RU2772126C2 - Food ingredients of stevia rebaudiana - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: method for producing a composition of non-steviolic glycosides is proposed, including stages of preparation of plant material of Stevia rebaudiana, preparation of an extracting solvent, mixing of plant material of Stevia rebaudiana and the extracting solvent for obtaining a mixture of stevia plant material and the solvent, separation of the mixture of stevia plant material and the solvent in order to obtain filtrate containing steviolic glycoside molecules and non-steviolic glycoside molecules. In this case, the separation includes filtration through diatomaceous earth. Then, steviolic glycoside molecules are isolated or separated from the filtrate in order to obtain a composition of non-steviolic glycosides.

EFFECT: obtained compositions can be used as flavors, sweeteners, antioxidants and other functional ingredients.

3 cl, 8 tbl, 14 ex, 2 dwg

Description

Related Applications

This application claims priority to US Patent Application No. 62/427,539, filed Nov. 29, 2016, which is incorporated herein by reference in its entirety. As applied to the United States of America, this application is also a continuation in part of U.S. Patent Application No. 15/284265, filed October 3, 2016, which is a continuation of U.S. Patent Application No. 14/677538, filed April 2, 2015, now filed as U.S. Patent No. 9456626 , which is a continuation of US patent application number 13/993415, filed on June 12, 2013, now registered as US patent number 9029426, which is an international application number PCT/US2011/064343, entered into the national phase, filed on December 12, 2011, which claims to priority of US Patent Application No. 61/424,798, filed December 20, 2010, and US Patent Application No. 61/422,403, filed December 13, 2010, each of which is incorporated herein by reference in its entirety. This application also includes by reference each of the following applications in its entirety: US patent application number 13/530113, filed June 22, 2012, now registered as US patent number 8530527; U.S. Patent Application No. 13/580098, filed November 6, 2012, now registered as U.S. Patent No. 8981081; US patent application number 13/943776, filed July 16, 2013; U.S. Patent Application No. 13/957098, filed August 1, 2013, now registered as U.S. Patent No. 9510611; US patent application number 14/195812, filed March 3, 2014; and U.S. Patent Application No. 14/829127, filed August 18, 2015, now registered as U.S. Patent No. 9771434.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a method for obtaining food ingredients from the plant Stevia rebaudiana and their use in food, beverages and other consumable products.

Description of the Prior Art

High intensity sweeteners have a degree of sweetness many times that of sucrose. They are essentially calorie-free and are widely used in the production of dietary and low-calorie foods. Although natural caloric sweeteners such as sucrose, fructose and glucose have a desirable taste for consumers, they are also high in calories. High intensity sweeteners do not affect blood glucose levels and have little or no nutritional value.

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

Currently, there are more than 230 types of stevia with pronounced sweetening properties. The plant is successfully grown in various climatic conditions: from subtropics to cold northern latitudes.

The extract from the plant Stevia rebaudiana contains a mixture of various sweet diterpene glycosides that have a common base - steviol - and differ in the presence of carbohydrate residues in positions C13 and C19. These glycosides accumulate in stevia leaves and make up approximately 10%-20% of the total dry matter weight. Generally, on a dry weight basis, 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.

Steviol glycosides have zero calories and can be used in place of sugar. They are ideal for diabetic and low calorie diets.

On the other hand, it should be noted that in the preparation process involving aqueous extraction, along with steviol glycosides, many other components of the stevia plant are also extracted. These associated components are mostly separated during further processing and released into the environment.

Little is known about these other components of the Stevia rebaudiana plant. Some authors report on phenolic compounds, free amino acids, etc., but information on the identity of these components is extremely scarce, and their possible use in foods, drinks and other consumables remains undescribed (Karaköse, et al., 2011, 2015; Wölwer-Rieck, 2012; Periche et al., 2014).

To date, it is not known whether other extracted components of the stevia plant can be processed into any food ingredient. Thus, if such information is obtained, it could open up opportunities for the almost waste-free processing of stevia and the use of the whole plant in the food chain, which will provide significant economic and environmental benefits.

Therefore, there is a need to develop a method for efficiently isolating these components from the Stevia rebaudiana plant and using them as ingredients in various consumable products.

The essence of the invention

The purpose of the present invention was to eliminate the shortcomings of existing schemes for the industrial processing of stevia. The invention describes a method for obtaining food ingredients from the plant Stevia rebaudiana and their use in various consumable products, including food and beverages.

The invention particularly relates to compositions containing phenolic and other non-steviol glycosidic compounds derived from the plant Stevia rebaudiana .

In this document, the term "steviol glycoside (steviol glycosides)", or "steviol glycoside (steviol glycosides)" means steviol glycosides naturally occurring in Stevia rebaudiana , including, but not limited to, steviol monoside, steviol bioside, 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 their combinations.

In this document, 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.

As used herein, the terms "Stev", "Sbio", "DulA", "Rub" refer to stevioside, steviolbioside, dulcoside A, and rubusoside.

In this document, the term "TSG content" means the total content of steviol glycosides (TSG), TSG content can 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 matter basis.

As used herein, the term "CGA" means chlorogenic acids and their derivatives naturally occurring in plants, including, but not limited to, neo-chlorogenic acid (neo-CGA; 5-O-caffeylquinic acid or 5-CQA), crypto- chlorogenic acid (crypto-CGA; 4-O-caffeylquinic acid or 4-CQA), n-chlorogenic acid (n-CGA; 3-O-caffeylquinic acid or 3-CQA), isochlorogenic acid A (iso-CGA A; 3 ,5-dicofeylquinic acid), isochlorogenic acid B (iso-CGA B; 3,4-dicofeylquinic acid), isochlorogenic acid C (iso-CGA C; 4,5-dicofeylquinic acid), and combinations thereof.

In this document, the term "TCGA content" means the total content of chlorogenic acids (TCGA), TCGA content can 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 basis.

In the present invention, Stevia rebaudiana plant material, in particular leaves and/or shoots, is used as a starting material.

The plant material is subjected to extraction with water or water-alcohol solvents.

The resulting aqueous or hydroalcoholic extract was further processed to separate the fraction of steviol glycosides. The residual fraction, termed "non-steviol glycoside composition" (NSGC), is a composition predominantly containing compounds other than steviol glycosides found in aqueous or hydroalcoholic extracts of the Stevia rebaudiana plant (hereinafter referred to as "non-steviol glycoside molecules"). Non-limiting examples of "non-steviol glycoside molecules" include phenolic compounds, polyphenols, flavonoids, quinic and caffeic acids and their derivatives, neo-chlorogenic acid (neo-CGA; 5-O-caffeylquinic acid or 5-CQA), crypto-chlorogenic acid (crypto -CGA; 4-O-caffeylquinic acid or 4-CQA), n-chlorogenic acid (n-CGA; 3-O-caffeylquinic acid or 3-CQA), isochlorogenic acid A (iso-CGA A; 3,5-dicofeylquinic acid), isochlorogenic acid B (iso-CGA B; 3,4-dicofeylquinic acid), isochlorogenic acid C (iso-CGA C; 4,5-dicofeylquinic acid), other chlorogenic acids and iso-chlorogenic acids known in the art , retinoids, pigments, polysaccharides, oligosaccharides, disaccharides, monosaccharides, essential oil components, sterols, terpenoids, sesquiterpenoids, diterpenes, triterpenes, coumarins, fatty acids and their derivatives, amino acids and their derivatives, dipeptides, oligopeptides, polypeptides, proteins, austroinulin, quercetin, sterebins, spatulenol, decans th acid, 8,11,14-eicosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, stigmasterol, sitosterol, α- and β-amirin, lupeol, β-amirin acetate, pentacyclic triterpene and/or their glycosides.

Compositions obtained in some embodiments of the present invention and referred to as NSGC, may also contain some residual amounts of steviol glycosides.

Some NSGCs may be further purified and/or otherwise processed by any food ingredient processing method known in the art to produce other NSGCs.

The NSGCs of the present invention can be used in various consumables, foods and beverages as flavoring agents, flavor modifiers, flavor enhancers, sweeteners, preservatives, antioxidants, emulsifiers, texturizers, bulking agents, stabilizing agents, solubilizing agents, and other food ingredients.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to further explain the claims.

Brief description of the drawings

The accompanying drawing is included for a better 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.

In FIG. 1 shows the structures of some CGAs.

In FIG. 2 shows HPLC chromatograms of CGA from Stevia rebaudiana .

Detailed description of the present invention

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

Those skilled in the art will also appreciate that one or more additional steps of the process described below may be omitted. Those skilled in the art will also appreciate that while the process described below assumes a certain order of the described steps, in some cases this order may be changed.

Extraction

The invention uses Stevia rebaudiana plant material as a starting material, in particular leaves and/or shoots of Stevia rebaudiana plant. Stevia rebaudiana plant material extract can be obtained by any method, such as, but not limited to, the extraction methods described in US patent number 7862845, the entire content of which is incorporated herein by reference, as well as membrane filtration, supercritical fluid extraction, enzymatic extraction , microorganism extraction, ultrasonic extraction, microwave extraction, etc.

In one embodiment, Stevia rebaudiana plant material (eg, leaves) can be dried at a temperature of from about 20°C to about 100°C until the moisture content reaches about 5%-15%. In a specific embodiment, the plant material can be dried at a temperature of from about 20°C to about 60°C for from about 1 hour to about 240 hours.

In other specific embodiments, the implementation of the plant material can be dried at a temperature of from about 20°C to about 35°C to prevent decomposition.

In some embodiments, Stevia rebaudiana plant material may be dried under vacuum or under reduced pressure.

In some embodiments, Stevia rebaudiana plant material may be dried in the presence of an inert gas such as N ₂ .

In some embodiments, the Stevia rebaudiana plant material may be lyophilized.

In some embodiments, the dried plant material is optionally ground. The particle sizes can be from about 0.1 to about 20 mm.

The plant material (whether ground or not ground) can be extracted by any suitable extraction method such as, for example, continuous or multi-stage reflux extraction, supercritical fluid extraction, enzymatic extraction, extraction with microorganisms, ultrasonic extraction, microwave extraction, etc. The solvent used for the extraction may be any suitable solvent such as, for example, polar organic solvents (degassed, vacuum, pressurized or distilled), non-polar organic solvents, water (degassed, vacuum, pressurized, deionized, distilled treated with carbon or subjected to reverse osmosis) or a mixture thereof. In a specific embodiment, the solvent contains 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 may be selected from, for example, methanol, ethanol, n-propanol, 2-propanol, 1-butanol, 2-butanol and mixtures thereof.

In a specific embodiment, the plant material is extracted with water in a continuous extractor with a reflux system. One skilled in the art will appreciate that the ratio of extractive solvent to plant material will vary depending on the identity of the solvent and the amount of plant material to be extracted. Typically, the ratio of extraction solvent to dry plant material is about 20-25 liters of extraction solvent per kilogram of leaves.

The pH of the extraction solvent may range from about pH 2.0 to about pH 7.0, for example, from about pH 2.0 to about pH 5.0, from about pH 2.0 to about pH 4.0, or from about pH 2.0 to about pH 3.0. In a specific embodiment, the extraction solvent is aqueous, such as water, and optionally contains an amount of acid and/or base required to achieve a pH value in the range of about pH 2.0 to about pH 7.0, such as about pH 2.0 to about pH 5.0, about pH 2.0 to about pH 4.0, or about pH 2.0 to about pH 3.0. Any suitable acid or base may be used to achieve the desired pH of the extraction solvent, such as HCl, NaOH, citric acid, and the like.

Extraction can be carried out at a temperature of from about 25°C to about 100°C, for example, from about 30°C to about 80°C, from about 35°C to about 75°C, from about 40°C to about 70°C , from about 45°C to about 65°C or from about 50°C to about 60°C.

In embodiments in which the extraction process is multi-stage, the duration of the extraction may vary from about 0.5 hours to about 24 hours, such as from about 1 hour to about 8 hours, or from about 1 hour to about 6 hours.

In an embodiment in which the extraction process is continuous, the duration of the extraction may vary from about 1 hour to about 5 hours, such as from about 2.5 hours to about 3 hours.

After extraction, insoluble plant material can be separated from the solution by filtration to obtain a filtrate containing steviol glycosides and other molecules described above as "non-steviol glycoside molecules". Such solid/liquid separation may be accomplished by any suitable means including, but not limited to, gravity filtration, frame filter press, crossflow filters, screen filters, suction filters, belt filters, ceramic filters, membrane filters, micro filters. , nanofilters, ultrafilters or centrifugation. Optionally, various filter aids such as diatomaceous earth, bentonite, zeolite, etc. may also be used in this process.

Pretreatment of the filtrate containing steviol glycosides and "non-steviol glycoside molecules"

In some embodiments, the filtrate containing steviol glycosides and "non-steviol glycoside molecules" is optionally pretreated prior to contact with the macroporous polymeric adsorbent. Said pre-treatment may be carried out, for example, with at least one agent selected from the group including, but not limited to, diatomaceous earth, diatomite, kieselguhr, celite, bentonite, activated carbon, any filter additives suitable for food processing applications, any flocculating agents, any chelating agents, any acids, any bases/alkalis, any ion exchange resins known in the art, or combinations thereof. In some embodiments, the pre-treatment may be additional filtration via ultrafiltration and/or nanofiltration and/or reverse osmosis membrane systems known in the art.

Adsorption of steviol glycosides

In some embodiments, a filtrate containing steviol glycosides and "non-steviol glycoside molecules" is contacted with a macroporous polymeric adsorbent. The macroporous polymeric adsorbent may be neutral, acidic or alkaline macroporous polymeric adsorbent resins capable of adsorbing steviol glycosides, for example, Amberlite® XAD series resins (Rohm and Haas), Diaion® HP series resins (Mitsubishi Chemical Corp), Sepabeads® SP series resins (Mitsubishi Chemical Corp), Cangzhou Yuanwei YWD series resins (Cangzhou Yuanwei Chemical Co. Ltd., China) or equivalents. The columns can be filled with adsorbent to about 75%-100% of the total volume.

Steviol glycosides and some "non-steviol glycoside molecules" are adsorbed by the macroporous polymeric adsorbent, while other "non-steviol glycoside molecules" are not adsorbed, pass through the column and enter the bypass.

Elution from macroporous adsorbent resins can be carried out with various concentrations of aqueous ethanol in order to obtain different eluate fractions enriched in steviol glycosides and/or "non-steviol glycoside molecules".

In some embodiments, the macroporous adsorbent resin may be eluted with eluting solvents of various pH values.

Optional precipitation of impurities

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 about 8.5-10.0 by adding a base such as, for example, calcium oxide or hydroxide (about 1.0% by volume of the filtrate) with slow agitation.

Treatment of the filtrate with base as above results in a slurry whose pH can be adjusted to between about 3.0 and about 4.0 by treatment with any suitable flocculating/coagulating agent. Suitable flocculating/coagulating agents include, for example, potassium alum, aluminum sulfate, aluminum hydroxide, alumina, CO ₂ , H ₃ PO ₄ , P ₂ O ₅ , MgO, SO ₂ , anionic polyacrylamides, quaternary ammonium compounds with long chain substituents fatty acids, bentonite, diatomaceous earth, KemTab Sep series, Superfloc series, KemTab Flote series, Kemtalo Mel series, Midland PCS-3000, Magnafloc LT-26, Zuclar 100, Prastal 2935, Talofloc, Magox, iron salts, or combinations thereof. Examples of iron salts include, but are not limited to, FeSO ₄ , FeCl ₂ , Fe(NO ₃ ) ₃ , Fe ₂ (SO ₄ ) ₃ , FeCl ₃ and combinations thereof. In a particular embodiment, the ferric salt is FeCl ₃ . The filtrate may be treated with a flocculating/coagulating agent for from about 5 minutes to about 1 hour, such as from about 5 minutes to about 30 minutes, from about 10 minutes to about 20 minutes, or from about 10 minutes to about 15 minutes. To facilitate processing, it can be carried out with stirring or with slow stirring. Optionally, the pH of the resulting mixture can be adjusted to about 8.5-9.0 by adding a base such as, for example, calcium oxide or sodium hydroxide. The duration of treatment with the base, optionally with agitation, is from about 5 minutes to about 1 hour, 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 specific embodiment, the base is calcium oxide, which is treated for about 15-40 minutes with slow stirring.

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

Precipitated compounds and insoluble particles are separated from the filtrate to obtain a composition containing "non-steviol glycoside molecules". The precipitate may be separated by any suitable means, including, but not limited to, gravity filtration, frame filter press, cross-flow filters, mesh filters, suction filters, belt filters, ceramic filters, membrane filters, microfilters, nanofilters, ultrafilters or centrifugation. Optionally, various filter aids such as diatomaceous earth, bentonite, zeolite, etc. may also be used in this process.

Deionization

The filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" may be deionized by any suitable method, including, for example, electrodialysis, filtration (nano- or ultrafiltration), reverse osmosis, ion exchange, mixed bed ion exchange, or a combination of these. ways. In one embodiment, the filtrate containing "non-steviol glycoside molecules" is deionized with one or more ion exchange resins to produce 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 weakly basic anion exchange resin. In yet another embodiment, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" is passed first through a strong acid cation exchange resin and then through a weak base anion exchange resin. In yet another embodiment, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" is passed first through a weak base anion exchange resin and then through a strong acid cation exchange resin.

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

The anion exchange resin can be any weakly basic anion resin, such as one with a tertiary amine as a functional group. 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. B In a specific embodiment, Amberlite-FPA53 (OH-) resin is used as the strong acid cation exchange resin. Those skilled in the art will be aware of other suitable commercially available weak base anion exchange resins for use in embodiments of this invention.

In a specific embodiment, to obtain a resin-treated filtrate, the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" is passed first through a strong acid cation exchange resin, such as Amberlite® 10 FPC22H (H+) resin, and then through a weakly basic anion exchange resin, such as , through Amberlite-FPA53 (OH-) resin. The specific velocity (SV) of passage through one or more ion exchange columns may be from about 0.01 to about 5 hours ^-1 , for example, from about 0.05 to about 4 hours ^-1 , from about 1 to about 3 hours ^-1 or from about 2 to about 3 hours ^-1 . In a specific embodiment, the specific throughput through one or more ion exchange columns may be about 0.8 hours ^-1 . After passing the second filtrate containing steviol glycosides through one or more ion exchange columns, one or more ion exchange columns are washed with water, preferably reverse osmosis (RO) water. The solution resulting from washing the column with water and the resin-treated filtrate can be combined before the next multi-stage step.

Bleaching

Decolorization of the filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" can be carried out in any way, for example by contacting with activated carbon. The amount of activated charcoal may vary from about 0.1% (w/v) to about 0.8% (w/v). In a specific embodiment, the amount of activated charcoal is from about 0.25% (w/v) to about 0.30% (w/v). The suspension may be continuously stirred. Processing can be carried out at a temperature of from about 20°C to about 30°C, for example, at about 25°C. The treatment may be continued for any time sufficient to discolor the eluted solution, for example, from about 20 minutes to about 3 hours, from about 20 minutes to about 2 hours, from about 30 minutes to about 1.5 hours, or from about 1 hour to about 1 hour. ,5 o'clock. After treatment, the used carbon can be separated by any known separation methods, such as, for example, gravity filtration or suction filtration, centrifugation or using a frame filter press.

Alternatively, a filtrate containing steviol glycosides and/or "non-steviol glycoside molecules" may be passed through an activated carbon column.

It should also be understood that treatment with charcoal or other bleaching agent can lead not only to discolouration, but also to an improvement in taste, odour, aroma and other organoleptic characteristics.

Concentration and/or drying

Water or alcohol can be removed from the filtrate containing "non-steviol glycoside molecules" by any suitable means, including, but not limited to, evaporation under reduced pressure or vacuum nanofiltration, lyophilization, flash drying, spray drying, or a combination thereof, to obtain concentrated or a dried composition containing "non-steviol glycoside molecules". Dried compositions may optionally be agglomerated and/or granulated by compact or wet granulation methods.

Chromatography

The non-steviol glycosides and NSGC molecules of the present invention can be further purified and separated using various chromatographic methods, 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, exclusion chromatography, affinity chromatography, supercritical fluid chromatography, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, adsorption chromatography on an expanded sorbent bed, reverse phase chromatography, normal phase chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography, two-dimensional chromatography, pseudo-moving bed chromatography (SMBC), countercurrent chromatography and chiral chromatography, each of which can be carried out in analytical, preparative, semi-industrial or industrial scale.

In one embodiment, a chromatographic system is used consisting of a column packed with adsorbent resin and elution is performed with a concentration gradient of an alcoholic solvent (e.g., ethanol) to separate fractions enriched in either steviol glycosides or "non-steviol glycoside molecules".

In another embodiment, a chromatographic system is used, consisting of a column filled with an ion exchange resin, and the elution is carried out with an acidic or alkaline solvent to separate fractions enriched in either steviol glycosides or "non-steviol glycoside molecules".

In another embodiment, a chromatographic system consisting of a plurality of series-connected columns filled with adsorbing and/or ion exchange resins is used, similar to the chromatographic system described in US patent number 8,981,081, which is incorporated herein by reference in its entirety.

In yet another embodiment, the separation is carried out using an HPLC system having the following configuration:

Agilent 1200 series HPLC system - equipped with binary pump, autosampler, column thermostat and DAD detector;

HPLC column - Poroshell 120 SB-C18, 4.6×150 mm, 2.7 µm at 40°C;

The volume of the injected sample - 5 μl;

Detector - UV 210 nm;

Mobile phase A - 25:75 (vol.%) Acetonitrile and buffer (10 mmol/l sodium phosphate buffer pH 2.6);

Mobile phase B - 32:68 (vol.%) Acetonitrile and buffer (10 nmol/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;

Chromatography time - 45 min;

Break time - 10 min.

Crystallization

The non-steviol glycosides and NSGC molecules of the present invention can be further purified and separated using various crystallization methods, including, but not limited to, cooling crystallization, evaporation crystallization, fractional crystallization, salting out, etc.

In some embodiments, crystallization can be carried out at concentrations from 0.1% to 99% (w/w).

In some embodiments, the crystallization is from a solvent containing at least one solvent selected from the group consisting of 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 their combinations.

In another embodiment, crystallization is achieved by adding a base, or an alkali, or a salt, or an acid, or any other agent with which the non-steviol glycoside molecules form derivatives having less solubility, while in the next step, the derivatized non-steviol glycoside molecule can be converted back to the native state .

In other embodiments, the implementation of the crystallization temperature may vary from -20°C to 80°C. In some embodiments, the temperature increase and/or decrease may be performed in a gradient manner.

In other embodiments, the polarity of the solvent or mixture of solvents used in the crystallization ranges from non-polar to polar. Including solvents with a dielectric constant from 1 to 88.

In some embodiments, the ionic strength of the crystallization solution ranges from 0 mol/L to 20 mol/L.

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

Extraction of liquid by liquid and solid by liquid

NSGCs containing the non-steviol glycoside molecules of the present invention or derivatives thereof can be further purified and separated using a variety of solid-liquid and liquid-liquid extraction methods, including, but not limited to, dispersed liquid-liquid extraction, direct organic extraction, continuous countercurrent extraction, multi-stage continuous countercurrent extraction, centrifugation extraction, aqueous two-phase extraction, polymer-polymer extraction, polymer-salt extraction, etc.

Suitable solvents include water and organic solvents selected from the group consisting of 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.

Separation of impurities with a membrane

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

Consumable Products with NSGC

One embodiment of the present invention is an NSGC containing at least one non-steviol glycoside molecule.

In some embodiments, the NSGC imparts a sweet taste.

In one embodiment, the present invention is a sweetener composition containing NSGC.

In another embodiment, the present invention is an NSGC that is used in consumable foods as a source of antioxidants, dietary fiber, fatty acids, vitamins, minerals, preservatives, hydrating agents, probiotics, prebiotics, weight management agents, osteoporosis treatment agents, phytoestrogens. , long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

In another embodiment, the present invention is a flavor enhancing composition comprising NSGC, wherein the NSGC is present in an amount sufficient to provide a concentration at or below the NSGC taste threshold when the flavor enhancing composition is added to the consumable product. In a specific embodiment, the NSGC is present in an amount sufficient to provide a concentration below the NSGC taste threshold when the flavor enhancing composition is added to the consumable product. In one embodiment, the NSGC is present in an amount sufficient to provide a concentration below 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, of the threshold level of NSGC for taste when adding the flavor enhancing composition to the consumed product.

In yet another embodiment, the present invention is a sweetness enhancing composition comprising an NSGC, wherein the NSGC is present in an amount sufficient to provide a concentration at or below the NSGC sweetness threshold when the sweetening composition is added to the consumable product. In a specific embodiment, the NSGC is present in an amount sufficient to provide a concentration below the NSGC sweetness threshold when the sweetening composition is added to the consumable product. In one embodiment, the NSGC is present in an amount sufficient to provide a concentration below at least about 1%, at least about 5%, at least about 10%, at least about 15 % by at least about 20%, or at least about 25% or more, of the NSGC threshold level for the sensation of sweetness when the sweetening composition is added to the consumable product.

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

In a specific embodiment, the present invention is a beverage containing NSGC. In a particular embodiment, the NSGC is present in the beverage at a concentration that is above, at or below the sweetness threshold NSGC concentration.

In another specific embodiment, the present invention is a beverage product containing NSGC. In a specific embodiment, the NSGC is present in the beverage product at a concentration that is above, at, or below the NSGC taste threshold concentration.

In another aspect, the present invention is a method for producing a consumable product comprising (i) preparing a consumable product base and (ii) adding NSGC to the consumable product base to form a consumable product. In a specific embodiment, the NSGC is present in the consumable product at a concentration that is above, at, or below the sweetness threshold NSGC concentration. In another specific embodiment, the NSGC is present in the consumable product at a concentration that is above, at, or below the threshold NSGC concentration for taste.

In a particular embodiment, the present invention is a method for preparing a beverage comprising (i) preparing a beverage base and (ii) adding NSGC to a consumable product base to produce a beverage. In a specific embodiment, the NSGC is present in the consumable product at a concentration that is above, at, or below the sweetness threshold NSGC concentration. In another specific embodiment, the NSGC is present in the consumable product at a concentration that is above, at, or below the threshold NSGC concentration for taste.

In another aspect, the present invention is a method for increasing the sweetness of a consumable comprising (i) preparing a consumable containing at least one sweetening ingredient and (ii) adding NSGC to the consumable to provide a consumable with increased sweetness at a concentration of at or below the threshold concentration of NSGC for the sensation of sweetness.

In a specific embodiment, the present invention is a method for enhancing the sweetness of a beverage, comprising (i) providing a beverage containing at least one sweetening ingredient, and (ii) adding NSGC to the beverage to produce a beverage with increased sweetness at a concentration below a threshold concentration. NSGC for a sweet sensation. In one embodiment, the NSGC concentration in the sweetened beverage is at least about 1% lower, at least about 5% lower, at least about 10% lower, at least about 15% lower, by at least about 20%, or at least about 25% or more, of the sweetness threshold NSGC concentration.

In another aspect, the present invention is a method of enhancing the taste of a consumable product, comprising (i) preparing a consumable product containing at least one flavoring ingredient and (ii) adding NSGC to the consumable product to obtain a consumable product with improved taste at a concentration level or below the threshold concentration of NSGC for taste sensation.

In a particular embodiment, the present invention is a method for enhancing the taste of a beverage, comprising (i) preparing a beverage containing at least one flavoring ingredient, and (ii) adding NSGC to the beverage to produce an enhanced beverage at or below a concentration threshold. NSGC concentrations for taste sensation. In one embodiment, the NSGC concentration in the sweetened beverage is at least about 1% lower, at least about 5% lower, at least about 10% lower, at least about 15% lower, by at least about 20%, or at least about 25% or more, of the NSGC taste threshold concentration.

In the above methods, NSGC as such or compositions containing NSGC can be added. When NSGC is provided in the form of a composition containing NSGC, when the composition is added to consumable products such as foods or drinks, the concentration of NSGC in the composition is sufficient to provide a concentration above, at or below the threshold concentration of the NSGC composition to impart flavor or sweetness.

In some embodiments, the compositions of the present invention also contain one or more mogrosides, wherein the mogroside is selected from the group consisting of Luo han guo extract, byproducts of isolation and purification of other mogrosides, commercially available Luo han guo extract, mogroside IIE, mogroside IIB, mogroside III, mogroside IV, mogroside V, 11-oxo-mogroside V, mogroside VI, siamenoside I, grosmomoside I, neomogroside and other mogrolic and oxomogrolic glycosides found in the fruits of Siraitia grosvenorii , and combinations thereof, but not limited to this.

In other embodiments, the compositions of the present invention also contain one or more sweeteners or additional sweeteners. In one embodiment, the additional sweetener is a natural sweetener or a synthetic sweetener. In a specific embodiment, the additional sweetener is a high intensity sweetener. In a particular embodiment, the additional sweetener is mogrozide.

In some embodiments, the compositions of the present invention also contain one or more additives. In a specific embodiment, the additive is selected from the group consisting of carbohydrates, polyols, amino acids and their respective salts, polyamino acids and their respective salts, sugar acids and their respective salts, nucleotides, organic acids, inorganic acids, organic salts, including salts of organic acids and organic base salts, inorganic salts, bitter compounds, flavoring and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

In some embodiments, the compositions of the present invention also contain 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, hydrating agents, probiotics, prebiotics, weight management agents, osteoporosis treatment agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

In a specific embodiment, the present invention is a consumable product containing an NSGC and one or more sweeteners, additional sweeteners, additives, or functional ingredients.

In another specific embodiment, the present invention is a beverage containing NSGC and one or more sweeteners, additional sweeteners, additives or functional ingredients.

NSGCs can be used alone or in combination with at least one other sweetener in consumable products such as food, beverages, pharmaceutical composition, tobacco, nutraceutical composition, oral hygiene composition or cosmetics. Other sweeteners are selected from the group consisting of sucrose, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, allulose, galactose, glucose, gulose, idose, mannose, talose, psicose, sorbose, tagatose, mannoheptulose, sedoheptulose, octolose, fucose, rhamnose, arabinose, turanose, sialose, inulin, inulooligosaccharides, fructooligosaccharides, high fructose corn syrup (HFCS), maltodextrin, binding 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 found in the plant Stevia rebaudiana , biosynthetic steviol glycosides, glycosylated steviol glycosides icosides, glycosylated steviol glycosides (GSG), mogroside IV, mogroside V, mogroside VI, Luo han guo, siamenoside, other mogrosides found in fruits of Siraitia grosvenorii , monatin and its salts, curculin, glycyrrhizic acid and its salt, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, bayunoside, osladin, polypodoside A, pterokaryoside A, pterokaryoside B, mucurosioside, flomysoside I, periandrin I, abruzoside A and cyclokaryoside I, sugar alcohols, sucralose, acesulfame potassium acid and its salts, aspartame, alitame, saccharin and its salts, neohesperidin, dihydrochalcone, cyclamate, cyclamic acid and its salts, neotame, advantam and combinations thereof.

The following examples illustrate preferred embodiments of the invention. It is clear that the invention is not limited to the materials, proportions, conditions and methods set forth in the examples, which are only illustrative.

Examples

Example 1

Get NSGC

Five kilograms of dry leaves of Stevia rebaudiana (with a moisture content of about 8% (w/w) and a total content of steviol glycosides of about 10% (w/w, based on dry matter)) were crushed to a particle size of 10-20 mm. The dried leaf plant material was loaded into an extraction tank and extracted with 100 liters of reverse osmosis (RO) water at 65° C. for 10 minutes. The insoluble matter was removed by filtration. The yellowish filtrate was collected and applied to a column packed with 8.5 L of polymeric macroporous adsorption resin (YWD-03, Cangzhou Yuanwei, China) at a flow rate of about 50 L/h. After passing the filtrate through the column, the column was further washed with 45 L of water and both washes were combined. The combined solution was evaporated in vacuo at 30° C. to 35° C. to a final volume of 10 L.

Most of the steviol glycosides adsorbed onto the macroporous adsorbent resin and were eluted with about 45 L of 70% aqueous ethanol. The water-ethanol eluate was further processed to obtain about 400 g of stevia extract with a total content of steviol glycosides of about 96% (w/w).

The above 10 liters of the combined and evaporated solution was mixed with 90 liters of pure ethanol and this mixture was incubated for 10 minutes with slow stirring. The resulting precipitate was removed by vacuum filtration. The filtrate was collected and further subjected to vacuum evaporation (at 30-35° C.) to remove ethanol and further concentration of NSGC to about 4 L to a syrup with a solids content of 22% (w/w). According to HPLC analysis, the content of residual steviol glycosides in this solution was approximately 1.3%, and the ratio (in%) of individual steviol glycosides was as follows: 0.41% rebaudioside E, 10.52% rebaudioside O, 5.95% rebaudioside D , 1.49% rebaudioside N, 3.07% rebaudioside M, 56.98% rebaudioside A, 11.66% stevioside, 0.89% rebaudioside F, 4.37% rebaudioside C, 0.11% dulcoside A and 0 35% rebaudioside B. The concentrate was dried by evaporation in a vacuum followed by drying in a vacuum oven at 30-35°C.

Example 2

Extraction of NSGC with aqueous ethanol

50 kg dry leaves of Stevia rebaudiana (with a moisture content of about 8% (w/w) and a total content of steviol glycosides of about 8.5% (w/w) were crushed to a particle size of 10-20 mm. According to HPLC analysis, the total content of CGA in of these leaves was approximately 3.2% (w/w), with a CGA content of 1.34% (neo-CGA, n-CGA and crypto-CGA) and iso-CGA of 1.86% (iso-CGA-B , iso-CGA-A and iso-CGA-C).The dried plant material from the leaves was loaded into an extraction tank and extracted with 800 liters of 30% (v/v%) ethanol at 65°C for 30 minutes. g diatomaceous earth The yellowish filtrate was collected and cooled to 30° C. EtOH was removed from the filtrate with a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) at 45° C. under a pressure of 0.5-0.8 MPa.

320 L of the retentate obtained after nanofiltration, which was estimated to contain a total of approximately 1.57 kg of CGA and 4.1 kg of steviol glycosides, was applied to a column filled with 125 L of polymeric macroporous adsorption resin (YWD-03, Cangzhou Yuanwei, China) , with a flow rate of approximately 125 l/h. After passing the retentate through the column, an additional 62.5 L of water was washed and both washes were combined. This resulted in a product run-through fraction which was further concentrated using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) until the solids content was 20%. Flocculation in the concentrated running fraction with the product was carried out by adding 9 volumes of ethanol. The flocculated precipitate was separated by filtration and ethanol was removed using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) as described above. The concentrate was dried using a lyophilizer. In the purified flow-through fraction with the product, the total content of CGA was 12.24% (w/w) in terms of dry matter, while the content of neo-CGA was 2.65%, n-CGA - 7.46%, crypto-CGA - 1.84%, iso-CGA-B - 0.07%, iso-CGA-A - 0.16% and iso-CGA-C - 0.06%, and the total content of steviol glycosides was found to be 0.70% ( in weight ratio).

The adsorbed CGAs were eluted from the macroporous adsorbent resin with 690 liters of 25% (v/v%) ethanol. The solution was passed through a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) to remove ethanol and then dried with a lyophilizer as described above to obtain a 25% ethanol product. The total content of CGA (in a weight ratio) in the 25% ethanol product was 19.52%, while the content of neo-CGA was 0.78%, n-CGA - 3.59%, crypto-CGA - 1.04% , iso-CGA B - 2.64%, iso-CGA-A - 4.14% and iso-CGA-C - 7.33%, and the total content of steviol glycosides was 12.28% (w/w), while the content of rebaudioside E was 1.43%, rebaudioside D - 1.54%, rebaudioside A - 5.31%, stevioside - 2.49%, etc.

The remaining steviol glycosides were eluted from the macroporous adsorbent resin with about 380 liters of 70% aqueous ethanol and further processed to obtain a stevia extract with a TSG content of 71%.

Example 3

Extraction of NSGC with water

50 kg of dried Stevia rebaudiana leaf plant material similar to that used in Example 2 was loaded into an extraction tank and extracted with 800 L of water at 90° C. for 30 minutes. The mixture was filtered through 800 g of diatomaceous earth. The yellowish filtrate was collected and cooled to 30° C. and applied to a column filled with 125 L of polymeric macroporous adsorbent resin (YWD-03, Cangzhou Yuanwei, China). The subsequent steps were similar to those described in example 2.

In the purified flow-through fraction with the product, the total content of CGA was 14.47% (in a weight ratio), while the content of neo-CGA was 4.67%, n-CGA - 5.67%, crypto-CGA - 3.484%, iso- CGA-B - 0.24%, iso-CGA-A - 0.21% and iso-CGA-C - 0.24%, and the total content of steviol glycosides was found to be 0.59% (w/w). The total content of CGA (in a weight ratio) in the 25% ethanol product was 17.97%, while the content of neo-CGA was 0.78%, n-CGA - 1.56%, crypto-CGA - 0.91%, iso -CGA B - 5.36%, iso-CGA-A - 3.21% and iso-CGA-C - 6.15%, and the total content of steviol glycosides was found to be 13.83% (w/w), while the content of rebaudioside E was 2.89%, rebaudioside D - 1.52%, rebaudioside A - 5.31%, stevioside - 2.49%, etc.

Example 4

Extraction of NSGC with water

50 kg of dried Stevia rebaudiana leaf plant material similar to that used in Example 2 was loaded into an extraction tank and extracted with 800 L of water at 65° C. for 30 minutes. The mixture was filtered through 800 g of diatomaceous earth. The yellowish filtrate was collected and cooled to 30° C. and applied to a column filled with 125 L of polymeric macroporous adsorbent resin (YWD-03, Cangzhou Yuanwei, China). The subsequent steps were similar to those described in example 2.

The total content of CGA (in a weight ratio) in the 25% ethanol product was 19.90%, while the content of neo-CGA was 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 the total content of steviol glycosides was 13.47% (w/w), while the content of rebaudioside D was 1.86%, rebaudioside A - 7.45%, stevioside - 2.13%, etc.

Example 5

NSGC cleaning

6 grams of the dried 25% ethanol product from example 3 was dissolved in 20 ml of water. 180 ml of pure ethanol was added to the mixture, and stirred at room temperature for 1 hour. The resulting suspension was filtered, 0.72 g of activated carbon was added to the filtrate and left to stir at room temperature for another 1 hour. The mixture was again filtered to remove activated charcoal. The resulting filtrate was concentrated using a rotary evaporator at 40°C and dried in a vacuum oven at 40°C. This gave 3.6 g of a treated 25% ethanol product. The total content of CGA (in a weight ratio) in the 25% ethanol product thus treated was 22.32%, while the content of neo-CGA was 0.84%, n-CGA - 1.26%, crypto-CGA - 0.91 %, iso-CGA B - 6.85%, iso-CGA-A - 4.99% and iso-CGA-C - 7.48%, and the total content of steviol glycosides was found to be 23.26% (w/w) , while the content of rebaudioside D was 2.03%, rebaudioside A - 12.18%, stevioside - 6.70%, etc.

Example 6

Membrane cleaning NSGC

One gram of the treated 25% ethanol product from Example 5 was dissolved in 100 ml of RO water and this solution was fed into a Sterlitech HP4750 filtration system equipped with a stirred cuvette for high pressure operation (Sterlitech Corporation, USA) at 20°C until 50 ml of permeate has been collected. Both retentate and permeate were lyophilized and analyzed by HPLC.

The experiment was repeated using various 47 mm membrane discs obtained from Sterlitech Corporation (USA). In particular, 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).

The results of the permeate and retentate analysis are shown in Table 1.

Table 1

Results of HPLC analysis of permeate and retentate

Sample Initially Membrane type GE 2000 UFGH GE 1000 Synder NFG Microdyn Nadir NP010 Evonik Duramem 900 SynderXT, PES, UF Ret* Per* Reth Per Reth Per Reth Per Reth Per Reth Per HPLC analysis CGA (% dry matter) neo-CGA 0.84 0.87 1.23 0.88 1.15 0.88 0.07 1.09 0.62 1.08 0.14 0.88 0.62 n-CGA 1.26 1.26 2.22 1.28 2.15 1.33 0.11 1.41 1.07 1.41 0.15 1.33 0.96 crypto-CGA 0.91 0.91 1.55 0.93 1.52 0.96 0.08 1.02 0.81 1.02 0.13 0.93 0.72 iso-CGA-B 6.85 7.14 6.88 6.96 5.96 7.09 0.03 7.47 2.89 6.99 0.44 7.54 2.13 iso-CGA-A 4.99 4.99 7.95 5.03 7.17 5.21 0.02 5.27 3.04 5.11 0.38 5.32 2.17 iso-CGA-C 7.48 7.77 7.11 7.63 5.94 7.76 0.03 7.92 2.65 7.60 0.38 7.93 1.83 Total CGA 22.32 22.94 26.93 22.71 23.90 23.23 0.34 24.18 11.08 23.22 1.62 23.93 8.44 HPLC analysis SG (% dry matter) Reb E 0.32 0.35 0.00 0.96 0.00 1.15 0.00 0.51 0.00 0.80 0.31 0.89 0.47 Reb O 0.20 0.21 0.05 0.15 0.00 0.18 0.00 0.10 0.00 0.12 0.22 0.14 0.00 Reb D 2.03 2.02 0.12 1.80 0.10 1.73 0.15 2.01 0.00 1.70 0.22 1.81 0.23 Reb N 0.34 0.38 0.00 0.43 0.00 0.43 0.06 0.27 0.00 0.35 0.04 0.39 0.03 Reb M 0.36 0.39 0.00 0.38 0.00 0.34 0.00 0.26 0.00 0.36 0.00 0.39 0.04 Reb A 12.18 13.31 1.79 12.65 1.10 12.71 1.00 12.93 1.23 12.62 1.51 12.87 1.71 Stev 6.70 7.29 1.91 6.92 1.30 6.97 0.59 6.98 1.57 6.80 0.97 6.91 1.17 Reb F 0.16 0.20 0.07 0.18 0.04 0.18 0.11 0.18 0.05 0.16 0.21 0.18 0.03 Reb C 0.81 0.87 0.11 0.83 0.08 0.83 0.08 0.83 0.12 0.81 0.09 0.78 0.12 Dul A 0.04 0.05 0.05 0.05 0.09 0.04 0.12 0.05 0.02 0.04 0.15 0.05 0.02 Rub 0.03 0.03 0.19 0.02 0.17 0.03 0.02 0.03 0.04 0.02 0.04 0.03 0.01 Reb B 0.06 0.08 0.02 0.09 0.00 0.07 0.02 0.14 0.19 0.05 0.02 0.09 0.01 Sbio 0.04 0.04 0.03 0.03 0.00 0.03 0.02 0.06 0.07 0.03 0.01 0.06 0.00 Total SG 23.26 25.22 4.34 24.49 2.89 24.69 2.17 24.34 3.30 23.86 3.79 24.58 3.86

* Note: Ret: Retentate; Per: Permeate

The GE 1000 membrane was chosen for further experiments.

Example 7

Purification of NSGC with Membrane and Diafiltration

One gram of the treated 25% ethanol product from Example 5 was mixed with 100 ml of RO water and this solution was fed into a Sterlitech HP4750 filtration system equipped with a high pressure stirred cuvette (Sterlitech Corporation, USA) and a GE 1000 membrane ( Sterlitech Cat No YMGESP475) at 20°C until 50 ml of permeate has been collected. After 50 ml of permeate had been collected, 50 ml of RO water was added to the cuvette, filtration was repeated and a further 50 ml of permeate was collected. In this way, 10 permeate fractions were obtained. Both retentate and permeates were lyophilized and analyzed by HPLC.

The total content of CGA (in a weight ratio, in terms of dry matter) in the retentate was 16.51%, while the content of neo-CGA was 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 content of steviol glycosides was found to be 37.69% (w/w), with in this case, the content of rebaudioside D was 2.75%, rebaudioside A - 20.48%, stevioside - 10.39%, rebaudioside C - 1.35%, etc. The total content of CGA (in weight ratio, in terms of dry matter) in the sample combined from 10 permeates was 30.33%, while the content of neo-CGA was 0.97%, n-CGA - 2.52%, crypto-CGA - 2.28%, iso-CGA B - 8.23% , iso-CGA-A - 8.24% and iso-CGA-C - 8.08%, and the total content of steviol glycosides was 5.99% (w/w), while the content of rebaudioside A was 2.66% , stevioside - 2.89%, etc.

Example 8

Purification of NSGC using an adsorption chromatography system

100 ml of an aqueous solution containing 10.07 g of the 25% ethanol product from Example 4 was applied to a column packed with 300 ml of polymeric macroporous adsorbent resin (TF3, Cangzhou Yuanwei, China) at a flow rate of about 300 ml/hour at room temperature. temperature. After application, the column was additionally washed with 600 ml of water, both washes were combined. As a result, a product flow-through fraction was obtained, which was further concentrated using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then dried using a spray dryer to obtain 1.9 g of a dry product flow-through fraction, with a total CGA content of 27 .82% (in a weight ratio, in terms of dry matter), while the content of neo-CGA was 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 the total content of steviol glycosides was found to be 0.12% (w/w).

The adsorbed CGAs were eluted from the macroporous adsorbent resin with 1500 ml of 20% (v/v%) ethanol. The solution was passed through a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) to remove ethanol and concentrate. The concentrate was dried using a spray dryer to obtain 4.3 g of dry 20% ethanol product, with a total CGA content of 21.99% (w/w), while the content of neo-CGA was 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 the total content of steviol glycosides was found to be 2.18% (w/w).

The remaining steviol glycosides adsorbed on the macroporous resin were eluted with about 900 ml of 60% aqueous ethanol and further processed to obtain a stevia extract with a total content of steviol glycosides of 60.50% (w/w).

Example 9

Purification of NSGC using an adsorption chromatography system

100 ml of an aqueous solution containing 9.0 g of the 25% ethanol product from Example 4 was applied to a column filled with 300 ml of polymeric macroporous adsorbent resin (TF3, Cangzhou Yuanwei, China) at a flow rate of about 300 ml/hour at room temperature. temperature. After application, the column was additionally washed with 900 ml of water, both washings were combined. As a result, a product flow-through fraction was obtained, which was further concentrated using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then dried using a spray dryer to obtain 1.51 g of a dry product flow-through fraction, with a total CGA content of 31 .27% (in a weight ratio, in terms of dry matter), while the content of neo-CGA was 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 the total content of steviol glycosides was found to be 0.01% (w/w).

The macroporous adsorbent resin was further washed successively with 900 ml of 15% (vol.%) ethanol, 900 ml of 20% (vol.%) ethanol, 900 ml of 25% (vol.%) ethanol, and 900 ml of 60% (vol.%) 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 and 60 % ethanol product, respectively.

The results of HPLC analysis of these fractions are shown in Table 2.

table 2

Results of HPLC analysis of eluate fractions

Fraction HPLC analysis (% dry matter) neo-CGA n-CGA crypto CGA iso-CGA-B iso-CGA-A iso-CGA-C Total CGA Total SG flowing 4.19 19.35 7.10 0.11 0.43 0.10 31.27 0.01 15% ethanol 0.04 0.11 0.06 4.04 14.43 3.97 22.65 0.11 20% ethanol 0.01 0.02 0.01 0.77 9.84 17.24 27.89 0.93 25% ethanol 0.01 0.03 0.01 0.13 1.66 16.62 18.46 9.30 60% ethanol 0.00 0.00 0.00 0.02 0.10 0.18 0.30 48.81

Example 10

Purification of NSGC with an Adsorption Chromatography System

100 ml of an aqueous solution containing 9.0 g of the 25% ethanol product from Example 4 was applied to a column filled with 300 ml of polymeric macroporous adsorbent resin (TF3, Cangzhou Yuanwei, China) at a flow rate of about 300 ml/hour at room temperature. temperature. After application, the column was additionally washed with 600 ml of water, both washes were combined. As a result, a product flow-through fraction was obtained, which was further concentrated using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then dried to obtain 1.45 g of a dry product flow-through fraction, with a total CGA content of 18.04% ( in a weight ratio, in terms of dry matter), while the content of neo-CGA was 2.12%, n-CGA - 13.40%, crypto-CGA - 2.47%, iso-CGA-A - 0.04 % and iso-CGA-C - 0.01%, and the total content of steviol glycosides was equal to 0.74% (w/w).

The macroporous adsorbent resin was further washed successively with 1500 ml of 20% (vol.%) ethanol and 900 ml of 60% (vol.%) ethanol. All collected solutions were concentrated using a nanofiltration membrane (NF90-400, Dow Chemical Company, USA) and then dried to obtain a 20% ethanol product and a 60% ethanol product, respectively.

The results of HPLC analysis of these fractions are shown in Table 3.

Table 3

Results of HPLC analysis of eluate fractions

Fraction HPLC analysis (% dry matter) neo-CGA n-CGA crypto-CGA iso-CGA-B iso-CGA-A iso-CGA-C Total CGA Total SG flowing 2.12 13.40 2.47 0.00 0.04 0.01 18.04 0.74 20% ethanol 0.07 0.87 0.27 1.97 15.85 5.31 24.34 0.21 60% ethanol 0.00 0.01 0.00 0.06 0.42 0.70 1.20 48.51

Example 11

Purification of NSGC with an Adsorption Chromatography System

A 500 mg/L aqueous solution of the 25% ethanol product from Example 4 was applied to a column packed with 150 ml polymeric macroporous adsorption resin (TF3, Cangzhou Yuanwei, China) at a flow rate of about 150 ml/hour at room temperature. The effluent was periodically collected and the level of sweetness in it was analyzed using a sensor. The application to the column was stopped when the level of sweetness in the effluent reached the recorded value. The effluents were combined, concentrated and dried using a spray dryer to obtain a flow-through fraction with the product, with a total CGA content of 21.65% (weight ratio, based on dry matter), while the content of neo-CGA was 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 the total content of steviol glycosides was found to be 0.42% (w/w).

The macroporous adsorbent resin was further washed successively with 300 ml of 20% (vol.%) ethanol and 450 ml of 60% (vol.%) 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 a 20% ethanol product and a 60% ethanol product, respectively.

The results of HPLC analysis of these fractions are shown in Table 4.

Table 4

Results of HPLC analysis of eluate fractions

Fraction HPLC analysis (% dry matter) neo-CGA neo-CGA neo-CGA neo-CGA neo-CGA neo-CGA neo-CGA neo-CGA flowing 1.37 5.42 1.97 1.9 6.12 4.87 21.65 0.42 20% ethanol 0.43 2.39 0.81 1.23 5.08 5.37 15.31 3.37 60% ethanol 0 0.01 0 0.03 0.04 0.08 0.16 67.79

Example 12

Purification of NSGC by Crystallization

1 gram of the dried 25% ethanol product from example 3 was dissolved in 20 ml of water. 10 mg Ca(OH) ₂ was added and the resulting mixture was incubated for 1 hour until a precipitate formed. The resulting suspension was filtered, the precipitate was resuspended in water and titrated with acetic acid until dissolved, after which it was applied to a column filled with 100 ml of polymeric macroporous adsorbing resin (YWD-03, Cangzhou Yuanwei, China). The subsequent steps were similar to those described in example 2.

The total content of CGA (in a weight ratio) in the 25% ethanol product was 42.90%, while the content of neo-CGA was 0.51%, n-CGA - 1.30%, crypto-CGA - 0.34% , iso-CGA B - 5.78%, iso-CGA-A - 6.89% and iso-CGA-C - 28.08%, and the total content of steviol glycosides was found to be 0.3% (w/w), while the content of rebaudioside A was 0.2%, stevioside - 0.1%, etc.

Example 13

Consumed Products Containing NSGC

Samples of carbonated drinks were prepared according to the recipe presented in table 5.

Table 5

carbonated drink recipe

Ingredients Quantity, % Cola flavor 0.340 Orthophosphoric acid 0.100 sodium citrate 0.310 sodium benzoate 0.018 Lemon acid 0.018 Sweetener composition 0.050 Water saturated with carbon dioxide up to 100

The following samples were used as the "sweetener composition" in the recipe (i) a commercial sample of Rebaudioside A (97% purity) and (ii) a mixture (w/v 95:5) of commercial Rebaudioside A (97% purity) and NSGC prepared in accordance with example 3. The organoleptic characteristics of the beverage samples were evaluated by 20 experts. The results are shown in table 6.

Table 6

Sensory evaluation of carbonated beverage samples

The "sweetener composition" used in the recipe Prolonged sensation of sweetness* Bitterness* Delayed sensation of sweetness* Licorice Flavor* General sense of taste RebA 97+NSGC (95:5) 2 one one 2 nice Pure RebA 97 5 5 5 5 unpleasant *Prolonged sweetness, bitterness, delayed sweetness, and licorice taste were rated by participants on a scale of 1 to 5, with a lower score indicating a more pleasant taste sensation

The results showed that beverages made with the sweetener composition containing NSGC had better organoleptic characteristics.

Example 14

Consumable Products Containing New NSGCs

Chocolate samples were prepared according to the recipe presented in Table 7.

Table 7

Chocolate samples recipe

Ingredients Quantity, % grated cocoa 30.0 Cocoa butter 11.5 Powdered milk 14.0 Sorbitol 44.0 Salt 0.1 Sweetener composition 0.1 Lecithin 0.3

The cocoa liquor, cocoa butter, milk powder, sorbitol, salt, and "sweetener composition" were thoroughly mixed, then the mixture was placed in a rolling machine for grinding for 24 hours. Thereafter, the contents were transferred to a conche, lecithin was added and the resulting composition was stirred at 50° C. for 48 hours. The contents were then placed in a molding machine and left to solidify.

Samples from Table 3 were used as the "sweetener composition" in the recipe. (i) a commercial sample of rebaudioside A (97% purity) and (ii) a blend (95:5 w/v ratio) of commercial rebaudioside A (97% purity) and NSGC obtained in accordance with example 3. The organoleptic characteristics of the chocolate samples were evaluated by 20 experts. The results are shown in table 8.

Table 8

Organoleptic evaluation of chocolate samples

The "sweetener composition" used in the recipe Prolonged sensation of sweetness* Bitterness* Licorice Flavor* General sense of taste RebA 97+NSGC (95:5) one 2 2 nice Pure RebA 97 5 5 5 unpleasant *Longer sweetness, bitterness, and licorice taste characteristics were rated by participants on a scale of 1 to 5, with a lower score representing a more pleasant taste sensation

The results showed that the chocolate samples prepared with the sweetener composition containing NSGC had the best organoleptic characteristics.

While one or more embodiments of the present invention have been described above, those skilled in the art will appreciate that various changes and modifications are possible and that elements or compositions thereof may be substituted for equivalents without departing from the true scope of the invention. invention. Therefore, it is intended that this invention is not limited to the specific embodiment disclosed, and that the invention includes all embodiments covered by the scope of the appended claims.

Claims (9)

1. A method for producing a composition of non-steviol glycosides, including the steps: a. preparation of plant material Stevia rebaudiana; b. preparation of the extracting solvent; c. mixing the Stevia rebaudiana plant material and the extracting solvent to obtain a mixture of the Stevia plant material and the solvent; d. separating a mixture of stevia plant material and a solvent to obtain a filtrate containing steviol glycoside molecules and non-steviol glycoside molecules, the separation comprising filtration through diatomaceous earth; and e. isolating or separating steviol glycoside molecules from the filtrate in order to obtain a composition of non-steviol glycosides, wherein the resulting composition of non-steviol glycosides contains at least one molecule of non-steviol glycosides selected from phenolic compounds, polyphenols, flavonoids, quinic and caffeic acids and their derivatives, non-chlorogenic acid (neo-CGA; 5-O-caffeylquinic acid or 5- CQA), crypto-chlorogenic acid (crypto-CGA; 4-O-caffeylquinic acid or 4-CQA), n-chlorogenic acid (n-CGA; 3-O-caffeylquinic acid or 3-CQA), isochlorogenic acid A (iso -CGA A; 3,5-dicapheylquinic acid), isochlorogenic acid B (iso-CGA B; 3,4-dicapheylquinic acid), isochlorogenic acid C (iso-CGA C; 4,5-dicapheylquinic acid), retinoids, pigments, polysaccharides, oligosaccharides, disaccharides, monosaccharides, components of essential oils, sterols, terpenoids, sesquiterpenoids, diterpenes, triterpenes, coumarins, fatty acids and their derivatives, amino acids and their derivatives, dipeptides, oligopeptides, polypeptides, proteins, austroinulin, quercetin, sterebins, spatulenol , dean voic acid, 8,11,14-eicosatrienoic acid, 2-methyloctadecane, pentacosane, octacosane, stigmasterol, sitosterol, α- and β-amirin, lupeol, β-amirin acetate, pentacyclic triterpene and/or their glycosides and combinations thereof. 2. A consumable product containing a non-steviol glycoside composition obtained by the method of claim 1, wherein the consumable product is selected from pharmaceutical compositions, food gel mixtures and compositions, dental compositions, food, beverages and beverage products. 3. A method for producing a consumable product, comprising the step of adding the non-steviol glycoside composition obtained by the method of claim 1 to the consumable product.

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