Method for extracting rubusoside including nades

Abstract

The present invention relates to a method for producing a plant extract comprising rubusoside, to a method for glycosylating rubusoside, to the use of a natural deep eutectic solvent in such a method and to the use of a glycosyltransferase in such a method.

Description

[0001] Eisenfuhr Speiser

[0002] Munich, 15 January 2025

[0003] Our Ref.: SM 7082-01 WO SOE / DRE

[0004] Applicant: Symrise AG

[0005] Serial Number: New Application

[0006] Symrise AG

[0007] MiihlenfeldstraBe 1 , 37603 Holzminden, Germany

[0008] Method for extracting rubusoside including NADES

[0009] The present invention relates to a method for producing a plant extract comprising rubusoside, to a method for glycosylating rubusoside, to the use of a natural deep eutectic solvent in such a method and to the use of a glycosyltransferase in such a method.

[0010] Consumers generally have a strong preference for foodstuffs or indulgence foods, which 5 have a large amount of high caloric sugar, in particular sucrose (saccharose), glucose, fructose or mixtures thereof, due to the pleasant sweetness and sweetness profile associated therewith. On the other hand, it is generally known that a large content of readily metabolizable carbohydrates causes a steep rise in blood sugar levels, leads to the formation of fat deposits and ultimately can result in health problems such as overweight, obesity, 0 insulin resistance, age-onset diabetes and complications thereof. Another particular aggravating factor is that many of the above-mentioned carbohydrates can also have an adverse effect on dental health, as they are decomposed by specific types of bacteria in the oral cavity into lactic acid, for example, and can attack the enamel of milk teeth or adult teeth (caries).

[0011] 5 Therefore, it has long been an objective to reduce the high caloric sugar content of food or beverage products and replace it partly or entirely by other substances that impart a sweet taste or which can positively affect the sweet taste in a low concentration without exhibiting sweet taste itself at these low concentration (taste modulators).The use of rubusoside and its glycosides as taste modulator for sugar-reduced products, flavoring mixtures, and methods for producing such products is already known.

[0012] Rubusoside is a natural compound occurring e.g. in plants of the genus Rubus, Stevia, or Hydrangea, such as plants of the species Rubus chingii, Stevia rebaudiana, or Hydrangea strigosa, in particular of the variety Rubus chingii suavissimus. Rubusoside cannot be economically produced by chemical synthesis or biotechnological approaches.

[0013] Several methods have been described for extracting rubusoside from the leaves of e.g. Rubus chingii suavissimus.

[0014] CN105061526 describes a method for extracting rubusoside with microwave counter current extraction. The extracted rubusoside is then purified by recrystallization in organic solvents.

[0015] CN106632540 describes a method for extracting rubusoside with cellulases and microwaves together with ethanol. The extracted rubusoside is then purified with macroporous resins and polyamide columns.

[0016] CN106243165 describes a method for extracting rubusoside with ethanol. The obtained extract is then filtered and precipitated with EuAIO3.

[0017] Further, methods for extracting rubusoside with e.g. methanol are described in the prior art.

[0018] However, when used in products for nutrition or pleasure or in pharmaceutical products, the solvents must be non-toxic and several regulatory conditions need to be met. In this regard, the obtained extract must not contain detectable amounts of certain substances, such as chloroform or methanol, which are frequently used as solvents for extraction.

[0019] Additionally, it is highly desired to use solvents, which can be obtained from renewable and natural sources.

[0020] Furthermore, since the extracted rubusoside shall be used in products for nutrition or pleasure or in pharmaceutical products, it is necessary that the sensory characteristics of rubusoside are not negatively affected by the extraction process or the compounds additionally present in the obtained extract. In the currently available extraction methods, several compounds are extracted as well, which provide significant off-notes in the obtained extracts.Additionally, the obtained extracted rubusoside is frequently glycosylated to obtain rubu-soside glycosides or a mixture or rubusoside and rubusoside glycosides, which provides advantageous taste characteristics. However, since the extraction of rubusoside in water or highly aqueous solutions provides minor yields and since the enzymatic glycosylation of rubusoside is very difficult, since the enzymes are not or less active in organic solvents such as ethanol or methanol, the extraction and glycosylation is currently a two-step approach using different solvents. A two-step approach is typically more cost-intense and time consuming than a single approach, particularly in case a solvent has to be exchanged. It is thus also desired to find advantageous options for saving time and costs when extracting and glycosylating rubusoside.

[0021] Therefore, a great need exists to provide methods for producing an extract containing rubusoside, wherein the extract can be used for products for nutrition or pleasure or for pharmaceutical products and wherein the sensory characteristics of rubusoside are acceptable or even advantageous.

[0022] The primary object of the present invention was thus to provide a method for producing an extract containing a possibly high amount of rubusoside with a non-toxic and non-expen-sive solvent, preferably wherein the extract provides acceptable or even advantageous sensory characteristics.

[0023] The primary object of the present invention is solved by a method for producing a plant extract comprising rubusoside, wherein the method comprises or consists of the following steps

[0024] i) providing plant material comprising rubusoside,

[0025] preferably wherein the plant material predominantly consists of plant leafs, preferably wherein the plant material is of or contains material from a plant of the genus Rubus, Stevia, and / or Hydrangea, preferably of the species Rubus chingii, Stevia rebaudiana, and / or Hydrangea strigosa, more preferably selected from the group consisting of the variety Rubus chingii suavissimus,

[0026] ii) drying the provided plant material,iii) subjecting the dried plant material of step ii) to an extraction with a natural deep eutectic solvent, at a temperature in the range of from 0° C to the boiling point of the respective solvent,

[0027] wherein the natural deep eutectic solvent comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,

[0028] wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, choline chloride and lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L-Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid, citric acid and 1 ,2-propane diol, citric acid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose,

[0029] iv) optionally: collecting the supernatant (S1) of the extraction of step iii) and

[0030] optionally: repeating step iii), collecting the supernatant (S2) and combining supernatants S1 and S2, and

[0031] v) filtering the mixture obtained after step iii) or the supernatant or combined supernatants obtained after step iv) and collecting the filtrate.

[0032] It was found that many natural deep eutectic solvents are suitable for extracting rubusoside and that the extraction yield is surprisingly different depending on the particular natural deep eutectic solvent, wherein the above natural deep eutectic solvents provided particularly high yields. Further, it was found that such extracts provided particularly good sensory characteristics.The term “rubusoside”, as used herein, is preferably described by the CAS number 64849-39-4 (as listed by the European Chemicals Agency) and preferably corresponds to the following chemical structure

[0033]

[0034] Preferably the term “rubusoside” as used herein, refers to the compound according to the chemical structure above, as well as its stereoisomers, and / or their pharmaceutically and cosmetically acceptable salt(s).

[0035] Preferably, the term “drying”, as used herein, refers to a reduction of the water content by at least 20 wt.-%, preferably at least 40 wt.-%, further preferably at least 50 wt.-%, even more preferably at least 60 wt.-%, particularly preferably at least 70 wt.-%, especially preferably at least 80 wt.-%, based on the total weight of the water content before drying.

[0036] Preferably, the term “drying” as used herein, refers to a process, in which the water content of the material or mixture is reduced to a residual water content of less than 5 wt.-%, preferably less than 2.5 wt.-%, particularly preferably less than 1 wt.-%, based on the total weight of the material or mixture. Preferably, the step of drying in step ii) of the method according to the invention is performed by a method selected from the group consisting of heat drying, sun drying, hot air drying, combined air and heat pump drying, vacuum oven drying, freeze drying.Preferably, a water content, as described herein, is determined by dry-loss-method or Karl-Fischer titration.

[0037] It is preferred that the dry matter content of the dried material or mixture obtained after step ii), i.e. the portion, which is not water, is at least 95 wt.-% preferably at least 97.5 wt.-%, particularly preferably at least 99 wt.-%, based on the total weight of the dried material or mixture.

[0038] The term “wherein the plant material predominantly consists of’ as used herein refers to an amount of at least 90 wt.-%, preferably at least 95 wt.-%, particularly preferably at least 98 wt.-%, especially preferably at least 99 wt.-%, based on the total weight of the plant material.

[0039] Natural deep eutectic solvents are mixtures of at least two components where the mixture shows a melting point (much) lower than either of the individual components. Preferably natural components such as sugars, alcohols, amines, organic acids or amino acids are used as such solvents.

[0040] Natural deep eutectic solvents are mixtures of typical substances in laboratories and are easily available at lower costs. Furthermore, natural deep eutectic solvents are biologically degradable and regenerative.

[0041] The term “boiling point” as used herein describes the boiling point at standard conditions, i.e. a pressure of 1013 mbar. In case it is referred to the boiling point of a mixture of substances, the skilled person knows that the boiling points of the respective single substances are known in literature and a boiling point can be calculated accordingly for the mixture. In this case, the term “boiling point” describes the calculated boiling point.

[0042] Preferably, the term “boiling point” refers to the degradation temperature or, respectively, the decomposition point of the applied natural deep eutectic solvent.

[0043] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,2-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,3-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0044] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and lactic acid, preferably wherein the molar ratio of choline chloride to lactic acid is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0045] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and lactic acid, preferably wherein the molar ratio of L-Proline to lactic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0046] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glycerol, preferably wherein the molar ratio of L-Proline to glycerol is in a range of from 1 :1 to 1 :5, preferably of from 1 :2 to 1 :3.

[0047] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.2-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0048] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.3-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0049] It is preferred that the natural deep eutectic solvent is or comprises citric acid and adonitol, preferably wherein the molar ratio of citric acid to adonitol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0050] It is preferred that the natural deep eutectic solvent is or comprises betaine and malic acid, preferably wherein the molar ratio of betaine to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0051] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,2-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,2-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,3-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,3-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0052] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and glucose, preferably wherein the molar ratio of lactic acid to glucose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0053] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and xylitol, preferably wherein the molar ratio of choline chloride to xylitol is in a range of from 1 :1 to 1 :10, preferably of from 1 :2.5 to 1 :5.

[0054] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,2-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0055] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,3-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0056] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and glycerol, preferably wherein the molar ratio of choline chloride to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0057] It is preferred that the natural deep eutectic solvent is or comprises betaine and citric acid, preferably wherein the molar ratio of betaine to citric acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0058] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,2-propane diol, preferably wherein the molar ratio of citric acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0059] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,3-propane diol, preferably wherein the molar ratio of citric acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises L-Proline and sucrose, preferably wherein the molar ratio of L-Proline to sucrose is in a range of from 7.5:1 to 1 :1, preferably of from 5:1 to 2:1.

[0060] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glucose, preferably wherein the molar ratio of L-Proline to glucose is in a range of from 5:1 to 1 :2, preferably of from 2:1 to 1 :1.

[0061] It is preferred that the natural deep eutectic solvent is or comprises betaine and glycerol, preferably wherein the molar ratio of betaine to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0062] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and xylitol, preferably wherein the molar ratio of L-Proline to xylitol is in a range of from 2:1 to 1 :3, preferably of from 1 :1 to 1 :2.

[0063] It is preferred that the natural deep eutectic solvent is or comprises citric acid and maltose, preferably wherein the molar ratio of citric acid to maltose is in a range of from 10:1 to 1 :1 , preferably of from 5:1 to 2.5:1.

[0064] It is preferred that the natural deep eutectic solvent is or comprises citric acid and fructose, preferably wherein the molar ratio of citric acid to fructose is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0065] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and malic acid, preferably wherein the molar ratio of L-Proline to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0066] It is preferred that the natural deep eutectic solvent is or comprises and lactic acid and sucrose, preferably wherein the molar ratio of lactic acid to sucrose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0067] Preferably the natural deep eutectic solvent further comprises water. Preferably, the natural deep eutectic solvent is a natural deep eutectic solvent as described above and with the preferred molar ratios and further comprises water. Preferably, the amount of water in the natural deep eutectic solvent is in a range of from 40 to 20 wt.-%, preferably of from 35 to25 wt.-%, particularly preferably of from 32.5 to 27.5 wt.-%, based on the total weight of the natural deep eutectic solvent.

[0068] It is preferred in step iii) that the molar ratio of the or of two components (preferably not referring to water, if present) of the natural deep eutectic solvent is in the range of from 10:1 to 1 :10, preferably in the range of from 7.5:1 to 1 :7.5, particularly preferably in the range of from 5:1 to 1 :5, even further preferably in the range of from 2.5:1 to 1 :2.5.

[0069] It is preferred in step iii) that the mixture comprising the dried plant material of step ii) and the natural deep eutectic solvent is stirred.

[0070] It is preferred in step iii) that the extraction is performed while stirring.

[0071] It is preferred that step iii) of the method according to the invention is performed at normal pressure, i.e. a pressure of 1013 mbar (preferably including a deviation of up to 10%). Alternatively, it is preferred that step iii) is performed at reduced pressure. Alternatively, it is preferred that step iii) is performed at elevated pressure.

[0072] Preferably, the term “reduced pressure” describes a pressure lower than normal pressure, i.e. lower than 1013 mbar, preferably lower than 911.7 mbar (i.e. 1013 mbar minus 10%).

[0073] Preferably, the term “elevated pressure” describes a pressure higher than normal pressure, i.e. higher than 1013 mbar, preferably higher than 1114.3 mbar (i.e. 1013 mbar plus 10%).

[0074] It is further preferred that the extraction in step iii) of the method according to the invention is performed by a method comprising or consisting of a step selected from the group consisting of counter-current, percolation and maceration.

[0075] The repeated step iii) as described in step iv) of the method according to the invention does not necessarily need to be performed the same temperature and time of the extraction and with the same solvent, provided that the temperature and time and solvent of the extraction in the repeated step are still within the limits described for (initial) step iii). Preferably, the repeated step iii) is performed at the same temperature as (initial) step iii). Preferably, the repeated step iii) is performed for the same time as (initial) step iii). Preferably, the repeated step iii) is performed at the same temperature and for the same time as (initial) step iii). Preferably, the repeated step iii) is performed with the same solvent as (initial) step iii). Preferably, the repeated step iii) is performed at the same temperature and with the samesolvent as (initial) step iii). Preferably, the repeated step iii) is performed for the same time and the same solvent as (initial) step iii). Preferably, the repeated step iii) is performed at the same temperature and for the same time and with the same solvent as (initial) step iii).

[0076] Preferably, step iii) is repeated once. Preferably, step iii) is repeated twice. Preferably, step iii) is repeated three times. Preferably, step iii) is repeated four times. Preferably, step iii) is repeated five times. Preferably, step iii) is not repeated.

[0077] It is preferred for the method according to the invention that the extraction in step iii) and / or repeated step iii) is performed for a time in the range of from 0.25 to 10 hours, preferably 0.5 to 5 hours, particularly preferably from 0.75 to 2 hours.

[0078] It is preferred for the method according to the invention that the temperature of the extraction in step iii) and / or in repeated step iii) is in the range of from 10 to 40 °C, preferably of from 15 to 30 °C, particularly preferably of from 20 to 25 °C.

[0079] Preferably, steps iv) and v) are performed simultaneously. In this case, the supernatant corresponds to the filtrate and in case step iii) is repeated, the retentate is subjected to the extraction in repeated step iii).

[0080] Preferably, the filtration in step v) is performed with a filter having a mesh size of at most 15 pm, preferably at most 10 pm. Additionally or alternatively, the term “filtration” or “filtering step” preferably refers to or includes nanofiltration or ultrafiltration.

[0081] It is preferred for the method according to the invention that the method further comprises the step

[0082] vi) purifying the mixture obtained after step iii) and / or supernatant and / or combined supernatants obtained after step iv) and / or the filtrate obtained after step v),

[0083] preferably by a method selected from the group consisting of precipitation, centrifugation, crystallization, solid-phase adsorption, reverse osmosis and combinations thereof,

[0084] particularly preferably by solid-phase adsorption.The term “purifying”, as used herein, may but does not necessarily refer to an isolation of the respective compound(s). Preferably, the term “purifying”, as used herein, refers to an increase of the concentration of the respective compound(s). Preferably, the described purification is conducted in addition to the filtration in step v).

[0085] Additionally in the above case, it is also preferred that the adsorbent used in the solidphase adsorption is selected from the group consisting of polystyrene, aliphatic methyl acrylate and mixtures thereof,

[0086] preferably wherein the adsorbent is polystyrene or a mixture of polystyrene and aliphatic methyl acrylate and wherein the polystyrene is cross-linked, preferably with a divinyl benzene,

[0087] particularly preferably wherein the adsorbent is polystyrene or a mixture of polystyrene and aliphatic methyl acrylate and wherein the polystyrene is cross-linked, preferably with a divinyl benzene, and wherein the polystyrene is macroporous and / or wherein the aliphatic methyl acrylate is microporous.

[0088] The term “macroporous” as used herein describes a material with pores having an average pore size of more than 2 nm, preferably a pore size of from 5 to 110 nm. The average pore size is the measured or calculated average over the size of all pores in the material. Methods for determining the pore size are well-known and include e.g. the Hg intrusion method.

[0089] The term “microporous” as used herein describes a material with pores having an average pore size of equal or less than 2 nm. The average pore size is the measured or calculated average over the size of all pores in the material. Methods for determining the pore size are well-known and include e.g. the Hg intrusion method.

[0090] Preferably, the term “a divinyl benzene” describes a divinyl benzene selected from the group consisting of 1 ,2-Divinylbenzol, 1 ,3-Divinylbenzol, 1 ,4-Divinylbenzol and mixtures thereof.

[0091] It is clear to a skilled person that after a solid-phase adsorption, as described above, an eluate is obtained. Preferably, the purified filtrate is eluted with ethanol, methanol, propanol, isopropanol, n-butanol, ethylacetate, supercritical water, supercritical carbon dioxide, particularly preferably with ethanol.It is further preferred that the method according to the invention comprises the step

[0092] diluting the filtrate collected in step v) with a diluent,

[0093] preferably wherein the diluent is selected from the group consisting of water; protic solvents, such as alcohols, preferably methanol, ethanol or isopropanol, and carbonic acids, preferably formic acid, acetic acid, propanoic acid; and mixtures thereof. Preferably, in case the filtrate is diluted, subsequent steps performed with the filtrate, as described herein, are performed with the diluted filtrate, if applicable.

[0094] It is preferred that the method according to the invention comprises the step

[0095] vii) removing natural deep eutectic solvent from the mixture obtained after step iii) and / or the supernatant and / or combined supernatants obtained after step iv) and / or from the filtrate obtained after step v) and / or from the purified filtrate obtained after step vi),

[0096] preferably by a method comprising an evaporation of natural deep eutectic solvent

[0097] particularly preferably wherein the method comprises step vi), wherein the purification includes a solid-phase adsorption, wherein the removal in step vii) is performed by a method comprising an evaporation of natural deep eutectic solvent with a rotary evaporator.

[0098] Preferably, the term “removing natural deep eutectic solvent” describes a partial reduction of the natural deep eutectic solvent, preferably by at least 10 wt.-%, preferably at least 20 wt.-%, further preferably at least 30 wt.-%, particularly preferably at least 40 wt.-%, even further preferably at least 50 wt.-%, based on the total weight of the natural deep eutectic solvent present in the filtrate obtained after step v) and / or from the purified filtrate obtained after step vi). Particularly preferably the term “removing natural deep eutectic solvent” describes an essentially complete reduction of the natural deep eutectic solvent, preferably by at least 85 wt.-%, further preferably at least 90 wt.-%, particularly preferably at least 95 wt.-%, even further preferably at least 98 wt.-%, more preferably at least 99 wt.-%, based on the total weight of the natural deep eutectic solvent present in the filtrate obtained after step v) and / or from the purified filtrate obtained after step vi).The method according to the invention may comprise step vi), as well as step vii). However, the method may also comprise step vi) but no step vii). Likewise, the method may also comprise step vii) but no step vi). The method according to the invention may also not comprise step vi) or step vii).

[0099] The present invention further relates to a method for glycosylating rubusoside, comprising the steps

[0100] a) providing rubusoside, preferably by a method according to the invention,

[0101] b) providing a natural deep eutectic solvent and a glycosyl donor,

[0102] wherein the natural deep eutectic solvent comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,

[0103] wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, choline chloride and lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L-Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid, citric acid and 1 ,2-propane diol, citric acid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose,

[0104] c) providing a glycosyltransferase,

[0105] d) mixing the rubusoside provided in step a), the natural deep eutectic solvent provided in step b) and the glycosyltransferase provided in step c),

[0106] e) incubating the mixture for at least 10 hours, preferably at least 10 hours, further preferably at least 15 hours, particularly preferably at least 20 hours, even further preferably at least 24 hours,at a temperature in the range of from 10 to 60 °C, preferably of from 20 to 50 °C, further preferably of from 30 to 40 °C, particularly preferably of from 32 to 39 °C, more preferably of from 35 to 38 °C.

[0107] It was surprisingly found that the respective natural deep eutectic solvents can not only be used in a method for producing a plant extract, but also in a method for glycosylating rubu-soside. It was thus surprisingly found that no change of solvent is required between the method for producing a plant extract and the subsequent method for glycosylating the obtained rubusoside. Thus, preferably no change of solvent is performed in or between steps a) to e) of the above method. However, the same solvent may be added in one or more of the steps. Preferably the term “no change of solvent” is understood such that a solvent as described for the method for producing a plant extract may be changed by another solvent as described for the method for producing a plant, preferably by a solvent as described for the method for method for glycosylating rubusoside.

[0108] What was said above with regard to the method for producing a plant extract according to the invention applies accordingly to the method for glycosylating rubusoside according to the invention, if applicable, particularly the preferred features.

[0109] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,2-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0110] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,3-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0111] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and lactic acid, preferably wherein the molar ratio of choline chloride to lactic acid is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0112] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and lactic acid, preferably wherein the molar ratio of L-Proline to lactic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glycerol, preferably wherein the molar ratio of L-Proline to glycerol is in a range of from 1 :1 to 1 :5, preferably of from 1 :2 to 1 :3.

[0113] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.2-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0114] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.3-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0115] It is preferred that the natural deep eutectic solvent is or comprises citric acid and adonitol, preferably wherein the molar ratio of citric acid to adonitol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0116] It is preferred that the natural deep eutectic solvent is or comprises betaine and malic acid, preferably wherein the molar ratio of betaine to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0117] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,2-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,2-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0118] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,3-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,3-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0119] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and glucose, preferably wherein the molar ratio of lactic acid to glucose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0120] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and xylitol, preferably wherein the molar ratio of choline chloride to xylitol is in a range of from 1 :1 to 1 :10, preferably of from 1 :2.5 to 1 :5.It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,2-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0121] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,3-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0122] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and glycerol, preferably wherein the molar ratio of choline chloride to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0123] It is preferred that the natural deep eutectic solvent is or comprises betaine and citric acid, preferably wherein the molar ratio of betaine to citric acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0124] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,2-propane diol, preferably wherein the molar ratio of citric acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0125] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,3-propane diol, preferably wherein the molar ratio of citric acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0126] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and sucrose, preferably wherein the molar ratio of L-Proline to sucrose is in a range of from 7.5:1 to 1 :1 , preferably of from 5:1 to 2:1.

[0127] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glucose, preferably wherein the molar ratio of L-Proline to glucose is in a range of from 5:1 to 1 :2, preferably of from 2:1 to 1 :1.

[0128] It is preferred that the natural deep eutectic solvent is or comprises betaine and glycerol, preferably wherein the molar ratio of betaine to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises L-Proline and xylitol, preferably wherein the molar ratio of L-Proline to xylitol is in a range of from 2:1 to 1 :3, preferably of from 1 :1 to 1 :2.

[0129] It is preferred that the natural deep eutectic solvent is or comprises citric acid and maltose, preferably wherein the molar ratio of citric acid to maltose is in a range of from 10:1 to 1 :1 , preferably of from 5:1 to 2.5:1.

[0130] It is preferred that the natural deep eutectic solvent is or comprises citric acid and fructose, preferably wherein the molar ratio of citric acid to fructose is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0131] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and malic acid, preferably wherein the molar ratio of L-Proline to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0132] It is preferred that the natural deep eutectic solvent is or comprises and lactic acid and sucrose, preferably wherein the molar ratio of lactic acid to sucrose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0133] Preferably the natural deep eutectic solvent further comprises water. Preferably, the natural deep eutectic solvent is a natural deep eutectic solvent as described above and with the preferred molar ratios and further comprises water. Preferably, the amount of water in the natural deep eutectic solvent is in a range of from 40 to 20 wt.-%, preferably of from 35 to 25 wt.-%, particularly preferably of from 32.5 to 27.5 wt.-%, based on the total weight of the natural deep eutectic solvent.

[0134] It is preferred in step b) and / or d) and / or e) that the molar ratio of the or of two components (preferably not referring to water, if present) of the natural deep eutectic solvent is in the range of from 10:1 to 1 :10, preferably in the range of from 7.5:1 to 1 :7.5, particularly preferably in the range of from 5:1 to 1 :5, even further preferably in the range of from 2.5:1 to 1 :2.5.

[0135] Preferably, the term “providing a natural deep eutectic solvent and a glycosyl donor", as used herein, refers to or includes providing a natural deep eutectic solvent and a saccharide donor separately. Preferably, the term “providing a natural deep eutectic solvent and a glycosyl donor", as used herein, refers to or includes providing a natural deep eutectic solventand a saccharide donor as a mixture comprising the natural deep eutectic solvent and the saccharide donor.

[0136] Preferably, the natural deep eutectic solvent may already comprise a saccharide donor. In these cases, the term “providing a natural deep eutectic solvent and a glycosyl donor", as used herein, may refer to providing the natural deep eutectic solvent or to providing the natural deep eutectic solvent and an additional saccharide donor.

[0137] Preferably, the term “glycosyl donor”, as used herein, refers to or includes a carbohydrate, such as a mono- or oligosaccharide, that will react with another compound (glycosyl acceptor) to form a new glycosidic bond.

[0138] Preferably, the term “glycosyl donor”, as used herein, refers to or includes alpha- or betaglucans, maltodextrin, alpha-, beta-, gamma-cyclodextrin, sucrose, inulin, cellulose, hemicellulose, maltose, maltobiose, starch, amylose, amylopectine, starch derivatives, maltotriose, raffinose, fructose, lactose, ribose, ribulose, arabinose, xylose, glucose, mannose, galactose, glycerol, rhamnose, hesperidose, rutinose, and further homogenous or mixed oligosaccharides based on the aforementioned monosaccharides and mixtures thereof.

[0139] Preferably, the term “glycosyl donor”, as used herein, refers to or includes a carbohydrate with at least one glucose unit and preferably includes one or more selected from alphaglucans, maltodextrin, alpha-, beta-, gamma-cyclodextrin, sucrose, maltose, maltobiose, starch, amylose, amylopectine, starch derivatives, maltotriose, raffinose, lactose, glucose, hesperidose, rutinose, and further homogenous or mixed oligosaccharides based on the aforementioned monosaccharides and mixtures containing at least one glucose unit.

[0140] Preferably, the term “glycosyl donor” refers to or includes a glucosyl donor. Preferably, the term “glucosyl donor”, as used herein, refers to or includes a carbohydrate, such as a mono-or oligosaccharide, that will react with another compound (glucosyl acceptor) to form a new glucosidic bond.

[0141] It is preferred that the glycosyltransferase provided in step c) refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 2.4 (https: / / enzyme.expasy.Org / EC / 2.4.-.-), preferably with the EC class E.C. 2.4.1 , particularly preferably a glucosyltransferase (preferably with the EC class E.C. 2.4.1), e.g. fructosyl-transferase, dextransucrase, 1 ,4-a-glucan 6-a-glucosyltransferase, alternansucrase, cy-clomaltodextrin glucanotransferase (alpha-CGTase), 4-alpha-glucanotransferase, 1 ,4-al-pha-glucan 6-alpha-glucosyltransferase, 1 ,4-alpha-glucan branching enzyme. Further preferably, the glycosyltransferase is or comprises a cyclodextrin glycosyltransferase, even further preferably a cyclodextrin glucanotransferase, more preferably liquid cyclodextrin glucanotransferase (CGTase (IUBMB No. 2.4.1 .19)) from Geobacillus stearothermophilus.

[0142] It is preferred that the glycosylation refers to a glucosylation.

[0143] It is preferred that step e) of the method according to the invention is performed at normal pressure, i.e. a pressure of 1013 mbar (preferably including a deviation of up to 10%). Alternatively, it is preferred that step e) is performed at reduced pressure. Alternatively, it is preferred that step e) is performed at elevated pressure.

[0144] Preferably, the term “reduced pressure” describes a pressure lower than normal pressure, i.e. lower than 1013 mbar, preferably lower than 911.7 mbar (i.e. 1013 mbar minus 10%).

[0145] Preferably, the term “elevated pressure” describes a pressure higher than normal pressure, i.e. higher than 1013 mbar, preferably higher than 1114.3 mbar (i.e. 1013 mbar plus 10%).

[0146] In step d) of the method according to the invention, the rubusoside provided in step a), the natural deep eutectic solvent provided in step b), the glycosyl donor provided in step b), and the glycosyltransferase provided in step c) are mixed. However, the sequence of mixing may be any possible sequence. Thus, the rubusoside provided in step a) and the natural deep eutectic solvent provided in step b) may be mixed first and then the glycosyl donor provided in step b) and / or the glycosyltransferase provided in step c) is admixed. Likewise, the rubusoside provided in step a) and the glycosyltransferase provided in step c) may be mixed first and then the glycosyl donor provided in step b) and / or the natural deep eutectic solvent provided in step b) is admixed. Likewise, the natural deep eutectic solvent provided in step b) and the glycosyltransferase provided in step c) may be mixed first and then the glycosyl donor provided in step b) and / or the rubusoside provided in step a) is admixed. Likewise, all components may be mixed at the same time. Preferably, the glycosyl donor provided in step b) is admixed before or together with the glycosyltransferase provided in step c).

[0147] It is preferred for the method according to the invention that the glycosyltransferase is provided in step b) in a cell lysate of cells expressing the glycosyltransferase.The term “cell lysate of cells expressing the glycosyltransferase” refers to a lysate of cells, which expressed the glycosyltransferase before lysis. Preferably, the expression is a homologous expression. Preferably, the expression is a heterologous expression. Preferably, the expression is a homologous expression combined with a heterologous expression.

[0148] Preferably, the incubation in step e) of the method according to the invention may be stopped by inactivating the provided glycosyltransferase, such as by increasing the temperature to at least 40 °C, preferably at least 50 °C, particularly preferably at least 60 °C, further preferably at least 70 °C, more preferably at least 80 °C. Particularly preferably, inactivating the provided glycosyltransferase is achieved by spray drying the mixture of step e). Further preferably, in case the glycosyltransferase is inactivated by increasing the temperature, the temperature for inactivating the glycosyltransferase is higher than the temperature in step e).

[0149] The present invention further relates to the use of a natural deep eutectic solvent in a method for glycosylating rubusoside according to the invention,

[0150] wherein the natural deep eutectic solvent comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,

[0151] wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, choline chloride and lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L-Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid, citric acid and 1 ,2-propane diol, citric acid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose,

[0152] What was said above with regard to the method for producing a plant extract according to the invention and / or the method for glycosylating rubusoside according to the invention,applies accordingly to the use according to the invention, if applicable, particularly the preferred features.

[0153] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,2-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0154] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,3-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0155] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and lactic acid, preferably wherein the molar ratio of choline chloride to lactic acid is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0156] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and lactic acid, preferably wherein the molar ratio of L-Proline to lactic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0157] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glycerol, preferably wherein the molar ratio of L-Proline to glycerol is in a range of from 1 :1 to 1 :5, preferably of from 1 :2 to 1 :3.

[0158] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1 ,2-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0159] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1 ,3-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0160] It is preferred that the natural deep eutectic solvent is or comprises citric acid and adonitol, preferably wherein the molar ratio of citric acid to adonitol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises betaine and malic acid, preferably wherein the molar ratio of betaine to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0161] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,2-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,2-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0162] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,3-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,3-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0163] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and glucose, preferably wherein the molar ratio of lactic acid to glucose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0164] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and xylitol, preferably wherein the molar ratio of choline chloride to xylitol is in a range of from 1 :1 to 1 :10, preferably of from 1 :2.5 to 1 :5.

[0165] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,2-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0166] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,3-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0167] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and glycerol, preferably wherein the molar ratio of choline chloride to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0168] It is preferred that the natural deep eutectic solvent is or comprises betaine and citric acid, preferably wherein the molar ratio of betaine to citric acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,2-propane diol, preferably wherein the molar ratio of citric acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0169] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,3-propane diol, preferably wherein the molar ratio of citric acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0170] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and sucrose, preferably wherein the molar ratio of L-Proline to sucrose is in a range of from 7.5:1 to 1 :1 , preferably of from 5:1 to 2:1.

[0171] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glucose, preferably wherein the molar ratio of L-Proline to glucose is in a range of from 5:1 to 1 :2, preferably of from 2:1 to 1 :1.

[0172] It is preferred that the natural deep eutectic solvent is or comprises betaine and glycerol, preferably wherein the molar ratio of betaine to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0173] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and xylitol, preferably wherein the molar ratio of L-Proline to xylitol is in a range of from 2:1 to 1 :3, preferably of from 1 :1 to 1 :2.

[0174] It is preferred that the natural deep eutectic solvent is or comprises citric acid and maltose, preferably wherein the molar ratio of citric acid to maltose is in a range of from 10:1 to 1 :1 , preferably of from 5:1 to 2.5:1.

[0175] It is preferred that the natural deep eutectic solvent is or comprises citric acid and fructose, preferably wherein the molar ratio of citric acid to fructose is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0176] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and malic acid, preferably wherein the molar ratio of L-Proline to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises and lactic acid and sucrose, preferably wherein the molar ratio of lactic acid to sucrose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0177] Preferably the natural deep eutectic solvent further comprises water. Preferably, the natural deep eutectic solvent is a natural deep eutectic solvent as described above and with the preferred molar ratios and further comprises water. Preferably, the amount of water in the natural deep eutectic solvent is in a range of from 40 to 20 wt.-%, preferably of from 35 to 25 wt.-%, particularly preferably of from 32.5 to 27.5 wt.-%, based on the total weight of the natural deep eutectic solvent.

[0178] It is preferred in step b) and / or d) and / or e) that the molar ratio of the or of two components (preferably not referring to water, if present) of the natural deep eutectic solvent is in the range of from 10:1 to 1 :10, preferably in the range of from 7.5:1 to 1 :7.5, particularly preferably in the range of from 5:1 to 1 :5, even further preferably in the range of from 2.5:1 to 1 :2.5.

[0179] The present invention further relates to a method for glycosylating rubusoside, comprising the steps

[0180] 1) performing the method for producing a plant extract according to the invention,

[0181] 2) providing and admixing a glycosyltransferase

[0182] wherein step 2) is performed before step ii), wherein in step ii) a mixture of the plant material provided in step i) and the glycosyltransferase provided in step 2) is dried, and wherein in step iii) the dried mixture is subjected to the extraction, and / or

[0183] wherein step 2) is performed before step iii) and in step iii) a mixture of the dried plant material obtained after step ii) and the glycosyltransferase provided in step 2) is subjected to the extraction, and / or

[0184] wherein step 2) is performed before repeated step iii) and in repeated step iii) a mixture of the dried plant material obtained after step ii) and the glycosyltransferase provided in step 2) is subjected to the extraction, and / orwherein step 2) is performed before step v) and in step v) a mixture of the supernatant or combined supernatants obtained after step iv) and the glycosyltransferase provided in step 2) is filtered, and / or

[0185] wherein step 2) is performed before step vi) and in step vi) a mixture of the filtrate obtained after step v) and the glycosyltransferase provided in step 2) is purified, and / or

[0186] wherein step 2) is performed before step vii) and in step vii) natural deep eutectic solvent is removed from a mixture of the filtrate obtained after step v) and the glycosyltransferase provided in step 2), and / or

[0187] wherein step 2) is performed before step vii) and in step vii) natural deep eutectic solvent is removed from a mixture of the purified filtrate obtained after step vi) and the glycosyltransferase provided in step 2).

[0188] What was said above with regard to the method for producing a plant extract according to the invention and / or the method for glycosylating rubusoside according to the invention and / or the use according to the invention, applies accordingly to the above method for glycosylating rubusoside according to the invention, if applicable, particularly the preferred features.

[0189] As described above, in the above method for glycosylating rubusoside according to the invention, the glycosyltransferase may be admixed in any step of the method for producing a plant extract according to the invention.

[0190] It was found that a glycosylation of rubusoside is obtained if at any of the steps of the method for producing a plant extract according to the invention a glycosyltransferase is added.

[0191] Preferably, the term “step 2) is performed before step iii)” describes that step 2) is performed before step iii) but after step ii). The same applies accordingly to the further steps described.

[0192] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,2-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,3-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0193] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and lactic acid, preferably wherein the molar ratio of choline chloride to lactic acid is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0194] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and lactic acid, preferably wherein the molar ratio of L-Proline to lactic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0195] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glycerol, preferably wherein the molar ratio of L-Proline to glycerol is in a range of from 1 :1 to 1 :5, preferably of from 1 :2 to 1 :3.

[0196] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.2-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0197] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.3-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0198] It is preferred that the natural deep eutectic solvent is or comprises citric acid and adonitol, preferably wherein the molar ratio of citric acid to adonitol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0199] It is preferred that the natural deep eutectic solvent is or comprises betaine and malic acid, preferably wherein the molar ratio of betaine to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0200] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,2-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,2-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,3-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,3-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0201] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and glucose, preferably wherein the molar ratio of lactic acid to glucose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0202] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and xylitol, preferably wherein the molar ratio of choline chloride to xylitol is in a range of from 1 :1 to 1 :10, preferably of from 1 :2.5 to 1 :5.

[0203] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,2-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0204] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,3-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0205] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and glycerol, preferably wherein the molar ratio of choline chloride to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0206] It is preferred that the natural deep eutectic solvent is or comprises betaine and citric acid, preferably wherein the molar ratio of betaine to citric acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0207] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,2-propane diol, preferably wherein the molar ratio of citric acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0208] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,3-propane diol, preferably wherein the molar ratio of citric acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises L-Proline and sucrose, preferably wherein the molar ratio of L-Proline to sucrose is in a range of from 7.5:1 to 1 :1 , preferably of from 5:1 to 2:1.

[0209] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glucose, preferably wherein the molar ratio of L-Proline to glucose is in a range of from 5:1 to 1 :2, preferably of from 2:1 to 1 :1.

[0210] It is preferred that the natural deep eutectic solvent is or comprises betaine and glycerol, preferably wherein the molar ratio of betaine to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0211] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and xylitol, preferably wherein the molar ratio of L-Proline to xylitol is in a range of from 2:1 to 1 :3, preferably of from 1 :1 to 1 :2.

[0212] It is preferred that the natural deep eutectic solvent is or comprises citric acid and maltose, preferably wherein the molar ratio of citric acid to maltose is in a range of from 10:1 to 1 :1 , preferably of from 5:1 to 2.5:1.

[0213] It is preferred that the natural deep eutectic solvent is or comprises citric acid and fructose, preferably wherein the molar ratio of citric acid to fructose is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0214] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and malic acid, preferably wherein the molar ratio of L-Proline to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0215] It is preferred that the natural deep eutectic solvent is or comprises and lactic acid and sucrose, preferably wherein the molar ratio of lactic acid to sucrose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0216] Preferably the natural deep eutectic solvent further comprises water. Preferably, the natural deep eutectic solvent is a natural deep eutectic solvent as described above and with the preferred molar ratios and further comprises water. Preferably, the amount of water in the natural deep eutectic solvent is in a range of from 40 to 20 wt.-%, preferably of from 35 to25 wt.-%, particularly preferably of from 32.5 to 27.5 wt.-%, based on the total weight of the natural deep eutectic solvent.

[0217] It is preferred in step iii) that the molar ratio of the or of two components (preferably not referring to water, if present) of the natural deep eutectic solvent is in the range of from 10:1 to 1 :10, preferably in the range of from 7.5:1 to 1 :7.5, particularly preferably in the range of from 5:1 to 1 :5, even further preferably in the range of from 2.5:1 to 1 :2.5.

[0218] It is preferred that the glycosyltransferase provided in step c) refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 2.4 (https: / / enzyme.expasy.Org / EC / 2.4.-.-), preferably with the EC class E.C. 2.4.1 , particularly preferably a glucosyltransferase (preferably with the EC class E.C. 2.4.1), e.g. fructosyl-transferase, dextransucrase, 1 ,4-a-glucan 6-a-glucosyltransferase, alternansucrase, cyclomaltodextrin glucanotransferase (alpha-CGTase), 4-alpha-glucanotransferase, 1 ,4-al-pha-glucan 6-alpha-glucosyltransferase, 1 ,4-alpha-glucan branching enzyme. Further preferably, the glycosyltransferase is or comprises a cyclodextrin glycosyltransferase, even further preferably a cyclodextrin glucanotransferase, more preferably liquid cyclodextrin glucanotransferase (CGTase (IUBMB No. 2.4.1.19)) from Geobacillus stearothermophilus.

[0219] It is preferred that the glycosylation refers to a glucosylation.

[0220] It is preferred in the method for glycosylating rubusoside according to the invention that the method further comprises the step

[0221] 3) incubating the respective mixture comprising the glycosyltransferase provided in step 2)

[0222] for at least 10 hours, preferably at least 10 hours, further preferably at least 15 hours, particularly preferably at least 20 hours, even further preferably at least 24 hours,

[0223] at a temperature in the range of from 10 to 60 °C, preferably of from 20 to 50 °C, further preferably of from 30 to 40 °C, particularly preferably of from 32 to 39 °C, more preferably of from 35 to 38 °C,

[0224] wherein step 3) is performed at any time after a mixture comprising the glycosyltransferase, as mentioned in the method for glycosylating rubusoside according to the invention is obtained.In the above method for glycosylating rubusoside according to the invention, the glycosyltransferase is added at a given step, as described above. After adding the glycosyltransferase as described, a mixture comprising the glycosyltransferase is obtained. Thus, the term “wherein step c) is performed at anytime after a mixture comprising the glycosyltransferase, as mentioned in claim 10, is obtained” refers to such a mixture. Thus, step 3) is necessarily performed simultaneously with or after step 2), wherein one or more of the steps ii) to vii) may be performed after step 2) and before or together with step 3).

[0225] It is preferred that step 3) is performed before step ii), wherein in step ii) a mixture of the plant material provided in step i) and the obtained glycosylated rubusoside and optionally the glycosyltransferase provided in step 2) is dried, and wherein in step iii) the dried mixture is subjected to the extraction.

[0226] Additionally or alternatively, it is preferred that step 3) is performed before step iii) and in step iii) a mixture of the dried plant material obtained after step ii) and the obtained glycosylated rubusoside and optionally the glycosyltransferase provided in step 2) is subjected to the extraction.

[0227] Additionally or alternatively, it is preferred that step 3) is performed before repeated step iii) and in repeated step iii) a mixture of the dried plant material obtained after step ii) and the obtained glycosylated rubusoside and optionally the glycosyltransferase provided in step 2) is subjected to the extraction.

[0228] Additionally or alternatively, it is preferred that step 3) is performed before step v) and in step v) a mixture of the supernatant or combined supernatants obtained after step iv) and the obtained glycosylated rubusoside and optionally the glycosyltransferase provided in step 2) is filtered.

[0229] Additionally or alternatively, it is preferred that step 3) is performed before step vi) and in step vi) a mixture of the filtrate obtained after step v) and the obtained glycosylated rubusoside and optionally the glycosyltransferase provided in step 2) is purified.

[0230] Additionally or alternatively, it is preferred that step 3) is performed before step vii) and in step vii) natural deep eutectic solvent is removed from a mixture of the filtrate obtained after step v) and the obtained glycosylated rubusoside and optionally the glycosyltransferase provided in step 2).Additionally or alternatively, it is preferred that step 3) is performed before step vii) and in step vii) natural deep eutectic solvent is removed from a mixture of the purified filtrate obtained after step vi) and the obtained glycosylated rubusoside and optionally the glycosyltransferase provided in step 2).

[0231] Preferably, the term “step 3) is performed before step iii)” describes that step 3) is performed before step iii) but after or simultaneous with step ii). The same applies accordingly to the further steps described herein.

[0232] It is preferred in the above method for glycosylating rubusoside according to the invention that the glycosyltransferase is provided in a cell lysate of cells expressing the glycosyltransferase.

[0233] The term “cell lysate of cells expressing the glycosyltransferase” refers to a lysate of cells, which expressed the glycosyltransferase before lysis. Preferably, the expression is a homologous expression. Preferably, the expression is a heterologous expression. Preferably, the expression is a homologous expression combined with a heterologous expression.

[0234] Preferably, the incubation in step 3) of the method according to the invention may be stopped by inactivating the provided glycosyltransferase, such as by increasing the temperature to at least 40 °C, preferably at least 50 °C, particularly preferably at least 60 °C, further preferably at least 70 °C, more preferably at least 80 °C. Particularly preferably, inactivating the provided glycosyltransferase is achieved by spray drying the mixture of step 3). Further preferably, in case the glycosyltransferase is inactivated by increasing the temperature, the temperature for inactivating the glycosyltransferase is higher than the temperature in step 3).

[0235] The present invention further relates to the use of a natural deep eutectic solvent in a method for glycosylating rubusoside according to the invention,

[0236] wherein the natural deep eutectic solvent in step iii) comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,

[0237] wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, choline chlorideand lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L-Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid, citric acid and 1 ,2-propane diol, citric acid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose.

[0238] What was said above with regard to the method for producing a plant extract according to the invention and / or the methods for glycosylating rubusoside according to the invention and / or the use according to the invention, applies accordingly to the above use according to the invention, if applicable, particularly the preferred features.

[0239] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,2-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0240] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and 1 ,3-propane diol, preferably wherein the molar ratio of lactic acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1 .5:1 to 1 :1.5.

[0241] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and lactic acid, preferably wherein the molar ratio of choline chloride to lactic acid is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0242] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and lactic acid, preferably wherein the molar ratio of L-Proline to lactic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0243] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glycerol, preferably wherein the molar ratio of L-Proline to glycerol is in a range of from 1 :1 to 1 :5, preferably of from 1 :2 to 1 :3.It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.2-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0244] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and 1.3-propane diol, preferably wherein the molar ratio of choline chloride to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1 .5.

[0245] It is preferred that the natural deep eutectic solvent is or comprises citric acid and adonitol, preferably wherein the molar ratio of citric acid to adonitol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0246] It is preferred that the natural deep eutectic solvent is or comprises betaine and malic acid, preferably wherein the molar ratio of betaine to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0247] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,2-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,2-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0248] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and 1 ,3-pro-pane diol, preferably wherein the molar ratio of L-Proline to 1 ,3-propane diol is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0249] It is preferred that the natural deep eutectic solvent is or comprises lactic acid and glucose, preferably wherein the molar ratio of lactic acid to glucose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0250] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and xylitol, preferably wherein the molar ratio of choline chloride to xylitol is in a range of from 1 :1 to 1 :10, preferably of from 1 :2.5 to 1 :5.

[0251] It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,2-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.It is preferred that the natural deep eutectic solvent is or comprises betaine and 1 ,3-pro-pane diol, preferably wherein the molar ratio of betaine to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0252] It is preferred that the natural deep eutectic solvent is or comprises choline chloride and glycerol, preferably wherein the molar ratio of choline chloride to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0253] It is preferred that the natural deep eutectic solvent is or comprises betaine and citric acid, preferably wherein the molar ratio of betaine to citric acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0254] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,2-propane diol, preferably wherein the molar ratio of citric acid to 1 ,2-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0255] It is preferred that the natural deep eutectic solvent is or comprises citric acid and 1 ,3-propane diol, preferably wherein the molar ratio of citric acid to 1 ,3-propane diol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0256] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and sucrose, preferably wherein the molar ratio of L-Proline to sucrose is in a range of from 7.5:1 to 1 :1 , preferably of from 5:1 to 2:1.

[0257] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and glucose, preferably wherein the molar ratio of L-Proline to glucose is in a range of from 5:1 to 1 :2, preferably of from 2:1 to 1 :1.

[0258] It is preferred that the natural deep eutectic solvent is or comprises betaine and glycerol, preferably wherein the molar ratio of betaine to glycerol is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0259] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and xylitol, preferably wherein the molar ratio of L-Proline to xylitol is in a range of from 2:1 to 1 :3, preferably of from 1 :1 to 1 :2.It is preferred that the natural deep eutectic solvent is or comprises citric acid and maltose, preferably wherein the molar ratio of citric acid to maltose is in a range of from 10:1 to 1 :1 , preferably of from 5:1 to 2.5:1.

[0260] It is preferred that the natural deep eutectic solvent is or comprises citric acid and fructose, preferably wherein the molar ratio of citric acid to fructose is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0261] It is preferred that the natural deep eutectic solvent is or comprises L-Proline and malic acid, preferably wherein the molar ratio of L-Proline to malic acid is in a range of from 2:1 to 1 :2, preferably of from 1.5:1 to 1 :1.5.

[0262] It is preferred that the natural deep eutectic solvent is or comprises and lactic acid and sucrose, preferably wherein the molar ratio of lactic acid to sucrose is in a range of from 2:1 to 1 :5, preferably of from 1 :1 to 1 :3.

[0263] Preferably the natural deep eutectic solvent further comprises water. Preferably, the natural deep eutectic solvent is a natural deep eutectic solvent as described above and with the preferred molar ratios and further comprises water. Preferably, the amount of water in the natural deep eutectic solvent is in a range of from 40 to 20 wt.-%, preferably of from 35 to 25 wt.-%, particularly preferably of from 32.5 to 27.5 wt.-%, based on the total weight of the natural deep eutectic solvent.

[0264] It is preferred in step iii) that the molar ratio of the or of two components (preferably not referring to water, if present) of the natural deep eutectic solvent is in the range of from 10:1 to 1 :10, preferably in the range of from 7.5:1 to 1 :7.5, particularly preferably in the range of from 5:1 to 1 :5, even further preferably in the range of from 2.5:1 to 1 :2.5.

[0265] The present invention further relates to the use of a glycosyltransferase or a composition comprising a glycosyltransferase in a method for glycosylating rubusoside according to the invention, preferably wherein the composition is or comprises a cell lysate.

[0266] What was said above with regard to any method or use according to the invention applies accordingly to the above use according to the invention, if applicable, particularly the preferred features.It is preferred that the glycosyltransferase provided in step c) refers to an enzyme classified according to the Enzyme Commission number (EC number), with the EC class E.C. 2.4 (https: / / enzyme.expasy.Org / EC / 2.4.-.-), preferably with the EC class E.C. 2.4.1 , particularly preferably a glucosyltransferase (preferably with the EC class E.C. 2.4.1), e.g. fructosyl-transferase, dextransucrase, 1 ,4-a-glucan 6-a-glucosyltransferase, alternansucrase, cyclomaltodextrin glucanotransferase (alpha-CGTase), 4-alpha-glucanotransferase, 1 ,4-al-pha-glucan 6-alpha-glucosyltransferase, 1 ,4-alpha-glucan branching enzyme. Further preferably, the glycosyltransferase is or comprises a cyclodextrin glycosyltransferase, even further preferably a cyclodextrin glucanotransferase, more preferably liquid cyclodextrin glucanotransferase (CGTase (IUBMB No. 2.4.1 .19)) from Geobacillus stearothermophilus.

[0267] It is preferred that the glycosylation refers to a glucosylation.

[0268] The term “cell lysate of cells expressing the glycosyltransferase” refers to a lysate of cells, which expressed the glycosyltransferase before lysis. Preferably, the expression is a homologous expression. Preferably, the expression is a heterologous expression. Preferably, the expression is a homologous expression combined with a heterologous expression.

[0269] It is preferred in the methods and uses according to the invention that 1 ,2-propane diol or, respectively, 1 ,3-propane diol may refer to a mixture comprising 1 ,2-propane diol and 1 ,3-propane diol. In this case, any reference (e.g. molar ratios) to 1 ,2-propane diol or, respectively 1 ,3-propane diol preferably refers to the mixture comprising 1 ,2-propane diol and 1 ,3-propane diol (e.g. the summed mol of 1 ,2-propane diol and 1 ,3-propane diol). Alternatively, any reference (e.g. molar ratios) to 1 ,2-propane diol or, respectively 1 ,3-propane diol preferably still refers to 1 ,2-propane diol or, respectively 1 ,3-propane diol only.

[0270] Further aspects and advantages of the invention result from the subsequent description of preferred examples.Examples

[0271] Example 1 : Preparation of a natural deep eutectic solvent

[0272] The natural deep eutectic solvents were produced as follows:

[0273] The single components (e.g. 1 ,2-propane diol, 1 ,3-propane diol, betaine, malic acid, choline chloride, citric acid, fructose) are weighed and mixed with each other. The mixture is heated to approximately 70 °C under stirring. The mixture is stirred until a clear and homogeneous mixture is obtained.

[0274] Example 2: Extraction of rubusoside

[0275] Leafs of Rubus chingii var. suavissimus were shredded and added to the natural deep eutectic solvent, as produced as in Example 1. The weight ratio of leafs and solvent was approx. 1 :20, however other ratios are also functional.

[0276] The leafs were extracted in the natural deep eutectic solvent for 1 h under stirring at room temperature. Subsequently, the obtained mixture was filtered and the retentate was mixed with fresh natural deep eutectic solvent and extracted under the same conditions as above. In the second extraction step, the natural deep eutectic solvent corresponded to the natural deep eutectic solvent as used in the first extraction steps, however other natural deep eutectic solvents as described herein could have been used as well.

[0277] Example 3: Extraction of rubusoside, comparison of different solvents

[0278] The extraction of rubusoside was conducted as described in Example 2. However, different natural deep eutectic solvents were prepared, as described in Example 1 , and the yield of rubusoside was compared. The water content in the prepared natural deep eutectic solvents was 30 wt.-%, which was kept consistent between the tested natural deep eutectic solvents to analyse effects of the particular selection of the other components (HBA and HBD in the below table).

[0279] The quantitative determination of the rubusoside yields was measured by a HPLC-UV-method with external calibration.

[0280] The following yields were obtained with the respective natural deep eutectic solvents:

[0281]

[0282] ‘similar results were also obtained with 1 ,3-propane diol

[0283]

[0284]

[0285] It was found that the extraction of rubusoside with natural deep eutectic solvents was possible and provided good yields. Particularly, the yields were in a comparable range as the other solvents tested (methanol and ethanol, as well as methanol or ethanol with water).

[0286] It was surprisingly found that strong differences between the natural deep eutectic solvents were observed. The natural deep eutectic solvents described herein provided a much higher yield of rubusoside than other tested natural deep eutectic solvents.

[0287] Example 4: Purification steps

[0288] For purification, two extracts were prepared as described in example 1 , wherein the natural deep eutectic solvent

[0289] lactic acid, 1 ,2-propane diol, molar ratio 1 / 1 , 30% water (extract 1) or

[0290] betaine, malic acid, molar ratio 1 / 1 , 30% water (extract 2)

[0291] was used. The respective extracts were filtered and the obtained filtrates were diluted with water (1 :10 v / v). The obtained mixtures were subsequently centrifuged at 8,000 rpm for 5 min. The supernatants (100 ml) were added on columns (250 x 25 mm; poly(styrene-divi-nylbenzene)). Rubusoside was then eluted with ethanol. Remaining solvent or eluent was evaporated with a rotary evaporator and rubusoside was precipitated. The obtained crystals (crystals 1 from extract 1 ; crystals 2 from extract 2) were then dried.

[0292] The crystals were measured fortheir rubusoside content by HPLC-UV.

[0293] Crystals 1 had a rubusoside content of approx. 172 mg / g, crystals 2 had a rubusoside content of approx. 161 mg / g.

[0294] Example 5: Sensory evaluation

[0295] The crystals prepared in Example 4 were dissolved in glycerol and diluted in water as described below. The obtained mixtures (mixture 1 from crystals 1 ; mixture 2 from crystals 2) were then tested for their sensory profile and compared with commercially available rubusoside, as described below, with a set of panellists (n = 7 panellists).

[0296]

[0297] It was found that with the method for producing a plant extract comprising rubusoside as described herein, a product with desired sensory profile was obtained, which may be used in e.g. food or beverage. The products could be used in reasonable amounts to achieve the same perceived sweetness as with the dilution of commercially available rubusoside (comparison).

[0298] Example 6: Glycosylation

[0299] An extract containing rubusoside was prepared from leafs of Rubus chingii var. suavissi-mus according to Example 2.

[0300] A natural deep eutectic solvent (L-Proline, Sucrose, molar ratio: 3:1 , 30 wt.-% water) was prepared as described in Example 1 and mixed with the extract such that the mixture contains 3mM rubusoside.

[0301] A glycosyltransferase (glucan sucrase GTF180-Q1140E-AN) was expressed in Lactobacillus reute and a cell lysate was prepared.

[0302] The cell lysate was admixed with the above mixture containing natural deep eutectic solvent and rubusoside. The resulting mixture was incubated at 37 °C for 24 h.

[0303] Subsequently, the presence of glycosylated rubusoside was determined by LC-MS.

[0304] It was found that single- and multiple-glycosylated derivatives of rubusoside could be detected.Example 7: Glycosylation, one-pot approach

[0305] A natural deep eutectic solvent (L-Proline, Sucrose, molar ratio: 3:1 , 30 wt.-% water) was prepared as described in Example 1 and mixed with 4 wt.-% of dried leafs of Rubus chingii (containing 5 wt.-% rubusoside) and 10 % of a cell lysate containing a glycosyltransferase (glucan sucrase GTF180-Q1140E-AN; as described in Example 6).

[0306] The resulting mixture was incubated at 37 °C for 24 h. Thus, the extraction and the glycosylation steps were performed simultaneously and at 37 °C.

[0307] Subsequently, the presence of glycosylated rubusoside was determined by LC-MS.

[0308] It was found that single- and multiple-glycosylated derivatives of rubusoside could be de-tected.

Claims

Claims1. Method for producing a plant extract comprising rubusoside, wherein the method comprises or consists of the following stepsi) providing plant material comprising rubusoside,preferably wherein the plant material predominantly consists of plant leafs, preferably wherein the plant material is of or contains material from a plant of the genus Rubus, Stevia, and / or Hydrangea, preferably of the species Rubus chingii, Stevia rebaudiana, and / or Hydrangea strigosa, more preferably selected from the group consisting of the variety Rubus chingii suavissimus,ii) drying the provided plant material,iii) subjecting the dried plant material of step ii) to an extraction with a natural deep eutectic solvent, at a temperature in the range of from 0° C to the boiling point of the respective solvent,wherein the natural deep eutectic solvent comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, choline chloride and lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L- Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid, citric acid and 1 ,2-propane diol, citric acid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose,iv) optionally: collecting the supernatant (S1) of the extraction of step iii) andoptionally: repeating step iii), collecting the supernatant (S2) and combining supernatants S1 and S2, andv) filtering the mixture obtained after step iii) or the supernatant or combined supernatants obtained after step iv) and collecting the filtrate.

2. Method according to claim 1 , wherein the extraction in step iii) and / or repeated step iii) is performed for a time in the range of from 0.25 to 10 hours, preferably 0.5 to 5 hours, particularly preferably from 0.75 to 2 hours.

3. Method according to any of the preceding claims, wherein the temperature of the extraction in step iii) and / or in repeated step iii) is in the range of from 10 to 40 °C, preferably of from 15 to 30 °C, particularly preferably of from 20 to 25 °C.

4. Method according to any of the preceding claims, further comprising the stepvi) purifying the mixture obtained after step iii) and / or supernatant and / or combined supernatants obtained after step iv) and / or the filtrate obtained after step v),preferably by a method selected from the group consisting of precipitation, centrifugation, crystallization, solid-phase adsorption, reverse osmosis and combinations thereof,particularly preferably by solid-phase adsorption.

5. Method according to claim 4, wherein the purification includes a solid-phase adsorption and wherein the adsorbent used in the solid-phase adsorption is selected from the group consisting of polystyrene, aliphatic methyl acrylate and mixtures thereof,preferably wherein the adsorbent is polystyrene or a mixture of polystyrene and aliphatic methyl acrylate and the polystyrene is cross-linked, preferably with a divinyl benzene,particularly preferably wherein the adsorbent is polystyrene or a mixture of polystyrene and aliphatic methyl acrylate and the polystyrene is cross-linked, preferably with a divinyl benzene, and the polystyrene is macroporous.

6. Method according to any of the preceding claims, further comprising the stepvii) removing natural deep eutectic solvent from the mixture obtained after step iii) and / or the supernatant and / or combined supernatants obtained after step iv) and / or from the filtrate obtained after step v) and / or from the purified filtrate obtained after step vi),preferably by a method comprising an evaporation of natural deep eutectic solventparticularly preferably wherein the method comprises step vi), wherein the purification includes a solid-phase adsorption, wherein the removal in step vii) is performed by a method comprising an evaporation of natural deep eutectic solvent with a rotary evaporator.

7. Method for glycosylating rubusoside, comprising the stepsa) providing rubusoside, preferably by a method according to any of the preceding claims,b) providing a natural deep eutectic solvent and a glycosyl donor,wherein the natural deep eutectic solvent comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, choline chloride and lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L- Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid, citric acid and 1 ,2-propane diol, citricacid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose,c) providing a glycosyltransferase,d) mixing the rubusoside provided in step a), the natural deep eutectic solvent provided in step b) and the glycosyltransferase provided in step c),e) incubating the mixture for at least 10 hours, preferably at least 10 hours, further preferably at least 15 hours, particularly preferably at least 20 hours, even further preferably at least 24 hours,at a temperature in the range of from 10 to 60 °C, preferably of from 20 to 50 °C, further preferably of from 30 to 40 °C, particularly preferably of from 32 to 39 °C, more preferably of from 35 to 38 °C.

8. Method for glycosylating rubusoside according to claim 7,wherein the glycosyltransferase is provided as a cell lysate of cells expressing the glycosyltransferase.

9. Use of a natural deep eutectic solvent in a method according to any of claims 7 or 8,wherein the natural deep eutectic solvent comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, choline chloride and lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L-Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid,citric acid and 1 ,2-propane diol, citric acid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose.

10. Method for glycosylating rubusoside, comprising the steps1) performing the method according to any of claims 1 to 6,2) providing and admixing a glycosyltransferasewherein step 2) is performed before step ii), wherein in step ii) a mixture of the plant material provided in step i) and the glycosyltransferase provided in step 2) is dried, and wherein in step iii) the dried mixture is subjected to the extraction, and / orwherein step 2) is performed before step iii) and in step iii) a mixture of the dried plant material obtained after step ii) and the glycosyltransferase provided in step 2) is subjected to the extraction, and / orwherein step 2) is performed before repeated step iii) and in repeated step iii) a mixture of the dried plant material obtained after step ii) and the glycosyltransferase provided in step 2) is subjected to the extraction, and / orwherein step 2) is performed before step v) and in step v) a mixture of the supernatant or combined supernatants obtained after step iv) and the glycosyltransferase provided in step 2) is filtered, and / orwherein step 2) is performed before step vi) and in step vi) a mixture of the filtrate obtained after step v) and the glycosyltransferase provided in step 2) is purified, and / orwherein step 2) is performed before step vii) and in step vii) natural deep eutectic solvent is removed from a mixture of the filtrate obtained after step v) and the glycosyltransferase provided in step 2), and / orwherein step 2) is performed before step vii) and in step vii) natural deep eutectic solvent is removed from a mixture of the purified filtrate obtained after step vi) and the glycosyltransferase provided in step 2).

11. Method for glycosylating rubusoside according to claim 10, further comprising the step3) incubating the respective mixture comprising the glycosyltransferase provided in step 2)for at least 10 hours, preferably at least 10 hours, further preferably at least 15 hours, particularly preferably at least 20 hours, even further preferably at least 24 hours,at a temperature in the range of from 10 to 60 °C, preferably of from 20 to 50 °C, further preferably of from 30 to 40 °C, particularly preferably of from 32 to 39 °C, more preferably of from 35 to 38 °C,wherein step 3) is performed at any time after a mixture comprising the glycosyltransferase, as mentioned in claim 10, is obtained.

12. Method for glycosylating rubusoside according to any of claims 10 to 11 ,wherein the glycosyltransferase is provided as a cell lysate of cells expressing the glycosyltransferase.

13. Use of a natural deep eutectic solvent in a method according to any of claims 1 to 6 and 10 to 12,wherein the natural deep eutectic solvent in step iii) comprises at least 2 components selected from the group consisting of 1 ,2-propane diol, 1 ,3-propane diol, adonitol, betaine, choline chloride, citric acid, fructose, glucose, glycerol, lactic acid, L-Proline, malic acid, maltose, sucrose, and xylitol,wherein the natural deep eutectic solvent is selected from the group consisting of lactic acid and 1 ,2-propane diol, lactic acid and 1 ,3-propane diol, cholinechloride and lactic acid, L-Proline and lactic acid, L-Proline and glycerol, choline chloride and 1 ,2-propane diol, choline chloride and 1 ,3-propane diol, citric acid and adonitol, betaine and malic acid, L-Proline and 1 ,2-propane diol, L- Proline and 1 ,3-propane diol, lactic acid and glucose, choline chloride and xylitol, betaine and 1 ,2-propane diol, betaine and 1 ,3-propane diol, choline chloride and glycerol, betaine and citric acid, citric acid and 1 ,2-propane diol, citric acid and 1 ,3-propane diol, L-Proline and sucrose, L-Proline and glucose, betaine and glycerol, L-Proline and xylitol, citric acid and maltose, citric acid and fructose, L-Proline and malic acid, and lactic acid and sucrose.

14. Use of a glycosyltransferase or a composition comprising a glycosyltransferase in a method according to any of claims 7 to 8 and 10 to 12,preferably wherein the composition is or comprises a cell lysate.

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