Technical field of the invention

The present invention relates to a process for isolating taxane compounds, like taxol, from plant material comprising a taxane compound.

Background of the invention

Throughout the development of paclitaxel, one of the most successful anticancer drugs in the past 50 years, adequate supply of taxane compound, in particular taxol (in the present context the terms "paclitaxel" and "taxol" may be used interchangeably), has been a major challenge. This drug has been very complex to synthesize economically from first principles and cumbersome to isolate from natural sources. In addition, paclitaxel represents only a minor proportion of the total taxoid content of Taxus species.

The nomenclature for paclitaxel is structured on a tetracyclic 17-atom skeleton and there is a total of 11 stereocenters in the molecule.

Since the discovery of the anticancer activities of paclitaxel (an taxol), a sustainable increase of its extraction was the principal goal of the industry.

Taxol has mainly been obtained from the bark of taxus trees, like Taxus brevifolia also called Yew trees. However, extraction showed to have serious obstacle because of the low proportion of taxol, even in the most productive species, Taxus brevifolia, the content of taxol is only 0.001-0.05%. Consequently, treatment of one cancer patient consumes approximately eight 60-year old yew trees. Scientists have found that the substance can be extracted from the needles of the trees. However, the taxol is found in only one-eighth as concentrated compared to the bark.

To overcome the low yield and the sacrifice of many and old taxus trees, scientists tried to synthesise taxol, but again only low yields were obtained and high complexity of the biosynthesis hampered its industrial applicability.

Scientists tried an alternative by producing taxol by semisynthesis via intermediates from the needles of the European yew. The semisynthesis involves partly synthesis in the taxus tree and followed by chemical synthesis.

One approach of commercial semisynthesis of paclitaxel is starting from 10- deacetylbaccatin III (isolated from the European yew) is based on tail addition of the so- called Ojima lactam (an organic compound) to the free hydroxyl group of the taxol skeleton.

Another commercial approach for semisynthesising taxol, relies on the isolation of a group of paclitaxel derivatives isolated from primary ornamental taxanes. These derivatives have the same skeleton as paclitaxel except for the organic residue of the terminal tail amide group which can be phenyl, or propyl or pentyl (among others) whereas in paclitaxel it is an explicit phenyl group. The semisynthesis consists of conversion of the amide group to an amine with Schwartz's reagent through an imine followed by acidic workup and a benzoylation.

The challenge of obtaining taxol, or taxol derivatives for semisynthesis of taxol, from taxus plants according to the methods described and used today is that growth of the taxus plants (location of the growth) is limited to being processed for isolating the taxane compounds, like taxol, to its original location, as well as being subjected to seasonality and weather conditions.

Taxol-producing microorganisms has been found, isolated from various taxus species, i.e. a fungus was found in Taxus, for producing taxol. However, these fermentation processes also showed to provide very low yields of paclitaxel.

Of the mentioned methods for providing and isolating taxol, the currently most practiced process is by way of acquiring taxus cuttings from commercial companies and voluntary organizations who offer to trim taxus hedges in governmental parks and private gardens. The cuttings are swept up from the ground and gathered in bags. Brought to a drying facility where the cuttings are chopped in smaller pieces and dried with hot air from a burner installation to come to less than 12% moisture content. After the dried and chopped taxus cuttings are packed again and chipped to a facility to extract the taxane compounds out of them.

Traditionally, there is little influence or control on the time of cutting, quality of the cuttings and how clean they get to the dryer (sweeping from the ground includes soil, other plant cuttings growing next to or even entangled in the taxus hedge, etc.) Thus, obtaining taxane compounds, like taxol, from taxus plants involves various disadvantages, like sacrificing the taxus plant, complexity of the taxol molecule, very low yields, slow growth rate, and limitations on the isolation process to specific treatment periods (just following harvest), location of isolating the taxol, dependent of weather etc..

Hence, an improved process for providing a taxane compound, like taxol, would be advantageous, and in particular a new process which provides more flexible periods and locations for isolating the taxane compounds, longer periods for isolating taxane compounds from the plant material after being harvested, more efficient, more reliable, with improved productivity and more a repeatably process would be advantageous.

Summary of the invention

Thus, an object of the present invention relates to a process for isolating taxane compounds, like taxol, from plant material.

In particular, it is an object of the present invention to provide a process for isolating taxane compounds, in particular taxol, from plant materials that solves the above mentioned problems of the prior art with sacrificing the taxus plant, complexity of the taxol molecule, very low yields, slow growth rate, and limitations on the isolation process to specific treatment periods (just following harvest), location of isolating the taxol, dependent of weather etc..

Thus, one aspect of the invention relates to a process for providing an isolated taxane compound from a plant material comprising a taxane compound, the process comprises the steps of:

(i) providing the plant material comprising a taxane compound;

(ii) adding an inoculum comprising one or more lactic acid bacteria strains to the plant material comprising the taxane compound, providing a fermenting medium;

(iii) allowing the fermenting medium to ferment for at least 1 hour providing a fermented composition; and (iv) isolating the taxane compound from the fermented composition, providing the isolated taxane compound.

Another aspect of the present invention relates to a fermented composition comprising a plant material comprising a taxane compound; and one or more lactic acid bacteria strain(s).

Yet another aspect of the present invention relates to an isolated product comprising the isolated taxane compound obtained from the process according to the present invention.

Still another aspect of the present invention relates to a composition comprising the isolated product according to the present invention for use as a medicament.

A further aspect of the present invention relates to a composition comprising the isolated product according to the present invention for use in chemotherapy, in particular, for use in the treatment of a condition selected from the group consisting of ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi sarcoma, cervical cancer, and pancreatic cancer.

The present invention will now be described in more detail in the following.

Detailed description of the invention

Accordingly, the inventor of the present invention surprisingly found a process for conserving the plant material providing a more flexible period and for isolating the taxane compounds, allowing a greater flexibility on the location for isolating taxane compounds and longer periods for isolating taxane compounds from the plant material between harvest and isolation. Furthermore, the inventor of the present invention surprisingly found that a more stable taxane compound, when present in the plant material may be provided and/or an improved solubility.

Hence, a preferred embodiment of the present invention relates to a process for providing an isolated taxane compound from a plant material comprising a taxane compound, the process comprises the steps of:

(i) providing the plant material comprising a taxane compound; (ii) adding an inoculum comprising one or more lactic acid bacteria strains to the plant material comprising the taxane compound, providing a fermenting medium;

(iii) allowing the fermenting medium to ferment for at least 1 hour providing a fermented composition; and

(iv) isolating the taxane compound from the fermented composition, providing the isolated taxane compound.

The fermenting medium provided may preferably comprises the plant material and the inoculum comprising one or more lactic acid bacteria strains.

In an embodiment of the present invention the plant material comprising a taxane compound provided in step (i) may be provided as a fresh and clean plant material comprising a taxane compound.

Taxanes compounds are a class of diterpenes. Taxanes compounds feature a taxadiene core.

The isolated taxane compound may preferably be selected from the group consisting of taxol, paclitaxel, 10-deacetylbaccatin III, baccatin III, paclitaxel C, 7-epipaclitaxel, docetaxel, taxotere, and cabazitaxel.

In the context of the present invention the term "plant material comprising a taxane compound" relates to naturally occurring plant materials, genetically modified plant materials, where taxane compounds may be found in the entire plant material or in part of the plant material.

In an embodiment of the present invention the plant material comprising taxane compounds may be selected from the group consisting of plant materials from the Taxaceae family, the Corylaceae family and/or the Betulaceae family.

Preferably, the plant materials from the Taxaceae family may be selected from the species Taxus.

In an embodiment of the present invention the plant material comprising the taxane compound may be selected from taxus and/or hazel. In the context of the present invention the terms "plant material" and "plant material comprising a taxane compound" may be used interchangeably and relates to a plant material comprising a taxane compound, preferably a plant material selected from the Taxaceae family, the Corylaceae family and/or the Betulaceae family.

While the entire plant may be used the plant material may preferably comprises the (top) leaves or needles of the plant material.

In an embodiment of the present invention the plant material provided in step (i) may comprise leafy needles of the plant material. Preferably, the plant material provided in step (i) comprises light green leafy needles of the plant material.

In an embodiment of the present invention the plant material provided in step (i) consist essentially of leafy needles of the plant material

In the context of the present invention, the term "consisting essentially of", relates to a limitation of the scope of a claim to the specified features or steps and those features or steps, not mentioned and that do not materially affect the basic and novel characteristic(s) of the claimed invention.

In an embodiment of the present invention the plant material provided in step (i) may be subjected to a step of pre-treatment before the one or more lactic acid bacterial strain(s) is added in step (ii) and/or before the plant material is subjected to fermentation, step (Hi).

In a further embodiment of the present invention the inoculum added in step (ii) consist essentially of one or more lactic acid bacteria strains.

Step (ii) of the process according to the present invention relates to adding one or more lactic acid bacteria strains to the plant material, providing a fermenting medium.

Lactic acid bacterial are important bacteria and are widely used, in particular in the food, beverage and feed industry. The importance of the lactic acid bacteria may be evidenced by their generally regarded as safe (GRAS) status, due to their ubiquitous appearance in food, beverages, and feeds and their contribution to the healthy microflora of human mucosal surfaces. The genera that comprise the lactic acid bacteria, and which may be used in the present invention, are Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella; these genera belong to the order Lactobacillales.

In an embodiment of the present invention the one or more lactic acid bacterial strain added in step (ii) and/or the one or more lactic acid bacterial strain present in the fermented composition and/or the one or more lactic acid bacterial strain present in the isolated cannabinoid composition, may be selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus, and Weisella.

Preferably, the one or more lactic acid bacterial strain provided in step (ii) and/or the one or more lactic acid bacterial strain present in the fermented composition and/or the one or more lactic acid bacterial strain present in the isolated cannabinoid composition is one ore more lactic acid bacteria of the genus Enterococcus, Lactobacillus, Pediococcus, Lactococcus, or a combination thereof.

In an even further embodiment of the present invention the one or more lactic acid bacteria stain provided in step (ii) and/or the one or more lactic acid bacterial strain present in the fermented composition and/or the one or more lactic acid bacterial strain present in the isolated cannabinoid composition may be selected from the group consisting of one or more Enterococcus spp., Lactobacillus spp., Lactococcus spp., Pediococcus spp., and a combination hereof. Preferably, the one or more lactic acid bacterial strain is selected from the group consisting of one or more of Enterococcus faecium, Lactobacillus rhamnosus, Lactobacillus plantarum, Pediococcus acidililactili, Pediococcus pentosaceus, Lactococcus Lactis, Lactococcus Cremoris, Lactococcus Diacetylactis, Leuconostoc Cremoris and a combination hereof.

In yet a further embodiment of the present invention, the main lactic acid bacterial strain provided in step (ii) and/or the one or more lactic acid bacterial strain present in the fermented composition and/or the one or more lactic acid bacterial strain present in the isolated cannabinoid composition may be Pediococcus pentosaceus; Pendiococcus acidilactici; Lactobacillus plantarum; Lactobacillus rhamnosus; or Enterococcus faecium. Preferably, the main lactic acid bacteria present in the composition may be Lactobacillus plantarum.

In another embodiment of the present invention the one or more lactic acid bacteria strain(s) provided in step (ii) and/or the one or more lactic acid bacterial strain present in the fermented composition and/or the one or more lactic acid bacterial strain present in the isolated cannabinoid composition may be selected from the group consisting of one or more of Enterococcus faecium MCIMB 30122, Lactobacillus rhamnosus NCIMB 30121, Pediococcus pentosaceus HTS (LMG P-22549), Pendiococcus acidilactici NCIMB 30086 and/or Lactobacillus plantarum LSI (NCIMB 30083).

In order to increase productivity and effectivity two or more lactic acid bacterial strains may be provided, such as three or more lactic acid bacterial strains, e.g. four or more lactic acid bacterial strains, such as 7 or more lactic acid bacterial strains, e.g. 10 or more lactic acid bacterial strains, such as 15 or more lactic acid bacterial strains, e.g. 20 or more lactic acid bacterial strains, such as 25 or more lactic acid bacterial strains, e.g. 30 or more lactic acid bacterial strains, such as 35 or more lactic acid bacterial strains, e.g. 40 or more lactic acid bacterial strains.

In order to provide the desired effects, the fermenting medium should have a high content of viable lactic acid bacteria. In an embodiment of the present invention the fermented composition comprises one or more lactic acid bacterial strain(s) in a total amount in the range of 10 ⁴ -10 ¹² CFU per gram, such as in the range of 10 ⁵ -10 ¹¹ CFU per gram, e.g. in the range of 1O ⁶ -1O ¹⁰ CFU per gram, such as in the range of 10 ⁶ -10 ⁸ CFU per gram, e.g. in the range of 10 ⁶ -10 ⁷ CFU per gram.

In yet an embodiment of the present invention a starter culture or an inoculum may be provided comprising one or more lactic acid bacterial strains defined herein.

The term "inoculum" relates to a source material, such as the one or more lactic acid bacterial strain, used for the inoculation of a new culture. The inoculum may be employed to prime a process of interest.

"Inoculation" refers to the placement of a microorganism (e.g. one or more lactic acid bacterial strain) that will grow when implanted in a culture medium such as a fermentation tank comprising media to be fermented, e.g. the plant material selected from the Taxaceae family, the Corylaceae family and/or the Betulaceae family.

A primary inoculum may be provided and refers to the generation of an initial inoculum in a series of repeated similar of essentially identical inoculation process, for example one or more repetitions of a fermentation process. An aliquot of the fermenting medium may be used to inoculate a new process of fermentation of plant material. Thus, the inoculum may be a portion of the fermenting medium which comprises viable lactic acid producing bacteria in sufficient amount to prime a lactic acid fermentation process of another plant material, to be fermented. The inoculum according to the present invention may be a in a liquid form, dry form, or essentially dry form. The moisture content of the inoculum may be adjusted in order to optimize the fermentation process.

In one embodiment of the present invention, the inoculum may be provided as essentially pure viable bacteria (such as bacteria in freeze dried form) or bacteria suspended in a suitable aqueous medium prior to the being added to the plant material provided in step (i).

The inoculum comprising the one or more lactic acid producing bacteria strain may be added to the plant material providing a fermenting medium.

The fermenting medium provided in step (ii) may be allowed to ferment for at least 1 hour providing a fermented composition (step (iii)), such as for at least 5 hours; such as for at least 10 hours; e.g. for at least at least 15 hours; such as at least 24 hours; e.g. for at least at least 2 days; such as at least 3 days; e.g. for at least 5 days; such as at least 10 days; e.g. for at least 20 days; such as at least 30 days; e.g. for at least 50 days; such as at least 100 days; e.g. for at least 200 days.

In an embodiment of the present invention, the fermentation may be performed in step (iii) may be performed at a temperature during the steady state fermentation below 50°C, such as below 47°C, e.g. below 45°C, such as below 43°C, e.g. below 40°C, such as about 35°C. Preferably, the fermentation process in step (iii) may be conducted at a temperature (a steady state temperature) in the range of 15-50°C, preferably in the range of 25-43°C, more preferably in the range of 30-40°C, even more preferably, about 35°C.

In an embodiment of the present invention the starting temperature may be in the range of 10-35°C, such as in the range of 15-30°C, e.g. in the range of 18-25°C.

In yet an embodiment of the present invention the temperature of the fermenting medium during storage may be allowed to fluctuate together with fluctuations of the surrounding temperature, in particular when the fermentation is performed as an ensilation of the plant material provided in step (i).

The temperature increase of the fermenting medium of the present invention may preferably be a slow temperature increase from the starting temperature to the steady state fermentation temperature. In an embodiment of the present invention the temperature increase is provided without addition of heat. In the present context, the term "without addition of heat" relates to a fermentation temperature increase wherein the heat provided is produced by the fermentation itself without the use of electrical, mechanical or fuel-based heat.

In a further embodiment of the present invention the fermentation process is a solid-state fermentation.

In the context of the present invention the term "solid state fermentation" relates to a process where fermenting microorganisms are added to a plant material and allowed to ferment at a low moisture content under controlled conditions for several days. A low moisture content may be conditions where the plant material during fermentation is not pumpable.

In an embodiment of the present invention the plant material provided in step (i) may have a moisture content above 10% (w/w), e.g. above 12% (w/w), such as above 15% (w/w), e.g. above 20% (w/w), such as above 30% (w/w), e.g. above 40% (w/w).

In yet an embodiment of the present invention the plant material provided in step (iii) may have a moisture content below 95% (w/w), such as below 90% (w/w), e.g. below 85% (w/w), such as below 82% (w/w), e.g. below 80% (w/w), such as below 75% (w/w), e.g. below 70% (w/w), such as below 65% (w/w), e.g. below 60% (w/w).

The plant material provided in step (iii) may have a moisture content in the range of 10- 90% (w/w); such as in the range of 20-80% (w/w); e.g. in the range of 30-70% (w/w); such as in the range of 40-60% (w/w), e.g. in the range of 45-55% (w/w).

It may be advantageous to have a fast decrease in pH because the low pH may prevent growth of undesirable and unwanted microorganisms in the plant material and in the fermenting medium.

In an embodiment of the present invention the fermentation process provided in step (iii) introduce a decrease in pH from about pH 7.0 to about pH 5.0 in 1-10 days; such as in 2-8 days; e.g. in 3-7 days; such as in 4-6 days, e.g. in 2-4 days.

In yet an embodiment of the present invention the fermentation process provided in step (iii) introduce a decrease in pH from about pH 7.0 to about pH 4.5 in 1-10 days; such as in 2-8 days; e.g. in 3-7 days; such as in 4-6 days; e.g. in 2-4 days. In another embodiment of the present invention the fermentation process provided in step (iii) introduce a decrease in pH from about pH 7.0 to about pH 4.0 in 1-10 days; such as in 2-8 days; e.g. in 3-7 days; such as in 4-6 days; e.g. in 2-4 days.

In an embodiment of the present invention the fermentation process as provided in step (iii) may be continued for at least 5 hours after the pH of the fermenting medium has reached a pH in the range of pH 3.0-5.5; such as for at least 10 hours; e.g. for at least at least 15 hours; such as at least 24 hours; e.g. for at least at least 2 days; such as at least 3 days; e.g. for at least 5 days; such as at least 10 days; e.g. for at least 20 days; such as at least 30 days; e.g. for at least 50 days; such as at least 100 days; e.g. for at least 200 days.

In yet an embodiment of the present invention the fermentation process as provided in step (iii) may be continued for at least 5 hours after the pH of the fermenting medium has reached a pH in the range of pH 3.0-5.0; such as for at least 10 hours; e.g. for at least at least 15 hours; such as at least 24 hours; e.g. for at least at least 2 days; such as at least 3 days; e.g. for at least 5 days; such as at least 10 days; e.g. for at least 20 days; such as at least 30 days; e.g. for at least 50 days; such as at least 100 days; e.g. for at least 200 days.

In another embodiment of the present invention the fermentation process as provided in step (iii) may be continued for at least 5 hours after the pH of the fermenting medium has reached a pH in the range of pH 3.0-4.5; such as for at least 10 hours; e.g. for at least at least 15 hours; such as at least 24 hours; e.g. for at least at least 2 days; such as at least 3 days; e.g. for at least 5 days; such as at least 10 days; e.g. for at least 20 days; such as at least 30 days; e.g. for at least 50 days; such as at least 100 days; e.g. for at least 200 days.

In an embodiment of the present invention the process involves no addition of acid compound to the fermenting medium.

In the context of the present invention the term "addition of acid compound" relates to the action of adding an isolated or partly isolated acid compound to the fermenting medium. The reduction provided in the fermenting medium according to the present invention may preferably be provided by the acids formed by the microorganisms present in the fermenting medium. In a preferred embodiment of the present invention the acid compound present in the composition originates from the lactic acid bacterial strain. Preferably the acid compound present in the composition originating from the lactic acid bacterial strain is lactic acid. The step of isolating the taxane compound, step (iv), may involve a step of extraction of the taxane compound providing an extracted taxane compound.

Extraction may relate to the process of selectively removing a compound of interest from a mixture (such as the fermented composition) using a solvent. For an extraction to be successful the compound must be more soluble in the solvent than in the mixture. Additionally, the solvent and mixture must be immiscible (not soluble in one another).

The step of extraction of the taxane compound, according to the present invention, may be a solid phase extraction where the fermented composition comprising the plant material, the taxane compound and the lactic acid bacterial strain(s) may be forming a stationary phase. In an embodiment of the present invention the stationary phase may be contacted with a mobile phase (e.g. a solvent).

Traditionally, taxane compounds are considered being poorly soluble in water, however, the inventors of the present invention surprisingly found that water solubility of taxane compounds are improved when the plant material is subjected to a fermentation process using one or more lactic acid bacteria strains.

The mobile phase may be a liquid. Preferably, the mobile phase may be an aqueous liquid. Preferably the aqueous liquid may be water.

In an embodiment of the present invention the step of extraction of the taxane compound may be performed using water (the extractant).

The step of isolating the taxane compound, step (iv), may involve a step of flocculating removing impurities from the fermented composition or from the extracted taxane compound, providing the isolated taxane compound.

Preferably, the isolation of the taxane compound may involves:

- a step of extraction of the taxane compound providing an extracted taxane compound, followed by;

- a step of flocculation of the extracted taxane compound, resulting in the isolated taxane compound.

In an embodiment of the present invention the isolated taxane compound obtained from the step of flocculation may be subjected a further chromatographic separation in order to further purify isolated taxane compound, or in order to purify individually isolated taxane compounds.

Individually isolated taxane compounds may be a taxane compound selected from the group consisting of taxol, paclitaxel, 10-deacetylbaccatin III, baccatin III, paclitaxel C, 7- epipaclitaxel, docetaxel, taxotere, and cabazitaxel. Preferably, the isolated taxane compound is 10-deacetylbaccatin III.

In a preferred embodiment of the present invention the plant material may be provided in step (i) as a fresh plant material.

In the context of the present invention, the term "fresh" relates to a plant material which has not been subjected to freezing; heat processing; or drying.

In a further embodiment of the present invention the plant material may be provided in step (i) within 1 month from harvest; such as within 20 days from harvest; e.g. within 10 days from harvest; such as within 5 days from harvest; e.g. within 3 days from harvest; such as within 1 days from harvest; e.g. within 20 hours from harvest; such as within 15 hours from harvest; e.g. within 10 hours from harvest; such as within 5 hours from harvest; e.g. within 3 hours from harvest; such as within 2 hours from harvest.

The fermentation process may preferably be an ensiling process or an ensiling-like process. The ensiling according to the present invention may involve packing fermenting medium according to the present invention (comprising plant material and inoculum comprising one or more lactic acid bacteria strains) in plastic bags which are then allowed to ferment.

Ensilation may relate to the process of making silage from field crops where by placing the plant material in a silo or pit, by piling it in a large heap and compressing it down so as to leave as little oxygen as possible and then covering it with a plastic sheet, or by wrapping large round or square bales tightly in plastic film, or pressing it into a plastic sausage-like packaging whereby the plant material is left for semi-anaerobic fermentation.

In an embodiment of the present invention, the fermenting medium may be packed in plastic bags and left for fermentation as described in step (iii).

One advantage of fermenting the plant material using an ensiling process may be that the weather conditions before and during harvesting may be less relevant since the plant material may be wet during fermentation (during ensiling the plant material). In an embodiment of the present invention water may be drained, decanted, filtered and/or pressed from the plant material.

In a further embodiment of the present invention the plant material may preferably not be subjected to a drying process before it is allowed to ferment as described in step (iii).

In order to improve fermentation of the plant material, the plant material may be pretreated before subjected to the fermentation process as defined in step (iii).

In an embodiment of the present invention, the pre-treatment may involve grinding, cutting, chopping, slicing, and/or fractionizing of the plant material.

The pre-treatment may result in a plant material having an average particle size of 50 mm or less, such as an average particle size of 30 mm or less, such as an average particle size of 20 mm or less, such as an average particle size of 10 mm or less such as an average particle size in the range 1-40 mm; such as in the range of 10-30 mm, e.g. in the range of 20-25 mm.

When adding the inoculum comprising one or more lactic acid bacteria strains to the plant material, the inoculum provided in step (ii) and the plant material provided in step (i) may be mixed in order to distribute the inoculum in the plant material providing the fermenting medium.

Following mixing of the inoculum provided in step (ii) and the plant material provided in step (i) providing the fermenting medium, the fermenting medium may be stored for fermentation.

In an embodiment of the present invention the storing for fermentation may be packing the fermentation medium in a fermentation tank or by ensiling the fermentation medium as described previously.

In an embodiment of the present invention, the inoculum may be provided with a concentration of lactic acid bacteria in the inoculum sufficient to outgrow other non-lactic acid bacteria, yeast or moulds, present in or on the plant material.

In a further embodiment of the present invention the inoculum provided may be of a sufficient amount in order to be sufficiently mixed with, and sufficiently distributed in, the plant material and forming the fermentation medium.

Accordingly, in one embodiment of the invention, the proportion of the inoculums in the combination of the plant material and the one or more lactic acid material as defined in step (iii) is in the range of 0.01 to 50 vol%; e.g. in the range of 0.05 to 25 vol%; such as 0.1 to 10 vol-%; e.g. 0.5 to 5 vol-%; such as 0.7 to 2.5 vol-%; or around 1 to 2 vol-%.

In order to provide the desired effects, the fermenting medium should have a high content of viable lactic acid bacteria. In an embodiment of the present invention the fermented composition comprises one or more lactic acid bacterial strain(s) in a total amount in the range of 10 ⁴ -10 ¹² CFU per gram, such as in the range of 10 ⁵ -10 ¹¹ CFU per gram, e.g. in the range of 1O ⁶ -1O ¹⁰ CFU per gram, such as in the range of 10 ⁶ -10 ⁸ CFU per gram, e.g. in the range of 10 ⁶ -10 ⁷ CFU per gram.

A preferred embodiment of the present invention relates to a fermented composition comprising a plant material comprising a taxane compound; and one or more lactic acid bacteria strain(s).

In an embodiment of the present invention the fermented composition may comprise a pH value of below 6.5, e.g. a pH value below 6.0, such as a pH value below 5.0 e.g. a pH value below 4.5 such as a pH value below 4.0.

In an embodiment of the present invention the fermented composition may comprise a concentration of lactic acid of at least 10 mM lactic acid, such as at least 50 mM lactic acid, such as 50-1000 mM lactic acid, such as 100-500 mM lactic acid, such as 100-300 mM lactic acid, such as 100-200 mM lactic acid, such as 150-500 mM lactic acid, such as 200- 500 mM lactic acid; or such as 300-500 mM lactic acid.

In the present context the term "mM" relates to mmol/l, where the term "I" relates to a volume of the fermented composition and is not limited to only relate to the liquid part of the fermented composition.

In an embodiment of the present invention the fermented composition may comprise a concentration of acetic acid of 5 mM or less, such as 2 mM or less, e.g. 1 mM or less, such as 0.1 mM or less, preferably no detectable acetic acid.

The fermented composition may comprise a concentration of acetic acid of in the range of O.OlmM to 5 mM acetic acid, such as in the range of 0.05 to 2 mM acetic acid, e.g. in the range of 0.1 mM to 1 mM or less, preferably the fermented composition comprises no detectable acetic acid.

In an embodiment of the present invention the fermented composition may comprise at least 25% of the fibres originally present in the plant material, when supplied in step (i), such as at least 50% of the fibres, e.g. at least 75% of the fibres, such as at least 90% of the fibres, e.g. at least 95% of the fibres, such as at least 98% of the fibres, e.g. 100% of the fibres.

The fermented composition according to the present invention may be provided by a process according to step (iii) of the present invention.

The fermented composition may comprise a liquid phase and a solid phase.

The taxane compound may be found in the solid phase (resulting in captured taxane compound) and/or the taxane compound may be found in the liquid phase (resulting in a solubilized or dissolved taxane compound). The captured taxane compound may be bound to and/or entrapped in the solid phase.

The taxane compound may be isolated from the fermented composition, from the solid phase and/or the liquid phase, resulting in the isolated taxane compound.

Preferably, the taxane compound may be isolated from the fermented composition, from the solid phase and/or the liquid phase, by subjecting the fermented composition, the solid phase and/or the liquid phase to an extraction process resulting in the isolated taxane compound.

In order to improve isolation of the taxane compound, in particular isolating the taxane compound from the solid phase the fermented composition may be subjected to sonification. The inventors of the present invention found that by subjecting the fermented composition and the solid phase to sonification, providing a sonificated fermented composition, the taxane compound becomes increasingly liberated and available to isolation, e.g. by extraction.

In an embodiment of the present invention the amount of extractant, e.g. water, to be used when isolating the taxane compound from a sonificated fermented composition is 80% (w/w) or less relative to the amount of extractant to be used when isolating the taxane compound from a non-sonificated fermented composition; such as 70% (w/w) or less, e.g. 60% (w/w) or less, such as 50% (w/w) or less, e.g. in the range of 30-80% (w/w), such as in the range of 40-60% (w/w); e.g. in the range of 45-55% (w/w) relative to the amount of extractant to be used when isolating the taxane compound from a non- sonificated fermented composition.

As mentioned the sonification of the fermented composition may improve and/or increase isolation of the taxane compound. In a further embodiment of the present invention, the amount of taxane compound obtained from sonificated fermented composition may comprise at least 75% of the taxane compound present in the plant material, such as at least 80%, e.g. at least 85%, such as at least 90%, e.g. at least 95%, such as at least 98%.

In yet an embodiment of the present invention the amount of extractant, e.g. water, to be used when isolating at least 75% of the taxane compound from a sonificated fermented composition (such as at least 80%, e.g. at least 85%, such as at least 90%, e.g. at least 95%, such as at least 98%) is 80% (w/w) or less relative to the amount of extractant to be used when isolating the taxane compound from a non-sonificated fermented composition; such as 70% (w/w) or less, e.g. 60% (w/w) or less, such as 50% (w/w) or less, e.g. in the range of 30-80% (w/w), such as in the range of 40-60% (w/w); e.g. in the range of 45-55% (w/w) relative to the amount of extractant to be used when isolating the taxane compound from a non-sonificated fermented composition

The isolated taxane compound may be subjected to a further purification step. The further purification step may include flocculation, chromatography or the combination flocculation and chromatography, preferably starting by subjecting the extracted fermented composition to a flocculation process followed by subjecting it to a chromatographic separation process.

The flocculation process may involve addition of an organic compound and/or an adjustment of the pH. Preferably, the organic compound may be a polysaccharide compound, preferably a chitosan compound, that may be added to the fermented composition, or the isolated taxane compound. The addition of the organic compound, such as a chitosan compound may be suitable for removing impurities from the isolated taxane compound. The flocculation process using the organic compound, such as chitosan, may be improved by adjusting the pH, preferably by increasing the pH, e.g. using lye (a metal hydroxide).

The flocculation process may remove 50% (w/w) or more of the contaminants (in particular the non-soluble contaminants) from the isolated taxane compound, such as 60% (w/w) or more, e.g. 70% (w/w) or more, such as 75% (w/w) or more, e.g. in the range of 50-95% (w/w), such as in the range of 60-90% (w/w), e.g. in the range of 70-80% (w/w). The chromatographic separation process may include a liquid mobile phase, which is carried through a system, like a column, a capillary tube, a plate, or a sheet, comprising a stationary phase. The taxane compound to be isolated may travel with the liquid phase through the stationary phase resulting in one or more fractions comprising the isolated taxane compound.

The chromatographic separation process may be selected from gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, reversed phase chromatography, affinity chromatography or a combination hereof.

In an embodiment of the present invention the taxane compound may be captured when subjecting the isolated taxane compound to at least one chromatographic separation process. Following capturing, the taxane compound may be eluted obtaining an isolated taxane compound with high purity.

In an embodiment of the present invention the isolated taxane compound with high purity comprises at least 60% (w/w) taxane compound, such as 10-DAB, on a dry matter basis, such as at least 70% (w/w), e.g. at least 80% (w/w), such as at least 90% (w/w), e.g. at least 95% (w/w), such as at least 98% (w/w), e.g. at least 99% (w/w), such as in the range of 90-99% (w/w), e.g. in the range of 95-99% (w/w), such as in the range of 97- 98% (w/w), e.g. in the range of 98-99% (w/w).

In an embodiment of the present invention the fermentation process as described in step (iii) of the present invention, comprising the plant material provided in step (i) with the one or more lactic acid bacterial strains provided in step (ii) may be ensiled in bags, e.g. plastic bags, 25 kg -250 tons, such as 3-200 tons, e.g. 5-150 tons, such as 10-100 tons, e.g. 50-75 tons.

In an embodiment of the present invention, the fermentation process provided in step (iii) may be a one-step fermentation of the plant material.

In the present context, the term "one-step fermentation" relates to a fermentation process wherein the same type of plant material may be subjected to the same fermentation conditions, or substantially the same fermentation conditions. Hence, the term "one-step fermentation", exclude the option of taking out a part of the plant material during fermentation, leaving the remaining plant material to be further fermented and followed by mixing the part which was taken out with the further fermented composition. In a further embodiment of the present invention, the fermenting medium does not involve subsequent supplementation of plant material and/or subsequent supplementation of one or more lactic acid bacterial strain(s) to the fermenting medium.

The fermentation process according to the present invention, may preferably be essentially homofermentative. "Essentially homofermentative" means, that the predominant bacterial flora driving the fermentation are essentially homofermentative. In the present context, the term "essentially homofermentative" relates to a fermentation process where, 70% or more of the bacteria are homofermentative, such as 80% or more of the bacteria are homofermentative, e.g. 85% or more of the bacteria are homofermentative, such as 90% or more of the bacteria are homofermentative, e.g. 95% or more of the bacteria are homofermentative, such as 98% or more of the bacteria are homofermentative, e.g. 99% or more of the bacteria are homofermentative.

In an embodiment of the present invention the fermentation is essentially a homofermentation, such as a homolactic fermentation.

The term "homolactic fermentation" when used according to the present invention indicates that the major fermentation product may be lactic acid, and the levels of e.g. acetic acid and ethanol are either below taste-threshold, around taste threshold or slightly above taste threshold, preferably below taste-threshold. The term "essentially homofermentative" may indicates a ratio of lactic acid to acetic acid or lactic acid to ethanol in (mM/mM) of more than 1 : 1, such as 2: 1 or more, e.g. 10: 1 or more, such as 20: 1 or more, e.g. 50: 1 or more, or such as 100: 1 or more.

In an embodiment of the present invention the fermentation process (step (iii)) may be an anaerobic fermentation process or a semi-anaerobic fermentation process. Preferably, the fermentation process is a semi-anaerobic fermentation process.

A semi-anaerobic fermentation process relates to a fermentation process without the addition of oxygen and/or air to the fermenting medium. However, the oxygen and/or air present in and surrounding the fermenting medium are maintained and may be available and used by the one or more lactic acid bacteria strains.

In order to promote growth of the lactic acid bacteria the plant material the fermenting medium may be further supplemented with one or more further ingredient. In an embodiment of the present invention a carbon source may be added to the plant material provided in step (i), or to the fermenting medium. Preferably, the carbon source may be a carbohydrate, a carbohydrate containing material or a combination hereof.

Preferably, the carbohydrate may be sugar, syrup, dextrose, molasses or other sugar containing sources.

Preferably, the carbohydrate containing material may be selected from cereals, (e.g. wheat, barley, rye, rice, maize (cob maize silage (CCM) or ripe), triticale, oat); vegetables (e.g. potatoes, beans, peas, maize, soy); carbohydrate ingredient (such as whey, curd, skim milk and the like); or any combination hereof.

By introducing the fermentation process according to the present invention of fresh plant materials, like fresh Taxus, the moment of harvesting may be disconnected from the moment of further processing of the fermented composition.

In the context of the present invention, the term "disconnected" relates to extending the time from harvesting to further processing, in order to make the process more cost effective and allow the individual farmer/producer to process larger amounts of plant materials in a single season. Hence, the process according to the present invention the time may be extended from a couple of days to e.g. 12 months where the taxane compounds, such as taxol, can be isolated from the fermented composition.

In an embodiment of the present invention the fermented composition provided in step (iii) may be stored for period between 10 hours and 12 months before being subjected to at least one processing step , providing the isolated taxane composition, such as isolated taxol,; e.g. for a period of 24 hours and 10 months; such as for a period of 36 hours to 9 months; e.g. for a period of 48 hours and 8 months; such as for a period of 60 hours to 7 months; e.g. for a period of 72 hours and 6 months; such as for a period of 96 hours to 5 months; e.g. for a period of 120 hours and 3 months.

A preferred embodiment of the present invention relates to an isolated product comprising the isolated taxane compound obtained from the process according to the present invention.

The isolated taxane compound according to the present invention may have medical and pharmaceutical applications. An embodiment of the present invention relates to a composition comprising the isolated product according to the present invention for use as a medicament.

An embodiment of the present invention relates to a pharmaceutical composition comprising the isolated product according to the present invention, in combination with a pharmaceutical acceptable carrier, for use as a medicament.

A further embodiment of the present invention relates to a composition comprising the isolated product according to the present invention for use in chemotherapy, in particular, for use in the treatment of a condition selected from the group consisting of ovarian cancer, esophageal cancer, breast cancer, lung cancer, Kaposi sarcoma, cervical cancer, and pancreatic cancer.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.