rebaudioside M

Abstract

This work aims to review and showcase the unique properties of rebaudioside M as a natural non-caloric potential sweetener in food and beverage products. To determine the potential of rebaudioside M, isolated from Stevia rebaudiana Bertoni, as a high potency sweetener, we examined it with the Beidler Model. This model estimated that rebaudioside M is 200–350 times more potent than sucrose. Numerous sensory evaluations of rebaudioside M’s taste attributes illustrated that this steviol glycoside possesses a clean, sweet taste with a slightly bitter or licorice aftertaste. The major reaction pathways in aqueous solutions (pH 2–8) for rebaudioside M are similar to rebaudioside A. Herein we demonstrate that rebaudioside M could be of great interest to the global food industry because it is well-suited for blending and is functional in a wide variety of food and beverage products.

Discovery of Rebaudioside M

Work to elucidate the chemical structures of S. rebaudiana sweeteners began in the early twentieth century, but proceeded slowly. The structures 1 and 8 () were not fully determined until 1970 [,,]. During the 1970s, additional sweet components, including rebaudiosides A–E, were isolated from S. rebaudiana leaves and characterized by Osamu Tanaka and co-workers at Hiroshima University in Japan []. Several novel steviol glycosides have been reported from the commercial extracts of the leaves of S. rebaudiana in the last few years [,,,,,,,,]. Recently we reported the structure elucidation and isolation of rebaudioside M from S. rebaudiana Bertoni (7) [,]. In addition, rebaudioside M received a Letter of No Objection concerning its Generally Recognized as Safe (GRAS) status from US FDA [].

2.1. Purification of Rebaudioside M

Rebaudioside M (7) was obtained from Stevia rebaudiana Bertoni cultivar AKH L1 leaves []. Two kilograms of S. rebaudiana leaves were extracted by 40 L water at 40 °C for two hours. The filtrate was separated and treated with a flocculant (calcium oxide) to remove the mechanical particles, proteins, polysaccharides and coloring agents. The resulting precipitate was separated by filtration and the filtrate was deionized by Amberlite FCP22 (H+) and Amberlite FPA53 (OH−) ion-exchange resins. The deionized filtrate was fractionated by seven columns, packed with Diaion HP20 (Mitsubishi Chemical, Japan). The columns with higher rebaudioside M content were eluted using aqueous ethanol. The obtained glycoside eluate was treated with activated carbon, deionized, evaporated and dried in vacuo. The obtained material was recrystallized twice from aqueous methanol to afford about 1.1 g of rebaudioside M with >98% purity by HPLC []. The highly purified stevia extract meets the international Joint Expert Committee on Food Additives (JECFA) purity requirement for steviol glycosides, which requires ≥95% total steviol glycosides [].

2.2. Properties of Rebaudioside M

Pure rebaudioside M (7) (>95%) crystallizes out as a crystalline form which is slightly soluble in water (0.1 g/100 mL at 25 °C in 5 min) and in ethanol. Its amorphous material has a solubility of 1.1%–1.3% in water at 25 °C. The thermodynamic equilibrium solubility in water is 0.26% at 25 °C [].

2.3. Stability of Rebaudioside M

As a dry powder, rebaudioside M (7) is stable for at least one year at ambient temperature and under controlled humidity conditions. In solution, it is most stable in pH 4–8 and noticeably less stable below pH 2. As expected, the stability decreases with increasing temperature. Its stability is very similar to rebaudioside A [].

2.3. Stability of Rebaudioside M

In aqueous solutions (pH 2–8), the major reaction pathways leading to loss of rebaudioside M are as follows (): isomerization of the C-16 olefin to form the C-15 isomer (12), hydration of the C-16 olefin to yield compound 13, hydrolysis of the glycosidyl ester at C-19 to form compound 2, and isomerization of the C-16 olefin to form the C-15 isomer in 2 to form compound 16. All of these compounds (2, 12, 13 and 16) are sweet. Compound 12 is sweeter than compounds 2, 13 and 16, and has similar taste properties as that of compound 7. Upon metabolism in vitro, rebaudioside M (7), just like rebaudioside A (1), is primarily converted to its aglycone steviol (14) and to the glucuronic acid of steviol, generally known as steviol glucuronide (15) [].

2.4. Sensory Panel for Rebaudioside M and Other Sweeteners

To determine the sensory attributes of rebaudioside M (7) we compared it to rebaudioside A (1), which possesses well defined characteristics. In addition, blending techniques with rebaudioside M and other sweetener were examined to demonstration the potential of this sweetener. Herein we describe our sensory panel testing. Concentration-response (C/R) function work was done with 60 in-house trained panelists (Two-Alternative Forced Choice, 2-AFC method), with the panelists specifying which of the two samples was perceptually sweeter. Sweetener blend work was done with 8–10 descriptive analysis (DA) panelists that have been highly trained. DA panelists go through 10–12 weeks of extensive descriptive training. The panel was calibrated at the beginning of each test session. In addition the panel also received anchor references representing early (sucrose), middle (aspartame) and late (thaumatin) AT every three test samples. Samples were given to the panel members sequentially and coded with triple digit numbers. The order of sample presentation was randomized to avoid the order of presentation bias. Water and unsalted crackers were provided in order to cleanse the palate. The panel members were asked to rate different attributes including sweetness onset, total sweetness, rounded sweetness, bitterness, acidity, leafy note, licorice, astringency, mouthfeel, mouth coating, sweet lingering, and bitter lingering. Samples were rated on a scale of zero (0) to ten (10), with zero indicating immediate onset, no intensity, watery/low viscosity, or very sharp peak, and ten indicating very delayed onset, high intensity, thick/high viscosity, or very round peak. One-way single factor ANOVA was used to analyze sensory results, where α = 0.05.

3.1. Rebaudioside M Sensory Attributes

In water solution, rebaudioside M (7) showed (A) the reduced perception of bitterness, astringency, bitter lingering compared to rebaudioside A (1), and similar sweetness intensity. A much higher sweetness perception of rebaudioside M than rebaudioside A in acidified water was established in B. Rebaudioside M displayed faster sweetness onset, reduced non-sweet taste (bitterness, sour, astringency) and bitterness lingering.

3.1. Rebaudioside M Sensory Attributes

To expand the descriptive taste profile of rebaudioside M (7) a set of trained panelist () examined 10% sucrose, aspartame at 531 mg/L, and rebaudioside M at 563 mg/L in water at 4 °C. Unlike other steviol glycosides including rebaudioside A (1), trained descriptive panel did not detect any significant bitter or licorice off taste when evaluated rebaudioside M in water at approximately 10% SE levels (). Rebaudioside M had a slightly more intense sweet aftertaste then aspartame. Based on our study rebaudioside M and aspartame have similar high potency sweetener profiles.

3.1. Rebaudioside M Sensory Attributes

With these samples in hand panelist examined the AT and ET of the three sweeteners described in . The sweetness temporal profiles of aspartame at 531 mg/L, rebaudioside M at 563 mg/L, and sucrose at 10% in water at room temperature were compared over a period of 3 min (samples were swallowed at 5 s). The AT maximum was the shortest for sucrose, slightly longer for aspartame and longest for rebaudioside M. The ET was longest for rebaudioside M, followed by aspartame and then sucrose.

3.2. Blending

To study the interactions between rebaudioside M (7) and other natural ingredients, blends were made with rebaudioside A (1), rebaudioside D (4), rebaudioside B (2), and erythritol at various concentrations in acidified water and sensory evaluation were performed.

3.2. Blending

illustrates the average response from sensory panelists that tasted the acidified solution with rebaudioside M (300 ppm) and rebaudioside A (100 ppm) or rebaudioside D (100 ppm) blends. The di-blends showed improvement in total sweetness, overall sweetness profile (peak), and leafy note. The blend with rebaudioside M and rebaudioside D showed higher improvement in sweetness intensity, overall sweetness profile, bitter lingering and sweet lingering.

3.2. Blending

The average response from sensory panelists that tasted the acidified solution with rebaudioside M and rebaudioside B blends (100 ppm or 50 ppm) is presented in . The rebaudioside M and rebaudioside B di-blend exhibited additional rounded sweetness profile with slight improvement in sweetness intensity, onset and bitterness perception.

3.2. Blending

The taste description of rebaudioside M and erythritol is displayed in based on the average response from sensory panelists that tasted the acidified solution di-blend. The blend with erythritol helps in reducing acidity, bitterness, astringency and bitter lingering. At a higher level (above 1%, 100 ppm) erythritol contributes additional sweetness, rounded sweet profile and earlier onset.