**1. Introduction**

The concept of nondigestible oligosaccharides (NDOs) came from the observation that the human body does not have the necessary enzymes to hydrolyze β-glycosidic linkages present in some sugars of the human diet. Thus, these carbohydrates can arrive intact in the intestine where they are fermented selectively stimulating the growth and/or activity of bacteria in the colon acting as prebiotics [1]. In this context, nondigestible oligosaccharides have received much attention since they have important biological properties promoting health beneficial effects. Stimulation of the intestinal microbiota growth associated with low cariogenic and caloric value are some of these properties. Also noteworthy is a stimulation of the immune system leading to a reduced risk of diarrhea and other infections. The benefits are obtained by a decrease in intestinal pH due to the fermentation of NDOs, decreasing the proliferation of

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pathogenic microorganisms, and an increase of the bifidobacteria population [2]. The bioactive properties of NDOs can be influenced by monosaccharide composition, type of glycosidic linkage, and degree of polymerization [2].

Nondigestible oligosaccharides can be produced using chemical or enzymatic processes. The synthesis using chemical methods are complicated, with numerous protection and deprotection steps required in order to achieve regioselectivity [3]. Other challenges of chemical synthesis are the low yields, expensive chemicals, and impossibility for scaleup. For those reasons with few exceptions, most of the NDOs are produced by enzymatic processes.

The enzymatic production of NDOs can be achieved by two different approaches, the use of glycosyltransferases or glycosidases. Glycosyltransferases catalyze the stereospecific and regiospecific transfer of a monosaccharide from a donor substrate (glycosyl nucleotide) to an acceptor substrate. Some of the difficulties associated with the application of glycosyltransferases are availability of enzymes and sugar nucleotide donors, product inhibition, and reagent costs. These factors decrease the applications of these enzymes in the production of NDOs [4]. The glycosidases offer a good alternative for enzymatic production of NDOs, where they can be synthetized from monosaccharides using transglycosylation reactions, or formed by controlled enzymatic hydrolysis of polysaccharides. Some advantages of the glycosidases in relation to glycosyltransferases are availability, good stability, and the fact that they act on easily found substrates and do not need cofactors [3].

The transglycosylation route can be performed by the use of a good glycosyl donor that can be a disaccharide, in high concentrations. This donor will form an intermediate glycosyl-enzyme that can be intercepted by an acceptor to give a new glycoside or oligosaccharide [3]. When the substrate is a monosaccharide, it will be acting as a donor and acceptor. Some glycosidases used to produce NDOs using this approach are α-galactosidases, β-fructofuranosidase, cyclomaltodextrin glucanotransferase, and α-glucosidase [4].

The production of NDOs by controlled hydrolysis of polysaccharides involves the break of glycosidic bonds, the reaction is acid base catalyzed by an oxocarbenium ion-like transition state and involves two carboxylic groups at the active site [5]. The glycosidases can be divided into inverting or retaining depending on the configuration of the glycosidic linkage after the hydrolysis. Inverting glycosidases operate through direct displacement of the leaving group by water. The two carboxylic groups are responsible for the reaction, one provides base catalytic assistance to the attack of water and the other provides acid catalytic assistance to cleavage of the glycosidic bond. Retaining glycosidases use a double displacement mechanism involving the formation of a covalent glycosyl enzyme intermediate, where one carboxylic group acts as acid catalyst for the glycosylation step and base catalyst for the deglycosylation step [3]. The second carboxylic group acts as a nucleophile and a leaving group. The enzymes inulinase, pullulanase, amylase, xylanase, endogalactanase, rhamnogalacturonase, endogalacturonase, and chitosanase are used for NDOs production using the controlled hydrolysis approach [4].
