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Advances in Food Biotechnology

Edited by Ravishankar Rai V.

Advances in Food Biotechnology

Edited by

RAVISHANKAR RAI V. Department of Studies in Microbiology, University of Mysore, Mysore, India

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23 Biotechnological Production of

Oligosaccharides: Advances and Challenges

Diana B. Muñiz-Márquez1, Juan C. Contreras1, Raúl Rodríguez1, Solange I. Mussatto2, José A. Teixeira3 and Cristóbal N. Aguilar1

1 Food Research Department, School of Chemistry, University Autonomous of Coahuila, 25280, Saltillo, Coahuila, Mexico

2 Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands 3 IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus

Gualtar, 4710-057, Braga, Portugal

23.1 Introduction

Oligosaccharides are compound-type carbohydrates formed by the union of 2–10 monosaccharides through glycosidic bonds (Mussatto & Mancilha 2007). These sugars are called ‘non-digestible oligosaccharides’ because they cannot be metabolized by digestive enzymes in the organism due to the presence of osidic bonds in their structure. However, these are used by gut microbiota and are therefore considered as prebiotics. The term ‘prebiotic’ was introduced in 1995 by Gibson and Roberfoid as an alternative approach to the stimulation of growth and activityofsomeselectivebacteria (particularly Bifidobacteria and Lactobacilli) that can improve the host health by suppressing or inhibition of reproduction of entero-pathogenic microorganisms (Yildiz 2011; Charalampopoulos & Rastrall 2012; Panesar et al. 2014).

Prebiotics and probiotics are actually used in symbiotic form for formulation of functional foods. Of the prebiotics ingredients that can be used in food and pharmaceutical industries the main components are oligosaccharides, especially fructooligosaccharides (FOS), galactooligosaccharides (GOS), isomaltooligosaccharides (IMO), xilooligosaccharides (XOS), soybean oligosaccharides,

lactusucrose, lactulose and cyclodextrins (Mussatto & Mancilha 2007; Charalampopoulos & Rastrall 2012). On the other hand, probiotics are microorganisms present in their natural form in the intestine and are important because they provide beneficial effects to health. Among all considered probiotics, lactic acid bacteria (L. acidophilus, L. casei, L. reuteri, L. plantarum, Lactococcus and Streptococcus thermophilus) and Bifidobacterium species (e.g. B. brevis, B. longum, B. infantis and B. animalis) are the most common class and important of microbes (Fig. 23.1; Tormo 2006). The aim of this chapter is to summarize the main advances in the biotechnological production of oligosaccharides (prebiotics) and the associated challenges with the recovery and purification of these compounds.

23.2 Beneficial Effects of Oligosaccharides

The beneficial effects of oligosaccharides in human host are related to the normal intestinal flora as there exists a symbiotic relationship between pre- and probiotics. The prebiotics are fermented by probiotics in the colon to produce metabolites that play an important role in health, such as short-chain fatty acids (SCFA) and enzymes (Lam & Cheung 2013). Among the beneficial effects of

Advances in Food Biotechnology, First Edition. Edited by Ravishankar Rai V. � 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd.

382 Advances in Food Biotechnology

Figure 23.1 Mechanism of action of probiotics and prebiotics.

the oligosaccharides (prebiotics) the most important are the inhibition of pathogenic and putrefactive microorganisms, minerals absorption, cancer prevention, decreased levels of cholesterol and triglycerides and stimulation of the immune system (Fig. 23.2; Serban 2013).

23.2.1 Stimulating Effect on Activity of Probiotic Microorganisms

Oligosaccharides serve as fermentable substrate for the growth or activity of gut microflora, occurring during selective fermentation. This biochemical process occurs in the large intestine and the results of fermentation between

pre- and probiotics allow the production of microbial metabolites that play a central role in promoting human health. Prebiotic substances such as low-molecular-weight oligosaccharides are more rapidly fermented by intestinal microflora (especially by Bifidubacteria and Lactobacilli) than high-molecular-mass compounds such as polysaccharides (Lam & Cheung 2013).

23.2.2 Cancer Prevention or Therapy

Oligosaccharides are more efficacious when are used in symbiotic form with the probiotics. There have been many in vitro and in vivo studies that have revealed that

Figure 23.2 Biological properties of oligosaccharides.

Biotechnological Production of Oligosaccharides: Advances and Challenges 383

oligosaccharides are potential compounds that reduce the risk of cancer, acting as a substrate for the probiotics in the intestinal tract. Symbiotics are effective mainly for the prevention of colon cancer because the biochemical mechanisms occur in the intestinal zone (Rastall & Maitin 2002). Among the beneficial effects of the pre- and probiotics for control of colon cancer are the generation of short-chain fatty acids (SCFA) for probiotics. Some pathogenic microbes do not survive in acidic media; in this case, the microbial enzymes that act on various substrates, possibly producing carcinogenetic agents, are inhibited (e.g. sulphatases, reductases, β-glucuronidase, decarboxylases etc.). For example, Campylobacter jejuni has been related to the development of lymphoma of the small intestine. In mice, Clostridium butyricum and Mitsuokella multiacida have been linked to the incidence of colonic adenoma. On the other hand, the most important SCFA is butyrate because it inhibits colon carcinoma cells and also induces apoptosis in these cells, including colon adenoma and other colon cell lines (Serban 2013).

23.2.3 Decreased Levels of Cholesterol and Triglycerides

Other benefits attributable to oligosaccharides are related to their capacity for decreasing levels of cholesterol and triglycerides. The effects of the oligosaccharides have been demonstrated using biological models, particularly rats or hamsters, resulting in changes in either serum triacylglycerol accumulation in the liver or serum lipids. Other mechanisms involved in the control of lipids are due to short-chain fatty acids (SCFA) produced by intestinal microflora in the process of fermentation, and could play a role in the equilibrium of lipid metabolism (Dominguez et al. 2014).

23.3 Types of Oligosaccharides

The majority of prebiotics are non-digestible oligosaccharides, including fructoologosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), isomaltosaccharides (IMOS) lactulose, lactusucrose and inulin (Otieno & Ahring 2012; Al-Sheraji et al. 2013; Lam & Cheung 2013).

23.3.1 Fructooligosaccharides (FOS)

Fructooligosaccharides (FOS) are compounds formed by 1–3 fructose units bound to the β-2, 1 position of sucrose (Yoshikawa et al. 2006; Ganaie et al. 2013), are are mainly understood to be composed of 1-kestose (GF2), 1-nystose (GF3) and 1-β-fructofuranosyl-nystose (GF4) (Fig. 23.3; Ganaie et al. 2013). FOS are actually considered as important

Figure 23.3 Fructooligosaccharides synthesized by fructosyltransferases.

prebiotic compounds because they are small dietary fibres which are non-digestible by the hydrolytic activity of human digestive enzymes; they are therefore fermented by selective intestinal microflora, causing a reduction of pathogens due to the decrease of intestinal pH (Mussatto & Mancilha 2007; Mussatto et al. 2013).

These sugars are formed from sucrose by microbial enzymes called fructosyltransferase (FTase, EC2.4.1.9) and β-fructofuranosidase (FFase, EC3.2.1.26) with high transfructosylating activity (Ganaie et al. 2013).

23.3.1.1 Fructosyltransferases (FTases)

These enzymes belong to the family of glycoside hydrolases (GH32), and are responsible for transferring a fructosyl unit to a molecule of sucrose or FOS, which acts as acceptors in the reaction (Fig. 23.4; Antos ová & Polakovic2001; Maiorano et al. 2008).

Figure 23.4 Mechanism of action of fructosyltransferases enzymes.


Figure 23.5 Mechanism of action of fructofuranosidases enzymes.

23.3.1.2 β-Fructofuranosidases (FFases)

β-Fructofuranosidases also belong to the family of glycoside hydrolases and are enzyme-type hydrolases as they act directly on sucrose by cleaving the linkage, releasing glucose and fructose in the reaction. However, a transfructosylating activity is exhibited by these enzymes when a higher sucrose concentration is used (Fig. 23.5; Antos ová & Polakovic 2001; Maiorano et al. 2008).

23.3.2 Galactooligosaccharides (GOS)

Galactooligosaccharides (GOS) are another type of prebiotic compounds that constitute the major part of oligosaccharides in milk human (Rodriguez-Colinas et al. 2013). These compounds are molecules formed from glucose and galactose through β-galactosidases enzymes according to the formula Galn-Glc where n = 2 to 20 units (Rodriguez-Colinas et al. 2013; Kovács et al. 2014). GOS, FOS and other prebiotic compounds have obtained the generally recognized as safe (GRAS) status by the US Food and Drug Administration. For example, GOS have been applied in children’s products as well as in other uses. The inclusion of GOS in infant formula is believed to provide a metabolic

activity similar to that described in breastfed infants (Sierra et al. 2015).

23.3.2.1 β-Galactosidases

β-Galactosidases (Galactohydrolases EC3.2.1.23), also called lactases, are members of GH1 and GH2 families of the glycoside hydrolases. β-Galactosidases catalysed the GOS formation from lactose (Gal (β1-4) Gal (β1-4) Glc); however, the efficiency of the synthesis of GOS depends on the conditions used as well as on the enzyme of choice. On the other hand, these enzymes are also responsible for the hydrolysis of lactose (Kovács et al. 2014; Sen et al. 2014); depending on the lactose concentration, the reaction is either focused on hydrolysis or transglycosylation. For example, when the amount of water in the process (expressed as water activity aw) is high, the hydrolysis of lactose is predominant (Manera et al. 2012).

23.3.3 Xylooligosaccharides (XOS)

Xylooligosaccharides (XOS) have been reported for their prebiotic effects when ingested as a part of the diet (Driss et al. 2014). These compounds are obtained from the


enzymatic hydrolysis of xylan and are oligomeric compounds with a degree of polymerization (DP) of 2-6 xylose units linked by β, (1-4) bonds with a variety of substituents such as uronic acids, acetyl groups and arabinose molecules (Chapla et al. 2012; Bian et al. 2013; Jayapal et al. 2013). Kovács et al. (2014) mentioned that an interesting growth of some intestinal flora has been demonstrated by in vitro fermentations using XOS as substrate.

23.3.3.1 Xylanases

Xylanase enzymes are the responsible for catalysis of XOS from various xylan-rich agroresidues that have been classified as one of the glycoside hydrolases (GH) 5, 7, 8, 10, 11 and 43 on the basis of their amino acidic sequences, catalytic process and structural fold (Gonçalves et al. 2012). With low levels of exoxylanase or β-xylosidase activity, the xylanases produce a high concentration of xylose, having an inhibitory effect on the generation of XOS (Chapla et al. 2012). The endo-1, 4-β-xylanases enzymes cleave the β-1, 4 glycosidic bonds between xylose residues (Gonçalves et al. 2012).

23.3.4 Isomaltooligosaccharides (IMOS)

Isomaltooligosaccharides (IMOS) have attracted special attention because of their high stability, low cost and high availability (Zhang et al. 2010). IMOS consists mainly of isomaltose, panose and isomaltotetraose and are obtained from starch through an enzymatic mechanism (Li et al. 2009). Dextran can be employed as a substrate to generate higher-molecular-weight IMOS (Rastall 2010).

23.3.4.1 α-Amylases and α-Glucosidases

IMOS are commercially manufactured from starch by enzymatic transfer reactions; starch is hydrolysed to a mixture of maltooligosaccharides through α-amylase (EC3.2.1.1) and pollulanase. The maltodextrins are then used as substrates in a glycosyl transfer reaction by α-glucosidase (EC3.2.1.20) which converts the α- 1, 4 linked maltodextrins into α- 1,6 linked isomaltooligosaccharides (Rastall 2010).

23.3.5 Inulins

Inulins are another type of dietary fibre produced by extraction chicory roots; due to their various properties, they are used in foods as a texturizing agent or sugar replacement (Tungland & Meyer 2002). Inulin-type fructans are linear fructose polymers that mostly (or exclusively) consist of β-(2-1) fructosyl–fructose linkages terminated by a glucose

residue through a sucrose-type linkage at the reducing end (Roberfroid 2005; Paixão et al. 2013; Apolinário et al. 2014).

23.3.5.1 Inulinases (Fructofuranosyl Hydrolases)

Inulin is hydrolysed by two types of enzymes called endoor exoinulinases. The endoinulinases act on the internal linkages in inulin and release inolooligosaccharides and the exoinulinases remove the terminal fructose residues from the non-reducing end of the chain (Apolinário et al. 2014). The microbial exoinulinases have been used for the production of high-fructose syrups while the endoinulinases have been proposed as the most promising approach for the generation of oligosaccharides with added value (Paixão et al. 2013).

23.3.6 Pectic Oligosaccharides (POS)

Pectic oligosaccharides (POS) are a new class of prebiotics which result from the hydrolysis of pectin obtained from pectin-rich products such as apple pomace or citrus peel. Structurally, POS are oligomers substituted or not, including oligogalacturonides (OGalA), galactooligosaccharides (GalOS), arabinooligosaccharides (AraOS), rhamnogalacturonoligosaccharides (RhaGalAOS), xylooligogalacturonides (XylOGalA) and arabinogalactooligosaccharides (AraGalOS), and are considered as novel candidate prebiotics due to their industrial potential (Gullón et al. 2013). They are produced by enzymatic, chemical or combined methods. For example, Manderson et al. (2005) obtained POS from orange albedo by a chemical method utilizing HNO3, whereas Mandalari et al. (2007) used pectynolitic enzymes for the POS production from bergamot peel. More recently, Martínez-Sabajanes et al. (2012) studied the production of POS from orange peel waste by enzymatic hydrolysis.

As well as prebiotic effects, POS have other beneficial properties such as antibacterial agents and stimulation of apoptosis in human colonic adenocarcinoma (Collins & Rastrall 2008).

23.4 Other Enzymes used for the Biosynthesis of Oligosaccharides

23.4.1 Glycosidases (GH)

Glycosidases or glycoside hydrolases are enzymes that have been extensively studied for the production of oligo- or polysaccharides. These enzymes are degrading catalysts responsible for the hydrolysis of glycosidic bonds, are stable


enzymes, easy to produce and their hydrolytic reaction can be reversed under certain conditions (Faijes & Planas 2007). Glycosidases are less specific in their capacity to catalyse certaincleavagescomparedwithglycosyltransferases;GHare also available from a larger number of sources such as microorganism, plant and animal cells (Playne & Crittenden 2009).

23.4.2 Glycosyltransferases (GTs)

Glycosyltransferase enzymes are also used for the production of oligosaccharides, glycoconjugates and their analogues. These enzymes catalyse the formation of glycosidic bonds by the transfer of a saccharide, in particular, a monosaccharide from a donor substrate to an acceptor substrate (Palcic 2011). Glycosyltransferases have been classified into different families on the basis of the active molecule which acts as donor, the type of saccharide that transfers and whether the enzyme forms an α- or β-glycosidic bond (Keegstra & Raikhel 2001). However, the instability of GTs as synthetic agents has led some studies to evaluate immobilized GTs for oligosaccharides biosynthesis (Playne & Crittenden 2009).

23.5 Microbial Production of Prebiotic Oligosaccharides

Extensive research on microbial production of oligosaccharides, related to the value of these compounds for their biological properties (particularly their prebiotic effects), has been published. For example, β-galactosidases from fungal species such as Aspergillus niger and Aspergillus oryzae are used for the production of galactooligosaccharides (Playne & Crittenden 2009). On the other hand, Kluyveromyces lactis produces β-galactosidase for the production of galactooligosaccharides. This mesophile yeast has been studied due to its preferential dairy growth environment and the good hydrolysis activity of lactose (Park & Oh 2010). Barthomeuf & Pourrat (1995) evaluated the production of fructooligosaccharides from sucrose by Penicillum rugulosum, demonstrating that the crude enzyme extract acts as a mixed system of fructosyltransferase and glycosidases. Production of inulooligosaccharides from inulin by

Escherichia coli recombinant was reported by Yun et al. (1999) using an inulinase gene (inu1) of Psuedomonas sp. Recently, Sathish & Prakasham (2013) investigated the production of fructosyltransferase and fructooligosaccharides by Aspergillus awamori GHRTS using organic solid substrates such as rice bran and

corncobs. Other species have been reported for the production of oligosaccharides such as Aerobasidium pollulans, Fusarium oxysporium, Phytophthora parasitica, Claviceps purpurea, Scopulariopsis sp. and Saccharomyces cerevisiae and different species of the genus Aspergillus (Mutanda et al. 2014).

23.6 Yeast Strains used in Galactooligosaccharide Production from Lactose

Some yeasts have been used for the microbial production of oligosaccharides; however, more often fungi and bacteria are employed. Enzymes such as β-galactosidase (also known as lactase or β-D-galactoside galactohydrolase) are responsible for catalysing the hydrolysis of the β-Dgalactoside linkage to D-glucose and D-galactose followed by transgalactosylation of galactooligosaccharides. Lactose in this reaction is a substrate which serves both as a galactosyl donor and acceptor to yield di-, tri- or higher oligosaccharides (Kim et al. 2001). Cho et al. (2003) purified and characterized a β-galacto

sidase, produced by the yeast Bullera singularis, using a crude extract which has shown high transgalactosylation activity resulting in the galactooligosaccharide conversion of over 34% using lactose as substrate. On the other hand, Onishi et al. (1995) studied the production of galactooligosaccharides from lactose using bacteria and yeasts. The best results were found with Sterigmatomyces elviae CBS8119, Rhodotorula minuta IFO879 and Sirobasidium magnum CBS6803, which were shown to be efficient producers of Gal-OS from lactose.

Recently, Fai et al. (2014) evaluated the production of galactooligosaccharides from lactose by Pseudozyma tsukubaensis and Pichia kluyveri, obtaining yields of 14–15% (w/w) with a solution of 40% lactose.

23.7 Analysis of Oligosaccharides

23.7.1 Thin-Layer Chromatography (TLC)

This is a classic simple technique for the analysis of oligosaccharides that has been utilized by several authors (e.g. Reifová et al. 2003; Reifová & Nemcová 2006). TLC is a type of chromatography involving the use of a solvent mixture and glass-backed TLC plates. Compared to HPLC and GC, TLC has the advantages of simple sample preparation, low cost, analysis of many samples from a single plate and analysis of crude samples (Reifová & Nemcová 2006).


23.7.2 High-Performance Liquid Chromatography (HPLC)

HPLC is widely used for the detection and quantification of several class of compounds. Oligosaccharides have also been analysed by this methodology, using a different column as stationary phase such as alkyl-bonded silica phases (C18), aminoalkyl-bonded silica gel phases, cyclodextrin-bonded phases, anion-exchange phases and size-exclusion phases. On the other hand, detectors used in HPLC for the identification and quantification of oligosaccharides are refractive index (RI) detectors because sugar itself does not contain a special chromophore of fluorophore (Sanz et al. 2009). HPLC is a methodology with certain sample preparation protocols to eliminate impurities as a filtration, for example using 0.45 μm nylon filters. Compared to other methods, HLPC is a sensitive, efficient and fast technique.

23.7.3 Gas Chromatography (GC)

Gas chromatography is applicable to the determination of sugars after an initial step of derivatization due to the polar structure. The most-used derivatization process is silylation using bis (trimethylsilyl) trifluoroacetamide (BSTFA), which produces multiple derivatives for sugar compounds with aldehyde and -OH groups from unwanted silylation of the aldehyde group (Wan & Yu 2006). This technique uses a capillary column that improves the resolution, reducing analysis time. Dimensions of capillary columns employed for sugar GC analysis can vary over the range 1–50 m in length, 0.1–05 mm in diameter and 0.02–2 μm with flame ionization detection (FID) (Sanz et al. 2009).

23.7.4 Liquid Chromatography Mass Spectrometry (LC-MS)

Liquid chromatography mass spectrometry is also a sensitive method for sugar determination. The LC-MS system consists of a separation of the compounds via liquid chromatography, using a column for separation such as Prevail carbohydrate ES packed with 5 μm spherical polymer beads coated with amino-based proprietary bonding material. In the second step, the analytes are determined by electrospray ionization mass spectrometry (ESI-MS) in positive or negative mode (Wan & Yu 2006).

23.7.5 MALDI-TOF-MS Analysis

MALDI-TOF-MS is a versatile and sensitive tool used for the characterization of sugars such as oligosaccharides. This technique allows the determination of the molecular

mass, giving the maximum DP, linkages and branching for the sugars present in the mixture. The derivatization process for oligosaccharides has been developed with the aim of increasing the sensitivity of MALDI-MS analysis. Matamoros et al. (2007) determined the characterization of oligosaccharides present in an enzyme-treated industrial fermentation residue using MALDI-TOF-MS, resulting in identification of molecular ions corresponding to sodiated hexose and pentose oligo/polysaccharides.

23.8 New Approaches for Purification of Oligosaccharides

Purification of oligosaccharides is an important stage in their commercialization or application. Today, various techniques such as liquid chromatography or filtration with membranes have been used. The following sections describe some procedures for the purification of oligosaccharides.

23.8.1 Gel Chromatography

Gel chromatography is a methodology used for the purification of many compounds such as oligosaccharides. Many types of chromatographic packings such as Bio-gel P2, Superdex 30, Shepadex G-10 and G-25 have been used in technique. Shepadex LH-20 has been less used, however; Mei et al. (2013) were first to publish results of the purification of neoagarooligosaccharides using a column Shepadex LH-20, finding two kinds of oligosaccharides.

23.8.2 Ethanol Precipitation

Ethanol precipitation is an interesting methodology for the purification of bioactive compounds such as oligosaccharides. This simple methodology involved the use of ethanol at various concentrations, using various temperatures of incubation (Swennen et al. 2005). Sen et al. (2011) purified galactooligosaccharides

through precipitation with ethanol at various concentrations; ethanol solutions of 90% concentration were found to induce the precipitation of the saccharides.

23.8.3 Membrane-Based Techniques

Membrane technology is a strategy for the purification of molecules and has advantages including low-energy


requirements, easy scale-up and easy modification of the operational variables (Pinelo et al. 2009). The general principle is based on the selective permeability of the membrane to allow the substances of interest penetrate through the membrane, while unwanted compounds are normally rejected (Li & Chase 2010).

23.8.4 Nanofiltration

Nanofiltration is a procedure of purification and concentration of products through membranes. This process is widely used in food processing applications because it provides purities of 85–90%, depending on the compound of interest. Kuhn et al. (2010) studied the process of purification of fructooligosaccharides by nanofiltration in two stages – diafiltration and nanofiltration – obtaining purities of 90% with yields of 80%.

23.8.5 Electrofiltration

Electrofiltration is a recent procedure that uses electric fields in the membrane to lower the concentration polarization. This technique provides a new alternative for the fractionation of certain sugars, particularly pectins because charge density and distribution can be quickly modified by enzymatic and chemical process (Pinelo et al. 2009).

23.8.6 Ultrafiltration

Ultrafiltration is other procedure based on size exclusion to separate molecules according to their molecular weight. In the ultrafiltration process, membranes such as MWCO (molecular weight cut-off) polyethersulphone (PES) may be used for the concentrationandpurificationofmolecules(Li&Chase2010). Thistypeoftechniqueissuitableforseparatingmacromolecules in terms of relative molecular weight, ranging from several hundred to several thousand grams per mole, corresponding to molecular size of nano- to micrometres (Li & Chasse 2010).

23.9 Emerging Trends in the Production of Novel Oligosaccharides

The interest in developing novel forms of oligosaccharides has been increasing as a result of their huge potential as prebiotic molecules. Other emerging oligosaccharides such as gentiooligosaccharides (GeOS) and glucooligosaccharides (GlcOS) (see following sections) have been described as important prebiotics.

23.9.1 Gentiooligosaccharides (GeOS)

These are composed of β-(1-6) linked D-glucose units and are produced commercially by Nippon Shokuhin Kako in Japan (Collins & Rastall 2008). Gentiooligosaccharides have been identified as prebiotic molecules, showing bifidogenetic activity which allows the production of the highest levels of SCFA (Collins & Rastall 2008; Fujimoto et al. 2009). They have been obtained from partial hydrolysis of the lichen polysaccharide pustulan but with low yields (Fujimoto et al. 2009); further research on these compounds is therefore necessary.

23.9.2 Glucooligosaccharides (GluOS)

These are derived from maltodextrins by dextran dextrinase enzymes through Gluconobacter oxydans (Collins & Rastall 2008). Other bacterium such as Lactobacillus, Leuconostoc, Streptococcus, Pediococcus and Weissella also produce extracellular α-glucooligosaccharides (Shukla et al. 2014). Glucooligosaccharides have in their structure glucose residues linked by glycosidic bonds and have demonstrated potential prebiotic activity (Grimoud et al. 2010).

23.10 Concluding Remarks

Oligosaccharides are important prebiotics with several applications in areas including foods, pharmacy and agriculture. In this chapter we have reviewed the biosynthesis of oligosaccharides formed by microbial or vegetal enzymes, including some mechanisms of product formation. In addition, functional properties of oligosaccharides are included in this chapter. Some analytical techniques for the determination of oligosaccharides as well as purification methods have also been described.

Acknowledgements

Author DB Muñiz-Márquez thanks the Mexican Council for Science and Technology (CONACYT) for the financial support for the postgraduate program (doctorate) in Food Science and Technology offered by Autonomous University of Coahuila (UAdeC).

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