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Increasing the dietary fiber contents in isomaltooligosaccharides by dextransucrase reaction with sucrose as a glucosyl donor
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Increasing the dietary fiber contents in isomaltooligosaccharides by dextransucrase reaction with sucrose as a glucosyl donor Ji Young Songa, Young-Min Kimb, Byung-Hoo Leec,*, Sang-Ho Yooa,* a
Department of Food Science & Biotechnology, and Carbohydrate Bioproduct Research Center, Sejong University, Seoul 05006, Republic of Korea Department of Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea c Department of Food Science & Biotechnology, Gachon University, Seongnam 13120, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Isomaltooligosaccharides (IMOs) Dextransucrase Non-digestible oligosaccharides Transglucosidase Dietary fiber (DF)
Isomaltooligosaccharides (IMOs) have been widely used as alternative sweeteners owing to their stabilities, low calorigenic, and prebiotic properties. The aim of this research was to improve the functionality of conventionally produced IMOs by increasing dietary fiber (DF) content with newly synthesized α-(1,6)-linkages through the dextransucrase reaction. To optimize the reaction conditions, various combinations of IMO and sucrose concentrations were applied as acceptor and donor molecules, respectively. Soluble DF content in the enzymaticallymodified IMOs increased significantly with the initial substrate mixture of 10 % sucrose and 20 % IMOs; both DF and IMO contents increased to 35 % and 54 %, respectively. It was clearly suggested a simple dextransucraseinvolved bioprocess could be applied to increase the DF content to the IMOs produced via a conventional process without scarifying the original IMO contents. Thus, it will be expected that the DF-enhanced IMO products are potentially applicable as functional ingredients as sugar substitutes in the food industry.
1. Introduction Dietary fiber (DF) is a health-functional food material, and is defined as edible plant-derived food stuff that is resistant to digestion by human digestive enzymes (Hou, Abraham, & El-Serag, 2011; Mudgil & Barak, 2013). Based on the definition proposed by the American Association of Cereal Chemists International (AACCI), DFs include polysaccharides, oligosaccharides, lignin, and associated plant substances having beneficial physiological effects such as easing laxation and attenuating blood glucose and cholesterol levels (Slavin, 2005). DF intake is well known to have health benefits, including weight regulation and prevention of diseases, such as cardiovascular disease, diabetes, and cancer (Cheung, 2013). DFs are classified into soluble and insoluble DFs (Prosky et al., 1994); soluble DFs are responsible for increase in viscosity and reduce postprandial glycemic response and plasma cholesterol (Haskell, Spiller, Jensen, Ellis, & Gates, 1992; Yokoyama et al., 1997), while insoluble DFs are helpful in increasing fecal bulk and decreasing intestinal transit (Bliss et al., 2001; Fuller, Beck, Salman, & Tapsell, 2016). Another on-going trial for pursuing the healthy life is to reduce daily sugar consumption. Excess consumption of mono- and di-saccharides, such as fructose, glucose, and sucrose, is considered one of the major factors influencing chronic metabolic diseases, such as obesity and type
⁎
II diabetes (Gibson, 2008; Malik et al., 2010). Thus, new types of alternative sweeteners, which have similar taste and levels of sweetness compared to sucrose, with attenuated glycemic response and low calories, have gained wide interest and have been studied (Lina, Jonker, & Kozianowski, 2002; O’Brien-Nabors, 2016). Isomaltooligosaccharides (IMOs) are one of low digestible oligosaccharide mixtures of which the individual compounds contain at least one α-1,6 glycosidic linkage in its malto-oligomeric structure (e.g., isomaltose, panose, isopanose, isomaltotriose, and isomaltotetratose) (Kaneko et al., 1994; Mussatto & Mancilha, 2007). Structurally, IMOs are very stable in low pH and moderately high temperature conditions during food processing (Seo et al., 2007). Many studies have shown that the functional properties of IMOs are related to human health: 1) as a prebiotic to proliferate bifidobacteria in the gastrointestinal tract (Kohmoto et al., 1988; Kohmoto, Fukui, Takaku, & Mitsuoka, 1991), 2) to improve constipation (Chen, Lu, Lin, & Ko, 2001), and 3) to reduce total serum cholesterol and triglyceride (Wang et al., 2001). Due to these physiologically beneficial effects, the IMOs are widely used as functional sweeteners in the food industry. Commercial IMOs are produced from native starch materials by the combination of α-hydrolytic enzymes (α- and β-amylases) and transglucosidases (Crittenden & Playne, 1996). However, the current industrial production method for commercial IMOs contains significant proportions of rapidly digestible α-1,4 linked maltooligosaccharides
Corresponding authors. E-mail addresses: [email protected] (B.-H. Lee), [email protected] (S.-H. Yoo).
https://doi.org/10.1016/j.carbpol.2019.115607 Received 16 August 2019; Received in revised form 1 October 2019; Accepted 9 November 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Ji Young Song, et al., Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2019.115607
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2.3. Application of dextransucrase to increase dietary fiber contents in IMOs
and glucose (Qinlu et al., 2011). These maltooligosaccharides could be easily degraded by human digestive enzymes, and would affect postprandial blood glucose levels (Lee et al., 2014). Therefore, the reduction of digestible carbohydrate in IMOs poses a challenge to enhancement of their health functionality. Dextransucrase (EC. 2.4.1.5), a type of transglucosylating enzyme, transfers a glucose unit from sucrose (as a glucosyl donor) to other preexisting accepting molecules by creating new glycosyl linkages to produce the α-(1,6)-linked α-dextrans (Robyt, Kimble, & Walseth, 1974). When the dextransucrase catalyzes reactions with maltose and isomaltose as acceptors, it produces iso-maltooligosyl products (McCabe & Smith, 1978; Robyt, Yoon, & Mukerjea, 2008) which are not hydrolyzed to glucose by small intestinal α-glucosidases. Currently, commercial IMO products have a drawback that the α-(1,6)-branched structure in the IMOs may not avoid the digestive enzyme attack and may be partially but steadily hydrolyzed by these enzymes, thus the IMOs cannot be used as effective dietary fibers or prebiotics. Here, dextransucrase was applied to improve the physiological functionality of IMOs by increasing DF content via introducing new α-(1,6)-linkages at various concentrations of sucrose and IMO substrates as acceptor and donor molecules, respectively. In our study, it was intended to establish a potential bioprocess for the production of novel IMO-based food ingredients capable of regulating postprandial glycemic response and improving dietary fiber properties, as well as possibly enhancing prebiotic effects.
The recombinant dextransucrase from Streptococcus mutans UA159 was prepared based on our previous research (Lee et al., 2017). One unit of purified dextransucrase was defined as the amount of enzyme that catalyzes the release of 1 μmol of fructose per min from 100 mM of sucrose at 30 °C in 50 mM sodium acetate buffer (pH 5.2). The released amount of fructose was assayed using the DNS method by comparing with a standard curve which was generated with fructose (Sengupta, Jana, Sengupta, & Naskar, 2000). To determine the reaction patterns of dextransucrase, time course study at 30 °C in 50 mM sodium acetate buffer, pH 5.2, with 100 mM sucrose was conducted, and the enzymatic products were analyzed by thin layer chromatography. A silica gel K5F TLC plate (Whatman, UK) was activated by placing it for 1 h in an oven adjusted to 110 °C. Prepared samples were spotted on a silica gel plate with a pipette and placed in a TLC chamber containing a solvent mixture of isopropyl alcohol: ethyl acetate: water (3: 1: 1, v/v/v) and developed at room temperature. The reducing sugar was detected via the naphtol-H2SO4 method (Robyt & Mukerjea, 1994). To increase the DF contents in IMOs enzymatically, different concentrations (5 %–40 %, w/w) of sucrose as a substrate were reacted with dextransucrase (2.0 U/mL) on the IMO substrate [dextrose equivalent (DE) = 5] as glucosyl acceptors for 0, 1, 3, 6, 12, 24, and 48 h. The reactants collected at different reaction time intervals were applied as a dietary fiber-enhanced IMO samples for further studies.
2. Materials and methods
2.4. α-1,4 and α-1,6 linkage ratio analysis
2.1. Materials
The relative abundance of α-1, 4 and α-1, 6 linkages in the IMOs was determined by proton nuclear magnetic resonance (1H-NMR) spectroscopy (DMX-500 spectrometer, Bruker, Karlsruhe, Germany). Freeze-dried enzyme-modified starch samples (20 mg/mL) were first dissolved in deuterium oxide (D2O), and then boiled with stirring for 30 min. The samples were freeze-dried again, and samples in D2O (20 mg/mL) were analyzed by 1H-NMR analysis. 1H-NMR spectra were collected at 75 °C and the percentages (%) of the linkages were calculated to measure the branching ratio of samples (Gidley, 1985).
Thermostable α-amylase (Liquozyme® Supra, Fungamyl 800 L, Novozyme, Franklinton, NC), β-amylase (Optimalt BBA, Dupont™ Genencor® Science Rochester, NY), and transglucosidase (Transglucosidase L-500, Dupont™ Genencor®) were used in this study. Sucrose was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), and normal corn starch (NCS) was obtained from Daesang Co. (Icheon, Korea) 2.2. Production of IMOs from liquefied normal corn starch
2.5. Carbohydrate analysis by HPAEC
NCS (30 %, w/v) was suspended and pre-gelatinized at 55 °C for 30 min in water. Liquefaction of the slurry mixture was initiated by adding thermostable α-amylase (0.3 mg/g starch) and incubated at 95 °C for 15 min. The liquefied starch was treated with α-amylase (0.15 mg/g starch), β-amylase (0.15 mg/g starch), and transglucosidase (0.3 mg/g starch) for transglucosylation processes to produce the IMO mixture at 55 °C and pH 5.0. IMO and DF contents in the products synthesized by dextransucrase were measured and changes in the IMO and DF contents were analyzed at 0, 6, 12, 24, 36, 48, and 72 h of the IMO production reaction. To determine the quantitative and qualitative analyses of the IMO product sample, high-performance liquid-chromatography with a refractive index detector (HPLC-RID; Thermo Fischer Scientific, Sunnyvale, CA) was used. The IMO sample was pre-filtered with a 0.2μm nylon-membrane filter before injection. The chromatographical separation was achieved using μBondapak NH2 (Waters Associates, Milford, MA) and Aminex HPX-42A (Bio-Rad Laboratories, Hercules, CA) columns for qualitative and quantitative analyses, respectively. The eluent condition for characterization of each IMO was achieved by 70 % (v/v) acetonitrile with 0.7 mL/min at 35 °C. The IMO content (%, w/v) was determined with H2O as an isocratic eluent (flow rate: 0.6 mL/min) at 80 °C, and the separated peaks among DP 2–7 without linear chains were summed for quantitative analysis. Also, the soluble DF content in IMOs was determined via the official AOAC, 2001.03 method (AOAC, 2001).
Fructose and sucrose were analyzed via high-performance anionexchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD; Thermo Fischer Scientific) using a CarboPac™ PA1 analytical column (Thermo Fischer Scientific). Samples were diluted properly with distilled water and filtered through a 0.45-μm syringe filter. The filtrate was eluted isocratically by 150 mM NaOH for 15 min at a flow rate of 1.0 mL/min at 30 °C and washed by 600 mM sodium acetate (in 150 mM NaOH). 2.6. Statistical analysis All measurements were made in triplicate. The analysis of variance (ANOVA) was used to statistically analyze the experimental results, followed by Tukey HSD tests. All statistical analyses were carried out with SAS software (version 9.4, SAS institute, Cary, NC). Statistical significance was indicated at a confidence level of 95 %. 3. Results and discussion 3.1. Enzymatic production of IMO & DF Three enzymes, α-amylase, β-amylase, and transglucosidase, were applied to produce IMOs from the liquefied starch. First, the liquified starch was partially hydrolyzed to DE 5 by α-amylase for application as a substrate for IMO production. To determine the effects of 2
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Fig. 1. Time course study of saccharification and transglucosylation reactions of the liquefied starch substrate in terms of isomaltooligosaccharide (A) and dietary fiber (B) contents (%).
lower than that of G2 during the entire reaction period. Decreases in the proportions of G2 and G3 during the reaction coincided with increases in isomaltose (IG2), panose (Pan), and isomaltotriose (IG3), the newly α-1,6-glucosylated products. Among these products, Pan did not increase consistently, but within 24 h it increased up to 19 %, then gradually decreased to 14 % by 72 h. This result was in agreement with the previous study showing that increase in Pan eventually became overturned along with the decrease in G2 proportion, when G2 was applied as a substrate for transglucosidase reaction (Duan, Sheu, & Lin, 1995). Pan could either be hydrolyzed by transglucosidase again or used as an acceptor molecule for the transglycosyl reaction. It was reported that transglucosidase could utilize up to DP13 as an acceptor for transglucosylation (Chiba, 1997), and the DP of the products increased as transglucosidase reaction time increased with DP4 as an initial substrate (Shimba et al., 2009). This result suggests that the simultaneous reaction time duration for optimizing the DF and IMO contents were between 48 and 72 h. Furthermore, a reaction time of 48 h would be considered better for improving the DF content of IMO products by utilizing dextransucrase treatment since the proportion of glucose in the reaction mixture at 48 h were
saccharification and transglucosylation durations on IMO synthesis, a time course study of the simultaneous reaction by the other two enzymes, β-amylase and transglucosidase, was conducted up to 72 h (Fig. 1). As the reaction time increased, up to 36 h, the IMO content tended to increase noticeably to 55 % (w/w), beyond which no significant change was observed (Fig. 1A). Also, the DF content substantially increased to 20 % (w/w) along with the IMO content during the initial 24-h reaction, and then the increasing rate slowed down noticeably to a plateau after 36 h (Fig. 1B). During the enzymatic production of IMOs by three different enzymes, the change in sugar compositions was also analyzed in the reaction mixture (Fig. 2). Glucose (G1) proportions in the reaction mixture consistently increased from 0.45 % in the liquefied starch (DE 5) substrate to 19.0 % in the final IMO product after 72 h. Although the maltose (G2) content rapidly increased to a maximum of 20 % at the initial stage, up to 6 h, it decreased to 10 % within 24 h. Then, the amount of G2 decreased more slowly to 7 % by 72 h. Maltotriose (G3) generally showed a similar change in proportion pattern as G2, with a maximum of 16 % after 6 h and a negligible amount of < 2 % by the end of the reaction; thus, the absolute amount of G3 was constantly
Fig. 2. Effect of simultaneous β-amylase and transglucosidase treatments on the sugar composition in the IMO mixture from the liquefied starch (30 %, w/v) with dextrose equivalent (DE) 5 at different reaction time points. G1, glucose; G2, maltose; IG2, isomaltose; G3, maltotriose; Pan, panose; IG3, isomaltotriose. 3
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Fig. 3. Effect of reaction time and sucrose concentration (5 %–40 %) on the IMO content.
38.2 % with 5, 10, 20, and 40 % sucrose, respectively, after completion of the dextransucrase reaction (Table 1). Theoretically, the final reaction product should display ca. 40.4 % of DF content based on total solid content of 40 % sucrose with 20 % IMO mixture as the glucose originated from sucrose was totally transferred to the IMO mixture. However, 38.2 % DF was produced from 40 % sucrose under this condition. The actual produced amount of DF was different from the theoretical estimate, probably due to side reaction of dextransucrase reaction on fructose as an acceptor. This byproduct reaction pathway produces a leucrose, a sucrose isomer consisting of α(1,5) linked glucose and fructose, has a potential to regulate the physiological properties such as slowly digestible glycemic response and regulating the adipogenesis in the cell (Fig. 4) (Lee et al., 2017). However, another pathway to produce leucrose by dextransucrase might result in the decrease in DF content since DP2 could not be accounted for in the DF fraction. In this study, we exclusively determined soluble DF content by adopting the official AOAC, 2001.03 method. Therefore, the insoluble glucan fraction that should be included in the total DF content might be excluded. Further studies should be conducted to identify the structure and property of the insoluble glucans produced.
lower and that of G2 was higher than at 72 h. The G1 has been known not to be a good acceptor for transglycosylating enzymes, while G2 is relatively better substrate for the same type of enzymes (Buchholz & Monsan, 2003). Thus, the sugar component composition at 48 h was more suitable for transglucosylating reaction of dextransucrase. 3.2. Effect of dextransucrase treatment on the DF content in IMOs IMO products containing G2 and IG2 could be effective dextransucrase substrates for improving its DF content without losing the amount of IMO compounds (Robyt et al., 2008). To determine the reaction efficiency of this enzymatic process, the residual amount of sucrose was also analyzed via HPAEC during the reaction time period. No detectable amount of sucrose remained from the reaction mixtures with 5, 10, 20, and 40 % of initial sucrose concentrations within 1, 1, 3, and 24 h of reaction, respectively (data not shown). These complete consumptions of sucrose in each reaction mixture suggested that no substrate for the dextransucrase reaction remained, and no further reaction occurred. As expected, no significant change in the IMO and DF contents in the products was observed after sucrose was completely consumed (Fig. 3). The IMO contents of dextransucrase-treated products were comparable to that of the untreated control (56.3 %) for a 48h reaction with 5 and 10 % sucrose. At relatively higher sucrose concentrations (20 and 40 %), the IMO contents decreased to 49.5 and 45.3 %, respectively. From 21.1 % DF content of the initial IMO substrate mixture, the DF contents significantly increased to 27.6, 34.8, 37.9, and
3.3. Structural characterization of high-DF IMO products by linkage analysis NMR analysis was pursued to determine the ratio of α-(1,4)- and α-
Table 1 Effect of dextransucrase reaction time and sucrose concentration on dietary fiber contents (%, w/v). Reaction time (h)
Sucrose (%) 5
0 1 3 6 12 24 48 1
19.12 26.91 27.45 26.14 27.63 – –
10 ± ± ± ± ±
0.20az1 0.04by 1.29by 1.56bz 0.20bz
19.77 31.11 31.99 33.18 31.80 34.80 34.40
20 ± ± ± ± ± ± ±
0.61az 2.25by 1.47bx 0.54byx 1.12bzy 0.53by 1.20by
12.27 27.41 35.47 36.62 37.27 37.91 37.56
40 ± ± ± ± ± ± ±
0.65ay 1.18by 0.90cx 0.65cy 1.64cx 0.57cyx 0.31cyx
Means of triplicates. Means with different letters in the same column (a–e) and row (x–z) differ significantly at α = 0.05 by Tukey’s HSD. 4
7.90 ± 0.30ax 14.55 ± 0.02bx 23.07 ± 0.56cy 30.46 ± 0.87dx 36.75 ± 1.14eyx 38.18 ± 0.16ex 38.00 ± 0.43ex
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Fig. 4. Comparison of low-molecular-weight sugar composition in the IMO product mixture before and after dextransucrase treatment by HPAEC analysis. Glc, glucose; Fru, fructose; Leu, leucrose; IG2, isomaltose.
Acknowledgement
Table 2 Glycosidic linkage ratio analysis of dextransucrase-treated IMO product by 1HNMR. Reaction conditions
Panose IMO mixture 40 % sucrose, 40 % sucrose, 40 % sucrose, 40 % sucrose,
0h 1h 6h 48 h
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2017R1D1A1B04036119)
Glycosidic linkage ratio α-1,2
α-1,4
α-1,6
– 0.00 1.88 1.89 0.60 –
1.00 1.00 1.00 1.00 1.00 1.00
1.00 0.56 0.59 1.11 2.00 2.00
References AOAC (2001). Dietary fiber containing supplemented resistant maltodextrin (RMD). AOAC official method 2001.03. Washington DC: Association of Official Agricultural Chemists. Bliss, D. Z., Jung, H.-J., Savik, K., Lowry, A., LeMoine, M., Jensen, L., et al. (2001). Supplementation with dietary fiber improves fecal incontinence. Nursing Research, 50(4), 203–213. Buchholz, K., & Monsan, P. F. (2003). Dextransucrase. In J. R. Whitaker, A. G. J. Voragen, & D. W. S. Wong (Eds.). Handbook of food enzymology (pp. 589–603). Boca Raton, FL: CRC Press. Chen, H.-L., Lu, Y.-H., Lin, J., Jr., & Ko, L.-Y. (2001). Effects of isomalto-oligosaccharides on bowel functions and indicators of nutritional status in constipated elderly men. Journal of the American College of Nutrition, 20(1), 44–49. Cheung, P. C. K. (2013). Mini-review on edible mushrooms as source of dietary fiber: Preparation and health benefits. Food Science and Human Wellness, 2(3), 162–166. Chiba, S. (1997). Molecular mechanism in α-glucosidase and glucoamylase. Bioscience, Biotechnology, and Biochemistry, 61(8), 1233–1239. Crittenden, R. G., & Playne, M. J. (1996). Production, properties and applications of foodgrade oligosaccharides. Trends in Food Science & Technology, 7(11), 353–361. Duan, K.-J., Sheu, D.-C., & Lin, C.-T. (1995). Transglucosylation of a fungal α-glucosidase. Annals of the New York Academy of Sciences, 750(1), 325–328. Fuller, S., Beck, E., Salman, H., & Tapsell, L. (2016). New horizons for the study of dietary fiber and health: A review. Plant Foods for Human Nutrition, 71(1), 1–12. Gibson, G. R., Probert, H. M., Loo, J. V., Rastall, R. A., & Roberfroid, M. B. (2004). Dietary modulation of the human colonic microbiota: Updating the concept of prebiotics. Nutrition Research Reviews, 17(2), 259–275. Gibson, S. (2008). Sugar-sweetened soft drinks and obesity: A systematic review of the evidence from observational studies and interventions. Nutrition Research Reviews, 21(2), 134–147. Gidley, M. J. (1985). Quantification of the structural features of starch polysaccharides by n.m.r. spectroscopy. Carbohydrate Research, 139, 85–93. Haskell, W. L., Spiller, G. A., Jensen, C. D., Ellis, B. K., & Gates, J. E. (1992). Role of watersoluble dietary fiber in the management of elevated plasma cholesterol in healthy subjects. The American Journal of Cardiology, 69(5), 433–439. Hou, J. K., Abraham, B., & El-Serag, H. (2011). Dietary intake and risk of developing inflammatory bowel disease: A systematic review of the literature. The American Journal of Gastroenterology, 106, 563. Kaneko, T., Kohmoto, T., Kikuchi, H., Shiota, M., Iino, H., & Mitsuoka, T. (1994). Effects of isomaltooligosaccharides with different degrees of polymerization on human fecal bifidobacteria. Bioscience, Biotechnology, and Biochemistry, 58(12), 2288–2290. Kohmoto, T., Fukui, F., Takaku, H., Machida, Y., Arai, M., & Mitsuoka, T. (1988). Effect of isomalto-oligosaccharides on human fecal flora. Bifidobacteria and Microflora, 7(2),
-: not detected.
(1,6)-linkages in dextransucrase-treated products. From the final product with 40 % sucrose and 20 % IMO substrates, relative α-(1,6)linkage ratio increased along with the dextransucrase reaction time. After 48 h reaction of 40 % sucrose, the α-1,6 linkage ratio increased from 0.56 to 2.0 and the peak corresponding to an α-1,2 linkage of sucrose decreased steadily and completely disappeared. This result confirmed that DF content was highly interconnected with the proportion of α-1,6 linkages in dextransucrase bio-converted products. The higher the ratio of α-1,6 linkage in IMOs, the more the increase in DF in IMOs occurred (Table 2). Thus, the IMO produced by dextransucrase is potentially applicable as prebiotic food ingredients and dietary fibers for improving the gut health after passing the small intestine (Gibson, Probert, Loo, Rastall, & Roberfroid, 2004; Kaneko et al., 1994). 4. Conclusion In this study, we tried to develop a health-functional food material by increasing the soluble dietary fiber content of conventionally produced IMO products and the simultaneous addition of α-amylase, βamylase, and transglucosidase. The results clearly showed that the amount of soluble DF in IMOs increased significantly with dextransucrase treatment. These DF-improved IMOs are structurally stable under acidic conditions and heating processes, and are low calorigenic, making them potentially applicable as health functional foods, as well as sugar substitutes. 5
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Qinlu, L., Huaxi, X., Jian, Z., Lihui, L., engxiang, Y. F., Xing, L., et al. (2011). Production of isomalto‐oligosaccharide syrup from rice starch using an one‐step conversion method. International Journal of Food Science & Technology, 46(6), 1194–1200. Robyt, J. F., Kimble, B. K., & Walseth, T. F. (1974). The mechanism of dextransucrase action: Direction of dextran biosynthesis. Archives of Biochemistry and Biophysics, 165(2), 634–640. Robyt, J. F., & Mukerjea, R. (1994). Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography. Carbohydrate Research, 251, 187–202. Robyt, J. F., Yoon, S.-H., & Mukerjea, R. (2008). Dextransucrase and the mechanism for dextran biosynthesis. Carbohydrate Research, 343(18), 3039–3048. Sengupta, S., Jana, M. L., Sengupta, D., & Naskar, A. K. (2000). A note on the estimation of microbial glycosidase activities by dinitrosalicylic acid reagent. Applied Microbiology and Biotechnology, 53(6), 732–735. Seo, E.-S., Nam, S.-H., Kang, H.-K., Cho, J.-Y., Lee, H.-S., Ryu, H.-W., et al. (2007). Synthesis of thermo- and acid-stable novel oligosaccharides by using dextransucrase with high concentration of sucrose. Enzyme and Microbial Technology, 40(5), 1117–1123. Shimba, N., Shinagawa, M., Hoshino, W., Yamaguchi, H., Yamada, N., & Suzuki, E.-i. (2009). Monitoring the hydrolysis and transglycosylation activity of α-glucosidase from Aspergillus niger by nuclear magnetic resonance spectroscopy and mass spectrometry. Analytical Biochemistry, 393(1), 23–28. Slavin, J. L. (2005). Dietary fiber and body weight. Nutrition, 21(3), 411–418. Wang, H.-F., Lim, P.-S., Kao, M.-D., Chan, E.-C., Lin, L.-C., & Wang, N.-P. (2001). Use of isomalto-oligosaccharide in the treatment of lipid profiles and constipation in hemodialysis patients. Journal of Renal Nutrition, 11(2), 73–79. Yokoyama, W. H., Hudson, C. A., Knuckles, B. E., Chiu, M. C. M., Sayre, R. N., Turnlund, J. R., et al. (1997). Effect of barley β‐glucan in durum wheat pasta on human glycemic response. Cereal Chemistry, 74(3), 293–296.
61–69. Kohmoto, T., Fukui, F., Takaku, H., & Mitsuoka, T. (1991). Dose-response test of isomaltooligosaccharides for increasing fecal bifidobacteria. Agricultural and Biological Chemistry, 55(8), 2157–2159. Lee, B.-H., Lin, A. H.-M., Nichols, B. L., Jones, K., Rose, D. R., Quezada-Calvillo, R., et al. (2014). Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation. Molecular Nutrition & Food Research, 58(5), 1111–1121. Lee, D., Lee, J., Hong, M.-G., Lee, B.-H., Kim, Y.-M., Chang, P.-S., et al. (2017). Optimization of leucrose production by dextransucrase from Streptococcus mutans and its application as an adipogenesis regulator. Journal of Functional Foods, 39, 238–244. Lina, B. A. R., Jonker, D., & Kozianowski, G. (2002). Isomaltulose (Palatinose®): A review of biological and toxicological studies. Food and Chemical Toxicology, 40(10), 1375–1381. Malik, V. S., Popkin, B. M., Bray, G. A., Després, J.-P., Willett, W. C., & Hu, F. B. (2010). Sugar-sweetened beverages and risk of metabolic syndrome and type 2 diabetes: A meta-analysis. Diabetes Care, 33(11), 2477–2483. McCabe, M. M., & Smith, E. E. (1978). The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R. Carbohydrate Research, 63, 223–239. Mudgil, D., & Barak, S. (2013). Composition, properties and health benefits of indigestible carbohydrate polymers as dietary fiber: A review. International Journal of Biological Macromolecules, 61(Supplement C), 1–6. Mussatto, S. I., & Mancilha, I. M. (2007). Non-digestible oligosaccharides: A review. Carbohydrate Polymers, 68(3), 587–597. O’Brien-Nabors, L. (2016). Alternative sweeteners: An overviwew. In L. O'Brien-Nabors (Ed.). Alternative sweeteners (pp. 1–12). Boca Raton, FL: CRC Press. Prosky, L., Asp, N., Schweizer, T., DeVries, J., Furda, I., & Lee, S. (1994). Determination of insoluble, soluble, and total dietary fiber in foods and food products: Interlaboratory study. Journal of the Association of Analytical Chemistry International, 71(5), 1017–1023.
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Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Increasing the dietary fiber contents in isomaltooligosaccharides by dextransucrase reaction with sucrose as a glucosyl donor Ji Young Songa, Young-Min Kimb, Byung-Hoo Leec,*, Sang-Ho Yooa,* a
Department of Food Science & Biotechnology, and Carbohydrate Bioproduct Research Center, Sejong University, Seoul 05006, Republic of Korea Department of Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea c Department of Food Science & Biotechnology, Gachon University, Seongnam 13120, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Isomaltooligosaccharides (IMOs) Dextransucrase Non-digestible oligosaccharides Transglucosidase Dietary fiber (DF)
Isomaltooligosaccharides (IMOs) have been widely used as alternative sweeteners owing to their stabilities, low calorigenic, and prebiotic properties. The aim of this research was to improve the functionality of conventionally produced IMOs by increasing dietary fiber (DF) content with newly synthesized α-(1,6)-linkages through the dextransucrase reaction. To optimize the reaction conditions, various combinations of IMO and sucrose concentrations were applied as acceptor and donor molecules, respectively. Soluble DF content in the enzymaticallymodified IMOs increased significantly with the initial substrate mixture of 10 % sucrose and 20 % IMOs; both DF and IMO contents increased to 35 % and 54 %, respectively. It was clearly suggested a simple dextransucraseinvolved bioprocess could be applied to increase the DF content to the IMOs produced via a conventional process without scarifying the original IMO contents. Thus, it will be expected that the DF-enhanced IMO products are potentially applicable as functional ingredients as sugar substitutes in the food industry.
1. Introduction Dietary fiber (DF) is a health-functional food material, and is defined as edible plant-derived food stuff that is resistant to digestion by human digestive enzymes (Hou, Abraham, & El-Serag, 2011; Mudgil & Barak, 2013). Based on the definition proposed by the American Association of Cereal Chemists International (AACCI), DFs include polysaccharides, oligosaccharides, lignin, and associated plant substances having beneficial physiological effects such as easing laxation and attenuating blood glucose and cholesterol levels (Slavin, 2005). DF intake is well known to have health benefits, including weight regulation and prevention of diseases, such as cardiovascular disease, diabetes, and cancer (Cheung, 2013). DFs are classified into soluble and insoluble DFs (Prosky et al., 1994); soluble DFs are responsible for increase in viscosity and reduce postprandial glycemic response and plasma cholesterol (Haskell, Spiller, Jensen, Ellis, & Gates, 1992; Yokoyama et al., 1997), while insoluble DFs are helpful in increasing fecal bulk and decreasing intestinal transit (Bliss et al., 2001; Fuller, Beck, Salman, & Tapsell, 2016). Another on-going trial for pursuing the healthy life is to reduce daily sugar consumption. Excess consumption of mono- and di-saccharides, such as fructose, glucose, and sucrose, is considered one of the major factors influencing chronic metabolic diseases, such as obesity and type
⁎
II diabetes (Gibson, 2008; Malik et al., 2010). Thus, new types of alternative sweeteners, which have similar taste and levels of sweetness compared to sucrose, with attenuated glycemic response and low calories, have gained wide interest and have been studied (Lina, Jonker, & Kozianowski, 2002; O’Brien-Nabors, 2016). Isomaltooligosaccharides (IMOs) are one of low digestible oligosaccharide mixtures of which the individual compounds contain at least one α-1,6 glycosidic linkage in its malto-oligomeric structure (e.g., isomaltose, panose, isopanose, isomaltotriose, and isomaltotetratose) (Kaneko et al., 1994; Mussatto & Mancilha, 2007). Structurally, IMOs are very stable in low pH and moderately high temperature conditions during food processing (Seo et al., 2007). Many studies have shown that the functional properties of IMOs are related to human health: 1) as a prebiotic to proliferate bifidobacteria in the gastrointestinal tract (Kohmoto et al., 1988; Kohmoto, Fukui, Takaku, & Mitsuoka, 1991), 2) to improve constipation (Chen, Lu, Lin, & Ko, 2001), and 3) to reduce total serum cholesterol and triglyceride (Wang et al., 2001). Due to these physiologically beneficial effects, the IMOs are widely used as functional sweeteners in the food industry. Commercial IMOs are produced from native starch materials by the combination of α-hydrolytic enzymes (α- and β-amylases) and transglucosidases (Crittenden & Playne, 1996). However, the current industrial production method for commercial IMOs contains significant proportions of rapidly digestible α-1,4 linked maltooligosaccharides
Corresponding authors. E-mail addresses: [email protected] (B.-H. Lee), [email protected] (S.-H. Yoo).
https://doi.org/10.1016/j.carbpol.2019.115607 Received 16 August 2019; Received in revised form 1 October 2019; Accepted 9 November 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Ji Young Song, et al., Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2019.115607
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2.3. Application of dextransucrase to increase dietary fiber contents in IMOs
and glucose (Qinlu et al., 2011). These maltooligosaccharides could be easily degraded by human digestive enzymes, and would affect postprandial blood glucose levels (Lee et al., 2014). Therefore, the reduction of digestible carbohydrate in IMOs poses a challenge to enhancement of their health functionality. Dextransucrase (EC. 2.4.1.5), a type of transglucosylating enzyme, transfers a glucose unit from sucrose (as a glucosyl donor) to other preexisting accepting molecules by creating new glycosyl linkages to produce the α-(1,6)-linked α-dextrans (Robyt, Kimble, & Walseth, 1974). When the dextransucrase catalyzes reactions with maltose and isomaltose as acceptors, it produces iso-maltooligosyl products (McCabe & Smith, 1978; Robyt, Yoon, & Mukerjea, 2008) which are not hydrolyzed to glucose by small intestinal α-glucosidases. Currently, commercial IMO products have a drawback that the α-(1,6)-branched structure in the IMOs may not avoid the digestive enzyme attack and may be partially but steadily hydrolyzed by these enzymes, thus the IMOs cannot be used as effective dietary fibers or prebiotics. Here, dextransucrase was applied to improve the physiological functionality of IMOs by increasing DF content via introducing new α-(1,6)-linkages at various concentrations of sucrose and IMO substrates as acceptor and donor molecules, respectively. In our study, it was intended to establish a potential bioprocess for the production of novel IMO-based food ingredients capable of regulating postprandial glycemic response and improving dietary fiber properties, as well as possibly enhancing prebiotic effects.
The recombinant dextransucrase from Streptococcus mutans UA159 was prepared based on our previous research (Lee et al., 2017). One unit of purified dextransucrase was defined as the amount of enzyme that catalyzes the release of 1 μmol of fructose per min from 100 mM of sucrose at 30 °C in 50 mM sodium acetate buffer (pH 5.2). The released amount of fructose was assayed using the DNS method by comparing with a standard curve which was generated with fructose (Sengupta, Jana, Sengupta, & Naskar, 2000). To determine the reaction patterns of dextransucrase, time course study at 30 °C in 50 mM sodium acetate buffer, pH 5.2, with 100 mM sucrose was conducted, and the enzymatic products were analyzed by thin layer chromatography. A silica gel K5F TLC plate (Whatman, UK) was activated by placing it for 1 h in an oven adjusted to 110 °C. Prepared samples were spotted on a silica gel plate with a pipette and placed in a TLC chamber containing a solvent mixture of isopropyl alcohol: ethyl acetate: water (3: 1: 1, v/v/v) and developed at room temperature. The reducing sugar was detected via the naphtol-H2SO4 method (Robyt & Mukerjea, 1994). To increase the DF contents in IMOs enzymatically, different concentrations (5 %–40 %, w/w) of sucrose as a substrate were reacted with dextransucrase (2.0 U/mL) on the IMO substrate [dextrose equivalent (DE) = 5] as glucosyl acceptors for 0, 1, 3, 6, 12, 24, and 48 h. The reactants collected at different reaction time intervals were applied as a dietary fiber-enhanced IMO samples for further studies.
2. Materials and methods
2.4. α-1,4 and α-1,6 linkage ratio analysis
2.1. Materials
The relative abundance of α-1, 4 and α-1, 6 linkages in the IMOs was determined by proton nuclear magnetic resonance (1H-NMR) spectroscopy (DMX-500 spectrometer, Bruker, Karlsruhe, Germany). Freeze-dried enzyme-modified starch samples (20 mg/mL) were first dissolved in deuterium oxide (D2O), and then boiled with stirring for 30 min. The samples were freeze-dried again, and samples in D2O (20 mg/mL) were analyzed by 1H-NMR analysis. 1H-NMR spectra were collected at 75 °C and the percentages (%) of the linkages were calculated to measure the branching ratio of samples (Gidley, 1985).
Thermostable α-amylase (Liquozyme® Supra, Fungamyl 800 L, Novozyme, Franklinton, NC), β-amylase (Optimalt BBA, Dupont™ Genencor® Science Rochester, NY), and transglucosidase (Transglucosidase L-500, Dupont™ Genencor®) were used in this study. Sucrose was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), and normal corn starch (NCS) was obtained from Daesang Co. (Icheon, Korea) 2.2. Production of IMOs from liquefied normal corn starch
2.5. Carbohydrate analysis by HPAEC
NCS (30 %, w/v) was suspended and pre-gelatinized at 55 °C for 30 min in water. Liquefaction of the slurry mixture was initiated by adding thermostable α-amylase (0.3 mg/g starch) and incubated at 95 °C for 15 min. The liquefied starch was treated with α-amylase (0.15 mg/g starch), β-amylase (0.15 mg/g starch), and transglucosidase (0.3 mg/g starch) for transglucosylation processes to produce the IMO mixture at 55 °C and pH 5.0. IMO and DF contents in the products synthesized by dextransucrase were measured and changes in the IMO and DF contents were analyzed at 0, 6, 12, 24, 36, 48, and 72 h of the IMO production reaction. To determine the quantitative and qualitative analyses of the IMO product sample, high-performance liquid-chromatography with a refractive index detector (HPLC-RID; Thermo Fischer Scientific, Sunnyvale, CA) was used. The IMO sample was pre-filtered with a 0.2μm nylon-membrane filter before injection. The chromatographical separation was achieved using μBondapak NH2 (Waters Associates, Milford, MA) and Aminex HPX-42A (Bio-Rad Laboratories, Hercules, CA) columns for qualitative and quantitative analyses, respectively. The eluent condition for characterization of each IMO was achieved by 70 % (v/v) acetonitrile with 0.7 mL/min at 35 °C. The IMO content (%, w/v) was determined with H2O as an isocratic eluent (flow rate: 0.6 mL/min) at 80 °C, and the separated peaks among DP 2–7 without linear chains were summed for quantitative analysis. Also, the soluble DF content in IMOs was determined via the official AOAC, 2001.03 method (AOAC, 2001).
Fructose and sucrose were analyzed via high-performance anionexchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD; Thermo Fischer Scientific) using a CarboPac™ PA1 analytical column (Thermo Fischer Scientific). Samples were diluted properly with distilled water and filtered through a 0.45-μm syringe filter. The filtrate was eluted isocratically by 150 mM NaOH for 15 min at a flow rate of 1.0 mL/min at 30 °C and washed by 600 mM sodium acetate (in 150 mM NaOH). 2.6. Statistical analysis All measurements were made in triplicate. The analysis of variance (ANOVA) was used to statistically analyze the experimental results, followed by Tukey HSD tests. All statistical analyses were carried out with SAS software (version 9.4, SAS institute, Cary, NC). Statistical significance was indicated at a confidence level of 95 %. 3. Results and discussion 3.1. Enzymatic production of IMO & DF Three enzymes, α-amylase, β-amylase, and transglucosidase, were applied to produce IMOs from the liquefied starch. First, the liquified starch was partially hydrolyzed to DE 5 by α-amylase for application as a substrate for IMO production. To determine the effects of 2
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Fig. 1. Time course study of saccharification and transglucosylation reactions of the liquefied starch substrate in terms of isomaltooligosaccharide (A) and dietary fiber (B) contents (%).
lower than that of G2 during the entire reaction period. Decreases in the proportions of G2 and G3 during the reaction coincided with increases in isomaltose (IG2), panose (Pan), and isomaltotriose (IG3), the newly α-1,6-glucosylated products. Among these products, Pan did not increase consistently, but within 24 h it increased up to 19 %, then gradually decreased to 14 % by 72 h. This result was in agreement with the previous study showing that increase in Pan eventually became overturned along with the decrease in G2 proportion, when G2 was applied as a substrate for transglucosidase reaction (Duan, Sheu, & Lin, 1995). Pan could either be hydrolyzed by transglucosidase again or used as an acceptor molecule for the transglycosyl reaction. It was reported that transglucosidase could utilize up to DP13 as an acceptor for transglucosylation (Chiba, 1997), and the DP of the products increased as transglucosidase reaction time increased with DP4 as an initial substrate (Shimba et al., 2009). This result suggests that the simultaneous reaction time duration for optimizing the DF and IMO contents were between 48 and 72 h. Furthermore, a reaction time of 48 h would be considered better for improving the DF content of IMO products by utilizing dextransucrase treatment since the proportion of glucose in the reaction mixture at 48 h were
saccharification and transglucosylation durations on IMO synthesis, a time course study of the simultaneous reaction by the other two enzymes, β-amylase and transglucosidase, was conducted up to 72 h (Fig. 1). As the reaction time increased, up to 36 h, the IMO content tended to increase noticeably to 55 % (w/w), beyond which no significant change was observed (Fig. 1A). Also, the DF content substantially increased to 20 % (w/w) along with the IMO content during the initial 24-h reaction, and then the increasing rate slowed down noticeably to a plateau after 36 h (Fig. 1B). During the enzymatic production of IMOs by three different enzymes, the change in sugar compositions was also analyzed in the reaction mixture (Fig. 2). Glucose (G1) proportions in the reaction mixture consistently increased from 0.45 % in the liquefied starch (DE 5) substrate to 19.0 % in the final IMO product after 72 h. Although the maltose (G2) content rapidly increased to a maximum of 20 % at the initial stage, up to 6 h, it decreased to 10 % within 24 h. Then, the amount of G2 decreased more slowly to 7 % by 72 h. Maltotriose (G3) generally showed a similar change in proportion pattern as G2, with a maximum of 16 % after 6 h and a negligible amount of < 2 % by the end of the reaction; thus, the absolute amount of G3 was constantly
Fig. 2. Effect of simultaneous β-amylase and transglucosidase treatments on the sugar composition in the IMO mixture from the liquefied starch (30 %, w/v) with dextrose equivalent (DE) 5 at different reaction time points. G1, glucose; G2, maltose; IG2, isomaltose; G3, maltotriose; Pan, panose; IG3, isomaltotriose. 3
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Fig. 3. Effect of reaction time and sucrose concentration (5 %–40 %) on the IMO content.
38.2 % with 5, 10, 20, and 40 % sucrose, respectively, after completion of the dextransucrase reaction (Table 1). Theoretically, the final reaction product should display ca. 40.4 % of DF content based on total solid content of 40 % sucrose with 20 % IMO mixture as the glucose originated from sucrose was totally transferred to the IMO mixture. However, 38.2 % DF was produced from 40 % sucrose under this condition. The actual produced amount of DF was different from the theoretical estimate, probably due to side reaction of dextransucrase reaction on fructose as an acceptor. This byproduct reaction pathway produces a leucrose, a sucrose isomer consisting of α(1,5) linked glucose and fructose, has a potential to regulate the physiological properties such as slowly digestible glycemic response and regulating the adipogenesis in the cell (Fig. 4) (Lee et al., 2017). However, another pathway to produce leucrose by dextransucrase might result in the decrease in DF content since DP2 could not be accounted for in the DF fraction. In this study, we exclusively determined soluble DF content by adopting the official AOAC, 2001.03 method. Therefore, the insoluble glucan fraction that should be included in the total DF content might be excluded. Further studies should be conducted to identify the structure and property of the insoluble glucans produced.
lower and that of G2 was higher than at 72 h. The G1 has been known not to be a good acceptor for transglycosylating enzymes, while G2 is relatively better substrate for the same type of enzymes (Buchholz & Monsan, 2003). Thus, the sugar component composition at 48 h was more suitable for transglucosylating reaction of dextransucrase. 3.2. Effect of dextransucrase treatment on the DF content in IMOs IMO products containing G2 and IG2 could be effective dextransucrase substrates for improving its DF content without losing the amount of IMO compounds (Robyt et al., 2008). To determine the reaction efficiency of this enzymatic process, the residual amount of sucrose was also analyzed via HPAEC during the reaction time period. No detectable amount of sucrose remained from the reaction mixtures with 5, 10, 20, and 40 % of initial sucrose concentrations within 1, 1, 3, and 24 h of reaction, respectively (data not shown). These complete consumptions of sucrose in each reaction mixture suggested that no substrate for the dextransucrase reaction remained, and no further reaction occurred. As expected, no significant change in the IMO and DF contents in the products was observed after sucrose was completely consumed (Fig. 3). The IMO contents of dextransucrase-treated products were comparable to that of the untreated control (56.3 %) for a 48h reaction with 5 and 10 % sucrose. At relatively higher sucrose concentrations (20 and 40 %), the IMO contents decreased to 49.5 and 45.3 %, respectively. From 21.1 % DF content of the initial IMO substrate mixture, the DF contents significantly increased to 27.6, 34.8, 37.9, and
3.3. Structural characterization of high-DF IMO products by linkage analysis NMR analysis was pursued to determine the ratio of α-(1,4)- and α-
Table 1 Effect of dextransucrase reaction time and sucrose concentration on dietary fiber contents (%, w/v). Reaction time (h)
Sucrose (%) 5
0 1 3 6 12 24 48 1
19.12 26.91 27.45 26.14 27.63 – –
10 ± ± ± ± ±
0.20az1 0.04by 1.29by 1.56bz 0.20bz
19.77 31.11 31.99 33.18 31.80 34.80 34.40
20 ± ± ± ± ± ± ±
0.61az 2.25by 1.47bx 0.54byx 1.12bzy 0.53by 1.20by
12.27 27.41 35.47 36.62 37.27 37.91 37.56
40 ± ± ± ± ± ± ±
0.65ay 1.18by 0.90cx 0.65cy 1.64cx 0.57cyx 0.31cyx
Means of triplicates. Means with different letters in the same column (a–e) and row (x–z) differ significantly at α = 0.05 by Tukey’s HSD. 4
7.90 ± 0.30ax 14.55 ± 0.02bx 23.07 ± 0.56cy 30.46 ± 0.87dx 36.75 ± 1.14eyx 38.18 ± 0.16ex 38.00 ± 0.43ex
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Fig. 4. Comparison of low-molecular-weight sugar composition in the IMO product mixture before and after dextransucrase treatment by HPAEC analysis. Glc, glucose; Fru, fructose; Leu, leucrose; IG2, isomaltose.
Acknowledgement
Table 2 Glycosidic linkage ratio analysis of dextransucrase-treated IMO product by 1HNMR. Reaction conditions
Panose IMO mixture 40 % sucrose, 40 % sucrose, 40 % sucrose, 40 % sucrose,
0h 1h 6h 48 h
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2017R1D1A1B04036119)
Glycosidic linkage ratio α-1,2
α-1,4
α-1,6
– 0.00 1.88 1.89 0.60 –
1.00 1.00 1.00 1.00 1.00 1.00
1.00 0.56 0.59 1.11 2.00 2.00
References AOAC (2001). Dietary fiber containing supplemented resistant maltodextrin (RMD). AOAC official method 2001.03. Washington DC: Association of Official Agricultural Chemists. Bliss, D. Z., Jung, H.-J., Savik, K., Lowry, A., LeMoine, M., Jensen, L., et al. (2001). Supplementation with dietary fiber improves fecal incontinence. Nursing Research, 50(4), 203–213. Buchholz, K., & Monsan, P. F. (2003). Dextransucrase. In J. R. Whitaker, A. G. J. Voragen, & D. W. S. Wong (Eds.). Handbook of food enzymology (pp. 589–603). Boca Raton, FL: CRC Press. Chen, H.-L., Lu, Y.-H., Lin, J., Jr., & Ko, L.-Y. (2001). Effects of isomalto-oligosaccharides on bowel functions and indicators of nutritional status in constipated elderly men. Journal of the American College of Nutrition, 20(1), 44–49. Cheung, P. C. K. (2013). Mini-review on edible mushrooms as source of dietary fiber: Preparation and health benefits. Food Science and Human Wellness, 2(3), 162–166. Chiba, S. (1997). Molecular mechanism in α-glucosidase and glucoamylase. Bioscience, Biotechnology, and Biochemistry, 61(8), 1233–1239. Crittenden, R. G., & Playne, M. J. (1996). Production, properties and applications of foodgrade oligosaccharides. Trends in Food Science & Technology, 7(11), 353–361. Duan, K.-J., Sheu, D.-C., & Lin, C.-T. (1995). Transglucosylation of a fungal α-glucosidase. Annals of the New York Academy of Sciences, 750(1), 325–328. Fuller, S., Beck, E., Salman, H., & Tapsell, L. (2016). New horizons for the study of dietary fiber and health: A review. Plant Foods for Human Nutrition, 71(1), 1–12. Gibson, G. R., Probert, H. M., Loo, J. V., Rastall, R. A., & Roberfroid, M. B. (2004). Dietary modulation of the human colonic microbiota: Updating the concept of prebiotics. Nutrition Research Reviews, 17(2), 259–275. Gibson, S. (2008). Sugar-sweetened soft drinks and obesity: A systematic review of the evidence from observational studies and interventions. Nutrition Research Reviews, 21(2), 134–147. Gidley, M. J. (1985). Quantification of the structural features of starch polysaccharides by n.m.r. spectroscopy. Carbohydrate Research, 139, 85–93. Haskell, W. L., Spiller, G. A., Jensen, C. D., Ellis, B. K., & Gates, J. E. (1992). Role of watersoluble dietary fiber in the management of elevated plasma cholesterol in healthy subjects. The American Journal of Cardiology, 69(5), 433–439. Hou, J. K., Abraham, B., & El-Serag, H. (2011). Dietary intake and risk of developing inflammatory bowel disease: A systematic review of the literature. The American Journal of Gastroenterology, 106, 563. Kaneko, T., Kohmoto, T., Kikuchi, H., Shiota, M., Iino, H., & Mitsuoka, T. (1994). Effects of isomaltooligosaccharides with different degrees of polymerization on human fecal bifidobacteria. Bioscience, Biotechnology, and Biochemistry, 58(12), 2288–2290. Kohmoto, T., Fukui, F., Takaku, H., Machida, Y., Arai, M., & Mitsuoka, T. (1988). Effect of isomalto-oligosaccharides on human fecal flora. Bifidobacteria and Microflora, 7(2),
-: not detected.
(1,6)-linkages in dextransucrase-treated products. From the final product with 40 % sucrose and 20 % IMO substrates, relative α-(1,6)linkage ratio increased along with the dextransucrase reaction time. After 48 h reaction of 40 % sucrose, the α-1,6 linkage ratio increased from 0.56 to 2.0 and the peak corresponding to an α-1,2 linkage of sucrose decreased steadily and completely disappeared. This result confirmed that DF content was highly interconnected with the proportion of α-1,6 linkages in dextransucrase bio-converted products. The higher the ratio of α-1,6 linkage in IMOs, the more the increase in DF in IMOs occurred (Table 2). Thus, the IMO produced by dextransucrase is potentially applicable as prebiotic food ingredients and dietary fibers for improving the gut health after passing the small intestine (Gibson, Probert, Loo, Rastall, & Roberfroid, 2004; Kaneko et al., 1994). 4. Conclusion In this study, we tried to develop a health-functional food material by increasing the soluble dietary fiber content of conventionally produced IMO products and the simultaneous addition of α-amylase, βamylase, and transglucosidase. The results clearly showed that the amount of soluble DF in IMOs increased significantly with dextransucrase treatment. These DF-improved IMOs are structurally stable under acidic conditions and heating processes, and are low calorigenic, making them potentially applicable as health functional foods, as well as sugar substitutes. 5
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Qinlu, L., Huaxi, X., Jian, Z., Lihui, L., engxiang, Y. F., Xing, L., et al. (2011). Production of isomalto‐oligosaccharide syrup from rice starch using an one‐step conversion method. International Journal of Food Science & Technology, 46(6), 1194–1200. Robyt, J. F., Kimble, B. K., & Walseth, T. F. (1974). The mechanism of dextransucrase action: Direction of dextran biosynthesis. Archives of Biochemistry and Biophysics, 165(2), 634–640. Robyt, J. F., & Mukerjea, R. (1994). Separation and quantitative determination of nanogram quantities of maltodextrins and isomaltodextrins by thin-layer chromatography. Carbohydrate Research, 251, 187–202. Robyt, J. F., Yoon, S.-H., & Mukerjea, R. (2008). Dextransucrase and the mechanism for dextran biosynthesis. Carbohydrate Research, 343(18), 3039–3048. Sengupta, S., Jana, M. L., Sengupta, D., & Naskar, A. K. (2000). A note on the estimation of microbial glycosidase activities by dinitrosalicylic acid reagent. Applied Microbiology and Biotechnology, 53(6), 732–735. Seo, E.-S., Nam, S.-H., Kang, H.-K., Cho, J.-Y., Lee, H.-S., Ryu, H.-W., et al. (2007). Synthesis of thermo- and acid-stable novel oligosaccharides by using dextransucrase with high concentration of sucrose. Enzyme and Microbial Technology, 40(5), 1117–1123. Shimba, N., Shinagawa, M., Hoshino, W., Yamaguchi, H., Yamada, N., & Suzuki, E.-i. (2009). Monitoring the hydrolysis and transglycosylation activity of α-glucosidase from Aspergillus niger by nuclear magnetic resonance spectroscopy and mass spectrometry. Analytical Biochemistry, 393(1), 23–28. Slavin, J. L. (2005). Dietary fiber and body weight. Nutrition, 21(3), 411–418. Wang, H.-F., Lim, P.-S., Kao, M.-D., Chan, E.-C., Lin, L.-C., & Wang, N.-P. (2001). Use of isomalto-oligosaccharide in the treatment of lipid profiles and constipation in hemodialysis patients. Journal of Renal Nutrition, 11(2), 73–79. Yokoyama, W. H., Hudson, C. A., Knuckles, B. E., Chiu, M. C. M., Sayre, R. N., Turnlund, J. R., et al. (1997). Effect of barley β‐glucan in durum wheat pasta on human glycemic response. Cereal Chemistry, 74(3), 293–296.
61–69. Kohmoto, T., Fukui, F., Takaku, H., & Mitsuoka, T. (1991). Dose-response test of isomaltooligosaccharides for increasing fecal bifidobacteria. Agricultural and Biological Chemistry, 55(8), 2157–2159. Lee, B.-H., Lin, A. H.-M., Nichols, B. L., Jones, K., Rose, D. R., Quezada-Calvillo, R., et al. (2014). Mucosal C-terminal maltase-glucoamylase hydrolyzes large size starch digestion products that may contribute to rapid postprandial glucose generation. Molecular Nutrition & Food Research, 58(5), 1111–1121. Lee, D., Lee, J., Hong, M.-G., Lee, B.-H., Kim, Y.-M., Chang, P.-S., et al. (2017). Optimization of leucrose production by dextransucrase from Streptococcus mutans and its application as an adipogenesis regulator. Journal of Functional Foods, 39, 238–244. Lina, B. A. R., Jonker, D., & Kozianowski, G. (2002). Isomaltulose (Palatinose®): A review of biological and toxicological studies. Food and Chemical Toxicology, 40(10), 1375–1381. Malik, V. S., Popkin, B. M., Bray, G. A., Després, J.-P., Willett, W. C., & Hu, F. B. (2010). Sugar-sweetened beverages and risk of metabolic syndrome and type 2 diabetes: A meta-analysis. Diabetes Care, 33(11), 2477–2483. McCabe, M. M., & Smith, E. E. (1978). The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R. Carbohydrate Research, 63, 223–239. Mudgil, D., & Barak, S. (2013). Composition, properties and health benefits of indigestible carbohydrate polymers as dietary fiber: A review. International Journal of Biological Macromolecules, 61(Supplement C), 1–6. Mussatto, S. I., & Mancilha, I. M. (2007). Non-digestible oligosaccharides: A review. Carbohydrate Polymers, 68(3), 587–597. O’Brien-Nabors, L. (2016). Alternative sweeteners: An overviwew. In L. O'Brien-Nabors (Ed.). Alternative sweeteners (pp. 1–12). Boca Raton, FL: CRC Press. Prosky, L., Asp, N., Schweizer, T., DeVries, J., Furda, I., & Lee, S. (1994). Determination of insoluble, soluble, and total dietary fiber in foods and food products: Interlaboratory study. Journal of the Association of Analytical Chemistry International, 71(5), 1017–1023.
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