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Modification of stevioside using transglucosylation activity of Bacillus amyloliquefaciens α-amylase to reduce its bitter aftertaste
LWT - Food Science and Technology 51 (2013) 524e530
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Modification of stevioside using transglucosylation activity of Bacillus amyloliquefaciens a-amylase to reduce its bitter aftertaste Fayin Ye a, b, Ruijin Yang a, b, *, Xiao Hua a, c, Qiuyun Shen a, b, Wei Zhao b, Wenbin Zhang b a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Wuxi 214122, China c Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, 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 10 February 2012 Received in revised form 3 December 2012 Accepted 9 December 2012
Stevioside is a natural high-intensity sweetener with low calorie. However, the intrinsic bitter aftertaste limits its application in food products. Transglucosylation of stevioside by a-amylase from Bacillus amyloliquefaciens in the starch solution to produce transglucosylated steviosides with reduced bitter aftertaste was investigated. Under the optimal conditions, the conversion yield of stevioside attained 38.3%. Two major transglucosylated steviosides, which accounted for 96 g/100 g of the total transglucosylated steviosides, were identified to be 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-(a-D-glucopyranosyl)-b-D-glucopyranosyl ester, and 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-[(4- O-a-D-glucopyranosyl)-a-Dglucopyranosyl]-b-D-glucopyranosyl ester by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Sensory evaluation demonstrated that, by enzymatic modification, the sweetness of two major transglucosylated steviosides was significantly improved and the bitter aftertaste was significantly reduced. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Stevioside a-Amylase Transglucosylation Stevia rebaudiana Enzymatic modification
1. Introduction Stevioside (13-[2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy] kaur-16-en-19-oic acid b-D-glucopyranosyl ester) is the major steviol glycoside extracted from Stevia rebaudiana Bertoni, a small shrub originally grown in Brazil and Paraguay. Since stevioside tastes about 300 times sweeter than sucrose in a 0.4 g/100 mL solution (Geuns, 2003), this compound is considered as a promising sugar substitute and low-caloric sweetener, and has been used in variety of foods, such as soft drinks, fruits, chocolates, soy sauce, chewing gum, yogurt, and other products (Garcia-Noguera, Weller, Oliveira, Rodrigues, & Fernandes, 2010; Kroyer, 2010; Shah, Jones, & Vasiljevic, 2010). A number of studies have suggested that stevioside may also exert therapeutic benefits, for its anti-hyperglycaemic (Gregersen, Jeppesen, Holst, & Hermansen, 2004), anti-hypertensive (Chan et al., 2000), anti-inflammatory (Boonkaewwan, Toskulkao, & Vongsakul, 2006), anti-tumour (Yasukawa, Kitanaka, & Seo, 2002), anti-diarrhoeal (Pariwat, Homvisasevongsa, Muanprasat, & Chatsudthipong, 2008), and immunomodulatory properties (Sehar, Kaul, Bani, Pal, & Saxena, 2008). * 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 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.12.005
Besides sweetness, stevioside has some undesirable bitter aftertaste, which partially restricts its use for human consumption (DuBois & Stephenson, 1985). To solve this problem, studies have been focused on producing debittered stevioside by enzymatic modification. An early work described the modification of stevioside with cyclodextringlucosyltransferase in the presence of soluble starch resulting in a mixture of a-glucosylated products, and the quality of taste of the products was improved (Fukunaga et al., 1989). Cyclodextrin glucanotransferases (CGTases) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli were used for transglycosylating stevioside with cyclodextrins or starch as glucosyl donor (Abelyan, Balayan, Ghochikyan, & Markosyan, 2004; Kochikyan, Markosyan, Abelyan, Balayan, & Abelyan, 2006). Jung, Kim, Lee, and Lee (2007) studied the transglycosylation of stevioside using CGTase from Bacillus sp. BL-12 with maltodextrin as the glucosyl donor, and the conversion yield of stevioside was 76%. Other approaches include enzymatic modification by pullanase, b-amylase (Lobov, Kasai, Ohtani, Tanaka, & Yamasaki, 1991), dextrin dextranase (Yamamoto, Yoshikawa, & Okada, 1994) and bfructofuranosidases (Ishikawa, Kitahata, Ohtani, Ikuhara, & Tanaka, 1990; Suzuki, Fukumura, Shibasaki-Kitakawa, & Yonemoto, 2002; Xu, Li, Wang, Yang, & Ning, 2009). However, modification of stevioside by new enzymes which can improve its intensity and quality of the sweetness is nevertheless still required.
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a-Amylases (1, 4-a-D-glucan glucanohydrolase; EC 3.2.1.1) are widely distributed in nature and belong to a family of glycoside hydrolases catalysing the cleavage of a-1, 4 glycosidic bonds in starch and related carbohydrates (MacGregor, 1988). a-Amylases are versatile catalysts that not only serve the primary function of hydrolysis, but also catalyse the transglucosylation reaction for glycosides or oligosaccharides synthesis. By selecting the acceptors added to the reaction medium, a-amylase is promising for synthesis of glycosides which are difficult to synthesise in other ways. Alkyl glucosides were synthesised from starch and alcohols using Aspergillus oryzae a-amylase as the catalyst (Larsson, Svensson, & Adlercreutz, 2005). Chitradon, Mahakhan, and Bucke (2000) reported the oligosaccharide synthesis by the reversed catalytic reaction using a-amylase in the presence of soluble starch and added b-methylglucoside, cellobiose and mannose. Moreno et al. (2010) investigated the synthesis of neotrehalose by Thermotoga maritima a-amylase. Mótyán et al. (2011) studied the transglycosylation by barley a-amylase 1 using maltoheptaose and maltopentaose donors and different chromophore containing acceptors. The production of oligosaccharide and glycosides by various a-amylases has been relatively well-studied. However, the transglucosylation of stevioside with a-amylases has received little research attention. Starch and a-amylases are both commercially available at relatively low cost, and starch is easy to remove after reaction. Based on the above considerations, a-amylase may find application in modifying stevioside in a more effective and economical manner. The objective of this study was to modify stevioside using the transglucosylation activity of a-amylase from Bacillus amyloliquefaciens. The structure of new compounds was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The sensory evaluation of the modified stevioside was conducted. 2. Materials and methods 2.1. Enzyme and chemicals BAN 480L (a-amylase from B. amyloliquefaciens) with a declared activity of 480 KNU/g was from Novozymes A/S (Bagsvaerd, Denmark) and was used without further purification. Stevioside standard (>99% by HPLC) and rebaudioside A standard (>97% by HPLC) were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. Stevioside (purity 95%) was purified from a commercial steviol glycosides extract (stevioside >55%; Jining Aoxing Stevia Products Co., Ltd, Shandong, China) in our laboratory (see Supplementary material). Soluble starch was from Sino-Pharm Chemical Reagent Co., Ltd., Shanghai, China. 2.2. Transglucosylation reactions For examining the glucosyl acceptor specificity of a-amylase BAN 480L, rebaudioside A and stevioside were served as glucosyl acceptors, respectively, and the following reaction conditions were used: the reaction mixture (total 100 mL) consisting of glucosyl acceptor (2.0 g) in the absence and presence of soluble starch as glucosyl donor (8.0 g) was incubated with BAN 480L (16 KNU) in 0.050 mol/L sodium phosphate buffer (pH 6.5) at 70 C for 12 h. Experimental design for optimisation of stevioside modification was outlined in Table 1. Effects of reaction temperature, pH, weight ratio of stevioside-to-starch, and substrates and enzyme concentrations as single factors on the yield of transglucosylated steviosides were investigated while other reaction conditions were kept constant. Samples were withdrawn at appropriate time intervals and immediately immersed in boiling water for 10 min to inactivate
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Table 1 Experimental design for optimisation of modification of stevioside. Level
Reaction temperature ( C)
Reaction pHa
Weight ratio of steviosideto-starchb
Initial starch concentration (g/100 mL)c
Enzyme concentration (KNU/g starch)d
1 2 3 4 5 6 7 8
40 50 60 70 80 90
4.0 5.0 6.0 6.5 7.5 8.5 9.5 10.5
1:10 1:8 1:6 1:4 1:2 1:1 2:1 4:1
2.0 4.0 8.0 10 16 24
1.0 2.0 5.0 7.0 10.0
a pH 4.0, 5.0 and 6.0: 0.050 mol/L sodium acetate buffer; pH 6.5 and 7.5: 0.050 mol/L sodium phosphate buffer; pH 8.5: 0.050 mol/L TriseHCl buffer; pH 9.5 and 10.5: 0.050 mol/L glycine-NaOH buffer. b Starch: at a fixed concentration of 10 g/100 mL and stevioside concentration: varied from 1.0 g/100 mL to 40 g/100 mL. c Weight ratio of stevioside-to-starch was kept at 1:4 and initial starch concentration was varied from 2.0 g/100 mL to 24 g/100 mL. d Optimisation of the enzyme concentration was conducted by reacting stevioside (2.0 g/100 mL) with starch (8.0 g/100 mL) at 70 C, pH 6.5 with 1.0e10.0 KNU/g starch BAN 480L for 12 h.
the enzyme, and then analysed by high performance liquid chromatography (HPLC). The data presented were the mean values of triplicate analyses. The data were subjected to one-way analysis of variance using SPSS 17.0 for windows software (SPSS Inc., Chicago, Illinois, USA). Comparisons of group means were obtained using Duncan’s multiple range test. Statistical significance was accepted at a level of p 0.05. The conversion yield of stevioside was calculated from the decrease in stevioside at each time point compared with the initial stevioside concentration. The optimal reaction conditions were employed to produce the modified stevioside in 2000 mL scale for further study. 2.3. Separation and purification of transglucosylated steviosides The reaction mixture was filtrated and loaded directly onto a column (1.6 cm i.d. 150 cm length) filled with macroporous adsorption resin DM301 (Anhui Sanxing Resin Co., Anhui, China). The column was eluted with 1000 mL deionized water, followed by 2000 mL of aqueous ethanol solution (85 mL/100 mL) at a flow rate of 5.0 mL/min. The collected eluate was condensed using a rotary evaporator (IKA RV 10, IKA-Werke GmbH & Co. KG, Staufen, Germany) and lyophilized (LabconcoÒ stoppering tray dryer, Labconco Co., Kansas, MO, USA), affording the modified stevioside (containing stevioside and transglucosylated steviosides). Two major transglucosylated steviosides (I and II) were isolated on a Shodex Asahipak NH2P-50 10E column (10.0 mm i.d. 250 mm, 5 mm; Showa Denko K. K, Tokyo, Japan) fitted with a Shodex Asahipak NH2P-50 7G guard column (Showa Denko K. K, Tokyo, Japan) with aqueous acetonitrile solution (70 mL/100 mL) acetonitrile aqueous as mobile phase at the flow rate of 3.0 mL/min at 35 C. 2.4. HPLC analysis The steviol glycosides in the reaction mixture were analysed on a Hitachi LaChrom Elite L-2000 series HPLC System (Hitachi Ltd., Tokyo, Japan) equipped with a diode-array detector and a Shodex Asahipak NH2P-50 4E column (Showa Denko K. K, Tokyo, Japan). The column was eluted at 35 C with aqueous acetonitrile solution (75 mL/100 mL) at a flow rate of 1.0 mL/min. The absorbance
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measurements were performed at 210 nm. Quantification of each steviol glycoside was performed by an external calibration curve. 2.5. LC-MS/MS analysis The reaction mixtures were analysed on a Waters Acquity ultraperformance liquid chromatography (UPLC) system coupled to a Waters MALDI SYNAPT Q-TOF mass spectrometer (Waters Co., Milford, MA, USA)，equipped with electrospray ionization (ESI) source in both ESI()-MS and ESI()-MS/MS modes. The steviol glycosides were separated in Acquity UPLC BEH amide column (2.1 mm 100 mm, 1.7 mm; Waters Co., Milford, MA, USA) with Milli-Q water containing 0.020 mol/L ammonium acetate (A) and acetonitrile (B) as eluents, at a flow rate of 0.3 mL/min with gradient elution: 0e10 min, 10% A; 10e15 min, 10e20% A; 15e 17 min, 20e30% A; 17e20 min, 30e50% A; 20e25 min, 50% A. Column oven set at 45 C, and diode-array detector scanned from 190 to 400 nm. MS parameters were ESI source block temperature of 100 C, desolvation temperature of 300 C, capillary voltage of 2.8 kV, and desolvation gas (nitrogen) flow of 500 L/h. In tandem mass spectrometry mode, the collision gas (argon) flow was 0.3 mL/ min, and the mass spectrometer was scanned from 100 to 2000 m/z. The whole system was controlled by MassLynx 4.1 software (Waters Co., Milford, MA, USA). 2.6. NMR analysis 1
H, 13C NMR, DEPT-135, HSQC and HMBC spectra were recorded in pyridine-d5 as solvent at 25 C with a Bruker Avance III Digital NMR Spectrometer (Bruker, Karlsruhe, Germany). Data acquisition and processing were done with Bruker Topspin 2.1 (Bruker, Karlsruhe, Germany). Coupling constants (J) are expressed in Hertz, and chemical shifts are given on a d (ppm) scale with TMS (tetramethylsilane) or solvent signals as an internal standard. 2.7. Sensory evaluation The sensory evaluation was carried out according to the literature (Pangborn, 1963; Parpinello, Versari, Castellari, & Galassi, 2001) with some modifications. The relative sweetness to 2 g/ 100 mL sucrose standard was evaluated by a human sensory panel. The modified stevioside (obtained from Section 2.3) and stevioside (purity 95%) were dissolved in drink water and diluted to different concentrations: 0.0040, 0.0050, 0.0064, 0.0071, 0.0080, 0.0091, 0.0100, 0.0111, 0.0117, 0.0133, 0.0154, 0.0182, and 0.0200 g/100 mL. The samples were served as 20 mL aliquots in 30 mL odour-free paper cups at room temperature (25 C) and analyses were performed in individual tasting booths under normal lighting conditions. Seventeen panelists (eight males and nine females at age 23e 34) were invited for the assessment of the taste of the products. They had been trained in the sensory analysis and had former experience in accessing sweetener samples. The panelist was presented with a pair of cups containing sucrose standard and the sample in one session. For each pair of cups, panelists were asked which of the two was sweeter and were instructed to record the sweetness potency rating by making the appropriate point on a 150 mm 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 panelist. Finally, the sample of which concentration elicited the same perceived sweetness intensity as the sucrose standard could be estimated. The relative sweetness was calculated according to the following formula.
The relative sweetness ¼ A=B where A: concentration (g/100 mL) of sucrose standard; B: concentration (g/100 mL) of the sample solution with the same intensity of sweetness as the sucrose standard. To determine the threshold for bitter aftertaste of stevioside and the modified stevioside, samples were dissolved in drink water to make solutions with varied concentrations: 0.0100, 0.0120, 0.0160, 0.0200, 0.0240, 0.0280, 0.0320, 0.0360, 0.0400, 0.0480, and 0.0600 g/100 mL. Eleven pairs of solutions, in which one member of each pair was always a drink water control, were presented in randomized order in each session. Panelists were asked to indicate in which sample bitter aftertaste was perceivable. The value corresponding to 75% of correct responses indicated the panel threshold. Data analysis was performed using SPSS 17.0 for windows software (SPSS Inc., Chicago, Illinois, USA). Student’s t-tests were applied to determine significant differences between the stevioside and the modified stevioside. Statistical significance was accepted at a level of p 0.01. 3. Results and discussion 3.1. Transglucosylation of rebaudioside A by BAN 480L Rebaudioside A, the least astringent and bitter, has the least persistent aftertaste and the greatest potency of sweetness (Phillips, 1989). Researches have been focused on the enzymatic modification of rebaudioside A with aiming to improve its taste (Kochikyan et al., 2006). In the present study, a preliminary experiment was conducted to evaluate the conversion yield of rebaudioside A when it is added as the glucosyl acceptor under the catalysis of BAN 480L. The results showed that rebaudioside A underwent neither hydrolysis nor transglucosylation reaction. In the chromatogram of the reaction mixtures without starch, no additional signals were observed, suggesting rebaudioside A remained intact in the presence of BAN 480L. By incubating rebaudioside A and starch with BAN 480L, a new peak was found in the chromatogram, which represents transglucosylated product. However, this signal was extremely weak and the calculated conversion yield was less than 1% (data not presented). It could be regarded that, due to its relatively low conversion yield, rebaudioside A kept intact when the modification of stevioside was performed using mixtures of steviol glycosides (containing rebaudioside A) as the starting material. 3.2. Transglucosylation of stevioside By using the Shodex Asahipak NH2P-50 4E column coupled with a ultraviolet detector, three new peaks (IeIII) appeared which belonged to the transglucosylation products of stevioside. I and II were the major products, which judged by the peak areas. Five transglucosylated steviosides were presented in the UPLC chromatogram, with retention times of 3.07, 5.61, 8.97, 9.46 and 9.76 min, respectively. III contained a mixture of three compounds. In the negative ion current chromatogram, the corresponding peaks were recorded at 3.13, 5.66, 8.97, 9.47, and 9.74 min, respectively (Fig. 1a). Since the formula weight of stevioside is 804.88, and m/z 641.3, 479.2, and 317.2 were the typical fragment ions of stevioside in negative-ion mode (Zimmermann, 2011), the quasi-molecular ion peaks [M H] at m/z 1289.5, 1127.4, 965.3, and 803.3 can be assigned as stevioside-(glucosyl) n ¼ 3e0. Compound I eluted at 3.13 min gave a deprotonated molecular ion at m/z 965.3 (Fig. 1b). Further fragmentation of this ion at m/z 965.3 yielded at least three product ions at m/z 803.3, m/z 641.3, and m/z 479.2 (Fig.1c), indicating
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Fig. 1. The negative ion current chromatogram and the MS spectra of steviol glycosides in the mixture of transglucosylation to stevioside. (a) Total ion chromatogram of the steviol glycosides. (b) MS spectrum of I. (c) MS/MS spectrum of I. (d) MS spectrum of II. (e) MS/MS spectrum of II.
the loss of a glucose unit. These results implied that compound I was a stevioside-(glucosyl) 1. Compound II assigned to the peak at a retention time of 5.66 min gave a deprotonated molecular ion at m/z 1127.4 (Fig. 1d); further fragmentation of this ion generated product ions at m/z 965.4 and m/z 641.3 in the MS/MS spectrum (Fig. 1e), implying that compound II was a stevioside-(glucosyl) 2 consisting of one stevioside and two glucose molecules. The compounds eluted at 8.97, 9.47, and 9.74 min belonged to stevioside-(glucosyl) 3, for they gave the deprotonated molecular ions at m/z 1289.5. The tandem mass spectrometric fragmentation patterns of these three compounds were different from each other (data not shown), suggesting that they were isomers with glucosyl moieties attached to stevioside via different types of glycosidic linkages. 3.3. Identification of I and II I and II were isolated by semi-preparative HPLC and characterized by NMR. Structure identification of III has not been carried out since their sum content is only 4 g/100 g. Assignment of the NMR data as shown in Supplementary material was made on the basis of HSQC and HMBC correlations and by the comparison of their chemical shifts with the reported data. The 1H NMR spectrum of I (Fig. 2a) exhibited four anomeric proton signals at d 6.11 (H-10 ), d 5.16 (H-100 ), d 5.24 (H-1000 ), and d5.91 (H-10000 ), and the 13C NMR spectrum of I showed four anomeric
carbon signals at d 96.4 (C-10 ), 98.5 (C-100 ), 107.4 (C-1000 ), and 103.5 (C-10000 ), which indicated that I was a mono-glucosylated stevioside. The large coupling constants of the three anomeric protons at d 6.11 (1H, d, J ¼ 8.2 Hz), 5.16 (1H, d, J ¼ 7.8 Hz), and 5.24 (1H, d, J ¼ 7.8 Hz) suggested their b-orientation, whereas the coupling constant for the fourth anomeric proton (H-1) at d 5.91(1H, d, J ¼ 3.8 Hz) suggested its a-orientation(Lobov et al., 1991). The downfield chemical shift value (þ7.2 ppm) of the C-2 position suggested the possible placement of the fourth glucosyl moiety which was supported by the HMBC correlations: H-20 /C-10000, C-10, C-30 ; H-10000 /C-20 , C-20000 , C-100. Based on the results from spectral studies, structure of I was assigned as 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl] oxy} ent-kaur-16-en-19-oic acid 2-O-(a-D-glucopyranosyl)b-D-glucopyranosyl ester (Fig. 3b). It was noteworthy that the structure of I was similar to that of rebaudioside E. The only difference between them was in the orientation of the fourth anomeric carbon, the fourth anomeric carbon of I was a-oriented, but the fourth anomeric carbon of rebaudioside E was b-oriented (Tanaka, Yamasaki, & Sakamoto, 1977). The 1H NMR spectrum of II (Fig. 2b) exhibited five anomeric proton signals at d 6.11 (1H, d, J ¼ 8.2 Hz, Glc0 ), 5.16 (1H, d, J ¼ 7.7 Hz, Glc00 ), 5.29 (1H, d, J ¼ 7.8 Hz, Glc000 ), 5.91 (1H, d, J ¼ 3.8 Hz, Glc0000 ) and 5.83 (1H, d, J ¼ 3.8 Hz, Glc’00000 ), and the 13C NMR and DEPT-135 NMR spectra showed five anomeric carbon signals at d 96.3 (C-10 ), 98.6 (C-100 ), 107.3 (C-1000 ), 103.3 (C-10000 ), and 103.7 (C-100000 ), and five
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Fig. 2. 1H NMR spectra of (a) I, and (b) II measured at 400.13 MHz in pyridine-d5 at 25 C.
carbinol methylene signals at d63.2 (C-60 ), 63.3 (C-600 ) 62.5 (C-6000 ) 62.0 (C-60000 ) 62.4 (C-600000 ), suggesting the presence of five glucosyl moieties in the structure. The 13C NMR data of glucosyl moieties Glc0 , Glc00 , Glc000 and Glc0000 of II were quite similar to that of I. Only a downfield chemical shift of C-40000 (þ7.0 ppm) was observed in the fourth glucosyl moiety (Glc0000 ) of II. The HMBC correlations H-40000 /C-100000, C-30000 , C-50000 ; H-1000000 /C-40000 , C-2000000 , C-500000 suggested the placement of the fifth glucosyl moiety at the C-40000 position of Glc0000 . Therefore, the structure of II was established as 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl] oxy} ent-kaur16-en-19-oic acid 2-O-[(4- O-a-D-glucopyranosyl)-a-D-glucopyranosyl]-b-D-glucopyranosyl ester (Fig. 3c). 3.4. Selecting reaction conditions for transglucosylated steviosides synthesis Fig. 4aed showed the effects of four factors on the synthesis of transglucosylated steviosides catalysed by BAN 480L in the starch
solution. The effect of temperature was examined and the results were shown in Fig. 4a. As the reaction temperature increased from 40 C to 70 C, the conversion yield increased significantly (Fig. 4a). A further increase in the reaction temperature resulted in a decline of the conversion yield, possibly because of the thermal-induced inactivation of the enzyme. As a result, the reaction temperature 70 C was selected for further study. The reaction was favoured at pH values ranging from 6.0 to 9.5, and the maximal conversion yield was attained at pH 6.5 (Fig. 4b). However, at lower pH (4.0 and 5.0) or higher pH (10.5), the conversion yield reduced to 5.9%, 13.2%, and 6.6%, respectively. As shown in Fig. 4c, the weight ratios of stevioside-to-starch of 1:10, 1:8, and 1:6 showed little effect on transglucosylated steviosides synthesis (p > 0.05), suggesting that the donor (starch) was sufficient for transglucosylation. However, the conversion yield decreased significantly (p 0.05) when the stevioside concentration was more than 2.5 g/100 mL. Fig. 4d presented the results on the effect of substrate concentration on transglucosylated steviosides synthesis. The substrate concentration had a significant effect on the conversion yield. A marked reduction in the conversion yield was observed when the initial starch concentration was increased from 2.0 g/100 mL to 24 g/100 mL, presumably due to substrate inhibition and the inefficiency of substrate diffusion at high concentrations. In the transglucosylation reactions, performed at an enzyme load of 1 KNU/g starch, the conversion yield of stevioside was 26.9%. The conversion yield increased with increasing the enzyme concentration until the maximal conversion yield 38.3% was reached at 2 KNU/g starch. Two major transglucosylated steviosides (I and II), which corresponded to 96 g/100 g of the total amount of transglucosylation products, was obtained. Further increases in the enzyme concentration resulted in no increase but a slight decrease in the conversion yield, probably due to the formation of enzyme aggregate or the partly hydrolysis of transglucosylation products. Previously, transglycosylation of stevioside had been conducted using various carbohydrases. Among these carbohydrases, a conversion yield of stevioside as high as 90% was obtained by using CGTase from Bacillus firmus with b-cyclodextrin as donor under microwave conditions (Jaitak et al., 2009). In this study, although the conversion yield of stevioside (38.3%) is relatively low, BAN 480L cannot glycosylated rebaudioside A but does transglycosylate stevioside. It is therefore possible that commercial steviol glycosides may be directly involved in the production of modified stevioside. Additionally, starch is a cheap and abundant material and BAN 480L is a commercial industrial enzyme. Thus, this approach has a potential application in industry.
Fig. 3. Structures of (a) stevioside; (b) I: 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-(a-D-glucopyranosyl)-b-D-glucopyranosyl ester; and (c) II: 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-[(4- O-a-D-glucopyranosyl)-a-D-glucopyranosyl]-b-D-glucopyranosyl ester.
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Fig. 4. Effects of physicochemical factors on the conversion yield of stevioside. Data plots are means of triplicate determinations. Vertical bars represent standard deviations. (a) Effect of temperature. Reaction conditions: stevioside 2.0 g/100 mL, starch 8.0 g/100 mL, enzyme load 1.0 KNU/g starch, deionized water as reaction media (pH 6.8) and incubation for 12 h. (b) Effect of pH. Reactions were carried out at 70 C in 0.050 mol/L buffers (sodium acetate buffer: pH 4.0e6.0, sodium phosphate buffer: pH 6.5e7.5, TriseHCl buffer: pH 8.5, glycine-NaOH buffer: pH 9.5e10.5) containing 2.0 g/100 mL stevioside and 8.0 g/100 mL starch, and enzyme load 1.0 KNU/g starch for 12 h. (c) Effect of weight ratio of stevioside-to-starch. The reactions were performed in 0.050 mol/L sodium phosphate buffer pH 6.5 at 70 C for 12 h with 1.0 KNU/g starch of enzyme. Different alphabets at the tops of the bars indicate significant differences (p 0.05). (d) Effect of initial starch concentration. The reaction mixtures with enzyme load 1.0 KNU/g starch were incubated in 0.050 mol/L sodium phosphate buffer pH 6.5 at 70 C for 12 h.
3.5. Relative sweetness and bitter aftertaste threshold of the modified stevioside
modified stevioside was significantly reduced. This research has provided a new method to modify stevioside.
According to the literature, the relative sweetness is strongly dependent on sucrose equivalency level for all high-potency sweeteners. For instance, rebaudioside A tastes 200 times sweeter than 6 g/ 100 mL sucrose (Walters, Orthoefer, & DuBois, 1991); however, the sweetness potency of rebaudioside A is 400 times that of 2 g/100 mL sucrose (Schiffman, Booth, Losee, Pecore, & Warwick, 1995). In this study, 2 g/100 mL sucrose was used to determine the relative sweetness of the modified stevioside. The sensory data showed that stevioside and the modified stevioside were, respectively, 173 19.0 times and 202 16.2 times sweeter than 2 g/100 mL sucrose, suggesting a significant improvement of the sweetness potency for the modified stevioside (p 0.01). In comparison with the threshold for bitter aftertaste of stevioside (0.0172 0.0050 g/100 mL), nevertheless, the threshold for bitter aftertaste of the modified stevioside (0.0263 0.0056 g/100 mL) was significantly increased (p 0.01). The results indicated that the modified stevioside was sweeter and more pleasant-tasting than stevioside.
Acknowledgements
4. Conclusions
a-Amylase BAN 480L from B. amyloliquefaciens has been studied to catalyse the transglucosylation of stevioside with starch as glucosyl donor. It was possible to achieve the conversion yield of stevioside up to 38.3%. Two major transglucosylated steviosides, which accounted for 96 g/100 g of the total transglucosylation products, were isolated and identified. The bitter aftertaste of
This study was supported by the National Key Technology R&D Program in the 12th Five year Plan of China (2011BAD23B03), the Key Project of National Natural Science Fund (31230057), the Open Research Project of Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of Education (KLCCB-KF201206) and the Natural Science Foundation of Jiangsu Province (BK2011149). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2012.12.005. 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. Boonkaewwan, C., Toskulkao, C., & Vongsakul, M. (2006). Anti-inflammatory and immunomodulatory activities of stevioside and its metabolite steviol on THP-1 cells. Journal of Agriculture and Food Chemistry, 54, 785e789. Chan, P., Tomlinson, B., Chen, Y. J., Liu, J. C., Hsieh, M. H., & Cheng, J. T. (2000). A double blind placebo-controlled study of the effectiveness and tolerability of oral stevioside in human hypertension. British Journal of Clinical Pharmacology, 50, 215e220. Chitradon, L., Mahakhan, P., & Bucke, C. (2000). Oligosaccharide synthesis by reversed catalysis using a-amylase from Bacillus licheniformis. Journal of Molecular Catalysis B: Enzymatic, 10, 273e280.
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DuBois, G. E., & Stephenson, R. A. (1985). Diterpenoid sweeteners. Synthesis and sensory evaluation of stevioside analogues with improved organoleptic properties. Journal of Medicinal Chemistry, 28, 93e98. Fukunaga, Y., Miyata, T., Nakayasu, N., Mizutani, K., Kasai, R., & Tanaka, O. (1989). Enzymatic transglucosylation product of stevioside: separation and sweetness evaluation. Agricultural and Biological Chemistry, 53, 1603e1607. Garcia-Noguera, J., Weller, C. L., Oliveira, F. I. P., Rodrigues, S., & Fernandes, F. A. N. (2010). Dual-stage sugar substitution in strawberries with a Stevia-based sweetener. Innovative Food Science and Emerging Technologies, 11, 225e230. Geuns, J. M. C. (2003). Stevioside. Phytochemistry, 64, 913e921. Gregersen, S., Jeppesen, P. B., Holst, J. J., & Hermansen, K. (2004). Antihyperglycemic effects of stevioside in type 2 diabetic subjects. Metabolism, 53, 73e76. Ishikawa, H., Kitahata, S., Ohtani, K., Ikuhara, C., & Tanaka, O. (1990). Production of stevioside and rubusoside derivatives by transfructosylation of b-fructofuranosidase. Agricultural and Biological Chemistry, 54, 3137e3143. Jaitak, V., Kaul, V. K., Bandna, Kumar, N., Singh, B., Savergave, L. S., Jogdand, V. V., et al. (2009). Simple and efficient enzymatic transglycosylation of stevioside by b-cyclodextrin glucanotransferase from Bacillus firmus. Biotechnology Letters, 31, 1415e1420. Jung, S. W., Kim, T. K., Lee, K. W., & Lee, Y. H. (2007). Catalytic properties of bcyclodextrin glucanotransferase from alkalophilic Bacillus sp. BL-12 and intermolecular transglycosylation of stevioside. Biotechnology and Bioprocess Engineering, 12, 207e212. Kochikyan, V. T., Markosyan, A. A., Abelyan, L. A., Balayan, A. M., & Abelyan, V. A. (2006). Combined enzymatic modification of stevioside and rebaudioside A. Applied Biochemistry and Microbiology, 42, 31e37. Kroyer, G. (2010). Stevioside and stevia-sweetener in food: application, stability and interaction with food ingredients. Journal für Verbraucherschutz und Lebensmittelsicherheit, 5, 225e229. Larsson, J., Svensson, D., & Adlercreutz, P. (2005). a-Amylase-catalysed synthesis of alkyl glycosides. Journal of Molecular Catalysis B: Enzymatic, 37, 84e87. Lobov, S. V., Kasai, R., Ohtani, K., Tanaka, O., & Yamasaki, K. (1991). Enzymatic production of sweet stevioside derivatives: transglucosylation by glucosidases. Agricultural and Biological Chemistry, 55, 2959e2965. MacGregor, E. A. (1988). a-Amylase structure and activity. Journal of Protein Chemistry, 7(4), 399e415. Moreno, A., Damian-Almazo, J. Y., Miranda, A., Saab-Rincon, G., Gonzalez, F., & Lopez-Munguia, A. (2010). 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. Pangborn, R. M. (1963). Relative taste intensities of selected sugars and organic acids. Journal of Food Science, 28, 726e733.
Pariwat, P., Homvisasevongsa, S., Muanprasat, C., & Chatsudthipong, V. (2008). A natural plant-derived dihydroisosteviol prevents cholera toxin-induced intestinal fluid secretion. Journal of Pharmacology and Experimental Therapeutics, 324, 798e805. Parpinello, 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. Phillips, K. C. (1989). Stevia: steps in developing a new sweetener. In T. H. Grenby (Ed.), Developments in sweeteners, Vol. 3 (pp. 1e43). London: Elsevier Applied Science. 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, 505e513. Sehar, I., Kaul, A., Bani, S., Pal, H. C., & Saxena, A. K. (2008). Immune up regulatory response of a non-caloric natural sweetener, stevioside. Chemico-Biological Interactions, 173, 115e121. Shah, A. B., Jones, G. P., & Vasiljevic, T. (2010). Sucrose-free chocolate sweetened with Stevia rebaudiana extract and containing different bulking agents-effects on physicochemical and sensory properties. International Journal of Food Science and Technology, 45, 1426e1435. Suzuki, K., Fukumura, T., Shibasaki-Kitakawa, N., & Yonemoto, T. (2002). Kinetic model for synthesis of fructosyl-stevioside using suspended b-fructofuranosidase. Biochemical Engineering Journal, 10, 207e215. Tanaka, O., Yamasaki, K., & Sakamoto, I. (1977). Application of 13C NMR spectroscopy to chemistry of plant glycosides: rebaudioside-D and -E, new sweet diterpeneglucosides of Stevia rebaudiana Bertoni. Chemical & Pharmaceutical Bulletin, 25, 3437e3439. Walters, E., Orthoefer, F. T., & DuBois, G. E. (1991). Sweeteners: discovery, molecular design, and chemoreception. In Symposium sponsored by the Division of Agricultural and Food Chemistry at the 199th National Meeting of the American Chemical Society, April 22e27, 1990,. Boston. ACS Symposium Series, Vol. 450 (pp. 261e276). Washington, DC: American Chemical Society (ACS). Xu, Z. W., Li, Y. Q., Wang, Y. H., Yang, B., & Ning, Z. X. (2009). Production of bfructofuranosidase by Arthrobacter sp. and its application in the modification of stevioside and rebaudioside A. Food Technology and Biotechnology, 47, 137e143. Yamamoto, K., Yoshikawa, K., & Okada, S. (1994). Effective production of glycosylstevioside a-1, 6-transglucosylation by dextrin dextranase. Bioscience, Biotechnology, and Biochemistry, 58, 1657e1661. Yasukawa, K., Kitanaka, S., & Seo, S. (2002). Inhibitory effect of stevioside on tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin. Biological and Pharmaceutical Bulletin, 25, 1488e1490. Zimmermann, B. F. (2011). Tandem mass spectrometric fragmentation patterns of known and new steviol glycosides with structure proposals. Rapid Communications in Mass Spectrometry, 25, 1575e1582.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Modification of stevioside using transglucosylation activity of Bacillus amyloliquefaciens a-amylase to reduce its bitter aftertaste Fayin Ye a, b, Ruijin Yang a, b, *, Xiao Hua a, c, Qiuyun Shen a, b, Wei Zhao b, Wenbin Zhang b a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Wuxi 214122, China c Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, 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 10 February 2012 Received in revised form 3 December 2012 Accepted 9 December 2012
Stevioside is a natural high-intensity sweetener with low calorie. However, the intrinsic bitter aftertaste limits its application in food products. Transglucosylation of stevioside by a-amylase from Bacillus amyloliquefaciens in the starch solution to produce transglucosylated steviosides with reduced bitter aftertaste was investigated. Under the optimal conditions, the conversion yield of stevioside attained 38.3%. Two major transglucosylated steviosides, which accounted for 96 g/100 g of the total transglucosylated steviosides, were identified to be 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-(a-D-glucopyranosyl)-b-D-glucopyranosyl ester, and 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-[(4- O-a-D-glucopyranosyl)-a-Dglucopyranosyl]-b-D-glucopyranosyl ester by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Sensory evaluation demonstrated that, by enzymatic modification, the sweetness of two major transglucosylated steviosides was significantly improved and the bitter aftertaste was significantly reduced. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Stevioside a-Amylase Transglucosylation Stevia rebaudiana Enzymatic modification
1. Introduction Stevioside (13-[2-O-b-D-glucopyranosyl-b-D-glucopyranosyl) oxy] kaur-16-en-19-oic acid b-D-glucopyranosyl ester) is the major steviol glycoside extracted from Stevia rebaudiana Bertoni, a small shrub originally grown in Brazil and Paraguay. Since stevioside tastes about 300 times sweeter than sucrose in a 0.4 g/100 mL solution (Geuns, 2003), this compound is considered as a promising sugar substitute and low-caloric sweetener, and has been used in variety of foods, such as soft drinks, fruits, chocolates, soy sauce, chewing gum, yogurt, and other products (Garcia-Noguera, Weller, Oliveira, Rodrigues, & Fernandes, 2010; Kroyer, 2010; Shah, Jones, & Vasiljevic, 2010). A number of studies have suggested that stevioside may also exert therapeutic benefits, for its anti-hyperglycaemic (Gregersen, Jeppesen, Holst, & Hermansen, 2004), anti-hypertensive (Chan et al., 2000), anti-inflammatory (Boonkaewwan, Toskulkao, & Vongsakul, 2006), anti-tumour (Yasukawa, Kitanaka, & Seo, 2002), anti-diarrhoeal (Pariwat, Homvisasevongsa, Muanprasat, & Chatsudthipong, 2008), and immunomodulatory properties (Sehar, Kaul, Bani, Pal, & Saxena, 2008). * 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 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.12.005
Besides sweetness, stevioside has some undesirable bitter aftertaste, which partially restricts its use for human consumption (DuBois & Stephenson, 1985). To solve this problem, studies have been focused on producing debittered stevioside by enzymatic modification. An early work described the modification of stevioside with cyclodextringlucosyltransferase in the presence of soluble starch resulting in a mixture of a-glucosylated products, and the quality of taste of the products was improved (Fukunaga et al., 1989). Cyclodextrin glucanotransferases (CGTases) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli were used for transglycosylating stevioside with cyclodextrins or starch as glucosyl donor (Abelyan, Balayan, Ghochikyan, & Markosyan, 2004; Kochikyan, Markosyan, Abelyan, Balayan, & Abelyan, 2006). Jung, Kim, Lee, and Lee (2007) studied the transglycosylation of stevioside using CGTase from Bacillus sp. BL-12 with maltodextrin as the glucosyl donor, and the conversion yield of stevioside was 76%. Other approaches include enzymatic modification by pullanase, b-amylase (Lobov, Kasai, Ohtani, Tanaka, & Yamasaki, 1991), dextrin dextranase (Yamamoto, Yoshikawa, & Okada, 1994) and bfructofuranosidases (Ishikawa, Kitahata, Ohtani, Ikuhara, & Tanaka, 1990; Suzuki, Fukumura, Shibasaki-Kitakawa, & Yonemoto, 2002; Xu, Li, Wang, Yang, & Ning, 2009). However, modification of stevioside by new enzymes which can improve its intensity and quality of the sweetness is nevertheless still required.
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a-Amylases (1, 4-a-D-glucan glucanohydrolase; EC 3.2.1.1) are widely distributed in nature and belong to a family of glycoside hydrolases catalysing the cleavage of a-1, 4 glycosidic bonds in starch and related carbohydrates (MacGregor, 1988). a-Amylases are versatile catalysts that not only serve the primary function of hydrolysis, but also catalyse the transglucosylation reaction for glycosides or oligosaccharides synthesis. By selecting the acceptors added to the reaction medium, a-amylase is promising for synthesis of glycosides which are difficult to synthesise in other ways. Alkyl glucosides were synthesised from starch and alcohols using Aspergillus oryzae a-amylase as the catalyst (Larsson, Svensson, & Adlercreutz, 2005). Chitradon, Mahakhan, and Bucke (2000) reported the oligosaccharide synthesis by the reversed catalytic reaction using a-amylase in the presence of soluble starch and added b-methylglucoside, cellobiose and mannose. Moreno et al. (2010) investigated the synthesis of neotrehalose by Thermotoga maritima a-amylase. Mótyán et al. (2011) studied the transglycosylation by barley a-amylase 1 using maltoheptaose and maltopentaose donors and different chromophore containing acceptors. The production of oligosaccharide and glycosides by various a-amylases has been relatively well-studied. However, the transglucosylation of stevioside with a-amylases has received little research attention. Starch and a-amylases are both commercially available at relatively low cost, and starch is easy to remove after reaction. Based on the above considerations, a-amylase may find application in modifying stevioside in a more effective and economical manner. The objective of this study was to modify stevioside using the transglucosylation activity of a-amylase from Bacillus amyloliquefaciens. The structure of new compounds was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The sensory evaluation of the modified stevioside was conducted. 2. Materials and methods 2.1. Enzyme and chemicals BAN 480L (a-amylase from B. amyloliquefaciens) with a declared activity of 480 KNU/g was from Novozymes A/S (Bagsvaerd, Denmark) and was used without further purification. Stevioside standard (>99% by HPLC) and rebaudioside A standard (>97% by HPLC) were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. Stevioside (purity 95%) was purified from a commercial steviol glycosides extract (stevioside >55%; Jining Aoxing Stevia Products Co., Ltd, Shandong, China) in our laboratory (see Supplementary material). Soluble starch was from Sino-Pharm Chemical Reagent Co., Ltd., Shanghai, China. 2.2. Transglucosylation reactions For examining the glucosyl acceptor specificity of a-amylase BAN 480L, rebaudioside A and stevioside were served as glucosyl acceptors, respectively, and the following reaction conditions were used: the reaction mixture (total 100 mL) consisting of glucosyl acceptor (2.0 g) in the absence and presence of soluble starch as glucosyl donor (8.0 g) was incubated with BAN 480L (16 KNU) in 0.050 mol/L sodium phosphate buffer (pH 6.5) at 70 C for 12 h. Experimental design for optimisation of stevioside modification was outlined in Table 1. Effects of reaction temperature, pH, weight ratio of stevioside-to-starch, and substrates and enzyme concentrations as single factors on the yield of transglucosylated steviosides were investigated while other reaction conditions were kept constant. Samples were withdrawn at appropriate time intervals and immediately immersed in boiling water for 10 min to inactivate
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Table 1 Experimental design for optimisation of modification of stevioside. Level
Reaction temperature ( C)
Reaction pHa
Weight ratio of steviosideto-starchb
Initial starch concentration (g/100 mL)c
Enzyme concentration (KNU/g starch)d
1 2 3 4 5 6 7 8
40 50 60 70 80 90
4.0 5.0 6.0 6.5 7.5 8.5 9.5 10.5
1:10 1:8 1:6 1:4 1:2 1:1 2:1 4:1
2.0 4.0 8.0 10 16 24
1.0 2.0 5.0 7.0 10.0
a pH 4.0, 5.0 and 6.0: 0.050 mol/L sodium acetate buffer; pH 6.5 and 7.5: 0.050 mol/L sodium phosphate buffer; pH 8.5: 0.050 mol/L TriseHCl buffer; pH 9.5 and 10.5: 0.050 mol/L glycine-NaOH buffer. b Starch: at a fixed concentration of 10 g/100 mL and stevioside concentration: varied from 1.0 g/100 mL to 40 g/100 mL. c Weight ratio of stevioside-to-starch was kept at 1:4 and initial starch concentration was varied from 2.0 g/100 mL to 24 g/100 mL. d Optimisation of the enzyme concentration was conducted by reacting stevioside (2.0 g/100 mL) with starch (8.0 g/100 mL) at 70 C, pH 6.5 with 1.0e10.0 KNU/g starch BAN 480L for 12 h.
the enzyme, and then analysed by high performance liquid chromatography (HPLC). The data presented were the mean values of triplicate analyses. The data were subjected to one-way analysis of variance using SPSS 17.0 for windows software (SPSS Inc., Chicago, Illinois, USA). Comparisons of group means were obtained using Duncan’s multiple range test. Statistical significance was accepted at a level of p 0.05. The conversion yield of stevioside was calculated from the decrease in stevioside at each time point compared with the initial stevioside concentration. The optimal reaction conditions were employed to produce the modified stevioside in 2000 mL scale for further study. 2.3. Separation and purification of transglucosylated steviosides The reaction mixture was filtrated and loaded directly onto a column (1.6 cm i.d. 150 cm length) filled with macroporous adsorption resin DM301 (Anhui Sanxing Resin Co., Anhui, China). The column was eluted with 1000 mL deionized water, followed by 2000 mL of aqueous ethanol solution (85 mL/100 mL) at a flow rate of 5.0 mL/min. The collected eluate was condensed using a rotary evaporator (IKA RV 10, IKA-Werke GmbH & Co. KG, Staufen, Germany) and lyophilized (LabconcoÒ stoppering tray dryer, Labconco Co., Kansas, MO, USA), affording the modified stevioside (containing stevioside and transglucosylated steviosides). Two major transglucosylated steviosides (I and II) were isolated on a Shodex Asahipak NH2P-50 10E column (10.0 mm i.d. 250 mm, 5 mm; Showa Denko K. K, Tokyo, Japan) fitted with a Shodex Asahipak NH2P-50 7G guard column (Showa Denko K. K, Tokyo, Japan) with aqueous acetonitrile solution (70 mL/100 mL) acetonitrile aqueous as mobile phase at the flow rate of 3.0 mL/min at 35 C. 2.4. HPLC analysis The steviol glycosides in the reaction mixture were analysed on a Hitachi LaChrom Elite L-2000 series HPLC System (Hitachi Ltd., Tokyo, Japan) equipped with a diode-array detector and a Shodex Asahipak NH2P-50 4E column (Showa Denko K. K, Tokyo, Japan). The column was eluted at 35 C with aqueous acetonitrile solution (75 mL/100 mL) at a flow rate of 1.0 mL/min. The absorbance
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measurements were performed at 210 nm. Quantification of each steviol glycoside was performed by an external calibration curve. 2.5. LC-MS/MS analysis The reaction mixtures were analysed on a Waters Acquity ultraperformance liquid chromatography (UPLC) system coupled to a Waters MALDI SYNAPT Q-TOF mass spectrometer (Waters Co., Milford, MA, USA)，equipped with electrospray ionization (ESI) source in both ESI()-MS and ESI()-MS/MS modes. The steviol glycosides were separated in Acquity UPLC BEH amide column (2.1 mm 100 mm, 1.7 mm; Waters Co., Milford, MA, USA) with Milli-Q water containing 0.020 mol/L ammonium acetate (A) and acetonitrile (B) as eluents, at a flow rate of 0.3 mL/min with gradient elution: 0e10 min, 10% A; 10e15 min, 10e20% A; 15e 17 min, 20e30% A; 17e20 min, 30e50% A; 20e25 min, 50% A. Column oven set at 45 C, and diode-array detector scanned from 190 to 400 nm. MS parameters were ESI source block temperature of 100 C, desolvation temperature of 300 C, capillary voltage of 2.8 kV, and desolvation gas (nitrogen) flow of 500 L/h. In tandem mass spectrometry mode, the collision gas (argon) flow was 0.3 mL/ min, and the mass spectrometer was scanned from 100 to 2000 m/z. The whole system was controlled by MassLynx 4.1 software (Waters Co., Milford, MA, USA). 2.6. NMR analysis 1
H, 13C NMR, DEPT-135, HSQC and HMBC spectra were recorded in pyridine-d5 as solvent at 25 C with a Bruker Avance III Digital NMR Spectrometer (Bruker, Karlsruhe, Germany). Data acquisition and processing were done with Bruker Topspin 2.1 (Bruker, Karlsruhe, Germany). Coupling constants (J) are expressed in Hertz, and chemical shifts are given on a d (ppm) scale with TMS (tetramethylsilane) or solvent signals as an internal standard. 2.7. Sensory evaluation The sensory evaluation was carried out according to the literature (Pangborn, 1963; Parpinello, Versari, Castellari, & Galassi, 2001) with some modifications. The relative sweetness to 2 g/ 100 mL sucrose standard was evaluated by a human sensory panel. The modified stevioside (obtained from Section 2.3) and stevioside (purity 95%) were dissolved in drink water and diluted to different concentrations: 0.0040, 0.0050, 0.0064, 0.0071, 0.0080, 0.0091, 0.0100, 0.0111, 0.0117, 0.0133, 0.0154, 0.0182, and 0.0200 g/100 mL. The samples were served as 20 mL aliquots in 30 mL odour-free paper cups at room temperature (25 C) and analyses were performed in individual tasting booths under normal lighting conditions. Seventeen panelists (eight males and nine females at age 23e 34) were invited for the assessment of the taste of the products. They had been trained in the sensory analysis and had former experience in accessing sweetener samples. The panelist was presented with a pair of cups containing sucrose standard and the sample in one session. For each pair of cups, panelists were asked which of the two was sweeter and were instructed to record the sweetness potency rating by making the appropriate point on a 150 mm 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 panelist. Finally, the sample of which concentration elicited the same perceived sweetness intensity as the sucrose standard could be estimated. The relative sweetness was calculated according to the following formula.
The relative sweetness ¼ A=B where A: concentration (g/100 mL) of sucrose standard; B: concentration (g/100 mL) of the sample solution with the same intensity of sweetness as the sucrose standard. To determine the threshold for bitter aftertaste of stevioside and the modified stevioside, samples were dissolved in drink water to make solutions with varied concentrations: 0.0100, 0.0120, 0.0160, 0.0200, 0.0240, 0.0280, 0.0320, 0.0360, 0.0400, 0.0480, and 0.0600 g/100 mL. Eleven pairs of solutions, in which one member of each pair was always a drink water control, were presented in randomized order in each session. Panelists were asked to indicate in which sample bitter aftertaste was perceivable. The value corresponding to 75% of correct responses indicated the panel threshold. Data analysis was performed using SPSS 17.0 for windows software (SPSS Inc., Chicago, Illinois, USA). Student’s t-tests were applied to determine significant differences between the stevioside and the modified stevioside. Statistical significance was accepted at a level of p 0.01. 3. Results and discussion 3.1. Transglucosylation of rebaudioside A by BAN 480L Rebaudioside A, the least astringent and bitter, has the least persistent aftertaste and the greatest potency of sweetness (Phillips, 1989). Researches have been focused on the enzymatic modification of rebaudioside A with aiming to improve its taste (Kochikyan et al., 2006). In the present study, a preliminary experiment was conducted to evaluate the conversion yield of rebaudioside A when it is added as the glucosyl acceptor under the catalysis of BAN 480L. The results showed that rebaudioside A underwent neither hydrolysis nor transglucosylation reaction. In the chromatogram of the reaction mixtures without starch, no additional signals were observed, suggesting rebaudioside A remained intact in the presence of BAN 480L. By incubating rebaudioside A and starch with BAN 480L, a new peak was found in the chromatogram, which represents transglucosylated product. However, this signal was extremely weak and the calculated conversion yield was less than 1% (data not presented). It could be regarded that, due to its relatively low conversion yield, rebaudioside A kept intact when the modification of stevioside was performed using mixtures of steviol glycosides (containing rebaudioside A) as the starting material. 3.2. Transglucosylation of stevioside By using the Shodex Asahipak NH2P-50 4E column coupled with a ultraviolet detector, three new peaks (IeIII) appeared which belonged to the transglucosylation products of stevioside. I and II were the major products, which judged by the peak areas. Five transglucosylated steviosides were presented in the UPLC chromatogram, with retention times of 3.07, 5.61, 8.97, 9.46 and 9.76 min, respectively. III contained a mixture of three compounds. In the negative ion current chromatogram, the corresponding peaks were recorded at 3.13, 5.66, 8.97, 9.47, and 9.74 min, respectively (Fig. 1a). Since the formula weight of stevioside is 804.88, and m/z 641.3, 479.2, and 317.2 were the typical fragment ions of stevioside in negative-ion mode (Zimmermann, 2011), the quasi-molecular ion peaks [M H] at m/z 1289.5, 1127.4, 965.3, and 803.3 can be assigned as stevioside-(glucosyl) n ¼ 3e0. Compound I eluted at 3.13 min gave a deprotonated molecular ion at m/z 965.3 (Fig. 1b). Further fragmentation of this ion at m/z 965.3 yielded at least three product ions at m/z 803.3, m/z 641.3, and m/z 479.2 (Fig.1c), indicating
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Fig. 1. The negative ion current chromatogram and the MS spectra of steviol glycosides in the mixture of transglucosylation to stevioside. (a) Total ion chromatogram of the steviol glycosides. (b) MS spectrum of I. (c) MS/MS spectrum of I. (d) MS spectrum of II. (e) MS/MS spectrum of II.
the loss of a glucose unit. These results implied that compound I was a stevioside-(glucosyl) 1. Compound II assigned to the peak at a retention time of 5.66 min gave a deprotonated molecular ion at m/z 1127.4 (Fig. 1d); further fragmentation of this ion generated product ions at m/z 965.4 and m/z 641.3 in the MS/MS spectrum (Fig. 1e), implying that compound II was a stevioside-(glucosyl) 2 consisting of one stevioside and two glucose molecules. The compounds eluted at 8.97, 9.47, and 9.74 min belonged to stevioside-(glucosyl) 3, for they gave the deprotonated molecular ions at m/z 1289.5. The tandem mass spectrometric fragmentation patterns of these three compounds were different from each other (data not shown), suggesting that they were isomers with glucosyl moieties attached to stevioside via different types of glycosidic linkages. 3.3. Identification of I and II I and II were isolated by semi-preparative HPLC and characterized by NMR. Structure identification of III has not been carried out since their sum content is only 4 g/100 g. Assignment of the NMR data as shown in Supplementary material was made on the basis of HSQC and HMBC correlations and by the comparison of their chemical shifts with the reported data. The 1H NMR spectrum of I (Fig. 2a) exhibited four anomeric proton signals at d 6.11 (H-10 ), d 5.16 (H-100 ), d 5.24 (H-1000 ), and d5.91 (H-10000 ), and the 13C NMR spectrum of I showed four anomeric
carbon signals at d 96.4 (C-10 ), 98.5 (C-100 ), 107.4 (C-1000 ), and 103.5 (C-10000 ), which indicated that I was a mono-glucosylated stevioside. The large coupling constants of the three anomeric protons at d 6.11 (1H, d, J ¼ 8.2 Hz), 5.16 (1H, d, J ¼ 7.8 Hz), and 5.24 (1H, d, J ¼ 7.8 Hz) suggested their b-orientation, whereas the coupling constant for the fourth anomeric proton (H-1) at d 5.91(1H, d, J ¼ 3.8 Hz) suggested its a-orientation(Lobov et al., 1991). The downfield chemical shift value (þ7.2 ppm) of the C-2 position suggested the possible placement of the fourth glucosyl moiety which was supported by the HMBC correlations: H-20 /C-10000, C-10, C-30 ; H-10000 /C-20 , C-20000 , C-100. Based on the results from spectral studies, structure of I was assigned as 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl] oxy} ent-kaur-16-en-19-oic acid 2-O-(a-D-glucopyranosyl)b-D-glucopyranosyl ester (Fig. 3b). It was noteworthy that the structure of I was similar to that of rebaudioside E. The only difference between them was in the orientation of the fourth anomeric carbon, the fourth anomeric carbon of I was a-oriented, but the fourth anomeric carbon of rebaudioside E was b-oriented (Tanaka, Yamasaki, & Sakamoto, 1977). The 1H NMR spectrum of II (Fig. 2b) exhibited five anomeric proton signals at d 6.11 (1H, d, J ¼ 8.2 Hz, Glc0 ), 5.16 (1H, d, J ¼ 7.7 Hz, Glc00 ), 5.29 (1H, d, J ¼ 7.8 Hz, Glc000 ), 5.91 (1H, d, J ¼ 3.8 Hz, Glc0000 ) and 5.83 (1H, d, J ¼ 3.8 Hz, Glc’00000 ), and the 13C NMR and DEPT-135 NMR spectra showed five anomeric carbon signals at d 96.3 (C-10 ), 98.6 (C-100 ), 107.3 (C-1000 ), 103.3 (C-10000 ), and 103.7 (C-100000 ), and five
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Fig. 2. 1H NMR spectra of (a) I, and (b) II measured at 400.13 MHz in pyridine-d5 at 25 C.
carbinol methylene signals at d63.2 (C-60 ), 63.3 (C-600 ) 62.5 (C-6000 ) 62.0 (C-60000 ) 62.4 (C-600000 ), suggesting the presence of five glucosyl moieties in the structure. The 13C NMR data of glucosyl moieties Glc0 , Glc00 , Glc000 and Glc0000 of II were quite similar to that of I. Only a downfield chemical shift of C-40000 (þ7.0 ppm) was observed in the fourth glucosyl moiety (Glc0000 ) of II. The HMBC correlations H-40000 /C-100000, C-30000 , C-50000 ; H-1000000 /C-40000 , C-2000000 , C-500000 suggested the placement of the fifth glucosyl moiety at the C-40000 position of Glc0000 . Therefore, the structure of II was established as 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl] oxy} ent-kaur16-en-19-oic acid 2-O-[(4- O-a-D-glucopyranosyl)-a-D-glucopyranosyl]-b-D-glucopyranosyl ester (Fig. 3c). 3.4. Selecting reaction conditions for transglucosylated steviosides synthesis Fig. 4aed showed the effects of four factors on the synthesis of transglucosylated steviosides catalysed by BAN 480L in the starch
solution. The effect of temperature was examined and the results were shown in Fig. 4a. As the reaction temperature increased from 40 C to 70 C, the conversion yield increased significantly (Fig. 4a). A further increase in the reaction temperature resulted in a decline of the conversion yield, possibly because of the thermal-induced inactivation of the enzyme. As a result, the reaction temperature 70 C was selected for further study. The reaction was favoured at pH values ranging from 6.0 to 9.5, and the maximal conversion yield was attained at pH 6.5 (Fig. 4b). However, at lower pH (4.0 and 5.0) or higher pH (10.5), the conversion yield reduced to 5.9%, 13.2%, and 6.6%, respectively. As shown in Fig. 4c, the weight ratios of stevioside-to-starch of 1:10, 1:8, and 1:6 showed little effect on transglucosylated steviosides synthesis (p > 0.05), suggesting that the donor (starch) was sufficient for transglucosylation. However, the conversion yield decreased significantly (p 0.05) when the stevioside concentration was more than 2.5 g/100 mL. Fig. 4d presented the results on the effect of substrate concentration on transglucosylated steviosides synthesis. The substrate concentration had a significant effect on the conversion yield. A marked reduction in the conversion yield was observed when the initial starch concentration was increased from 2.0 g/100 mL to 24 g/100 mL, presumably due to substrate inhibition and the inefficiency of substrate diffusion at high concentrations. In the transglucosylation reactions, performed at an enzyme load of 1 KNU/g starch, the conversion yield of stevioside was 26.9%. The conversion yield increased with increasing the enzyme concentration until the maximal conversion yield 38.3% was reached at 2 KNU/g starch. Two major transglucosylated steviosides (I and II), which corresponded to 96 g/100 g of the total amount of transglucosylation products, was obtained. Further increases in the enzyme concentration resulted in no increase but a slight decrease in the conversion yield, probably due to the formation of enzyme aggregate or the partly hydrolysis of transglucosylation products. Previously, transglycosylation of stevioside had been conducted using various carbohydrases. Among these carbohydrases, a conversion yield of stevioside as high as 90% was obtained by using CGTase from Bacillus firmus with b-cyclodextrin as donor under microwave conditions (Jaitak et al., 2009). In this study, although the conversion yield of stevioside (38.3%) is relatively low, BAN 480L cannot glycosylated rebaudioside A but does transglycosylate stevioside. It is therefore possible that commercial steviol glycosides may be directly involved in the production of modified stevioside. Additionally, starch is a cheap and abundant material and BAN 480L is a commercial industrial enzyme. Thus, this approach has a potential application in industry.
Fig. 3. Structures of (a) stevioside; (b) I: 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-(a-D-glucopyranosyl)-b-D-glucopyranosyl ester; and (c) II: 13-{[2-O-(b-D-glucopyranosyl)-b-D-glucopyranosyl]oxy} ent-kaur-16-en-19-oic acid 2-O-[(4- O-a-D-glucopyranosyl)-a-D-glucopyranosyl]-b-D-glucopyranosyl ester.
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Fig. 4. Effects of physicochemical factors on the conversion yield of stevioside. Data plots are means of triplicate determinations. Vertical bars represent standard deviations. (a) Effect of temperature. Reaction conditions: stevioside 2.0 g/100 mL, starch 8.0 g/100 mL, enzyme load 1.0 KNU/g starch, deionized water as reaction media (pH 6.8) and incubation for 12 h. (b) Effect of pH. Reactions were carried out at 70 C in 0.050 mol/L buffers (sodium acetate buffer: pH 4.0e6.0, sodium phosphate buffer: pH 6.5e7.5, TriseHCl buffer: pH 8.5, glycine-NaOH buffer: pH 9.5e10.5) containing 2.0 g/100 mL stevioside and 8.0 g/100 mL starch, and enzyme load 1.0 KNU/g starch for 12 h. (c) Effect of weight ratio of stevioside-to-starch. The reactions were performed in 0.050 mol/L sodium phosphate buffer pH 6.5 at 70 C for 12 h with 1.0 KNU/g starch of enzyme. Different alphabets at the tops of the bars indicate significant differences (p 0.05). (d) Effect of initial starch concentration. The reaction mixtures with enzyme load 1.0 KNU/g starch were incubated in 0.050 mol/L sodium phosphate buffer pH 6.5 at 70 C for 12 h.
3.5. Relative sweetness and bitter aftertaste threshold of the modified stevioside
modified stevioside was significantly reduced. This research has provided a new method to modify stevioside.
According to the literature, the relative sweetness is strongly dependent on sucrose equivalency level for all high-potency sweeteners. For instance, rebaudioside A tastes 200 times sweeter than 6 g/ 100 mL sucrose (Walters, Orthoefer, & DuBois, 1991); however, the sweetness potency of rebaudioside A is 400 times that of 2 g/100 mL sucrose (Schiffman, Booth, Losee, Pecore, & Warwick, 1995). In this study, 2 g/100 mL sucrose was used to determine the relative sweetness of the modified stevioside. The sensory data showed that stevioside and the modified stevioside were, respectively, 173 19.0 times and 202 16.2 times sweeter than 2 g/100 mL sucrose, suggesting a significant improvement of the sweetness potency for the modified stevioside (p 0.01). In comparison with the threshold for bitter aftertaste of stevioside (0.0172 0.0050 g/100 mL), nevertheless, the threshold for bitter aftertaste of the modified stevioside (0.0263 0.0056 g/100 mL) was significantly increased (p 0.01). The results indicated that the modified stevioside was sweeter and more pleasant-tasting than stevioside.
Acknowledgements
4. Conclusions
a-Amylase BAN 480L from B. amyloliquefaciens has been studied to catalyse the transglucosylation of stevioside with starch as glucosyl donor. It was possible to achieve the conversion yield of stevioside up to 38.3%. Two major transglucosylated steviosides, which accounted for 96 g/100 g of the total transglucosylation products, were isolated and identified. The bitter aftertaste of
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