METHOD FOR PRODUCING PURIFIED STEVIOL PRODUCT USING SIMULATED MOVING BED CHROMATOGRAPHY
20210379508 · 2021-12-09
Assignee
Inventors
- Babu Siddegowda Antharavally (Caledonia, IL)
- Anil R. Oroskar (Oak Brook, IL)
- Asha A. Oroskar (Oak Brook, IL)
Cpc classification
A23V2002/00
HUMAN NECESSITIES
B01D2311/04
PERFORMING OPERATIONS; TRANSPORTING
B01D61/026
PERFORMING OPERATIONS; TRANSPORTING
B01D15/1842
PERFORMING OPERATIONS; TRANSPORTING
C07H15/24
CHEMISTRY; METALLURGY
B01D2311/08
PERFORMING OPERATIONS; TRANSPORTING
B01D15/185
PERFORMING OPERATIONS; TRANSPORTING
International classification
A23L5/20
HUMAN NECESSITIES
B01D15/36
PERFORMING OPERATIONS; TRANSPORTING
B01D61/02
PERFORMING OPERATIONS; TRANSPORTING
B01D61/14
PERFORMING OPERATIONS; TRANSPORTING
Abstract
Disclosed is a continuous process for the purification of steviol glycosides such as Rebaudioside D and/or Rebaudioside M extracted from the dried stevia leaves or extracted from a fermentation broth using continuous simulated moving bed processes and nanofiltration without the addition of organic solvents to obtain a purified steviol product comprising sweet steviol glycosides. The sweet steviol glycosides can be used as substitutes for caloric sweeteners in beverages and in other food items.
Claims
1. A method of purifying one or more steviol glycosides from a mixture, the mixture including the one or more steviol glycosides and at least one impurity, the method comprising: passing the mixture through a first adsorbent with a first solvent, the first adsorbent arranged in an SMB configuration to form a first SMB zone, wherein passing the mixture through the first adsorbent with the first solvent comprises passing the mixture through the first SMB zone, the first adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange resins to provide a first eluate stream, the first eluate stream having the first solvent and a higher purity of the one or more steviol glycosides than in the mixture as measured by weight percentage of the solid content, and optionally removing at least a portion of the first solvent from the first eluate stream to provide a reduced first eluate stream.
2. The method of claim 1, the method further comprising: passing the first eluate stream or the reduced first eluate stream through a second adsorbent with a second solvent, the second adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange resins to provide a second eluate stream, the second eluate stream having the second solvent and a higher purity of the one or more steviol glycosides than in the first eluate stream or the reduced first eluate stream as measured by weight percentage of the solid content, and optionally removing at least a portion of the second solvent from the second eluate stream to provide a reduced second eluate stream.
3. The method of claim 1, wherein the one or more steviol glycosides are selected from the group consisting of Dulcoside A, Rebaudioside C, Rebaudioside A, Stevioside, Rebaudioside B, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside M, Rebaudioside I, Steviolbioside, and Rubusoside.
4. The method of claim 1, wherein the one or more steviol glycosides are Rebaudioside D and Rebaudioside M.
5. The method of claim 1, wherein the one or more steviol glycosides is Rebaudioside D.
6. The method of claim 1, wherein the one or more steviol glycosides is Rebaudioside M.
7. The method of claim 1, wherein the mixture is a stevia plant extract, a stevia fermentation broth, a yeast fermentation broth, an Escherichia coli fermentation broth, or an enzyme-converted mixture.
8. The method of claim 7, wherein the stevia plant is Stevia rebaudiana Bertoni.
9. The method of claim 2, wherein the first solvent and the second solvent comprise water.
10. The method of claim 9, wherein the first solvent and the second solvent comprise greater than 95% v/v water.
11. The method of claim 10, wherein the first solvent and the second solvent comprise greater than 99% v/v water.
12. The method of claim 11, wherein the first solvent and the second solvent are water.
13. The method of claim 1, wherein said at least one impurity comprises at least one of tri-terpenes, sterols, flavonoids, volatile oils, pigments, gums, proteins, carotenoids, chlorophyll, vitamins, phospholipids, saccharides, solid insoluble, salts, fermentation byproducts, or N-acetyl glucosamine.
14. The method of claim 4, wherein said at least one impurity comprises a second steviol glycoside selected from Dulcoside A, Rebaudioside C, Rebaudioside A, Stevioside, Rebaudioside B, Rebaudioside E, Rebaudioside F, Rebaudioside I, Steviolbioside, Rubusoside, and combinations thereof.
15. The method of claim 2, wherein the first adsorbent comprises a porous hydrophobic interaction resin with a particle size of 70-200 microns.
16. The method of claim 2, wherein the first adsorbent comprises a phenylated polymethacrylate polymer resin.
17. The method of claim 16, wherein the phenylated polymethacrylate polymer resin has a particle size of from about 75 μm to about 200 μm.
18. The method of claim 2, wherein the second adsorbent is one or more ion exchange resins.
19. The method of claim 18, wherein the one or more ion exchange resins is a combination of a cationic resin and an anionic resin.
20. The method of claim 19, wherein the cationic resin and anionic resin are arranged in series.
21. The method of claim 19, wherein the cationic resin is a styrene-divinylbenzene copolymer resin and the anionic resin is an acrylic-divinylbenzene copolymer resin.
22. The method of claim 2, wherein the second adsorbent is arranged in a SMB configuration to form a second SMB zone, and wherein passing the first eluate stream or the reduced first eluate stream through the second adsorbent with the second solvent comprises passing the first eluate stream or the reduced first eluate stream through the second SMB zone.
23. The method of claim 1, the method further comprising: preconditioning the first adsorbent prior to passing the mixture through a first adsorbent with a first solvent.
24. The method of claim 23, wherein preconditioning comprises washing with acid, base, water, or a combination thereof.
25. The method of claim 2, the method further comprising: preconditioning the first adsorbent prior to passing the mixture through a first adsorbent with a first solvent; preconditioning the second adsorbent prior to passing the first eluate stream or the reduced first eluate stream through a second adsorbent with a second solvent.
26. The method of claim 25, wherein preconditioning comprises washing with acid, base, water, or a combination thereof.
27. The method of claim 2, the method further comprising: drying the second eluate stream or the reduced second eluate stream.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The accompanying drawings are included to provide a further understanding of the disclosure. The drawings illustrate embodiments of the disclosure and together with the description serve to explain the principles of the embodiments of the disclosure.
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DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0050] The sweet herb of Paraguay, Stevia rebaudiana Bertoni, produces an alternative sweetener with the added advantage that stevia sweeteners are natural plant products. The extract of the Stevia rebaudiana Bertoni plant contains a mixture of different sweet diterpene glycosides which have a single base, steviol, and differ by the presence of carbohydrate residues at positions C13 and C19. These steviol glycosides accumulate in stevia leaves and compose approximately 10%-20% of the total dry weight. Typically, on a dry weight basis, the four major glycosides found in the leaves of stevia are Dulcoside A (0.3%), Rebaudioside C (0.6-1.0%), Rebaudioside A (3.8%) and Stevioside (9.1%). Other steviol glycosides identified in Stevia extract include Rebaudiosides B, D, E, F, M, N, and O, Steviolbioside and Rubusoside.
[0051] As used herein, the term “stevia” refers to the plant stevia rebaudiana, commonly known as sweetleaf, sweet leaf, sugarleaf, or simply stevia unless otherwise indicated. The phrase “stevia extract” refers to a sweetener-rich extract derived from the leaves of the stevia rebaudiana plant.
[0052] As used herein, the phrase “hot water” in the context of an extraction solvent refers generally to water having a temperature of 50° C. to 100° C.
[0053] As used herein, the term “steviol glycoside(s)” refers to glycosides of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside M, Rebaudioside I, Stevioside, Steviolbioside, Dulcoside A, Rubusoside, etc. or synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.
[0054] As used herein, the term “total steviol glycosides” (TSG) is calculated as the sum of the content of all steviol glycosides on a dry (anhydrous) basis, including, for example, Rebaudioside A (Reb A), Rebaudioside B (Reb B), Rebaudioside C (Reb C), Rebaudioside D (Reb D), Rebaudioside E (Reb E), Rebaudioside F (Reb F), Rebaudioside G (Reb G), Rebaudioside M (Reb M), Stevioside, Steviolbioside, Dulcoside A and Rubusoside.
[0055] Applicant discovered a method of purifying one or more steviol glycosides from a mixture, the mixture including the one or more steviol glycosides and at least one impurity, the method comprising: passing the mixture through a first adsorbent with a first solvent, the first adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange resins to provide a first eluate stream, the first eluate stream having the first solvent and a higher purity of the one or more steviol glycosides than in the mixture as measured by weight percentage of the solid content, and optionally removing at least a portion of the first solvent from the first eluate stream to provide a reduced first eluate stream. In some embodiments, the method further comprising: passing the first eluate stream or the reduced first eluate stream through a second adsorbent with a second solvent, the second adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange resins to provide a second eluate stream, the second eluate stream having the second solvent and a higher purity of the one or more steviol glycosides than in the first eluate stream or the reduced first eluate stream as measured by weight percentage of the solid content, and optionally removing at least a portion of the second solvent from the second eluate stream to provide a reduced second eluate stream. In certain embodiments, the method includes one or more of a micro filtration zone, first swing bed simulated moving bed zone, a first nanofiltration zone, second swing bed simulated moving bed zone, a polishing stage simulated moving bed zone, and a second nanofiltration zone provided a scheme wherein water could be employed exclusively as the mobile phase desorbent without requiring the addition of any organic solvent to provide a high purity steviol glycoside product.
[0056] In some embodiments, the methods described herein comprise removing one or more solvents. For example, the method can include removing at least a portion of the first solvent from the first eluate stream to provide a reduced first eluate stream and/or removing at least a portion of the second solvent from the second eluate stream to provide a reduced second eluate stream. The solvent can be removed by any suitable method. For example, the solvent can be removed by evaporation (e.g., under reduced pressure, elevated temperature, or a combination thereof), membrane permeation (e.g., nano-filtration), extraction, or a combination thereof.
[0057] The methods described herein include purifying one or more steviol glycosides from a mixture. Generally, the one or more steviol glycosides are selected from the group consisting of Dulcoside A, Rebaudioside C, Rebaudioside A, Stevioside, Rebaudioside B, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside M, Rebaudioside I, Steviolbioside, and Rubusoside. In preferred embodiments, the one or more steviol glycosides to be purified are Rebaudioside D and/or Rebaudioside M.
[0058] The purity of a steviol glycoside (e.g., Rebaudioside D and/or Rebaudioside M) can be measured by any suitable means known to a person of ordinary skill in the art. In some embodiments, the purity of a steviol glycoside (e.g., Rebaudioside D and/or Rebaudioside M) is measured using high performance liquid chromatography (HPLC). In some embodiments, the purity of a steviol glycoside (e.g., Rebaudioside D and/or Rebaudioside M) is assessed using the Joint FAO/WHO Expert Committee on Food Additives (JECFA) method for determining purity. In some embodiments, the purity of a steviol glycoside (e.g., Rebaudioside D and/or Rebaudioside M) is measured using weight percentage of the solid content. If the weight percentage of a constituent in the solid content increases, the constituent is considered to be more pure. If the weight percentage of a constituent in the solid content decreases, the constituent is considered to be less pure. To illustrate, a constituent having a weight percentage of 15% is more pure than if it had a weight percentage of 10%. Similarly, a constituent having a weight percentage of 90% is more pure than if it had a weight percentage of 75%.
[0059] As used herein, the term “solid concentration” refers to the mass of solids per volume of liquid in a given stream and is expressed as grams/Liter. The mass of the solids content in a stream is determined by subjecting a fixed volume of the sample, typically 1 ml, to an effective amount of heat, up to 80° C., at atmospheric pressure for a time sufficient to fully evaporate the sample to dryness, typically 1-2 hours.
[0060] Methods of the disclosure can use normal-phase chromatography and/or reversed-phase chromatography. In some embodiments, the methods of the disclosure employ a process known as reversed-phase chromatography. As used herein, the term “reversed-phase chromatography” employs a polar (aqueous) mobile phase. As a result, hydrophobic molecules in the polar mobile phase tend to adsorb to the hydrophobic stationary phase, and hydrophilic molecules in the mobile phase will pass through the column and are eluted first. Accordingly, any suitably stationary phase adsorbent (i.e., chromatographic resin) can be used in methods of the disclosure.
[0061] In some embodiments, the method comprises passing the mixture through a first adsorbent with a first solvent, the first adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange (i.e., a cationic resin and/or and anionic resin) resins to provide a first eluate stream. In certain embodiments, the method further comprises passing the first eluate stream or the reduced first eluate stream through a second adsorbent with a second solvent, the second adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange resins to provide a second eluate stream. Accordingly, the method can include passing a mixture through one or more hydrophobic interaction resins followed by one or more ion exchange (i.e., a cationic resin and/or and anionic resin) resins or passing a mixture through one or more ion exchange (i.e., a cationic resin and/or and anionic resin) resins followed by one or more hydrophobic interaction resins. In preferred embodiments, the first adsorbent is one or more hydrophobic and the second adsorbent is one or more ion exchange (i.e., a cationic resin and/or and anionic resin) resins.
[0062] The stationary phase adsorbents may be disposed in a single adsorbent bed or may be disposed in a single column or series of single columns containing multiple adsorbent bed zones. Embodiments of the instant disclosure employ separate stationary phase adsorbents in carrying out the overall process of the disclosure. A list of exemplary stationary phases (i.e., chromatographic resins) for use in various embodiments of the methods of the disclosure are as follows.
[0063] The hydrophobic interaction resin can be any suitable hydrophobic interaction resin. In some embodiment, the hydrophobic interaction resin is a porous hydrophobic interaction resin with a particle size of 70-200 microns. In certain embodiments, the hydrophobic interaction resin is a phenylated polymethacrylate polymer resin. Examples of suitable hydrophobic interaction resins having a particle size of about 70-200 microns include TOYOPEARL PHENYL-650C (Available from Tosoh Bioscience, Tokyo, Japan) or RELISORB PH400 (Available from Mitsubishi Chemical Company, Tokyo, Japan). The hydrophobic interaction resin can be used to remove lipids and saccharides as impurities from the steviol glycosides. In certain embodiments, the hydrophobic interaction resin is used in the polishing bed simulated moving bed (SMB) zone, and the lipid and saccharide impurities are rejected into a polishing bed raffinate stream and steviol glycosides are extracted into a polishing bed extract stream.
[0064] The ion exchange resin can be any suitable ion exchange (i.e., a cationic resin and/or and anionic resin) resin. In some embodiments, the ion exchange resin is a combination of a cationic resin and an anionic exchange resin. In certain embodiments, the cationic resin and the anionic exchange resin are arranged in series in fluid communication such that the mixture to be purified passes through the cationic resin then the anionic exchange resin or the anionic exchange resin then the cationic exchange resin. The ion exchange resin can be used to remove impurities such as ionic salts, metal ions, flavonoids, pigments, carotenoids, vitamins, proteins, and color bodies. In some embodiments, the ion exchange resin is used in the second swing bed simulated moving bed (SMB) chromatography zone. In the second swing bed simulated moving bed zone, the impurities are retained on the resin and the steviol glycosides are rejected.
[0065] In some embodiments, the cationic resin is a styrene-divinylbenzene copolymer resin. In certain embodiments, the cationic resin has an 8 percent cross-linkage with an effective particle size of about 0.5 mm. Suitable examples of the cationic resin include: AMBERLITE IR-118 (Available from Dow Chemical Company, Midland, Mich.), or DIAION PK216LH (Available from Mitsubishi Chemical Company, Tokyo, Japan).
[0066] In some embodiments, the anionic resin is an acrylic-divinylbenzene copolymer resin. In certain embodiments, the anionic resin has an effective particle size of about 0.6 mm. Suitable examples of the anionic resin include AMBERLITE IRA-70RF (Available from Dow Chemical Company, Midland, Mich.) or RELITE RAM2 (Available from Mitsubishi Chemical Company, Tokyo, Japan).
[0067] In some embodiments, the methods described herein further comprise passing a mixture through an aromatic non-polar copolymer of styrene-divinyl benzene adsorbent resin with an effective particle size of 0.25 mm and effective surface area of 590 m.sup.2/g. Examples of suitable styrene-divinyl benzene adsorbent resins can be selected from the AMBERLITE XAD resin series (Available from Dow Chemical Company, Midland, Mich.), DIAION HP-20 (Available from Mitsubishi Chemical Company, Tokyo, Japan), or Stratosphere PL-PS/DVB (Available from Sigma-Aldrich, St. Louis, Mo.). The styrene-divinyl benzene adsorbent resin matrix provides an aromatic non-polar surface with selectivity for hydrophobic areas of molecules. In some embodiments, the styrene-divinyl benzene adsorbent is used in a first swing bed simulated moving bed zone and the steviol glycosides are retained on the resin and are subsequently recovered in a first swing bed extract stream. Impurities such as tri-terpenes, sterols, flavonoids and some of the pigments are rejected into a first swing bed raffinate stream. The stationary phase adsorbents may be disposed in a single adsorbent bed or may be disposed in within a single column or series of single columns containing multiple adsorbent bed zones.
[0068] In some embodiments, the methods described herein further include preconditioning an adsorbent prior to use. For example, the methods can include preconditioning the first adsorbent prior to passing the mixture through a first adsorbent with a first solvent and/or preconditioning the second adsorbent prior to passing the first eluate stream or the reduced first eluate stream through a second adsorbent with a second solvent. As used herein, the term preconditioning refers to a method of washing an adsorbent and/or equilibrating an adsorbent at a particular temperature or flow rate. In some embodiments, preconditioning comprises washing with acid, base, water, or a combination thereof.
[0069] In some embodiments, the method comprises purifying a steviol glycoside (e.g., Rebaudioside D and/or Rebaudioside M) with simulated moving bed (SMB) chromatography. In some embodiments, the method relates to a continuous process for the purification of steviol glycosides, specifically Rebaudioside D and/or Rebaudioside M using a sequence of purification steps and a continuous simulated moving bed process and downstream recovery steps to separate Rebaudioside D and/or Rebaudioside M.
[0070] The SMB system of the current invention was arranged for maximum selectivity. The simulated moving bed operation is achieved by use of a plurality of adsorbent beds connected in series or portions in series or parallel and a complex valve system, whereby the complex valve system facilitates switching at regular intervals the feed entry in one direction, the mobile phase desorbent entry in the opposite direction, while changing the extract and raffinate takeoff positions as well. The SMB system is a continuous process. Feed enters and extract and raffinate streams are withdrawn continuously at substantially constant compositions. The overall operation is equivalent in performance to an operation wherein the fluid and solid are contacted in a continuous countercurrent manner, without the actual movement of the solid, or stationary phase adsorbent.
[0071] The SMB system may be operated such that the adsorbent beds are operated individually or in parallel using a single rotary valve and associated control system. A column may comprise one or several beds containing chromatographic media. Those feed tanks, filters, piping connecting flow between columns and/or beds where so connected, pumps, valving, pressure regulators, metering equipment, flow control and microprocessor equipment utilized in the embodiment are well known in construction and function to those of ordinary skill in the art.
[0072] In some embodiments, a simulated moving bed (SMB) system is arranged for maximum selectivity. The simulated moving bed operation is achieved by use of a plurality of adsorbent beds connected in series or portions in series or parallel and a complex valve system, whereby the complex valve system facilitates switching at regular intervals the feed entry in one direction, the mobile phase desorbent entry in the opposite direction, while changing the extract and raffinate takeoff positions as well. The SMB system is a continuous process in some embodiments. Feed and mobile phase desorbent enter, while extract and raffinate streams are withdrawn continuously at substantially constant compositions. The overall operation is similar in performance to an operation wherein the fluid and solid are contacted in a continuous countercurrent manner, without the actual movement of the solid, or stationary phase adsorbent.
[0073] In some embodiments, the method comprises passing a mixture through a SMB zone to provide a primary raffinate stream. The SMB zone comprises a plurality of adsorbent beds (e.g., columns comprising a stationary phase). The SMB zone can comprises any suitable number of adsorbent beds. For example, the SMB zone can comprise 2 or more adsorbent bed, e.g., 3 or more adsorbent beds, 4 or more adsorbent beds, 5 or more adsorbent beds, 6 or more adsorbent beds, 10 or more adsorbent beds, or 20 or more adsorbent beds. In some embodiments, the plurality of adsorbent beds are arranged in serial fluid communication such that fluid introduced at a top of any adsorbent bed (n) passes to the next highest adsorbent bed (n+1). In such embodiments, the method can further comprise advancing each adsorbent bed, such that adsorbent bed n+1 becomes adsorbent bed n after advancing, and adsorbent bed n prior to advancing becomes adsorbent bed n+x after advancing, wherein adsorbent bed n+x is the highest adsorbent bed in the serial fluid communication arrangement.
[0074] In some embodiments, the SMB zone comprises eight adsorbent beds. The eight adsorbent beds can be broken down into four zones referring to a desorption zone, a rectification zone, an adsorption zone, and a concentration zone. The adsorbent beds can be in any suitable arrangement (e.g., 2-2-2-2, 3-2-2-1, 2-3-2-1, 2-2-3-1, 1-3-3-1, 3-3-1-1, 3-1- 3-1, or 2-2-3-1, etc.), wherein each number refers to one of the four zones. In certain embodiments, the SMB zone is in a 2-3-2-1 arrangement, wherein two adsorbent beds are operated in a desorption zone, three adsorbent beds are operated in a rectification zone, two adsorbent beds are operated in an adsorption zone, and one adsorbent bed is operated in a regeneration zone, respectively.
[0075] In some embodiments, the first adsorbent is arranged in a SMB configuration to form a first SMB zone, and wherein passing the mixture through the through the first adsorbent with the first solvent comprises passing the mixture through the first SMB zone.
[0076] In some embodiments, the second adsorbent is arranged in a SMB configuration to form a second SMB zone, and wherein passing the first eluate stream or the reduced first eluate stream through the second adsorbent with the second solvent comprises passing the first eluate stream or the reduced first eluate stream through the second SMB zone.
[0077] The mobile phase desorbent or solvent (e.g., first solvent and second solvent) of the present invention for use in all of the purification methods comprises water, preferably deionized water. Accordingly, the mobile phase desorbent or solvent can comprise an additional solvent such as ethanol, acetone, ethyl acetate, acetonitrile, pentanes, hexanes, heptanes, methanol, propanol, or a combination thereof. In some embodiments, the mobile phase desorbent (e.g., the first solvent and the second solvent) comprise greater than 95% v/v water. In certain embodiments, the mobile phase desorbent (e.g., the first solvent and the second solvent) comprise greater than 99% v/v water. In preferred embodiments, the mobile phase desorbent (e.g., the first solvent and the second solvent) are water (i.e., do not contain an additional solvent).
[0078] The mixture of steviol glycosides can be obtained by any suitable means. For example, the mixture can be obtained from a stevia plant extract, a stevia fermentation broth, a yeast fermentation broth, an Escherichia coli fermentation broth, or an enzyme-converted mixture. Accordingly, in addition to obtaining a naturally occurring mixture from extraction of a stevia plant such as Stevia rebaudiana Bertoni, the mixture of steviol glycosides can be obtained from recombinant production systems or enzymatic conversion reactions. Without wishing to be bound by any particular theory, it is believed that recombinant production systems or enzymatic conversion reactions can be utilized to produce an increased abundance of steviol glycosides and/or a preferred distribution of particular steviol glycosides.
[0079] An exemplary procedure for purification of the extract from a stevia plant such as Stevia rebaudiana Bertoni is as follows: following harvesting and processing, the grinded and dried stevia leaves are extracted with an appropriate GRAS solvent or hot water. A number of different parameters can influence the overall yield, quality and/or purity of the desired final product. These parameters include, but are not limited to, the identity of the chosen GRAS solvent; the temperature and time at which the chosen natural solvent is used; the ratio of raw material to solvent (raw material:solvent (v/v)) that is employed; the number of successive extractions performed; the chosen method of purification of the desired products and the conditions related thereto. The skilled person will understand that these parameters are not necessarily mutually exclusive, and that a particular choice relating to one parameter may or may not affect the choice of other parameters. For example, the identity of the chosen natural solvent, and the temperature thereof, can affect the optimal ratio of raw material to solvent that is required to obtain the desired results. Following the extraction of the steviol glycosides from the stevia leaves, an extract stream comprising crude steviol glycosides is withdrawn from the extraction zone. Preferably, the crude steviol glycosides are admixed with water to provide a crude extract stream which comprises from about 34 wt. % to about 40 wt. % total steviol glycosides in the aqueous mixture. More preferably, the crude extract stream comprises from about 34 wt. % to about 37 wt. % total steviol glycosides in the aqueous mixture. The concentration of solids in the crude extract stream varies from about 60 to about 80 g/L and is preferably about 75 g/L.
[0080] In some embodiments, the crude extract stream (i.e., mixture to be purified) is obtained from a yeast fermentation broth. In certain embodiments where the crude extract stream (i.e., mixture to be purified) is obtained from a yeast fermentation broth, the mixture comprises from about 15 wt. % to about 30 wt. % total steviol glycosides. Alternatively, or additionally, the crude extract stream (i.e., mixture to be purified) obtained from a yeast fermentation broth can have a concentration of solids from 20 mg/mL to about 50 mg/mL. In some embodiments, the crude extract stream (i.e., mixture to be purified) is obtained from an Escherichia coli fermentation broth. In certain embodiments where the crude extract stream (i.e., mixture to be purified) is obtained from an Escherichia coli fermentation broth, the mixture comprises from about 5 wt. % to about 15 wt. % total steviol glycosides. Alternatively, or additionally, the crude extract stream (i.e., mixture to be purified) obtained from an Escherichia coli fermentation broth can have a concentration of solids from 20 mg/mL to about 50 mg/mL.
[0081] In some embodiments, a mixture described herein (e.g., the crude extract stream) is passed to a microfiltration zone to remove any solid particles to provide a filtered extract stream. The microfiltration can be carried at a microfiltration temperature ranging from about 50° C. to about 60° C. and the microfilter in the microfiltration zone can range from about 0.2 μm to about 0.5 μm. Preferably, the microfilter pore size in the microfiltration zone comprises a microfilter pore size about 0.2 μm.
[0082] In some embodiments, a mixture described herein (e.g., the crude extract stream) is nanofiltered. Nanofiltration is a membrane filtration-based method that uses nanometer sized cylindrical through-pores that pass through the membrane at 90°. Nanofiltration membranes have pore sizes from 1-10 nanometers, smaller than that used in microfiltration and ultrafiltration, but just larger than that in reverse osmosis. Membranes used are predominantly created from polymer thin films. Materials that are commonly use include polyethylene terephthalate or metals such as aluminum. The nano filter pore size employed in the nanofiltration zones of present invention range from about 100 to 300 Da (Dalton). The pore size of a nanofiltration membrane in the nanofiltration zone characterized by a cut-off value. This cut-off value is consistent with the molecular weight of the smallest molecule that can be 90% restricted by the top layer of the membrane. The cut-off value of the nanofiltration membrane is typically expressed in Dalton (Dalton=weight in grams of mole of the molecule). More preferably, the pore size in the first nanofiltration zones is less than 150 Da. The nanofiltration is employed to remove excess water from extract streams withdrawn from simulated moving bed zones to obtain a target concentration of steviol glycosides in the effluent from the nanofiltration zone.
[0083] According to one embodiment of the invention and with reference to
[0084] In some embodiments, and with reference to
[0085] In certain embodiments, the first swing bed simulated moving bed zone has a plurality of first swing adsorbent beds which are disposed in one or more clockwise, sequentially disposed sections. Each section comprises five first swing adsorbent beds and separated into 3 zones, wherein a zone 1 comprises the first two first swing adsorbent beds which are desorbed with a portion of the first mobile phase desorbent to provide a portion of the first swing extract stream, a zone 2 comprising the third first swing adsorbent bed, which is loaded with a portion of the first mobile phase desorbent to provide a portion of an extract recycle stream. The portion of an extract recycle stream is admixed with the filtered crude extract stream and passed to the fourth and fifth first swing adsorbent beds to load the first swing adsorbent and to provide a portion of the primary first swing bed raffinate stream during a first portion of a simulated moving bed step and a portion of the secondary first swing bed raffinate stream during a second or remaining portion of a simulated moving bed step. At the completion of each of the steps of the first swing bed SMB cycle, a rotary valve is sequenced to increment each first swing adsorbent bed by one position to the left in a counterclockwise manner.
[0086] In some embodiments, and with reference to
[0087] In some embodiments of the second swing bed SMB cycle, the nanofiltered elute stream in line 110 is introduced to SB2-5 via lines 110, 113 and 114 and to SB2-7 via lines 110, 111, and 115 and 120 for a portion of each step in a loading step, after which for the remainder of each step, the adsorbent beds (SB2-5-SB2-10) are serially and sequentially washed with the second mobile phase desorbent comprising or consisting of water introduced in lines 112 and 114 to adsorbent bed SB2-5 to provide a second swing bed extract stream in line 130. The SB2 regeneration section (SB2-1-SB2-4) operates essentially in parallel carrying out a series of regeneration steps which include: [0088] (1) a serial water wash zone wherein a adsorbent beds SB2-1 (cation) and SB2-2 (anion) pair of adsorbent beds are water washed by passing a second water wash stream in line 128 to the top of adsorbent bed SB2-1, withdrawing the wash effluent from adsorbent bed SB2-1 in line 142 and passing the wash effluent to the top of adsorbent bed SB2-2 and withdrawing a first SB2 waste water stream I line 134; [0089] (2) a basic/acidic regeneration zone wherein adsorbent bed SB2-3, containing the strongly acidic cation adsorbent is subjected to a four part regeneration procedure wherein (a) adsorbent bed SB2-3 is washed with an aqueous basic solution such as sodium hydroxide having an NaOH concentration of from about 2 grams/100 ml water to about 4 grams/100 ml water NaOH (a NaOH concentration ranging from a 0.5N to IN NaOH solution), (b) washed with a water wash stream in a first regeneration wash step, (c) washed with an aqueous mild acid solution such as HCl having an HCl concentration of from about 2 ml HCl per 100 ml water to about 4 ml HCl per 100 ml water (an HCl concentration of from 0.2 to 0.6 N HCl solution) to reactivate the adsorbent, and (d) washed in a second water wash with the water wash stream; and [0090] (3) an basic regeneration zone wherein adsorbent bed SB2-4, containing the weakly basic anion adsorbent is subjected to a two part regeneration procedure wherein (a) adsorbent bed SB2-4 is washed with an aqueous basic solution such as sodium hydroxide having an NaOH concentration of from about 2 grams/100 ml water to about 4 grams/100 ml water NaOH (a NaOH concentration ranging from a 0.5N to IN NaOH solution), and (b) washed in a second water wash with the water wash stream.
[0091] All of the parts of the above series of four part regeneration streams are introduced to adsorbent bed SB2-3 sequentially in line 138 and a regeneration zone effluent stream is withdrawn in line 140 as a second SB2 waste water stream. Second swing adsorbent bed SB2-4 is sequentially washed by passing a basic solution via line 132 to the top of second swing adsorbent bed SB2-4, followed by washing the second swing bed adsorbent bed SB-2-4 with a water wash stream introduced in line 132 to provide a third SB2 waste water stream line 144. The basic solution in line 132 is an aqueous basic solution wherein the base is an alkali metal such as sodium, and the base is sodium hydroxide having a concentration ranging between about 2 grams/100 ml water to about 4 grams/100 ml water (a NaOH concentration ranging from a 0.5N to 1N NaOH solution). The first waste water stream in line 134, the second SB2 waste water stream in line 140, and the third waste water stream in line 144 are collected in line 146 as a total SB2 waste water stream and passed to the waste water recovery zone (not shown). In the operation of the second swing bed simulated moving bed system (SB2), the second swing adsorbent beds are moved in cation/anion pairs. Thus, at the completion of each step of the second swing bed SMB cycle, the rotary valve is sequenced to increment each second swing adsorbent bed by two positions (n+2) to the left in a counterclockwise manner, whereby second swing adsorbent beds SB2-3 and SB2-4 are moved to the position of second swing adsorbent beds SB2-1 and SB2-2, and so on; and second swing adsorbent beds SB2-1 and SB2-2 are moved to the positions previously occupied by second swing adsorbent beds SB2-9 and SB2-10.
[0092] According to one embodiment of the invention and with reference to
[0093] In one embodiment, in the polishing bed zone, the plurality of polishing bed adsorbent beds is disposed in one or more clockwise, sequentially disposed sections. Each section has a desorption zone, a separation zone and a loading zone. In the operation of each section of the polishing bed zone; a portion of the third mobile phase desorbent is passed to the desorption zone to provide a portion of the polishing bed extract stream; a portion of the polishing bed extract stream is passed to the separation zone to provide a separation zone effluent stream; and, a portion of the polishing bed extract stream is admixed with the second swing bed elute stream to provide a portion of the polishing bed raffinate stream. At the completion of each of step of a polishing bed SMB cycle, a rotary valve is sequenced to increment each polishing bed adsorbent bed by one position to the left in a counterclockwise manner.
EMBODIMENTS
[0094] Principles of the present disclosure are incorporated in the following embodiments:
[0095] Embodiment (1) A method of purifying one or more steviol glycosides from a mixture, the mixture including the one or more steviol glycosides and at least one impurity, the method comprising: passing the mixture through a first adsorbent with a first solvent, the first adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange resins to provide a first eluate stream, the first eluate stream having the first solvent and a higher purity of the one or more steviol glycosides than in the mixture as measured by weight percentage of the solid content, and optionally removing at least a portion of the first solvent from the first eluate stream to provide a reduced first eluate stream.
[0096] Embodiment (2) The method of embodiment (1), the method further comprising: passing the first eluate stream or the reduced first eluate stream through a second adsorbent with a second solvent, the second adsorbent comprising one or more hydrophobic interaction resins or one or more ion exchange resins to provide a second eluate stream, the second eluate stream having the second solvent and a higher purity of the one or more steviol glycosides than in the first eluate stream or the reduced first eluate stream as measured by weight percentage of the solid content, and optionally removing at least a portion of the second solvent from the second eluate stream to provide a reduced second eluate stream.
[0097] Embodiment (3) The method of embodiment (1), wherein the one or more steviol glycosides are selected from the group consisting of Dulcoside A, Rebaudioside C, Rebaudioside A, Stevioside, Rebaudioside B, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside M, Rebaudioside I, Steviolbioside, and Rubusoside.
[0098] Embodiment (4) The method of embodiment (1), wherein the one or more steviol glycosides are Rebaudioside D and Rebaudioside M.
[0099] Embodiment (5) The method of embodiment (1), wherein the one or more steviol glycosides is Rebaudioside D.
[0100] Embodiment (6) The method of embodiment (1), wherein the one or more steviol glycosides is Rebaudioside M.
[0101] Embodiment (7) The method of embodiment (1), wherein the mixture is a stevia plant extract, a stevia fermentation broth, a yeast fermentation broth, an Escherichia coli fermentation broth, or an enzyme-converted mixture.
[0102] Embodiment (8) The method of embodiment (7), wherein the stevia plant is Stevia rebaudiana Bertoni.
[0103] Embodiment (9) The method of embodiment (2), wherein the first solvent and the second solvent comprise water.
[0104] Embodiment (10) The method of embodiment (9), wherein the first solvent and the second solvent comprise greater than 95% v/v water.
[0105] Embodiment (11) The method of embodiment (10), wherein the first solvent and the second solvent comprise greater than 99% v/v water.
[0106] Embodiment (12) The method of embodiment (11), wherein the first solvent and the second solvent are water.
[0107] Embodiment (13) The method of embodiment (1), wherein said at least one impurity comprises at least one of tri-terpenes, sterols, flavonoids, volatile oils, pigments, gums, proteins, carotenoids, chlorophyll, vitamins, phospholipids, saccharides, solid insoluble, salts, fermentation byproducts, or N-acetyl glucosamine.
[0108] Embodiment (14) The method of embodiment (4), wherein said at least one impurity comprises a second steviol glycoside selected from Dulcoside A, Rebaudioside C, Rebaudioside A, Stevioside, Rebaudioside B, Rebaudioside E, Rebaudioside F, Rebaudioside I, Steviolbioside, Rubusoside, and combinations thereof.
[0109] Embodiment (15) The method of embodiment (2), wherein the first adsorbent is one or more hydrophobic interaction resins.
[0110] Embodiment (16) The method of embodiment (15), wherein the one or more hydrophobic interaction resins is a phenylated polymethacrylate polymer resin.
[0111] Embodiment (17) The method of embodiment (16), wherein the phenylated polymethacrylate polymer resin has a particle size of from about 75 μm to about 200 μm.
[0112] Embodiment (18) The method of embodiment (15), wherein the second adsorbent is one or more ion exchange resins.
[0113] Embodiment (19) The method of embodiment (18), wherein the one or more ion exchange resins is a combination of a cationic resin and an anionic resin.
[0114] Embodiment (20) The method of embodiment (19), wherein the cationic resin and anionic resin are arranged in series.
[0115] Embodiment (21) The method of embodiment (19), wherein the cationic resin is a styrene-divinylbenzene copolymer resin and the anionic resin is an acrylic-divinylbenzene copolymer resin.
[0116] Embodiment (22) The method of embodiment (1), wherein the first adsorbent is arranged in a SMB configuration to form a first SMB zone, and wherein passing the mixture through the through the first adsorbent with the first solvent comprises passing the mixture through the first SMB zone.
[0117] Embodiment (23) The method of embodiment (2), wherein the second adsorbent is arranged in a SMB configuration to form a second SMB zone, and wherein passing the first eluate stream or the reduced first eluate stream through the second adsorbent with the second solvent comprises passing the first eluate stream or the reduced first eluate stream through the second SMB zone.
[0118] Embodiment (24) The method of embodiment (1), the method further comprising: preconditioning the first adsorbent prior to passing the mixture through a first adsorbent with a first solvent.
[0119] Embodiment (25) The method of embodiment (24), wherein preconditioning comprises washing with acid, base, water, or a combination thereof.
[0120] Embodiment (26) The method of embodiment (2), the method further comprising: preconditioning the second adsorbent prior to passing the first eluate stream or the reduced first eluate stream through a second adsorbent with a second solvent.
[0121] Embodiment (27) The method of embodiment (26), wherein preconditioning comprises washing with acid, base, water, or a combination thereof.
[0122] Embodiment (28) The method of embodiment (2), the method further comprising: drying the second eluate stream or the reduced second eluate stream.
[0123] The foregoing exemplary embodiments of the disclosure numbered 1-28 are non-limiting. Other exemplary embodiments are apparent from the entirety of the description herein. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered aspects.
EXAMPLES
[0124] The following examples are provided to illustrate the present disclosure. These examples are shown for illustrative purposes, and any disclosures embodied therein should not be limited thereto.
Example 1—First Swing Bed Simulated Moving Bed Zone
[0125] The removal of a portion of the impurities, such as tri-terpenes, sterols, flavonoids and some of the pigments, from the filtered crude extract stream was demonstrated in an SMB unit in a configuration operating as the first swing bed simulated moving bed zone. A lab scale SMB unit (OCTAVE-300 unit, available from Semba Biosciences, Inc. Madison, Wis.) was used for separation of steviol glycosides from impurities including: tri-terpenes, sterols, flavonoids, and some of the pigments. The Semba Octave-300 Chromatography System is a bench top automated liquid chromatography platform designed for preparative-scale purification of chemical and biological compounds. The Octave System carries eight column positions arranged in series and connected through a proprietary pneumatic valve array. The independently working and programmable 72-valve array contains no moving parts, occupies only 3 μl per valve, and responds within 100 ms. Fluid flow is controlled by four independent pumps. The valve switching and pump flow rates are controlled via the SembaPro Software. Five adsorbent beds, each comprising a SS316 column having an inside diameter of 50 mm and a length of 600 mm were packed with about 960 grams of DIAION HP-20 resin, styrene-divinyl benzene adsorbent resin (Available from Mitsubishi Chemical Company, Tokyo, Japan).
[0126] According to the configuration in
TABLE-US-00002 TABLE 2 SMB Operating Parameters-First Swing Bed SMB PARAMETER VALUE UNIT Step Time 18.0 Minutes Bath Temperature 62 ° C. Feed Rate (line 10)* 1.5 L/HR Desorbent (line 28) 8.0 L/HR Desorbent (line 30) 7.1 L/HR Zone 2 (SB1-3, line 56) 3.4 L/HR Extract (line 44) 4.7 L/HR Extract (line 48) 7.1 L/HR Primary Raffinate (line 68, 55) 2.4 L/HR Secondary Raffinate (line 68, 70) 2.4 L/HR *Line numbers refer to FIG. 2
[0127] The desorbent or mobile phase was low conductivity RO (reverse osmosis) water (20-50 micro Siemens). The adsorbent beds were arranged in a water bath having a temperature range of 55-70° C., and was maintained at an average bath temperature of ranging from 62-65° C. Analysis of the feed and products was carried out by high performance liquid chromatography. The samples were analyzed on a 3 μm, OROSIL C18, a reverse phase C18 column having an interior diameter of 4.6 mm and a length of 150 mm (Available from Orochem Technologies, Inc., Naperville, Ill.), at 40° C. using a mobile phase comprising a solution of 10 mM Sodium phosphate, a pH of 2.6, acetonitrile (69:31, v/v), and a flow rate of 0.6 ml/min. The concentration of sweet steviol glycosides (Rebaudioside A (A), Stevioside(S), and Rebaudioside C(C)) in the total SB1 extract was 66 wt. % on an anhydrous basis, and the recovery of total steviol glycosides based on the feed to the First Swing Bed SMB was 90% on a weight basis (w/w).
[0128]
[0129]
Example 2—Second Swing Bed Simulated Moving Bed Zone
[0130] The first swing bed (SB1) extract stream withdrawn from the first swing bed simulated moving bed zone of Example 1 was concentrated by nanofiltration to provide a concentrated extract having a dry mass of about 75 g/l. The first swing bed extract stream comprises essentially all of the steviol glycosides in the crude steviol plant extract, nonionic impurities, and ionic impurities such as salts and proteins. The second swing bed employed alternating cationic and anionic resin zones to separate the ionic impurities from steviol glycosides. The second swing bed (SB2) consisted of a plurality of adsorbent beds connected in series, wherein every other adsorbent bed contained a cation or an anion resin. Six chlorinated polyvinyl chloride columns, each having an inside diameter of 100 mm (4 inches) and a length of 914 mm (36 inches) were each loaded alternately in pairs with about 5600 grams of a strongly acidic cation exchange porous styrene-divinylbenzene copolymer bead resin, DIAION PK216LH (Available from Mitsubishi Chemical Company, Tokyo, Japan), or with a weakly basic anion exchange porous acrylic-divinylbenzene copolymer bead resin, RELITE RAM2 (Available from Mitsubishi Chemical Company, Tokyo, Japan) and are connected in series fluid communication as shown in
[0131]
Example 3—Polishing Bed Simulated Moving Bed Zone
[0132] The polishing bed zone consisted of a plurality of adsorbent beds connected in series as shown in
TABLE-US-00003 TABLE 3 SMB Operating Parameters- Second Swing Bed SMB PARAMETER VALUE UNITS Step Time 10 Minutes Bath Temperature 60 ° C. Feed Rate (SB2 Elute) 25.0 ml/minute Desorbent (DI Water) 170 ml/minute Polishing Bed Extract 64 ml/minute Polishing Bed Raffinate 131 ml/minute
[0133] The polishing bed extract stream had a total steviol glycoside purity of 97 wt. % on an anhydrous basis, and the recovery of the polishing bed extract represented a total steviol glycoside recovery of 75% on a weight basis, relative to the feed to the polishing bed simulated moving bed zone. Thus, the purity of the second swing bed (SB2) elute was enhanced from a concentration of 85 wt. % of sweet steviol glycosides (Rebaudioside A (A), Stevioside(S), and Rebaudioside C(C)) on an anhydrous basis, to an enhanced concentration of 97 wt. % sweet steviol glycosides (Rebaudioside A (A), Stevioside(S), and Rebaudioside C(C)) on an anhydrous basis.
[0134]
Example 4—Polishing Bed Simulated Moving Bed Zone
[0135] Using the same lab scale SMB unit, OCTAVE-300 (Available from Semba Biosciences, Inc., Madison, Wis.) of Example 3, the same process configuration shown in
TABLE-US-00004 TABLE 4 SMB Operating Parameters - Polishing Bed SMB PARAMETER VALUE UNITS Step Time 6 Minutes Bath Temperature 60 ° C. Feed Rate (Elute) 1.0 ml/minute Desorbent (DI Water) 25 ml/minute Polishing Bed Extract 4.7 ml/minute Polishing Bed Raffinate 21.3 ml/minute
[0136] The resulting polishing bed extract of Example 4 showed enrichment of the feed to provide a polishing bed extract stream having a total steviol glycoside purity of 98 wt. % on an anhydrous basis, and having a ratio of Rebaudioside A (A), to Stevioside (S) ranging from 2.4 to 2.5 (w/w, %) (See
[0137]
Example 5—Overall Process Improvement of Composition
[0138] A steviol extract prepared from the extraction of stevia leaves using conventional hot water extraction was subjected to the continuous process of the present invention according to the process flow as shown in
TABLE-US-00005 TABLE 5 Steviol Glycoside Composition of SMB Zone Products Rebaudioside Stevioside Rebaudioside Total % Process Stream A, % (w/w) % (w/w) C % (w/w) (w/w) Steviol 28.25 9.25 3.0 40.50 Glycoside Extract First Swing 43.50 15.14 5.72 64.36 Bed Extract Second Swing 59.64 20.74 6.25 86.63 Bed Elute Polishing 62.56 26.09 6.62 95.27 Bed Extract
Example 6—Purification of Rebaudioside D and Rebaudioside M from Yeast Fermentation Broth
[0139] A 22×300 mm stainless steel column (114 mL) packed with hydrophobic interaction resin RELISORB PH400 (Available from Mitsubishi Chemical Company, Tokyo, Japan) was maintained at a temperature of 60° C. The resin was washed in sequential order with (1) two column volumes (CV) of 0.1 N NaOH at 5 mL/min, (2) two CVs of deionized water at 5 mL/min, (3) one CV of 0.1 N HCl at 5 mL/min, and (4) five CVs of deionized water at 5 mL/min to equilibrate at pH 5. Yeast fermentation broth (Available from Cargill, PF59A) was centrifuged and the resulting supernatant (20 mL at 20 mg/mL) was loaded on the column at 1.5 mL/min, and eluded with deionized water. The elution of steviol glycosides were monitored by high performance liquid chromatography using a C18 column. The pooled fractions were analyzed using HPLC with the following parameters: [0140] Column: Orosil C18, 3 μm, 4.6×150 mm [0141] Temperature: 40° C. [0142] Mobile Phase: 69:31 10 mM Sod phosphate, pH 2.6, flow rate 0.6 ml/min
[0143] The hydrophobic interaction resin removes the majority of front end impurities as demonstrated by the HPLC traces shown in
Example 7—Purification of Rebaudioside D and Rebaudioside M on Ion Exchange Resins
[0144] A 10×250 mm stainless steel column (20 mL) packed with cationic resin DIAION PK216LH (Available from Mitsubishi Chemical Company, Tokyo, Japan) was washed in sequential order with (1) 1.5 column volumes (CV) of 0.2 N HCl at 1.5 mL/min and (2) 1.5 CVs of deionized water at 1.5 mL/min. A second 10×250 mm stainless steel column (20 mL) packed with anionic resin RELITE RAM2 (Available from Mitsubishi Chemical Company, Tokyo, Japan) was washed in sequential order with (1) 1.5 CVs of 0.5 N NaOH at 1.5 mL/min and (2) 1.5 CVs of deionized water at 1.5 mL/min. The two columns were connected in series and washed with 20 CVs of deionized water at 1 mL/min until the anionic column effluent had a pH of 7.2.
[0145] The purified fractions from Example 6 (18 mL) were loaded at 1 mL/min and eluded with deionized water. The elution of steviol glycosides were monitored by high performance liquid chromatography using a C18 column. The pooled fractions were analyzed using HPLC with the following parameters: [0146] Column: Orosil C18, 3 μm, 4.6×150 mm [0147] Temperature: 40° C. [0148] Mobile Phase: 69:31 10mM Sod phosphate, pH 2.6, flow rate 0.6 ml/min.
[0149] The combination of cationic and anionic resins removes the majority of impurities remaining after purification with the hydrophobic interaction resin, as demonstrated by
[0150] The purity of the sample obtained after purification with the cationic and anionic resins was further assessed using the Joint FAO/WHO Expert Committee on Food Additives (JECFA) method for determining purity. The results are set forth in Table 6.
TABLE-US-00006 TABLE 6 Purification of Rebaudio sides D and Rebaudioside M Rebaudioside Rebaudioside Rebaudioside Purity* A D M Sample (wt. %) (wt. %) (wt. %) (wt. %) Fermentation 22.5 ND 7.1 15.4 Broth Purified 95.0 1.4 17.5 76.0 Sample *Purity was determined as described in JECFA method
[0151] As demonstrated by Table 6, the purity of Rebaudioside D and Rebaudioside M increases significantly from a purity of 22.5 wt. % to a purity of 95 wt. % upon purification with the combination of a hydrophobic interaction resin, cationic resin, and anionic resin.
[0152] Although the systems and processes described herein have been described in detail, it should be understood that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the invention as defined by the following claims. Those skilled in the art may be able to study the preferred embodiments and identify other ways to practice the invention that are not exactly as described herein. It is the intent of the inventors that variations and equivalents of the invention are within the scope of the claims, while the description, abstract and drawings are not to be used to limit the scope of the invention. The invention is specifically intended to be as broad as the claims below and their equivalents.
[0153] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0154] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. As used herein, the term “exemplary” indicates an example thereof and does not suggest a best or optimal of the recited item. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0155] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.