WO2014150127A1

WO2014150127A1 - Redistribution of mogrol glycoside content

Info

Publication number: WO2014150127A1
Application number: PCT/US2014/022320
Authority: WO
Inventors: Yuqing ZHOU; Richard W. Armentrout; Ryan David WOODYER; John R. BRIDGES; Timothy C. SCHUNK; Joshua N. FLETCHER
Original assignee: Tate & Lyle Ingredients Americas, LLC
Priority date: 2013-03-15
Filing date: 2014-03-10
Publication date: 2014-09-25
Also published as: GB201310060D0

Abstract

Fruit extracts containing mogrol glycosides substituted with five or more sugar residues per molecule such as mogroside V may be treated with acid or enzymes to increase the content of mogrol glycosides such as siamenoside I and mogroside IIIE containing three or four sugar residues per molecule. The modified fruit extracts thereby obtained are useful sweeteners and flavor enhancers for food and beverage compositions and the like.

Description

REDISTRIBUTION OF MOGROL GLYCOSIDE CONTENT

FIELD OF THE INVENTION

The invention relates to methods of preparing or modifying mogrol glycoside- containing compositions useful as sweeteners and flavor enhancers in food and beverage products.

BACKGROUND

Fruits of certain members of the Cucurbitaceae family, such as Siraitia grosvenorii (also known as Momordica grosvenorii, Luo Han Guo or monkfruit), are known to contain certain sweet compounds, some of which have been recognized as being much sweeter than sugar (sucrose). These intensely sweet compounds are triterpenoid glycosides also known as mogrol glycosides or mogrosides. The primary sweet component of Luo Han Guo fruit is mogroside V, a mogrol glycoside containing five sugar residues per molecule. In recent years, various extraction and purification methods have been developed which are capable of providing Luo Han Guo fruit extracts having a relatively high content of mogroside V (e.g ., at least 30% by weight or more). Such extracts are now being marketed as non-caloric natural sweeteners in some countries.

However, the taste and temporal profiles of these Luo Han Guo fruit extracts may not be ideal. For example, the extracts may have certain off-flavors or a lingering aftertaste or may take longer than desired to develop a sweet taste after being consumed (i.e., a delayed onset of sweetness). Accordingly, methods for improving or enhancing the performance of such fruit extracts as sweeteners would be desirable. BRIEF SUMMARY OF THE INVENTION

Certain mogrol glycosides found as minor naturally occurring components of Luo Han Guo fruit possess desirable taste characteristics. Siamenoside I, for example, tastes sweeter than mogroside V (the main sweet constituent of Luo Han Guo fruit) at 200 and 350 ppm. Mogroside HIE, contrary to previously published reports, has now been found to also be intensely sweet. The present invention provides ways of enriching the content of such minor mogrol glycosides in Luo Han Guo fruit extracts, thereby making possible improved sweeteners and flavor enhancers based on Luo Han Guo fruit as well as Luo Han Guo fruit-based sweeteners and flavor enhancers having taste and temporal profiles different from those exhibited by conventional Luo Han Guo fruit extract products.

One aspect of the invention provides a method of modifying the mogrol glycoside profile of a fruit extract (such as a Luo Han Guo fruit extract) comprised of one or more mogrol glycosides containing five or more sugar residues per molecule (such as mogroside V and mogroside VI), comprising contacting the fruit extract with at least one acid or at least one enzyme capable of cleaving β-1,6 glycosidic bonds under conditions effective to increase the amount of mogrol glycoside containing three or four sugar residues per molecule (e.g., siamenoside I, mogroside IV and/or mogroside IIIE) which is present in the fruit extract. The enzyme may, for example, be selected from the group consisting of cellulases, dextranases, galactosidases, glucanases, glucosidases, pustulanases and combinations thereof.

Another aspect of the invention provides a method of improving the taste or temporal profile of a Luo Han Guo fruit extract comprised of mogroside V, comprising contacting the Luo Han Guo fruit extract with at least one acid or at least one enzyme capable of cleaving β-1,6 glycosidic bonds under conditions effective to increase the total siamenoside I and mogroside IIIE content of the Luo Han Guo fruit extract.

Contrary to previous reports in the literature, mogroside IIIE (which may be obtained by reaction of mogroside V, siamenoside I or mogroside IV with acid or certain enzymes as described herein) has now been discovered to have an intensely sweet, non-bitter taste and thus is suitable for use as a sweetener and flavor enhancer. Accordingly, one aspect of the invention provides a food or beverage composition, comprising at least one food or beverage ingredient and an amount of mogroside IIIE effective to increase the perceived sweetness of the food or beverage composition. Also provided by the present invention is a method of sweetening a food or beverage composition, comprising combining the food or beverage composition with an amount of mogroside IIIE effective to increase the perceived sweetness of the food or beverage composition. A method of enhancing the flavor of a food or beverage composition, comprising combining the food or beverage composition with an amount of mogroside IIIE which is below the sweetness detection threshold but which is effective to enhance the flavor of the food or beverage composition, is provided in another aspect of the invention.

A modified fruit extract is also provided in another aspect of the invention, wherein the modified fruit extract comprises mogrol glycosides and the modified fruit extract is derived from a fruit having a naturally occurring mogrol glycoside distribution and the modified fruit extract has a mogrol glycoside distribution which is enriched in at least one of siamenoside I, mogroside IV or mogroside IIIE as compared to the naturally occurring mogrol glycoside distribution.

In yet another aspect of the invention, mogroside IIIE is prepared by a method comprising contacting a composition comprised of one or more of mogroside V, mogroside IV or siamenoside I with at least one acid or enzyme capable of cleaving β- 1,6 glycosidic bonds, such as an enzyme or combination of enzymes selected from the group consisting of cellulases, dextranases, galactosidases, glucanases, glucosidases and pustulanases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A, IB and 1C show the chemical structures of various mogrol glycosideFigure 2 shows the H NMR spectrum of a sample of mogroside IIIE produced in accordance with one aspect of the invention.

Figure 3 shows the ¹³C NMR spectrum of a sample of mogroside IIIE produced in accordance with one aspect of the invention.

Figure 4 shows the resonance shift assignments derived from the NMR spectra of Figures 2 and 3.

Figure 5 is a graph showing the release of free dextrose (by YSI) from a Luo Han Guo extract over time under acid hydrolysis conditions.

DETAILED DESCRIPTION OF THE INVENTION

Figure 1 shows the chemical structures of various mogrol glycosides. As may be seen from Figure 1, these mogrol glycosides differ with respect to how many sugar residues are attached to the triterpene nucleus as well as the types of linkages between the sugar residues and the locations/positions of the sugar residues within the mogrol glycoside molecule. For example, mogroside V contains five sugar residues per molecule. Mogroside VI contains six sugar residues per molecule. Mogroside IIIE contains three sugar residues per molecule. Siamenoside I and mogroside IV each contain four sugar residues per molecule.

The fruit extract comprised of one or more mogrol glycosides containing five or more sugar residues per molecule may be obtained from any suitable source. The fruit extract may, for example, be an extract from the fruit of a member of the

Cucurbitaceae family, such as Luo Han Guo fruit. Methods for extracting, purifying and concentrating the sweet mogrol glycoside components of such fruit are well known in the art. For example, the fruit extract may be obtained by extracting the fruit with hot water and/or alcohol and then subjecting the liquid extract thereby produced to subsequent filtration, ion exchange and/or adsorbent treatment steps. Suitable methods for preparing fruit extracts useful in the present invention are described, for example, in US Patent Publication Nos. 2011/0021456 and 2012/0264831, each of which is incorporated herein by reference in their entirety for all purposes. The fruit extract may be in dry (e.g., powdered) form or may be in the form of a solution (e.g., an aqueous solution) of varying concentration. Where the fruit extract starting material is in dry form, it will generally be desirable to combine it with water or other liquid medium prior to contacting it with an acid or enzyme, as will be described hereinafter in more detail. Where the fruit extract starting material is already in liquid form (e.g., an aqueous solution), it may be desirable to adjust the solids concentration by adding or removing solvent (e.g., water) and/or to adjust the pH by adding acid, base and/or buffering agent.

In one embodiment of the invention, the fruit extract employed as the starting material has a high content of mogrol glycosides containing five or more sugar residues. The mogrol glycosides containing five or more sugar residues per molecule may also be characterized as containing one or two β-1,6 glycosidic bonds per molecule. For example, mogrol glycosides containing five or more sugar residues per molecule (e.g., mogroside V, mogroside VI) may constitute at least about 40% by weight, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight or even at least about 80% by weight of the total amount of mogrol glycosides present in the fruit extract. In addition to mogrol glycosides, the fruit extract starting material may contain some amount of substances other than mogrol glycosides and water. For example, the fruit extract may contain about 15 to about 30% by weight or about 20 to about 25% by weight (on a dry solids basis) of non- mogrol glycoside substances, such as polyphenols, flavonoids, melanoidins, terpenes, proteins, sugars, aromatic glycosides and semi-volatile organic compounds. In certain embodiments, however, the fruit extract starting material comprises less than 10% by weight or less than 5% by weight of substances other than mogrol glycosides and water.

In one embodiment of the invention, the fruit extract is contacted with at least one enzyme capable of converting the mogrol glycosides containing five or more sugar residues per molecule to mogrol glycosides containing three or four sugar residues per molecule. Suitable enzymes for such purpose include cellulases, dextranases, galactosidases (e.g. β-galactosideases), glucanases (e.g., -(l-3)-D-glucanases), glucosidases (e.g ., β-glucosidases) and pustulanases and combinations thereof. In one embodiment, the enzyme or combination of enzymes is selected such that it has activity or selectivity for cleaving (hydrolyzing) the beta l->6 (β-1,6) glycosidic linkages (bonds) present in the initially present mogrol glycosides containing five or more sugar residues per molecule. Particularly suitable commercially available enzymes include OptiFLOW RC 2.0 (sold by Genencor), which is described as a complex cellulase enzyme preparation produced in a controlled fermentation of Trichoderma reesei. Other commercially available enzymes suitable for use in the present invention include, but are not limited to, Cellulase DS-K (sold by Amano), AlternaFuel CMAX-P "algae cell wall break" (sold by Dyadic), CelluStar CL cellulose (sold by Dyadic),

Cellulase CP COIMC (sold by Dyadic), DEXC dextranase from Penicilliam sp. (sold by Worthington), CEL (sold by Worthington), 31571 β-glucosidase from Bacillus (sold by Lucigen), Cel 136 pustulanase (sold by Prokazyme), 190097 dextranase from

Penicillum sp. (sold by MPBio), 150583 (sold by MPBio), D4668 (sold by Sigma), G8798 (sold by Sigma), C0615 cellulase from Trichoderma viride (sold by Sigma), G5160 β- galactoside from Aspergillus oryzae (sold by Sigma), 67138 -(l-3)-D-glucanase from Helix pomatia (sold by Sigma), 22178 cellulase from Aspergillius niger (sold by Sigma), 49291 glucosidase from Aspergillus niger (sold by Sigma), E0025 β-glucosidase from Clostridium thermocellum (sold by Prozomix), E0110 β-glucosidase from Rhizoium etli (sold by Prozomix) and E0105 β-glucosidase from Bacteroides fragilis (sold by

Prozomix). Such commercially available enzymes typically do not have a single enzyme activity, but rather have multiple types of activity (for example, a "cellulase" may have activity in catalyzing reactions other than cellulolysis (the hydrolysis of cellulose)) .

The enzyme(s) are contacted with the fruit extract for a time and under conditions effective to achieve the desired redistribution of mogrol glycosides. That is, the reaction conditions are selected to provide the desired extent of conversion of mogrol glycosides having five or more sugar residues per molecule (such as mogroside V and mogroside VI) to mogrol glycosides having just three or four sugar residues per molecule (such as siamenoside I, mogroside IV and mogroside HIE). Generally speaking, shorter reaction times will tend to favor the production of siamenoside I and mogroside IV over mogroside IIIE. As will be demonstrated subsequently in the

Examples, certain enzymes favor the production of siamenoside I over the production of mogroside IV while other enzymes favor the production of mogroside IV over the production of siamenoside I. Still other enzymes yield products containing

approximately equal amounts of these mogrol glycosides. If a particular ratio of siamenoside I to mogroside IV is desired in the final product, the enzyme may be selected such that it is capable of yielding the desired result. Siamenoside I is generally considered to be better tasting than mogroside IV, when each compound is used by itself as a sweetener. However, the presence of at least some amount (e.g., about 5% by weight to about 40% by weight of the total mogrol glycoside content) of mogroside IV in a blend of mogrol glycosides also containing mogroside V has been found to actually improve the taste of such a blend as compared to an analogous composition not containing mogroside IV.

In one embodiment of the invention, a mogrol glycoside composition is provided which comprises mogroside V and at least one mogrol glycoside selected from the group consisting of siamenoside I, mogroside IV and mogroside IIIE, wherein mogroside V is from about 60% to about 85% by weight of the total amount of (mogroside V + siamenoside I + mogroside IV and mogroside IIIE), siamenoside I is from 0 to about 40% by weight of the total amount of (mogroside V + siamenoside I + mogroside IV and mogroside HIE), mogroside IV is from 0 to about 20% by weight of the total amount of (mogroside V + siamenoside I + mogroside IV and mogroside HIE), and mogroside HIE is from 0 to about 40% by weight of the total amount of (mogroside V + siamenoside I + mogroside IV and mogroside IIIE) and wherein mogroside IV, if present, is present in an amount not greater than the total amount of siamenoside I and mogroside IIIE.

The reaction may be stopped (e.g., by separating the enzyme from the fruit extract or deactivating the enzyme) when the desired mogrol glycoside profile is attained. It has now been discovered that further reaction of mogroside IIIE generally does not take place upon prolonged contact of a fruit extract with an enzyme as described herein. That is, once mogroside IIIE is formed as a result of the cleavage of sugar residues from mogrol glycosides containing five or more sugar residues per molecule, it tends to be stable under reaction conditions in accordance with the present invention. This makes possible the production of compositions useful as sweeteners and flavor enhancers having a desirably high content of mogroside IIIE relative to other mogrol glycosides. As previously mentioned, mogroside IIIE has been found to be intensely sweet, contrary to earlier literature reports that it was "tasteless" (see Table II of Matsumoto et al., Chem. Pharm. Bull. 38(7) 2030-2032 (1990)) and that mogrol glycosides having fewer than four sugar residues per molecule are weakly sweet with a bitter taste (U.S. Pat. No. 4,084,010).

Typically, the reaction of enzyme with the fruit extract is carried out at a temperature of from about 20°C to about 80°C, a pH which is from about 3 to about 10, and for a time of from about 1 hour to about 24 hours. In other embodiments, the pH may be weakly acidic to neutral (i.e., from about 4 to about 7). For certain enzymes, higher rates of conversion may be attained at an acidic pH of from about 3.5 to about 6. As will be illustrated in more detail in the Examples, the mogrol glycoside profile or distribution of the product thereby obtained can be varied and controlled as may be desired by selecting different reaction conditions within these ranges.

Generally speaking, enzyme concentrations of from about 1 to about 100 Mg/mL will be suitable. The total concentration of solids (e.g., mogrol glycosides) in the liquid medium which is contacted with the enzyme(s) may be, for example, from about 10% by weight to about 50% by weight.

In another embodiment of the invention, the fruit extract is contacted with at least one acid under conditions effective to increase the amount of mogrol glycoside containing three or four sugar residues per molecule which is present in the fruit extract. Suitable acids for such purpose include, but are not limited to, protic inorganic ~J~ and organic acids such as sulfuric acid, hydrochloric acid, phosphoric acid,

trifluoroacetic acid, trichloroacetic acid, acidic resins and the like and combinations thereof. Contacting the fruit extract with the acid may generally be carried out with the fruit extract being dissolved in an aqueous solution. The acid may be also dissolved in the aqueous solution, for example at a concentration of from about 0.05 to about 1 M. The use of solid phase acid catalysts, such as acidic resins, is also possible, with an aqueous solution of fruit extract containing the starting mogrol glycoside(s) being passed over or through a bed of the solid phase acid catalyst. Contacting the fruit extract with the acid may be carried out for a time and at a temperature effective to achieve the desired conversion of at least a portion of the mogrol glycosides having five or more sugar residues per molecule to mogrol glycosides having three or four sugar residues per molecule, while avoiding where possible the further hydrolysis of the mogrol glycosides having three or four sugar residues per molecule. Suitable conditions for this purpose will vary depending upon the acid selected, its

concentration, the concentration of mogrol glycoside(s) and other factors, but typically contact temperatures of from about 40°C to about 90°C and contact times of from about 0.5 hours to about 12 hours may be employed. Once the acid digestion has reached the desired state of completion, the acid may be neutralized or removed.

The product obtained from the aforedescribed enzymatic or acid treatment (hereinafter sometimes referred to as a "modified fruit extract") may be subjected to further processing and/or purification steps, such as filtration, treatment with adsorbent, concentration and/or drying. For example, the product may be in the form of an aqueous mixture containing the desired distribution of different mogrol glycoside isomers which is concentrated by removal of water or membrane treatment to provide a more concentrated syrup useful as a sweetening agent or flavor enhancer or dried

(by spray-drying, for example) to provide a solid composition (in the form of a powder, for example) which is also useful as a sweetening agent or flavor enhancer. The modified fruit extract may be combined with one or more additional sweeteners or other food ingredients (such as a bulking agent or carrier) prior to such further processing.

By using the enzymatic and acid treatment methods described herein, modified fruit extracts having mogrol glycoside distributions different from those found in the naturally occurring fruit may be prepared. In particular, the amounts of mogroside HIE, siamenoside I and/or mogroside IV relative to the amounts of mogroside V and/or mogroside VI may be increased. In one embodiment of the invention, the modified fruit extract has a mogrol glycoside distribution as follows: mogroside V 0 to about 85% by weight, mogroside IV 0 to about 35% by weight, mogroside HIE 0 to 100% by weight, and siamenoside I 0 to about 65% by weight, wherein the weight percentages are based on the total mogrol glycoside content of the modified fruit extract and wherein the amount of at least one of mogroside V, mogroside IV, mogroside IIIE or siamenoside I is greater than 0% by weight.

The conditions under which the fruit extract starting material is contacted with acid or enzyme may, in various embodiments of the invention, be selected to provide a modified fruit extract product wherein the mogroside IIIE content is increased at least five fold, at least ten fold, at least fifteen fold or at least twenty fold as compared to the mogroside IIIE content of the fruit extract starting material. In other embodiments of the invention, the conditions under which the fruit extract starting material is contacted with acid or enzyme may be selected to provide a modified fruit extract product wherein the siamenoside I content is increased at least five fold, at least ten fold, or at least fifteen fold as compared to the siamenoside I content of the fruit extract starting material.

Fruit extract reaction conditions may be selected such that the product obtained is enriched in mogroside IIIE (i.e., the modified fruit extract has a mogrol glycoside content such that at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or even 100% by weight of the mogrol glycoside present is mogroside IIIE). The treatment methods described herein thus may be employed to obtain pure mogroside IIIE. The mogroside IIIE thereby obtained may be used as a high intensity sweetener, alone or in combination with one or more other high intensity sweeteners or conventional sweeteners such as sucrose. Mogroside IIIE may also be utilized as a flavor enhancer at sub-sweetening concentrations in food and beverage products and the like.

The modified fruit extracts and mogroside IIIE obtained in accordance with the present invention may be incorporated into any type of food or beverage composition as sweeteners or flavor enhancers. Non-limiting examples of such food and beverage compositions include baked goods, soups, sauces, processed meats, canned fruits, canned vegetables, dairy products, frozen confections, carbonated soft drinks, sports drinks, ready to drink teas, dairy drinks, alcoholic beverages, energy drinks, flavored waters, vitamin drinks, fruit drinks, fruit juices, powdered soft drinks, candy, confections, chewing gum, nutraceutical products and the like. The modified fruit extracts and mogroside IIIE may also be used in products such as medicines, pharmaceutical products and tobacco products. The modified fruit extract and/or mogroside IIIE is included in an amount effective to impart the desired amount of sweetness to the sweetened product. The product may contain one or more additional sweeteners, e.g ., a caloric sweetener such as sugar or another high intensity sweetener (either natural or synthetic) or may be free of any sweetening component other than the modified fruit extract or mogroside HIE of the present invention. The modified fruit extracts and mogroside HIE described herein may also find utility as taste enhancers, wherein they are included in a food or beverage at a concentration below the threshold where they impart a sweet taste to the product but in sufficient amount that they improve, modify or enhance the taste of the product.

In some embodiments, the modified fruit extract or mogroside HIE obtained in accordance with the invention is present in the foodstuff or beverage at a concentration of at least 100, at least 200, at least 500, at least 1000, at least 1500 or at least 2000 ppm (based on weight, as calculated on a dry solids basis) . At such concentrations, the modified fruit extract or mogroside HIE tends to function as a sweetener, i.e., it imparts a sweet taste to the foodstuff or beverage or increases the perceived sweetness of a foodstuff or beverage that already has (prior to the incorporation of the modified fruit extract or the mogroside IIIE) some degree of sweetness. In other embodiments, the modified fruit extract or mogroside IIIE is present in a foodstuff or beverage at a lower concentration, e.g., below the

concentration at which the modified fruit extract or mogroside IIIE imparts any perceived sweetness. The maximum sub-sweetening concentration (sometimes referred to as the "sweetness detection threshold") will vary somewhat depending upon the mogrol glycoside content of the modified fruit extract or the purity of the mogroside IIIE, but typically sub-sweetening concentrations of the modified fruit extract or mogroside IIIE will be more than about 1 ppm but less than about 60 ppm.

Concentrations of from about 10 to about 50 ppm, for example, may be effective to improve the taste or flavor of a foodstuff or beverage without increasing the perceived sweetness of such foodstuff or beverage.

Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the invention.

EXAMPLES

Example 1 : Enzyme Digestion

In this example, a variety of different enzymes were screened to assess their effectiveness in achieving redistribution of the mogrol glycoside content of a Luo Han Guo fruit extract.

Experimental Methods

Dinitrosalicylic Acid (DNS) Colorimetric Assay:

Solution 1 : 1% DNS (dinitrosalicyclic)

0.2% Phenol

0.05. Sodium Sulfite

1% NaOH

Solution 2 :

40% Potassium sodium tartrate

Assay:

1. Add 50μΙ_ sample to be tested to a 96 well plate.

2. Add 100μΙ_ Solution 1 to each well containing sample.

3. Place 96 well plate in PCR machine and heat for 15min at 95C.

4. Remove from PCR machine and immediately add 15μΙ_ Solution 2.

5. Allow to cool for 15min at room temperature.

6. Read at 575nm (optional).

Moqroside V sample preparation : Prepared a 1L 30% Monk Fruit Extract solution (300g/L; 300000ppm) in DI water. Then 500ml_ of the 30% Monk Fruit Extract solution was added to 150g of carbon to perform carbon extraction. This mixture was stirred and heated for 5 min. The carbon was removed over Whatman 1 filter paper, and the filtrate was passed over a 0.22μηΊ filter to remove residual carbon. This treated material was labeled "Carbon Treated Monk Fruit Extract" and was the source of mogroside V used as a starting material throughout the screening process.

Enzyme Preparation : Enzymes that were received as a solution were used as is, while solid products were made to a 50mg/mL solution (unless noted otherwise in Table 1 below) using DI water. If particulate remains at this concentration, the tube was quickly spun down before use in the screening reactions.

DNS and Monk Fruit Extract: The Carbon Treated Monk Fruit Extract was diluted (2, 4, 8, 16 and 32X) and run in a DNS assay. It was found that >8X dilutions gave little to no DNS activity. For subsequent screening reactions, Carbon Treated Monk Fruit Extract was always used at a final dilution of 8X.

DNS and Enzyme: All enzymes were tested for the presence of reducing sugar through the DNS assay. A 50pL sample of 1 : 50 (20pL/mL final) enzyme dilution in DI water was used as an initial screen for reducing sugars. If the enzyme contained enough reducing sugar to cause distinct color change in the DNS assay then the enzyme concentration was reduced to 2μΙ7ηΊΐ. for subsequent screening reactions. This is noted in Table 1 below.

Screening Reactions:

1. 150μί of 4X diluted Carbon Treated Monk Fruit Extract was placed into 96 well plates. water was used as an initial screen for reducing sugars. If the enzyme contained enough reducing sugar to cause distinct color change in the DNS assay then the enzyme concentration was reduced to 2pL/mL for subsequent screening reactions. This is noted in Table 1 below.

Screening Reactions:

1. 150μΙ- of 4X diluted Carbon Treated Monk Fruit Extract was placed into 96 well plates.

2. 150μΙ_ of lOOmM buffer (varying pH) was added to each sample.

3. Enzyme was added to start the reaction (either at 20pL/mL or 2pL/mL final concentration).

4. 96 well plate mats were used to prevent evaporation and the plates were placed at the screening temperature.

5. 80μ1_ samples were taken at varying time points and placed at -20°C until DNS/HPLC testing.

Final reactions contained 8X diluted Carbon Treated Monk Fruit Extract, 50mM buffer and 20μΙ_ or 2pL/mL enzyme.

HPLC method :

Method for analyzing mogrosides

Method Name: "LHG7"

Column : Restek Allure C18 5μητι 250x4.6mm (Catalog # 9164575) Mobile Phase: A = H₂0; B = ACN; Needle Flush = 20%B

System : Perkin Elmer Series 225 Autosampler, Series 200 Pump, Series 200 UV/Vis Detector, Series 200 Column Oven and Series 200 Vacuum Degasser.

Retention Times: Mogroside VI ( 11.6), Mogroside V (11.9), Siamenoside I

(12.6), Mogroside IV (13.3), Mogroside HIE (14.4) and Mogroside II ( 15.5).

Siamenoside I and Mogroside HIE each have two concomitantly produced peaks that often make split peaks which vary in size depending on the enzyme used. Step Time (min) Flow (mL/min) %A %B Curve

Equil 0.1 1.0 80 20 N/A

Run 7.0 1.0 70 30 6.0

Run 5.0 1.0 50 50 6.0

Run 1.0 1.0 20 80 6.0

Run 3.0 1.0 20 80 6.0

Run 1.0 1.0 80 20 6.0

Run 7.0 1.0 80 20 6.0 Results

A total of 62 different enzymes were collected and screened by DNS assay and the reaction products thereby obtained subsequently analyzed by HPLC. Promising enzymes were further analyzed under variable temperature and pH conditions. Table 1 summarizes the enzymes screened and the conditions tested. Enzymes marked with an asterisk (*) did not cause a release of glucose from the substrate, indicating that at least under the screening conditions used they were likely not effective in converting mogrol glycosides with five or more sugar residues per molecule to mogrol glycosides with just three or four sugar residues per molecule.

Table 1 : Listing of Enzymes and Test Conditions Used

Enzyme # Enzyme Name Stock Enzyme cone, Reaction

Cone. used (Mg/mL) conditions tested

Zl Genencore N/A 20 50°C and

OptiFLOW RC2.0 70^OC; pH 4-8

Z2* Genencore N/A 2 50°C and

OptiMAX L-1000 70°C; pH 5

Z3* Amano N/A 2 50°C and

Kleistase ESC 70°C; pH 5

Z4* Amano N/A 2 50°C and

Kleistase T10S 70°C; pH 5

Z5* Amano ~30 20 50^OC and

Kleistase SD80 mg/mL 70°C; pH 5

Z6 Amano ~50 2 50°C and

Cellulase DS-K mg/mL 70°C; pH 4-8

Z7 Dyadic ~50 20 50°C; pH 5

AlternaFuel CMAX-P mg/mL

Z8 Dyadic N/A 2 50°C; pH 4, 5

CelluStar CL and 6

Z9 Dyadic ~50 20 50°C; pH 5

Cellulase CP CONC mg/mL

Z10* MPBio ~8 20 37°C; pH 7

153487 mg/mL

Zl l* MPBio ~10 20 37⁰C; pH 7

194123 9 mg/mL

Z12* MPBio ~25 20 37⁰C; pH 5

100348 mg/mL

Z13* MPBio ~10 20 37°C; pH 5

195197 mg/mL

Z14* Worthington ~25 20 25°C; pH 7

BG mg/mL

Z15 Worthington ~50 20 370C; pH 5

CEL mg/mL

Z16* Worthington ~50 20 370C; pH 7

CEPM mg/mL Z17 Worthington ~25 20 37⁰C; pH 6, 7

DEXC mg/mL and 8

Z18* Biomatik ~50 2 37⁰C; pH 5

A4147 mg/mL

Z19 Lucigen 20m 20 70°C; pH 6

31571 g/mL

Z20* Lucigen 20m 20 70°C; pH 6

31592 g/mL

Z21* Lucigen 20m 20 70°C; pH 6

31591 g/mL

Z22* Lucigen lmg 20 70°C; pH 6

30650 /mL

Z23* Noor N/A 2 70°C; pH 6

Cellulase ACEL

Z24* Prokazyme ~25 20 70°C; pH 6

Bglul lO mg/mL

Z25 Prokazyme ~25 20 70°C; pH 6

Cel 136 mg/mL

Z26 MPBio ~5 20 37⁰C; pH 4, 5

190097 mg/mL and 6

Z27* MPBio ~5 20 25⁰C; pH 7

104939 mg/mL

Z28 MPBio ~50 20 and 2 37⁰C; pH 4, 5

150583 mg/mL and 6

Z29* Sigma P2986 400 20 37⁰C; pH 5 units/mL

Z30* Sigma D0443 N/A 2 50°C; pH 5

Z31 Sigma D4668 ~50 20 37°C; pH 5 mg/mL

Z32* Sigma G0395 ~50 20 37°C; pH 5 mg/mL

Z33 Sigma G8798 ~20 20 70⁰C; pH 6 mg/mL

Z34* Sigma G8548 ~20 20 70°C; pH 6 mg/mL

Z35* Sigma G8673 ** 20 70^OC; pH 6 Z36 Sigma C0615 ~25 20 37⁰C; pH 5 mg/mL

Z37* Sigma C9499 ** 20 70°C; pH 6

Z38* Sigma C0660 ~50 20 25°C; pH 6 mg/mL

239* Sigma G9259 1.5 20 37⁰C; pH 5 mg/mL

Z40 Sigma G5160 ~50 20 37°C; pH 5 mg/mL

Z41 Sigma 67138 ~25 20 37°C; pH 5 mg/mL

Z42 Sigma 22178 ~50 20 37°C; pH 5 mg/mL

Z43* Sigma 10113 ~50 20 50°C; pH 5 mg/mL

Z44 Sigma 49291 ~50 20 37⁰C; pH 5 mg/mL

Z45* Roche ~50 20 37°C; pH 7

11641735 mg/mL

Z46* Roche ~50 2 25°C; pH 6

11636987 mg/mL

Z47* Roche ~50 20 25°C; pH 6

11116541 mg/mL

Z48* Roche ~50 20 37°C; pH 7

11520857 mg/mL

Z49* Roche ~50 20 37°C; pH 6

10129046 mg/mL

Z50 Prozomix 0.6 20 60^OC; pH 6

E0025 mg/mL

Z51* Prozomix 0.5 20 60°C; pH 6

E0005 mg/mL

Z52* Prozomix 0.6 20 50°C; pH 7

E0009 mg/mL

Z53* Prozomix 2 20 50°C; pH 7

E0004 mg/mL

Z54* Prozomix 3.5 20 37°C; pH 7 E0008 mg/mL

Z55* Prozomix 0.6 20 60°C; pH 6

E0003 mg/mL

Z56* Prozomix 2 20 60°C; pH 5

E0020 mg/mL

Z57* Prozomix 2 20 600C; pH 7

E0021 mg/mL

Z58 Prozomix 1.0 20 37°C; pH 5

E0110 3 mg/mL

Z59 Prozomix 3.6 20 37°C; pH 5

E0105 3 mg/mL

Z60* Prozomix 2.8 20 25⁰C; pH 7

E0410 9 mg/mL

Z61* Prozomix 6.6 20 37⁰C; pH 5

E0401 mg/mL

Z62* Roche ~50 20 37°C; pH 6

11626329 mg/mL

* Enzyme did not cause a release of glucose from the substrate

** Supposed to be 100pL solution, but nothing was in the vial as received from the supplier. Added 100pL of water and used for reaction.

The remaining enzymes (those with the enzyme identifying numbers not marked with an asterisk) showed varying degrees of activity and specificity which are detailed further in the table below (Table 2). The enzymes listed in the first section of Table 2 exhibited higher specificity for producing siamenoside I than mogroside IV, whereas the enzymes listed in section 2 of Table 2 exhibited higher specificity for producing mogroside IV than siamenoside I. The enzymes listed in section 3 of Table 2 were found to be capable of producing mogroside HIE selectively within a relatively short period of time. Table 2: Specificity of Enzymes Screened

Section 1 - Enzymes exhibiting higher specificity for producing siamenoside I than mogroside IV

Section 2 - Enzymes exhibiting higher specificity for producing mogroside IV than siamenoside I

Section 3 - Enzymes selectively producing mogroside HIE

Enzymes that are not mentioned in Table 2 produced an approximately equal ratio of siamenoside I and mogroside IV.

It should be noted that a majority of the enzymes listed in Table 2 will eventually convert mogroside V to mogroside IIIE if the reactions are carried out for a sufficiently long period of time. The enzymes that showed the highest activity and produced mogroside IIIE quickly (<2hrs) are mentioned in the above table. Diluting these enzymes 10-100 fold was needed to determine their specificity, which is also mentioned in the above Table 2 if it was not found to be 1 : 1 siamenoside I to mogroside IV.

Conditional effects of time, pH and temperature were investigated, and it was found that the composition (mogrol glycoside profile) of the final products could be varied by changing the reaction conditions used for most of the enzymes investigated, as shown in Tables 3 and 4. In Tables 3 and 4, MogVI = mogroside VI; MogV = mogroside V; Siam = siamenoside I; MoglV = mogroside IV; MoglllE = mogroside IIIE; and Mogll = mogroside II.

Table 3 : Effect of pH on enzyme activity.

% Products

Enzyme PH Hrs MogVI MogV Siam MoglV MoglllE Mogll

Z6 4 4 0 2 11 16 70 0

Z6 5 4 2 5 14 21 59 0

Z6 6 4 9 42 17 23 9 0

Z6 7 4 15 85 0 0 0 0

Z6 8 4 15 85 0 0 0 0

Z6 4 8 0 0 0 0 100 0

Z6 5 8 0 0 3 11 86 0

Z6 6 8 8 42 17 24 10 0

Z6 7 8 15 85 0 0 0 0

Z6 8 8 15 85 0 0 0 0

Table 4: Effect of active enzymes on mogrol glycoside distribution.

Example 2: Acid Digestion

Experiments were designed to evaluate if acid digestion conditions could be used to convert mogroside V to other mogrol glycosides. The starting materials containing about 50 weight % mogroside V were all semi-purified and purified extracts of Luo Han Guo ("LHG") fruit obtained from BioVittoria. The LHG extracts were incubated with 0.5 M H₂S0₄ for the times and at the temperatures indicated below. Samples were taken during the course of digestion and analyzed for release of dextrose (YSI). Samples were analyzed by HPLC to determine decreases in mogrosides V and increases in siamenoside I and other mogrol glycosides.

Acid digestion was performed at 78°C. Samples were taken over a 6 hr period.

The rate of release of free dextrose (by YSI) from the LHG extract during the acid hydrolysis was measured (Figure 5). There was a progressive release of dextrose and a decrease in mogroside V during the acid digestion. There was an increase in siaminoside I, mogroside IV, and mogroside Hie as measured by HPLC during the acid digestion (Table 5).

Table 5. Changes in concentrations of mogroside compounds during acid hydrolysis (% of total compounds).

Minutes at 78°C Mogroside V Siamenoside I Mogroside IV Mogroside Ille

0 68.6 2.8 2.4 1.0 60 52.8 4.5 3.3 1.0

120 41.2 4.5 3.9 1.5

180 31.4 4.7 3.9 1.3

240 23.9 4.6 3.7 1.5

300 18.2 4.2 3.7 1.9 360 13.8 5.0 3.1 2.9

Example 3 : Characterization of Mogroside HIE

Mogroside HIE has been previously isolated from the fruit of Siraitia grosvenori and found to have the chemical structure set forth in Figure 1C [Matsumoto et al. Chem. Pharm. Bull. 38(7) 2030-2032 (1990)] .

A sample of purified mogroside HIE was obtained for further characterization and use as a reference standard by the following procedure. Luo Han Guo extract dry solid (1.0 kg) was dissolved in DI water to a final volume of 4.0 L at 60°C. To this mixture 870 g of powdered activated carbon (Texas Natural Supply) was added and mixed for 1 hour. After cooling to 45°C, the carbon was removed using Whatman # 1 filter paper. The mogrol glycoside composition of the filtrate (which initially contained 7% by weight mogroside V as determined by HPLC) was modified with OptiFLOW RC 2.0 enzyme (Genencor). To each 1 L of filtrate in 2.8 L foil-capped baffled flasks was added 2 ml_ of OptiFLOW RC 2.0 and the mixture incubated at 50°C for 15.5 hr with shaking. The resulting mogrol glycoside mixture was freeze dried.

The freeze dried mogrol glycoside mixture was dissolved in DI water at 16% by weight solids and fractionated using preparative HPLC. Preparative HPLC was performed on a Phenomenex 5 μιη particle packing, 21.2 x 250 mm Phenomenex Gemini C18 column at ambient temperature using a Waters Breeze 25P HPLC operated at 22.5 mL/min. Separation of repetitive injections of 5 mL of the mogrol glycoside mixture were monitored at 208 nm using a Waters 2487 detector and timed fractions collected. The gradient program of linear segments was:

The fraction eluting between 23 and 25.5 minutes was accumulated, vacuum stripped to remove acetonitrile, and freeze dried. The resulting solid mogrol glycoside mixture was dissolved at 50 mg/mL in DI water and separated a second time by preparative HPLC using the same system with the exception of the solvent program shown below.

The resulting fraction eluting between 40 and 45 minutes was accumulated from multiple injections, vacuum stripped to remove acetonitrile, and freeze dried. The resulting purified mogroside HIE (94% pure by HPLC) was subjected to H and ¹³C-NMR for structure characterization.

The Η and ¹³C NMR spectra obtained are shown in Figures 2 and 3,

respectively. Figure 4 sets forth the resonance shift assignments, which establish that the sample has a chemical structure corresponding to that of mogroside HIE. Contrary to the previously mentioned literature article, which states that mogroside IIIE is "tasteless," pure mogroside HIE as prepared in accordance with the present invention was found to be intensely sweet in taste.

Example 4: Sensory Evaluations of Various Mogrol Glycoside Compositions

When used as a single agent mogroside IIIE was found to be sweeter than mogroside V. The highest concentration that this was confirmed at was 500 ppm. Siamenoside I and mogroside IV were found to be sweeter than mogroside V at 350 ppm but not at 500 ppm.

Various mogroside mixtures were evaluated for sweetness at 350 ppm total mogrosides in aqueous solution. The results are presented in Table 6 below. The general trend found was that blends that are high in mogroside IIIE and siamenoside I relative to mogroside IV are preferred.

Table 6: Mogrol Glycoside Sensory Evaluations

Additional mogroside solutions were tested at 350 and 500 ppm with mogroside V making up 35% of the blend on a dry solids basis. The general conclusion from this study was that mixtures with more combined mogroside IIIE and siamenoside I than mogroside IV were preferred.

Claims

What is claimed is:

1, A method of modifying the mogrol glycoside profile of a fruit extract comprised of one or more mogrol glycosides containing five or more sugar residues per molecule, comprising contacting the fruit extract with at least one acid or at least one enzyme capable of cleaving β-1,6 glycosidic bonds under conditions effective to increase the amount of mogrol glycoside containing three or four sugar residues per molecule which is present in the fruit extract.

2. The method of claim 1, wherein the fruit extract is an extract of Luo Han Guo fruit.

3. The method of claim 1 or 2, wherein mogrol glycosides containing five or more sugar residues per molecule comprise at least about 50% by weight of the mogrol glycosides present in the fruit extract.

4. The method of any one of claims 1 to 3, wherein mogroside V comprises at least about 70% by weight of the mogrol glycosides present in the fruit extract.

5. The method of any one of claims 1 to 4, wherein the fruit extract is contacted with at least one enzyme that is selective for cleaving β-1,6 glycosidic bonds.

6. The method of any one of claims 1 to 4, wherein the fruit extract is contacted with at least one enzyme selected from the group consisting of cellulases, dextranases, galactosidases, pustulanases and combinations thereof.

7. The method of any one of claims 1 to 4, wherein the contacting is with at least one enzyme and is carried out in an aqueous medium at a pH of from about 3 to about 10.

8. The method of any one of claims 1 to 7, wherein the contacting is carried out at a temperature of from about 20°C to about 80°C.

9. The method of any one of claims 1 to 8, wherein the contacting is carried out for a time of from about 1 hour to about 24 hours.

10. The method of any one of claims 1 to 9, wherein the contacting is carried out under conditions effective to increase the total amount of siamenoside I and mogroside IIIE, relative to other mogrol glycosides, present in the fruit extract.

11. The method of any one of claims 1 to 9, wherein the contacting is carried out under conditions effective to increase the amount of mogroside IIIE, relative to other mogrol glycosides, in the fruit extract.

12. The method of any one of claims 1 to 9, wherein the contacting is carried out under conditions effective to increase the amount of siamenoside I, relative to other mogrol glycosides, in the fruit extract.

13. A method of improving the taste or temporal profile of a Luo Han Guo fruit extract comprised of mogroside V, comprising contacting the Luo Han Guo fruit extract with at least one enzyme that is selective for cleaving β-1,6 glycosidic bonds under conditions effective to increase the total siamenoside I and mogroside HIE content of the Luo Han Guo fruit extract.

14. A modified fruit extract comprising mogrol glycosides, wherein the modified fruit extract is derived from a fruit having a naturally occurring mogrol glycoside distribution and the modified fruit extract has a mogrol glycoside distribution which is enriched in at least one of siamenoside I, mogroside IV or mogroside HIE as compared to the naturally occurring mogrol glycoside distribution.

15. A food or beverage composition, comprising at least one food or beverage ingredient and an amount of mogroside HIE effective to increase the perceived sweetness of the food or beverage composition.

16. A method of sweetening a food or beverage composition, comprising combining the food composition with an amount of mogroside HIE effective to increase the perceived sweetness of the food or beverage composition.

17. A method of making mogroside HIE, comprising contacting a composition comprised of one or more mogrol glycosides containing four or more sugar residues per molecule with at least one acid or enzyme that is capable of cleaving β-1,6 glycosidic bonds.

18. A method of enhancing the flavor of a food or beverage composition, comprising combining the food or beverage composition with an amount of mogroside HIE which is below the sweetness threshold but which is effective to enhance the flavor of the food or beverage composition.