Trays Drum dryer Trays Figure 1: Tray dryer. Figure 2: Fluid-bed drying process. Olive residues are spread over the surface of a heated drum. The drum rotates and the residues remain on the drum surface during the major part of the rotation, while the drying process takes place, and then they are scraped off. The following table compares tray drying and drum drying processes used with olive processing residues: Technique Initial investment Drum drying easy long Fluid bed dryer Energy consumption Operating easiness Drying time Tray drying + + easy long The feed of wet material is dried by intimate contact with hot air when the material is in a fluidized state Milling and Homogenisation of the olive processing residues Sometimes it may be necessary to reduce the size of the particles (by milling) and to mix the residues by a homogenisation process in order to improve the extraction process. Milling of the dried olive processing residues Milling is used to convert the shred material into fine particles. One possible milling technique for the pre-treatment of the olive processing residues is the hammermill. A hammermill is essentially a steel drum containing a vertical or horizontal crossshaped rotor on which pivoting hammers are mounted (see figure 3). The hammers are free to swing on the ends of the cross. The rotor is spun at a high speed inside the drum while material is fed into a feed hopper. The material is impacted by the hammers on the ends of the rotating cross and thereby is shredded and expelled through screens in the drum. The dryer comprises: A top fluidising chamber. A bottom air distribution chamber. A specially designed perforated plate. Figure 3: Hammermill

9 Homogenisation of the dried olive processing residues Homogenisation is a process that makes a mixture the same throughout the entire substance. A mixing phase is required and enough in order to homogenise the dried and milled residues. The most relevant parameter to control in sonicated assisted extraction is frequency, because small changes of this parameter can affect dramatically the yield of extraction. Ultrasounds cause a greater penetration of solvent into cellular matrices improving mass transfer Extraction of the dried and homogeneous olive processing residues The operation of extraction consists of the separation of one or more species from a solid or liquid matrix based on the different relative solubility that such substance or substances present in a certain solvent respect to the rest of the components of the matrix. In other words, extraction works according to the principle that soluble components can be separated from insoluble or less soluble components by dissolving them in a suitable solvent. Raw materials that are suitable for extraction may contain solids only, solids and a solution, or solids and a liquid Solvent extraction Ultrasounds Fresh or dry biomass Extract Solvent extraction Filtration Drying Solvent Solvent Evaporation Extract Conventional solid-liquid extraction This technique implies the contact of the plant solid matrix with a liquid solvent. The selection of the solvent will be determined by the chemical and physical properties of the target substances. In particular, the thermal stability and the polar character of the substance have special relevance. The solvent temperature must be chosen accurately depending on the raw material and on the thermal resistance of the solutes we want to recover. In order to facilitate the transfer of the target substances to the liquid, the plant feedstock is normally treated mechanically. This process is used to extract oils. It is not suitable for thermolabile substances. Some organic solvents that may be used as extraction agent are toxic and can leave traces in the end product. Ethanol can be used to replace some toxic or dangerous organic solvents. In addition, all the solvent extraction requires a purification stage after extraction, such as filtration or centrifugation. Ultrasound and microwave assisted extraction are similar to conventional extraction, with the addition of ultrasound or microwave in order to increase the yields, reduce the volume of solvent and reduce the working time. Sonicated-assisted extraction (ultrasound) Sound waves with frequencies higher than 20 khz can improve the extraction yield of plant material because they involve alternative expansions and compressions of matter inducing the creation of bubbles in liquids. Spent biomass Figure 4: Sonicated-assisted extraction process diagram. Ultrasound-assisted extraction has been used to extract nutraceutical such as essential oils, lipids, antioxidants, steroids and terpenoids. It allows process conditions to be milder compared to traditional solvent extraction, so it is recommended for thermolabile substances. Microwave-assisted extraction (MAE) Microwaves are electromagnetic waves that interact with matter, in particular with polar molecules to generate heat. They can therefore, penetrate water and biological matrices heating up the whole at homogeneous rate. Radiation produces superheating of water within plant cells and causes the rupture of the cellular wall facilitating the transfer of interesting substances to the bulk phase inside the extraction vessel and the penetration of the solvent into the plant matrix. Microwaves can therefore improve the extraction yields of nutraceuticals. The volume of solvent needed, and the extraction time are reduced. The effectiveness of MAE depends strongly on the polarity of the solvent and on the particle size and the distribution of the vegetable material. It can be applied to extract polar components, but it is not suitable for dry materials or too wet matrices using nonpolar solvents. (Water, methanol and ethanol are polar enough to be employed). In addition, MAE requires a purification stage after extraction, such as filtration or centrifugation

10 Accelerated solvent extraction (ASE) Accelerated solvent extraction is a solid-liquid extraction carried out at high temperatures (that improve the diffusivity of the solvent accelerating the extraction) and pressures (to maintain the solvent in liquid phase), below the critical point of the solvent. Most of the solvents used in conventional solid-liquid extraction are suited for ASE (including water) to recover polar compounds from plant material Supercritical Fluid Extraction (SFE) SC-CO 2 extraction The supercritical state is reached by bringing the fluid to a temperature and pressure beyond its critical point. Supercritical fluids present characteristics of both gases and liquids and properties that make them especially suitable for extraction processes. Supercritical fluids have higher diffusion coefficients and lower viscosity and surface tension than conventional solvents. The dissolving capacity of supercritical fluids depends on its density, so the selectivity of extraction can be changed by adjusting the temperature and/or the pressure of extraction. After the extraction time, the pressure is reduced, or the temperature is increased, so the solubility of the extract decreases and it can be separated. The most used solvent is CO 2, which is cheap, safe, non-toxic and its supercritical conditions may be fairly easily reached. It can be used to extract polyphenols such as resveratrol and other natural antioxidants from grape peels and stalks. It is suitable for thermolabile substances and it can be also used for polar substances if some modifiers are added to it (methanol, ethanol, water, acetone ). The following table compares the different extraction techniques exposed. Extraction method Compounds extracted Humidity tolerance Purification requirements Conventional Polar & Non-polar + High 3.3 Purification of the extracts After the extraction processes, recovering a biological product from the interferences and the impurities requires some purification steps, to obtain the product according to the final specifications. The purification simply aims at obtaining the target molecule pure enough in the shortest possible time Chromatographic techniques Chromatography is a very special purification process because, it can separate complex mixtures with great precision (even very similar components can be separated). In fact, chromatography can purify basically any soluble or volatile substance. It can be used to separate delicate products because the conditions are not typically hard. For these reasons, it can be used to separate mixtures of olive bioactive compounds. Another advantage of these techniques is that the separated compounds are immediately available for identification or quantification. On the other hand, some instrumentation is expensive and not easily portable and some work is needed to avoid the contamination of the column. Separation by chromatography depends on the differential partition of compounds between a stationary phase (the adsorbent) and a mobile phase (the buffer solution). Normally, the stationary phase is packed into a vertical column of plastic, glass or stainless steel, and the buffer is pumped through this column. The sample to be fractionated is pumped on the top of the column and the various sample components travel with different velocities through the column and are subsequently detected and collected at the bottom of the column. In general, bio-molecules are purified using purification techniques that separate them according to differences in their specific properties, as shown in following table. Ultrasound Polar & Non-polar + High Molecular property exploited Type of chromatography Microvawe Polar + High Size Gel filtration (sometimes called size-exclusion) CO 2 supercritical Non-polar Low Charge Ion exchange chromatography Subcritical Polar ++ Medium-high Ligand specificity Adsorption chromatography 16 17

11 Partition chromatography The stationary phase is usually a liquid, which can be mechanically coated or chemically bonded on a comparatively inert solid support. The molecules to separate are held in this stationary phase (shown in figure 5). Reverse phase (RP) chromatography, when the stationary phase is less polar than the mobile phase, is a good example of liquid-liquid chromatography. The disadvantages of this simple and effective method are its low capacity and the fact that it does not work very well for crude mixtures, so this process can be applied in the final polishing step. Ion exchange chromatography The basis for ion exchange chromatography is the competitive binding of compounds with differences in charge, to an oppositely charged chromatographic medium, the ion exchanger. Sample molecules carried by mobile phase Sample molecules held in sorbed solvent Figure 5: partition chromatography. The advantages of this technique are the high recovery, the large volumes and the easiness to scale-up. Size-exclusion or gel filtration The separation in gel filtration depends on the different abilities of the sample molecules to enter inside the pores which contain the stationary phase. Ver y large molecules do not enter and move through the chromatographic bed faster. Smaller molecules, which can enter inside the gel pores, move slower through the column, because they spend a part of their time in the stationary phase. Molecules are eluted according to their decreasing molecular size. Figure 7: Ion-exchange chromatography mechanism. By using this technology, large volumes can be processed. Adsorption chromatography A bio specific adsorbent is prepared by coupling a specific ligand, on a solid surface, that will only interact with the molecules that can selectively bind to it, (the ones that are needed to be separated). Molecules that are not bound elute unretained. The retained compound can later be released in a purified state. This kind of technique is used in Fine Chemistry. The following table compares the main characteristics of the different chromatography technologies that have been explained before. Figure 6: Size-Exclusion chromatography mechanism

12 Technique Laboratory scale Partition chromatography X Size-exclusion chromatography Ion exchange chromatography Adsorption chromatography X X X Large scale X X X X Selectivity High Low High High Resolution High Low High High Capacity High Low High High Recovery yields Operating easiness High (close to 100%) High Low (50-60%) High Easy Simple, fast Lengthy procedure Easy Costs Fine chemistry applications Industrial chemistry applications X NO X X X X X X Chromatographic methods such as reverse phase, ion-exchange and adsorption chromatography compete reasonably well with affinity-based purifications, especially when high purity is not required Membranes filtration techniques Membranes selectively filter gases or liquids in solutions or mixtures into their different components. The membrane micropores are sized to allow some molecules and particles to go through and block others. Thus membranes are very specific, with their molecular structure tailored according to the particular species to be separated. Membrane filtration is regarded as BAT in the BREF (Best Available Techniques Reference Document) for the Food, Drink and Milk Industries due to the reduced water consumption and waste water pollution that its use entails. There are three main technological processes depending on the size of the components to retain: microfiltration, ultrafiltration and reverse osmosis. Microfiltration Microfiltration is a low-pressure cross-flow membrane process for separating colloidal and suspended particles in the diameter range µ m. Microfiltration is a purely physical process in which particles are captured on the surface on the membrane. Any particle larger than the pore size of the membrane cannot squeeze through. Membrane filters are widely used in biotechnology and food and beverage applications where sterile product is required. Ultrafiltration Ultrafiltration membranes retain particles in the range µ m and operate in the pressure range bar. It has become the best method to concentrate, largely replacing size-exclusion chromatography in these applications. UF membranes are highly used in biopharmaceutical applications. The major advantages of ultrafiltration over competing purification techniques such as chromatography are: High throughput of product. Relative easiness of scale-up. The equipments are easy to clean and sanitise. Reverse osmosis Osmosis explains the phenomenon whereby if a semi-permeable membrane separates two salt solutions of different concentration, water will migrate from the weaker solution, through the membrane, to the more concentrated solution, until the solutions have the same salt concentration. Reverse osmosis involves applying pressure to reverse the natural flow of water, forcing the water to move from the more concentrated solution to the weaker. The semi-permeable membrane is porous, allowing water to pass through, but blocking the passage of the bulkier salt molecules. The result is water without salt on one side of the membrane. Reverse osmosis mainly remove water and molecular compounds smaller in size than water molecules. Reverse osmosis is a high-efficient technique for concentrating/separating low-molecular-weight substances in solution. For this purposes, it requires an energy source and is fairly expensive

13 Crystallization Crystallization is a technique used to purify solid compounds. It is based on the principles of solubility. As a general rule, compounds (solutes) tend to be more soluble in hot liquids (solvents) than they are in cold liquids. If a saturated hot solution is allowed to cool, the solute is no longer soluble in the solvent and it forms crystals of pure compound. Impurities are excluded from the growing crystals and the pure solid crystals can be separated from the dissolved impurities by filtration. High purity products are obtained so, this process is particularly used at pharmaceutical level. The following table compares the purification techniques explained in the present chapter. Scale-up Equipment Pressure sensitive compounds Temperature sensitive compounds Relatively easy Flexible Suitable Suitable More difficult Relatively inexpensive More difficult Expensive More difficult Complex and expensive Suitable Suitable Suitable Relatively easy Simple, reliable, easy to maintain Not suitable Relatively easy Simple, reliable, easy to maintain Not suitable Reverse osmosis Relatively easy Simple, reliable, easy to maintain Not suitable Technique Partition Sizeexclusion exchange tion Ion Adsorp- chromato chromato chromato chromato Microfiltration Ultrafiltration Crystallization Used Suitable Suitable Suitable Suitable Suitable Suitable Suitable Suitable Selectivity High Low High High Low Low Low High Freeze Drying It is also known as lyophilization. It is used to preserve a perishable material or to make it easier to transport. Freeze-dryed product can be rehydrated quickly and easily. This process includes the following steps: Freezing the material. Reducing the surrounding pressure. Adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas Spray Drying Spray drying is the most widely used industrial process, involving particle formation and drying. It is highly suited for the continuous production of dry solids in powder, granulated or agglomerated form from a liquid feedstock. It is an ideal process when the final product must comply with precise quality standards regarding particle size distribution, residual moisture content, bulk density, and particle shape. Spray drying involves the atomization of a liquid feedstock into a spray of droplets and contacting the droplets with hot air in a drying chamber. Evaporation of moisture from the droplets and formation of dry particles proceed under controlled temperature and airflow conditions (it is shown in the following drawing). Particle Purification time Dependent Dependent Independent Dependent Independent Independent Dependent Independent (0,1-10 µ m) (0,01-0,1µ m) Fast Fast Low Long Long Long Long Costs Energy requirements Low Low Low Low Quite low Quite low Fairly costly Efficiency High Low High High High Medium High ++ Low Figure 8: Spray drying process 3.4. Drying of the purified bioactive extracts The bioactive compounds need to be dried enough to be safely stored until they are required for further processing. The whole drying process needs to be gentle to reduce the risk of bioactive compounds degradation. Different drying methods can be used. Drying occurs very fast, so this process is very useful for materials that can be damaged by long time exposures to heat. Spray drying has been identified in the Best Available Techniques Reference Document for the Food, Drink and Milk Sectors as best available technique for drying because of its reduced energy and water consumption and its reduced emissions of dust

14 Rotary Vacuum Drying Wet feed is loaded as a batch and is heated indirectly while being agited by a paddle. The operation is normally carried out under vacuum. Solvent recovery is possible by condensing the vapours generated during the drying operation. Other advantages of this process are: Granular / pasty wet materials can be handled. Low temperature operation is possible: it is ideal for materials that would be damaged or changed if they were exposed to high temperatures. The vacuum removes moisture while preventing the oxidation or explosions that can happen when some materials are mixed with air. Mode of heating is indirect. High energy efficiency. Closed operation: solvents can be recovered, it is safer and minimizes the product loss caused by atmospheric contaminants, dusting, oxidation, discoloration, and chemical change. Commission Directive 95/45/EC laying down specific purity criteria concerning colours for use in foodstuffs. Commission Directive 95/31/EC laying down specific criteria of purity concerning sweeteners for use in foodstuffs. Commission Directive 96/77/EC laying down specific purity criteria on food additives other than colours and sweeteners. Cosmetic products Council Directive 76/768 of 27 July 1976 on the approximation of the laws of the Member States relating to cosmetic products. Restrictions and prohibitions on ingredients that can be used in cosmetics are included in various lists under the EU Cosmetics Directive. Decision 96/335/EC updated by Commission Decision 2006/257/EC, establish an inventory and a common nomenclature of ingredients employed in cosmetic products. The inventory is purely indicative and shall not constitute a list of substances authorized for use in cosmetic products Olive polyphenols 4. Application fields for BIOACTIVE COMPOUNDS in food and COSMETIC PRODUCTS 4.1. Legislation From a legislative point of view, natural ingredients are regulated as Food additives and/or Cosmetic products. Food additives Food additives used as ingredients during the manufacture or preparation of food and which are part of the finished product are covered by the scope of the Directive 89/107/EEC. Prior to their authorisation, food additives are evaluated for their safety by the Scientific Committee on Food, an expert panel that advises the European Commission in questions relating to food. All authorised food additives have to fulfil purity criteria which are set out in detail in three Commission Directives: State of the competition The following companies have been identified as bulk olive polyphenols suppliers: Product OLIVEPURE 35% polyphenols PROLIVOLSTM Company Naturex SEPPIC Origin of polyphenols Purity Price per kg Olive fruits >35% $/kg Margines (water soluble part of olives, obtained from the pulp of olives during the olive oil process) >35% $/kg Possible application Italian botanicals manufacturer Indena and Japanese cosmetics giant Kanebo have joined forces to develop an olive fruit extract designed to provide moisturizing and anti-ageing properties for the skin care category. This is one example of the application of olive polyphenols in the cosmetic industry

15 Olive polyphenols can also be incorporated in health food products (biscuits, bakery products, dietetic products) and nutraceuticals Hydroxytyrosol State of the competition Two companies offering bulk supply of hydroxytyrosol have been found: Lachifarma in Italy and Genosa in Malaga (Spain). Possible application Hydroxytyrosol can be used in food (as natural additives alternatively to synthesis compounds), chemical, pharmaceutical and cosmetic industries Oleuropein State of the competition The supply of bulk oleuropein is not exploited by a lot of companies so far, as shown in the following Table where only one company is represented. 5. Assessment of the ECONOMIC FEASIBILITY for the extraction of OLIVE BIOACTIVE COMPOUNDS from processing residues The objective of this chapter is to demonstrate the net benefit of the extraction of bioactive compounds from olive processing residues in light of the costs for the industrial extraction of bioactive compounds and the revenues deriving from their selling. Two extraction processes have been used as calculation example to determine the economic feasibility of the extraction of bioactive compounds from olive processing residues: Example 1: Supercritical Fluid Extraction (SFE)/SC-CO 2 extraction. Solvent extraction. Considering the SFE and Solvent extraction, the following flow-charts show the process steps required to extract compounds and the necessary equipments. Product OLIVEPURE leaf 15% Company Naturex Origin of oleuropein Purity Price per kg Olive leaves >15% 42.12$/kg Example 1: Olive processing residues (Pomace) Olive processing residues (Pomace) Possible application Oleuropein can be used in the food, chemical, pharmaceutical and cosmetic industries for its antibacterial properties. Belt Dryer Hammermill Drying Milling Belt Dryer Hammermill Drying Milling 4.5. Existing and potential markets for natural ingredients from olive processing residues During the market observation only one enterprise was located which concerns itself with the subject of the natural production of olive polyphenols from olive processing residues and not from olive fruits. This method of approach is a world novelty. Supercritical fluid extractor Spray Dryer Extraction Drying Homogenisator Stirred Tank Decanter Ultrafiltration Evaporator Homogenisation Extraction Decantation Filtration Evaporation Different companies are currently extracting and marketing oleuropein from olive leaves. It would be interesting to estimate the part of leaves gained during the washing stage before the milling of the olives to evaluate the economic interest of the extraction of oleuropein out of the washing residues. Spray Dryer Drying 26 27

16 It is interesting to underline that the pre-treatment (drying and milling of the pomace) must be necessarily done during harvesting season (from November to February, 4 months) to preserve the product. The extraction and the drying are two operations that can be done all year over (either 110 days in the minimum hypothesis; 200 days in the intermediate hypothesis or 330 days in the maximum hypothesis; 24 hours a day), reducing in this way the effort during the short and hard period of the olive harvesting and partially overlapping the problem of the seasonality of the extracts obtainable from olive. It is also very important to note that the calculations developed in this chapter include only labour, energy, maintenance, quality control and consumable costs. Costs such as costs of sales and marketing, shipping, handling and storing, recovery costs of solvent have not been considered. However, even if not exhaustive, the following evaluations give a good idea of the economic feasibility of two different extraction methods Extraction of bioactive compounds from olive processing residues Starting from the production data of olive processing residues provided by the European industrial Associations of the sector participating to the project, three hypotheses have been chosen (minimum, intermediate and maximum) to provide more adapted information to companies with different production sizes. Example 1: PRE-TREATMENT (during harvesting season): 7.3t Pomace / day (moisture ~55%) Belt dryer 3.6t dry Pomace / day (moisture ~10%) Hammermill 3.6t dry milled Pomace / day (all year over=110 days) 2.5t fibre/day EXTRACTION (all year=110days): Supercritical fluid extractor 145 kg polyphenols containing oil / day DRYING (all year over): Spray dryer PRE-TREATMENT (during harvesting season): 7.3t Pomace / day (moisture ~55%) Belt dryer 3.6t dry Pomace / day (moisture ~10%) Hammermill 47.3t dry milled Pomace / day Addition water solvent 1:12 (all year over=110 days) Homegenisator 2.5t fibre / day EXTRACTION (all year=110days): Solvent Extractor Decanter kg polyphenols containing oil / day The minimum hypothesis considers a quantity of 800 tons/year of olive processing residues to treat in single olive processing companies. Small olive mills have a yearly work of 110 days corresponding to the harvesting season lasting from November to February (4 months). 101 kg polyphenols powder / day 101 kg polyphenols powder / day DRYING (all year over): Spray dryer With SFE it is possible to extract, during the 110 days of yearly production, around 145kg of oil containing polyphenols and 2 500kg of fibre per day. The oil can be sold as such or further processed into polyphenol powder leading to 101kg/day. With solvent extraction it is possible to extract, during the 110 days of yearly production, around 145kg of oil containing polyphenols and 2 500kg of fibre per day. The oil can be sold as such or further processed into polyphenol powder leading to 101kg/day

17 The intermediate hypothesis considers a quantity of tons/year of olive processing residues to treat in single cooperatives. Cooperatives are working 200 days a year. The olive processing residues have to be pre-treated during the harvesting season lasting from November to February (4months) before the extraction that can be performed during the 200 days of yearly production. 5.2 Economic analysis of the extraction of olive bioactive compounds from processing residues Extraction of Polyphenol containing oil and olive fibres Cost analysis The costs of the different treatment steps have been evaluated. With SFE it is possible to extract, during the 200 days of yearly production, around 5t of oil containing polyphenols and 87.5t of fibre per day. The oil can be sold as such or further processed into polyphenol powder leading to 3.5t/day. With solvent extraction it is possible to extract, during the 200 days of yearly production, around 5t of oil containing polyphenols and 87.5t of fibre per day. The oil can be sold as such or further processed into polyphenol powder leading to 3.5t/day. Drying costs The drying phase is the same in both processes (SFE and solvent). The production costs have been calculated for different capacities of belt dryer depending on the hypothesis considered. The labour costs, the energy costs and the quality control analysis costs are proportional to the olive residues amount to treat yearly The maximum hypothesis considers an amount of tons/year of olive processing residues to pre-treat during the harvesting season lasting from November to February (4months). The maximum hypothesis for the cost calculation considers that the extraction of bioactive compounds is performed by a big and centralized extractor working 330 days a year for the extraction of the processing residues of the whole region. Yearly DRYING costs () Capacity of the Dryer: kg/day Initial Investment: Capacity of the Dryer: kg/day Initial Investment: Yearly DRYING costs () Capacity of the Dryer: kg/day Initial Investment: Capacity of the Dryer: kg/day Initial Investment: With SFE it is possible to extract, during the 330 days of yearly production, around 6t of oil containing polyphenols and 106t of fibre per day. The oil can be sold as such or further processed into polyphenol powder leading to 4.2t/day. With solvent extraction it is possible to extract, during the 330 days of yearly production, around 6t of oil containing polyphenols and 106t of fibre per day. The oil can be sold as such or further processed into polyphenol powder leading to 4.2t/day. Capacity of the Dryer: 2x kg/day Initial Investment: Capacity of the Dryer: 2x kg/day Initial Investment: Milling costs The milling phase is the same in both processes (SFE and solvent) and requires a Hammermill

18 Yearly MILLING costs () Yearly MILLING costs () Yearly EXTRACTION costs () 2: Yearly EXTRACTION costs () Capacity of the Mill: kg/day Initial Investment: Capacity of the Mill: 3x kg/day Initial Investment: Capacity of the Mill: 4x kg/day Initial Investment: Capacity of the Mill: kg/day Initial Investment: Capacity of the Mill: 3x kg/day Initial Investment: Capacity of the Mill: 4x kg/day Initial Investment: Capacity of the SFE Extractor: 3x kg/day Initial Investment: Capacity of the SFE Extractor: 95x kg/day Initial Investment: Capacity of the SFE Extractor: 115x kg/day Initial Investment: Capacity of the SFE Extractor: 2x kg/day Initial Investment: and Capacity of the SFE Extractor: 2x kg/day Initial Investment: Homogenisation costs The homogenization is a phase necessary only in the solvent extraction process. It requires the investment in a Homogenisator. No homogenisation necessary!! Yearly HOMOGENIZING costs () Capacity of the Homogenisator: kg/day Initial Investment: and Capacity of the Homogenisator: kg/day Initial Investment: Extraction costs The initial investment of the two extraction processes is extremely different: to for the solvent extraction and to for the SFE extraction. Besides, the solvent consumption is higher for the non-supercritical extraction. A CO 2 consumed toward sample extracted ratio of 30 and a CO 2 recycling of 80% have been considered. For the solvent extraction 90% solvent recovery has been considered. Decantation costs The decantation is a phase necessary only in the solvent extraction process. No decantation necessary!! Yearly DECANTATION costs () Capacity of the Decanter: 2x kg/day Initial Investment: and Capacity of the Decanter: 2x kg/day Initial Investment: Other costs The cost of supervision and an average transport of 15km have been considered for the maximum hypothesis. Yearly OTHER costs () Yearly OTHER costs ()

19 Total investment and production costs Summing the pre-treatment, the extraction and the purification costs, the following investment in equipments and operation costs are required. TOTAL yearly cost () TOTAL initial investment () TOTAL yearly cost () TOTAL initial investment () Benefit analysis The process provides different kinds of natural extracts which can be sold in raw state as food integrator or for cosmetic formulations. Market price of polyphenol oil has been estimated around 30 /kg while olive fibres are estimated at 4.50 /kg Extraction of Polyphenol powder and olive fibres To obtain polyphenol powder, the polyphenol containing oil have to be further processed. The olive fibres obtained from SFE or solvent extraction can be sold as such Cost analysis The costs related to a further drying phase were estimated. Drying costs A carrier is needed to spray dry an oil. Yearly SPRAY DRYING costs () Capacity of the Spray Dryer: 500 kg/day Initial Investment: Capacity of the Spray Dryer: 2x kg/day Initial Investment: Yearly SPRAY DRYING costs () Capacity of the Spray Dryer: 500 kg/day Initial Investment: Capacity of the Spray Dryer: 2x kg/day Initial Investment: Products of the extraction (kg) Polyphenols Fibres Polyphenols Fibres Produits de l extraction (kg) Produits de l extraction (kg) Products of the extraction (kg) Capacity of the Spray Dryer: 3x kg/day Initial Investment: Capacity of the Spray Dryer: 3x kg/day Initial Investment: Yearly revenue () Hypothèse Maximale (2 500t/an) Yearly revenue ()

20 Total investment and production costs By adding the pre-treatment, the extraction, the purification costs and the further drying costs, the following investment in equipments and operation costs are required. TOTAL yearly cost () TOTAL initial investment TOTAL yearly cost () TOTAL initial investment Benefits analysis Considering an average price of about /kg for polyphenol powder and the described incomes related to selling olive fibres, the following annual revenues can be estimated. Products of the extraction () Polyphenols Fibres Products of the extraction () Polyphenols Fibres Produits de l extraction (kg) Produits de l extraction (kg) Yearly profit and break-even point Example 1: SUPERCRITICAL FLUID EXTRACTION SOLVENT EXTRACTION Polyphenol containing oil; Olive fibres Polyphenol powder; Olive fibres Polyphenol containing oil; Olive fibres Polyphenol powder; Olive fibres Yearly profit ( ) without amortization Yearly profit ( ) including a 5 years amortization Yearly profit ( ) without amortization Yearly profit ( ) including a 5 years amortization Yearly profit ( ) without amortization Yearly profit ( ) including a 5 years amortization Yearly profit ( ) without amortization Yearly profit ( ) including a 5 years amortization The financial loss for the minimum hypotheses for the supercritical fluid extraction demonstrate the non-feasibility of this process. The other cases studied proved to be economically feasible. Nevertheless, the advantage of SFE over solvent extraction in term of residual solvent in the end product and also the cost of building the treatment center, the transport for conveyance of the by-products, the packaging, and the environment have to be considered Yearly revenue () Yearly revenue () ACKNOWLEDGEMENTS The BIOACTIVE-NET members gratefully acknowledge the financial support of the European Commission for the completion of the Bioactive-net manual. The publication of this handbook would have not been possible without the inputs of all experts contributing from the eight BIOACTIVE-NET member organizations and from external collaborators who kindly supported the consortium with their valuable contributions