The present invention relates generally to the field of food supplements and/or food products for cosmetic purpose. More specifically, the present invention aims to provide an ingredient containing a mixture of roasted and green coffee beans for preventing and/or treating hyper- pigmentation of skin, skin color imperfections such as age-spots and other skin disorders characterized by abnormal pigments. The present invention also aims at improving skin tone as well as providing a skin lightening or whitening agent. Background of the invention

Skin color is primarily determined by the amount and type of melanin, a brown pigment present in the skin. Lower amounts of melanin result in lighter skin color while higher amounts result in darker skin color. Also, hyper-pigmentation in the skin is caused by the over expression or accumulation of melanin in the skin. As a result, the pathway involved in melanin production has been the target for many inhibitors so as to reduce the levels produced. One of the principal enzymes involved in the melanin pathway is tyrosinase.

The synthesis of melanin is a process under hormonal control, including the melanocyte stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) peptides that are produced from the precursor proopiomelanocortin. It is stimulated by the DNA damages that are caused by UVB-radiations as well.

Then, exposure to the sun over time can induce many biochemical reactions in the skin, leading to sunburn and tanning, for example. Other consequences of exposure to the sun accumulate over time. These changes can result in the development of age spots and create an uneven, mottled skin tone. Unfortunately, many of the commercially available products in today's market are either only marginally effective, or contain active agents that are unstable and lose their potency when incorporated into a final formula.

The ability to modify the expression of pigment content in the skin, to promote an evenness skin tone or lightening skin tone, is highly desired in today's society. Many people desire to modify their skin tone, to reduce aging spots, etc., or for purely cosmetic reasons.

As a result, efforts to develop effective compositions have focused on agents that inhibit the activity of tyrosinase. For example, a variety of tyrosinase inhibitors, such as hydroquinone, vitamin C, cystein, kojic acid, arbutin and glutathione among others have been proposed in topical compositions. Also, a variety of dermatological compositions have been suggested for improving the appearance of pigment disorders such as that observed in melasma, freckles, vitiligo, piebaldism, phenylketonuria, and the like, and/or for cosmetic purposes.

Also, the use of skin bleaching compositions is widely expanded. However, they either destroy melanin or inhibit its formation. Many of these contain harsh chemicals such as peroxides, acids or formaldehyde, or thiolated materials. Less stringent therapies have other disadvantages.

Topical retinoid and topical corticosteroids have been suggested as hypo-pigmenting agents, as have laser treatment and chemical peels, but these fall short of desirable responses. Other compositions suggested the use on the skin of natural materials, which have in some cases been used for centuries in Asia or Europe to bleach skin and skin areas, or enhance the appearance of fair skin. These include the use of lemon, orange, cucumber, ginkgo, carob, rose fruit, geranium herb, cinnamon, sweet marjoram, rosemary, etc... In order to combat disorders related to abnormal pigment or to lighten skin tone, various compounds which, when applied topically to the skin, are capable of reducing tyrosinase activity and consequently limiting melanin production, have thus been proposed. Unfortunately, the treatments currently available are not entirely satisfactory, in particular in terms of the side effects which are frequently associated therewith, such as irritant side effects with certain topical agents.

It would thus be highly desirable to have alternative preparations that do not have the drawbacks of those described in the prior art. In particular, it would be highly desirable to develop nutritional cosmetic compositions to be administered via oral route that have improved stability and efficacy to promote an evenness skin tone or to lighten skin tone.

There also remains a need to active agents that are effective for treating and/or preventing skin pigmentation disorders, in particular those due to environmental factors or aging. The object of the present invention is to meet these needs. Summary of the invention

The present inventors could achieve this object by providing a food or food supplement composition that comprises at least an ingredient containing a mixture of roasted and green coffee beans.

Thus, according to a first subject, the invention relates to the cosmetic use of an effective amount of at least an ingredient containing roasted and green coffee portions as an active agent for treating and/or preventing skin pigmentation disorders. Such skin disorder are in particular those due to age or to environmental factors (e.g. UV), such as age-spots. It may also be skin disorders that are observed in melasma, freckles, vitiligo, piebaldism, phenylketonuria, and the like.

The present inventors have discovered that mixture of unroasted and roasted coffee beans effectively suppress the formation of melanin, melanogenesis, despite the fact that the extracts show little to no inhibition of tyrosinase activity. This result is surprising and unexpected considering the pivotal role of tyrosinase in melanogenesis and the focus of development efforts in the art to inhibit this enzyme. For the purpose of the present invention, the term "skin" is intended to mean the skin of the face or of the body.

For the purpose of the present invention the term "effective amount", is intended to mean an amount sufficient to obtain the expected effect.

For the purpose of the present invention the term "prevent" is intended to mean the fact of reducing the risk of occurrence of the manifestation of the disorder under consideration.

The present invention is also directed towards the cosmetic use of the abovementioned ingredient, as an active agent for treating and/or preventing the skin pigment imperfections. As a result, the complexion becomes brighter and more homogeneous, without areas of dyschromia, or of dryness.

The present invention is also directed towards the cosmetic use of an effective amount of at least an ingredient containing unroasted and roasted coffee portions according to the invention, as an active agent for whitening or lightening skin tone. The present inventors have also discovered that the ingredient according to the invention further improves hydration and/ or skin barrier function. A use in accordance with the present invention may also comprises the use of at least an ingredient containing green and roasted coffee portions, in combination with an effective amount of at least one active agent for further improving skin hydration or skin ageing, in particular as described hereinafter. According to another of its aspects, the subject of the invention is a method, in particular a cosmetic method, for treating and/or preventing skin tone imperfections and the disorders associated with hyper-pigmentation, in particular aesthetic disorders, in an individual, comprising at least one step of administering, to said individual, at least an ingredient containing unroasted and roasted coffee portions according to the invention.

Compositions according to the present invention are orally administrable. This has the advantage of acting globally on the entire skin, in its deep layers (dermis, hypodermis), by means of a rapid and relatively non-restrictive mode of administration. Specifically, the metabolites and other active nutriments are in particular distributed within the dermal matrix by means of the bloodstream. Oral administration also has the advantage of a rapid and relatively non-restrictive mode of administration.

Detailed description of the invention According to a first object, the present invention provides the use of a coffee product which depends upon the blending of coffee beans, a portion of which are roasted and portion of which are unroasted or green.

In the text which follows" coffee beans" must be understood to mean the bean obtained by the moist route (fermentation, washing) or by the dry route (drying followed by mechanical husking) starting from the coffee "cherry", after husking as described above.

"Extract" must be understood to mean all of the compounds obtained starting from an alcoholic or aqueous-alcoholic extraction of a crude product, in this instance decaffeinated coffee beans, roasted or unroasted. The species of coffee trees selected for the preparation of the extracts of coffee beans used in the compositions are advantageously selected from the Coffea species.

Coffee trees are small trees with smooth-margined, perennial, coriaceous, glossy leaves (10- 15 x 4-6 cm). The white, fragrant flowers are grouped in whorls at the axil of the leaves. The fruit is a green drupe, which becomes red at maturity and usually contains two planar-convex berries which are made contiguous through their planar face. Although only two species supply the essential needs of the coffee market (C. arabica and C. canephora), many species of coffee trees exist in the wild state in the tropical forests of East Africa. The coffee "bean" is obtained by the moist route (fermentation, washing) or the dry route (drying, followed by mechanical decortication) starting from the coffee"cherry", i.e. from the drupes. The reduction to pulp removes the red pericarp and the fleshy mesocarp; it leads to the coffee "husk". It is after husking (removal of the lignified endocarp) that the coffee "berry" (or bean) is obtained.

In a particular embodiment, the green coffee portion may be a blend of green Arabica and green Robusta beans. However, it is particularly preferred that the green coffee portion is substantially or wholly derived from Robusta coffee beans. Thus, preferably at least 65%, more preferably 75%, even more preferably 85%, most preferably 95%, e.g. 100% by weight of the green coffee beans are Robusta.

The roasted coffee portion may be a blend of Arabica and Robusta, although it is particularly preferred that the roasted portion is substantially or wholly derived from Arabica beans. This is because Arabica provides a richer taste and aroma profile, which is associated with coffee of higher quality.

The roasted coffee may have different roasting levels form light to dark, preferably CTN from 60 to 100.

The green coffee portion and/or the roasted coffee portion may be at least partially decaffeinated. This is to provide products the caffeine level of which can be modulated from caffeine-free to regular caffeine level. However, it is particularly preferred that the green coffee portion is from decaffeinated coffee.

In particular, a coffee bean extract can be obtained by an aqueous- alcoholic or alcoholic extraction of coffee beans, and preferably by an extraction with the aid of methanol, ethanol or propanol. Preferably, it does not contain the fractions of coffee beans extractable by non- polar solvents. Methods for the preparation of decaffeinated coffee extracts are well known in the art.

In one aspect of the present invention, the coffee product is derived from a mixture of green and roasted coffee beans in which the weight ratio of the green portion and roasted portion in the coffee product is from 1 :99 to 90:10, preferably from 30:70 to 50:50, more preferably from 35:65 to 45:55, most preferably from 37:63 to 42:58. Such coffee product is described in EP1871173 which is included by reference.

In particular, the coffee product preferably comprises:

(i) a first portion consisting of unroasted ground and/or unground coffee, in an amount of from 1 to 90 % by wei g ht based o n the total wei g ht of the coffee prod uct, and (ii) a second portion consisting of ground coffee which has been roasted to a higher degree of roast than said first portion, in an amount of from 99 to 10% by weight based on the total weight of the coffee product,

wherein the chlorogenic acids are present in an amount of at least 2g per 100g of the coffee product, more preferably at least 4g, even more preferably at least 6g, most preferably at least 8g, e.g. at least 9g.

The compositions according to the invention may be in any of the galenical forms normally available for the method of administration selected. The carrier may be of diverse nature depending on the type of composition under consideration.

The composition may be any food or pharmaceutical product, or a cosmetic product for oral application. Examples for food or pharmaceutical carriers nutritional complete formula, dairy product such as milk, yogurt, cheese, fermented milks, milk-based fermented products, icecreams, cereal-based products or fermented cereal-based products, milk-based powders, infant and baby formulas, chilled or shelf stable beverages, food products of confectionary, chocolate or cereal type, animal feed, in particular for domestic animals. In particular, the chicory or extract thereof may be incorporated into any other form of food supplement or enriched food , for example food bars or compacted or non-compacted powders. The powders may be diluted in water, soda, milk products or soya bean derivatives, or be incorporated into food bars. The food product may also further comprise a protein source, a carbohydrate source, a lipid source, a mineral source and/or a vitamin source. The presence of proteins, carbohydrates, lipids, minerals and/or vitamins may have several advantages. These compounds generally contribute to the taste and mouthfeel of the final product. They also provide the body with nutrients that it may need urgently when it is affected by skin disorders. They also allow formulating the product of the present invention as a complete nutritional formula, so that no additional nutrition is needed.

The individual ingredients may comprise any kind of edible compound. Typical ingredients for food compositions, in particular drinks, are well known in the art, e.g. milk, cream, coffee whiteners, and coffee creamers. Alternatively, ingredients could also be a salad dressing or parts thereof, for example. Such compounds are used by consumers to modify e.g. the aroma, appearance and texture of a composition. The ingredients may be in liquid or dry form, e.g. as powders, that are dissolved and/or suspended in a drink.

The invention also provides a coffee beverage product, such as (a) a roast and ground coffee product, (b) a soluble coffee product, (c) a single portion coffee pad, tablet or capsule (d) a ready to drink coffee product or (e) any other suitable coffee product, comprising the coffee product as defined herein as an ingredient. Soluble coffee or instant coffee as it is otherwise known, is very well known and has been produced commercially for many decades. It is produced from roast and ground coffee in a vari ety of ways , al l of wh i ch a re we l l kn own to th e pe rso n s ki l l ed i n th e a rt .

The present invention is particularly suitable for use as a soluble coffee product or any product, which is based on or derived from a soluble coffee product. For instance, the coffee product may also be used in so-called ready-to-drink beverages. Examples of ready-to-drink beverages for which the product of the present invention are suited include two-in-one beverages which comprise a coffee component together with a natural or artificial sweetener component, the components optionally being pre-diluted with a liquid such as water or milk. In this case, the coffee component can comprise the coffee product of the present invention.

Three-in-one beverages, which comprise coffee, a sweetener and a whitener such as milk, a liquid creamer or a solid (e.g. powdered) creamer, may comprise the coffee product of the present invention.

Furthermore, any beverage, which comprises soluble coffee as an ingredient, may comprise the coffee product of the present invention.

The composition may further comprise further ingredient suitable for inclusion in a food composition. Usual ingredients may e.g. be sugars, artificial sweeteners, emulsifiers, stabilizers, thickeners, flowing agents, colors, flavors, aromas, and the like. Suitable artificial sweeteners include saccharin, cyclamates, acetosulfame, L-aspartyl based sweeteners such as aspartame, and mixtures of these. Suitable emulsifiers include monoglycerides, diglycerides, lecithin, diacetyl tartaric acid esters of mono-diglycerides, emulsifying starches, and mixtures thereof. Suitable stabilisers include dipotassium phosphate and sodium citrate. A suitable flowing agent is sodium silica aluminate. In one embodiment the composition comprises milk protein and/or vegetable protein. In a further embodiment the composition comprises milk fat and/or vegetable fat.

Additionally, the present invention can be used as an ingredient for standard brew, espresso, coffee-based beverage preparations to be used as bulk or in single beverage portion for in- home preparation.

If the product is a nutritional supplement for oral administration it may be present in capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes or pastilles, gums, or drinkable solutions or emulsions, a syrup or a gel. Such a supplement might also include a sweetener, a stabilizer, an antioxidant, an additive, a flavoring agent and/or a colorant. The formulation thereof is carried out by means of the usual methods for producing sugar-coated tablets, gel capsules, gels, hydrogels for controlled release, emulsions, tablets or capsules. In this respect the adjuvant for oral compositions, in particular for dietary supplements, are known to the specialist. Mention may be made, among others and for purely illustrative purposes , of lubricants such as magnesi um stearate, products for instantaneous solubilisation, gelling agents, thickeners, moisturizers, fatty and/or aqueous compounds, preservatives, texturizing, flavoring and/or coating agents, anti-oxidants and coloring materials usually used in foods.

The composition according to the present invention may contain, in addition, lipids, polyphenols, taurine, probiotic microorganisms, vitamins and/or oligo-elements. If probiotics are used, they may be included in a live form, semi-active or in a desactivated form, e. g., as a lyophilized powder. Also culture supernatants of the micro-organisms may be included in the composition. They may be selected from the group consisting of Lactic acid bacteria, in particular Lactobacilli and/or Bifidobacteria and are more preferably selected among the group consisting of Lactobacillus johnsonii, Lactobacilus reuteri , Lactobacillus rhamnosus, Lactobacillus paracasei , Lactobaci ll us casei , Bifidobacteri um bifidu m , Bifidobacteri u m breve, Bifidobacterium animais, Bifidobacterium infantis, Bifidobacterium dolescentis and Bifidobacterium pseudocatenulatum. According to a most preferred embodiment the strains used are Lactobacillus johnsonii (La1) deposited on June 30, 1992, under Budapest Treaty with the Institute Pasteur and receiving the deposit no. CNCM 1-1225 or Lactobacillus paracasei (ST1 1) deposited on January 12, 1999, with the Institute Pasteur according to the Budapest Treaty and receiving the deposit no. CNCM I- 2116. The following compounds may for example be used alone or in combination: zinc and its salts including zinc sulfate and zinc glucanate, the vitamins B5, B6, B8, C, E or PP, p-carotene and the carotenoids, extracts of garl ic in particular in the form of allyl sulfide or oi l garlic, selenium , curcumi n, the curcuminoids, niacin, lithospermic acid and adenosine. It is understood that the specialist will select such active compounds and, where possible, combine them in such a manner as to improve the effects expected of the composition which is the object of the invention by preventing the desired activity of interest from being inhibited or attenuated. Use

The products of the invention may be efficiently used for treating or preventing skin pigmentation disorders or cosmetically lightening skin tone e.g. by decreasing the production of melanin. Indeed blends of roasted and unroasted (green) coffee beans were shown to decrease in vitro the synthesis of melanin (Example 1 , Figure 1 ) . The production of tyrosinase was also decreased but to a lim ited extent (Figure 2), suggesting that the decrease in melanin was not due to tyrosinase inhibition but rather to mechanisms acting upstream or downstream of this enzyme. The ingredients according to the present invention have further a positive effect on strengthening skin barrier and maintaining skin hydration.

As a result, the pigment imperfections are reduced, the complexion becomes brighter and more homogeneous, without areas of dyschromia, or of dryness.

Thus, according to one subject, the invention relates to the cosmetic use of an effective amount of at least one ingredient containing roasted and unroasted coffee beans as an active agent for treating and/or preventing skin pigmentation disorders, in particular those due to age or environmental factors such as UV. The present invention is also directed towards the cosmetic use of an effective amount of at least one ingredient containing roasted and unroasted coffee beans as an active agent for whitening or lightening skin tone, which is particularly desirable for asian population.

A use in accordance with the present invention may also comprises the use of at least one ingredient containing roasted and unroasted coffee beans, in combination with an effective amount of at least one active agent for improving skin hydration or skin ageing, in particular as described hereinafter.

According to another of its aspects, the subject of the invention is a method, in particular a cosmetic method, for treating and/or preventing skin tone imperfections and/or disorders associated with hyper-pigmentation, in particular aesthetic disorders, in an individual , comprising at least one step of administering, to said individual, at least one ingredient containing roasted and unroasted coffee beans according to the invention. The cosmetic treatment method of the invention may be carried out in particular by orally administering at least an effective amount of at least one ingredient containing roasted and unroasted coffee beans in accordance with the invention. Oral administration comprises ingesting, in one or more intakes, an oral composition as defined above. It may comprise a single application. According to another embodiment, the application is repeated, for example, 2 to 3 times a day, for one day or more, and generally for a sustained period of at least 4, or even 1 to 15, weeks.

I n addition, combinations of treatment with, optionally, oral or topical forms may be envisaged in order to supplement or reinforce the activity of the ingredients as defined by the invention.

Thus, a topical or oral treatment with a composition containing roasted and unroasted coffee beans in accordance with the invention, combined with an oral or topical composition optionally containing another active ingredient, in particular a probiotic microorganism, or other probiotics in dead, live or semi-active form or an hydrating or anti-ageing agent could be imagined as a kit. The ingredients are mixed, before they are formulated, in the order and under conditions readily determined by those skilled in the art.

The ingredients are mixed, before they are formulated, in the order and under conditions readily determined by those skilled in the art.

The proportion of coffee bean extract in the composition will of course be determined as a function of the desired effect and on the mode of administration of the composition. The daily doses of coffee bean extract administered by the oral route may preferably be comprised between 0.01 and 5000 mg/day depending on the caffeine content. Preferentially, the coffee bean blend is present in the composition according to the invention in a quantity permitting its administration at a dose comprised between 0.5 and 1000 mg/day. The present inventors have found that the effectiveness of the coffee bean blend according to the present invention is generally dose dependant and follows a dose response curve. If generally mild skin disorders or damages are to be prevented and the product will be used frequently, very small amounts will be sufficient to achieve the desired effect. If a severe skin pigment disorder is to be treated, larger amounts will be more appropriate, although also small amounts will produce an effect.

Preferably, the composition intended for oral administration contains an extract of coffee beans in a quantity ranging from 10% to 100% by weight of the composition and preferably from 20 % to 80% by weight of the composition depending on the product form (soluble coffee, RTD, coffee mixes, food product or food supplement).

Further advantages and features of the present invention are apparent from the following Examples and Figures. The examples hereinafter are thus presented by way of non-limiting illustration of the field of the invention. In these examples, unless otherwise indicated, the percentages are percentages by weight and the ranges of values written in the form "between ... and ..." include the upper and lower limits specified.

Figures

Figure 1 : Assessment of melanin production by murine melanocytes pre-treated with coffee blends.

Figure 2: Assessment of tyrosinase production by murine melanocytes pre-treated with coffee blends. Figure 3: Assessment of filaggrin synthesis by human primary epidermal keratinocytes pre- treated with coffee blends.

Figure 4: Assessment of hyaluronic acid secretion by human primary epidermal keratinocytes pre-treated with coffee blends.

EXEMPLES Exemple 1 : Effect on skin pigmentation

In order to evaluate the potential beneficial effect of ingredients towards skin de- or pro- pigmentation we used 2D culture of murine melanocytes (B16) and we performed 2 tests: 1- assessment of melanin production and 2- assessment of tyrosinase production. 1. The cell culture conditions.

B16 cells were cultured in DMEM 1 g/L glucose without phenol red supplemented with 10 % foetal calf serum, in a humidified chamber at 37 °C and containing 5% C02. 2. The production of melanin by B16 murine melanocyte cell line.

Cells were incubated with the selected ingredients or the test references (Kojic acid at 40C^g/ml_) for 72 hours, in the presence or absence of NDP-MSH an analog of MSH. The total quantity of melanin (extracellular and intracellular) was evaluated by measurement of the optical density at 405 nm of each sample against melanin standards in presence or in absence of NDP-MSH.

3. The production of tyrosinase by B16 murine melanocyte cell line. Cells were incubated with the selected ingredients or the test references (Kojic acid at 400μg/mL) for 48 hours. The production of tyrosinase was evaluated by immunolabeling.

INGREDIENTS: We have tested coffee blends including PROTECT® extract (Nestle Green blend) as well as other blends e.g Stevol (provided by Naturex) and Oryza's Extract-P. The tested concentrations for each coffee extract is indicated in Table 1. Highest Highest

Tested

non non Tested

cone.

cytotoxic cytotoxic cone, on

Ingredient on

cone, on cone, on HDF

HPEK

HDF HPEK (mg/mL)

(mg/mL)

(mg/mL) (mg/mL)

Svetol 0.016 0.04 0.04 0.04

Oryza 0.04 1 0.04 0.04

Robusta light roast, brew 0.04 0.2 0.04 0.04

Robusta medium roast brew 0.04 0.2 0.04 0.04

Robusta dark roast brew 0.04 0.2 0.04 0.04

PROTECT ® coffee 0.1 0.1 0.1 0.1

Green coffee leaves 0.004 0.004 0.004 0.004

Table 1

We have worked with the highest non cytotoxic concentration, with respect to normalize as much as possible the conditions in between the different extracts to further be able to compare them. Thus, coffee extracts have been tested at a concentration of 40μg/mL.

Results Results are expressed in percentage relative to the control. Test reference (Kojic acid) induced, as expected, a decrease in melanin content. Figure 1 shows the melanin production by B16 melanocytes treated for 72 hours with the selected ingredients.

We have observed overall that coffee extracts had some potential for skin de-pigmentation as they were able to decrease the production of melanin and tyrosinase in vitro compared to control conditions (Figures 1 and 2). Some extracts including Protect and roasted coffee brews, decreased the melanin content without affecting tyrosinase production, suggesting mechanisms acting upstream or downstream tyrosinase.

Exemple 2: Effect on skin barrier function and hydration The potential beneficial effect of the Extracts of Example 1 towards skin barrier function and skin hydration was evaluated by using 2D culture of human primary epidermal keratinocytes and we performed 2 tests: 1- assessment of the synthesis of filaggrin and 2- assessment of the secretion of hyaluronic acid by human primary epidermal keratinocytes.

The cell culture conditions.

Human epidermal keratinocytes were cultured in control keratinocytes-SFM medium, in a humidified chamber at 37 °C and containing 5% C02. The synthesis of filaggrin by human epidermal keratinocytes.

Cells were incubated with the selected ingredients or the test references (CaCI2 at 1.5mM) for 144 hours. The production of filaggrin by was evaluated by immunolabeling.

The secretion of hyaluronic acid by human epidermal keratinocytes.

Cells were incubated with the selected ingredients or the test references (retinoic acid at 10- 7M) for 72 hours. The release of hyaluronic acid by human epidermal keratinocytes in supernatants was quantify by ELISA assay.

Results

The results are showed in Figure 3 and 4. Pre-treatment of the cells with coffee blends resulted in an increase of filaggrin of nearly 200%, suggesting that these extracts could strengthen skin barrier (Figure 3). Protect and light roasted coffee brew also increased the secretion of hyaluronic acid (Figure 4), glycosaminglycan secreted by skin cells in vivo which help maintain a proper hydration by attracting and trapping water into the extracellular spaces. A stronger skin barrier ensures a better protection of the body from the environment and pathogens' attack. It also limits the loss of water through the epidermis, thus ensuring an appropriate skin hydration.

Example 3

Preparation of a roasted extract of Coffee robusta 0.5 kg of roasted coffee beans is reduced to a powder by grinding with the Turrax apparatus at 24000 rev/min for 1 minute at 4°C (ice bath). The powder obtained is mixed with 5 litres of 0.05M phosphate buffer at pH 8.5. The entire mixture is stirred for 30 minutes at 4 C, then centrifuged at 10 000 G at 4°C. The supernatant is filtered through a 0.22, urn filter (sterilizing filtration). The extract is then fractionated by ultrafiltration through a Sartorius type membrane in order to remove from it oxidation phenomena. The extract is then lyophilized. 29.5 grams of active extract called "lyophilized extract" are thus obtained.

Caffeine is then removed by supercritical chromatography (C02 is used as carrier gas). 25.5 grams of active extract called "decaffeinated lyophilized extract" are thus obtained.

Example 4: Powder stick

One stick can be taken per day.