Use of solid residue from the olive industry

La investigación de nuevos aprovechamientos del olivar y en particular de lo subproductos del proceso de producción del aceite, tiene gran relevancia tanto en la economía como en el medio ambiente de los pueblos donde se desarrolla este cultivo. Son numerosos los trabajos publicados sobre la composición química de la aceituna y el aceite de oliva, en cambio sólo existen algunos estudios sobre el aislamiento e identificación de los compuestos de la hoja de olivo, en esta contribución se revisan los conocimientos sobre la composición química de la hoja de olivo, así como, las utilizaciones de los residuos sólidos -hoja de olivo y hueso de aceitunaque se originan en la producción del aceite de oliva y de aceitunas de mesa, como energía renovable y para la obtención de compuestos de alto valor añadido. Estos últimos, compuestos bioactivos dirigidos a los mercados de los aditivos alimentarios naturales, farmacéutico y cosmético, todos ellos muy receptivos en la actualidad a los productos de origen natural.


INTRODUCTION
Olive tree cultivation in the Mediterranean goes back to ancient times, ever since the Roman Age

Use of solid residue from the olive industry
By Ángeles Guinda Instituto de la Grasa (C.S.I.C.), Avda. Padre García Tejero, 4, 41012-Sevilla, Spain. E-mail: guinda@cica.es) (Melillo, 1994), when olive cultivation spread throughout the entire Mediterranean basin. This longevous tree (Olea europaea, Oleaceae) integrates and identifies economically, socially, and culturally the inhabitants of this basin; olive oil constitutes the main source of nutritional fats and is a very valuable product for exportation. Today, Mediterranean countries account for around 98% of the world's olive cultivation (approximately ten million hectares); they produce about 1,9 million metric tonnes per annum of olive oil, in addition to 1,1 million t of table olives (mean production during the four-season reference period from 1999/00 to 2002/03, http://www.internationaloliveoil.org/ ). The use of solid residue, such as leaves and stones, is of great economic and social importance for the Mediterranean area, as these by-products accumulate in large amounts during olive oil production and the elaboration of table olives. At present, it is possible to obtain activated carbon from olive stones (Rivera-Utrilla et al., 1986;Galiatsatou et al., 2001;Feng-Chin and Ru-Ling, 2000;Khalil et al., 2001;Blázquez et al., 2005), and the new biomass boiler system designs facilitate the use of olive stones as fuel for the generation of heat and electricity (Ortega-Jurado et al., 2004;Vlyssides et al., 2004). However, olive leaves, which are produced in large amounts, render scarce profits at present. Nevertheless, the market for natural ingredients and additives is rapidly growing, with such products obtaining high prices.
In the last few years, the possible toxicity of synthetic compounds (Fukushima and Tsuda, 1985;Fukushima et al., 1986;Stone, 2003), has increased interest in natural products and the food additives, cosmetics and pharmaceutical industries have improved their efforts in preparing bioactive compounds from natural products by extraction, fractionation, and purification.

BIOACTIVE COMPOUNDS IN OLIVE LEAVES
Olive leaves are agricultural residue resulting from the pruning of olive trees. The leaves may also be considered an easily available industrial byproduct, as they find their way to the olive mills in appreciable quantities. Olive leaves represent around 10% of the total weight of olives arriving to the mill (Espínola, 1996). Currently this by-product is not very profitable, given that in many countries the leaves have traditionally been used only as feed for livestock (Gómez-Cabrera et al., 1992;Sánchez et al., 2002;Ruíz et al., 2004). Often the leaves are disposed of by simply burning them together with the branches from pruning.

Phenolic Components
The leaves of Olea europaea L. have been studied, among other reasons, for their therapeutic effects. Interest in the potential health benefits of olive leaf extract dates back to the mid-nineteenth century, when reports were made of the ability of an extract (made from boiling the leaves) to reduce fever, including the ability to prevent or cure malaria symptoms even more effectively than quinine (Hanbury, 1854;Capretti and Bonaconza, 1949).
This singular physiological effect also encouraged identification of the principal agent involved. In the early 20 th century, the phenolic compound which is most abundant and characteristic in the fruit of the olive was isolated by Bourquelot and Vintilesco (1908). In 1960 Panizzi et al. established that the compound was a heterosidic ester of elenolic acid and hydroxytyrosol. Oleuropein is the principal member of the family of secoiridoids in the olive tree (Olea europaea L.). In 1974, Inouye et al. confirmed its structure by mass spectrometry.
For over thirty years since the isolation of oleuropein, extracts from various parts of the olive tree have been studied. The glucoside has been found in all of them: leaves, shoots, olive (pulp, husk and stone), wood, bark and roots Juven, 1968). Le Tutour and Guedon (1992) point out that oleuropein content in olive leaves is very high, around 60-90 mg per gram (dry material), although they also contain other compounds.Through hydrolysis, other phenolic compounds have been isolated and identified in olive leaves, although they are present in lesser quantity. These compounds include demethyloleuropein, ligstroside (Ragazzi et al., 1973), verbascoside (Fleuriet et al., 1984;Soler-Rivas et al., 2000) oleoside dimethylester (Panizzi et al. 1960), a non-glucosidic secoiridoid (Gariboldi et al., 1986), and oleuroside, an isomer of oleuropein (Kuwajima et al., 1988).
Additionally, olive leaves contain a significant amount of flavonoids. Found in the leaf are luteolin-7-O-glucoside, luteolin-7-O-rutinoside, apigenin-7-O-glucoside, rutin, luteolin, and apigenin, all of which have been identified by HPLC (De Laurentis, 1997;Ficarra and Ficarra 1991;Heimler et al., 2002). The purpose of this group of phenolic compounds present in the olive leaf seems to be, by virtue of their absorption spectrum, that of limiting the effects of UV light and the entailing oxidative damage (Galli and Visioli, 1999). Flavonoids also have the capacity to inhibit oxidation caused by free radicals (Bors et al., 1990 a and b).
The complexity of the chromatographic profiles of the fraction extracted with polar solvents from the olive leaf clearly indicates the presence of other phenolic derivatives, which may be present in the natural matrix. On the other hand, it can not be ruled out that the procedures of extraction and treatments of the sample may alter the structure of some of the secondary metabolites originally present in the plant tissue. Therefore, the need exists for the development of reliable methods for the analysis of the alcoholic extract of the leaf, prior to any stage of purification. De Nino et al. (1997) employ a method of direct analysis of the leaf extract, whose objective is the direct characterization of metabolites of the olive leaf using mass spectrometry (MS). This methodology would permit the "chemical characterization" of a particular variety, or the identification of compounds of great biological value.
Among the group of phenolic compounds present in the olive leaf, oleuropein and the products of its hydrolysis are those of the greatest biological interest (Fleming et al., 1973).
Oleuropein has a vasodilator effect, increases blood flow in the coronary arteries and improves arrhythmia (Zarzuelo et al., 1991;Manna et al., 2004). This compound has proven to be a potent antioxidant with anti-inflammatory properties (Visioli and Galli, 2002). In 1970 several articles presented the antiviral and antibacterial activity of calcium enolate (obtained by hydrolysis from oleuropein) (Elliot et al., 1975;Soret, 1969). Subsequent publications show that oleuropein also engages in antimicrobial activity against viruses, retroviruses, bacteria, yeasts, fungus, molds and other parasites (Juven et al., 1972;Tassou and Nychas, 1995;Aziz et al., 1998;Zanichelli et al., 2005). Other clinical effects of oleuropein are the fortification of cellular and organism protection through macrophage response (Visioli et al., 1998), the inhibition of plaque aggregation and eicosanoid production (Petroni et al., 1995), and a reduction by nearly 30% in the level of low density lipoproteins (LDL) (Visioli and Galli, 1994;Andrikopoulos et al., 2002).

Terpenic components
Vázquez-Roncero and Janer (1969) contributed diverse qualitative results of the presence of terpenic acids in the olive leaf. Later, Mussini et al. (1975) isolated alpha-amyrine and confirmed the presence of maslinic acid. Bianchi et al. (1993Bianchi et al. ( , 1994 analyzed these compounds and some of those found by Vázquez-Roncero and collaborators, in three Italian varieties. Among the terpenic compounds in the olive leaf, one to take notice of is oleanolic acid (3-betahydroxy-28-carboxyolean). This compound is found extensively throughout the plant kingdom, and in the olive leaf it occurs in free acid form in considerable proportions, accounting for ≅ 3% of dry leaf weight (Albi et al., 2001). Oleanolic acid is biologically active with antiabortive, anti-cariogenic, anti-fertility, antihepatotoxic, anti-inflammatory, antisarcomic, cancerpreventive, cardiotonic, diuretic, hepatoprotective and uterotonic properties. Numerous studies have been published on the possible biological activity of this acid and its glucosides. It has been studied in its activity as an inhibitor of the proliferation of leucemic cells (Es-Saady et al., 1994), as a hypoglucemiant (Yoshikawa et al., 1994), as an anti-tumoral agent (Gerhauser et al., 2003), as a hepatoprotective agent (Yim et al., 2001), as an anti-inflammatory (Ismaili et al., 2002;Takeoka et al., 2000 andChoi et al., 2002), as an anti-bacterial (Sasazuka et al., 1995), as an inhibitor of the production of nitric acid (Honda et al., 1997), as an anti-HIV agent (Mengoni et al., 2002), in its anti-atherogenic activity (Somova et al., 2003) and as a cardiotonic (Somova et al., 2004). Also in existence are publications and patents for the preparation of functional foods with oleanolic acid (Hassel et al., 1998;Putte, 2000;Guinda et al., 2004).

Liposoluble compounds
In 1973, Janer isolated by Thin Layer Chromatography some liposoluble compounds present in olive leaf extract (hexane extract) and analyzed the fatty acids of the triacylglycerols from leaves. In recent years, Guinda et al. (2002) have isolated, identified and quantified numerous components of a hexane extract of olive leaves, consisting of: saturated hydrocarbons, squalene, ester waxes, alpha-tocopherol, triglycerides, betacarotene, linear alcohols, alpha-and beta-amyrine, and beta-sytosterol. These compounds have been found in five Spanish varieties (Picual, Hojiblanca, Empeltre, Arbequina and Cornicabra). The concentration (ppm) of the compounds ranged from the following: 050-3400 (saturated hydrocarbons), 38-152 (squalene), 915-1874, 28-100 (betacarotene), 832-1396 (triglycerides), 41-125 (alphatocopherol), 714-2500 (beta-sytosterol), 95-311 (alcohols) and 342-837 (terpenic dialcohols). The most numerous hydrocarbons in all of the varieties studied are C29, C31 and C33. The waxes are mainly constituted of fatty acids and the terpenic alcohols of the olive leaf (palmitic, oleic, and linoleic; alpha-and beta-amyrine). The terpenic dialcohols found in the olive leaf are uvaol and erythrodiol, with the uvaol content always higher than that of erythrodiol, which is the opposite case in olive oil. A large part of the compounds analyzed in the hexane extract of the olive leaf, such as alpha-tocopherol, beta-carotene, alpha-and beta-amyrine, squalene and beta-sytosterol, are bioactive compounds capable of modulating human physiological systems. Therefore, these compounds have multiple applications in the pharmaceutical, cosmetics and food additives industries. Numerous articles have been published on the antioxidant activity of alphatocopherol, as well as on the protective effects of alpha-tocopherol and of beta-carotene with regard to atherogenesis (Devaraj et al., 1998) and cancer (Virtamo et al., 2000;. Other publications examine the potential use of squalene (Kelly, 1999) and its preventive effect on colon cancer (Newmark, 1999;Rao et al., 1998). Still others examine the potential health benefits of the phytosterols: betasytosterol reduces cholesterol levels in the blood, as well as being effective at in vitro reduction of breast cancer cells (Awad et al., 2001;. Another area of study has been the anti-inflammatory activity of the triterpenic dialcohols of the olive leaf (alpha-and beta-amyrine) (Martín-Cordero et al., 2001;Puerta et al., 1997).

Other compounds
The olive tree, like other members of the family of Oleaceas, is distinguished by it capacity for the biosynthesis of mannitol and oligosaccharides of the rafinose family (raffinose and, above all, stachyose), as well as sucrose, as direct products of photosynthesis (Flora and Madore, 1993;Stoop et al., 1996). Mannitol, a hexitol derivative of mannose, offers a series of properties with advantageous applications in the food and pharmaceutical industries. Its sweetening potency is equivalent to 70% that of sucrose; it has a low caloric value (2 kcal/g), it does not cause caries and its metabolism in humans is not dependent on insulin, which makes it suitable for consumption by diabetics. Additionally, it has healthful effects as an antioxidant (Wisselink et al., 2002) and is of therapeutic use in severe head injuries, as it is effective in the reduction of intracraneal pressure (Brain Trauma Foundation, 1996;Filipo et al., 1997;Unterberg et al., 1997). Its industrial preparation is achieved by means of catalytic hydrogenation of sugars. As this chemical process yields a scant quantity of mannitol, in recent years research has been conducted into alternative biotechnological processes. The use of olive leaves as raw material in obtaining mannitol offers a promising alternative, as they contain between 10 and 20 mg per gram of plant tissue of this carbohydrate (Flora and Madore, 1993;Sánchez, 1998).
Recently, Campeol et al., 2004, have isolated and identified through mass spectrometry (EIMS) and nuclear magnetic resonance (H and C RMN), a new compound: 1.5-Anhydroxylitol. This compound, never before detected in the plant kingdom, was obtained in the range of 0.1 to 1%.
For the purpose of obtaining basic data for the processing and manufacture of foods, Ok-Hwan et al. (2005) have analyzed the contents of the leaf as to moisture, ash, crude protein, fat, carbohydrates, the principal amino acids (glutamic and aspartic) and principal free sugar (sucrose), as well as the concentration of Ca and vitamins A and C. Neither vitamins B6 nor B12 were detected.

The Benefits of extracts
At present, there is a growing body of evidence for the beneficial role that the phytonutrients play in relation with the prevention of serious diseases (Arai, 1997;Ovesen, 1997). The enormous interest in these substances in recent years as additives or natural food ingredients has gone beyond food preparation and conservation, into the new area known as nutraceutics. This science studies the therapeutic effects of the components of foods. It is known that "functional foods" can modulate human physiological systems, such as the immunological, endocrinal, nervous, circulatory and digestive systems, with this last a proven object of study (Hassel, 1998). At present, several natural extracts, among them that of the olive leaf, are used in the manufacture of functional foods (Micol et al., 2003).
Diverse epidemiological studies have proven the association between diets rich in antioxidant compounds and a reduction in the risk of contracting certain types of cancer, cardiovascular and ocular diseases in which oxidative stress plays an important part in their development (Garewall, 1997;Visioli et al., 2000). As a result, nutritional recommendations generally include the consumption of food and drink rich in antioxidant compounds (Packer et al., 1999). It is also becoming more frequent to design foods with antioxidant functions (Hollingsworth, 1998). Among the antioxidants present in the olive leaf, the phenolic compounds are notable. Phenolic compounds are one of the main products of the secondary metabolism and comprise a wide range of structural classes and biological functions.
In the olive tree, phenolic compounds are more abundant in the fruit than in the leaf; even so, they are present in significant amounts (Le Tutour and Guedon, 1992;Paiva-Martins and Gordon, 2001), which makes utilization of the leaves worthwhile. In addition, the leaf contains a considerable amount of terpenic compounds, whose biological activity is well-known. A specific review of the pharmacological activity of oleanolic acid has been published (Tian et al., 2002), as well as a study of the anticarcinogenic effect of ursolic acid (Es-Saady et al., 1994, 1996ay 1996b. Basically, the leaf represents a by-product whose potential for use is still under development. Its use would facilitate the obtaining of natural products at a competitive cost.

Uses of the extracts
Up to now, use of the olive leaf has been made principally through formulations which include an extract of the same, without any previous separation of the active compounds. There are numerous preparations of this type on the market; they are used as complements to the treatment of certain diseases or as nutritional supplements. New patents appear daily for functional foods or cosmetics based on olive leaf extracts. The patents are quite varied in their formulation; for example, there are liquid preparations with alcohol and with glycerin (Oliveda Network SL, 2005 a y b). Equally diverse are the applications to which these formulations are destined: manufacture of dietetic bread for diabetics (Scheneder, 1985); preparations which contain lycopene and olive leaf extract for use in food or cosmetics (Coll et al., 1999); use of olive leaf extracts in dietetic cookies (Shtukatur, 2003); the manufacture of a nutritional supplement with physiological effects containing olive leaf extract (Stueckler 1998); an extract of olive leaves to combat free radicals, and their use in dietetic foods and in cosmetics (Amari 1998).

OBTAINING HIGH ADDED-VALUE COMPOUNDS FROM THE OLIVE LEAF
Works on obtaining bioactive compounds from the olive leaf are recent and not very numerous. The techniques employed range from biotransformation (Ryan et al., 2002;Verhe et al., 1992;Briante et al., 2001), solid/liquid extraction and subsequent fractionation of the extract (Albi et al., 2001), and supercritical fluid extraction (SFE) (Lucas et al., 2002;Tabera et al., 2004b). Briante et al., (2001Briante et al., ( , 2002Briante et al., ( and 2004 have proposed a bioreactor for obtaining large quantities of hydroxytyrosol of high purity (91-94% in weight), starting from olive leaf extract. The process consists of extraction with ethanol/water (1:1) from the leaf and subsequent biotransformation of the extract, by means of a hyperthermophilic beta-glucosidase, partially purified and immobilized in a support of chitosan (polysaccharide obtained from the shells of crustaceans). The bioreactor is separated into two chambers; the first column is filled with the matrix of the chitosan enzyme and is used for the hydrolysis of oleuropein into aglucone. The second column of the bioreactor is used as a second reaction chamber at high temperature and under pH-controlled conditions, permitting total hydrolysis of the aglucone of oleuropein into hydroxytyrosol. Lucas et al., (2002), obtain concentrates of alpha-tocopherol directly from the leaves, by means of supercritical fluid extraction (SFE). Tabera et al., (2004b), have patented a process for obtaining high added-value compounds from the olive leaf, consisting mainly of a first stage of solid/liquid extraction with solvents of decreasing polarity, and subsequent fine fractionation of the different extracts through extraction with supercritical CO2, in countercurrent (CC-SFE). With this method they have obtained concentrates of alpha-tocopherol (Tabera et al., 2004a) and hydroxytyrosol, among others. Guinda and colaborators, have developed a procedure for obtaining terpenic acids from the leaves of Olea europaea by means of extraction from the leaf with an organic polar solvent, and separation of this acid by crystallization. This patented process (Guinda et al., 2000) allows for obtaining oleanolic acid with approximately 93% purity. Subsequent purification by means of a simple thermal treatment allows for this acid to be obtained with a purity of 98-99%. The advantage of this procedure is even greater when taking into account that even though this triterpenic acid is found extensively throughout the plant kingdom, in the olive leaf, oleanolic acid occurs in free acid form in considerable proportions (≅ 3% dry leaf weight). Additionally, there is a considerable amount of leaves available annually, when including those coming from pruning and those separated from the olives in the mills.
At the present time, is being investigated in the Instituto de la Grasa (CSIC), (Spanish project AGL 2003-02795), a new and encouraging process that involves a continuous separation procedure, by means of molecular distillation (Short Path Distillation), of the natural compounds present in alcoholic olive leaf extract.

FINAL CONSIDERATIONS
To increase the economic performance of the olive and related industries, it is of great importance to make use of a by-product like the olive leaf. Bioactive compounds obtained from the leaves have dietetic, pharmaceutical and cosmetic applications. Using this residue not only meets with the requirement of its effective and innocuous elimination: as well as preventing environmental problems, it also creates new sources of wealth which contribute to higher profitability in the initial industrial process. Moreover, it should be taken into account that making the management of residue profitable generates all sorts of new industries, with the resulting social gains.