Extraction of stevioside using aqueous two-phase systems formed by choline chloride and K3PO4

food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 107–115

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Extraction of stevioside using aqueous two-phase systems formed by choline chloride and K3 PO4 T. Abolghasembeyk a , Sh. Shahriari b,∗ , M. Salehifar a a b

Department of Food Science and Technology, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran Department of Chemical Engineering, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran

a r t i c l e

i n f o

a b s t r a c t

Article history:

Limitations and disadvantages of artificial sweeteners in the food and beverage products act

Received 30 September 2016

as an incentive for paying attention to the extraction and purification of natural sweeteners.

Received in revised form 9

Aqueous two-phase systems (ATPSs) are believed to be a desirable method for the extraction

December 2016

and separation of biomolecules. In recent years, researchers have focused on the use of

Accepted 15 December 2016

innocuous, benign components having potential to form ATPSs. In this research, choline

Available online 24 December 2016

chloride, as a biocompatible and nutritious constituent, has been utilized to establish an

Keywords:

of choline chloride and potassium phosphate, the partitioning of stevioside was explored.

ATPS performing the extraction of stevioside. To assess the efficiency of the ATPS composed Aqueous two-phase systems

The effects of such parameters as the weight percents of choline chloride and potassium

Choline chloride

phosphate on the partitioning of stevioside were studied. All experiments were conducted at

Partitioning

four temperatures of 298 K, 303 K, 308 K, and 313 K. Also, the effect of pH on the partitioning

Extraction

of stevioside was investigated. Different regression models were adopted to correlate the

Stevioside

empirical results of the stevioside partition coefficient, and through statistical analyses the

Separation

most reliable regression model was chosen. © 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1.

Introduction

Advanced science of biotechnology has presented a new generation of natural sweeteners which can be widely utilized in industrial as well

In food industries, the first, and the most important deterrent to the use of natural sweeteners is their rather steep price. Separation and purification of biomolecules, which are considered as downstream processes, are costly and demanding. The downstream costs include

as domestic applications. One of these natural sweeteners is stevioside which is extracted from a plant called stevia, and is currently available for commercial use. Drawing considerable attention recently, stevio-

80% of the total costs for the purification of biomolecules (Freire et al., 2012; Lebovka et al., 2011; Waites et al., 2009). Since stevioside has numerous applications in food industry, phar-

side is regarded as one of the natural and well-known sweeteners in the food and pharmaceutical industries. Stevioside is said to be 300 times sweeter than beet-derived sucrose. This sweetener is comprised

maceutical and medical fields, its purity must be very high so as to meet

of complex molecules besides the building blocks of glucose (Geuns, 2003; Kroyer, 1999).

icance. The adopted method must be fast, selective, and must possess the ability to function economically on a large scale.

Known as a no-calorie sweetener, stevoside is not absorbed in the digestive system. Compared to other artificial sweeteners, it has sev-

major problems related to the extraction and separation of

eral other benefits such as lack of carcinogenic effects, non-toxicity, potential impact on reducing the obesity and the high blood pressure,

biomolecules. For this reason, researchers and industrialists constantly seek for methods which are suitable and sustainable. In 1950, Albertsson (1986) suggested the use of ATPSs as an alterna-

and marginal effect on blood glucose. Since the artificial sweeteners are suspected to be carcinogenic, the natural stevioside takes on economic importance in the production of beverages, sweet breads, and dairy (Kroyer, 1999; Stoyanova et al., 2011).

their strict standards. Therefore, the choice of a suitable method for the separation and purification in downstream processing is of great signif-

Food and pharmaceutical industries are frequently faced with

tive for the biphasic systems containing conventional organic solvents. Using the aqueous two phase systems (ATPSs), proposed in recent decades, are believed to be the most effective technique for the extraction and separation of biomolecules in one stage. This method seems to

∗

be selective and appropriate for the continuous processes on an industrial scale as well. ATPSs have no harmful impacts on the structure of

Corresponding author. biomolecules, and as reflected by the literature, have many advantages E-mail address: shahla [email protected] (Sh. Shahriari). http://dx.doi.org/10.1016/j.fbp.2016.12.011 0960-3085/© 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 107–115

The aim of this study is to assess the partitioning of stevioside in the ATPSs containing [Ch]Cl and tripotassium phosphate. The partition coefficient and recovery percent of stevioside have been taken into consideration to evaluate the extent of applicability of these favorable two-phase systems. Aiming at determining the most optimal conditions for the separation of the stevioside, the effects of the weight percent of [Ch]Cl, the weight percent of tripotassium phosphate, pH, and four different temperatures of (298 K, 303 K, 308 K, and 313 K) on the partition coefficient of stevioside have been investigated. The optimal recovery of stevioside in the bottom phase was determined in terms of the volume ratio of phases.

Fig. 1 – Chemical structure of stevioside.

2.

Materials and methods

over the other conventional extraction approaches (Freire et al., 2010; In et al., 2005; Wu et al., 2014).

2.1.

Materials

Researchers have shown that separation of a whole range of biomolecules such as pigments (Mageste et al., 2009; Wu et al., 2011), viruses (Liu et al., 1998), enzymes (Sarangi et al., 2011; Shahriari et al.,

Choline chloride and tripotassium phosphate, with a purity of 99%, were purchased from Alfa Aesar Company. Stevioside (>98%) was procured from Sigma-Aldrich Company, USA. Distilled water was provided by RO-LAB, DW65 equipment utilizing twice distillation reverse osmosis. The molecular structure of stevioside has been illustrated in Fig. 1.

2010), proteins (Li et al., 2012) and antibiotics (Shahriari et al., 2012; Shahriari et al., 2013) through ATPSs is quite practicable. ATPSs are formed by mixing two immiscible phases such as two hydrophilic polymers or a hydrophilic polymer and a strong salt (Johansson et al., 1998; Li et al., 2005; Walter, 2012). Polymer-based ATPSs suffer from such drawbacks as high viscosity and slow phase separation. Gutowski et al. (2003) for the first time, reported the possibility of the ATPSs formation using mineral salts and ionic liquids. Ionic liquids are categorized as salts that are liquid in lower temperatures and can be a good substitute for organic solvents in separation processes. Finding agents that are safer and cheaper than ionic liquids, with similar characteristics, is still a bottleneck. Because of the sensitivity of biomolecules and their applications, particularly in the food and pharmaceutical industries, certain restrictions on the ATPS ingredients should be set. Recently published articles introduced a new class of salts having the ability to overcome these limitations. The cholinium-based salts are promising candidates and seem to be viable alternatives to conventional salts used for the preparation

2.2.

Methods

2.2.1.

Phase diagrams and tie-lines

In this study, the phase diagram was obtained using the cloud point titration method at a temperature of 298 K (±1 K) and the ambient pressure in accordance with our previous work (Pourebrahimi et al., 2015). The experimental data of the binodal curve were correlated according to the following equation proposed by Merchuk et al. (1998). [ChCl] = A exp[(B ∗ [K3 PO4 ]0.5 ) − (C ∗ [K3 PO4 ]3 )]

(1)

of ATPSs (Pereira et al., 2013b; Shahriari et al., 2013). Cholinium chloride (choline chloride) was discovered by Adolph Strecker who separated the platinum salts from the pig bile (Strecker, 1862). He showed that choline is an integral part of the egg yolk lecithin. Choline is classified as a vitamin and belongs to the B-group vitamins, which acts similar to amino acids or essential fatty acids. Choline chloride ([Ch]Cl) is a tetravalent ammonium salt [(2-hydroxyethyl) trimethylammonium chloride, or vitamin B4 ] which is an essential nutrient for healthy growth of animals, particularly poultry, pigs and pets. Thanks to its role in the human organism, [Ch]Cl is also a pharmaceutical ingredient, as confirmed by the Institute of Medicine in 1998 (Zeisel and Da Costa, 2009). Its proven benefits are: ease of preparation, greater stability in water and air, biocompatibility, biodegradability, and being relatively cheap and more hydrophilic compared to other cholinebased salts (Shahriari et al., 2013). Also, Shahriari et al. showed that the viscosity of choline-chloride-rich phase is lower than that of corresponding phases with other choline-based salts. The lower viscosity benefits mass transfer and makes the phase separation procedure easier when it comes to scaling-up and industrial extraction (Shahriari et al., 2013). A review of the previous works reveals that a few papers on the use of [Ch]Cl-based ATPS have been published, aimed at extracting and separating biomolecule (Liu et al., 2013; Pereira et al., 2014). For instance, Freire et al. (2010) pointed out the safety of these systems for the extraction of the antibiotics. Hydrophilic choline chloride together with tripotassium phosphate are practical options to stablish an ATPS. It is worth noting that this salt is readily miscible in the aqueous phases (Zempleni et al., 2007).

where [ChCl] and [K3 PO4 ] are the choline chloride and the tripotassium phosphate weight percentages, and A, B, and C are constants which can be found by the regression of the experimental binodal data (Pourebrahimi et al., 2015). The tie lines (TLs) were measured using a gravimetric method presented by Merchuk et al. (1998). Also, the TLs were calculated by the application of a mass balance along with the data of phase diagram related to Eq. (1). The mixture concentrations of [Ch]Cl and potassium phosphate are determined by their corresponding values in the top and bottom phases, adopting the lever-arm rule (the mass balance expression). To determine each TL, the following four equations are solved: [ChCl]T = A exp[(B ∗ [K3 PO4 ]T 0.5 ) − (C ∗ [K3 PO4 ]T 3 )]

(2)

[ChCl]B = A exp[(B ∗ [K3 PO4 ]B 0.5 ) − (C ∗ [K3 PO4 ]B 3 )]

(3)

 [ChCl]T =

[K3 PO4 ]T =

[ChCl]M ˛

   1−˛ −

˛

× [ChCl]B

 [K PO ]   1 − ˛  4 M 3 ˛

−

˛

× [K3 PO4 ]B

(4)

(5)

Tripotassium phosphate is a strong, inexpensive salt which is frequently used in food processing. Thanks to its great potential for salting-out, it has drawn a lot of attention in the field of ATPSs (Shahriari et al., 2013).

In the above equations, B, T, and M refer to the bottom phase, the top phase, and the mixture, respectively. ˛ is the ratio between the bottom phase mass and the total mixture mass.

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food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 107–115

To calculate the tie line length (TLL), the following equation has been employed:

 2

TLL =

([K3 PO4 ]T − [K3 PO4 ]B ) + ([ChCl]T − [ChCl]B )

2.2.2.

Partitioning of stevioside

2

(6)

The experimental method for the measurement of the partitioning of biomolecules in ATPSs was described in other papers (Pereira et al., 2013a). An aqueous solution of stevioside was prepared with a concentration of 0.008 g L−1 , which in turn was mixed with specified concentrations of [Ch]Cl and tripotassium phosphate, and thoroughly stirred. The mass of components was weighed using a digital balance (with an accuracy of ±10−4 g). After 30 min, the mixture was left at room temperature without stirring for 24 h in order to make sure that the equilibrium conditions were attained. The temperature of the container was controlled in an incubator (Memmert, Germany) with an accuracy of ± 0.01 ◦ C. After reaching the perfect equilibrium and observing the two distinct phases, the top phase was discharged by means of a syringe to the point of the interface of two phases. Then, each phase was poured into the lidded plastic containers so as to be later analyzed. For the purpose of finding the stevioside concentration, the absorbance of stevioside in the top and bottom phases was determined through spectrophotometry (UV/vis Model: sp-2100uv, USA) at a wavelength of 208 nm where the maximum absorption of stevioside occurred. Subsequently, the concentration of stevioside in both phases were specified by the calibration curve. The following equation was employed to calculate the stevioside partition coefficient represented by the ratio of the stevioside concentration in [Ch]Cl-rich phase (top phase) to its concentration in salt-rich phase (bottom phase) (Pereira et al., 2013a): KStev

[Stev]ChCl = [Stev] K3 PO4

(7)

where KStev is the partition coefficient of stevioside, and [Stev]ChCl and [Stev]K3 PO4 are, respectively, stevioside concentration in the [Ch]Cl-rich phase and the salt-rich phase. The recovery percentage of stevioside in the bottom phase was obtained applying the following equation (Ebrahimi and Shahriari, 2016): [Stev]B RB = [Stev]T + [Stev]B

(8)

In this equation, RB is the recovery percentage of stevioside in the bottom phase, [Stev]T is the concentration of stevioside in the top phase, and [Stev]B is the concentration of stevioside in the bottom phase.

2.2.3.

pH determination

The pH measurement of the top and bottom phases was performed by a digital pH meter (AZ-86502, AZ instruments) with an accuracy of ±0.02. The pH meter was calibrated with the help of two buffer solutions having pH values of 4.00 and 7.00. The adopted compositions were similar to those utilized in the partitioning experiments. The preparation of mixtures was carried out gravimetrically within ±10−4 g. In order to adjust the pH of the systems, 85% food grade phosphoric acid (Sigma) was utilized.

2.3.

Statistical analysis

2.3.1.

Investigated variables

In order to obtain a proper pattern for the empirical procedure, statistical evaluation was carried out on the experimental data. On the basis of experimental results in this work, the independent and dependent variables have been defined. The temperature and the weight percent of the salt and [Ch]Cl were considered as the independent variables and the partition coefficient of steveioside was regarded as the dependent variable. The experiments have been accomplished in triplicate in the form of a completely randomized design.

2.3.2.

Regression analysis

Regression analysis is used for understanding scientific relations between a dependent variable and several independent variables. The experimental results were analyzed by the statistical software SPSS (16), and MATLAB (R 2008a) software. The different regression models were used for predicting the partition coefficient of stevioside. The optimal regression model was determined through minimizing the deviation (DEV%), coefficient of determination (R2 ) and root mean squared error (RMSE) defined by Eqs. (9–11), respectively: |KEXPT. − KMODEL | × 100 KEXPT.

DEV% =

N 

R2 =

2

(KiEXPT. − K) −

i=1

N 

(9)

(KiEXPT. − KiMODEL )

2

i=1 N 

(KiEXPT.

(10) − K)

2

i=1

⎡ ⎢ ⎢

RMSE = ⎢ ⎢

⎣

N 

⎤0.5 (KiEXPT.

2 − KiMODEL )

i=1

N

⎥ ⎥ ⎥ ⎥ ⎦

(11)

where N is the number of experimental data points; KiEXPT. is the ith experimental value of the partition coefficient; KiMODEL. is the partition coefficient estimated by the regression model and K is the mean of the experimental data.

3.

Results and discussion

3.1.

Experimental results

3.1.1.

Phase diagram, tie lines

To evaluate the phase behavior, the phase diagram, based on our previous work, was used for determining the concentration of the components of the two phases in equilibrium (Pourebrahimi et al., 2015). The phase diagram for the [Ch]Cl + K3 PO4 + H2 O system is shown in Fig. 2. The binodal curve was correlated with Eq. (1), and the respective parameters A, B, and C, along with the correlation coefficient (R2 ) have been reported in Table 1 (Pourebrahimi et al., 2015). To assess the TLL effect on partitioning, three points in the biphasic area of the phase diagram were chosen, which were related to the components concentration and the volume ratio of phases (VR ). Also, the affinity of biomolecule toward each phase could be altered by manipulating ATPS parameters such as TLL (as a function of phase composition) and the VR . Based

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food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 107–115

60

50

[Ch]Cl / wt%

40

30

20

10

0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

[K3PO4] / wt %

Fig. 2 – Phase diagram of the ATPS containing [Ch]Cl + K3 PO4 + H2 O at 298 K: () TL data; () binodal curve data; () critical point; (–) adjusted data through Eq. (1) (Pourebrahimi et al., 2015). Table 1 – Correlation parameters of Eq. (1) adjusted to the binodal experimental data (also correlation coefficients, R2 ) at 298 K (Pourebrahimi et al., 2015). Regression parameters A ± a

B ± a

K3 PO4 + [Ch]Cl + H2 O C ±  a (10−5 ) R2

163.700 ± 2.000−0.383 ± 0.0361.000 ± 0.2500.993 a

Standard deviation.

on our previous experience of [Ch]Cl + K3 PO4 + H2 O system, the following rules were adopted to determine the mixture composition (working point) in the biphasic area and plot the tie-lines (Pourebrahimi et al., 2015; Shahriari et al., 2013): (a) The working points were selected within the liquid–liquid area, not in the liquid–solid zone; (b) In order to facilitate phase separation, sampling, and partition coefficient determination, according to a general standard, the equal volumes of the top and bottom phases were put into use (Hatti-Kaul, 2000); (c) The phase separation time depends on the distance between the working point and the critical point. To achieve a shorter separation time, the working point was chosen in the middle. (d) If the volume of the more viscous phase (salt-rich phase) is greater than that of less viscous phase (upper phase), the phase separation time increases. The TLLs derived from experimental data together with the weight percents of the [Ch]Cl and K3 PO4 in the top and bottom phases are reported in Table 2. The unit of TLLs is the same as that of the components in the top and bottom phases, namely weight percent (wt%). An example of the TLs representation along with the correlation of the binodal data is presented in Fig. 2. Since the TLL implies the difference between the weight percent of [Ch]Cl and the weight percent of K3 PO4 in the top

and bottom phases, it is regarded as an essential parameter by which the properties of ATPS can be related to each other. In fact, it may serve as a standard to evaluate the difference between the two phases in equilibrium. The results reported in Table 2 indicate that for the ATPS with VR ≈ 1, the stevioside partition coefficient diminished as TLL increases. This behavior can be justified by a decline in [Ch]Cl concentration in the bottom phase, leading to enhanced interactions between salt and stevioside. Hence, the stevioside tends to migrate into the lower phase. The changes in [Ch]Cl and salt concentration influenced the stevioside partitioning and the TLL. Regarding the correlation between TLL and the system composition, the effect of changing the weight fraction of each component on the partitioning is investigated in the following to reach a deeper and more precise understanding.

3.1.2.

Stevioside partitioning

In conducting the experiments, a repeatable method was utilized with the aim of achieving the partition coefficient of stevioside in the [Ch]Cl + K3 PO4 + H2 O ATPS; In addition, this approach was adopted in order to study the influence of the effective parameters on the partition coefficient of stevioside, which was investigated by changing one parameter while keeping the other conditions constant. The weight fractions of salt and [Ch]Cl were selected in accordance with our previous experience and the general rules mentioned in the earlier section (3.1.1). The partition coefficients of stevioside were obtained using the ATPSs with the same volumes of top and bottom phases (VR ≈ 1). The partition coefficients of stevioside, presented as the mean value of three determinations, have been obtained together with the corresponding values of standard deviation which is the best statistical scale for analyzing the quantitative data scattering. In this study, the small values of the standard deviation confirm proper accuracy of the experimental data.

3.1.3. Effect of salt concentration on the stevioside partitioning A comparison has been made between the partition coefficients of stevioside in four different weight percents of potassium phosphate (25 wt%, 27 wt%, 29 wt%, and 31 wt%). For this purpose, the weight percent of [Ch]Cl was kept constant at 22 wt% in all experiments. The effect of the weight percent of potassium phosphate on the partition coefficient of stevioside at four different temperatures (298 K, 303 K, 308 K, and 313 K) is shown in Table 3. As it can be observed from the experimental data, stevioside prefers to stay in the bottom phase (K3 PO4 -rich phase). Therefore, the partition coefficient of stevioside is less than one (K < 1). The results show that the weight percent of K3 PO4 has a significant effect on the partition coefficient of stevioside; that is, with an increase in the concentration of tripotassium phosphate, the partition coefficient of stevioside decreases at each of the four temper-

Table 2 – Weight fraction composition (wt%) for the top (T) and bottom (B) phases, initial mixture composition (M), partition coefficient (K) and respective TLLs for the system composed of [Ch]Cl + K3 PO4 + H2 O at 298 K and atmospheric pressure. Weight fraction (wt%) [Ch Cl]M

[K3 PO4 ]M

[Ch Cl]T

[K3 PO4 ]T

[Ch Cl]B

[K3 PO4 ]B

21.981 22.023 22.117

24.986 26.974 28.914

30.932 49.149 52.029

17.689 9.719 8.849

13.839 12.786 9.205

31.631 32.851 37.576

TLL

˛

22.057 43.098 51.568

0.477 0.254 0.301

K

0.132 0.131 0.127

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Table 3 – Effect of K3 PO4 on the stevioside partitioning (K) at different weight percents of [Ch]Cl. wtChCl %

22

a

K ± a

wtK3 PO4 %

25 27 29 31

T = 298 K

T = 303 K

T = 308 K

0.132 ± 0.020 0.131 ± 0.002 0.127 ± 0.009 0.125 ± 0.000

0.266 ± 0.01 0.164 ± 0.004 0.161 ± 0.010 0.157 ± 0.000

0.676 ± 0.016 0.663 ± 0.009 0.639 ± 0.006 0.619 ± 0.007

T = 313 K 0.761 ± 0.002 0.752 ± 0.010 0.701 ± 0.006 0.670 ± 0.004

Standard deviation.

Table 4 – Effect of [Ch]Cl on the stevioside partitioning (K) at different weight percents of K3 PO4 . wtK3 PO4 %

K ± a

wtChCl % T = 298 K

30

a

24 26 28

0.117 ± 0.007 0.121 ± 0.001 0.138 ± 0.003

T = 303 K 0.222 ± 0.06 0.230 ± 0.005 0.233 ± 0.001

T = 308 K 0.235 ± 0.002 0.240 ± 0.03 0.247 ± 0.004

T = 313 K 0.242 ± 0.001 0.246 ± 0.004 0.252 ± 0.002

Standard deviation.

atures studied. The maximum partition coefficient has been observed at 25 wt% of K3 PO4 and 313 K. In general, increasing the weight percent of potassium phosphate can facilitate the migration of stevioside toward the bottom phase. By elevating the K3 PO4 concentration, the ionic strength of system intensifies, and consequently the tendency of stevioside toward the bottom phase is reinforced. The hydrophobicity of biomolecules plays an important role as long as partitioning is concerned. As a hydrophilic glycoside, stevioside opts to accumulate in the less hydrophobic phase, namely the salt-rich phase. The partition behavior of 12 different hydrophobic and hydrophilic proteins in the ATPS has been investigated by Andrews et al. The protein hydrophobicity was a contributing factor in the partitioning process in a way that amyloglucosidase enzyme, which is more hydrophilic than the other studied proteins, had a tendency to partition into the lower (salt-rich) phase (Andrews et al., 2005).

3.1.4. Effect of [Ch]Cl concentration on the stevioside partitioning The partition coefficients of stevioside were considered at three different weight percents (24 wt%, 26 wt%, and 28 wt%) of [Ch]Cl. To this end, the weight percent of tripotassium phosphate was kept constant at 30 wt % in all of the experiments. The results were investigated at four different temperatures of 298 K, 303 K, 308 K and 313 K. The effect of [Ch]Cl weight percent on the partition coefficient of stevioside is demonstrated in Table 4. It can be deduced from the statistical measurements that the concentration of [Ch]Cl has a significant effect on the partition coefficient, as the maximum partition coefficient of stevioside has been observed at 28 wt% of [Ch]Cl, which indicates the fact that by increasing the weight percent of [Ch]Cl in the initial feedstock, the partition coefficient of stevioside goes up. containing [Ch]Cl + K3 PO4 + H2 O have two ATPSs hydrophilic phases. Apparently, by raising the amount of [Ch]Cl (while holding the potassium phosphate concentration constant), the hydrophilicity of the top phase rises. Therefore, thanks to its high solubility in water, the hydrophilic stevoside is apt to migrate to the top phase. Furthermore, it seems that with a gradual increase in the [Ch]Cl concentration, the salting-out effect induced by PO4 3− anion becomes dominant.

Fig. 3 – Three dimensional plot (top) and counter plot (bottom) on the partition coefficients of stevioside with the combined effects of K3 PO4 weight percent and temperature.

3.1.5.

Effect of temperature on the stevioside partitioning

The concurrent effects of the independent variables – i.e. weight percent and temperature – on the stevioside partition coefficient are illustrated in Figs. 3 and 4 as 3D and counter plots, in which the mutual influence of variables as well as their optimum level to achieve the maximum response (partition coefficient) can be readily examined. The effect of four

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food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 107–115

Table 5 – Effect of [Ch]Cl on the recovery percentages of stevioside in the bottom phase (K3 PO4 -rich phase). wtK3 PO4 %

wtChCl %

T/K 298

303 RB ± 

30

a

24 26 28

89.512 ± 0.632 89.174 ± 0.099 87.860 ± 0.294

308

313

80.948 ± 0.157 80.643 ± 2.252 80.176 ± 0.316

80.484 ± 0.127 80.225 ± 0.318 79.850 ± 0.149

a

81.775 ± 0.415 81.244 ± 0.342 81.073 ± 0.099

Standard deviation.

temperatures (T = 298 K, 303 K, 308 K, and 313 K) on the partitioning of stevioside at the different weight percents of K3 PO4 is presented in Fig. 3, and also in Fig. 4 for the different weight percents of [Ch]Cl. The results indicate that the maximum partition coefficient of stevioside corresponds to the temperature of 313 K. Thus, by elevating the temperature of the system, the partition coefficient of stevioside increases. In addition, statistical analyses reveal that the temperature has a meaningful effect on the stevioside partition coefficient. In view of the fact that the phase composition, electrostatic as well as hydrophobic effects are associated with temperature, the temperature influence on the partitioning of biomolecules and enzymes seems to be complicated (Ebrahimi and Shahriari, 2016). It has been indicated in some sources that the temperature has no direct effect on the partition coefficient. Hey et al. (2005) showed that temperature did not have a significant impact on the behavior of steroid partitioning.

According to the results, it may be stated that the effect of temperature on the partition coefficient significantly depends on the dissolved substance and the type of triple system.

3.1.6.

Recovery percentage of stevioside

Employing ATPSs to recycle biomolecules in the downstream process is believed to be one of the economic and efficient bioseparation techniques. The phase components, the components concentration in the top and bottom phases, the volume ratio and temperature are the key factors that impact on the biomolecules recovery. The recovery percentages of stevioside in the bottom phase of the ATPS made up of [Ch]Cl + K3 PO4 + H2 O have been reported in Tables 5 and 6 at four different temperatures of 298 K, 303 K, 308 K, and 313 K. The effect of the alteration of the [Ch]Cl concentration, at fixed concentrations of K3 PO4 , on the stevioside recovery has been detailed in Table 5. It is evident that by raising the [Ch]Cl weight percent in the feed, the stevioside recovery in the bottom phase diminishes. The maximum recovery percentage of stevioside is 89.51 ± 0.63% achieved at 24 wt% of [Ch]Cl. Table 6 demonstrates the effect of K3 PO4 concentration on the recovery of stevioside at fixed concentrations of [Ch]Cl. Obviously, the stevioside recovery has a direct relationship with the K3 PO4 concentration. Stevioside exhibits a tendency toward the salt-rich phase; in this regard, by raising the salt concentration, the “salting in” effect intensifies, which in turn gives rise to a decline in the steviside partition coefficient. The highest recovery percentage of stevioside, i.e. 88.81 ± 0.01%, was obtained at 31 wt% of K3 PO4 . The recovery percentage has been calculated based on the equal volumes of top and bottom phases; however, to achieve higher values of recovery, the VR could be altered without producing any change in the K values. Eq. (12) was used to specify the recovery percentage and the optimum VR . RB =

100 KVR + 1

(12)

The stevioside recovery was calculated as 89.512% while the values of K and VR were 0.117 and 1.001, respectively. Interestingly, with a VR of 0.448, the recovery percentage would rise to 95.02%. The results implied that by increasing the volume of bottom phase (i.e. increasing the salt concentration), the stevioside recovery could be improved. This point to the significance of salt concentration in the system and its effect on the recovery enhancement.

3.1.7.

Fig. 4 – Three dimensional plot (top) and counter plot (bottom) on the partition coefficients of stevioside with the combined effects of [Ch]Cl weight percent and temperature.

Effect of pH on the stevioside partitioning

The effect of pH on the partition coefficient of stevioside in the [Ch]Cl + K3 PO4 + H2 O ATPS was studied at 298 K. Kroyer has pointed out that stevioside enjoys good stability under normal conditions applied in various food processing procedures. Nevertheless, in the extreme conditions of pH and temper-

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Table 6 – Effect of K3 PO4 on the recovery percentages of stevioside in the bottom phase ([Ch]Cl-rich phase). wtChCl %

wtK3 PO4 %

T/K 298

303 RB ± 

25 27 29 31

22

a

88.320 ± 2.290 88.351 ± 0.182 88.711 ± 0.732 88.815 ± 0.015

308

313

59.665 ± 0.596 60.103 ± 0.335 60.985 ± 0.228 61.759 ± 0.278

56.757 ± 0.085 57.066 ± 0.559 58.774 ± 0.230 59.846 ± 0.148

a

78.985 ± 0.735 85.910 ± 0.310 86.133 ± 0.856 86.395 ± 0.072

Standard deviation.

Table 7 – Effect of pH on the stevioside partitioning (K) at 298 K and atmospheric pressure. pH(Top)

pH(Bottom)

K

to be predominating, pushing the stevisode toward the top [Ch]Cl-rich phase.

RB

22 wt% [Ch]Cl + 31 wt% K3 PO4 8.32 8.4 9.42 9.32 10.45 10.32 13.73 13.65

0.981 0.975 0.96 0.125

50.458 50.61 51.01 88.815

24 wt% [Ch]Cl + 30 wt% K3 PO4 8.3 8.43 9.45 9.35 10.46 10.35 13.76 13.7

0.967 0.96 0.954 0.117

50.715 50.862 51.117 89.512

ature, its stability lessens (Kroyer, 2010). Both phases of the ATPSs containing [Ch]Cl + K3 PO4 + H2 O are alkaline due to the presence of the basic PO4 3− anion. The pH measurement of top and bottom phases revealed that the pH values were in the range of 13.46–13.66. For that reason, the addition of some droplets of diluted phosphoric acid into the ATPS made it possible to analyze the impact of pH changes in the range of 8.32–10.45. It is worth mentioning that stevioside is quite stable over the pH range from 2 to 10 and the temperatures up to 80 ◦ C (Kroyer, 2010). The pH values of the coexisting phases of the systems were shown in Table 7. Having the highest values of stevioside recovery in the bottom phase, these two sets of composition were chosen for the pH study: 22 wt% of [Ch]Cl + 31 wt% K3 PO4 and 24 wt% of [Ch]Cl + 30 wt% K3 PO4 . The results reported in Table 7 suggest that the stevioside partition coefficient declines with increasing pH. According to a general rule, a change in pH leads to a change in solute solubility. For pH values around 13, it seems that the stevioside becomes positively charged and tends to move toward the lower phase which has more negative charge density due to the presence of PO4 3− anion. For pH values below 8, it can be assumed that the electrostatic interactions between the mineral salt ions and the charged stevioside are of less importance in a way that the stevioside prefers to migrate into the [Ch]Cl-rich phase. Moreover, the increase in the concentration of PO4 3− anion (as a consequence of the pH decrease and the addition of phosphoric acid) may cause the salting-out effect induced by the anion

3.2.

Statistical analysis

3.2.1.

Regression analysis

To determine the relationship between the partition coefficient of stevioside and the weight percents of the [Ch]Cl and potassium phosphate as well as temperature, a regression analysis was conducted. Three different regression models, correlated with experimental data, are detailed in Table 8. Considering the probability plot, the scatter pattern of residuals closely follows the 45-degree reference line, which indicates a normal distribution of the residuals. In order to verify the accuracy of the proposed models, the residuals’ behavior was assessed; the outcomes demonstrated that the residuals were normally distributed with a constant variance. The optimal regression model was defined through minimizing the deviation (DEV%), the coefficient of determination (R2 ), and the root mean squared error (RMSE). The coefficient of determination (R2 ) is a statistical measure evaluating the capability of a model to describe and predict the outcomes. The more the value of R2 approximates to 1, the stronger the correlation between the experimental data of the stevioside partition coefficients and the regression model results is expected to be. The root mean squared error (RMSE) is used as a criterion to represent the deviation of the regression model results from the experimental data. The smaller the RMSE value, the less the results of the regression model deviate from the experimental data, and the model can predict the trend more accurately. As regards the regression models detailed in Table 8, it can be deduced that the second regression model is able to fit the experimental data with a high accuracy. A comparison between experimental data and those derived from the second regression model can be found in Table 9 in terms of the [Ch]Cl weight percents and the salt weight percents. In regard to the error percentages, it could be inferred that the regression model no. 2 has a good potential to correlate the experimental data.

Table 8 – The regression models for the partition coefficient of stevioside. Regression model

RMSE

R2

⁄K = 8.917 − 0.369 × [T] + 0.114 × [wtChCl %] + 0.170 × [wtK3 PO4 %] ln K = −1.304 + 0.091 × [T] − 0.067 × [wtChCl %] − 0.048 × [wtK3 PO4 %] √ K = 0.713 + 0.025 × [T] − 0.024 × [wtChCl %] − 0.014 × [wtK3 PO4 %]

0.116 0.090 0.087

0.791 0.981 0.719

Model 1 2 3

1

Deviation 28.103 30.284 37.144

114

food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 107–115

Table 9 – Comparison between the experimental partition coefficients and the results obtained from the regression model no. 2. T = 298 K EXPT

wtChCl % 24% 26% 28% wtK3 PO4 % 25% 27% 29% 31%

4.

K

KMODEL

0.117 0.121 0.138

0.125 0.11 0.096

0.133 0.132 0.127 0.126

0.182 0.165 0.15 0.137

Conclusions

Choline chloride-based ATPSs are considered as valuable, biocompatible systems for separating biomolecules. In this study, to achieve a high quality extraction of stevioside, an ATPS containing [Ch]Cl + K3 PO4 + H2 O has been utilized. The partition coefficient of stevioside and the recovery of stevioside were estimated in this system. The purpose of performing this research was to study the effective parameters contributing to the partitioning of stevioside. The effect of such parameters as the concentration of [Ch]Cl and tripotassium phosphate on the partition coefficient of stevioside was measured. All the examined parameters have shown their particular impact on the extraction of stevioside. The results indicated that the stevioside tended toward the bottom phase (K < 1). The dependence of the partition coefficient of stevioside on the weight percent of the [Ch]Cl, the weight percent of the tripotassium phosphate, and temperature was presented using an acceptable regression model. The optimum ATPS composition was 24 wt% of [Ch]Cl + 30 wt% K3 PO4 at a temperature of 298 K and a pH of 13.7; the results for a one-stage process showed that the maximum recovery has been 95.02% in the bottom phase when the VR was set to 0.448. Overall, with regard to the obtained results, the usage of the [Ch]Cl-based aqueous two-phase systems can be quite successful for the extraction of stevioside.

References Albertsson, P.Å., 1986. Partition of Cell Particles and Macromolecules: Separation and Purification of Biomolecules, Cell Organelles, Membranes, and Cells in Aqueous Polymer Two-phase Systems and Their Use in Biochemical Analysis and Biotechnology. Wiley, New York. Andrews, B.A., Schmidt, A.S., Asenjo, J.A., 2005. Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge. Biotechnol. Bioeng. 90, 380–390. Ebrahimi, T., Shahriari, S., 2016. Extraction of betanin using aqueous two-phase systems. Bull. Chem. Soc. Jpn. 89, 565–572. Freire, M.G., Claudio, A.F.M., Araujo, J.M.M., Coutinho, J.A.P., Marrucho, I.M., Lopes, J.N.C., Rebelo, L.P.N., 2012. Aqueous biphasic systems: a boost brought about by using ionic liquids. Chem. Soc. Rev. 41, 4966–4995. Freire, M.G., Neves, C.M.S.S., Marrucho, I.M., Canongia Lopes, J.N., Rebelo, L.P.N., Coutinho, J.A.P., 2010. High-performance extraction of alkaloids using aqueous two-phase systems with ionic liquids. Green Chem. 12, 1715–1718. Geuns, J.M.C., 2003. Stevioside. Phytochemistry 64, 913–921. Gutowski, K.E., Broker, G.A., Willauer, H.D., Huddleston, J.G., Swatloski, R.P., Holbrey, J.D., Rogers, R.D., 2003. Controlling the aqueous miscibility of ionic liquids: aqueous biphasic systems

of water-miscible ionic liquids and water-structuring salts for recycle, metathesis, and separations. J. Am. Chem. Soc. 125, 6632–6633. Hatti-Kaul, R., 2000. Methods in biotechnology. In: Aqueous Two-Phase Systems. Humana Press. Hey, D.L., Urban, L.S., Kostel, J.A., 2005. Nutrient farming: the business of environmental management. Ecol. Eng. 24, 279–287. In, M.-J., Kim, D.C., Chae, H.J., 2005. Downstream process for the production of yeast extract using brewer’s yeast cells. Biotechnol. Bioprocess Eng. 10, 85–90. Johansson, H.-O., Karlström, G., Tjerneld, F., Haynes, C.A., 1998. Driving forces for phase separation and partitioning in aqueous two-phase systems. J. Chromatogr. B: Biomed. Sci. Appl. 711, 3–17. Kroyer, G., 2010. Stevioside and stevia-sweetener in food: application, stability and interaction with food ingredients. J. Verbrauch. Lebensm. 5, 225–229. Kroyer, G.T., 1999. The low calorie sweetener stevioside: stability and interaction with food ingredients. LWT — Food Sci. Technol. 32, 509–512. Lebovka, N., Vorobiev, E., Chemat, F., 2011. Enhancing Extraction Processes in the Food Industry. CRC Press. Li, S., He, C., Liu, H., Li, K., Liu, F., 2005. Ionic liquid-based aqueous two-phase system, a sample pretreatment procedure prior to high-performance liquid chromatography of opium alkaloids. J. Chromatogr. B 826, 58–62. Li, Z., Liu, X., Pei, Y., Wang, J., He, M., 2012. Design of environmentally friendly ionic liquid aqueous two-phase systems for the efficient and high activity extraction of proteins. Green Chem. 14, 2941–2950. Liu, C.-l., Kamei, D.T., King, J.A., Wang, D.I.C., Blankschtein, D., 1998. Separation of proteins and viruses using two-phase aqueous micellar systems. J. Chromatogr. B: Biomed. Sci. Appl. 711, 127–138. Liu, X., Li, Z., Pei, Y., Wang, H., Wang, J., 2013. (Liquid + liquid) equilibria for (cholinium-based ionic liquids + polymers) aqueous two-phase systems. J. Chem. Thermodyn. 60, 1–8. Mageste, A.B., de Lemos, L.R., Ferreira, G.M.D., da Silva, M.d.C.H., da Silva, L.H.M., Bonomo, R.C.F., Minim, L.A., 2009. Aqueous two-phase systems: an efficient, environmentally safe and economically viable method for purification of natural dye carmine. J. Chromatogr. A 1216, 7623–7629. Merchuk, J.C., Andrews, B.A., Asenjo, J.A., 1998. Aqueous two-phase systems for protein separation: studies on phase inversion. J. Chromatogr. B: Biomed. Sci. Appl. 711, 285–293. Pereira, J.F.B., Kurnia, K.A., Cojocaru, O.A., Gurau, G., Rebelo, L.P.N., Rogers, R.D., Freire, M.G., Coutinho, J.A.P., 2014. Molecular interactions in aqueous biphasic systems composed of polyethylene glycol and crystalline vs. liquid cholinium-based salts. Phys. Chem. Chem. Phys. 16, 5723–5731. Pereira, J.F.B., Ventura, S.P.M., e Silva, F.A., Shahriari, S., Freire, M.G., Coutinho, J.A.P., 2013a. Aqueous biphasic systems composed of ionic liquids and polymers: a platform for the purification of biomolecules. Sep. Purif. Technol. 113, 83–89. Pereira, J.F.B., Vicente, F., Santos-Ebinuma, V.C., Araújo, J.M., Pessoa, A., Freire, M.G., Coutinho, J.A.P., 2013b. Extraction of tetracycline from fermentation broth using aqueous two-phase systems composed of polyethylene glycol and cholinium-based salts. Process Biochem. 48, 716–722. Pourebrahimi, F., Shahriari, S., Salehifar, M., Mozafari, H., 2015. Partitioning of vanillin in aqueous two-phase systems formed by cholinium chloride and K3 PO4 . Bull. Chem. Soc. Jpn. 88, 1494–1499. Sarangi, B.K., Pattanaik, D.P., Rathinaraj, K., Sachindra, N.M., Madhusudan, M.C., Mahendrakar, N.S., 2011. Purification of alkaline protease from chicken intestine by aqueous two phase system of polyethylene glycol and sodium citrate. J. Food Sci. Technol. 48, 36–44. Shahriari, S., Doozandeh, S.G., Pazuki, G., 2012. Partitioning of cephalexin in aqueous two-phase systems containing poly(ethylene glycol) and sodium citrate salt at different temperatures. J. Chem. Eng. Data 57, 256–262.

food and bioproducts processing 1 0 2 ( 2 0 1 7 ) 107–115

Shahriari, S., Taghikhani, V., Vossoughi, M., Safe kordi, A.A., Alemzadeh, I., Pazuki, G.R., 2010. Measurement of partition coefficients of ␤-amylase and amyloglucosidase enzymes in aqueous two-phase systems containing poly(ethylene glycol) and Na2 SO4 /KH2 PO4 at different temperatures. Fluid Phase Equilib. 292, 80–86. Shahriari, S., Tome, L.C., Araujo, J.M.M., Rebelo, L.P.N., Coutinho, J.A.P., Marrucho, I.M., Freire, M.G., 2013. Aqueous biphasic systems: a benign route using cholinium-based ionic liquids. RSC Adv. 3, 1835–1843. Stoyanova, S., Geuns, J., Hideg, É., Van Den Ende, W., 2011. The food additives inulin and stevioside counteract oxidative stress. Int. J. Food Sci. Nutr. 62, 207–214. Strecker, A., 1862. Ueber einige neue Bestandtheile der Schweinegalle. Justus Liebigs Ann. Chem. 123, 353–360. Waites, M.J., Morgan, N.L., Rockey, J.S., Higton, G., 2009. Industrial Microbiology: An Introduction. John Wiley & Sons.

115

2012. Partitioning in Aqueous Two-Phase System: Theory, Methods, Uses, and Applications to Biotechnology. Elsevier. Wu, X., Liang, L., Zou, Y., Zhao, T., Zhao, J., Li, F., Yang, L., 2011. Aqueous two-phase extraction, identification and antioxidant activity of anthocyanins from mulberry (Morus atropurpurea Roxb.). Food Chem. 129, 443–453. Wu, Y., Wang, Y., Zhang, W., Han, J., Liu, Y., Hu, Y., Ni, L., 2014. Extraction and preliminary purification of anthocyanins from grape juice in aqueous two-phase system. Sep. Purif. Technol. 124, 170–178. Zeisel, S.H., Da Costa, K.-A., 2009. Choline: an essential nutrient for public health. Nutr. Rev. 67, 615–623. Zempleni, J., Rucker, R., McCormick, D., Suttie, J., 2007. Vitamin E. In: Traber, M.G. (Ed.), Handbook of Vitamins.