br Materials and methods br Acknowledgments br Introduction

Materials and methods
Acknowledgments
Introduction Gentiooligosaccharides are novel functional oligosaccharides composed of glucose units linked through β-1,6 glycosidic bonds, examples of which include gentiobiose, gentiotriose and gentiotetraose (Barreteau et al., 2006, Kim et al., 2003, Smaali et al., 2004). Gentiooligosaccharides cannot be broken down by digestive enzymes in the human body, and are therefore low in terms of calories, making them useful for people with obesity, high blood lipids, high blood pressure and diabetes (Rycroft, Jones, Gibson, & Rastall, 2001). They have a high moisture content and hygroscopicity, and are thus conducive for maintaining food moisture and preventing aging of starch (Gibson & Roberfroid, 1984). They have a soft, refreshing, bitter taste and thus can be used as food additives to diversify taste. At present, gentiooligosaccharides are widely used in chocolate, ice cream, coffee, spices, baked goods and beverages (Unno et al., 2006). Various methods for preparing gentiooligosaccharides have been reported. In early studies, they were extracted from the roots and stems of gentian plants, or purified from by-products of bitter almond benzene reduction or English name hydrolysis of starch (Zhu & Kong, 2010). However, these extraction methods are subject to constraints, such as the availability of raw materials, and the process is complex, inefficient, and not well suited to industrial-scale production (Nanjo, Usui, & Suzuki, 1984). By comparison, enzymatic conversion methods have many advantages, including mild reaction conditions, strong controllability and lower pollution levels (Fujimoto, Hattori, Uno, Murata, & Usui, 2009). Gentiooligosaccharides can now be prepared by bioenzymatic methods, using β-glucosidase, which affords products of different specification that are relatively easily separated and purified (Ajisaka & Yamamoto, 2010). β-Glucosidase is referred to as β-d-glucosidase, gentiobiose enzyme and cellobiose enzyme (Palmeri & Spagna, 2007). It is widely found in plants, animals and microorganisms, including prokaryotic microorganisms, such as Escherichia coli, and eukaryotic microbes, such as yeast and Aspergillus niger (James and Asim, 2010, Kong et al., 2015). β-Glucosidase can hydrolyse β-glucosidic bonds in a non-reducing manner to free β-glucosidic bonds and various ligands (Peralta et al., 1997, Zhao et al., 2015). β-Glucosidase also has transglycoside activity, and can transfer the ligand or glucose released from hydrolysis to other sugar substrates via the formation of a β-1,6 glycosidic bond (Chang et al., 2011, Kono et al., 1999, Park et al., 2005). Some β-glucosidase genes from bacteria and fungi have been cloned and expressed. In a study by Liang Zhang et al., a β-glucosidase gene bgl1 from T. viride was cloned into the Puc19 vector and integrated into the genome of Saccharomyces cerevisiae (Zhang, Hong, & Shi, 2006). In another study by Ping Chen et al., β-glucosidase gene bgl1 from T. viride was cloned into the pPIC9 vector and integrated into the genome of P. pastoris GS115 (Chen, Fu, Ng, & Ye, 2011). To date, research on the production of gentiooligosaccharides via enzyme conversion has focused on reverse hydrolysis by β-glucosidase using high concentrations of glucose as the raw material (Gao et al., 2014, He et al., 2013, Qin et al., 2011, Yang et al., 2013). Because β-glucosidase generally has a high gentiobiose hydrolysis activity, the reaction can be shifted in the direction of reverse hydrolysis to synthesise gentiobiose by increasing the concentration of glucose (Guo et al., 2015). Previously, β-glucosidase has been used to synthesise gentiooligosaccharides from high concentrations of glucose mainly derived from fungi, among which A. niger CMI CC324262 achieved the highest yield of 50 g/l (Liu et al., 2009). To date, Japan Food and Chemical Co., Ltd. is the only company to achieve industrial production of gentiooligosaccharides (Nakakuki, 2010). β-glucosidase also possesses transglycosylation activity, which bestows β-glucosidase with the capability to cleave β-1,4-glycosidic bonds and transfer substrates to glucose via the formation of β-1,6-glycosidic bonds, as occurs in the synthesis of gentiooligosaccharides (Ramani, Meera, Rajendhran, & Gunasekaran, 2015). Few studies have reported the production of gentiooligosaccharide by β-glucosidase-based transglycosylation (Guo et al., 2015).