**3. Galactooligosaccharides**

operation, and both free and immobilized enzyme produced FOS from sucrose with a yield around 57% [58]. Similarly, production of FOS using the enzyme preparation immobilized onto epoxy-activated Sepabeads EC (Sepabeads EC-EP5) reached a yield of 61% after 36 h of

Synthesis of FOS by dried alginate entrapped enzymes (DALGEEs) was recently reported [60]. FTase from *Aspergillus aculeatus*, contained in Pectinex Ultra SP-L, was entrapped in alginate gel beads, which were then submitted to dehydration. The dried alginate biocatalysts were evaluated for the synthesis of FOS from sucrose in a continuous fixed-bed reactor. A 40-fold enhancement of the space-time-yield of the fixed-bed bioreactor was observed when using DALGEEs compared with conventional gel beads. The fixed-bed reactor packed with DAL‐ GEEs presented excellent operational stability since the composition of the outlet was nearly

A partially purified β-fructofuranosidase from the commercial enzyme preparation Visco‐ zyme L was covalently immobilized on glutaraldehyde-activated chitosan particles [61]. Thermal stability of the immobilized biocatalyst was around 100-fold higher at 60°C when compared to the free enzyme. The biocatalyst also showed a high operational stability, which allowed its reuse for at least 50 cycles without significant loss of activity. The average yield of

An alternative to the enzymatic production of FOS is the use of either free or immobilized whole cells in bioreactors [62]. Production of these oligosaccharides via fermentation processes has the advantage of obviating purification of FOS-producing enzymes from the cell extracts

An integrated one-stage method for production of FOS via sucrose fermentation by *Aureoba‐ sidium pullulans* was developed and optimized with experimental design tools. To maximize production of FOS, temperature and agitation speed were optimized. A production yield of FOS from sucrose of 64% was obtained in 48 h of fermentation under the optimum conditions

Two filamentous fungi, *Cladosporium cladosporioides* and *Penicillium sizovae*, with myceliumbound transfructosylating activity were recently isolated. *C. cladosporioides* and *P. sizovae* provided maximum FOS yields of 56% and 31%, respectively. *C. cladosporioides* synthesized a

(blastose)) with different glycosidic linkages, which could afford certain benefits regarding

Two food companies in Japan and Korea use different commercial processes for the continuous production of FOS with immobilized cells of *Aspergillus niger* and *Aureobasidi‐ um pullulans*, respectively, both entrapped in calcium alginate gel [27,63]. Calcium algi‐ nate has also been employed to immobilize mycelia of *A. japonicus* aiming to establish FOS-

Immobilization of whole cells of *Aspergillus japonicus* ATCC 20236 onto different lignocellulosic materials was also undertaken to produce fructooligosaccharides. Cells immobilized in the

G-FOS, including a non-conventional disaccharide

constant during at least 700 h, with an average FOS concentration of 275 g/L.

reaction [59].

36 Food Production and Industry

[29,63,64].

(32°C and 385 rpm) [62].

mixture of FOS (1

their bioactivity [41].

producing processes [65,66].

FOS production from sucrose was 55%.

F-FOS, 6

F-FOS and 6

Lactose is a disaccharide formed by the condensation of glucose and galactose molecules, and is the most important component of mammalian milk, present in a concentration range from 2.0% to 10%. Lactose can be obtained at industrial scale from whey during cheese production, with dry weight around 80-85%, using crystallization techniques [76-78]. In the past, whey was considered a waste, although, nowadays, it is used to produce whey powders products, improving economic and environmental aspects of the by-products [79].

Lactose presents a great importance for food and pharmaceutical industries, being used in various food products such as chocolate, confectionary and other processed products, as well as carrier of medicines in dry powder inhalation preparations, excipient of tablets [80]. In humans, lactose can cause abdominal discomfort due to its maldigestion, which reaches approximately 70% of the world's adult population [81]. β-Galactosidase (β-D-galactoside galactohydrolase, E.C. 3.2.1.23) plays an important role in human health because it is able to catalyze the hydrolysis of lactose in glucose and galactose, and because of that, it is often referred to as lactase. In addition, the transglycosylation reaction can also occur, in which galactooligosaccharides (GOS) are produced, and their structures can differ in regiochemistry of glycosidic linkage and degree of polymerization (Figures 3-5; Tables 1 and 2) [82,83].

**Figure 3.** Structure of a galactooligosaccharide (GOS) derived from lactose, a β-(1,4) linked galactosyl oligomer (n=1-4), attached to a terminal glucosyl residue by a β-(1,4) bond. GOS are synthesized by the reverse action of β-galactosidases on lactose in higher concentrations.

Despite the fact that enzymes such as β-glycosidases and β-glucosidases, which also hydrolyze carbohydrates, are able to catalyze transglycosylation reactions, β-galactosidase is the most used enzyme in dairy industry to produce GOS. β-galactosidases from *Kluyveromyces* sp. and *Aspergillus* sp. are the most used in industry because products from those microorganisms are considered as GRAS [84].

Galactooligosaccharides can be defined as a mixture of substances produced from lactose, with two to eight saccharide units, in which one of the units is a terminal glucose and the remaining units are galactose and disaccharides comprising two units of galactose [85]. Several of these GOS are recognized as prebiotics, because they are non-digestible saccha‐ rides and can be used selectively by bifidobacteria and lactobacilli in human intestine, and thus improve host health [86, 87].

Conversion of lactose into GOS is catalyzed by β-galactosidases in a kinetically controlled reaction that involves competition between hydrolysis and transgalactosylation. The thermo‐ dynamically favored hydrolysis of lactose, which generates D-galactose and D-glucose, competes with the transferase activity that produces a complex mixture of galactose-based diand oligosaccharides. Transgalactosylation involves direct galactosyl transfer (intramolecular reaction) to D-glucose yielding regio-isomers of lactose, and indirect transgalactosylation (intermolecular) giving rise to disaccharides, trisaccharides, and tetrasaccharides, and eventually longer GOS. The interaction in the active site of the enzyme differs with the acceptor. When the acceptor is water, glucose and galactose are formed; whereas if the acceptor is a sugar, reaction results in GOS [86, 87].

Biotechnological Production of Oligosaccharides — Applications in the Food Industry http://dx.doi.org/10.5772/60934 39

as carrier of medicines in dry powder inhalation preparations, excipient of tablets [80]. In humans, lactose can cause abdominal discomfort due to its maldigestion, which reaches approximately 70% of the world's adult population [81]. β-Galactosidase (β-D-galactoside galactohydrolase, E.C. 3.2.1.23) plays an important role in human health because it is able to catalyze the hydrolysis of lactose in glucose and galactose, and because of that, it is often referred to as lactase. In addition, the transglycosylation reaction can also occur, in which galactooligosaccharides (GOS) are produced, and their structures can differ in regiochemistry of glycosidic linkage and degree of polymerization (Figures 3-5; Tables 1 and 2) [82,83].

**Figure 3.** Structure of a galactooligosaccharide (GOS) derived from lactose, a β-(1,4) linked galactosyl oligomer (n=1-4), attached to a terminal glucosyl residue by a β-(1,4) bond. GOS are synthesized by the reverse action of β-galactosidases

Despite the fact that enzymes such as β-glycosidases and β-glucosidases, which also hydrolyze carbohydrates, are able to catalyze transglycosylation reactions, β-galactosidase is the most used enzyme in dairy industry to produce GOS. β-galactosidases from *Kluyveromyces* sp. and *Aspergillus* sp. are the most used in industry because products from those microorganisms are

Galactooligosaccharides can be defined as a mixture of substances produced from lactose, with two to eight saccharide units, in which one of the units is a terminal glucose and the remaining units are galactose and disaccharides comprising two units of galactose [85]. Several of these GOS are recognized as prebiotics, because they are non-digestible saccha‐ rides and can be used selectively by bifidobacteria and lactobacilli in human intestine, and

Conversion of lactose into GOS is catalyzed by β-galactosidases in a kinetically controlled reaction that involves competition between hydrolysis and transgalactosylation. The thermo‐ dynamically favored hydrolysis of lactose, which generates D-galactose and D-glucose, competes with the transferase activity that produces a complex mixture of galactose-based diand oligosaccharides. Transgalactosylation involves direct galactosyl transfer (intramolecular reaction) to D-glucose yielding regio-isomers of lactose, and indirect transgalactosylation (intermolecular) giving rise to disaccharides, trisaccharides, and tetrasaccharides, and eventually longer GOS. The interaction in the active site of the enzyme differs with the acceptor. When the acceptor is water, glucose and galactose are formed; whereas if the acceptor is a

on lactose in higher concentrations.

38 Food Production and Industry

considered as GRAS [84].

thus improve host health [86, 87].

sugar, reaction results in GOS [86, 87].

**Figure 4.** Examples of structures of galactooligosaccharides: 4-galactosyl lactose (top) and 6-galactosyl lactose (bottom) are represented, showing usual regiochemistry differences in galactosyl linkages.

Therefore, high lactose concentrations and low water contents are favorable for GOS synthesis, being the initial lactose concentrations the most important factor, independently of the enzyme source. In general, higher lactose concentrations than 30% are necessary to favor synthesis over hydrolysis [87]. However, at the same lactose concentrations, different yields of GOS can be obtained, because β-galactosidases from different sources, with different structures and/or mechanisms, exhibit different selectivity for water and saccharides. Moreover, GOS yields depend on process conditions, such as temperature, reaction time, pH and enzyme/substrate ratio [88]. However, GOS production can be affected by glucose and/or galactose that are recognized as inhibitors of hydrolysis for many β-galactosidases [89,90].

The reaction time and initial concentration of lactose are considerably important to favor GOS production, since they are simultaneously synthesized and hydrolyzed by β-galactosidase, being regulated by the kinetics of synthesis and hydrolysis. Additionally, lactose concentration can increase formation of GOS due to increased availability of galactosyl and decreased availability of water [82,91]. Additionally, reverse micelle systems, in which the enzyme is entrapped in an aqueous micelle surrounded by organic solvent, provide decrease of the thermodynamic activity of water [92,93]. Chen et al. 2003 [93] reported that the transgalacto‐ sylation capability of low concentrations of β-galactosidase and lactose, operating in reverse micelles system, was similar to high concentrations of enzyme and substrate in an aqueous system. Authors also showed that GOS production decreases with the increase in water content.

**Figure 5.** Enzymatic synthesis of GOS by transgalactosylation reactions. Transgalactosylation is the transfer of the gal‐ actosyl residue, after the cleavage of lactose, to an acceptor molecule containing a hydroxyl group. When the acceptor is water (A), a galactose is formed by lactose hydrolysis, whereas if the acceptor is a sugar, a disaccharide or a GOS may be formed. In intramolecular transgalactosylation (B), galactosyl donor and acceptor are the same (glucose), only linkage position changes. In intermolecular transgalactosylation (C), there is an enzymatic transfer to another nucleo‐ philic acceptor (Y), which can be all the sugars present in the reaction media, resulting in GOS mixtures.

Production of GOS can be improved increasing the reaction temperature. Lactose has relatively low solubility at room temperature, which increases with increasing temperature. Therefore high temperatures are desirable since they allow the increase of lactose concentration [94,95]. Besides the possibility to increase the solubility of subtracts and products, high temperature is advantageous due to the reduced risk of microbial contamination, lower viscosity and improved transfer rates [96]. However, this is not a general rule, Boon et al. (1998) [97] reported that the increase of initial lactose concentration achieved at high temperature does not influence GOS yield using β-galactosidase from *Pyrococcus furiosus*. Another problem of carrying out GOS synthesis at high temperature is the occurrence of Maillard reaction and enzyme inactivation. Bruins et al. (2003) [95] noted that in addition to enzyme inactivation with the increase of temperature (80°C or above), Maillard reactions almost doubled the rate of enzyme inactivation. Therefore, the development of new thermostable enzymes, through recombinant DNA technology, has been undertaken in order to improve the GOS yield *figure\_5*

[98-102]. Hansson et al (2001) [103] verified an increase of GOS yield due to an increase of transgalactosylation/hydrolysis ratio by changing a phenylalanine residue to tyrosine in βglucosidase from *Pyrococcus furiosus*, using site directed mutagenesis.

Another strategy to decrease water activity, and carry out catalysis with both high lactose concentration and temperature, demonstrated by Maugard *et al.* (2003) [104], is the use of microwave irradiation. GOS was produced using immobilized β-galactosidase from *Kluyver‐ omyces lactis* along with organic solvents. In these conditions the selectivity for GOS synthesis was increased 217-fold, compared to a reaction carried out under conventional heating.

Similarly to temperature, pH value can affect the GOS yield, possibly through the control of synthesis and degradation [105] According to Huber *et al.* (1976) [106], that studied β-galac‐ tosidase from *Escherichia coli* K-12, higher pH values than 7.8 increased transgalactosylation/ hydrolysis ratio, which decreased at lower pH values than 6.0. In contrast, Hsu *et al.* (2006) [107] observed that β-galactosidase from *Bifidobacterium longum* CCRC 15708 exhibits its maximum activity at pH 7.0. This enzyme was stable between pH 6.5-7.0, and after three hours in these conditions, 20% of its activity was lost.

In general, oligosaccharides, including galactooligosaccharides, are produced using sucrose or starch, whey, among other substrates with high quality and low cost. The process designed to convert raw material into oligosaccharides must be inexpensive and focused on increasing the productivity and stability of enzymes. In this context, immobilization of biocatalysts can reduce the process costs due to some advantages; such as possibility to reuse the biocatalyst, applying a series of batchwise or continuous reactions; the biocatalyst can exhibit more stability than the native counterpart; besides this, immobilization can reduce costs of downstream, since separation of the biocatalyst from the product can be minimized [108-110]. Recently, several authors have employed immobilized β-galactosidase to produce GOS, applying different strategies with promising results [111-114]. Urrutia et al (2013) [111] immobilized *Bacillus circulans* β-galactosidase in glyoxyl agarose. The enzyme did not lose the synthetic capacity, and retained 92% of its activity along 10 reaction batches, producing 1956 g GOS/g protein at the end of 10 batches. Palai et al (2014) [112] immobilized β-galactosidase in hydrophobic polyvinylidene fluoride and the reaction for GOS production was carried out with partial recirculation loop. Both GOS concentration and selectivity for GOS production increased with increasing initial lactose concentrations, with maximum GOS production of 30% at 50°C, and feed flow rate of 0.5 mL/min. A novel economic and efficient method to produce GOS through cellulose-binding fusion β-galactosidase was developed by Lu et al (2012) [113]. A fusion protein, formed by β-galactosidase from *Lactobacillus bulgaricus* L3 and a cellulose binding domain were employed for immobilization by adsorption onto microcrystalline cellulose. The immobilization was conducted with efficiency of 61% and the maximum GOS yield was 49% (w/w). Moreover, enzymatic activity of 85% and yield over 40% (w/w) were maintained after twenty batches. Warmerdam *et al.* (2014) [114] carried out GOS production in a packed-bed reactor using commercial β-galactosidase (Biolacta N5) immobilized on Eupergit C250L. GOS productivity was six-fold higher in one run in the packed-bed reactor than observed in one run in a batch reactor.

Production of GOS can be improved increasing the reaction temperature. Lactose has relatively low solubility at room temperature, which increases with increasing temperature. Therefore high temperatures are desirable since they allow the increase of lactose concentration [94,95]. Besides the possibility to increase the solubility of subtracts and products, high temperature is advantageous due to the reduced risk of microbial contamination, lower viscosity and improved transfer rates [96]. However, this is not a general rule, Boon et al. (1998) [97] reported that the increase of initial lactose concentration achieved at high temperature does not influence GOS yield using β-galactosidase from *Pyrococcus furiosus*. Another problem of carrying out GOS synthesis at high temperature is the occurrence of Maillard reaction and enzyme inactivation. Bruins et al. (2003) [95] noted that in addition to enzyme inactivation with the increase of temperature (80°C or above), Maillard reactions almost doubled the rate of enzyme inactivation. Therefore, the development of new thermostable enzymes, through recombinant DNA technology, has been undertaken in order to improve the GOS yield

philic acceptor (Y), which can be all the sugars present in the reaction media, resulting in GOS mixtures.

40 Food Production and Industry

**Figure 5.** Enzymatic synthesis of GOS by transgalactosylation reactions. Transgalactosylation is the transfer of the gal‐ actosyl residue, after the cleavage of lactose, to an acceptor molecule containing a hydroxyl group. When the acceptor is water (A), a galactose is formed by lactose hydrolysis, whereas if the acceptor is a sugar, a disaccharide or a GOS may be formed. In intramolecular transgalactosylation (B), galactosyl donor and acceptor are the same (glucose), only linkage position changes. In intermolecular transgalactosylation (C), there is an enzymatic transfer to another nucleo‐

*figure\_5*

Smart polymers have been studied to develop GOS production processes. Poly-N-isopropyl acrylamide is a thermo-responsive poly-N-isopropyl acrylamide (PNIPAAm), which presents good solubility in water and distinct phase transition at its lower critical solution temperature (LCST). It is applied in different areas, such as medicine, biotechnology, and engineering [115,116]. Based on these advantages, Palai et al (2014) [117] developed a useful bioconjugate between PNIPAAm and β-galactosidase. The constructed PNIPAAm-β-galactosidase (PNbG) can be used in catalysis and, after that; it can be easily separated from the solution by heating at a temperature above its LCST. Further on, Palai et al (2015) [118] continued the GOS production research using this bioconjugate. A maximum GOS yield of 35 % was obtained at pH 6 and 40°C. An increase in GOS yield was observed when the temperature was risen from 30 to 40°C. At 45°C or above, after prolonged time, enzyme deactivation occurred. Moreover, bioconjugates could be reutilized at least ten times; and the separation was done by simple decantation after addition of 0.05 M NaCl and heating at 40°C.

The use of resting or living cells for GOS production appears to be interesting due to its low cost when compared to the use of purified enzyme. Despite the complexity of biocatalysis processes involving whole cells, glucose and galactose can be consumed by them. The consumption of the monosaccharides is interesting because their presence in foods is unde‐ sirable, since they do not exhibit prebiotic effect, increase caloric value of food, and can inhibit the activity of certain β-galactosidases [119].

Nevertheless, the use of whole cells can be exploited in order to selectively improve GOS production [120]. Beta-galactosidase form *Aspergillus oryzae* was used to produce GOS from lactose, followed by fermentation with *Kluyveromyces marxianus* cells, that consumed mono and disaccharides. GOS with 95% purity containing mostly tri- and tetrasaccharides were obtained [120]. Association of β-galactosidase and cells can be applied to develop GOS enriched food products. During yogurt manufacturing, GOS was produced by addition of a commercial β-galactosidase, since starter and probiotic culture were not able to provide it. Thus, this yogurt with low lactose content can be useful for lactose intolerant people. Moreover, GOS was stable during storage, probably because it was not metabolized by microbial culture and enzyme was inactivated by yogurt pH [121].

Products containing GOS were launched for the first time in Japan in the 1980s. Due to their various and important health benefits, applications of GOS gradually increased worldwide. These oligosaccharides can be found in diverse products such as yogurt, bakery products, beverages, snack bars among others [122]. GOS are able to stimulate the growth of bifidobac‐ teria and lactobacilli in the lumen despite other members of the microbiota that were consid‐ ered potentially harmful. These oligosaccharides can prevent bacterial adherence due to their properties of mimicking host cell receptors in which bacterial adhesion occurs [123]. GOS can hinder the development of colon cancer, effect which can be attributed to their capacity of delaying fermentation processes, and reducing the activity of genotoxic bacterial enzymes associated with this disease [124]. Mineral absorption can be stimulated by GOS administra‐ tion, and their effect on calcium absorption was verified. GOS can be used to alleviate constipation, which is relatively common in elderly people and pregnant women. It occurs due to increased bacterial growth and fecal weight; besides this, short fatty acids stimulate Biotechnological Production of Oligosaccharides — Applications in the Food Industry http://dx.doi.org/10.5772/60934 43

**Figure 6.** Structures of chitooligosaccharides (n= 3-7), A – Chitin (β-1,4-linked N-acetyl-D-glucosamine residues); B – Chitosan (β-1,4-linked D-glucosamine polymer).

intestinal peristalsis and increase osmotic pressure of fecal weight. Moreover, GOS have been reported as indirectly acting on mucosal and systemic immune activity, and also as having protective effects against allergic manifestations [125].
