"HIGH POLYPHENOLS COCOA POWDER BASED PRODUCTS, USES AND METHODS FOR THE MANUFACTURE THEREOF"

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FIELD OF THE INVENTION

The present invention relates to cocoa powder based products with a high content in polyphenols and uses and method for the manufacture thereof.

The present invention origins in the field of food industry and of nutritional products.

In particular, the present invention concerns with a process for the production of cocoa based food products, especially cocoa powder, and healthy food products having a high polyphenol contents and with nutritional, nutraceutical, medical or cosmetics uses of these cocoa based products.

BACKGROUND ART

Edible products containing cocoa derivatives such as chocolate, cocoa butter, cocoa liquor and other cocoa solids are widely consumed for their taste and mouthfeel.

Typically cocoa powder is used in the food industry for the preparation of a variety of food products, amongst which chocolate, bakery products, desserts and beverages.

The discovery that cocoa contains certain naturally occurring antioxidant substances such as flavonoids and flavanols, especially catechins and epicatechins and other nutritional substances such as theobromine, theophylline and minerals whose consumption has been associated with certain health benefits, has increased the potential applications of cocoa-based food products. For example, the consumption of chocolate has been associated with the certain health benefits such as improvement in the endothelial vascular function, including regulation of blood pressure and with certain anti-inflammatory activities.

However, at present the low content of effective antioxidant substances because of traditional industrial processes in most of the commercially available cocoa- based products, limits the possibility of achieving real health benefits derived from the consumption of chocolate and its derivatives.

Therefore, there is the need to have cocoa-based food products with a content of specific antioxidant substances which is sufficient to obtain certain health benefits. One of the general objects of the present invention is to provide a cocoa powder and cocoa based products having a high content of antioxidant substances especially polyphenols such as epicatechins and catechins.

Another object of the invention is to provide a method for the preparation of a cocoa powder with a high content in selected polyphenols.

Yet another object of the invention is the provision of nutritional or healthy products such as chocolate, beverages or chocolate derivatives with a high content of a selected polyphenol having antioxidant properties.

SUMMARY OF THE INVENTION

The present invention originates from the finding that a cocoa powder or cocoa- based product highly rich in selected polyphenols can be obtained by using certain specific conditions in the industrial production of cocoa powder.

The inventors found that certain steps of the conventional methods for producing cocoa powders, which are highly desirable to provide the typical aroma and flavour to cocoa powder and chocolate, negatively affect the content of specific polyphenols contained in the starting cocoa beans.

Specifically, the inventors found that the amount of selected naturally occurring polyphenolic components contained in the starting cocoa beans is retained or only partially affected, if certain specific treatments, which are typical of the traditional methods for producing cocoa powder, are avoided or substantially changed. In addition, there are evidences that certain polyphenols contained in the cocoa- based products/powders obtained by the process of the invention may have a bioavailability in the human body which is increased with respect to the naturally occurring polyphenols contained in the starting cocoa beans.

Thus, in accordance with certain aspects of the present invention, the denaturation or oxidation of polyphenols that occurs during processing of cocoa beans is substantially avoided or reduced and selected polyphenolic monomeric components having an improved bioavailability are made available.

In accordance with certain aspects, the present invention provides cocoa components, especially cocoa powder, having high levels of selected cocoa polyphenols.

In accordance with a first aspect, the present invention provides a cocoa-based product, especially a cocoa powder, having an epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg.

In accordance with a second aspect the present invention concerns with a composition comprising the cocoa-based product of the invention, especially in the form of cocoa powder, having an epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg.

In certain embodiments, the cocoa-based product of the invention is a chocolate. The composition may be a nutritional composition, a food supplement, a medicament or medicine useful for ameliorating or maintaining the health of a human being.

In particular, the cocoa-based product or a composition containing the cocoa- based product of the invention are useful in the treatment and/or prevention of oxidative stress of the human body.

In accordance with a fourth aspect the present invention concerns with a cocoa- based product, especially a cocoa powder or an extract thereof having an epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg for use in the treatments of an inflammatory disease, a metabolic disorder or a cardiovascular disease.

In accordance with a fifth aspect, the present invention provides a process for the production of a cocoa-based product, especially cocoa powder, having an epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg, the process comprising providing unfermented or underfermented cocoa beans having a moisture or water content of less than 15% by weight and heating the cocoa beans at a temperature of 70 to 130 °C for a period of time of 1 to 30 minutes.

Typically, the process of the invention excludes a roasting step of the cocoa beans or nibs.

With roasting step it is intended a conventional heating treatment different from an IR heating.

In accordance with certain embodiments, the process for the production of cocoa- based products or powders especially having an epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg, said process comprising the following steps:

a) Providing unfermented or underfermented cocoa beans;

b) Draining the unfermented or underfermented cocoa beans to reduce the action of microorganisms that initiate the fermentation process and to reduce loss or damage of flavanols by the action of oxidase enzymes;

c) Drying the drained cocoa beans, typically by exposure to sunlight

d) Heating the cocoa beans preferably at a temperature of 70 to 130 °C for a period of time of 1 to 30 minutes especially by infrared treatment to facilitate the elimination or removal of the shell of the beans.

e) Winnowing the heated beans to separate the cocoa nibs from the cocoa shell;

f) Grinding the nibs to give a cocoa liquor typically containing the ground particle of cocoa solids suspended in a cocoa butter;

g) Pressing the cocoa liquor to separate the cocoa solids from cocoa butter. Typically, the degree of fermentation of the cocoa beans is determined by changing of the colour grains and may be measured by applying the so-called cutting test according to ISO International Standard No. 1 1 14. 30.

In addition thereto, the inventors have investigated that a cocoa powder rich in certain selected polyphenols especially epicatechins, finds application in the preparation of either a nutritional or medical product which is useful in the prevention and/or treatment of certain conditions or affections of the human organisms.

BRIEF DESCRIPTION OF THE DRAWINGS The features and the advantages of the present invention will become apparent from the following detailed description and from the examples provided as an illustrative and non limiting process and from the accompanying Figures, in which: Fig. 1 shows a bar graph illustrating the results of a NF-kB p65 DNA binding activity assay (O.D.) carried out in cultured macrophages with LPS (Lipopolysaccharide), LPS + CE (commercial cocoa powder), LPS + CCP (Colombian Cocoa Powder of the invention) stimulation according to Example 2A. The comparison on the bar graphs show that CCP is more effective in reducing NF-kB p65 DNA binding activity compared to CE;

Fig. 2 shows comparative bar graphs illustrating the cytokines IL-1 b, IL-6 and TNF-a expression on macrophages pre-treated with CE (commercial cocoa powder), CCP (Colombian Cocoa Powder of the invention) and exposed to LPS. The blank bars refer to the basal conditions of the macrophages (i.e. untreated cells), according to Example 2A. The exposure of macrophages to LPS induces maximal secretion of cytokines whereas the exposure to CCP the minimal.

Fig. 3 shows graphs summarizing the results of a heme oxygenase activity assay by real time PCR determinations carried out according the Example 2D of the present application. The graphs show the ability of CCP of the invention to activate HO-1 gene expression in a more efficient way compared to traditional CE.

Fig. 4 shows the results of a Western blot for Nrf2 as illustrated in Example 2E. The bar graphs prove that the treatment with CCP of the invention provides a greater time-dependent increase in Nrf2 protein expression in the nuclear extracts compared to conventional CE.

Fig. 5 shows bar graphs illustrating the effects of CCP of the invention in inducing NO production using cultured human umbilical vein endothelial cells (HUVEC) according to Example 2F.

Fig. 6 shows a diagram with peaks of (-)-Epicatechin, and of 3'-O-methyl-(-)- epicatechin, and 4'-O-methyl-(-)-epicatechin which are derived from the metabolism of epicatechin, as evidenced in Example 3.

Fig. 7 shows a graph reporting the variation of the level of (-)-Epicatechin in plasma (nM) vs. the time (h) after administration in subjects undergoing the protocol according Example 4. Fig. 8 shows a graph reporting the variation in the level of 3'-O-methyl-(-)- epicatechin in plasma (nM) vs. the time (h) after administration in subjects of the protocol according Example 4.

Fig. 9 shows a graph reporting the variation in the level of 4'-O-methyl-(-)- epicatechin in plasma (nM) vs. the time (h) after administration in subjects undergoing the protocol according Example 4.

Fig. 10 shows three bars reporting the changes of total cholesterol in the subjects treated with cocoa according to the Protocol of Example 6. White, grey and black circle are the different dosages of cocoa group during the treatment. The graph shows a significant differences between the curves of 8 pills/day group and the other two groups (p 0.04).

Fig. 1 1 shows the changes of LDL-cholesterol in the subjects treated with cocoa according to the Protocol of Example 6. White, grey and black circle are shown in cocoa group. P < 0.05 for the comparison with the baseline.

Fig. 12 shows graph bars illustrating the plasma HDL concentrations in subjects of the Protocol of Example 6 who showed an increase in total amount of epicatechin metabolites in the urine. P < 0.05 compared to their counterparts.

Fig. 13 shows graph bars illustrating the change of oxidized LDL (Ox-LDL) values within each group after 4 weeks of intervention of the Protocol of Example 6

Data are expressed as mean ± SD. ^* Significant p < 0.05 from baseline.

Fig. 14 shows bar graphs illustrating the variation in the ratio omega6/omega3

(AA/EPA) after assumption of the cocoa with high contents of flavanols according to Protocol of Example 6.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that the amount of (-)epicatechin available in the naturally occurring cocoa beans is retained or preserved during cocoa beans processing for producing cocoa based products such as cocoa powder, if certain specific process conditions are met and combined.

In a general aspect the invention concerns with isolated cocoa based products, especially isolated cocoa powder having an (-)epicatechin content which is far close to the amount available in the starting material represented by unfermented or underfermented cocoa beans. The inventors have also ascertained that (-) epicatechin contained in the cocoa based product of the invention, when administered by the oral route, has a greater bioavailability with respect to the other polyphenols available in the naturally occurring cocoa beans.

Thus, in a general aspect, the invention provides a cocoa based product which is rich in (-) epicatechin, a selected polyphenol, and which finds application in the food, nutritional and medical field by virtue of its antioxidative activity and bioavailability.

In accordance with a first aspect, the present invention provides an cocoa-based product, especially an cocoa powder, having an epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg.

Typically the epicathechin is (-) -epicatechin. In the present description the term epicathechin should be intended (-)epicatechin.

In certain embodiments the cocoa-based product of the invention has an epicatechin or (-) -epicatechin content from 15 to 45 g/Kg, from 18 to 36 g/Kg, preferably from 21 to 33 g/Kg, from 28 to 31 g/kg.

One of the advantages of the cocoa-based products of the invention is that the (-) epicatechin contained therein, in contrast with most of the polyphenols contained in the cocoa beans, are monomers which having a bioavailability greater that polyphenols available from traditional cocoa product and for this reason may be easily absorbed in the gastrointestinal tract of a subject.

In certain embodiments, the cocoa products of the invention has an amount of (-) epicatechin which is from 5 to 60%, preferably from 10 to 40% by weight greater than the content of (-)epicatechin contained in naturally occurring cocoa beans.

With the term naturally occurring cocoa beans it is intended unfermented cocoa beans extracted from naturally occurring cocoa pods. The cocoa-based products of the invention may be in solid form or in liquid or semi-liquid form. The terms cocoa based product in solid form and cocoa solids, as used herein, substantially have the same meaning.

Examples of cocoa-based product in solid form or cocoa solids include cocoa powder, cocoa bean or portions thereof, cocoa nibs, or solid products obtained by processing cocoa beans with the method of the present invention.

In accordance with preferred embodiments, the cocoa-based product of the invention is solid and more preferably is a cocoa powder.

In a second aspect the present invention provides a composition comprising the cocoa-based product of the invention, especially in the form of cocoa powder, having an (-)epicatechin content > 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg.

The composition of the invention may be a food product, a nutritional product a dietetic product or a composition certain activity influencing certain activities or metabolism of the human body.

The compositions of the invention may be solid, semi-solid or liquid.

Typically, the composition of the invention comprises a chocolate based product.

The term chocolate based product as used herein means a composition containing cocoa solids dispersed in a fat phase. Examples of chocolate based products in solid form include sweet semisweet or bittersweet-chocolates, chocolate flavoured products, chocolate flavoured confections, chocolate sweetens, chocolate bars, chocolate fat coatings, low fat chocolate. Examples of chocolate based products in liquid form include chocolate muss, milk or buttermilk chocolate, chocolate based beverages or drinks.

The fat phase of the chocolate includes cocoa butter, milkfat, vegetable or substituted fats, replacer-equivalent fats and in general conventional fats used in the confectionary industry. In certain embodiments the chocolate based product contains sweeteners such as natural sugars such as sucrose, fructose, dextrose, maltose, glucose syrup, honey, invert sugar or artificial sweeteners and its blends such as saccharine, aspartame, acesulphame-k cyclamates or natural sweetener such as stevia or polyalcohols such as mannitol, xylitol, sorbitol, maltitol.

In certain embodiments the chocolate based product contain edible emulsifiers such as lecithin, mono and dyglycerides, sorbitan monostearate, polyglycerol esters of fatty acids.

In certain embodiments the composition of the invention is a nutritional composition. In certain embodiments the nutritional composition contains cocoa solids of the invention or an extract thereof having an epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg and optional nutrients or micronutrients such as a source of carbohydrates, a source of lipids, and optionally other nutrients or micronutrients.

In accordance with certain aspects the invention provides a non-therapeutical or therapeutical uses of the cocoa-based product or cocoa powder for reducing the cellular oxidative stress in an organism, especially a human being.

The term oxidative stress is understood herein to refer to an imbalance between oxidizing substances formed in the body by the action of breathing or sunlight and other factors and natural antioxidants produced by the body or in other words, an imbalance between the production of free radicals and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants.

Since the oxidative stress is at the basis of the developments of certain conditions in the body such as Parkinson's disease, Alzheimer's disease, gene mutations, cancers, chronic fatigue syndrome, fragile X syndrome, heart and blood vessel disorders, atherosclerosis, heart failure, heart attack and inflammatory diseases, the cocoa-based products of the invention find application in the prevention or treatment of the above conditions. In accordance with a fourth aspect, the present invention provides a process for the production of a cocoa-based product, especially cocoa powder, having a (-) epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg, said process comprising a heating treatment of unfermented or underfermented cocoa beans wherein said heating treatment comprises or consist essentially of an infrared treatment.

Typically, the process of the invention exclude a roasting step of the beans for example including a heating at a temperature of 120 to 150 °C, 125 to 145 °C for a time of 1 to 120 minutes.

The term roasting step as used herein means a conventional heating which is different from a heating treatment by infrared.

The infrared heating of the unfermented or underfermented cocoa beans rapidly heat and expand the shell of the beans facilitating the elimination or removal of the shell of the beans limiting or avoiding damages or denaturalization of polyphenol substances, which occurs when the beans or nibs are roasted in accordance with conventional processes.

Typically, the process of the invention excludes a roasting step wherein the beans or nibs contained in the shell are roasted. In certain embodiments the process excludes a roasting step wherein the

In some embodiments the unfermented or underfermented cocoa beans before the heat treatment by infrared have a moisture level or water content of less than 20% by weight of the weight, typically from 5 to 12%, especially from 7 to 8% by weight.

In certain embodiments the infrared heating treatment is carried out at a temperature of 90 to 1 18 °C, of 105 to 1 15 °C for a time of 1 to 5 minutes, preferably of 2 to 4 minutes.

In another embodiment, the heating step is carried out by infrared heating at a temperature of 80 to 95 °C for a time of 2 to 10 minutes, typically from 2 to 5 minutes.

Typically the heating treatment of the invention is made by a commercial infrared equipment such as that commercialized by the Barth company.

Typically, the infrared heating treatment is carried out by exposing the beans to an infrared radiation having a wavelength of 2 to 6 microns, which means a frequency from 0.7 to 1 .2x10 ⁸ megacycles/sec.

In accordance with certain embodiments the process for the production of cocoa- based products, especially cocoa powder, having an (-)epicatechin content greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg said process comprising the following steps:

a) Providing unfermented or underfermented wet cocoa beans;

b) Draining the wet unfermented or underfermented cocoa beans

c) Drying the drained cocoa beans;

d) Heating by infrared the dried cocoa beans at a temperature of 70 to 120 °C for a period of time of 1 to 30 minutes to facilitate the elimination or removal of the shell of the beans.

e) Winnowing the heated beans to separate the nib from the shell;

f) Grinding or milling the cocoa nibs.

In certain embodiments the process of the invention comprises grinding or milling the cocoa nibs into a fluid dark brown cocoa liquor containing the ground/milled particles of cocoa solids suspended in a cocoa butter and

g) Separating the cocoa solids from the cocoa butter, for example by screw pressing.

Typically, the starting material of the process of the invention comprises freshly harvested unfermented or underfermented cocoa beans which are obtained from the pods derived from cocoa trees which typically belong to species of Theobroma cacao. In accordance with certain embodiments, the cocoa trees are selected clones of cocoa trees of Colombian origins having a high polyphenol content.

Typically, the unfermented cocoa beans have a fermentation degree from 35 to 45% of grains. The grains are extracted by the pod and processed according to the invention without carrying out a fermentation step.

In cases where the cocoa bean are not subjected to a fermentation step and nevertheless they have a degree of fermentation from 3 to 8% as grain well fermented, this is the consequence of a naturally occurring fermentation between harvesting of pods and processing of cocoa beans contained in the pods.

Typically, the underfermented cocoa beans have a fermentation degree from 55 to 65% of grain with violet or purple colour. In certain embodiments underfermented cocoa beans are obtained subjecting the extracted cocoa beans to a partial fermentation step (during the draining process) which for example is carried out for a period of time of 20 minutes to 5 hours, of 30 minutes to one hour.

Typically, the degree of fermentation of the cocoa bean or the change of the colour of the beans is determined by applying the so-called cut test a method in accordance with the International standard ISO cut test No. 1 1 14. 300. According to this test method, cocoa beans are opened or cut lengthwise through the middle, so as to expose the maximum cut surface of cotyledons. Both halves of visually examine each bean in full daylight or equivalent artificial light. Count the number of cocoa bean that shows a violet or purple colored on at least half of the surface of the cotyledons exposed by the cut test. Alternatively, it is used the ISO International Standard N ° 2451 for cocoa beans specifications and ISO 2292 Cocoa beans -Sampling for the number of bags to be sampled in order to avoid variation on results of the cutting test .

By changing the fermentation degree changes the colour of the cocoa beans.

Exemplarily, according to the colouration, the beans may be divided in four different typologies: 1 . fully fermented beans wherein the predominant colour is brown/dark brown, 2. partially fermented beans wherein the predominant colour is brown-purple, 3. underfermented beans wherein the predominant colour is purple- violet and 4. unfermented bean wherein the predominant colour is slaty-grey. Tipically, 1 and 2 are added.

The term "fermentation degree" as used herein is a numerical value of percentage (%) of the degree of fermentation of a batch of cocoa beans. In certain embodiments, the fermentation degree may vary from 100 to 400 where lower values represents unfermented beans and upper represents fully fermented beans.

The "fermentation degree" may be calculated by using a grading scale in % for characterizing the fermentation degree of the 300 grains analysed (divided in 3 groups of 100 grains each one) taken from the sample (about one kilogram) of General batch. In accordance to the present invention the slaty/grey coloration means completely unfermented cocoa beans and, purple-violet coloration means under fermented cocoa beans.

Typically, the starting material of the process of the invention comprises or consists essentially of underfermented or unfermented beans or mixtures thereof. The beans are extracted from pods from cocoa trees.

Typically, in the process of the invention a fermentation step of the cocoa beans according to the conventional processes of production of cocoa powder/product is not carried out, which means that is absent.

Typically, the starting unfermented or underfermented cocoa beans may be wet. In accordance with step b) of the process of the invention, the available unfermented or underfermented cocoa beans are drained.

The draining step b) comprises the removal of the free water within the cocoa beans to delay the start of the fermentation and to reduce the rate of reduction of flavanols contained in the beans by oxidase enzymes action.

According to some embodiments step b) takes from 5 to 12 or from 12 to 20 hours, for example 14 to 18 hours. Typically, during step b) the leached water is in an amount of 12 to 16% by weight in respect of the total weight of the beans.

Step c) of the process comprises drying the beans to reduce the water content. In certain embodiments, the total content of the moisture of the beans is reduced to an amount of 7 to 8% by weight to prevent mold growth during storage of cocoa beans, thereby preventing damage to the cocoa butter and the appearance of unusual flavors or odors.

The drying step may be carried out gradually in order to prevent the closure of the small holes placed on the surface of the cocoa bean thereby allowing moisture and volatile substances such as acetic acid to outflow while the amount of polyphenols remain unchanged.

In certain embodiments the drying step comprises the following drying times: (i) First day 3 hours; (ii) Second day: 3-4 hours; (iii) Third day 4-5 hours (iv) Fourth day 5-6 hours and (iv) Fifth day: all day.

Optionally, after drying the cocoa beans may be cleaned to remove extraneous non-cocoa materials such as dust particles, metals and nonmetals, rocks, stem, cluster beans, broken beans and other parts of the tree.

The average of impurities may be 1 % by weight of the total weight after cleaning.

The cleaning step may be carried out using one or more of the following apparatuses: densimetric table, metal detector, dust extractor.

In accordance with certain embodiments the heating treatment of step d) is carried out by infrared.

In certain embodiments the shell is removed by contacting with boiling water in spray way during the IR process, which makes a swell of the shell and facilitate the separation between the nib and the shell. Another advantage of some embodiments of the process is represented by the fact that the cocoa butter doesn ^' t go from the kernel to coat seed.

Typically, the heating temperature of step is lower than traditional roasting with the whole bean, the lower temperature and time 3 min at 1 10-120 °C reduce possible impairment in the polyphenolics content of the beans.

One of the aim of step d) is the preparation of the cocoa beans to make easy take off the shell.

In the winnowing step e) of the process the outer portion of the bean referred as shell is separated by the inner portion referred as nib.

The term nibs refers to small pieces of cocoa beans of different sizes without shell obtained in the winnowing process.

Typically, the beans are cracked and carry to a vibrating screening where the shell is removed for the air action and is graduated depending on the size of the bean. For example, the sieve of the sieving apparatus has holes with different diameters: (i) 8,5 mm, the biggest; (ii) 6,0 mm, flow 44%; 4,0 mm, flow 30,6%; (iv) 2,2 mm, flow 18,1 %; (v) 1 ,0 mm flow 4,9 %.

The difference in holes and flow allow to make a classification of the nib by the size. Typically, the range admitted is 0, 3 to 0, 6 % nibs in shells, and 1 ,5 -1 ,8 % of shell in nibs. For example in the fine cocoa least 1 mm, they are approximate 1 ,8%. In certain embodiments, after the winnowing step, the nibs are passed through sieves and over magnets to remove non-cocoa materials such as metal materials. After that, the nibs undergo to grinding step f) of the process. The grinding step may consist of or comprises a milling step. In certain embodiments, the grinding step may include a pre-milling step carried out in pre-mills for example in an apparatus with blades wherein the ribs are ground.

Typically, the grinding step carried out with a mill is referred to as milling.

In certain embodiments the milling step comprises the milling of cocoa nibs into a liquefied product which is referred to as cocoa liquor which typically is a fluid dark brown liquor. In certain embodiments during milling the cocoa nibs reach a temperature of 70 to 90 °C for example 80 °C. In these conditions the nibs are liquefied to a cocoa liquor.

In certain embodiments the fat content of the cocoa liquor is from 50 to 60% for example about 54% and the particle size may be 1 .2% retained on 200 microns mesh.

The cocoa liquor produced by the milling step is collected and in certain embodiments is stored in a tank for preconditioning for example at a temperature of 100 -1 10 °C.

The resulting mass is pumped to a tank, for example having a capacity of 180- 200kg, and the temperature may be arranged in the range of 105 to 1 15, for example of about 1 10 "C.

In step g) the cocoa solids are separated from cocoa butter. Typically, the cocoa solids are partially defatted.

In certain embodiments, the separation step comprises or consists of a pressing step h) of the cocoa liquor containing both cocoa solids and cocoa butter.

During pressing step the liquor is pressed in a press, for example under a pressure of 1 1 ,000 psi with a cycle time from 8 to 12 min or 16 min, depending on the fat content.

At the end of the pressing step, the press is opened and two products are separated: cocoa solids according to the invention, which typically are in form of a cocoa cake, and a cocoa butter in liquid or semifluid form. Typically, the cocoa butter is a high-fat extract from the cocoa liquor obtained by grilling or milling the nibs.

In certain embodiments, the cocoa cake obtained by the pressing step may be crushed and or a milled to yields a cocoa powder with a high content of polyphenols according to the invention.

Typically, the cocoa butter may be purified for example by filtering and crystalizing. The term cocoa solids as used herein refers to the extract of cocoa beans which contains flavonoids, flavanols such as catechins and epicatechins. Cocoa solids may also refer to the cocoa powder.

In accordance with certain aspects the invention provides, chocolate liquor and/or cocoa solids or powder and/or cocoa butter obtained by the process according to one or more of the embodiments herein described.

In certain embodiments the cocoa powder or chocolate liquor or cocoa butter obtained by the process of the invention has a epicatechin content of greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg.

The inventors also founds quite unexpectedly that the epicatechin amount of the cocoa powder obtained in accordance with one or more embodiments of the invention is greater than the epicatechin amount contained in the starting cocoa beans.

This finding is evidenced by the data contained in the following Table

In certain aspects the invention provides a chocolate comprising a cocoa based product or a cocoa powder according to anyone of the above referred embodiments. The chocolates may be prepared starting from the suitable cocoa- based product of the invention using conventional techniques.

In certain embodiments, the chocolate of the invention has a content of cocoa powder of greater than 20%, 30%, 40%, 50%, 60%, 70% 80%, 90%.

In a further aspect the present invention provides food or nutritional products containing a cocoa powder or cocoa solids having an epicatechin or (-) - epicatechin content of greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg.

In certain embodiments the food product is chocolates comprising a cocoa powder and/or cocoa solids and/or chocolate liquor according to anyone of the above referred embodiments.

In other embodiments the food product is a chocolate flavoured product comprising a cocoa component referred hereinabove.

In certain aspects the invention provides the use of the cocoa based product of the invention, including cocoa powder, cocoa liquor, cocoa solids referred hereinabove for ameliorating or maintaining the health conditions of a mammal, typically a human being, by administering a nutritionally effective amount of cocoa based product having an epicatechin content of from greater or equal than 15 g/kg, typically in the range from 15 to 45 g/Kg, from 18 to 36 g/Kg, from 21 to 33 g/Kg of the product. The following examples are illustrative of the cocoa-based products of the invention and of a process for its production.

Example 1

In this example the amount of total and specific polyphenols especially (-) epicatechin contained in cocoa powders of the invention are compared with those contained in traditional cocoa powders, products available in the market.

The following bioactive compounds available in cocoa were measured by HPLC method: theobromine, caffeine, (-)epicatechin, procyanidin B2, gallic acid, theophylline by. A simultaneous determination of these compounds by adding known amounts of reference standard was carried out.

The comparison between the cocoa powder of the invention with other commercial cocoa powders provided the following results: Comparative data between

cocoa powder of invention

s.

commercial cocoa-based products

Total

polyphenols

(g/Kg)gallic Gallic Procyanidin theobro

Batch acid acid theofylline Caffeine B2 epicatechin mine

Colombia

Invention cocoa

3870 powder 55,50 1,8800 1,4000 6,3800 11,7000 21,7070 28,0000

Maranta

535 61% 13,39 0,0266 0,0871 1,1311 0,8361 1,1337 7,5246

Atlantico

536 33,5% 2,08 0,0044 0,0237 0,4354 0,2462 0,2506 2,1087

537 Huila 65 % 16,15 0,0373 0,1337 1,2202 1,2918 1,3014 9,4935

Noche

538 40% cocoa 3,56 0,0060 0,0417 0,6012 0,2833 0,2714 3,1312

Macondo

539 60% 12,74 0,0169 0,0861 1,0487 0,7732 0,9856 7,8932

Milk

Colombia

540 45% 4,45 0,0220 0,0263 0,4655 0,1693 0,5353 0,4391

Venezuela

cocoa

541 powder 8,63 0,0102 0,1387 0,1396 2,4766 1,8808 1,9822

Cocoa

388 A21 SRB 14,99 0,0082 0,0405 1,2380 1,3186 0,9373 27,5639

Cocoa

389 10/12 AK 18,16 0,1110 0,1925 1,5506 1,4422 0,9953 26,8084

390 0,51 0,0021 0,0048 0,1860 0,0038 0,0107 0,0583

Cocoa butter IC

Cocoa

butter

391 Colombia 0,03 0,0017 0,0061 0,4595 0,0051 0,0211 0,1876

Commerci

al cocoa

Colombia 30.28 < 0.1 < 0.1 0.15 11.7 1.685 2.15

The comparative data contained in Table 1 makes evident that cocoa powder batch according to the invention has a content of procyandin B2, gallic acid, teofillin, polyphenols and especially (-) epicathechin, higher than the batches of the products available in the market.

Example 2

REPORT: Cellular experimental models to assess biological activities of a Colombian Cocoa Powder with high polyphenols concentration.

Example 2A

Colombian Cocoa Powder (CCP) reduce NF-kB p65 DNA binding activity in cultured macrophages

Many studies found that the NF-kB pathway play a pivotal role in the genesis of inflammatory chronic diseases. The NF-kB family of transcriptional factors plays a critical role on regulation the expression of a wide variety of genes, particularly in the inflammatory process, included cytokines such as IL-1 β, IL-6 and TNF-a.

To evaluate the ability of CCP to inhibit nf-kb activity in macrophages we have used the NF-kB p65 DNA Binging Activity Assay by TransAM p65 transcription factor ELISA kit (Active Motif, Carlsbad, CA). Immortalized cell line of macrophages were stimulated with 500 ng/ml LPS. Cells were pretreated or not for 1 h with CCP 10 μηη or a commercial cocoa powder (CE) 10 μιη, for 12 hours. Then, DNA binding activity of NF-kB p65 was determined. 12 hours after, LPS stimulation markedly promoted NF-kB p65 DNA binding activity and expression of proinflammatory cytokines IL-1 β, IL-6 and TNF-a. However, a lower DNA binding activity was observed in macrophages with LPS stimulation in the presence of both CCP and CE, as shown in figure 1 . CCP was extremely more effective in the ability to reduce NF-kB p65 DNA binding activity compared to CE, demonstrating a stronger anti-inflammatory potential for CCP.

Coherently, similar effects have been shown in terms of cytokines expression (figure 2). Cells were pretreated or not for 1 h with CCP 10 μιη or CE 10 μιη, and then exposed to 500 ng/ml LPS for another 24 h. IL-1 b, IL-6 and TNF-a were detected by ELISA kits purchased from Abeam, Cambridge, (MA) USA. Data are expressed as mean ±SEM, n=4; ^* p<0.001 vs. LPS, ^** p<0.001 CCP vs. CE.

24-h exposure of macrophages to 500 ng/mL LPS induced the maximal secretion of IL-1 β, IL-6 and TNF-a, which are all released in small amounts in basal conditions, as shown in fig.2. Lipopolysaccharide strongly increased cytokines secretion. Preincubation (1 h) with CCP 10 μιη or CE 10 μιη significantly reduced LPS-stimulated cytokines secretion. CE dramatically reduced the secretion of IL- 1 β, IL-6 and TNF-a, demonstrating a stronger anti-inflammatory activity compared to CE.

Example 2B

Effect of cocoa CCP and CE (conventional) on Nrf2/ARE pathway in cultured macrophages

This part of the study has shown the ability of CCP to strongly induce the expression of heme oxygenase-1 (HO-1 ), a highly active protein in oxidative stress defense (Poon HF, Calabrese V, Scapagnini G, Butterfield DA (2004) J. Gerontol A Biol Sci Med Sci 59(5): 478-493), in treated cells and accordingly to protect cells from the damage caused by the exposure to free radicals. Its activation appears to represent an important defense mechanism for several cells and tissues exposed to oxidative damage in acute situations, such as ischemic accidents, or chronic conditions, such as COPD (chronic obstructive pulmonary disease) or neurodegenerative diseases, such as Alzheimer's disease. The study has also shown that the induction of HO- 1 by CCP is related to its ability to activate Nrf2. This transcription factor is capable of strongly inducing the expression of other protective genes, such as type-2 detoxifying enzymes, having high anti-oxidant and antitumor activity.

Example 2C

Up-regulation of HO-1 and phase II detoxification enzymes in different cells.

ATCC human immortalized macrophages were exposed for 6 h to different concentrations of CCP (1 , 10, 25, and 50 μΜ). Treatment resulted in a significant (p < 0.05) increase of HO-1 imRNA, measured by quantitative real-time PCR, with a maximal value at 25 μΜ. CCP elicits a dose-dependent increase of HO-1 imRNA, measured with respect to the not-inducible HO-2 paralog gene, which reaches the maximum (about 6 fold) at 25 μΜ, and decreases subsequently at 50 μΜ. CE is instead much less active in inducing HO-1 gene expression at the same concentrations, and reaches the maximum again at 25 μΜ (figure 3).

To confirm that HO-1 gene expression measured at the imRNA level corresponded to an equivalent increase of heme oxygenase activity, it was measured this activity at the same CCP and CE doses after 6 and 24 hours from the administration.

Example 2D

Heme oxygenase activity assay

Heme oxygenase activity was determined at the end of each treatment as described previously by our group (Scapagnini et al. Molecular Pharmacology 2002). Briefly, microsomes from harvested cells were added to a reaction mixture containing NADPH, glucose-6-phosphate dehydrogenase, rat liver cytosol as a source of biliverdin reductase, and the substrate hemin. The mixture was incubated in the dark at 37 °C for 1 h and the reaction was stopped by the addition of 1 ml of chloroform. After vigorous vortex and centrifugation, the extracted bilirubin in the chloroform layer was measured by the difference in absorbance between 464 and 530 nm (e = 40 m/W_1 cm_1 ).

The pattern of increase of activity was comparable to the results obtained looking at the cellular imRNAs, confirming the functional significance of the data obtained by the real time PCR determinations (figure 3).

Results highlight the ability of CCP to activate HO-1 gene expression, in a more efficient way compared to CE.

Example 2E

Activation of Nrf2 expression in different cells.

Cultured macrophages, were exposed to CCP or to CE at the final concentration of 25 μΜ to evaluate the expression of Nrf2 protein over time.

Preparation of nuclear extract and Western blot for Nrf2

Cells were washed twice with PBS 1 X. Cells were then harvested in 1 ml PBS 1 X and centrifuged at 3000 rpm for 3 min at 4°C. The cell pellet was carefully resuspended in 200 μΙ of cold buffer containing 10 mM HEPES (pH 7.9), 10 mM KCI, 0.1 mM EDTA, 0.1 mM EGTA, 1 μΜ DTT, and complete protease inhibitor cocktail (Roche, Mannheim, Germany). The pellet was then incubated on ice for 15 min to allow cells to swell. After this time, 15 μΙ of 10% NP-40 was added and the tube was vortexed for 10 s. The homogenate was then centrifuged at 3000 rpm for 3 min at 4 ^Q C and the nuclear pellet was resuspended in 30 μΙ of cold buffer consisting of 20 mM HEPES (pH 7.9), 0.4 M NaCI, 1 mM EDTA, 1 mM EGTA, 1 μ/W DTT, and protease inhibitors. The pellet was then incubated on ice for 15 min and vortexed for 10-15 s every 2 min. The nuclear extract was finally centrifuged at 13,000 rpm for 5 min at 4 ^Q C. The supernatant containing the nuclear proteins was resolved by SDS-polyacrylamide gel and submitted to immunoblot analysis using anti-Nrf2 (1 :500 dilution) and anti-Sp1 (1 :500 dilution) antibodies

As shown in figure 4, treatment with CCP caused a significant time-dependent increase in Nrf2 protein expression in the nuclear extracts. Quantification of three independent western blots showed that after 1 h exposure to 25 μΜ CCP, Nrf2 expression significantly increased and remained up-regulated till 12h, whereas the levels of the housekeeping transcription factor Sp1 were stable. CE induced a lower increment of nuclear Nrf2.

Example 2F

Nitric oxide production induced by CCP

A key modulator of endothelial cell activity is nitric oxide (NO), which under physiological conditions is mainly produced by the endothelial nitric oxide synthase (eNOS) isoform. NO regulates vascular tone, proliferation of vascular smooth muscle cells, and hemostasis, among other important functions. Disruptions in the physiological production of NO triggers endothelial cell dysfunction, resulting in an increased susceptibility to CVD. Therefore, strategies aimed at "physiologically" increasing NO bioavailability are promising for the prevention and therapy of CVD, such as of others diseases related to vascular disfunctions. To evaluate the ability of CCP to effectively induce NO production we used cultured human umbilical vein endothelial cells (HUVEC). After treatment with different concentrations of CCP we measured NO amounts produced by the cells. This step was performed by measuring the accumulation of nitrites, a stable end product of NO metabolism, in the supernatant of HUVEC. Cells were seeded into 24-well plates at a density of 5X10 ⁴ cells/well, allowed to grow to subconfluence and then incubated for 12 h in medium alone (controls) or medium added with CCP at the noted concentrations. The amount of nitrites was determined spectrophotometrically by the Griess reaction adapted for a 96- well plate reader (Quattrone S et al., Molecular Human Reproduction 2004), by conversion of nitrates to nitrites by a 30 min incubation at 37°C with 100 mlU/ml nitrate reductase (Sigma) and 20 pg/ml NADPH (Sigma). In brief, 100 μΙ of sample was added to 100 μΙ of Griess reagent (1 % sulphanilamide and 0.1 % N-[1 -naphtylethylendiamine in 5% phosphoric acid). The optical density at a wavelength of 546 nm was measured with a Bio Rad 550 micro plate reader. Nitrite concentrations in the supernatants were calculated by comparison with standard concentrations of NaNO2 dissolved in culture medium and expressed as nmol/mg of proteins, the latter measured spectrophotometrically by the bicinchoninic acid method. The reported values are the mean (6 SEM) of three separate experiments, each performed in duplicate. CCP added to the culture medium for 12 h caused significant increase of nitrites at all the concentration used with maximal activity at 10, 25 and 50 μΜ.

Example 3

Determination of (-)-epicatechin metabolites after ingestion of cocoa powder according to an embodiment of the invention by healthy humans

The product tested in this analysis is a Colombian cocoa sample according to the invention. Firstly, the starting product was subjected to a qualitative and quantitative analyses of its key components. The results are shown in Tables 1 and 2.

Table 1 . Content of purinic alkaloids

Sample preparation for the identification and quantification of (-)-epicatechin and its two metabolites in human plasma

The sample preparation method was adapted from Unno et al. [1 ]. Two milliliters of plasma were spiked with 100 μΙ_ of a solution containing 10 μΜ of 3'-O-Methyl-(-)- epicatechin and 10 μΜ of 4'-O-Methyl-(-)-epicatechin (internal standards) and diluted with 4 imL of 3.4% (w/v) phosphoric acid. Thereafter, samples were loaded onto solid-phase extraction (SPE) cartridges (Water, 60 mg, 3 cc) previously conditioned with 1 mL of Ν,Ν-dimethyl formamide (DMF) : methanol (7 : 3) and 0.5% (v/v) acetic acid in water.

The washing steps consisted of 5 mL of 0.5% (v/v) acetic acid in water, 2 mL of water : methanol : acetic acid (80 : 20 : 0.5), and 2 mL of 0.5% (v/v) acetic acid in acetonitrile.

For elution, cartridges were dried and eluted with the addition of 5 mL of DMF : methanol (7: 3). The eluate was collected in tubes containing 500 μί of 0.5% (v/v) acetic acid in methanol. The total volume was reduced to approximately 50 μί at low pressure. Samples were analyzed by HPLC within 24 h.

To determine the percentage of recovery of epicatechin and its metabolites plasma samples were spiked with increasing concentrations of 3'-O-Methyl-(-)- epicatechin and 4'-O-Methyl-(-)-epicatechin to generate final concentrations ranging from 10 to 1000 nM. These samples were analyzed applying the method described above.

The recoveries obtained for this series of (-)-epicatechin metabolite standards ranged from 90 to 94% (Table 3).

Table 3. Recovery of Epicatechin and its metabolites from plasma samples using SPE as above described.

Hydrolysis of human plasma

After absorption in the intestinal tract, (-)-epicatechinis rapidly metabolized into structurally related (-)-epicatechin metabolites by O-glucuronidation, O- sulfonation, O-methylation, and combinations thereof catalyzed by uridine-50- diphosphate glucuronosyl-transferases, sulfo-transferases, and catechol-O- methyltransferases, respectively. The net result of the extensive first-pass metabolism is the presence of glucuronides, sulfates, and/or methyl conjugates in the blood stream. These metabolites chemically different from the aglycone forms originally present in foods, are the compounds that reach the target organs.

Taking into account the lack of standards present commercially available, it was necessary to synthesize two compounds such as 3'-O-methyl-(-)-epicatechin, and 4'-O-methyl-(-)-epicatechin.

These compounds are directly derived from the metabolism of epicatechin and obtained by the hydrolysis of the corresponding glucuronyl or sulfate derivatives, according to the diagram illustrated in Fig 6.

In this first phase of the study, it was decided to carry out the qualitative and quantitative evaluations of two of the most important metabolites obtained from the epicatechin.

The quantification of (-)-epicatechin, 3'-O-methyl-(-)-epicatechin, and 4'-O-methyl- (-)-epicatechin in plasma was determined in 0.5 imL of plasma acidified with 50 plot 1 .2 M acetic acid and incubated for 40 min at 37 °C in the presence of 5000 IU of β-glucuronidase and at least 150 IU of arylsulfatase from H. pomatia. Thereafter, samples were put on ice, diluted with 1 .3 imL of 0.5% (v/v) acetic acid in water, and centrifuged for 15 min at 16,500 g at 4 °C. Finally, samples were loaded onto SPE cartridges following the same protocol as for the determination of individual metabolites.

-)-Ep catech n

Chromatographic conditions for the quantification of (-)-epicatechin metabolites (-)-Epicatechin and its related metabolites were resolved and quantified using an Agilent HPLC 1 100 series equipped with a quaternary pump, autosampler, column and sample thermostat, UV/Vis (UVD) and fluorescence detectors (FLD), and an ESA Coulochemarray detector (ECD), Model 5600A, equipped with a Cell Model 6210 with an array of four electrodes. Chromatography was based on a Phenomenex Luna C18(2) column (150x4.6 mm, 3-μιη particle size) with guard column.

The separation was performed at 40 °C. Solvent A was composed of water with 0.1 % acetic acid and solvent B of acetonitrile with 0.1 % acetic acid. Gradient conditions were as follows: time 0 min, 8% solvent B; time 0.5 min, 33% solvent B; time 5 min, 40% solvent B; time 5.1 min, 100% solvent B; time 6 min, 100% solvent B; time 6.1 min, 33% solvent B; time 8 min, 33% solvent B. The injection volume was 10 imL and the flow rate was 0.5 imL/min. The best signal and resolution in the MS system were achieved using negative-ion mode under the following conditions: capillary 2.00 kV, source temperature 150 °C, desolvation temperature 500 °C, and desolvation gas flow 1000 L/h.

The identification of (-)-epicatechin metabolites was conducted with the following m/z transition: 305→ 109 (O-Methylepicatechins), and 289→ 109 (Epicatechin). The ion spray and orifice voltages, temperature, and collision energy were set at -4200 V, -65 V, 500 °C, and -36 V, respectively. The collision gas (nitrogen) was maintained at the medium setting. Data acquisition was conducted using multiple- reaction monitoring with a 75-ms dwell time per transition.

Additional information on the polyphenolic content

In general the polyphenols contained in the cocoa based products of the invention can be divided into three groups:

1 ) catechins (about 37%)

2) anthocyanins (4%)

3) proanthocyanidins (58%)

The main catechin is the epicatechin (up to 35% of the total phenolic content), followed by catechin, gallocatechin, epigallocatechin. Anthocyanins are represented essentially by cianidin-3-a-L-arabinoside and cianidin-3-p-D- galactoside. The most represented in the cocoa procyanidins are the flavan-3,4- diols which, through links condense to form dimers, trimers or oligomers with high molecular weight.

Among glycosides flavonols, quercetin is found. Recently studies of HPLC confirmed the presence of luteolin, apigenin, naringenin and their glucosides in cocoa products. Cocoa also contains hydroxybenzoic and hydroxycinnamic acids, whose concentrations are influenced by roasting.

During fermentation according to conventional processes, polyphenols diffuse pigmented cells and oxidize, giving rise to compounds with high molecular weight, largely insoluble. The hydrolysable tannins are essentially made gallotannins.The processing technology of cocoa during the production of chocolate (roasting, grinding, refining, conching, ...) affect decreasing the polyphenol content.

Example 4

Clinical study

Participants

We screened two healthy male volunteers of 28 and 33 years of age that gave their written consent and participated in the study. They showed a body mass index higher than 30 kg/m2, without cardiovascular, renal, or liver disease, or gastrointestinal disorders; smoking within the 3 years before the initialization of the study; current consumption of herbal, antioxidant, or vitamin supplements. To reduce the contribution of flavanols from the diet, the two volunteers were instructed on how to follow a low-flavanol diet on the day before and during the study days (Flavanols and procyanidins are present in a variety of foods and beverages, but the main sources in the context of a European diet are cocoa and chocolate products, tea, grape wine, apples, certain berries, and a couple of other fruit and vegetables). They received a list of suggested foods containing low or negligible amounts of flavanols. Volunteers were asked to restrain from consuming alcohol, coffee, or other caffeine-containing beverages on the day before and during the study visits. Volunteers were asked to fast for 12 h before each study day (water ad libitum).

Study design

Upon arrival, volunteers received 4 g/kg bw drinking water to control for the level of hydration. One hour later, volunteers were given the two cocoa pills (amount 1 g)-

Blood samples were collected using EDTA-containing Vacutainers (?) at 0 h (before intake) and 1 , 2, and 4 h after the ingestion. Immediately after collection, the blood samples were placed on ice and centrifuged for 10 min at 3000 g at 4 °C. After centrifugation, the plasma was separated into aliquots, spiked with ascorbic acid (final concentration: 1 img/mL), and stored at -80 °C until analysis. Calibration curves

Two calibration standards were prepared by adding appropriate amounts of standards stock solutions to plasma. Blank plasma was collected and pooled from healthy volunteers who followed a diet free of polyphenols for 2 days.

Pharmacokinetic analysis of (-)-epicatechin metabolites

A volume of 3 mL of blood was taken to the two volunteers 1 , 2 3, and 4 hours after the administration of one gram of cocoa, under the conditions previously described.

The samples were treated according to the protocols previously described and subjected to HPLC analysis. The analysis of plasma samples identified (-)- epicatechin, 3'-O-methyl-(-)-epicatechin, and 4'-O-methyl-(-)-epicatechin such as some of the major constituents of the metabolome of (-)-epicatechin in humans (Figure 1 ).

As evidenced in figure 2, the (-)-epicatechin reached a maximal plasma level of 578 ± 61 nM at 2 h after consumption.

In addition, as evidenced in Figure 3 the use of hydrolysis permitted the identification and quantification of 3'- and 4'-O-methyl-(-)-epicatechin. In this case, the maximal level of 3'-O-methyl-(-)-epicatechin were 389 ± 36 nM always at 2 h after consumption, and as shown in Fig. 4, the 4'-O-methyl-(-)-epicatechin presented plasma levels of 125 ± 15 nM (maximal level after 2 h).

Experimental

Acetonitrile, dioxane, n-hexane, methanol, methyl-t-butyl ether HPLC plus grade were from Sigma-Aldrich. Water was treated in a milliQ water purification system (TGI Pure Water Systems, USA). All other chemicals and solvents were of analytical grade and purchased from common sources.

Statistical analysis

Experiments were executed in five replicates and the results were expressed as the average ± standard deviation (Microsoft Excel 2002 Analysis Toolpak). The statistical analysis was carried out with Student's t-test (Microsoft Excel). Correlation analyses were made by using the regression analysis module of software Microsoft Excel.

Example 5 Process for the preparation of cocoa powder having a polyphenolic high content

A preliminary step included harvesting of cocoa pod from blend of cocoa clones of Colombian cocoa plants, gathering of the pods in the field and bean extraction from the pods.

The wet cocoa beans obtained from the preliminary step were unfermented beans having a predominant colouration slaty-grey with some beans having a colour prurple violet. The degree of fermentation of the beans was in the range of 1 10 to 130 calculated by applying the cut test in accordance with ISO cut test No. 1 1 14.

The wet beans were subjected to a draining step having a duration of around 15 hours.

From 6 cocoa pods of around 650g each one, where obtained around 1 kg of fresh cocoa bean plus pulp containing 0,12 to 0,16 kg of leachates and 0,84 to 086 kg of fresh cocoa beans with a moisture content of around 60% which after draining gave from 0,50 to 0,516 kg of partially dried cocoa beans.

After draining the cocoa beans were subjected to the drying step under sunlight for reducing the water content.

During drying step almost 50% of the weight is lost. 100 kg of drained cocoa beans with a moisture content of 57% after exposure to sun light for six days lead to 49,5 kg of beans having a mosture content of 7,5%. The beans were violet beans around 73% and slaty beans around 21 %, the remaining having a brown purple colour.

The dried cocoa beans were then transported and stored in a factory for further processing.

Before processing, the cocoa bean were cleaned to remove the impurities or extraneous material such as dust particles, metals and nonmetallic byproducts, rocks, stem, cluster bean and other remaining part of the tree. The average weight of the impurity was around 1 % of the starting weight of beans. In the cleaning step were used densimetric table, metal detector, and duty extractors. After cleaning the dried cocoa beans underwent to a heating treatment by infrared Barth. The aim of this IR treatment was to make easy the take off of coat seed or shell. During IR treatment the temperature and time of heating is lower than traditional roasting step thus reducing the damages to the polyphenolic components.

100 kg of dried cleaned cocoa beans having a moisture content of 7.5% and a cnontent of cocoa butter of 52% by weight were subjected to IR treatment at a temperature of 130 °C for 3 minutes. At the end of the IR heating were obtained 96.5 kg of dried cocoa beans having a moisture content of 3.5% and a content of cocoa butter of 51 .9% by weight. The content of polyphenols (g/kg) was 40 catechins equivalent.

After the IR heating the dried beans were subjected to a winnowing treatment to separate the shell from nibs. The cocoa beans are cracked and carry on a vibrating screening of a sieving apparatus and the shell is removed by air action.

The sieve of the apparatus had different holes: (i) 8,5 mm, the biggest; (ii) 6,0 mm flow 44%; 4,0 mm flow 30,6%; (iv) 2,2 mm flow 18,1 %; (v) 1 ,0 mm flow 4,9 %. The difference in holes and flow allow to make a classification of nib by size. The range admitted is 0, 3 and 0, 6 % nibs in shells, and 1 ,5 - 1 ,8 % of shell in nibs. In the fine cocoa least 1 mm, they are approximate 1 ,8%.

In the winnowing step 100 kg of dried cocoa beans having a moisture content of 3.5% and a content of cocoa butter of 51 .9% by weight on winnowing gave 88,5 kg of nibs having a moisture content of 3.5% and a content of cocoa butter of 51 .9%.The content of polyphenols (g/kg) was 40 catechins equivalent.

After the winnowing step the nibs were subjected to a milling step. After the winnowing process, the nibs ride to the pre-mills where they are ground. The mills was an apparatus provided with a blade, and the nib were passed through sieves and over strong magnets to remove. In this step the nibs are liquefied and became cocoa liquor, for the temperature and fat content (52%).

In the milling step 100 kg of dried cocoa nibs having a moisture content of 3.5% and a content of cocoa butter of 51 .9% by weight gave 99,5 kg of cocoa liquor having a moisture content of 1 .5% and a content of cocoa butter of 51 .9%.

The content of polyphenols (g/kg) was 40 catechins equivalent.

After the milling process, the cocoa liquor was store in a big tank for preconditioning at a temperature of 100 -1 10 °C. Aftertaht the mass was pumped to a small tank with a capacity 180-200kg, and the temperature should be1 10 °C. This liquor went to the press. The press pressure was 1 1 ,000 psi with a cycle time between 8 and 12 min ( depending of the fat content). At the end of the press step, the machine was opened and the final products were:

- Cake, solid part, it was crushed, passes to milling and finally the cake produce a high polyphenols cocoa powder;

- The cocoa butter, liquid part, it passed a purification step where it was filtered and crystalized.

On entry in the press step 100 kg of cocoa liquor had a moisture content of 1 .5% and a content of cocoa butter of 51 .9% by weight the pH was 5.4. After press were obtained:

-a cocoa powder 68 kg with a moisture of 1 ,9%, cocoa butter 20,6 and a polyphenol content g/kg of 68.9 catechins equivalent, flavanol 34 g/kg catechin equivalent

-Cocoa butter 32 kg with a moisture of 0,1 %, free fatty acid 1 ,5% a peroxide Index max 1 meq/kg.

Example 6

Clinical study

A clinical study was carried out on 20 healthy volunteers recruited from the Inter- University Consortium "SannioTech", Benevento, Italy

The health status of the participants was assessed and they were selected according to the following inclusion criteria: inclusive age 25-50 years, good general health, and body mass index between 22 and 30 kg/m ² .

The main data are summarized in the following Table 1

Table 1 shows the baseline characteristics of the participants. None of the subjects had a history of chronic diseases. Exclusion criteria included individuals who were taking anti-inflammatory drugs, cardiovascular medications, lipid-altering drugs, and hormone replacement therapy. We also excluded individuals engaged in vigorous exercise, vegetarians, and people who routinely took multivitamins or herbal supplements. From the initial 20 subjects enrolled in the study, 18 met the inclusion criteria, and were divided into 3 groups of 6: the first group took 2 tablets (500 mg) of cocoa/day (1 grams of cocoa/day), the second group took four pills/day (2 grams in total) and the last group took 8 pills/day (4 grams total). The samples were collected at baseline, at 2 hours after the first pills ingestion, at 2 weeks and at the end of the intervention period (figure 1 ). The participants were instructed to maintain their usual dietary intakes during the trial, and to avoid high polyphenols food starting from 3 days before the beginning of the treatment. All subjects declared that they did not change their diets during the course of the study and they attended our medical facility every week to assess clinical conditions and adherence to the protocol. Table 2

Polyphenol-free

Daily treatment with CHFC

diet

2h 15 days 15 days

3 days TO Tl T2 T3

Blood samples were collected in EDTA-containing vials that were immediately stored at -80 C until analysis. Triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDLc), and low-density lipoprotein cholesterol (LDLc) were measured using colorimetric enzymatic tests (Thermo Scientific, Waltham, MA).

The plasma concentration of oxidized LDL (OxLDL) was measured by a sandwich enzyme-linked immuno-sorbent assay method using a commercially available kit (Immundiagnostik AG, Bensheim, Germany) described previously by Licastro et al.

In summary, compared with baseline, 8 pills/day of cocoa produced a statistically significant reduction in total cholesterol, LDL, HDL and oxidized LDL.