Stevia- life without sugar

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EXTRACTION OF STEVIA GLYCOSIDES WITHCO2 + WATER, CO2 + ETHANOL, AND CO2 +

WATER + ETHANO

A. Pasquel1,2, M.A.A. Meireles1*, M.O.M. Marques3 and A.J. Petenate4

1LASEFI, Departamento de Engenharia de Alimentos (FEA), Unicamp, Cx. Postal 6121,13083-970

Campinas - SP, Brazil Phone: 55 19 788-4033; Fax 55 19 788-4027,E-mail:[email protected];

2Departamento de Ingeniera de Alimentos, Universidad Nacional de laAmazona Peruana, Iquitos, Peru.

3LPN, Centro de Gentica, Biologia Molecular e Fitoqumica,Instituto Agronmico, Campinas - SP, Brazil

4

Departamento de Estatstica (IMECC), Unicamp, Cx.Postal 6065, 13083-970 Campinas, SP - Brazil

(Received: March 29, 2000 ; Accepted: April 23, 2000)

Abstract - Stevia leaves are an important source of natural sugar substitute. There are

some restrictions on the use of stevia extract because of its distinctive aftertaste. Someauthors attribute this to soluble material other than the stevia glycosides, even though itis well known that stevia glycosides have to some extent a bitter taste. Therefore, thepurpose of this work was to develop a process to obtain stevia extract of a better quality.The proposed process includes two steps: i) Pretreatment of the leaves by SCFE; ii)Extraction of the stevia glycosides by SCFE using CO2 as solvent and water and/orethanol as cosolvent.The mean total yield for SCFE pretreatment was 3.0%. The yields for SCFE withcosolvent of stevia glycosides were below 0.50%, except at 120 bar, 16C, and 9.5%(molar) of water. Under this condition, total yield was 3.4%. The quality of the glycosidicfraction with respect to its capacity as sweetener was better for the SCFE extract ascompared to extract obtained by the conventional process. The overall extraction curves

were well described by the Lack extended model.Keywords: stevia, supercritical extraction, mass transfer, stevioside, rebaudioside-A,glycoside, cosolvent, water, ethanol.

INTRODUCTION

In 1900, the Paraguayan chemist Ovidio Rebaudi, after whom Bertoni named the plant,studied the major characteristics of stevia. He succeeded in isolating two types ofsubstances: one extremely sweet and the other bitter, resembling a digestive appetizer.Of the two, it was the sweetening principle that attracted more attention at that time, as
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is still true today. The Stevia rebaudiana Bertoni contains a complex mixture of labdanediterpenes, triterpenes, stigmasterol, tannins, volatile oils, and eight diterpenenicglycosides: stevioside, steviobioside, dulcoside, and rebaudiosides A, B, C, D, and E. Themost abundant substances are stevioside and rebaudioside A. Of the stevia glycosidesrebaudioside A is the sweetest and the most stable, and it is less bitter than stevioside.Rebaudioside E is as sweet as stevioside, and rebaudioside D is as sweet as rebaudioside

A, while the other glycosides are less sweet than stevioside (Cramer and Ikan, 1987).

A combined process involving a solid/liquid extraction step, followed by a liquid/liquid-purifying step, is traditionally used to extract the glycosides from stevia. However, theglycosidic extract has a pronounced bitter aftertaste that is responsible for many of therestrictions on the use of stevia as a sweetener. There are several hypotheses in regardto the source of the bitter aftertaste of stevia glycosides. Phillips (1987) described aEuropean patent held by the Stevia Company, which attributes the bitter aftertaste tothe presence of essential oils, tannins, and flavonoids. Soejarto et al. (1983) believedthat the sesquiterpene lactones are responsible for the bitter aftertaste. Tsanava et al.(1991) suggested that caryophyllene and spathulenol contribute decisively to theaftertaste. Nevertheless, as pointed out by Phillips (1987), stevioside and rebaudioside A

are partially responsible for the aftertaste, even though the contribution of rebaudiosideA is significantly less than that of stevioside.

Tan et al. (1988) hold a Japanese patent for the production of stevia glycosides bysupercritical fluid extraction (SCFE) with CO2 and a cosolvent. Methanol, ethanol, andacetone were used as cosolvents. The purification step is accomplished by adsorption.Kienle (1992) holds a similar patent in the USA. Pasquel et al. (1999) studied the SCFEof the nonglycoside fraction of stevia leaves. The following substances were identified inthe extracts: spathulenol; decanoic acid; 8, 11, 14-ecosatrienoic acid; 2-methyloctadecane; pentacosane; octacosane; stigmasterol; -sitosterol, - and -amyrine;lupeol; -amyrin acetate; and pentacyclic triterpene. These substances represent 56%of the total extracts; therefore, 44% of the substances present in the extract still need

to be identified.

All the conventional extraction processes described in the literature follow a similarmethodology (Phillips, 1987). The stevia leaves are extracted with hot water or alcohols.In some cases, the leaves are pretreated with nonpolar solvents such as chloroform orhexane to remove the essential oils, lipids, chlorophyll, and other nonpolar substances.The extract is clarified by precipitation with salt or alkaline solutions. The extract isconcentrated and redissolved in methanol for crystallization of the glycosides. Thecrystals are formed almost by pure stevioside.

Using the information just discussed, the objectives of the present work were to producea stevia sweetener employing a two-step process: i) pretreatment of stevia leaves by

SCFE with CO2 and ii) extraction of the stevia glycosides by SCFE using the followingmixtures: CO2 + water, CO2 + ethanol, and CO2 + water + ethanol. Using informationfrom Pasquel et al. (1999) pretreatment conditions were set at 200 bar and 30oC. Theglycosides were obtained at 120 and 200 bar at 16, 30, and 45oC. The composition ofthe SCFE glycosidic extract was compared to the composition of stevia extract obtainedby conventional low-pressure extraction.

MATERIAL AND METHODS

The Raw Material


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Stevia leaves from the crop of 1995 were bought in Maring (Paran, Brazil). The solidmaterial was cleaned, selected, packed in plastic bags, and stored at room temperature(20to 32oC). The humidity of the raw material was determined using the toluenedistillation method (Jacobs, 1973). The glycoside content was determined according tothe phenol sulfur method for total carbohydrates (Alvarez et al., 1986).

Particles and Bed Characterization

The real density of the stevia particles was determined by picnometry with gas helium(Multivolume Picnometer 1305) at the Central Analtica, IQ Unicamp. Apparent densitywas calculated from the mass used to fill the extraction cell. Bed porosity was definedusing the real density of the particles and the apparent density of the bed. The meandiameter of the particles was evaluated using the methodology described by Corra(1994).

The Experimental Unit For the SCFE

The experimental unit used was that described by Pasquel et al. (1999) for thepretreatment of stevia leaves. A cosolvent pump was added to the system (Figure 1)

Experimental Procedure: SCFE

The mass of solid used varied from 69.10-3 to 82.10-3 kg. The triturated solid was packedinside the extraction cell (SS 316, with a length of 0.375 m and an inside diameter of
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0.0283 m). The extraction cell was adapted to the SCFE unit and the heating and/orcooling system was turned on. Once the system reached a temperature of 30oC(approximately 3 hours), valves 2a, 2b, 2c, and 2h were opened. As soon as the systempressure reached 200 bar, valves 2j, 2m, and micrometering valve 15 were opened. Theextracts were collected in 20mL glass flasks. An adsorption column containing Porapak Q(80 /100 mesh, Waters Associates Inc., USA) was adapted to prevent losses of volatile

substances in the pretreatment step at the solvent outlet. The solvent flow rate wascontinuously monitored. Samples of the extract were collected every hour. Pretreatmentwas carried out at 200 bar, 30oC, and an average solvent flow rate of 4.82.10-5 kg/s fora period of 12 hours. The extraction cell containing the pretreated stevia leaves wasstored in a domestic refrigerator.

For extraction of the glycosides, the extraction cell was readapted in the SCFE unit. Theexperimental procedure was similar to the one described above. Samples of the extractwere collected every 30 minutes and the total extraction time was 12 hours. Theexperimental runs were conducted at 120 and 200 bar at 16, 30, and 45oC. Thecosolvents used were 9.5% (molar) water, ethanol, or an equimolar mixture of waterand ethanol. Because the experiments were very long (12 hours for the pretreatment,

12 hours for the glycoside extraction plus setup time), the experimental plan was afractional factorial design and only one-third of the total was selected.

Experimental Procedure: Conventional Extraction

Stevia leaves subjected to the SCFE pretreatment and stevia leaves with nopretreatment were used. The method described by Alvarez and Couto (1984) and Goto(1997) was used. One liter of boiling water was added to fifty grams of stevia leaves.The infusion was kept at room temperature (25 to 30oC) for one hour. The aqueousextract was vacuum filtered. In a separation funnel the aqueous extract was mixed withisobutyl alcohol (Merck P.A., 99.99%) maintaining the 40:60 (v/v) proportion. Thesystem was allowed to rest until complete phase separation was achieved. The butanolic

extract was centrifuged at 3500 rpm (Solvall, RT 600D) for 15 minutes. The extract washeated up to 80oC, and percolated through a bed of activated carbon (1 g of activatedcarbon for every 100 mL of extract). The extract was concentrated in a rota-evaporator(Tecnal, TE 120) and allowed to rest for 24 hours to achieve crystallization of theglycosides. The crystals were washed with methanol (Merck P.A. 99.9%) and dried in anair-circulating oven. The crystallization mother liquor was concentrated and extractedwith acetone (Merck P.A., 99.8%). The crystals were washed with anhydrous acetoneand dried in an air-circulating oven.

Analysis of the Glycosidic Extracts

(a) Identification of the Glycosides

The preliminary identification of the stevioside in the extracts was made by thin-layerchromatography (TLC) using silica gel plates (Merck, lot PF254336) and the extractswere eluted with chloroform:methanol:water (30:20:4). The spots were developed byspraying with methanol:sulfuric acid (1:1) and heating to 110oC. The extracts werediluted with ethanol and hexane. The standard stevioside (95%, Steviafarma Industrial,Maring, Paran, Brazil) was diluted in the elution solvent. The chloroform, methanol,sulfuric acid, ethanol, and hexane were from Merck and were of chromatographic grade.

Identification of the glycosides was accomplished by HPLC (CG Instrumentos Cientficos,Model CG-480C) with a UV detector (Jasco, model 970 UV) at a wavelength of 210 nm,using a NH2 Licrosorb column (5 m, 220 x 4.6 mm, Technology Techsphere).Acetonitrile and methanol (85:15) (Merck, HPLC grade) were used as mobile phase at aflow rate of 1.5 mL/min. Stevioside and rebaudioside A (95% and 85%, respectively,


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from Steviafarma Industrial, Maring, Paran, Brazil) were used as standard. Theanalyses were performed in the Chemical Engineering Department, UEM, Paran.

(b) Quantification of the Glycosides

The aqueous extracts were concentrated and redissolved in water at knownconcentrations. The calibration curve was made using a commercial sample of stevioside(95% from Steviafarma Industrial, Maring, Paran, Brazil) diluted in water.Quantification of the glycosides was done using a spectrophotometer (Hitachi, model U-2000, double bean) at a wavelength of 210 nm (Nikolova-Damyanova et al., 1994).

Calculation of the Kinetic Parameters

The experimental data on mass of extract as a function of time of extraction were usedto calculate the kinetic parameters for the constant extraction rate period (CER): themass transfer rate, MCER; the duration of the CER period, tCER; the mass ratio of solute inthe fluid phase at the bed outlet, YCER, and the yield (mass of extract/mass of feed),

RCER. The methodology of Rodrigues et al. (2000) was used.

RESULTS AND DISCUSSION

The humidity of the stevia leaves was 7.0 0.1%. The real density of the leaves was1370 30 kg/m3 and the bed apparent density was 400 20 kg/m3. The average particlediameter was (0.98 0.03).10-3 m. The mean bed porosity was 0.77 0.01. The glycosidecontent of the leaves was determined as 5.0 0.1% and was small compared to valuesfound in the literature which vary from 7.8 to 14.5% (Phillips, 1987). This can beexplained by the fact that besides the leaves the raw material also contained some

stevia flowers.

SCFE Pre-Treatment of the Stevia Leaves

Figure 2shows the overall extraction curves for the pretreatment step.Table 1presentsthe kinetic parameters for the CER period. The average rate of mass transfer was MCER =(7.9 0.7).10-8 kg/s, the average mass ratio in the fluid phase was YCER =(1.6 0.2).10

-

3 kg-extract/kg-CO2, and the average CER and total yields were RCER = 1.7 0.8% andRTOTAL = 3.0 0.1%. About 63% of the total yield was obtained during the CER period.
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It is well known that a typical overall extraction curve for SCFE from a solid substratumhas three distinctive steps (Frana et al., 1999): i) the constant extraction rate period(CER), characterized by the predominance of convective effects; ii) the falling ordecreasing rate period (FER), for which both convective as well as diffusional effects areimportant; and iii) the diffusion-controlled rate period that is characterized by thediffusion of solvent and the solute/solvent mixture in the solid substratum. InFigure 2,

these three periods can be easily identified; nevertheless, the asymptotic behaviorexpected for the third step was not achieved. This can be explained in terms of thecomposition of the extract obtained in the pretreatment (Pasquel et al., 1999).

SCFE of the Lycosides From Stevia Leaves

Ethanol, water, and an equimolar mixture of these two solvents were chosen ascosolvents. Ethanol was selected based on legal restrictions on the residual amount oforganic solvents present in inputs for the food industry and also on information given byKienle (1992) that attributes an increase in extraction yield to alcohol of up to 4 carbonsused as cosolvents. Water was chosen due to its capability of solubilizing rebaudioside A,the sweetest stevia glycoside with the least aftertaste.Table 2shows the kinetic

parameters for SCFE of stevia glycosides. Because the fractional factorial design hadthree levels of temperature and cosolvent, and two levels of pressure, only sixexperiments were done; thus, no analysis of variance was performed.Figure 3showsthat the overall extraction curves for the SCFE of glycosides have the expected behavior.The yields were in general very low, except for the assay using water as cosolventcarried out at 120 bar and 16oC (Figure 4).
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The results indicated a possible combined effect of temperature, pressure, andcosolvent. In fact, the solvation power of a solvent or a solvent mixture is directlyrelated to mixture polarity as well as to intermolecular interactions of the followingkinds: solvent/cosolvent, solvent/solute, and cosolvent/solute (McHugh and Krukonis,1994). Water is highly polar compared to ethanol and has a larger dipole moment;therefore, its molecules should appreciably increase the CO2 polarity, resulting in animportant increase in glycosides solubility. In addition, the increase in temperaturenegatively affects the attractive forces such as dipole-dipole between water and

glycoside molecules (McHugh and Krukonis, 1994). This explains the behavior of thesystem at 120 bar and 16oC: The rate of mass transfer, MCER, was up to 35 times higher


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than that for the other experiments. A similar behavior was observed for YCER.Nevertheless, no pattern could be drawn for tCER. The yield (0.12%) for the assay at16oC using ethanol as cosolvent and carried out at 65 bar was smaller than that for theother two experiments using ethanol as cosolvent, indicating the combined effect oftemperature and pressure. Comparison of the yields for the experiments using water ascosolvent showed that regardless of whether it was used with or without ethanol, water

definitively increased the solubility of the glycosides.

Yields for the conventional process were 2.60 0.01% for pre-treated leaves and2.10 0.01% for stevia leaves with no pretreatment. These yields were appreciablysmaller than those obtained for SCFE using water as cosolvent. The behavior of thepretreated leaves was as expected, since during pretreatment several substances wereremoved and therefore did not get the chance to compete with the glycosides for thesolvent during glycoside extraction.

The Composition of the Extracts

The thin-layer chromatography showed that the pretreatment extracts had noglycosides. The SCFE glycosidic extracts were quantified by UV spectrophotometry asstevioside. Nevertheless, the HPLC analysis showed that the extracts were indeed amixture of stevioside and rebaudioside A.

Comparison of the chromatogram for the SCFE glycosidic extract using water ascosolvent with the glycosidic extract obtained by conventional extraction from pretreatedstevia leaves (200 bar, 30oC) clearly showed the larger amount of rebaudioside Aobtained by SCFE. For the assay at 120 bar, 16oC and 9.5% (molar) of water, therelationship between stevioside/rebaudioside A was approximately 3 to 1 (Table 3). Thisresult corroborates the fact that the CO2+water mixture is capable of extracting largeramounts of rebaudioside A than the conventional process using water and organicsolvents. Considering that rebaudioside A is about 50% sweeter than stevioside and hasless aftertaste, the glycosidic extract obtained is a much better sweetener than thestevia extract produced by the conventional process.

Modeling the Mass Transfer for the Pretreatment Step

Among the various models presented in the literature to describe SCFE from a solidsubstratum, the extended Lack model developed by Sovov (1994) was chosen. Themodel was selected based on the shape of the overall extraction curves that shows a

predominance of the constant extraction rate period. As discussed before, during theCER period as much as 63% of the extract was obtained (Table 1). The model developed
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by Sovov (1994) assumes that the solvent flows axially at a constant superficialvelocity through a fixed bed of cylindrical shape. The solvent is pure at the entrance ofthe extractor; temperature and pressure are kept constant throughout the fixed bed. Inaddition, the bed is homogeneous in terms of solute distribution as well as particle size.All these conditions were fulfilled by the SCFE from the bed of stevia. Therefore, themass balance for a bed element is given by

Solid Phase:

(1)

Fluid phase:

(2)

where X and Y are the solute mass ratio for the solid and fluid phases, respectively; t isthe time (s); is the bed porosity; U is the superficial solvent velocity(m/s); s and fare the solid and fluid phase densities respectively, and J(X,Y) is therate of interfacial mass transfer. Usually, for the SCFE process the fluid phase is a diluted

solution; therefore, the solvent density, , can adequately be used as f.

The solution to the system ofError! Reference source not found. and (2 given bySovov (1994) is described by the following system of equations:

For the CER period, t < tCER

(3)

For the falling extraction rate period (FER)

(4)

For the diffusion-controlled rate period, t tFER

(5)

with the following restrictions:

(6)


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(7)

(8)

(9)

(10)

where mext is the mass of extract (kg), N is the mass of inert solid (kg), Y* is theoperational solubility (kg/kg), kYa is the fluid-phase mass transfer coefficient (s

-1), kXa isthe solid-phase mass transfer coefficient (s-1), tFER is the duration of the falling extractionrate period (s), X0 is the initial solute mass ratio in the solid phase, and Xk is the solutemass ratio for the unruptured cells in the solid phase.

To use the model it is necessary to know the model parameters. Several authors haveobtained the parameters by directly fitting the overall extraction curves to the modelequations. This procedure is very helpful if experimental data, including operationalsolubility data, are available. A database for SCFE from a solid substratum is still beingbuilt; therefore, the required data can be found in the literature for only a few systems.We will now discuss a slightly different approach to evaluating model parameters, based

on the theory of similarity used over the years in other areas of process engineering. Themethodology used data from the literature and just one overall extraction curve toevaluate the parameters required to test the model proposed by Sovov (1994). kYaandY* were calculated using the kinetic and operational solubility data measured byMonteiro (1999) for the ginger/CO2 system in the same equipment used to obtain thedata for the present work. The model parameters were evaluated under the assumptionthat there was a close similarity between the system of interest and a reference systemfor which experimental data were available. Therefore, the systems should be similarhydrodynamically as well as thermodynamically. The hydrodynamic similarity wasguaranteed since both systems were studied in the same equipment. Thethermodynamic similarity was a bit more difficult to achieve. Nevertheless, the amountof CO2 soluble material in ginger (3%) is approximately equal to the CO2 soluble material

in stevia leaves. Thus, the hypothesis of thermodynamic similarity was assumed to bevalid. Therefore, the fluid-phase mass transfer coefficient for the stevia/CO2 system was


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assumed to be equal to that of the ginger/CO2 system. The ginger/CO2 fluid-phase masstransfer coefficient was calculated using the following definition:

(11)

where S is the cross-sectional bed area (m2), H the bed length (m), the solventdensity (kg/m3), and Y the average logarithmic mean for the solute mass ratio in thefluid phase (Ferreira et al., 1999):

(12)

Yin and Yout are the solute mass ratio at the bed entrance and exit, respectively. The dataof Monteiro (1999) measured at 200 bar and 30oC were used. For the kinetic

experiments, the solvent flow rate ranged from 4.82.10-5

to 4.98.10-5

kg/s. while for theoperational equilibrium experiments the solvent flow rate was 1.67.10-5kg/s. Bedporosity varied from 0.76 to 0.74 and mean particle diameter from 0.98.10-3 to 1.02.10-3 m. The ginger/CO2 mass transfer coefficient was calculated as KYa 3.019.10

-4 s-1. Usingthe mean value for MCER(Table 1), the operational solubility of the stevia extract inCO2 was estimated as Y* = 2.27.10

-3 kg/kg.

The solid-phase mass transfer coefficient was evaluated using the following definition:

(13)

where

(14)

X* is the mass ratio of solute to the inert solid in equilibrium with Y*. XP is the massratio of easily accessible solute in the solid phase, and Xk is the mass ratio of soluteinside the cells in the solid phase, as defined by Sovov (1994). The initial solute content

was estimated from the highest yield for the pretreatment.Table 4presents the values ofthe model parameters used to simulate the overall extraction curve shown inFigure 5.The model systematically overestimates the overall extraction curves for the CER period.In spite of that, the overall yield of the process is only marginally different from theexperimental value. Therefore, the procedure described here can be used to get first-hand information for projects of SCFE systems. Pasquel (1999) estimated the solubilityof the stevia extract in CO2, using the Sherwood number calculated by Monteiro (1999).Using the fluid-phase mass transfer coefficient of Monteiro (1999) he fitted the overallextraction curve to the model discussed by Sovov (1994). His results were better thosethe ones presented here mainly due to the fact that his solid-phase mass transfer wasobtained by fitting the experimental data to the model equations.
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CONCLUSIONS

The mean total yield for SCFE pretreatment of stevia leaves at 200 bar and 30oC was3.0% (m/m). About 63% of this total was obtained during the CER period. Conversely,yields for SCFE with cosolvent of stevia glycosides were below 0.50%, except at 120 bar,

16o

C, and 9.5% (molar) water. For this condition, the total yield was 3.40.3% and asmuch as 70% of the total glycosidic fraction was obtained during the CER period. The


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yields for the conventional process were approximately equal regardless of whetheruntreated or pretreated leaves were used. The quality of the glycosidic fraction withrespect to its capacity as a sweetener was better for the SCFE in terms of the relativeamount of stevioside and rebaudioside A. The overall extraction curves were welldescribed by the Lack extended model using operational solubility and the mass transfercoefficient estimated with data from the literature.

ACKNOWLEDGEMENTS

This work was financed by FAPESP under grant 95/05262-3. A. Pasquel thanks FAPESPfor the Ph. D. assistantship (95/03390-4). The authors also thank Silvania R. M.Moreschi and DEQ/UEM for their assistance in the glycoside analysis and SteviafarmaIndustrial for the stevioside and rebaudioside standards.

REFERENCES

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