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The present invention relates to a process which allows to obtain extracts having a high polyphenol content from leaves, preferably olive tree leaves, characterized by a drying stage of the olive tree leaves by microwave and by an extraction stage of the polyphenols in acidic aqueous solution.

Current European (Capone et al., 2014; Giampietro, 2009) and international (Rose, 2002) policies are strongly pointing on the sustainability of agro-food productions and on the reuse of by-products. A representative example is, undoubtedly, the olive-oil production chain. The advent of innovative continuous crushing and production equipment has definitely optimized the production process in terms of productivity increasing and labor reduction (Beghi et al., 2017). However, it has not solved the serious problem of the by-products and waste disposal, particularly olive mill waste water (OMWW), where polyphenols represent the most impactful substances for the environment.

The amount of OMWW generated vary according to the olive's type (and cultivar), the extraction system, and the process conditions. The concentrations of solutes are variable and depend on the type of cultivar, the ripeness of the fruit, and the crushing process. However, their pollutant load, expressed in terms of COD (Chemical Oxygen Demand), remains on average of 150 g/L, equivalent to a BOD (Biological Oxygen Demand) of about 80 g/L (Incelli et al., 2016). The pollutant load of 1 m3 of OMWW corresponds to about 100-200 m3 of domestic waste. The potential reuse of olive mill by-products and wastes has been investigated in several studies and researches for many years with the aim to reduce the pollutant load. Nevertheless, very poor results, also due to the excessive costs that olive milling industries were not willing to bear (Incelli et al., 2016). In the past, OMWW and pomace were disposed directly in the field, following only partially the national environmental regulations, causing damage to agriculture, groundwater, and the environment.

In this direction, important efforts have been made to identify optimized processes for the disposal of olive pomace.

However, it is required the development of new processes for the management and disposal of wastes from agro-food productions, in particular for the reuse of the different by-products of the olive-oil industry. For example, it is strongly desirable to separate and purify the phenolic compounds, particularly oleuropein and its derivatives, contained in the leaves and drupe of the olive tree, to destine these bioactive molecules to the production of extracts which can be used in various sectors (biomedical, pharmaceutical, nutraceutical and food). Polyphenols have widely known antioxidant properties and are considered to be the main responsible for the beneficial properties of the Mediterranean diet based on extra virgin olive oil. In this context and with improved approaches, the by-products of the food industry can therefore become inestimable resources.

WO 2005/123603 provides a method for the treatment of OMWW generated by oil mills, in order to solve the problem of waste disposal and by-products reuse; the method includes an initial stage of OMWW acidification with 1% citric acid and subsequent stages of filtration. This method represents a solid opportunity to reuse by-products and wastes of the olive oil industry, with a considerable environmental impact. However, the acidification stage of OMWW cause the degradation of most oleuropein, which is present in a very low concentration in the final phenolic concentrates compared to hydroxytyrosol and tyrosol (see table A which shows the concentration in mg/ml of the main phenolic compounds in permeates and concentrates ( Pizzichini et al . ,2006).

This method requires high costs for machines and plants, which are further increased in case of polyphenols fractionation or in the case of the production of polyphenolic powder extracts. In addition, there are high probability of severe fouling and clogging of the membranes. Finally, the results obtained in this method are variable due to the variability of the OMWW chemical composition.

The development of processes for the management and disposal of wastes different from olive pomace, e.g. olive tree leaves, and for the recovery of bioactive molecules has progressed to a much lesser extent.

Some methods involve the extraction of polyphenols from olive oil industry by-products with ethanol to enrich the oleuropein content of the final product.

Typically polyphenols and oleuropein are thus extracted using alcoholic solvents.

US 10,258,660 discloses the extraction of oleuropein from olive leaves using ethanol.

Several methods for leaves drying, are reported in the literature to prepare the material for the extraction. Typically, drying takes place outdoors, at room temperature, or in oven at high temperatures. Kamran M. et al. (2015), describe the recovery of polyphenols from dried olive tree leaves by several methods: in air, at room temperature, by freeze-drying, or in an oven at temperatures between 60°C and 105°C; followed by extraction with 80% methanol. The highest yields of polyphenols were obtained by the extraction with 80% methanol from oven- dried leaves at 105°C: As a results, almost 140 mg of oleuropein per gram of dried leaves (Frantoio cultivar) were extracted.

Ansari et al. (2011) described a procedure for extract oleuropein from olive tree leaves, dried at room temperature and pulverized, in an acidic aqueous solution. This method avoids the use of alcoholic and potentially toxic solvents. However, the content of oleuropein in the extract is only about 30 mg per gram of dried and pulverized leaves.

The aim of the present invention is to provide an improved process for the extraction of polyphenols, notably oleuropein, from olive tree leaves, which allows to obtain higher amounts of oleuropein compared to the other methods available in the literature, with a degree of purity of at least 45% or higher and which does not employ alcoholic solvents.

The present invention enables the achievement of this purpose with a relatively simple and low-cost approach.

The process of the present invention includes a first drying step of the olive tree leaves by microwave and a subsequent step of polyphenols extraction, preferably oleuropein, in an acidic aqueous solution. Surprisingly, the combination of microwave drying and polyphenols extraction in an acidic aqueous solution, according the process described in this invention, allows to extract significantly higher amounts of polyphenols from the leaves, compared to the processes reported in the literature.

Additional features and advantages of the invention will be clarified in the following description of the preferred, but not exclusive, realization forms of the process of the invention and from the examples. The results of the examples are illustrated in the attached tables and figures, wherein:

Figure 1 shows an HPLC chromatogram of an extract obtained according to a preferred form of the process of the invention from leaves of the cultivar Coratina;

Figure 2 shows an HPLC chromatogram obtained according to a preferred form of the process of the invention from leaves of cultivar Frantoio.

The process of the present invention comprises the following steps in succession: a) supply the leaves, preferably previously cleaned by washing and dryied to remove other waste materials b) drying the leaves by microwave drying c) pulverize the dried leaves d) extract the polyphenols from the dried and pulverized leaves by mixing the powdered leaves in an acidic aqueous solution e) recover the extract comprising polyphenols, in particular oleuropein.

The process is applicable to any type of leaf containing polyphenols; particularly preferred are the olive tree leaves. Examples of olive tree leaves cultivars that might be used in the present invention, individually or in combination of two or more, include: Lucca, Mission, Nevadillo Blanco, Manzanillo, Amellenque, Arbequina, Ascolana Tenera, Ascolana, Azapa, Barnea, Barouni, Biancolilla, Bidh El Hamman, Blanqueta, Caillet Blanc, Carolea, Cayonne, Chemilali, Chitoni, Cipressino, Coratina, Cornicabra, Correggio, Correggiola, Cucco, Gigante di Cerignola, Frantoio, Glappolo, Gordal, Hardy's Mammoth, Hojiblanca, Itrana, Jumbo Kalamata, Kalamata, Koroneiki, Leccino, Leccio del Corno, Liani, Lucques, Manzanilla, Maurino, Michellenque, Moraiolo, Nabali Mohassan, Nab Tamri, Negral, Nocellara del Belice, Obliza, Oblonga, Paragon, Pendolino, Picual, Redding, picholine, Redounan, Saurin a foglia larga, Saurin a foglia media, Saurin a foglia piccola, Sevillano, Sorani, South Australian Verdale, St. Catherin, Taggiasca, Tanche, Tiny Oil Kalamata, Tsunati, Verdale, Wagga Verdale, Zarza, Oliviere, FS17. In general, the process of the invention is applicable to the leaves of any variety of olive tree.

Preferably the microwave drying, according to the present invention, is carried out in a microwave oven at 350 - 900 W, more preferably at 600-800 W, for a time between 1 and 2.5 minutes.

The pulverization of the dried leaves can be carried out either by grinding the leaves manually, for example using a mortar, or mechanically, for example using a mill. Preferably, the temperature during the pulverization stage need to be controlled and kept about to room temperature.

Preferably, the acidic aqueous solution used for the extraction have a pH between 2.5 and 5, more preferably about equal to 3. Preferably the aqueous solution is acidified by the addition of a suitable amount of acid to the water. The following acids can be used in the present invention: hydrochloric acid, hydrogen sulfide acid, sulfuric acid, sulfurous acid, phosphoric acid, nitric acid, hydroiodic acid, hydrobromic acid, perchloric acid, lactic acid, oxalic acid, tartaric acid, hydrofluoric acid, citric acid, ascorbic acid, acetic acid. More preferably, the aqueous solution is acidified by adding an appropriate amount of hydrochloric acid.

In a preferred form, the aqueous solution is acidified without the addition of potentially toxic products, e.g. by adding an appropriate amount of citric acid.

Preferably the extraction step comprises the mixing of the dried leaf powder in the acidic aqueous solution, preferably for an extraction time between 2 and 8 hours, more preferably between 3 and 6 hours, at a controlled temperature, preferably between 30°C and 65°C, more preferably between 50°C and 65°C. Preferably, the extraction mixture comprising the dried leaf powder and the acidified water is kept in continuous agitation or mixed regularly during the extraction.

Preferably, the ratio between leaves powder and acid solution (grams of leaves powder: ml of acid solution) is within 1:7 and 1:20, more preferably between 1:7 and 1:15.

At the end of the extraction step, the extract is recovered from the supernatant of the mixture including the dried leaf powder in acidic aqueous solution.

The recovery of the extract might be accomplished according to the methods reported in the literature. For example, the extract may be recovered by separating the liquid fraction of the extraction mixture, comprising the extracted polyphenols in suspension, from the solid fraction, including the leaves residues, using a fluted filter.

Successively, the liquid extract including polyphenols is thus preferably centrifuged and then filtered, in order to remove other undesired substances such as chlorophylls, to increase the degree of purity of the final extract.

For example, the liquid fraction of the extraction mixture including the polyphenols is centrifuged within 5000 to 12000 rpm for a time preferably between 6 and 15 minutes. Once that centrifugation is completed, the supernatant is separated from the pellet and subjected to filtration, preferably with a filter having a pore size between 0.20 and 0.60 μm. The extracted polyphenols are thus recovered as a purified extract obtained as filtration permeate.

Preferably, the extract including the polyphenols, optionally purified, is freeze-dried, more preferably using a freeze-drying technique named "spray drying" which involves the use of a spray dryer. This type of freeze- drying allows to further increase the degree of purity of the final extract, as well as to obtain a more stable final product which can be stored for longer periods of time compared to traditionally freeze-dried products obtained using standard equipment.

EXAMPLES

EXAMPLE 1

Extracts from olive leaves powder of Coratina and Frantoio cultivars were prepared according to a particularly preferred form of the method of the invention. Olive leaves powder was obtained by drying the olive tree leaves in a microwave at 750 W for 2 minutes. Successively, the dried leaves were pulverized using a small mill. The temperature was kept within 35°C. Polyphenols, particularly oleuropein, were extracted from the leaves powder in a solution of distilled water acidified with 1 millimolar (mM) HC1 at pH 3. In particular, the dried olive leaves powder was mixed with the acidified water solution using a 1:10 ratio. Successively, the extraction was performed for approximately 4 hours at 60°C. At the end of the extraction step, the extraction mixture was filtered using a fluted filter and, successively, the liquid permeate was centrifuged for 10 minutes at 12000 RPM. After centrifugation, the obtained supernatant was filtered using a filter with a pore size of 0.2 μm. Finally, the permeate was freeze-dried using a conventional freeze-dryer.

A representative HPLC chromatogram of one of the obtained extract is shown in Figure 1 for the Coratina cultivar and in Figure 2 for the Frantoio cultivar.

Table 1 shows the results obtained with the leaves of the two cultivars, assessing each extract with HPLC. The amount of oleuropein was quantified in accordance with the HPLC obtained chromatograms which were compared with an analytical standard of oleuropein (Sigma-Aldrich) at 98% purity.

Table 1 The process of the present invention, compared to known processes reported in the literature, makes it possible, in a relatively simple and low-cost manner, to obtain vegetable extracts having a high polyphenols and oleuropein content. The aforementioned result was not predictable on the basis of the techniques reported in the literature. In particular, the high concentration of oleuropein in the extracts obtained by the process of the present invention demonstrates a synergistic and unexpected effect of the leaves drying method (using microwaves) and of the polyphenols extraction method (in acidic aqueous solution). As a result, it was not expected that the extraction in acidic aqueous solution would allow to obtain high amounts of oleuropein, since, usually, this procedure determines the degradation of oleuropein in its by-products (such as hydroxytyrosol and tyrosol). It was even less predictable that the combination of this extraction method with the preliminary microwave drying would allow to reach the surprising results reported in example 1.

References:

Capone, R., Bilali, H. E., Debs, P., Cardone, G., &

Driouech, N. (2014). Food system sustainability and food security: connecting the dots. Journal of Food Security, 2(1), 13-22.

Giampietro, F. (2009). Commento alia direttiva 2008/98/CE sui rifiuti. Quali modifiche al codice dell'ambiente (Vol. 6).

Wolters Kluwer Italia.

Incelli, M., Cordiner, S., Tosti, S., Borgognoni, F., Sansovini, M., & Santucci, A. (2016). Trattamento delle acque di vegetazione do oleifici: tecnologie a membrana e processi termochimici. Chim. Ind, 3(10.17374).

Pizzichini M., Russo C., Pizzichini D., Tasselli P. (2006). Trattamento delle acque di vegetazione con tecnologie di membrana per il recupero di bio-fenoli nel rispetto ambientale ENEA RT/2006/21/BIOTEC.

Rose, G. (2002). International Law of Sustainabel Agriculture in the 21st Century: The International Treaty on Plant Genetic Resources for Food and Agriculture. Geo. Int'l Envt1. L. Rev., 15, 583.

Kamran M., Hamlin A. S., Scott C. J., Hassan K. (2015) Obied Drying at high temperature for a short time maximizes the recovery of olive leaf biophenols. Industrial Crops and Products 78, 29-38.