Wolfberry - commercially called Goji berry - is the common name for the fruit of two very closely related species: Lycium barbarum and Lycium chinense, two species of boxthorn. Since Lyci m barbarum L. is the only species approved for use in China the present invention is especially directed to the fruits of Lycium barbarum L., the "Goji berries".

Goji berries have the highest content of zeaxanthin of all known food sources. The berries have been cultivated in China for decades and are used as food and in Traditional Chinese Medicine. The content of carotenoids in Goji berries can range from 600 to 900 ppm. The main carotenoid is zeaxanthin in its natural ester form.

The inventors of the present invention have now succeeded in developing a process for the extraction of the dried Goji berries with a lipophilic solvent and obtained in high yield an oily oleoresin, rich in zeaxanthin (2 - 11 weight-% content). Further purification of the oleoresin could be achieved by crystallization from a solvent such as ethanol yielding an orange-red powder with a zeaxanthin dipalmitate content of 15 - 60 weight-% corresponding to a zeaxanthin content of 9 - 34 weight-%. In addition the zeaxanthin dipalmitate containing oleoresin could be treated to cleave the esters to give a powder with a zeaxanthin content of 30-95 weight % and in 30 - 95% yield.

Therefore, the present invention is directed to a process for the manufacture of an extract of Goji berries containing zeaxanthin and/or its esters, comprising the following steps:

a) obtaining dried Goji berries or size reduced pieces thereof; and

b) treating the dried Goji berries or the size reduced pieces thereof with an alkali solution; and

c) extracting the Goji berries or the size reduced pieces thereof obtained in step b) with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether, acetone, butane, ethyl methyl ketone, methanol, propan-2-ol and mixtures thereof; and d) removing the solvent from the extracts obtained in step c); and

e) optionally cleaving the esters in the dried extracts obtained in step d); and

f) optionally crystallizing the dried extracts obtained in step d) or e) in an aliphatic straight or branched C alcohol. Any person skilled in the art should understand, steps e) and f) are optional, so they may be performed or not in the process according to the present invention. Preferably the process according to the present invention consists only of these steps a) to f) and does not comprise any additional steps.

One preferred embodiment according to the present invention is directed to a process for the manufacture of an extract of Goji berries containing zeaxanthin (di)esters comprising the following steps:

a) obtaining dried Goji berries or size reduced pieces thereof; and

b) treating the dried Goji berries or the size reduced pieces thereof with an alkali solution; and

c) extracting the Goji berries or the size reduced pieces thereof obtained in step b) with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether, acetone, butane, ethyl methyl ketone, methanol, propan-2-ol and mixtures thereof; and d) removing the solvent from the extracts obtained in step c); and

f) optionally crystallizing the dried extracts obtained in step d) in an aliphatic straight or branched Ci^ alcohol.

The other preferred embodiment according to the present invention is directed to a process for the manufacture of an extract of Goji berries containing zeaxanthin consisting of the following steps: a) obtaining dried Goji berries or size reduced pieces thereof; and

b) treating the dried Goji berries or the size reduced pieces thereof with an alkali solution; and

c) extracting the Goji berries or the size reduced pieces thereof obtained in step b) with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether, acetone, butane, ethyl methyl ketone, methanol, propan-2-ol and mixtures thereof; and d) removing the solvent from the extracts obtained in step c); and

e) cleaving the esters in the dried extracts obtained in step d); and

f) optionally crystallizing the dried extracts obtained in step e) in an aliphatic straight or branched Ci_4 alcohol.

Processes to obtain zeaxanthin are already known in the prior art, but have several disadvantages. CN-A 1 158 263 e.g. uses an extraction solvent which contains water, so that also the sugars contained in the berries are extracted.

The process according to CN-A 1 086 730 focuses on the preservation of the natural nutritious components and special flavour of the Goji berries.

The process of US 2007/0161826 needs a washing step with a polar solvent such as an ethanol- water or an acetone-water mixture (see page 2, paragraphs [0021] and [0024], as well as example 3). The process of US 2005/0038271 involves either an extraction step with tetrahydrofuran (see examples 2 and 3) which easily forms peroxides under the reaction conditions or a crystallization step in water (see example 4), which is much more difficult to remove than the aliphatic straight or branched C alcohol used in step e) of the process of the present invention. According to example 3 of WO 97/23436 the berries were first put in water and then dried again after having removed the sugars.

US 6,191,293 also describes a process where Chinese wolfberries are pre-extracted with water to eliminate water-soluble gums (see example 4).

The aim of the process of US 2008/0124416 (see the examples) is to obtain an extract rich in polysaccharides and in carotenoids, so that an extraction step with water is mandatory, whereas the aim of the present invention is to obtain an extract highly enriched in zeaxanthin and preferably in the form of a powder and not a paste, since powders are much more easily manageable.

A further advantage of the present invention is that the process of the present invention is cost- efficient, of high yield and can be used in industrial scale to obtain natural zeaxanthin and/or its esters, especially its dipalmitate esters, in a large amount (especially in an amount of several tons). The process of the present invention is now described in more detail. Preferably all steps a) to f) of the processes according to the present invention are performed under an inert gas atmosphere such as e.g. under nitrogen or argon or mixtures thereof.

The Goji berries can either be of the species Lyci m barbarum and/or of the species Lycium chinense, preferably they are of the species Lycium barbarum L.

The term "zeaxanthin esters" encompasses zeaxanthin monoesters, as well as zeaxanthin diesters. In Goji berries the zeaxanthin esters are found mainly in form of their dipalmitate diesters. When only steps a), b), c) and d) of the processes according to the present invention are performed, a so-called "oleoresin" is obtained. The consistence of this oleoresin is pasty-like at room- temperature, and it mainly consists of lipophilic compounds such as fats. The content of zeaxanthin in form of its esters in this oleoresin is usually in the range of 3 to 20 weight-%, based on the total weight of the oleoresin.

When only steps a), b), c), d) and f) of the processes according to the present invention are performed, an extract is obtained, which has a zeaxanthin(di)ester content in the range of 15 to 60 weight-%, based on the total weight of the extract. This extract is pulverous which makes its handling very easy.

When steps a), b), c), d) and e) of the processes according to the present invention are performed, an extract is obtained, which has a zeaxanthin content in the range of 30 to 95 weight-% in form of free zeaxanthin, based on the total weight of the extract. This extract is pulverous which makes its handling also very easy.

The extracts obtained by the processes according to the present invention can be used as food supplements e.g. for eye health. Furthermore the extracts and food supplements containing them may be used as antioxidants and/or blue light absorber. The single steps of the processes according to the present invention are now described in more detail below, Step a): Obtaining dried Goii berries or size reduced piece thereof

The dried Goji berries are usually commercially available. The commercial purchased Goji berries in dried form may be used in the present invention directly.

Optionally, the size of the dried Goji berries may be reduced. It was found out that the extraction in the step c), as described in the content below, leads to extracts with higher enrichments in zeaxanthin if the dried Goji berries have a reduced size before the extraction. The size reduction may be preformed by any method known to the person skilled in the art. Preferred methods are mechanical reduction such as crushing, smashing, chopping and milling, whereby crushing and milling and their combination are preferred.

Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step a) as listed above are combined.

Step b): Treatment with alkali solution

The dried Goji berries or the size reduced pieces thereof obtained in step a) are treated with an alkali solution in step b).

The alkali solution used in step b) may be any alkali solution with pH < 12, which is known in the art. The example of such alkali solution includes but not limited to aqueous solution of Na ₂ C0 ₃ , K2CO3, Ca(OH) ₂ , aHC0 ₃ and KHCO3, ammonia, and NH ₃ , etc. A preferred alkali solution according to the present invention is aqueous Na ₂ C0 ₃ solution, for example, 0.3 moI/L-0.5 mol/L Na ₂ C0 ₃ solution.

The alkali solution may be added in an amount of 1 to 5 liters, preferably 1 to 4 liters, more preferably 1 to 3 liters, even more preferably 1.5 to 2.5 liters, and most preferably 2 liters, per 1 kg of the dried Goji berries or the size reduced pieces thereof obtained in step a). The treatment is preformed one or more times, for example, one, two or three times. Each treatment may proceed for at least 10 minutes, preferably 20 minutes to 1 hour, most preferably 30 minutes.

After the treatment, an acid may be added into the mixture containing the dried Goji berries or the size reduced pieces thereof for neutralization. Such neutralization is not compulsory, so it may be performed or not according to the present invention. However, it was found that the acid added for the neutralization increased the zeaxanthin content and yield in the extracts obtained in step c), as described in the content below. Any person skilled in the art could understand that any acid known in the art may be used for this purpose. A preferred acid used in this step is mineral acid such as HC1. The mixture containing the dried Goji berries or the size reduced pieces thereof is preferably neutralized to pH 7-8, more preferably pH 7.5.

After the treatment, the Goji berries or the size reduced pieces thereof may be filtrated out from the mixture and then used into the next step directly or after dried.

Preferably step b) is performed at a temperature in the range of 10 °C to 30 °C, more preferably at a temperature in the range of 15 °C to 25 °C, most preferably at a room temperature.

It was found that step b) can remove more than 70% byproducts from the dried Goji berries or the size reduced pieces thereof, so the residue can be easily extracted in step c), as described in the content below, and also the treatment can make the extraction efficiency doubled and the used solvent decreased by 70% in step c).

Step c): Extraction

The Goji berries or the size reduced pieces thereof obtained from step b) are extracted in step c).

Extraction of step c) may be performed several times, preferably three times. Of course the extraction may also be performed only one or two times, but with a longer extraction time.

Usually the every extraction is carried out for 30 mins - 2 hours, preferably 45 mins - 1 hour. The most efficient time needed for the extraction may be found out easily by the person skilled in the art. Preferred extraction solvents used in step c) are a mixture of ethyl acetate and hexane in a volume ratio of 1 : 1, ethyl acetate alone and hexane alone. The most preferred extraction solvent is ethyl acetate.

Preferably the amount of solvent used in step c) is in the range of 0.3 to 2 liters, more preferably in the range of 0.3 to 1.2 liters, even more preferably in the range of 0.3 to 0.6 liters, per 1 kg of the dried Goji berries or the size reduced pieces thereof obtained in step a). Preferably step c) is performed at a temperature in the range of 20°C to 80°C, more preferably at a temperature in the range of 30°C to 70°C, even more preferably at a temperature in the range of 40°C to 70°C, most preferably at a temperature in the range of 50°C to 65°C.

If the extract obtained in step c) contains non-soluble parts, they are removed, preferably by filtration.

Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step c) as listed above are combined, Step dV Solvent removal

The solvent of the extract obtained in step c) is removed to obtain an "oleoresin" in step d).

The solvent of the extract obtained in step c) may be removed by any method known in the art, for example, by evaporation under reduced pressure.

Preferably the solvent removal is preformed at a temperature in the range of 20°C to 78°C, more preferably at a temperature in the range of 30°C to 50°C, most preferably at a temperature in the range of 40°C to 45°C.

The solvent may be removed batch wise, continuously or in a serial manner. Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step d) as listed above are combined.

Step e): Ester cleavage

In the oleoresin obtained in step d) most of the zeaxanthin present is esterified and occurs mainly as its dipalmitate ester. In order to obtain free zeaxanthin the oleoresin has to be treated with a base, such as OH and/or NaOH, to cleave the esters. If this is done with an aqueous base zeaxanthin is obtained together with fatty acid salts (saponification). If this is done with a base (for example with solid KOH and/or solid NaOH) in an aliphatic straight or branched C 1.4 alcohol zeaxanthin is obtained together with fatty acid esters (trans-esterification).

Optionally, the oleoresin obtained in step d) may experience a saponification or trans-esterification procedure to obtain free zeaxanthin.

Preferably the cleavage of the zeaxanthin esters is performed by trans-esterification. More preferably this trans-esterification is performed at a temperature in the range of 20°C to 120°C, preferably at a temperature in the range of 60°C to 100X, more preferably at a temperature in the range of 70°C to 90°C. Depending on the temperature chosen, the reaction can be performed at ambient pressure or at a higher pressure.

Most preferably the trans-esterification is performed by reaction of the zeaxanthin esters with an aliphatic straight or branched Ci- alcohol in the presence of a base. Examples of the aliphatic straight or branched Ci _-4 alcohol are methanol, ethanol, iso-propanol, n- propanol, n-butanol, iso-butanol and tert-butanol. Preferred alcohols are methanol and ethanol and any mixture thereof. The most preferred alcohol is ethanol.

Preferably the amount of said alcohol is in the range of 2 to 12 liter per kg of dried extract, more preferably in the range of 4 to 10 liter per kg of dried extract, most preferably in the range of 5 to 8 liter per kg of the oleoresin obtained in step d). Examples of bases are alkali metal hydroxides such as KOH and NaOH and any mixtures thereof. Preferably the amount of said base is in the range of stoichiometrical or catalytic amounts, such as 25-40% weight-% KOH based on the oleoresin obtained in step d). Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step e) as listed above are combined.

Step f): Crystallization Optionally, the oleoresin obtained in step d) or e) may be crystallized in an aliphatic straight or branched C1- alcohol to enrich zeaxanthin or its ester in step f .

If step e) is performed by trans-esterification, the aliphatic straight or branched alcohol used in step f) is the same as used in step e). Here the same preferences for this alcohol apply as for step e). Thus, the most preferred solvent used in step f) is ethanol.

When the crystallization is done in ethanol, preferably step f) is performed at a temperature in the range of room temperature to 78 °C, more preferably at a temperature in the range of 40 °C to 60 ^a C, most preferably at a temperature around 50 °C.

When step e) is not performed, the amount of ethanol used in step f) is preferably in the range of 1 to 10 liter, more preferably the amount of ethanol used in step f) is in the range of 2 to 7 liter, most preferably the amount of ethanol used in step f) is in the range of 3 to 5 liter, per 1 kg of the oleoresin obtained in step d).

Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step f) as listed above are combined.

Further preferred embodiments of the present invention are embodiments where one or more of the preferred embodiments of step a) and/or step b) and/or step c) and/or step d) and/or step e) and/or step f) as listed above are combined. The most preferred embodiment of the present invention is a process for the manufacture of an extract of Goji berries containing zeaxanthin, comprising the following steps:

a) obtaining dried Goji berries or size reduced pieces thereof; and

b) treating the dried Goji berries or the size reduced pieces thereof with an Na ₂ COj solution; and

c) extracting the Goji berries or the size reduced pieces thereof obtained in step b) with ethyl acetate at a temperature in the range of 40 °C to 50 °C; and

d) removing the solvent from the extracts obtained in step c); and

f) cleaving the esters in the dried extracts obtained in step d) by trans-esterification with ethanol in the presence of a base.

Another most preferred embodiment of the present invention is a process for the manufacture of an extract of Goji berries containing zeaxanthin (di)esters, comprising the following steps:

a) obtaining dried Goji berries or size reduced pieces thereof; and

b) treating the dried Goji berries or the size reduced pieces thereof with an Na ₂ C0 ₃ solution; and

c) extracting the Goji berries or the size reduced pieces thereof obtained in step b) with ethyl acetate at a temperature in the range of 40 °C to 50 °C; and

d) removing the solvent from the extracts obtained in step c); and

e) crystallizing the dried extracts obtained in step d) in ethanol.

The invention will now further be illustrated by the following non-limiting examples.

Examples

For analytical purposes, samples of all products obtained were saponified and thus the zeaxanthin esters contained therein measured as free zeaxanthin. Thus, the zeaxanthin contents given below always refer to free zeaxanthin and not to esterified zeaxanthin.

All examples were performed at an inert gas atmosphere, even if not explicitly mentioned. Example 1: Preparation of Oleoresin by treatment with NagCOVHCl and extraction with ethyl acetate

The dried Goji berry was purchased from Zhaokang Goji berry Inc. (Ningxia province, China). 100.28 g Goji berries were pulverized and pretreated with 100 mL NaiCO _. i (0.3 mol/L), stirring at room temperature for 30 min, then adjusted pH to 7.5 with 1 mol L HC1. The mixture were treated with 200 mL ethyl acetate for 3 times, stirring at 50°C for 1 h. 1.8861 g of oleoresin were obtained. The zeaxanthin content is 5.11%. The yeild is 90.50 %. Yield calculated formula is: yield= ((content x weight of product)/ (content x weight of oleoresin)) xlOO

Example 2: Preparation of Oleoresin by treatment with Na?COVHgQ and extraction with hexane

The dried Goji berry was purchased from Zhaokang Goji berry Inc. (Ningxia province, China). 101.1 g of powder of the dried Goji berries was put into a 500 ml flask, 200 ml of 0.3 M of Na ₂ C0 ₃ was added and stirred for 30 min at room temperature. The solids were filtered out by pressure filter under 1.5 bars, and extracted with 200 ml of ethyl acetate for 45 mins under stirring at 45 "C. The extraction repeated three times, and the extracts were combined. 20 g Na ₂ S0 ₄ was added to dehydrate the extracts for 1 hour and then filtered out. After removal of the solvent of the extract using rotational evaporator in vacuum, 1.2681 g of the deep red oleoresin was obtained. The yield was 39.98% and the content was 2.19%. Yield calculated formula is: yield= ((content x weight of product)/ (content x weight of oleoresin)) xlOO

Example 3: Preparation of Oleoresin by extraction with less ethyl acetate than example 1 The Goji berry was purchased from Zhaokang Gojiberry Inc. (Ningxia province, China). 100.6 g of powder of the dried Goji berries was put into a 500 ml flask. 200 ml of 0.3 M of Na ₂ C03 was added and stirred for 30 min at room temperature. 1 M of HC1 was added into the flask carefully under shaking till to the pH of mixture was adjusted to 7.5. The solids were filtered out by pressure filter under 1.5 bars, and extracted with 100 ml of ethyl acetate for 45 mins under stirring at 45 °C. The extraction repeated three times, and the extracts were combined. 20 g Na ₂ S0 was added to dehydrate the extracts for 1 hour and then filtered out. After removal of the solvent of the extract using rotational evaporator in vacuum, 0.9365 g of the deep red oleoresin was obtained. The yield was 77.51% and the content was 5.72%, Yield calculated formula is: yield= ((content x weight of product)/ (content x weight of oleoresin)) xlOO

Example 4: Purification of the oleoresin by crystallisation in ethanol to prepare a natural zeaxanthin enriched powder

1.65 g with content of 51,1 g/kg of oleoresin of example 1 was distributed into 8 ml of ethanol. The mixture was stirred at 50T for 30 min, then cool to room temperature for 2 h. The formed solid was filtered off, washed with cool ethanol (4°C, 5 ml, 3 times), dried under high vaccum. 211.7 mg of products were obtained. The content zeaxanthin is 307.4 g/Kg analyzed by HPLC. The yeild is 77.18 %. Yeild calculated formula is: yeild= ((content x weight of product)/ (content x weight of oleoresin)) xlOO.

Example 5: Trans-esterification of the Zeaxanthin Esters in the Oleoresin to free zeaxanthin crystals

2.43 g of Oleoresin of example 1 was mixed with 29 ml of ethanol, and heated to reflux (oil bath 130T), then add 20 ml of KOH(0.32 g) ethanol solution in 5 min and refluxed for 20 min. Subsequently, the oil bath was removed, 0.8 ml of acetic acid was added dropwise over a period of 10 min, the mixture was then allowed to cool to room temperature for 3 h. The formed solid was washed with ethanol (20 ml), ethanol : water=l:l (20 ml), water (40 ml) and ethanol (20 ml), then dried under high vacuum. 53.5 mg product was obtained. The content of free zeaxanthin is 182.6 g/Kg. The yield is 13.14 %.