2015 Volume 21 Issue 2 Pages 193-205
Processing of olive fruits using a three-phase extraction system for the production of virgin olive oil was analyzed to clarify the material balance of olive fruit components, i.e., oil, water, carbohydrates, proteins, minerals and polyphenols. The estimated total olive material balance based on mass flow indicated a final oil extraction yield of around 83.3%, with an initial olive fruit mass flow of 500 kg/h. The remaining oil was discharged as a by-product, mainly in olive cake (8.9%) and olive mill water (7.8%). The material balance of carbohydrates showed that the majority were discharged in olive cake (73.3 kg/h) and olive mill water (27.7 kg/h). The material balance of polyphenols, the most important functional compounds in olives, was determined because of their importance to olive oil chemical stability. Results showed an increase in polyphenols content during malaxation operation (17.6%), which was correlated with several enzymatic reactions directly reflected in the composition of virgin olive oil.
Virgin olive oil (VOO) is the foremost edible oil in the Mediterranean region diet. VOO is produced from the whole olive fruits of Olea europaea, a traditional crop in the Mediterranean region. The fruit is an oval-shaped drupe, consisting essentially of the following three parts: epicarp, mesocarp, and endocarp. The epicarp (skin) and the mesocarp (pulp) account for about 65 – 83% of the total weight, while the endocarp (stone) may vary from 13% to 30%. The average chemical composition of olive fruit is 50% water, 1.5% protein, 22% oil, 24% carbohydrates, and 1.5% minerals. Olive fruit is also high in polyphenols, in the range of 1% of fresh weight (IOOC, 2002).
VOO is produced via industrial extraction, which represents one of the most traditional food processing activities in the Mediterranean region (Vlyssides et al., 2004). The process involves only mechanical operations, which can be sub-divided in two main parts: (1) preparation of a homogeneous paste based on crushing and malaxing operations, and (2) oil extraction. The objective of any oil extraction method is to extract as much oil as possible without altering the quality.
The following three types of extraction systems are currently in use: (1) a system of hydraulic presses used to press the olive paste; (2) a continuous three-phase system in which the oil is separated from the mass by centrifugation; and (3) a continuous two-phase system in which centrifugation is used to separate olive oil from a mixture of olive cake and olive mill water in one phase, which is characterized by a pasty consistency and termed two-phase olive cake.
Currently, the three-phase extraction system is the most commonly used method for VOO extraction in Tunisia. The flow diagram comprises four main operations: fruit cleaning; preparation of the paste (crushing, malaxation); separation of solid and liquid phases; and finally, the clarification of the collected VOO.
Fruit cleaning consists of two successive operations: leaf removal using defoliators and a vibrating screen.
Defoliation is required to prevent the intense green color that causes high bitterness in VOO. During the washing operation, olive fruits are washed in a current of water. This water is recycled after decanting, and fresh water is constantly mixed in pre-set proportions.
The crushing operation is designed to break the cell wall, thereby releasing oil from the inner cavity (vacuole). Not all oil can be released owing to the difficulty of lacerating all of the cells.
Malaxation consists of the low and continuous kneading of olive paste at a carefully monitored temperature, with the aim of achieving high and satisfactory VOO extraction yields. In fact, this critical operation allows the small droplets of oil formed during crushing to merge into larger drops, which can then be easily separated by centrifugation. The increase in temperature during malaxation, either by using a heating system or by adding hot water, serves to break down the emulsion and improve the VOO separation from the olive paste (Inarejos-García et al., 2009; Koutsaftakis et al., 1999).
Centrifugation is used to separate oil from the water and solids in the paste; this exploits differences in density between the three phases. Normally, a centrifugal decanter is used for this purpose. Three outlets are present: an oily must (VOO with varying amount of fine solid particles and water) and two residues: olive mill water (OMW) and olive cake (mainly consisting of stones and pulp) with varying residual oil content.
The collected oily must and OMW undergo one final centrifugation to enhance the extraction yield and clarify the VOO. A disc stack centrifuge separates oil from water, with simultaneous removal of fine solids that are periodically discharged.
Actually, the addition of water (temperature 20 – 25°C) is commonly used during the three-phase extraction process to increase oil extraction efficiency. During malaxation, water is added at approximately 25 kg/100 kg of olives. During centrifugal decanting, a large volume of water is added, ranging from 40 to 60 kg/100 kg olives, in order to enhance oil extraction during horizontal centrifugation, depending on the initial moisture content of the olive paste. For vertical centrifugation, water consumption of approximately 20 kg/100 kg olives is used to increase the oil extraction yield.
During olive processing, many chemical and enzymatic reactions take place, resulting in the release and mixture of cellular components. These reactions may include examples such as the hydrolysis of triglycerides and carbohydrates by lipases and hydrolases; the oxidation of polyphenols by oxidases, and the condensation of free polyphenols by polymerases (Ryan and Robards, 1998). In addition, the partition phenomena of minor olive constituents in oil and water phases after centrifugation is responsible for the unequal distribution of those compounds between OMW and VOO (Amirante et al., 2009).
Polyphenols account for 1 – 2% of olive fresh mass, and represent some of the most important minor olive constituents for VOO chemical stability because of their antioxidant activities (Tsimidou et al., 1992). Olive polyphenols, especially oleuropein and hydroxytyrosol, have been studied with respect to their potential preventive effects, including anticarcinogenic, antiatherogenic, and antimicrobial properties (Ryan et al., 2002). Their chemical structures are extremely variable, ranging from simple molecules to highly polymerized compounds, with molecular weights ranging from 100 Da to more than 30 kDa. Their concentrations in VOO depend on their partition coefficients (Kp) and the temperature (Artajo et al., 2007). According to Rodis et al. (2002), the partition coefficient (Kp) is the ratio of concentrations in the oil and water phases at equilibrium. This coefficient was estimated to be as low as 0.0006 for oleuropein to a maximum of 1.5 for oleuropein aglycone. The oleuropein molecule, which is an ester of hydroxytyrosol and elenolic acid glucoside (Takaç and Karakaya, 2009), represents a typical major compound among polyphenols in olive fruits, with high amounts depending on the cultivar and olive ripeness (60 ∼ 90 mg/g dry weight) (36 ∼ 2400 mg/kg fresh olives) (Le Tutour and Guedon, 1992). It has been reported that oleuropein extracted from olive leaves has related biological effects, such as antioxidant, antibiotic, antihypertensive and radical scavenging activities (Le Tutour and Guedon, 1992). During malaxation, hydroxytyrosol can be produced by the enzymatic hydrolysis of oleuropein, verbascoside and secoiridoid aglycones by β-glucosidase and esterase (Ryan and Robards, 1998).
Material balance is an important principal in calculating mass flow composition, process yields, and separation efficiency during processing. Understanding material balance during VOO processing would clarify the mass flow of main olive fruit components during processing, and is potentially useful for process optimization. However, there have been few detailed reports dealing with material balance during VOO processing. Vlyssides et al. (2004) described the material balance in relation to the olive oil extraction method; water, total solids and oil were assessed. Nonetheless, a detailed material balance was not described for the three-phase extraction method.
In order to more fully understand the process, we investigated the total material balance of olive fruits and their main components during continuous three-phase extraction. Further, we interpreted changes in component material balance and distribution based on partition phenomena and enzymatic reactions.
Sampling and unit operations of the olive oil factory Experimental work was performed in a VOO producing plant located in Sfax (southern Tunisia) during olive oil crop season (November, 2011). A three-phase cycle modular machine (Gruppo Pieralisi, Jesi, Italy) was used for extracting VOO. A scale was used to measure the weight of the olive oil and all the processing intermediates, which are presented in Fig. 1. The following operations were carried out successively during continuous operating mode:
Quantitative flow chart of the three-phase olive oil extraction system.
Harvested olives (Chemlali cultivar) were defoliated using a mechanical defoliator (Optima model, Gruppo Pieralisi), which sucks leaves, twigs and dirt through a powerful airflow generated by an exhaust fan, with a mass flow of about 500 kg/h. Mass flow was calculated using the estimated mass in the outlet over the time of each operation and was reported on a per-hour basis. The olive fruits were maintained at an ambient temperature (∼ 24°C) during defoliation and washing operations.
Washed olive fruits were crushed using a metal hammer mill at 2200 rpm (FP HP 40 model, Gruppo Pieralisi). The temperature of olive paste in the outlet was raised to around 28°C due to the high rotation speed of the hammer mill.
The obtained paste was mixed in a horizontal tank kneader (Dulpex model, Gruppo Pieralisi), with a horizontal shaft equipped with a heating jacket for temperature monitoring. This operation was carried out at 20 – 30 rpm and 30°C for 1 h, volume of 4 m3 after malaxation.
The paste was then pumped into a centrifugal decanter (Jumbo 4 model, Gruppo Pieralisi) and centrifuged at 2,400×g. Inside the rotating conical drum of the centrifuge is a coil that rotates a few rpm slower, pushing the solid materials out of the system.
The separated oily must was then passed through a disk-stack centrifuge (designated as centrifugation 1), (Valente model, Gruppo Pieralisi) at a speed of 9100×g for the additional removal of residual water from the oily must. The system contains a second disk-stack centrifuge (designated as centrifugation 2), (Valente model, Gruppo Pieralisi), set at a speed of 9100×g, for further recovery of the remaining olive oil in OMW, collected after centrifugal decanting. During centrifugal decanting and both vertical centrifugations 1 and 2, the temperature was 28 – 30°C.
Duplicate samples were collected during the different operations: homogenized samples were collected before and after olive defoliation/washing operations, olive paste samples were taken from the output of the malaxation tank, the centrifugal decanter and disk-stack centrifuges 1 and 2 outputs. Each sample was kept frozen at −20°C before analysis to avoid any chemical changes.
Chemicals HPLC-grade methanol, hexane, ethyl acetate and acetonitrile were purchased from RiedeldeHaen (Gallen, Switzerland), oleuropein (85%) from Extrasynthese (Genay, France), hydroxytyrosol (99%) from Sigma-Aldrich (Chemie Gmbh, Germany), and Folin-Ciocalteu reagent from Fluka (Gallen, Switzerland).
Oil content Oil content was analyzed using the Soxhlet method as described in the official method (AOAC Method 920.39C).
Sample pre-treatment for protein determination Samples were prepared as follows: the finely ground solid sample (50 g) was defatted using hexane at room temperature. After evaporation of the hexane residue, the defatted sample was dried at 105°C until reaching a constant weight.
Protein content Total organic nitrogen was analyzed using the Kjeldahl method as described in the official method (AOAC Method 992.23).
Sample preparation for carbohydrate determination The samples were first dried at 105°C. The material was then ground into a fine powder and defatted using hexane at room temperature (AOAC Method 982.14). After evaporation of the hexane residue, the defatted sample was bleached using polyvinylpolypyrrolidone (PVPP). Briefly, approximately 8 g of defatted sample was added to 25 mL of 3% (w/w) PVPP in water. After shaking overnight on a reciprocal shaker at 4°C, the solution was stored at 4°C in the dark until use (BeMiller and Whistler, 2009; Brummer and Cui, 2005; Bonn, 1998).
Carbohydrate content The method of Dubois (Dubois et al., 1956) was used to determine the total carbohydrate content.
Reducing sugars were evaluated using the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959); absorbance was determined at 550 nm.
The analyses of maltotriose, cellobiose and glucose were carried out using a Shimadzu apparatus (Kyoto, Japan) equipped with LC-10AT pump and refractometer detector (model RID-10Avp; Shimadzu). A Fast Carbohydrate column (100 × 7.8 mm) from Bio-Rad (California, U.S.) was employed, and the temperature was maintained at 80°C. Water was used as the mobile phase at a flow rate of 0.3 mL/min. Sample concentrations were calculated based on peak areas in comparison to external standards.
Mineral content Mineral content was determined by sample incineration in an open inert vessel and destruction of the combustible (organic) portion of the sample by thermal decomposition at 700°C within 2 h using a muffle furnace (FO100 Yamato, Tokyo, Japan; AOAC Method 900.02).
Sample preparation for polyphenols determination
(1) Preparation of olive fruit, olive paste and olive cake samples
The olive fruit samples were crushed using a domestic blender (AEG, model SB4600-U, FR) and the resulting paste (0.1 kg) was extracted according to the method described by Bouaziz et al. (2006). Briefly, 250 mL of methanol and water (80/20, v/v) was added to the olive paste (0.1 kg) and the mixture was left to stand under agitation for 24 h. The solution was filtered using filter paper and rinsed twice with 300 mL of hexane to remove the oil. After evaporation of the hydro-methanolic extract to dryness at room temperature, the residue was stored at 4°C in the dark until use.
(2) Preparation of OMW and olive oil samples
Initially, samples were washed with hexane to remove the oil: 10 mL of the samples were mixed with 15 mL of hexane. The mixture was vigorously shaken and centrifuged at 6,000×g for 5 min. The hexane phase was separated out and the procedure was repeated twice. Extraction of polyphenols was then carried out with ethyl acetate: the previously washed samples were mixed with 10 mL of ethyl acetate; then, the mixture was vigorously shaken and centrifuged for 5 min at 6,000×g. The phases were separated and the extraction was repeated four times. After evaporation of ethyl acetate under vacuum, the dry residue was dissolved in 3 mL of methanol and used for characterization and quantification of polyphenols.
Polyphenols content Polyphenol content was analyzed using the Folin-Ciocalteu assay according to the method of Singleton and Rossi (1965).
HPLC analysis of oleuropein and hydroxytyrosol The assays were performed on a Shimadzu apparatus (Kyoto, Japan). A C-18 column (4.6×250 mm; Shimpack VP-ODS) was employed, and its temperature was maintained at 40°C. The mobile phase used was 0.1% (v/v) phosphoric acid in water (A) versus 70% (v/v) acetonitrile in water (B) for a total running time of 50 min.
Sample pre-treatment for determination of enzymatic activity The enzyme extraction procedure was modified from the method of Britsch and Grisebach (1985). Olive fruits were ground with a mortar and pestle in 2 mL/g tissue of extraction medium containing 0.1 mol/L Tris -Hel (pH 7.2), 10% (v/v) glycerol and 30 mmol/L sodium ascorbate. The slurry was filtered through cotton and centrifuged at 10,000×g for 20 min. The supernatant was used for enzymatic assay.
Determination of β-glucosidase and esterase activities β-Glucosidase activity was determined according to Norkrans (1950) by measuring the hydrolysis of p-nitrophenyl-β-glucoside. The amount of para-nitrophenol released was determined spectrophotometrically by measuring the absorbance at 405 nm. One unit (IU) of β-glucosidase activity was defined as the amount of enzyme that produced 1 µmol of p-nitrophenol per min under the assay conditions.
Esterase activity was measured according to the method of Mackness (1983) by measuring the hydrolysis of p-acetylnitrophenol. Absorbance was determined at 405 nm.
The activity flow of both β-glucosidase and esterase was calculated using estimated mass flow in the outlet of each operation, and the determined β-glucosidase and esterase activities were reported on a per-hour basis.
Total material balance In the olive oil factory visited, water was not added during the malaxation and centrifugation operations. The specific addition of water by olive oil producers is used to enhance the oil extraction yield.
After weight measurement, data reconciliation was adopted to adjust the measured data so that the adjusted values obey the mass conservation law and other constraints.
The flowchart for the production of VOO at each step is shown in Fig. 1. The mass flow of washed olives was around 475 kg/h. After crushing and malaxation of washed fruits, the olive paste was then treated using the centrifugal decanter (without water addition) and three phases in the outlet were obtained: olive cake, oily must, and OMW with mass flows of 236.5, 70.5, and 168.0 kg/h, respectively. The oily must was then clarified using vertical centrifugation 1 (without water addition), and the VOO and OMW mass flows in the outlet were 61.4 and 9.1 kg/h, respectively. After obtaining the OMW from the centrifugal decanter, VOO was recovered using vertical centrifugation 2; the mass flows of VOO and OMW in the outlet were 23.4 and 144.6 kg/h, respectively.
The maximum operating capacity of the factory depended on the equipment size, and could process up to 100 tons/day. The material balance of total weight indicates that raw olive generates approximately 17% of VOO, with 83.3% of the initial weight transformed into by-products, i.e., olive leaves (5.4%), olive cake (47.3%) and OMW (30.7%).
The oil extraction yield of VOO is 83.8% of the initial oil content in the feed (initially 100 kg/h). Loss of oil to the olive cake and OMW represents 8.9 and 7.8% of the initial oil content, respectively. Not all of the oil in the olives can be extracted, as some remains enclosed in unruptured cells, some is spread through the colloidal system (micro gels) of the olive paste, and some is bound in an emulsion with OMW. The main difficulty in recovering this “bound” oil is that the dispersed or emulsified oil droplets are surrounded by a lipoprotein membrane (phospholipids and proteins), which stabilizes the oil dispersion. The smaller the size of the droplets, the greater their stability, meaning that they are prevented from coalescing to form larger droplets (Petrakis, 2006).
Material balance of oil After defoliation, washing, crushing and malaxation, the material balance of oil in the olive fruit was generally maintained without change (Fig. 2). Figure 3 represents the material balance for separation of olive paste components in the centrifugal decanter. The yield of oil extracted in the outlet represents 66.6% of the oil content in the feed of the centrifugal decanter; the remaining oil in OMW and olive cake is 24.5% and 8.9%, respectively. The churning effect of the centrifuge, which rotates the olive paste, leads to the formation of an emulsion from the two immiscible phases (oil/water), causing a small proportion of oil to be lost in the OMW. The collected oily must contained 3.5% water, 94.6% oil, and 0.8% solid impurities, which were mostly composed of carbohydrates and minerals. To maintain oil stability, it is necessary for these impurities to be removed before storage.
Schematic presentation of the material balance during malaxation.
Schematic presentation of the material balance during centrifugal decanting (all values are given in kg/h).
Similarly, the generated OMW, representing the water phase, contained 14.6% oil as an oil-in-water emulsion. Thus, both liquids were further treated by high-speed centrifugation to increase the oil extraction yield and enhance the purity of the final VOO product. Regarding the residual oil in olive cake (3.8%), a second extraction with organic solvents is performed in the extractor industry for the production of olive-pomace oil.
The low separation yield in the centrifugal decanter was related to the low applied centrifugal speed (3000×g), which was unable to separate out oil droplets less than 30 µm in size from the olive paste (Boskou et al., 2006). Further, the proportion of oil droplets in olive paste reaching this value depends on the malaxation step, which is used to coalesce oil droplets and increase their size. After crushing, only 45% of the droplets had a diameter greater than 30 µm (minimum size for separation of oil); this percentage rises to 80% after malaxation, with an accompanying large increase in the number of larger diameter droplets (Di Giovacchino, 1996).
Figure 4a shows the material balance for separation of oily must components using vertical centrifugation 1. The oil extraction yield shows 92% of oil in the oily must. The VOO produced by vertical centrifugation 1 contained 0.34% impurities, which were mainly composed of water and minerals.
Schematic presentation of the material balance during vertical centrifugations 1 and 2 (all values are given in kg/h).
The oil extraction yield by vertical centrifugation 2 showed 90.2% of oil in the initial OMW was obtained from the centrifugal decanter, with 5.7% impurities composed mostly of water (Fig. 4b).
Vertical centrifugations 1 and 2 produced 72.4% and 27.6% of the final extracted VOO, respectively.
The oil content in the final OMW was 5.1%, representing 7.8% of the initial oil content.
The VOO acidity value ranged from 0.4 to 0.6% in the three-phase extraction method; by definition, VOO must have an acidity value below 2.0% (Giovacchino et al., 2002).
Material balance of carbohydrates, proteins and minerals The chemical composition of olive fruit, i.e., carbohydrates, proteins, and minerals, was around 25, 1.5, and 1.7%, respectively. Most parts of the olive fruit (pulp and stones), which are mainly composed of arabinose-rich pectic polysaccharides, are found in olive mill by-products (Niaounakis and Halvadakis, 2006) (Fig. 5).
Olive pulp cell walls main structural composition of arabino-rich pectic polysaccharide, GLc: glucose, RH: rhamnose, HT: hydroxytyrosol, CA: caffeic acid, EA: elenolic acid.
After oil extraction processing, the majority of carbohydrates, proteins, and minerals end up in the olive cake and OMW. After centrifugal decanting, 72.3, 43.8, and 54.3% of olive paste content of carbohydrates, proteins, and minerals, respectively, was present in olive cake. The mineral and protein mass flows showed similar behavior during separation in accordance with their hydrophilicity.
The majority of the remaining carbohydrates, proteins, and minerals in the oily must were relocated from the VOO to the OMW after vertical centrifugation; only trace amounts remained in the VOO.
The rest of the carbohydrates, proteins, and minerals were discharged into OMW at 27.7%, 56.1%, and 45.6%, respectively.
Notably, the generated olive cake can be utilized as a foodstuff for livestock (ovine, bovine, and camelids), a solid fuel for burning, or an organic fertilizer.
The material balance of reducing sugars following malaxation was unequal (Fig. 2); the generation of reducing sugars was due to hydrolytic reactions, where reducing sugars were released from initial complex molecules.
Table 1 shows the variation in saccharides during VOO extraction. There was an increase, by a factor of 1.9, of reducing sugars caused by activation of hydrolytic enzymes during malaxation. Moreover, several chemical reactions took place due to the exposure of olive components to air and endogenous enzymes released from olive fruit after crushing (Di Giovacchino, 2002). The amount of maltotriose, a trimer of dextrose, decreased by 22.1% compared to the initial maltotriose concentration, and was related to α-glucosidase activity (Fig. 6a). Cellobiose, a dimer of dextrose, showed a decrease of 20.7% compared to the initial concentration, likely in response to the catalytic activity of β-glucosidase (Fig. 6b). As a consequence, the amount of glucose increased by a factor of 1.42 during malaxation.
| Step | Mass flow of total carbohydrates [kg/h] | Mass flow of reducing sugars [kg/h] | Mass flow of maltotriose [g/h] | Mass flow of cellobiose [g/h] | Mass flow of glucose [g/h] |
|---|---|---|---|---|---|
| Raw olives | 125.00 | 2.90 | 16.30 | 8.20 | 16.30 |
| Washed and defoliated olives | 113.60 | 2.90 | 16.30 | 8.20 | 16.30 |
| Olive paste before malaxation | 113.60 | 2.90 | 16.30 | 8.20 | 16.30 |
| Olive paste after malaxation | 115.50 | 5.50 | 12.70 | 6.50 | 23.10 |
| Olive cake | 83.50 | 0.70 | 3.40 | 0.05 | 9.70 |
| Oily must | 0.50 | 0.00 | 0.00 | 0.00 | 0.30 |
| OMW from centrifugal decanter | 31.50 | 4.80 | 7.10 | 6.50 | 12.80 |
| OMW from centrifugations 1 and 2 | 32.00 | 4.80 | 8.60 | 6.50 | 13.40 |
| Virgin olive oil | 0.00 | 0.00 | 0.70 | 0.00 | 0.00 |
Hydrolysis of maltotriose by α-glucosidase (a). Hydrolysis of cellobiose by β-glucosidase (b).
Material balance of polyphenols The chromatogram in Fig. 7 shows that olive fruits contained oleuropein and hydroxytyrosol as the major polyphenols. We determined the material balance of both molecules during olive oil processing. Table 2 presents the mass flow of total polyphenols and the phenolic monomers, oleuropein and hydroxytyrosol.
Chromatogram (280 nm) of polyphenols extracted from the olive fruits before processing. HT: hydroxytyrosol, OLP: oleuropein.
| Step | Mass flow of total polyphenols [kg/h] | Mass flow of oleuropein [kg/h] | Mass flow of hydroxytyrosol [kg/h] |
|---|---|---|---|
| Raw olives | 11.00 | 5.08 | 1.61 |
| Washed and defoliated olives | 10.80 | 4.06 | 1.55 |
| Olive paste before malaxation | 10.80 | 4.06 | 1.55 |
| Olive paste after malaxation | 12.70 | 3.15 | 2.85 |
| Olive cake | 4.18 | 1.27 | 0.83 |
| Oily must | 0.90 | 0.53 | 0.44 |
| OMW from centrifugal decanter | 7.62 | 1.35 | 1.65 |
| OMW from vertical centrifugations 1 and 2 | 8.12 | 1.73 | 2.02 |
| Virgin olive oil | 0.40 | 0.15 | 0.00 |
During defoliation and washing, total polyphenols mass flow showed a slight decrease of 1.8%. Oleuropein mass flow dropped by about 20% after this operation, due to the removal of olive leaves, which are rich in oleuropein.
After crushing and malaxation, 1.9 kg/h of total polyphenols was generated. The material balance of oleuropein and hydroxytyrosol monomers showed an opposite feature: oleuropein mass flow decreased to 0.9 kg/h, whereas hydroxytyrosol increased to 1.3 kg/h. This suggests that malaxation involves not only the mechanical process separating olive oil droplets from the solid phase, but also a series of complex biochemical reactions due to various enzymatic activities. Some researchers have reported that hydroxytyrosol was released from oleuropein by the activities of β-glycosidases and esterases (Boskou et al., 2006). Caponio and Gomes (2001) observed that total polyphenols and levels of hydroxytyrosol, tyrosol and caffeic acid were higher when the paste was subjected to malaxation. Thus, our results show that hydrolytic enzymes were activated during malaxation, thereby releasing polyphenols from the olive tissue. Oleuropein and other olive polyphenols (e.g. verbascoside and hydroxytyrosol glucoside) contained hydroxytyrosol moieties in their chemical structure, which could be released by the action of β-glucosidase (Fig. 8).
Oleuropein and its derivatives after enzymatic hydrolysis by β-glycosidase and esterase (a) Verbascoside and its derivatives after enzymatic hydrolysis by β-glycosidase (b).
After centrifugal decanting, 7.1% of total polyphenols in the mixed olive paste were found in the oily must, as shown in Fig. 3. Servili et al. (2004) reported that polysaccharides were linked with hydrophilic polyphenols in the uncrushed tissue, thus reducing their release to the oil during centrifugation. In this regard, it was reported that the use of technical enzymatic preparations containing cell wall degrading enzymes during malaxation can improve the polyphenols concentration of oil (Ranalli et al., 2003; Siniscalco et al., 1989; Siniscalco and Montedoro, 1988).
The vertical centrifugations 1 and 2 differed in the partition of polyphenols between the two separated phases.
Table 2 indicates that VOO has the lowest concentration of polyphenols; only 3.6% of the initial polyphenols content of raw olive was in the final VOO.
The partition coefficient (Kp) is defined as the ratio of polyphenols concentrations in the oil and water phases at equilibrium (Rodis et al., 2002). Therefore, a hydrophobic compound has a Kp value greater than unity, and the inverse is true for hydrophilic molecules.
The calculated Kp values for oleuropein were 0.006 and 0.003 for vertical centrifugations 1 and 2, respectively. This result indicates the hydrophilic character of oleuropein. For hydroxytyrosol, Kp values of 0.77 and 0.68 were found for vertical centrifugations 1 and 2, respectively. The total polyphenols partition coefficients were 0.08 and 0.06 for vertical centrifugations 1 and 2, respectively.
Table 3 shows the changes in β-glucosidase and esterase activities during olive oil extraction. These are intrinsic activities, and were released upon crushing of the olive fruit. Malaxation aided the homogenization of the olive paste as well as the interaction between enzymes and substrates. Both enzyme activities are related to the bioconversion of oleuropein into hydroxytyrosol. The activity flows of both β-glucosidase and esterase decreased during olive processing because of protein denaturation during processing and the resultant enzyme dysfunction. Alterations in enzyme activity flows were observed during malaxation. Indeed, the activity flow values after malaxation were 93% for β-glucosidase and 74% for esterase, likely due to the effect of olive oil processing on protein stability. Subsequently, about half of β-glucosidase and esterase activity flows were discharged in the olive cake and OMW. Thus, 30% of β-glucosidase activity flow was discharged in the olive cake and 23% into the final OMW. Moreover, 26.8% of esterase flow activity was discharged into olive cake and 20.6% into the final OMW. The mass flow of water presented in Table 3 showed the water distribution during olive processing in the different by-products; thereby, 56% and 42% of the initial water mass flows were discharged in olive cake and OMW, respectively. The VOO product retained only 0.5% of the initial water mass flow, which must be removed in order to maintain oil stability during storage. These results clarify that polyphenols are abundant in OMW due to their hydrophilic property. The majority of the initial total polyphenols content was found in the by-products after centrifugation, with 74% and 38% in OMW and olive cake, respectively.
| Step | Mass flow of water [kg/h] | Mass flow of minerals [kg/h] | Mass flow of proteins [kg/h] | Activity flow of β-glucosidase [kIU/h] | Activity flow of esterase [kIU/h] |
|---|---|---|---|---|---|
| Raw olives | 235.60 | 8.50 | 7.34 | 85000 | 120000 |
| Washed and defoliated olives | 235.60 | 7.66 | 7.34 | 76500 | 91700 |
| Olive paste before malaxation | 235.60 | 7.66 | 7.34 | 76500 | 91700 |
| Olive paste after malaxation | 231.80 | 7.66 | 7.34 | 78850 | 88350 |
| Olive cake | 132.54 | 4.16 | 3.22 | 25550 | 32170 |
| Oily must | 2.46 | 0.04 | 0.00 | 2285 | 720 |
| OMW from centrifugal decanter | 96.80 | 3.46 | 4.12 | 38640 | 31100 |
| OMW from vertical centrifugations 1 and 2 | 98.15 | 3.49 | 4.12 | 19700 | 24750 |
| Virgin olive oil | 1.11 | 0.01 | 0.00 | 500 | 610 |
In this work, we clarified the detailed material balances of olives during processing. The three-phase extraction method enabled a relatively high VOO extraction yield of around 83.3%. Large amounts of by-products, including liquid and solid residues, were formed during the extraction process. Large amounts of carbohydrates (115.5 kg/h) were discharged during olive processing, with the majority in olive cake (83.5 kg/h) and OMW (32.0 kg/h). The results indicated that OMW had a high polyphenol content, and thus represents an inexpensive source of natural antioxidants. Moreover, the polyphenols content of OMW was more than 20 times greater than that of VOO. This is a particular advantage in that OMW, which is an undesirable by-product of the olive oil manufacturing process, can be used productively.
Acknowledgements We thank Mr. Kamel Kachaou, owner of the olive oil factory; Ms. Samira Abidi for her technical assistance in the HPLC analysis; Dr. Maher Boukhris, Dr. Gaith Rigane, and Mr. Slim Loukil from CBS, Tunisia; and Dr. Zheng Wang, Dr. Miyuki Hara and Dr. Kana Miyata from the University of Tsukuba, Japan, for their help during this work. This research was funded by the Science and Technology Research Partnership for Sustainable Development-SATREPS project, from JICA / JST, Japan.
