Patent Description:
Olive oil, an edible oil extracted from olives, i.e. the fruits of the olive tree (Olea europaea), has aroused particular interest thanks to the high level of polyphenols contained in it. These compounds are natural antioxidants of plant origin capable of inhibiting the formation of free radicals.

The beneficial properties of olive oil have led to a considerable increase, above all in Italy, in olive cultivation and olive oil production. Consequently, there has also been a large increase in by-products of olive oil production, mainly the vegetation waters and pomace, which are characterised by a high pollutant load and thus generate a considerable environmental impact.

The disposal of this material is strictly regulated at both a national and regional level and entails heavy costs for producers, who are unable to derive any advantage from these waste products.

However, vegetation waters and pomace are a rich source of compounds that are useful for human health; in particular, they include hydroxytyrosol, verbascoside and <NUM>,<NUM> DHPEA-EDA (oleuropein-aglycone di-aldehyde). Verbascoside exerts an anti-inflammatory activity, typically on the oral mucosa. Furthermore, verbascoside is capable of blocking the activity and reducing the presence of iNOS and COX-<NUM>, thereby preventing oxidative stress.

<NUM>,<NUM> DHPEA-EDA is an oleuropein derivative with marked antibacterial, anti-inflammatory and antioxidant properties.

Hydroxytyrosol is one of the most studied molecules owing to the wide range of biochemical processes in which it exerts its antioxidant activity. Hydroxytyrosol is in fact a potent inhibitor of the oxidative damage deriving from lipid peroxidation in cells, considered to be the main process of tissue damage by free radicals.

Various techniques and processes for the disposal of olive vegetation waters are described in the literature. For example, <CIT> describes a process for treating vegetation waters with membrane technologies comprising filtration steps that go from microfiltration to reverse osmosis. However, the process described in <CIT> allows a water purified from pollutants to be obtained at the end of the process, but does not allow the various polyphenolic components present in the vegetation waters to be separated, nor does it allow fractions with high concentrations of polyphenols to be obtained. <CIT> discloses a concentrate of vegetation waters and/or olive pomace obtained by a process comprising the steps of (i) microfiltering a sample of vegetation waters and/or olive pomace and (ii) concentrating by reverse osmosis the microfiltration permeate obtained in step (i).

Thus, there is a strongly felt need for a process which makes it possible to obtain polyphenol-enriched fractions using olive vegetation waters and/or olive pomace. In particular, it would be very advantageous to develop a process that enables fractions with a high content of hydroxytyrosol, <NUM>,<NUM> DHPEA-EDA and verbascoside to be obtained, while at the same time enabling the vegetation waters to be purified of pollutants for the correct disposal thereof.

A first aspect of the present invention relates to a process for obtaining a concentrate of olive vegetation waters or olive pomace enriched in polyphenols, said process comprising the steps of:.

A second aspect of the present invention relates to a concentrate that is obtained/obtainable according to the process described above. The concentrate preferably comprises hydroxytyrosol and tyrosol. In one embodiment, the concentrate further comprises <NUM>,<NUM>-DHPEA-EDA.

A third aspect of the present invention relates to the use of the concentrate as described above as a nutritional supplement.

In the context of the present invention, "<NUM>,<NUM>-DHPEA-EDA" means the polyphenolic compound also known as "<NUM>,<NUM>-DHPEA-Elenolic acid di-aldehyde" or "Oleuropein-aglycone di-aldehyde", with a molecular weight of <NUM>.

A first aspect of the present invention relates to a process for obtaining concentrate of olive vegetation waters or olive pomace enriched in polyphenols, said process comprising the steps of:.

In a preferred embodiment of the invention, the olive vegetation waters and/or olive pomace are subjected to step (a) of the process without any pre-treatment, i.e. the vegetation waters or olive pomace derive directly from olive pressing processes, without undergoing any treatment, such as pH adjustment, enzymatic hydrolysis reactions or filtration. The microfiltration step (a) is preferably carried out within a time of between <NUM> and <NUM> hours after the generation of the vegetation waters or pomace in the mill, in order to avoid an excessive oxidation of the polyphenolic compounds that will be recovered in the various steps of the process.

In one embodiment, the microfiltration step (a) is carried out with a porous membrane, preferably a ceramic membrane, with a pore size between <NUM> and <NUM>, preferably between <NUM> and <NUM>. In one embodiment, the microfiltration step (a) is carried out at a maximum pressure between <NUM> and <NUM> bar, preferably between <NUM> and <NUM> bar.

In one embodiment, the ultrafiltration step (b) is carried out with a membrane, preferably a polymeric membrane, with a molecular cut-off between <NUM> and <NUM> kDa, preferably between <NUM> and <NUM> kDa. The polymeric membrane is preferably made of one of the following materials: polysulfone, polyethersulfone, polyamide or cellulose acetate. In a preferred embodiment of the invention, the polymeric membrane is made of polysulfone. In one embodiment, the ultrafiltration step (b) is carried out at a maximum pressure between <NUM> and <NUM> bar, preferably between <NUM> and <NUM> bar.

In one embodiment, the nanofiltration steps (c) and (d) are carried out using membranes, preferably polymeric membranes, with a molecular cut-off between <NUM> and <NUM> kDa, preferably between <NUM> and <NUM> Da.

The polymeric membranes used in the nanofiltration steps (c) and (d) are preferably shaped as a wound spiral and are made of polyamide or nylon or polyethersulfone, preferably polyamide.

In one embodiment, the nanofiltration step (c) is carried out at a temperature between <NUM> and <NUM>, preferably between <NUM> and <NUM>, and at a maximum pressure between <NUM> and <NUM> bar, preferably between <NUM> and <NUM> bar.

The nanofiltration step (d) is preferably carried out at a temperature between <NUM> and <NUM>, preferably between <NUM> and <NUM>, and at a maximum pressure between <NUM> and <NUM> bar, preferably between <NUM> and <NUM> bar.

In one embodiment, the reverse osmosis step (e) is carried out with a membrane, preferably a high salt rejection polymeric membrane. The polymeric membrane is preferably made of polyamide or nylon, preferably polyamide. The reverse osmosis step (e) is preferably carried out at a maximum pressure between <NUM> and <NUM> bar, preferably between <NUM> and <NUM> bar.

In one embodiment, after each step (a)-(d) a diafiltration step is performed by feeding each membrane with water to obtain a retentate which is combined with each permeate obtained in steps (a)-(d). The water used for diafiltration is preferably water obtained from the reverse osmosis step (e).

The CNF1 and CNF2 are preferably combined after being obtained in order to have a single concentrate.

As shown in <FIG>, the raw vegetation waters undergo <NUM> membrane filtration steps with increasingly small pore sizes and molecular cut-offs, performed under different conditions with membranes suitable for each type of filtration. From each step one obtains a permeate and a concentrate rich in polyphenols (indicated at the bottom in <FIG> as CMF, CUF, CNF1, CNF2 and CRO) which can be used to prepare both supplements and other preparations for cosmetic or medical use. The permeates obtained from each step, indicated in <FIG> as PMF, PUF, PNF1 and PNF2 undergo a subsequent filtration step, up to the final step, where a reverse osmosis concentrate (CRO) and pure water are obtained. After each filtration step, with the exception of the reverse osmosis, a diafiltration step is carried out, i.e. each membrane is fed with water, preferably water obtained from the reverse osmosis in the last step, in order to obtain a retentate that is combined with each permeate. Each step is preferably carried out immediately at the end of the preceding step, i.e. the process is carried out continuously until the reverse osmosis concentrate and pure water are obtained.

The Applicant has advantageously found that the process of the present invention makes it possible to obtain liquid fractions comprising various polyphenolic families that can be profitably used both in the field of nutritional supplements and in preparations having biomedical characteristics. In particular, thanks to a double nanofiltration step under different temperature and pressure conditions and the use of untreated vegetation waters, i.e. which derive directly from the mill and do not undergo any treatment prior to the microfiltration step (a), one obtains polyphenol-rich concentrates. In particular, one obtains polyphenol-rich concentrates with a high molecular weight, e.g. hydroxytyrosol, tyrosol and <NUM>,<NUM>-DHPEA-EDA. In fact, the two nanofiltration concentrates combined together (CNF1+ CNF2) comprise a percentage of hydroxytyrosol and hydroxytyrosol glucoside that is higher than <NUM>% relative to the total polyphenol percentage in the two concentrates. Furthermore, the reverse osmosis concentrate (CRO) comprises a percentage of hydroxytyrosol and hydroxytyrosol glucoside that is higher than <NUM>% relative to the total polyphenols of the reverse osmosis concentrate.

A second aspect of the present invention relates to a concentrate that is obtained/obtainable according to the process described above in detail. In a preferred embodiment, the concentrate is obtained after the nanofiltration step (c) (CNF1) and/or after the nanofiltration step (d) (CNF2 or CNF1+CNF2), or else after the reverse osmosis step (CRO). The concentrate is preferably obtained by combining CNF1 and CNF2, or it is CRO. The concentrate preferably comprises hydroxytyrosol and tyrosol. In a preferred embodiment, the concentrate comprises a percentage of hydroxytyrosol and hydroxytyrosol glucoside higher than <NUM>% relative to the total polyphenols present in the concentrate. The concentrate preferably comprises a percentage of hydroxytyrosol and hydroxytyrosol glucoside higher than <NUM>% relative to the total polyphenols present in the concentrate.

In one embodiment, the concentrate further comprises <NUM>,<NUM>-DHPEA-EDA. The concentrate preferably further comprises β-hydroxyverbascoside, verbascoside, caffeic acid, glucose and fructose.

The amount of hydroxytyrosol preferably ranges between <NUM> and <NUM> grams per litre of concentrate (g/L), more preferably between <NUM> and <NUM>/L.

The amount of the hydroxytyrosol glucoside preferably ranges between <NUM> and <NUM>/L, more preferably between <NUM> and <NUM>/L.

The amount of <NUM>,<NUM>-DHPEA-EDA is preferably between <NUM> and <NUM>/L, more preferably between <NUM> and <NUM>/L.

The amount of tyrosol is preferably between <NUM> and <NUM>/L, more preferably between <NUM>/L and <NUM>/L.

The amount of β-hydroxyverbascoside is preferably between <NUM> and <NUM>, more preferably between <NUM> and <NUM>/L.

The amount of verbascoside is preferably between <NUM> and <NUM>/L, more preferably between <NUM> and <NUM>/L.

The amount of caffeic acid is preferably between <NUM> and <NUM>/L, more preferably between <NUM> and <NUM>/L.

The amount of glucose and fructose is preferably between <NUM> and <NUM>/L, more preferably between <NUM> and <NUM>/L.

A third aspect of the present invention relates to the use of the concentrate as described above in detail as a nutritional supplement. In fact, the concentrate of the present invention can be used as a dietary supplement, for example to reduce the effects of oxidative stress in an individual.

Vegetation waters just generated in a mill by cold pressing of organic olives from biodynamic agriculture were treated with a microfiltration (MF) process that uses porous ceramic membranes with a pore size of <NUM> micron. A raw extract of polyphenols, proteins, sugars, mineral salts and a soluble cellulose fraction were obtained from the permeate of this process. The MF filtered product had a dark red colour but appeared perfectly clear. In order to improve the polyphenol content of this matrix, the microfiltration permeate was subjected to ultrafiltration (UF), a double nanofiltration step (NF1 and <NUM>) and finally a reverse osmosis step (RO) on the second permeate of NF2.

A diagram of the process is shown in <FIG>.

As can be seen from <FIG>, the raw vegetation waters underwent <NUM> membrane filtration steps with increasingly tight molecular cut-offs, which were carried out under different conditions and with membranes suitable for each type of filtration. The technical features of the membranes used are shown in table <NUM>.

About <NUM> of fresh vegetation waters were fed to the MF system; the permeate produced was stored in the feed tank of the subsequent membrane filtration section (UF), whilst the vegetation waters from which the permeate was removed were concentrated in the feed tank. During the filtration, the flow value of the permeate produced, graphically represented over time in <FIG>, decreased, following a typical trend; to avoid excessively rapid clogging and thus an excessively marked decline in productivity, the system is operated with in-line backwashing (back pulse). Once the desired value of the concentration ratio (VCR) was reached, a diafiltration step (Dia) with demineralised water was carried out in order to further extract the products of interest from the concentrated fraction. The volume of the feed tank is reduced to <NUM> when the permeate produced is about <NUM> (VCR = <NUM>/<NUM> = <NUM>); then <NUM> of demineralised water are added, bringing the load volume to <NUM>, and operation continues for enough time to permeate another <NUM> of product. The total permeate produced and available for the next UF step was about <NUM>.

Overall, for the MF step, a mean specific flow of about <NUM>/m<NUM>h is assumed. The MF permeate is clear and has a pH of <NUM> and an electrical conductivity of <NUM>/cm at <NUM>.

During filtration, a system for controlling the temperature by cooling with mains water allows the temperature to be maintained below the set values, thus avoiding the normal increasing trend; this serves not so much to protect the membrane, which, being made of ceramic in this section would not need it, but rather to protect the product and the components of interest thereof, which, as known, are thermolabile.

About <NUM> of microfiltration permeate are refrigerated in order to start the UF from an initial temperature of about <NUM>. The aim is to obtain about <NUM> of PUF thanks to the addition of the diafiltration volume. The inlet operating pressure of the membrane is about <NUM> bar and the feed flow rate of the system is about <NUM>/h, whereas the recirculation flow rate over the membrane with the second pump is about <NUM><NUM>/h. The productivity in UF is initially very high (about <NUM>/h) but then decreases suddenly as expected.

The permeability pattern over time is shown in the graph in <FIG>. Overall, for the UF step, a specific mean flow of about <NUM>/m<NUM>h is assumed.

Once the desired value of the concentration ratio (VCR) is reached, a diafiltration step (Dia) with demineralised water is carried out in order to further extract the products of interest from the concentrated fraction. The volume of the feed tank is reduced to <NUM> when the permeate produced is about <NUM> (VCR = <NUM>/<NUM> = <NUM>); then <NUM> of demineralised water are added, bringing the load volume to <NUM>, and operation continues for enough time to permeate another <NUM> of product The total permeate produced and available for the next UF step is about <NUM>.

The aim was to produce a sufficient amount of permeate to guarantee the next steps of the process. All the permeate produced was used to feed the 1st NF step. The UF permeate has a light yellow-orange colour, a pH of <NUM> and an electrical conductivity of <NUM>/cm at <NUM>.

In this test the NF was divided into two successive steps in series on the permeate (see diagram in <FIG>). A diafiltration step (Dia) was carried out on the concentrate to increase the amount of hydroxytyrosol that passes in the permeate. The second NF stage was performed at a low temperature and at a high pressure in order to maximise the performance of the membrane, which, in this manner, will retain the polyphenols as much as possible and allow hydroxytyrosol to pass.

Overall, for the first NF step, a mean specific flow of about <NUM>/m<NUM>h is assumed.

The total volume of PNF of the first step, about <NUM>, was fed to the second NF step. The mean temperature was lower than in the first NF step, since refrigeration is activated during the test, plus the operating pressure was higher. The filtration specifications are summed up below:
NF2 Pressure <NUM>-<NUM> bar, Temperature <NUM> - <NUM>.

Feed <NUM>, Dia <NUM>, Permeate <NUM>, Concentrate <NUM>, VCR <NUM>.

At this point the product was already treated by NF and therefore, as was expected, the test proceeded with rather high permeability values.

Out of the about <NUM> fed (including the <NUM> for diafiltration), about <NUM> of permeate were produced, reaching a VCR equal to about <NUM>.

A graph of the specific permeate flow over time (expressed in L/h/m<NUM>) is shown in <FIG>.

Overall, for the second NF step, a mean specific flow of about <NUM>/m<NUM>h is assumed.

The NF permeate was treated by reverse osmosis (RO) to obtain, in the concentrate, the polyphenolic fractions with a lower molecular weight, i.e. the ones that passed the NF barrier, mainly hydroxytyrosol. One starts from a volume of NF permeate of about <NUM>. The RO was conducted at high pressure and at a low temperature to increase the selectivity of the membrane vis-à-vis the polyphenols present in the NF permeate. The maximum RO pressure is about <NUM> bar; the RO permeate is clear in appearance. The test is pushed further by gradually lowering the operating temperature (from an initial <NUM> to a final temperature of about <NUM>) until a final value of VCR equal to about <NUM>. The electrical conductivity value of the permeate remains low during the entire test, <NUM>-<NUM>/cm, and in the final part the value begins to increase, as expected, up to a maximum of about <NUM>/cm.

At the end of the RO filtration about <NUM> of concentrate are obtained in the feed tank. A graph of the specific permeate flow over time is shown in <FIG>. Overall, for the RO step, a mean specific flow of about <NUM>/m<NUM>h is assumed.

The RO permeate is about <NUM>; this clear permeate is used in part to carry out the diafiltrations described in the previous sections.

During the various steps, samples are collected and direct intermediate conductivity analyses are performed; the data are shown in table <NUM>.

During the various filtration steps (MF, UF, NF and RO) samples were collected for chemical analyses in a laboratory; the results are shown in table <NUM>.

The results are expressed in g of Tyrosol per litre of sample solution, internal standard: Syringic Acid. The quantification limit is <NUM>/L; therefore, values below <NUM> are to be considered only approximate.

The value indicated as % of hydroxytyrosol and derivatives thereof, relative to the total polyphenols determined by HPLC, comprises not only hydroxytyrosol and tyrosol, but also hydroxytyrosol glucoside and caffeic acid. In practical terms, it is a % of acid polyphenols relative to the complex ones but determined with HPLC, hence the data are reliable. The carbohydrates were determined with ionic chromatography techniques.

The membrane filtration tests were carried out with various pilot membrane units, which allowed the volumes of the different fractions to be treated as shown in the diagram in <FIG>.

The stated volumes are indicative of the invention, whereas the membranes used are commercial ones that can be freely used in an industrial process. Therefore, the invention is scalable upwards in terms of production.

Claim 1:
A process for obtaining a concentrate of olive vegetation waters and/or olive pomace enriched in polyphenols, said process comprising the steps of:
(a) microfiltering a sample of olive vegetation waters and/or olive pomace to obtain a concentrate (CMF) and a microfiltration permeate (PMF);
(b) ultrafiltering the PMF obtained in step (a) to obtain a concentrate (CUF) and an ultrafiltration permeate (PUF);
(c) nanofiltering the PUF obtained in step (b) to obtain a first concentrate (CNF1) and a first nanofiltration permeate (PNF1);
(d) nanofiltering the PNF1 obtained in step (c) to obtain a second concentrate (CNF2) and a second nanofiltration permeate (PNF2); and
(e) concentrating by reverse osmosis the PNF2 obtained from step (d) to obtain a reverse osmosis concentrate (CRO) and water.