FIELD OF THE INVENTION

The invention relates to a comprehensive process for the separation, isolation and characterization of steviol glycosides from the extract of theStevia rebaudianaplants and their use in sweetening compositions.

BACKGROUND OF THE INVENTION

Nowadays, sugar alternatives are receiving increasing attention due to awareness of many diseases in conjunction with consumption of high-sugar foods and beverages. However, many artificial sweeteners such as dulcin, sodium cyclamate and saccharin were banned or restricted in some countries due to concerns on their safety. Therefore, non-caloric sweeteners of natural origin are becoming increasingly popular.

Stevia rebaudianaBertoni is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. The leaves of the plant contain from 10 to 20% of diterpene glycosides, which are around 150 to 450 times sweeter than sugar. The leaves have been traditionally used for hundreds of years in Paraguay and Brazil to sweeten local teas and medicines.

Typical practice is to grow plants during the warm part of the year, then harvest before the plants flower. David J. Midmore and Andrew H. Rank (“A new rural industry—Stevia—to replace imported chemical sweeteners. A report for the Rural Industries Research and Development Corporation, August 2002; RIRDC Web Publication No. W02/022, RIRDC Project No UCQ-16A) report multiple crops per year are possible in warmer climates such as Brazil, Paraguay, India or Indonesia. “Commercial yield figures are not reported other than in general terms, e.g. 1,600-2,000 kg dried leaves/ha/year. Experimental yields suggest that systems with multiple harvests a year will give higher yields.” This yield refers to mass of dried plant, not to optimization of steviol glycoside per hectare per harvest. By leveraging improved analytical methods, and good agronomic development of plants selected for improved steviol glycoside yield per hectare, and by coordinating plant maturation within a given geographic region with staggered harvesting, improvements in the post-harvest steviol glycoside yield per harvest season can be accomplished.

Midmore and Rank go on to report “The usual procedure is to harvest the whole crop green and transport it to drying facilities: sun drying or (artificial) drying kilns. With low humidity, sun drying of a thin layer of cut plants can be quite rapid (9-10 hours) to reduce plant moisture from approximately 80% to 10% [116]. Kiln drying can take two days [37]. Fast drying is likely to give ‘better quality’ dried leaves. If cut plant material is not dried quickly leaf quality can deteriorate by oxidation, losing up to one third of stevioside content after three days [116]. High temperature during artificial drying can also lead to loss of content. A green dried leaf colour is desirable and represents good quality”. We have enabled improvement in this practice by staggering harvest and timing it to extractive processing of wet (i.e., fresh, undried) leaves that are not subjected to any drying process. This improvement confers the dual benefits of reducing glycoside loss during drying and the savings in processing costs by obviating the need for a drier.

DriedStevialeaves are reported to contain approximately 5% to 9% moisture, 10% to 20% protein, 35% to 62% carbohydrates, 3% to 5% fats (principally palmitic, linolenic and linoleic acids), 7% to 13% ash and several volatile components including spathulenol, beta-pinene, beta-caryophyllene, and caryophyllene oxide.Steviais also a rich source of oxalic acid (see Wölwer-Rieck U. The Leaves ofStevia rebaudiana(Bertoni), Their Constituents and the Analysis Thereof: A Review. J. Agric. Chem. 2012, 60, 886-895). These components can affect the flavor aroma of the sweetener if not removed or reduced to a substantially low level. As a result, the isolation of individual high-puritySteviasweeteners has entailed extensive processing including treatment with various resins, separation using organic solvents, and crystallization to render a product of suitable taste and quality.

At present there are more than 230Steviaspecies with significant sweetening properties. The plant has been successfully grown under a wide range of conditions from its native subtropics to the cold northern latitudes. The composition of steviol glycosides can be modified by directed plant breeding. Whereas native plant species had stevioside as a predominant steviol glycosides modern cultivars have been selected for increased Rebaudioside A content, which is considered a better quality sweetener than stevioside.Stevia rebaudianaproduces a number of other steviol glycosides that also feature high intensity sweetness and sensory properties, in some cases superior to those of many other naturally occurring high potency sweeteners. Steviol glycosides are not metabolized for energy in the human digestive system, and can be used for sweetening foods without adding calories wherever sugar is used. They are suitable for diabetic and low calorie diets.

The above-mentioned glycosides have a common aglycone, steviol, and differ by the number and type of carbohydrate residues at the C13and C19positions (R2 and R1 respectively inFIG. 1). The leaves ofSteviaare able to accumulate up to 10-20% (on dry weight basis) steviol glycosides. The major glycosides found in nativeStevialeaves are Rebaudioside A (2-10%), Stevioside (2-10%), and Rebaudioside C (1-2%). Other glycosides, such as Rebaudioside B, D, E, and F, Steviolbioside and Rubusoside, are also found at lower levels (less than 1%). Still other glycosides have been recently reported by Ohta, et al. in J. Appl. Glycosci. (2010) 57:199-209.

Two major glycosides—Stevioside and Rebaudioside A, were extensively studied and characterized in terms of their suitability as commercial high intensity sweeteners. For example, stability studies in carbonated beverages confirmed their heat and pH stability. Chang S. S. et al. (1983) Stability studies of Stevioside and Rebaudioside A in carbonated beverages. J. Agric. Food Chem. 31: 409-412. Even in a highly purified state, these steviol glycosides still possess undesirable taste attributes such as bitterness, sweet aftertaste, licorice flavor, etc. One of the main obstacles for the successful commercialization ofSteviasweeteners are these undesirable taste attributes. It was shown that these flavor notes become more prominent as the concentration of steviol glycoside increases (Prakash et al. (2008) Development of Rebiana®, a natural, non-caloric sweetener. Food Chem. Toxicol., 46, S75-S82).

Rebaudioside A has shown the least astringent, the least bitter, and the least persistent aftertaste thus possessing favorable sensory attributes as compared to known steviol glycosides (Tanaka O. (1987) Improvement of taste of natural sweeteners. Pure Appl. Chem. 69:675-683; Phillips K. C. (1989)Stevia: steps in developing a new sweetener. In: Grenby T. H. ed. Developments in sweeteners, vol. 3. Elsevier Applied Science, London. 1-43).

U.S. Published Application No. 2013347140 to Wang discloses a method of breedingSteviaplants having high content of Rebaudioside A.

U.S. Pat. Nos. PP23,164 and PP23,728 disclose cultivars ofStevianamed AKH L1 and AKH L4. The cultivars have high rebaudioside A content.

Methods for the extraction and purification of sweet glycosides from theStevia rebaudianaplant using water or organic solvents are described in, for example, U.S. Pat. Nos. 4,361,697; 4,082,858; 4,892,938; 5,972,120; 5,962,678; 7,838,044 and 7,862,845.

Dobberstein and Ahmed (U.S. Pat. No. 4,361,697) and Morita et al. (U.S. Pat. No. 4,082,858) disclose the silica chromatography of steviol glycosides. Giovenatto (U.S. Pat. No. 4,892,938) employs calcium hydroxide to clarify a crude extract ofSteviasweeteners, followed by filtration or centrifugation. This precipitate is treated with a strongly acidic ion exchange resin and subsequently with a weakly basic ion exchange resin, filtered and dried. Magomet and others (U.S. Pat. No. 7,862,845) also teach the isolation of partially purified steviol glycosides by treatment of a crude extract with calcium salts, filtration of the crude slurry, followed by crystallization of Rebaudioside A from a methanol-water mixture. Abelyan et al. (U.S. Pat. No. 7,838,044) teaches the extraction of sweet glycosides from theStevia rebaudianaBertoni in the presence of pectinase, and purification by contacting the extract with cyclodextrin and bentonite and ion-exchange resins, followed by crystallization and recrystallization from ethanol. Kutowy et al. (U.S. Pat. No. 5,972,120) disclose a process for the extraction of sweet compounds fromStevia rebaudiana(Bertoni) in a vertical extraction column, followed by purification by filtration, by using microfiltration as a pre-treatment step to clarify the extract, then ultrafiltration followed by nanofiltration. Payzant et al. (U.S. Pat. No. 5,962,678) uses two ion exchange columns to remove impurities from sweet glycosides extracted from theStevia rebaudiana, methanol being used to elute sweet glycosides from the second column, and cooling the solution to crystallize Stevioside. The filtrate is further concentrated and cooled to crystallize out Rebaudioside A.

U.S. Pat. No. 4,612,942 discloses diterpene glycosides, including Rebaudioside D, and their use in foodstuffs, medical compositions, oral hygiene compositions, chewing compositions and smoking compositions.

It has been determined that some of these undesirable properties can be reduced or eliminated by subjecting steviol glycosides to the reaction of intermolecular transglucosylation, when new carbohydrate residues are attached to initial molecule at C13and C19positions. Depending on the number of carbohydrate residues in these positions the quality and potency of the compounds taste will vary. Table 1 illustrates how sweetness quality and intensity vary with the number and position of glycosides. Glucose moieties (designated as Glue or G in the table) are connected by a beta-glycosidic linkage. Pullulanase, isomaltase (Lobov S. V. et al. (1991) Enzymatic production of sweet stevioside derivatives: transglycosylation by glucosidases. Agric. Biol. Chem. 55: 2959-2965), 3-galactosidase (Kitahata S. et al. (1989) Production of rubusoside derivatives by transglycosylation of various β-galactosidase. Agric. Biol. Chem. 53: 2923-2928), and dextran saccharase (Yamamoto K. et al. (1994) Effective production of glucosyl-stevioside by α-1,6-transglucosylation of dextran dextranase. Biosci. Biotech. Biochem. 58: 1657-1661) have been used as transglycosylating enzymes, together with pullulan, maltose, lactose, and partially hydrolyzed starch, respectively, as donors of glycosidic residues to extend linearly the carbohydrate portion on the steviol glycoside core with alpha-linked glucose units.

The transglucosylation of steviol glycosides was also performed by action of cyclodextrin glucanotransferases (CGTase) produced byBacillus stearothermophilus(U.S. Pat. Nos. 4,219,571, and 7,807,206). As a result, α-1,4-glucosyl derivatives were formed with a degree of polymerization up to 10.

It was shown that the taste profile and sweetness power of glucosyl derivatives are largely dependent on a number of additional glucosyl derivatives, i.e., the degree of polymerization of the α-1,4-glucosyl chain. The increase in number of α-1,4-glucosyl residues improved the taste quality but at the same time reduced the sweetness level. (Tanaka, 1987).

It is to be noted also that many glucosylSteviaproducts contain up to 20% residual dextrins which do not possess significant functional properties and reduce the content of steviol glycosides in the product, further reducing sweetness intensity.

Therefore, it is necessary to develop a simple and efficient process of preparation of high puritySteviaglycosides.

China Published Application No. CN103012516 to Wuxi KingboonSteviaInternat Trade Co. Ltd. discloses methods of preparing stevioside that include a step of soakingStevialeaves in water; a step of coarse filtration and ultrafiltration to obtain an ultrafiltration solution; a step of introduction of the ultrafiltration solution to a nano micro guard column; and at least two steps of alcoholysis on the nano micro guard column.

China Published Application No. CN102127129 to Liaoning Qianqian Biolog Technology Co. discloses a method of preparing aSteviaextract that includes a step of mechanical crushingStevialeaves; a step of filtering theStevialeaves; a step of ultrasonic extraction; a step of flocculation; a step of adsorption; a step of analysis via ethanol elution; a step of desalting and decolorizing; a step of concentrating; and a step of spray drying.

China Published Application No. CN102406113 to Ningbo Green Health Pharmaceutical Co. Ltd. discloses a method to prepare a rebaudioside A/rebaudioside D preparation that includes a step of extracting pulverizedStevialeaves with water; a step of passing the filtrate through a macroporous resin column; and a step of eluting with an alcoholic solvent using HPLC analysis detecting rebaudioside A/rebaudioside D content.

U.S. Published Application No. 20130071537 to E.P.C Plant Pharmaceutical Technology Co., Ltd. discloses compositions ofSteviabased sweeteners that include a salt form of Rebaudioside B.

U.S. Published Application No. 20140243514 to Cargill, Incorporated discloses a method of preparing an enriched composition comprising at least one of rebaudioside B, rebaudioside D, or a mixture thereof that includes use of a macroporous neutral porous resin.

U.S. Published Application No. 20130309389 to Cargill, Incorporated discloses a composition that contains specified amounts of rebaudioside D and rebaudioside B.

U.S. Published Application No. 20140004248 to LGL Life Tech Corporation discloses a process for producing a natural sweetener composition comprising at least one of steviolbioside extract, rebaudioside B extract and rebaudioside D extract, comprising the use of a porous adsorption column having specified parameters. U.S. Published Application No. 20130071339 to Markosyan discloses methods of preparing highly purified steviol glycosides, particularly Rebaudioside D.

U.S. Published Application No. 20150017284 to Markosyan discloses a rebaudioside M and rebaudioside D crystalline composition containing at least 75% rebaudioside M.

U.S. Published Application No. 20130071339 to Markosyan discloses a method for purifying steviol glycosides that includes the use of consecutively connected columns packed with an adsorbent resin capable of adsorbing steviol glycosides.

U.S. Pat. No. 8,299,224 to PureCircle Sdn Bhd discloses a method for purifying Rebaudioside D fromSteviaextract that includes providing an extract ofStevia; dissolving the extract in a first aqueous solution of organic solvent to result in a first mixture of steviol glycosides, wherein the organic solvent is selected from the group consisting of methanol, ethanol, 1-propanol, isopropanol, and a mixture thereof, and the organic solvent is 75-99 vol. %; inducing crystallization in the first mixture; filtering the mixture from to obtain a first precipitate and a first filtrate; dissolving the first precipitate in a second aqueous solution of organic solvent to result in a second mixture, wherein the organic solvent is selected from the group consisting of methanol, ethanol, 1-propanol, isopropanol, and a mixture thereof, and the organic solvent is 70-80-vol. %; inducing crystallization in the second mixture; filtering the mixture to obtain a second precipitate and a second filtrate; dissolving the second precipitate in a third aqueous solution of organic solvent to result in a third mixture, wherein the organic solvent is selected from the group consisting of methanol, ethanol, 1-propanol, isopropanol, and a mixture thereof, and the organic solvent is 10-80 vol. %; inducing crystallization in the third mixture; and filtering the mixture to obtain a third precipitate and a third filtrate; whereby drying the third precipitate yields purified Rebaudioside D.

U.S. Published Applications Nos. 20140335253, 20140335254, 20140335264 and 20140335265 to EPC Beijing Natural Products Co. Ltd. disclose non-natural rebaudioside D compositions that contain an increased amount of purified rebaudioside D, wherein the increased amount of purified rebaudioside D is added as purified rebaudioside D to the compositions.

U.S. Published Application No. 20120269954 to Tate & Lyle Ingredients Americas LLC discloses aSteviaextract comprising Rebaudioside B.

U.S. Published Application No. 20110256588 to Lee et al. discloses a method of producing Rebaudioside A in a high yield by recycling by-products produced when Rebaudioside A is produced from leaves ofStevia RebaudianaBertoni.

U.S. Published Application No. 20110183056 to Morita et al. discloses isolated steviol glycosides having the structures disclosed therein. The reference discloses that the steviol glycosides, which have structures which present more glycosidic moieties than on stevioside or Rebaudioside A, may provide a subtle improvement toSteviasweetener taste.

U.S. Pat. No. 8,520,527 to PureCircle Sdn Bhd discloses a process for producing aSteviafood ingredient that includes a step of soaking aSteviabiomass in water to remove soluble components; a step of incubating a water-insolubleSteviabiomass in alkaline solution to produce a pulp; and further processing steps to achieve aSteviafood ingredient that contains microcrystalline cellulose.

U.S. Pat. No. 8,337,928 to Concentrate Manufacturing Company of Ireland discloses a beverage product that comprises at least one steviol glycoside sweetener and anisic acid.

U.S. Pat. Nos. 8,318,459 and 8,257,948 to PureCircle USA disclose a process for producing a highly purified glucosylSteviacomposition using starch as a source of the glucose residues.

U.S. Pat. No. 8,299,224 to PureCircle Sdn Bhd discloses a method for purifying Rebaudioside D fromSteviaextract.

U.S. Pat. No. 8,277,862 to Concentrate Manufacturing Company of Ireland discloses a beverage product that comprises Rebaudioside A, erythritol and an acid component.

U.S. Pat. No. 8,277,862 to Concentrate Manufacturing Company of Ireland discloses a beverage product that comprises a steviol glycoside and an acid component.

U.S. Pat. No. 7,964,232 to PepsiCo, Inc. discloses steviol glycoside isomers wherein the exo-cyclic double bond of formula I therein has been moved to an endo-cyclic position within the five-membered ring.

U.S. Pat. No. 7,927,851 to Vineland Research and Innovation Centre discloses a method of producing a steviol glycoside in a plant or plant cell comprising, a) selecting a plant or plant cell that produces ent-kaurenoic acid; b) transforming the plant or plant cell with a first nucleotide sequence encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity, and at least one other nucleotide sequence encoding one or more glucosyltransferases to catalyse the addition of one or more glucose molecules to steviol, or glucosyl-steviol; and c) expressing the polypeptide having ent-kaurenoic acid 13-hydroxylase and said one or more glucosyltransferases in the cell to convert ent-kaurenoic acid to one or more steviol glycosides.

Ohta et al., Characterization of novel steviol glycosides from leaves ofStevia rebaudianamorita, J. Appl. Glycosci., 57, 199-209 (2010), discloses the structures of steviol glycosides extracted from leaves ofS. rebaudianaMorita, which was produced by selection and breeding ofS. rebaudianaBertoni.

Zimmerman, Tandem mass spectrometric fragmentation patterns of known and new steviol glycosides with structure proposals, Rapid Commun. Mass. Spectrom. 2011, 25, 1575-1582, discloses the use of tandem mass spectrometry to identify 12 previously unknown steviol glycosides.

Brandle et al. (2002) Plant Molecular Biology 50: 613-622; Richman et al. (1999) The Plant Journal 19(4), 411-421; and Richman et al., (2005) The Plant Journal 41, 55-67, disclose metabolic pathways for the production of steviol and the conversion of steviol to various steviol glycosides.

Existing methods deal with isolation and purification of a steviol glycoside from an initial extract and do not show a way for the further treatment of residual solution or purification of minor compounds, individually or collectively. Thus, there remains a need for an efficient and economical method for comprehensive retreatment of extract produced fromStevia rebaudianaplants.

SUMMARY OF THE INVENTION

The invention relates to a comprehensive process for separation, isolation, characterization and use of two or more minor steviol glycosides (and, in particular, those naturally occurring at less than 1% dry leaf weight) fromStevia rebaudianaplants.

According to an embodiment, an efficient method of separating, isolating and characterizing combinations of two or more steviol glycosides fromSteviaextract is provided, thus minimizing waste.

The combinations of two or more steviol glycosides, alone or in the combination with other sweeteners and/or other ingredients, are useful as non-caloric sweeteners in edible and chewable compositions such as beverages, confectionaries, bakeries, cookies, chewing gums, pharmaceuticals and the like.

According to an embodiment, the method includes:1. the use of a chromatographic method for the identification and quantitation of combinations of two or more minor steviol glycosides (occurring at less than 5% dry leaf weight) as a means of informing a breeding program to optimize for the production of that defined set of steviol glycosides, which are not currently used commercially as sweeteners,2. the method of extracting, including by staged extraction, ofStevialeaves, fresh leaves or desiccated for preservation, to optimize recovery of the two or more minor steviol glycosides of molecular weight greater than about 900 g/mol, and minimize the need for subsequent processing steps,3. the use of chromatography to isolate a collection of two or more steviol glycosides of molecular weight greater than about 900 g/mol, preferably rebaudiosides D, I, O M and/or N, all of which individually occur at levels below 5% dry leaf weight basis,4. a process which avoids the need for crystallization in the isolation of steviol glycosides D, I, O M and/or N, which involves gradient elution using 2 or more solvents at a constant temperature, or at a temperature gradient with the eluting solvent,5. the use as a sweetener of a combination of two or more steviol glycosides, preferably rebaudiosides D, I O, M and/or N, which individually occur at levels below 5% dry leaf weight basis,6. the two or more steviol glycosides can be used alone or in combination with more predominant steviol glycosides, including, but not limited to, Rebaudioside A, Rebaudioside B, Rebaudioside D, in amounts less than about 25% predominant steviol glycoside, less than about 15% predominant steviol glycoside, less than about 10% predominant steviol glycoside, less than about 5% predominant steviol glycoside. Table 9, taken from Ohta et al. (2010), provides the relative amounts of steviol glycosides from the leaves ofS. rebaudianaMorita andS. rebaudianaBertoni.

Continuous counter current ion exchange and adsorption such as SepTor technology by Outotec, Espoo, Finland, is a technique that could be employed to remove contaminants. Continuous chromatography is different from conventional (single or multi-column) stationary solid phase chromatography in that the multiple columns employed in continuous chromatography are operated as if they were a single column, but with multiple feed points and discharge points between the columns to enable progressively advancing the feed points for the mixture and eluting solvent, and the take-off points for faster and slower eluting components in the mixture. This mode of operation allows each component column segment to be operated autonomously with respect to eluant composition or flow rate. The simulation of continuous separations process is described by Johan Samuelsson in “Simulation of continuous chromatographic processes” and by Niklas Andersson in “Simulation of continuous preparative chromatography: A case study in MCSGP.” (2009) for the use of a solvent gradient (SG) in a multi column (MC) system. “Preparative” (P) relates to effecting a separation at a scale larger than for analytical purposes, up to commercial production scale. The application of continuous chromatographic separation of sugars is well developed in the beet sugar refining industry and is described by Bubnik and co-authors in “Application of continuous chromatographic separation in sugar processing”, Journal of Food Engineering 61 (2004) 509-513. The application of continuous separation methods must be customized to the type of separation undertaken. For example, a separation strategy for the isolation of high purity rebaudioside A will be different from the operation of the system for a custom mixture of other steviol glycosides. Up to the present, continuous separation methods have not been applied to the isolation of custom steviol glycoside mixtures comprising at least two steviol glycosides selected from rebaudiosides A, D, I, O, M, and N.

Another important distinction in continuous chromatography is the use of a persistent inventory of material to be separated residing in the multi-column system and which is continually circulated in the system. This inventory is advantageously employed in aiding the separation by adjusting the inventory of individual components to favor a desired composition. For example, if a feed mixture has a low level of a set of desired components (e.g., 3-5% rebaudiosides A, D, I, O, M, and N) but a dominant portion (>20%) of rebaudioside A, the column can be operated initially by taking off mainly the predominant component while allowing the lesser components to remain inventory in the column. This may allow the lesser components in the feed to be better resolved in the column inventory from other components enabling the take-off of a stream enriched in the desired (minor) components.

The ability to operate each column independently is of crucial importance in separations involving two or more inputs to the separation system (e.g., process feed, eluent A and eluent B), and two or more take-offs (e.g., Product 1, Product 2, Product 3, etc.) where the flow rates of take-off are different for each. The column flow balance is calculated as
Fin(preceding column)+Fin(feed)=Fout(Product)+Fout(to next column)

Where Fin(feed)may be the process feed or an eluting solvent and Fout(product)is a product take-off.

Simulated Moving Bed

In manufacturing, the simulated moving bed (SMB) process is a highly engineered process for implementing chromatographic separation. It is used to separate one chemical compound or one class of chemical compounds from one or more other chemical compounds to provide significant quanitites of the purified or enriched material at a lower cost than could be obtained using simple (batch) chromatography. It cannot provide any separation or purification that cannot be done by a simple column purification. The process is rather complicated. An advantage that it brings to a chromatographic purification is that it allows the production of large quantities of highly purified material at a dramatically reduced cost. The cost reductions come about as a result of: the use of a smaller amount of chromatographic separation media stationary phase, a continuous and high rate of production, and decreased solvent and energy requirements. This improved economic performance is brought about by a valve-and-column arrangement that is used to lengthen the stationary phase indefinitely and allow very high solute loadings to the process.

In the conventional moving bed technique of production chromatography the feed entry and the analyte recovery are simultaneous and continuous, but because of practical difficulties with a continuously moving bed, simulated moving bed technique was proposed. In the simulated moving bed technique instead of moving the bed, the feed inlet, the solvent or eluent inlet and the desired product exit and undesired product exit positions are moved continuously, giving the impression of a moving bed, with continuous flow of solid particles and continuous flow of liquid in the opposite direction of the solid particles.

Another form of continuous chromatographic separation is annular chromatography. In a published review, Frank Hilbrig and Ruth Freitag (“Continuous annular chromatography”, Journal of Chromatography B, 790 (2003) 1-15) report “The principle of continuous annular chromatography (CAC) has been known for several decades. CAC is a continuous chromatographic mode, which lends itself to the separation of multi-component mixtures as well as of bi-component ones. In CAC, the mobile and stationary phases move in a crosscurrent fashion, which allows transformation of the typical one-dimensional batch column separation into a continuous two-dimensional one. With the exception of linear gradient elution, all chromatographic modes have at present been applied in CAC.” Earlier, Bart and co-authors (Continuous chromatographic separation of fructose, mannitol and sorbitol. Chemical Engineering and Processing 35 (1996) 459-471) applied the method to separation of simple sugars, but this method has not been employed for the separation of steviol glycosides.

