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Modification of steviol glycosides using α-amylase
LWT - Food Science and Technology 57 (2014) 400e405
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Modification of steviol glycosides using a-amylase Fayin Ye a, b, Ruijin Yang b, *, Xiao Hua a, c, Qiuyun Shen b, Wei Zhao b, Wenbin Zhang b a
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Wuxi 214122, China c State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 April 2012 Received in revised form 16 November 2013 Accepted 30 December 2013
Stevia extract usually contains a high content of stevioside which produces a significant bitter aftertaste, thus limiting its commercial application as a high-potency sweetener in foods. In order to improve its organoleptic properties, the stevia extract (containing 90 g/100 g stevioside) was modified by a-amylase from Aspergillus oryzae (TAKA), with soluble starch as substrate. Stevioside was found to be an efficient acceptor substrate and has been transglycosylated during the reaction. The mono-glycosylated stevioside was the major product. The influences of reaction conditions on the conversion yield of stevioside, including pH, reaction temperature, weight ratio of stevioside to starch, substrate and enzyme concentrations, were investigated. With 2 g/100 mL stevioside and 20 g/100 mL soluble starch in 0.05 mol/L potassium phosphate buffer, pH 6.4 and 50 U/mL TAKA, 48.2% conversion yield of stevioside was obtained in 0.5 h at 70 C. For the enzymatically modified steviol glycosides, the bitter aftertaste was significantly decreased and the sweetness potency was significantly improved. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Steviol glycosides Stevioside a-Amylase Enzymatic modification Organoleptic properties
1. Introduction The increasing consumption of sucrose has resulted in growing incidence of obesity, diabetes and cardiovascular diseases. Diterpene glycosides produced by Stevia rebaudiana Bertoni, known as steviol glycosides, are natural, high-potency low-calorie sweeteners, which has been used as an alternative to sucrose (Geuns, 2003). Steviol glycosides preparations, or stevia extracts, are commonly obtained by aqueous or solvent extraction from the leaves of the plant (Puri, Sharma, & Tiwari, 2011). Most commercial products have stevioside as the major component, followed by rebaudioside A and smaller amounts of other glycosides of steviol. Rebaudioside A, which has an extra glucose unit relative to stevioside, imparts the greatest potency of sweetness and less bitter aftertaste. Stevioside is 250e300 times sweeter than 0.4 g/100 g sucrose solutions but produces a significant bitter aftertaste (Lemus-Mondaca, Vega-Gálvez, Zura-Bravo, & Ah-Hen, 2012). As a result of the dominance of unpleasant bitter aftertaste due to stevioside, the use of stevia extracts for sweetening foods has to some extent been restricted. Transglycosylation has been used to modify the organoleptic or physicochemical properties of bioactive compounds in foods to
* Corresponding author. School of Food Science and Technology, Jiangnan University, No. 1800 Lihu Road, 214122 Wuxi, China. Tel./fax: þ86 510 85919150. E-mail address: [email protected] (R. Yang). 0023-6438/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2013.12.045
improve their use (Lee et al., 1999; Shin, Cheong, Lee, & Kim, 2009). Particularly, enzymatic transglycosylation was an effective tool for improving taste quality and sweetness potency of stevioside (Ohtani & Yamasaki, 2002). Kusama, Kusakabe, Nakamura, Eda, and Murakami (1986) studied the transglucosylation into stevioside by the enzyme system from Streptomyces sp. W19-1 with curdlan (or b-1, 3-glucan) as substrate, and the bitter taste of products was reduced. Fukunaga et al. (1989) studied the modification of stevioside with cyclodextringlucosyltransferase in the presence of soluble starch resulting in a mixture of a-glucosylated products, and the taste quality and sweetness potency of some products were claimed to be improved substantially. Ishikawa, Kitahata, Ohtani, Ikuhara, and Tanaka (1990) investigated the production of fructosyl-stevioside by transfructosylation of b-fructofuranosidase, and the taste quality of fructosyl-stevioside was found to be superior to that of rebaudioside A. Lobov, Kasai, Ohtani, Tanaka, and Yamasaki (1991) studied transglycosylation of stevioside by commercial pullanase and crude b-amylase, with pullulan and maltose as glycosyl donor, respectively. Transglycosylation of stevioside using cyclodextrin glucanotransferases (CGTases) from various microorganisms has been well investigated. In these CGTasescatalysed reactions, cyclodextrins or starches were employed as donor substrates (Abelyan, Balayan, Ghochikyan, & Markosyan, 2004; Jung, Kim, Lee, & Lee, 2007; Kochikyan, Markosyan, Abelyan, Balayan, & Abelyan, 2006) and the highest conversion yield of stevioside (90%) was obtained by microwave-assisted transglycosylation reaction (Jaitak et al., 2009).
F. Ye et al. / LWT - Food Science and Technology 57 (2014) 400e405
a-Amylases (EC 3.2.1.1) are a class of glycoside hydrolases widely distributed in nature and have recently attracted considerable research interest for their applications in food, textile, fermentation and pharmaceutical industries (Van der Maarel, van der Veen, Uitdehaag, Leemhuis, & Dijkhuizen, 2002). In addition to starch hydrolysis, some a-amylases were also used in transglucosylation reactions in the presence of various acceptor molecules (Mótyán et al., 2011; Rivera, López-Munguía, Soberón, & Saab-Rincón, 2003). Termamyl, a commercial a-amylase from Bacillus licheniformis, was found to catalyse the synthesis of oligosaccharide with starch and non-starch sugars as substrates (Chitradon, Mahakhan, & Bucke, 2000). Aspergillus oryzae a-amylase was capable of catalysing the synthesis of alkyl glucosides with starch and alcohols as substrates (Larsson, Svensson, & Adlercreutz, 2005). The transglucosylation activity of a-amylase from Trichoderma viride JCM22452 had been applied in the synthesis of flavonoid monoglucoside which showed higher heat stability and solubility and lower astringency and astringent stimulation than its aglycon (Noguchi, Inohara-Ochiai, Ishibashi, & Fukami, 2008). Moreno et al. (2010) reported the versatility of Thermotoga maritima a-amylase in transglucosylation reactions and its capacity to generate neotrehalose and alkyl-glucosides. In the present study, enzymatic modification of steviol glycosides was attempted using a commercial a-amylase from A. oryzae (TAKA) in the presence of soluble starch. To increase the conversion yield of stevioside, the reaction conditions, including pH, temperature, and substrate and enzyme concentrations, were studied. In addition, the organoleptic properties of the enzymatically modified steviol glycosides (EMSGs) were examined.
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molecular weight of the modified products was determined by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) mentioned in Section 2.4. 2.2.3. Transglycosylation reactions of stevioside In a 250-mL enzymatic reactor, stevia extract and soluble starch dissolved with buffer solution and TAKA were mixed, giving final reaction volume of 100 mL, and incubated for 0.5e12 h. The reaction mixture was deactivated by boiling and the resulting transglycosylated steviol glycosides were analysed by HPLC. The effects of pH (4.0e10.0), temperature (30e80 C), initial stevioside concentration (0.5e6.67 g/100 mL), initial starch concentration (2.5e 30 g/100 mL), and enzyme concentration (10e300 U/mL), were investigated. The detailed reaction conditions were described in the figure legends of Figs. 4e8 in Section 3. The optimal reaction conditions were considered in terms of obtaining the maximal conversion yield of stevioside. The conversion yield of stevioside was calculated from the decrease in stevioside at each time point compared with that at time t ¼ 0 min. Under optimal reaction conditions, a preparative scale production (2000 mL) of the EMSGs was then conducted. 2.2.4. Separation and purification of the EMSGs The aforedescribed product mixtures were filtered through a Büchner funnel and the filtrate was loaded on a 2.6 cm 100 cm column packed with macroporous adsorption resin DM 301 (Anhui Sanxing Resin Co., Anhui, China) and eluted with 1000 mL deionised water (1 BV/h) to remove the saccharides. Thereafter, the EMSGs were eluted with 80 mL/100 mL ethanol (1 BV/h), followed by concentration and lyophilization.
2. Materials and methods 2.3. High performance liquid chromatography (HPLC) 2.1. Materials
a-Amylase from A. oryzae (TAKA) was purchased from Sigmae Aldrich (Shanghai, China). Stevia extract (containing 90 g/100 g stevioside and 5 g/100 g rebaudioside A) was from Jining Aoxing Stevia Product Co., Ltd. (Shandong, China). Stevioside (>99 g/100 g by HPLC) and rebaudioside A (>97 g/100 g by HPLC) were from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Soluble starch, ethanol and other chemicals were all of analytical grade and from Sino-Pharm Chemical Co. (Beijing, China). Sucrose for sweetness potency determination was of food grade and from Nanjing Ganzhiyuan Sugar Co., Ltd. (Jiangsu, China). 2.2. Methods 2.2.1. Enzyme assay The activity of TAKA was assayed with 1.0 g/100 mL soluble starch prepared in 0.05 mol/L potassium phosphate buffer (pH 6.0) at 60 C using the 3, 5-dinitrosalicylic acid method (Bernfield, 1955). The absorbance of the reducing sugars liberated at 540 nm was measured using a UVevisible spectrophotometer (Unico UV2102C, WI, USA). One unit was defined as the amount of enzyme that released 1 mmol of reducing sugar equivalent to maltose per minute at pH 6.0 and 60 C. 2.2.2. Acceptor specificity Stevioside and rebaudioside A were used to assay their ability to function as acceptor substrates. Reactions were performed by incubating 1 g/100 mL acceptor substrate (stevioside or rebaudioside A), 10 g/100 mL soluble starch and 20 U/mL TAKA in 0.05 mol/L potassium phosphate buffer (pH 6.0, 100 mL) at 60 C for 1 h. The transglycosylation to the acceptor was evaluated by the peak area of the modified products shown on the HPLC chromatogram. The
The analysis of the modified products was conducted by an HPLC system (Hitachi L-2000, Tokyo, Japan) equipped with Hitachi L-2495 diode array detector and a Shodex Asahipak NH2P-50 4E chromatographic column (length 250 mm; inner diameter 4.6 mm). The column was eluted at 35 C with a 75:25 mixture of acetonitrile and Milli-Q water at 1.0 mL/min. The chromatograms were recorded at 210 nm. 2.4. UHPLC-MS Analysis UHPLC-MS analysis of the reaction mixtures was performed on a Waters Acquity ultra-high-performance liquid chromatography (UHPLC) system coupled to a Waters MALDI SYNAPT Q-TOF mass spectrometer (Waters Co., Ltd., Milford, MA, USA) with ESI (electrospray ionisation) source. Each sample was separated by an Acquity UPLC BEH amide column (2.1 mm 100 mm, 1.7 mm; Waters Co., Ltd., Milford, MA, USA). The injection volume was 1.0 mL, and elution was performed using an 80:20 mixture of acetonitrile and Milli-Q water (containing 0.02 mol/L ammonium acetate) at a flow rate of 0.3 mL/min. The column was maintained at 45 C. The spectrum at 210 nm was recorded. Mass spectra were obtained by ESI in the negative mode under the following conditions: ESI source block and desolvation temperature: 100 C and 300 C, respectively; capillary voltage: 2.8 kV; desolvation gas (N2) flow: 500 L/h and scan range m/z 100e2000. 2.5. Determination of relative sweetness and detection threshold for bitter aftertaste The determination of sweetness potency was performed according to the method as described by Ishikawa et al. (1990) and Parpmello, Versari, Castellari, and Galassi (2001). Seventeen
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Fig. 1. HPLC chromatogram of the transglycosylation reaction between stevioside and soluble starch showing the transglycosylated steviosides formation (reaction conditions: 1 g/ 100 mL stevioside, 10 g/100 mL soluble starch, and 20 U/mL TAKA in 0.05 mol/L potassium phosphate buffer, pH 6.0, temperature 60 C, incubation time 1 h). Peak identification: 1, stevioside; 2, mono-glycosylated stevioside; 3, di-glycosylated stevioside; 4e6, tri-glycosylated stevioside; 7, tetra-glycosylated stevioside.
panellists (eight males and nine females, aged 23 to 34 y with average age 28 y) were invited based on interest, availability, and knowledge of basic tastes (sweet, sour, salty, and bitter) as well as previous sensory analysis experience in assessing sweetener samples. Each panelist had greater than 300 h of training in proficiency testing of sweet tastes. The stevia extract and the purified EMSGs (described in Section 2.2.4) were dissolved in potable water to make appropriate concentrations: 0.0040, 0.0060, 0.0080, 0.0100, 0.0120, 0.0150, 0.0180, 0.0210, 0.0240, 0.0300, 0.0360, 0.0420, 0.0480, 0.0540 and 0.0600 g/100 mL. The test solutions were served as 20 mL aliquots in 30 mL odour-free paper cups and analyses were conducted at room temperature (25 C) in individual tasting booths under normal lighting conditions. Each panelist was presented with a pair of cups containing the sucrose standard (2 g/100 mL) and the test solution in each session. For each pair of cups, panellists were asked which of the two was sweeter and were instructed to record the sweetness potency rating by making the appropriate point on a 15 cm continuous line scale. The mid-point of the scale was the sucrose standard. They spat out the solutions without swallowing and rinsed their mouths between each test. The concentration of sample in next session was chosen depending on the response of the panellist. Finally, they pick out the test solution where the sweetness was equivalent to the sucrose standard. The relative sweetness of the test solution was calculated according to the following formula.
2.6. Statistical analysis The enzymatic assays and analytic measurements (except for UHPLC-MS analysis) were performed in triplicate and the results were expressed as mean values standard deviations. Data from determination of sweetness potency and bitter aftertaste threshold were statistically analysed using Student’s t-tests to determine significant differences between the stevia extract and the EMSGs. Statistical significance was accepted at a level of p 0.01. 3. Results and discussion 3.1. Acceptor specificity of transglycosylation reactions 3.1.1. Stevioside as the acceptor substrate In the reaction of stevioside as acceptor substrate, thirty percent of stevioside was depleted (calculation based on peak area of stevioside in the HPLC chart) and six modified products were detected by chromatographic analysis, with retention times of 10.01, 15.78, 25.58, 26.95, 28.27 and 43.57 min, respectively (Fig. 1). The peak 1 at 6.40 min was confirmed to be stevioside by
The relative sweetness ¼ A=B where A: concentration (g/100 mL) of the sucrose standard; B: concentration of the test solution (g/100 mL) which sweetness equivalent to the sucrose standard. To determine the threshold for bitter aftertaste of the stevia extract and the purified EMSGs (described in Section 2.2.4) were dissolved in potable water to make appropriate concentrations from 0.0160 g/100 mL to 0.0600 g/100 mL with intervals of 0.0040 g/100 mL. Twelve pairs of test solutions, in which one member of each pair was always a potable water control, were presented in randomised order in each session. Panellists were asked to indicate in which sample bitter aftertaste was perceivable. The value corresponding to 75% of positive responses indicated the panel threshold.
Fig. 2. Mass spectra of (a) mono-glycosylated stevioside and (b) di-glycosylated stevioside, respectively.
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Fig. 3. HPLC analysis of the products by treating rebaudioside A with TAKA in the presence of soluble starch (reaction conditions: 1 g/100 mL rebaudioside A, 10 g/100 mL soluble starch, and 20 U/mL TAKA in 0.05 mol/L potassium phosphate buffer, pH 6.0, temperature 60 C, incubation time 1 h). Peak identification: 8, rebaudioside A; 9, mono-glycosylated rebaudioside A; 10, di-glycosylated rebaudioside A; 11, tri-glycosylated rebaudioside A.
comparing its retention time with that of the standard. Molecular weight of the modified products was determined by UHPLC-MS. The compound corresponding to peak 2 was a mono-glycosylated stevioside, since it showed an [MeH] ion at m/z 965 in the extracted ion chromatogram (set m/z values 803, 965, 1127, 1289 and 1451, data not shown). The mono-glycosylated stevioside was the main modified product and its mass spectrum was presented in Fig. 2a, which gave an [MeH] ion at m/z 965.1 and [M þ Cl35] and [M þ Cl37] ion at m/z 1001.1 and 1003.1, respectively. The compound corresponding to peak 3 was a diglycosylated stevioside, which showed an [MeH] ion at m/z 1127.2 and [M þ Cl35] ion at m/z 1163.2 (Fig. 2b). Compared with the mono-glycosylated stevioside, the di-glycosylated stevioside gave a much smaller peak area (about 30% of the total modified products). Tri-glycosylated steviosides (peaks 4, 5 and 6) and tetra-glycosylated stevioside (peak 7) constituted about 9% (peak area) of the total modified products, which indicated that transferring more glucosyl residues onto the glycoside became more difficult. In the present study, stevioside was found be to an efficient acceptor substrate with TAKA, giving rise to a group of mono-, di-, and tri-glycosylated stevioside derivatives. 3.1.2. Rebaudioside A as the acceptor substrate Fig. 3 showed the HPLC profile of modified products formed by treating rebaudioside A with TAKA in the presence of soluble starch. By a comparison of the retention time with the standard, peak 8 at 8.99 min was assigned to rebaudioside A. After 1 h of reaction, 13% of rebaudioside A vanished and three new but weak peaks, respectively, at retention times of 14.47, 24.15 and 40.64 min appeared on the chromatogram. UHPLC-MS analysis of the modified products gave deprotonated molecular ions [MeH] at m/z 1127, 1289 and 1451, respectively, which implied that rebaudioside A was transformed to mono-, di-, and tri-glycosylated products. By calculating the peak area of the modified products shown on the HPLC chromatograms (Figs. 1 and 3), transglycosylation reaction performed with rebaudioside A as the acceptor substrate presented rather lower conversion yield (conversion yield of rebaudioside A: 13%) than that with stevioside (conversion yield of stevioside: 30%). Therefore, it can be concluded that rebaudioside A was a relatively poor transglycosylation acceptor.
3.2. Influence of reaction conditions on the conversion yield of stevioside In this section, stevia extract (stevioside 90 g/100 g) was used to investigate the influence of reaction conditions on the conversion yield of stevioside. The transformation of rebaudioside A was not considered due to its low conversion yield and low content in the stevia extract (rebaudioside A 5 g/100 g). 3.2.1. pH The conversion yield at different pH values indicated that reaction was favoured in the pH range of 5.0e7.0 (Fig. 4). The maximal conversion yield was observed at pH 6.4, which was used in further experiments. However, there was a marked decline in the conversion yield in basic pH and only 4.0% conversion yield was attained at pH 8.0. 3.2.2. Temperature The stability and activity of a-amylase are strongly associated with the reaction temperature (Apar & Özbek, 2004). As shown in
Fig. 4. Influence of pH on the conversion yield of stevioside. Reaction conditions: stevioside, 1 g/100 mL; soluble starch, 10 g/100 mL; TAKA, 20 U/mL; incubation of the mixture (total 100 mL) at 60 C for 12 h. Buffers and pH values: sodium acetate buffer (pH 4.0e5.8), potassium phosphate buffer (pH 6.0e8.0), TriseHCl buffer (pH 8.0e9.0), and glycine-NaOH buffer (pH 9.0e10.0). Molarity of the buffer was 0.05 mol/L. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
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Fig. 5. Influence of temperature on the conversion yield of stevioside. Reaction conditions: stevioside, 1 g/100 mL; soluble starch, 10 g/100 mL; TAKA, 20 U/mL; incubation of the mixture (total 100 mL, pH 6.4) for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
Fig. 7. Influence of initial starch concentration on the conversion yield of stevioside. Reaction conditions: substrate weight ratio (stevioside/starch), 1:10; TAKA, 20 U/mL; incubation of the mixture (total 100 mL) at 70 C, pH 6.4 for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
Fig. 5, the conversion yield increased with rising the reaction temperature up to 70 C. At lower test temperatures (30e50 C), the conversion yield increased slowly. Higher test temperatures (>50 C) facilitated the substrate diffusion and fastened the reaction velocity. Approximately an 88% increase in the conversion yield was obtained as the test temperature increased from 50 C to 70 C. However, the conversion yield dramatically decreased when the test temperature was above 70 C, which may have been caused by the thermal-induced inactivation of TAKA.
conversion yield up to 43.1% as the initial starch concentration increased from 2.5 g/100 mL to 20 g/100 mL (Fig. 7). A decrease of conversion yield was found as the initial starch concentration above 20 g/100 mL, which was mainly caused by the inefficiency of reaction due to limitation of substrate and enzyme diffusion at high viscosities (North, 1966; Slaughter, Ellis, & Butterworth, 2001).
3.2.3. Substrate weight ratio (stevioside/starch) Fig. 6 showed the influence of substrate weight ratios on the conversion yield of stevioside. It was found that the conversion yield decreased from 40.9% at the weight ratio 1:20 to 7.6% at the weight ratio 1:1.5. This decreasing trend may be due to substrate inhibition (López, Torrado, Fuciños, Guerra, & Pastrana, 2006). 3.2.4. Initial substrate concentration The initial substrate concentration is the most significant factor for transglycosylation reaction (Moreno et al., 2010; Mótyán et al., 2011). To investigate the influence of starch concentration on the conversion yield, the reactions were performed using different initial starch concentrations, in which the weight ratio of stevioside to starch was fixed at 1:10. The result showed a 2.1-fold increase in
Fig. 6. Influence of substrate weight ratio (stevioside/starch) on the conversion yield of stevioside. Reaction conditions: soluble starch, 10 g/100 mL; stevioside, 0.5e6.67 g/ 100 mL; TAKA, 20 U/mL; incubation of the mixture (total 100 mL) at 70 C, pH 6.4 for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
3.2.5. Enzyme concentration Fig. 8 showed the influence of enzyme concentration on the conversion yield of stevioside. Initially, the increase of enzyme loading could accelerate the reaction rate and enhance the conversion yield. At lower enzyme loading (10 U/mL and 20 U/mL), the reaction took more than 10 h to reach the maximal conversion yield of 28.2% and 43.1%, respectively. At 50 U/mL of TAKA, however, the reaction rate was much faster and the conversion yield was 48.2% in 0.5 h. The maximal conversion yield decreased marginally when the enzyme loading was above 50 U/mL, which might be caused by the formation of enzyme aggregate when excess enzyme was added. The enzyme molecules on the external surface of the aggregate were easy to react with substrate but the accessibility of enzyme molecules present inside the aggregate to substrate was reduced (Yadav & Devendran, 2012). Hence, the optimal conditions obtained were incubation of 2 g/ 100 mL stevioside and 20 g/100 mL soluble starch with 50 U/mL of TAKA in 0.05 mol/L potassium phosphate buffer (pH 6.4) at 70 C.
Fig. 8. Influence of enzyme concentration on the conversion yield of stevioside. Enzyme concentration：- 10 U/mL; B 20 U/mL; 50 U/mL; 7 75 U/mL; ◄ 150 U/ mL; < 300 U/mL. Reaction conditions: stevioside, 2 g/100 mL; soluble starch, 20 g/ 100 mL; incubation of the mixture (total 100 mL) at 70 C, pH 6.4 for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
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Under these conditions, the conversion yield of stevioside achieved 48.2% in 0.5 h and 50.2% after a reaction period of 12 h. 3.3. Relative sweetness and bitter aftertaste threshold of the modified steviol glycosides Sucrose equivalency level had a significant effect on the relative sweetness of high-potency sweeteners (Parpmello et al., 2001; Schiffman, Booth, Losee, Pecore, & Warwick, 1995). The evaluation of the sweetness potency of stevioside becomes complex at high concentration due to the intensifying of nonsweet taste and aftertaste (Cardello, Da Silva, & Damasio, 1999). A low sucrose equivalency level (2 g/100 mL) was therefore chosen to evaluate the sweetness potency of the stevia extract and the EMSGs. The results showed that the stevia extract and the EMSGs were, respectively, 171 15.4 times and 208 13.8 times sweeter than 2 g/100 mL sucrose, suggesting a significant increase in the sweet potency of the modified steviol glycosides (p 0.01). In comparison with the threshold for bitter aftertaste of stevioside (0.0124 0.0042 g/ 100 mL), the threshold for bitter aftertaste of the EMSGs (0.0241 0.0061 g/100 mL) was significantly increased (p 0.01). It was noteworthy that the bitter aftertaste of the stevia extract was perceived even at the concentration it exhibited similar sweetness to 2 g/100 mL sucrose; while the EMSGs were sweeter and exhibited less bitter aftertaste than stevioside at the same concentration. 4. Conclusions This work reported the improvement of organoleptic properties of steviol glycosides by enzymatic modification using a-amylase from A. oryzae (TAKA) in the presence of soluble starch. It was found that stevioside in stevia extract was an efficient acceptor substrate and has been transglycosylated during the reaction, giving rise to a group of mono-, di-, and tri-glycosylated stevioside derivatives. Under optimal conditions, 48.2% of stevioside was transformed within 0.5 h and the mono-glycosylated stevioside was the major product. The study suggested that, in comparison with the stevia extract, the EMSGs had a significant improvement in sweetness potency and a remarkable decrease in bitter aftertaste. Acknowledgements Financial support from the Key Project of National Natural Science Fund (31230057) and the “Five-twelfth” National Science and Technology Support Program (2011BAD23B03) are appreciated. We also acknowledge the support from the Natural Science Foundation of Jiangsu Province (BK2011149) and the Research Program of State Key Laboratory of Food Science and Technology, Jiangnan University (SKLF-TS-200903). References Abelyan, V. A., Balayan, A. M., Ghochikyan, V. T., & Markosyan, A. A. (2004). Transglycosylation of stevioside by cyclodextrin glucanotransferases of various groups of microorganisms. Applied Biochemistry and Microbiology, 40, 129e134. Apar, D. K., & Özbek, B. (2004). a-Amylase inactivation by temperature during starch hydrolysis. Process Biochemistry, 39, 1137e1144. Bernfield, P. (1955). Amylases a/b. Methods in Enzymology, 1, 149e158. Cardello, H. M., Da Silva, M. A., & Damasio, M. H. (1999). Measurement of the relative sweetness of Stevia extract, aspartame and cyclamate/saccharin blends as compared to sucrose at different concentrations. Plant Foods for Human Nutrition, 54, 119e130.
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Transglycosylation reactions of Thermotoga maritima a-amylase. Enzyme and Microbial Technology, 46, 331e337. Mótyán, J. A., Fazekas, E., Mori, H., Svensson, B., Bagossi, P., Kandra, L., et al. (2011). Transglycosylation by barley a-amylase 1. Journal of Molecular Catalysis B: Enzymatic, 72, 229e237. Noguchi, A., Inohara-Ochiai, M., Ishibashi, N., & Fukami, H. (2008). A novel glucosylation enzyme: molecular cloning, expression, and characterization of Trichoderma viride JCM22452 a-Amylase and enzymatic synthesis of some flavonoid monoglucosides and oligoglucosides. Journal of Agricultural and Food Chemistry, 56(24), 12016e12024. North, A. M. (1966). Diffusion-controlled reactions. Quarterly Reviews, Chemical Society, 20(3), 420e440. Ohtani, K., & Yamasaki, K. (2002). Methods to improve the taste of the sweet principles of Stevia rebaudiana. In A. D. Kinghorn (Ed.), Stevia: the genus Stevia (Vol. 19); (pp. 138e159). London: Taylor & Francis. Parpmello, G. P., Versari, A., Castellari, M., & Galassi, S. (2001). Stevioside as a replacement of sucrose in peach juice: sensory evaluation. Journal of Sensory Studies, 16, 471e484. Puri, M., Sharma, D., & Tiwari, A. K. (2011). Downstream processing of stevioside and its potential applications. Biotechnology Advances, 29, 781e791. Rivera, M. H., López-Munguía, A., Soberón, X., & Saab-Rincón, G. (2003). a-Amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity. Protein Engineering, 16, 505e514. Schiffman, S. S., Booth, B. J., Losee, M. L., Pecore, S. D., & Warwick, Z. S. (1995). Bitterness of sweeteners as a function of concentration. Brain Research Bulletin, 36(5), 505e513. Shin, M. H., Cheong, N. Y., Lee, J. H., & Kim, K. H. (2009). Transglucosylation of caffeic acid by a recombinant sucrose phosphorylase in aqueous buffer and aqueoussupercritical CO2 media. Food Chemistry, 115, 1028e1033. Slaughter, S. L., Ellis, P. R., & Butterworth, P. J. (2001). An investigation of the action of porcine pancreatic a-amylase on native and gelatinised starches. Biochimica et Biophysica Acta - General Subjects, 1525(1e2), 29e36. Van der Maarel, M. J. E. C., van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H., & Dijkhuizen, L. (2002). Properties and applications of starch-converting enzymes of the a-amylase family. Journal of Biotechnology, 94, 137e155. Yadav, G. D., & Devendran, S. (2012). Lipase catalysed synthesis of cinnamyl acetate via transesterification in non-aqueous medium. Process Biochemistry, 47, 496e502.
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Modification of steviol glycosides using a-amylase Fayin Ye a, b, Ruijin Yang b, *, Xiao Hua a, c, Qiuyun Shen b, Wei Zhao b, Wenbin Zhang b a
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Wuxi 214122, China c State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 April 2012 Received in revised form 16 November 2013 Accepted 30 December 2013
Stevia extract usually contains a high content of stevioside which produces a significant bitter aftertaste, thus limiting its commercial application as a high-potency sweetener in foods. In order to improve its organoleptic properties, the stevia extract (containing 90 g/100 g stevioside) was modified by a-amylase from Aspergillus oryzae (TAKA), with soluble starch as substrate. Stevioside was found to be an efficient acceptor substrate and has been transglycosylated during the reaction. The mono-glycosylated stevioside was the major product. The influences of reaction conditions on the conversion yield of stevioside, including pH, reaction temperature, weight ratio of stevioside to starch, substrate and enzyme concentrations, were investigated. With 2 g/100 mL stevioside and 20 g/100 mL soluble starch in 0.05 mol/L potassium phosphate buffer, pH 6.4 and 50 U/mL TAKA, 48.2% conversion yield of stevioside was obtained in 0.5 h at 70 C. For the enzymatically modified steviol glycosides, the bitter aftertaste was significantly decreased and the sweetness potency was significantly improved. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Steviol glycosides Stevioside a-Amylase Enzymatic modification Organoleptic properties
1. Introduction The increasing consumption of sucrose has resulted in growing incidence of obesity, diabetes and cardiovascular diseases. Diterpene glycosides produced by Stevia rebaudiana Bertoni, known as steviol glycosides, are natural, high-potency low-calorie sweeteners, which has been used as an alternative to sucrose (Geuns, 2003). Steviol glycosides preparations, or stevia extracts, are commonly obtained by aqueous or solvent extraction from the leaves of the plant (Puri, Sharma, & Tiwari, 2011). Most commercial products have stevioside as the major component, followed by rebaudioside A and smaller amounts of other glycosides of steviol. Rebaudioside A, which has an extra glucose unit relative to stevioside, imparts the greatest potency of sweetness and less bitter aftertaste. Stevioside is 250e300 times sweeter than 0.4 g/100 g sucrose solutions but produces a significant bitter aftertaste (Lemus-Mondaca, Vega-Gálvez, Zura-Bravo, & Ah-Hen, 2012). As a result of the dominance of unpleasant bitter aftertaste due to stevioside, the use of stevia extracts for sweetening foods has to some extent been restricted. Transglycosylation has been used to modify the organoleptic or physicochemical properties of bioactive compounds in foods to
* Corresponding author. School of Food Science and Technology, Jiangnan University, No. 1800 Lihu Road, 214122 Wuxi, China. Tel./fax: þ86 510 85919150. E-mail address: [email protected] (R. Yang). 0023-6438/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2013.12.045
improve their use (Lee et al., 1999; Shin, Cheong, Lee, & Kim, 2009). Particularly, enzymatic transglycosylation was an effective tool for improving taste quality and sweetness potency of stevioside (Ohtani & Yamasaki, 2002). Kusama, Kusakabe, Nakamura, Eda, and Murakami (1986) studied the transglucosylation into stevioside by the enzyme system from Streptomyces sp. W19-1 with curdlan (or b-1, 3-glucan) as substrate, and the bitter taste of products was reduced. Fukunaga et al. (1989) studied the modification of stevioside with cyclodextringlucosyltransferase in the presence of soluble starch resulting in a mixture of a-glucosylated products, and the taste quality and sweetness potency of some products were claimed to be improved substantially. Ishikawa, Kitahata, Ohtani, Ikuhara, and Tanaka (1990) investigated the production of fructosyl-stevioside by transfructosylation of b-fructofuranosidase, and the taste quality of fructosyl-stevioside was found to be superior to that of rebaudioside A. Lobov, Kasai, Ohtani, Tanaka, and Yamasaki (1991) studied transglycosylation of stevioside by commercial pullanase and crude b-amylase, with pullulan and maltose as glycosyl donor, respectively. Transglycosylation of stevioside using cyclodextrin glucanotransferases (CGTases) from various microorganisms has been well investigated. In these CGTasescatalysed reactions, cyclodextrins or starches were employed as donor substrates (Abelyan, Balayan, Ghochikyan, & Markosyan, 2004; Jung, Kim, Lee, & Lee, 2007; Kochikyan, Markosyan, Abelyan, Balayan, & Abelyan, 2006) and the highest conversion yield of stevioside (90%) was obtained by microwave-assisted transglycosylation reaction (Jaitak et al., 2009).
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a-Amylases (EC 3.2.1.1) are a class of glycoside hydrolases widely distributed in nature and have recently attracted considerable research interest for their applications in food, textile, fermentation and pharmaceutical industries (Van der Maarel, van der Veen, Uitdehaag, Leemhuis, & Dijkhuizen, 2002). In addition to starch hydrolysis, some a-amylases were also used in transglucosylation reactions in the presence of various acceptor molecules (Mótyán et al., 2011; Rivera, López-Munguía, Soberón, & Saab-Rincón, 2003). Termamyl, a commercial a-amylase from Bacillus licheniformis, was found to catalyse the synthesis of oligosaccharide with starch and non-starch sugars as substrates (Chitradon, Mahakhan, & Bucke, 2000). Aspergillus oryzae a-amylase was capable of catalysing the synthesis of alkyl glucosides with starch and alcohols as substrates (Larsson, Svensson, & Adlercreutz, 2005). The transglucosylation activity of a-amylase from Trichoderma viride JCM22452 had been applied in the synthesis of flavonoid monoglucoside which showed higher heat stability and solubility and lower astringency and astringent stimulation than its aglycon (Noguchi, Inohara-Ochiai, Ishibashi, & Fukami, 2008). Moreno et al. (2010) reported the versatility of Thermotoga maritima a-amylase in transglucosylation reactions and its capacity to generate neotrehalose and alkyl-glucosides. In the present study, enzymatic modification of steviol glycosides was attempted using a commercial a-amylase from A. oryzae (TAKA) in the presence of soluble starch. To increase the conversion yield of stevioside, the reaction conditions, including pH, temperature, and substrate and enzyme concentrations, were studied. In addition, the organoleptic properties of the enzymatically modified steviol glycosides (EMSGs) were examined.
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molecular weight of the modified products was determined by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) mentioned in Section 2.4. 2.2.3. Transglycosylation reactions of stevioside In a 250-mL enzymatic reactor, stevia extract and soluble starch dissolved with buffer solution and TAKA were mixed, giving final reaction volume of 100 mL, and incubated for 0.5e12 h. The reaction mixture was deactivated by boiling and the resulting transglycosylated steviol glycosides were analysed by HPLC. The effects of pH (4.0e10.0), temperature (30e80 C), initial stevioside concentration (0.5e6.67 g/100 mL), initial starch concentration (2.5e 30 g/100 mL), and enzyme concentration (10e300 U/mL), were investigated. The detailed reaction conditions were described in the figure legends of Figs. 4e8 in Section 3. The optimal reaction conditions were considered in terms of obtaining the maximal conversion yield of stevioside. The conversion yield of stevioside was calculated from the decrease in stevioside at each time point compared with that at time t ¼ 0 min. Under optimal reaction conditions, a preparative scale production (2000 mL) of the EMSGs was then conducted. 2.2.4. Separation and purification of the EMSGs The aforedescribed product mixtures were filtered through a Büchner funnel and the filtrate was loaded on a 2.6 cm 100 cm column packed with macroporous adsorption resin DM 301 (Anhui Sanxing Resin Co., Anhui, China) and eluted with 1000 mL deionised water (1 BV/h) to remove the saccharides. Thereafter, the EMSGs were eluted with 80 mL/100 mL ethanol (1 BV/h), followed by concentration and lyophilization.
2. Materials and methods 2.3. High performance liquid chromatography (HPLC) 2.1. Materials
a-Amylase from A. oryzae (TAKA) was purchased from Sigmae Aldrich (Shanghai, China). Stevia extract (containing 90 g/100 g stevioside and 5 g/100 g rebaudioside A) was from Jining Aoxing Stevia Product Co., Ltd. (Shandong, China). Stevioside (>99 g/100 g by HPLC) and rebaudioside A (>97 g/100 g by HPLC) were from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Soluble starch, ethanol and other chemicals were all of analytical grade and from Sino-Pharm Chemical Co. (Beijing, China). Sucrose for sweetness potency determination was of food grade and from Nanjing Ganzhiyuan Sugar Co., Ltd. (Jiangsu, China). 2.2. Methods 2.2.1. Enzyme assay The activity of TAKA was assayed with 1.0 g/100 mL soluble starch prepared in 0.05 mol/L potassium phosphate buffer (pH 6.0) at 60 C using the 3, 5-dinitrosalicylic acid method (Bernfield, 1955). The absorbance of the reducing sugars liberated at 540 nm was measured using a UVevisible spectrophotometer (Unico UV2102C, WI, USA). One unit was defined as the amount of enzyme that released 1 mmol of reducing sugar equivalent to maltose per minute at pH 6.0 and 60 C. 2.2.2. Acceptor specificity Stevioside and rebaudioside A were used to assay their ability to function as acceptor substrates. Reactions were performed by incubating 1 g/100 mL acceptor substrate (stevioside or rebaudioside A), 10 g/100 mL soluble starch and 20 U/mL TAKA in 0.05 mol/L potassium phosphate buffer (pH 6.0, 100 mL) at 60 C for 1 h. The transglycosylation to the acceptor was evaluated by the peak area of the modified products shown on the HPLC chromatogram. The
The analysis of the modified products was conducted by an HPLC system (Hitachi L-2000, Tokyo, Japan) equipped with Hitachi L-2495 diode array detector and a Shodex Asahipak NH2P-50 4E chromatographic column (length 250 mm; inner diameter 4.6 mm). The column was eluted at 35 C with a 75:25 mixture of acetonitrile and Milli-Q water at 1.0 mL/min. The chromatograms were recorded at 210 nm. 2.4. UHPLC-MS Analysis UHPLC-MS analysis of the reaction mixtures was performed on a Waters Acquity ultra-high-performance liquid chromatography (UHPLC) system coupled to a Waters MALDI SYNAPT Q-TOF mass spectrometer (Waters Co., Ltd., Milford, MA, USA) with ESI (electrospray ionisation) source. Each sample was separated by an Acquity UPLC BEH amide column (2.1 mm 100 mm, 1.7 mm; Waters Co., Ltd., Milford, MA, USA). The injection volume was 1.0 mL, and elution was performed using an 80:20 mixture of acetonitrile and Milli-Q water (containing 0.02 mol/L ammonium acetate) at a flow rate of 0.3 mL/min. The column was maintained at 45 C. The spectrum at 210 nm was recorded. Mass spectra were obtained by ESI in the negative mode under the following conditions: ESI source block and desolvation temperature: 100 C and 300 C, respectively; capillary voltage: 2.8 kV; desolvation gas (N2) flow: 500 L/h and scan range m/z 100e2000. 2.5. Determination of relative sweetness and detection threshold for bitter aftertaste The determination of sweetness potency was performed according to the method as described by Ishikawa et al. (1990) and Parpmello, Versari, Castellari, and Galassi (2001). Seventeen
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Fig. 1. HPLC chromatogram of the transglycosylation reaction between stevioside and soluble starch showing the transglycosylated steviosides formation (reaction conditions: 1 g/ 100 mL stevioside, 10 g/100 mL soluble starch, and 20 U/mL TAKA in 0.05 mol/L potassium phosphate buffer, pH 6.0, temperature 60 C, incubation time 1 h). Peak identification: 1, stevioside; 2, mono-glycosylated stevioside; 3, di-glycosylated stevioside; 4e6, tri-glycosylated stevioside; 7, tetra-glycosylated stevioside.
panellists (eight males and nine females, aged 23 to 34 y with average age 28 y) were invited based on interest, availability, and knowledge of basic tastes (sweet, sour, salty, and bitter) as well as previous sensory analysis experience in assessing sweetener samples. Each panelist had greater than 300 h of training in proficiency testing of sweet tastes. The stevia extract and the purified EMSGs (described in Section 2.2.4) were dissolved in potable water to make appropriate concentrations: 0.0040, 0.0060, 0.0080, 0.0100, 0.0120, 0.0150, 0.0180, 0.0210, 0.0240, 0.0300, 0.0360, 0.0420, 0.0480, 0.0540 and 0.0600 g/100 mL. The test solutions were served as 20 mL aliquots in 30 mL odour-free paper cups and analyses were conducted at room temperature (25 C) in individual tasting booths under normal lighting conditions. Each panelist was presented with a pair of cups containing the sucrose standard (2 g/100 mL) and the test solution in each session. For each pair of cups, panellists were asked which of the two was sweeter and were instructed to record the sweetness potency rating by making the appropriate point on a 15 cm continuous line scale. The mid-point of the scale was the sucrose standard. They spat out the solutions without swallowing and rinsed their mouths between each test. The concentration of sample in next session was chosen depending on the response of the panellist. Finally, they pick out the test solution where the sweetness was equivalent to the sucrose standard. The relative sweetness of the test solution was calculated according to the following formula.
2.6. Statistical analysis The enzymatic assays and analytic measurements (except for UHPLC-MS analysis) were performed in triplicate and the results were expressed as mean values standard deviations. Data from determination of sweetness potency and bitter aftertaste threshold were statistically analysed using Student’s t-tests to determine significant differences between the stevia extract and the EMSGs. Statistical significance was accepted at a level of p 0.01. 3. Results and discussion 3.1. Acceptor specificity of transglycosylation reactions 3.1.1. Stevioside as the acceptor substrate In the reaction of stevioside as acceptor substrate, thirty percent of stevioside was depleted (calculation based on peak area of stevioside in the HPLC chart) and six modified products were detected by chromatographic analysis, with retention times of 10.01, 15.78, 25.58, 26.95, 28.27 and 43.57 min, respectively (Fig. 1). The peak 1 at 6.40 min was confirmed to be stevioside by
The relative sweetness ¼ A=B where A: concentration (g/100 mL) of the sucrose standard; B: concentration of the test solution (g/100 mL) which sweetness equivalent to the sucrose standard. To determine the threshold for bitter aftertaste of the stevia extract and the purified EMSGs (described in Section 2.2.4) were dissolved in potable water to make appropriate concentrations from 0.0160 g/100 mL to 0.0600 g/100 mL with intervals of 0.0040 g/100 mL. Twelve pairs of test solutions, in which one member of each pair was always a potable water control, were presented in randomised order in each session. Panellists were asked to indicate in which sample bitter aftertaste was perceivable. The value corresponding to 75% of positive responses indicated the panel threshold.
Fig. 2. Mass spectra of (a) mono-glycosylated stevioside and (b) di-glycosylated stevioside, respectively.
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Fig. 3. HPLC analysis of the products by treating rebaudioside A with TAKA in the presence of soluble starch (reaction conditions: 1 g/100 mL rebaudioside A, 10 g/100 mL soluble starch, and 20 U/mL TAKA in 0.05 mol/L potassium phosphate buffer, pH 6.0, temperature 60 C, incubation time 1 h). Peak identification: 8, rebaudioside A; 9, mono-glycosylated rebaudioside A; 10, di-glycosylated rebaudioside A; 11, tri-glycosylated rebaudioside A.
comparing its retention time with that of the standard. Molecular weight of the modified products was determined by UHPLC-MS. The compound corresponding to peak 2 was a mono-glycosylated stevioside, since it showed an [MeH] ion at m/z 965 in the extracted ion chromatogram (set m/z values 803, 965, 1127, 1289 and 1451, data not shown). The mono-glycosylated stevioside was the main modified product and its mass spectrum was presented in Fig. 2a, which gave an [MeH] ion at m/z 965.1 and [M þ Cl35] and [M þ Cl37] ion at m/z 1001.1 and 1003.1, respectively. The compound corresponding to peak 3 was a diglycosylated stevioside, which showed an [MeH] ion at m/z 1127.2 and [M þ Cl35] ion at m/z 1163.2 (Fig. 2b). Compared with the mono-glycosylated stevioside, the di-glycosylated stevioside gave a much smaller peak area (about 30% of the total modified products). Tri-glycosylated steviosides (peaks 4, 5 and 6) and tetra-glycosylated stevioside (peak 7) constituted about 9% (peak area) of the total modified products, which indicated that transferring more glucosyl residues onto the glycoside became more difficult. In the present study, stevioside was found be to an efficient acceptor substrate with TAKA, giving rise to a group of mono-, di-, and tri-glycosylated stevioside derivatives. 3.1.2. Rebaudioside A as the acceptor substrate Fig. 3 showed the HPLC profile of modified products formed by treating rebaudioside A with TAKA in the presence of soluble starch. By a comparison of the retention time with the standard, peak 8 at 8.99 min was assigned to rebaudioside A. After 1 h of reaction, 13% of rebaudioside A vanished and three new but weak peaks, respectively, at retention times of 14.47, 24.15 and 40.64 min appeared on the chromatogram. UHPLC-MS analysis of the modified products gave deprotonated molecular ions [MeH] at m/z 1127, 1289 and 1451, respectively, which implied that rebaudioside A was transformed to mono-, di-, and tri-glycosylated products. By calculating the peak area of the modified products shown on the HPLC chromatograms (Figs. 1 and 3), transglycosylation reaction performed with rebaudioside A as the acceptor substrate presented rather lower conversion yield (conversion yield of rebaudioside A: 13%) than that with stevioside (conversion yield of stevioside: 30%). Therefore, it can be concluded that rebaudioside A was a relatively poor transglycosylation acceptor.
3.2. Influence of reaction conditions on the conversion yield of stevioside In this section, stevia extract (stevioside 90 g/100 g) was used to investigate the influence of reaction conditions on the conversion yield of stevioside. The transformation of rebaudioside A was not considered due to its low conversion yield and low content in the stevia extract (rebaudioside A 5 g/100 g). 3.2.1. pH The conversion yield at different pH values indicated that reaction was favoured in the pH range of 5.0e7.0 (Fig. 4). The maximal conversion yield was observed at pH 6.4, which was used in further experiments. However, there was a marked decline in the conversion yield in basic pH and only 4.0% conversion yield was attained at pH 8.0. 3.2.2. Temperature The stability and activity of a-amylase are strongly associated with the reaction temperature (Apar & Özbek, 2004). As shown in
Fig. 4. Influence of pH on the conversion yield of stevioside. Reaction conditions: stevioside, 1 g/100 mL; soluble starch, 10 g/100 mL; TAKA, 20 U/mL; incubation of the mixture (total 100 mL) at 60 C for 12 h. Buffers and pH values: sodium acetate buffer (pH 4.0e5.8), potassium phosphate buffer (pH 6.0e8.0), TriseHCl buffer (pH 8.0e9.0), and glycine-NaOH buffer (pH 9.0e10.0). Molarity of the buffer was 0.05 mol/L. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
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Fig. 5. Influence of temperature on the conversion yield of stevioside. Reaction conditions: stevioside, 1 g/100 mL; soluble starch, 10 g/100 mL; TAKA, 20 U/mL; incubation of the mixture (total 100 mL, pH 6.4) for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
Fig. 7. Influence of initial starch concentration on the conversion yield of stevioside. Reaction conditions: substrate weight ratio (stevioside/starch), 1:10; TAKA, 20 U/mL; incubation of the mixture (total 100 mL) at 70 C, pH 6.4 for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
Fig. 5, the conversion yield increased with rising the reaction temperature up to 70 C. At lower test temperatures (30e50 C), the conversion yield increased slowly. Higher test temperatures (>50 C) facilitated the substrate diffusion and fastened the reaction velocity. Approximately an 88% increase in the conversion yield was obtained as the test temperature increased from 50 C to 70 C. However, the conversion yield dramatically decreased when the test temperature was above 70 C, which may have been caused by the thermal-induced inactivation of TAKA.
conversion yield up to 43.1% as the initial starch concentration increased from 2.5 g/100 mL to 20 g/100 mL (Fig. 7). A decrease of conversion yield was found as the initial starch concentration above 20 g/100 mL, which was mainly caused by the inefficiency of reaction due to limitation of substrate and enzyme diffusion at high viscosities (North, 1966; Slaughter, Ellis, & Butterworth, 2001).
3.2.3. Substrate weight ratio (stevioside/starch) Fig. 6 showed the influence of substrate weight ratios on the conversion yield of stevioside. It was found that the conversion yield decreased from 40.9% at the weight ratio 1:20 to 7.6% at the weight ratio 1:1.5. This decreasing trend may be due to substrate inhibition (López, Torrado, Fuciños, Guerra, & Pastrana, 2006). 3.2.4. Initial substrate concentration The initial substrate concentration is the most significant factor for transglycosylation reaction (Moreno et al., 2010; Mótyán et al., 2011). To investigate the influence of starch concentration on the conversion yield, the reactions were performed using different initial starch concentrations, in which the weight ratio of stevioside to starch was fixed at 1:10. The result showed a 2.1-fold increase in
Fig. 6. Influence of substrate weight ratio (stevioside/starch) on the conversion yield of stevioside. Reaction conditions: soluble starch, 10 g/100 mL; stevioside, 0.5e6.67 g/ 100 mL; TAKA, 20 U/mL; incubation of the mixture (total 100 mL) at 70 C, pH 6.4 for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
3.2.5. Enzyme concentration Fig. 8 showed the influence of enzyme concentration on the conversion yield of stevioside. Initially, the increase of enzyme loading could accelerate the reaction rate and enhance the conversion yield. At lower enzyme loading (10 U/mL and 20 U/mL), the reaction took more than 10 h to reach the maximal conversion yield of 28.2% and 43.1%, respectively. At 50 U/mL of TAKA, however, the reaction rate was much faster and the conversion yield was 48.2% in 0.5 h. The maximal conversion yield decreased marginally when the enzyme loading was above 50 U/mL, which might be caused by the formation of enzyme aggregate when excess enzyme was added. The enzyme molecules on the external surface of the aggregate were easy to react with substrate but the accessibility of enzyme molecules present inside the aggregate to substrate was reduced (Yadav & Devendran, 2012). Hence, the optimal conditions obtained were incubation of 2 g/ 100 mL stevioside and 20 g/100 mL soluble starch with 50 U/mL of TAKA in 0.05 mol/L potassium phosphate buffer (pH 6.4) at 70 C.
Fig. 8. Influence of enzyme concentration on the conversion yield of stevioside. Enzyme concentration：- 10 U/mL; B 20 U/mL; 50 U/mL; 7 75 U/mL; ◄ 150 U/ mL; < 300 U/mL. Reaction conditions: stevioside, 2 g/100 mL; soluble starch, 20 g/ 100 mL; incubation of the mixture (total 100 mL) at 70 C, pH 6.4 for 12 h. Data plots are means of triplicate determinations. Vertical bars represent standard deviations.
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Under these conditions, the conversion yield of stevioside achieved 48.2% in 0.5 h and 50.2% after a reaction period of 12 h. 3.3. Relative sweetness and bitter aftertaste threshold of the modified steviol glycosides Sucrose equivalency level had a significant effect on the relative sweetness of high-potency sweeteners (Parpmello et al., 2001; Schiffman, Booth, Losee, Pecore, & Warwick, 1995). The evaluation of the sweetness potency of stevioside becomes complex at high concentration due to the intensifying of nonsweet taste and aftertaste (Cardello, Da Silva, & Damasio, 1999). A low sucrose equivalency level (2 g/100 mL) was therefore chosen to evaluate the sweetness potency of the stevia extract and the EMSGs. The results showed that the stevia extract and the EMSGs were, respectively, 171 15.4 times and 208 13.8 times sweeter than 2 g/100 mL sucrose, suggesting a significant increase in the sweet potency of the modified steviol glycosides (p 0.01). In comparison with the threshold for bitter aftertaste of stevioside (0.0124 0.0042 g/ 100 mL), the threshold for bitter aftertaste of the EMSGs (0.0241 0.0061 g/100 mL) was significantly increased (p 0.01). It was noteworthy that the bitter aftertaste of the stevia extract was perceived even at the concentration it exhibited similar sweetness to 2 g/100 mL sucrose; while the EMSGs were sweeter and exhibited less bitter aftertaste than stevioside at the same concentration. 4. Conclusions This work reported the improvement of organoleptic properties of steviol glycosides by enzymatic modification using a-amylase from A. oryzae (TAKA) in the presence of soluble starch. It was found that stevioside in stevia extract was an efficient acceptor substrate and has been transglycosylated during the reaction, giving rise to a group of mono-, di-, and tri-glycosylated stevioside derivatives. Under optimal conditions, 48.2% of stevioside was transformed within 0.5 h and the mono-glycosylated stevioside was the major product. The study suggested that, in comparison with the stevia extract, the EMSGs had a significant improvement in sweetness potency and a remarkable decrease in bitter aftertaste. Acknowledgements Financial support from the Key Project of National Natural Science Fund (31230057) and the “Five-twelfth” National Science and Technology Support Program (2011BAD23B03) are appreciated. We also acknowledge the support from the Natural Science Foundation of Jiangsu Province (BK2011149) and the Research Program of State Key Laboratory of Food Science and Technology, Jiangnan University (SKLF-TS-200903). References Abelyan, V. A., Balayan, A. M., Ghochikyan, V. T., & Markosyan, A. A. (2004). Transglycosylation of stevioside by cyclodextrin glucanotransferases of various groups of microorganisms. Applied Biochemistry and Microbiology, 40, 129e134. Apar, D. K., & Özbek, B. (2004). a-Amylase inactivation by temperature during starch hydrolysis. Process Biochemistry, 39, 1137e1144. Bernfield, P. (1955). Amylases a/b. Methods in Enzymology, 1, 149e158. Cardello, H. M., Da Silva, M. A., & Damasio, M. H. (1999). Measurement of the relative sweetness of Stevia extract, aspartame and cyclamate/saccharin blends as compared to sucrose at different concentrations. Plant Foods for Human Nutrition, 54, 119e130.
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