BR112019011036B1 - METHOD FOR PREPARING NON-STEVIOL GLYCOSIDE COMPOSITION, CONSUMABLE INCLUDING SAID COMPOSITION AND METHOD FOR PREPARING CONSUMABLE - Google Patents

Abstract

The invention relates to a process for producing food ingredients from the Stevia rebaudiana plant and their use in food products, beverages and other inputs. The obtained compositions are useful as flavorings, sweeteners, antioxidants and other functional ingredients.

Description

RELATED ORDERS

[001] This application claims priority over U.S. Patent Application At the. 62/427,539, filed November 29, 2016, which is incorporated herein by reference in its entirety. For United States of America purposes, this application is also a continuation in part of U.S. Patent Application At the. 15/284,265, filed October 3, 2016, which is a continuation of U.S. Patent Application At the. 14/677,538, filed April 2, 2015, now granted as a U.S. Patent At the. 9,456,626, which is a continuation of U.S. Patent Application At the. 13/993,415, filed June 12, 2013, now granted as a U.S. Patent At the. 9,029,426, which is a U.S. of National Phase of International Application No. PCT/US2011/064343, filed December 12, 2011, which claims priority over U.S. Patent Application At the. 61/424,798, filed December 20, 2010, and U.S. Patent Application At the. 61/422,403, filed December 13, 2010, each of these applications is incorporated by reference herein in its entirety. This application also incorporates by reference each of the following applications in their entirety: U.S. Patent Application At the. 13/530,113, filed June 22, 2012, now issued as a U.S. Patent At the. 8,530,527; U.S. Patent Application At the. 13/580,098, filed November 6, 2012, now issued as a U.S. Patent. At the. 8,981,081; U.S. Patent Application At the. 13/943,776, filed July 16, 2013; U.S. Patent Application At the. 13/957,098, filed August 1, 2013, now issued as a U.S. Patent At the. 9,510,611; U.S. Patent Application At the. 14/195,812, filed March 3, 2014; and U.S. Patent Application At the. 14/829,127, filed August 18, 2015, now granted as a U.S. Patent At the. 9,771,434.

FIELD OF INVENTION

[002] The invention relates to a process for the production of food ingredients from the Stevia rebaudiana plant and its use in food products, beverages and other inputs.

DESCRIPTION OF THE RELATED TECHNIQUE

[003] High intensity sweeteners have a level of sweetness many times higher than that of sucrose. They are essentially non-caloric and widely used in the manufacture of low-calorie and diet foods. Although natural caloric sweeteners such as sucrose, fructose and glucose provide the most desirable taste to consumers, they are high in calories. High-intensity sweeteners do not affect the blood glucose level and provide little or no nutritional value.

[004] Stevia rebaudiana Bertoni is an evergreen shrub of the Asteraceae family (Compositae), native to certain regions of South America. The leaves of the plant contain 10 to 20% diterpene glycosides, which are about 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.

[005] Currently, there are over 230 species of Stevia with significant sweetening properties. The plant has been successfully cultivated under a wide range of conditions, from its native subtropical latitudes to the cold northern latitudes.

[006] Stevia rebaudiana plant extract 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 glycosides accumulate in Stevia leaves and make up approximately 10% - 20% of the total dry weight. Typically, based on dry weight, the four main glycosides found in Stevia leaves are Dulcoside A (0.3%), Rebaudioside C (0.6-1.0%), Rebaudioside A (3.8%) and Stevioside (9.1%). Other glycosides identified in Stevia extract include Rebaudioside B, C, D, E and F, Steviolbioside and Rubusoside.

[007] Steviol glycosides have zero calories and can be used wherever sugar is used. They are ideal for diabetic and hypocaloric diets.

[008] On the other hand, it should be noted that in the manufacturing process, along with the steviol glycosides, large amounts of other constituents of the Stevia plant are also extracted with water. These other constituents are mostly separated during downstream processing and discarded into the environment.

[009] Little is known about these other constituents of the Stevia rebaudiana plant. Few authors reported phenolic compounds, free amino acids, etc. however, information on the identity of these constituents remains scarce and their possible uses in food, beverages and other inputs are not described (Karakose, et al, 2011, 2015; Wolwer-Rieck, 2001). 2012; Periche et al, 2014).

[010] There are no reports to date on the processing of the other components extracted from the Stevia plant in any food ingredient. Therefore, if carried out on a large scale, this could provide significant economic and environmental benefits, as it could provide an opportunity for the inclusion of the entire stevia plant in the food chain, creating virtually wasteless stevia processing.

[011] Therefore, it is necessary to develop a method to effectively isolate the constituents of the Stevia rebaudiana plant and use them as ingredients in different consumables.

SUMMARY OF THE INVENTION

[012] The present invention aims to overcome the disadvantages of existing industrial processing schemes for Stevia. The invention describes a process for producing food ingredients from the Stevia rebaudiana plant and their use in various consumables, including food and beverage products.

[013] The invention, in part, relates to compositions comprising phenolics and other glycosidic compounds without steviol, derived from the Stevia rebaudiana plant.

[014] Hereinafter the term "steviol glycoside(s)" shall mean steviol glycosides naturally occurring in Stevia rebaudiana, including but not limited to steviolmoside, and steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G , stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O and combinations thereof.

[015] Below, the terms “RebA”, “RebB”, “RebC”, “RebD”, “RebE”, “RebF”, “RebM”, “RebN” and “RebO” refer to Rebaudiosides A, B, C, D, E, F, M, N and O.

[016] In the following, the terms “Stev”, “Sbio”, “DulA”, “Rub”, refer to Stevioside, Steviolbioside, Dulcoside A and Rubusoside.

[017] Hereafter, the term "TSG content" shall mean the total content of Steviol Glycosides (TSG), and will be calculated as the sum of the concentrations of Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside M, Rebaudioside N, Rebaudioside O, Stevioside, Steviolbioside, Dulcoside A and Rubusoside on a dry weight/weight basis.

[018] Hereinafter the term "CGA(s)" shall mean chlorogenic acids and their derivatives that occur naturally in plants, including but not limited to neochlorogenic acid (neo-CGA; 5-O-caffeoylquinic acid or 5-CQA), cryptochlorogenic acid (crypto-CGA; 4-O-caffeoylquinic acid or 4-CQA), n-chlorogenic acid (n-CGA; 3-O-caffeoylquinic acid or 3-CQA), iso-chlorogenic acid A (iso- CGA A; 3,5-dicafoylquinic acid) iso-CGA B (iso-CGA B; 3,4-dicafoylquinic acid), iso-chlorogenic acid C (iso-CGA C; 4,5-dicafoylquinic acid) and combinations thereof .

[019] From now on, the term "TCGA content" means the content of Total Chlorogenic Acids (TCGA), and will be calculated as the sum of the concentrations of neo-CGA, crypto-CGA, n-CGA, iso-CGA A , iso-CGA B and iso-CGA C on a dry weight/weight basis.

[020] In the invention, plant material from Stevia rebaudiana, particularly the leaves and/or stems, was used as a starting material.

[021] The plant material was subjected to extraction using water or alcoholic aqueous solvent.

[022] The obtained water or aqueous alcoholic extract was further processed to separate the fraction of steviol glycosides. The remaining fraction was designated as a “non-steviol glycoside composition” (NSGC) - meaning a composition predominantly comprising compounds, other than steviol glycosides, which occur in water or aqueous alcoholic extracts of the Stevia rebaudiana plant (hereinafter “non-steviol glycoside molecules ”). Non-limiting examples of “steviol-free glycoside molecules” include phenolic compounds, polyphenols, flavonoids, quinic and caffeic acids and their derivatives, neochlorogenic acid (neo-CGA; 5-O-caffeoylquinic acid or 5-CQA), crypto-chlorogenic acid (crypto-CGA acid; 4-O-caffeoylquinic acid or 4-CQA), n-chlorogenic acid (n-CGA acid; 3-O-caffeoylquinic acid or 3-CQA), iso-chlorogenic acid A (iso-CGA A 3,5-dicaffeoylquinic acid) iso-chlorogenic acid B (iso-CGA B; 3,4-dicaffeoylquinic acid), iso-chlorogenic acid C (iso-CGA C; 4,5-dicaffeoylquinic acid), other chlorogenic acids and acids art-known iso-chlorogens, retinoids, pigments, polysaccharides, oligosaccharides, disaccharides, monosaccharides, volatile oil components, sterols, terpenoids, sesquiterpenoids, diterpenes, triterpenes, coumarins, fatty acids and their derivatives, amino acids and their derivatives, dipeptides, oligopeptides , polypeptides, proteins, austroinulin, quercetin, steribines, spathulenol, decanoic acid, 8,11,14-ecosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, stigmasterol, bsitosterol, a- and b-amyrin, lupeol, b-acetate amyrin, pentacyclic triterpenes and/or glycosides thereof.

[023] Compositions prepared in some embodiments of the present invention and designated as NSGC may also contain some residual amounts of steviol glycosides.

[024] Some NSGCs can be further purified and/or processed by any food ingredient processing method known in the art to obtain other NSGCs.

[025] The NSGCs of the present invention are applicable in various consumables, foods and beverages such as flavorings, flavor modifiers, flavor enhancers, sweeteners, preservatives, antioxidants, emulsifiers, texturizing agents, bulking agents, stabilizers, solubilizers and other ingredients food.

[026] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[027] The attached drawing is included to provide an additional understanding of the invention. The drawing illustrates embodiments of the invention and together with the description serves to explain the principles of the embodiments of the invention.

[028] FIG. 1 shows the structures of some CGAs.

[029] FIG. 2 shows the HPLC chromatogram of Stevia rebaudiana CGA.

DETAILED DESCRIPTION OF THE INVENTION

[030] Advantages of the present invention will become more apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are presented by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. technique of this detailed description.

[031] Those skilled in the art will also recognize that one or more of the process steps described below may be omitted. Those skilled in the art will also understand that while the process described below assumes a certain order of the steps described, that order may be altered in some cases.

Extraction

[032] In the invention, plant material from Stevia rebaudiana, particularly the leaves and/or stems of the Stevia rebaudiana plant, were used as the starting material. Stevia rebaudiana plant material extract can be obtained using any method such as, but not limited to, the extraction methods described in U.S. Patent No. 7,862,845, the entire contents of which are incorporated herein by reference, as well as membrane filtration, supercritical fluid extraction, enzyme-assisted extraction, microorganism-assisted extraction, ultrasound-assisted extraction, microwave-assisted extraction, etc.

[033] In one embodiment, Stevia rebaudiana plant material (e.g., leaves) may be dried at temperatures between about 20°C and about 100°C until a moisture content of between about 5% and about of 15%. In a particular embodiment, the plant material can be dried at between about 20°C and about 60°C for a time period of from about 1 to about 240 hours.

[034] In other particular embodiments, the plant material may be dried at temperatures between about 20 °C and about 35 °C to prevent decomposition.

[035] In some embodiments, the plant material of Stevia rebaudiana can be dried under vacuum or reduced pressure.

[036] In some embodiments, the plant material of Stevia rebaudiana can be dried in the presence of an inert gas such as N2.

[037] In some embodiments, plant material from Stevia rebaudiana can be freeze-dried.

[038] In some embodiments, the dried plant material is optionally ground. Particle sizes can be between about 0.1 and about 20 mm.

[039] The plant material (ground or unground) can be extracted by any suitable extraction process, such as, for example, continuous or batch reflux extraction, supercritical fluid extraction, enzyme-assisted extraction, microorganism-assisted extraction , ultrasound-assisted extraction, microwave-assisted extraction, etc. The solvent used for the extraction can be any suitable solvent, for example polar organic solvents (degassed, vacuolated, pressurized or distilled), non-polar organic solvents, water (degassed, vacuolated, pressurized, deionised, distilled, carbon treated or reverse osmosis) or a mixture of these. In a particular embodiment, the solvent comprises water and one or more alcohols. In another embodiment, the solvent is water. In another embodiment, the solvent is one or more alcohols. The alcohol can be selected from, for example, methanol, ethanol, n-propanol, 2-propanol, 1-butanol, 2-butanol and mixtures thereof.

[040] In a particular embodiment, the plant material is extracted with water in a continuous reflux extractor. One skilled in the art will recognize that the ratio of extraction solvent to plant material will vary based on the identity of the solvent and the amount of plant material to be extracted. Generally, the ratio of extraction solvent to kilogram of dry plant material is about 20 liters to about 25 liters for about one kilogram of leaves.

[041] The pH of the extraction solvent may be between about pH 2.0 and 7.0, such as, for example, between about pH 2.0 and about pH 5.0, between about pH 2, 0 and about pH 4.0 or between about pH 2.0 and about pH 3.0. In a particular embodiment, the extraction solvent is aqueous, for example, water and, optionally, acid and/or base in an amount to provide a pH between about pH 2.0 and 7.0, such as, for example, between about pH 2.0 and about pH 5.0, between about pH 2.0 and about pH 4.0 or pH 2.0 and about pH 3.0. Any suitable acid or base can be used to provide the desired pH for the extraction solvent, such as, for example, HCl, NaOH, citric acid and the like.

[042] The extraction can be carried out at temperatures between about 25 °C and about 100 °C, such as, for example, between about 30 °C and about 80 °C, between about 35 °C and about from about 75°C, between about 40°C and about 70°C, between about 45°C and about 65°C, or between about 50°C and about 60°C.

[043] In embodiments where the extraction process is a batch extraction process, the duration of the extraction may vary from about 0.5 hour to about 24 hours, such as, for example, from about 1 hour to about 12 hours, from about 1 hour to about 8 hours, or from about 1 hour to about 6 hours.

[044] In embodiments where the extraction process is a continuous process, the duration of the extraction can vary from about 1 hour to about 5 hours, such as, for example, from about 2.5 hours to about 3 hours.

[045] After extraction, the insoluble plant material can be separated from the solution by filtration to provide a filtrate containing steviol glycosides and other molecules, described above as “non-steviol glycoside molecules”. This solid-liquid separation may be accomplished by any suitable means including, but not limited to, gravity filtration, a plate and frame filter press, cross flow filters, screen filters, Nutsche filters, belt filters, ceramic filters, membrane filters, microfilters, nanofilters, ultrafilters or centrifugation. Optionally, various filter aids such as diatomaceous earth, bentonite, zeolite, etc. can also be used in this process. Pre-treatment of filtrate containing steviol glycosides and “non-steviol glycoside molecules”

[046] In some embodiments, the filtrate containing steviol glycosides and "non-steviol glycoside molecules" is optionally pretreated before coming into contact with the macroporous polymeric adsorbent. Said pretreatment may be achieved, for example, by at least one agent selected from the group including but not limited to diatomaceous earth, diatomite, kieselgur/kieselguhr, Celite®, bentonite, activated carbon, any grade of filtration aids food, any flocculating agent, any chelating agent, any acid, any base/alkaline, any ion exchange resin known in the art, or combinations thereof. In some embodiments, pretreatment can be achieved by additional filtration through ultrafiltration and/or nanofiltration and/or art-known RO-filtration membrane systems.

Adsorption of steviol glycosides

[047] In certain embodiments, the filtrate containing steviol glycosides and "non-steviol glycoside molecules" is placed in contact with a macroporous polymeric adsorbent. The macroporous polymeric adsorbent can be any neutral, acidic or alkaline macroporous polymeric adsorption resin capable of adsorbing steviol glycosides, such as the Amberlite® XAD series (Rohm and Haas), Diaion® HP series (Mitsubishi Chemical Corp), Sepabeads® SP series (Mitsubishi Chemical Corp.), Cangzhou Yuanwei YWD series (Cangzhou Yuanwei Chemical Co. Ltd., China), or the equivalent. The adsorbent can be packed into columns to about 75% to about 100% of their total volume.

[048] Steviol glycosides and some “non-steviol glycoside molecules” are adsorbed by the macroporous polymeric adsorbent, while other “non-steviol glycoside molecules” are not adsorbed and pass through the column in the effluent bypass.

[049] The macroporous adsorption resin can be eluted by varying concentrations of aqueous ethanol to obtain various eluate fractions enriched in steviol glycosides and/or "non-steviol glycoside molecules".

[050] In some embodiments, the macroporous adsorption resin can be eluted by varying the pH of the eluting solvent.

Optional Precipitation of Impurities

[051] The pH of the filtrate containing steviol glycosides and/or “non-steviol glycoside molecules” can be adjusted to remove additional impurities. In one embodiment, the pH of the filtrate can be adjusted to between about 8.5 and about 10.0 by treatment with a base, such as, for example, calcium oxide or hydroxide (about 1.0% by volume). of filtrate) with slow stirring.

[052] Treatment of the filtrate with the base, as set out above, results in a suspension, the pH of which can be adjusted to about 3.0 to about 4.0 by treatment with any suitable flocculating/coagulating agent. Suitable flocculating/coagulating agents include, for example, potassium alum, aluminum sulfate, aluminum hydroxide, aluminum oxide, CO2, H3PO4, P2O5, MgO, SO2, anionic polyacrylamides, quaternary ammonium compounds with fatty acid substituents of long chain, bentonite, diatomaceous earth, KemTab Sep series, Superfloc series, KemTab Flote series, Kemtalo Honey series, Midland PCS-3000, Magnafloc LT-26, Zuclar 100, Prastal 2935, Talofloc, Magox, iron salts or a combination thereof same. Exemplary iron salts include, but are not limited to, FeSO4, FeCl2, Fe(NO3)3, Fe2(SO4)3, FeCl3, and combinations thereof. In a particular embodiment, the ferric salt is FeCl3. The filtrate may be treated with the flocculating/coagulating agent for a period of time between about 5 minutes to about 1 hour, such as, for example, from about 5 minutes to about 30 minutes, from about 10 minutes. about 20 minutes or about 10 minutes to about 15 minutes. Agitated or slow agitation can also be used to facilitate treatment. Optionally, the pH of the resulting mixture can then be adjusted to between about 8.5 and about 9.0 with a base, such as, for example, calcium oxide or sodium hydroxide. The duration of time for treatment with base and optionally with agitation is from about 5 minutes to about 1 hour, such as, for example, from about 10 minutes to about 50 minutes, from about 15 minutes to about 45 minutes, from about 20 minutes to about 40 minutes, or from about 25 minutes to about 35 minutes. In a particular embodiment, the base is calcium oxide used for between about 15 and about 40 minutes with slow agitation.

[053] In one embodiment, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules", may be mixed with at least one alcohol to precipitate some impurities.

[054] The precipitated compounds and insoluble particles are separated from the filtrate to provide a composition comprising "non-steviol glycoside molecules". Precipitation separation may be accomplished by any suitable means including, but not limited to gravity filtration, a plate and frame filter press, cross flow filters, screen filters, Nutsche filters, belt filters, ceramic filters, membrane, microfilters, nanofilters, ultrafilters or centrifugation. Optionally, various filter aids such as diatomaceous earth, bentonite, zeolite, etc. can be used in this process.

Deionization

[055] The filtrate containing steviol glycosides and/or “non-steviol glycoside molecules” may be subjected to deionization by any suitable method including, for example, electrodialysis, filtration (nano or ultrafiltration), reverse osmosis, ion exchange, ion exchange mixed bed or a combination of such methods. In one embodiment, the filtrate containing "non-steviol glycoside molecules" is deionized by treatment with one or more ion exchange resins to provide a resin-treated filtrate. In one embodiment, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" is passed through a strong acid cation exchange resin. In another embodiment, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" is passed through a weak base anion exchange resin. In yet another embodiment, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" is passed through a strong acid cation exchange resin followed by a weak base anion exchange resin. In yet another embodiment, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" is passed through a weak base anion exchange resin followed by a strong acid cation exchange resin.

[056] The cation exchange resin can be any strong acid cation exchanger in which the functional group is, for example, sulfonic acid. Suitable strong acid cation exchange resins are known in the art and include, but are not limited to, Amberlite® 10 FPC22H resin from Rohm & Haas, which is a sulfonated divinyl benzene styrene copolymer, Dowex® ion exchange resins from Dow Chemical Company, 15 Serdolit® ion exchange resins available from Serva Electrophoresis GmbH, strong acidic cation exchange resin T42 and strong base A23, an ion exchange resin available from Qualichem, Inc. and strong Lewatit ion exchange resins available from Lanxess. In a particular embodiment, the strong acid cation exchange resin is Amberlite® 10 FPC22H (H+) resin. As would be known to those skilled in the art, other strong acid cation exchange resins suitable for use in embodiments of this invention are commercially available.

[057] The anion exchange resin can be any weak base anion exchanger, in which the functional group is, for example, a tertiary amine. Suitable weak base anion exchange resins are known in the art and include, but are not limited to, resins such as Amberlite-FPA53 (OH-), Amberlite IRA-67, Amberlite IRA-95, Dowex 67, Dowex 77 and Diaion WA 30 can be used. In a particular embodiment, the strong acid cation exchange resin is Amberlite-FPA53 (OH-) resin. As would be known to those skilled in the art, other weak base anion exchange resins suitable for use with the embodiments of this invention are commercially available.

[058] In a particular embodiment, the filtrate containing steviol glycosides and/or “non-steviol glycoside molecules” is passed through a strong acid cation exchange resin, for example, Amberlite® Resin 10 FPC22H (H+), followed by by a weak base anion exchange resin, for example, Amberlite-FPA53 (OH-), to provide a resin-treated filtrate. The specific velocity (SV) through one or more ion exchange columns can be between about 0.01 to about 5 hours -1, such as, for example, between about 0.05 to about 4 hours -1, between about 1 and about 3 hours -1 or between about 2 and about 3 hours -1. In a particular embodiment, the specific velocity through one or more ion exchange columns is about 0.8 hour-1. Upon completion of passing the second filtrate containing steviol glycosides through one or more ion exchange columns, the one or more ion exchange columns are washed with water, preferably reverse osmosis (RO) water. The solution obtained from the water wash and resin-treated filtrate can be combined before proceeding to the multi-column step.

Discoloration

[059] Discoloration of the filtrate containing steviol glycosides and/or “non-steviol glycoside molecules” can be obtained with any known method, such as, for example, contact with activated charcoal. The amount of activated carbon can be from about 0.1% (w/vol) to about 0.8% (w/vol). In a particular embodiment, the amount of activated carbon is from about 0.25% (w/vol) to about 0.30% (w/vol). The suspension can be continuously stirred. The treatment temperature can be between about 20°C and about 30°C, such as, for example, about 25°C. The treatment can be for any duration sufficient to discolor the eluted solution, such as, for example, between about 20 minutes and about 3 hours, between 20 minutes and about 2 hours, between about 30 minutes and 1.5 hours or between about 1 hour and about 1.5 hours. After the treatment, the separation of the used carbon can be carried out by any known means of separation, such as, for example, gravity or suction filtration, centrifugation or plate and frame filter presses.

[060] Alternatively, the filtrate containing steviol glycosides and/or “non-steviol glycoside molecules” can be passed through the column packed with activated carbon.

[061] It should also be understood that treatment with carbon or other bleaching agent can result not only in the bleaching effect, but also provide improvement in taste, flavor, aroma and other organoleptic characteristics.

Concentration and/or Drying

[062] The water or alcohol in the filtrate containing "non-steviol glycoside molecules" may be removed by any suitable means, including, but not limited to, evaporation under reduced pressure or vacuum nanofiltration, lyophilization, flash drying, spray drying or a combining these to provide a concentrated or dry composition comprising "non-steviol glycoside molecules". The dry compositions can optionally be agglomerated and/or granulated by compact or wet granulation techniques.

chromatography

[063] Non-steviol glycoside molecules and NSGCs of the present invention can be further purified and separated using various chromatographic techniques including paper chromatography, thin layer chromatography, column chromatography, liquid chromatography LC, medium pressure LC (MPLC) , high performance LC (HPLC), ultra high performance LC (UHPLC), flash column chromatography, displacement chromatography, affinity chromatography, supercritical fluid chromatography, ion exchange chromatography, size exclusion chromatography, adsorption chromatography , expanded bed adsorption chromatography, reversed phase chromatography, normal phase chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography, two-dimensional chromatography, simulated moving bed chromatography (SMBC), countercurrent chromatography and chiral chromatography conducted on an analytical, preparative, pilot or industrial scale.

[064] In one embodiment, a chromatography system comprising a column packed with adsorption resin and elution achieved by applying an alcoholic solvent (for example, ethanol) with a concentration increase gradient is used to separate fractions enriched with steviol glycosides or ”non-steviol glycoside molecules”.

[065] In another modality, a chromatography system comprising a column packed with ion exchange resin and the elution achieved by applying an acidic or alkaline solvent is used to separate fractions enriched with steviol glycosides or "non-steviol glycoside molecules" .

[066] In another embodiment, a chromatography system comprising a plurality of consecutively connected columns packed with adsorption and/or ion exchange resins, similar to that described in US Patent 8,981,081 which is hereby incorporated in its entirety by reference, is used.

[067] In yet another mode, the separation is conducted by an HPLC system with the following configuration: • Agilent 1200 series HPLC - equipped with binary pump, automatic sampler, column oven and DAD detector; • HPLC Column - Poroshell 120 SB-C18, 4.6x150 mm, 2.7 μm at 40 °C; • Injection volume - 5 μL; • Detector - UV 210 nm; • Mobile phase A - 25:75 (% v/v) acetonitrile and buffer (10 mmol/L sodium phosphate buffer pH 2.6); • Mobile phase B - 32:68 (% v/v) acetonitrile and buffer (10 mmol/L sodium phosphate buffer pH 2.6); • Mobile phase gradient: 0 min - 100% A, 0% B 12 min - 100% A, 0% B 12.5 min - 50% A, 50% B 13 min - 0% A, 100% B 40 min - 0% A, 100% B • Flow Rate - 0.5 mL/min; • Running time - 45 minutes; • Post Hour - 10 minutes.

Crystallization

[068] Non-steviol glycoside molecules and NSGCs of the present invention can be further purified and separated using various crystallization techniques including, but not limited to, cooling crystallization, evaporative crystallization, fractional crystallization, salting out (re-extension), etc.

[069] In some embodiments, crystallization can be conducted at concentrations ranging from 0.1% to 99% (w/w).

[070] In some embodiments, the crystallization is carried out from a solvent comprising at least one solvent selected from the group including water, ethanol, methanol, n-propanol, isopropanol, n-butanol, chloroform, toluene, benzene, xylene, carbon tetrachloride, cyclohexane, 1,2-dichloroethane, dichloromethane, diethyl ether, dimethylformamide, ethyl acetate, heptane, hexane, methyl tert-butyl ether, pentane, 2,2,4-trimethylpentane, acetone, tetrahydrofuran, formic acid, acetic acid and combinations thereof.

[071] In yet another embodiment, crystallization is achieved by adding a base, or alkali, or salt, or acid or any other agent capable of forming less soluble derivatives of non-steviol glycoside molecules, and in which other process may include a step to convert the derivatized non-steviol glycoside molecule back to the native state.

[072] In other embodiments, the crystallization temperature can vary between -20 °C and 80 °C. In some embodiments, the temperature increase and/or decrease can be done by the gradient method.

[073] In other embodiments, the polarity of the solvent or mixture of solvents used in the crystallization varies from non-polar to polar. Including solvents whose dielectric constant ranges from 1 to 88.

[074] In some embodiments, the ionic strength of the crystallization solution varies between 0 mol/L and 20 mol/L.

[075] In another embodiment, the pH of the crystallization solution ranges from 1 to 12.

Liquid-liquid and solid extraction

[076] NSGCs comprising non-steviol glycoside molecules of the present invention, or derivatives thereof, can be further purified and separated using various solid-liquid and liquid-liquid extraction techniques including but not limited to dispersive liquid-liquid extraction, direct organic extraction , continuous countercurrent extraction, multistage continuous countercurrent extraction, centrifugal extraction, two-phase aqueous extraction, polymer-polymer extraction, polymer salt extraction etc.

[077] Suitable solvents include water and organic solvents selected from the group including ethanol, methanol, n-propanol, isopropanol, n-butanol, chloroform, toluene, benzene, xylene, carbon tetrachloride, cyclohexane, 1,2 -dichloroethane, dichloromethane, diethyl ether, dimethylformamide, ethyl acetate, heptane, hexane, methyl tert-butyl ether, pentane, 2,2,4-trimethylpentane, acetone, tetrahydrofuran, formic acid, acetic acid and combinations thereof.

membrane separation

[078] NSGCs comprising non-steviol glycoside molecules of the present invention, or derivatives thereof, can be further purified and separated using various membrane separation techniques including ultrafiltration, nanofiltration, reverse osmosis, dialysis, direct osmosis, electrodialysis, electrodeionization, electrofiltration, cross flow filtration, tangential flow filtration, dead end filtration, spiral would membrane filtration, hollow fiber membrane filtration, cartridge filtration, cascade membrane filtration etc.

Consumables with NSGCs

[079] One embodiment of the present invention is an NSGC comprising at least one non-steviol glycoside molecule.

[080] In some modalities, NSGC confers a sweet taste.

[081] In one embodiment, the present invention is a sweetener composition comprising NSGC.

[082] In another embodiment, the present invention is NSGC which is used in consumable products as a source of antioxidant, dietary fiber, fatty acids, vitamins, minerals, preservatives, moisturizing agents, probiotics, prebiotics, weight control agents, agents osteoporosis management agents, phytoestrogens, long-chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

[083] In another embodiment, the present invention is a flavor-enhancing composition comprising NSGC, wherein the NSGC is present in an amount effective to provide a concentration at or below the NSGC flavor recognition level when the flavor-enhancing composition is added to a consumable. In a particular embodiment, the NSGC is present in an amount effective to provide a concentration below the threshold aroma recognition level of the NSGC when the aroma enhancing composition is added to a consumable. In one embodiment, the NSGC is present in an amount effective to provide a concentration of at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20 % or at least about 25% or more below the NSGC Taste Recognition Threshold Level when the flavor enhancer composition is added to a consumable.

[084] In yet another embodiment, the present invention is a sweetness-enhancing composition comprising NSGC, wherein the NSGC is present in an amount effective to provide a concentration equal to or less than the threshold sweetness recognition level of the NSGC when the enhancer composition of sweetness is added to a consumable. In a particular embodiment, the NSGC is present in an amount effective to provide a concentration below the sweetness recognition threshold level of the NSGC when the sweetness enhancing composition is added to a consumable. In one embodiment, the NSGC is present in an amount effective to provide a concentration of at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20 % or at least about 25% or more below the NSGC sweetness recognition threshold level when the sweetness enhancing composition is added to a consumable.

[085] In yet another embodiment, the present invention is a consumable comprising NSGC. Suitable consumables include, but are not limited to, liquid or dry consumables, such as, for example, pharmaceutical compositions, edible mixtures and gel compositions, dental compositions, food products, beverages and beverage products.

[086] In a particular embodiment, the present invention is a drink comprising NSGC. In a particular embodiment, the NSGC is present in the beverage at a concentration that is above, at, or below the sweetness recognition threshold concentration of the NSGC.

[087] In another particular embodiment, the present invention is a beverage product comprising NSGC. In a particular embodiment, the NSGC is present in the beverage product at a concentration that is above or below the NSGC's taste recognition threshold concentration.

[088] In another aspect, the present invention is a method for preparing a consumable comprising (i) providing a consumable matrix and (ii) adding NSGC to the consumable matrix to provide a consumable. In a particular embodiment, the NSGC is present in the consumable at a concentration above, at or below the NSGC recognized sweetness threshold. In another particular embodiment, the NSGC is present in the consumable at a concentration above, equal to or below the NSGC threshold flavor recognition.

[089] In a particular embodiment, the present invention is a method of preparing a beverage comprising (i) providing a beverage matrix and (ii) adding NSGC to the consumable matrix to provide a beverage. In a particular embodiment, the NSGC is present in the consumable at a concentration above, at or below the sweetness recognition threshold of the NSGC. In another particular embodiment, the NSGC is present in the consumable at a concentration above, at or below the threshold flavor recognition concentration of the NSGC.

[090] In another aspect, the present invention is a method for increasing the sweetness of a consumable comprising (i) providing a consumable comprising at least one sweetened ingredient and (ii) adding NSGC to the consumable to provide a consumable with enhanced sweetness, in that the NSGC is present in the sweetened beverage at a concentration equal to or less than the sweetness recognition threshold concentration of the NSGC.

[091] In a particular embodiment, the present invention is a method for increasing the sweetness of a beverage comprising (i) providing a beverage comprising at least one sweet ingredient and (ii) adding NSGC to the beverage to provide a beverage with improved sweetness, wherein the NSGC is present in the beverage with enhanced sweetness at a concentration below the NSGC threshold sweetness recognition concentration. In one embodiment, the concentration of the NSGC is present in the sweetened beverage at a concentration that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 15% about 20%. %, or at least about 25% or more below the NSGC sweetness recognition threshold concentration.

[092] In another aspect, the present invention is a method for improving the taste of a consumable comprising (i) providing a consumable comprising at least one flavoring ingredient and (ii) adding NSGC to the consumable to provide a consumable with improved taste, wherein the NSGC present in the flavor-enhanced consumable at a concentration equal to or less than the NSGC taste recognition threshold concentration.

[093] In a particular embodiment, the present invention is a method of enhancing the flavor of a beverage comprising (i) providing a beverage comprising at least one flavoring ingredient and (ii) adding NSGC to the beverage to provide a beverage with improved taste, wherein the NSGC is present in the flavor-enhanced beverage at a concentration equal to or less than the NSGC taste recognition threshold concentration. In one embodiment, the concentration of the NSGC is present in the sweetened beverage at a concentration that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 15% about 20% or at least about 25% or more below the NSGC taste recognition threshold concentration.

[094] In the above methods, the NSGC can be added as such, or in the form of a composition comprising the NSGC. When the NSGC is provided as a composition, the concentration of the NSGC in the composition is effective to provide a concentration above, at or below the threshold flavor or sweetener composition of the NSGC when the composition is added to the consumable, e.g. the food or beverage. .

[095] In some embodiments, the compositions of the present invention further comprise one or more mogrosides, wherein the mogrosides are selected from, but not limited to, the group consisting of Luo han guo extract, by-products of other isolation and purification processes of mogrosides, a commercially available extract of Luo han guo, mogroside IIE, mogroside IIB, mogroside III, mogroside IV, mogroside V, 11-oxo-mogroside V, mogroside VI, siamenoside I, grosmomoside I, neomogroside, and other mogrol glycosides and oxo-mogrol occurring in Siraitia grosvenorii fruit and combinations thereof.

[096] In other embodiments, the compositions of the present invention further comprise one or more sweeteners or additional sweeteners. In one embodiment, the additional sweetener is a natural sweetener or a synthetic sweetener. In a particular embodiment, the additional sweetener is a high intensity sweetener. In a particular embodiment, the additional sweetener is a mogroside.

[097] In some embodiments, the compositions of the present invention further comprise one or more additives. In a particular embodiment, the additive is selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, salts organics including salts of organic acids and salts of organic bases, inorganic salts, bitter compounds, flavors and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

[098] In some embodiments, the compositions of the present invention further comprise one or more functional ingredients. In a particular embodiment, the functional ingredient is selected from the group consisting of caffeine, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, moisturizing agents, probiotics, prebiotics, weight, osteoporosis management agents, phytoestrogens, long-chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

[099] In a particular embodiment, the present invention is a consumable comprising an NSGC and one or more sweeteners, additional sweeteners, additives or functional ingredients.

[0100] In another particular embodiment, the present invention is a beverage comprising NSGC and one or more sweeteners, additional sweeteners, additives or functional ingredients.

[0101] NSGCs can be used alone or in combination with at least one other sweetener in consumables including food, beverage, pharmaceutical composition, tobacco, nutraceuticals, oral hygiene composition or cosmetics. The other sweeteners are selected from the group including sucrose, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lipose, ribose, xylose, ribulose, xylulose, allose, altrose, allulose, galactose, glucose, glutose, idose, mannose, thalose., fructose, psychose, sorbose, tagatose, mannoheptulose, sedoheltulose, octolose, fucose, rhamnose, arabinose, turanose, sialose, inulin, inulooligosaccharides, fructooligosaccharides, high fructose corn syrup (HFCS), maltodextrin, coupling sugar , honey, stevia, rebaudioside A , rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, steviolbioside, stevioside, other steviol glycosides occurring in the plant Stevia rebaudiana, biosynthetic steviol glycosides, glycosylated steviol glycosides, glycosylated steviol glycosides (GSGs), mogroside IV, mogroside V, mogroside VI, Luo han guo, siamenoside, other mogrosides present in the fruits of Siraitia grosvenorii, monatin and its salts, curculin, glycyrrhizic acid and its salts, thaumatin, monelin, mabinlin, brazein, hernandulcin, phyllodulcin, glycyphylline, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mucurozioside, flomisoside I, periandrin I, abrusoside A, and cyclocaryoside I, sugar alcohols, sucralose, acesulfame potassium, acesulfame acid and salts thereof, aspartame, alitame, saccharin and its salts, neohesperidin dihydrochalcone, cyclamate, cyclamic acid and its salts, neotame, advantame and combinations thereof.

[0102] The following examples illustrate preferred embodiments of the invention. It will be understood that the invention is not limited to the materials, proportions, conditions and procedures set forth in the examples, which are illustrative only.

EXAMPLE 1 NSGC preparation

[0103] Five kilograms of dried Stevia rebaudiana leaves (having about 8% (w/w) moisture content and about 10% (w/w, dry basis) total steviol glycosides) were ground into 10-20 mm. The dried leaf material was loaded into an extraction tank and the extraction was performed with 100 L of RO water at 65 °C for 10 min. Insoluble material was removed by filtration. The yellowish filtrate was collected and fed to a column packed with 8.5 L of polymeric macroporous adsorbent resin (YWD-03, Cangzhou Yuanwei, China), with a flow rate of approximately 50 L/hour. Upon completion of the filtrate, the column was further washed with 45 L of water and both effluents were combined. The combined solution was evaporated under vacuum at 30°C-35°C to a final volume of 10 L.

[0104] Most of the steviol glycosides were absorbed onto macroporous adsorbent resin and were eluted with about 45 L of 70% aqueous ethanol. The aqueous ethanol eluate was further processed to yield about 400g of stevia extract with about 96% w/w total steviol glycoside content.

[0105] The above-mentioned 10 L combined and evaporated solution was mixed with 90 L of pure ethanol and the mixture was kept for 10 min with slow stirring. The resulting precipitate was removed by vacuum filtration. The filtrate was collected and then subjected to vacuum evaporation (at 30°C - 35°C) to remove ethanol and then concentrated to about 4 L of NSGC syrup containing 22% w/w solids. HPLC assay of this solution shows about 1.3% residual steviol glycosides where the % ratio of individual steviol glycosides was: Rebaudioside E 0.41%, Rebaudioside O 10.52%, Rebaudioside D 5.95 %, Rebaudioside N 1.49%, Rebaudioside M 3.07%, Rebaudioside A 56.98%, Stevioside 11.66%, Rebaudioside F 0.89%, Rebaudioside C 4.37%, Dulcoside A 0.11% and Rebaudioside B 0.35%. The concentrate was dried using vacuum evaporation followed by drying in a vacuum oven at 30°C - 35°C.

EXAMPLE 2 NSGC extraction by aqueous ethanol

[0106] 50 kg of dried Stevia rebaudiana leaves (having about 8% (w/w) of moisture content and about 8.5% (w/w) of total steviol glycosides) were ground to particles of 10 -20 mm. The HPLC assay of this sheet also shows about 3.2% w/w of total CGA content, comprising 1.34% of CGAs (neo-CGA, n-CGA and crypto-CGA), and 1.86% of iso-CGAs (iso-CGA-B, iso-CGA-A and iso-CGA-C). The dried leaf material was loaded into an extraction tank and the extraction was performed with 800 L of 30% (v/v) ethanol at 65 °C for 30 min. The mixture was filtered through 800 g of diatomaceous earth. The yellowish filtrate was collected and cooled to 30°C. EtOH was removed from the filtrate by nanofiltration membrane (NF90-400, Dow Chemical Company, USA) at 45 °C under pressure of 0.5-0.8 MPa.

[0107] 320 L of the retentate obtained from nanofiltration estimated at about 1.57 kg of total CGAs and 4.1 kg of total steviol glycosides were fed to the column packed with 125 L of polymeric macroporous adsorbent resin (YWD- 03, Cangzhou Yuanwei, China), flow rate of 125 L/hour. After feeding the retentate, the column was further washed with 62.5 L of water and both effluents were combined to make a flow-through product, which was further concentrated using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) to 20% total solids content. Flocculation of the concentrated flux product was performed using 9 volumes of ethanol. The flocculated precipitate was separated by filtration and Ethanol was removed by nanofiltration membrane (NF90-400, Dow Chemical Company, USA) as mentioned above. The concentrate was dried using a lyophilizer. The purified flux product contained 12.24% w/w (dry basis) total CGA, which comprised 2.66% neo-CGA, 7.46% n-CGA, 1.87% crypto-CGA, 0.07% iso-CGA-B, 0.16% iso-CGA-A and 0.06% iso-CGA-C and 0.70% w/w of total steviol glycosides.

[0108] The adsorbed CGAs were eluted from the macroporous adsorbent resin using 690 L of 25% ethanol (v/v). The solution was passed through a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) to remove Ethanol and then dried by lyophilization as mentioned above to make 25% ethanol product. The 25% ethanol product contained 19.52% w/w total CGA, which included neo-CGA 0.78%, n-CGA 3.59%, croco-CGA 1.04%, iso-CGA B 2 .64%, iso-CGA-A 4.14. % and iso-CGA-C 7.33% and 12.28% w/w of total steviol glycosides including Rebaudioside E 1.43%, Rebaudioside D 1.54%, Rebaudioside A 5.31%, Stevioside 2.49 % and others.

[0109] The remaining steviol glycosides were eluted from the macroporous adsorbent resin with approximately 380 L of 70% aqueous ethanol and further processed to produce a stevia extract with a 71% TSG content.

EXAMPLE 3 NSGC extraction by water

[0110] 50 kg of dry leaf material of Stevia rebaudiana, similar to that used in Example 2, was loaded into an extraction tank and extraction was performed with 800 L of water at 90°C for 30 min. The mixture was filtered through 800 g of diatomaceous earth. The yellowish filtrate was collected and cooled to 30 °C, and fed to a column with 125 L of polymeric macroporous adsorbent resin (YWD-03, Cangzhou Yuanwei, China). Subsequent steps were similar to those described in Example 2.

[0111] The flow-through product contained 14.47% w/w of total CGA, which included neo-CGA 4.67%, n-CGA 5.67%, crypto-CGA 3.44%, iso-CGA -B 0.24%, iso-CGA-A 0.21% and iso-CGA-C 0.24% and 0.59% w/w of total steviol glycosides. The 25% ethanol product contained 17.97% w/w total CGA, consisting of 0.78% neo-CGA, 1.56% n-CGA, 0.91% crypto-CGA, iso-CGA -B 5.36%, iso-CGA-A. 3.21% and iso-CGA-C 6.15% and 13.83% w/w of total steviol glycosides, including Rebaudioside E 2.89%, Rebaudioside D 1.52%, Rebaudioside A 5.31%, Stevioside 2.49% and others.

EXAMPLE 4 NSGC extraction by water

[0112] 50 kg of dry leaf material of Stevia rebaudiana, similar to that used in Example 2, were loaded into an extraction tank and extraction was performed with 800 L of water at 65 °C for 30 min. The mixture was filtered through 800 g of diatomaceous earth. The yellowish filtrate was collected and cooled to 30 °C, and fed to a column packed with 125 L of polymeric macroporous adsorbent resin (YWD-03, Cangzhou Yuanwei, China). The subsequent process was similar to that described in Example 2.

[0113] The 25% ethanol product contained 19.90% w/w of total CGA, which comprised neo-CGA 0.35%, n-CGA 4.30%, crypto-CGA 1.23%, iso- CGA-B 0.81%, iso-CGA-A 8.19% and iso-CGA-C 5.02% and 13.47% w/w of total steviol glycosides including Rebaudioside D 1.86%, Rebaudioside 7.45%, Stevioside 2.13% and others.

EXAMPLE 5 NSGC purification

[0114] 6 grams of the dry 25% ethanol product of Example 3 was dissolved in 20 ml of water. 180 mL of neat ethanol was added to the mixture and stirred at room temperature for 1 hour. The obtained suspension was filtered and 0.72 g of activated carbon was added to the filtrate and allowed to stir at room temperature for another 1 hour. The mixture was filtered again to remove activated carbon. The obtained filtrate was concentrated using a rotary evaporator at 40 °C and dried in a vacuum oven at 40 °C to give 3.6 g of 25% ethanol product. The 25% ethanol processed product contained 22.32% w/w total CGA, including 0.84% neo-CGA, 1.26% n-CGA, 0.91% crypto-CGA, 0.91% iso-CGA B 6 .85%, iso-CGA-A 4.99% and iso-CGA-C 7.48% and 23.26% w/w of total steviol glycosides including Rebaudioside D 2.03%, Rebaudioside A 12.18% , Estevioside 6.70% and others.

EXAMPLE 6 NSGC Membrane Purification

[0115] One gram of the processed 25% ethanol product of Example 5 was dissolved in 100 mL of RO water and the solution was fed into the Sterlitech HP4750 high pressure stirred cell filtration system (Sterlitech Corporation, USA) at 20 °C, up to 50 °C mL of permeate was collected. Both the retentate and permeate were lyophilized and tested by HPLC.

[0116] The experiment was repeated using different 47 mm membrane discs obtained from Sterlitech Corporation (USA). Particularly GE 2000 UFGH (Sterlitech Cat. No. YMGHSP475), GE 1000 (Sterlitech Cat. No. YMGESP475), Synder NFG (Sterlitech Cat. No. YMNFG475), Microdyn Nadir NP010 (Sterlitech Cat. No. YMNP010475), Evonik Duramem 900 (Sterlitech Cat. No. 1120773) and Synder XT, PES, UF (Sterlitech Cat. No. YMXT475).

[0117] The results of the permeate and retentate analysis are summarized in Table 1. Table 1 * Note: Ret: Retentate; Per: Permeate

[0118] The GE 1000 membrane was selected for further experiments.

EXAMPLE 7 Purification of NSGC Using Membrane with Diafiltration Step

[0119] One gram of 25% processed ethanol product from Example 5 was mixed with 100 mL of RO water and fed to the Sterlitech HP4750 high pressure stirred cell filtration system (Sterlitech Corporation, USA) equipped with GE 1000 (Sterlitech Cat No YMGESP475) at 20 °C until 50 mL of permeate was collected. After collecting 50 mL of permeate, 50 mL of RO water was added to the cell and the filtration was repeated to collect an additional 50 mL of permeate. This process was repeated to obtain ten permeate fractions. The retentate and permeates were lyophilized and assayed by HPLC.

[0120] For the retentate, the total CGA was 16.51% (w/w, dry basis), composed of neo-CGA 0.35%, n-CGA 0.65%, crypto-CGA 1.11%, iso-CGA-B 5.52%, iso-CGA-A 2.75% and iso-CGA-C 6.13% and the total steviol glycoside content was 37.69% w/w, including Rebaudioside D 2.75%, Rebaudioside A 20.48%, Estevioside 10.39%, Rebaudioside C 1.35% and others. For the combined sample of 10 permeate samples, the total CGA was 30.33% (w/w, dry basis), composed of neocat-097%, n-CGA 2.52%, cryptocrac-CGA 2.28%, iso-CGA-B 8.23%, iso-CGA-A 8.24% and iso-CGA-C 8.08%, and the total steviol glycoside content was 5.99% w/w, including Rebaudioside A 2.66%, Stevioside 2.89% and others.

EXAMPLE 8 Purification of NSGC using adsorption chromatography system

[0121] 100 mL of aqueous solution containing 10.07 g of 25% Ethanol product from Example 4 were fed to a column packed with 300 mL of macroporous polymeric adsorbent resin (TF3, Cangzhou Yuanwei, China), at a rate of flow rate of 300 mL/hour at room temperature. Upon completion of loading, the column was further washed with 600 mL of water and both effluents were combined and collected as flow-through product. The flux product was concentrated by nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then dried using spray dryer to obtain 1.9 g of dry product flux containing 27.82% w/w (dry basis ) of the total CGAs, comprising neo-CGA 4.11%, n-CGA 17.21%, crypto-CGA 6.25%, iso-CGA-B 0.04%, iso-CGA-A 0.17 % and iso-CGA-C 0.04% and 0.12% w/w of total steviol glycosides.

[0122] The adsorbed CGAs were eluted from the macroporous adsorbent resin using 1,500 mL of 20% ethanol (v/v). The solution was passed through a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) to remove Ethanol and to concentrate. The concentrate was dried using spray dryer and 4.3 g of dried sample was collected as 20% ethanol product. This product contained 21.99% w/w total CGA, consisting of neo-CGA 0.03%, n-CGA 0.23%, crypto-CGA 0.16%, iso-CGA-B 2.39%, iso-CGA-A 11.93% and iso-CGA-C 7.25% and 2.18% w/w of total steviol glycosides.

[0123] The remaining steviol glycosides absorbed onto a macroporous resin were eluted with approximately 900 mL of 60% aqueous ethanol and further processed to provide 3.6 g of stevia extract containing 60.5% w/w of steviol glycosides total steviol.

EXAMPLE 9 Purification of NSGC using adsorption chromatography system

[0124] 100 mL of aqueous solution containing 9.0 g of 25% Ethanol from Example 4 was fed to a column packed with 300 mL of macroporous polymeric adsorbent resin (TF3, Cangzhou Yuanwei, China), with a flow rate of 300 mL/hour at room temperature. Upon completion of the load, the column was further washed with 900 mL of water and both effluents were combined and collected as a flow-through product. The product was concentrated using nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then spray dried to yield 1.51 g of dry flow product containing 31.27% w/w (dry basis) total CGA , composed of neo-CGA 4.19%, n-CGA 19.35%, crypto-CGA 7.10%, iso-CGA-B 0.11%, iso-CGA-A 0.43% and iso-CGA -C 0.10% and 0.01% w/w of total steviol glycosides.

[0125] The macroporous adsorbent resin was then sequentially washed with 900 mL of 15% ethanol (v/v), 900 mL of 20% ethanol (v/v), 900 mL of 25% ethanol (v/v) and 900 mL 60% (v/v) ethanol. All collected solutions were concentrated using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then dried using a spray dryer to obtain 15% ethanol product, 20% ethanol product, 25% ethanol product. ethanol and 60% ethanol product, respectively. The HPLC assay of these fractions is summarized in Table 2. Table 2 HPLC assay of eluate fractions

EXAMPLE 10 Purification of NSGC using adsorption chromatography system

[0126] 100 mL of aqueous solution containing 9.0 g of the 25% ethanol product of Example 4 was fed to a column packed with 300 mL of macroporous polymeric adsorbent resin (TF3, Cangzhou Yuanwei, China), with a rate of flow rate of 300 mL/hour at room temperature. Upon completion of the load, the column is further washed with 600 mL of water and both effluents are combined and collected as a flow-through product. The flow-through product was concentrated using nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then dried to yield 1.45 g of total product containing 18.04% w/w (dry basis) of total CGA, composed of neo-CGA 2.12%, n-CGA 13.40%, crypto-CGA 2.47%, iso-CGA-A 0.04% and iso-CGA-C 0.01% and 0.74% w/w of total steviol glycosides.

The macroporous adsorbent resin was then washed sequentially with 1500 mL of 20% (v/v) ethanol and 900 mL of 60% (v/v) ethanol. All collected solutions were concentrated using nanofiltration membranes (NF90-400, Dow Chemical Company, USA) and then dried to obtain 20% ethanol product and 60% ethanol product, respectively.

[0128] The HPLC assay of these fractions is summarized in Table 3. Table 3 HPLC assay of eluate fractions

EXAMPLE 11 Purification of NSGC using adsorption chromatography system

[0129] 500 mg/L of aqueous solution of 25% ethanol product of Example 4 was fed to a column packed with 150 mL of polymeric macroporous adsorbent resin (TF3, Cangzhou Yuanwei, China), with a flow rate of about of 150 mL/hour at room temperature. The effluent was collected and its sweetness was periodically analyzed by the sensorial method. Feeding was stopped when sweetness was detected in the effluent. The effluents were combined, concentrated and then dried using a spray dryer to obtain an effluent product containing 21.65% w/w (dry basis) total CGAs, composed of neo-CGA 1.37%, n-CGA 5, 42%, crypto-CGA 1.97%, iso-CGA-B 1.90%, iso-CGA-A 6.12% and iso-CGA-C 4.87% and 0.42% w/w of glycosides of total steviol.

[0130] The macroporous adsorbent resin was then washed sequentially with 300 ml of 20% ethanol (v/v) and 450 ml of 60% ethanol (v/v). All collected solutions were concentrated using nanofiltration membranes (NF90-400, Dow Chemical Company, USA) and then dried using a spray dryer to obtain 20% ethanol product and 60% ethanol product, respectively.

[0131] The HPLC assay of these fractions is summarized in Table 4. Table 4 HPLC assay of eluate fractions

EXAMPLE 12 Purification of NSGC using crystallization

[0132] 1 gram of the dry 25% ethanol product of Example 3 was dissolved in 20 mL of water. 100 mg of Ca(OH) 2 was added and the mixture was kept for 1 hour until a precipitate formed. The obtained suspension was filtered and the precipitate was resuspended in water and titrated with acetic acid until dissolution and fed to a column packed with 100 ml of polymeric macroporous adsorbent resin (YWD-03, Cangzhou Yuanwei, China). The subsequent process was similar to that described in Example 2.

[0133] The 25% ethanol product contained 42.90% w/w total CGA, which comprised 0.5% neo-CGA, 1.37% n-CGA, 0.37% crypto-CGA , 5.68% iso-CGA-B, 6.89% iso-CGA-A and 28.08% iso-CGA-C and 0.3% w/w of total steviol glycosides including, Rebaudioside A 0 .2%, Stevioside 0.1% and others.

EXAMPLE 13 Consumable comprising NSGC

[0134] Samples of carbonated beverages were prepared according to the formula shown in Table 5. Table 5 Formula for carbonated beverages

[0135] The following samples were used as a “sweetener composition” in the formula. (i) commercial sample of Rebaudioside A (97% pure) and (ii) mixture (95:5 w/v ratio) of commercial Rebaudioside A (97% pure) and NSGC prepared according to Example 3. Sensory evaluation of beverage samples were conducted by 20 speakers. The results are summarized in Table 6. Table 6 Sensory evaluation of carbonated beverage samples *For the characteristics of “Persistent sweetness”, “Bitterness”, “Delayed onset of sweetness”, and “Licorice taste” the panelists scored between 1 to 5, where the lowest score represents a more pleasant taste sensation by the panelists

[0136] The results showed that the beverages prepared using the sweetener composition comprising NSGC had the best organoleptic characteristics.

EXAMPLE 14 Consumable comprising the new NSGC

[0137] The chocolate samples were placed according to the formula in Table 7. Table 7

[0138] Chocolate liquor, cocoa butter, milk powder, sorbitol, salt and the “sweetening composition” were kneaded enough, and the mixture was then placed in a refiner to reduce its particle size for 24 hours. Subsequently, the contents were transferred to a ladle, lecithin was added and the composition was kneaded at 50 °C for 48 hours. Then, the content was placed in a modeling apparatus and solidified.

[0139] The following samples were used as a “sweetener composition” in the formula in Table 3. (i) Commercial sample of Rebaudioside A (97% pure) and (ii) mixture (with 95:5 w/v ratio) of Rebaudioside A commercial (97% pure) and NSGC prepared according to Example 3. Sensory evaluation of chocolate samples was carried out by 20 panelists. The results are summarized in Table 8. Table 8 Sensory evaluation of chocolate samples *For the characteristics of “Persistent sweetness”, “Bitterness” and “Licorice taste” the speakers scored between 1 and 5, where the lowest score represents a more pleasant taste sensation by the speakers

[0140] The results showed that the chocolate samples prepared using the sweetener composition comprising NSGC had the best organoleptic characteristics.

[0141] Although the foregoing has described one or more embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications can be made and equivalents can be substituted for elements or compositions thereof without departing from the true scope of the invention. Therefore, it is intended that this invention not be limited to a particular disclosed embodiment, but that the invention include all embodiments falling within the scope of the appended claims.

Claims (3)

1. Method for preparing non-steviol glycoside composition, CHARACTERIZED in that it comprises the steps of: a. supply plant material of Stevia rebaudiana; B. provide extraction solvent; w. mixing Stevia rebaudiana plant material and extraction solvent to provide stevia plant material and solvent mixture; d. separating stevia plant material and solvent mixture to obtain filtrate comprising steviol glycoside molecules and non-steviol glycoside molecules, wherein the separation comprises filtration through diatomaceous earth; and is. isolating or separating the steviol glycoside molecules from the filtrate to obtain a non-steviol glycoside composition, wherein the non-steviol glycoside composition obtained comprises at least one non-steviol glycoside molecule selected from phenolic compounds, polyphenols, flavonoids, quinic acids, neochlorogenic acid (neo-CGA; 5-O-caffeoylquinic acid or 5-CQA), crypto-chlorogenic acid (crypto-CGA; 4-O-caffeoylquinic acid or 4-CQA), n-chlorogenic acid (n-CGA; 3-O-caffeoylquinic acid or 3-CQA), iso-chlorogenic acid A (iso-CGA A; 3,5-dicaffeoylquinic acid), iso-chlorogenic acid B (iso-CGA B; 3,4-dicaffeoylquinic acid), iso-chlorogenic C (iso-CGA C; 4,5-dicaffeoylquinic acid), retinoids, pigments, polysaccharides, oligosaccharides, disaccharides, monosaccharides, volatile oil components, sterols, terpenoids, sesquiterpenoids, diterpenes, triterpenes, coumarins, fatty acids, amino acids, dipeptides, oligopeptides, polypeptides, proteins, austroinulin, quercetin, steribines, spatulenol, decanoic acid, 8,11,14-ecosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, stigmasterol, bsitosterol, a- and b-amyrin, lupeol , b-amyrin acetate, pentacyclic triterpenes and/or glycosides thereof, and combinations thereof. 2. Consumable CHARACTERIZED by the fact that it comprises the non-steviol glycoside composition prepared by the method as defined in claim 1, in which the consumable is selected from pharmaceutical compositions, mixtures and edible gel compositions, dental compositions, food, beverages and health products drinks. 3. Method for preparing a consumable CHARACTERIZED by the fact that it comprises the step of adding the non-steviol glycoside composition, prepared by the method as defined in claim 1, to the consumable.