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  • br Declarations of interest br Acknowledgements This work wa


    Declarations of interest
    Acknowledgements This work was supported by the National Natural Science Foundation of China (81673321, 21506099), the State Key Laboratory of Drug Research (SIMM1705KF-13), the Natural Science Foundation of Jiangsu Province (Grant No. BK20151541), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTC1812).
    Introduction The research in the optimization of the synthesis of galactooligosaccharides (GOS) is of ongoing interest as they exhibit prebiotic properties, and thus are beneficial compounds for infants [[1], [2], [3]] and adults [[4], [5], [6]] alike. GOS are generated in a side reaction called transgalactosylation during the hydrolysis of lactose by β-galactosidase (EC, if the acceptor molecule is another sugar instead of water [7]. As this sugar molecule can be any sugar in the reaction mixture, that is glucose, galactose, lactose, or GOS itself, the chain length (or degree of polymerization (DP)) of the generated GOS will increase with ongoing reaction time. However, GOS are also possible substrates for the enzyme and their hydrolysis will exceed their synthesis after a certain degree of lactose conversion is reached [8]. With the enzyme source being one of the most important factors influencing GOS yield [9], as well as GOS structure in terms of β-glycosidic linkage [10], we propose that the combination of two β-galactosidases from different origins, which thus have different preferences for transgalactosylation and hydrolysis, might lead to an enhanced GOS yield. However, research on this topic is very rare with only a handful of publications during a period of more than 30 years of GOS research. While Yakult Pharmaceutical Industry Co., Ltd uses two tgf beta receptor inhibitor from Sporobolomyces singularis and Kluyveromyces lactis during the production of Oligomate® [11], with the second enzyme aiming mainly for hydrolysis of unreacted lactose, it is not clear whether the second enzyme also contributes to the total GOS yield. Similar, Vitalus Nutrition Inc. [12] submitted a patent application for a consecutive combination of β-galactosidases from Aspergillus oryzae and K. lactis, which led to an increased GOS yield from about 32% to 41%. On the other hand, Moon et al. [13] reported a reduced synthesis of GOS tri- and tetrasaccharides (disaccharides were unchanged), when A. oryzae and Kluyveromyces fragilis were used simultaneously in comparison to K. fragilis alone, ultimately resulting in a decreased total GOS yield (37% compared to 27%). A consecutive application of A. oryzae followed by Bacillus circulans could increase GOS yield (DP3-DP6) from 26% to 34% [14]. However, this was considerably lower compared to using only the B. circulans enzyme (40% GOS yield). Investigating the consecutive (but without inactivation of the first enzyme) and simultaneous coupling of β-galactosidases from B. circulans with those from A. oryzae or K. lactis, also showed no increase in GOS yield (exemplified by the two main trisaccharides) compared to B. circulans alone [15]. This study aims to investigate the consecutive and simultaneous combination of different β-galactosidase enzymes and its effect on total GOS yield, as well as potential compositional changes.
    Materials and methods
    Results and discussion
    Funding sources
    Introduction β-galactosidases (EC are enzymes of glycoside hydrolase (GH) families that play important biological roles in living organisms by removing terminal non-reducing galactosyl residues from various carbohydrate polymers (Gantulga et al., 2009). The Carbohydrate-Active enZYmes (CAZy) database presently classifies these enzymes into GH-1, GH-2, GH-35, GH-42, GH-59 and GH-117 families (CAZy, Majority of the microbial β-galactosidases are from GH-2 and GH-35 families. Higher plants contain many β-galactosidases, all of which are members of GH-35 family (Hobson and Deyholos, 2013; Eda et al., 2016). The diversity in sequence and structure of plant β-galactosidases allows them to influence various biological processes including seed germination (Dean et al., 2007), early growth and development (Esteban et al., 2003; Martín et al., 2008), phloem differentiation (Roach and Deyholos, 2008; Martín et al., 2013), reproductive organ development (Hrubá et al., 2005; O'Donoghue et al., 2017) and fruit ripening (Smith and Gross, 2000). The primary function of β-galactosidase in most of these processes is degradation and remodeling of cell wall by cleaving galactosyl moieties from pectin and xyloglucan residues.