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 box- thorn.

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 berries with a lipophilic solvent and obtained in high yield an oily oleoresin, rich in zeaxanthin (6 - 12 weight-% content). Further purification of the oleoresin could be achieved by crystallization from hot ethanol yielding an orange -red powder with a zeaxanthin content of 22 - 36 weight-% corresponding to a zeaxanthin dipalmitate content of 41 - 64 weight-%. In addition the zeaxanthin dipalmitate containing oleoresin could be saponified with caustic in ethanol to give a powder in 70% yield and with a zeaxanthin content of 75 %. 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 reduced sized pieces of dried Goji berries; and

b) extracting the smaller Goji berries obtained in step a) with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether and mixtures thereof; and

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

MST, 25.08.201 1 d) optionally cleaving the esters in the dried extracts obtained in step c); and e) crystallizing the dried extracts obtained in step c) or d) in an aliphatic straight or branched C _1-4 alcohol.

Preferably the process according to the present invention consists only of these steps a) to e) and does not comprise any additional steps.

Thus, a preferred embodiment of the present invention is a process for the manufacture of an extract of Goji berries containing zeaxanthin and/or its esters, consisting of the following steps:

a) obtaining reduced sized pieces of dried Goji berries; and

b) extracting the smaller Goji berries obtained in step a) with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether and mixtures thereof; and

c) removing the solvent from the extracts obtained in step b); and d) optionally cleaving the esters in the dried extracts obtained in step c); and e) crystallizing the dried extracts obtained in step c) or d) in an aliphatic straight or branched C _1-4 alcohol.

Thus, one of these preferred embodiments is directed to a process for the manufacture of an extract of Goji berries containing zeaxanthin (di)esters consisting of the following steps:

- obtaining reduced sized pieces of dried Goji berries; and

- extracting the smaller (= size reduced) Goji berries obtained in the preceding step with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether and mixtures thereof; and

- removing the solvent from the extracts obtained in the preceding step; and

- crystallizing the dried extracts obtained in the preceding step in an aliphatic straight or branched C _1-4 alcohol;

the other preferred embodiment is directed to a process for the manufacture of an extract of Goji berries containing zeaxanthin consisting of the following steps: - obtaining reduced sized pieces of dried Goji berries; and

- extracting the smaller Goji berries obtained in the preceding step with a solvent selected from the group consisting of ethyl acetate, hexane, heptane, petroleum ether and mixtures thereof; and

- removing the solvent from the extracts obtained in the preceding step; and

- cleaving the esters in the dried extracts obtained in the preceding step; and

- crystallizing the dried extracts obtained in the preceding step in an aliphatic straight or branched C _1-4 alcohol.

Processes to obtain zeaxanthin are already known in the prior art, but have several disad- vantages.

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 _1-4 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. The present invention showed that it is not necessary to perform this additional step which is time and energy consuming. US 6,191,293 also describes a process where Chinese wolfberries are pre-extracted with water to eliminate water-soluble gums (see example 4). Such a pre-extraction step is not necessary according to the process of the present invention. 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 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 e) 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 used can either be of the species Lycium barbarum and/or of the species Lycium chinense.

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) and c) 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 11 to 23 weight-%, based on the total weight of the oleoresin.

When only steps a), b), c) and e) 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 25 to 80 weight-%, preferably a zeaxanthin(di)ester content in the range of 30 to 70 weight-%, more preferably a zeaxanthin(di)ester content in the range of 40 to 65 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 90 weight-% in form of free zeaxanthin, preferably a zeaxanthin content in the range of 45 to 90 weight-% in form of free zeaxanthin, more preferably a zeaxanthin content in the range of 60 to 90 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 of the present invention are now described in more detail below.

Step a): Size reduction of the Goji berries

The Goji berries are usually commercially available in dried form. The drying can be carried out by any method known to the person skilled in the art such as by sunshine drying or by oven drying. Preferably the dried Goji berries used in step a) have a water content in the range of 3 to 10 weight-%, preferably in the range of 5 to 7 weight-%, based on the total weight of the Goji berries.

It was found out that the extraction leads to extracts with higher enrichments in zeaxan- thin if the size of the commercially purchased Goji berries was reduced before the extraction. The size reduction can be done 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): Extraction Step b) is preferably performed several times, especially step b) is performed three times. Of course step b) could also be performed only two times, but with a longer extraction time.

Usually the extraction proceeds within 1 to 2 hours per extraction step. 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 b) 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 b) is in the range of 1 to 5 liters, more preferably in the range of 1 to 4 liters, even more preferably in the range of 1 to 3 liters, most preferably in the range of 1.5 to 2.5 liters, per 1 kg of size reduced dried Goji berries obtained in step a). Preferably step b) is performed at a temperature in the range of 20 °C to 80 °C, more preferably at a temperature in the range of 20 °C to 65 °C, even more preferably at a temperature in the range of 22 °C to 55 °C, most preferably at a temperature in the range of 40 °C to 50 °C. Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step b) as listed above are combined.

Step c): Solvent removal If the extract obtain in step b) contains non soluble parts, they are removed, preferably by filtration.

Then the solvent of the extract is evaporated under reduced pressure, preferably at a temperature in the range of 20 °C to 60 °C, more preferably at a temperature in the range of 30 °C to 60 °C, most preferably at a temperature in the range of 35 °C to 50 °C, whereby an "oleoresin" is obtained.

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 c) as listed above are combined.

Step d): Ester cleavage In the oleoresin from the Goji berry extraction 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 KOH 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).

Thus, the cleavage of the zeaxanthin esters in step d) can be performed by saponification or trans-esterification of the zeaxanthin esters of the dried extracts obtained in step c).

Preferably the cleavage of the zeaxanthin esters is performed by trans-esterification. More preferably this trans-esterification in step d) is performed at a temperature in the range of 20 °C to 150 °C, preferably at a temperature in the range of 60 °C to 100 °C, more preferably at a temperature in the range of 70 °C to 90 °C.

The reaction can be performed at ambient pressure or at a higher pressure depending on the temperature chosen.

Most preferably the trans-esterification is performed by reaction of the zeaxanthin esters with an aliphatic straight or branched C _1-4 alcohol in the presence of a base.

Examples of the aliphatic straight or branched C _1-4 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 5 to 30 liter per kg of dried extract, more preferably in the range of 5 to 25 liter per kg of dried extract, most preferably in the range of 5 to 20 liter per kg of dried extract. 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. Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step d) as listed above are combined.

Step e): Crystallization If a step d) is performed by trans-esterification, the aliphatic straight or branched C _1-4 alcohol used in step e) is the same as used in step d). Here the same preferences for this alcohol apply as for step d). Thus, the most preferred solvent used in step e) is ethanol.

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

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

Preferred embodiments of the present invention are also embodiments where several preferred embodiments of step e) 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) 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, with a zeaxanthin content of at least 60 weight-%, based on the total weight of the extract, consisting of the following steps:

a) obtaining reduced sized pieces of dried Goji berries with a water content of 3 to 10 weight-%, based on the total weight of the Goji berries; and b) extracting the smaller Goji berries obtained in step a) with ethyl acetate at a temperature in the range of 40 °C to 50 °C; and

c) removing the solvent from the extracts obtained in step b); and d) cleaving the esters in the dried extracts obtained in step c) by transesterifi- cation with ethanol in the presence of a base; 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 ze- axanthin 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 : Influence of the Milling on the Extraction Yield of Zeaxanthin To test the influence of the milling of the Goji berries on the extraction yield, two extractions were performed using either the whole berries or the milled berries as starting materials under exactly the same extraction conditions (200 g berries, 3 x 500 ml hexane, 45 °C for 1 hour, respectively). From the extraction of the whole berries 630 mg of a red oleoresin was obtained with a zeaxanthin content of 12.4 weight-%, corresponding to 78 mg zeaxanthin. The extraction of pulverized berries resulted in 1.35 g of a red oleoresin with a zeaxanthin content of 10.3 weight-%, corresponding to 139 mg zeaxanthin. Therefore milling improves the yield by 44 % under these extraction conditions. Example 2: Use of different solvents for the extraction

For the extraction of zeaxanthin from Goji berries, hexane and ethyl acetate were used, as well as a mixture thereof. Both solvents have the further advantage that they are both food approved and lipophilic.

2.1) The extraction of 209 g of pulverized Goji berries employing 700 ml ethyl acetate per extraction (1 hour at 45 °C) gave the following results (see Table 1):

Table 1 : Ethyl acetate extraction of zeaxanthin (3 x 700 ml)

These figures suggest that three extractions are required for a complete extraction of zeaxanthin from Goji berries if one extraction only takes 1 hour. Most likely the number of extractions could be reduced if the duration of the single extraction is prolonged. The av- erage zeaxanthin content of the obtained oleoresins was 75.8 g of zeaxanthin per kg of oleoresin.

2.2) In a second experiment a further sample of Goji berries (200 g) was extracted employing a mixture of ethyl acetate and hexane with a volume ratio of 1 : 1 (600 ml per extraction, 1 hour, 45 °C). The results are depicted in Table 2. These results are comparable to the ones shown in Table 1. Table 2: Ethyl acetate : hexane (1 : 1, volume ratio) extraction of zeaxanthin

2.3) The complete extraction of zeaxanthin from 200 g pulverized Goji berries using 600 ml pure hexane required also three extractions to give 1.29 g of a combined oleoresin with a zeaxanthin content of 11.0 weight-%.

These examples show that both hexane and ethyl acetate are suitable for the extraction of zeaxanthin from goji berries. The hexane extracts show a slightly higher content of zeaxanthin. However, ethyl acetate is the less toxic solvent and should therefore be preferred.

Example 3: Extraction of zeaxanthin from Goji berries to prepare an oleoresin with less amount of ethyl acetate used than in example 2.1

Goji-berries were milled using a food processor. 200 g of the pulverized berries were then transferred to a 1500 ml-flask and treated with 400 ml of ethyl acetate. The mixture was stirred at 45 °C for 1 hour under an inert gas atmosphere. The mixture was filtered and the solvent was evaporated under reduced pressure. 1.27 g of a red oleoresin were obtained which content of zeaxanthin was measured by HPLC and is given in Table 3 below.

The residue was extracted 2 further times following the same procedure, i.e. a total of 1.2 liter of ethyl acetate were used for the extraction. The content of zeaxanthin of the oleo- resins obtained in the second and third extraction is also given in Table 3 below. Table 3 : Ethyl acetate extraction of zeaxanthin (3 x 400 ml)

Example 4: Extraction of zeaxanthin from Goji berries to prepare an oleoresin with less amount of hexane than in example 2.3

Goji-berries were milled using a food processor. 200 g of the pulverized berries were then transferred to a 1500 ml-flask and treated with 400 ml of hexane. The mixture was stirred at 45 °C for 1 hour under an inert gas atmosphere. Subsequently, the mixture was filtered and the solvent of the filtrate was evaporated under reduced pressure.

The residue was extracted 2 further times following the same procedure, i.e. a total of 1.2 liter of hexane was used for the extraction. A total of 2.68 g of a red oleoresin was obtained with a zeaxanthin amount of 95.1 mg corresponding to a zeaxanthin content of 35.5 g per kg of oleoresin.

Example 5: Repetition of example 4 with less amount of Goji berries

Goji-berries were milled using a food processor. 100 g of the pulverized berries were then transferred to a 750 ml-flask and treated with 200 ml of hexane. The mixture was stirred at 45 °C for 1 hour. Subsequently, the mixture was filtered and the solvent was evaporated under reduced pressure. The residue was extracted 2 further times following the same procedure. 1.04 g of a red oleoresin were obtained with an amount of zeaxanthin of 106 mg corresponding to a ze- axanthin content of 102 g per kg of oleoresin.

Example 6: Extraction of zeaxanthin from Goji berries to prepare an oleoresin at room temperature

Goji berries were milled using a food processor. 100 g of the pulverized berries were then transferred to a 750 ml-flask and treated with 200 ml of hexane. The mixture was stirred at 23 °C for 1 hour. Subsequently, the mixture was filtered and the solvent was evapo- rated under reduced pressure.

The residue was extracted 2 further times following the same procedure. Thus, in total an amount of 600 ml of hexane was used for the extraction. 0.62 g of a red oleoresin were obtained with an amount of 76.26 mg of zeaxanthin corresponding to a zeaxanthin con- tent of 123 g per kg of oleoresin.

Example 7: Extraction of zeaxanthin from Goji berries to prepare an oleoresin at 65 °C

Goji berries were milled using a food processor. 100 g of the pulverized berries were then transferred to a 750 ml-flask and treated with 200 ml of hexane. The mixture was stirred at 65 °C for 1 hour. Subsequently, the mixture was filtered and the solvent was evaporated under reduced pressure.

The residue was extracted 2 further times following the same procedure. 1.01 g of a red oleoresin were obtained with an amount of 102 mg of zeaxanthin corresponding to a zeaxanthin content of 101 g per kg of oleoresin. Example 8: Purification of the oleoresin by crystallisation in ethanol to prepare a natural zeaxanthin enriched powder

To 2.55 g of the oleoresin obtained in example 4 12.8 ml of ethanol were added. The mixture was stirred at 50 °C for 30 minutes and subsequently it was allowed to cool to room temperature (within 2 hours). The formed solid was filtered off, washed with ethanol (10 ml) and dried under high vacuum. One obtained 251 mg of an orange powder with an amount of zeaxanthin of 88.6 mg corresponding to a zeaxanthin content of 353 g per kg. Thus, the crystallization resulted in a 10-fold enrichment in zeaxanthin. Example 9: Scale-Up

The extraction process could be successfully scaled-up. Batches of 4 kg of Goji berries were milled and extracted 3 times with either 10 liters of hexane or ethyl acetate, respectively. In total 68 kg of dried goji berries were extracted.

9.1) The extraction of 4 kg of milled goji berries employing hexane gave around 37 g of oleoresin with a zeaxanthin content of up to 90.3 g per kg of oleoresin.

9.2) Using ethyl acetate for the extraction of 4 kg of milled goji berries resulted in about 40 g of an oleoresin with a zeaxanthin content of up to 75.1 g per kg of oleoresin.

Example 10: Trans-esterification of the Zeaxanthin Esters in the Oleoresin (= dried extract obtained in step c) to free zeaxanthin crystals A mixture of 5.15 g of an oleoresin with a zeaxanthin content of 66.8 g per kg of the oleoresin (corresponding to an amount of 344 mg of zeaxanthin) and ethanol (100 ml) was heated to reflux (oil bath temperature: 130 °C). Then KOH pellets (1.5 g; = 27 mmol) were added and the mixture was refluxed for another 20 minutes. Subsequently, the oil bath was removed and acetic acid (1.6 ml; = 28 mmol) was added drop wise over a period of 10 minutes. The mixture was then allowed to cool to room tempera- ture within 3 hours. The formed precipitate was filtered off and washed with ethanol (40 ml), ethanol : water = 1 : 1 (volume ratio; 40 ml), water (80 ml) and ethanol (40 ml). The solid was dried under high vacuum. 320 mg of a dark orange solid were obtained with an all-E zeaxanthin content of 240 mg according to HPLC analysis which corresponds to a weight- % ratio of 75 %.