ITMI20132040A1 - RICH NATURAL EXTRACTS IN MELANOIDINS AND POLYPHENOLS OBTAINED FROM VIRGIN ROASTED COFFEE AND PROCEDURE FOR THEIR PRODUCTION - Google Patents

Description

Description of the invention: "Natural extracts rich in melanoidins and polyphenols obtained from virgin roasted coffee and the process for their production"

DESCRIPTION

The present invention relates to the field of natural extracts, in particular of coffee extracts.

More specifically, the present invention refers to a process for the production of a roasted coffee extract rich in melanoidins and polyphenols and the extract thus obtained.

BACKGROUND OF THE INVENTION

Various processes are known in the art for producing extracts of natural substances from coffee.

In the work "Spent coffee grounds as a valuable source of phenolic compounds and bioenergy." by Zuorro A., Lavecchia R., Journal of Cleaner Production (2012), 1-8, waste coffee residues were studied as a potential resource of phenolic compounds and a source of energy. The extraction procedure used involves the use of aqueous ethanol as a solvent. It is shown in this work that extracts containing phenols can be obtained from waste coffee residues.

US8226992 describes a coffee extract obtained from roasted or non-roasted coffee beans by means of a process which includes cavitation of the residues of the beans after their grinding; cavitation leads to the formation of micelles with a core of cafestol and kahweol (diterpenes) and an outer coating comprising polysaccharide, melanoidin or a combination thereof.

Recent studies have highlighted the presence in coffee of constituents with antioxidant activity.

For example, in the work of Daglia et al. (2000) (Daglia M., Papetti A., Gregotti C., Berte` F. and Gazzani G., In vitro antioxidant and ex vivo protective activities of green and roasted coffee, J. Agric. Food Chem., 2000) is reported the study of the antioxidant activity in vitro and the ex vivo protective activity of coffee constituents and it is observed that the fractions with higher molecular mass had antioxidant activity while the fractions with lower molecular mass had a protective activity.

One of the components of coffee with antioxidant activity are melanoidins.

Melanoidins are heterogeneous high molecular weight polymers that are formed mainly from the combination of carbohydrates and proteins (through the Maillard reaction) at high temperatures and in the presence of water. Their structure is extremely complex and still largely unknown. They are formed during the coffee roasting process and during this process they incorporate different components present in green coffee. In particular, the incorporation of chlorogenic acids, present in important quantities in green coffee, plays an important role in the formation of melanoidins in coffee infusions (Bekedam EK, Schols HA, Van Boekel MA, Smit G. Incorporation of chlorogenic acids in coffee brew melanoidins. J Agric Food Chem. 2008 Mar 26; 56 (6): 2055-63; Bekedam EK, Loots MJ, Schols HA, Van Boekel MA, Smit G. Roasting effects on formation mechanisms of coffee brew melanoidins. J Agric Food Chem.

2008 Aug 27; 56 (16): 7138-45; Vitaglione P, Fogliano V, Pellegrini N. Coffee, colon function and colorectal cancer. Food Funct. 2012 Sep; 3 (9): 916-22.). Chlorogenic acid is the ester of caffeic acid with quinic acid and shares the antioxidant properties of caffeic acid. It is believed that the inclusion in melanoidins of these chlorogenic acids, which are largely lost by green coffee when roasted, provides a significant contribution to their antioxidant activity, which is however present, to a lesser extent, even in the absence of chlorogenic acids.

Several processes are known for the extraction of melanoidins from various natural products.

For example, JP2001112439 describes a process for obtaining a cane sugar extract, in which said sugar is dissolved in water, adsorbed on a resin and eluted with a solvent, e.g. ethanol. The extract thus obtained contains melanoidins and has an antioxidant action. The extract is described as indicated for reducing body weight and improving skin conditions.

Melanoidins are present in green coffee in very low quantities, close to zero, while roasting increases the quantity in coffee infusions (Bekedam et al. 2008).

The quantity of melanoidins in green coffee is so low that it does not make their extraction and isolation interesting and economical from an industrial point of view.

Products containing extracts obtained from green coffee are available on the market. Here are some examples:

Green Coffee Puro 4750mg - Green Coffee, sold by WeightWorld Italy;

Green Coffee Pure Extract Capsules - BodyLine, sold by salutebio.it;

Green coffee seeds (Coffea arabica L.) tablets in dry extract, titrated and standardized in Chlorogenic Acid and caffeine produced by KOS Herbal Dietary Laboratory.

The use of these products is recommended by their respective manufacturers and sellers mainly to stimulate the metabolism, and in general to promote weight loss, and for their antioxidant action. These effects are mainly attributed to the presence in green coffee, together with caffeine, of chlorogenic acid. The presence of melanoidins in these extracts is not reported.

Melanoidins are the subject of recent scientific interest, in particular for their antioxidant activity (Borrelli RC, Visconti A, Mennella C, Anese M, Fogliano V. Chemical characterization and antioxidant properties of coffee melanoidins. J Agric Food Chem. 2002 Oct 23 ; 50 (22): 6527-33; Delgado-Andrade C, Morales FJ. Unraveling the contribution of melanoidins to the antioxidant activity of coffee brews. J Agric Food Chem. 2005 Mar 9; 53 (5): 1403-7).

For example, they seem to play a role in protecting the colon from the development of colorectal cancer (Vitaglione et al. 2012).

Furthermore, they have the ability to enhance the antioxidant activity of polyphenols.

Furthermore, melanoidins have anti-tumor and protective properties of the skin against ultraviolet rays (UV).

As mentioned above, roasting coffee causes the formation and increase of melanoidins in coffee beans.

However, when a common coffee infusion is obtained, only the water-soluble portion passes into the infusion, while the insoluble melanoidins remain in the filters.

An extract obtained from coffee that is particularly enriched in melanoidins is therefore highly sought after.

GB1073738 describes a process for recovering the melanoidins from the coffee grounds that remain after obtaining an aqueous extract from roasted coffee beans. In a variant of the process, the melanoidins are extracted from the aforementioned coffee grounds by acid-alcoholic extraction, in which a mixture of hydrochloric acid and an organic solvent (methanol or ethanol or acetone) is used; the extract thus obtained is then subjected to subsequent phases of adsorption, elution with an alkaline solution and pH adjustment, until a slightly acid aqueous solution is obtained, which can then be dried. The melanoidins thus recovered are then added to the aforementioned aqueous extract, obtained from roasted coffee beans, in aqueous or dry form to obtain an instant coffee containing melanoidins. The purpose of the process is to obtain an instant coffee with the properties of a "Turkish" coffee, or a coffee traditionally prepared in the same container that also contains the coffee grounds, in order to prevent physical ailments deriving from the consumption of the coffee. coffee obtained in the traditional way. The process described in this document is carried out only starting from the roasted coffee grounds, therefore not from the roasted coffee itself but from what remains after its aqueous extraction. Furthermore, the extraction of melanoidins is related to the pH, which must therefore be strictly controlled during the procedure.

Several processes are known for the extraction of melanoidins from coffee which have coffee grounds (exhausted coffee) as a starting material, such as the one described above,

In light, in particular, of the properties of the melanoidins described above, a process is highly sought that allows an extract rich in melanoidins to be obtained efficiently from coffee.

It has now been found by the inventors of the present invention that by applying an extraction process that uses a mixture of ethanol and water to a starting material consisting of virgin roasted coffee, an extract particularly rich in melanoidins and polyphenols is obtained.

In particular, it has surprisingly been found that this extract obtained from virgin roasted coffee contains a considerably higher quantity of melanoidins than the quantity present in a typical extract from exhausted coffee.

Furthermore, the extract obtained is also rich in polyphenols, in particular it contains a higher quantity of polyphenols than that typically present in an extract from exhausted coffee.

It was also surprisingly discovered that said extract obtained from virgin roasted coffee has a very high antioxidant power, in particular much higher than the antioxidant power of an extract typically obtained from exhausted coffee.

Said extract is also obtained with a process that has the advantages of being simple, quick and low-cost, with a high yield and which allows the recovery of residual biomass.

SUMMARY OF THE INVENTION

An object of the present invention is a composition obtained from virgin roasted coffee through an extraction process which includes the following steps:

a) adding to the starting material comprising virgin roasted coffee a solvent comprising a mixture of ethanol and water so as to obtain a suspension;

b) stirring the suspension obtained in step a) possibly heating;

c) remove the solid component from the suspension obtained at the end of step b),

and if desired

d) remove the liquid component from the solution remaining after step c).

A titrated solid extract is thus obtained, which is an object of the present invention.

A process for the production of the composition object of the invention including the following steps is also an object of the present invention:

a) adding to a starting material comprising virgin roasted coffee a solvent comprising a mixture of ethanol and water to obtain a suspension;

b) stirring the suspension obtained in step a) possibly heating;

c) remove the solid component from the suspension obtained at the end of step b)

and if desired d)

d) remove the liquid component from the solution remaining after step c).

Additional optional steps in the process described above are:

e) concentration of the final composition;

f) isolation of polyphenols and / or melanoidins from the final composition; g) purification of the final composition.

These optional steps can be performed, when desired, alternatively to each other or in sequence in any order.

The composition obtained with the process of the invention is characterized by a high content of melanoidins and polyphenols and a high antioxidant power.

It can be taken alone or in addition to food and drinks and be used in the food, nutraceutical, cosmetic, cosmeceutical and pharmaceutical fields.

In particular, a pharmaceutical composition comprising the composition of the invention and at least one pharmaceutically acceptable carrier or excipient is also an object of the present invention.

The use of said composition in the treatment and prevention of diseases that require antioxidant activity, in the adjuvant treatment of tumors and in the treatment of colorectal cancer are further objects of the present invention.

A further object of the present invention is the use of the composition of the invention in the treatment of skin diseases that require protection from ultraviolet rays.

A composition or a food product comprising the composition of the invention is also an object of the present invention.

The composition of the invention can also be included in a cosmetic composition, which is also an object of the present invention.

In particular, said cosmetic composition can be used for protection from ultraviolet rays.

DESCRIPTION OF THE INVENTION

Definitions

In the context of the present invention, by "roasted" or "toasted" coffee is meant the coffee obtained by roasting green coffee.

The roasting process can be any process known in the art. Preferably, roasting takes place through an industrial roasting process, where industrial roasting means any process commonly used in the industrial sector for the preparation of roasted coffee.

In the context of the present invention, virgin roasted coffee means coffee obtained by grinding roasted coffee.

In the context of the present invention, by green coffee is meant unroasted coffee.

In the context of the present invention, by extract is meant a preparation, generally concentrated, obtained from vegetable matter.

In the context of the present invention, by "solid" or "dry" extract is meant an extract from which the liquid component has been removed.

In the context of the present invention, by "titrated" solid or dry extract is meant an extract which contains a determined measurable quantity of one or more certain active components of interest (melanoidins and / or polyphenols).

Figures

Figure 1. Verification of the linearity of the response of the spectrophotometer in the determination of the melanoidin content.

Detailed description of the invention

The starting material used in the extraction process described here is virgin roasted coffee.

Preferably, said coffee is obtained by industrial roasting of green coffee and its subsequent grinding.

For example, commercially available ground roasted coffee can be used as the starting material.

Said starting material can be partially dried before its use in the process. Drying can take place using traditional methods well known in the art, for example controlled dehydration.

The starting material is then subjected to the extraction process comprising steps a) -d) as defined above.

Further details and preferred embodiments of the process will be described below.

The solvent used in step a) comprises a mixture of ethanol and water. The ethanol / water ratio of the mixture expressed in volumes is preferably between 20 and 70%. More preferably, said ratio is 50%.

The solvent is used in step a) of the process in a liquid / solid ratio (l / s) with respect to the starting material, preferably between 5 and 90 l / kg. Conveniently, said liquid / solid ratio is between 5 and 50 l / kg.

In a preferred embodiment, said ratio is comprised between 10 and 45 l / kg, more preferably between 15 and 30 l / kg.

In a preferred embodiment, said l / s ratio is 30 l / kg.

The possibility of carrying out the process of the invention in the presence of low liquid / solid ratios confers several advantages to this process from an industrial point of view. First of all, it allows you to process a greater amount of raw material in a unit of time. Furthermore, a low liquid / solid ratio allows to reduce solvent consumption and the size of the equipment used to carry out the process. These advantages are particularly relevant in the industrial use of the process.

Furthermore, the lower the solvent (liquid) used in the process, the lower the amount of energy required to carry out the removal of the liquid component in step d) of the process. This allows a considerable overall energy saving of the process and therefore also of its possible use in a production plant, with significant reductions in production costs.

If it is desired to carry out step b) at a temperature higher than the ambient one, the suspension obtained in step a) can be brought to a temperature between room temperature and 70 ° C, preferably at a temperature between 30 and 70 ° C. More preferably between 30 and 60 ° C. Even more preferably the temperature is 50 ° C or 60 ° C.

The desired temperature can be reached by heating the suspension obtained in step a) by any heating method commonly used in the art.

In an alternative embodiment, the suspension is brought to the desired temperature by preheating the ethanol / water mixture added in step a).

In step b) the suspension is subjected to stirring and possibly maintained at the above temperature for a time preferably comprised between 20 and 150 minutes.

Conveniently, said time is comprised between 30 and 90 minutes.

Preferably, said time is comprised between 30 and 80 minutes, for example it is 40 minutes.

In a preferred embodiment, said time is 60 minutes.

It should be noted that a short duration of the process gives a considerable advantage from an industrial point of view as it allows you to perform a greater number of daily processing cycles.

The parameters illustrated above can be combined and varied in the ranges indicated in order to optimize yields and for this the expert in the sector can use his / her own general knowledge.

The parameters can be varied in the indicated ranges also in order to obtain advantages from an industrial and / or commercial point of view. For example, the use of a low liquid / solid ratio confers several advantages from an industrial point of view, as indicated above. Or, a shorter process duration confers a commercial advantage by allowing the obtaining of a greater quantity of extract in the same period of time.

In a particularly preferred embodiment of the invention, the ethanol / water ratio of the mixture used in step a) is 50:50, the liquid / solid ratio in step a) is 30 l / kg of starting material, in step b) the temperature is 50 ° C and the stirring time is 60 minutes.

In step c) of the process, the removal of the solid component from the suspension can be carried out according to the methods commonly used in the sector. Conveniently, a filtration method is used.

A liquid solution is thus obtained which is an object of the present invention.

Steps a) -c) of the process can be repeated on the solid component removed in step c) in order to increase the yield of the final extract.

In one embodiment of the invention, the process is repeated on the solid component removed from time to time in step c) three times. The process parameters (liquid / solid ratio, ethanol / water ratio, temperature and duration of step b)) may vary at each repetition of the process or remain the same. The liquid solutions obtained at the end of step c) of each repetition of the process are collected in a single final liquid solution.

If desired, the liquid solution obtained at the end of step c) is subjected to step d) of removing the liquid to give the dry extract, which is another object of the present invention.

This removal of the liquid component, ie the solvent comprising a mixture of water and ethanol, can be carried out, for example, by evaporation of the liquid, in particular by evaporation under vacuum.

The removed solvent can be collected and subsequently reused in the process. In fact, it does not undergo such changes or alterations during the process as to prevent or limit its subsequent reuse. For example, the solvent can be removed by evaporation, collected and subsequently recondensed and reused in step a) of the process.

The possible recovery of the solvent is a further advantage of the present process.

If a further enrichment or increase in the titre of the dry extract is desired, the latter, as obtained after step d) of the process, can be subjected again to steps a) -c) of the extraction process.

Optionally, the process can comprise a further step e) of concentrating the obtained composition.

This step can be useful to obtain an extract with a higher polyphenol and melanoidin content.

The concentration can be carried out, for example, by repeating steps a) -c) of the process on the liquid solution obtained at the end of step c) or on the dry extract obtained at the end of step d) or on the solid component removed in step c) , as already described above.

A further optional step of the process is isolation (step f), which may be useful, for example, to isolate specific polyphenols and / or melanoidins or their desired groups from the composition obtained at the end of step c) or d).

Said isolation can be carried out by means of the common methods known in the field.

Any unwanted agents or impurities can be removed from the composition obtained with the process of the invention through a further optional purification step (step g).

Purification can take place according to any method known to the expert in the field for the removal of unwanted components from a liquid or solid composition.

As evident from the description, the process of the present invention has several advantages, which are summarized below:

1. High yield: the process of the invention allows to obtain a high quantity of solid extract per unit of raw material used (virgin roasted coffee);

2. Simplicity, since it includes few unitary operations;

3. Rapidity: the times of realization of the extraction cycle are short, thus allowing to carry out several daily cycles.

4. Low liquid / solid ratio, which translates into less solvent use, smaller device dimensions, lower consumption, less total space occupied by machinery;

5. Using low temperatures;

6. High content of melanoidins and polyphenols;

7. Use of a non-toxic extraction mixture and the possibility of its recovery;

8. Closed extraction cycle;

9. Starting product available all year round;

10. Recovery of biomass with production of heat and electricity:

plants with zero (or almost) waste;

11. Low overall energy consumption of the system.

The extract obtained with the extraction process described above is characterized by a high content of melanoidins and polyphenols.

In particular, the content of both is higher than in an extract typically obtainable from exhausted coffee.

A further advantage of the solid extract of the invention is to be "titrated" in melanoidins and / or polyphenols, or to contain a determined and measurable amount of the same.

Compositions according to the invention are preferred with a melanoidin content of between about 60 and about 140 mg / gram of dry extract (gs), preferably between about 70 and about 130 mg / gs, more preferably between about 80 and about 130 mg / gs, even more preferably between about 100 and about 130 mg / gs. Particularly preferred is a composition in which the melanoidin content is 118 mg / gs, 97 mg / gs or 125 mg / gs.

The above compositions are preferred which, in addition to the melanoidin content indicated above, also comprise a content of polyphenols (indicated as equivalents of gallic acid - GAE) comprised between about 30 and about 70 mg / (gs), preferably between about 40 and about 60 mg / (gs), more preferably between about 50 and about 60 mg / (gs).

The compositions according to the invention are preferred in which the combined recovery (RC) equal to the product of the extraction yields of the melanoidins and phenolic compounds is comprised between about 50 and 100 mg / (gs), preferably between about 60 and 100 mg / (gs), more preferably between about 70 and 60 mg / (gs), even more preferably between about 80 and 100, particularly between about 90 and 100. The composition in which RC is 100 is most preferred.

The extract object of the present invention is also characterized by a high antioxidant power, where antioxidant power means the ability to neutralize free radicals. In particular, the antioxidant power of the extract of the invention is much higher than that of an extract typically obtainable from exhausted coffee.

Possible uses of the composition

It is known that oxidative stress can be the cause of various diseases. Antioxidants can eliminate the damaging effects that free radicals have on cells. They are in fact widely used in food supplements to maintain physical well-being and assist in the prevention of some diseases, such as cancer and coronary heart disease.

In particular by virtue of its high antioxidant activity, the composition of the present invention can have application in the food, nutraceutical, cosmetic, cosmeceutical and pharmaceutical fields.

It can be used alone or in addition to a drink or food.

In particular, the composition of the invention can be included in a pharmaceutical composition, further comprising at least one carrier or a pharmaceutically acceptable excipient. The type of vehicle or excipient, the quantities and methods of administration may vary depending on the patient and the type of pathology and can be chosen by the expert in the field according to common knowledge in the art. A preferred form of administration is oral administration, in the form, for example, of tablets, hard or soft capsules, liquid preparations, for example solutions or suspensions, optionally flavored with conventional ingredients.

The composition of the invention can be used for the prevention and treatment of diseases due to oxidative stress and which can therefore be prevented or improved by an antioxidant action.

Oxidative stress, in fact, is believed to be responsible for premature aging and is associated with a series of very common diseases, such as arterial hypertension, diabetes, atherosclerosis, heart attack, stroke, Parkinson's disease, 'Alzheimer's, obesity, arthritis, some types of cancers, etc.

Therefore, in particular, an object of the invention is the use of the composition of the invention in the treatment of diseases that require an antioxidant activity. These diseases can be all diseases caused or worsened by oxidative stress such as, for example, premature aging diseases, degenerative or neurodegenerative diseases, chronic inflammatory diseases, some types of tumors.

The composition can also be used in the prevention of the aforementioned oxidative stress diseases, in particular as an adjuvant.

The composition of the invention can also be used in the adjuvant treatment of tumors, or in support and completion of a main anticancer therapy.

A further object of the present invention is the use of the composition of the invention in the prevention or treatment of colorectal cancer, thanks, in particular, to the presence of high quantities of melanoidins which play a role in protecting the colon from the development of colorectal cancer. .

The composition of the invention can also be used in the treatment of skin diseases that require protection from ultraviolet rays. An example of such a pathology is skin cancer, in particular melanoma.

In all of the aforementioned therapeutic applications, the methods of treatment, times and quantities to be administered will be determined by the expert in the field, in particular by the doctor, on the basis of his knowledge in the sector.

A composition or a food product comprising the composition of the invention is also an object of the present invention.

The food product can be, for example, a drink or a tablet that can be used as a food supplement.

The composition of the invention can also be included in a cosmetic composition, which is also an object of the present invention. In such a case, the cosmetic composition will also comprise a cosmetically and / or dermatologically acceptable vehicle and will preferably be in a form suitable for being applied to the skin.

Said cosmetic composition can be used, for example, for protection from ultraviolet rays. In particular, for the protection of the skin, lips and hair from ultraviolet rays.

The present invention will hereinafter be further illustrated by non-limiting examples.

EXAMPLES

Materials and methods

Roasted coffee was used commercially available in vacuum packs of 250 gr.

The chemicals used, with their respective degrees of purity and the suppliers, are shown in Table 1. All the products, of analytical grade, were used without being further purified.

The aqueous solutions were prepared using double distilled (Carlo Erba) or demineralized (Deterchimica) water. Filtration, when required, was carried out on Whatman paper or with 0.22 or 0.45 μm nylon filters.

Purity Product Supplier

Potassium Acetate> 99% Sigma-Aldrich

Glacial acetic acid> 99.8% Sigma-Aldrich

Hydrochloric acid fuming 37% 36.5–38% Carlo Erba

Chlorogenic acid> 95% Sigma-Aldrich

Gallic acid> 98% Sigma-Aldrich

Caffeine> 98% Sigma-Aldrich

Sodium carbonate> 99.5 Carlo Erba

Aluminum chloride> 99% Fluka

DPPH> 90% Sigma-Aldrich

Absolute ethanol> 99.8% Fluka

Quercetin> 95% Sigma-Aldrich

Reagent of Folin-Ciocalteu - Fluka

Table 1 - Chemicals, degree of purity and suppliers.

Stabilization of materials

The stabilization of the samples was carried out by controlled dehydration in a specially designed apparatus consisting of the following units:

• dryer / dehydrator with horizontal perforated plates (STOCKLI, Switzerland) equipped with an electrical resistance for heating the air and a fan for forced circulation;

• chemical hood (BICASA, Milan) with high air flow equipped with centrifugal extractor fan, to operate in depression, and filtering system;

• electric resistance thermometer (HD 2127.1, Delta OHM, Padua) equipped with a probe with Pt 100 platinum sensor for measuring temperatures between –50 and 200 ° C with an accuracy of ± 0.01 ° C.

Up to three perforated plates can be inserted into the dryer for depositing the material to be dried. Above the plates there is a cover plate with a central 55 mm hole for the air outlet and the insertion of measurement probes. This hole can also be used to pick up the material during drying.

The drying / dehydration procedure of the materials with the described apparatus is carried out through the following steps:

• switching on the heater and adjusting the heating power according to the desired temperature;

• deposition of the sample on one or more perforated plates;

• closing the dryer;

• activation of the fan for air circulation;

• control of the temperature and / or air speed during the entire duration of the process.

In the case of simultaneous use of the three perforated plates, especially for prolonged periods of the heating phase, it may be convenient to vary the stacking positions of the plates during the treatment to obtain a uniform degree of drying.

As regards the air speed, it has been seen that above 0.5–0.7 m / s this parameter has a limited effect on the final drying degree. The temperature adopted is linked to the drying duration and the need to avoid the degradation of thermolabile compounds.

The materials considered were dried at a temperature between 30 and 40 ° C for a time varying between 6 and 12 hours depending on the initial humidity value of the material.

Analytical extraction procedure

The multi-stage batch-type extraction procedure uses the 50% (v / v) ethanol-water mixture as a solvent and three extraction stages in sequence to ensure quantitative recovery of the compounds.

In a typical test, 0.2 g of sample and the desired amount of solvent were placed inside Duran glass containers. The vessels were transferred to a thermostated bath and magnetically stirred. At the end of each extraction phase, the suspension was filtered on paper, the solid was recovered for subsequent extraction and the liquid extract analyzed. The total amount of phenolic compounds and melanoidins was determined as the sum of the values obtained in each step.

The experimental conditions used (temperature T, liquid solid ratio L / S, and extraction time t) are for the virgin roasted coffee and for the typical exhausted coffee reported in the following table 2.

TYPE OF COFFEE TOPT (L / S) OPTtOPT

(° C) (l / kg) (min) Virgin roasted coffee 56.8 83.6 60 Typical exhausted coffee 56.8 83.6 60

Table 2. - Extraction conditions for virgin roasted coffee and typical used coffee.

The extraction tests were carried out at least in duplicate for each material and the individual extracts were stored at 4 ° C until the subsequent analytical determinations.

Determination of polyphenols

The total content of polyphenols in the extracts was determined using the Folin-Ciocalteu method according to the procedure described by Singleton et al. (Methods in Enzymology, Volume 299, 1999, 152-178) with some modifications. The method is based on the oxidation of phenolic compounds by the action of the Folin-Ciocalteu reagent. It consists of a mixture of phosphotungstenic acid (H3PW12O40) and phosphomolybdic acid (H3PMo12O40).

By reaction with phenols, these acids are reduced to blue tungsten and molybdenum oxides (W8O23 and Mo8O23) whose concentration, related to the quantity of phenolic compounds, can be measured using a spectrophotometer or a colorimeter.

The tests were carried out by placing, inside a 10 mL graduated glass cuvette, 5 mL of 0.1 N HCl, 200 μL of the extract sample and 144 μL of Folin-Ciocalteu reagent. After short stirring, a 10% (w / v) Na2CO3 solution is added until a final volume of 10 mL is reached. The vessel is kept in the dark at room temperature for 60 minutes. We then proceed to measure the absorbance of the samples at 525 nm through a colorimeter (HI 83742, Hanna Instruments, Italy), using a blank of distilled water.

The total content of phenolic compounds (q ') in the samples was expressed as equivalents of gallic acid (GAE) or chlorogenic acid (CAE) per unit mass of dry material (mg GAE / gso mg CAE / gs) through the following relation:

q '= (ceff · Vsolv) / [m (1 - UR / 100)]

where ce is the actual concentration of phenolic compounds in the sample as measured with the colorimeter, m is the amount of solid, RH is its relative humidity and Vsolv is the volume of solvent.

Determination of the melanoidin content in the extracts

A review of the literature on methods for determining the content of melanoidins in products or raw materials of the food industry made it possible to identify a spectrophotometric analytical method as the most appropriate for the measures in question. The method consists in the ultrafiltration of the liquid extracts containing melanoidins, in the measurement of the absorbance of these extracts at a fixed wavelength and in the transformation of the absorbance values into those of concentration through the use of an extinction coefficient based on weight it. In particular, reference was made to the analytical procedure described in the work of Budryn et al. (Budryn G., Nebesny E., Podsȩdek A., Zyzelewicz D., Materska M., Jankowski S., Janda B., Effect of different extraction methods on the recovery of chlorogenic acids, caffeine and Maillard reaction products in coffee beans, Eur. Food Res. Technol. 2009, 228, 913–922, DOI: 10.1007 / s00217-008-1004-x), where the melanoidins present in coffee (brown pigments or "brown pigments") are correlated to the absorbance values of extracted at 420 nm.

The samples to be examined were preliminarily diluted in distilled water to obtain absorbance values in the linearity range of the instrument used, consisting of a double beam UV / VIS spectrophotometer (Lambda 25, Perkin Elmer, USA). Distilled water was used as a blank. Figure 1 shows the effect of the dilution ratio on the absorbance at 420 nm.

The absorbance values at 420 nm (A420) were converted into the corresponding melanoidin concentration values (cmel) through the Beer-Lambert law:

A420 = ε420cmel (1)

where ε420 is the extinction coefficient at 420 nm. For the latter, the following value was used: ε420 = 1.1289 g <–1> L <–1> cm <–1> reported in the work of Tagliazucchi et al. (Tagliazucchi D., Verzelloni E., Conte A., Effect of dietary melanoidins on lipid peroxidation during simulated gastric digestion: their possible role in the prevention of oxidative damage, J. Agric. Food Chem. 2010, 58, 2513–2519, DOI: 10.1021 /) related to the extraction of high molecular weight melanoidins (> 10 kDa) from roasted coffee.

Finally, from the cmelsi values it is passed to the melanoidin content per unit of dry weight of the starting material (q'mel) through the following relationship: q'mel = (cmel · VL) / [mS · (1 - UR / 100 )] (2)

where VLe mS represent, respectively, the volume of solvent and the amount of coffee used in the extraction test and RH the relative humidity of the solid material. The q'mel values were expressed as mg of melanoidins per g of dry coffee or, equivalently, g of melanoidins per kg of dry coffee.

Measurement of antioxidant power

The antioxidant power of the extracts was determined by the DPPH method. DPPH, or 1,1-diphenyl-2-picrylylhydrazyl, is a very stable nitrogen radical with an intense purple color. In the presence of a substance with antioxidant capacity, the DPPH is reduced and the color of the solution in which it is present turns yellow. Consequently, the antioxidant power of the considered compound can be determined by means of a colorimeter or a spectrophotometer by measuring the change in absorbance of the solution at a certain wavelength. Typically, the measurement is carried out at 517 nm, the wavelength at which the radical form absorbs.

The antioxidant power is usually quantified through a parameter, conventionally referred to as EC50, which represents the concentration of the sample in the cuvette required to reduce the initial amount of DPPH by 50%. Therefore, the lower the EC50 value, the greater the antioxidant power of the tested substance.

The measurement of the antioxidant power of the phenolic extracts was performed by placing 3.9 mL of an ethanolic solution of the DPPH radical at a concentration of 0.05 g / L in a 4.5 mL optical glass cuvette and a 10 mm optical path. appropriate quantity of extract. The cuvette is shaken vigorously and after 30 minutes the absorbance is measured at 517 nm using a double beam UV-VIS spectrophotometer (Lambda 25, Perkin-Elmer). As a reference we consider a solution containing 3.9 mL of ethanol and the same amount of sample.

The EC50 is obtained from a diagram showing the EC values obtained at different sample concentrations. The EC is calculated through the following equation:

EC = 100 (A0 - Af) / Af

where A0 and Af represent the initial absorbance and after 30 min. Finally, from the correlation of the EC - concentration data, the sample concentration corresponding to an EC value of 50 is evaluated. It represents the EC50 of the tested sample.

During the development phase of the procedure, some tests were carried out with gallic acid as a reference sample. The EC50 was found to be 1.72 μg / mL.

RESULTS

Yield and phenolic title of dry extracts

The yields in dry solid extract and the phenolic title of the extracts obtained by evaporation under vacuum of solutions prepared with the analytical extraction procedure described above were determined.

In particular, the percentage yield in dry extract (R%), referred to the material as it is, was calculated as:

R% = 100 qES / m

or, in terms of dry material, such as:

R '% = 100 qES / mS

where qES is the quantity of dry extract, while m and mS are the quantities of starting material as is or dry. qES was evaluated as:

qES = q'ES · V / VR

where q ES is the quantity of dry extract determined gravimetrically after evaporation of the solvent, V is the volume of solvent used in the extractor and VR is that of the solvent recovered at the end of evaporation. Due to the inevitable losses of solvent that occur in the filtration of the suspension present in the extractor and in the subsequent evaporation of the liquid under vacuum, VR is lower than V. The phenolic content of the extracts (T%) was evaluated as follows:

T% = 100 mPOL / qES

where mPOL represents the quantity of extracted polyphenols, expressed in equivalents of gallic acid, and qES the quantity of dry extract.

From the results obtained it is possible to calculate the quantity of polyphenols recovered per unit of mass of the starting material (P):

P = R · T

The results obtained are presented in the following Tables 3 and 4.

R%

Coffee type (% w / w)

Roasted virgin coffee 28.77

Typical exhausted coffee 11.04

Table 3 - Yields in dry extract (R%).

P.

Type of coffee (mg GAE / gS)

Roasted virgin coffee 50.35

Typical exhausted coffee 25.89

Table 4 - Values of the parameter P (recovered polyphenols) expressed in equivalents of gallic acid

Melanoidin content of dry extracts

The melanoidin content of the dry extracts of roasted and exhausted coffee was measured with the methods described above. The results are contained in Table 5.

Type of coffee Total melanoidins (gr / Kg) Virgin roasted coffee 120

Typical exhausted coffee 20

Table 5 - Melanoidin content expressed as gr / kg per unit of raw material used

The results as a whole are summarized in the following table 6: Dry Extract Dry Extract

Titled By Titled Da

lementi Quantitative Exhausted Coffee Roasted Coffee Typical Ratio Typical Quantitative. Yield (wt%) of

produced per unit of 11 28 2.5 atery previously used

. Total polyphenols for

nity of raw material 25 50 2 m folded (g GAE / Kg)

. Total melanoidins

gr / kg) per unit of 20 120 6 atery previously used

. Antioxidant power

0.28 2.6 Approx.10 / EC50 (DPPH method)

Table 6: Summary of the results obtained with extraction tests on roasted coffee and used coffee.

The first element confirms that the yield (percentage by weight of product obtained per unit of weight of raw material used in the process) is typically, for roasted coffee, about 2.5 times that obtained from exhausted coffee.

The second element confirms that the overall quantity of polyphenols obtained without further refining (weight of product obtained per unit of raw material used multiplied by the polyphenol content of the product obtained) is in a ratio of two in favor of the product obtained from roasted coffee.

Note the increased content of melanoidins (about 6 times) in roasted coffee compared to the exhausted coffee extract.

The antioxidant power is about an order of magnitude in favor of the product obtained from roasted coffee. The antioxidant power is here expressed as the inverse of EC50, where the EC50 (calculated with the DPPH method) represents the amount of product needed to neutralize 50% of free radicals. As can be seen, the product obtained from roasted coffee requires a quantity of product approximately nine times lower than that obtained from exhausted coffee.

Study of the process variables in roasted coffee

The set of variables that influence the final result for roasted coffee has been thoroughly investigated. It emerged that the relevant variables are four: water / ethanol ratio, temperature, liquid solid ratio, process duration.

The extraction yield of melanoidins, polyphenols and their combined recovery in different temperature conditions, liquid-solid ratio, process duration was then analyzed (the water / ethanol ratio is always 50/50). The results are represented in the following table 7.

umer T L / S t q'mel q'pol RC i test (° C) (l / kg) (min) (mg / gs) (mg (-)

GAE / gs)

1 30 30 60 87.02 44.406 70.74 2 50 30 60 117.58 46.459 100.0 3 30 70 60 83.27 42.848 65.31 4 50 70 60 105.21 48.119 92.68 5 30 30 120 103.95 47.508 90.40 6 50 30 120 97.15 51.054 90.80 7 30 70 120 96.07 44.836 78.85 8 50 70 120 110.00 48.189 97.04 9 23.2 10 90 82.62 36.799 55.66 10 56.8 10 90 121.78 42.299 94.30 11 40 16.4 90 105.49 44.726 86.37 12 40 83.6 90 96.22 49.636 87.43 13 40 50 40 86.58 40.643 64.42 14 40 50 140 124.64 40.918 93.36 15 40 50 90 93.52 42.052 71.99 16 40 50 90 99.45 41.490 75.53 17 40 50 90 98.67 41.504 74.97 18 40 50 90 94.78 40.970 71.09 19 40 50 90 95.08 42.100 73.28 20 40 50 90 93.25 41.231 70.38

Table 7 - Extraction yields of melanoidins (q'mel), polyphenols (q'pol) and their combined recovery (RC) on 20 roasted coffee tests.

Where the combined recovery function (RC) equal to the product of the extraction yields of melanoidins and phenolic compounds is:

RC = q'mel · q'pol (3)

and the resulting values are normalized.

gs = gram of dry extract

GAE = Gallic Acid Equivalents

As can be seen from the table, the extraction yields are entirely satisfactory throughout the process. In particular, the maximum extraction yield for the combined recovery of melanoidins and polyphenols is obtained with the set of parameters reported in row 2 of table 7.

Claims (19)

CLAIMS 1. A composition obtained from virgin roasted coffee through an extraction process that includes the following steps: to. adding to the starting material comprising virgin roasted coffee a solvent comprising a mixture of ethanol and water so as to obtain a suspension; b. stirring the suspension obtained in step a) possibly by heating; c. remove the solid component from the suspension obtained at the end of step b), and if desired d. remove the liquid component from the remaining solution after step c). The composition according to claim 1 wherein said solvent of step a) comprises a mixture of ethanol and water in an ethanol / water ratio expressed in volumes comprised between 20 and 70%. 3. The composition according to claim 1 or 2 wherein said solvent is used in step a) in a liquid / solid ratio with respect to the starting material comprised between 5 and 90 l / kg. 4. The composition according to any one of claims 1-3 wherein in step b) said suspension is brought to a temperature comprised between 30 and 70 ° C. The composition according to any one of claims 1-4 wherein in step b) the suspension is subjected to stirring for a time comprised between 20 and 150 minutes. The composition according to any one of claims 1-5 wherein in step c) the removal of the solid component is carried out by filtration. The composition according to any one of claims 1-6 wherein in step d) the removal of the liquid component is carried out by evaporation under vacuum. The composition according to any one of claims 1-7 wherein the liquid component removed in step d) is collected and reused in step a). The composition according to any one of claims 1-8 wherein said solid component removed in step c) is subjected again to steps a) -c). The composition according to any one of the preceding claims wherein said composition is added to a drink or a food. A pharmaceutical composition comprising the composition of any one of the preceding claims and at least one pharmaceutically acceptable carrier or excipient. A composition or a food product comprising the composition of any one of claims 1 to 9. A cosmetic composition comprising the composition of any one of claims 1 to 9. 14. Use of the cosmetic composition of claim 13 for protection against ultraviolet rays. 15. Composition of any of claims 1-9 for use in the treatment or prevention of diseases that require antioxidant activity. 16. Composition of any one of claims 1-9 for use in the treatment of skin diseases that need protection from ultraviolet rays. 17. The composition of any one of claims 1-9 for use in the adjuvant treatment of tumors. 18. The composition of any one of claims 1-9 for use in the prevention or treatment of colorectal cancer. 19. A process for producing the composition of any one of claims 1-9 comprising the following steps: a) adding to the starting material comprising virgin roasted coffee a solvent comprising a mixture of ethanol and water so as to obtain a suspension; b) stirring the suspension obtained in step a) possibly heating; c) remove the solid component from the suspension obtained at the end of step b), and if desired d) remove the liquid component from the solution remaining after step c).