BACKGROUND OF THE INVENTION

Sugar alternatives are receiving increasing attention due to awareness of many diseases in conjunction with consumption of high-sugar foods and beverages. However, many artificial sweeteners such as dulcin, sodium cyclamate and saccharin were banned or restricted in some countries due to concerns on their safety. Therefore non-caloric sweeteners of natural origin are becoming increasingly popular. The sweet herb Stevia rebaudiana produces a number of diterpene glycosides which feature high intensity sweetness and sensory properties superior to those of many other high potency sweeteners.

Stevia rebaudiana is a plant species belonging to the Astracea family, and is native to South America and cultivated now in many parts of the world (Gardana et al., 2003; Koyama et al., 2003; Carakostas et al., 2008). Stevia leaves are naturally sweet, and have been used for sweetening food products for hundreds of years in South America (Soejarto et al., 1982). Extracts of Stevia rebaudiana have been used commercially to sweeten foods in Japan and other Southeast Asian countries for a number of years (Koyama et al., 2003). As a product of nature, the stevia plant leaves contain different sweet tasting components, called steviol glycosides. Reportedly, more than 40 steviol glycosides have been identified that are typically present in the stevia leaf extract (Ceunen and Geuns, 2013; Chaturvedula et al., 2011a,b,c; Chaturvedula and Prakash, 2011a,b; Ohta et al., 2010). Each of these steviol glycosides has its own unique taste profile and sweetness intensity, which can be up to 350 times sweeter than sugar, but all share a similar molecular structure where different sugar moieties are attached to aglycone steviol (an ent-kaurene-type diterpene). The generic structure of the steviol glycosides is presented in Figure 1.

Rebaudioside A and stevioside have garnered the most commercial interest and have been extensively studied and characterized in terms of their suitability as commercial high intensity sweeteners. Stability studies in carbonated beverages confirmed their heat and pH stability (Chang S. S., Cook, J. M. (1983) Stability studies of stevioside and rebaudioside A in carbonated beverages. J. Agric. Food Chem. 31 : 409-412.) Steviol glycosides differ from each other not only by molecular structure, but also by their taste properties. Usually stevioside is found to be 110-270 times sweeter than sucrose and rebaudioside A is between 150 and 320 times sweeter than sucrose. Rebaudioside A has the least astringent, the least bitter, and the least persistent aftertaste thus possessing the most favorable sensory attributes in major steviol glycosides (Tanaka O. (1987) Improvement of taste of natural sweeteners. Pure Appl. Chem.69:675-6&3; Phillips K. C. (1989) Stevia: steps in developing a new sweetener. In: Grenby T.H. ed. Developments in sweeteners, vol. 3. Elsevier Applied Science, London. 1-43.)

By the early 21st century, only a limited number of the chemical structures of steviol glycosides in Stevia rebaudiana have been characterized including stevioside, rebaudioside A-F, dulcoside A, and steviolbioside (Ceunen and Geuns, 2013). In recent years, many minor steviol glycosides with diverse chemical structures, have been reported from the leaves of Stevia rebaudiana (Chaturvedula et al., 2011a,b,c; Chaturvedula and Prakash, 2011 a,b). These diverse steviol glycosides, which are ent-kaurene-type diterpenes, are connected to various sugars such as glucose, rhamnose, xylose, fructose and deoxy glucose at C-13 and C-19 positions via 1,2-; 1,3-; 1,4- or 1,6- a or β-glycosidic linkages. The identity and grouping of various steviol glycosides is summarized in Table 1. Due to the large number of steviol glycosides, Table 1 lists the steviol glycosides in 5 groups depending on the sugar moieties connected to the steviol backbone and identified with abbreviated compositional names.

• Glucosyl steviol family: comprising only steviol and glucose residues. This group can be presented by general formula "SvGn", wherein Sv is the steviol and G is glucose.

• Rhamnosyl steviol family: comprising steviol, rhamose and glucose residues. This group can be presented by general formula "SvRIGn", wherein R is the Rhamnose.

• Xylosyl steviol family: comprising steviol, xylose and glucose residues. This group can be presented by general formula "SvXIGn", wherein X is the Xylose.

• Fructosyl steviol family: comprising steviol, fructose and glucose residues. This group can be presented by general formula "SvFIGn", wherein F is the Fructose. • Deoxyglucose steviol family: comprising steviol, deoxyglucose and glucose residue. This group can be presented by general formula SvdGlGn.

Table 1

Since 2008 several United States (U.S.) Food and Drug Administration (FDA) Generally Recognized as Safe (GRAS) notifications in relation to steviol glycoside preparations with major individual steviol glycosides (stevioside and rebaudiosides A, C, D, and M) received "No Objection" letter from the FDA (Rebaudioside A: e.g., U.S. FDA, 2008a,b, 2009a,b, 2011a, 2012a,b,c; Stevioside: U.S. FDA, 2011b, 2012d; Rebaudioside D: U.S. FDA, 2013a; Rebaudioside X or M: U.S. FDA, 2013b, 2014a; Rebaudioside C: U.S. FDA, 2015a). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) reviewed the safety of steviol glycosides at four separate meetings (51st, 63rd, 68th and 69th) in 1998, 2004, 2007 and 2008 (JECFA, 2000, 2006, 2007, 2009) and established specifications, and an acceptable daily intake (ADI) for stevia extract as a high potency sweetener. At the 68th meeting, JECFA added three more steviol glycosides (rubusoside, steviolbioside and rebaudioside B) to the existing specification (containing stevioside, rebaudioside A, rebaudioside C and dulcoside A) and deleted the requirement for 70% stevioside/rebaudioside A, which was published in FAO JECFA monograph 4 (JECFA, 2007). An ADI of 0-4 mg/kg body weight (bw) (expressed as steviol) for steviol glycosides was then established at the 69th JECFA (2009). The current specification contains rebaudiosides A, B, C, D, F, stevioside, dulcoside A, rubusoside, steviolbioside where rebaudioside A and stevioside are the primary sweetener molecules (JECFA, 2010). The European Commission permitted the use of steviol glycosides as a sweetening agent under Commission Regulation (EU) No 1131/2011 and established a steviol glycoside specification containing not less than 95% steviol glycosides, rebaudiosides A, B, C, D, E, F, stevioside, dulcoside A, rubusoside and steviolbioside, with a total rebaudioside A and/or stevioside content of not less than 75% (EU, 2011). In 2011, the Codex Alimentarius Commission (CAC) adopted proposed draft maximum levels for steviol glycosides in food stuffs (Weston, 2011). Codex standards are developed by committees from the United Nation members and these standards are often used by many countries to support their own approval process.

Critical to the initial establishment of the safety of rebaudioside A/stevioside was the consideration that both of these compounds were metabolized by gut microflora to the aglycone steviol (Renwick and Tarka, 2008). In vitro and ex vivo studies have confirmed that steviol glycosides are not hydrolyzed by digestive enzymes of the upper gastrointestinal tract and are not absorbed through the upper portion of the gastrointestinal tract (Hutapea et al., 1997; Geuns et al., 2003, 2007; Koyama et al., 2003a,b). Therefore, steviol glycosides enter the colon intact, where they are subject to microbial degradation by members of the Bacteroidaceae family, resulting in the release of the aglycone steviol (Renwick and Tarka, 2008). Several in vitro studies mimicking the anaerobic conditions of the colon have confirmed the ability of the gut microbiota from mice, rats, hamsters, and humans to hydrolyze steviol glycosides completely to steviol (Wingard et al., 1980; Hutapea et al., 1997; Gardana et al., 2003; Koyama et al., 2003a; Nikiforov et al., 2013; Purkayastha et al., 2014, 2015, 2016).

Since the initial commercial focus was on rebaudioside A and/or stevioside, it has become apparent that other steviol glycosides in Table 1 may play a role in the sweetening effect of steviol glycosides as a whole, and as such, may have commercial value. Recent publications point out the value proposition of rebaudioside D and M (Prakash et al 2014) as a major component in stevia sweetener preparations. However, since current regulatory specifications have not been developed for all these molecules in any specific steviol glycoside combination matrix, and given that it is impractical to test in animal toxicity studies, each and every steviol glycoside molecule and mixture combination, the commercialization of such minor steviol glycosides, either individually or as mixtures, has been very slow due to the regulatory barriers in various jurisdictions.

To expedite the introduction of new innovations in regards to steviol glycoside mixtures that simulate the sugar-like taste profile in food and beverage applications, the regulatory safety paradigm needs to be directed toward the safety of all the steviol glycosides as a group, rather than toward individual glycoside and mixtures. Currently, to be an acceptable food additive, many regulatory agencies require a steviol glycoside content of not less than 95%, calculated by the content of each of the 9 to 11 major steviol glycosides determined by the regulations. This quantification of the 9-11 major steviol glycosides is known as the "total steviol glycoside", or "TSG", content of the food additive. The regulatory approval of all steviol glycosides extracted from Stevia leaf will facilitate innovations with minor steviol glycosides that are currently excluded when calculating a 95% steviol glycoside concentration, which currently includes only the approved nine (JECFA) to eleven (EU) glycosides. Considering the requirement for the high purity (95%) of steviol glycosides as a food additive, this invention shows that these minor glycoside molecules as part of the steviol glycosides mixture provide superior taste and sugar like sensory attributes that combinations of major molecules may not be able to deliver.

SUMMARY OF THE INVENTION

The present invention is directed to providing steviol glycoside compositions that have properties similar to compositions having higher "total steviol glycoside" content (TSG) with 9 to 12 major steviol glycoside molecules. These compositions require less processing of the leaf extract, thereby reducing cost and increasing operational efficiency, but surprisingly perform similar to higher TSG compositions. The lower TSG compositions contain some of the major steviol glycosides (e,g. Rebaudiosides A, B, C, D, and Stevioside), but also contain minor steviol glycosides. These minor steviol glycosides, although present in small amounts, demonstrate statistically significant positive impacts on the sweetness profile of stevia sweeteners. As used herein, a minor steviol glycoside is one that is present in a composition in an amount less than 5%), or less than 3%.

DETAILED DESCRIPTION

The total steviol glycoside content of a steviol glycoside composition typically determines the efficacy and quality of the sweetness and flavor profile of the composition. Extracting steviol glycosides from stevia plants is a well-defined process, but refining the plant extracts down to certain TSG levels with selected steviol glycosides can be time consuming and costly.

It was unexpectedly discovered that certain "upstream" intermediate compositions obtained during the extract concentration process have surprisingly desirable properties. In some instances, these intermediate compositions perform comparably to the more concentrated final products, even though the TSG content with major molecules is lower in these intermediate compositions than in the final products. Using these less purified stevia leaf extract provides a resourceful way to efficiently provide steviol glycoside ingredients having desired organoleptic properties.

In one embodiment, a lower TSG composition with major Steviol glycosides was obtained from the stevia leaf extract in a process designed to obtain a high level of rebaudioside A and stevioside. Though the TSG of a PCRA50 sample with 9 steviol glycosides (as approved by JECFA) was less than 95%; additional analytical evaluation shows that several minor steviol glycosides (not approved by JECFA yet) are present in the complex mixture of the exact as shown in Table 2. Table 2 also lists a downstream, purified stevia extract product PCSG9 that has higher than 95% purity with 9 steviol glycosides.

Table 2: Steviol Gl coside Composition

Example 1 : 50% Sugar Reduced Carbonated Soft Drinks

A 50% sugar reduced cola-flavored carbonated soft drink beverage were prepared with 200ppm of PCRA50. Another Cola sample with 200 ppm of PCSG9 was prepared as a control sample (Table 2). A trained sensory panel evaluated the beverage samples, and the results are shown in Fig. 2.

A similar evaluation was done using a 50% reduced sugar lemon-lime flavored carbonated soft drink beverage containing 250ppm of PCRA50 composition in Table 2. A comparative control beverage was prepared using 250ppm of PCSG9. A trained sensory panel evaluated the beverage samples, and the results are shown in Fig. 3. Notably, in the lemon -lime beverage, the samples with minor glycosides outperformed the PCSG9 samples in bitter aftertaste, bitterness, and acidity.

As can be seen from the data, even though the steviol glycoside composition had a reduced TSG content with 9 approved steviol glycosides, it performed comparably to, and in some cases better than, a higher TSG-containing composition across many taste and flavor attributes. This result is particularly surprising given the relatively low concentration of the minor steviol glycoside components.

In another embodiment, a stevia leaf extract (PCA9) containing less than 95% TSG content with 1 1 recognized steviol glycosides (COMMISSION REGULATION (EU) 2016/1814) was compared with a more purified sample (PCRebDM) containing mainly Rebaudiosides D and M. PCA9 can be used as a source of purified Reb D and/or Reb M products. An exemplary steviol glycoside compositions according to this embodiment is described in Table 3. PCA9 contains several minor steviol glycosides in measurable quantities as outlined in Table 3.

Table 3: Steviol Glycoside Composition

Example 2: Acidified water with Sugar and steviol glycoside compositions

To test the contribution of minor glycosides to the taste and flavor profile of acidified water, three acidified samples were prepared with 300 ppm of citric acid and sweetened with 5% sugar, 200 ppm of PCA9, or PCRebDM. The samples were served in 2 oz cup and tasted by 15 trained panel members to identify and quantify different sensory attributes on a predefined relative scale of measurements. The panel members were instructed to take a sip to rate the mouth feel and taste/flavor of the samples and 15 seconds after ingestion they rated the sweetness linger and the aftertaste.

*= 80% confidence interval, **= 90% Confidence interval, ***=95% Confidence interval The sensory panel found that PCA9 and PCRebDM were parity in sweetness with 5% sugar solution. However, The highly purified sample PCRebDM had significantly higher astringency, acid, off-notes, and sweet aftertaste compared to PCA9. The PCA9 sample was closer to sugar solution in several taste attributes as shown in Table 3. In conclusion, the PcRebDM is significantly different from PCA9 and sugar at a 95% Confidence level across key attributes. This result is unexpected due to the relatively low concentration of the minor steviol glycosides in the composition.

As can be seen from the data, even though the steviol glycoside compositions of the invention have a reduced TSG content with major molecules approved as sweeteners, they performed comparably to, and in some cases better than, a higher TSG-containing composition across many taste and flavor attributes. In the embodiments described, the TSG content of the steviol glycoside ingredient can be from about 3% to about 7% less than the high TSG control ingredient, while providing similar or comparable sensory results. In other embodiments, the lower TSG content can be less than about 99% TSG, or between about 0.5% to about 75%, or 1%) to about 50%), less than a high TSG ingredient, while providing similar or comparable sensory results for the finished product.

In conclusion, the minor steviol glycosides as listed in Table 1 can contribute significant improvement in taste of steviol glycoside compositions with lower TSG made of 9 to 11 approved steviol glycosides as major sweetener compounds. Specifically, several minor compounds (Reb J, K, H, N, O, Stevioside A, Reb C2 and their isomers) , with individual concentration ranging between 0.1% and 5% can contribute significantly to an improved taste and flavor profile in food and beverage applications. Given the low concentration of these minor steviol glycosides in the composition, their significant favorable impact on the taste and flavor profile was unexpected.

It is to be understood that the foregoing descriptions and specific embodiments shown herein are merely illustrative of the best mode of the invention and the principles thereof, and that modifications and additions may be easily made by those skilled in the art without departing for the spirit and scope of the invention, which is therefore understood to be limited only by the scope of the appended claims.