**3. The use of enzymes in the extraction of olive oil**

The most commonly used method for the extraction of olive oil is the mechanical process, however some of the non-extracted oil remains in the solid residue or cake. The majority of the oil is located in the vacuoles as free oil, but oil dispersed in the cytoplasm is not extractable and is therefore lost in the waste (Najafian et al., 2009). Therefore the cell walls must be destroyed to effectively recover the oil enclosed in the cell. The use of enzymes has been studied for the hydrolysis of the different types of polysaccharides in the cell wall structure (Chiacchierini et al., 2007). The major polysaccharides found in the cell wall of the olive fruit were pectic polysaccharides and the hemicellulosic polysaccharides xyloglucan and xylan (Vierhuis et al., 2003).

Several innovating biotechnological techniques have been studied to obtain high-quality oils and/or improve product outputs. They include the use of microorganisms (Kachouri & Hamdi, 2004, 2006) or enzymes (Vierhuis et al., 2001) during different steps of the oil processing procedure. Several enzyme processing aids have been successfully tested for olives in recent years (De Faveri et al., 2006; García et al., 2001). Different enzymes are naturally present inside the olive fruit, but are strongly deactivated during the pressing phase, most likely due to the formation of oxidized phenols bonding to the enzyme prosthetic group (Vierhuis et al., 2001). In this case, the addition of suitable enzymes to the olive paste during the mixing step was proposed as a tool to replace the deactivated natural ones (Ranalli et al., 2001). Furthermore, the enzyme complexes are water-soluble and after the application, they are found in the olive mill waste waters, indicating that the oil composition is not modified (Chiacchierini et al., 2007).

Ranalli et al. (2003a) estimated the composition of three types of olive oil (Caroleo, Coratina and Leccino) extracted by the application of the Bioliva enzymatic complex. During extraction, the action of the enzymes on the fruit tissues resulted in the release of greater amounts of oil and other constituents, which dissolved in the oily phase (Ranalli et al., 2003b). The enzymatic application resulted in an increase in several key compounds, such as phenols, tocopherols, and flavour compounds, without changing the natural parameters related to product authenticity (waxes, sterols, triterpene alcohols, fatty acids and triacylglycerol composition).

The loss of phenols during processing can be attributed to interactions between the polysaccharides and phenolic compounds present in the olive pastes (Servili et al., 2004). Studies show that the addition of commercial enzyme preparations during the malaxation can reduce the complexation of hydrophilic phenols with polysaccharides. It increases the concentration of free phenols in the olive paste and their consequent release into the oil and waste waters during processing (De Faveri et al., 2008).

A mixture of three enzyme formulations was tested by Aliakbarian et al. (2008) to improve the yield and the quality of the olive oil obtained from the Italian cultivar Coratina. Since no single enzyme is adequate for the efficient maceration and extraction of oil from olives, pectinase, cellulase and hemicellulase were essential for this purpose (Chiacchierini et al., 2007; De Faveri et al., 2008). A homogeneous mixture of the three different enzyme formulations was used at the beginning of the malaxation step in the proportions 33.3:33.3:33.3% (v/v/v). This choice was suggested by the higher efficacy of these enzymes in releasing phenolics into the oil when working as a ternary system (A:B:C), rather than in binary combinations (A:B, A:C, B:C) (De Faveri et al., 2008). In summary, A is a complex formulation containing pectinase plus cellulase and hemicellulase; B shows equilibrated pectinase–hemicellulase activity; C is a pectolytic enzyme. The enzymes selected are naturally present inside the olive fruit, but are strongly deactivated during the critical pressing step, presumably because of the oxidation (Chiacchierini et al., 2007). The highest levels of total polyphenols (874 µgCAE/goil), antiradical power (25.1 µgDPPH/µLextract) and odiphenols (µgCAE/goil) were all reached at the highest enzyme concentration (25 mL/kgpaste). Moreover, the highest oil extraction yield (17.5 goil/100 gpaste) was reached with the longest malaxation time (t = 150 min), always with the highest enzyme concentration.

### **4. Enzymatic synthesis of structured lipids**

270 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

composition appeared predominantly under genetic control while the environmental effect explained 0.31 of the total variance. The oil content in phenolic compounds, instead, had lower heritability (0.29) and was more affected by the environment, which explained 0.50 of the total variance. Few genotypes were selected as the best for each location, but none performed best in all locations. This suggests that, in olives, the highest oil quality is difficult to achieve with a single genotype in different environments, due to a strong or even predominant effect of the environment on some quality traits. More likely, combinations of genotypes and territories can

The most commonly used method for the extraction of olive oil is the mechanical process, however some of the non-extracted oil remains in the solid residue or cake. The majority of the oil is located in the vacuoles as free oil, but oil dispersed in the cytoplasm is not extractable and is therefore lost in the waste (Najafian et al., 2009). Therefore the cell walls must be destroyed to effectively recover the oil enclosed in the cell. The use of enzymes has been studied for the hydrolysis of the different types of polysaccharides in the cell wall structure (Chiacchierini et al., 2007). The major polysaccharides found in the cell wall of the olive fruit were pectic polysaccharides and the hemicellulosic polysaccharides xyloglucan

Several innovating biotechnological techniques have been studied to obtain high-quality oils and/or improve product outputs. They include the use of microorganisms (Kachouri & Hamdi, 2004, 2006) or enzymes (Vierhuis et al., 2001) during different steps of the oil processing procedure. Several enzyme processing aids have been successfully tested for olives in recent years (De Faveri et al., 2006; García et al., 2001). Different enzymes are naturally present inside the olive fruit, but are strongly deactivated during the pressing phase, most likely due to the formation of oxidized phenols bonding to the enzyme prosthetic group (Vierhuis et al., 2001). In this case, the addition of suitable enzymes to the olive paste during the mixing step was proposed as a tool to replace the deactivated natural ones (Ranalli et al., 2001). Furthermore, the enzyme complexes are water-soluble and after the application, they are found in the olive mill waste waters, indicating that the oil

Ranalli et al. (2003a) estimated the composition of three types of olive oil (Caroleo, Coratina and Leccino) extracted by the application of the Bioliva enzymatic complex. During extraction, the action of the enzymes on the fruit tissues resulted in the release of greater amounts of oil and other constituents, which dissolved in the oily phase (Ranalli et al., 2003b). The enzymatic application resulted in an increase in several key compounds, such as phenols, tocopherols, and flavour compounds, without changing the natural parameters related to product authenticity (waxes, sterols, triterpene alcohols, fatty acids and triacylglycerol composition). The loss of phenols during processing can be attributed to interactions between the polysaccharides and phenolic compounds present in the olive pastes (Servili et al., 2004). Studies show that the addition of commercial enzyme preparations during the malaxation can reduce the complexation of hydrophilic phenols with polysaccharides. It increases the concentration of free phenols in the olive paste and their consequent release into the oil and

produce oils with high and typical quality.

and xylan (Vierhuis et al., 2003).

**3. The use of enzymes in the extraction of olive oil** 

composition is not modified (Chiacchierini et al., 2007).

waste waters during processing (De Faveri et al., 2008).

The enzymatic synthesis of structured lipids is relatively new in lipid modification. Although enzymes have been used for several years to modify the structure and composition of foods, they have only recently become available for large-scale use, mainly because of the high cost. Within this context lipases are reported for the enzymatic synthesis of structured lipids. They have the ability to carry out hydrolytic reactions, but the manipulation of the reaction at low water levels permits their use also for the synthesis of triacylglycerols. These enzymes can be successfully used in the production of lipids structured for medical purposes (De Araújo, 2011).

Enzymatic modification of olive oil triacylglycerols has been discussed by Boskou (2006, 2009). The development of techniques for the preparation of oils and fats from enzymemodified olive oil is an attractive prospect for the food industry, given the high oxidative stability of the product at frying temperatures and the health enhancing properties of this material (Criado et al., 2007).

Nunes et al. (2011) produced structured triacylglycerols containing medium chain fatty acids, by the acidolysis of virgin olive oil (VOO) with caprylic or capric acids in a solventfree media or in n-hexane, catalyzed by immobilized lipases from *Thermomyces lanuginosa, Rhizomucor miehei* and *Candida antarctica*. The results indicated that the incorporation was always greater for capric than for caprylic acid, but for both acids, higher incorporation was always attained in solvent-free media. All the biocatalysts presented 1,3-regioselectivity. The lipases from *Rhizomucor miehei* and *Candida antarctica* were the biocatalysts presenting the highest operational stability, together with high incorporation levels and low acyl migration in the batch production of structured lipids by the acidolysis of VOO with caprylic or capric acids. Therefore, these biocatalysts seem to be the most adequate for the implementation of a process aimed at the production of triacylglicerols containing medium and long fatty acids (MLM) rich in caprylic and capric acids. The structured triacylglycerol obtained from VOO has oleic acid at the sn-2 position, indicating a better absorption, whilst medium chain fatty acids will mainly be esterified at the external positions of the TAG molecules.

Genetic Improvement of Olives, Enzymatic Extraction and Interesterification of Olive Oil 273

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of copper ions. *Journal of Agriculture and Food Chemistry*. V.54, pp.4880–4887. Boskou D. (Ed.). (2009). *Olive Oil. Minor Constituents and Health*. CRC Press Taylor & Francis

Boskou D. (Ed.). (2006). *Olive Oil: Chemistry and Technology*. CRC Press Taylor & Francis Group. ISBN-13: 978-1-893997-88-2, ISBN-10: 1-893997-88-X, United States. Criado M.; Hernandez-Martín E., Lopez-Hernandez A.; Otero C. (2007) Enzymatic

Chiacchierini E, Mele G, Restuccia D, Vinci G. (2007) Impact evaluation of innovative and

De Araujo, M. E. M. B., Campos, P. R. B., Noso, T. M., Oliveira, R. M. A., Cunha, I. B. S.,

De Faveri D, Aliakbarian B, Avogadro M, Perego P, Converti A. (2008) Improvement of

Fabbri A, Lambardi M, Ozden-Tokatli Y. (2009). Olive Breeding. In:*Breeding Plantation Tree Crops: Tropical Species*, Springer Science+Business Media, LLC. Cp.12, p.423-465. Fitó M, Cladellas M, de la Torre R, Martí J, Alcántara M, Pujadas-Bastardes M, Marrugat J,

García A, Brenes M, Moyano MJ, Alba J, Garcia P, Garrido A. (2001) Improvement of

Guerfel M, Ouni Y, Taamalli A, Boujnah D, Stefanoudaki E, Zarrouk M. (2009) Effect of

Kachouri F, Hamdi M. (2004) Enhancement of polyphenols in olive oil by contact with

Kachouri F, Hamdi M. (2006) Use *Lactobacillus plantarum* in olive oil process and improvement of phenolic compounds content, *Journal of Food Engineering*. V.77, pp.746-752. León L, Beltrán G, Aguilera MP, Rallo L, Barranco D, De La Rosa R. (2011) Oil composition

León L, De la Rosa R, Barranco D, Rallo L. (2007) Breeding for early bearing in olive.

León L, Martín LM, Rallo L. (2004b) Phenotypic correlations among agronomic traits in olive

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*Journal of Lipid Science and Technology. V.*111, pp.926-932.

*Science and Technology.* V.113, pp.870–875.

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of extra virgin olive oil phenolics on oxidative stability in the presence or absence

Interesterification of Extra Virgin Olive Oil with a Fully Hydrogenated Fat: Characterization of the Reaction and Its Products. *Journal of American Oil Chemical* 

sustainable extraction technologies on olive oil quality. *Trends in Food Science and* 

Simas, R. C., Eberlin, M. N., Carvalho, P.O. (2011) Response surface modelling of the production of structured lipids from soybean oil using *Rhizomucor miehei* lipase.

olive oil phenolics content by means of enzyme formulations: effect of different enzyme activities and levels. *Biochemical Engineering Journal*, V.41, pp.149-156. De Faveri D, Torre P, Aliakbarian B, Perego P, Domínguez JM, Rivas Torres B. (2006) Effect

of different enzyme formulations on the improvement of phenolic compound content in olive oil, in: Proceedings of the IUFoST 13thWorld Congress of Food

Bruguera J, López-Sabater MC, Vila J, Covas MI. (2005) Antioxidant effect of virgin olive oil in patients with stable coronary heart disease: a randomized, crossover,

phenolic compound content in virgin olive oils by using enzymes during

location on virgin olive oils of the two main Tunisian olive cultivars. *European* 

fermented olive mill wastewater by *Lactobacillus plantarum*. *Process Biochemistry*.

of advanced selections from an olive breeding program. *European Journal of Lipid* 

Criado et al. (2007) have also studied the enzymatic interesterification of extra virgin olive oil with a fully hydrogenated palm oil to produce lipids with desirable chemical, physical and functional properties. The sn-1,3 non-specific immobilized lipases from Candida antarctica and two sn-1,3 specific immobilized lipases from *Thermomyces lanuginosus* and *Rhizomucor miehei* were employed as the biocatalysts. The authors concluded that the oxidative stability increased when the percentage of TAG containing multiple fully saturated residues increased. In all the cases studied, the stability of the physical blend was higher than that of the reaction products. The final products were considered as plastic over wider temperature ranges. The large amount of unsaturated residues present in these samples, primarily oleic acid residues, was the factor leading to the extended plasticity range of these interesterified mixtures.

### **5. Conclusion**

The improvement of the properties of extra virgin olive is crucial, considering the extensive number of functional compounds present in this oil. Genetic improvement techniques are the option showing the most promising results. The genetic modification of olive cultivar crossbreeding is focused on solving agronomic and commercial problems, such as the control of fruit ripening and increase in the oil content and quality. Another improvement suggested by different research groups is the use of enzymes added to the paste to improve extraction of the oil and the bioactive compounds it contains. Enzymes, such as microbial lipases, can also be used to synthesize different high-value products from tryacylglicerol of olive oil. It is possible to obtain structured lipids that have functional and technological properties suitable for applications in the food, pharmaceutical and oleochemical industries. This can be considered as a "green" industrial process, taking into account that these compounds are currently synthesized by chemical processes that use catalysts and generate byproducts.

#### **6. References**


Criado et al. (2007) have also studied the enzymatic interesterification of extra virgin olive oil with a fully hydrogenated palm oil to produce lipids with desirable chemical, physical and functional properties. The sn-1,3 non-specific immobilized lipases from Candida antarctica and two sn-1,3 specific immobilized lipases from *Thermomyces lanuginosus* and *Rhizomucor miehei* were employed as the biocatalysts. The authors concluded that the oxidative stability increased when the percentage of TAG containing multiple fully saturated residues increased. In all the cases studied, the stability of the physical blend was higher than that of the reaction products. The final products were considered as plastic over wider temperature ranges. The large amount of unsaturated residues present in these samples, primarily oleic acid residues,

was the factor leading to the extended plasticity range of these interesterified mixtures.

The improvement of the properties of extra virgin olive is crucial, considering the extensive number of functional compounds present in this oil. Genetic improvement techniques are the option showing the most promising results. The genetic modification of olive cultivar crossbreeding is focused on solving agronomic and commercial problems, such as the control of fruit ripening and increase in the oil content and quality. Another improvement suggested by different research groups is the use of enzymes added to the paste to improve extraction of the oil and the bioactive compounds it contains. Enzymes, such as microbial lipases, can also be used to synthesize different high-value products from tryacylglicerol of olive oil. It is possible to obtain structured lipids that have functional and technological properties suitable for applications in the food, pharmaceutical and oleochemical industries. This can be considered as a "green" industrial process, taking into account that these compounds are currently synthesized by chemical processes that use catalysts and generate byproducts.

Aguilera, M. P., Beltrán, G., Ortega, D., Fernández, A., et al. (2005) Characterization of virgin

Aliakbarian, B, De Faveri D, Converti A, Perego P. (2008) Optimisation of olive oil extraction

Ayton, J., Mailer, R. J., Haigh, A., Tronson, D., Conlan, D. (2007) Quality and oxidative

Baccouri, B., Ben Temime, S., Taamalli, W., Daoud, D. et al. (2007) Analytical characteristics

Baccouri, B., Zarrouk, W., Baccouri, O., Guerfel, M., et al. (2008) Composition, quality and

Belaj, A., Munõz-Diez, C., Baldoni, L., Satovic, Z.,Barranco, D. (2010) Genetic diversity and

europaea L. subsp. Oleaster). *Grasas y Aceites*, V.59, pp.346–351.

olive oil of Italian olive cultivars: 'Frantoio' and 'Leccino', grown in Andalusia.

by means of enzyme processing aids using response surface methodology.

stability of Australian olive oil according to harvest date and irrigation. *Journal of* 

of virgin olive oils from two new varieties obtained by controlled crossing on

oxidative stability of virgin olive oils from some selected wild olives (Olea

relationships of wild and cultivated olives at regional level in Spain. *Scientific* 

**5. Conclusion** 

**6. References** 

*Food Chemistry*. V.89, pp.387–391.

*Food Lipids*, V.14, pp.138–156.

*Horticulture*, V.124, pp.323–330.

*Biochemical Engineering Journal.*V. 42, pp.34-40.

Meski variety. *Journal of Food Lipids*, V.14, pp.19–34.


**15** 

*Spain* 

**Olive Oil Mill Waste Treatment:** 

**Digestion Technology** 

**Improving the Sustainability of the** 

Bárbara Rincón, Fernando G. Fermoso and Rafael Borja

*Instituto de la Grasa (Consejo Superior de Investigaciones Científicas), Sevilla,* 

The processes used to treat waste streams are chosen according to technical feasibility, simplicity, economics, societal needs and political priorities. However, the needs and priorities of a sustainable society undergo pressure which means a shift in the focus of wastewater treatment from pollution control to resource exploitation (Angenent et al., 2004). The organic fraction of agro-wastes (e.g. olive oil wastes, sugar beet pulp, potato pulp, potato thick stillage or brewer´s grains) has been recognized as a valuable resource that can be converted into useful products via microbially mediated transformations. Organic waste can be treated in various ways, of which bio-processing strategies resulting in the production of bioenergy (methane, hydrogen, electricity) are promising (Khalid et al., 2011). The aim of the present chapter is to discuss: firstly the quantities, characteristics and current treatments of the solid wastes and wastewaters from the continuous olive oil extraction industry for their exploitation and recovery; secondly, anaerobic digestion processes as an

The olive mill elaboration system has evolved over time for economic and environmental reasons. The traditional system or "pressing system" was replaced in the 70s by the threephase continuous centrifugation system. The centrifugation of the milled and beaten olives to obtain olive oil by the three-phase system produces 20% oil, 50% three-phase olive mill wastewaters (3POMWW) and 30% three-phase olive mill solid wastes (3POMSW). This three-phase system led to an increase in the processing capacity and consequently to an increase in the yield of the mills and the growth of the average mill size. However, large quantities of water needed for carrying out the three-phase process generate a high volume of olive mill wastewaters. The uncontrolled discharge of 3POMWW brought environmental problems. In some countries, technology manufacturers developed the "ecological" two-phase process. This system enables

alternative for waste treatment and valuable energy recovery

**2. Olive mill extraction wastes 2.1 Olive oil extraction technology** 

**1. Introduction** 

**Olive Oil Industry with Anaerobic** 

